Findings establish SSR42 as a major post-transcriptional regulator of S. aureus virulence and reveal a novel RNA-mediated mechanism that promotes α-toxin production through stabilization of the hla transcript.
Abstract
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 α-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 α-toxin production through stabilization of the hla transcript.
Methicillin-resistant Staphylococcus aureus (MRSA) is an opportunistic pathogen that colonizes a significant proportion of humans, contains numerous virulence factors promoting infection, and continues to threaten human lives and burden healthcare systems globally. Many MRSA virulence factors are known to be either secreted or anchored on the outer leaflet of the cell surface. Although many virulence factors have been studied intensively in MRSA, there remains a significant proportion of secreted and surface proteins that are unstudied for their potential as virulence factors. We began with identifying proteins secreted from MRSA in axenic culture using an unbiased mass-spectrometry based approach. 2 secreted proteins thus identified mapped to an operon of 6 genes, SAUSA300_1739 to SAUSA300_1744. Mutation of each of the individual genes in the operon resulted in attenuation in a mouse model of subcutaneous infection. We demonstrate that two genes in the operon, SAUSA300_1739, and SAUSA300_1740, encode nucleases with DNase activity. Genetic analysis of the SAUSA300_1739 to SAUSA300_1744 operon across several Staphylococcus aureus strains indicate that the operon is highly conserved, highlighting its importance for virulence. Summary Methicillin-resistant Staphylococcus aureus (MRSA) remains a serious human pathogen with a myriad of virulence factors supporting pathogenicity. From a MRSA exoproteome, we identified an operon of 6 genes with unknown function that are dispensable for growth in axenic culture but promote MRSA virulence during subcutaneous infection.
Alex Zilinskas, H. M. Ni, Zoe Netter et al.· bioRxiv· 0 citations
The global spread of methicillin-resistant Staphylococcus aureus (MRSA) underscores the urgent need for novel therapeutics. This study explores the anti-hemolytic activity and mechanism of MGS-3, a semi-synthetic xanthone derivative with potent anti-MRSA properties. Using a multi-tiered approach, we evaluated MGS-3's suppression of S. aureus α-hemolysin (Hla) through in vitro and in vivo models. Mechanistic studies combining gene editing, microscale thermophoresis (MST), and cellular thermal shift assays (CETSA) revealed that MGS-3 directly targets SarA, a key transcriptional regulator of hemolysin biosynthesis. MGS-3 exhibited dose-dependent inhibition of Hla virulence in strains ATCC29213, USA300, and NCTC8325 by repressing hla transcription and expression. Specifically, it downregulated sarA without affecting agrA, disrupting SarA's binding to the hla promoter and thereby reducing Hla-mediated cytotoxicity in A549 and A375 cells. In vivo, MGS-3 reduced skin abscess bacterial loads by 2.1-fold, comparable to ΔsarA (2.3-fold), while mitigating cytokine response and improving histopathology. These results establish SarA suppression as MGS-3's primary anti-virulence mechanism. Notably, MGS-3 maintained low hemolytic activity, highlighting its therapeutic potential. MGS-3 represents a promising candidate for anti-staphylococcal drug development, targeting virulence rather than bacterial viability to potentially curb resistance evolution. This study provides a pharmacodynamic foundation for novel anti-MRSA strategies. IMPORTANCE: The global spread of methicillin-resistant Staphylococcus aureus(MRSA) requires new treatments. The semi-synthetic xanthone derivative MGS-3 inhibits α-hemolysin production by targeting the SarA regulator, reducing virulence. In a murine skin abscess model, it significantly lowered bacterial load and mitigated inflammation. This anti-virulence strategy presents a promising approach to combat MRSA.
Han Bai, Yuehan Zhou, Wenchong Ye et al.· Microbial Pathogenesis· 0 citations
Staphylococcus aureus is a Gram-positive pathogen that is a major cause of soft tissue and bloodstream infections in humans. Previous studies have shown that S. aureus triggers myeloid cell-driven innate immune activation including inflammatory signaling and type I interferon (IFN) responses; however, the role type I IFNs remains controversial.
Primary human CD14+ monocytes were purified from PBMCs of healthy donors using anti-CD14 magnetic beads. Monocytes were infected with various doses of S. aureus and harvested for RT-qPCR, Western blotting, and integrated bulk RNA sequencing, ATAC-seq and CUT&RUN.
