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 . Importance: S. 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 .

Staphylococcus aureus SigS Induces Expression of a Regulatory Protein Pair That Modulates Its mRNA Stability.

SigS is the sole extracytoplasmic function sigma factor in Staphylococcus aureus and is necessary for virulence, immune evasion, and adaptation to toxic chemicals and environmental stressors. Despite the contribution of SigS to a myriad of critical phenotypes, the downstream effectors of SigS-dependent pathogenesis, immune evasion, and stress adaptation remain elusive. To address this knowledge gap, we analyzed the S. aureus transcriptome following transient overexpression of SigS. We identified a bicistronic transcript, upregulated 1,000-fold, containing two midsized genes, each containing single domains of unknown function (DUFs). We renamed these genes SigS-regulated orfA (sroA) and SigS-regulated orfB (sroB). We demonstrated that SigS regulation of the sroAB operon is direct by using in vitro transcription analysis. Using Northern blot analysis, we also demonstrated that SroA and SroB have opposing autoregulatory functions on the transcriptional architecture of the sigS locus, with SroA stimulating SigS mRNA levels and SroB stimulating s750 (SigS antisense) levels. We hypothesized that these opposing regulatory effects were due to a direct interaction. We subsequently demonstrated a direct interaction between SroA and SroB using an in vivo surrogate genetics approach via bacterial adenylate cyclase-based two-hybrid (BACTH) analysis. We demonstrated that the SroA effect on SigS is at the posttranscriptional 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 murine pneumonia model of infection. IMPORTANCE SigS is necessary for S. aureus virulence, immune evasion, and adaptation to chemical and environmental stressors. These processes are critically important for the ability of S. aureus to cause disease. However, the SigS-dependent transcriptome has not been identified, hindering our ability to identify downstream effectors of SigS that contribute to these pathogenic and adaptive phenotypes. Here, we identify a regulatory protein pair that is a major direct target of SigS, known as SroA and SroB. SroA also acts to stimulate SigS expression at the posttranscriptional level of RNA turnover, providing insight into intrinsically low levels of SigS. The discovery of SroA and SroB increases our understanding of SigS and the S. aureus pathogenesis process.

The novel two-component system AmsSR governs alternative metabolic pathway usage in Acinetobacter baumannii.

In this study, we identify a novel two-component system in Acinetobacter baumannii (herein named AmsSR for regulator of alternative metabolic systems) only present in select gammaproteobacterial and betaproteobacterial species. Bioinformatic analysis revealed that the histidine kinase, AmsS, contains 14 predicted N-terminal transmembrane domains and harbors a hybrid histidine kinase arrangement in its C-terminus. Transcriptional analysis revealed the proton ionophore CCCP selectively induces P amsSR expression. Disruption of amsSR resulted in decreased intracellular pH and increased depolarization of cytoplasmic membranes. Transcriptome profiling revealed a major reordering of metabolic circuits upon amsR disruption, with energy generation pathways typically used by bacteria growing in limited oxygen being favored. Interestingly, we observed enhanced growth rates for mutant strains in the presence of glucose, which led to overproduction of pyruvate. To mitigate the toxic effects of carbon overflow, we noted acetate overproduction in amsSR-null strains, resulting from a hyperactive Pta-AckA pathway. Additionally, due to altered expression of key metabolic genes, amsSR mutants favor an incomplete TCA cycle, relying heavily on an overactive glyoxylate shunt. This metabolic reordering overproduces NADH, which is not oxidized by the ETC; components of which were significantly downregulated upon amsSR disruption. As a result, the mutants almost exclusively rely on substrate phosphorylation for ATP production, and consequently display reduced oxygen consumption in the presence of glucose. Collectively, our data suggests that disruption of amsSR affects the function of the aerobic respiratory chain, impacting the energy status of the cell, which in turn upregulates alternative metabolic and energy generation pathways.

Mycobacterium abscessus DosRS two-component system controls a species-specific regulon required for adaptation to hypoxia.

