Small molecule DnaK modulators targeting the beta-domain.

The molecular chaperone DnaK is essential for the survival of bacterial pathogens in the hostile environment of the host. Hence, it is in principle a promising target for drug design but for which no current inhibitors are available apart from certain antimicrobial peptides. To this end, we have screened libraries of small molecules for their ability to interact with the substrate-binding domain of DnaK. The most promising hit from the screen was synthesized and along with its analogs subjected to further assays to determine their binding affinity and ability to interfere with bacterial growth. This work resulted in the identification of a number of compounds that bind with submicromolar affinity and capable of inhibiting Yersinia pseudotuberculosis growth more effectively than the previously characterized peptides.

Salmonella secreted factor L deubiquitinase of Salmonella typhimurium inhibits NF-kappaB, suppresses IkappaBalpha ubiquitination and modulates innate immune responses.

Salmonella enterica translocates virulent factors into host cells using type III secretion systems to promote host colonization, intracellular bacterial replication and survival, and disease pathogenesis. Among many effectors, the type III secretion system encoded in Salmonella pathogenicity island 2 translocates a Salmonella-specific protein, designated Salmonella secreted factor L (SseL), a putative virulence factor possessing deubiquitinase activity. In this study, we attempt to elucidate the mechanism and the function of SseL in vitro, in primary host macrophages and in vivo in infected mice. Expression of SseL in mammalian cells suppresses NF-kappaB activation downstream of IkappaBalpha kinases and impairs IkappaBalpha ubiquitination and degradation, but not IkappaBalpha phosphorylation. Disruption of the gene encoding SseL in S. enterica serovar typhimurium increases IkappaBalpha degradation and ubiquitination, as well as NF-kappaB activation in infected macrophages, compared with wild-type bacteria. Mice infected with SseL-deficient bacteria mount stronger inflammatory responses, associated with increased production of NF-kappaB-dependent cytokines. Thus, SseL represents one of the first bacterial deubiquitinases demonstrated to modulate the host inflammatory response in vivo.

Helicobacter pylori preferentially induces interleukin 12 (IL-12) rather than IL-6 or IL-10 in human dendritic cells.

Dendritic cells are potent antigen-presenting cells that are present in the gastrointestinal tract and are required for the induction of a Th1 T-cell acquired immune response. Since infection with the gastric pathogen Helicobacter pylori elicits a Th1 cell response, the interaction of these organisms with dendritic cells should reflect the Th1 bias. We incubated H. pylori with cultured human dendritic cells and measured the cytokine induction profile, comparing the response to that induced by Salmonella enterica serovar Typhimurium. We found that H. pylori induced little interleukin 6 (IL-6) and essentially no IL-10 in contrast to S. enterica. However, H. pylori induced levels of IL-12 that were 30% of those induced by S. enterica, indicating a Th1 response. An isogenic cagE mutant of H. pylori lost about 50% of its IL-12-inducing ability, suggesting a role for the cag type IV secretion system in the stimulation of dendritic cells.