Convergent evolution in the supercoiling of prokaryotic flagellar filaments.

The supercoiling of bacterial and archaeal flagellar filaments is required for motility. Archaeal flagellar filaments have no homology to their bacterial counterparts and are instead homologs of bacterial type IV pili. How these prokaryotic flagellar filaments, each composed of thousands of copies of identical subunits, can form stable supercoils under torsional stress is a fascinating puzzle for which structural insights have been elusive. Advances in cryoelectron microscopy (cryo-EM) make it now possible to directly visualize the basis for supercoiling, and here, we show the atomic structures of supercoiled bacterial and archaeal flagellar filaments. For the bacterial flagellar filament, we identify 11 distinct protofilament conformations with three broad classes of inter-protomer interface. For the archaeal flagellar filament, 10 protofilaments form a supercoil geometry supported by 10 distinct conformations, with one inter-protomer discontinuity creating a seam inside of the curve. Our results suggest that convergent evolution has yielded stable superhelical geometries that enable microbial locomotion.

Flagellin outer domain dimerization modulates motility in pathogenic and soil bacteria from viscous environments.

Flagellar filaments function as the propellers of the bacterial flagellum and their supercoiling is key to motility. The outer domains on the surface of the filament are non-critical for motility in many bacteria and their structures and functions are not conserved. Here, we show the atomic cryo-electron microscopy structures for flagellar filaments from enterohemorrhagic Escherichia coli O157:H7, enteropathogenic E. coli O127:H6, Achromobacter, and Sinorhizobium meliloti, where the outer domains dimerize or tetramerize to form either a sheath or a screw-like surface. These dimers are formed by 180° rotations of half of the outer domains. The outer domain sheath (ODS) plays a role in bacterial motility by stabilizing an intermediate waveform and prolonging the tumbling of E. coli cells. Bacteria with these ODS and screw-like flagellar filaments are commonly found in soil and human intestinal environments of relatively high viscosity suggesting a role for the dimerization in these environments.

EPEC-induced activation of the Ca2+ transporter TRPV2 leads to pyroptotic cell death.

The enteropathogenic Escherichia coli (EPEC) type III secretion system effector Tir, which mediates intimate bacterial attachment to epithelial cells, also triggers Ca2+ influx followed by LPS entry and caspase-4-dependent pyroptosis, which could be antagonized by the effector NleF. Here we reveal the mechanism by which EPEC induces Ca2+ influx. We show that in the intestinal epithelial cell line SNU-C5, Tir activates the mechano/osmosensitive cation channel TRPV2 which triggers extracellular Ca2+ influx. Tir-induced Ca2+ influx could be blocked by siRNA silencing of TRPV2, pre-treatment with the TRPV2 inhibitor SET2 or by growing cells in low osmolality medium. Pharmacological activation of TRPV2 in the absence of Tir failed to initiate caspase-4-dependent cell death, confirming the necessity of Tir. Consistent with the model implicating activation on translocation of TRPV2 from the ER to plasma membrane, inhibition of protein trafficking by either brefeldin A or the effector NleA prevented TRPV2 activation and cell death. While infection with EPECΔnleA triggered pyroptotic cell death, this could be prevented by NleF. Taken together this study shows that while integration of Tir into the plasma membrane activates TRPV2, EPEC uses NleA to inhibit TRPV2 trafficking and NleF to inhibit caspase-4 and pyroptosis.

A systematic review of Sec24 cargo interactome.

Endoplasmic reticulum (ER)-to-Golgi trafficking is an essential and highly conserved cellular process. The coat protein complex-II (COPII) arm of the trafficking machinery incorporates a wide array of cargo proteins into vesicles through direct or indirect interactions with Sec24, the principal subunit of the COPII coat. Approximately one-third of all mammalian proteins rely on the COPII-mediated secretory pathway for membrane insertion or secretion. There are four mammalian Sec24 paralogs and three yeast Sec24 paralogs with emerging evidence of paralog-specific cargo interaction motifs. Furthermore, individual paralogs also differ in their affinity for a subset of sorting motifs present on cargo proteins. As with many aspects of protein trafficking, we lack a systematic and thorough understanding of the interaction of Sec24 with cargoes. This systematic review focuses on the current knowledge of cargo binding to both yeast and mammalian Sec24 paralogs and their ER export motifs. The analyses show that Sec24 paralog specificity of cargo (and cargo receptors) range from exclusive paralog dependence or preference to partial redundancy. We also discuss how the Sec24 secretion system is hijacked by viral (eg, VSV-G, Hepatitis B envelope protein) and bacterial (eg, the enteropathogenic Escherichia coli type III secretion system effector NleA/EspI) pathogens.

