Salmonella enterica encompasses over 2400 unique serovars. Serovar Typhi is a human specific pathogen and the cause of typhoid fever. Analysis of molecular bacterial events that occur during human infection with serovar Typhi has been hampered by the absence of an animal model of typhoid fever using serovar Typhi, and the relative paucity of recoverable organisms from humans with typhoid fever. Much of our knowledge of possible pathogenic events during serovar Typhi infection of humans is extrapolated from analysis of serovar Typhimurium infection in mice. Serovar Typhimurium causes a typhoid-like illness in mice, but only gastroenteritis in humans. Our hypothesis in this high-risk developmental pilot R21 study is that high throughput proteomic technology can be used to identify Salmonella enterica antigens important during human infection. A number of regulatory systems that facilitate bacterial adaption and survival within eukaryotic hosts have been identified in S. enterica organisms, including the phoP/phoQ, pmrA/B, ropS, fur, and ompR-envZregulons. These regulons have also been shown to respond to in vitro environmental stimuli thought to mirror conditions bacteria encounter within infected hosts (including alterations in pH, cationic concentrations, low iron, among others). In order to preliminarily address whether high throughput proteomic technology can be used to better understand bacterial pathogenic events during infection with S. enterica organisms, we propose two specific aims.
In specific aim one, we propose to evaluate the complete organism proteome of serovar Typhimurium organisms grown under conditions that induce the PhoP regulon, a regulatory system expressed in vivo during infection. We have chosen this regulatory system as our model since it is perhaps the best-studied regulon in S. enterica organisms. We will compare the PhoP-induced proteome to that expressed by organisms grown under non-PhoP-inducing conditions. We will also repeat these experiments using a PhoP mutant strain of serovar Typhimurium. We will thus identify serovar Typhimurium proteins either expressed or repressed during conditions thought to mirror certain signaling events in vivo, and we will develop technologies and analysis tools that can subsequently be applied to serovar Typhi organisms.
In specific aim two, we will use proteomic technology (proteomic- based expression library screening; PELS) to directly identify serovar Typhi antigens expressed in vivo during human infection, using convalescent sera of humans recovering from serovar Typhi bacteremia and typhoid fever in Bangladesh. Preliminary data gathered during this high-risk developmental pilot R21 project would be critical to developing tools required for high throughput proteomic analysis to rapidly and efficiently identify Salmonella enterica proteins expressed in vivo during human infection, and may assist in the development of novel therapeutic, diagnostic and preventative technologies. ? ? ? ?
