No abstract

DNA sequences similar to those of the Escherichia coli hemolysin genes were detected among uropathogenic isolates of Proteus vulgaris and Morganella morganii by using the Southern blotting technique and hly gene-specific DNA probe. Immunoblotting revealed that among the hemolytic P. vulgaris and M. morganii isolates there was expressed a polypeptide species similar in molecular size (110 kilodaltons) and antigenicity to Escherichia coli HlyA. A plasmid-mediated P. vulgaris hemolysin determinant identified by Southern blotting analysis was molecularly cloned, and the recombinant plasmid (pWPV100) was characterized by restriction endonuclease fragment mapping. A second recombinant library of genomic DNA prepared from a hemolytic, urinary tract isolate of Proteus mirabilis was constructed in E. coli. A 5.5-kilobase XhoI fragment encoding an extracellular hemolytic activity was molecularly cloned (pWPM100), and this plasmid was subjected to transposon-mediated mutagenesis with TnphoA. The P. mirabilis hemolytic phenotype was determined to be encoded by a polypeptide species (HpmA) with an estimated molecular size of 140 kilodaltons based on minicell polypeptide analysis of pWPM100 and its mutant derivatives. Southern blotting analysis with a HpmA-specific DNA probe revealed that this novel determinant is commonly found in both Proteus species but is not present in hemolytic isolates of M. morganii, E. coli, Citrobacterfreundii, and Serratia marcescens.

Proteus mirabilis, a common agent of nosocomially acquired and catheter-associated bacteriuria, can cause acute pyelonephritis. In ascending infections, bacteria colonize the bladder and ascend the ureters to the proximal tubules of the kidney. We postulate that Proteus species uses the HpmA hemolysin and urease to elicit tissue damage that allows entry of these bacteria into the kidney. To study this interaction, strains of Proteus mirabilis and P. vulgaris and their isogenic hemolysin-negative (hpmA) or isogenic urease-negative (ureC) constructs were overlaid onto cultures of human renal proximal tubular epithelial cells (HRPTEC) isolated from kidneys obtained by immediate autopsy. Cytotoxicity was measured by release of soluble lactate dehydrogenase (LDH). Two strains of P. mirabilis inoculated at 106 CFU caused a release of 80% of total LDH after 6 h, whereas pyelonephritogenic hemolytic Escherichia coli CFT073 released only 25% at 6 h (P < 0.012). Ten P. mirabilis isolates and five P. vulgaris isolates were all hemolytic and cytotoxic and produced urease which was induced by urea. The HpmA hemolysin is apparently responsible for the majority of cytotoxicity in vitro since the hemolysin-negative (hpmA) mutants of P. mirabilis and P. vulgaris were significantly less cytotoxic than wild-type strains. P. mirabilis WPM111 (hemolysin negative) was used to test the effect of urease-catalyzed urea hydrolysis on HRPTEC viability. In the presence of 50 mM urea, WPM111 caused the release of 42% of LDH versus 1% at 6 h in the absence of substrate (P = 0.003). We conclude that the HpmA hemolysin of Proteus species acts as a potent cytotoxin against HRPTEC. In addition, urease apparently contributes to this process when substrate urea is available.

Recent genomic analyses of Escherichia coli O157:H7 strain EDL933 revealed two loci encoding urease gene homologues (ureDABCEFG), which are absent in nonpathogenic E. coli strain K-12. This report demonstrates that the cloned EDL933 ure gene cluster is capable of synthesizing urease in an E. coli DH5␣ background. However, when the gene fragment is transformed back into the native EDL933 background, the enzymatic activity of the cloned determinants is undetectable. We speculate that an unidentified trans-acting factor in enterohemorrhagic E. coli (EHEC) is responsible for this regulation of ure expression. In addition, Fur-like recognition sites are present in three independent O157:H7 isolates upstream of ureD and ureA. Enzymatic assays confirmed a difference in urease expression of cloned EHEC ure clusters in E. coli MC3100⌬fur. Likewise, interruption of fur in O157:H7 isolate IN1 significantly diminished urease activity. We propose that, similar to the function of Fur in regulating the acid response of Salmonella enterica serovar Typhimurium, it modulates urease expression in EHEC, perhaps contributing to the acid tolerance of the organism.

Enterohemorrhagic E. coli (EHEC) are human bacterial pathogens that have caused numerous outbreaks of foodborne illness worldwide. EHEC infection causes hemorrhagic colitis that in 5–10% of cases can progress to the potentially fatal hemolytic uremic syndrome (HUS). Although EHEC is not considered a classical inflammatory pathogen, clinical data suggest that inflammation may play a role during intestinal infection and in kidney damage during HUS. Our aim is to understand the molecular mechanisms used by EHEC to elicit a pathologic host immune response. Using our well‐established in vitro model of transepithelial migration of neutrophils, we found that EHEC induce migration of neutrophils across polarized epithelial monolayers. We further demonstrated that secretion of StcE during EHEC infection promotes neutrophil transepithelial migration. StcE is a large zinc metalloprotease, involved in cleavage of the mucin‐type O‐glycosylated proteins CD43 and CD45 at the neutrophil surface. CD43 provides an antiadhesive force in cell‐cell interactions and regulates leukocyte adhesion and migration. Treatment with purified StcE rendered neutrophils more adherent in vitro. Our results suggest that during EHEC infection, modulation of neutrophil migration by StcE may play a pro‐inflammatory role that contributes to a dysregulated immune response and the subsequent development of HUS.