Mycobacterium abscessus (Mab), an emerging opportunistic pathogen, predominantly infects individuals with underlying pulmonary diseases such as cystic fibrosis (CF). Current treatment outcomes for Mab infections are poor due to Mab's inherent antibiotic resistance and unique host interactions that promote phenotypic tolerance and hinder drug access. The hypoxic, mucus-laden airways in the CF lung and antimicrobial phagosome within macrophages represent hostile niches Mab must overcome via alterations in gene expression for survival. Regulatory mechanisms important for the adaptation and long-term persistence of Mab within the host are poorly understood, warranting further genetic and transcriptomics study of this emerging pathogen. DosRS Mab , a two-component signaling system (TCS), is one proposed mechanism utilized to subvert host defenses and counteract environmental stress such as hypoxia. The homologous TCS of Mycobacterium tuberculosis (Mtb), DosRS Mtb , is known to induce a ~50 gene regulon in response to hypoxia, carbon monoxide (CO) and nitric oxide (NO) in vitro and in vivo. Previously, a small DosR Mab regulon was predicted using bioinformatics based on DosR Mtb motifs however, the role and regulon of DosRS Mab in Mab pathogenesis have yet to be characterized in depth. To address this knowledge gap, our lab generated a Mab dosRS knockout strain (MabΔdosRS) to investigate differential gene expression, and phenotype in an in vitro hypoxia model of dormancy. qRT-PCR and lux reporter assays demonstrate Mab_dosR and 6 predicted downstream genes are induced in hypoxia. In addition, RNAseq revealed induction of a much larger hypoxia response comprised of >1000 genes, including 127 differentially expressed genes in a dosRS mutant strain. Deletion of DosRS Mab led to attenuated growth under low oxygen conditions, a shift in morphotype from smooth to rough, and down-regulation of 216 genes. This study provides the first look at the global transcriptomic response of Mab to low oxygen conditions encountered in the airways of CF patients and within macrophage phagosomes. Our data also demonstrate the importance of DosRS Mab for adaptation of Mab to hypoxia, highlighting a distinct regulon (compared to Mtb) that is significantly larger than previously described, including both genes conserved across mycobacteria as well as Mab-specific genes.

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. This resilience is often attributed to a proclivity for biofilm formation, which facilitates a higher tolerance toward external stress, desiccation, and antimicrobials. Despite this, little is known regarding the mechanisms orchestrating A. baumannii biofilm formation. Here, we performed RNA sequencing (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), had a highly mucoid colony phenotype, enhanced tolerance to polysaccharide degradation, altered antibiotic tolerance, and diminished adherence to abiotic surfaces. RNA-seq of the cspC mutant revealed 201 genes with altered expression, including the downregulation of pili and fimbria genes and the upregulation of multidrug efflux pumps. Using transcriptional arrest assays, it appears that CspC mediates its effects, at least in part, through RNA chaperone activity, influencing the half-life of several important transcripts. Finally, we show that CspC is required for survival during challenge by the human immune system and is key for A. baumannii dissemination and/or colonization during systemic infection. Collectively, our work identifies a cadre of new biofilm-associated genes within A. baumannii and provides unique insight into the global regulatory network of this emerging human pathogen.

Stochastic activation of a family of TetR type transcriptional regulators controls phenotypic heterogeneity in Acinetobacter baumannii.

Phenotypic heterogeneity is an important mechanism for regulating bacterial virulence, where a single regulatory switch is typically activated to generate virulent and avirulent subpopulations. The opportunistic pathogen Acinetobacter baumannii can transition at high frequency between virulent opaque (VIR-O) and avirulent translucent subpopulations, distinguished by cells that form opaque or translucent colonies. We demonstrate that expression of 11 TetR-type transcriptional regulators (TTTRs) can drive cells from the VIR-O opaque subpopulation to cells that form translucent colonies. Remarkably, in a subpopulation of VIR-O cells, four of these TTTRs were stochastically activated in different combinations to drive cells to the translucent state. The resulting translucent subvariants exhibited unique phenotypic differences and the majority were avirulent. Due to their functional redundancy, a quadruple mutant with all four of these TTTRs inactivated was required to observe a loss of switching from the VIR-O state. Further, we demonstrate a small RNA, SrvS, acts as a "rheostat," where the levels of SrvS expression influences both the VIR-O to translucent switching frequency, and which TTTR is activated when VIR-O cells switch. In summary, this work has revealed a new paradigm for phenotypic switching in bacteria, where an unprecedented number of related transcriptional regulators are activated in different combinations to control virulence and generate unique translucent subvariants with distinct phenotypic properties.

