Metabolic and fitness determinants for in vitro growth and intestinal colonization of the bacterial pathogen <i>Campylobacter jejuni</i>

<div><p><i>Campylobacter jejuni</i> is one of the leading infectious causes of food-borne illness around the world. Its ability to persistently colonize the intestinal tract of a broad range of hosts, including food-producing animals, is central to its epidemiology since most infections are due to the consumption of contaminated food products. Using a highly saturated transposon insertion library combined with next-generation sequencing and a mouse model of infection, we have carried out a comprehensive genome-wide analysis of the fitness determinants for growth in vitro and in vivo of a highly pathogenic strain of <i>C</i>. <i>jejuni</i>. A comparison of the <i>C</i>. <i>jejuni</i> requirements to colonize the mouse intestine with those necessary to grow in different culture media in vitro, combined with isotopologue profiling and metabolic flow analysis, allowed us to identify its metabolic requirements to establish infection, including the ability to acquire certain nutrients, metabolize specific substrates, or maintain intracellular ion homeostasis. This comprehensive analysis has identified metabolic pathways that could provide the basis for the development of novel strategies to prevent <i>C</i>. <i>jejuni</i> colonization of food-producing animals or to treat human infections.</p></div

Colonization determinants of <i>C</i>. <i>jejuni</i> 81–176 identified by insertion sequencing (INSeq) analyses.

<p>Mice rendered dysbiotic by antibiotic treatment were orally infected with wild-type <i>C</i>. <i>jejuni</i> 81–176 (A) or a library of transposon-insertion mutants (B), and the number of colony-forming units (CFUs) in the ceca of infected animals was determined as indicated in “Materials and methods.” (C) Histogram depicting the number of genes (<i>y</i> axis) that exhibited the indicated log₂ (fold change [output/input]) change (<i>x</i> axis) in the numbers of transposon insertions recovered from infected mice relative to the number of transposon insertions in the original inoculum. Areas colored with red represent genes whose number of transposon insertions showed a significant decrease after mouse infection. The calculation of log₂ (fold change [output/input]) is based on the read numbers of insertional mutants of each gene presented in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001390#pbio.2001390.s020" target="_blank">S3 Table</a>. (D) Functional categories of the <i>C</i>. <i>jejuni</i> 81–176 genes whose inactivation led to mouse intestinal colonization defects as measured by INseq analysis. The genes belonging to each functional category can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001390#pbio.2001390.t001" target="_blank">Table 1</a>. p.i., post infection.</p

Role of amino acid and peptide metabolism in <i>C</i>. <i>jejuni</i> intestinal colonization.

<p>(A and B) Impact of the mutations in genes involved in the transport, metabolism (A), or biosynthesis (B) of amino acids and peptides that are important for <i>C</i>. <i>jejuni</i> colonization as determined by insertion sequencing (INSeq) analyses. The log₂ (fold change [intestine/inoculum]) in the number of transposon insertions within the depicted <i>C</i>. <i>jejuni</i> genes are shown, and when depicted in red, the value indicates a statistically significant colonization defect. *: denotes genes showing a limited number of insertions within the library and no insertions within the pooled of mutants recovered from the intestine. The red arrows in panel (B) denote that the number of insertions within the gene involved in the indicated reaction was significantly reduced within the pooled of mutants recovered from the mouse intestine (relative to the inoculum). The log₂ (fold change [intestine/inoculum]) depicted beside each gene is taken from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001390#pbio.2001390.s020" target="_blank">S3 Table</a>. Asp, asparagine; Cys, cysteine; Gln, glutamine; Glu, glutamic acid; Gly, glycine; Ile, isoleucine; Leu, leucine; Met, methionine; OAA, oxaloacetate; SAM, S-adenosyl methionine; Ser, serine; TCA, tricarboxylic acid; Thr, threonine; Val, valine.</p

The contribution of CO₂ metabolism to <i>C</i>. <i>jejuni</i> intestinal colonization.

