We previously showed that hpmA is the hemolysin determinant most commonly found among Proteus isolates. To assess the potential contribution of HpmA to virulence, we first characterized the toxic activities of this hemolysin. Hemolytic activity was present in total cell cultures and cell-free supernatants ofProteus clinical isolates as well as Escherichia coli containing cloned hpm genes. HpmA also possesses cytotoxic activity which was detected by a chromium release assay against a variety of target cell lines (Daudi, Raji, T24, U937, and Vero). Analysis of the dose response of bacterial cells against both T24 cells and erythrocytes showed that E. coli containing cloned hpm genes was 30-fold more cytotoxic than Proteus mirabilis BA6163. Also, 105-fold more bacterial cells were needed to lyse T24 cells than to lyse erythrocytes. HpmA-mutants of two Proteus strains in which the central portion of hpmA was deleted were constructed. These HpmA-mutants, which have lost the hemolytic and cytotoxic activities exhibited by their respective parent strains, demonstrate that HpmA is needed for both of these activities. In an ascending model of murine urinary tract infection, the hpmA mutant strain WPM111 behaved no differently from its parent strain, BA6163, with respect to either the level of kidney colonization or histopathological changes in the kidney. However, WPM111 had a sixfold higher 50% lethal dose than BA6163 when injected intravenously into C3H mice.

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.

Two different hemolysins, HpmA and HlyA, have been reported in Proteus spp. To study the distribution of these hemolysins among Proteus strains, isolates from various infections and normal feces were screened for hemolysin production. All 63 Proteus mirabilis strains and 23 of the 24 Proteus vulgaris strains produced a calcium-independent hemolytic activity detectable in cell-free supernatants. The calcium-independent activity was due to HpmA; this activity correlated with the presence of hpmA sequences and the production of an extracellular 166-kilodalton (kDa) protein that reacted with anti-HpmA antiserum. HpmA-mutants, constructed by deletion of the central portion of the hpmA gene, did not produce the 166-kDa protein and were no longer hemolytic when compared with their respective parent strains. Among the 87 P. mirabilis and P. vulgaris isolates examined, calcium-dependent hemolytic activity was produced by only two P. vulgaris strains. These strains produced a 110-kDa protein which comigrated with the Escherichia coli hemolysin (HlyA) in 10% polyacrylamide gels and reacted with both polyclonal and monoclonal anti-E. coli HlyA antibodies in immunoblots. These studies show that Proteus spp. produce two distinct extracellular hemolysins, with nearly all strains producing the calcium-independent hemolysin, HpmA, but only an occasional P. vulgaris isolate producing EIyA.