MOLECULAR MECHANISMS OF LISTERIA MONOCYTOGENES INVASION OF THE
INTESTINAL EPITHELIUM
A DISSERTATION SUBMITTED TO THE DEPARTMENT OF MICROBIOLOGY &
IMMUNOLOGY AND THE COMMITTEE ON GRADUATE STUDIES OF STANFORD
UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
Mickey Joseph Pentecost September 2009
ii
Copyright by Mickey Joseph Pentecost 2009 All Rights
Reserved
iii
I certify that I have read this dissertation and that, in my
opinion, it is fully adequate in scope and quality as a
dissertation for the degree of Doctor of Philosophy.
________________________________ (Manuel R. Amieva) Principal
Advisor
I certify that I have read this dissertation and that, in my
opinion, it is fully adequate in scope and quality as a
dissertation for the degree of Doctor of Philosophy.
________________________________ (Stanley Falkow)
I certify that I have read this dissertation and that, in my
opinion, it is fully adequate in scope and quality as a
dissertation for the degree of Doctor of Philosophy.
________________________________ (Julie A. Theriot)
I certify that I have read this dissertation and that, in my
opinion, it is fully adequate in scope and quality as a
dissertation for the degree of Doctor of Philosophy.
________________________________ (W. James Nelson)
Approved for the Stanford University Committee on Graduate
Studies. ________________________________
iv
ABSTRACT
Listeria monocytogenes causes invasive disease by crossing the
intestinal epithelial barrier. This process depends on the
interaction between the bacterial surface protein Internalin A
(InlA) and the host protein E-cadherin. A second L. monocytogenes
invasin Internalin B (InlB) promotes invasion of numerous
non-phagocytic cell types, but has not been shown to promote oral
infection. The receptor for InlB is c-Met, a receptor tyrosine
kinase and the endogenous receptor for Hepatocyte Growth Factor
(HGF). E-cadherin and c-Met are localized to the basolateral side
of polarized epithelial cells and are not thought to be accessible
to the apical (lumenal) side across functional tight junctions. We
used polarized MDCK cells as a model epithelium to determine how L.
monocytogenes gain access to basolateral receptors. We found that
L. monocytogenes do not actively disrupt the tight junctions, but
find E-cadherin at a morphologically distinct subset of
intercellular junctions. We identified these sites as naturally
occurring regions where single senescent cells are extruded from
the epithelium. The surrounding cells reorganize to form a
multicellular junction (MCJ) that maintains epithelial continuity.
We found that E-cadherin is transiently exposed to the lumenal
surface at MCJs during and after cell extrusion. We hypothesized
that L. monocytogenes utilize analogous extrusion sites for
epithelial invasion in vivo. By infecting rabbit ileal loops, we
found that the MCJs at the cell extrusion zone of villus tips are
the specific target for InlA-mediated L. monocytogenes adhesion and
invasion. L. monocytogenes expressing a modified InlA capable of
binding murine E-cadherin (InlAm) specifically invade and replicate
within villous tips of orally infected of mice. We hypothesized
that InlB functions synergistically with InlA to promote intestinal
invasion. Utilizing L. monocytogenes expressing InlAm, we found
that InlB promotes oral infection of mice and colonization of mouse
villous tips.
v We investigated the mechanism by which InlB mediates Listeria
invasion at MCJs using polarized MDCK monolayers. Following
InlA-mediated attachment at MCJs, cMet activation by InlB increases
the rate of bacterial uptake. The efficiency of invasion is also
controlled by intrinsic epithelial properties since MCJs undergo
rapid remodeling and are naturally more endocytic than other
junctional sites; MCJs endocytose fluorescent dextran, a fluid
phase marker, from the apical surface into unique cytoplasmic
puncta containing both tight- and adherens junction proteins.
Apical HGF or InlB increase the number and size of dextran puncta
at MCJs, but do not increase endocytosis at other junctions,
suggesting that c-Met is apically exposed at MCJs and that L.
monocytogenes can modulate cellular endocytosis during invasion of
this specific site. Thus, L. monocytogenes exploit the dynamic
nature of junctional remodeling and epithelial renewal to target
exposed receptors and hijack host cell processes for epithelial
invasion and intestinal barrier breach.
vi
ACKNOWLEDGMENTS
Throughout this thesis I use the pronoun we rather than I when
discussing results because this work has been possible only though
the assistance and intellectual contributions of many people.
First, I thank my Advisor Manuel Amieva. His patience and personal
generosity have made my graduate career enjoyable and rewarding. I
will rely on him throughout my life for both personal and
professional guidance. Manuel has also created a very interactive,
collaborative and friendly lab environment. I thank my friends and
colleagues Michael Howitt, Josephine Lee, Shumin Tan, Lee
Shaughnessy, Brooke Lane, Fabio Bagnoli, Roger Vogelman and
Elizabeth Joyce for experimental and intellectual help and
guidance, as well as for the coffee breaks, costume parties, soccer
games, travels, and baked goods. I wish to thank the rest of my
Thesis Committee. Stanley Falkow, James Nelson and Julie Theriot
are dedicated educators and wonderful people who have always been
constructive, available and generous with their time and expertise.
In addition to my committee, Denise Monack, Glen Otto, Susanne
Rafelski, Alex Nielsen, Ana Bakardjiev and Jonathan Hardy have been
mentors and collaborators. I acknowledge the people who made my
academic and intellectual growth possible. My parents Lyn and Dave
always provided love and encouragement, but more importantly, they
let me pursue all of my interests. They also gave me my younger
brother and Best Man Will, a chef/artists/musician/handball champ
who I admire. I inherited an interest in science from my
grandfathers, Joe Pentecost and Bill Tiefenbacher, and inherited a
love of literature and a sense of civic responsibility from my
grandmothers, Maxene Pentecost and Sally Tiefenbacher. My aunts and
uncles, especially Wendy, Ken, Moira, Tom and June have always
given love and support in every possible way, including housing,
warm meals and emotional support during my schooling.
vii I would like to thank a scientific mentor, role model, and
dear friend, Issar Smith. Smitty and his former graduate student
Ben Gold introduced me to biological research and began my training
microbiology. They also showed me that scientists dont have to
sacrifice a full life and dedication to family and friends to be
professionally successful and admired. Finally, I thank my best
friend and gorgeous wife, Tina Huang. Tina is the most talented,
warm and generous person in the world. I thank her for love and
support through all the successes, challenges, and changes of homes
we have had. I also thank her for keeping art and excitement in my
life.
viii DEDICATION
The author wishes to dedicate this dissertation to Grandma
Sally.
ix TABLE OF CONTENTS
List of Tables
...............................................................................................................
xiii List of
Figures..............................................................................................................
xiv List of Videos
.............................................................................................................
xvii Chapter 1 : Introduction and Literature Review
............................................................... 1
Research Rationale
....................................................................................................
1 History
......................................................................................................................
2 Listeria monocytogenes, an Enteroinvasive Bacterial
Pathogen............................ 2 Identification
.......................................................................................................
3 Listeriosis: a Disease of Humans and
Animals..................................................... 5
Pathogenesis and
Epidemiology...........................................................................
6 Listeria in Basic Biological Research
..................................................................
8 Intracellular
Parasitism............................................................................................
10 Stages and Mechanisms of Listerias Intracellular
Life-Cycle............................ 10 Genetic Regulation of
Intracellular Infection
..................................................... 12 Adherence
to the Cell
Surface............................................................................
16 Internalin A, Internalin B and the Internalin
Family........................................... 16 Internalin A
Co-opts the Epithelial Junctions
..................................................... 20 Internalin
B Co-opts Growth Factor Signaling
................................................... 23 Synergy
Between Internalin A and Internalin B
................................................. 26 Species
Specificities of Internalin A and Internalin B
........................................ 27 Animal Models
Permissive for Internalin A and Internalin
B............................. 29 Tissue Specificities of
Internalin A and Internalin B
.......................................... 30 Challenging the
Internalin Tissue Specificity
Dogma......................................... 30 Anatomical and
Subcellular Site of Epithelial Invasion: The Polarity
Paradox......... 31 The Epithelial Barrier, Cell Renewal and
Gastrointestinal Pathogens................. 31 E-cadherin and c-Met
are Basolateral Receptors
................................................ 32 The Peyers
Patch Paradigm
..............................................................................
33 Challenging the Peyers Patch
Paradigm............................................................
34
x Chapter 2 : Internalin A Targets Listeria monocytogenes to
Epithelial Junctions at Sites of Cell
Extrusion...................................................................................................
36
Introduction.............................................................................................................
36 Materials and Methods
............................................................................................
38 Results
....................................................................................................................
43 L. monocytogenes Invade The Epithelium at Distinct Multicellular
Junction
Sites.............................................................................................................
43 Apical Attachment to Multicellular Junctions is Dependent on
Internalin A....... 48 Quantitative Analysis of L. monocytogenes
Adhesion Sites ............................... 50 Multicellular
Junctions Form and Persist at Sites of Cell
Extrusion.................... 52 L. monocytogenes Attachment Sites
are Sites of Cell Extrusion ......................... 55 L.
monocytogenes Adhere to Transiently Exposed E-cadherin at Sites of
Cell
Extrusion..............................................................................................
59
Discussion...............................................................................................................
65 Chapter 3 : Internalin A targets L. monocytogenes to the Villus
Tip Extrusion Zone...... 69
Introduction.............................................................................................................
69 Materials and Methods
............................................................................................
72 Results
....................................................................................................................
80 L. monocytogenes Invade Multicellular Junctions at the Villus
Tip Extrusion Zone
............................................................................................
80 Analysis of L monocytogenes Infection of the Villous Tip
Extrusion Zone by Transmission Electron Microscopy
......................................................... 87
Rational Design of Internalin A variants Predicted to Bind Murine
Ecadherin.......................................................................................................
90 Mutations in Internalin Differentially Affect the Tropism of
Listeria for Epithelial Cells of Different Species
............................................................ 90
InlA S192N Y369S (InlAm) Permits Oral Infection of
Mice............................... 95 InlAm Specifies Invasion of
the Villus Tips, But Not of Peyers Patches ............ 98
Discussion.............................................................................................................
101 Chapter 4 : InlB Targets c-Met and Modulates Endocytosis at
Multicellular Junctions
.....................................................................................................................
103
xi
Introduction...........................................................................................................
