RNase III CLASH in MRSA uncovers sRNA regulatory networks coupling metabolism to toxin expression.

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen responsible for significant human morbidity and mortality. Post-transcriptional regulation by small RNAs (sRNAs) has emerged as an important mechanism for controlling virulence. However, the functionality of the majority of sRNAs during infection is unknown. To address this, we performed UV cross-linking, ligation, and sequencing of hybrids (CLASH) in MRSA to identify sRNA-RNA interactions under conditions that mimic the host environment. Using a double-stranded endoribonuclease III as bait, we uncovered hundreds of novel sRNA-RNA pairs. Strikingly, our results suggest that the production of small membrane-permeabilizing toxins is under extensive sRNA-mediated regulation and that their expression is intimately connected to metabolism. Additionally, we also uncover an sRNA sponging interaction between RsaE and RsaI. Taken together, we present a comprehensive analysis of sRNA-target interactions in MRSA and provide details on how these contribute to the control of virulence in response to changes in metabolism.

Diversity and pathogenesis of Staphylococcus aureus from bovine mastitis: current understanding and future perspectives.

Staphylococcus aureus is a leading cause of bovine mastitis worldwide. Despite some improved understanding of disease pathogenesis, progress towards new methods for the control of intramammary infections (IMI) has been limited, particularly in the field of vaccination. Although herd management programs have helped to reduce the number of clinical cases, S. aureus mastitis remains a major disease burden. This review summarizes the past 16 years of research on bovine S. aureus population genetics, and molecular pathogenesis that have been conducted worldwide. We describe the diversity of S. aureus associated with bovine mastitis and the geographical distribution of S. aureus clones in different continents. We also describe studies investigating the evolution of bovine S. aureus and the importance of host-adaptation in its emergence as a mastitis pathogen. The available information on the prevalence of virulence determinants and their functional relevance during the pathogenesis of bovine mastitis are also discussed. Although traits such as biofilm formation and innate immune evasion are critical for the persistence of bacteria, the current understanding of the key host-pathogen interactions that determine the outcome of S. aureus IMI is very limited. We suggest that greater investment in research into the genetic and molecular basis of bovine S. aureus pathogenesis is essential for the identification of novel therapeutic and vaccine targets.

Evolutionary and Functional Analysis of Coagulase Positivity among the Staphylococci.

The bacterial genus Staphylococcus comprises a large group of pathogenic and nonpathogenic species associated with an array of host species. Staphylococci are differentiated into coagulase-positive or coagulase-negative groups based on the capacity to promote clotting of plasma, a phenotype historically associated with the ability to cause disease. However, the genetic basis of this important diagnostic and pathogenic trait across the genus has not been examined to date. Here, we selected 54 representative staphylococcal species and subspecies to examine coagulation of plasma derived from six representative host species. In total, 13 staphylococcal species mediated coagulation of plasma from at least one host species including one previously identified as coagulase negative (Staphylococcus condimenti). Comparative genomic analysis revealed that coagulase activity correlated with the presence of a gene (vwb) encoding the von Willebrand binding protein (vWbp) whereas only the Staphylococcus aureus complex contained a gene encoding staphylocoagulase (Coa), the classical mediator of coagulation. Importantly, S. aureus retained vwb-dependent coagulase activity in an S. aureus strain deleted for coa whereas deletion of vwb in Staphylococcus pseudintermedius resulted in loss of coagulase activity. Whole-genome-based phylogenetic reconstruction of the Staphylococcus genus revealed that the vwb gene has been acquired on at least four different occasions during the evolution of the Staphylococcus genus followed by allelic diversification via mutation and recombination. Allelic variants of vWbp from selected coagulase-positive staphylococci mediated coagulation in a host-dependent manner indicative of host-adaptive evolution. Taken together, we have determined the genetic and evolutionary basis of staphylococcal coagulation, revealing vWbp to be its archetypal determinant. IMPORTANCE The ability of some species of staphylococci to promote coagulation of plasma is a key pathogenic and diagnostic trait. Here, we provide a comprehensive analysis of the coagulase positivity of the staphylococci and its evolutionary genetic basis. We demonstrate that the von Willebrand binding protein rather than staphylocoagulase is the archetypal coagulation factor of the staphylococci and that the vwb gene has been acquired several times independently during the evolution of the staphylococci. Subsequently, vwb has undergone adaptive diversification to facilitate host-specific functionality. Our findings provide important insights into the evolution of pathogenicity among the staphylococci and the genetic basis for a defining diagnostic phenotype.

