Biology subjects

Shaw, L. N.

Publications and source records attributed to Shaw, L. N..

16 recordsLinked to original sources

bioRxiv2026

SSR42 Enhances the Hemolytic Capacity of Staphylococcus aureus by Stabilizing the hla mRNA

Staphylococcus aureus utilizes a complex regulatory network to precisely control a vast array of virulence factors and facilitate infection. One such regulator, the long regulatory RNA SSR42, is emerging as a key modulator of virulence factor abundance and pathogenesis. Previous work has shown that a primary role for SSR42 is controlling hemolytic behavior through the positive regulation of -toxin (hla/Hla). Herein, we confirm that loss of SSR42 limits hemolytic capacity due to reduced Hla production. Others have suggested that this occurs through the SaeRS two-component system; however, using epistasis experiments, we reveal that SSR42 and SaeRS function independently to control Hla activity. Using a two-plasmid system optimized to detect regulatory RNA-mediated control in Gram-positive bacteria, we demonstrate that SSR42 directly enhances Hla production through the hla 5 untranslated region. Using interaction prediction and targeted mutagenesis, we identify a discrete site upstream of the hla ribosome-binding site that is required for SSR42 binding and hemolytic activity. Transcriptional arrest experiments further show that SSR42 stabilizes the hla mRNA, increasing its half-life and promoting downstream toxin production. Finally, electrophoretic mobility shift assays confirm a specific interaction between SSR42 and the hla 5 UTR. Collectively, these findings establish SSR42 as a major post-transcriptional regulator of S. aureus virulence and reveal a novel RNA-mediated mechanism that promotes -toxin production through stabilization of the hla transcript.

microbiology↗

bioRxiv2026

SroA links SigS-dependent stress signaling to metabolic remodeling in Staphylococcus aureus

Staphylococcus aureus encounters diverse environmental conditions during colonization and infection, including fluctuations in nutrient availability, oxidative stress, and oxygen limitation. Adaptation to these environments requires regulatory systems that coordinate stress responses with metabolic remodeling. The extracytoplasmic function sigma factor SigS contributes to stress adaptation and virulence in S. aureus and directly activates expression of the sroAB operon, which encodes the small proteins SroA and SroB. While previous work demonstrated that SroA participates in feedback regulation of sigS expression, the broader physiological role of SroA has remained unclear. To define the regulatory functions of SroA, we performed RNA sequencing following inducible overexpression of sroA in S. aureus. Transcriptome analysis revealed extensive remodeling of gene expression, with approximately 200 transcripts significantly altered. Transcriptome analysis revealed coordinated repression of metabolic pathways (including nitrate respiration and nucleotide biosynthesis) alongside activation of stress-response and nutrient acquisition genes. Northern blot and quantitative RT-PCR analysis confirmed repression of narG and narJ transcripts following SroA overexpression. Consistent with these transcriptional changes, nitrate reduction assays demonstrated that SroA overexpression reduces nitrate respiration activity. In addition to repression of nitrate respiration genes, SroA overexpression broadly suppressed genes involved in de novo purine and pyrimidine biosynthesis. In contrast, transcripts associated with stress responses and nutrient acquisition, including the SOS-associated gene sosA and the phosphate transport gene pstS, were upregulated. Together, these findings identify SroA as a regulator that links stress-responsive signaling to metabolic remodeling in S. aureus, particularly through modulation of nitrate respiration pathways. ImportanceStaphylococcus aureus must rapidly adapt its metabolism to survive the diverse environments encountered during colonization and infection, including conditions where oxygen availability is limited. In this study, we identify a previously uncharacterized role for the small protein SroA in regulating metabolic adaptation in S. aureus. Transcriptome analysis revealed that SroA strongly represses genes involved in nitrate respiration, a pathway that enables bacteria to maintain energy production when oxygen is scarce. Consistent with these transcriptional changes, SroA overexpression reduced nitrate respiration activity. These findings reveal a regulatory link between stress-responsive signaling pathways and respiratory metabolism, expanding our understanding of how S. aureus adapts to oxygen-limited environments encountered during infection.

microbiology↗

bioRxiv2025

A single transcriptional regulator is crucial for the adaptation of Staphylococcus aureus to diverse niches