103 Materials and Methods
..........................................................................................
105 Results
..................................................................................................................
111 Construction of GFP-expressing Listeria
Strains.............................................. 111 inlB GFP
L. monocytogenes Express InlA
..................................................... 113 inlB L.
monocytogenes Recruit E-cadherin and -catenin to Sites of Bacterial
Attachment
.................................................................................
116 Internalin B promotes Apical Invasion of Multicellular
Junctions .................... 118 InlB Targets c-Met Locally
During Apical Invasion of Polarized MDCK
Monolayers................................................................................................
120 InlB Does Not Influence Listeria Intracellular Replication and
Cell-to-cell Spread Within a Polarized
Epithelium........................................................
122 Junctional Remodeling at Multicellular Junctions Correlates
with Increased Apical Endocytosis
....................................................................................
124 InlB and HGF Modulate Dextran Endocytosis at Multicellular
Junctions......... 128 Apical Endocytosis AND L. monocytogenes
Invasion at Multicellular Junctions Require Common Endocytic
Machinery ..................................... 128
Discussion.............................................................................................................
132 Chapter 5 : InlB Promotes L. monocytogenes Oral Infection and
Colonization of the Villus Tip Extrusion Zone
.....................................................................................
136
Introduction...........................................................................................................
136 Materials and Methods
..........................................................................................
139 Results
..................................................................................................................
145 Development and Verification of L. monocytogenes Strains
expressing InlAm, InlB and GFP
..................................................................................
145 InlB Promotes Oral Infection of
Mice..............................................................
148 InlB Promotes Colonization of the Villus Tip Extrusion Zone
......................... 150
Discussion.............................................................................................................
154 Chapter 6 : Questions for Future
Research...................................................................
157 Do Other Enteric Bacteria Utilize Cell Extrusion
Zones?....................................... 157
xii Are Multicellular Junctions Inherently Permissive for
Bacterial Uptake In Vivo?
...............................................................................................................
159 What are the Cellular Molecular Mechanisms of Listeria Invasion
or Junctional endocytosis at Multicellular Junctions?
........................................... 159 How are Basolateral
Receptors Exposed to the Lumenal Surface at Multicellular
Junctions?...................................................................................
161 How Do Listeria and Host Cells Interact at the Villous Tips?
................................ 162 Are the Villus Tips a Site of
Colonization or Asymptomatic Carriage?.................. 162
Bibliography
...............................................................................................................
165
xiii LIST OF TABLES
Number......................................................................................................................Page
Table 3.1: Oligonucleotides Used in Chapter
3.............................................................. 78
Table 3.2: L. monocytogenes Strains Used in Chapter
3................................................. 79 Table 4.1:
Oligonucleotides Used in Chapter
4............................................................ 110
Table 4.2: L. monocytogenes Strains Used in Chapter
4............................................... 110 Table 5.1:
Oligonucleotides Used in Chapter
5............................................................ 143
Table 5.2: L. monocytogenes Strains Used in Chapter
5............................................... 144
xiv LIST OF FIGURES
Number......................................................................................................................Page
Figure 1.1: Schematic Representation of the Pathophysiology of
Listeria Infection......... 2 Figure 1.2: Molecular and Genetic
Requirements for Listerias Intracellular LifeCycle (Following Page)
...............................................................................
14 Figure 1.3: Domain Organization of Internalins and Sequence
Alignments of Internalin LRR Domains (Following Page)
................................................. 18 Figure 1.4:
Structural and Molecular Aspects of InlA/E-cadherinmediated
Listeria Invasion (Following Page)
.............................................................. 21
Figure 1.5: Structural Aspects of InlB/c-Met-mediated Listeria
Invasion (Following Page)
.........................................................................................
24 Figure 1.6: Listeria versus The Apical Junctional Complex of
Polarized Epithelia ........ 35 Figure 2.1: Preservation of Barrier
Function During L. monocytogenes Infection of MDCK Cells Polarized
on Transwell Filters (Following Page)..................... 44
Figure 2.2: Invasion and Replication of L. monocytogenes at
Multicellular Junction Sites (Following
Page).................................................................................
46 Figure 2.3: Internalin A-dependent Apical Adhesion and Invasion
of Polarized Epithelia
......................................................................................................
49 Figure 2.4: Tropism of L. monocytogenes for Multicellular
Junctions............................ 51 Figure 2.5: Multicellular
Junctions Created by Cell Extrusion
....................................... 53 Figure 2.6: L.
monocytogenes Adhesion to Sites of Cell Extrusion
................................ 56 Figure 2.7: E-cadherin
Associated with L. monocytogenes at Multicellular
Junctions......................................................................................................
57 Figure 2.8: L. monocytogenes Attachment to Accessible E-cadherin
at Multicellular Junctions of Cell Extrusion
Sites............................................. 60 Figure 2.9:
Increased E-cadherin Exposure and L. monocytogenes Adhesion in
Calcium Depleted MDCK Monolayers
........................................................ 63 Figure
2.10: L. monocytogenes Adherence to Single Cell Polarity Defects
.................... 64
xv Figure 3.1: L. monocytogenes Invasion of the Intestinal
Epithelium at the Villustip Extrusion Zone (Following
Page)............................................................
81 Figure 3.2: Lack of Association of L. monocytogenes Invasion
with the Intestinal Crypts or the Peyers Patches; inlA-mutant is
Noninvasive (Following Page)
...........................................................................................................
83 Figure 3.3: L. monocytogenes Associate with Intercellular
Junctions Prior to Villus Tip
Invasion.................................................................................................
88 Figure 3.4: L. monocytogenes Infect Cells Adjacent to Apoptotic
Cells at the Villous
Tip...................................................................................................
89 Figure 3.5: Internalin Variants with Altered Tropism for Canine
and Murine Cells ....... 93 Figure 3.6: InlA R168S E170G Q190G
Promotes Invasion of the Murine Intestine....... 94 Figure 3.7:
InlA S192N Y369S Reconstitutes Oral Infection in Mice (Following
Page)
...........................................................................................................
96 Figure 3.8: Functional InlA Targets Listeria to the Villus Tip
Epithelium (Following Page)
.........................................................................................
99 Figure 4.1: Construction of GFP Expression Constructs and
Verification of InlA and InlB Expression in GFP Listeria Strains
.............................................. 112 Figure 4.2:
Activation of c-Met and MDCK Cell Scattering by Purified InlB
.............. 115 Figure 4.3: Recruitment of E-cadherin and
-catenin by Listeria to Sites of Attachment
................................................................................................
117 Figure 4.4: InlB Mediated Apical Invasion of Polarized MDCK
Monolayers. ............. 119 Figure 4.5: Invasion of Listeria
Through Local c-Met Activation ................................ 121
Figure 4.6: Lack of a Role for InlB in Intracellular Replication
and Cell-to-Cell Spread
.......................................................................................................
123 Figure 4.7: Unique Para-endocytosis of Junctional Components at
Sites of Cell Extrusion (Following Page)
......................................................................
125 Figure 4.8: Enhancement of Dynamin-dependent Endocytosis at
Multicellular Junctions by HGF and InlB (Following Page)
............................................ 130 Figure 5.1:
Construction of Expression Constructs and Verification of InlAm and
InlB Expression in Listeria
........................................................................
147
xvi Figure 5.2: Analysis of the role of InlB in Listeria Oral
Infections by Bioluminescence
Imaging..........................................................................
149 Figure 5.3: Single Infection of Mice with inlAB inlAmB gfp or
inlAB InlAm gfp........ 151 Figure 5.4: InlB-mediated Invasion of
the Intestinal Villus Tips.................................. 152
Figure 5.5: Coinfection with inlAB inlAmB and inlAB inlAm
gfp............................... 153
xvii LIST OF VIDEOS
Number......................................................................................................................Page
Video 2.1: Cell
Extrusion..............................................................................................
54 Video 2.2: L. monocytogenes Invasion at a Multicellular
Junction................................. 58 Video 3.1: Villus Tip
Infected with Wild Type Listeria monocytogenes
........................ 85 Video 3.2: Villus Tip Infected with
inlA L. monocytogenes......................................... 86
Video 4.1: Junctional Endocytosis at Cell
Extrusion....................................................
127
1
CHAPTER 1 : INTRODUCTION AND LITERATURE REVIEW
RESEARCH RATIONALE In the United States, food-borne pathogens
have been estimated to be responsible for 76 million illnesses,
323,914 hospitalizations, and 5,194 deaths each year (Mead et al.,
1999). Invasive microorganisms that infect the intestinal
epithelium and breach the intestinal barrier cause more than 75% of
these deaths. Listeria monocytogenes has emerged as a significant
cause of mortality due to food-borne illness in the United States
since it was responsible for 27.6% of deaths from enteric infection
(Mead et al., 1999). Other important pathogens that invade the
epithelium include rotavirus, Salmonella, Shigella, Yersinia,
Enteroinvasive E. coli, and Campylobacter. How these organisms
breach the gastrointestinal epithelial barrier is not fully
understood. Specific interactions between microbial adhesins and
cell surface receptors are known to be critical for invasion (Boyle
and Finlay, 2003). Paradoxically, many of the known
adhesin-receptor interactions involve cellular receptors that are
not typically present on the apical (lumenal) side of the
gastrointestinal epithelium because intact intercellular junctions
prevent the diffusion of these molecules from the basolateral to
the apical side of the epithelial cells. For example rotavirus,
Shigella and Yersinia are known to use integrin receptors for
attachment and entry through the basolateral (interstitial)
surfaces of epithelial cells (Ciarlet et al., 2002; Graham et al.,
2003; Guerrero et al., 2000; Hewish et al., 2000; Isberg and Leong,
1990; Mounier et al., 1992; Watarai et al., 1996). Similarly,
Listeria monocytogenes use the basolateral junction protein
E-cadherin and the basolateral signaling protein c-Met for
epithelial cell invasion (Mengaud et al., 1996; Shen et al., 2000).
How and where L. monocytogenes find receptors for attachment and
entry are important questions in the pathogenesis and clinical
outcomes of microbial gastroenteritis and enteric fever.
