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-κB activation downstream of IκBα kinases and impairs IκBα ubiquitination and degradation, but not IκBα phosphorylation. Disruption of the gene encoding SseL in S. enterica serovar typhimurium increases IκBα degradation and ubiquitination, as well as NF-κB 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-κB-dependent cytokines. Thus, SseL represents one of the first bacterial deubiquitinases demonstrated to modulate the host inflammatory response in vivo.
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.
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. Keywordsallostery; chaperone; DnaK; drug discovery; NMR Chaperones provide crucial cellular functions across all domains of life (1). Hsp70 homologs are involved in such essential processes as protein folding, translocation, degradation, and even gene expression (2-7). The multiple homologs and the wide array of co-chaperones for Hsp70 underscore their importance and functional diversity (1,8,9). Hsp70s, including DnaK, the Escherichia coli Hsp70 (10-14) investigated here, have been extensively studied and have greatly increased our understanding of allostery. DnaK is composed of two structural domains (Figure 1A and B), an N-terminal nucleotide-binding ATPase domain (NBD) and a C-terminal substrate-binding domain (SBD), which can be further subdivided into an all β-strand region that binds substrate (β-domain) and an α-helical lid region that can close over bound substrate (11,12,15). DnaK cycles between two main states: an ATP-bound state with low affinity for substrate that is characterized by a tight interaction of the two domains, and an ADP-bound state that has high substrate affinity with less interaction between the domains (16-18). Structural and biochemical data have shown that residues from both domains (including loop L2,3 of the β-domain), as well as from the linker between them are required for communicating the nucleotide or substrate occupancy to the other respective domain (10,12,14,19,20 Figure 1A and B). We report structure-activity relationship (SAR) studies, in silico docking, isothermal titration calorimetry (ITC), and antimicrobial activity of the most promising compounds of the series. Given the dire need for new classes of antibiotics, we hope that these compounds can eventually stimulate further research in this area may lead to clinically useful drugs. Materials and Methods Protein expression and purificationThe gene coding for the E. coli DnaK substrate-binding (β) domain (393-507) was amplified by PCR and subcloned into pET21a using the NdeI and BamHI cloning sites. The resulting protein contains 17 extra amino acid residues (MGSSHHHHHHGLVPRGS) at the N-terminus. The protein was expressed in the E. coli strain BL21(DE3) pLysS and purified using Ni 2 + affinity chr...
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