Raghuveer Parthasarathy Department of Physics, University of Oregon, U.S.A. Glimpses of Gut Microbes in their Physical World IIT Madras, 5 November 2019
Raghuveer ParthasarathyDepartment of Physics, University of Oregon, U.S.A.
Glimpses of Gut Microbes in their Physical World
IIT Madras, 5 November 2019
Raghuveer Parthasarathy University of Oregon November 2019
Gut Bacterial CommunitiesYou: human + bacteria• As many bacterial as human cells • hundreds of species per individual (lots
of variation)
Animal-associated microbes: • Digestion• Immunity• Development • Disease (complex diseases, e.g.
diabetes; resistance to pathogens)Aug. 2012
Raghuveer Parthasarathy University of Oregon November 2019
Gut Bacterial Communities
A difficult, confusing topic.
Unknown: • What determines community
compositions?• How can we alter community
compositions – changing what species are present, or their abundances?
Can physics help?
Raghuveer Parthasarathy University of Oregon November 2019
Physical LandscapesUnderstanding ecosystems requires understanding their physical environment
Parthasarathy Labhttps://pages.uoregon.edu/raghu/
Nearly all gut microbiome studies: Sequencing, fecal samples
Raghuveer Parthasarathy University of Oregon November 2019
• Spatial niches for coexistence, competition, ...?• Timescales for fluctuations, responses to perturbations? • Nucleation, growth of bacterial colonies?
Need model system, imaging.
We know little about the structure and dynamics of gut microbial communities
To answer biophysical questions…
Raghuveer Parthasarathy University of Oregon November 2019
ZebrafishZebrafish as a model system• Usual reasons (vertebrate,
transparent, genetically tractable...)
Adult (75 days)
250 micronsintestinal bulb vent
Larval zebrafish, 6 days post-fertilization. (Red dye in gut, for illustration.)
Raghuveer Parthasarathy University of Oregon November 2019
Zebrafish + bacteria
Karen Guillemin (UO, Biology)
Zebrafish as a model system• Usual reasons (vertebrate,
transparent, genetically tractable...)• Gnotobiotic embryos / larvae.
Germ-free; add well-defined, engineered microbial populations.
Raghuveer Parthasarathy University of Oregon November 2019
How can we observe gut bacteria?
John McNamee, The New Yorker, June 9, 2017
Raghuveer Parthasarathy University of Oregon November 2019
Imaging zebrafish + bacteria4D, quantitative data on host, microbes requires • Large fields of view• High speed 3D imaging• Low photodamage
DICPeristalsis: Roughly 1/min.
250 μm
Raghuveer Parthasarathy University of Oregon November 2019
Imaging zebrafish + bacteria4D, quantitative data on host, microbes requires • Large fields of view• High speed 3D imaging• Low photodamage
Why is 3D microscopy difficult? Out of focus light!
Raghuveer Parthasarathy University of Oregon November 2019
Imaging zebrafish + bacteria4D, quantitative data on host, microbes requires • Large fields of view• High speed 3D imaging• Low photodamage
Raghuveer Parthasarathy University of Oregon November 2019
Imaging zebrafish + bacteria4D, quantitative data on host, microbes requires • Large fields of view• High speed 3D imaging• Low photodamage
Confocal microscopy: NO• High resolution! 3D!But...• Slow• Inefficient (high photodamage)
Raghuveer Parthasarathy University of Oregon November 2019
Imaging zebrafish + bacteria4D, quantitative data on host, microbes requires • Large fields of view• High speed 3D imaging• Low photodamageLight sheet fluorescence microscopy:
Especially Ernst Stelzer, EMBL (Heidelberg)Review (RP): Curr. Opin. Microbiol. 43:31 (2018)
Raghuveer Parthasarathy University of Oregon November 2019
Imaging zebrafish + bacteria4D, quantitative data on host, microbes requires • Large fields of view• High speed 3D imaging• Low photodamageLight sheet fluorescence microscopy:
Review (RP): Curr. Opin. Microbiol. 43:31 (2018)
Raghuveer Parthasarathy University of Oregon November 2019