2 HISTORY , AN ENTEROINVASIVE BACTERIAL PATHOGEN The
Gram-positive, facultative intracellular bacterium is a cause of
human and animal food-borne infection (Vazquez-Boland et al.,
2001). The initial steps in the pathogenesis of Listeriosis involve
colonization and growth in the intestinal tissue, followed by
spread to other organs via the lymphatics or blood stream (Figure
1.1) (Vazquez-Boland et al., 2001). Invasive Listeriosis is one of
the most deadly bacterial infections with a mortality of ~30%, and
the ability of to survive within hosts is attributed to the
organisms sophisticated intracellular infection cycle: hijacks
endocytic machinery to invade cells, escapes from the vacuole to
replicate within the host cell cytosol, and recruits components of
the host cell cytoskeleton to translocate to neighboring cells, all
the while avoiding the humoral immune system (Figure 1.2A) (Pamer,
2004; Portnoy et al., 2002; Vazquez-Boland et al., 2001).
Figure 1.1: Schematic Representation of the Pathophysiology of
Figure from (Vazquez-Boland et al., 2001).
Infection
3
IDENTIFICATION Listeria monocytogenes was identified and
described in two independent reports in 1926 and 1927. In the
first, Murray, Webb and Swann isolated the bacterium during
epidemic outbreak of lethal disease in their rabbit colony in
Cambridge, England (Murray et al., 1926). The disease, affecting
young animals within the first few months of age and pregnant
animals, was characterized by development of a distended belly,
rapid weight loss and lethargy interrupted by convulsive struggles.
Necropsies revealed edema of subcutaneous tissues, accumulation of
fluids in pleural, pericardial and peritoneal cavities, enlarged
and edematous mesenteric lymph nodes, foci of necrosis in the
liver, and enlarged spleens. These are now well-known pathologies
caused by invasive Listeria. Although the gastrointestinal tract
would not be recognized as a site of L. monocytogenes host invasion
until the 1960s and 1970s, Murray et al. made at least two
important observations that could have put the field in the right
direction. First, the authors stated that the that outbreaks had
always occurred at times when the fresh food upon which the
breeding establishment largely depended either became scarce or
rank, and noted that adequate food would terminate the epidemic.
Second, they identified the location of L. monocytogenes invasion
within the gastrointestinal tract since they could trace the most
affected mesenteric lymph nodes to terminal ileum via the
connecting mesenteries. However, they did not understand the
significance of these observations since recreating infection in
rabbits and guinea pigs through the oral route was inefficient. The
strength of the gastrointestinal tract as a barrier to Listeria
invasion hampered the recognition of this site as the natural route
of infection, and continues to hamper research into the
gastrointestinal phase of Listeriosis. The feature of the disease
that was most compelling to Murray et al. was the ability of the
bacterium to elicit a huge increase in the number of circulating
monocytes in the blood. For this reason, they named the organism
Bacterium monocytogenes. Unfortunately, this characteristic also
resulted in the erroneous belief, held through the 1960s, that
Listeria was the (or a) cause of infectious mononucleosis despite
evidence
4 of viral etiology since the late 1930s and despite the fact
that monocytosis was never a marked feature of human Listeriosis
(Gray and Killinger, 1966; Murray, 1955; Schultz, 1945). Without
knowledge of Bacterium monocytogenes, in 1927 Pirie identified the
bacterium from the livers of a South African gerbil (known as the
African Jumping Mouse) suffering from a plaque-like disease with
necrotizing hepatic infection (Gray and Killinger, 1966; Pirie,
1927). Pirie named the bacterium Listerella hepatolytica in honor
of Joseph Lister (1827 1912), the British surgeon and one time
president of the Royal Society who promoted sterile surgery. In
1940, Pirie suggested the use of the current name Listeria
monocytogenes, since Bacterium monocytogenes and Listerella
hepatolytica were identified as the same, and since the genus
Listerella had already been assigned to a group of slime molds in
1906 (Pirie, 1940). In retrospect, L. monocytogenes was probably
identified a number of times prior to 1926. In the 1890s,
Gram-positive rods were isolated from tissue sections from patients
who died of Listeriosis-like disease in Germany and In 1911 an
organism named Bacterium hepatitis was isolated from necrotic foci
in the liver of a rabbit in Sweden (Gray and Killinger, 1966;
Murray, 1955). In 1917 a diptheroid with L. monocytogenes
characteristics was isolated from 5 children with meningitis in
Australia and in 1921 a bacterium later confirmed as L.
monocytogenes was isolated from the cerebrospinal fluid of an
Italian soldier (Schultz, 1945). Even after 1926, the differences
in symptoms and diseased hosts, and the difficulty of strain
characterization led identification of a number of species only
later confirmed as L. monocytogenes. These include hemolytic
Corenybacterium, Listerella hepatolytica, Listerella monocytogenes
hominis, Corenybacterium parvulum, Listerella ovis, Corenybacterium
infantisepticum, Listeria infantiseptica, Listerella bovina, L.
gallinarium, L. cuniculi, L. suis, and L. gerbilli (Gray and
Killinger, 1966; Schultz, 1945).
5 LISTERIOSIS: A DISEASE OF HUMANS AND ANIMALS In the three
decades following Murray et al. and Pirie, only sporadic cases of
human Listeriosis were reported (Murray, 1955). Rather, Listeriosis
was a curious disease of animals. Listeria was found associated
with numerous species including rabbit, hare, guinea pig, gerbil,
lemming, mouse, rat, hamster, vole, sheep, goat, cattle, pig,
horse, dog, ferret, raccoon, fox, chicken, canary duck, goose and
eagle (Gray and Killinger, 1966; Murray, 1955; Schultz, 1945). It
should be noted that a number of these associations are based on
fecal shedding as in mice and rats rather than disease. Despite the
initial identification of L. monocytogenes in rabbits, domestic
livestock were recognized as the major victims of Listeriosis,
though with some differences in symptoms and pathology. In contrast
to rabbits and some other monogastric animals, ruminants do not
develop monocytosis. Rather, young sheep and cattle can develop
septicemia with or without encephalitis (Gray and Killinger, 1966).
Listeriosis of ruminants was often called circling disease since
encephalitic animals were observed walking in circles
(Vazquez-Boland et al., 2001). Between the first confirmed
identification of neonatal human Listeriosis in 1933 and the early
1950s, encephalitis, meningitis and meningoencephalitis in
non-pregnant humans and animals was given the majority of medical
and experimental attention (Gray and Killinger, 1966). However,
pregnancy-associated Listeria infection soon became a great concern
when in the early 1950s, hundreds of tragic reports of neonatal
deaths came from hospitals of bombed cities in East Germany being
reconstructed after the war. Gray and Killinger wrote that, life,
or even existence, was difficult. Food was poor, meager, and
rationed, and essentials, such as milk for pregnant women, were
found only in the black markets. Among the many who died were the
yet unborn. Some of these stillborn infants showed characteristic,
distinctive focal necrosis throughout their tiny bodies (Gray and
Killinger, 1966). This generalized infection with extensive focal
necrosis of the liver, infection of the lungs, central nervous
system and skin was named granulomatosis infantiseptica. It is now
known that some infants can also be born apparently well and
develop disease within days or weeks post partum. Although the
majority of cases of Listeriosis have been associated
6 with pregnancy, attention is returning to adult disease, which
is on the increase due to immune suppression by HIV and immune
suppressant therapies associated with organ transplants (Farber and
Peterkin, 1991).
PATHOGENESIS AND EPIDEMIOLOGY Prior to the use of antibiotics,
mortality of invasive Listeriosis was at least 70% (Gray and
Killinger, 1966). By the late 1960s, that had dropped to roughly
50% and now stands at roughly 30% (Farber and Peterkin, 1991; Gray
and Killinger, 1966; Mead et al., 1999; Vazquez-Boland et al.,
2001). We note that the statistics are skewed by the high mortality
rates in the very young and very old; The case fatality rates is
thought to be as high as 50% for infants and as high as 20% for
people over 60 years of age (Bortolussi, 2008). Despite the
decrease in percent mortality, Listeriosis remains one of the most
deadly food-borne illnesses. Furthermore, over the decades there
has been an increase in both incidence of infection and in total
death, even when accounting for improved detection and diagnosis
(Farber and Peterkin, 1991). By the 1960s only ~ 500 human deaths
due to L. monocytogenes were identified throughout the world (Gray
and Killinger, 1966). More recent estimates suggest approximately
500 deaths per year in the U.S. (Mead et al., 1999). Factors
contributing to the increase in Listeriosis include industrialized
farming and industrialized food production which has increased
prevalence of L. monocytogenes in the environment and in food.
Because of industrialization of cattle production, cows now
represent 80-90% of all animal Listeriosis, and livestock
infections can be traced to contaminated feed, notably poorly
fermented silage (Fenlon, 1985, 1986). The epidemiological
connection between silage feed and infection had been made by the
early 1960s, although confirmation of the food-borne route of
infection would wait nearly 20 years with the advent of human
epidemics (Farber and Peterkin, 1991; Gray and Killinger, 1966;
Vazquez-Boland et al., 2001). Infected animals perpetuate the
expansion of L. monocytogenes in the environment and at least 10%
of asymptomatic animals are known to shed L. monocytogenes in their
feces (Esteban et al., 2009; Husu, 1990; Unnerstad et al., 2000)
Thus, livestock amplify and shed L.
7 monocytogenes in the farm environment, leading to new or
sustained infections and potential contamination of animal and
human foodstuffs (Farber and Peterkin, 1991; Gray and Killinger,
1966; Nightingale et al., 2005; Nightingale et al., 2004).
Industrialized food processing of ready to eat food products has
led to increased exposure because L. monocytogenes grows at
refrigeration temperatures and is highly resistant to food
preservation techniques such as smoking, curing or added chemical
preservatives. A study surveying luncheon meats, deli salads, fresh
soft "Hispanicstyle" cheeses, bagged salads, blue-veined and soft
mold-ripened cheeses, smoked seafood, and seafood salads detected
Listeria in nearly all food types as high as 106 CFU / g (Gombas et
al., 2003). L. monocytogenes contamination has resulted in numerous
disease epidemics and costly food recalls (Gottlieb et al., 2006).
