Biology subjects

Tomlinson, B. R.

Publications and source records attributed to Tomlinson, B. R..

6 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↗

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↗

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

Characterization of a novel endopeptidase Cwl0971 that hydrolyzes peptidoglycan of Clostridioides difficile involved in pleiotropic cellular processes

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 (R20291{Delta}0971) 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.

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↗