Phosphate Transporter PstSCAB of Campylobacter jejuni Is a Critical Determinant of Lactate-Dependent Growth and Colonization in Chickens

Abstract

Campylobacter jejuni causes acute gastroenteritis worldwide and is transmitted primarily through poultry, in which it is often a commensal member of the intestinal microbiota. Previous transcriptome sequencing (RNA-Seq) experiment showed that transcripts from an operon encoding a high-affinity phosphate transporter (PstSCAB) of C. jejuni were among the most abundant when the bacterium was grown in chickens. Elevated levels of the pstSCAB mRNA were also identified in an RNA-Seq experiment from human infection studies. In this study, we explore the role of PstSCAB in the biology and colonization potential of C. jejuni. Our results demonstrate that cells lacking PstSCAB survive poorly in stationary phase, in nutrient-limiting media, and under osmotic conditions reflective of those in the chicken. Polyphosphate levels in the mutant cells were elevated at stationary phase, consistent with alterations in expression of polyphosphate metabolism genes. The mutant strain was highly attenuated for colonization of newly hatched chicks, with levels of bacteria at several orders of magnitude below wild-type levels. Mutant and wild type grew similarly in complex media, but the pstS::kan mutant exhibited a significant growth defect in minimal medium supplemented with l-lactate, postulated as a carbon source in vivo. Poor growth in lactate correlated with diminished expression of acetogenesis pathway genes previously demonstrated as important for colonizing chickens. The phosphate transport system is thus essential for diverse aspects of C. jejuni physiology and in vivo fitness and survival. IMPORTANCE Campylobacter jejuni causes millions of human gastrointestinal infections annually, with poultry a major source of infection. Due to the emergence of multidrug resistance in C. jejuni, there is need to identify alternative ways to control this pathogen. Genes encoding the high-affinity phosphate transporter PstSCAB are highly expressed by C. jejuni in chickens and humans. In this study, we address the role of PstSCAB on chicken colonization and other C. jejuni phenotypes. PstSCAB is required for colonization in chicken, metabolism and survival under different stress responses, and during growth on lactate, a potential growth substrate in chickens. Our study highlights that PstSCAB may be an effective target to develop mechanisms for controlling bacterial burden in both chicken and human.

Fitness Factor Genes Conserved within the Multi-species Core Genome of Gram-negative Enterobacterales Species Contribute to Bacteremia Pathogenesis

“…This transporter was predicted as a potential fitness factor in 3 species. It is well known that pst mutants are attenuated in other species [Proteus mirabilis in a biofilm and UTI model (36,48) and Campylobacter jejuni in the murine gut model of infection (49)]. Thus, we attempted to validate this finding for bacteremia by testing pstSCABphoU mutants of all five species in the murine model of bacteremia and found that two, E. coli CFT073 and E. hormaechei UM_CRE_14, were attenuated (Fig…”

Gut metabolite L-lactate supportsCampylobacter jejunipopulation expansion during acute infection

“…The C. jejuni lctP :: kan mutant was constructed by inserting a kanamycin resistance cassette into the gene as previously described (51). Briefly, 500 bp of both upstream and downstream regions of the lctP locus was amplified by PCR with primers containing Sma I restriction sites.…”

“…The strains were restreaked and grown for 16 h. The bacteria were suspended in MH broth and inoculated in 25 ml of MH broth containing the desired antibiotics to an initial optical density at 600 nm (OD 600 ) of approximately 0.05. Bacterial numbers were measured at different times up to 48 h. To determine the growth of C. jejuni , strains were grown in MCLMAN minimal medium containing 0.5% partially purified gastric mucin and various carbon sources, including sodium pyruvate, sodium butyrate and L-lactate (28, 51), as noted. Growth was measured via absorbance at OD 600 up to 48 h after inoculating at an initial OD 600 of 0.05.…”

“…After digestion with TURBO Dnase treatment, cDNA was made by SuperScript III first-strand synthesis supermix (Invitrogen). Then real-time PCR was performed by the Syber green method (51). Expression of different targeted genes was measured by the 2 −ΔΔ CT method as previously described.…”

Trivalent outer membrane vesicles-based combination vaccine candidate induces protective immunity against Campylobacter and invasive non-typhoidal Salmonella in adult mice

“…CJ load in different organs was determined by plating the serially diluted samples on Campylobacter selective Mueller-Hinton (MH) agar plates supplemented with 10% sheep's blood, 40 µg/mL cefoperazone, 100 µg/mL cycloheximide, 10 µg/mL trimethoprim and 100 µg/mL vancomycin. Plates were incubated at 37° C for 48 h under microaerobic conditions and the number of colonies was subsequently counted [32]. ST and SE counts were enumerated by plating the serially diluted samples on Xylose Lysine Deoxycholate (XLD) agar plates followed by incubation at 37° C for 24 h and subsequent counting of the colonies in plates.…”