A case report: insights into reducing plastic waste in a microbiology laboratory.

Single-use plastics have often replaced more sustainable materials in microbiology laboratories. Keeping in mind that one of the objectives of the United Nations Sustainable Development Goals is responsible consumption and production, we wanted to document how many single-use plastic items could be saved by taking reduction and reuse approaches in a microbiology laboratory. After taking 4 weeks to document the baseline levels of single-use plastic waste being generated in our laboratory and identifying ways to reduce our reliance on them, we implemented various reduction and reuse approaches and then documented our plastic use over a 7-week period. Reduction approaches included moving to sustainable materials, such as reusable wooden sticks for patch plating and metal loops for inoculation. Reuse approaches focused on reusing plastic tubes via a chemical decontamination station and autoclaving, facilitating the reduction of single-use plastics and a decrease in the amount of waste generated. By utilizing reduction and reuse strategies, which could be implemented in other microbiology laboratories, substantial single-use plastic savings were achieved. These savings had an impact on the amount of biohazard waste being autoclaved and incinerated, as well as generating substantial cost savings for the research institute. The reductions in waste documented in this study could act as a benchmark for others wanting to implement the changes described.

The Role of Gram-Positive Surface Proteins in Bacterial Niche- and Host-Specialization.

Gram-positive bacterial pathogens have an array of proteins on their cell surface that mediate interactions with the host environment. In particular, bacterial cell wall-associated (CWA) proteins play key roles in both colonization and pathogenesis. Furthermore, some CWA proteins promote specialization for host-species or mediate colonization of specific anatomical niches within a host. In this mini review, we provide examples of the many ways by which major pathogens, such as Staphylococci, Streptococci and Listeria monocytogenes, utilize CWA proteins for both host- and niche-specialization. We describe different biological mechanisms mediated by CWA proteins including: the acquisition of iron from hemoglobin in the bloodstream, adherence to and invasion of host cells, and innate immune evasion through binding to the plasma proteins fibrinogen, immunoglobulin G, and complement. We also discuss the limitations of using animal models for understanding the role of specific CWA proteins in host-specialization and how transformative technologies, such as CRISPR-Cas, offer tremendous potential for developing transgenic models that simulate the host environment of interest. Improved understanding of the role of CWA proteins in niche- or host-specificity will allow the design of new therapeutic approaches which target key host-pathogen interactions underpinning Gram-positive bacterial infections.

Population Analysis of Staphylococcus aureus Reveals a Cryptic, Highly Prevalent Superantigen SElW That Contributes to the Pathogenesis of Bacteremia.