Imaging zebrafish + bacteria4D, quantitative data on host, microbes requires • Large fields of view• High speed 3D imaging• Low photodamageLight sheet fluorescence microscopy:
Review (RP): Curr. Opin. Microbiol. 43:31 (2018)
Matt Jemielita
Raghuveer Parthasarathy University of Oregon November 2019
Initially germ-free Zebrafish … + bacteria …
100 μm
Anterior Posterior
Single plane, real time
Raghuveer Parthasarathy University of Oregon November 2019
Initially germ-free Zebrafish + Vibrio ZWU0020, imaged 24 hours post-inoculation
100 μm
Anterior Posterior
Single plane, real time
Some species: motile individuals
Raghuveer Parthasarathy University of Oregon November 2019
Initially germ-free Zebrafish + Aeromonas veronii (Red + GreenFluor. Protein), imaged 12 hours post-inoculation
Some species: mostly aggregated
max. intensity projection of 3D scan
100 μm
Raghuveer Parthasarathy University of Oregon November 2019
Initially germ-free Zebrafish + Aeromonas veronii (Red + GreenFluor. Protein), imaged 12 hours post-inoculation
3D Scan
Some species: mostly aggregated
max. intensity projection of 3D scan
100 μm1 μm/framescan subset: 1-84 μm
Raghuveer Parthasarathy University of Oregon November 2019
A variety of bacterial behaviors
Different bacterial species:Different aggregation behaviors, Different locations in the gut
Brandon H Schlomann, …, Raghuveer Parthasarathy, Biophysical Journal115: 1-7 (2018).
Raghuveer Parthasarathy University of Oregon November 2019
OutlineModel system + imaging → examples of physical processes orchestrating gut microbial dynamics
[1] SL Logan, ..., R Parthasarathy, Proc. Natl. Acad. Sci. 115: E3779-E3787 (2018). [2] BH Schlomann, TJ Wiles, … R Parthasarathy, Proc. Natl. Acad. Sci.116: 21392-21400 (2019) (also bioRxiv 565556)
1 Cholera and intestinal invasion [1]2 Antibiotic perturbations of gut microbes [2]3 General themes revisited, and glimpses of other stories
Raghuveer Parthasarathy University of Oregon November 2019
1 Cholera and Intestinal Invasion
S. L. Logan, ..., R. Parthasarathy, Proc. Natl. Acad. Sci. 115: E3779-E3787 (2018).
Savannah Logan
Raghuveer Parthasarathy University of Oregon November 2019
Vibrio cholerae and T6SSVibrio cholerae (Vc):• Causes cholera (20,000-140,000
deaths/year, still!)• Unknown: how does it invade the gut?• Vc has a Type VI Secretion System (T6SS):
Punctures adjacent cells, inserts toxins• What is the T6SS doing in vivo?
w/ Brian Hammer (Georgia Tech)Joao Xavier (Sloan Kettering)
Raghuveer Parthasarathy University of Oregon November 2019
V. cholerae T6SS strains
T6SS+ : T6SS constitutively expressed (i.e. always on)
Details; Other strains (incl. Wild Type, Immunity Mutants): See S. L. Logan, ..., R. Parthasarathy, PNAS115: E3779 (2018).
T6SS– : Defective syringe apparatus
Engineered strains:
Raghuveer Parthasarathy University of Oregon November 2019
Vibrio cholerae in ZebrafishWill human-derived Vibrio choleraecolonize zebrafish?• Dissection and plating [yes]• Light sheet imaging [yes]≈ 104 bacteria/gut (≈109-10 /ml)
Data: 6 dpf, 24h after mono-association of germ-free fish
T6SS
–
T6SS
+
motile
50 μm
Raghuveer Parthasarathy University of Oregon November 2019
InvasionCan Vc use T6SS to defeat another species?• Target species: Aeromonas; native to zebrafish,
abundant.• Aeromonas: individual bacteria + large aggregates
Raghuveer Parthasarathy University of Oregon November 2019
Aeromonas post-Invasion“Challenge” Aeromonas at +1 day w/ a Vibrio cholerae strain
Aeromonas fluorescence; after introduction of T6SS– V. cholerae
With T6SS– Vc, Aeromonas exists at large (normal) numbers
Raghuveer Parthasarathy University of Oregon November 2019
Aeromonas post-Invasion“Challenge” Aeromonas at +1 day w/ a Vibrio cholerae strain
Aeromonas fluorescence; after introduction of T6SS+ V. cholerae
With T6SS+ Vc, Aeromonas almost completely removed
Raghuveer Parthasarathy University of Oregon November 2019
Aeromonas post-InvasionAeromonas abundance 24h after invasion by...