Protection of food from L. monocytogenes is of such great financial
and public health importance that the FDA recently approved
bacteriophage (listeriocidal virus) as a food additive to ready to
eat meat and poultry products (Lang, 2006). Frequent and severe
outbreaks of Listeriosis from ready to eat foods beginning in the
1980s provided the first unequivocal epidemiological evidence that
Listeria is a foodborne pathogen. Previously proposed routes of
invasion included inhalation, ocular inoculation, cutaneous
infection (either by tick bite or by handling contaminated animal
material), through sexual transmission, and from mothers to
newborns through the vaginal canal during child-birth (Gray and
Killinger, 1966). Because many thought that Listeria had to be
transmitted directly from animal to human, Gray and Killinger made
the off-color joke: it is fortunate that L. monocytogenes has not
been isolated from a stork, or surely this poor bird would be
blamed not only for his big bill but also for transmitting the
bacterium to newborn infants (Gray and Killinger, 1966). The link
to food was made in 1981 after an epidemic in Canada involving 41
people (34 perinatal and 7 adult) was linked to consumption of
prepackaged ready-to-eat coleslaw. A sample of coleslaw from a
patients refrigerator was contaminated with the epidemic strain,
and the cabbage was traced to a farm that fertilized with manure
from a flock of sheep that had two members die of Listeriosis
(Schlech et al., 1983).
8 More recently, an outbreak of Listeriosis in Canada in the
summer of 2008 resulted in 57 confirmed cases and 22 deaths. The
outbreak was traced to contaminated meat from the processing plant
of Maple Leaf food products and caused a massive nationwide recall
of 220 products from the company (Austen, 2008; Canada, 2009). In
addition to epidemiological correlations, molecular genetics and
phylogenetics show that L. monocytogenes evolved to invade the
intestinal epithelium (see below). The emergence of the AIDS
epidemic was also a major factor influencing the increase in
incidence of Listeriosis in the 1980s. As an intracellular
pathogen, L. monocytogenes avoids a humoral immune response, and
antibodies are not protective against L. monocytogenes (Cerny et
al., 1988; Miki and Mackaness, 1964). Rather clearance of L.
monocytogenes requires components of cell-mediated immunity
including neutrophils and activated macrophages, and protective
immunity requires CD8 T-cells (Pamer, 2004). A majority of adults
with Listeriosis have underlying conditions that suppress T-cell or
other cellular immune responses (Farber and Peterkin, 1991;
Vazquez-Boland et al., 2001). These include leukemias, lymphomas,
chemotherapy, immunosuppressant therapy, cirrhosis of the liver,
alcoholism, kidney disease, diabetes, lupus, advanced age, and HIV
infection. HIV as a predisposing factor accounts for as much as 20%
of adult Listeriosis (Farber and Peterkin, 1991; Vazquez-Boland et
al., 2001). That it is not higher is probably due to frequent
treatment of AIDS patients with antimicrobials for numerous
infections (Farber and Peterkin, 1991). These data also suggests
that L. monocytogenes should be considered an opportunistic
pathogen, targeting the very young, the very old and the immune
compromised. The corollary is that invasive disease may be a
distraction from the real or evolved natural biology of Listeria
infection, which probably includes subclinical carrier states in as
yet unrecognized natural hosts.
LISTERIA IN BASIC BIOLOGICAL RESEARCH Immunologists were
interested in L. monocytogenes long before the emergence of the
organism as a public health risk. Initially, L.
monocytogenes-induced monocytosis in rabbits was used to
investigate the origin and development of monocytes (Gray and
9 Killinger, 1966). Since the 1960s, L. monocytogenes has been
used as a model intracellular parasite and was instrumental in
understanding innate and protective cell mediated immunity,
including the roles of T-cells and activated macrophages in
intracellular parasite clearance (Mackaness, 1962, 1969; Miki and
Mackaness, 1964; North, 1970, 1978; Pamer, 2004; Shaughnessy et
al., 2007). In the past 20 years, with the increasing power of
biochemical and genetic approaches, L. monocytogenes has
contributed to molecular dissection of intracellular (e.g. TLRs,
NODs, NFB, Caspase-1, Myd88) and intercellular (e.g. CCL2, TNF,
IFN-, IFN-) immune signaling (Pamer, 2004). In the 1990s and 2000s,
L. monocytogenes emerged as a tool for studying cell biology (Hamon
et al., 2006). (There are now numerous books and journals devoted
to this approach; e.g. Cellular Microbiology and Cell Host and
Microbe.) Stanley Falkow has stated that bacteria are the worlds
best cell biologists and Julie Theriot writes on her lab website,
we spy on them [L. monocytogenes] as they've spied on cells, trying
to learn what they know. For example, the ability of L.
monocytogenes ActA to polymerize actin forming a propulsive actin
comet tail has shed great light on mechanisms of eukaryotic cell
motility and cytoskeletal force generation, the biomechanics and
biochemistry of actin polymerization, and the physical properties
of the eukaryotic cytosol (Auerbuch et al., 2003; Cameron et al.,
2000; Chakraborty et al., 1995; Dabiri et al., 1990; Domann et al.,
1992; Kocks et al., 1992; Lacayo and Theriot, 2004; Niebuhr et al.,
1997; Rafelski and Theriot, 2002, 2004; Robbins et al., 1999;
Shenoy et al., 2007; Skoble et al., 2000; Tilney and Portnoy,
1989). The ability of L. monocytogenes InlA to bind the junctional
protein E-cadherin has shed light on the components and function of
the intercellular junctions. For example, ARHGAP10 (Rho
GTPase-activating protein 10) was found to be necessary for InlA
mediated bacterial invasion and then shown to be a novel regulator
of the epithelial junctions (Sousa et al., 2005a). A second
Invasin, InlB targets c-Met, a receptor kinase, to induce bacterial
uptake by Clathrin-mediated endocytosis and has been used to study
c-Met trafficking (Li et al., 2005; Veiga and Cossart, 2005). We
believe that the study
10 of InlA/InlB mediated Listeria invasion will provide a fuller
understanding of how intercellular junctions are regulated by
endocytosis.
INTRACELLULAR PARASITISM Although some normally extracellular
bacteria are capable of survival and replication within the cytosol
of cells, e.g. when given access by microinjection, it should not
be assumed that the cytosol is necessarily hospitable (Goetz et
al., 2001). Although little is known about the specific chemical
makeup of the cytosol, intracellular bacteria have taught us that
the cytosol is a reducing environment limiting in free iron and
aromatic amino acids (Ray et al., 2009). Nor is the cytosol
necessarily a protective environment. Intracellular bacteria must
find a new niche before significant intracellular immune detection
or host cell killing that would expose the bacteria to humoral and
inflammatory host responses. Thus, intracellular bacteria like
Shigella flexneri, Burkholderia pseudomallei, Listeria
monocytogenes, Francisella tularensis and Rickettsia species have
evolved mechanisms to invade cells, escape the primary vacuole,
acquire nutrients, modulate intracellular immune detection, and in
some cases spread directly to neighboring cells, avoiding exposure
to the extracellular milieu (Ray et al., 2009).
STAGES AND MECHANISMS OF LISTERIAS INTRACELLULAR LIFE-CYCLE L.
monocytogenes is capable of infecting phagocytic and nonphagocytic
cells. Surface proteins like Internalin A (InlA) and Internalin B
(InlB) bind host cell receptors and induce internalization of
bacteria by nonphagocytic cells. Internalization of Listeria occurs
through a so-called zipper-like mechanism where host cell plasma
membrane is closely opposed to the bacterium during internalization
(Figure 1.2A) (Karunasagar et al., 1994). Following initial
internalization, cytosolic bacteria escape from the
vacuole/endosome by secreting enzymes that disrupt the vacuolar
membrane. Listeria uses the enzymes listeriolysin O (LLO) and two
type C phospholipases (Figure 1.2A) (Portnoy et al., 1988; Portnoy
et al., 1994; Portnoy et al., 1992; Smith et al., 1995a; Smith and
Portnoy, 1993). LLO binds cholesterol in the vacuolar membrane and
forms
11 pores, which serve to prevent vacuolar maturation into a
lysosome and to destabilize the membrane for bacterial escape
(Beauregard et al., 1997; Bielecki et al., 1990; Henry et al.,
2006; Portnoy et al., 1992; Shaughnessy et al., 2006). hly,
encoding LLO, is transcribed only after cell invasion (see below)
and LLO is activated by acidification of the vacuole and by the
host derived enzyme gamma-interferoninducible lysosomal thiol
reductase (Glomski et al., 2002; Singh et al., 2008). Following
vacuolar disruption LLO is rapidly degraded in the cytosol (Glomski
et al., 2003; Glomski et al., 2002; Schnupf et al., 2006; Schnupf
et al., 2007). This temporal and spatial compartmentalization of
LLO expression and activity prevents disruption of cell plasma
membranes that would cause cytotoxicity and expose Listeria to the
extracellular environment (Glomski et al., 2003; Schnupf and
Portnoy, 2007). The secreted phosphatidylinositol-specific
phospholipase C (PI-PLC, encoded by plcA) functions with LLO to
disrupt the single membrane primary vacuole (Figure 1.2A). Some
cytosolic bacteria like Listeria monocytogenes, Shigella flexneri,
Burkholderia pseudomallei, Rickettsia spp., Mycobacterium marinum
and viruses like Vaccinia virus have evolved mechanisms to hijack
the host cell cytoskeleton for intracellular and intercellular
motility. Either by expressing proteins that directly bind actin,
or by expressing proteins that bind actin nucleators, these
microbes polymerize and elongate actin filaments to generate
propulsive actin comet tails (Cudmore et al., 1995; Ray et al.,
2009; Stamm et al., 2003). Upon entry into the cytosol L.
monocytogenes expresses the surface protein ActA, which directly
binds actin (Figure 1.2A) (Kocks et al., 1992; Theriot et al.,
1992; Tilney and Portnoy, 1989). Listeria actin comet tail
formation requires ActA concentrated on a single pole of the
bacterium (Kocks and Cossart, 1993; Kocks et al., 1993; Smith et
al., 1995b). This occurs only after intracellular L. monocytogenes
have undergone a few rounds of replication since polarization of
ActA is linked to new ActA synthesis and to cell wall growth (Moors
et al., 1999; Rafelski and Theriot, 2005, 2006). Thus, ActA
expression and actin tail formation is also a good indication of
bacterial viability in the cytosol. When actin comet tails propel
the bacterium into host cell plasma membrane, a neighboring cell
may internalize the resulting protrusion, or listeriopod
(Figure
12 1.2A). Presented but unpublished research from Keith Iretons
lab suggests that the virulence factor InlC may promote
cell-to-cell spread by interacting with the apical junctions and
making them more slack and permissive for protrusion formation
(Engelbrecht et al., 1996; Rajabian, 2008). Successful uptake of
the protrusion also requires cooperation of the recipient cell and
may depend on the state of cell-cell adhesion and/or the
organization of the submembranous cytoskeleton (Robbins et al.,
1999). Once the protrusion is fully internalized in the recipient
cell, the resultant double membrane vacuole is disrupted by LLO and
phosphatidylcholine-specific phospholipase C (PC-PLC, encoded by
plcB) (Camilli et al., 1993; Marquis and Hager, 2000; Smith et al.,
1995a). A Listeria metalloprotease (Mpl) regulates this process by
proteolytically activating PC-PLC upon acidification of the
secondary vacuole (Marquis et al., 1997). Free bacteria can now
repeat the intracellular infectious cycle, which is critical for
Listeria pathogenesis. Loss of any stage of the intracellular
infectious cycle severely attenuates Listeria pathogenicity
(Portnoy et al., 2002).
