A new family of secreted toxins in pathogenic Neisseria species

Abstract

The genus Neisseria includes both commensal and pathogenic species which are genetically closely related. However, only meningococcus and gonococcus are important human pathogens. Very few toxins are known to be secreted by pathogenic Neisseria species. Recently, toxins secreted via type V secretion system and belonging to the widespread family of contact-dependent inhibition (CDI) toxins have been described in numerous species including meningococcus. In this study, we analyzed loci containing the maf genes in N. meningitidis and N. gonorrhoeae and proposed a novel uniform nomenclature for maf genomic islands (MGIs). We demonstrated that mafB genes encode secreted polymorphic toxins and that genes immediately downstream of mafB encode a specific immunity protein (MafI). We focused on a MafB toxin found in meningococcal strain NEM8013 and characterized its EndoU ribonuclease activity. maf genes represent 2% of the genome of pathogenic Neisseria, and are virtually absent from non-pathogenic species, thus arguing for an important biological role. Indeed, we showed that overexpression of one of the four MafB toxins of strain NEM8013 provides an advantage in competition assays, suggesting a role of maf loci in niche adaptation.

Figure 1. Organization and location of maf genomic islands in pathogenic Neisseria species.

A) Schematic depiction of maf genomic island (MGI). By definition, a GI containing a mafBI module is a MGI. Each mafBI module is composed of two genes: mafB (green) and mafI (red). Additional genes (on a grey background) are mafA (pink), ORFs encoding alternative C-terminal domains of MafB (mafB-CT, green) associated with their cognate mafI gene (red). Black box at the 5′ end of mafB-CT indicates a region potentially involved in antigenic variation of MafB. In class 1 MafBs, this region encodes a bacterial intein-like (BIL) domain whereas in class 3 MafBs, this region encodes a DWVKN motif. B) Simplified genomic organizations and flanking genes of the 5 MGIs found in pathogenic Neisseria. MGIs 1, 2 and 3 are found in N. meningitidis and N. gonorrhoeae while MGIs 4 and 5 are only found in N. gonorrhoeae. MGI-1 flanking genes encode an Anhydro-N-acetylmuramic acid kinase (AnmK) on the 5′end of the island and a hypothetical periplasmic protein on the 3′end. MGI-2 flanking genes encode a Proline tRNA on the 5′end of the island and Trk system potassium uptake protein on the 3′end of the island. A cluster of genes, which is only present in some strains, is represented on a grey background. MGI-3 flanking genes encode an uridylate kinase (PyrH) and a truncated IS1016 element. MGI-4 is flanked by two truncated IS1016 elements. MGI-5 flanking genes encode an IS1016 and a ppGpp synthase (RelA). Color code: conserved flanking genes (white), mafA (pink), mafB and mafB-CT cassettes (green), mafI (red), location of sequence encoding BIL domain (blue box), location of sequence encoding DWVKN motif (salmon rose box). The number (1, 2 or 3) inside the 5′ end of mafB genes indicates the corresponding class of MafB (Class 1, 2 or 3 respectively). The dotted outline indicates a mafB-CT gene without initiation codon. C) A schematic representation of the 3 classes of MafB proteins. All MafBs contain a signal peptide (SP, light grey), a N-terminal conserved domain named DUF1020 (white) and a C-terminal variable region (dark grey). Class 1 MafBs contain a VSGDF motif at the end of the N-terminal conserved domain, and between the conserved and variable regions a bacterial intein-like (BIL) domain can be inserted (blue box). Class 2 MafBs contain a VKYDT motif at the end of the N-terminal conserved domain. Class 3 MafBs contain a DWVKN motif (salmon rose box) at the end of the N-terminal conserved domain.

Figure 2. Pairwise comparison of the genetic organization of MGI-1, MGI-2 and MGI-3 loci in 6 representative Neisseria genomes.