We observed a dose-dependent dichotomous regulation of the IFN response, which was induced by low doses and suppressed at high doses of S. aureus. Only a subset of interferon-stimulated genes (ISGs) was induced despite intact IFNβ production following S. aureus infection. Intriguingly, S. aureus selectively abrogated the activation and nuclear translocation of STAT1, while activation and nuclear translocation of the other ISGF3 components STAT2 and IRF9 were preserved. Furthermore, RNA profiling revealed a disparate regulation of gene expression, with a distinct subset of genes exhibiting significantly enhanced expression upon combined S. aureus and IFNβ treatment. Epigenomic profiling showed a complete repression of chromatin accessibility as well as STAT1 binding at a subset of ISG loci upon S. aureus infection, whereas another subset of ISGs displayed increased chromatin accessibility and transcriptional activation upon infection.
Our findings suggest that S. aureus alters activation of the canonical type I IFN-mediated Jak-STAT pathway through suppression of STAT1 activation and altering chromatin remodeling . This change in signaling is associated with gene-specific reprogramming of the type I IFN response by S. aureus that may alter the host response to this pathogen in a context-dependent manner and contribute to difficulty in clearing S. aureus infection.
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Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)
Qiong Zhang, Ruoxi Yuan, Lionel B Ivashkiv· Journal of Immunology· 0 citations
Staphylococcus aureus
(
S. aureus
)is an important foodborne pathogen. Host-derived catecholamines released under stress are known to influence bacterial pathogenic processes, including growth, virulence, biofilm formation. However, the role of dopamine in this context remains less well understood, particularly in
S. aureus
. In this study, we investigated the effects of dopamine on
S. aureus
ATCC 25,923 using a combination of phenotypic assays and multi-omics analyses. Dopamine was taken up by the bacteria and was associated with enhanced pathogenic phenotypes, including increased proliferation, toxin production, biofilm formation, motility, adhesion, and multidrug resistance. In a mouse infection experiment, dopamine treatment further increased bacterial colonization and organ invasion. Mechanistically, multi-omics analyses indicated that dopamine activates the SaeRS two-component system and is associated with the upregulation of virulence-related factors, adhesion-associated proteins, and membrane lipid synthesis pathways, along with increased activity in key metabolic processes such as the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. Importantly, deletion of
saeS
markedly attenuated these dopamine-associated effects. Taken together, these findings suggest that dopamine likely influences
S. aureus
pathogenicity primarily through the SaeRS signaling pathway and provide insight into how host stress signals may contribute to bacterial infection.
Ben Xu, Ziyue Wang, Jinmei Chai et al.· BMC Microbiology· 0 citations
Simple Summary The high pathogenicity of methicillin-resistant Staphylococcus aureus (MRSA) is mainly attributed to elevated hemolysin expression. The current study explored the effects of functional membrane microdomains (FMMs) on hemolysin expression and virulence in MRSA, aiming to find new clues for the prevention and treatment of MRSA infections. Our findings demonstrate that FMMs function as a negative regulator of MRSA virulence. FMMs repress hemolysin expression and excessive host inflammatory responses through modulating the VraS/R-Agr signaling axis. This provides novel mechanistic insights into FMM-mediated regulation of hemolysis and offers a theoretical basis for developing anti-MRSA therapeutic strategies targeting FMMs.
Bing-Tian Jin, Chang-Zhen Wang, Tiantian Liu et al.· Veterinary Sciences· 0 citations
Toxin-antitoxin (TA) systems are bacteria-specific components involved in maintaining mobile genetic elements that may carry antibiotic resistance determinants (ARDs), among other functions. PemIK-Sa1 is a plasmid-encoded TA system found in Staphylococcus aureus, a dangerous pathogen affecting humans and animals. The PemK-Sa1 toxin is a sequence-specific ribonuclease inhibited by its cognate antitoxin, PemI-Sa1. Using recombinant multidrug-resistant strains, we demonstrated that expression of the toxin reduces resistance to antibiotics from various classes, including β-lactams, fluoroquinolones, aminoglycosides, and chloramphenicol. Transcriptomics revealed that, in addition to downregulation of genes encoding ARDs, nearly one-third of the staphylococcal transcriptome is altered. Furthermore, proteomic analysis revealed that toxin expression results in the downregulation of several proteins involved in nucleotide biosynthesis, carbohydrate metabolism, and energy production. This indicates parallel mechanisms in which the toxin not only degrades ARD transcripts, thereby directly impacting their function, but also alters the expression of genes encoding proteins crucial to essential cell processes. The ability to reduce resistance to a broad range of antibiotics makes the PemK-Sa1 toxin a promising tool for combating multidrug-resistant bacteria through the artificial activation of the PemIK-Sa1 TA system.
Emilia Bonar, Kinga Chlebicka, Michał Bukowski et al.· Frontiers in Microbiology· 0 citations