GENETIC REGULATION OF INTRACELLULAR INFECTION Many of the genes
required for the intracellular infection cycle of L. monocytogenes
are organized in a genetic island, LIPI-1 (Figure 1.2B). Following
cell invasion, this core set of genes is upregulated by the master
regulator of virulence genes, Positive Regulatory Factor A (PrfA).
(Chakraborty et al., 1992). PrfA also regulates some virulence
genes outside LIPI-1, such as the inlAB locus, inlC and hpt (Figure
1.2B) (Chico-Calero et al., 2002; Engelbrecht et al., 1996). InlC
is required for full virulence and may promote cell-to-cell spread
(Engelbrecht et al., 1996; Rajabian, 2008). hpt encodes a
glucose-6-phosphate translocase that allows pathogenic Listeria
species to use hexose phosphates from the host cell cytosol as a
carbon energy source (ChicoCalero et al., 2002). To regulate genes,
PrfA dimers bind a 14-bp (7-bp invariant) consensus sequence or a
PrfA-box directly upstream of promoters (Figure 1.2B). PrfAs
activity is regulated on numerous levels including autoregulation
of its own transcription and allosteric regulation of DNA binding
activity by either a cofactor or a repressor (Scortti et al.,
13 2007). In addition, translation of prfA is
temperature-dependent since a transition to 37 dissolves a
secondary structure in prfA RNA that otherwise prevents ribosome
binding (Scortti et al., 2007). The degree of PrfAs rgulation of a
given promoter is in part determined by the degree of homology of
the PrfA-box to the canonical PrfA-box sequence. For example the
PrfA-box upstream of inlAB has mutations that result in only weak
regulation and InlA is nearly undetectable during intracellular
growth (Engelbrecht et al., 1996; Kazmierczak et al., 2003; Lingnau
et al., 1995; McGann et al., 2007a; McGann et al., 2008; Scortti et
al., 2007). In contrast, actA has a canonical PrfA-box and is
upregulated by as much as 300-fold following cell invasion, making
ActA the most abundant surface or secreted protein during
intracellular growth (Figure 1.2B) (Brundage et al., 1993; Moors et
al., 1999; Scortti et al., 2007; Shetron-Rama et al., 2002).
Although InlA and InlB are only weakly regulated by PrfA, they are
strongly regulated by the general stress response sigma factor, B
(Figure 1.2B) (Kazmierczak et al., 2003; Kim et al., 2004; Kim et
al., 2005). Sigma factors are dissociable protein subunits of
prokaryotic RNA polymerase (RNAP) that provide promoter recognition
specificity to the RNAP holoenzyme and contribute to DNA strand
separation during transcription initiation. Most transcription in
exponentially growing Listeria is mediated by an RNAP holoenzyme
carrying the housekeeping sigma factor A, which is similar in
function to E. coli 70. In contrast, B is activated is response to
a variety of stresses including heat, high osmolarity, high ethanol
concentrations, high and low pH, and oxidizing agents leading to
transcription of the B regulon (van Schaik and Abee, 2005). B
increases InlA expression in response to acid and osmotic stress
simulating the intestinal environment, and B is also required for
InlA/InlB expression and cell invasion in the absence of a specific
stress (Kim et al., 2005; McGann et al., 2008; McGann et al.,
2007b; Sue et al., 2004). prfA is partially regulated by B. A-RNAP
transcribes prfA from both prfA promoters, while B-RNAP shares one
prfA promoter (Figure 1.2B). In addition, PrfA regulates prfA
transcription from a PrfA-box upstream of plcA (Figure 1.2B).
Another gene regulated by both PrfA and B is bsh encoding a bile
salt hydrolase that contributes to
14 L. monocytogenes survival within the intestinal lumen and
fecal shedding in a guinea pig model of oral infection (Dussurget
et al., 2002; Kazmierczak et al., 2003). Thus, it appears that the
core virulence genes are regulated as two sets. First, the genes
required for survival in the gastrointestinal tract and needed in
preparation for cell invasion (inlA, inlB, bsh) are regulated by B
and partially regulated by PrfA. The second set includes the genes
required for intracellular parasitism (hly, mpl, plcA, plcB, actA,
hpt, inlC), which are strongly regulated by PrfA, but not influence
by a stress response. Given this model, it is tempting to speculate
that a third, independent set regulated by B, but not PrfA, might
be important for L. monocytogenes in an environmental reservoir or
during noninvasive persistence in the gastrointestinal tract. This
set includes the genes encoding the surface internalins InlC2,
InlD, and InlE, which have not been found to be important for
invasive disease (Dramsi et al., 1997; Kazmierczak et al.,
2003).
Figure 1.2: Molecular and Genetic Requirements for Listerias
Intracellular LifeCycle (Following Page) (A). Inside, a cartoon
depicting key Listeria proteins and stages in the intracellular
life-cycle of L. monocytogenes, which include entry, escape from a
vacuole, actin nucleation, actin-based motility, and cell-to-cell
spread. Outside, electron micrographs from which the cartoon was
derived (Tilney and Portnoy, 1989). Figure from (Portnoy et al.,
2002). (B) Physical and transcriptional organization of Listeria
pathogenicity island-1 (LIPI-1), genes the inlAB operon, and the
inlC and hpt monocistrons. PrfAboxes are indicated by black
squares, known promoters indicated by P and transcripts are
indicated by dotted lines. Adapted from a figure in (Scortti et
al., 2007) and data in (Garner et al., 2006; Kim et al., 2005;
McGann et al., 2008; McGann et al., 2007b; Ollinger et al., 2009;
Ollinger et al., 2008).
15
16 INVASION OF NONPHAGOCYTIC CELLS ADHERENCE TO THE CELL SURFACE
From the bacterial perspective, phagocytic cells are not
necessarily a preferential cell type to infect since immune cells,
especially activated macrophages, can kill Listeria (Shaughnessy
and Swanson, 2007). ActA- mediated cell-to-cell spread allows
Listeria to translocate between cell types and Listeria infects
nonphagocytic cells such as hepatocytes, endothelial cells,
fibroblasts and neurons. At least in tissue culture, Listeria can
directly invade many non-phagocytic cell types through the
interaction of surface adhesins with host cell surface receptors.
Putative Listeria adhesins include Internalin A (InlA), Internalin
B (InlB), InlJ, ActA, Listeria adhesion protein (LAP), P60 (Iap),
Ami, FbpA, and Vip (Cabanes et al., 2005; Dramsi et al., 2004;
Jaradat et al., 2003; Milohanic et al., 2001; Pilgrim et al., 2003;
Sabet et al., 2005; Suarez et al., 2001; Wampler et al., 2004).
Only Internalin A (InlA), Internalin B (InlB) efficiently promote
invasion, while the other proteins appear to function as adhesins
primarily in the absence of InlA and InlB or if overexpressed. In
addition, some have known functions or spatiotemporal patterns of
expression that suggests that they evolved for purposes other than
invasion (e.g. p60, Ami, ActA, FbpA) (Domann et al., 1992; Dramsi
et al., 2004; Kocks et al., 1992; Milohanic et al., 2001; Pilgrim
et al., 2003). InlA is necessary and sufficient for invasion of
epithelial cells (Gaillard et al., 1991; Lecuit et al., 1997;
Mengaud et al., 1996). While InlB promotes invasion of multiple
cell types including epithelial cells, endothelial cells,
fibroblasts and hepatocytes (Banerjee et al., 2004; Copp et al.,
2003; Dramsi et al., 1995; Greiffenberg et al., 1998; Ireton et
al., 1999; Li et al., 2005; Lingnau et al., 1995; Marino et al.,
2002; Marino et al., 1999; Niemann et al., 2007; Parida et al.,
1998; Shen et al., 2000). INTERNALIN A, INTERNALIN B AND THE
INTERNALIN FAMILY InlA and InlB are expressed from adjacent genes
transcribed both independently and biciscronically from the inlAB
locus (Figure 1.2B) (Gaillard et al., 1991). They were identified
in a genetic screen of L. monocytogenes transposon-insertion
mutants unable
17 to invade the enterocyte-like colon carcinoma cell line
Caco-2 (Gaillard et al., 1991). In the study, InlA was found to be
necessary for attachment and invasion, and InlA was sufficient to
reconstitute invasion when expressed in the non-invasive species L.
innocua. Southern Blot analysis with an inlA-based probe suggested
that inlA and inlB were members of a larger highly homologous
family (Gaillard et al., 1991). The family now includes at least
eight additional members: inlC, inlC2, inlE, inlF, inlG, inlH,
inlI, and inlJ. In addition, there are also at least 15
Internalin-like genes identified through genomic analyses (Figure
1.3A) (Bierne and Cossart, 2007; Bierne et al., 2007; Cabanes et
al., 2002; Domann et al., 1997; Dramsi et al., 1997; Engelbrecht et
al., 1996; Lingnau et al., 1996; Raffelsbauer et al., 1998; Sabet
et al., 2008). Only InlA and InlB are well understood. The defining
characteristic of Internalins is a leucine rich repeat (LRR) domain
of 3 to 28 repeats of 22 amino acids each (Figure 1.3A, 1.3B). Each
repeat contains a short strand and a spatially larger 310-helix and
each LRR wraps in a right-handed direction to stack upon one
another. The entire LRR domain takes a solenoid sickle shape with
parallel stacked -strands forming the concave face and stacked
310-helices forming the convex face (Figure 1.3B, Figure 1.4A,
1.4B, Figure 1.5B) (Bierne et al., 2007; Marino et al., 1999, 2000;
Schubert and Heinz, 2003). In addition, each repeat is rotated ~5
degrees with respect to its predecessor giving the sickle-shaped
solenoid a superhelical twist (Figure 1.4A, 1.4B, Figure 1.5B
(Bierne et al., 2007; Marino et al., 1999, 2000; Schubert et al.,
2001; Schubert and Heinz, 2003; Schubert et al., 2002). An
N-terminal cap and an Ig-Like IR domain always flank the LRR domain
and it is thought that these domains stabilize the LRR domain by
shielding the hydrophobic core from an aqueous environment
(Schubert and Heinz, 2003). Internalin and Internalin-like proteins
all have an N-terminal signal sequence suggesting that these
proteins are processed to the bacterial surface by the general
secretory pathway (Figure 1.3A) (Bierne et al., 2007; Rafelski and
Theriot, 2006). All but InlC, a secreted Internalin, are attached
to the bacterial surface, generally through a Cterminal
peptidoglycan-anchoring sequence (e.g. LPXTG) or C-terminal domains
that
18 associate noncovalently with the bacterial cell wall (e.g. GW
domains that bind lipoteichoic acid) (Figure 1.3A) (Engelbrecht et
al., 1996).