Type III secretion system effector subnetworks elicit distinct host immune responses to infection.

Citrobacter rodentium, a natural mouse pathogen which colonises the colon of immuno-competent mice, provides a robust model for interrogating host-pathogen-microbiota interactions in vivo. This model has been key to providing new insights into local host responses to enteric infection, including changes in intestinal epithelial cell immunometabolism and mucosal immunity. C. rodentium injects 31 bacterial effectors into epithelial cells via a type III secretion system (T3SS). Recently, these effectors were shown to be able to form multiple intracellular subnetworks which can withstand significant contractions whilst maintaining virulence. Here we highlight recent advances in understanding gut mucosal responses to infection and effector biology, as well as potential uses for artificial intelligence (AI) in understanding infectious disease and speculate on the role of T3SS effector networks in host adaption.

The type III secretion system effector EspO of enterohaemorrhagic Escherichia coli inhibits apoptosis through an interaction with HAX-1.

Many enteric pathogens employ a type III secretion system (T3SS) to translocate effector proteins directly into the host cell cytoplasm, where they subvert signalling pathways of the intestinal epithelium. Here, we report that the anti-apoptotic regulator HS1-associated protein X1 (HAX-1) is an interaction partner of the T3SS effectors EspO of enterohaemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium, OspE of Shigella flexneri and Osp1STYM of Salmonella enterica serovar Typhimurium. EspO, OspE and Osp1STYM have previously been reported to interact with the focal adhesions protein integrin linked kinase (ILK). We found that EspO localizes both to the focal adhesions (ILK localisation) and mitochondria (HAX-1 localisation), and that increased expression of HAX-1 leads to enhanced mitochondrial localisation of EspO. Ectopic expression of EspO, OspE and Osp1STYM protects cells from apoptosis induced by staurosporine and tunicamycin. Depleting cells of HAX-1 indicates that the anti-apoptotic activity of EspO is HAX-1 dependent. Both HAX-1 and ILK were further confirmed as EspO1-interacting proteins during infection using T3SS-delivered EspO1. Using cell detachment as a proxy for cell death we confirmed that T3SS-delivered EspO1 could inhibit cell death induced during EPEC infection, to a similar extent as the anti-apoptotic effector NleH, or treatment with the pan caspase inhibitor z-VAD. In contrast, in cells lacking HAX-1, EspO1 was no longer able to protect against cell detachment, while NleH1 and z-VAD maintained their protective activity. Therefore, during both infection and ectopic expression EspO protects cells from cell death by interacting with HAX-1. These results suggest that despite the differences between EHEC, C. rodentium, Shigella and S. typhimurium infections, hijacking HAX-1 anti-apoptotic signalling is a common strategy to maintain the viability of infected cells. TAKE AWAY: EspO homologues are found in EHEC, Shigella, S. typhimurium and some EPEC. EspO homologues interact with HAX-1. EspO protects infected cells from apoptosis. EspO joins a growing list of T3SS effectors that manipulate cell death pathways.

Structural basis for species-selective targeting of Hsp90 in a pathogenic fungus.

New strategies are needed to counter the escalating threat posed by drug-resistant fungi. The molecular chaperone Hsp90 affords a promising target because it supports survival, virulence and drug-resistance across diverse pathogens. Inhibitors of human Hsp90 under development as anticancer therapeutics, however, exert host toxicities that preclude their use as antifungals. Seeking a route to species-selectivity, we investigate the nucleotide-binding domain (NBD) of Hsp90 from the most common human fungal pathogen, Candida albicans. Here we report structures for this NBD alone, in complex with ADP or in complex with known Hsp90 inhibitors. Encouraged by the conformational flexibility revealed by these structures, we synthesize an inhibitor with >25-fold binding-selectivity for fungal Hsp90 NBD. Comparing co-crystals occupied by this probe vs. anticancer Hsp90 inhibitors revealed major, previously unreported conformational rearrangements. These insights and our probe's species-selectivity in culture support the feasibility of targeting Hsp90 as a promising antifungal strategy.