The adaptation of versatile multi-host pathogens to various hosts and various niches within hosts is often still poorly understood. The alternative Sigma factor B (SigB) is the master regulator of the general stress response of most gram-positive bacteria, which is a classic case of adaptive plasticity. In Staphylococcus aureus, SigB appears co-opted to function as a switch between intracellular and extracellular niches. During bovine mastitis, low SigB-activity confers an advantage in the milk-rich extracellular niche of the bovine udder. We show that narrowly adapted SigB-deficient strains evolved repeatedly from phenotypically plastic SigB-wildtype strains during persistent mastitis. This genetic assimilation appears driven by the cost of phenotypic plasticity: long time lags in adapting to milk and slow growth. Surprisingly, we observe that mutations causing SigB-deficiency allow even human isolates to grow in milk. While host adaptation often proceeds by mobile genetic elements exchanged between strains, we show how a master regulator in the core genome can drive niche adaptation.

microbiology↗

bioRxiv2025

Genomic and Phenotypic Characterization of Mupirocin Resistant Staphylococcus aureus Clinical Isolates

BackgroundColonization with Staphylococcus aureus is a risk factor for subsequent infection. Decolonization with the topical antibiotic mupirocin is effective and reduces the risk of subsequent S. aureus infection for both methicillin-sensitive (MSSA) and methicillin-resistant (MRSA) strains but may select for mupirocin-resistant isolates. MethodsWe characterized oxacillin and mupirocin susceptibility amongst 384 S. aureus strains isolated from clinical samples isolated 2017-2023 in Tampa, Florida, spanning strains collected before and after the onset of the COVID-19 pandemic. Whole genome sequencing of bacterial isolates was conducted in parallel and correlated with drug susceptibility profiles. ResultsMupirocin resistance (MupR) was nearly exclusively present in MRSA strains (103/106 97.1% of MupR; 103/299 34.4% of MRSA). Although our hospital protocol for decolonization shifted to povidone iodine in the Post-COVID period, the overall prevalence of MupR did not change in Pre-COVID and Post-COVID samples (28.9% vs 26%). Genotype correlated with antibiotic susceptibility with low level MupR (MupLR), linked to mutations in ileS and high level MupR (MupHR), linked to the presence of mupA. Genome analysis revealed that most MupR strains fell into three sequence types (ST) falling into two major clonal complexes (CC): CC8 ST8 (including Community-Associated MRSA strains USA300 and USA500), CC5 ST5 (associated with Healthcare-Associated MRSA such as USA100), and CC5 ST3390. ST3390 isolates had the highest prevalence of MupR (30/36 83%; MupHR 20/36 55.6%; MupLR 10/36 27.8%). ConclusionsMupirocin resistance was prevalent in our hospital MRSA strains. We also found evidence for emergence and persistence of ST3390 MRSA-MupR strains in Florida. Key pointsO_LIIn a survey of clinical isolates in Florida, 34.4% of MRSA strains were mupirocin resistant. C_LIO_LIMupirocin resistance correlated with mutations in ileS or carriage of mupA. C_LIO_LIWe found evidence for emergence of MRSA mupirocin-resistant strains that were sequence type ST3390. C_LI

microbiology↗

bioRxiv2025

Emergence of ST3390: A non-pigmented HA-MRSA clone with enhanced virulence

BackgroundOne of the most successful and widely-distributed hospital-associated lineages of MRSA is CC5. These strains are known from widespread antibiotic resistance, but less severe disease than CA-MRSA counterparts. Recently, CC5 descendant lineages have appeared globally with hypervirulent properties. MethodsHerein we use genomic analyses to study the epidemiology of a rare CC5 MRSA sequence type, ST3390, circulating within Tampa General Hospital (TGH). We employ genetic tools alongside in vitro and in vivo models of virulence to study the pathogenic capabilities of strains. ResultsTo date, there have only been 50 recorded instances of infection caused by ST3390 globally, with 36 of those occurring at TGH. Genomic analysis of strains identified numerous spa-types, with a t010 cluster found only at TGH. Exploration of AMR genes detected the presence of unique hybrid SCCmec types, with [~]90% of TGH strains possessing components of SCCmecIa, SCCmecIIa and/or SCCmecVIII. Phenotypically, all ST3390 strains lack the staphyloxanthin pigment, which is mediated by a conserved 6 aa in frame deletion within the staphyloxanthin biosynthesis protein CrtN. ST3390 strains display high levels of cytotoxicity towards human neutrophils compared to other CC5 lineages, with several isolates displaying hypervirulence in animal models of infection. ConclusionsThis is the first study to characterize the pathogenicity and genomic architecture of the rare MRSA lineage ST3390. Our work provides a deeper understanding of the clonal expansion of CC5, and the wider diversification of S. aureus isolates within patient populations.

