Research Center Borstel Leibniz-Center for Medicine and Biosciences Priority Area Infections Program Director: Prof. Dr. Ulrich Schaible Cellular Microbiology Group Mycobacterium tuberculosis - Host-Cell Interactions in the Phagosome Dissertation for Fulfillment of Requirements for the Doctoral Degree of the University of Lübeck from the Department of Natural Sciences Submitted by Anna Christina Geffken from Büschelskamp/Scheeßel
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maturation and signal for the production of pro-inflammatory cytokine secretion [63].
1.3.4 The cell biology of phagocytosis in professional phagocytes
Phagocytosis of particles such as pathogens is the exclusive effector mechanism of
macrophages, neutrophils and DCs. As explained in detail above, phagocytic
receptors tether bacteria as M. tuberculosis to the phagosomal membrane and initiate
uptake. Overall the process of phagocytosis can be divided into three main steps: (i)
particle recognition, (ii) particle internalization and (iii) maturation of the particle-
containing phagosome to the phago-lysosome. Several PRRs engage in the process
of particle binding and thus multiple signaling cascades are triggered concomitantly.
Introduction
19
Herein, the process of FcγR-mediated phagocytosis is representatively explained in
greater detail since it is the best understood model of phagocytosis.
In order to trigger phagocytosis, FcγR have to sense and bind their ligand. Onwards
several receptors must cluster to elicit cellular responses and particle uptake [64]. This
spatial convergence brings together the cytosolic ITAM domains of the receptors.
Subsequently, Src-family kinases (SFK) phosphorylate the ITAM motifs.
Figure 1.4: Signalling events leading to actin polymerization during FcγR–mediated phagocytosis. Modified from [36]. See text for details.
The spleen tyrosine kinase (Syk) is able to bind the double phosphorylated ITAM-motif
and recruits several additional signaling proteins [65]: First, the linker of activated T
cells (LAT) is phosphorylated by Syk inducing docking of Grb2 and in turn Gab2 [66].
Additionally Gab2 is recruited to the receptor-complex by its interaction with
phosphatidylinositol-3,4,5-triphosphat (PI(3,4,5)P3), which is an important membrane-
lipid in the cytoplasmic lipid bilayer leaflet around receptor clusters. Phosphorylation
of Syk also recruits the CrkII adaptor proteins to the phagocytic cup important for
downstream signaling of FcγR [67].
At this time point after particle binding, lipids have an important role in orchestrating
phagocytosis. On the inner leaflet of the phagosomal membrane, phosphatidylinositol-
4,5-bisphosphat (PI(4,5)P2) is involved in pseudopod generation to form the
phagocytic cup [68]. Together with other anionic phospholipids such as
Introduction
20
phosphatidylserine (PS) and PI(3,4)P2, it creates a negative charge at the inner leaflet
of the plasma membrane. This attracts phosphatidyl-kinases such as PI4P-5K which
is essential for the lipid-homeostasis. Shortly after the transient synthesis of PI(4,5)P2,
it disappears allowing particle internalization probably by actin disassembly [69]. The
decrease of PI(4,5)P2 is mediated by PI-specific phospholipase Cγ and PI-3-kinase,
which are recruited to the phagocytic cup and hydrolyze PI(4,5)P2 to diacylglycerol
(DAG) or phosphorylate it to PI(3,4,5)P3 respectively [71],[73]. To further support
phagocytic cup formation, in addition phospholipase D is attracted to hydrolyze
phosphatidylcholine (PC) to phosphatidic acid (PA). With its cone shape, PA promotes
curvature of the membrane.
The formation of pseudopods and the phagocytic cup depend on the correct
orchestration of actin assembly and disassembly. This is mediated by small GTPases
of the Rho family. During phagocytosis these comprise Cdc42 and Rac1/2 which
stimulate formation of filopodia and lamellopodia, respectively [71]. Both cellular
protrusions are important for the formation of the phagocytic cup and the nascent
phagosome. Additionally the GTPase ARF6 delivers endosome membranes to
nascent phagosomes. Downstream of these GTPases, the assembly and disassembly
of (branched) actin filaments at the phagocytic cup is controlled by actin binding
proteins (APBs) and nucleation-promoting-factors (NPFs). Here the Arp2/3 complex
and the NPFs Wiskott-Aldrich syndrome proteins (WASP)/N-WASP or Scar/WAVE-
family proteins have an important role. Together with PI(4,5)P2, Cdc42 activates the
NPFs, which in turn activate Arp2/3 to promote actin assembly [72]. Furthermore,
several proteins such as myosins interact with actin filaments during phagocytosis. It
has been shown that myosin II, IXb and IC are important for particle engulfment and
that myosin X has a role in phagosome formation [73].
Introduction
21
Figure 1.5: Stages of phagosome maturation. Modified from [36]. After sealing of the nascent phagosome, maturation starts immediately. Through highly orchestrated fusion and fission events with early (EE), intermediate (IE), late (LE) and lysosomes (LY), the phago-lysosome matures. The phago-lysosome is a hostile organelle with a low pH of 4.5 containing hydrolytic enzymes as cathepsins, AMPs and reactive oxygen- and reactive nitrogen-intermediates. Specific phago-lysosomal markers are LAMP1 and LAMP2, high contents of the v-H+-ATPase and the absence of early endosome markers such as Rab5 or EEA1. Phagosomes of M. tuberculosis (Mtb) pause at an early phagosomal stage.
Introduction
22
After scission from the plasma membrane, the nascent phagosome interacts with early
endosomes thereby initiating phagosome maturation. These membrane fusion events
are mediated by Rab GTPases. Vps34 is targeted to Rab5-positive membranes via
Vps15 and catalyzes the conversion of PI to PI(3)P which is essential for progression
to the late phagosomal stage [74]. PI(3)P together with Rab5 recruit EEA1. The latter
has a fusion-promoting function as it interacts with syntaxin13, a soluble N-
ethylmaleimide-sensitive factor attachment protein (SNARE). SNARE-proteins are
universal mediators of membrane fusion. They form complexes composed of R-
SNARE (localized at donor membranes as early endosomes) and Q-SNARE (localized
at acceptor membranes as early phagosomes) proteins. These hairpin-like protein
complexes bring donor- and acceptor membranes in close proximity, thereby reducing
the free energy barrier for membrane fusion [36].
Despite fusion with early endosomes, the volume of the early phagosome remains
constant. This is due to the fission of endosomes recycling from phagosomes to e.g.
retrieve cell-surface proteins as the transferrin-receptor (TfR) back to the plasma
membrane. Endosomal fission from phagosomes is mediated by an elaborate network
of signaling, budding and tubulating components as Rab4, Rab11, Eps15, the hetero-
oligomeric complex (COPI) and the retromer complex [36]. Another mechanism
maintaining phagosome size is the formation of intraluminal vesicles (ILVs). At the
intermediary phagosomal stage ILVs develop via invagination and pinching of the
phagosomal membrane with the help of the endosomal sorting complex for transport
(ESCRT) machinery and PI(3)P binding proteins Hrs and SNX3 [75].
With ongoing fusion and fission of the phagosome with intermediary and late
endosomes, phagosome maturation proceeds. At the late stage, the phagosome
differs significantly from its early stage. It loses early markers such as Rab5, gains late
markers like Rab7, acidifies and acquires acid hydrolases. The exchange between
Rab5 and Rab7 is an essential step towards maturation. Simultaneously, late
phagosomes acquire the lysosome-associated membrane proteins 1 and 2
(LAMP1/2). Apart from providing membrane integrity and protection against
membrane-active enzymes, LAMP1 and 2 were recently shown to be important for
phagosome maturation by recruiting Rab7. Another characteristic of late phagosomes
is the presence of the unique lipid lysobisphosphatidic acid (LBPA) inside ILVs.
Furthermore, acidification of the maturing phagosome is a consequence of the
constant accumulation of proton (H+) pumps as the vacuolar ATPase (v-H+-ATPase).
Introduction
23
This multimeric protein complex translocates H+ across the phagosomal membrane at
the expense of ATP lowering the early pH of 6.3 to 4.5 [36].
Finally, the late phagosome fuses with lysosomes to form phago-lysosomes. This final
fusion event is, in part, coordinated by the formation of a specific SNARE complex
made of syntaxin7 and VAMP7. The phago-lysosome is the intracellular compartment
that is specifically designed to digest proteins, lipid-membranes, carbohydrates and
thus also microbes [36]. The v-H+-ATPase-mediated acidification of the late
phagosome starts a well-defined process of activation of microbicidal effectors. Due
to the low pH, microbial growth is inhibited, the enzymatic activity of proteases such
as cathepsin B, D and L is optimized at pH of 4.5 and the natural-resistance-associated
macrophage protein1 (NRAMP) is recruited. NRAMP is able to export metal ions such
as iron (Fe3+) from the phago-lysosome to sequestrate ions essential for growth of
intra-phagosomal bacteria [76]. The phago-lysosome harbors several bactericidal
peptides and proteins: lysozyme is able to hydrolyze peptidoglycan; the main structural
components of bacterial cell-walls. Furthermore, cationic antimicrobial peptides
(cAMPs) interact with anionic bacterial membranes and generate pores allowing the
diffusion of ions across the cell-wall. With high proton concentrations and the activation
by pro-inflammatory cytokines as IFN-γ, the macrophage is also capable of producing
reactive oxygen intermediates (ROI) by the NADPH oxidase (NOX2) and reactive
nitrogen intermediates (RNI) by nitrous oxide synthase 2 (iNOS) (Figure 1.6). ROI and
RNI contribute to the elimination of pathogens in phago-lysosomes by damaging
proteins, lipids and DNA/RNA.
Figure 1.6: Production of ROI and RNI in the phagosome. Modified from [77]. The membrane-standing proteins iNOS and NOX2 catalyse the production of NO-and O2-radicals from oxygen. These highly reactive compounds further react to form peroxinitrite (ONOO-) or nitrogen dioxide radicals (NO2
-) and hydrogen peroxide (H2O2), respectively. All compounds are able to damage proteins, lipids, DNA and RNA.
Introduction
24
1.4 Host-pathogen interactions in the phagosome
As outlined, M. tuberculosis infects the host by engaging phagocytic receptors to enter
professional phagocytes as macrophages to abuse as host-cells. After invasion, the
pathogen ensures survival and colonization by deviating the microbicidal mechanism
of phagosome maturation. For that purpose, M. tuberculosis expresses an array of
virulence factors. The main reservoir of M. tuberculosis virulence factors is their unique
and highly complex cell-wall. Here lipids and proteins synergize to disturb the
macrophage microbicidal properties. This chapter gives a detailed overview about the
secreted virulence factors as well as the components of the mycobacterial cell-wall
and, as far as known, their molecular function in virulence.
Per definition, virulence factors are peptides, proteins or lipids of pathogens, whose
inactivation leads to a significant loss in pathogenicity or virulence but fails to impair
the bacterial growth in standard growing media [77]. This criterion comprises a very
large spectrum of candidates including the genes and proteins required for expression,
transport and positioning of virulence factors. In the present work, the focus lies on
lipids that are at the molecular forefront of direct interaction with the host-cell.
The reference laboratory strain M. tuberculosis H37Rv harbors 14 regions of difference
in its genome (RD 1-14) which are absent in the vaccine strain M. bovis BCG and are
thought to be related to pathogenicity. Together with 6 regions termed H37Rv deletion
1-5 (RvD1-5) and the M. tuberculosis specific deletion (TbD1), these regions are
believed to code for the virulence factors of the MTBC. However, the virulence-
associated genes of M. tuberculosis are not classically concentrated on pathogenicity
islands as in other bacteria such as Salmonella but rather are widely distributed
throughout the genome.
1.4.1 The role of cell envelope and secreted proteins in M. tuberculosis
virulence
Proteomic studies of the mycobacterial cell envelope revealed more than 500 proteins
[77]. Most of them are thought to be important for cell-wall homeostasis but 5 % might
have a role in virulence. Cell-wall proteins include the outer membrane proteins
(OMPs) localized in the mycobacterial outer membrane (MOM), cell-wall-associated
or secreted lipo- and glycoproteins.
The delivery of virulence proteins across the cell envelope during infection of host-
cells is mediated via specialized type 1-4 secretion systems (T1-4SS) [78]. The
Introduction
25
genome of M. tuberculosis encodes for at least four types of secretion systems but
only T2SS and the Mycobacteria specific T7SS have a role in virulence. The ESAT6
secretion system 1 (ESX-1) is a specialized T7SS for secretion of virulence relevant
proteins ESAT6 (ESXA), CPF10 (ESXB), EspA-D and EspR. ESAT6 and CFP10,
encoded on RD1 and indispensable for virulence of M. tuberculosis, are small proteins
of 9 kDa and 10 kDa respectively, which form heterodimers [78]. Their virulence
function has been linked to M. tuberculosis escape from the phagosome into the
cytoplasm and spread to uninfected cells as well as inhibition of apoptosis [79].
OMPs are MOM-associated and therefore can directly interact with the host-cell [77].
Lipo- and glycoproteins are exported into the cell-wall or the host-cell cytosol via T2SS
or general Sec secretory pathways. The fibronectin binding proteins are a complex of
three proteins (FbpA-C), better known as the antigen 85 complex (Ag85). Besides
being a mycolic acid transferase, this complex is the major secreted protein of
Mycobacteria and has been shown to mediate the adhesion of the pathogen to
fibronectin on mucosal surfaces, thereby facilitating entry to the host. The six Mce
(mammalian cell entry) proteins are secreted or surface exposed. It has been shown
that Mce1 supports entry of mycobacterial pathogens into mammalian cells and
survival inside macrophages [80]. Adhesion of M. tuberculosis to epithelial cells is also
accomplished via HbhA (heparin-binding protein). HbhA tethers Mycobacteria to
epithelial cells and promotes bacterial aggregation and primary biofilm formation [81].
