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Previously, our group demonstrated a role for the small RNA (sRNA) Teg41 in regulating production of the alpha phenol-soluble modulin toxins (αPSMs) in Staphylococcus aureus. Overexpressing Teg41 increased αPSM production while deleting the 3' end of Teg41 (Teg41Δ3' strain) resulted in a decrease in αPSM production, reduced hemolytic activity of S. aureus culture supernatants, and attenuated virulence in a murine abscess model of infection. In this study, we further explore the attenuation of virulence in the Teg41Δ3' strain. Using both localized and systemic models of infection, we demonstrate that the Teg41Δ3' strain is more severely attenuated than an ΔαPSM mutant, strongly suggesting that Teg41 influences more than the αPSMs. Proteomic and transcriptomic analysis of the wild-type and Teg41Δ3' strains reveals widespread alterations in transcript abundance and protein production in the absence of Teg41, confirming that Teg41 has pleiotropic effects in the cell. We go on to investigate the molecular mechanism underlying Teg41-mediated gene regulation. Surprisingly, results demonstrate that certain Teg41 target genes, including the αPSMs and βPSMs, are transcriptionally altered in the Teg41Δ3' strain, while other targets, specifically spa (encoding surface protein A), are regulated at the level of transcript stability. Collectively, these data demonstrate that Teg41 is a pleiotropic RNA regulator in S. aureus that influences expression of a variety of genes using multiple different mechanisms.

Staphylococcus aureus is a pathogenic bacterium but also a commensal of skin and anterior nares in humans. As S. aureus transits from skins/nares to inside the human body, it experiences changes in temperature. The production and content of S. aureus extracellular vesicles (EVs) have been increasingly studied over the past few years, and EVs are increasingly being recognized as important to the infectious process. Nonetheless, the impact of temperature variation on S. aureus EVs has not been studied in detail, as most reports that investigate EV cargoes and host cell interactions are performed using vesicles produced at 37°C. Here, we report that EVs in S. aureus differ in size and protein/RNA cargo depending on the growth temperature used. We demonstrate that the temperature-dependent regulation of vesicle production in S. aureus is mediated by the alpha phenol-soluble modulin peptides (αPSMs). Through proteomic analysis, we observed increased packaging of virulence factors at 40°C, whereas the EV proteome has greater diversity at 34°C. Similar to the protein content, we perform transcriptomic analysis and demonstrate that the RNA cargo also is impacted by temperature. Finally, we demonstrate greater αPSM- and alpha-toxin-mediated erythrocyte lysis with 40°C EVs, but 34°C EVs are more cytotoxic toward THP-1 cells. Together, our study demonstrates that small temperature variations have great impact on EV biogenesis and shape the interaction with host cells. IMPORTANCE Extracellular vesicles (EVs) are lipid bilayer spheres that contain proteins, nucleic acids, and lipids secreted by bacteria. They are involved in Staphylococcus aureus infections, as they package virulence factors and deliver their contents inside host cells. The impact of temperature variations experienced by S. aureus during the infectious process on EVs is unknown. Here, we demonstrate the importance of temperature in vesicle production and packaging. High temperatures promote packaging of virulence factors and increase the protein and lipid concentration but reduce the overall RNA abundance and protein diversity in EVs. The importance of temperature changes is highlighted by the fact that EVs produced at low temperature are more toxic toward macrophages, whereas EVs produced at high temperature display more hemolysis toward erythrocytes. Our research brings new insights into temperature-dependent vesiculation and interaction with the host during S. aureus transition from colonization to virulence.