microbiology↗

bioRxiv2024

Development of a New N-Terminomic Method to Study the Pathodegradome of the Staphylococcus aureus V8 Protease in Human Neutrophils

Staphylococcus aureus is a notorious human pathogen that relies on an array of virulence factors to engender infection and evade the host-immune system. Among these are the secreted proteases, which promote pathogenesis by degrading host proteins and modulating host-defenses. Human neutrophils play a pivotal role in these defenses, acting as the first responders against invading bacteria. While many S. aureus effectors of virulence have been shown to target leukocytes, there is limited knowledge on how the extracellular proteases modulate neutrophil fate. Typically, protease substrates have been identified in isolated settings using one at a time approaches; with neutrophil targets few and far between. Herein, we have developed a novel N-terminomic methodology termed TAGS-CR that can facilitate global substrate characterization in streamlined manner. We thus present the application of TAGS-CR to unravelling the human neutrophil pathodegradome of the S. aureus V8 protease. In so doing, we captured [~]350 V8 targets, revealing critical insight into how this virulence factor can modulate neutrophil functionality on various levels relevant to S. aureus disease progression. We recorded cleavage of proteins necessary for neutrophil adhesion and migration, a fundamental process necessary for pathogen clearance. Furthermore, we highlight V8 cleavage of proteins involved in important neutrophil defense tactics, such as degranulation and reactive oxygen species production. This protease may also facilitate bacterial dissemination via the intentional activation of neutrophil apoptosis. Collectively, this work deepens our understanding of host-pathogen interaction and begins to unravel how S. aureus proteases can induce immune dysregulation through the targeting of leukocytes. ImportanceDuring infection Staphylococcus aureus must engage and evade the host immune system in order to successfully cause disease. As neutrophils represent the frontline of defense against invading S. aureus cells, it becomes increasingly important to decode how this bacterium subverts their host-defense tactics. While the contributing role to neutrophil engagement for many S. aureus virulence factors have been elucidated, the effects of their proteases remain largely unclear. Here, we present a novel method for global protease substrate identification, TAGS-CR, and use it to identify S. aureus V8 protease targets in human neutrophils. These include factors that not only govern general neutrophil function but moreover, their defense mechanisms, such as migration, degranulation, oxidative defense, phagocytosis and apoptosis.

microbiology↗

bioRxiv2024

A Simplified Method for Comprehensive Capture of the Staphylococcus aureus Proteome

Staphylococcus aureus is a major human pathogen causing myriad infections in both community and healthcare settings. Although well studied, a comprehensive exploration of its dynamic and adaptive proteome is still somewhat lacking. Herein, we employed streamlined liquid- and gas-phase fractionation with PASEF analysis on a TIMS-TOF instrument to expand coverage and explore the S. aureus dark proteome. In so doing, we captured the most comprehensive S. aureus proteome to date, totaling 2,231 proteins (85.6% coverage), using a significantly simplified process that demonstrated high reproducibility with minimal input material. We then showcase application of this library for differential expression profiling by investigating temporal dynamics of the S. aureus proteome. This revealed alterations in metabolic processes, ATP production, RNA processing, and stress-response proteins as cultures progressed to stationary growth. Notably, a significant portion of the library (94%) and proteome (80.5%) was identified by this single-shot, DIA-based analysis. Overall, our study shines new light on the hidden S. aureus proteome, generating a valuable new resource to facilitate further study of this dangerous pathogen.

