Emergence of a new Neisseria meningitidis clonal complex 11 lineage 11.2 clade as an effective urogenital pathogen

Abstract

Neisseria meningitidis (Nm) clonal complex 11 (cc11) lineage is a hypervirulent pathogen responsible for outbreaks of invasive meningococcal disease, including among men who have sex with men, and is increasingly associated with urogenital infections. Recently, clusters of Nm urethritis have emerged primarily among heterosexual males in the United States. We determined that nonencapsulated meningococcal isolates from an ongoing Nm urethritis outbreak among epidemiologically unrelated men in Columbus, Ohio, are linked to increased Nm urethritis cases in multiple US cities, including Atlanta and Indianapolis, and that they form a unique clade (the US Nm urethritis clade, US_NmUC). The isolates belonged to the cc11 lineage 11.2/ET-15 with fine type of PorA P1.5-1, 10-8; FetA F3-6; PorB 2-2 and express a unique FHbp allele. A common molecular fingerprint of US_NmUC isolates was an IS1301 element in the intergenic region separating the capsule ctr-css operons and adjacent deletion of cssA/B/C and a part of csc, encoding the serogroup C capsule polymerase. This resulted in the loss of encapsulation and intrinsic lipooligosaccharide sialylation that may promote adherence to mucosal surfaces. Furthermore, we detected an IS1301-mediated inversion of an ∼20-kb sequence near the cps locus. Surprisingly, these isolates had acquired by gene conversion the complete gonococcal denitrification norB-aniA gene cassette, and strains grow well anaerobically. The cc11 US_NmUC isolates causing urethritis clusters in the United States may have adapted to a urogenital environment by loss of capsule and gene conversion of the Neisseria gonorrheae norB-aniA cassette promoting anaerobic growth.

Keywords: IS1301; Neisseria meningitidis; capsule; denitrification; meningococcal urethritis.

Fig. S1.

Core genome similarity dendrogram of US N. meningitidis urethritis clade (US_NmUC) isolates with respect to lineage 11 (ST-11 clonal complex) isolates presented by Lucidarme et al. (12), isolates identified in Table S1 as well as more recent urethral isolates from PubMLST. The dendrogram is rooted between sublineages 11.1 and 11.2. Isolates are marked with colored circles representing serogroup as documented in the PubMLST. The outer circle contains the PubMLST identifier and additional information about the sources of the isolates. The serogroup W clade in sublineage 11.1 is represented by a red triangle and contains 470 isolates, including one urethral isolate. A total of 812 isolates were included in the analysis.

Fig. 1.

The cps locus of the US_NmUC isolates. (A) The IS1301 insertion and deletion of the capsule biosynthesis genes in the US Nm urethritis clade isolates. Based on overlapping PCR products and sequencing of the cps locus in clade isolates, an 844-bp insertion element (IS1301) has inserted into the IGR with a simultaneous 3,625-bp deletion of cssA/B/C and a partial csc deletion. When amplified across the deleted region using primers located in ctrA and csc (blue arrows), shorter PCR products (∼1.6 kb) were obtained from clade isolates compared with that (4.4 kb) of the serogroup C WT FAM18 strain (examples shown in C). The 1.6-kb PCR product was sequenced to determine the insertion/deletion junctions. (B) Genomic segments of ∼38 kb surrounding the cps locus of the US_NmUC isolates. A genome configuration between two IS1301 elements corresponding to the ctrA-IS1301-csc linkage in the US_NmUC isolates as determined by PCR and sequencing is shown on top, and the genome configuration of the single-contig PacBio genome of CNM10 is shown below. The ∼20-kb genome inversion between two IS1301 elements is depicted by red triangles. Genes are color-coded: ctrB/C/D and ctrG in cyan, ctrA in dark blue, ctrE/ctrG in green, truncated csc in purple, tex in gray, dnaJ in light blue, and pykA in red. The IS1301 elements are shown as yellow rectangles. (C) Gel pictures of the ctrA to csc (Left) and the ctrA to pykA (Right) PCR amplifications. The presence of both ctrA-IS1301-csc (red line in B) and ctrA-IS1301-pykA (blue line in B) orientations was determined by PCR amplification across IS1301 and sequencing of the resulting products. Lanes: 1, CNM3; 2, CNM10; 3, CNM26; 4, FAM18. Cell suspensions from single colonies were used as templates. The expected product sizes are 1,614 bp and 2,316 bp for ctrA-IS1301-csc and ctrA-IS1301-pykA, respectively.

