Mammalian cells employ a variety of innate immune mechanisms to recognize and eliminate invading microorganisms, including autophagy of intracellular particles and pro-inflammatory cytokine mediated recruitment of inflammatory cells to the site of infection. Within minutes of Shigella spp. or Salmonella spp. entry into cells, autophagy is activated and inflammasome receptors and adaptors are recruited around intracellular bacteria. A substantial subpopulation of intracellular bacteria avoids association with autophagy markers. How this subpopulation of bacteria evades recognition by these markers has been unclear. Our preliminary data suggest a model in which S. flexneri actively protects itself from autophagy markers. We find that the subpopulation of S. flexneri not associated with certain autophagy markers is universally associated with the host multifunctional protein Toca-1. Based on our findings and on published data, we hypothesize that in the setting of vacuolar membrane remnants, which normally induce innate immune responses, Toca-1 serves as a scaffold that (1) is anchored to membrane fragments through IcsB and IpaB, and (2) coordinates repression of autophagy and restriction of pro-inflammatory cytokine production. Specifically, we hypothesize that (i) IpaB, IcsB, OspC2/3 proteins, and Toca-1 form a complex associated with vacuolar membrane remnants;(ii) the complex assembles on IpaB that has been inserted into the plasma membrane soon after contact of bacteria with the membrane;and, (iii) the IpaB-IcsB-Toca-1-OspC2/3 protein complex per se interferes with pro- inflammatory cytokine production by promoting OspC3-mediated inhibition of caspase activation. The exploratory investigations proposed in this R21 application will test aspects of this hypothesis.
Aim 1 : Investigate whether IcsB, OspC2/3 proteins, IpgD, and/or Toca-1 form a complex associated with vacuolar membrane, and if they do, characterize how.
Aim 2 : Investigate whether the IpaB-IcsB-Toca-1-OspC2/3 protein complex promotes interference with caspase activation. The proposed investigations are highly likely to lead to new insights into mechanisms of S. flexneri manipulation of innate immune responses early during infection. The results obtained from these investigations are likely to have important implications for infection by all intracellular microorganisms and for modulation of innate immune responses more generally.

Public Health Relevance

Human cells are able to eradicate invading microorganisms via a variety of mechanisms. We propose to investigate how the intracellular bacterium Shigella inhibits the cell's ability to recognize it and trigger these responses.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21AI113484-01
Application #
8772174
Study Section
Special Emphasis Panel (ZRG1-IDM-B (80))
Program Officer
Mills, Melody
Project Start
2014-06-01
Project End
2016-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
1
Fiscal Year
2014
Total Cost
$261,000
Indirect Cost
$111,000
Name
Massachusetts General Hospital
Department
Type
DUNS #
073130411
City
Boston
State
MA
Country
United States
Zip Code
02199