The Gram-negative bacterium Vibrio cholerae, the causative agent of cholera, is a facultative pathogen that resides in both human and aquatic environments. Extensive in vitro studies have identified a number of virulence factors required to produce disease during infection. However, how V. cholerae alters its gene expression upon transition from its marine ecosystem to the human host and along progression of infection is largely unknown. It has become clear in recent years that bacteria actively sense their surroundings. But how, specifically, do bacterial pathogens "know" that they are in the infectious setting? The inside of the human body is a different niche world around it: in the case of the gastrointestinal tract, the oxygen tension is lower, there are high densities of other bacteria, an perhaps most important, there are adaptive and innate mammalian immune effectors designed specifically to kill bacteria or inhibit their toxicity. Thus, the act of infection requires a dramtic physiologic shift that must be orchestrated by the bacteria at the appropriate time. Remarkably few bacterial sensors of the host environment are known. During the first funding period, we made the discovery that the transcription factor AphB possesses a cysteine residue that undergoes modification in response to the microoxic conditions of the intestines, leading to activation of virulence. We now have further evidence that diverse cysteine modifications in AphB lead to diverse effects on cell physiology through changes in gene transcription and protein stability. We hypothesize that AphB is the central processor of environmental information relevant to infection for V. cholerae. Specifically, we hypothesize that AphB uses thiol modifications to integrate the presence of reductive, oxidative and nitrosative reactants into the gene expression decisions necessary to guide the organism in and out of the human gut. We will use a mix of biochemical and genetic techniques to define mechanistically how AphB orchestrates the pathogenic life cycle of V. cholerae and what other factors are involved at this cysteine-based sensory hub. We believe that by comprehensively defining the way AphB monitors the chemical microenvironment for V. cholerae, we then may be able to extend the paradigm of cysteine-based environmental sensing to other V. cholerae proteins and other pathogens. We will first investigate how V. cholerae senses the low oxygen tension in the gut as a signal to reduce AphB. We will also investigate the molecular mechanisms of AphB modification, oligomerization, and activation under oxygen-limiting conditions using a series of genetic and biochemical approaches. We will then conduct genetic screens to identify regulatory factors that control temporal expression of AphB reduction-related genes during infection. Next we will examine how AphB is modified by reactive nitrogen species (RNS), such as nitric oxide (NO), and oxidative stress from reactive oxygen species (ROS) generated in vivo and how these modifications affect AphB functionality, including posttranslational modification-induced AphB degradation. We hope that by delineating the effect of various modifications on AphB function we can outline how this critical regulatory protein samples the local chemical microenvironment to inform cellular processes. Finally, as V. cholerae is capable of tightly regulating the timing of its gene expression during infection in response to host stimuli, we hypothesize that the role of AphB, having already been identified as necessary for activating virulence, extends generally to organizing the infectious life cycle of V. cholerae, affecting myriad processes that require timely expression for pathogenesis. Our preliminary data indicate that AphB also regulates both RNS detoxification and chemotaxis, both processes central to the V. cholerae life cycle, in response to diverse stimuli. We will study the broad effects of AphB modification on cell physiology, focusing specifically chemotaxis and ROS/RNS stress management with the following model in mind: upon entry to the gut, reduced AphB activates virulence in response to low oxygen tension~ in response to increasing chemical stress in the gut, modified AphB deactivates virulence while up-regulating ROS and RNS detoxification and chemotaxis, thus preparing the cell for survival in the aquatic environment. By understanding V. cholerae gene regulation and posttranslational modification in vivo, we hope that our studies will hopefully lead to novel therapies that can target these most basic of V. cholerae in vivo survival strategies and shed new light on the host-pathogen interactions that underpin infections by related enteric bacterial pathogens.

Public Health Relevance

Cholera is a devastating diarrheal illness currently taking lives in poverty-stricken countries around the world, including Haiti and the causative agent of cholera Vibrio cholerae has figured prominently in the history of infectious diseases as a cause of periodic global pandemics. Despite major improvements in medical treatment as well as a better understanding of the molecular processes involved in the virulence of V. cholerae, cholera still occurs widely in epidemic form particularly in South Asia, Africa, and Latin America. This study proposed will shed light on importance of V. cholerae's in vivo gene regulations in host-pathogen interactions, with the goal of better understanding V. cholerae pathogenesis and, potentially, discovering novel treatment options for the cholera disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
High Priority, Short Term Project Award (R56)
Project #
2R56AI072479-06A1
Application #
8703959
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
Hall, Robert H
Project Start
2007-01-01
Project End
2014-07-31
Budget Start
2013-08-08
Budget End
2014-07-31
Support Year
6
Fiscal Year
2013
Total Cost
$376,000
Indirect Cost
$141,000
Name
University of Pennsylvania
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Fan, Fenxia; Liu, Zhi; Jabeen, Nusrat et al. (2014) Enhanced interaction of Vibrio cholerae virulence regulators TcpP and ToxR under oxygen-limiting conditions. Infect Immun 82:1676-82