Furthermore, the 15 kDa lipoprotein was shown to interact with TLR2 possibly
regulating immune responses in favor of mycobacterial survival in the phagosome.
The 27 kDa lipoprotein AG P27 (LprG) has been shown to have a role in infection by
suppression of the host immune response and to bind DC-SIGN mediating adhesion
[77].
M. tuberculosis harbours also three cell-wall associated secretory glycoproteins: the
MPT83 antigen, the 45-47-kDa alanine-proline-rich antigen (Apa, Rv1860) and LqpH
(19 kDa antigen). All three are believed to be potential adhesins and thus have role in
host-cell attachment. Additionally LqpH binds MR, is able to inhibit antigen
presentation of MHC class II molecules in a TLR2-dependent manner and induces
apoptosis for the cell to cell spread of the pathogen [82]. Furthermore, Psts-1 (38 kDa
glycoprotein) was also shown be secreted and to interact with TLR2 and TLR4
resulting in induction pro-inflammatory cytokines [77].
Introduction
26
Pathogens of the MTBC harbor several secreted virulence proteins which increase the
resistance to host toxic compounds as ROI and RNI, arrest phagosome maturation
and prevent apoptosis. M. tuberculosis employs at least six proteins that are secreted
into the phagosomal lumen via SecA2 to directly interfering with ROI and RNI. SodC,
KatG, AhpC, TpX, Mel2 and putatively Acr2 have superoxide dismutase activity that
either inhibits production of O2 radicals or detoxifies H2O2. Further, M. tuberculosis
possesses several proteins suggested to be involved in inhibition of phagosome
maturation. These include Ndk, PtpA and PE_PGRS30. Ndk was shown to inhibit
recruitment of Rab7-GTP and Rab5-GTP to phagosomes and inactivate their function
by dephosphorylation [77]. Furthermore the phosphatase PtpA dephosphorylates
VPS33B, a host protein involved in regulation of membrane fusion. PtpA also binds to
the v-H+-ATPase machinery, thereby inhibiting luminal acidification. The detailed role
of PE_PGRS30 is unknown, although its deletion renders mutants unable to inhibit to
phago-lysosome fusion [77]. Recently the secreted acid phosphatase SapM was found
to dephosphorylate PIP(3)P, that has an essential role in phagosome maturation [83].
Inhibition of apoptosis is another strategy to survive inside professional phagocytes
since the programmed cell death is one of the major mechanisms of the innate immune
response to contain the spread of pathogens. Virulent Mycobacteria modulate host-
cell death by switching from apoptosis to necrosis. This is achieved by controlling the
production of ROI and RNI-mediated apoptosis by NuoG, SecA2/SodA, Rv3600-3653c
and protein kinase PknE.
1.4.2 The role of cell envelope lipids in M. tuberculosis virulence
The mycobacterial cell envelope is composed of three layers: (i) the outermost layer
composed of the capsule and the mycobacterial outer membrane (MOM), (ii) the cell-
wall core composed of arabinogalactan (AG) covalently linked to peptidoglycan (PG)
and (iii) the plasma membrane (PM) (Figure 1.7). The mycobacterial cell-wall
contributes to virulence also by being highly impermeable impeding entry of toxic
molecules as antibiotics.
Introduction
27
Figure 1.7: Schematic representation of the composition of the mycobacterial cell-wall. Modified from [84]. The mycobacterial cell-wall is partitioned in three layers. The capsule constitutes the outermost surface, followed by the mycobacterial outer membrane (MOM) or mycolic acid layer. Peptidoglycan (PG) and arabinogalactan (AG) build the core of the cell-wall. The plasma membrane or cell membrane (CM) surrounds the lumen of the pathogen.
(i) The plasma membrane (PM) is an asymmetric bilayer composed of phospholipids
as phosphatidylglycerol (PG), P2G, phosphatidylethanolamine (PE) and proteins.
Mannosylated lipoarabinomannan (ManLAM) and phosphatidyl-inositol mannosides
(PIMs) are anchored here via their phosphatidyl-myo-inositol residue.
(ii) The cell-wall core is composed of arabinogalactan (AG) and peptidoglycan (PG).
AG is a polymer made of arabinose and galactose monosaccharides that tethers the
mycolic acid layer to peptidoglycan (PG). The PG polymer is orientated orthogonal to
the plasma membrane and consists of sugars as N-acetyl-α-D-glucosamine and
modified muramic acid and the amino acids L-alanyl-D-isoglutaminyl-meso-
diaminopimelyl-D-alanine (L-ala-D-glu-A2pm-D-Ala) similar to Gram-positive and
Gram-negative bacteria. The peptide chain can be cross linked to the peptide chain of
another strand forming the 3D mesh-like layer [23]. The cell-wall core forms a rigid
layer outside the PM providing cellular shape and strength as well as scaffold for AG
and the MOM.
(iii) The mycobacterial outer membrane (MOM) is composed of a bilayer membrane of
Figure 1.8: Acyltrehaloses TMM and TDM of the mycobacterial outer membrane. Modified from [82]. TMM and TDM are made of a headgroup of the sugar trehalose that can be esterified at position 6 and/or 6´ to one or two mycolic acids, respectively.
Introduction
29
The exact function of SL in mycobacterial virulence, in particular in intracellular
trafficking of Mycobacteria, remains to be fully understood. However, DAT, TAT, PAT
lipids, although non-essential for mycobacterial viability in vitro, probably contribute to
the properties of the cell surface and the permeability barrier formed by the cell
envelope [77].
TDM, the most abundant lipid in the mycobacterial cell-wall, is composed of trehalose
esterified with one (TMM, precursor of TDM) or two (TDM) mycolic acids at position 6
and/or 6’ (Trehalose-6,6-dimycolate) (Figure 1.8). Mycolic acids are long-chain fatty
acids, α-branched and β-hydroxylated with different chain lengths (from 60 to 90
carbon atoms in Mycobacteria) and chemical functional groups, such as double bonds,
cyclopropanes or oxygenated functions located at two defined positions of the
meromycolic chain [87]. TDM is also referred to as “cord factor” since its presence
alters the colony morphology to rope-like forms [23]. Alike ManLAM, TDM has various
functions relevant for M. tuberculosis early in infection as well as in granuloma
formation [89]. TDM has been proposed to be the main glycolipid interfering with
phagosome maturation. In 2003, Indrigo et al. showed that TDM restores virulence
function of delipidated Mycobacteria [90]. Moreover beads coated with TDM are able
to delay phagosome maturation and retain a close proximity to the phagosomal
membrane, prerequisite to inhibit phagosome maturation [91]. However, in IFN-γ-
activated macrophages, the inhibitory effect of TDM on phagosome maturation is
abolished by NO [91]. Furthermore, TDM alone is sufficient to induce granuloma
formation when intravenously injected on oil droplet formulations into mice. TDM-
mediated granuloma formation is mediated by Mincle. Engagement of Mincle activates
the Syk-Card9 signalling pathway in macrophages, which is required for activation of
macrophages in vitro and for granuloma formation in vivo following injection [92].
However, the exact molecular function of TDM remains unknown.
The mycerosate-containing lipids are phtiocerol dimycerosates (PDIM) and phenolic
glycolipids (PGL). Besides the importance of PDIM in multiplication of M. tuberculosis,
the lipid has been shown to inhibit secretion of pro-inflammatory cytokines and was
proposed to inhibit phagosome maturation by incorporating into host membranes
disturbing their organization [36],[93]. Also, cell surface associated PDIM were shown
to mask underlying PAMPs avoiding activation of pro-inflammatory processes [94].
PGL from M. marinum has been found to inhibit maturation of bead phagosomes [95].
Introduction
30
(iv) The capsule is a loosely-bound, non-covalently linked structure on the outermost
compartment of the cell. In M. tuberculosis it is composed of proteins, such as porins
for nutrient uptake, oligosaccharides and only small amounts of lipids such as PIMs
[82]. Capsular components are prone to release upon contact with the host-cell or
within the phagosome. The oligosaccharides comprise α-D-glucan, D-arabino-D-
mannan and D-mannan and were described to mediate phagocytosis via non-opsonic
binding to CR3 [82].
1.5 Objectives
The aim of this PhD-thesis was to investigate how TDM exerts its virulence function
inside the phagosome of macrophages. We hypothesize that TDM manipulates host-
cell biology through interaction with targets at the phagosome interface, in order to
inhibit phagosome maturation. To identify putative interaction partners, the lipid-coated
bead model should be used. Therefore, the following aims had to be accomplished:
i. establishment of a protocol for isolation and purification of magnetic lipid-
coated bead phagosomes from macrophages suitable for mass spectrometry
based proteomic and lipidomic analysis,
ii. analysis of control and TDM bead phagosomes proteomes and lipidomes to
identify potential host-cell-derived direct or indirect interaction partners,
iii. evaluation of selected potential host-cell targets of TDM for their role in TDM-
and M. tuberculosis-mediated inhibition of phagosome maturation and
intracellular survival.
Material and Methods
31
2 Material and Methods
2.1 Material
2.1.1 Consumables
Table 2-1: Consumables
Designation Manufacturer
75 cm2 flask Corning
5 ml screw top vial Chromacol
Dynabeads M-280 tosylactivated Life Technologies
50 ml tube, blue screw cap Greiner Bio-One
15 ml centrifuge tubes Corning
Filtropur S plus 0.2 Sarstedt
1000 µl pipette tips Sarstedt
200 µl pipette tips Sarstedt
10 µl pipette tips Molecular Bio Products
25 ml stripette Costar Corning
10 ml stripette Costar Corning
5 ml stripette Costar Corning
1 ml stripette Costar Corning
25 cm cell scraper Sarstedt
Syringe, plastipak 1 ml Becton Dickinson
Canula, microlance 3, 23 G1, Nr. 16 Becton Dickinson
FACS tube, 5 ml Becton Dickinson
1.5 ml, 2 ml reaction-tube Sarstedt
96-well plate, flat bottom Corning
2 ml protein lobind tube Eppendorf
24-well plate, flat bottom Corning
4-10 % criterion-TGX-precast gel Biorad
LC-vial 100 µl Chromacol
Coverslips, 10 mm, round VWR
Canula, microlance 3, 37G 3/4, Nr. 20 Becton Dickinson
acetatel, 2-propanol). For flow injection analysis, 20 µl of the mixture were injected into
the ESI Qq-TOF-MS via an Agilent 1100 HPLC system. The solvent used and flow
rates are shown in Table 2-17.
Material and Methods
52
Table 2-17: Flow rates used for the analysis of cholesteryl acetate by ESI Qq-TOF-MS
Time [min] Flow [µl/min] Pressure [bar]
0 5 400
8 5 400
8.01 20 400
Solvent: MS-mix
Source temperature was set to 150 °C and instrumental parameters were optimized
prior analysis following recommendations of the manufacturer. PRM scans of m/z 446
and m/z 443 were performed using 1 Da isolations width and CID voltages of 10 V and
10.3 V for Chol-D7 and Chol, respectively.
2.2.4.6 Data processing and analysis
µLC-FT-ICR-MS data files were processed using Data Analysis 4.0 software. Mass
spectra were averaged in the retention time ranges of lipid classes of interest (defined
by the elution time of the internal standards). Each mass spectrum was smoothed,
baseline subtracted and peaks lists were extracted (m/z values and corresponding
intensities). Lipids were identified by their monoisotopic masses with an accuracy
better 16 ppm (LBPA) and 3 ppm (all others) using LipidXplorer [97] and their
respective retention time window.
Intensity ratios of lipid species and their corresponding internal standard were used for
quantification of lipids in the sample. All further calculations were performed with
Microsoft Excel 2010. First samples were grouped to BSA (control), TDM and negative
controls. Lipids with intensities higher than 10 % in negative controls compared to BSA
and TDM samples were deleted. For minimal occupation requirements, lipids had to
be present in at least half of the BSA or half of the TDM samples. To calculate the
amount of lipids in pmol, intensities of identified lipids were divided by the intensity of
the corresponding internal standard and multiplied by the amount of the internal
standard added.
For further analysis, values for all identified species obtained from both experiments
were combined. To standardize all samples to the amount of bead phagosomes used
in the experiment, pmol values were divided by the amount of iron in µM in the
corresponding sample. Then, the amount of all lipids in one sample was summed up.
For calculation of the mol percentages (mol %), the amount of every lipid species in
Material and Methods
53
pmol was divided by the sum of all lipids identified in the samples and multiplied by
100.
ESI Qq-TOF-MS data were processed using the MassLynx 4.0 software. Intensity
values were obtained by manually combining mass spectra observed above half peak
height (spectra #3-59) in PRM scans of m/z 446 and m/z 443, respectively. Intensities
of peaks corresponding to the product ion masses of cholesteryl acetate (m/z 369) and
the deuterated standard (m/z 376) were picked and used for calculations. To calculate
the amount of free cholesterol, the intensity ratio of cholesteryl acetate and the
corresponding deuterated internal standard was used.
