Respiratory proteins contribute differentially to Campylobacter jejuni’s survival and in vitro interaction with hosts’ intestinal cells

Abstract

BackgroundThe genetic features that facilitate Campylobacter jejuni’s adaptation to a wide range of environments are not completely defined. However, whole genome expression studies showed that respiratory proteins (RPs) were differentially expressed under varying conditions and stresses, suggesting further unidentified roles for RPs in C. jejuni’s adaptation. Therefore, our objectives were to characterize the contributions of selected RPs to C. jejuni’s i- key survival phenotypes under different temperature (37°C vs. 42°C) and oxygen (microaerobic, ambient, and oxygen-limited/anaerobic) conditions and ii- its interactions with intestinal epithelial cells from disparate hosts (human vs. chickens).ResultsC. jejuni mutant strains with individual deletions that targeted five RPs; nitrate reductase (ΔnapA), nitrite reductase (ΔnrfA), formate dehydrogenase (ΔfdhA), hydrogenase (ΔhydB), and methylmenaquinol:fumarate reductase (ΔmfrA) were used in this study. We show that only the ΔfdhA exhibited a decrease in motility; however, incubation at 42°C significantly reduced the deficiency in the ΔfdhA’s motility as compared to 37°C. Under all tested conditions, the ΔmfrA showed a decreased susceptibility to hydrogen peroxide (H2O2), while the ΔnapA and the ΔfdhA showed significantly increased susceptibility to the oxidant as compared to the wildtype. Further, the susceptibility of the ΔnapA to H2O2 was significantly more pronounced at 37°C. The biofilm formation capability of individual RP mutants varied as compared to the wildtype. However, the impact of the deletion of certain RPs affected biofilm formation in a manner that was dependent on temperature and/or oxygen concentration. For example, the ΔmfrA displayed significantly deficient and increased biofilm formation under microaerobic conditions at 37°C and 42°C, respectively. However, under anaerobic conditions, the ΔmfrA was only significantly impaired in biofilm formation at 42°C. Additionally, the RPs mutants showed differential ability for infecting and surviving in human intestinal cell lines (INT-407) and primary chicken intestinal epithelial cells, respectively. Notably, the ΔfdhA and the ΔhydB were deficient in interacting with both cell types, while the ΔmfrA displayed impairments only in adherence to and invasion of INT-407. Scanning electron microscopy showed that the ΔhydB and the ΔfdhA exhibited filamentous and bulging (almost spherical) cell shapes, respectively, which might be indicative of defects in cell division.ConclusionsWe conclude that the RPs contribute to C. jejuni’s motility, H2O2 resistance, biofilm formation, and in vitro interactions with hosts’ intestinal cells. Further, the impact of certain RPs varied in response to incubation temperature and/or oxygen concentration. Therefore, RPs may facilitate the prevalence of C. jejuni in a variety of niches, contributing to the pathogen’s remarkable potential for adaptation.

Formate simultaneously reduces oxidase activity and enhances respiration in Campylobacter jejuni

“…C. jejuni 81–176 (wildtype)24 was cultured on Mueller-Hinton (MH) agar at 42 °C under microaerobic conditions (85% N 2 , 10% CO 2 , 5% O 2 ) or under <1% O 2 (hereafter referred to as anaerobic conditions) which was generated using the BD GasPak Sachets system (BD Diagnostics, NJ, USA)11. The Campylobacter selective supplement (SR155E, Oxoid, KS, USA), sodium formate, sodium succinate, sodium fumarate, sodium cyanide, sodium azide and sodium nitrate (Sigma, MO, USA) were added to the MH medium when necessary.…”

“…The impact of formate on motility and biofilm formation was determined as described previously1127. To evaluate motility, C. jejuni cultures were adjusted to OD 600 of 0.02.…”

“…Biofilm formation was assessed by using crystal violet staining1127. C. jejuni was suspended in MH broth containing formate (0, 5, 10, 25 and 50 mM) to achieve an OD 600 of 0.05.…”

“…Similarly, it has been shown that formate plays an important role in C. jejuni ’s pathobiology. For example, several studies reported that disparate C. jejuni mutant strains that were deficient in formate metabolism exhibited: (1)- reduced ability to infect human intestinal cells as well as primary chicken intestinal cells in vitro 1011, (2)- deficiency in the colonization of 1-day old chickens1213, and (3)- impairments in motility, resistance to oxidative stress, and biofilm formation and defects in cell shape11. This is not surprising, because C. jejuni exhibits a strong chemoattraction towards formate1415, which is also a major energy source for the bacterium16.…”

Host Hydrogen Rather than That Produced by the Pathogen Is Important for Salmonella enterica Serovar Typhimurium Virulence

“…These NiFe hydrogenases have been shown or predicted to enable a number of pathogens to use molecular H 2 within the host as a respiratory energy-yielding substrate (16)(17)(18)(19)(20)(21)(22)(23)(24). Previous studies in our laboratory have shown that each of the membrane-associated uptaketype respiratory hydrogenases contributes to the survival and growth of S. Typhimurium within the host.…”