Figure 1.
A proteomics approach to profile intracellular C. jejuni.
A A schematic diagram of the experimental approach used in this study. B SDS-PAGE analysis of extracted proteins from C. jejuni samples. Proteins obtained from intracellular bacteria isolated following the protocol described above (intracellular), from equal number of bacteria obtained after growth in Brain Heart Infusion broth (BHI grown) or from uninfected cells subjected to the same protocol (mock), were separated by SDS-PAGE and stained with Coomassie brilliant blue.
Table 1.
Levels of C. jejuni proteins associated with oxidative stress.
Figure 2.
Functional categorization of C. jejuni proteins whose expression was lower in samples obtained 20 hours after infection.
Proteins were assigned to the different functional groups based on genome annotation and additional curation based on published literature.
Figure 3.
Cellular overview of C. jejuni metabolic pathways.
Metabolic pathways are grouped into different general functional categories as indicated. Protein transporters are located at the double peripheral lines representing the bacterial membrane structure. Within individual pathways, symbols represent metabolites and connecting lines denote respective enzymes that catalyze the inter-conversion of those metabolites. Most enzymes are color-coded in the following fashion: blue indicates mapped proteins in pathways present in the C. jejuni proteome, grey indicates those not found in the annotated genome, bold green represents those proteins whose expression levels were lower at 20 h of infection relative to 2 h of infection, and bold red corresponds to up-regulated proteins at 20 h of infection. Description of individually numbered pathways is as follows: 1. a, flavin biosynthesis; b, methylerythritol phosphate pathway; c, tetrapyrrole biosynthesis; d, ubiquinone-8 biosynthesis; e, di-trans, poly-cis-undecaprenyl phosphate biosynthesis; f, pantothenate biosynthesis I; g, chlorophyllide a biosynthesis I; h, TCA cycle; i, tetrahydrofolate biosynthesis; j, thiamine biosynthesis I; k, pyridoxal 5′-phosphate biosynthesis; l, coenzyme M biosynthesis; m, 6-hydroxymethyl-dihydropterin diphosphate biosynthesis; n, NAD biosynthesis I; o, folate transformations p. phosphate acquisition I; q, NAD phosphorylation and dephosphorylation; r, acyl carrier protein metabolism; s, formyl THF biosynthesis I; t, thioredoxin pathway; u, NAD salvage pathway II; v, biotin biosynthesis; w, menaquinone-8 biosynthesis; x, biotin biosynthesis; y, heme biosynthesis from uroporphyrinogen-III II; z, trans, trans-farnesyl diphosphate biosynthesis; aa, glutathionylspermidine biosynthesis; ab, NAD salvage pathway I; ac, geranyldiphosphate biosynthesis. 2. a, histidine biosynthesis; b, lysine biosynthesis I; c, valine biosynthesis; d, isoleucine biosynthesis I; e, threonine biosynthesis from homoserine; f, homocysteine biosynthesis; g, homoserine biosynthesis; h, ornithine biosynthesis; i, tryptophan biosynthesis; j, proline biosynthesis I; k, S-adenosyl-L-methionine cycle I; l, serine biosynthesis; m, alanine biosynthesis; n, tyrosine biosynthesis I; o, alanine biosynthesis III; p, selenocysteine biosynthesis I; q, glutamate biosynthesis; r, protein citrullination; s, cysteine biosynthesis I; t, L-glutamine biosynthesis II; u, glutamine biosynthesis I; 3. a, palmitate biosynthesis II; b, CMP-KDO biosynthesis I; c, biotin-carboxyl carrier protein assembly; d, fatty acid biosynthesis initiation III; e, CDP-diacylglycerol biosynthesis I; f, fatty acid biosynthesis initiation II; g, cyclopropane fatty acid (CFA) biosynthesis; h, CDP-diacylglycerol biosynthesis II; i, cis-vaccenate biosynthesis; j, phospholipid biosynthesis I; k, stearate biosynthesis II; l, fatty acid elongation-saturated. 4. a, (5R)-carbapenem biosynthesis; b, hyperforin biosynthesis; c, myo-inositol biosynthesis. 5. a, respiration (anaerobic); b, aerobic respiration-electron donor II. 6. a. homolactic fermentation; b. (S)-acetoin biosynthesis; c. (R)-acetoin biosynthesis I d. mixed acid fermentation; e. pyruvate fermentation to acetate; f. pyruvate fermentation to lactate. 7. a, 5-aminoimidazole ribonucleotide biosynthesis I; b, uridine-5′-phosphate biosynthesis c, adenosine nucleotides de novo biosynthesis; d, guanosine nucleotides de novo biosynthesis; e, pyrimidine deoxyribonucleotides de novo biosynthesis; f, salvage pathways of pyrimidine ribonucleotides; g, salvage pathways of guanine, xanthine, and their nucleosides; h, salvage pathways of purine and pyrimidine nucleotides; i, salvage pathways of purine and pyrimidine nucleotides; j, salvage pathways of purine and pyrimidine nucleotides; k, salvage pathways of purine and pyrimidine nucleotides; l, salvage pathways of purine and pyrimidine nucleotides. 