Figure 1.
Resistance to manganese toxicity is a conserved trait in Meningococcus.
Disk assay of the sensitivity of bacterial growth to 1M MnCl2 in GCB agar media was carried out for several clinical isolates of N. gonorrhoeae and N. meningitidis. (*** p<0.01)
Figure 2.
Search for Mn2+-regulated bacterial factor(s) in a simple model organism.
A) Xanthomonas campestris MntR controls Mn transport: genetic studies of bacterial sensitivity to 1M MnCl2, measured by disk assay, revealed a novel Mn resistance factor, as mntR disruption increased Mn sensitivity independent of MntH import (*** p<0.01 compare to the wild type sensitivity). This experiment done in triplicates is representative of several experiments. B) Multiple sequence alignments of MntR binding sites found in the promoters of genes which contribute to Mn homeostasis (mntH, mntR, sitA) in γ-proteobacterial plant or animal pathogens (respectively Escherichia coli, Salmonella typhimurium, Shigella flexneri and Xylella fastidiosa, X. campestris, X. axonopodis). C) Sequence alignment of the MntR binding site detected upstream of one gene (yebN or XCC4075 herein referred to as mntX), using the PredictRegulon web site, and that is conserved γ-proteobacteria.
Figure 3.
MntX which confers Mn resistance, is often not functional in N. gonorrhoeae.
Disk assay of bacterial sensitivity to 1M MnCl2 was carried out for A) X. campestris wild type and mutants lacking mntH and mntR or mntX. B) N. meningitidis MC58 wild type, lacking mntX or back complemented (insertion of p5Y3 by double recombination). C) N. gonorrhoeae 16626 wild type or transformed with mntX from N. meningitidis. These experiments done in triplicates are representative of several experiments (*** p<0.01 compare to the wild type sensitivity).
Figure 4.
MntX identifies a family of predicted bacterial metal transporters.
A) MntXXc hydrophobicity plot based on the Goldman, Engelman, Steitz hydropathy scale, using core and wedge windows of 14 and 4 a.a. residues, respectively. B) Proposed model of structure-function relationships in MntX deduced from transmembrane topology prediction and evolutionary sequence analyses. MntX inverted structural symmetry and pattern of sequence conservation suggest a functional interface that may act as a conformational switch for metal transport. The black lightening form represents the localization of the truncation of MntXNg.
Figure 5.
A) MntX depletes E. coli intracellular metal pools. The E. coli K-12 strain Δfur PmntH::luc emits light upon intracellular metal depletion with the divalent metal chelator 2,2′-Dipyridyl (DP). This process is suppressed by co-incubation of DP with divalent metals, therefore allowing monitoring potential metal export activity. Plasmid-driven expression of MntX was induced with L-arabinose. Each bar represents the average of four independent measurements and is representative of several experiments B and C) ICP-MS quantification of divalent metals contents (Mg, Mn, and Fe), for strains of N. meningitidis and N. gonorrhoeae harboring mntXNm (black bars) or not (white bars) and grown in rich medium. The data are expressed in ratio Mn/Mg in B and Mn/Fe in C. Each bar represents the average of three independent measurements for N. meningitidis and two for N. gonorrhoeae (*** p<0.01).
Figure 6.
MntX protects N. meningitidis from Mn cytotoxicity exacerbated in low iron condition.
A) Light emission normalized with OD, and done in triplicates, of N. meningitidis harboring PmntX::luc and grown in GCB agar for 4 hours containing indicated concentration of Desferal and manganese. (*** p<0.01, ** p<0.05 in comparison to the same Desferal concentration without Mn; +++ p<0.01 in comparison to the same Mn concentration without Desferal). B) Serial dilution plate assays were performed to compare the tolerance to iron chelation (Desferal) of N. meningitidis wild type strain, ΔmntX mutant and the complemented strain, in the presence of increased concentrations of MnCl2. C) Percentage of CFU growing on Desferal and manganese compared to those growing in the same concentration of Desferal but without manganese. Each bar represents the mean of three measurements. (*** p<0.01, ** p<0.05)
Figure 7.
MntX is expressed during infection and is important to survive in serum.
A) Expression of selected genes in bacteria from blood 6h post IP infection of hTf mice (black) or wild type mice (white), quantified by qRT-PCR. gyrA was used as endogenous house keeping gene whereas the reference is MC58 growing on GCB. Each bar represents the mean of three infections and representative of several experiments (*** p<0.001, ** p<0.005). B) Standard human serum bactericidal assays (50%) were performed for N. meningitidis wild type, ΔmntX or complemented. Each point represents a measure done in three independent experiments. (*** p<0.01) C) BALB/c mice expressing the human transferrin were infected intraperitoneally with 106 bacteria from GCB agar. The graph represents the number of CFUs recovered from blood 6h post-infection compared to 2h p.i. and expressed in %. D) Similarly, BALB/c mice were infected intraperitoneally with 106 bacteria from GCB agar containing 12.5 µM Desferal. The number of CFU recovered from blood 4h after infection was compared to 2h. For C and D, the graph presents the median % of CFU from two pooled independent experiments (* p<0.05).
Table 1.
Oligonucleotide primers used in this study.