Nucleotide comparison of MGI-1 (A), MGI-2 (B) and MGI-3 (C) from meningococcal strains Z2491, MC58 and NEM8013, gonococcal strains FA1090 and NCCP11945 and N. cinerea strain ATCC 14685. Genome comparisons were generated using BLASTn implemented in Easyfig 2.1 with a cutoff value of 80%. Grey vertical blocks indicate regions of shared similarity shaded according to BLASTn identity. The level of nucleotide identity is shown in the gradient scale for each MGI. Genes are indicated with arrows colored according to their predicted functions with the color code illustrated in the legend. Double-headed arrows connect mafB-CT genes that are identical to the 3′ region of a full-length mafB gene. The orientation of genes is as in the published genomic sequences, except for MGI-1_NEM8013, MGI-1_MC58, MGI-2_NEM8013, MGI-3_Z2491 and MGI-3_FA1090, which have been reversed for clarity purpose. Genomic regions found in N. cinerea at the location of MGI-2 and MGI-3 are not considered as MGIs because they do not encode a full-length MafB toxin.

Figure 3. Toxic effect of MafB proteins of NEM8013 strain on E. coli growth.

A) Effect of NEM8013 MafB putative toxins over-expression on E. coli grown in the presence of 0.2% L-arabinose (Ara+) or without arabinose (Ara−). BL21(DE3) cells were transformed with vector pBAD33 carrying mafB genes found in NEM8013 strain (mafB_MGI-1NEM8013, mafB1_MGI-2NEM8013, mafB2_MGI-2NEM8013 and mafB_MGI-3NEM8013). B) Inhibition of growth due to the toxin is counteracted by cognate immunity protein co-expression on LB agar plates (0.2% L-arabinose) and in LB broth. LB agar plates and LB broth contain 0.01 mM IPTG to induce expression of mafI. Toxin expression was induced by adding 0.2% L-arabinose in LB broth 2 h after inoculation (arrow). mafB_MGI-1NEM8013 and mafB_MGI-3NEM8013 are cloned in pBAD33, mafI_MGI-1NEM8013 is cloned in pET28 and mafI_MGI-3NEM8013 is cloned in pET15. Control strains (Ctrl) contain empty vectors. C) Co-expression of a non-cognate immunity protein does not confer protection against MafB toxicity. LB agar plates contain 0.01 mM IPTG and 0.2% L-arabinose. The results shown are from one of three independent experiments. D) MafB_MGI-1NEM8013 and MafI_MGI-1NEM8013 copurify. mafB and mafI from MGI-1_NEM8013 were cloned under the control of two independent T7 promoters in plasmid pcolaDUET. Upon induction with IPTG, E. coli BL21(DE3) carrying pcolaDuet-mafBmafI expressed His₆- MafI_MGI-1NEM8013 and MafB_MGI-1NEM8013, as evidenced by immunoblotting of whole-cell lysates (WC). E) His₆-tagged MafI_MGI-1NEM8013 and MafB_MGI-1NEM8013 were recovered from Ni-NTA-agarose column eluate (Eluate). Copurified proteins were analysed by immunoblotting. Antibodies used for immunoblotting of WC and Ni-NTA eluate were Anti- MafB_MGI-1NEM8013 and Anti- MafI_MGI-1NEM8013. Ni-NTA, Nickel-nitrilotriacetic acid.

Figure 4. MafB1_MGI-2NEM8013 provides an advantage in competition assay.

Competition assays were performed with an initial inhibitor to target cell ratio of 10 to 1. Putative inhibitory cells were NEM8013 overexpressing each of the four mafB toxins and their cognate mafI immunity genes using pGCC4 constructs. Putative target cells were an unencapsulated derivative of NEM8013 with a transposon insertion in ctrA gene (NEM8013Δcaps) or this unencapsulated derivative overexpressing MafI1_MGI-2NEM8013. The overexpressed toxin of the inhibitor cells is indicated (otherwise a – indicates that there is no overexpressed toxin) and the overexpressed immunity of the target cells is indicated (otherwise a – indicates that there is no overexpressed immunity protein). Mixed cultures were spotted on a membrane filter placed on GCB agar plate containing 1 mM IPTG and incubated overnight. Filters recovered after overnight incubation were used to perform viable counts and the competitive index was calculated as the inhibitor/target ratio in the output divided by the initial inhibitor/target ratio. The data from three independent experiments were examined for significance using a two-tailed Student's t-test. * p-value p<0.05.

