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ABSTRACTAmong genes present in all group A streptococci (GAS), those encoding M-fibril and T-pilus proteins display the highest levels of sequence diversity, giving rise to the two primary serological typing schemes historically used to define strain. A new genotyping scheme for the pilin adhesin and backbone genes is developed and, when combined with emm typing, provides an account of the global GAS strain population. Cluster analysis based on nucleotide sequence similarity assigns most T-serotypes to discrete pilin backbone sequence clusters, yet the established T-types correspond to only half the clusters. The major pilin adhesin and backbone sequence clusters yield 98 unique combinations, defined as “pilin types.” Numerous horizontal transfer events that involve pilin or emm genes generate extensive antigenic and functional diversity on the bacterial cell surface and lead to the emergence of new strains. Inferred pilin genotypes applied to a meta-analysis of global population-based collections of pharyngitis and impetigo isolates reveal highly significant associations between pilin genotypes and GAS infection at distinct ecological niches, consistent with a role for pilin gene products in adaptive evolution. Integration of emm and pilin typing into open-access online tools (pubmlst.org) ensures broad utility for end-users wanting to determine the architecture of M-fibril and T-pilus genes from genome assemblies.IMPORTANCEPrecision in defining the variant forms of infectious agents is critical to understanding their population biology and the epidemiology of associated diseases. Group A Streptococcus (GAS) is a global pathogen that causes a wide range of diseases and displays a highly diverse cell surface due to the antigenic heterogeneity of M-fibril and T-pilus proteins which also act as virulence factors of varied functions. emm genotyping is well-established and highly utilized, but there is no counterpart for pilin genes. A global GAS collection provides the basis for a comprehensive pilin typing scheme, and online tools for determining emm and pilin genotypes are developed. Application of these tools reveals the expansion of structural-functional diversity among GAS via horizontal gene transfer, as evidenced by unique combinations of surface protein genes. Pilin and emm genotype correlations with superficial throat vs skin infection provide new insights on the molecular determinants underlying key ecological and epidemiological trends.

Abstract Background Indigenous children in colonised nations experience high rates of health disparities linked to historical trauma resulting from displacement and dispossession, as well as ongoing systemic racism. Skin infections and their complications are one such health inequity, with the highest global burden described in remote-living Australian Aboriginal and/or Torres Strait Islander (hereafter respectfully referred to as Aboriginal) children. Yet despite increasing urbanisation, little is known about the skin infection burden for urban-living Aboriginal children. More knowledge is needed to inform service provision, treatment guidelines and community-wide healthy skin strategies. In this pilot study, we aimed to test the feasibility and design of larger multi-site observational studies, provide initial descriptions of skin disease frequency and generate preliminary hypotheses of association. Methods This project has been co-designed with local (Noongar) Elders to provide an Australian-first description of skin health and disease in urban-living Aboriginal children. In collaboration with an urban Aboriginal Community Controlled Health Organisation (Derbarl Yerrigan Health Service), we conducted a week-long cross-sectional observational cohort study of Aboriginal children (0–18 years) recruited from the waiting room. Participants completed a questionnaire, skin examination, clinical photos, and swabs and received appropriate treatment. We assessed the feasibility and impact of the pilot study. Results From 4 to 8 October 2021, we recruited 84 Aboriginal children of whom 80 (95%) were urban-living. With a trusted Aboriginal Health Practitioner leading recruitment, most parents (or caregivers) who were approached consented to participate. Among urban-living children, over half (45/80, 56%) of parents described a current concern with their child’s skin, hair and/or nails; and one-third (26/80, 33%) reported current itchy skin. Using a research-service model, 27% (21/79) of examined urban-living participants received opportunistic same-day treatment and 18% (14/79) were referred for later review. Conclusions This co-designed pilot study to understand skin health in urban-living Aboriginal children was feasible and acceptable, with high study participation and subsequent engagement in clinical care observed. Co-design and the strong involvement of Aboriginal people to lead and deliver the project was crucial. The successful pilot has informed larger, multi-site observational studies to more accurately answer questions of disease burden and inform the development of healthy skin messages for urban-living Aboriginal children.

There is increasing evidence for metabolic processes mediating antimicrobial resistance. Here, the authors present a mechanism of sulfamethoxazole resistance in Group A Streptococcus that is dependent on acquiring end products of the host folate biosynthesis pathway.

The pathogenesis of Streptococcus pyogenes (GAS) causing scarlet fever has been associated with the presence of prophages, such as ΦHKU.vir, and their products. Here, the authors characterize the exotoxins SpeC and Spd1 of ΦHKU.vir and show these to act synergistically to facilitate nasopharyngeal colonization in mice.

