Loss of regulatory protein RfaH attenuates virulence of uropathogenic Escherichia coli

Abstract

RfaH is a regulatory protein in Escherichia coli and Salmonella enterica serovar Typhimurium. Although it enhances expression of different factors that are proposed to play a role in bacterial virulence, a direct effect of RfaH on virulence has not been investigated so far. We report that inactivation of rfaH dramatically decreases the virulence of uropathogenic E. coli strain 536 in an ascending mouse model of urinary tract infection. The mortality rate caused by the wild-type strain in this assay is 100%, whereas that of its isogenic rfaH mutant does not exceed 18%. In the case of coinfection, the wild-type strain 536 shows higher potential to colonize the urinary tract even when it is outnumbered 100-fold by its rfaH mutant in the inoculum. In contrast to the wild-type strain, serum resistance of strain 536rfaH::cat is fully abolished. Furthermore, we give evidence that, besides a major decrease in the amount of hemin receptor ChuA (G. Nagy, U. Dobrindt, M. Kupfer, L. Emody, H. Karch, and J. Hacker, Infect. Immun. 69:1924-1928, 2001), loss of the RfaH protein results in an altered lipopolysaccharide phenotype as well as decreased expression of K15 capsule and alpha-hemolysin, whereas levels of other pathogenicity factors such as siderophores, flagella, Prf, and S fimbriae appear to be unaltered in strain 536rfaH::cat in comparison to the wild-type strain. trans complementation of the mutant strain with the rfaH gene restores wild-type levels of the affected virulence factors and consequently restitutes virulence in the mouse model of ascending urinary tract infection.

FIG. 1.

Number of E. coli 536 (♦) and 536rfaH::cat (░⃞) cells eliminated with the urine following intravesical coinjection of bacteria into mice. The inoculum contained 5 × 10² CFU of wild-type strain 536 complemented with either 5 × 10³ CFU (A) or 5 × 10⁴ CFU (B) of its isogenic rfaH mutant. Graphs represent means ± standard errors of the means of values originated from nine (A) and five (B) animals.

FIG. 2.

Viable counts in different organs on 21st day postinfection. Mice were inoculated intravesically with a mixture of 5 × 10² CFU of wild-type strain 536 complemented with either 5 × 10³ CFU (A) or 5 × 10⁴ CFU (B) of its isogenic rfaH mutant. Means ± standard errors of the means of values originated from nine (A) and five (B) mice are shown. Dark bars represent CFU of wild-type strain 536, and the light bar represents that of mutant strain 536rfaH::cat. L., left; R., right.

FIG. 3.

Serum resistance of E. coli strain 536 (♦), its isogenic mutant 536rfaH::cat (░⃞), and trans-complemented strain 536rfaH::cat (pSMK1) (▴). Bacteria were incubated in 50% serum obtained from a healthy individual. The graph represents means ± standard errors of the means of values originated from three similar assays.

FIG. 4.

(A) Hemolytic activities derived from E. coli strain 536 (♦), its isogenic mutant 536rfaH::cat (░⃞), and trans-complemented strain 536rfaH::cat (pSMK1) (▴). Values represent the percentage of total hemolysis relative to that induced by Triton X-100. (B) Growth curves of strain 536 (♦), 536rfaH::cat (░⃞), and 536rfaH::cat (pSMK1) (▴).

FIG. 5.

LPS structures obtained from wild-type strain 536 (A), mutant 536rfaH::cat (B), and its trans-complemented variant 536rfaH::cat (pSMK1) (C). Equal quantities of purified LPS were silver stained following separation by SDS-PAGE.

FIG. 6.

Reactivity of E. coli strain 536 (O6:K15:H31) and its variants to different concentrations of a K15-specific serum. E. coli strains RZ430 (O6:K⁻:H31) and RZ451 (O6:K18/22:H31) were used as negative controls. The graph represents means ± standard errors of the means of values originated from two similar assays.