Figure 1.3: Domain Organization of Internalins and Sequence
Alignments of Internalin LRR Domains (Following Page)
(A) The three families of internalins by reference to their
association with the bacterial surface are as follows: I,
LPXTG-internalins; II, GW- or WxL-internalins; III, secreted
internalins. InlH results from a recombination event between InlC2
and InlD. Figure from (Bierne et al., 2007). (B) Sequence
alignments of L. monocytogenes Internalin LRR regions for InlB,
InlA, InlC, InlC2, InlD, InlE, InlF, InlG, and InlH. Asterisks show
conserved Internalin LRR residues, and bars show the position and
extent of bstrands 310-helices. Hydrophobic, negatively charged,
and positively charged residues predicted to be surface exposed are
highlighted in yellow, red, and cyan, respectively. Figure from
(Marino et al., 2000).
19
20
INTERNALIN A CO-OPTS THE EPITHELIAL JUNCTIONS Using affinity
chromatography E-cadherin was identified as the cellular receptor
for InlA (Mengaud et al., 1996). E-cadherin is a transmembrane cell
surface glycoprotein and the dominant adhesion molecule of
epithelial adherens junctions (Figure 1.4A) (D'Souza-Schorey, 2005;
Hartsock and Nelson, 2008). Like other classical cadherens (N, P,
and R-cadherin), E-cadherin contains five Ig-like extracellular
domains (ECs), and the most N-terminal E-cadherin EC1 makes a
trans-pairing interaction with Ecadherin on adjacent cells
(Hartsock and Nelson, 2008). Internalin A co-opts Ecadherin by
binding EC1 within the concave face the LRR domain (Figure 1.4A,
1.4B) (Schubert et al., 2002). The E-cadherin-E-cadherin
interaction is Ca2+ dependent as is the interaction between InlA
and E-cadherin (Schubert et al., 2002). Each Ecadherin-E-cadherin
interaction is relatively weak, Kd = 720 M (Haussinger et al.,
2004). The InlA-E-cadherin interaction is ~100X stronger with a Kd
= 8+/-4 M (Wollert et al., 2007a; Wollert et al., 2007b). The total
strength of cell-cell or Listeriacell adherence is due to the high
density of the individual protein-protein interactions, like a
molecular Velcro. Although InlA binds the extracellular domain of
E-cadherin, it is the function of the intracellular domain that is
required for bacterial uptake. It appears that many, if not all of
the intracellular components required for maintaining the
integrity, tension or endocytic recycling of the intercellular
junctions are also involved in generating the forces that
reorganize cell membrane and internalize the bacterium. Experiments
with cytochalasin first demonstrated that Listeria internalization
requires a functional actin cytoskeleton (Wells et al., 1998). The
cytoplasmic domain of E-cadherin dynamically interacts with the
actin cytoskeleton through interactions with - and -catenin and
both of these proteins are recruited to the site of bacterial
attachment (the endocytic cup) and are required for internalization
(Drees et al., 2005; Hartsock and Nelson, 2008; Lecuit et al.,
2000; Yamada et al., 2005). p120, which binds the juxtamembrane
region of E-cadherin and regulates E-cadherin stability at the
junctions, is also recruited to the endocytic cup (Hartsock and
Nelson, 2008; Lecuit et al., 2000). A study of InlA-dependent
invasion identified ARHGAP10 as a novel regulator of -
21 and -catenin at cell-cell junctions, possibly through
regulation of RhoA and CDC42 (Sousa et al., 2005a). Myosin VIIA and
its ligand Vezatin, which generate tension required to hold cells
together, were found to be involved in Listeria internalization
(Sousa et al., 2004). Hakai, a ubiquitin ligase involved in
Clathrin-dependent Ecadherin internalization is recruited to the
site of Listeria invasion and is required for InlA mediated
internalization (Bonazzi et al., 2008; Fujita et al., 2002).
Finally, Ecadherin can be internalized through Clathrin-dependent
and Caveolin-dependent pathways and InlA mediated invasion also
utilizes both for efficient invasion (Bonazzi et al., 2008). All
combined, these results suggest that regulation of E-cadherin
stability at the membrane and Listeria binding and internalization
via E-cadherin are mechanistically related.
Figure 1.4: Structural and Molecular Aspects of
InlA/E-cadherinmediated Listeria Invasion (Following Page) (A)
Bacterial surface attached InlA binds the N-terminal EC1 domain of
the transmembrane junctional protein E-cadherin. Figure from
(Schubert et al., 2002). (B) Ribbon and Space fill model of Wt
InlA- human E-cadherin. Figure from (Schubert and Heinz, 2003). (C)
Detailed View of the Interactions between InlA and hEC1. All
residue side chains involved in direct interactions or as ligands
to bridging ions/water are indicated in ball-and-stick
representation. InlA strands and adjacent coils are shown in
violet. Figure from (Schubert et al., 2002). (D) View of the
hydrophobic pocket in InlA, which accommodates P16 of hEC1.
Hydrogen bonds are indicated by green dotted lines. In mice,
E-cadherin P16 is replaced by glutamate (yellow model). Figure from
(Schubert et al., 2002). (E) Superposition of InlA-hEC1 and
InlAm-mEC1 complexes. Figure from (Wollert et al., 2007b). (F) The
carboxylate of E16 mEC1 (yellow) occupies the same hydrophobic
pocket of InlAm as P16 hEC1 (violet) in InlA. Figure from (Wollert
et al., 2007b). (G) Partial alignment of E-cadherin sequences from
various species. Note critical residue 16. (H) Cartoon diagram of
molecular components involved in formation of the adherens junction
(AJ). Figure from (Ireton, 2007). (I) Cartoon diagram of molecular
components involved in InlA-mediated Listeria entry. Proteins or
domains known to contribute to both uptake of Listeria and AJ
formation appear in yellow. Molecules that promote bacterial entry,
but are not yet known to participate in AJ formation are in orange.
Proteins that regulate AJ assembly, but have not yet been directly
demonstrated to participate in internalization of Listeria, are in
green. Figure from (Ireton, 2007).
22
23
INTERNALIN B CO-OPTS GROWTH FACTOR SIGNALING InlB promotes L.
monocytogenes invasion of cells by binding the extracellular domain
of c-Met, a receptor tyrosine kinase (Banerjee et al., 2004; Copp
et al., 2003; Dramsi et al., 1995; Greiffenberg et al., 1998;
Ireton et al., 1999; Li et al., 2005; Lingnau et al., 1995; Marino
et al., 2002; Marino et al., 1999; Niemann et al., 2007; Parida et
al., 1998; Shen et al., 2000). Although InlB is unrelated by
sequence or structure to the endogenous c-Met ligand, Hepatocyte
Growth Factor (HGF), it acts as an exogenous c-Met agonist. Binding
of InlB to c-Met results in phosphorylation and ubiquitination of
c-Met, leading to recruitment of Clathrin, protein adaptors such as
Gab1, Shc or Cbl, and activation of type I phosphatydylinositol
3-kinase (PI3K) (Ireton, 2007; Ireton et al., 1996; Ireton et al.,
1999; Li et al., 2005; Shen et al., 2000; Veiga and Cossart, 2005;
Veiga et al., 2007). PI3K can regulate Rac, Cofilin and the Arp2/3
complex, which control cytoskeletal dynamics and are required for
bacterial uptake (Bierne et al., 2001; Ireton et al., 1999). c-Met
is a disulfide-linked two-chain heterodimer that is initially
translated as a 1390 amino acid precursor. The c-Met precursor
polypeptide is cleaved between residues 307 and 308 to yield a
small, extracellular chain and a large, multidomain transmembrane
chain. The chain and amino acids 308514 of the chain form the
N-terminal semaphorin (Sema) domain, which is the binding site for
HGF. The extracellular portion of the chain also contains a small
cysteine-rich domain and four Ig-like domains (Ig1Ig4). A
transmembrane helix links the extracellular portion of cMet to the
cytoplasmic juxtamembrane and tyrosine kinase domains (Niemann et
al., 2007). Biochemical studies of InlB / c-Met binding
demonstrated that InlB it does not compete with HGF for c-Met
binding, yet both agonists result in seemingly identical kinase
signaling and endocytic trafficking of c-Met (Li et al., 2005; Shen
et al., 2000). A recent crystal structure of InlB bound to c-Met
shows that InlB acts as a molecular clamp that forces the flexible
Met receptor into a signaling-competent conformation (Figure 1.5A)
(Niemann et al., 2007). Like InlA, the concave surface of the LRR
domain is the binding interface in InlB (Figure 1.5B). InlB makes
two important
24 interactions with c-Met via two interfaces: The InlB LRR
region and Met Ig1 are the primary interface (Figure 1.5B, 1.5C,
1.5D), while a secondary less extensive contact involves the InlB
IR region and the Sema domain of c-Met (Figure 1.5B). The primary
binding interface approximately encompasses residues 599-660 in
c-Met. The residues in c-Met thought to be critical for binding are
indicated in Figure 1.5C, 1.5D (red and pink) and shown in
alignments with arrows in Figure 1.5E. Other receptors for InlB
have been identified, but play only supporting roles in promoting
invasion. Negatively charged cell surface heparin sulfate
proteoglycans (HGPGs) can bind the InlB GW domains and promote
invasion. The GW domains noncovalently anchor InlB to lipoteichoic
acid in the bacterial cell wall (Jonquieres et al., 1999; Marino et
al., 2002). It is thought that HSPGs on the host cell surface might
locally displace InlB from adherent bacteria, thereby presenting
InlB to c-Met (Banerjee et al., 2004; Ireton, 2007). Using affinity
chromatography gC1qR, the receptor for complement component C1q,
was also found as a receptor for the InlB GW domains (Braun et al.,
2000). gC1qR may also present InlB to c-Met. However, there is no
conclusive role for gC1qR in Listeria invasion (Ireton, 2007).