microbiology↗

bioRxiv2024

SSR42 is a Novel Regulator of Cytolytic Activity in Staphylococcus aureus

SSR42 is the longest noncoding RNA in the S. aureus cell and the second-most abundant transcript in the stationary phase transcriptome, second only to RNAIII. It is highly conserved across strains and exhibits pronounced stability in stationary phase, however the mechanism behind its regulatory role has yet to be fully elucidated. Herein, we used transcriptomic and proteomic approaches to probe the role of SSR42, revealing that it is a powerful, novel activator of the primary leukocidin LukAB. SSR42 is required for cytotoxicity towards, and escape from within, human neutrophils, and also mediates survival within human blood. We show that SSR42 wields this role via derepression by the peroxide repressor PerR in response to the presence of human neutrophils and governs lukAB induction in this niche. Importantly, this regulation is driven by direct RNA-RNA interaction, as we show binding of the 5 UTR of the lukAB transcript with the 3 end of SSR42, which ultimately modulates transcript stability as well as translational activity. Finally, we demonstrate that this behavior is absolutely required for full virulence of S. aureus in murine models of both pneumonia and sepsis. Collectively, we present SSR42 as a pleiotropic regulatory RNA that acts as a nexus between environmental sensing and the regulation of pathogenesis, responding to environmental stimuli and host immune factors to bolster cytotoxic behavior and facilitate infection in S. aureus. ImportanceS. aureus is a master pathogen due to its formidable collection of virulence factors. These are tightly controlled by a diverse group of regulators that titrate their abundance to adapt to unique infectious niches. The role of regulatory RNAs in stress adaptation and pathogenesis is becoming increasingly more relevant in S. aureus. In this study, we provide the most comprehensive global analysis to date of just such a factor, SSR42. Specifically, we uncover that SSR42 is required for mediating cytotoxicity - one of the pillars of infection - in response to phagocytosis by human neutrophils. We find that SSR42 is induced by components of the host immune system and facilitates downstream activation of cytotoxic factors via RNA-RNA interactions. This illustrates that SSR42 forms a pivotal link between sensing the external environment and mediating resistance to oxidative stress while promoting virulence, solidifying it as a major global regulator in S. aureus.

microbiology↗

bioRxiv2024

Culture-dependent identification of rare marine sediment bacteria from the Gulf of Mexico and Antarctica

Laboratory-viable cultivars of previously uncultured bacteria further taxonomic understanding. Despite many years of modern microbiological investigations, the vast majority of bacterial taxonomy remains uncharacterized. While many attempts have been made to decrease this knowledge gap, culture-based approaches parse away at the unknown and are critical for improvement of both culturing techniques and computational prediction efficacy. To this end of providing culture-based approaches, we present a multi-faceted approach to recovering marine environmental bacteria. We employ combinations of nutritional availability, inoculation techniques, and incubation parameters in our recovery of marine sediment-associated bacteria from the Gulf of Mexico and Antarctica. The recovered biodiversity spans several taxa, with 16S-ITS-23S rRNA gene-based identification of multiple isolates belonging to rarer genera increasingly undergoing phylogenetic rearrangements. Our modifications to traditional culturing techniques have not only recovered rarer taxa, but also resulted in the recovery of biotechnologically promising bacteria. Together, we propose our stepwise combinations of recovery parameters as a viable approach to decreasing the bacterial knowledge gap.

microbiology↗

bioRxiv2022

Staphylococcus aureus SigS induces expression of a regulatory protein pair that modulate its mRNA stability

SigS is the sole extracytoplasmic function sigma in S. aureus and is necessary for virulence, immune evasion, as well as surviving exposure to toxic chemicals and environmental stressors. Despite the contribution of SigS to a myriad of critical phenotypes, the downstream effectors of the SigS-dependent S. aureus pathogenesis, immune evasion, and stress response remain elusive. To address this knowledge gap, we analyzed the S. aureus transcriptome following transient over-expression of SigS. We identified a bi-cistronic transcript, up-regulated by 1000-fold, containing two mid-sized genes each containing single domains of unknown function (DUF). We renamed these genes sroA (SigS regulated orfA) and sroB (SigS regulated orfB). We demonstrated that the SigS regulation of the sroAB operon is direct using in vitro transcription analysis. Using northern blot analysis, we also demonstrated that SroA and SroB have opposing auto- regulatory functions on the transcriptional architecture of the sigS locus; with SroA stimulated SigS mRNA levels and SroB stimulating s750 (SigS antisense) levels. We hypothesized that these this opposing regulatory effects were due to a direct interaction. We demonstrated an interaction between SroA and SroB using an in-vivo surrogate genetics approach via Bacterial Two Hybrid. We demonstrated that the SroA effect on SigS is at the post-transcriptional level of mRNA stability, highlighting a mechanism likely used by S. aureus to tightly control SigS levels. Finally, we demonstrate that the sroAB locus promotes virulence in a female murine pneumonia model of infection.

microbiology↗

bioRxiv2021

Phenogenomic characterization of a newly domesticated and novel species from the genus Verrucosispora