2.2.4.7 Statistical analysis and graphic representation
For statistical analysis and graphic representation, the Graph Pad Prism 5 software
was used. For statistical analysis of lipid classes, the mol % of every lipid class was
summarized. Values for every sample pair were analyzed with a paired TTest and
depicted with a before-and-after plot.
For statistical analysis of lipid species, as well, mol % values were used. For every
lipid class and corresponding samples pair, p-values were calculated using a paired
TTest. Further, the ratios between every sample pair were calculated by dividing the
mol % of TDM samples by the mol % of control samples. The mean of all seven p-
values for every lipid species was transformed to a log10-value and multiplied by -1.
The mean of all seven ratios for every lipid species was transformed to a log2-value.
For volcano-plot analysis, the transformed ratios were plotted against the transformed
p-values.
Results
54
3 Results
3.1 Method development: Isolation and purification of bead
phagosomes for mass spectrometry analysis
Inhibition of phagosome maturation by TDM is well established, but the underlying
mechanisms at the phagosomal interface remain unknown [90], [91], [100].
Consequently an experimental system to study the effect of single lipid species on
phagosome maturation and concomitantly to identify potential host-cell interaction
partners was required. In 2008, Axelrod et al. developed a reductionist lipid-coated
bead model [91]. Lipids such as TDM were coated to beads and fed to macrophages
to monitor bead phagosome maturation using stage specific markers.
To achieve the prime aim of this study that is identification of host-cell derived putative
interaction partners, bead phagosome proteomes and lipidomes had to be analyzed
by mass spectrometry. Based on the model system described by Axelrod et al., an
optimized protocol to isolate and purify bead phagosomes from macrophages was
developed.
To identify TDM specific interaction partners, control and TDM bead phagosomes were
isolated and purified for comparative proteomic and lipidomic analysis. Thus, magnetic
beads were coated with BSA (control) and TDM and used for “bead infection” of
macrophages. After 30 min, bead phagosome maturation was stopped by scraping the
cells in ice-cold buffer and disruption using a metal douncer. The disruption process
released un-cleaved DNA which caused formation of aggregated vesicles. Therefore,
as a first purification step, samples were treated with DNase to eliminate DNA. Further,
samples were washed three times using a magnet. Since the bead phagosomes still
stuck together, samples were additionally treated with trypsin. To separate bead
phagosomes from cell debris, a Ficoll gradient was applied. Differences in phagosome
maturation were controlled by measuring lysososmal β-galactosidase activity in the
samples prior continuing the purification procedure. This step had to be performed
prior sorting, because after sorting process bead phagosome concentrations were too
low to yield a signal in the lysososmal β-galactosidase assay. As final step, bead
phagosomes were sorted by FACS to separate bead phagosomes from residual cell
debris. Bead phagosomes were isolated from the FACS supernatant using a magnet
and proteins remaining in the supernatant were precipitated using StrataClean beads.
A scheme of the overall practical procedure for isolation and purification of bead
phagosomes is shown in Figure 3.1.
Results
55
Figure 3.1: Scheme of the procedure for isolation and purification of bead phagosomes from macrophages for mass spectrometry analysis. After coating of magnetic beads with TDM (step 2), macrophages were “bead infected” with control or TDM beads (step 3). Following 30 min of bead phagosome maturation, cells were disrupted using a douncer at 4 °C (step 4). Bead phagosomes were purified by DNase digestion (step 5) and freed from crude cell debris by magnetic isolation (step 6) and further, trypsin (step 7) treatment and Ficoll gradient centrifugation (step 8). Finally bead phagosomes were freed from residual cell debris by sorting by FACS (step 9) and analyzed for their protein content by mass spectrometry. β-galactosidase activity of control and TDM bead phagosomes was determined prior sorting by FACS.
3.1.1 Quality control of purified bead phagosomes
Potential interaction partners were predicted to be found exclusively in or associated
with bead phagosomes that halted a non-mature stage by TDM. Thus TDM mediated
inhibition of bead phagosome maturation had to be tested prior analysis of samples by
mass spectrometry. As mentioned above prior sorting by FACS, β-galactosidase
activity was used to reveal the phagosomal stage. Active β-galactosidase is only found
in late endosomal/lysosomal compartments. Thus, phago-lysosomes contain more
enzymatic activity than early, intermediate or late phagosomes. Consequently, due to
TDM-mediated inhibition of phagosome maturation, TDM bead phagosomes should
have less β-galactosidase activity compared to control ones. The β-galactosidase
activity of all four sample pairs analyzed in this study is shown in Figure 3.2. TDM bead
phagosomes of samples 1 to 4 (A-D), showed less β-galactosidase activity (89.22 %,
87.46 %, 60.85 % and 54.15 %) compared to controls. Although TDM was able to
inhibit maturation of bead phagosomes compared to control ones, the differences
between TDM and control bead phagosomes differed significantly between
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preparations. All four sample pairs were used further purification and mass
spectrometry analysis.
Figure 3.2: β-galactosidase assay of purified control versus TDM bead phagosomes. A-D: Macrophages were “bead infected” with control and TDM beads at a MOI of 10. Bead phagosome maturation was allowed for 30 min. Afterwards, cells were harvested and bead phagosomes were isolated and purified as described in detail in section 2.2.1. Prior to sorting by FACS, 150 µl of control and TDM bead phagosomes in PBS were saved for verification of β-galactosidase activity as described in 2.2.1.7. The β-galactosidase activity was standardized with the number of beads per sample.
3.1.2 Purification of bead phagosomes via sorting by FACS
The efficiency of the final purification step by FACS is shown in Figure 3.3. After
sorting, TDM and control bead phagosome samples were reanalysed for purity and
presence of remaining contaminating cell debris. Representatively, prior sorting, in
control bead isolation number 4 and in TDM bead phagosome isolation number 3,
86.23 % and 93.22 % of all detectable particles were bead phagosomes, respectively.
However, sorting by FACS increased the purity of the samples to 97.72 % and
98.88 %, respectively. Concluding, sorting by FACS was suitable to exclude
contaminants and increase purity of isolated bead phagosomes.
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Figure 3.3: Control and TDM bead phagosomes prior to and after sorting by FACS. Representative fraction of bead phagosomes enriched by a FACSAriaII cell sorter from TDM bead phagosome isolation #3 (lower) and control bead isolation #4 (upper). The Area of Forward (FSC-A) and Side Scatter (SSC-A) was used to determine the bead fraction. Before sorting, the portion of bead was around 93.22 % (TDM) and 86.23 % (control) (left scatter plot), after sorting and re-analyzing the sorted fraction, the enrichment was increased up to 98.22 % and 97.72 % purity of bead phagosomes (right scatter plot), respectively.
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3.2 The TDM bead phagosome proteome
Processing of all sample pairs for mass spectrometry analysis was performed in the
laboratory of our collaboration partners Prof. Dr. Dörte Becher and Dr. Andreas Otto
in Greifswald by Jürgen Bartel as described in detail in 2.2.2. The data obtained that
way, were received as “Scaffold file” and further processed with the “Scaffold 4”
software.
In total 1054 proteins were detected in all four sample pairs (Table 0-1). In control and
TDM bead phagosome samples, 919 and 843 proteins were detected, respectively.
As shown in Figure 3.4, 708 proteins were shared between both groups while 211 and
135 proteins were detected exclusively either in control or TDM bead phagosome
samples, respectively.
Figure 3.4: Intersection diagram of total numbers of identified proteins in proteomes of control versus TDM bead phagosome samples. Four pairs of isolated and purified control and TDM bead phagosomes samples were analyzed for their protein content by mass spectrometry. Results were processed using the “Scaffold 4” software. 1054 proteins were identified in total. 708 proteins were shared between control and TDM bead phagosome proteomes whereas 211 proteins were found exclusively in control and 135 in TDM bead phagosomes, respectively.
Using the Scaffold 4 software, the NCBI annotations were downloaded that provide
current information about each identified protein. Figure 3.5 depicts all proteins
grouped according to their cellular localization according to the NCBI definition.
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Figure 3.5: Identified proteins in control and TDM beads phagosomes grouped according to their cellular localization. Four pairs of isolated and purified control and TDM bead phagosomes samples were analyzed for their protein composition by mass spectrometry. Results were processed using the “Scaffold 4” software. Using the “quantify” mode, all identified proteins were sorted according to their cellular localization. The numbers shown above for each category exceed the total numbers of 1054 identified proteins because some proteins were allocated to several cellular localizations.
Accordingly, 684 and 512 proteins were associated with intracellular organelles and
the organelle part (same as intracellular organelle, term should not be used any more
according NCBI but has not been replaced at the time this thesis was prepared),
respectively. 632 were cytoplasmic proteins. Of the 478 membrane proteins, 294 and
229 were allocated to organelle or plasma membranes, respectively. 288 proteins
were grouped to the nucleus. 173 mitochondrial proteins were discovered. Further, of
the endoplasmic reticulum and the cytoskeleton 123 proteins were enriched whereas
87 proteins were assigned to the Golgi apparatus and the endosome. 21 ribosomal
and 83 extracellular proteins were identified. Finally, 264 proteins had unknown
localization.
Proteins specified with the categories ribosome, extracellular region, Golgi apparatus,
cytoskeleton, ER, mitochondria, nucleus and cytoplasm are potential contaminant
proteins, because per definition of the gene ontology (GO) terms, these groups do not
comprise proteins that localize in intracellular vesicles or their membranes. In contrast,
categories endosome, intracellular organelle, organelle part or membrane and
organelle membrane can contain, per GO term definition, proteins within organized
structure of distinctive morphology and function, occurring within the cell or associated
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with membranes thereof. These categories may be those containing phagosomal or
lysosomal proteins.
Next, the percentage of proteins according to their cellular localization in control versus
TDM bead phagosome was compared. Figure 3.6 shows that there were no major
differences between both samples.
Figure 3.6: Percentage distribution of all proteins identified in control versus TDM bead phagosome samples grouped according to their cellular localization. Four pairs of isolated and purified control and TDM bead phagosome samples were analyzed for their protein content by mass spectrometry. Results were processed using the “Scaffold 4” software. The “quantify” mode of the software sorts all identified proteins according to their cellular localization and calculates the percentages of identified proteins that can be classified to a cellular compartment. The numbers shown above for every category exceed 100 because some proteins have more than one cellular localization.
Identified proteins were quantified by spectral counting. Using the Scaffold 4 software,
the exclusive unique spectrum count (EUSC) mode was selected to assess the
“number of unique spectra associated only with this protein”. For further calculations,
the EUSC of all proteins identified in all four sample pairs was exported from Scaffold
4 to a Microsoft Excel file. In order to find proteins that were more abundant in TDM
bead phagosome samples compared to controls, and thus are potential TDM host-cell
derived interaction partners, first all proteins with an overall EUSC less than 10 were
excluded. For minimal occupation requirements, proteins had to be identified in at least
4 of the 8 control or TDM bead phagosome samples. Subsequently the EUSC for all
proteins identified in the 8 control and TDM bead phagosomes samples were summed
up and the value of all EUSC from TDM phagosomes was divided by the number of
all EUSC from control phagosomes. Proteins with ratios of ≥2 were considered
enriched to TDM bead samples. Thus, the list of TDM specific proteins comprised 34
(Table 3-1). Proteins with ratios of ≤0.5 were considered enriched in control bead
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samples (Table 3-2). Table 3-3 gives an overview about the cellular localization and
biological process of the 34 proteins enriched to TDM bead phagosome samples.
Table 3-1: List of proteins enriched in TDM bead phagosomes compared to controls. 1054 proteins were identified in control and TDM bead phagosomes. To unravel potential host-cell derived TDM interaction partners, all proteins with an overall EUSC less than 10 were excluded. Next, for minimal occupation requirements, proteins had to be identified in at least 4 of 8 control or TDM bead phagosomes samples. Finally the EUSCs of all control and all TDM bead phagosome samples were added and TDM/control ratios were calculated. 34 proteins with ratios ≥2 were considered enriched in TDM bead phagosomes and thus potential interaction partners.
Identified protein Accession number
EUSC control
EUSCTDM
Ratio
Rab22B OS tr|Q3TXV4|Q3TXV4 2 13 6.5
Valine--tRNA ligase sp|Q9Z1Q9|SYVC 2 11 5.5
Vesicle-associated membrane protein 7
sp|P70280|VAMP7 3 15 5.0
Ras-related protein Rab-11A sp|P62492|RB11A 2 9 4.5
Protein 5730469M10Rik tr|Q3U125|Q3U125 3 12 4.0
Annexin A1 sp|P10107|ANXA1 5 17 3.4
Cofilin-1 tr|F8WGL3|F8WGL3 4 13 3.3
Profilin-1 sp|P62962|PROF1 7 22 3.1
Annexin A6 tr|F8WIT2|F8WIT2 8 25 3.1
TOM34 sp|Q9CYG7|TOM34 3 9 3.0
60S ribosomal protein L22 sp|P67984|RL22 7 18 2.6
Vesicle-associated membrane protein 3
sp|P63024|VAMP3 8 20 2.5
Synaptosomal-associated protein 23
tr|Q9D3L3|Q9D3L3 7 17 2.4
Cytochrome c oxidase subunit 4 isoform 1, mitochondrial
sp|P19783|COX41 5 12 2.4
Protein ERGIC-53 sp|Q9D0F3|LMAN1 5 12 2.4
Voltage-dependent anion-selective channel protein 3
sp|Q60931|VDAC3 5 12 2.4
MKIAA1699 protein (Fragment) tr|Q69ZD1|Q69ZD1 5 12 2.4
Ras-related protein Rab-8A sp|P55258|RAB8A 12 26 2.2
Lysosomal protective protein tr|G3X8T3|G3X8T3 6 13 2.2
Ras-related protein Rab-10 sp|P61027|RAB10 6 13 2.2
Nucleolar protein 58 sp|Q6DFW4|NOP58 6 13 2.2
Histone H4 sp|P62806|H4 13 28 2.2
Uncharacterized protein tr|E9PZV5|E9PZV5 11 22 2.0
YLP motif-containing protein 1 tr|D3YWX2|D3YWX2
6 12 2.0
Major facilitator superfamily domain-containing protein 1
sp|Q9DC37|MFSD1 4 8 2.0
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Synaptic vesicle membrane protein VAT-1 homolog
sp|Q62465|VAT1 6 12 2.0
ATP-dependent RNA helicase DDX18
sp|Q8K363|DDX18 6 12 2.0
Unconventional myosin-Ie sp|E9Q634|MYO1E 6 12 2.0
V-type proton ATPase subunit F
sp|Q9D1K2|VATF 4 8 2.0
Table 3-2: List of proteins enriched in control bead phagosomes. 1054 proteins were identified in control and TDM bead phagosomes. To unravel potential host-cell derived TDM interaction partners, all proteins with an overall EUSC less than 10 were excluded. Next, for minimal occupation requirements, proteins had to be identified in at least 4 of 8 control or TDM bead phagosomes samples. Finally the EUSCs of all control and all TDM bead phagosome samples were added and TDM/control ratios were calculated. Proteins with ratios ≤0.5 were considered enriched in control bead samples.
