Extreme genetic diversity in the type VII secretion system of Listeria monocytogenes suggests a role in bacterial antagonism

Abstract

The type VII protein secretion system (T7SS) has been characterized in members of the phyla Actinobacteria and Firmicutes. In mycobacteria the T7SS is intimately linked with pathogenesis and intracellular survival, while in Firmicutes there is mounting evidence that the system plays a key role in interbacterial competition. A conserved membrane-bound ATPase protein, termed EssC in Staphylococcus aureus, is a critical component of the T7SS and is the primary receptor for substrate proteins. Genetic diversity in the essC gene of S. aureus has previously been reported, resulting in four protein variants that are linked to specific subsets of substrates. Here we have analysed the genetic diversity of the T7SS-encoding genes and substrate proteins across Listeria monocytogenes genome sequences. We find that there are seven EssC variants across the species that differ in their C-terminal region; each variant is correlated with a distinct subset of genes for likely substrate and accessory proteins. EssC1 is most common and is exclusively linked with polymorphic toxins harbouring a YeeF domain, whereas EssC5, EssC6 and EssC7 variants all code for an LXG domain protein adjacent to essC. Some essC1 variant strains encode an additional, truncated essC at their T7 gene cluster. The truncated EssC, comprising only the C-terminal half of the protein, matches the sequence of either EssC2, EssC3 or EssC4. In each case the truncated gene directly precedes a cluster of substrate/accessory protein genes acquired from the corresponding strain. Across L. monocytogenes strains we identified 40 LXG domain proteins, most of which are encoded at conserved genomic loci. These loci also harbour genes encoding immunity proteins and sometimes additional toxin fragments. Collectively our findings strongly suggest that the T7SS plays an important role in bacterial antagonism in this species.

Keywords: Listeria monocytogenes; bacterial antagonism; immunity protein; toxin; type VII secretion.

Figure 1. The firmicutes T7b secretion system.

A. Loci encoding the functional components of the T7b secretion system in Staphylococcus aureus, Listeria monocytogenes and Bacillus subtilis. The gene numbers for L. monocytogenes relate to the reference strain EGDe. B. Subcellular location and topology of the T7b components. The two forkhead associated domains (FHA1/FHA2) and the four ATPase domains (D0, D1, D2 and D3) of EssC are indicated. C. The ess (T7) gene cluster of S. aureus strain NCTC8325. Genes encoding functional components are shaded in blue, secreted substrates in yellow, a chaperone protein in white and EsaG family immunity proteins in turquoise. D. Schematic representation of L. monocytogenes EssC. Amino acid positions of the predicted domains are shown and the region that is variable across strains is indicated. Amino acid 771, shown boxed in red, is the position at which the truncated EssC proteins encoded in some L. monocytogenes essC1 strains start to align with the full-length protein.

Figure 2. Phylogenetic distribution of essC variants across strains of L. monocytogenes.

Maximum-likelihood phylogenetic tree showing the presence of multiple essC variants across the four evolutionary lineages (I – IV) of L. monocytogenes. (CC= clonal complex; ST = sequence type). The scale bar represents the number of substitutions per site.

Figure 3. Two genetically variable regions can be identified downstream of essC.

A. Schematic representation of the T7-encoding region on the chromosome of L. monocytogenes highlighting the positions of two genetically variable regions. Genes shown in blue encode functional components of the T7SS and genes in black are invariant. The gene shown in dashed lines encodes a tRNA. B and C. Three genetic arrangements of variable region 1 from B; essC1 and C; essC2 strains. Genes encoding candidate toxins are shaded yellow with the cognate immunity gene in green. Probable orphan immunity proteins are shown in peach (with likely immunity indicated where identified) and genes encoding toxin fragments in hatched grey and yellow shading. Genes numbered 21-28 are conserved in the same position across all essC2 strains. Gene 24 in this cluster is predicted to encode an immunity protein and is also found in the EGDe essC1 strain. Genes numbered 44-47 are common to essC4 strains. Gene sizes and intergenic regions not to scale.

Figure 4. Genetic variability of region 1 in essC3 and essC4 strains.

A and B. Three genetic arrangements of variable region 1 from A; essC3 and B; essC4 strains. Genes encoding candidate toxins are shaded yellow. Probable orphan immunity proteins are shown in peach (with likely immunity indicated where identified) and genes encoding toxin fragments in hatched grey and yellow shading. Genes numbered 41-47 are common across all essC4 strains. Genes numbered 24-28 are common to essC2 strains, and genes numbered 61 and 62 are common to essC6 strains. Gene sizes and intergenic regions not to scale.

Figure 5. An LXG toxin is encoded in variable region 1 of essC5, essC6 and essC7 strains.

A and C. Two genetic arrangements of variable region 1 from A; essC5 and C; essC7. B. A single genetic arrangement is seen for essC6 strains. Genes encoding candidate toxins are shaded yellow with the cognate immunity gene in green. Probable orphan immunity proteins are shown in peach (with likely immunity indicated where identified). Genes numbered 61-64 are common to essC6 strains. Genes numbered 24-28 are common to essC2 strains and those numbered 44-47 are common to essC4 strains. Gene sizes and intergenic regions not to scale.

Figure 6. An additional truncated EssC protein is encoded in some essC1 strains.

A Genetic arrangement of some example essC1 strains encoding truncated EssCs derived from essC2, essC3 and essC4 strains as indicated. B. Genetic arrangement of some example essC1 strains that harbour conserved T7 genes from essC2 and essC4 strains but no truncated EssC. and B. Genes encoding toxins are shaded yellow with the cognate immunity gene in green. Probable orphan immunity proteins are shown in peach (with likely immunity indicated where identified) and genes encoding toxin fragments in hatched grey and yellow shading. Genes numbered 21-28 are common across all essC2 strains; genes numbered 41-47 are common across all essC4 strains. Gene sizes and intergenic regions not to scale.

Figure 7. LXG toxins are often encoded in variable region 2.

Three genetic arrangements of variable region 2. Genes encoding candidate toxins are shaded yellow with the cognate immunity gene in green. Probable orphan immunity proteins are shown in peach (with likely immunity indicated where identified). Gene sizes and intergenic regions not to scale.

Figure 8. Chromosomal positions of LXG-protein encoding genes in L. monocytogenes.

The positions of the loci at which LXG proteins may be encoded is shown on a genomic map of strain EGDe. LXG protein encoding in EDGe are shown as blue arrows, with the toxin assignment indicated. ** indicates the truncated variant of Toxin α. The position of the T7 locus is indicated in green.