T6SS+ Vibrio cholerae; mean >100x lower!
T6SS– Vibrio cholerae
Raghuveer Parthasarathy University of Oregon November 2019
Aeromonas post-Invasion
Aeromonas fluorescence; after introduction of T6SS+ Vibrio cholerae
Aeromonas fluorescence; after introduction of T6SS– Vibrio choleraeTotal time: 12h 40m
Total time: 16h 40m
200 μm
Raghuveer Parthasarathy University of Oregon November 2019
Aeromonas: collapsesInvaded by T6SS+ V cholerae, Aeromonas collapsesBacterial aggregates are expelled from the gut.
Stochastic collapse dynamics:Relating population mean, variance to collapse magnitude, probabilities.• Computational: Wiles,
Jemielieta et al, PLoS Bio 2016;• Analytic solutions in Schlomann
Journal Theor. Bio. 2018.
Aero
mon
as p
opul
atio
n +1
w/ T6SS– Vc+ w/ T6SS+ Vc+
Raghuveer Parthasarathy University of Oregon November 2019
What is Vibrio cholerae’s T6SS doing?
Killing Aeromonas, right?Let’s keep looking...
Raghuveer Parthasarathy University of Oregon November 2019
T6SS and the zebrafish gut
T6SS+
T6SS-
Raghuveer Parthasarathy University of Oregon November 2019
Measures of gut motility
Image velocimetry…: Frequency, Amplitude
Raghuveer Parthasarathy University of Oregon November 2019
T6SS induces large gut contractions
T6SS-Germ-Free
Nor
mal
ized
Gut
Mot
ility
Am
plitu
de
Raghuveer Parthasarathy University of Oregon November 2019
T6SS induces large gut contractions
T6SS-Germ-Free
T6SS+Nor
mal
ized
Gut
Mot
ility
Am
plitu
de
T6SS increases the strength of host gut motility! (>100%!)
Raghuveer Parthasarathy University of Oregon November 2019
Type VI Secretion System
Marek Basler, University of Basel, Biozentrum
• Eukaryotes: actin cytoskeleton; Prokaryotes: No actin.• T6SS “syringe” contains an actin crosslinking domain
(ACD). (Kills amoebas, e.g.)• Delete the actin crosslinking domain!• T6SS+ACD– Vc kills Aeromonas in vitro (as expected). • In vivo?...
Raghuveer Parthasarathy University of Oregon November 2019
T6SS ACD and gut contractions
T6SS increases the strength of hostgut motility via actin crosslinking!
T6SS-Germ-Free
T6SS+Nor
mal
ized
Gut
Mot
ility
Am
plitu
de
T6SS+ACD–
Raghuveer Parthasarathy University of Oregon November 2019
T6SS ACD and native bacteria
Bacterial competition?
Aeromonas has normal abundance after invasion by T6SS+ ACD– V. cholerae
Raghuveer Parthasarathy University of Oregon November 2019
T6SS and InvasionCan Vibrio cholerae use T6SS to defeat another species?Yes! ... by altering the host environment!• Aeromonas: aggregated, sensitive to intestinal
contractions. V cholerae: motile individuals; insensitive.
• A specific, unexpected mechanism for influencing gut communities. (The first instance of T6SS influencing animal physiology.)
