Respiratory hydrogen use by Salmonella enterica serovar Typhimurium is essential for virulence

Abstract

Based on available annotated gene sequence information, the enteric pathogen salmonella, like other enteric bacteria, contains three putative membrane-associated H2-using hydrogenase enzymes. These enzymes split molecular H2, releasing low-potential electrons that are used to reduce quinone or heme-containing components of the respiratory chain. Here we show that each of the three distinct membrane-associated hydrogenases of Salmonella enterica serovar Typhimurium is coupled to a respiratory pathway that uses oxygen as the terminal electron acceptor. Cells grown in a blood-based medium expressed four times the amount of hydrogenase (H2 oxidation) activity that cells grown on Luria Bertani medium did. Cells suspended in phosphate-buffered saline consumed 2 mol of H2 per mol of O2 used in the H2-O2 respiratory pathway, and the activity was inhibited by the respiration inhibitor cyanide. Molecular hydrogen levels averaging over 40 microM were measured in organs (i.e., livers and spleens) of live mice, and levels within the intestinal tract (the presumed origin of the gas) were four times greater than this. The half-saturation affinity of S. enterica serovar Typhimurium for H2 is only 2.1 microM, so it is expected that H2-utilizing hydrogenase enzymes are saturated with the reducing substrate in vivo. All three hydrogenase enzymes contribute to the virulence of the bacterium in a typhoid fever-mouse model, based on results from strains with mutations in each of the three hydrogenase genes. The introduced mutations are nonpolar, and growth of the mutant strains was like that of the parent strain. The combined removal of all three hydrogenases resulted in a strain that is avirulent and (in contrast to the parent strain) one that is unable to invade liver or spleen tissue. The introduction of one of the hydrogenase genes into the triple mutant strain on a low-copy-number plasmid resulted in a strain that was able to both oxidize H2 and cause morbidity in mice within 11 days of inoculation; therefore, the avirulent phenotype of the triple mutant is not due to an unknown spurious mutation. We conclude that H2 utilization in a respiratory fashion is required for energy production to permit salmonella growth and subsequent virulence during infection.

FIG. 1.

Dual-channel amperometric recording of simultaneous H₂ and O₂ use by whole cells of S. enterica serovar Typhimurium. H₂ and O₂ were injected into the (5.5-ml-volume) amperometric chamber from gas-saturated solutions, and the gases were monitored continuously by amplified amperometric electrode signals on a chart recorder. The chamber contained 6.8 × 10⁸ washed S. enterica serovar Typhimurium wild-type cells obtained from blood agar plates; the cells were suspended in PBS, and the assay was conducted in PBS. Oxygen is exhausted at about the 5-min time point, and the minimum detectable level is about 20 nM. The O₂ pen was offset in the figure to record approximately 30 s behind the H₂ pen.

FIG. 2.

Virulence of S. enterica serovar Typhimurium strains for mice. The data shown are for a total of 30 mice each for the wild type and the triple mutant strain, based on the combined data from two separate experiments. Values on the y axis indicate the percent survival. The inoculant was 10⁶ cells introduced orally. Twenty mice were used for strain JSG319 (group II and III mutant), and 10 mice were used for strain JSG315 (group I and II mutant). Data for another double mutant strain (JSG317, group I and III mutant) are not shown but were similar to those of strain JSG315. All mice that survived to day 30 were still alive at day 40. Both the triple mutant (strain JSG321) and the double mutants (the group I and II mutant and the group I and III mutant strains but not the group II and III mutant strain) were significantly less virulent than the parent strain, according to the alternative Wilcoxon test (see Table G in reference 10), with a γ (confidence interval) of 0.992 (for the wild type versus the triple mutant) or 0.988 (for the wild type versus the double mutants). Still, the triple mutant was significantly less virulent than the double mutants, with no morbidity observed by the triple mutant strain over a 40-day postinoculation period.