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. 2004 Aug;186(16):5332-41.
doi: 10.1128/JB.186.16.5332-5341.2004.

Role of an inducible single-domain hemoglobin in mediating resistance to nitric oxide and nitrosative stress in Campylobacter jejuni and Campylobacter coli

Karen T Elvers  1 , Guanghui Wu, Nicola J Gilberthorpe, Robert K Poole, Simon F Park
Affiliations

Role of an inducible single-domain hemoglobin in mediating resistance to nitric oxide and nitrosative stress in Campylobacter jejuni and Campylobacter coli

Karen T Elvers et al. J Bacteriol. 2004 Aug.

Abstract

Campylobacter jejuni expresses two hemoglobins, each of which exhibits a heme pocket and structural signatures in common with vertebrate and plant globins. One of these, designated Cgb, is homologous to Vgb from Vitreoscilla stercoraria and does not possess the reductase domain seen in the flavohemoglobins. A Cgb-deficient mutant of C. jejuni was hypersensitive to nitrosating agents (S-nitrosoglutathione [GSNO] or sodium nitroprusside) and a nitric oxide-releasing compound (spermine NONOate). The sensitivity of the Cgb-deficient mutant to methyl viologen, hydrogen peroxide, and organic peroxides, however, was the same as for the wild type. Consistent with the protective role of Cgb against NO-related stress, cgb expression was minimal in standard laboratory media but strongly and specifically induced after exposure to nitrosative stress. In contrast, the expression of Cgb was independent of aeration and the presence of superoxide. In the absence of preinduction by exposure to nitrosative stress, no difference was seen in the degree of respiratory inhibition by NO or the half-life of the NO signal when cells of the wild type and the cgb mutant were compared. However, cells expressing GSNO-upregulated levels of Cgb exhibited robust NO consumption and respiration that was relatively NO insensitive compared to the respiration of the cgb mutant. Based on similar studies in Campylobacter coli, we also propose an identical role for Cgb in this closely related species. We conclude that, unlike the archetypal single-domain globin Vgb, Cgb forms a specific and inducible defense against NO and nitrosating agents.

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Figures

FIG. 1.
FIG. 1.
(a) Comparison of Cgb from C. jejuni NCTC 11168 and C. coli UA585 with hemoglobins from other bacterial and yeast species. Hemoglobin sequences from C. jejuni NCTC 11168 (SwissProt Q9PM89), C. coli UA585, V. stercoraria (sp P04252), B. subtilis (sp P49852), S. cerevisiae (SwissProt [sp] P39676), S. enterica serovar Typhimurium (sp P26353), and E. coli (sp P24232) from BLASTP results were aligned by using the CLUSTAL W algorithm (www.ebi.ac.uk). Alignments for the last four species are truncated. The amino acid residues that are identical in all sequences are shaded in black. (b) Alignment of intergenic nucleotide sequences from C. jejuni NCTC 11168 and C. coli UA585. CAT and ATG code for methionine for the start of the Cj1585c putative oxidoreductase and of the Cj1586 putative bacterial hemoglobin Cgb, respectively. Identical nucleotides in C. coli UA585 and C. jejuni NCTC 11168 are shown by an asterisk.
FIG. 2.
FIG. 2.
Effects of nitrosative stress on the viability of C. jejuni. Growing cultures of C. jejuni NCTC 11168 (▴) and CJCGB01 (○) were challenged with the indicated concentrations of GSNO (a), SNP (b), and spermine NONOate (c) for 1 h and then plated on MH agar. The results are a mean of two independent experiments. Bars indicate one standard error.
FIG. 3.
FIG. 3.
Cgb expression, as monitored from transcriptional astA fusions, is induced by nitrosative stress in C. coli. Cells of CCSF1 sodB-astA (open symbols) and CCCF1 cgb-astA (solid symbols) were grown with no additions (○, •) or in the presence of GSNO at 0.05 mM (♦), 0.1 mM (▪, □), or 0.25 mM (▴, ▵) (a); SNP at 0.005 mM (♦, ⋄), 0.01 mM (▪, □), or 0.05 mM (▴) (b); and methyl viologen at 1 μM (♦, ⋄), 5 μM (▴, ▵), or 10 μM (▪, □) (c). The agents were added at time zero, and arylsulfatase activity was assessed and is expressed as micrograms of p-nitrophenol released/hour/OD600. The data shown are representative of the results obtained from three independent experiments.
FIG. 4.
FIG. 4.
GSNO induces the expression of Cgb in C. jejuni. Different concentrations of GSNO were added to growing cultures of C. jejuni. The expression of Cgb was detected by using the anti-Cgb antibody. Each lane of the 15% SDS gels was loaded with 10 μg of protein extract.
FIG. 5.
FIG. 5.
Inhibition of respiration by NO. Sodium formate (2 mM) was incubated with MOPS buffer at 37°C until the traces were stable. Cells (160 μg of cell protein) were added to initiate respiration of C. jejuni NCTC 11168 (a) and CJCGB01 (b). NO (5 μΜ), indicated by the black line, was added when the oxygen tension (gray line) was ∼110 μM. The experiments were repeated twice, and the assay was repeated three to six times, yielding essentially the same results.
FIG. 6.
FIG. 6.
(a) Adherence to and invasion of Caco-2 cells by C. jejuni NCTC 11168 and CJCGB01. The numbers of adhered and invaded cells (gray shaded bars) and invasive cells only (open bar) were determined by viable counts after 3 h. The results are a mean ± the standard error of three replicates. (b) Nitrite production by Caco-2 cells in response to C. jejuni infection. Nitrite production was measured after 3 h of infection with C. jejuni NCTC 11168 and CJCGB01. Values are means ± the standard error of 16 replicates for Caco-2 cells and 4 replicates for media.
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