S. L. Logan, ..., R. Parthasarathy, PNAS 115: E3779-E3787 (2018).
Questions: Regulation of T6SS, native bacterial T6SS, engineering T6SS activity, effects on other species (and multiple species), ways to reduce intestinal contractions…
Raghuveer Parthasarathy University of Oregon November 2019
Outline
BH Schlomann, TJ Wiles, … R Parthasarathy, Proc. Natl. Acad. Sci. 116: 21392-21400 (2019) (also bioRxiv 565556)
1 Cholera and intestinal invasion2 Antibiotic perturbations of gut microbes*3 General themes revisited, and glimpses of other stories
Raghuveer Parthasarathy University of Oregon November 2019
2 Antibiotics
Travis Wiles
Brandon Schlomann
Karen Guillemin
Raghuveer Parthasarathy University of Oregon November 2019
Antibiotics• Induce large, long-lasting changes to the gut microbiome…• … even at low (sublethal) concentrations (such as are
often found in the environment)• How?
Let’s see…
Raghuveer Parthasarathy University of Oregon November 2019
Antibiotic: Model Systems
Antibiotic: ciprofloxacin (inhibits DNA replication). Widely used; often found in environmental samples.
100 μm
Raghuveer Parthasarathy University of Oregon November 2019
Antibiotic: Model Systems
Antibiotic: ciprofloxacin (inhibits DNA replication). Widely used; often found in environmental samples.Bacteria: mono-association of zebrafish with• Vibrio Z20 (highly planktonic), or• Enterobacter (highly aggregated)
100 μm
Raghuveer Parthasarathy University of Oregon November 2019
Antibiotics + Vibrio Z20
10 ng/ml cipro:In water: ≈10x lower densityIn the gut: → ≈100x lower!
Raghuveer Parthasarathy University of Oregon November 2019
Cipro + Vibrio Z20In vivo: filamentation, loss of motility
10 ng/ml cipro, 5 hrs/
→ expulsion of (live) bacteria
Vibrio Z2050 um
200 um
Raghuveer Parthasarathy University of Oregon November 2019
Cipro + Vibrio Z20In vitro: filamentation, loss of motility (stress response)
No cipro 10 ng/ml cipro
10 um10 um
Antibiotic effects “amplified” in the gut due to the coupling of aggregation, transport
Raghuveer Parthasarathy University of Oregon November 2019
Antibiotic: Model Systems
Bacteria: mono-association with• Vibrio (highly planktonic)• Enterobacter (highly aggregated)
Cipro → aggregation, expulsion. What do sublethal antibiotics do to a species that’s normally aggregated?
200 um
Raghuveer Parthasarathy University of Oregon November 2019
Cipro + Enterobacter
25 mg/ml cipro In water: ≈20x lower densityIn the gut: → ≈1000x lower (!!)
Raghuveer Parthasarathy University of Oregon November 2019
Cipro + EnterobacterIn vivo: With sublethal cipro, suppression of small clusters
Raghuveer Parthasarathy University of Oregon November 2019
Aggregated species: homeostasisA general question: How can aggregating species persist in the gut, despite intestinal transport?
Nucleation, growth of new aggregates is crucial!
Enterobacter, without antibiotics
200 um
A quantitative model of cluster dynamics?
Raghuveer Parthasarathy University of Oregon November 2019
Aggregated species: homeostasis
A kinetic model:Assumptions:Anm = αFnm = β for m=1, 0 otherwiseGn = rn(1 – n/K) [logistic growth]Enm = λ [collapse rate]
Similar to polymer & colloidal physics models → sol/gel transitions, cluster size distributions, etc.
But with growth, expulsionAlso: Number of clusters is small →stochastic simulations
Raghuveer Parthasarathy University of Oregon November 2019
Aggregated species: homeostasis
A kinetic model (similar to sol/gel polymer physics models):
BH Schlomann, TJ Wiles, … R Parthasarathy, Proc. Natl. Acad. Sci. 116: 21392-21400 (2019)
Five parameters:Anm = αFnm = β for m=1, 0 otherwiseGn = rn(1 – n/K) [logistic growth]Enm = λ [collapse rate]
λ [collapse rate] – measured from time-series imaging.r [growth rate] – measured from time-series imaging.α/β and K – fit to the mean, variance of static (single time-point) abundance data→ predict cluster size distribu on with no free parameters!
