Host hydrogen rather than that produced by the pathogen is important for Salmonella enterica serovar Typhimurium virulence

Abstract

Salmonella enterica serovar Typhimurium utilizes molecular hydrogen as a substrate in various respiratory pathways, via H2-uptake enzymes termed Hya, Hyb, and Hyd. A different hydrogenase, the hydrogen-evolving Hyc enzyme, removes excess reductant during fermentative growth. Virulence phenotypes conferred by mutations in hyc genes, either alone or in combination with mutations in the H2-uptake enzyme genes, are addressed. Anaerobically grown ΔhycB or ΔhycC single-deletion strains were more sensitive to acid than the wild-type strain, but the Δhyc strains were like the virulent parent strain with respect to both mouse morbidity and mortality and in organ burden numbers. Even fecal-recovery numbers for both mutant strains at several time points prior to the animals succumbing to salmonellosis were like those seen with the parent. Neither hydrogen uptake nor evolution of the gas was detected in a hydrogenase quadruple-mutant strain containing deletions in the hya, hyb, hyd, and hyc genes. As previously described, a strain lacking all H2-uptake ability was severely attenuated in its virulence characteristics, and the quadruple-mutant strain had the same (greatly attenuated) phenotype. While H2 levels were greatly reduced in ceca of mice treated with antibiotics, both the ΔhycB and ΔhycC strains were still like the parent in their ability to cause typhoid salmonellosis. It seems that the level of H2 produced by the pathogen (through formate hydrogen lyase [FHL] and Hyc) is insignificant in terms of providing respiratory reductant to facilitate either organ colonization or contributions to gut growth leading to pathogenesis.

FIG 1

Acid sensitivity of S. enterica serovar Typhimurium strains JSG210 (wild type [WT]), RLK5 (ΔhycB), and RLK7 (ΔhycC). Cells were grown for 20 h in LBK medium supplemented with glucose and either MOPS (pH 7; black bars) or formate (pH 5.5; white bars), diluted, and spread.

FIG 2

Virulence of S. enterica serovar Typhimurium strains JSG210 (WT), RLK5 (ΔhycB), RLK7 (ΔhycC), RLK9 (quadruple mutant), and ALZ43 (triple mutant) on BALB/c mice. The results shown are for 15 mice infected as described for the ALZ43 strain and 8 mice each as described for the WT, RLK5, RLK7, and RLK9 strains. Another experiment yielded a similar result.

FIG 3

Recovery of S. enterica serovar Typhimurium from feces of mice infected with strains JSG210 (WT), RLK5 (ΔhycB), and RLK7 (ΔhycC). Fresh fecal pellets were obtained from each mouse (n = 6 per strain) at 2.5 days after inoculation. Fecal pellets were homogenized, diluted, and plated on Brilliant Green agar plates, and CFU were counted. Each point represents the CFU count from the feces of one mouse, and the solid lines represent the geometric means of the colonization numbers for each group.

FIG 4

Putative and known roles of the four hydrogenases in S. enterica serovar Typhimurium. Numbers in parentheses represent reference citations.