Capturing Salmonella SspH2 Host Targets in Virus-Like Particles

In the context of host-pathogen interactions, gram-negative bacterial virulence factors, such as effectors, may be transferred from bacterial to eukaryotic host cytoplasm by multicomponent Type III protein secretion systems (T3SSs). Central to Salmonella enterica serovar Typhimurium (S. Typhimurium) pathogenesis is the secretion of over 40 effectors by two T3SSs encoded within pathogenicity islands SPI-1 and SPI-2. These effectors manipulate miscellaneous host cellular processes, such as cytoskeleton organization and immune signaling pathways, thereby permitting host colonization and bacterial dissemination. Recent research on effector biology provided mechanistic insights for some effectors. However, for many effectors, clearly defined roles and host target repertoires—further clarifying effector interconnectivity and virulence networks—are yet to be uncovered. Here we demonstrate the utility of the recently described viral-like particle trapping technology Virotrap as an effective approach to catalog S. Typhimurium effector-host protein complexes (EH-PCs). Mass spectrometry-based Virotrap analysis of the novel E3 ubiquitin ligase SspH2 previously shown to be implicated in modulating actin dynamics and immune signaling, exposed known host interactors PFN1 and−2 besides several putative novel, interconnected host targets. Network analysis revealed an actin (-binding) cluster among the significantly enriched hits for SspH2, consistent with the known localization of the S-palmitoylated effector with actin cytoskeleton components in the host. We show that Virotrap complements the current state-of-the-art toolkit to study protein complexes and represents a valuable means to screen for effector host targets in a high-throughput manner, thereby bridging the knowledge gap between effector-host interplay and pathogenesis.

Typhoid toxin sorting and exocytic transport from Salmonella Typhi infected cells

Typhoid toxin is an essential virulence factor for Salmonella Typhi, the cause of typhoid fever in humans. This toxin has an unusual biology in that it is produced by Salmonella Typhi only when located within host cells. Once synthesized, the toxin is secreted to the lumen of the Salmonella-containing vacuole from where it is transported to the extracellular space by vesicle carrier intermediates. Here we report the identification of the typhoid toxin sorting receptor and the cellular machinery that packages the toxin into vesicle carriers, and exports it to the extracellular space. We found that the cation-independent mannose-6-phosphate receptor serves as typhoid toxin sorting receptor and that the coat protein COPII and the GTPase Sar1 mediate its packaging into vesicle carriers. Formation of the typhoid toxin carriers requires the specific environment of the Salmonella Typhi-containing vacuole, which is determined by the activities of specific effectors of its type III protein secretion systems. We also found that Rab11B and its interacting protein Rip11 control the intracellular transport of the typhoid toxin carriers, and the SNARE proteins VAMP7, SNAP23, and Syntaxin 4 their fusion to the plasma membrane. Typhoid toxin's cooption of specific cellular machinery for its transport to the extracellular space illustrates the remarkable adaptation of an exotoxin to exert its function in the context of an intracellular pathogen.

Functional self-nonamerization of the Type III translocase chaperone/exported protein receptor

The Type III protein secretion (T3S) pathway is widespread in bacterial Gram-negative pathogens. It comprises the injectisome with a cytoplasm-facing inner membrane translocase and a surface-exposed needle. The translocase comprises a conical SctR5S4T1 export channel, decorated by SctU, and enveloped by SctV. The large cytoplasmic domain (C-domain) of SctV binds T3S chaperone/exported protein and forms a putative ante-chamber leading to the membrane translocase. Here we probed the mechanism of assembly and function of SctV. Using live cell imaging, SctV was shown to assemble in peripheral oligomeric clusters in both EPEC and a non-T3SS harbouring E.coli strain. Non-ionic detergents extracted SctV homo-nonamers from membranes of both strains. His-SctV9 reconstituted in peptidiscs revealed an elongated, tripartite particle of ~22nm with a membrane domain and a narrower linker connecting to a C-domain. The C-domain assembles in a hollow asymmetric ring with a 5-6 nm-wide inner opening. SctV9 is necessary and sufficient to act as a receptor for two different chaperone/exported protein pairs by binding them at distinct C-domain sites identified by immobilized peptide arrays. Binding sites are not only important for binding but also essential for secretion suggesting a close mechanistic link between the receptor and secretion activities. These findings advance structural understanding of injectisome assembly and reveal that chaperone/exported protein targeting is mechanistically uncoupled from the succeeding translocation step.Author summaryThe export apparatus of the Type III secretion pathway is conserved in flagellar and virulence injectisomes. Its major component SctV, is essential for T3S substrate targeting and translocation. Here, we analysed SctV assembly and function as a receptor for targeting T3S exported proteins. SctV was shown to self-nonamerize, in a structure that is sufficient for functional binding of chaperone/exported protein complexes. Nonameric SctV reconstituted in peptidiscs and its nonameric ring-forming cytoplasmic domain reveal structural features and lay the foundation for high-resolution cryoEM. These tools set the stage for mechanistic dissection of the structural interactions of the export apparatus with the exported proteins, independently of the transmembrane crossing reaction.