Raghuveer Parthasarathy University of Oregon November 2019
Aggregated species: phase diagramRates estimated from experiments predict the cluster size distribution of Enterobacter…
… and the phase diagram of intestinal abundance
No fit parameters!
Raghuveer Parthasarathy University of Oregon November 2019
Aggregated species + Antibiotics
Enterobacter + Low-dose cipro → stalled growth, reduced fragmentation/aggregation → extinction(expulsion)
Raghuveer Parthasarathy University of Oregon November 2019
Aggregated species + Antibiotics
Vibrio + Low-dose cipro → stalled growth, aggregation → reduced population (not exctinction)
Raghuveer Parthasarathy University of Oregon November 2019
Aggregated species + Antibiotics
General features• Sublethal antibiotics and bacteria: stress,
filamentation, loss of motility• Vertebrate intestines: Transport!Suggests a general mechanism for antibiotic effects on the gut microbiome.
Raghuveer Parthasarathy University of Oregon November 2019
Aggregated species + Antibiotics
… a general mechanism for antibiotic effects on the gut microbiome.And…• Useful metrics for humans
and other animals?
And…• Most expelled bacteria are
alive!• Transmission of antibiotic
resistance?!
Raghuveer Parthasarathy University of Oregon November 2019
Outline
1 Cholera and intestinal invasion2 Antibiotic perturbations of gut microbes3 General themes revisited, and glimpses of other stories
Raghuveer Parthasarathy University of Oregon November 2019
Transport-mediated dynamics
Strong connections between• the spatial structure of gut bacteria• intestinal mechanics / transport• bacterial population dynamics
Raghuveer Parthasarathy University of Oregon November 2019
And more…Externally inducible genetic switches: turn motility (flagellar gene expression), chemotaxis (cheA), etc. on / off.
100 μm
Motility loss of function:Motile (magenta) and non-motile (cyan) commensal Vibrio Z20 six hours after inducing loss of flagellar gene function.
Raghuveer Parthasarathy University of Oregon November 2019
And more…Immune responses: Dynamics of immune cells and signaling pathways. (Green: TNF-alpha+ cells, esp. macrophages; Magenta: bacteria)
100 μm
Raghuveer Parthasarathy University of Oregon November 2019
Multi-species communities
Teddy Hay Dylan Martins Deepika Sundarraman
Raghuveer Parthasarathy University of Oregon November 2019
Multi-species interactions
Beyond 1 or 2 species: Multi-species communities• Rules of community assembly?• “Model” N>2 species community• How does spatial structure matter [spoiler: we don’t
know…]
Raghuveer Parthasarathy University of Oregon November 2019
Conclusions
• Populations are spatially heterogeneous and dynamic, influenced by the physical environment of the gut…
• Aggregation + Gut motility → susceptibility to expulsion• Low-dose antibiotics → enhanced aggregation
• … and influencing it themselves! • T6SS and intestinal contractions
https://pages.uoregon.edu/raghu/
• Imaging → in vivo gut microbial population dynamics.
More generally: There’s interesting biophysics to uncover in the world of gut microbes!
Raghuveer Parthasarathy University of Oregon November 2019
https://pages.uoregon.edu/raghu/
Parthasarathy LabSavannah LoganBrandon SchlomannTeddy HayPhil JahlJulia NgoDeepika SundarramanDrew ShieldsDylan MartinsChristopher Dudley
FundingKaren Guillemin Travis WilesBrian Hammer (GT)Joao Xavier (SK)
People!
Raghuveer Parthasarathy University of Oregon November 2019
Conclusions
• Populations are spatially heterogeneous and dynamic, influenced by the physical environment of the gut…
• Aggregation + Gut motility → susceptibility to expulsion• Low-dose antibiotics → enhanced aggregation
• … and influencing it themselves! • T6SS and intestinal contractions
https://pages.uoregon.edu/raghu/
• Imaging → in vivo gut microbial population dynamics.
More generally: There’s interesting biophysics to uncover in the world of gut microbes!