8. a, autoinducer AI-2 biosynthesis I; b, PRPP biosynthesis I. 9. ppGpp biosynthesis. 10. a, chorismate biosynthesis I; b, 3-dehydroquinate biosynthesis I. 11. IAA biosynthesis V. 12. putrescine biosynthesis II. 13. a, UDP-N-acetylmuramoyl-pentapeptide biosynthesis I (generic); b, superpathway of KDO2-lipid A biosynthesis; c, peptidoglycan biosynthesis III; d, UDP-N-acetylmuramoyl-pentapeptide biosynthesis III (meso-DAP); e, enterobacterial common antigen biosynthesis; f, O-antigen biosynthesis; g, dTDP-L-rhamnose biosynthesis I; h, UDP-N-acetyl-D-glucosamine biosynthesis I. 14. a, purine degradation II (anaerobic); b, purine degradation III (anaerobic); c, formaldehyde oxidation V (tetrahydrofolate pathway); d, reductive monocarboxylic acid cycle; e, formaldehyde assimilation I (serine pathway). 15. a, aspartate-glutamate-proline degradation; b, tyrosine degradation I; c, arginine degradation IV; d, aspartate degradation II; e, proline degradation II; f, L-serine degradation; g, lysine degradation I; h, glutamine degradation II; i, citrulline degradation; j, proline degradation I; k, phenylalanine degradation (aerobic); l, asparagine degradation I. 16. tRNA charging pathway. 17. a, gluconeogenesis I; b, colanic acid building blocks biosynthesis; c, colanic acid building blocks biosynthesis; d, GDP-mannose biosynthesis; e, ADP-L-glycero-beta-D-manno-heptose biosynthesis; f, CMP-KDO biosynthesis II; g, GDP-glucose biosynthesis; h, GDP-D-rhamnose biosynthesis; i, glycogen degradation I; j, CMP-N-acetylneuraminate biosynthesis II. 18. a, protocatechuate degradation I; b, methylgallate degradation; c, protocatechuate degradation III; d, orthanilate degradation; e, cyanurate degradation; f, anthranilate degradation I (aerobic). 19. a, galactose degradation I; b, glycogen degradation II; c, glucose and glucose-1 phosphate degradation. 20. a, glycolate and glyoxylate degradation I; b, pyruvate fermentation to acetate VII; c, glycolate and glyoxylate degradation II; d, acetate conversion to acetyl-CoA. 21. a, pentose phosphate pathway (non-oxidative branch); b, pentose phosphate pathway (partial). 22. a, glycolysis I b. glycolysis II. 23. a, seed germination protein turnover; b, wound-induced proteolysis I. 24. fatty acid and lipids degradation. 25. a, sulfate activation for sulfonation, b, nitrate reduction III; c, nitrate reduction IV. Open triangles: Amino acids; open squares: carbohydrates; open rhomboids: proteins; vertical ovals: purines; horizontal ovals: pyrimidines; inverted triangles: cofactors; T:tRNAs; open circles: other; closed symbols indicate phosphorylated forms. (Note: this data has been deposited in the BioCyc home page [http://biocyc.org/] where it can be seen in an interactive fashion).
Table 2.
Levels of C. jejuni proteins associated with respiration/electron transport chains.
Table 3.
SRM measurements of a subset of C. jejuni metabolic enzymes.
Figure 4.
Protein abundance of respiration/electron transport chain components of C. jejuni in four distinct environments.
Protein abundance (expressed as spectral counts) in C. jejuni samples obtained after growth in BHI, after 2 and 20 hs after infection, and after exposure to oxygen limiting conditions subsequently to their recovery 20 hs after infection (indicated as O2-limiting). A Protein abundance of representative enzymes involved in aerobic respirations. B Protein abundance of representative enzymes involved in anaerobic respirations. C Protein abundance of enzymes relevant to fumarate respiration. Values represent the mean ± standard error of the mean of three determinations.
Figure 5.
Intracellular survival of C. jejuni mutant strains defective in specific respiration pathways.
Cultured mammalian cells were infected with the wild type C. jejuni 81–176 strain (WT) and the indicated mutant derivatives at a MOI of 100 for 2 hs, followed by 2 and 18 hs incubation in the presence of gentamicin. For each strain, levels of intracellular bacteria at 20 hs after infection are shown relative to the levels obtained at 2 hs after infection. The value of the ratio in wild type was set at 100%. The error bars represent the standard deviation of three independent determinations. The survival values at 20 hs of the aspA, frdA, or frdA mfrA double mutant strains were statistically significantly different (P<0.05, Student t test) from those of wild type.