The concept of bacterial dark matter stems from our inability to culture most microbes and represents a fundamental hole in our knowledge of microbial diversity. Herein we present the domestication of such an organism: a previously uncultured, novel species from the rare-Actinomycetes genus Verrucosispora. Although initial recovery took >4 months, isolation of phenotypically distinct, domesticated generations occurred within weeks. Two isolates were subjected to phenogenomic analyses, revealing domestication correlated with enhanced growth rates in nutrient-rich media, but diminished capacity to metabolize diverse amino acids. This is seemingly mediated by genomic decay through the pseudogenization of amino acids metabolism genes. Conversely, later generational strains had enhanced spore germination rates, potentially through the reversion of a sporulation-associated kinase from pseudogene to true gene status. We observed that our most wild-type isolate had the greatest potential for antibacterial activity, which correlated with extensive mutational attrition of biosynthetic gene clusters in domesticated strains. Comparative analyses revealed wholesale genomic reordering in strains, with widespread SNP, indel and pseudogene mutations observed. We hypothesize that domestication of this previously unculturable organism resulted from the shedding of genomic flexibility required for life in a dynamic marine environment, parsing out genetic redundancy to allow for a newfound cultivable amenability.

microbiology↗

bioRxiv2021

Assessing the role of cold-shock protein C: A novel regulator of Acinetobacter baumannii biofilm formation and virulence

Acinetobacter baumannii is a formidable opportunistic pathogen that is notoriously difficult to eradicate from hospital settings and can spread quickly via healthcare personnel despite preventive measures. This resilience is often attributed to a proclivity for biofilm formation, which grants A. baumannii a higher tolerance towards external stress, desiccation, and antimicrobials. Despite this, little is known regarding the mechanisms orchestrating A. baumannii biofilm formation. Herein, we performed RNA-seq on biofilm and planktonic populations for the multidrug resistant isolate, AB5075, and identified 438 genes with altered expression. To assess the potential role of genes upregulated within biofilms, we tested the biofilm forming capacity of their respective mutants from an A. baumannii transposon library. In so doing, we uncovered 24 genes whose disruption led to reduced biofilm formation. One such element, cold shock protein C (cspC), produced a mucoidal, non-mucoviscous colony phenotype. RNA-sequencing of this mutant revealed the down regulation of pili and fimbriae in the cspC mutant, which would explain the decreased biofilm observed. Using MIC assays, we note that the mutant displayed increased antibiotic tolerance that we hypothesize is mediated by overexpression of multi-drug efflux pumps and altered mRNA stability of their corresponding transcriptional repressor. Finally, we show that CspC is required for survival during oxidative stress and challenge by the human immune system, and plays a pivotal role during systemic infection. Collectively, our work identifies a cadre of new biofilm associated genes within A. baumannii and provides insight into the global regulatory network of this emerging human pathogen.

microbiology↗

bioRxiv2020

A Global Transcriptomic Analysis of Staphylococcus aureus Biofilm Formation Across Diverse Clonal Lineages

