Salmonella is a Gram-negative pathogenic bacterium that causes significant morbidity, mortality, and economic loss worldwide. Salmonellosis represents a broad spectrum of clinical diseases, ranging from gastroenteritis to typhoid fever, and is responsible for 1.4 billion illnesses and nearly 4 million deaths annually. Additionally, th treatment of Salmonella infection is increasingly complicated by multidrug resistance. In order to cause disease, pathogenic bacteria in general and Salmonella in particular must sense, respond to, and limit the considerable stresses imposed by innate host defense, including oxidative and nitrosative stress that accompanies the host response to infection. Preliminary data presented in this application identifies the bacterial RNA polymerase regulatory protein DksA as a direct sensor of oxidative and nitrosative stress. We hypothesize that the Zn finger of DksA forms a novel redox sensor capable of distinguishing amongst, and differentially responding to, specific host-derived reactive oxygen and nitrogen species, thereby endowing Salmonella with a rapid and reversible mechanism to affect transcriptional responses. This """"""""smart switch"""""""" concept would allow DksA to integrate nutritional, oxidative, and nitrosative signals into a coordinated regulatory output capable of effectively tailoring bacterial metabolism and defense programs to best address the dynamic and hostile microenvironments Salmonella encounters during infection. The investigations proposed herein will determine the molecular mechanism used by DksA to sense reactive oxygen and nitrogen species, and the role this regulatory protein plays in Salmonella pathogenicity.
In Aim 1, biochemical analysis and transcriptional evaluation will define disulfide bond formation in response to reactive species, and the role this bonding plays in DksA structural rearrangement and changes in regulatory function.
Aim 2 will determine the molecular mechanism by which DksA promotes antioxidative and antinitrosative defenses;the role of DksA-mediated redox sensing and response in Salmonella pathogenesis will also be examined using macrophages and a murine model of salmonellosis. Collectively, the proposed investigations will characterize a previously unknown sensory role for DksA and greatly expand our understanding of biological redox sensors that mediate pleiotropic roles in diverse pro- and eukaryotic processes. These studies will also yield novel insights into Salmonella pathogenesis and the molecular mechanisms of infectious diseases. Conservation of dksA by Gram-negative bacteria indicates the proposed investigations are likely relevant to a range of medically important bacteria. A mechanistic understanding of bacterial pathogenesis and the processes used by bacteria to sense, respond to, and limit host defense will provide an important foundation for the identification of unique, broad-spectrum therapeutic targets and aid in the development of innovative strategies for effectively treating infections caused by pathogenic, potentially multidrug-resistant bacteria.
In order to cause disease, pathogenic bacteria in general and Salmonella in particular must sense, respond to, and limit the considerable stresses imposed by innate host defense. Understanding the mechanisms by which these sensory events occur and subsequently coordinate adaptive responses will provide novel insight into the molecular mechanisms of infectious diseases and aid in the development of innovative avenues for treating infections caused by pathogenic, potentially multidrug-resistant bacteria. The proposed studies will determine how the Salmonella protein DksA senses reactive oxygen and nitrogen species, prominent effectors of innate immunity, and converts this recognition into a global transcriptional response essential to pathogenesis.
|Lomonaco, Sara; Crawford, Matthew A; Lascols, Christine et al. (2018) Resistome of carbapenem- and colistin-resistant Klebsiella pneumoniae clinical isolates. PLoS One 13:e0198526|
|Tapscott, Timothy; Kim, Ju-Sim; Crawford, Matthew A et al. (2018) Guanosine tetraphosphate relieves the negative regulation of Salmonella pathogenicity island-2 gene transcription exerted by the AT-rich ssrA discriminator region. Sci Rep 8:9465|
|Crawford, Matthew A; Fisher, Debra J; Leung, Lisa M et al. (2017) CXC Chemokines Exhibit Bactericidal Activity against Multidrug-Resistant Gram-Negative Pathogens. MBio 8:|
|Crawford, Matthew A; Tapscott, Timothy; Fitzsimmons, Liam F et al. (2016) Redox-Active Sensing by Bacterial DksA Transcription Factors Is Determined by Cysteine and Zinc Content. MBio 7:e02161-15|