HreP, an In Vivo - Expressed Protease of Yersinia enterocolitica , Is a New Member of the Family of Subtilisin/Kexin-Like Proteases

Abstract

The role of proteases in pathogenesis is well established for several microorganisms but has not been described for Yersinia enterocolitica. Previously, we identified a gene, hreP, which showed significant similarity to proteases in a screen for chromosomal genes of Y. enterocolitica that were exclusively expressed during an infection of mice. We cloned this gene by chromosome capture and subsequently determined its nucleotide sequence. Like inv, the gene encoding the invasin protein of Y. enterocolitica, hreP is located in a cluster of flagellum biosynthesis and chemotaxis genes. The genus Yersinia consists of several species, three of which are considered to be pathogens for mammals. Yersinia pestis is the etiologic agent of plague, while Yersinia pseudotuberculosis and Yersinia enterocolitica primarily cause gastrointestinal syndroms. Yersinia enterocolitica is a pathogen for humans and can cause a variety of syndromes, including acute enteritis, mesenteric lymphadenitis, and enterocolitis (15). All three species have a tropism for lymphoid tissues and contain a 70-kb virulence plasmid which encodes a system that delivers antiphagocytic effector proteins into the cytosol of eukaryotic cells (16).In contrast to the virulence plasmid, relatively little is known about chromosomally borne virulence genes of Y. enterocolitica. In addition to the genes inv and ail, which encode proteins that enable the bacterium to adhere to and invade eukaryotic cells (29,39), genes for the acquisition of iron and the synthesis of the O antigen component of lipopolysaccharide and an enterotoxin gene have been described as chromosomally borne virulence-associated genes (1, 13, 27, 52, 53, 59). By signature-tagged transposon mutagenesis, chromosomal genes of Y. enterocolitica were identified that were previously not described as required for in vivo survival. They include genes for the synthesis of outer-membrane components, stress response, and nutrient acquisition (17). One transposon insertion was localized in pspC, and the corresponding strain was severely attenuated for virulence (17).Recently, in vivo expression technology (IVET) was established as a powerful tool to select for and identify genes that are expressed during an infection (35,36). To overcome the discrepancy between the availability of information about chromosomally and virulence plasmid-borne virulence genes, IVET was used with Y. enterocolitica in a mouse model of infection (58). This study led to the identification of 45 different chromosomal loci, designated hre for host responsive elements, that are expressed early during an infection but not under standard laboratory conditions. The identified hre loci were grouped according to their predicted function or property and comprise genes important for stress response, iron starvation response, or cell envelope maintenance. Another group contains hre loci with noncategorized functions, including those with unknown function or no similarity to genes in the database (58). The same IVET pool was also used to identif...

A Regulatory Network Controls Expression of the In Vivo-Expressed HreP Protease of Yersinia enterocolitica

“…Bacteria normalized to an optical density at 600 nm of 0.25 (corresponding to approximately 1.2 ϫ 10 6 bacteria) were resuspended in 4ϫ protein loading buffer, boiled, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose membranes by Western blotting and incubated with an anti-HreP prosequence antiserum as described previously (19).…”

“…A further study characterizes hreP, which codes for a protease with similarity to eukaryotic subtilisin/kexin-like proteases. Y. enterocolitica hreP mutant strains have a defect in virulence in the mouse model of infection (19,44). The HreP protease is expressed as a proprotein, where the amino-terminal prosequence serves as an intramolecular chaperone, which is autocatalytically cleaved off after folding.…”

“…The HreP protease is expressed as a proprotein, where the amino-terminal prosequence serves as an intramolecular chaperone, which is autocatalytically cleaved off after folding. The hreP gene is specific for Y. enterocolitica and cannot be found in the genomes of the other pathogenic Yersinia species, Y. pseudotuberculosis and Y. pestis, implying that HreP might play a role during infection that is specific for the pathogenesis of Y. enterocolitica (19).…”

Unraveling the Secret Lives of Bacteria: Use of In Vivo Expression Technology and Differential Fluorescence Induction Promoter Traps as Tools for Exploring Niche-Specific Gene Expression

“…Mutational analysis revealed a strong reduction of competitiveness and virulence of the hreP mutant (300). The HreP protease shows significant similarity with eukaryotic subtilisin/kexin-like proprotein convertases and was probably acquired by horizontal gene transfer (100). Although mutational studies unequivocally demonstrated the importance of HreP in virulence, its precise role has yet to be elucidated.…”

PathogenicYersinia enterocoliticaO:3 isolated from a hunted wild alpine ibex

“…(pYV) and five virulence genes (ail, ystA, ystB, myfA, hreP) and rfbC for O :3 serotype located in the chromosome [17][18][19][20]. The DNA was released from bacterial colonies by heating at 97 xC for 10 min, and 1 ml of this liquid was added to 19 ml of the mastermix (iQ TM SYBR Green Supermix ; Bio-Rad, USA).…”