A key characteristic of S. aureus infections, and one that also varies phenotypically between clones, is that of biofilm formation, which aids in bacterial persistence through increased adherence and immune evasion. Though there is a general understanding of the process of biofilm formation - adhesion, proliferation, maturation, and dispersal - the tightly orchestrated molecular events behind each stage, and what drives variation between S. aureus strains, has yet to be unraveled. Herein we measure biofilm progression and dispersal in real-time across the 5 major S. aureus CDC-types (USA100-USA500) revealing adherence patterns that differ markedly amongst strains. To gain insight into this, we performed transcriptomic profiling on these isolates at multiple time points, compared to planktonically growing counterparts. Our findings support a model in which eDNA release, followed by increased positive surface charge, perhaps drives initial abiotic attachment. This is seemingly followed by cooperative repression of autolysis and activation of PNAG production, which may indicate a developmental shift in structuring the biofilm matrix. As biofilms mature, diminished translational capacity was apparent, with 53% of all ribosomal proteins downregulated, followed by upregulation of anaerobic respiration enzymes. These findings are noteworthy because reduced cellular activity and an altered metabolic state have been previously shown to contribute to higher antibiotic tolerance and bacterial persistence. In sum, this work is, to our knowledge, the first study to investigate transcriptional regulation during the early, establishing phase of biofilm formation, and to compare global transcriptional regulation both temporally and across multiple clonal lineages. ImportanceStaphylococcus aureus is a highly virulent, opportunistic pathogen and a leading cause of both nosocomial and community acquired infections. Biofilms are associated with persistent, chronic infections and the capacity to form a biofilm varies phenotypically amongst S. aureus clonal lineages. The molecular regulation of biofilm formation has been a popular area of study for this pathogen, however, a comprehensive mapping of the regulatory processes and factors driving strain-specific variation has yet to be elucidated. This study presents transcriptomic analyses of five diverse methicillin-resistant S. aureus isolates during various stages of biofilm formation, tracked in real time. The transcriptomic profiles of all five isolates were compared to identify both core and unique networks of regulation. Importantly, much of what we currently know about biofilms is based on mature, preformed biofilm populations, with little known of the transient regulation driving attachment, proliferation, maturation, and dissemination. We address this issue by investigating transcriptional regulation during the early, establishing phase of biofilm formation, and compare global transcriptional regulation both temporally and across multiple clonal lineages. This study provides a launching point towards understanding the highly orchestrated regulation driving each phase of biofilm development and may inform on future strategies to combat biofilm-mediated infections. Data SummaryO_LIRNA sequencing results have been deposited to the NCBI Gene Expression Omnibus; GEO submission GSE163153 (url - https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE163153) C_LIO_LIOverview and comparisons of gene expression within biofilm and planktonic cell populations for the various Staphylococcus aureus strains are shown in Table S1, S2, S3. Validation and visual depiction of these data can be found as Figure S2 and S3 respectively (available in supplementary material). C_LI

microbiology↗

bioRxiv2020

Suppressors of YpsA-mediated cell division inhibition in Bacillus subtilis

Although many bacterial cell division factors have been uncovered over the years, evidence from recent studies points to the existence of yet to be discovered factors involved in cell division regulation. Thus, it is important to identify factors and conditions that regulate cell division to obtain a better understanding of this fundamental biological process. We recently reported that in the Gram-positive organisms Bacillus subtilis and Staphylococcus aureus, increased production of YpsA resulted in cell division inhibition. In this study, we isolated spontaneous suppressor mutations to uncover critical residues of YpsA and the pathways through which YpsA may exert its function. Using this technique, we were able to isolate four unique intragenic suppressor mutations in ypsA (E55D, P79L, R111P, G132E) that rendered the mutated YpsA non-toxic upon overproduction. We also isolated an extragenic suppressor mutation in yfhS, a gene that encodes a protein of unknown function. Subsequent analysis confirmed that cells lacking yfhS were unable to undergo filamentation in response to YpsA overproduction. We also serendipitously discovered that YfhS may play a role in cell size regulation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/946632v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1098d63org.highwire.dtl.DTLVardef@216f86org.highwire.dtl.DTLVardef@121050eorg.highwire.dtl.DTLVardef@d2ef3a_HPS_FORMAT_FIGEXP M_FIG C_FIG ABBREVIATED SUMMARYIn Bacillus subtilis, we discovered that increased expression of ypsA results in cell division inhibition and impairment of colony formation on solid medium. Colonies that do arise possess compensatory suppressor mutations. Analysis of one such suppressor mutation led us to a protein of unknown function, YfhS, which appears to play a role in regulating cell length and cell width.

microbiology↗

bioRxiv2019

Mapping the Global Network of Extracellular Protease Regulation in Staphylococcus aureus

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 accumulation, or lack thereof, of individual virulence factors. Herein, we comprehensively map this system, exploring regulation of the four protease loci by known and novel factors. In so doing, we determine 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, whilst 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 appear 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.\n\nImportanceThe complex regulatory role of the proteases necessitates very tight coordination and control of their expression. Whilst this process has been well studied, a major oversight has been the consideration of proteases as a single entity, rather than 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 producing a comprehensive regulatory circuit that emphasizes the complexity of protease regulation in Staphylococcus aureus.

microbiology↗

bioRxiv2019

MroQ is a Novel Abi-domain Protein That Influences Virulence Gene Expression in Staphylococcus aureus via Modulation of Agr Activity

Numerous factors have to date been identified as playing a role in the regulation of Agr activity in S. aureus, including transcription factors, antisense RNAs, and host elements. Herein we investigate 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 datasets for both mroQ and agr mutants reveal 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 is not wielded at the level of AgrD processing. Virulence assessment demonstrated that mroQ and agr mutants both 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.

microbiology↗