Identified protein accession number
EUSC control
EUSC TDM
ratio
Cytochrome b-245 heavy chain sp|Q61093|CY24B 14 7 0.5
Table 3-3: Cellular localization and biological process of proteins enriched to TDM bead over control phagosomes.
Identified protein Cellular compartment
Biological process
Rab22B OS early endosome / late endosome / phagocytic vesicle / trans-Golgi network / membrane
small GTPase mediated signal transduction / protein transport / receptor internalization / Rab protein signal transduction / cellular response to insulin stimulus / Golgi to plasma membrane protein transport / regulated secretory pathway / Golgi vesicle transport / phagosome maturation
Valine--tRNA ligase
mitochondrium valyl-tRNA aminoacylation
VAMP7 Golgi apparatus / ER membrane / late endosome
vesicle-mediated transport/ Golgi to plasma membrane
mitotic cytokinesis / neural crest cell migration / neural fold formation / protein phosphorylation / protein import into nucleus / cytoskeleton organization / actin filament organization / response to virus / regulation of cell morphogenesis / negative regulation of cell size / regulation of dendritic spine morphogenesis / response to amino acid / positive regulation of actin filament depolymerisation /
Profilin-1 cytoplasm / cytoskeleton / extracellular region / nucleus
neural tube closure / sequestering of actin monomers
Annexin A6 cytoplasm / mitochondrium / lysosomal membrane / cytosol / focal adhesion / membrane / late endosome membrane / melanosome / perinuclear region of cytoplasm / extracellular exosome /
calcium ion transport / regulation of muscle contraction / ion transmembrane transport / protein homooligomerization / mitochondrial calcium ion homeostasis / apoptotic signalling pathway
histamine secretion by mast cell / transport / exocytosis / protein transport / synaptic vesicle priming / synaptic vesicle fusion to presynaptic membrane
Cytochrome c oxidase subunit 4 isoform 1, mitochondrial
small GTPase mediated signal transduction / cellular response to insulin stimulus / polarized epithelial cell differentiation / Golgi to plasma membrane protein transport / axonogenesis /
Nucleolar protein 58
cytoplasm/ nucleolus / Cajal body
snRNP protein import into nucleus /
Histone H4 actin cytoskeleton / nucleoplasm /
negative regulation of megakaryocyte differentiation / nucleosome assembly /
3.2.1 Introduction of selected proteins and STRING analysis
To see whether and how the 34 enriched proteins interact, they were analyzed with
the STRING 9.1 online tool. STRING (Search Tool for the Retrieval of Interacting
Genes/Proteins) is a database of direct (physical) and indirect (functional) predicted
protein interactions. One protein could not be identified since its entry in the source
(NCBI entries) used by STRING was deleted.
Figure 3.7 shows the interaction network of 33 of the 34 selected proteins. Identified
proteins are illustrated by dots, identified genes by smaller dots with their
corresponding names. Interaction between proteins is illustrated by colour-coded
connecting lines. In general, more lines indicate more profound interaction. The
arrangement of proteins without known interactions in the network is purely random
and does not correspond to their potential function or the genetic organisation.
Figure 3.7: STRING network of proteins enriched in TDM bead phagosomes. 1054 proteins were identified in control and TDM bead phagosomes. To unravel potential host-cell derived TDM interaction partners, all proteins with an overall EUSC less than 10 were excluded. Next, for minimal occupation requirements, proteins had to be identified in at least 4 of 8 control or TDM bead phagosomes samples. Finally the EUSCs of all control and all TDM bead phagosome samples were added and TDM/control ratios were calculated. 34 proteins with ratios equal or greater than 2 were considered enriched in TDM and thus potential interaction partners. 33 proteins were analyzed regarding their interaction using the online tool STRING 9.1.
The STRING network shows 6 (numbers 1-6) major interacting networks. Network 1
comprises 7 proteins and 1 gene which function in membrane organization and protein
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localization to membranes. Network 2 includes 3 proteins that have a role in protein
localization to organelles and are ribonucleoprotein complex binding proteins. Network
3 is made of 3 proteins that take part in cellular respiration and energy metabolism.
Network 4 comprises 2 proteins that interact with the actin cytoskeleton. Network 5
involves 3 proteins that are known to be important for hemopoiesis. Finally, network 6
includes 2 proteins involved in fatty acid biosynthesis.
Of the 34 potential TDM interacting proteins, 6 were chosen for further analysis
regarding localization and function in trafficking and M. tuberculosis infection in
macrophages using fluorescence microscopy, Western blot and siRNA knock-down
experiments, respectively (Table 3-4). Selection of those 6 proteins was based on 3
criteria, (i) known association with endosomes/phagosomes and/or (ii) component of
the 6 major networks (Figure 3.7) and (iii) availability of appropriate antibodies for
analysis with immunofluorescence microscopy and Western blot.
Selected proteins are listed in Table 3-4. AnnexinA1 and A6 are important for
calcium/phospholipid-binding, which promotes membrane fusion during phagosome
maturation. They were also found on mycobacterial phagosomes and were described
to link actin filaments to the phagosome during phagocytosis [101], [102]. Profilin1 and
cofilin1 are known to regulate the actin cytoskeleton dynamics important for
phagosome biogenesis [103]. The synaptosomal-associated protein 23 (Snap23) was
recently shown to regulate phagosome formation and maturation in macrophages
[104]. VAMP3 is s SNARE-protein involved in vesicle transport to and from endosomes
and phagosomes.
Table 3-4: Identified proteins selected for further analysis as potential host-cell derived TDM interaction partners.
Identified proteins Accession Number EUSC control
EUSC TDM
Ratio
Annexin A1 sp|P10107|ANXA1 5 17 3.4
Annexin A6 tr|F8WIT2|F8WIT2 8 25 3.1
Cofilin-1 tr|F8WGL3|F8WGL3 4 13 3.3
Profilin-1 sp|P62962|PROF1 7 22 3.1
Synaptosomal-associated
protein (SNAP23)
tr|Q9D3L3|Q9D3L3 7 17 2.4
Vesicle-associated
membrane protein 3 (VAMP3)
sp|P63024|VAMP3 8 20 2.5
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3.3 Evaluation of TDM interaction partners
First, we analyzed the intracellular localization of the selected proteins with respect to
bead and M. tuberculosis phagosomes in macrophages. Therefore, RAW264.7
macrophages were incubated with control beads, TDM beads or M. tuberculosis GFP.
Bead phagosome maturation was stopped after 30 min and M. tuberculosis GFP after
2 h. Subsequently, infected macrophages were stained using antibodies against
annexinA1, annexinA6, cofilin1, profilin1, SNAP23 and VAMP3. To determine the
maturation status of bead and M. tuberculosis GFP phagosomes, macrophages were
co-stained for LAMP1. LAMP1 is an integral membrane protein highly abundant in
matured phagosomes, late endosomes and lysosomes [36]. Thus it is commonly used
as a marker for late phago-/endosomes and phago-lysosomes. Infected cells stained
for cofilin1 and profilin1 were additionally stained for actin in order to study co-
localization of actin with the nucleation-promoting-factors (NPFs).
Notably, not all antibodies available could be used for immunofluorescence staining of
RAW264.7 macrophages infected with M. tuberculosis GFP because they cross-
reacted with M. tuberculosis GFP (results not shown). According to information
obtained from the manufacturers, these polyclonal antibodies were generated in
rabbits using adjuvants containing mycobacterial components. Consequently, the
antibody to annexinA1 was not used because it stained free M. tuberculosis GFP.
Specific binding of antibodies was tested by incubating RAW264.7 macrophages and
M. tuberculosis GFP with the secondary antibody alone. This revealed that neither
Alexa405 anti-rabbit, Cy2 anti-rabbit, Cy3 anti-rat nor Cy5 anti-rabbit displayed
unspecific binding to RAW264.7 macrophages or M. tuberculosis GFP.
In all experiments, TDM bead phagosomes showed significantly decreased LAMP1
association compared to controls. Thus, TDM successfully inhibited maturation of
bead phagosomes.
3.3.1 Evaluation of the presence of candidate proteins on control bead, TDM
bead and M. tuberculosis phagosomes by immunofluorescence staining
AnnexinA1: Figure 3.8 shows representative immunofluorescence pictures of control
(A-D) and TDM (E-H) bead phagosomes in RAW264.7 macrophages stained for
annexinA1 (green) and LAMP1 (red). Quantitative analysis showed that the annexinA1
staining pattern for both bead types was similar, neither TDM nor control bead
phagosomes accumulated annexinA1 as assessed by immunofluorescence staining
(Figure 3.8, I).
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Figure 3.8: Immunofluorescence staining of control and TDM bead infected RAW264.7 macrophages for annexinA1 and LAMP1. Macrophages were “bead infected” with control and TDM beads (*) in a MOI of 3. 30 mpi, phagosome maturation was stopped and macrophages were stained using primary antibodies to annexinA1 and LAMP1 followed by secondary fluorescent antibodies to either rabbit or rat IgG, respectively. A & E: Staining pattern of annexinA1 in macrophages with control and TDM bead phagosomes. B & F: Staining pattern of LAMP1 in macrophages with control and TDM bead phagosomes. C & G: Merged images with DAPI stain for nucleus (blue). D & H: Phase contrast. I: Quantitative analysis of annexinA1 and LAMP1 association with control and TDM bead phagosomes. TTest, n=3 biological replicates, p-value * >0.05, ** >0.01, *** >0.001, bar=5 µm
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AnnexinA6: Figure 3.9 shows representative immunofluorescence pictures of control
(A-D), TDM (E-H) bead and M. tuberculosis GFP (I-L) phagosomes in RAW264.7
macrophages stained for annexinA6 (green) and LAMP1 (red). Quantitative analysis
revealed that neither bead nor M. tuberculosis GFP phagosomes accumulated
annexinA6 as assessed by immunofluorescence staining (Figure 3.9, M)
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Figure 3.9: Immunofluorescence staining of control, TDM bead and M. tuberculosis GFP infected RAW264.7 macrophages for annexinA6 and LAMP1. Macrophages were “bead infected” with control and TDM beads (*) in a MOI of 3 or M. tuberculosis GFP with a MOI of 5. 30 mpi and 2 hpi, respectively, phagosome maturation was stopped and macrophages were stained using primary antibodies to annexinA6 and LAMP1 followed by secondary fluorescent antibodies to either rabbit or rat IgG, respectively. A, E & J: Staining pattern of annexinA6 in macrophages with control, TDM bead and M. tuberculosis GFP phagosomes. B, F & K: Staining pattern of LAMP1 in macrophages with control, TDM bead and M. tuberculosis GFP phagosomes. C & G: Merged images with DAPI stain for nucleus (blue). D, H & L: Phase contrast. I: M. tuberculosis GFP. M: Quantitative analysis of annexinA6 and LAMP1 association with control, TDM bead and M. tuberculosis GFP phagosomes. TTest, n=3 biological replicates, p-value * >0.05, **
>0.01, *** >0.001, bar=5 µm
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Cofilin1: Figure 3.10 shows representative immunofluorescence pictures of control
bead (A-D), TDM (E-H) bead and M. tuberculosis (I-L) phagosomes in RAW264.7
macrophages stained for cofilin1 (green) and LAMP1 (red). Quantitative analysis
showed that the cofilin1 staining pattern for both bead types was similar since
punctuate pattern were absent from control and TDM bead phagosomes. In contrast,
phagosomes of M. tuberculosis GFP show a significant punctuate association with
cofilin1 when LAMP1-positive (arrow). Further, TDM bead and M. tuberculosis
phagosomes were significantly associated with actin compared to controls (not
shown). However, actin did not co-localize with cofilin1. In conclusion,
immunofluorescence staining revealed association of cofilin1 only with LAMP1-
positive M. tuberculosis but not with bead phagosomes or actin.
