Analysis of the LIV system of Campylobacter jejuni reveals alternative roles for LivJ and LivK in commensalism beyond branched-chain amino acid transport

Abstract

Campylobacter jejuni is a leading cause of diarrheal disease in humans and an intestinal commensal in poultry and other agriculturally important animals. These zoonotic infections result in significant amounts of C. jejuni present in the food supply to contribute to disease in humans. We previously found that a transposon insertion in Cjj81176_1038, encoding a homolog of the Escherichia coli LivJ periplasmic binding protein of the leucine, isoleucine, and valine (LIV) branched-chain amino acid transport system, reduced the commensal colonization capacity of C. jejuni 81-176 in chicks. Cjj81176_1038 is the first gene of a six-gene locus that encodes homologous components of the E. coli LIV system. By analyzing mutants with in-frame deletions of individual genes or pairs of genes, we found that this system constitutes a LIV transport system in C. jejuni responsible for a high level of leucine acquisition and, to a lesser extent, isoleucine and valine acquisition. Despite each LIV protein being required for branched-chain amino acid transport, only the LivJ and LivK periplasmic binding proteins were required for wild-type levels of commensal colonization of chicks. All LIV permease and ATPase components were dispensable for in vivo growth. These results suggest that the biological functions of LivJ and LivK for colonization are more complex than previously hypothesized and extend beyond a role for binding and acquiring branched-chain amino acids during commensalism. In contrast to other studies indicating a requirement and utilization of other specific amino acids for colonization, acquisition of branched-chain amino acids does not appear to be a determinant for C. jejuni during commensalism.

Fig. 1.

Organization of the liv loci of C. jejuni and E. coli and analysis of constructed C. jejuni liv mutants. The LIV locus of C. jejuni 81-176 contains six consecutive genes. In E. coli, livJ is separated from the other liv genes by the gene yhhK (light gray arrow) (1). The genes of the LIV system in these bacteria include livJ and livK (encoding the LIV binding proteins; black arrows), livH and livM (encoding the inner membrane permeases; dark gray arrows), and livG and livF (encoding the cytoplasmic ATPases; white arrows). The triangle indicates the site of the insertion of the signature-tagged Tn in the 81-176 mutant that was previously identified to have a reduced ability to colonize the chick ceca (14). (B) Qualitative reverse transcriptase PCR analysis of expression of liv genes in wild-type (WT) C. jejuni and ΔlivJ, ΔlivK, and ΔlivJK mutants. RNAs from C. jejuni strains were used in reactions with or without reverse transcriptase (RT) to generate cDNA. Each gene was amplified from cDNA using gene-specific primers. A positive control for amplification of each gene was performed by PCR with wild-type C. jejuni 81-176 genomic DNA (g).

Fig. 2.

Analysis of in vitro growth rates of wild-type C. jejuni and mutant strains in different liquid media. C. jejuni strains were grown in three different types of liquid media over 24 h at 37°C under microaerobic conditions. The liquid media included Mueller-Hinton (MH) broth (A), Campylobacter defined medium supplemented with all amino acids (CDM+) (B), or Campylobacter-defined medium supplemented with all amino acids except leucine (CDM-L) (C). Final OD₆₀₀ measurements were taken at 24 h postinoculation. Strains include wild-type C. jejuni 81-176 Sm^r (DRH212) and in-frame deletions of each liv gene or pairs of liv genes encoding proteins with similar functions. Growth of 81-176 Sm^r ΔilvE (SMS270) was also measured. Data are reported as the averages of three experiments ± standard errors.

Fig. 3.

Involvement of C. jejuni LivJ and LivK in transport of various amino acids. After growth on CDM-LIV, strains were assayed for transport of ³H-labeled amino acids over a 5-min period by measuring the amount of radioactivity (as pmol per mg [dry weight] of cells) associated with each strain. Each strain was assayed in triplicate. The data presented represent an average of three assays. Standard errors were calculated for each data point and are too small to noticeably appear in graphs. y axis values were adjusted as appropriate for the level of amino acid transported for each graph. Data for transport assays for leucine (A), isoleucine (B), valine (C), serine (D), glutamic acid (E), proline (F), and asparagine (G) are shown. Strains include wild-type C. jejuni 81-176 Sm^r (DRH212) and ΔlivJ (SMS301), ΔlivK (SMS241), and ΔlivJK (SMS263) mutants.

Fig. 4.

Specificity of leucine-mediated transport by the LIV system of C. jejuni. Competition assays were performed to measure the specificity of leucine transport by adding unlabeled amino acids in excess to potentially compete for ³H-labeled leucine for transport via the LIV system. Unlabeled amino acids were added at 100 (A)- or 10 (B)-fold-higher concentrations than ³H-labeled leucine. Assays were stopped after 2 min, and the total [³H]leucine associated with C. jejuni was measured. The amount of transport of ³H-labeled leucine in samples with competing unlabeled amino acid is reported as a percentage relative to the amount of ³H-labeled leucine transported in respective strains without competing unlabeled amino acid. Competition experiments were performed with wild-type C. jejuni 81-176 Sm^r (DRH212; black bars) and ΔlivJ strain (SMS301; gray bars). Bars represent standard errors of data points.

Fig. 5.

Expression of liv operon upon growth of C. jejuni in the presence or absence of leucine. Quantitative real-time RT-PCR was performed on RNA isolated from C. jejuni grown on Mueller-Hinton (MH) agar or Campylobacter defined medium with all amino acids (CDM+) or without LIV (CDM-LIV). Expression of livJ or ilvE is reported relative to expression of these genes in wild-type C. jejuni 81-176 Sm^r (DRH212) grown on MH agar and has been set to a baseline of 1. Bars represent standard errors of data points.

Fig. 6.

Chick colonization phenotypes of wild-type C. jejuni and isogenic liv or ilvE mutants. One-day-old chicks were orally inoculated with C. jejuni strains with approximately 10⁴ CFU (A) or 100 CFU (B). At 7 days postinfection, chicks were sacrificed and the C. jejuni loads in the ceca of each chick were determined as the number of CFU per gram of cecal content. Each circle represents the number of C. jejuni bacteria from one chick. Bars represent the geometric means of bacterial loads from chicks infected with specific strains. Asterisks indicate statistically significant differences in colonization of mutants compared to wild-type C. jejuni using the Mann-Whitney U test (P < 0.05). Strains include wild-type C. jejuni 81-176 Sm^r (DRH212) and in-frame deletions of each liv gene or pairs of liv genes encoding proteins with similar functions. The colonization capacity of 81-176 Sm^r ΔilvE (SMS270) is also included.