Genomic Characterisation of Mushroom Pathogenic Pseudomonads and Their Interaction with Bacteriophages

Bacterial diseases of the edible white button mushroom Agaricus bisporus caused by Pseudomonas species cause a reduction in crop yield, resulting in considerable economic loss. We examined bacterial pathogens of mushrooms and bacteriophages that target them to understand the disease and opportunities for control. The Pseudomonastolaasii genome encoded a single type III protein secretion system (T3SS), but contained the largest number of non-ribosomal peptide synthase (NRPS) genes, multimodular enzymes that can play a role in pathogenicity, including a putative tolaasin-producing gene cluster, a toxin causing blotch disease symptom. However, Pseudomonasagarici encoded the lowest number of NRPS and three putative T3SS while non-pathogenic Pseudomonas sp. NS1 had intermediate numbers. Potential bacteriophage resistance mechanisms were identified in all three strains, but only P. agarici NCPPB 2472 was observed to have a single Type I-F CRISPR/Cas system predicted to be involved in phage resistance. Three novel bacteriophages, NV1, ϕNV3, and NV6, were isolated from environmental samples. Bacteriophage NV1 and ϕNV3 had a narrow host range for specific mushroom pathogens, whereas phage NV6 was able to infect both mushroom pathogens. ϕNV3 and NV6 genomes were almost identical and differentiated within their T7-like tail fiber protein, indicating this is likely the major host specificity determinant. Our findings provide the foundations for future comparative analyses to study mushroom disease and phage resistance.

A protein secreted by the Salmonella type III secretion system controls needle filament assembly

Type III protein secretion systems (T3SS) are encoded by several pathogenic or symbiotic bacteria. The central component of this nanomachine is the needle complex. Here we show in a Salmonella Typhimurium T3SS that assembly of the needle filament of this structure requires OrgC, a protein encoded within the T3SS gene cluster. Absence of OrgC results in significantly reduced number of needle substructures but does not affect needle length. We show that OrgC is secreted by the T3SS and that exogenous addition of OrgC can complement a ∆orgC mutation. We also show that OrgC interacts with the needle filament subunit PrgI and accelerates its polymerization into filaments in vitro. The structure of OrgC shows a novel fold with a shared topology with a domain from flagellar capping proteins. These findings identify a novel component of T3SS and provide new insight into the assembly of the type III secretion machine.

Visualization and characterization of individual type III protein secretion machines in live bacteria

Type III protein secretion machines have evolved to deliver bacterially encoded effector proteins into eukaryotic cells. Although electron microscopy has provided a detailed view of these machines in isolation or fixed samples, little is known about their organization in live bacteria. Here we report the visualization and characterization of theSalmonellatype III secretion machine in live bacteria by 2D and 3D single-molecule switching superresolution microscopy. This approach provided access to transient components of this machine, which previously could not be analyzed. We determined the subcellular distribution of individual machines, the stoichiometry of the different components of this machine in situ, and the spatial distribution of the substrates of this machine before secretion. Furthermore, by visualizing this machine inSalmonellamutants we obtained major insights into the machine’s assembly. This study bridges a major resolution gap in the visualization of this nanomachine and may serve as a paradigm for the examination of other bacterially encoded molecular machines.