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Figure 3.10: Immunofluorescence staining of control, TDM bead and M. tuberculosis GFP infected RAW264.7 macrophages for cofilin1 and LAMP1. Macrophages were “bead infected” with control and TDM beads (*) in a MOI of 3 or M. tuberculosis GFP with a MOI of 5. 30 mpi and 2 hpi, respectively, phagosome maturation was stopped and macrophages were stained using primary antibodies to cofilin1 and LAMP1 followed by secondary fluorescent antibodies to either rabbit or rat IgG, respectively. A, E & J: Staining pattern of cofilin1 in macrophages with control, TDM bead and M. tuberculosis GFP phagosomes. B, F & K: Staining pattern of LAMP1 in macrophages with control, TDM bead and M. tuberculosis GFP phagosomes. C & G: Merged images with DAPI stain for nucleus (blue). D, H & L: Phase contrast. I: M. tuberculosis GFP. M: Quantitative analysis of cofilin1 and LAMP1 association with control, TDM bead and M. tuberculosis
Profilin1: Figure 3.11 shows representative immunofluorescence pictures of control (A-
D), TDM (E-H) bead and M. tuberculosis GFP (I-L) phagosomes in RAW264.7
macrophages stained for profilin1 (green) and LAMP1 (red). The profilin1 staining
pattern for both bead types was similar, as the candidate protein was completely
absent from control and TDM bead phagosomes as analyzed by immunofluorescence
staining. However, as with cofilin1, phagosomes of M. tuberculosis GFP show a
significant association with profilin1 when LAMP1-positive (arrow). Further, TDM bead
and M. tuberculosis phagosomes were significantly associated with actin compared to
controls (not shown). However, actin did not co-localize with profilin1. In conclusion,
immunofluorescence staining revealed association of profilin1 only with LAMP1-
positive M. tuberculosis but not with bead phagosomes or actin.
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Figure 3.11: Immunofluorescence staining of control, TDM bead and M. tuberculosis GFP infected RAW264.7 macrophages for profilin1 and LAMP1. Macrophages were “bead infected” with control and TDM beads (*) in a MOI of 3 or M. tuberculosis GFP with a MOI of 5. 30 mpi and 2 hpi, respectively, phagosome maturation was stopped and macrophages were stained using primary antibodies to profilin1 and LAMP1 followed by secondary fluorescent antibodies to either rabbit or rat IgG, respectively. A, E & J: Staining pattern of profilin1 in macrophages with control, TDM bead and M. tuberculosis GFP phagosomes. B, F & K: Staining pattern of LAMP1 in macrophages with control, TDM bead and M. tuberculosis GFP phagosomes. C & G: Merged images with DAPI stain for nucleus (blue). D, H & L: Phase contrast. I: M. tuberculosis GFP. M: Quantitative analysis of profilin1 and LAMP1 association with control, TDM bead and M. tuberculosis GFP phagosomes. TTest, n=3 biological replicates, p-value * >0.05, ** >0.01, ***
>0.001, bar=5 µm
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SNAP23: Figure 3.12 shows representative immunofluorescence pictures of control
(A-D), TDM (E-H) bead and M. tuberculosis GFP (I-L) phagosomes in RAW264.7
macrophages stained for SNAP23 (green) and LAMP1 (red). Quantitative analysis
revealed that neither bead nor M. tuberculosis GFP phagosomes accumulated
SNAP23 (Figure 3.12, M). In conclusion, as assessed by immunofluorescence
staining, SNAP23 did not reveal a significant association with bead or M. tuberculosis
GFP phagosomes.
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Figure 3.12: Immunofluorescence staining of control, TDM bead and M. tuberculosis GFP infected RAW264.7 macrophages for SNAP23 and LAMP1. Macrophages were “bead infected” with control and TDM beads (*) in a MOI of 3 or M. tuberculosis with a MOI of 5. 30 mpi and 2 hpi, respectively, phagosome maturation was stopped and macrophages were stained using primary antibodies to SNAP23 and LAMP1 followed by secondary fluorescent antibodies to either rabbit or rat IgG, respectively. A, E & J: Staining pattern of SNAP23 in macrophages with control, TDM bead and M. tuberculosis GFP phagosomes. B, F & K: Staining pattern of LAMP1 in macrophages with control, TDM bead and M. tuberculosis GFP phagosomes. C & G: Merged images with DAPI stain for nucleus (blue). D, H & L: Phase contrast. I: M. tuberculosis GFP. M: Quantitative analysis of SNAP23 and LAMP1 association with control, TDM bead and M. tuberculosis phagosomes. TTest, n=3 biological replicates, p-value * >0.05, ** >0.01, *** >0.001, bar=5 µm
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VAMP3: Figure 3.13 shows representative immunofluorescence pictures of control (A-
D), TDM (E-H) bead and M. tuberculosis GFP (I-L) phagosomes in RAW264.7
macrophages stained for VAMP3 (green) and LAMP1 (red). Quantitative analysis
revealed that neither bead nor M. tuberculosis phagosomes were significantly
associated with candidate protein VAMP3 as assessed by immunofluorescence
staining.
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Figure 3.13: Immunofluorescence staining of control, TDM bead and M. tuberculosis GFP infected RAW264.7 macrophages for VAMP3 and LAMP1. Macrophages were “bead infected” with control and TDM beads (*) in a MOI of 3 or M. tuberculosis with a MOI of 5. 30 mpi and 2 hpi, respectively, phagosome maturation was stopped and macrophages were stained using primary antibodies to VAMP3 and LAMP1 followed by secondary fluorescent antibodies to either rabbit or rat IgG, respectively. A, E & J: Staining pattern of VAMP3 in macrophages with control, TDM bead and M. tuberculosis GFP phagosomes. B, F & K: Staining pattern of LAMP1 in macrophages with control, TDM bead and M. tuberculosis GFP phagosomes. C & G: Merged images with DAPI stain for nucleus (blue). D, H & L: Phase contrast. I: M. tuberculosis GFP. M: Quantitative analysis of VAMP3 and LAMP1 association with control, TDM bead and M. tuberculosis phagosomes. TTest,
3.3.2 Evaluation of the presence of candidate proteins on isolated and purified
control and TDM bead phagosomes by Western blot
To further validate the differential abundances of annexinA1, annexinA6, cofilin1,
profilin1, SNAP23 and VAMP3 found by proteomics, control and TDM bead
phagosomes were isolated and purified from RAW264.7 macrophages and analyzed
by Western blot. For this purpose, the isolation and purification protocol did not include
sorting by FACS. This was due to the fact that, after sorting by FACS, protein
concentrations of candidate proteins were below the Western blot detection limit.
The β-galactosidase assay of three independent isolations revealed 60 %, 43 % and
46 % remaining β-galactosidase activity in TDM bead phagosomes compared to
control ones (results not shown). Figure 3.14 shows representative pictures of the
amount of proteins annexinA1, annexinA6, profilin1, SNAP23 and VAMP3 on control
versus TDM bead phagosomes. The bands below represent the loading control signal
for α-tubulin. Quantification of three independent experiments via densitometry
revealed that average annexinA1 was 35 %, annexinA6 75 %, profilin1 31 %, SNAP23
72 % and VAMP3 16 % more abundant on TDM versus control bead phagosomes.
Cofilin1 could not be detected.
Figure 3.14: Quantification of candidate proteins annexinA1, annexinA6, profilin1, SNAP23 and VAMP3 on isolated control versus TDM bead phagosomes via Western blot. Macrophages were “bead infected” with control and TDM beads with an MOI of 10. 30 mpi, bead phagosome maturation was stopped. Bead phagosomes were subsequently isolated and purified as described in chapter 2.2.1. Control and TDM bead phagosomes isolated and purified in three independent experiments were analyzed by Western blot and quantified by densitometry as described in 2.2.3. Figure 3.14 shows representative pictures of one experiment. Cofilin1 could not be detected by Western Blot. Average, annexinA1 was 35 %, annexinA6 75 %, profilin1 31 %, SNAP23 72 % and VAMP3 16 % more abundant on TDM versus control bead phagosomes.
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3.3.3 Evaluation of the role of candidate proteins for intracellular survival of M.
tuberculosis by small interfering RNA/CFUs analysis
Ultimately, the relevance of candidate proteins in early processes of M. tuberculosis-
mediated inhibition of phagosome maturation and consequently survival in
macrophages was tested. Therefore, candidate proteins were knocked-down in
RAW264.7 macrophages using the small interfering RNA (siRNA) technology followed
by infection with M. tuberculosis and analysis of colony-forming-units (CFUs) at 2 hpi,
4 hpi, 1 dpi, 2 dpi and 3 dpi (hours/days post infection). As control, untreated (negative
control, NK) and RAW264.7 macrophages transfected with non-targeting-RNA (NTR)
were used. Of note, due to technical difficulties only candidate proteins annexinA6,
cofilin1, profilin1 and VAMP3 could be analyzed.
The efficiency of siRNA-mediated knock-down of candidate proteins was tested via
Western blot. In this experimental set-up, the role of candidate proteins early in
infection should be tested. Thus only their absence on the day of infection was of
interest and evaluated by Western blot as shown in Figure 3.15. The upper bands
show the signals for annexinA6, profilin1, cofilin1 and VAMP3, 1 day (profilin1,
VAMP3) and 2 days (annexinA6, cofilin1) post transfection (dpt) with NTR and
targeting-siRNA in RAW264.7 macrophages, respectively. The bands below represent
the loading control signal for α-tubulin. Densitometry analysis revealed that, compared
to NTR controls, the amount of annexinA6 was reduced to 80 %, of cofilin1 to 5 %, of
profilin1 to 55 % and of VAMP3 to 2 %.
Figure 3.15: Control of siRNA-mediated knock-down of candidate proteins annexinA6, cofilin1, profilin1 and VAMP3 via Western blot. Macrophages were transfected with siRNA for annexinA6, profilin1, cofilin1 and VAMP3 and NTR as control. 1 dpt or 2 dpt, cells were infected with M. tuberculosis or harvested and destroyed by a freeze (N2 (l)) and thaw (37 °C) cycle. Samples were treated with DNAse and the protein concentration was determined using the Pierce 660 nm protein assay. Afterwards, 10 µg of protein were loaded onto a SDS-PAGE. Gels were blotted, blocked and incubated with antibodies against annexinA6, cofilin1, profilin1 and VAMP3 in
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parallel with an antibody against α-tubulin as loading control. Subsequently, blots were incubated with anti-rabbit coupled to HRP. Blots were washed, developed with an ECL-solution and exposed to Hyperfilm 10 sec to 2 min corresponding to band strength. Densitometry analysis revealed that, compared to NTR controls, the amount of annexinA6 was reduced to 80 %, of cofilin1 to 5 %, of profilin1 to 55 % and of VAMP3 to 2 % as assessed by Western blot.
Figure 3.15 shows the CFUs of M. tuberculosis from macrophages with reduced
amounts of (A) annexinA6, (B) cofilin1, (C) profilin1 and (E) VAMP3. Compared to NTR
controls, reduction of annexinA6 significantly lowered CFUs at 2 and 4 hpi. Similarly,
knock-down of cofilin1 revealed significantly decreased M. tuberculosis CFUs at 2 and
4 hpi and 1 dpi whereas reduced amounts of profilin1 decreased CFUs at 4 hpi and
1 dpi. Finally, the CFU of cells with reduced VAMP3 is significantly increased at 2 hpi.
Figure 3.16: Survival of M. tuberculosis inside RAW264.7 macrophages after knock-down of annexinA6, cofilin1, profilin1 and VAMP3 using RNA interference. Macrophages were transfected with NTR or siRNA for annexinA6, cofilin1, profilin1 and VAMP3. 1 dpi or 2 dpt, macrophages were infected with M. tuberculosis with an MOI of 0.5. As negative control, untreated macrophages were infected as well. CFUs were analyzed 2 hpi, 4 hpi, 1 dpi, 2 dpi and 3 dpi. The CFU assay was performed once with three technical replicates and analyzed with Two-Way-ANOVA with Bonferroni post test. NTR served as control for statistical analysis. n=3 technical replicates, p-values *< 0.5 *, < 0.01 **, < 0.001 ***.
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3.4 A role for the β-actin cytoskeleton in TDM-mediated inhibition
of phagosome maturation
A certain number of proteins identified in both control and bead phagosomes were
actin-interacting proteins including profilin1 and cofilin1. Additionally, actin was one of
the most abundant proteins identified with over 100 EUSC in both samples. (Table 0-1,
#571) Consequently, the localization of actin on control and TDM bead phagosomes
was examined.
Therefore, RAW264.7 macrophages were “bead infected” with control and TDM
beads. Bead phagosome biogenesis was stopped 30 min after phagocytosis and
macrophages were stained for β-actin using Phalloidin Alexa 488 (Figure 3.17).
Confocal fluorescence microscopy revealed that a certain percentage of TDM bead
phagosomes were decorated with β-actin (B) while control bead phagosomes were
devoid of β-actin (A). Quantitative analysis showed that circa 12 % of TDM bead
(arrows) but only 1 % of the control phagosomes were associated with β-actin rims at
30 min after phagocytosis (C).
Figure 3.17: Accumulation of β-actin around control and TDM bead phagosomes in RAW264.7 macrophages. Macrophages were “bead infected” with control and TDM beads with a MOI of 10. 30 mpi, bead phagosome maturation was stopped and macrophages were stained for β-actin using Phalloidin Alexa 488 (green). Bead phagosomes are indicated with *. β-actin accumulation around TDM bead phagosomes is marked with arrows. A & B: Representative pictures of control and TDM bead phagosomes in macrophages stained for β-actin, respectively C: For quantification, 100 control and TDM bead phagosomes from three independent experiments were counted and analyzed for the presence of β-actin. n=3 biological replicates, TTest, p-value * < 0.05, ** < 0.01, *** < 0.001.
