Molecular Analysis of Bacterial Adaptive Response to Host Reactive Species

Background and Innovation: Diarrheal and invasive infections caused by non-typhoidal Salmonella account for the highest burden among all foodborne diseases. Non-typhoidal Salmonella infections cause diarrhea in healthy individuals and are associated with life-threatening disseminated infections in immunocompromised patients with defects in neutrophils, macrophages, or CD4 T cells due to HIV or cancer comorbidities. Investigations on the precise activation of virulence programs crucial to Salmonella pathogenesis will lead to the development of novel antibiotics. Our research has demonstrated that DksA and Gre factors regulate vital aspects of Salmonella pathogenesis. Specifically, our investigations have uncovered a conserved pocket within the DksA coiled-coil domain that interacts closely with RNA polymerase and is necessary for the intracellular lifestyle of Salmonella. A better understanding of the interactions between conserved charged residues in the DksA pocket will help us improve the design of drugs against this transcription factor. Gre factors compete with DksA for binding to the secondary channel of RNA polymerase, and like DksA, Gre factors are essential for Salmonella pathogenesis. We have discovered a hitherto unsuspected, yet essential, link between the regulatory actions of Gre factors and the activation of Salmonella pathogenicity island-2 (SPI-2) gene transcription. The Gre-mediated expression of the SPI-2 type III secretion system is likely to play a significant role in Salmonella pathogenesis, as the horizontally acquired SPI-2 gene cluster is indispensable for Salmonella's growth in humans and experimental animal models. Our research aims to elucidate the biochemical mechanism through which Gre factors activate SPI-2 transcription that is essential for Salmonella pathogenesis. Significance and Impact to Veteran's Healthcare: Acute gastroenteritis associated with non-typhoidal Salmonella infection is a frequent occurrence among inpatients at VA hospitals. The highest incidence of non- typhoidal Salmonella cases among veterans and their families is observed in the very young and the elderly. The emergence of antibiotic resistance poses a significant clinical problem of epic proportions among Gram- negative rods such as Salmonella. The dwindling number of therapeutic compounds in development compounds antibiotic resistance. The knowledge gain in our analysis of the conserved DksA pocket will inform the rational development of future therapies against a variety of Gram-negative, antibiotic-resistant bacteria, which pose significant risks of morbidity and mortality among active servicemen, women, veterans, and their families. Path to translation/implementation: Future prophylactic and therapeutic strategies against drug-resistant bacteria stand to benefit greatly from the identification of novel molecular targets in pathogenic bacteria. Our characterization of critical charged residues in the conserved DksA pocket will inform our medicinal chemistry to improve anti-DksA antibiotics.