Characterization of RP 333, a gene encoding CapD of Rickettsia prowazekii with UDP-glucose 4-epimerase activity.

The genetic mechanism of membrane assembly in Rickettsia prowazekii, the causative agent of epidemic typhus, is unknown. Recent progress in genomics of this small intracellular bacterium prompted us to explore membrane biogenesis as it pertains to identification and characterization of gene products that are of importance in polysaccharide biosynthesis. We chose to define the function of the R. prowazekii capD gene (RP 333) product designated as a putative epimerase. We detected a capD transcript in yolk sacs infected with R. prowazekii at ten days post-infection. The capD gene was PCR amplified and cloned into the entry vector pENTR/SD/D-TOPO and transferred to a low copy arabinose-inducible pBAD/Myc-HisA expression plasmid constructed in our laboratory. The corresponding recombinant protein (CapD) was expressed in PL2, a galE28 mutant of Escherichia coli, and has been shown to epimerize UDP-galactose to UDP-glucose without the addition of NAD+. CapD characterization is important for defense strategies targeted towards design of an R. prowazekii vaccine, generation of new antibiotics, and opening the way for research in membrane biogenesis with respect to the parasitic lifestyle of Rickettsia.

Development of Rickettsia prowazekii DNA vaccine: cloning strategies.

Rickettsia prowazekii, the etiologic agent of louse-borne typhus, is listed as a category B agent under the select agent list of the United States Centers for Disease Control and Prevention. R. prowazekii was placed on the select agent list due to its potential to cause epidemic, high mortality in untreated and/or misdiagnosed cases, and ease of spread in vulnerable populations. Historically, R. prowazekii vaccines using crude antigen and/or inactivated rickettsia were partially protective but have been accompanied with undesirable toxic reactions and difficulties in standardization. The availability of the genome sequence of R. prowazekii allowed us to select genes that encode proteins with potential in immuno-protection against this human pathogen. We successfully PCR-amplified a group of genes involved in invasion (invA), cell division (fts), protein secretion (sec gene family), and virulence (ompA and ompB, virB gene family, cap and tlyA and tlyC). The generated PCR products were cloned into the Gateway cloning system and the cloned products will be introduced into Vical VR 1020-DV and VR 1012-DV DNA vaccine plasmids. Twenty-four target genes from R. prowazekii have been PCR amplified, of which fifteen have been introduced into the pENTR/SD/D-TOPO entry cloning vector.

The Helicobacter pylori flbA flagellar biosynthesis and regulatory gene is required for motility and virulence and modulates urease of H. pylori and Proteus mirabilis.

Helicobacter pylori and Proteus mirabilis ureases are nickel-requiring metallo-enzymes that hydrolyse urea to NH3 and CO2. In both H. pylori and in an Escherichia coli model of H. pylori urease activity, a high affinity nickel transporter, NixA, is required for optimal urease activity, whereas the urea-dependent UreR positive transcriptional activator governs optimal urease expression in P. mirabilis. The H. pylori flbA gene is a flagellar biosynthesis and regulatory gene that modulates urease activity in the E. coli model of H. pylori urease activity. All flbA mutants of eight strains of H. pylori were non-motile and five had a strain-dependent alteration in urease activity. The flbA gene decreased urease activity 15-fold when expressed in E. coli containing the H. pylori urease locus and the nixA gene; this was reversed by disruption of flbA. The flbA gene decreased nixA transcription. flbA also decreased urease activity three-fold in E. coli containing the P. mirabilis urease locus in a urea- and UreR-dependent fashion. Here the flbA gene repressed the P. mirabilis urease promoter. Thus, FlbA decreased urease activity of both H. pylori and P. mirabilis, but through distinct mechanisms. H. pylori wild-type strain SS1 colonised gerbils at a mean of 5.4 x 10(6) cfu/g of antrum and caused chronic gastritis and lesions in the antrum. In contrast, the flbA mutant did not colonise five of six gerbils and caused no lesions, indicating that motility mediated by flbA was required for colonisation. Because FlbA regulates flagellar biosynthesis and secretion, as well as forming a structural component of the flagellar secretion apparatus, two seemingly unrelated virulence attributes, motility and urease, may be coupled in H. pylori and P. mirabilis and possibly also in other motile, ureolytic bacteria.