Next, phagosomes of M. tuberculosis were analyzed for β-actin accumulation.
Therefore, RAW264.7 macrophages were infected with M. tuberculosis GFP and
stained for β-actin with Phalloidin Alexa 594. In contrast to the β-actin rims observed
on TDM bead phagosomes, the β-actin staining in M. tuberculosis GFP infected cells
revealed a punctate accumulation decorating the phagosome as shown in picture B
and D of Figure 3.18 (arrows).
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Figure 3.18: Localization of β-actin and WASH1 around M. tuberculosis GFP phagosomes in RAW264.7 macrophages. Macrophages were infected with M. tuberculosis GFP with a MOI of 3. 2 hpi, phagosome maturation was stopped and macrophages were stained for β-actin and WASH1 using Phalloidin Alexa 549 (red) and a primary antibody against WASH1 and secondary anti-rabbit IgG fluorescent antibody (white), respectively. β-actin and WASH1 accumulation around M. tuberculosis GFP phagosomes is marked with arrows. A: M. tuberculosis GFP. B: β-actin staining pattern in macrophages. C: WASH1 staining pattern in macrophages. D: merged picture.
Spontaneous polymerization of monomeric cytoplasmic actin is relatively inefficient.
Thus, polymerization is facilitated by several proteins acting in synergy. Actin
polymerization on target membranes is mediated by the Arp2/3 complex in cooperation
with nucleating-promoting-factors (NPFs). Recently, the WASH-complex was
discovered to be the major actin polymerisation-promoting complex on endosomes.
The WASH-complex is constituted by five proteins: strumpellin, SWIP, WASH1,
Fam21 and CCDC53. To analyze whether β-actin on phagosomes of M. tuberculosis
is associated with the WASH-complex, RAW264.7 macrophages infected with M.
tuberculosis GFP and stained for β-actin were co-stained for WASH1 (Figure 3.18).
Indeed, WASH1 co-localized with β-actin to the M. tuberculosis GFP phagosome as
indicated with arrows in pictures C and D [103].
To test whether the accumulation of β-actin around the phagosome of M. tuberculosis
is involved in inhibition of phagosome maturation, 30 min after M. tuberculosis GFP
infection, β-actin was removed with Latrunkulin A (LatA), a drug that depolymerises F-
actin. After 2 hpi and 4 hpi, phagosomes of M. tuberculosis GFP were analyzed for
their association with lysotracker (A) and the lysosomal marker LAMP1 (B) (Figure
3.19). Upon LatA treatment of RAW264.7 macrophages, the numbers of M.
tuberculosis GFP in mature phagosomes as indicated by lysotracker positive staining
were significantly increased at 2 hpi (A). In contrast, after 4 hpi LatA did not alter
lysotracker association with M. tuberculosis GFP. LAMP1 association with M.
tuberculosis was also significantly enhanced at 2 hpi but not 4 hpi (B).
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Figure 3.19: Co-localization of lysotracker and LAMP1 with M. tuberculosis GFP in RAW264.7 macrophages after removal of β-actin with LatrunkulinA (LatA). Macrophages were infected with M. tuberculosis GFP with an MOI of 0.5. 1 hpi, cells were treated with LatA for 30 min. 2 hpi and 4 hpi, infection was stopped and macrophages were stained for LAMP1 using a primary antibody against LAMP1 and secondary anti-rat IgG fluorescent antibody. 30 mpi macrophages were loaded with Lysotracker. The experiment was performed with three biological replicates. For quantification, 100 M. tuberculosis GFP phagosomes from three independent experiments were counted and analyzed for their association with Lysotracker and LAMP1. Statistical analysis was performed using a Two-Way-ANOVA with Bonferroni post test. n=3 biological replicates, p-value * < 0.05, ** < 0.01, *** < 0.001.
Removal of β-actin by LatA from M. tuberculosis GFP phagosomes in RAW264.7
macrophages was further analyzed for its influence on survival of M. tuberculosis.
Compared to controls (DMSO), the CFU in treated cells declined from 2 dpi to 3 dpi
on. At 6 dpi, the CFU was significantly decreased.
Figure 3.20: Survival of M. tuberculosis GFP in RAW264.7 macrophages after removal of β-actin with LatrunkulinA (LatA). Macrophages were infected with M. tuberculosis GFP with an MOI of 0.5. 1 hpi, cells were treated with LatA for 30 min. After 2 hpi, 4 hpi, 1 dpi, 2 dpi, 3 dpi and 6 dpi, CFUs were analyzed. The experiment is a representative one out of three. Statistical analysis was performed using a Two-Way-ANOVA with Bonferroni post test. n=3 technical replicates, p-value * < 0.05, ** < 0.01, *** < 0.001.
To determine whether WASH-complex mediated β-actin accumulation is important for
survival of M. tuberculosis phagosomes, WASH1 was knocked-down via small
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interference RNA in RAW264.7 macrophages. The efficiency of siRNA-mediated
knock-down of WASH1 was tested via Western blot. In this experimental set-up, the
role of the WASH-complex early in infection should be tested. Thus only its absence
on the day of infection was of interest. The upper bands show the amount of protein
of WASH1, 2 day post transfection (dpt) in RAW264.7 macrophages treated with NTR
and WASH1-targeting siRNA. The bands below represent loading control with α-
tubulin. Overall the amount of WASH1 was decreased successfully by siRNA knock-
down. Compared to 2 dpt NTR control, the amount of WASH1 was significantly
reduced to 0 % as assessed by Western blot.
Figure 3.21: Control of siRNA-mediated knock-down of WASH1 via Western blot. Macrophages were transfected with siRNA targeting WASH1 and NTR as control. 2 dpt, cells were infected with M. tuberculosis or harvested and destroyed by a freeze (N2 (l)) and thaw (37 °C) cycle. Samples were treated with DNAse and the protein concentration was determined using the Pierce 660 nm protein assay. Afterwards, 10 µg of protein were loaded onto a SDS-PAGE. Gels were blotted, blocked and incubated with antibodies against WASH1 in parallel with an antibody against α-tubulin as loading control ON at 4 °C. Subsequently, blots were incubated with anti-rabbit coupled to HRP. Blots were washed, developed with an ECL-solution and exposed to Hyperfilm 10 sec to 2 min corresponding to band strength. As assessed by Western blot, the amount of WASH1 could be reduced to 0 %.
2 dpt with WASH1-targeting siRNA, macrophages were infected with M. tuberculosis
and CFUs were analyzed 2 hpi, 4 hpi, 1 dpi, 2 dpi and 3 dpi. Over time, knock-down
of WASH1 had no major impact on CFUs when compared to controls although CFUs
were significantly increased compared to controls at 2 dpi.
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Figure 3.22: Survival of M. tuberculosis inside RAW264.7 macrophages after knock-down of WASH1 using RNA interference. Macrophages were transfected with NTR and siRNA targeting WASH1. 2 dpt, macrophages were infected with M. tuberculosis with an MOI of 0.5. As negative control, untreated macrophages were infected as well. CFU were analyzed 2 hpi, 4 hpi, 1 dpi, 2 dpi and 3 dpi. The CFU assay was performed once with three technical replicates and analyzed with Two-Way-ANOVA with Bonferroni post test. NTR served as control for statistical analysis. n=3 technical replicates, p-values *< 0.05 *, < 0.01 **, < 0.001 ***.
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3.5 The lipid coated bead phagosome lipidome
In order to analyze whether TDM affects the lipid composition of bead phagosomes,
RAW264.7 macrophages were “bead infected” with control and TDM beads.
Subsequently control and TDM bead phagosomes were isolated and purified, internal
standards were added and lipids were extracted. Samples were analyzed by mass
spectrometry for the lipid composition as described in 2.2.4. Comparison of control and
TDM bead phagosomes should then reveal TDM-mediated changes in the bead
phagosome lipidome.
Seven pairs of control and TDM bead phagosomes were compared. Prior mass
spectrometry analysis, the β-galactosidase activity of all pairs was determined. TDM
bead phagosome preparations 1 and 4 showed remaining β-galactosidase activities
of 55 %, 3 of 28 %, 2, 5, 6 and 7 of 62 %, 64 %, 69 % and 64 %, respectively.
Figure 3.23: β-galactosidase activity of all seven paired samples used for lipidomic analysis of bead phagosomes. Macrophages were “bead infected” with control and TDM beads with a MOI of 10. 30 mpi, bead phagosome maturation was stopped and bead phagosomes were isolated and purified as described in 2.2.1. β-galactosidase activity was determined as described in 2.2.1.7. Percentages are shown above bars.
We analysed the different classes of ceramides (Cer), cholesterol (Chol),
phosphatidylinositol (PI), phosphatidylserine (PS) and sphingomyelins (SM).
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For a first general overview about the lipid composition of bead phagosomes, Figure
3.24 shows the lipid class composition of matured control bead phagosomes.
Accordingly, with 24 %, 23 % and 19 %, PC, LBPA and cholesterol were the most
prominent lipids in the bead phagosome lipidome, respectively, followed by SM (9 %),
PC-O (6 %), PI (5 %), PS (5 %), PE-O (4 %), PE (3%), Cer (1 %), PG (1 %) and LPC
(0.3 %).
Figure 3.24: Identified lipid classes in control bead phagosomes. Macrophages were “bead infected” with control and TDM beads with a MOI of 10. 30 mpi, bead phagosome maturation was stopped and bead phagosomes were isolated and purified as described in 2.2.1. Subsequently, internal standards were added, lipids were extracted and pairs of control and TDM bead phagosomes samples were analyzed for their lipid content by mass spectrometry as described in 2.2.4. Results were processed using Microsoft Excel and Prism 6.0. The pie chart shows the composition of lipid classes identified in matured control bead phagosomes.
The lipid profile was compared between control and TDM bead phagosomes.
Therefore, mol % of lipid classes of matching pairs were analyzed with a paired TTest.
Figure 3.25 and Figure 3.26, show the befor-and-after-plot of the 12 lipid classes
identified in all seven control (before) and TDM (after) bead phagosome pairs. Overall,
the lipid composition of control and TDM bead phagosomes was comparable.
However, three lipid classes were significantly different in TDM samples compared to
controls. While amounts of LBPA (Figure 3.25, C) were lower in TDM samples
compared to controls, amounts of cholesterol (Figure 3.25, B) and PS (Figure 3.26, E)
were significantly enhanced.
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Figure 3.25: Before-and-after-plot of Cer, Chol, LBPA, LPC, PC and PC-O identified in control versus TDM bead phagosomes. Macrophages were “bead infected” with control and TDM beads with a MOI of 10. 30 mpi, bead phagosome maturation was stopped and bead phagosomes were isolated and purified as described in 2.2.1. Subsequently, internal standards were added, lipids were extracted and pairs of control and TDM bead phagosomes samples were analyzed for their lipid content by mass spectrometry as described in 2.2.4. Results were processed using Microsoft Excel and Prism 6.0. Statistical analysis was performed with a paired TTest. n=7 biological replicates, p-value * < 0.05, ** < 0.01, *** < 0.001.
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Figure 3.26: Before-and-after-plot of PE, PE-O, PG, PI, PS and SM identified in control versus TDM bead phagosomes. Macrophages were “bead infected” with control and TDM beads with a MOI of 10. 30 mpi, bead phagosome maturation was stopped and bead phagosomes were isolated and purified as described in 2.2.1. Subsequently, internal standards were added, lipids were extracted and pairs of control and TDM bead phagosomes samples were analyzed for their lipid content by mass spectrometry as described in 2.2.4. Results were processed using Microsoft Excel and Prism 6.0. Statistical analysis was performed with a paired TTest. n=7 biological replicates, p-value * < 0.05, ** < 0.01, *** < 0.001.
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The TDM-induced changes in lipid class composition of bead phagosomes were
further analyzed on lipid species level. Summarizing, 16 lipid species were identified
for ceramides, 1 for cholesterol, 48 for LBPA, 5 for LPC, 38 for PC, 34 for PC-O, 30
for PE, 38 for PE-O, 31 for PG, 23 for PI, 27 for PS and 21 for SM (Table 0-2)
For graphic representation of single lipid species, a volcano-plot was chosen (Figure
3.27). Therefore the mean of p-values calculated from the mol % of all identified lipid
species in sample pairs was transformed to a negative log10 value and plotted against
the mean of the mol % fold change (ratio of TDM/control) of all sample pairs
transformed to a log2 value. This gives a volcano-plot as shown in Figure 3.27. Every
spot represents a single lipid species. Single lipid species shift left when decreased
and right when increased in TDM samples compared to controls. If single lipid species
have a paired p-value <0.05 and thus are significantly altered, they have a -log10 value
>1.13 (dashed horizontal line). If the fold change is >1.5 or >2, log2 values are >0.58
or >1 (dashed red vertical lines), respectively. If the fold change is <0.75 or <0.5, log2
values are <-0.41 or <-1 (dashed green vertical lines), respectively. Lipid species that
center around 0 are equally distributed in control versus TDM bead phagosome
samples.
