Diversity of the type VI secretion systems in the Neisseria spp

Abstract

Complete Type VI Secretion Systems were identified in the genome sequence data of Neisseria subflava isolates sourced from throat swabs of human volunteers. The previous report was the first to describe two complete Type VI Secretion Systems in these isolates, both of which were distinct in terms of their gene organization and sequence homology. Since publication of the first report, Type VI Secretion System subtypes have been identified in Neisseria spp. The characteristics of each type in N. subflava are further investigated here and in the context of the other Neisseria spp., including identification of the lineages containing the different types and subtypes. Type VI Secretion Systems use VgrG for delivery of toxin effector proteins; several copies of vgrG and associated effector / immunity pairs are present in Neisseria spp. Based on sequence similarity between strains and species, these core Type VI Secretion System genes, vgrG, and effector / immunity genes may diversify via horizontal gene transfer, an instrument for gene acquisition and repair in Neisseria spp.

Keywords: Commensal Neisseria; Neisseria spp; T6SS; Toxin antitoxin system; Type 6 secretion system; Type VI secretion system.

Fig. 1.

A circular representation of the complete N. subflava strain KU1003-01 genome created using DNAPlotter [68]. Annotated CDSs are shown in the outer two rings (light blue, dark blue, red) and tRNA loci within the inner grey circle (green). The inner circle (purple and gold plots) represents chromosomal GC content, purple indicates regions that are below the chromosomal average and gold are those above the chromosomal average. The T6SS-A core gene clusters (red) are in two locations on the chromosome (a and b) with a duplication of one of the clusters (c). The vgrG EI clusters (dark blue) are associated with one of the core gene clusters (b) and elsewhere on the genome. The genomic region marked ‘dif’ represents the gene clusters where dif sequences are present.

Fig. 2.

The Neisseria spp. Type VI Secretion System core gene clusters. (a) Organisation of the T6SS-A core genes in N. subflava strain KU1003-01. (b) The T6SS-B in the reference genome sequence of N. subflava strain ATCC49276. (c) The T6SS-Bi subtype in N. animaloris strain NCTC12227 and (d) N. oralis strain 21 044. N. subflava strain KU1003-02 and RH3002-v2g have identically organised T6SS to that shown in panel b.

Fig. 3.

Neighbour Joining tree constructed following alignment of 11 concatenated Neisseria spp. T6SS core gene products using Ugene [75]. The tree image was formatted using the online Interactive Tree of Life (iTOL) tool [158]. (a) Eleven T6SS-A genome sequences believed to be N. sicca . (b) Six T6SS-A genome sequences believed to be N. mucosa . (c) Twelve T6SS-B genome sequences believed to be N. sicca . (d) Twelve T6SS-B genome sequences believed to be N. mucosa . (e) Evidence of sharing of T6SS-B core components between N. bergeri, N. lactamica, and N. polysaccharae. (f) Evidence of sharing of T6SS-B core components between N. cinerea, N. lactamica, and N. polysaccharae. (g) Lone N. subflava (strain 42060) with T6SS-B gene products 99 % similar to those in N. polysacchareae and N. lactamica . * Genome sequences with both T6SS types A and B. Geographical origin not available.

Fig. 4.

Protein domains of the Neisseria spp. T6SS VgrG. VgrG domains with structures similar to bacteriophage proteins. Phage hub proteins, GPD in P2 bacteriophage or gene product 27 (gp27) of bacteriophage T4. The oligonucleotide/oligosaccharide-binding (OB) domains resemble regions within the T4 gene product 5 (gp5) [29, 133]. T6SS_Vgr domains are associated with some VgrG types [122] and are followed by Domains of Unknown Function (DUF2345). (a) VgrG domains, predicted using Genomic SMART [77] and Phyre² [78], as well as their approximate locations within VgrGs 1–10 of N. subflava strain KU1003-01. The percent similarity of each domain in comparison to the VgrG1 copy, which is encoded at the core cluster (see Fig. 1, location B) are shown highlighted in grey above each image. VgrG4, VgrG6, and VgrG8 each lack one or more domains seen in the complete Type VI Secretion System tip protein, VgrG. (b) The approximate domain locations predicted for the single VgrG present in the T6SS-B in N. subflava strains KU1003-02, RH3002-v2g and ATCC 49275. These VgrG are between 98–100 % similar to one another but only around 25 % similar overall to VgrG1 of N. subflava strain KU1003-01. The domains of the T6SS-B type VgrG are shown in different colours to the T6SS-A type to highlight these being different VgrG types.

Fig. 5.

N. subflava strain KU1003-01 vgrG 1–10 and downstream EI gene clusters. The vgrG (orange), putative T6SS effectors (grey with a red border), hypothetical genes (hyp) downstream of vgrG and putative T6SS effectors (grey with a black border), non-T6SS genes (blue), transposases (yellow), and short sequences (vertical green and blue bars highlighting sequences within both the N-terminal Gp5 OB fold regions of vgrG as well as homologous sequences located outside of the vgrG CDS) were analysed. In particular, locations of DNA Uptake Sequence variants (DUSvar), Inverted repeats (IR), Inverted repeats predicted to be transcriptional terminators (IRT), and dif recombinase sequences (dif) where all present associated with vgrG regions. Duplications were identified associated with vgrG 6 (d, i and ii, iii and iv) and vgrG 7 (e, v and vi). The vgrG 9 copy (g) is predicted to be on a short, circular sequence, based on data from both combined MinION/Illumina enhanced genome sequencing.