Staphylococcal superantigens (SAgs) are a family of secreted toxins that stimulate T cell activation and are associated with an array of diseases in humans and livestock. Most SAgs produced by Staphylococcus aureus are encoded by mobile genetic elements, such as pathogenicity islands, bacteriophages, and plasmids, in a strain-dependent manner. Here, we carried out a population genomic analysis of >800 staphylococcal isolates representing the breadth of S. aureus diversity to investigate the distribution of all 26 identified SAg genes. Up to 14 SAg genes were identified per isolate with the most common gene selw (encoding a putative SAg, SElW) identified in 97% of isolates. Most isolates (62.5%) have a full-length open reading frame of selw with an alternative TTG start codon that may have precluded functional characterization of SElW to date. Here, we demonstrate that S. aureus uses the TTG start codon to translate a potent SAg SElW that induces Vß-specific T cell proliferation, a defining feature of classical SAgs. SElW is the only SAg predicted to be expressed by isolates of the CC398 lineage, an important human and livestock epidemic clone. Deletion of selw in a representative CC398 clinical isolate, S. aureus NM001, resulted in complete loss of T cell mitogenicity in vitro, and in vivo expression of SElW by S. aureus increased the bacterial load in the liver during bloodstream infection of SAg-sensitive HLA-DR4 transgenic mice. Overall, we report the characterization of a novel, highly prevalent, and potent SAg that contributes to the pathogenesis of S. aureus infection.IMPORTANCEStaphylococcus aureus is an important human and animal pathogen associated with an array of diseases, including life-threatening necrotizing pneumonia and infective endocarditis. The success of S. aureus as a pathogen has been linked in part to its ability to manipulate the host immune response through the secretion of toxins and immune evasion molecules. The staphylococcal superantigens (SAgs) have been studied for decades, but their role in S. aureus pathogenesis is not well understood, and an appreciation for how SAgs manipulate the host immune response to promote infection may be crucial for the development of novel intervention strategies. Here, we characterized a widely prevalent, previously cryptic, staphylococcal SAg, SElW, that contributes to the severity of S. aureus infections caused by an important epidemic clone of S. aureus CC398. Our findings add to the understanding of staphylococcal SAg diversity and function and provide new insights into the capacity of S. aureus to cause disease.

Staphylococcus aureus Lipase 1 Enhances Influenza A Virus Replication.

Influenza A virus (IAV) causes annual epidemics of respiratory disease in humans, often complicated by secondary coinfection with bacterial pathogens such as Staphylococcus aureus Here, we report that the S. aureus secreted protein lipase 1 enhances IAV replication in vitro in primary cells, including human lung fibroblasts. The proviral activity of lipase 1 is dependent on its enzymatic function, acts late in the viral life cycle, and results in increased infectivity through positive modulation of virus budding. Furthermore, the proviral effect of lipase 1 on IAV is exhibited during in vivo infection of embryonated hen's eggs and, importantly, increases the yield of a vaccine strain of IAV by approximately 5-fold. Thus, we have identified the first S. aureus protein to enhance IAV replication, suggesting a potential role in coinfection. Importantly, this activity may be harnessed to address global shortages of influenza vaccines.IMPORTANCE Influenza A virus (IAV) causes annual epidemics and sporadic pandemics of respiratory disease. Secondary bacterial coinfection by organisms such as Staphylococcus aureus is the most common complication of primary IAV infection and is associated with high levels of morbidity and mortality. Here, we report the first identified S. aureus factor (lipase 1) that enhances IAV replication during infection via positive modulation of virus budding. The effect is observed in vivo in embryonated hen's eggs and greatly enhances the yield of a vaccine strain, a finding that could be applied to address global shortages of influenza vaccines.

Host-specialized fibrinogen-binding by a bacterial surface protein promotes biofilm formation and innate immune evasion.

Fibrinogen is an essential part of the blood coagulation cascade and a major component of the extracellular matrix in mammals. The interface between fibrinogen and bacterial pathogens is an important determinant of the outcome of infection. Here, we demonstrate that a canine host-restricted skin pathogen, Staphylococcus pseudintermedius, produces a cell wall-associated protein (SpsL) that has evolved the capacity for high strength binding to canine fibrinogen, with reduced binding to fibrinogen of other mammalian species including humans. Binding occurs via the surface-expressed N2N3 subdomains, of the SpsL A-domain, to multiple sites in the fibrinogen α-chain C-domain by a mechanism analogous to the classical dock, lock, and latch binding model. Host-specific binding is dependent on a tandem repeat region of the fibrinogen α-chain, a region highly divergent between mammals. Of note, we discovered that the tandem repeat region is also polymorphic in different canine breeds suggesting a potential influence on canine host susceptibility to S. pseudintermedius infection. Importantly, the strong host-specific fibrinogen-binding interaction of SpsL to canine fibrinogen is essential for bacterial aggregation and biofilm formation, and promotes resistance to neutrophil phagocytosis, suggesting a key role for the interaction during pathogenesis. Taken together, we have dissected a bacterial surface protein-ligand interaction resulting from the co-evolution of host and pathogen that promotes host-specific innate immune evasion and may contribute to its host-restricted ecology.