The Salmonella typhi melittin resistance gene pqaB affects intracellular growth in PMA-differentiated U937 cells, polymyxin B resistance and lipopolysaccharide

Abstract

Salmonella typhi is the causative agent of typhoid fever in humans. A cellculture based assay involving the human monocyte macrophage cell line U937 has been developed to examine S. typhi invasion and survival. An 5. typhi PhoP' (null) mutant was shown to be restricted in net growth in phorbol myristate acetate (PMA) differentiated U937 (PMA4937) cells, and an 5. typhi PhoPC (constitutive) mutant showed a defect in invasion. Neither of the phoPIQ mutants were growth impaired in HeLa cells, however the PhoPc mutant was impaired in invasion. As opposed to what was found for S. typhi, Salmonella typhimurium wild-type, PhoP' and PhoPc mutants grew equally well in PMA4937 cells, indicating that the PhoP--mediated net growth restriction in the PMA4937 cells was S. typhi specific. An S. typhi mutation, pqaB::MudJ, recently shown t o be a PhoP-activated locus, was shown to have a net growth defect in PMA4937 cells. Sequencing of the S. typhipqaB gene revealed it had 98% identity to the fifth gene in a S. typhimurium PmrA/B regulated operon necessary for 4-aminoarabinose lipid A modification and polymyxin B resistance. The pqaB locus was regulated by PmrA/B (whose activity is modulated by PhoP-PhoQ) and the pqaB transposon mutant was sensitive to polymyxin B. The lipopolysaccharides (LPS) of 5. @phi and S. typhimurium wildtype, PhoP' and PhoPc mutants, were compared by SDS-PAGE and silver staining. Differences in the LPS profile between the two Salmonella species were observed, and shown to be affected differently by the PhoPc mutation. Additionally, the pqaB:: MudJ mutation affected S. typhi LPS. The effects on LPS may have ramifications for the difference between S. typhi and S. typhimurium infection of hosts.

Host adapted serotypes of Salmonella enterica

“…In itro studies have demonstrated that host-specific pathogenesis of Salmonella serotypes may depend on the selective recognition of complement receptor (CR) types on the macrophages membrane [206]. Typhi and Typhimurium induced their own uptake by micropinocytosis in both human and murine macrophages, but only Typhi was capable of growth in human macrophages.…”

“…Strikingly, Typhi and Typhimurium recognized, respectively, CR1 and CR3 on human macrophages, whereas they recognized, respectively, CR3 and CR1 on murine macrophages. Baker and Morona have recently observed that phorbol myristate acetate ( PMA) differentiated U937 (PMA-U937, human) cells restricted the net growth of Typhi but not Typhimurium phoP mutants, suggesting that the phoP\Q locus may control expression of genes involved in host specificity, particularly affecting differential effects on Typhi and Typhimurium LPS [206]. The relevance of the phoP\Q regulatory system in host specificity can also be recognized by the recent finding that pagK, a positively regulated gene in a trasposon-like element, is found only in broad host range Salmonella spp.…”

Genetic modulation of Shigella flexneri 2a lipopolysaccharide O antigen modal chain length reveals that it has been optimized for virulence

“…General DNA manipulation, plasmid DNA isolation, dye terminator DNA sequencing, PCR, DNA transformation and electroporation were performed as described previously (Baker et al, 1999;Daniels & Morona, 1999). The location of mini-Tn5-Cm R in pRMCD72 was determined by digestion with SmaI, and cloning three SmaI fragments (~2?8,~1?95 and 0?4 kb) into the SmaI site of pBluescript-SK (Stratagene).…”

Functional cloning and characterization of a UDP- glucuronic acid decarboxylase: The pathogenic fungus Cryptococcus neoformans elucidates UDP-xylose synthesis

“…PmrE encodes a UDP-Glc dehydrogenase. The UDP-GlcA synthesized by this enzyme could serve as a precursor to UDP-aminoarabinose (16)(17)(18), via initial reactions similar to those that convert UDP-GlcA to UDP-Xyl in eukaryotic systems (Fig. 2).…”

“…PmrF is located in a putative operon with several genes whose sequences suggest roles in sugar metabolism; Gunn et al (16) have proposed that these genes also may participate in aminoarabinose addition. Extending this model, the groups of Raetz and Morona (17,18) independently hypothesized a pathway for UDP-aminoarabinose synthesis based on sequence analysis of the genes in the pmrF cluster. They suggested that the protein product of one sequence, ORF3, might catalyze oxidation of the 4-position of UDP-GlcA.…”