Raghu NCBS IITM 2019 · Title: Microsoft PowerPoint - Raghu NCBS IITM 2019 Author: Raghu Created Date: 11/6/2019 6:09:18 PM

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

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?

Physical LandscapesUnderstanding ecosystems requires understanding their physical environment

Parthasarathy Labhttps://pages.uoregon.edu/raghu/

Nearly all gut microbiome studies: Sequencing, fecal samples

• 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…

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.)

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.

How can we observe gut bacteria?

John McNamee, The New Yorker, June 9, 2017

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

Why is 3D microscopy difficult? Out of focus light!

Confocal microscopy: NO• High resolution! 3D!But...• Slow• Inefficient (high photodamage)

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)

Review (RP): Curr. Opin. Microbiol. 43:31 (2018)

Matt Jemielita

Initially germ-free Zebrafish … + bacteria …

100 μm

Anterior Posterior

Single plane, real time

Initially germ-free Zebrafish + Vibrio ZWU0020, imaged 24 hours post-inoculation

100 μm

Anterior Posterior

Single plane, real time

Some species: motile individuals

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

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

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).

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

1 Cholera and Intestinal Invasion

S. L. Logan, ..., R. Parthasarathy, Proc. Natl. Acad. Sci. 115: E3779-E3787 (2018).

Savannah Logan

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)

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:

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

motile

50 μm

InvasionCan Vc use T6SS to defeat another species?• Target species: Aeromonas; native to zebrafish,

abundant.• Aeromonas: individual bacteria + large aggregates

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

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

Aeromonas post-InvasionAeromonas abundance 24h after invasion by...

T6SS+ Vibrio cholerae; mean >100x lower!

T6SS– Vibrio cholerae

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

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.

w/ T6SS– Vc+ w/ T6SS+ Vc+

What is Vibrio cholerae’s T6SS doing?

Killing Aeromonas, right?Let’s keep looking...

T6SS and the zebrafish gut

Measures of gut motility

Image velocimetry…: Frequency, Amplitude

T6SS induces large gut contractions

T6SS-Germ-Free

T6SS induces large gut contractions

T6SS-Germ-Free

T6SS+Nor

T6SS increases the strength of host gut motility! (>100%!)

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?...

T6SS ACD and gut contractions

T6SS increases the strength of hostgut motility via actin crosslinking!

T6SS-Germ-Free

T6SS+Nor

T6SS+ACD–

T6SS ACD and native bacteria

Bacterial competition?

Aeromonas has normal abundance after invasion by T6SS+ ACD– V. cholerae

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…

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

2 Antibiotics

Travis Wiles

Brandon Schlomann

Karen Guillemin

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…

Antibiotic: Model Systems

Antibiotic: ciprofloxacin (inhibits DNA replication). Widely used; often found in environmental samples.

100 μm

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

Antibiotics + Vibrio Z20

10 ng/ml cipro:In water: ≈10x lower densityIn the gut: → ≈100x lower!

Cipro + Vibrio Z20In vivo: filamentation, loss of motility

10 ng/ml cipro, 5 hrs/

→ expulsion of (live) bacteria

Vibrio Z2050 um

200 um

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

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

Cipro + Enterobacter

25 mg/ml cipro In water: ≈20x lower densityIn the gut: → ≈1000x lower (!!)

Cipro + EnterobacterIn vivo: With sublethal cipro, suppression of small clusters

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?

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

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!

Aggregated species: phase diagramRates estimated from experiments predict the cluster size distribution of Enterobacter…

… and the phase diagram of intestinal abundance

No fit parameters!

Aggregated species + Antibiotics

Enterobacter + Low-dose cipro → stalled growth, reduced fragmentation/aggregation → extinction(expulsion)

Vibrio + Low-dose cipro → stalled growth, aggregation → reduced population (not exctinction)

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.

… 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?!

Outline

1 Cholera and intestinal invasion2 Antibiotic perturbations of gut microbes3 General themes revisited, and glimpses of other stories

Transport-mediated dynamics

Strong connections between• the spatial structure of gut bacteria• intestinal mechanics / transport• bacterial population dynamics

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.

And more…Immune responses: Dynamics of immune cells and signaling pathways. (Green: TNF-alpha+ cells, esp. macrophages; Magenta: bacteria)

100 μm

Multi-species communities

Teddy Hay Dylan Martins Deepika Sundarraman

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…]

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!

Parthasarathy LabSavannah LoganBrandon SchlomannTeddy HayPhil JahlJulia NgoDeepika SundarramanDrew ShieldsDylan MartinsChristopher Dudley

FundingKaren Guillemin Travis WilesBrian Hammer (GT)Joao Xavier (SK)

People!

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

• Imaging → in vivo gut microbial population dynamics.

More generally: There’s interesting biophysics to uncover in the world of gut microbes!

Raghu NCBS IITM 2019 · Title: Microsoft PowerPoint - Raghu NCBS IITM 2019 Author: Raghu Created Date: 11/6/2019 6:09:18 PM

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