Heat shock protein 90 localizes to the surface and augments virulence factors of Cryptococcus neoformans.

BACKGROUND: Thermotolerance is an essential attribute for pathogenesis of Cryptococcus as exemplified by the fact that only two species in the genus, which can grow at 37°C, are human pathogens. Species which have other virulence factors including capsule formation and melanisation, but lack the ability to propagate at 37°C are not pathogenic. In another related fungal pathogen, Candida albicans, heat shock protein 90 has been implicated to be a central player in commanding pathogenicity by governing yeast to hyphal transition and drug resistance. Exploring Hsp90 biology in Cryptococcus in context of thermotolerance may thus highlight important regulatory principles of virulence and open new therapeutic avenues. METHODOLOGY/PRINCIPAL FINDINGS: Hsp90 is involved in regulating thermotolerance in Cryptococcus as indicated by growth hypersensitivity at 37°C upon mild compromise of Hsp90 function relative to 25°C. Biochemical studies revealed a more potent inhibition of ATPase activity by pharmacological inhibitor 17-AAG at 37°C as compared to 25°C. Catalytic efficiency of the protein at 37°C was found to be 6.39×10-5µM-1. Furthermore, indirect immunofluorescence analysis using a specific antibody revealed cell surface localization of Hsp90 via ER Golgi classical secretory pathway. Hsp90 was found to be induced under capsule inducing conditions and Hsp90 inhibition led to decrease in capsular volume. Finally compromising Hsp90 function improved anidulafungin tolerance in Cryptococcus. CONCLUSIONS/SIGNIFICANCE: Our findings highlight that Hsp90 regulates pathogenicity of the fungus by myriad ways. Firstly, it is involved in mediating thermotolerance which implies targeting Hsp90 can abrogate thermotolerance and hence growth of the fungus. Secondly, this study provides the first report of biochemical properties of Hsp90 of a pathogenic fungus. Finally, since Hsp90 is localised at the cell wall, targeting cell surface Hsp90 can represent a novel strategy to combat this lethal infection.

Draft genome of a commonly misdiagnosed multidrug resistant pathogen Candida auris.

BACKGROUND: Candida auris is a multidrug resistant, emerging agent of fungemia in humans. Its actual global distribution remains obscure as the current commercial methods of clinical diagnosis misidentify it as C. haemulonii. Here we report the first draft genome of C. auris to explore the genomic basis of virulence and unique differences that could be employed for differential diagnosis. RESULTS: More than 99.5 % of the C. auris genomic reads did not align to the current whole (or draft) genome sequences of Candida albicans, Candida lusitaniae, Candida glabrata and Saccharomyces cerevisiae; thereby indicating its divergence from the active Candida clade. The genome spans around 12.49 Mb with 8527 predicted genes. Functional annotation revealed that among the sequenced Candida species, it is closest to the hemiascomycete species Clavispora lusitaniae. Comparison with the well-studied species Candida albicans showed that it shares significant virulence attributes with other pathogenic Candida species such as oligopeptide transporters, mannosyl transfersases, secreted proteases and genes involved in biofilm formation. We also identified a plethora of transporters belonging to the ABC and major facilitator superfamily along with known MDR transcription factors which explained its high tolerance to antifungal drugs. CONCLUSIONS: Our study emphasizes an urgent need for accurate fungal screening methods such as PCR and electrophoretic karyotyping to ensure proper management of fungemia. Our work highlights the potential genetic mechanisms involved in virulence and pathogenicity of an important emerging human pathogen namely C. auris. Owing to its diversity at the genomic scale; we expect the genome sequence to be a useful resource to map species specific differences that will help develop accurate diagnostic markers and better drug targets.