A novel variant of the Listeria monocytogenes type VII secretion system EssC component is associated with an Rhs toxin

The Type VIIb protein secretion system (T7SSb) is found in Bacillota (firmicute) bacteria and has been shown to mediate interbacterial competition. EssC is a membrane-bound ATPase that is a critical component of the T7SSb and plays a key role in substrate recognition. Prior analysis of available genome sequences of the foodborne bacterial pathogen Listeria monocytogenes has shown that although the T7SSb was encoded as part of the core genome, EssC could be found as one of seven different sequence variants. While each sequence variant was associated with a specific suite of candidate substrate proteins encoded immediately downstream of essC, many LXG-domain proteins were encoded across multiple essC sequence variants. Here we have extended this analysis using a diverse collection of 37,930 L. monocytogenes genomes. We have identified a rare eighth variant of EssC present in ten L. monocytogenes Lineage III genomes. These genomes also encode a large toxin of the rearrangement hotspot (Rhs) repeat family adjacent to essC8, along with a probable immunity protein and three small accessory proteins. We have further identified nine novel LXG-domain proteins, and four additional chromosomal hotspots across L. monocytogenes genomes where LXG proteins can be encoded. The eight L. monocytogenes EssC variants were also found in other Listeria species, with additional novel EssC types also identified. Across the genus, species frequently encoded multiple EssC types, indicating that T7SSb diversity is a primary feature of the genus Listeria. DATA SUMMARYAll genome sequences used in this study are available via Genbank, and the assembly accession numbers are provided in Table S1. This file also lists relevant metadata (name, source category, country, year and clonal complex). IMPACT STATEMENTListeria monocytogenes is a soil-borne saprophytic bacterium and a food-borne pathogen of humans. Decomposing plant matter and the human GI tract are rich in diverse microbial species and to colonise these niches L. monocytogenes must be able to compete with other bacteria. The type VII secretion system (T7SS) of Bacillota has been shown to secrete protein toxins that target other bacteria. In this study we have analysed a diverse collection of L. monocytogenes genome sequences to study the diversity of the Listeria T7SS and its putative effector proteins. We show that the EssC component of the L. monocytogenes T7SS is highly diverse, clustering into one of eight sequence variants. Each EssC variant is associated with a specific toxin candidate, and the EssC8 variant T7SS likely secretes a novel rearrangement hotspot (Rhs) repeat toxin. We also identify multiple new LXG-families of T7SS toxins and describe genomic hotspots where they are encoded. We find no link between EssC variants and clinical outcome. In agreement with this, analysis of EssC variability in available genomes of other Listeria species showed that all eight L. monocytogenes EssC variants are present in non-monocytogenes Listeria species.