Staphylococcus aureus possesses ten extracellular proteases with mostly unknown targets in the human proteome. To assist with bacterial protease target discovery, we have applied and compared two N-terminomics methods to investigate cleavage of human serum proteins by S. aureus V8 protease, discovering 85 host-protein targets. Among these are virulence-relevant complement, iron sequestration, clotting cascade, and host protease inhibitor proteins. Protein cleavage sites have been identified, providing insight into the disruption of host protein function by V8. Complement proteins are cleaved within peptidase and sushi domains, and host protease inhibitors are cleaved outside their protease-trapping motifs. Our data highlight the potential for further application of N-terminomics in discovery of bacterial protease substrates in other host niches and provide omics-scale insight into the role of the V8 protease in S. aureus pathogenesis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Peptidyl-prolyl cis/trans isomerases (PPIases) are enzymes that assist in protein folding around proline-peptide bonds, and they often possess chaperone activity. Staphylococcus aureus encodes three PPIases, i.e., PrsA, PpiB, and trigger factor (TF). Previous work by our group demonstrated a role for both PrsA and PpiB in S. aureus ; however, TF remains largely unstudied. Here, we identify a role for TF in S. aureus biofilm formation and demonstrate cooperation between TF and the cytoplasmic PPIase PpiB. Mutation of the tig gene (encoding TF) led to reduced biofilm development in vitro but no significant attenuation of virulence in a mouse model of infection. To investigate whether TF possesses chaperone activity, we analyzed the ability of a tig mutant to survive acid and base stress. While there was no significant decrease for a tig mutant, a ppiB tig double mutant exhibited significant decreases in cell viability after acid and base challenges. We then demonstrated that a ppiB tig double mutant had exacerbated phenotypes in vitro and in vivo , compared to either single mutant. Finally, in vivo immunoprecipitation of epitope-tagged PpiB revealed that PpiB interacted with 4 times the number of proteins when TF was absent from the cell, suggesting that it may be compensating for the loss of TF. Interestingly, the only proteins found to interact with TF were TF itself, fibronectin-binding protein B (FnBPB), and the chaperone protein ClpB. Collectively, these results support the first phenotype for S. aureus TF and demonstrate a greater network of cooperation between chaperone proteins in Staphylococcus aureus IMPORTANCE S. aureus encodes a large number of virulence factors that aid the bacterium in survival and pathogenesis. These virulence factors have a wide variety of functions; however, they must all be properly secreted in order to be functional. Bacterial chaperone proteins often assist in secretion by trafficking proteins to secretion machinery or assisting in proper protein folding. Here, we report that the S. aureus chaperone TF contributes to biofilm formation and cooperates with the chaperone PpiB to regulate S. aureus virulence processes. These data highlight the first known role for TF in S. aureus and suggest that S. aureus chaperone proteins may be involved in a greater regulatory network in the cell., (Copyright © 2021 American Society for Microbiology.)

Staphylococcus aureus controls the progression of infection through the coordinated production of extracellular proteases, which selectively modulate virulence determinant stability. This is evidenced by our previous finding that a protease-null strain has a hypervirulent phenotype in a murine model of sepsis, resulting from the unchecked accumulation of virulence factors. Here, we dissect the individual roles of these proteases by constructing and assessing the pathogenic potential of a combinatorial protease mutant library. When strains were constructed bearing increasing numbers of secreted proteases, we observed a variable impact on infectious capacity, where some exhibited hypervirulence, while others phenocopied the wild-type. The common thread for hypervirulent strains was that each lacked both aureolysin and staphopain A. Upon assessment, we found that the combined loss of these two enzymes alone was necessary and sufficient to engender hypervirulence. Using proteomics, we identified a number of important secreted factors, including SPIN, LukA, Sbi, SEK, and PSMα4, as well as an uncharacterized chitinase-related protein (SAUSA300_0964), to be overrepresented in both the aur scpA and the protease-null mutants. When assessing the virulence of aur scpA SAUSA300_0964 and aur scpA lukA mutants, we found that hypervirulence was completely eliminated, whereas aur scpA spn and aur scpA sek strains elicited aggressive infections akin to the protease double mutant. Collectively, our findings shed light on the influence of extracellular proteases in controlling the infectious process and identifies SAUSA300_0964 as an important new component of the S. aureus virulence factor arsenal. IMPORTANCE A key feature of the pathogenic success of S. aureus is the myriad virulence factors encoded within its genome. These are subject to multifactorial control, ensuring their timely production only within an intended infectious niche. A key node in this network of control is the secreted proteases of S. aureus , who specifically and selectively modulate virulence factor stability. In our previous work we demonstrated that deletion of all 10 secreted proteases results in hypervirulence, since virulence factors exist unchecked, leading to overly aggressive infections. Here, using a combinatorial collection of protease mutants, we reveal that deletion of aureolysin and staphopain A is necessary and sufficient to elicit hypervirulence. Using proteomic techniques, we identify the collection of virulence factors that accumulate in hypervirulent protease mutants, and demonstrate that a well-known toxin (LukA) and an entirely novel secreted element (SAUSA300_0964) are the leading contributors to deadly infections observed in protease-lacking strains., (Copyright © 2021 Gimza et al.)

Numerous factors have, to date, been identified as playing a role in the regulation of Agr activity in Staphylococcus aureus , including transcription factors, antisense RNAs, and host elements. Herein we investigated the product of SAUSA300_1984 (termed MroQ), a transmembrane Abi-domain/M79 protease-family protein, as a novel effector of this system. Using a USA300 mroQ mutant, we observed a drastic reduction in proteolysis, hemolysis, and pigmentation that was fully complementable. This appears to result from diminished agr activity, as transcriptional analysis revealed significant decreases in expression of both RNAII and RNAIII in the mroQ mutant. Such effects appear to be direct, rather than indirect, as known agr effectors demonstrated limited alterations in their activity upon mroQ disruption. A comparison of RNA sequencing data sets for both mroQ and agr mutants revealed a profound overlap in their regulomes, with the majority of factors affected being known virulence determinants. Importantly, the preponderance of alterations in expression were more striking in the agr mutant, indicating that MroQ is necessary, but not sufficient, for Agr function. Mechanism profiling revealed that putative residues for metalloprotease activity within MroQ are required for its Agr-controlling effect; however, this was not wielded at the level of AgrD processing. Virulence assessment demonstrated that both mroQ and agr mutants exhibited increased formation of renal abscesses but decreased skin abscess formation alongside diminished dermonecrosis. Collectively, we present the characterization of a novel agr effector in S. aureus which would appear to be a direct regulator, potentially functioning via interaction with the AgrC histidine kinase., (Copyright © 2019 American Society for Microbiology.)