<p>Illustrated are metabolic reactions in <i>C</i>. <i>jejuni</i> that utilize bicarbonate (H₂CO₃^-) (A) and the carbonic anhydrase CanB-catalyzed reaction that generates bicarbonate from CO₂ (B). Numbers indicate the log2 (fold change [intestine/inoculum]) in the number of insertions in the indicated genes and are derived from the raw data in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001390#pbio.2001390.s020" target="_blank">S3 Table</a>. Values below −6.2 indicate mutations that led to a statistically significant colonization defect. Green arrows indicate mutations that led to a statistically significant colonization defect. (C) Incorporation of CO₂ into amino acids after <i>C</i>. <i>jejuni</i> 81–176 cultivation in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with H¹³CO₃^-. Shown are the overall ¹³C-excess (%) (upper panel) and relative fractions of ¹³C-labeled isotopologues (lower panel) in protein-derived amino acids of <i>C</i>. <i>jejuni</i> 81–176 cultivated in DMEM supplemented with 44 mM ¹³C-labeled hydrogen carbonate. The colored boxes indicate the relative contributions (%) of isotopologues with 1, 2, 3, 4, 5, and 6 ¹³C-atoms (M+1, M+2, M+3, M+4, M+5, and M+6). Numbers are the means ± standard deviation (SD) of 6 measurements (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001390#pbio.2001390.s028" target="_blank">S11 Table</a>). (D) Heat map for the overall ¹³C-excess of labeled amino acids in <i>C</i>. <i>jejuni</i> 81–176 wild-type and the respective <i>pycA</i>::<i>Cm</i> mutant strain after growth with [¹³C]bicarbonate and 20 mM lactate (upper panel) or 20 mM Ser (lower panel) as carbon and energy sources. The values of the color map depict the mean of 2 biological experiments measured in triplicate (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001390#pbio.2001390.s028" target="_blank">S11 Table</a>). (E) Growth analysis of the <i>C</i>. <i>jejuni</i> 81–176 <i>pycA</i> mutant (grey column) compared to the wild type (black column) when cultivated in DMEM supplemented with 20 mM of different carbon and energy sources. Values represent the mean values ± SD of 3 independent experiments (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001390#pbio.2001390.s029" target="_blank">S12 Table</a>). Ala, alanine; Asp, asparagine; Glu, glutamic acid; Gly, glycine; Ile, isoleucine; Lac, lactate; Leu, leucine; Lys, lysine; Phe, phenylalanine; Pro, proline; Ser, serine; Thr, threonine; Tyr, tyrosine; Val, valine; w/o, without; WT, wild type.</p

Ion homeostasis in <i>C</i>. <i>jejuni</i> intestinal colonization.

<p>Overview of insertion sequencing (INSeq) data for putative transporters in <i>C</i>. <i>jejuni</i> 81–176 mediating the transport of transition metals (A) and facilitating the homeostasis of protons and sodium or the uptake of solutes by cotransport (B). (C) Comparison of the relative contribution of 2 predicted potassium transport systems (Ktr and Kdp) to <i>C</i>. <i>jejuni</i> mouse colonization. Blue and red bars indicate the normalized read number of each insertion site within the open reading frames (ORFs) in the input and output pool, respectively. (D) Role of the Ktr and Kdp potassium transport system in mouse colonization. Mice were inoculated with an equal number of wild-type <i>C</i>. <i>jejuni</i> and the indicated mutant or complemented mutant strains via oral gavage (<i>n</i> = 5 or 7). Competitive indices (CIs) were calculated as the ratio of the colony-forming units (CFUs) of the mutant over the CFUs of the wild-type strain recovered from the ceca of infected mice (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001390#pbio.2001390.s031" target="_blank">S14 Table</a>). Significance was determined by the unpaired <i>t</i> test. ***: <i>p</i> = 0.001; ****: <i>p</i> < 0.0001. (E) Growth of <i>C</i>. <i>jejuni ktr</i> and <i>kdp</i> mutants in defined rich medium supplemented with various K⁺ concentrations, as indicated. All strains were inoculated to culture medium at an OD₆₀₀ of 0.02, and the cell density of the cultures was measured at the indicated times over a 46-hour period. Pro, proline; Ser, serine.</p

List of <i>C</i>. <i>jejuni</i> 81–176 genes whose insertion mutants resulted in a significant mouse colonization defect.

<p>List of <i>C</i>. <i>jejuni</i> 81–176 genes whose insertion mutants resulted in a significant mouse colonization defect.</p

Impact of central carbon metabolism during <i>C</i>. <i>jejuni</i> intestinal colonization.

<p>The log₂ (fold change [intestine/inoculum]) in the number of transposon insertions within <i>C</i>. <i>jejuni</i> genes encoding enzymes in the tricarboxylic acid (TCA) cycle, gluconeogenesis, and the acetate switch pathways are shown and are derived from the raw data in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001390#pbio.2001390.s020" target="_blank">S3 Table</a>. Values below −6.2 indicate mutations that led to a statistically significant colonization defect. *: denotes genes showing a limited number of insertions within the library and no insertions within the pooled of mutants recovered from the intestine. The red arrows denote that the number of insertions within the gene involved in the indicated reaction was significantly reduced within the pool of mutants recovered from the mouse intestine (relative to the inoculum). Green arrows indicate that the enzyme that catalyzes the corresponding reaction does not have an insertional mutant in our mutant library. Enzymes not encoded in the <i>C</i>. <i>jejuni</i> genome are indicated with an “X.” The inset depicts the overall ¹³C-excess and relative fractions of ¹³C-labeled isotopologues in free and bound glucose or galactose (as indicated) derived from <i>C</i>. <i>jejuni</i> 81–176 cell surface carbohydrates after cultivation in Dulbecco’s Modified Eagle Medium (DMEM) with [3-¹³C₁]Ser. The colored boxes indicate the relative contributions (%] of isotopologues with 1, 2, and 3 ¹³C-atoms indicated as M+1, M+2, and M+3, respectively. Numbers are the means ± standard deviation (SD) of 6 measurements (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001390#pbio.2001390.s028" target="_blank">S11 Table</a>). Acetyl-CoA, acetyl coenzyme A; LOS, lipooligosaccharide; OAA, oxaloacetate; PEP, phosphoenolpyruvic acid; PPP, pentose phosphate pathway.</p