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Staphylococcus aureus is responsible for a substantial number of invasive infections globally each year. These infections are problematic because they are frequently recalcitrant to antibiotic treatment. Antibiotic tolerance, the ability of bacteria to persist despite normally lethal doses of antibiotics, contributes to antibiotic treatment failure in S. aureus infections. To understand how antibiotic tolerance is induced, S. aureus biofilms exposed to multiple anti-staphylococcal antibiotics are examined using both quantitative proteomics and transposon sequencing. These screens indicate that arginine metabolism is involved in antibiotic tolerance within a biofilm and support the hypothesis that depletion of arginine within S. aureus communities can induce antibiotic tolerance. Consistent with this hypothesis, inactivation of argH, the final gene in the arginine synthesis pathway, induces antibiotic tolerance. Arginine restriction induces antibiotic tolerance via inhibition of protein synthesis. In murine skin and bone infection models, an argH mutant has enhanced ability to survive antibiotic treatment with vancomycin, highlighting the relationship between arginine metabolism and antibiotic tolerance during S. aureus infection. Uncovering this link between arginine metabolism and antibiotic tolerance has the potential to open new therapeutic avenues targeting previously recalcitrant S. aureus infections., (© 2024. The Author(s).)

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.)

Staphylococcus aureus is a highly successful Gram-positive pathogen capable of causing both superficial and invasive, life-threatening diseases. Of the invasive disease manifestations, osteomyelitis or infection of bone, is one of the most prevalent, with S. aureus serving as the most common etiologic agent. Treatment of osteomyelitis is arduous, and is made more difficult by the widespread emergence of antimicrobial resistant strains, the capacity of staphylococci to exhibit tolerance to antibiotics despite originating from a genetically susceptible background, and the significant bone remodeling and destruction that accompanies infection. As a result, there is a need for a better understanding of the factors that lead to antibiotic failure in invasive staphylococcal infections such as osteomyelitis. In this review article, we discuss the different non-resistance mechanisms of antibiotic failure in S. aureus . We focus on how bacterial niche and destructive tissue remodeling impact antibiotic efficacy, the significance of biofilm formation in promoting antibiotic tolerance and persister cell formation, metabolically quiescent small colony variants (SCVs), and potential antibiotic-protected reservoirs within the substructure of bone., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gimza and Cassat.)

Staphylococcus aureus is an important human pathogen that causes a plethora of diverse infections within the human host that range in severity from the relatively minor to the severe. Of note, bloodstream infections caused by this organism result in high mortality rates, often following failed rounds of surgical and antibiotic intervention. The capacity for S. aureus to exist in blood is driven by myriad virulence factors that engage in a manipulation of various host responses to evade destruction and ensure survival. These include both secreted elements, such as coagulase and von Willebrand factor protein, as well as surface displayed factors, including clumping factor A and fibronectin binding protein A. In addition to this, there are a number of other loci within the S. aureus genome whose products have been shown to contribute to blood survival by more indirect means. Accordingly, ex vivo whole human blood survival assays are often used as a preliminary study to investigate host-bacterial interactions in an effort to delineate the pathogenicity of S. aureus strains. Herein we provide a detailed assessment of the protocol required to perform such studies., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Using DNA affinity chromatography we demonstrate that the S. aureus regulatory proteins MgrA, Rot, SarA, and SarS bind DNA baits derived from the promoter regions associated with the genes encoding aureolysin, ScpAB, SspABC, and SplA-F. Three of four baits also bound SarR and SarZ, the exception in both cases being the ScpAB-associated bait. Using the USA300, methicillin-resistant strain LAC and the USA200, methicillin-sensitive strain UAMS-1, we generated mutations in the genes encoding each of these proteins alone and in combination with sarA and examined the impact on protease production, the accumulation of high molecular weight proteins, and biofilm formation. These studies confirmed that multiple regulatory loci are involved in limiting protease production to a degree that impacts all of these phenotypes, but also demonstrate that sarA plays a predominant role in this regard. Using sarA mutants unable to produce individual proteases alone and in combination with each other, we also demonstrate that the increased production of aureolysin and ScpA is particularly important in defining the biofilm-deficient phenotype of LAC and UAMS-1 sarA mutants, while aureolysin alone plays a key role in defining the reduced accumulation of alpha toxin and overall cytotoxicity as assessed using both osteoblasts and osteoclasts.

A primary function of the extracellular proteases of Staphylococcus aureus is to control the progression of infection by selectively modulating the stability of virulence factors. Consequently, a regulatory network exists to titrate protease abundance/activity to influence the accumulation, or lack thereof, of individual virulence factors. Herein, we comprehensively map this system, exploring the regulation of the four protease loci by known and novel factors. In so doing, we determined that seven major elements (SarS, SarR, Rot, MgrA, CodY, SaeR, and SarA) form the primary network of control, with the latter three being the most powerful. We note that expression of aureolysin is largely repressed by these factors, while the spl operon is subject to the strongest upregulation of any protease loci, particularly by SarR and SaeR. Furthermore, when exploring scpA expression, we find it to be profoundly influenced in opposing fashions by SarA (repressor) and SarR (activator). We also present the screening of >100 regulator mutants of S. aureus , identifying 7 additional factors (ArgR2, AtlR, MntR, Rex, XdrA, Rbf, and SarU) that form a secondary circuit of protease control. Primarily, these elements serve as activators, although we reveal XdrA as a new repressor of protease expression. With the exception or ArgR2, each of the new effectors appears to work through the primary network of regulation to influence protease production. Collectively, we present a comprehensive regulatory circuit that emphasizes the complexity of protease regulation and suggest that its existence speaks to the importance of these enzymes to S. aureus physiology and pathogenic potential. IMPORTANCE The complex regulatory role of the proteases necessitates very tight coordination and control of their expression. While this process has been well studied, a major oversight has been the consideration of proteases as a single entity rather than as 10 enzymes produced from four different promoters. As such, in this study, we comprehensively characterized the regulation of each protease promoter, discovering vast differences in the way each protease operon is controlled. Additionally, we broaden the picture of protease regulation using a global screen to identify novel loci controlling protease activity, uncovering a cadre of new effectors of protease expression. The impact of these elements on the activity of proteases and known regulators was characterized by producing a comprehensive regulatory circuit that emphasizes the complexity of protease regulation in Staphylococcus aureus ., (Copyright © 2019 Gimza et al.)