Fig. 2.

Acquisition of the gonococcal norB-aniA gene cassette in the US Nm urethritis clade. (A) Schematics of the norB-aniA locus in the US_NmUC isolates and N. gonorrheae. Homologous genes are color-coded. A gpxA gene encoding a glutathione peroxidase adjacent to norB is present in the genomes of the US_NmUC clade isolates and other meningococci, but is absent in N. gonorrheae. On the 3′ side, a 53-bp sequence is present downstream of aniA in gonococci but absent in the US clade and other meningococci. The presence and the absence of these two genetic features (gpxA and the 53-bp element) in the US_NmUC genome indicated the recombination junctions (blue double-arrow lines). (B) The sequence between stop codons of norB and aniA (3808 bp) marked by the red line in A was used for the phylogenetic sequence analyses. Two isolates each from Columbus, Indianapolis, and Atlanta, together with three N. lactamica strains, two meningococcal urethral isolates, three meningococcal MSM isolates, and the FAM18 reference strain were included in the alignment by Clustal W. The US urethritis clade norB-aniA locus clustered with the gonococcal sequences. (C) A SNP density plot of the core genome alignment by Parsnp at the norB-aniA locus with FAM18 set as the reference genome. Each SNP that differed from FAM18 is shown as a single line, and multiple neighboring SNPs appear as thick lines. The sequence identity of four US_NmUC isolates to the gonococcal FA1090 sequence is compared with that of a meningococcal MSM isolate LNP27256 at this locus. The light-gray region indicates that sequence is absent in one or some of the aligned genomes (polymorphisms within the glycosylation motif of aniA). (D) Anaerobic growth of the US urethritis clade isolates on GC agar plates supplemented with sodium nitrite. The US clade isolate (CNM10) was compared with meningococcal strains with either an intact AniA (MC58) or a frame-shift inactivated AniA (FAM18) and the gonococcal reference strain FA1090. After incubating under anaerobic conditions for 24 h at 37 °C and another 24 h at room temperature, growth on the plates was recorded. Data shown are representative anaerobic growth experiments repeated five times. Additional CNM isolates showed anaerobic growth similar to that of CNM10.

Fig. S2.

NorB protein sequence alignment by clustalW. Three N. gonorrheae (Ng) strains (FA1090, F62, and MS11), two US_NmUC isolates (CNM10 and NM1), one Nm urethral isolate, one Nm MSM isolate, and one N. lactamica (Nl) strain are included in the alignment. Residues different from FA1090 are shaded in black.

Fig. S2.

NorB protein sequence alignment by clustalW. Three N. gonorrheae (Ng) strains (FA1090, F62, and MS11), two US_NmUC isolates (CNM10 and NM1), one Nm urethral isolate, one Nm MSM isolate, and one N. lactamica (Nl) strain are included in the alignment. Residues different from FA1090 are shaded in black.

Fig. S3.

AniA protein sequence alignment. The US urethritis clade isolate CNM10 is aligned to the gonococcal strain FA1090, the N. lactamica strain 020–60, the meningococcal urethral isolate PE5, and the MSM invasive isolate LNP27256 with intact AniA. Residues different from FA1090 are highlighted in yellow. Residues important for activity are marked with an asterisk.

Fig. S4.

The intergenic region between start codons of norB and aniA of CNM10 was aligned to gonococcal (FA1090) and meningococcal (LNP27256) sequences. The nucleotide different from that of strain FA1090 is shaded in black. The ATG start codons, the −10 element, and the transcriptional start site (+1) are underlined. The NsrR-binding motifs of norB and aniA are in red. Two NarP-binding inverted repeat sequences are marked with two sets of arrows and are in blue. The Fur-binding site is labeled with blue underline and the FNR-binding motif is boxed. The location of the poly-C tract is shown with a thick red line above. The SNPs shown to affect regulatory activities of FNR and NarP (44) are marked with asterisks.