Figure 1.5: Structural Aspects of InlB/c-Met-mediated Listeria
Invasion (Following Page)
A) HGF and InlB non-competitively bind and induce conformational
changes in c-Met promoting kinase signaling. Figure from (Veiga and
Cossart, 2007). (B) The GW domains of cell-dissociated InlB induce
clustering via interaction with heparin sulfate proteoglycans
(HSPGs) on the host cell. Adapted from a Figure in (Niemann et al.,
2007). (C) Close-up showing Y170 and Y214 of InlB interacting with
K599 and K600 of c-Met. Y170 makes hydrogen bonds (dotted orange
lines) to K599 and the R602 side chain. Intra- and intermolecular
salt bridges (dotted purple lines), hold the side chains of K599
and K600 in place. Figure from (Niemann et al., 2007). (D) Side
chains of residues from strands C, F, and G of the c-Met Ig1 domain
form a hydrophobic pocket into which W124 from the concave face of
the InlB LRR binds. Figure from (Niemann et al., 2007). (E) Clustal
X alignment of c-Met binding interface from C and D, with critical
residues indicated (arrows). Boxed residues represent differences
that may account for InlB insensitivity of Guinea pig and Rabbit
c-Met. (F) Phosphorylation of c-Met cytoplasmic tail recruits
adaptors and signaling molecules. Figure from (Ireton, 2007). (G)
InlB-mediated entry. Proteins or domains shown to be involved in
InlB-mediated bacterial uptake are in orange. PI 3-kinase and
25 its lipid product PIP3 might affect F-actin through (1) actin
polymerization, (2) recruiting WAVE2 (3) inducing membrane
association of a guanine nucleotide exchange factor (GEF) for Rac1.
Figure from (Ireton, 2007).
26
SYNERGY BETWEEN INTERNALIN A AND INTERNALIN B Because InlA is
covalently attached to the bacterial cell wall, it acts as an
adhesin (promotes adhesion) and an invasin (promotes bacterial
uptake). In contrast, InlB is only loosely associated with the
bacterial surface and appears to function as an invasin, but not an
adhesin (at least of epithelial cells) (Pentecost et al., 2006).
InlB acts synergistically with InlA during invasion of cultured
epithelial cells (Bergmann et al., 2002; Dramsi et al., 1995;
Lingnau et al., 1995). However, the mechanism of synergy between
the two proteins is poorly understood because the invasion pathways
are often investigated independent of one another. This is
generally accomplished by genetic deletion of one protein from L.
monocytogenes, expression of one protein in the closely related L.
innocua, which lacks internalins, or by use of beads coated with
only one protein at a time. In addition, most of what we know about
InlB signaling is the result of an experimental trick where InlB is
made adhesive for the bacterium by artificially linking the protein
to the bacterial cell wall through genetic addition of a cell wall
anchor sequence (Bierne et al., 2001; Bierne et al., 2005; Braun et
al., 1999; Dramsi and Cossart, 2003; Jonquieres et al., 2001;
Khelef et al., 2006; Seveau et al., 2004; Seveau et al., 2007;
Veiga and Cossart, 2005; Veiga et al., 2007). Yet, whether InlB
signals through c-Met local to the attached bacterium, and whether
InlB is dissociated, remains attached, or diffuses across an
epithelium is critical to understanding how and why InlB promotes
invasion. We hypothesize that InlB regulates uptake by imitating
the regulatory role of RTKs on endocytosis of the epithelial
junctions, and more specifically the role of c-Met in regulating
E-cadherin endocytosis. For example, growth factor activation of
receptor tyrosine kinases has been shown to induce macropinocytosis
of E-cadherin (Bryant et al., 2007). c-Met and E-cadherin are
co-endocytosed in HGF treated MDCK cells (Kamei et al., 1999). InlB
mimics HGF for c-Met activation and internalization (Li et al.,
2005). InlB can promote Clathrin-mediated internalization of
Listeria while HGF similarly promotes Clathrin-mediated
internalization of E-cadherin (Izumi et al., 2004; Veiga and
Cossart, 2005; Veiga et al., 2007). c-Met signaling regulates p120,
Hakai, and Clathrin, which in turn have been shown to regulate
E-cadherin endocytosis or
27 InlA-mediated Listeria invasion (Bonazzi et al., 2008;
Cozzolino et al., 2003; Fujita et al., 2002; Lecuit et al., 2000;
Veiga and Cossart, 2005). Thus InlA and InlB should be studied in
the same context to determine how Listeria invasion is
mechanistically related to growth factor regulation of E-cadherin
endocytosis.
SPECIES SPECIFICITIES OF INTERNALIN A AND INTERNALIN B
Internalin A Does Not Bind Murine E-cadherin Although L.
monocytogenes has been cultured in association with mice and rats,
these species have never been found to acquire natural disease
(Gray and Killinger, 1966; Lecuit et al., 1997; Murray, 1955).
Furthermore, it has long been recognized that mice and rats are not
easily or consistently infected via the oral route. Oral infections
have generally required extremely high doses, which often failed to
produce lethal infection (Gaillard et al., 1996; Huleatt et al.,
2001; MacDonald and Carter, 1980; Marco et al., 1992; Pron et al.,
1998; Roll and Czuprynski, 1990; Zachar and Savage, 1979). Although
internalins are critical for host cell invasion in tissue culture,
a role for internalins in intestinal invasion could not be
established until relatively recently (Lecuit et al., 2001). For
example, infections of mice found no role for InlA or InlB and
initial rates of translocation of the rat intestine by L.
monocytogenes is low, independent of inlAB, hly or actA, and is
similar to translocation by L. innocua, which lacks internalins
(Gaillard et al., 1991; Gaillard et al., 1996; Pron et al., 1998).
It was found that mouse epithelial cells were resistant to L.
monocytogenes invasion because InlA does not bind murine E-cadherin
(Lecuit et al., 1999). The species specificity of InlA was shown to
depend primarily on the difference of a single amino acid in
E-cadherin, the 16th, which is a proline in permissive species
(human, rabbit, guinea pig) but a glutamic acid in mice and rats
(Figure 1.4 G). A P16E mutation in human E-cadherin is sufficient
to prevent InlA binding (Figure 2.10C, 2.10D) (Lecuit et al.,
1999). Crystal structures of InlA bound with human E-cadherin then
revealed the nature of the specificity: P16 of EC1 adopts a
strained cis-conformation to fit in a hydrophobic pocket between
-strands 6 and 7 in InlA (Figure 1.4C, D) (Schubert et al., 2002).
The bulky and charged nature of glutamic acid at residue 16 in
mouse and
28 rat E-cadherin prevents a close association of InlA (Figure
1.4C, D) (Wollert et al., 2007a; Wollert et al., 2007b). A
transgenic mouse was developed where human Ecadherin is expressed
from the promoter of the intestinal fatty acid binding protein
(iFABP) gene, which is turned on in non-proliferative small
intestinal enterocytes. This model demonstrated that InlA could
promote L. monocytogenes invasion of the intestinal epithelium by
interacting with permissive E-cadherin (Lecuit et al., 2001). We
note that canine E-cadherin is also expected to bind InlA. Canine
E-cadherin is identical to human E-cadherin in the first 30 amino
acids, which also contains the critical proline at position 16
required for InlA interaction. Furthermore, the Ecadherin residues
in closest contact with InlA (V3, I4, P5, P6, K14, P16, F17, P18,
K19, Q23, K25, N27, V48, W59, E64, M92) are all conserved in the
human and canine sequences (Figure 1.4G) (Schubert et al., 2002).
Furthermore L. monocytogenes efficiently infects Madin Darby Canine
Kidney cells, and dogs, unlike mice and rats, are susceptible to
Listeriosis (Gray and Killinger, 1966; Robbins et al., 1999).
Internalin B Does Not Activate Guinea Pig or Rabbit c-Met InlB has
also been found to be species specific. Following intravenous
inoculation of guinea pigs and rabbits, an inlB mutant exhibits no
attenuation in the liver (Khelef et al., 2006). In contrast, InlB
appears to promote colonization of the livers of mice given a high
infectious dose intravenously (Dramsi et al., 1995; Dramsi et al.,
2004; Gaillard et al., 1996; Khelef et al., 2006). It was shown
that transfection of guinea pig and rabbit cells with human c-Met
restores the ability of InlB to stimulate c-Met and promote
Listeria invasion (Khelef et al., 2006). Thus InlB activates c-Met
on human, canine mouse and rat cells but not guinea pig or rabbit
(Khelef et al., 2006; Shen et al., 2000). Cow and sheep c-Met is
also permissive for InlB according to unpublished data in (Disson
et al., 2008). The structural basis for InlB species specificity is
not known. However, there are some intriguing differences between
the c-Met sequence from permissive and nonpermissive species within
the InlB binding region (Figure 1.5E). For example, I639 in human
c-Met is mutated to tyrosine in rabbit and leucine in guinea pig
and T646 is mutated to arginine in rabbit and is absent in guinea
pig. We
29 hypothesize that humanizing these residues will restore
binding to InlB by rabbit and guinea pig c-Met.