A hallmark of Listeria (L.) monocytogenes pathogenesis is bacterial escape from maturing entry vacuoles, which is required for rapid bacterial replication in the host cell cytoplasm and cell-to-cell spread. The bacterial transcriptional activator PrfA controls expression of key virulence factors that enable exploitation of this intracellular niche. The transcriptional activity of PrfA within infected host cells is controlled by allosteric coactivation. Inhibitory occupation of the coactivator site has been shown to impair PrfA functions, but consequences of PrfA inhibition for L. monocytogenes infection and pathogenesis are unknown. Here we report the crystal structure of PrfA with a small molecule inhibitor occupying the coactivator site at 2.0 Å resolution. Using molecular imaging and infection studies in macrophages, we demonstrate that PrfA inhibition prevents the vacuolar escape of L. monocytogenes and enables extensive bacterial replication inside spacious vacuoles. In contrast to previously described spacious Listeria-containing vacuoles, which have been implicated in supporting chronic infection, PrfA inhibition facilitated progressive clearance of intracellular L. monocytogenes from spacious vacuoles through lysosomal degradation. Thus, inhibitory occupation of the PrfA coactivator site facilitates formation of a transient intravacuolar L. monocytogenes replication niche that licenses macrophages to effectively eliminate intracellular bacteria. Our findings encourage further exploration of PrfA as a potential target for antimicrobials and highlight that intra-vacuolar residence of L. monocytogenes in macrophages is not inevitably tied to bacterial persistence. Author summary Listeria monocytogenes is a bacterial pathogen that causes severe, often fatal disease in vulnerable individuals. Listeria monocytogenes infects host cells, where it needs to escape from its entry vacuole to replicate inside the host cell cytoplasm and spread to neighboring cells. Inability of Listeria to escape from vacuoles has been associated with long-term bacterial survival inside infected immune cells and chronic infection. We discovered that inhibition of the master regulator of the bacterial factors that orchestrate the intracellular lifestyle of Listeria monocytogenes traps the bacteria inside their entry vacuoles and facilitates extensive bacterial replication in spacious vacuoles. Yet, the majority of infected host immune cells survive the infection and ultimately clear the bacteria. Thus, our data demonstrate that intra-vacuolar existence of Listeria monocytogenes in immune cells is not inevitably tied to bacterial persistence and chronic infection. We provide high resolution structural insights into how an effective inhibitory molecule interacts with the master regulator of Listeria virulence, which may be exploited for rational drug design.

PICO questionDo oral or minimally invasive cheek tooth extraction techniques reduce the incidence of post-operative complications in the horse when compared to repulsion methods?Clinical bottom lineThere is evidence that both oral and minimally invasive cheek tooth extraction techniques may reduce the incidence of post-operative complications in the horse when compared to repulsion methods. However, the published literature covering equine cheek tooth extraction techniques is sparse and of low evidential quality, so a definitive answer on the optimal methods available is not easily determined. Bearing this in mind, the results of this PICO suggest the use of oral extraction, after periodontal stretching, as the first line treatment due to its apparently more favourable success rate. Where an alternative approach is required, such as with fractured teeth or loss of clinical crown, which can prevent the use of routine oral extraction methods, results indicate that minimally invasive extraction techniques may result in a better outcome, with fewer complications, than repulsion methods. Further research that accurately categorises the various minimally invasive techniques available and compares them to repulsion and conventional oral extraction methods is needed.

Streptococcus pyogenes (Group A Streptococcus; GAS) commonly causes pharyngitis in children and adults, with severe invasive disease and immune sequelae being an infrequent consequence. The ability of GAS to invade the host and establish infection likely involves subversion of host immune defenses. However, the signaling pathways and innate immune responses of epithelial cells to GAS are not well-understood. In this study, we utilized RNAseq to characterize the inflammatory responses of primary human tonsil epithelial (TEpi) cells to infection with the laboratory-adapted M6 strain JRS4 and the M1T1 clinical isolate 5448. Both strains induced the expression of genes encoding a wide range of inflammatory mediators, including IL-8. Pathway analysis revealed differentially expressed genes between mock and JRS4- or 5448-infected TEpi cells were enriched in transcription factor networks that regulate IL-8 expression, such as AP-1, ATF-2, and NFAT. While JRS4 infection resulted in high levels of secreted IL-8, 5448 infection did not, suggesting that 5448 may post-transcriptionally dampen IL-8 production. Infection with 5448ΔcepA, an isogenic mutant lacking the IL-8 protease SpyCEP, resulted in IL-8 secretion levels comparable to JRS4 infection. Complementation of 5448ΔcepA and JRS4 with a plasmid encoding 5448-derived SpyCEP significantly reduced IL-8 secretion by TEpi cells. Our results suggest that intracellular infection with the pathogenic GAS M1T1 clone induces a strong pro-inflammatory response in primary tonsil epithelial cells, but modulates this host response by selectively degrading the neutrophil-recruiting chemokine IL-8 to benefit infection.

To invade epithelial cells, Salmonella enterica serovar Typhimurium (S. Typhimurium) induces macropinocytosis through the action of virulence proteins delivered across the host cell membrane via a type III secretion system. We show that after docking at the plasma membrane S. Typhimurium triggers rapid recruitment of cytosolic SNX18, a SH3-PX-BAR domain sorting nexin protein, to the bacteria-induced membrane ruffles and to the nascent Salmonella-containing vacuole. SNX18 recruitment required the inositol-phosphatase activity of the Salmonella effector SopB and an intact phosphoinositide-binding site within the PX domain of SNX18, but occurred independently of Rho-GTPases Rac1 and Cdc42 activation. SNX18 promotes formation of the SCV from the plasma membrane by acting as a scaffold to recruit Dynamin-2 and N-WASP in a process dependent on the SH3 domain of SNX18. Quantification of bacteria uptake revealed that overexpression of SNX18 increased bacteria internalization, whereas a decrease was detected in cells overexpressing the phosphoinositide-binding mutant R303Q, the ΔSH3 mutant, and in cells where endogenous levels of SNX18 were knocked-down. This study identifies SNX18 as a novel target of SopB and suggests a mechanism where S. Typhimurium engages host factors via local manipulation of phosphoinositide composition at the site of invasion to orchestrate the internalization process.

We describe a method using an inexpenive craft glue to routinely isolate specific areas of tissue as small as 1 mm2 from paraffin sections. The tissue may be digested to release nucleic acid suitable for PCR or reverse transcription PCR. The use of this procedure obviates the requirement for manual microdissection or ultraviolet light irradiation. Tissue remaining on the slide can be stained and analyzed, allowing the precision of the extraction to be determined. The slide can be stored as a permanent record of the material taken for analysis.