Transcript Document

Regulation of cellular antimicrobial activity by
a single microbiota metabolite
Zeni
1
1
Crisp ,
Nandita
2
Kohli ,
1
Mueller ,
Carrie
Arul
1,2
Jayaraman ,
Robert C.
1
Alaniz
Microbial & Molecular Pathogenesis, College of Medicine, Texas A&M Health Science Center
2 Chemical Engineering, College of Engineering, Texas A&M University
Abstract: The microbiota, commensal symbiotic microbes that naturally inhabit the gastrointestinal (GI) tract, contribute to immune & physiologic homeostasis in the host GI tract. The microbiota provide resistance
to enteric pathogen invasion and colonization; an important function termed “colonization resistance” (CR). Despite this observation, the mechanisms mediating CR are poorly understood. We hypothesized the
microbiota produce compounds that either directly or indirectly contribute to CR. Indole is an abundant GI tract metabolite produced from tryptophan strictly by the microbiota. In the GI tract, dendritic cells (DCs),
professional phagocytes that sense the microbiota and provide a link to host immunity, are continuously exposed to indole. Our previous work demonstrated that indole reduces pathogenic bacteria chemotaxis,
motility, and epithelial cell attachment. Here, we tested whether indole directly promotes DC antimicrobial properties. Our results demonstrate that DCs conditioned with indole restrict Salmonella typhimurium invasion,
revealing a potential novel mechanism regulating CR.
How do the bacteria that naturally live in our gut protect us from oral infection with pathogenic microbes?
Introduction
• Salmonellosis is one of the most
common foodborne disease in the world.
Methods
Phagocytes are immune cells
that digest and kill bacteria.
• 93 million cases per year of
M acrophage
Results
Cytotoxicity Assay
Stop Solution
Lysate from T =4 culture
+
Assay Buffer
Dendritic cell
gastroenteritis worldwide caused by
Plate Reader
OD at 490 nm
Salmonella.
Figure 6. Cytotoxicity is determined by the Lactase
Dehydrogenase release from the cytoplasm.
• The increase of multi-drug resistant
Salmonella strain highlights the need for
more research on this clinically relevant
pathogen and for the discovery of new
Results
Figure 3.
•
(Left) Macrophage is potent antimicrobial phagocyte.
•
(Right) Dendritic cells activate immune system (Tcells)
Figure 9. Indole-treated Dendritic cells reduce
Salmonella- induced Cytotoxicity.
treatment options.
Salmonella Pathogenesis
Hypothesis
Conclusions
Microbiota produce molecules that augment
phagocyte killing of bacteria.
• These data suggest that indole is an
important microbiota-derived endogenous
Dendritic Cell
Salmonella
Dendritic Cell
Salmonella
+
+
Indole
mediator
+
that
protects
against
gut
pathogens such as Salmonella.
Figure 7. Indole-treated
Salmonella invasion
[make this text bullets]
Dendritic
cells
Discussions
resist
• Indole is a natural product, derived from
Figure 1.
• Upon contact with intestinal epithelial cells, Salmonella activate Type 3
Secretion System I to translocate the virulence proteins (effectors) into
the host cytoplasm.
• The effectors modulate rearrangement of actin cytoskeleton, which
results in membrane ruffles and promote Salmonella uptake.
• After internalization, Salmonella modify host phagosome into
Salmonella-Containing Vacuole (SCV). Changes in microenvironment of
SCV trigger the activation of Type 3 Secretion System II (T3SSII).
• The effectors of T3SSII are necessary for positioning of SCV near the
Golgi network. This allow Salmonella to intercept host vesicular
trafficking and redirect nutrients to promote intracellular replication
Microbiota:
Beneficial microbes in our gut
microbiota, with low toxicity, and potential to
Figure 4. Host cells conditioned with Indole are more resistant to Salmonella
infection.
develop new therapeutic compounds.
Methods
• With chemical engineering, we may develop
indole analogues that may be superior to
Gentamicin Protection Assay
antibiotics, without the emergence of drug
+
resistance.
+
1 hour Incubation with Gentamicin
Gentamicin
1 hour Infection
MOI = 10
“Colonization Resistance”
Pathogens
What other microbiotaDependent mechanisms
Contribute to
Coloniza on Resistance?
•
•
•
Space
Nutrients
Short-chain fatty acid
eg. Butyrate
Acknowledgments
Lyse = Invasion
T=0
Figure 8. Indole-treated Dendritic cells restrict
Salmonella intracellular survival and replication.
Lyse = Survival/
Replication
Dr. Robert C. Alaniz
Nandita Kohli
Carrie Mueller
Dr. Arul Jayaraman
•
•
•
•
Madhu Katepalli
Shelby Steinmeyer
Dr. Andrews-Polymenis
NSF and NIH
T=4
M icrobiota
Figure 2. Colonization Resistance is a phenomenon
where microbiota protect the host from infection by
pathogens.
•
•
•
•
Figure 5.
• Dendritic cells (DC2.4) are conditioned with Indole over night. Infect DC
2.4 with S. typhimurium for 1 hour.
• Add Gentamicin to kill the extracellular Salmonella for 1 hour.
• At T=0 hr, lyse DC 2.4 and determine CFU/mL for Salmonella invasion.
• At T=4 hr, lyse DC 2.4 and determine CFU/mL for Salmonella survival and
intracellular replication.
References
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Tarun Bansal, Robert C. Alaniz, Thomas K. Wood, Arul Jayaraman. The bacterial signal indole increases epithelial-cell tight-junction resistance and attenuates indicators of inflammation. doi: 10.1073/pnas.0906112107.
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Andrea Haraga, Maikke B. Ohlson, and Samuel l. Miller. Salmonella interplay with host cells. 2008 January. Doi: 10.1038/nrmicro1788.
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Shannon E. Majowicz, Jennie Musto, Elaine Scallan. The Global Burden of Nontyphoidal Salmonella Gastroenteritis. International Collaboration on Enteric Disease “Burden of Illness” Studies.
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Terence A. Agbor, Beth A,. McCormick. Salmonella Effectors: Important players modulating host cell function during infection. Cell Microbiol. 2011 December; 13(12): 1858-1869. doi: 10.111/j.1462-5822.2011.01701.x.
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Susan L. Fink, Tessa Bergbaken, Brad T. Cookson. Anthrax lethal toxin and Salmonella elicit the common cell death pathway of caspase-1-dependent pyroptosis via distinct mechanisms. 2008 March. doi: 10.1073/pnas.0707370105.
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