Figure 5. MafB_MGI-1NEM8013 is a secreted toxin.

A) Schematic representation of the construct expressed under IPTG inducible promoter in NEM8013. This construction has been inserted in an intergenic region of NEM8013 chromosome using pGCC4 vector. B) C-terminal FLAG-tagged MafB_MGI-1NEM8013 (MafB) with or without its signal sequence (MafB-SP) are detected in the whole-cell lysates (WC) of NEM8013 strain expressing MafB or MafB-SP under an IPTG inducible promoter. NEM8013 parental strain is used as a control (ctrl). MafB is detected in the supernatant only when its signal sequence is present. As the production of MafB-SP was less efficient than the production of MafB, a larger quantity of supernatant was loaded as shown by the intensity of the nonspecific band (arrow) detected with Anti-FLAG antibody. Antibodies used for immunoblotting of WC and supernatants were Anti-NADP-GDH (NADP-dependent glutamate dehydrogenase, as a cytoplasmic marker protein) and Anti-FLAG to detect C-terminal, FLAG-tagged MafB.

Figure 6. MafB_MGI-1NEM8013 is a bacterial EndoU nuclease.

A) Partial sequence alignment of EndoU nuclease domain of Nidovirus Nsp15 protein (NendoU; NP_740619), Xenopus laevis XendoU (Q8JFY9), human placental protein PP11 (HendoU P21128) and MafB_MGI-1 from NEM8013 strain (C9X2Z7). The arrows indicate two conserved histidine residues, which are part of the catalytic site of previously characterized EndoU nucleases. Multiple alignment was performed using MUSCLE and shaded using the BoxShade server. Residues that are identical or similar in at least three of the four sequences are shaded with black or grey background respectively. B) Analysis of in vivo impact of MafB_MGI-1NEM8013 expression in E. coli. Total RNA from E. coli expressing MafB_MGI-1NEM8013 (pBAD33-mafB_MGI-1NEM8013) was isolated before induction (T0) and 30 min after addition of L-arabinose (T30). Samples were run on 5% denaturing polyacrylamide gels and stained with ethidium bromide. Positions of 23S, 16S, and 5S rRNAs and tRNAs are shown. -, empty vector control; B, E. coli expressing MafB_MGI-1NEM8013 from pBAD33 C) RNase activity of purified recombinant MafB_MGI-1NEM8013 was assessed by incubating MafB_MGI-1NEM8013 -His₆ alone or with MafI_MGI-1NEM8013 -His₆ with total RNA isolated from different sources (N. meningitidis NEM8013, E. coli TOP10 and human epithelial cells FaDu). Each reaction was performed for 30 min at 37°C with 4 µg of RNA in Tris-EDTA buffer. D) Synthetic mRNA of 43 bp was incubated with purified MafB_MGI-1NEM8013-His₆ alone or with MafI_MGI-1NEM8013 -His₆ in Tris-EDTA buffer for 15 min at 37°C. The cleavage products were separated by electrophoresis in a 14% polyacrylamide/8M urea gel and were visualized by ethidium bromide staining. E) Synthetic oligoribonucleotides containing several uridylates (U) or none (0) were incubated with purified MafB_MGI-1NEM8013-His₆ in Tris-EDTA buffer for 20 min at 37°C. The reaction products were analyzed by 14% polyacrylamide/8 M urea gel. Sequences of synthetic oligonucleotides used in this experiment are shown.