Pigs have been shown to be a reservoir for recently emerging livestock-associated Staphylococcus aureus (LA-SA), including methicillin resistant strains in many countries worldwide. However, there is sparse information about LA-SA strains circulating in Zambia. This study investigated the prevalence, phenotypic and genotypic characteristics of S. aureus from pigs and workers at farms and abattoirs handling pigs in Lusaka Province of Zambia. A total of 492 nasal pig swabs, 53 hand and 53 nasal human swabs were collected from farms and abattoirs in selected districts. Standard microbiological methods were used to isolate and determine antimicrobial susceptibility patterns of S. aureus. Polymerase Chain Reaction was used to confirm the species identity and detect antimicrobial resistance and virulence genes of isolates, whereas genetic diversity was evaluated using spa typing. Overall prevalence of S. aureus was 33.1%, 37.8% for pigs and 11.8% for humans. The isolates were resistant to several antibiotics with resistance ranging from 18% to 98% but were all susceptible to vancomycin. Typical LA-SA spa types were detected. The presence of plasmid mediated resistance genes such as tetM (12.8%), other resistance determinants and immune evasion cluster genes among the isolates is of great public health concern. Thus, continuous surveillance of S. aureus using a "One health" approach is warranted to monitor S.aureus infections and spread of antimicrobial resistance. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a Gram‐positive commensal that can also cause a variety of infections in humans. S. aureus virulence factor gene expression is under tight control by a complex regulatory network, which includes, sigma factors, sRNAs, and two‐component systems (TCS). Previous work in our laboratory demonstrated that overexpression of the sRNA tsr37 leads to an increase in bacterial aggregation. Here, we demonstrate that the clumping phenotype is dependent on a previously unannotated 88 amino acid protein encoded within the tsr37 sRNA transcript (which we named ScrA for S. aureusclumping regulator A). To investigate the mechanism of action of ScrA we performed proteomics and transcriptomics in a ScrA overexpressing strain and show that a number of surface adhesins are upregulated, while secreted proteases are downregulated. Results also showed upregulation of the SaeRS TCS, suggesting that ScrA is influencing SaeRS activity. Overexpression of ScrA in a saeR mutant abrogates the clumping phenotype confirming that ScrA functions via the Sae system. Finally, we identified the ArlRS TCS as a positive regulator of scrA expression. Collectively, our results show that ScrA is an activator of the SaeRS system and suggests that ScrA may act as an intermediary between the ArlRS and SaeRS systems. [ABSTRACT FROM AUTHOR]

• Staphylococcus aureus including methicillin-resistant S. aureus (MRSA) are prevalent in African pig and pork production systems. • The presence of antimicrobial, heavy metal resistance and virulence factor genes was noted. • The typical ST398 MRSA strains among pigs have been reported in Africa. • Other detected genotypes suggest an anthropogenic origin rather than zoonosis. To assess the emergence of livestock-associated Staphylococcus aureus including methicillin-resistant S. aureus (MRSA) in the pig and pork production systems in Africa for the past two decades. PubMed and African Journals OnLine were searched for relevant primary studies from 2000 to 2019 using standardized key words. In total, 19 eligible articles were included in this review. The prevalence of S. aureus including MRSA ranged from 0% to 55% among live pigs and raw pork, and from 9.4% to 30.8% among pig farm and abattoir workers. Risk factors associated with S. aureus carriage among workers were: male gender, working in an abattoir, and medical-related occupation of a household member. S. aureus and MRSA from pigs and pork production systems in Africa are potentially pathogenic with diverse spa types and clonal complexes, with genes encoding antimicrobial resistance, heavy metal resistance, and virulence factors including secreted and enterotoxins, proteases and immune evasion cluster. The typical livestock-associated S. aureus CC398 and mecC genes were reported in two studies. Pigs are a potential source of the emerging livestock-associated S. aureus in Africa. Continued monitoring using a 'One Health' approach is recommended for effective infection prevention and control of these infections in Africa. [ABSTRACT FROM AUTHOR]