Stepwise assembly and release of Tc toxins from Yersinia entomophaga.

Tc toxins are virulence factors of bacterial pathogens. Although their structure and intoxication mechanism are well understood, it remains elusive where this large macromolecular complex is assembled and how it is released. Here we show by an integrative multiscale imaging approach that Yersinia entomophaga Tc (YenTc) toxin components are expressed only in a subpopulation of cells that are 'primed' with several other potential virulence factors, including filaments of the protease M66/StcE. A phage-like lysis cassette is required for YenTc release; however, before resulting in complete cell lysis, the lysis cassette generates intermediate 'ghost' cells, which may serve as assembly compartments and become packed with assembled YenTc holotoxins. We hypothesize that this stepwise mechanism evolved to minimize the number of cells that need to be killed. The occurrence of similar lysis cassettes in diverse organisms indicates a conserved mechanism for Tc toxin release that may apply to other extracellular macromolecular machines.

Lysis Cassette-Mediated Exoprotein Release in Yersinia entomophaga Is Controlled by a PhoB-Like Regulator.

Secretion of exoproteins is a key component of bacterial virulence, and is tightly regulated in response to environmental stimuli and host-dependent signals. The entomopathogenic bacterium Yersinia entomophaga MH96 produces a wide range of exoproteins including its main virulence factor, the 2.46 MDa insecticidal Yen-Tc toxin complex. Previously, a high-throughput transposon-based screening assay identified the region of exoprotein release (YeRER) as essential to exoprotein release in MH96. This study defines the role of the YeRER associated ambiguous holin/endolysin-based lysis cluster (ALC) and the novel RoeA regulator in the regulation and release of exoproteins in MH96. A mutation in the ambiguous lysis cassette (ALC) region abolished exoprotein release and caused cell elongation, a phenotype able to be restored through trans-complementation with an intact ALC region. Endogenous ALC did not impact cell growth of the wild type, while artificial expression of an optimized ALC caused cell lysis. Using HolA-sfGFP and Rz1-sfGFP reporters, Rz1 expression was observed in all cells while HolA expression was limited to a small proportion of cells, which increased over time. Transcriptomic assessments found expression of the genes encoding the prominent exoproteins, including the Yen-Tc, was reduced in the roeA mutant and identified a 220 ncRNA of the YeRER intergenic region that, when trans complemented in the wildtype, abolished exoprotein release. A model for Y. entomophaga mediated exoprotein regulation and release is proposed. IMPORTANCE While theoretical models exist, there is not yet any empirical data that links ALC phage-like lysis cassettes with the release of large macro-molecular toxin complexes, such as Yen-Tc in Gram-negative bacteria. In this study, we demonstrate that the novel Y. entomophaga RoeA activates the production of exoproteins (including Yen-Tc) and the ALC at the transcriptional level. The translation of the ALC holin is confined to a subpopulation of cells that then lyse over time, indicative of a complex hierarchical regulatory network. The presence of an orthologous RoeA and a HolA like holin 5' of an eCIS Afp element in Pseudomonas chlororaphis, combined with the presented data, suggests a shared mechanism is required for the release of some large macromolecular protein assemblies, such as the Yen-Tc, and further supports classification of phage-like lysis clusters as type 10 secretion systems.

Identification of genes involved in exoprotein release using a high-throughput exoproteome screening assay in Yersinia entomophaga.

Bacterial protein secretion is crucial to the maintenance of viability and pathogenicity. Although many bacterial secretion systems have been identified, the underlying mechanisms regulating their expression are less well explored. Yersinia entomophaga MH96, an entomopathogenic bacterium, releases an abundance of proteins including the Yen-Tc into the growth medium when cultured in Luria Bertani broth at ≤ 25°C. Through the development of a high-throughput exoproteome screening assay (HESA), genes involved in MH96 exoprotein production were identified. Of 4,080 screened transposon mutants, 34 mutants exhibited a decreased exoprotein release, and one mutation located in the intergenic region of the Yen-Tc operon displayed an elevated exoprotein release relative to the wild-type strain MH96. DNA sequencing revealed several transposon insertions clustered in gene regions associated with lipopolysaccharide (LPSI and LPSII), and N-acyl-homoserine lactone synthesis (quorum sensing). Twelve transposon insertions were located within transcriptional regulators or intergenic regions. The HESA will have broad applicability for identifying genes associated with exoproteome production in a range of microorganisms.

Evolution of virulence in a novel family of transmissible mega-plasmids.

Some Serratia entomophila isolates have been successfully exploited in biopesticides due to their ability to cause amber disease in larvae of the Aotearoa (New Zealand) endemic pasture pest, Costelytra giveni. Anti-feeding prophage and ABC toxin complex virulence determinants are encoded by a 153-kb single-copy conjugative plasmid (pADAP; amber disease-associated plasmid). Despite growing understanding of the S. entomophila pADAP model plasmid, little is known about the wider plasmid family. Here, we sequence and analyse mega-plasmids from 50 Serratia isolates that induce variable disease phenotypes in the C. giveni insect host. Mega-plasmids are highly conserved within S. entomophila, but show considerable divergence in Serratia proteamaculans with other variants in S. liquefaciens and S. marcescens, likely reflecting niche adaption. In this study to reconstruct ancestral relationships for a complex mega-plasmid system, strong co-evolution between Serratia species and their plasmids were found. We identify 12 distinct mega-plasmid genotypes, all sharing a conserved gene backbone, but encoding highly variable accessory regions including virulence factors, secondary metabolite biosynthesis, Nitrogen fixation genes and toxin-antitoxin systems. We show that the variable pathogenicity of Serratia isolates is largely caused by presence/absence of virulence clusters on the mega-plasmids, but notably, is augmented by external chromosomally encoded factors.