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 gap in our knowledge of microbial diversity. Here, 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 atrophy through a mixed approach of pseudogenization and reversion of pseudogenization of amino acid 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 single nucleotide polymorphism, indel, and pseudogene-impactful 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. IMPORTANCE The majority of environmental bacteria cannot be cultured within the laboratory. Understanding why only certain environmental isolates can be recovered is key to unlocking the abundant microbial dark matter that is widespread on our planet. In this study, we present not only the culturing but domestication of just such an organism. Although initial recovery took >4 months, we were able to isolate distinct, subpassaged offspring from the originating colony within mere weeks. A phenotypic and genotypic analysis of our generational strains revealed that adaptation to life in the lab occurred as a result of wholesale mutational changes. These permitted an enhanced ability for growth in nutrient rich media but came at the expense of reduced genomic flexibility. We suggest that without dynamic natural environmental stressors our domesticated strains effectively underwent genomic atrophy as they adapted to static conditions experienced in the laboratory.

A global transcriptomic analysis of Staphylococcus aureus biofilm formation across diverse clonal lineages.

A key characteristic of Staphylococcus 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 unravelled. Herein we measure biofilm progression and dispersal in real-time across the five 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 timepoints, 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 poly-N-acetylglucosamine (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.

Cwl0971, a novel peptidoglycan hydrolase, plays pleiotropic roles in Clostridioides difficile R20291.

Clostridioides difficile is a Gram-positive, spore-forming, toxin-producing anaerobe that can cause nosocomial antibiotic-associated intestinal disease. Although the production of toxin A (TcdA) and toxin B (TcdB) contribute to the main pathogenesis of C. difficile, the mechanism of TcdA and TcdB release from cell remains unclear. In this study, we identified and characterized a new cell wall hydrolase Cwl0971 (CDR20291_0971) from C. difficile R20291, which is involved in bacterial autolysis. The gene 0971 deletion mutant (R20291Δ0971) generated with CRISPR-AsCpfI exhibited significantly delayed cell autolysis and increased cell viability compared to R20291, and the purified Cwl0971 exhibited hydrolase activity for Bacillus subtilis cell wall. Meanwhile, 0971 gene deletion impaired TcdA and TcdB release due to the decreased cell autolysis in the stationary/late phase of cell growth. Moreover, sporulation of the mutant strain decreased significantly compared to the wild type strain. In vivo, the defect of Cwl0971 decreased fitness over the parent strain in a mouse infection model. Collectively, Cwl0971 is involved in cell wall lysis and cell viability, which affects toxin release, sporulation, germination, and pathogenicity of R20291, indicating that Cwl0971 could be an attractive target for C. difficile infection therapeutics and prophylactics.

Interdependent YpsA- and YfhS-Mediated Cell Division and Cell Size Phenotypes 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, and G132E) that rendered the mutated YpsA nontoxic 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. Finally, we provide evidence showing a mechanistic link between YpsA and YfhS.IMPORTANCEBacillus subtilis is a rod-shaped Gram-positive model organism. The factors fundamental to the maintenance of cell shape and cell division are of major interest. We show 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. We have isolated multiple intragenic (within ypsA) mutants and an extragenic suppressor mutant. Further analysis of the extragenic suppressor mutation led to a protein of unknown function, YfhS, which appears to play a role in regulating cell size. In addition to confirming that the cell division phenotype associated with YpsA is disrupted in a yfhS-null strain, we also discovered that the cell size phenotype of the yfhS knockout mutant is abolished in a strain that also lacks ypsA This highlights a potential mechanistic link between these two proteins; however, the underlying molecular mechanism remains to be elucidated.

Regulatory networks important for survival of Acinetobacter baumannii within the host.

Acinetobacter baumannii is known for its intrinsic resistance to conventional antibiotic treatment and hypervirulence during infection. This coupled with its extraordinary capacity to survive in myriad harsh environments has led to increasing rates of infection in clinical settings. Numerous studies have characterized the virulence factors and resistance genes in A. baumannii responsible for the detrimental outcomes seen in patients; however, the role of regulatory factors in controlling the expression of these genes remains less well explored. Herein we discuss the latest and most influential findings on the regulatory network of A. baumannii, focusing on the transcription factors, two-component systems, and sRNAs. We place particular focus on those identified as being crucial for sensing and responding to continually changing environments, and influencing survival and virulence when engaging with the human host.

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 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.