The volcano-plot confirms the decrease of LBPA (turquoise dots) because most of the
LBPA species shift to the left. Species 44:10 but most prominently, species 36:2 is
significantly reduced in TDM bead compared to control phagosomes. Similarly, the
increase in PS in TDM samples compared to controls is revealed by the shift of several
PS (dark green) lipid species to the right. PS 37:1, PS 38:4 and PS 38:5 are
significantly accumulated in TDM samples compared to controls. The single light green
spot marked with “Chol” indicates a minor but significant increase in TDM bead
phagosome samples. Although increased without statistical significance on the lipid
class level, ceramides 32:1, 34:0, 34:1 and 41:2 are significantly accumulated in TDM
bead phagosomes. Similarly, SM species 40:1, 41:1, 42:2 are significantly increased
in TDM bead phagosomes.
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Figure 3.27: Volcano-plot - Distribution of identified lipid species in control versus TDM bead phagosome samples. Macrophages were “bead infected” with control and TDM beads with a MOI of 10. 30 mpi, bead phagosome maturation was stopped and bead phagosomes were isolated and purified as described in 2.2.1. Subsequently, internal standards were added, lipids were extracted and pairs of control and TDM bead phagosomes samples were analyzed for their lipid content by mass spectrometry as described in 2.2.4. For volcano-plot graphic representation, -log10 transformed p-values and log2 transformed fold changes of control versus TDM sample pairs were plotted. Every dot represents a single lipid species. Unchanged lipid species centre around 0. Altered lipid species shift left when decreased and right when increased in TDM bead phagosomes. Significant changes (p-value <0.05) appear above the horizontal black dashed line. Fold-changes >1.5 or >2 appear beyond red vertical dashed lines and fold-changes <0.75 or <0.5 appear beyond the green vertical dashed lines. Results were processed using Microsoft Excel and Prism 6.0. n=7 biological replicates
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4 Discussion
4.1 Resume
Localized in the mycobacterial outer membrane, the cell-wall lipid TDM is a major
virulence factor of M. tuberculosis. When coated to beads, TDM was shown to inhibit
bead phagosome maturation [90], [91]. However, the molecular function underlying
TDM-mediated inhibition of phagosome maturation remains elusive. We hypothesized,
that TDM exerts its virulence function by interacting with host-cell partners at the
phagosome interface. In the TDM determined vesicle, these interaction partners are
present either within the phagosome, inside the phagosomal membrane or linked to
the cytoplasmic side of the phagosome. The direct or indirect interaction of TDM with
these interactors putatively affects phagosome maturation. Thus the main aim of this
thesis was to identify TDM direct or indirect interaction partners. To study the effect of
TDM, a simplified lipid-coated bead model was used. Beads were coated with TDM
and used for “bead infection” of macrophages to isolate and purify bead phagosomes
for mass spectrometry based proteomics and lipidomics.
To identify and quantify potential interaction partners, a protocol had to be developed
that enabled isolation and purification of bead phagosomes for mass spectrometry
analysis. An existing protocol including (i) coating of beads with TDM, (ii) “bead
infection” of macrophages (iii) bead phagosome isolation from macrophages by
magnet and (iv) purification of bead phagosomes by DNase digestion and Ficoll-
gradient centrifugation was refined by addition of a protease digestion step and sorting
bead phagosomes by FACS.
Proteomics of control and TDM bead phagosomes identified 1054 proteins in total.
Grouping of identified proteins according to their cellular localization revealed 2916
proteins that could be assigned as phagosome-associated whereas 898 proteins were
potential contaminants indicating an enrichment of phagosomal proteins. After
application of exclusion criteria, 34 proteins were filtered that were enriched in TDM
bead phagosomes compared to controls and thus potential TDM interaction partners.
Of these, annexinA1, annexinA6, cofilin1, profilin1, SNAP23 and VAMP3 were chosen
for further analysis because they have been described to be associated with
phagosomes as part of a phagosome-interacting network.
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Immunofluorescence to verify mass spectrometry results, however failed to reveal
specific association with TDM bead or M. tuberculosis phagosomes. Notably, cofilin1
and profilin1 were found positive to LAMP1 M. tuberculosis phagosomes. In contrast
to the immunofluorescence approach, Western blot analysis of isolated and purified
bead phagosomes confirmed enrichment of all 6 candidate proteins on TDM bead
phagosomes compared to control ones. Preliminary siRNA experiments to knock-
down candidate proteins in macrophages for subsequent infection with M. tuberculosis
indicated that absence of annexinA6 and VAMP3 impairs phagocytosis and that
cofilin1 and profilin1 are important for survival of the pathogen in macrophages.
Furthermore, proteomics revealed enrichment of β-actin and β-actin-binding-proteins
in bead phagosome proteomes. Fluorescence staining using Phalloidin demonstrated
accumulation of β-actin on TDM bead and a punctate pattern on M. tuberculosis
phagosomes that co-localized with NPF WASH1. Removal of β-actin drove M.
tuberculosis in phago-lysosomes and impaired survival of M. tuberculosis in
macrophages. However, preliminary RNA interference experiments suggested that
WASH1 is dispensable for survival of M. tuberculosis in macrophages.
Differential lipidomics by mass spectrometry of control and TDM bead phagosomes
demonstrated that TDM bead phagosomes had significantly less LBPA and
significantly increased amounts of cholesterol and phosphatidylserine compared to
control ones. Analysis at the single lipid species level additionally revealed significant
increase of certain ceramide and sphingomyeline species in TDM bead phagosomes
when compared to control ones.
In conclusion, these results reflect that TDM and control bead phagosomes vary on
protein and lipid levels because each bead phagosome represents a different
maturation stage. However, identified TDM interactors may have an important direct
or indirect functional role in inhibition of phagosome maturation. Notably, there is a
specific association of the actin cytoskeleton network with non-maturing M.
tuberculosis and TDM bead phagosomes. A model for actin cytoskeleton function in
interference with phagosome biogenesis by TDM is proposed.
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4.2 Method development: Isolation and purification of bead
phagosomes from macrophages for mass spectrometry
analysis
Phagocytosis and subsequent phagosome maturation is a hallmark of innate immune
cells. The process of phagosome maturation is designed to ingest and eliminate
engulfed particles by a well orchestrated mechanism (Figure 1.5). Phagocytosis is not
only important for elimination of pathogens but links innate and acquired immunity
through antigen processing and presentation to T-cells. Nevertheless, several
intracellular pathogens have developed distinct strategies to interfere with phagosome
maturation and thus survive and thrive within their niche. Due to the fact that the
successful manipulation of phagocytosis and phagosome maturation determines the
outcome of infections, the underlying intracellular events are of outermost importance
for novel concepts of infection control.
In the past decades, several techniques for isolation and purification of phagosomes
were developed. These techniques underlie three different principles: (i) choosing the
host-cell and the phagocytic particle, (ii) infection, destruction and isolation of the
phagosome from host-cells and (iii) purification of phagosomes. Current methods are
mainly based on experiments performed by Wetzel and Korn in 1969 [105]. In principle,
polystyrene latex-beads were fed to amoeba, isolated by a Potter-Elvehjem-
homogenizer and purified by density gradient centrifugation using a continuous
sucrose gradient. Latex bead phagosomes (LBPs) can be prepared with high purity
because of their significantly distinct density compared to host-cell organelles.
In the following years, similar separation protocols have been applied to isolate and
purify LBPs from murine and human macrophages [106],[107] and amoeba
[108],[109]. Density gradient centrifugation was also applied to isolate pathogen-
containing vacuoles (PCVs) from macrophages [98], [110]–[112]. Nevertheless,
because PCVs have densities closer to certain organelles, multiple steps of purification
by differential density centrifugation are typically employed [113]. To circumvent the
time-consuming ultra-centrifugation steps, magnetic particles were introduced in the
field of phagosome cell biology [114]. In these protocols, both magnetic beads or
viable, magnetically labelled pathogens such as BCG or M. tuberculosis were studied
[115]–[117]. For phagosome isolation, whole cell homogenates containing
phagosomes were subjected to a strong magnetic field and further purified by several
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98
washing steps [114]–[117]. Upon removal of the magnet, the phagosomes were
collected and further enriched. Furthermore, organelle and free-flow electrophoresis
were used for purification of PCVs. Here PVCs were separated from other intracellular
organelles in homogenates of infected macrophages by electrophoresis through a
gradient according to their charge and size [111]. However, using the described
methods, phagosomes could never be perfectly purified since complete separation
from other cell constituents was never achieved due to the complex interaction of
phagosomes with other compartments in the cytoplasm [113]. Consequently,
phagosomes were rather enriched than purified.
In the present study, magnetic bead phagosomes were isolated and purified from
macrophages. The practical procedure included (i) bead infection of macrophages with
magnetic beads, (ii) homogenization of macrophages using a douncer, (iii) isolation of
magnetic bead phagosomes with a magnet and (iv) purification of bead phagosomes
by DNase and trypsin digestion steps, Ficoll-gradient centrifugation and final sorting
by FACS.
Magnetic (Dyna-) beads are polystyrene beads with enclosed magnetic particles. The
bead surface is tosylactivated to covalently bind proteins/peptides via primary amino-
or sulfhydryl groups. We covalently bound BSA to link TDM to the magnetic beads.
From previous experiments, we knew that this technique is suitable to coat beads with
TDM and that these TDM beads are able to inhibit phagosome maturation [91]. Binding
is most likely enabled by hydrophobic interactions between the mycolic acids of TDM
and hydrophobic pockets of BSA. Accordingly, the trehalose-moiety of TDM most likely
sticks out for interaction with host-cell “targets”.
Disruption of macrophages to isolate bead phagosomes is a delicate task since
phagosomes must remain intact. In many protocols for isolation of LBPs,
homogenization is achieved by passing cells through syringes with needle gauges
ranging from 25 to 28 [113]. This worked fine for LBPs since they have a much smaller
diameter (0.8-1.1 µm). We homogenized macrophages with a douncer. This method
allows destruction of macrophages without affecting phagosome integrity since the
gap between the pestle and the douncer chasses (13 µm) is wide enough for bead
phagosomes (Ø 2.8 µm) to stay intact.
Digestion of DNA with DNase is a very important step in the process of phagosome
purification because, due to their negative charge attracting cell debris, nucleotides
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99
(DNA in particular) can change a sample into a sticky gel impeding all further
purification efforts [98].
We noticed that, despite removal of DNA, bead phagosomes still stuck together,
probably due to protein-meshworks linking bead phagosomes together. Thus samples
were additionally treated with trypsin. Protease digestion was applied for phagosome
isolation procedures before [111]. However, it is still an unusual step, due to the fact
that proteases act non-targeted and therefore may also digest proteins of interest.
Thus, this step had to be performed very precise regarding incubation time and sample
protein to trypsin concentration and the enzyme had to be effectively removed
afterwards. However, maintained β-galactosidase activity in phagosome preparations
after trypsin treatment indicated bead phagosome integrity.
Sucrose gradient centrifugation takes advantage of the fact that latex beads have a
relatively low density and thus migrate in sucrose gradients differently from other cell
organelles [105]. Polystyrene beads including magnetic ones used herein have a
relatively high density, which precluded sucrose gradients from being applied for
further purification. Therefore, we used a Ficoll-gradient to separate phagosomes from
cellular debris. Ficoll-gradients are hydrophilic polysaccharide meshworks commonly
used to separate cell populations but also allowed us to separate magnetic bead
phagosomes from cellular debris and other organelles [98], [115], [118].
However, our own results and other reports on isolation of particle containing
phagosomes showed that contaminations with non-phagosomal host-cell material
from distinct compartments such as ER, mitochondrial or nucleus cannot be
completely avoided by the purification steps described above. Together with
increasing sensitivity of mass spectrometry methods in recent years, purity of
phagosome preparations is a significant bottleneck because high abundant
contaminating proteins can overwrite signals of phagosomal proteins with lower
abundance [119]. In 1998, Ramachandra et al. discussed sorting by flow cytometry as
an easy and rapid method for isolating phagosomes from cell lysates [120]. Sorting by
FACS was applied to isolate Salmonella and Staphylococcus aureus phagosomes
from infected cells [121]–[123]. We introduced FACS sorting as a new method to
further purify bead phagosomes. Bead phagosomes were sorted by size, re-isolated
using a magnet and analyzed by mass spectrometry. Proteins in the supernatant from
phagosomes which were destroyed during sampling into tubes after sorting were
precipitated using protein-binding beads [124]. Mass spectrometry analysis revealed
Discussion
100
that the majority (74 %) of identified proteins were present in bead phagosome
samples whereas only minor (26 %) amounts of proteins were found in the supernatant
precipitates. Thus, we are confident that most bead phagosomes remained intact
during sorting by FACS. Analysis of the cellular localization of identified proteins
showed strong accumulation of endosomal/phagosomal proteins (2916) compared to
non-intracellular vesicle proteins (898).
Taken together, the newly developed protocol is suitable for purification of bead
phagosomes for subsequent mass spectrometry analysis (Figure 3.1).
4.2.1 Quantification of phagosomal proteins by spectral counting is a label-free
and semi-quantitative approach
In the present work, quantification of proteins was accomplished by spectral counting.
According to the Scaffold 4 software used for evaluation of data, the EUSC (exclusive
unique spectrum count) describes the number of spectra that can be assigned to only
one specific peptide/protein and does not fit other peptides/proteins. Quantification by
spectral counting is classified as semi-quantitative because it is a label-free method.
Spectral counting is based on the empirical observation that the more of a particular
protein is present in a sample, the more tandem MS-spectra are collected for peptides
of the same protein [125]. In contrast, with label-based methods, mass spectrometry
can recognize mass differences between the labelled and unlabeled forms of a peptide
and quantification is achieved by comparing their respective signal intensities [125].
