Manganese regulation of virulence factors and oxidative stress resistance in Neisseria gonorrhoeae

Abstract

Neisseria gonorrhoeae has evolved a complex and novel network of oxidative stress responses, including defence mechanisms that are dependent on manganese (Mn). We performed systematic analyses at the transcriptomic and proteomic (1D SDS-PAGE and Isotope-Coded Affinity Tag [ICAT]) levels to investigate the global expression changes that take place in a high Mn environment, which results in a Mn-dependent oxidative stress resistance phenotype. These studies revealed that there were proteins regulated at the post-transcriptional level under conditions of increased Mn concentration, including proteins involved in virulence (e.g., pilin, a key adhesin), oxidative stress defence (e.g., superoxide dismutase), cellular metabolism, protein synthesis, RNA processing and cell division. Mn regulation of inorganic pyrophosphatase (Ppa) indicated the potential involvement of phosphate metabolism in the Mn-dependent oxidative stress defence. A detailed analysis of the role of Ppa and polyphosphate kinase (Ppk) in the gonococcal oxidative stress response revealed that ppk and ppa mutant strains showed increased resistance to oxidative stress. Investigation of these mutants grown with high Mn suggests that phosphate and pyrophosphate are involved in Mn-dependent oxidative stress resistance.

Figure 1

Representative 1D SDS-PAGE analyses comparing the soluble fractions of N. gonorrhoeae strain 1291 grown in the absence (−) and presence (+) of 40 M Mn(II) on (A) 1D 12.5% 7 cm, (B) 10% 18 cm gels. Total: total cell lysates; soluble: cytoplasmic and periplasmic proteins. CM: cytoplasmic membrane proteins; OM: outer membrane proteins. The numbers and protein names indicate examples of proteins showing differences in expression level between the control and Mn-treated sample.

Figure 2

(A) Coomassie stained SDS-PAGE, (B) Western blot and (C) Northern blot of Mn(II) regulated proteins in N. gonorrhoeae. Panel A, N. gonorrhoeae wild type (WT) cells were grown on BHI agar ±40 M MnSO₄. Identical loadings of total cell protein were run on a 12% SDS-PAGE gel and Coomassie stained. A band of 17.3 kDa (calc.) is indicated that displayed a four-fold decrease in the presence of 40 M Mn (determined by densitometry in several independent experiments). Panel B, Western blot analysis of whole cells of N. gonorrhoeae strain 1291. Samples were run on a 12% SDS-PAGE gel, transferred to nitrocellulose and probed with the pilin specific probe, SM1. Panel C, Northern blot analysis of RNA isolated from N. gonorrhoeae strain 1291 WT grown on BHI agar ± 40 μM MnSO₄, probed with a pilE specific probe (amplified by the PilEsignalF and PilEsignalR primers, see Table 1).

Figure 3

(A) Paraquat, (B) xanthine-xanthine oxidase and (C) hydrogen peroxide oxidative stress killing assays of N. gonorrhoeae strain 1291 (wild type) and the ppk mutant strains grown on BHI agar or BHI plus 100 M Mn (Mn). Experiments were performed in triplicate. Error bars indicate ±1 standard deviation of the mean. P values were determined using Students t-tests. Mn protected the wild type cells in all assays; PQ (on average approximately 2000-fold higher survival at 60 min), X/XO (approximately 6000-fold at 60 min) and H₂O₂ (approximately 5-fold at 60 min; P <0.05 at the final time point for all assays). The ppk mutant was slightly more resistant than the wild type to PQ with increased survival seen in the absence of Mn (2.1-fold at 30 min, P=0.016; equal at 60 min, P=0.37) and the presence of Mn (2.2-fold at 30 min, P=0.011; equal at 60 min, P=0.069). The ppk mutant was significantly more resistant to H₂O₂ than was the wild type with increased survival seen in both the absence (25-fold at 60 min, P=0.00005) and the presence of Mn (21-fold at 60 min, P=0.0004). Mn protected the ppk mutant in all assays; PQ (>3000-fold at 60 min, P=0.014), X/XO (>4000-fold at 60 min, P=0.041) and H₂O₂ (6-fold at 60 min, P=0.001).

Figure 4

(A) Paraquat, (B) xanthine-xanthine oxidase and (C) hydrogen peroxide oxidative stress killing assays of N. gonorrhoeae strain 1291 (wild type) and the ppa mutant strain. Experiments were performed in triplicate. Error bars indicate ±1 standard deviation of the mean. P values were determined using Students t-tests. The ppa mutant was more resistant than was the wild type strain to killing by PQ in the absence of Mn (>5000-fold at 60 min, P=0.001) and in the presence of Mn (8-fold at 60 min, P=0.004). Increased resistance was also seen in the ppa mutant in the X/XO assay in the absence of Mn (>1300-fold at 60 min, P=0.016) and presence of Mn (5-fold increase in survival at 60 min, P=0.011), and the H₂O₂ assay in the absence of Mn (2.4-fold at 60 min, P=0.0043) and presence of Mn (4.2-fold at 30 min, P=0.008; >5000-fold increase at 60 min, P=0.05). Growth of the ppa mutant in the presence of Mn did not provide protection (with respect to the ppa mutant on unsuplemented media) in the PQ assay (3-fold at 60 min, P= 0.001) or the X/XO assay (4.5-fold at 60 min, P=0.011) and the difference seen in the H₂O₂ assay is not significant as judged by a P-value > 0.5 (5-fold at 30 min, P=0.008; 4-fold at 60 min, P=0.055).

Figure 5

Gonococcal association with and intracellular survival within primary human cervical epithelial (pex) cells. The histogram shows the mean percent association, invasion and survival as a function of the original inoculum of the N. gonorrhoeae wild type strain (WT), and the ppk and ppa mutant strains. Data, determined from the number of colony forming units formed upon plating of the cervical cell lysates, were obtained from three experiments performed in triplicate. Y-error bars show +/− 1 variance. P-values were determined using a Kruskal-Wallis non-parametric analysis of variance.