Mammalian and prokaryotic high-temperature requirement A (HtrA) proteins are chaperones and serine proteases with important roles in protein quality control. Here, we describe an entirely new function of HtrA and identify it as a new secreted virulence factor from Helicobacter pylori, which cleaves the ectodomain of the cell-adhesion protein E-cadherin. E-cadherin shedding disrupts epithelial barrier functions allowing H. pylori designed to access the intercellular space. We then designed a small-molecule inhibitor that efficiently blocks HtrA activity, E-cadherin cleavage and intercellular entry of H. pylori.

BackgroundCampylobacter jejuni is one of the most important bacterial pathogens causing food-borne illness worldwide. Crossing the intestinal epithelial barrier and host cell entry by C. jejuni is considered the primary reason of damage to the intestinal tissue, but the molecular mechanisms as well as major bacterial and host cell factors involved in this process are still widely unclear.ResultsIn the present study, we characterized the serine protease HtrA (high-temperature requirement A) of C. jejuni as a secreted virulence factor with important proteolytic functions. Infection studies and in vitro cleavage assays showed that C. jejuni’s HtrA triggers shedding of the extracellular E-cadherin NTF domain (90 kDa) of non-polarised INT-407 and polarized MKN-28 epithelial cells, but fibronectin was not cleaved as seen for H. pylori’s HtrA. Deletion of the htrA gene in C. jejuni or expression of a protease-deficient S197A point mutant did not lead to loss of flagella or reduced bacterial motility, but led to severe defects in E-cadherin cleavage and transmigration of the bacteria across polarized MKN-28 cell layers. Unlike other highly invasive pathogens, transmigration across polarized cells by wild-type C. jejuni is highly efficient and is achieved within a few minutes of infection. Interestingly, E-cadherin cleavage by C. jejuni occurs in a limited fashion and transmigration required the intact flagella as well as HtrA protease activity, but does not reduce transepithelial electrical resistance (TER) as seen with Salmonella, Shigella, Listeria or Neisseria.ConclusionThese results suggest that HtrA-mediated E-cadherin cleavage is involved in rapid crossing of the epithelial barrier by C. jejuni via a very specific mechanism using the paracellular route to reach basolateral surfaces, but does not cleave the fibronectin receptor which is necessary for cell entry.

Background:The function of HtrA proteases in bacterial infections is widely unknown. Results: Secreted HtrA from various bacterial pathogens exhibits a conserved specificity for cleavage of E-cadherin. Conclusion: HtrA-mediated E-cadherin cleavage is a prevalent novel mechanism in bacterial pathogenesis. Significance: HtrA activity plays a direct role in the pathogenesis of different bacteria.

Secondary site mutations that restore sporulation to sporulation-defective spoOF or spoOB deletion mutants were found to reside in the spoOA gene. Sequence analysis of 23 such sofmutants showed that the sof mutations fell into six classes of missense codon changes, primarily in the conserved amino-terminal domain of the response regulator SpoOA protein. Changes were observed in codons 12, 14, 60, 92, and 121. The residues affected were predominantly located in the potential turn regions at one end of the amino-terminal conserved domain on the same topological face as the active site aspartate residues. The ability of sof mutations to suppress deficiencies in the transmitter kinases, KinA and KinB, of two-component regulatory systems was tested. AU of the sof mutations suppressed the sporulation deficiency of kinA mutants but only two classes among five tested suppressed kinB mutations. sof mutants segregated Spo-colonies at high frequency. Five of these Spo-mutants were found to result from mutations in the spoOA locus that reversed the effect of the sof mutation. One of these was sequenced and found to have the original sof mutation and a new mutation, sos, at codon 105. The accumulation of sos mutations in sof strains suggested that the sof mutations have a subtle, yet deleterious, effect on the growth of the cell. The results suggested that the sofmutations increase the avidity for or reactivity with transmitter kinases in an allele-specffic manner, although in some cases it is possible that the sofmutations obviate the need for phosphorylation to activate the SpoOA protein. An alternative hypothesis is presented in which the sof mutations play the role of bypass mutations for kinases.Sporulation is the result of complex interactions of developmental processes which lead to the formation of dormant, heat-resistant spores. In Bacillus subtilis, the products of the following eight genes appear to control the onset of sporulation: spoOA, spoOB, spoOE, spoOF, spoOH, spoOJ, spoOK, and spoOL (8,20; and Hoch, unpublished data). Mutations in each of these genes block sporulation at the earliest stage, stage 0, and cause a wide variety of pleiotropic phenotypes (4). Since mutations in the spoOA gene are the most pleiotropic among the spoO genes, it is possible that the spoOA gene product plays a major role in transcriptional activation of the differentiation process. The SpoOA and SpoOF proteins have homology to response regulators of two-component regulatory systems which presumably respond to environmental or metabolic stimuli (6,16,29). Unlike most two-component regulatory systems, the spoOA and spoOF genes are not linked to a gene with homology to the signal transmitter kinases, which activate the response regulators by phosphorylation (7, 15). Thus, the identity of the kinase(s) responsible for phosphorylation of SpoOF and SpoOA is open to speculation. Recently, a kinase gene (also known as kinA, spoIIJ), which has a sporulation defective phenotype when mutated, was cloned and sequenced (2,18 enzymatic and geneti...