Quantification by spectral counting has several disadvantages. First, the spectrum
count response for every peptide is different since e.g. the chromatographic behaviour
(retention time, peak width) varies. Furthermore, at higher spectral counts, saturation
effects will occur which are different for every peptide [125]. In addition, measurement
accuracy of the mass spectrometer varies prone to measurement errors.
Nevertheless, the correlation between amount of protein and number of tandem mass
spectra yields reliable results and quantification by spectral counting is applied
routinely in proteomics [126].
4.2.2 Evaluation of proteomics data by immunofluorescence staining and
Western blot revealed contradicting results
In the present study, proteomics data of isolated control and TDM bead phagosomes
were evaluated by IF-staining and Western blot. For Western blot experiments, the
isolation and purification protocol did not include sorting by FACS. We excluded this
Discussion
101
purification step, because protein concentration of samples (107 bead phagosomes)
after sorting by FACS was below the Western blot detection limit.
Evaluation of proteomics data by IF-staining and Western blot, gave very different
results. While Western blot experiments confirmed proteomics data, i.e. enrichment of
candidate proteins in TDM bead phagosomes, the respective proteins could not be
detected on bead phagosomes by IF-staining. These results may be due to the
epitopes of the antigens recognized by the antibodies in their native (IF-staining)
versus linearized (Western blot) form. However, the staining pattern of all candidate
proteins in macrophages resembled that shown in previous reports of those
antibodies. Another possibility, the antigens could be present in a different
conformation at phagosomes compared to other compartments.
Discussion
102
4.3 TDM induced changes on the bead phagosome proteome
A “proteome” is described as the complement of proteins in a sample as e.g. isolated
phagosomes. Up to date, searching “phagosome” and “proteome” on pubmed.com
revealed 68 hits. Probably one third of these results covered LBP studies, the gold
standard to study phagocytosis and phagosome maturation. Only one third dealt with
PCV-proteomes. Searching “Mycobacterium tuberculosis”, “phagosome” and
“proteome” only gave 5 results. Although analysis of isolated mycobacterial
phagosomes is reported several times, only 3 studies presented whole proteomes of
Mycobacteria containing phagosomes.
Mycobacterial phagosomes have been characterized before by various approaches
[119], [131], [132]. Due to the fact that Mycobacteria maintain early endosomal
characteristics, they acquire proteins of the early and intermediate phagosomes stage
such as the transferrin receptor (TfR), Rab5, EEA1, synthaxin4, synthaxin6 and
VAMP3 and carry limited amounts of v-H+-ATPase, lysosomal hydrolases including
cathepsinD, LAMP1/2 and Rab7 [129]–[131]. In a previous study with TDM beads, IF-
staining and Western blot experiments confirmed retention of TfR and exclusion of
LAMP1 and mature cathepsinD [91].
Our proteomics data corroborate that TfR and VAMP3 are enriched on TDM bead
phagosomes compared to controls (# 303, 166, Table 0-1). Rab5a and Rab5c were
equally distributed in both samples (# 208, 456, Table 0-1). Syntaxin4 was only found
in the TDM sample (#128, Table 0-1). In contrast, syntaxin6, EEA1 and LAMP2 were
not detected. In both samples, the v-H+-ATPase was one of the most abundant
proteins since plenty subunits were identified (# 16, 200, 262, 380, 389, 416, 426, 445-
447, 837, 1020, Table 0-1). Nevertheless, lower abundance of the v-H+-ATPase on
TDM bead phagosomes was not observed. In fact, many subunits were accumulated
in TDM samples. Levels of LAMP1 were almost similar in both samples with a trend to
lower amounts in TDM samples (# 612, Table 0-1). In contrast, Rab7a was strongly
accumulated at TDM bead phagosomes compared to controls (#233, Table 0-1).
These data indicate that TDM bead phagosomes share characteristics with M.
tuberculosis phagosomes but differ regarding other ones.
In the present study, 843 proteins were identified in TDM bead phagosomes.
Proteomic analysis of BCG containing phagosomes from the human monocyte cell line
THP-1 identified 447 proteins [127]. Similar to our results, proteins from several
Discussion
103
categories were identified. Table 4-1 provides a coarse overview about proteins
identified in BCG versus TDM bead phagosomes:
Table 4-1: Comparison of proteins identified in BCG versus TDM bead phagosomes. Modified from [127]. The left column provides a coarse overview of proteins identified in BCG phagosomes. The right column lists proteins identified in BCG phagosomes that were present in TDM phagosomes as well. Underlined proteins were exclusively present in BCG compared to latex bead phagosomes.
Fam3C, tumor protein D54, VAT1, transmembrane emp24 domain-containing protein 9
Fam3C, tumor protein D54
Others:
glutamate dehydrogenase 1, nucleobindin-1, vacuolar proton pump subunit C1, 60 S ribosomal protein L35, 60 S ribosomal protein L8, macrophage migration-inhibitory factor, translocon-associated protein subunit α, band 4.1-like protein 3, intracellular adhesion molecule 1
vacuolar proton pump subunit C1, 60 S ribosomal protein L35, 60 S ribosomal protein L8, macrophage migration-inhibitory factor, translocon-associated protein subunit α, band 4.1-like protein 3, intracellular adhesion molecule 1
In the study by Lee et al., BCG phagosomes were further compared to LBPs to detect
BCG phagosome specific proteins revealing 32 exclusive proteins in BCG
phagosomes (underlined in Table 4-1). Of these, 17 were not present in TDM bead
phagosomes indicating specificity for BCG. Overall, comparison with our data revealed
a large overlap between the TDM and BCG phagosome proteomes.
In another study performed by Rao et al., proteomes of phagosomes with either M.
tuberculosis H37Rv, attenuated strain M. tuberculosis H37Ra or BCG were compared
in a systems biology approach [132]. Principal component analysis revealed that
proteins Hsd17b12, Rpl38, Rpl6, nicastrin, Rab11b, Gnb1, Rps3a, Cdc42 and
C230096C10Rik were specifically enriched in M. tuberculosis H37Rv phagosomes. Of
these, only Hsd17b12 and Gnb1 were found in TDM bead preparations indicating
exclusive enrichment in phagosomes containing viable M. tuberculosis. In contrast,
the of proteins Cct8, Asns, Nomo1, Ptcd3, Rpl26, Rpl11, Cyb5b and Rab5a enriched
in BCG phagosomes, only Ptcd3, Rpl26 and Rpl11 were absent in TDM bead samples.
Discussion
105
This comparison indicates that TDM bead phagosomes may share more
characteristics with phagosomes containing BCG than those with M. tuberculosis. Of
note, the whole phagosome proteome data of M. tuberculosis H37Rv and BCG were
not available for comparison to the scientific community.
In experiments similar to ours, Shui et al. coated latex-beads with ManLAM from M.
tuberculosis, PILAM (phosphoinositol-capped LAM) from M. smegmatis as well as LPS
from E.coli [133]. All three bead types were fed to RAW264.7 macrophages and the
membrane fraction of bead phagosomes was isolated and analyzed in an iTRAQ-
labelled approach by mass spectrometry. 823 proteins were identified in total and 42
proteins were significantly up- or down-regulated (p > 1.25-fold, p < 0.05-fold) by
exposure of macrophages to ManLAM but not the other two lipoglycan beads:
Table 4-2: Comparison of proteins identified in ManLAM versus TDM bead phagosomes. Modified from [133]. The left column provides the list of proteins up- and down-regulated in ManLAM bead phagosomes compared to controls. The right column lists proteins identified in ManLAM bead that were present in TDM bead phagosomes as well.
ManLAM-specific phagosome proteome
TDM bead phagosome proteome
up-regulated:
transferrin receptor protein 1 vacuolar protein sorting-associated
protein 41 homologue Rab-5A Rab-5C Rab-14 isoform 1 of sequestosome-1 isoform 2 of sequestosome-1 ferritin H subunit calnexin heat-shock protein 90B1 tripeptidyl-peptidase 1 N-acetylglucosamine-6-sulfatase D-3-phosphoglycerate dehydrogenase alpha-N-acetylglucosaminidase vimentin titin 24 kDa protein Ddost
mannose-6-phosphate receptor CD63 scavenger receptor class B member 2 lysosomal membrane glycoprotein 1 lysosomal membrane glycoprotein 2 transmembrane protein 55A
mannose-6-phosphate receptor lysosomal membrane glycoprotein 1 transmembrane protein 55A
Discussion
106
early endosome antigen 1 vesicle transport through interaction with
subunit a vacuolar ATP synthase subunit B vacuolar ATP synthase subunit E1 vacuolar ATP synthase subunit S1 vacuolar ATP synthase subunit C vacuolar ATP synthase subunit F vacuolar ATP synthase subunit d vacuolar ATP synthase catalytic subunit
A isoform 1 of reticulon-4 cathepsin Z lysosomal acid phosphatase collectin subfamily member 12 zinc finger, FYVE domain containing 26
may also inactivate SNARE proteins responsible for membrane fusion events.
Moreover, retention of cholesterol itself might contribute to inhibition of phagosome
Discussion
121
maturation as accumulation of the lipid was shown to permit membrane fusion
processes [180].
Figure 4.1: Scheme of the potential function of TDM-mediated inhibition of phagosome maturation. TDM may (1) interact with host-cell proteins or lipids via its trehalose headgroup, (2) by intercalation of 1 mycolic acid with the phagosomal membrane or (3) by intercalation of the whole molecule to the phagosomal membrane. TDM may exert several mechanisms to inhibit phagosome maturation. TDM could influence the phagosomal membrane and retain phosphatidylserine (PS) and cholesterol (chol). PS and cholesterol are major binding partners for annexinA1 and annexinA6. Annexins then may induce actin nucleation at the phagosomal membrane by recruiting actin-binding-proteins and/or nucleation-promoting-factors as profilin1 (pfl1) to establish actin rims. Actin rims might inhibit fusion of the phagosome with multivesicular bodies (MVBs) and lysosomes. Furthermore TDM might retain cholesterol on the phagosomal membrane to inhibit fusion with MVBs and lysosomes because the lipid itself was shown to inhibit fusion processes. SNAREs SNAP23 and VAMP3 indicate the fusogenic properties of TDM bead phagosomes with early and intermediate endosomes but might also be manipulated and thus inactivated by TDM intercalating to the phagosomal membrane.
Discussion
122
4.7 Perspectives
In this PhD-thesis, a protocol for isolation and purification of magnetic lipid-coated
bead phagosomes was developed. Still, the protocol has its drawbacks. A critical step
is the trypsin digestion because it may remove potential TDM interaction partners from
the outer surface of the phagosomal membrane. Emphasis should be put on
development of another way to detach bead phagosomes from each other. In other
studies, bead phagosomes were freed by incubation with ATP that leads to
depolymerization of actin networks possibly gluing bead-phagosomes together.
Another critical step is the label-free approach for quantification of proteins. For the
future, macrophages should be metabolically labeled by stable-isotope-labeling-in-
cell-culture (SILAC) in order to establish a fully-quantitative method. Furthermore,
purification by FACS sorting should include IF-staining of bead phagosomes for
LAMP1 in order to sort bead phagosomes according to their maturation state in
addition to size.
Herein 34 proteins were discovered to be accumulated to TDM bead phagosomes
compared to controls. Of the latter, 6 were chosen for further analysis. An ongoing
attempt should be the analysis of remaining 27 proteins for their potential function in
TDM-mediated inhibition of phagosome maturation using IF-staining, Western blot and
siRNA knock-down/CFU experiments. Moreover, the siRNA knock-down/CFU
experiments shown herein were performed once. To further verify the results, the
experiment should be repeated at least three times, also for statistical reasons.
We could show actin accumulation on TDM bead and M. tuberculosis phagosomes
has a role in inhibition of phagosome maturation. SiRNA/CFU experiments suggested
that profilin1 and cofilin1 are important for survival of M. tuberculosis whereas WASH1,
annexinA6, SNAP23 and VAMP3 were not. Ongoing experiments must elucidate the
NPF of actin on phagosomes of TDM beads and M. tuberculosis. Potential candidates
are annexins as they are well known actin nucleators. Apart from analysing the survival
of M. tuberculosis in absence of candidate proteins, one must also check whether TDM
and the pathogen are still able to nucleate actin on their phagosomes and inhibit
phagosome maturation. This could be accomplished by siRNA knock-down and
subsequent IF-staining of phagosomes of TDM beads and M. tuberculosis by
lysotracker, LAMP1 and actin. The latter experiment is most important as it would
Discussion
123
directly show the link between the single virulence factor TDM and its interaction
partner.
Further experiments to evaluate the role of lipids in TDM-mediated inhibition of
phagosome maturation are difficult. In contrast to most proteins, depletion of lipids has
extensive impact on cell physiology. In addition, specific antibodies to certain lipids are
rare. However, ongoing experiments could include staining of TDM bead phagosome
membranes in macrophages using fluorescent dyes specific for cholesterol rich
domains. Moreover, for comparison with TDM bead phagosome samples, the lipid
composition of M. tuberculosis phagosomes should be analyzed.
To conclude, this thesis suggests that TDM inhibits phagosome maturation by
manipulating the cells actin cytoskeleton. However, experiments are required to further
corroborate this hypothesis. Moreover, this thesis provides several other interesting
TDM targets worth analyzing.
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124
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Supplementary material
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Supplementary material
Table 0-1: List of all proteins identified in control and TDM bead phagosomes. Identified protein Accession number EUSC