ANIMAL MODELS PERMISSIVE FOR INTERNALIN A AND INTERNALIN B The
humanized mouse expressing hE-cadherin in the intestinal epithelium
limits the study of InlA-mediated invasion to the intestine (Lecuit
et al., 2001). Furthermore, if InlB function requires InlA-mediated
adhesion, this mouse model could also be insufficient to establish
a role for InlB at extra-intestinal sites. For example, a role for
InlB in crossing the fetoplacental barrier was not found in mice
even though mice are permissive for InlB (Le Monnier et al., 2007).
However, a second humanized transgenic mouse was recently developed
by knocking-in murine E-cadherin with E16P mutation (Disson et al.,
2008). Both InlA and InlB were implicated in fetoplacental
infection in this model where InlA is adhesive. Although this model
appears to be sufficient to study InlA and InlB in the same
context, we are concerned by the fact that mE-cadherinE16P binds
InlA with a lower affinity (96 M) than hEcadherin (8 M) (Wollert et
al., 2007b). It was recently found that gerbils are permissive for
InlA and InlB functions (Disson et al., 2008). However, in contrast
to mice, this model lacks the power of forward genetics and also
lacks well characterized reagents. Rather than making permissive
mice, we were interested in generating L. monocytogenes strains
with InlA mutations that would allow binding to murine Ecadherin.
Our efforts are detailed in Chapter 3. In addition, a recent
independent effort in Germany recently succeeded in generating a
mouse-adapted InlA. In designing mutations that would increase the
binding of InlA to human E-cadherin, InlA S192N Y369S (InlAm) was
found to have an equivalent binding affinity for mouse E-cadherin
(Kd = 10+/-2 M) as wild type InlA has for human E-cadherin (8+/-4
M) (Figure 1.4E) (Wollert et al., 2007b). The S192N mutation
displaces a water molecule and introduces a direct hydrogen bond
between N192 in InlA and F17 in human EC1. As an unexpected
consequence, S192N allows E16 of mEC1 to adopt a relaxed trans
conformation and the carboxy group of E16 of mEC1 occupies the same
hydrophobic
30 pocket of InlA as P16 of hEC1 in InlA/hEC1 (Figure 1.4E,
1.4F) (Wollert et al., 2007a; Wollert et al., 2007b). The second
mutation improves overall binding at the major interface of the
InlA-E-cadherin interaction. Y369S replaces the bulky tyrosine
sidechain with a serine, which makes a water-mediated hydrogen bond
to N27 in EC1 (Wollert et al., 2007a; Wollert et al., 2007b). The
binding of InlAm and murine Ecadherin promotes oral infection of
mice through the intestinal epithelium (Wollert et al., 2007b).
TISSUE SPECIFICITIES OF INTERNALIN A AND INTERNALIN B Loss of
Internalin A abrogates intestinal invasion of guinea pigs, but has
no effect on pathogenesis when mutant bacteria are administered
intravenously (Lecuit et al., 2001). Whether InlB also promotes
efficient invasion of the intestine in permissive hosts has
implications for the success of Listeria invasion and colonization
at this site. However, InlB has not been found to be important for
intestinal invasion, but rather for colonization of mouse livers
after intravenous infection (Dramsi et al., 2004; Khelef et al.,
2006). Furthermore InlB did not appear to affect invasion of the
intestine of transgenic mice expressing human E-cadherin in the
intestine (Khelef et al., 2006). Thus, the current dogma is that
InlA and InlB have evolved to target different tissues at different
stages of infection: InlA is required for intestinal infection and
is subsequently dispensable for invasive disease in non-pregnant
animals, while InlB primarily mediates invasion of other tissues,
notably the liver (Ireton, 2007; Schubert and Heinz, 2003).
CHALLENGING THE INTERNALIN TISSUE SPECIFICITY DOGMA There is
reason to hypothesize a role for InlB in intestinal invasion.
First, c-Met is present on many tissues, including epithelia
suggesting that InlB may also promote infection of the
gastrointestinal tract or other barriers (Di Renzo et al., 1991;
Disson et al., 2008; Fukamachi et al., 1994; Ishikawa et al., 2001;
Kato et al., 1997a, b; Neo et al., 2005; Nusrat et al., 1994;
Sunitha et al., 1999; Wormstone et al., 2000). Indeed, as mentioned
above, InlB appears to play a role in crossing of fetoplacental
barrier after
31 intravenous inoculation, but only with the coexpression of a
functional InlA (Disson et al., 2008). Second, InlA and InlB are
coregulated and are upregulated in the intestinal tract and under
conditions simulating the gastrointestinal tract (Kim et al., 2005;
McGann et al., 2007b; Sue et al., 2004; Toledo-Arana et al., 2009).
Third, InlB promotes infection of cultured epithelial cells,
including primary intestinal epithelial cells from permissive
animal models, like gerbils (Disson et al., 2008).
ANATOMICAL AND SUBCELLULAR SITE OF EPITHELIAL INVASION: THE
POLARITY PARADOX THE EPITHELIAL BARRIER, CELL RENEWAL AND
GASTROINTESTINAL PATHOGENS The gastrointestinal epithelium fulfills
two seemingly incompatible tasks. On the one hand, it maintains a
tight epithelial barrier that controls fluid and solute transport,
separates the external (lumenal) environment from the internal
(interstitial) environment, and prevents invasion of potentially
harmful microbes. The tight and adherens junctions that comprise
the apical junctional complex (AJC) are the intercellular glue that
maintains this barrier (Anderson et al., 2004; Balda and Matter,
1998; Laukoetter et al., 2006). On the other hand, the epithelium
continuously disassembles the barrier in a conveyor belt of rapid
cell renewal and cell death. 1010 cells are shed per day and a new
epithelial monolayer is generated every 3-6 days as a continuous
flow of cell division, differentiation, migration and cell loss
along the crypt-villus axis (Bullen et al., 2006). Epithelial
renewal continuously threatens the integrity of the epithelial
barrier since dead cells must be removed and detached through
junction disassembly followed by epithelial junction reassembly.
The epithelium also needs to maintain its functions and integrity
in the face of continuous exposure to potentially invasive
microbes, and their metabolic products and toxins. Most
bacterial-epithelial relationships in the intestine are benign, and
in some cases symbiotic (the gastrointestinal lumen is thought to
be home to more bacterial cells than the total number of cells in
the body). However a number of invasive bacterial and viral
pathogens have evolved mechanisms to breach the
32 intestinal barrier. Interestingly, many of these invasive
microbes cross the epithelial barrier by utilizing cellular
receptors that are found only on the basolateral membrane of the
epithelial cell, and thus should not be available on the lumenal
surface of an intact epithelium since the AJC also allows cells to
separate the plasma membrane into distinct apical versus
basolateral domains. For example, rotaviruses and Yersiniae bind
integrins, a class of adhesion and signaling molecules found solely
on the basolateral sides of enterocytes (Guerrero et al., 2000;
Isberg and Leong, 1990).
E-CADHERIN AND C-MET ARE BASOLATERAL RECEPTORS L. monocytogenes
receptors E-cadherin and c-Met are also basolateral proteins that
co-localize at the adherens junction, below the epithelial tight
junction (Boller et al., 1985; Boyle and Finlay, 2003; Crepaldi et
al., 1994; Nusrat et al., 1994; Sousa et al., 2005b). Even prior to
identification of these receptors, it was known that L.
monocytogenes invades polarized epithelial cells most efficiently
from the basolateral side (Gaillard and Finlay, 1996; Temm-Grove et
al., 1994). For example, when cultured epithelial cells are plated
sparsely and grown as small islands of cells, the edge cells do not
have a continuous tight junction to prevent mixing of apical and
basolateral proteins and L. monocytogenes preferentially infects
these cells (Gaillard and Finlay, 1996; Temm-Grove et al., 1994).
Some cultured epithelial cell lines will become more differentiated
and polarized over time in culture and L. monocytogenes invasion of
confluent monolayers of Caco-2 cells decreases with epithelial
monolayer maturity. The epithelial cell-to-cell junctions are
Ca2+-dependent and disrupting the intercellular junctions with
calcium chelators, there by exposing lateral cell membranes,
increases L. monocytogenes invasion of polarized epithelial
monolayers (Gaillard and Finlay, 1996). These data present a
paradox that L. monocytogenes has evolved to use receptors that are
not accessible during infection of the gastrointestinal epithelium
from the lumen.
33 THE PEYERS PATCH PARADIGM The prevailing hypothesis to
explain the discrepancy between receptor localization and the site
of infection has been that enteric pathogens may not directly enter
enterocytes from the lumenal side. Rather, they first invade the
intestine by taking advantage of the function of M-cells (Clark and
Jepson, 2003). M-cells are modified epithelial cells overlying the
intestinal lymphoid follicles known as Peyers patches (PP). They
are capable of phagocytosis and also express basolateral receptors
on their lumenal surface. M-cells normally function in immune
surveillance by engulfing lumenal antigens and presenting them to
dendritic cells and macrophages found underneath the Peyers patch
epithelium. Some pathogens, like Salmonella typhi, which causes
enteric fever in humans, and Salmonella typhimurium which causes a
similar disease in mice, have been shown to enter through M-cells
before spreading systemically through the lymphatics and blood
stream (Jensen et al., 1998; Jones et al., 1994). Yersinia
pseudotuberculosis expresses the protein Invasin to attach to and
stimulate entry through integrin receptors (Marra and Isberg,
1997). Although integrins are found only on the basolateral surface
of enterocytes, they have been detected on the apical surface of
M-cells (Clark et al., 1998). In humans, Yersinia
pseudotuberculosis causes a self-limited enteritis and occasionally
mesenteric adenitis. In mice this pathogen is able to invade and
spread systemically and cause an enteric fever-like syndrome.
Similarly Shigella, which also use integrins for basolateral
enterocyte invasion has been proposed to first cross the intestinal
epithelial barrier through M-cells (Mounier et al., 1992;
Zychlinsky et al., 1994). The same model has been proposed for
Listeria monocytogenes (Gaillard and Finlay, 1996; Jensen et al.,
1998; Pron et al., 1998). For example, Jean-louis Gaillard and B.
Brett Finlay postulated: because invasion of these highly
differentiated cells [enterocytes] is thought to be exclusively
basolateral, L. monocytogenes must utilize another site of entry.
This could be the M cell, as reported for other bacterial
pathogens. This hypothesis is in agreement with previous results
showing that listeriae given to rodents penetrate mostly into the
Peyers patches (Gaillard and Finlay, 1996).
34 CHALLENGING THE PEYERS PATCH PARADIGM