Elucidating the potential of isorhapontigenin in targeting the MgrA regulatory network: a paradigm shift for attenuating MRSA virulence.

As methicillin-resistant Staphylococcus aureus (MRSA) exhibits formidable resistance to many drugs, the imperative for alternative therapeutic strategies becomes increasingly evident. At the heart of our study is the identification of a novel inhibitor through fluorescence anisotropy assays, specifically targeting the crucial multiple gene regulator A (MgrA) regulatory network in S. aureus. Isorhapontigenin (Iso), a natural compound, exhibits outstanding inhibitory efficacy, modulating bacterial virulence pathways without exerting direct bactericidal activity. This suggests a paradigm shift toward attenuating virulence instead of purely focusing on bacterial elimination. Through comprehensive in vitro and in vivo evaluations, we elucidated the complex interplay between Iso and MgrA, leading to reduced S. aureus adhesion, and overall virulence. At the cellular level, Iso offers significant protection to A549 cells infected with S. aureus, reducing cellular damage. Importantly, Iso augments the chemotaxis of neutrophils, curtailing the immune evasion capabilities of S. aureus. Furthermore, in vivo investigations highlight the notable effectiveness of Iso against MRSA-induced pneumonia and within the Galleria mellonella infection model, underscoring its pivotal role in the evolving realm of antibacterial drug discovery. Significantly, when Iso is used in combination with vancomycin, it outperforms its solo application, indicating a more pronounced therapeutic impact. This seminal research emphasizes Iso's potential as a primary defense against the surge of multidrug-resistant pathogens, heralding new prospects in antimicrobial therapy.

Exploring the potential of isorhapontigenin: attenuating Staphylococcus aureus virulence through MgrA-mediated regulation.

The emerging prevalence of drug-resistant Staphylococcus aureus isolates underscores the urgent need for alternative therapeutic strategies due to the declining effectiveness of traditional antibiotics in clinical settings. MgrA, a key virulence regulator in S. aureus, orchestrates the expression of numerous virulence factors. Here, we report the discovery of isorhapontigenin, a methoxylated analog of resveratrol, as a potential anti-virulence agent against S. aureus. Isorhapontigenin effectively inhibits the hemolytic activity of S. aureus in a non-bactericidal manner. Additionally, it significantly reduces the cytotoxicity of S. aureus and impairs its ability to survive in macrophages. Mechanistically, isorhapontigenin modulates the expression of virulence factors, dose-dependently downregulating hla and upregulating the MgrA-regulated gene spa. Electrophoretic mobility shift assays demonstrated that isorhapontigenin inhibits the binding of MgrA to the hla promoter in a dose-dependent manner. Thermal shift assays confirmed the direct interaction between isorhapontigenin and the MgrA protein. The in vivo experiments demonstrated that isorhapontigenin significantly reduced the area of skin abscesses and improved survival in a pneumonia model while decreasing bacterial burden and inflammation in the lungs. In conclusion, isorhapontigenin holds potential as a candidate drug for further development as an anti-virulence agent for treating S. aureus infections. IMPORTANCE: The emergence of antibiotic-resistant Staphylococcus aureus strains presents a formidable challenge to public health, necessitating novel approaches in combating these pathogens. Traditional antibiotics are becoming increasingly ineffective, leading to a pressing need for innovative therapeutic strategies. In this study, targeting virulence factors that play a crucial role in the pathogenesis of bacterial infections offers a promising alternative to circumvent resistance mechanisms. The discovery of isorhapontigenin as an inhibitor of S. aureus virulence represents a significant advance in anti-virulence therapy.

The Small RNA Teg41 Is a Pleiotropic Regulator of Virulence in Staphylococcus aureus.

Previously, our group demonstrated a role for the small RNA (sRNA) Teg41 in regulating production of the alpha phenol-soluble modulin toxins (αPSMs) in Staphylococcus aureus. Overexpressing Teg41 increased αPSM production while deleting the 3' end of Teg41 (Teg41Δ3' strain) resulted in a decrease in αPSM production, reduced hemolytic activity of S. aureus culture supernatants, and attenuated virulence in a murine abscess model of infection. In this study, we further explore the attenuation of virulence in the Teg41Δ3' strain. Using both localized and systemic models of infection, we demonstrate that the Teg41Δ3' strain is more severely attenuated than an ΔαPSM mutant, strongly suggesting that Teg41 influences more than the αPSMs. Proteomic and transcriptomic analysis of the wild-type and Teg41Δ3' strains reveals widespread alterations in transcript abundance and protein production in the absence of Teg41, confirming that Teg41 has pleiotropic effects in the cell. We go on to investigate the molecular mechanism underlying Teg41-mediated gene regulation. Surprisingly, results demonstrate that certain Teg41 target genes, including the αPSMs and ßPSMs, are transcriptionally altered in the Teg41Δ3' strain, while other targets, specifically spa (encoding surface protein A), are regulated at the level of transcript stability. Collectively, these data demonstrate that Teg41 is a pleiotropic RNA regulator in S. aureus that influences expression of a variety of genes using multiple different mechanisms.

SarA plays a predominant role in controlling the production of extracellular proteases in the diverse clinical isolates of Staphylococcus aureus LAC and UAMS-1.

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.

MgrA Activates Staphylococcal Capsule via SigA-Dependent Promoter.

Staphylococcus aureus capsule polysaccharide is an important antiphagocytic virulence factor. The cap genes are regulated at the promoter element (Pcap) upstream of the cap operon. Pcap, which consists of a dominant SigB-dependent promoter and a weaker upstream SigA-dependent promoter, is activated by global regulator MgrA. How MgrA activates capsule is unclear. Here, we showed that MgrA directly bound to the Pcap region and affected the SigA-dependent promoter. Interestingly, an electrophoretic mobility shift assay showed that MgrA bound to a large region of Pcap, mainly downstream of the SigA-dependent promoter. We further showed that the ArlRS two-component system and the Agr quorum sensing system activated capsule primarily through MgrA in the early growth phases.IMPORTANCE The virulence of Staphylococcus aureus depends on the expression of various virulence factors, which is governed by a complex regulatory network. We have been using capsule as a model virulence factor to study virulence gene regulation in S. aureus MgrA is one of the regulators of capsule and has a major effect on capsule production. However, how MgrA regulates capsule genes is not understood. In this study, we were able to define the mechanism involving MgrA regulation of capsule. In addition, we also delineated the role of MgrA in capsule regulatory pathways involving the key virulence regulators Agr and Arl. This study further advances our understanding of virulence gene regulation in S. aureus, an important human pathogen.

MgrA Negatively Impacts Staphylococcus aureus Invasion by Regulating Capsule and FnbA.

Virulence genes are regulated by a complex regulatory network in Staphylococcus aureus Some of the regulators are global in nature and affect many downstream genes. MgrA is a multiple-gene regulator that has been shown to activate genes involved in capsule biosynthesis and repress surface protein genes. The goal of this study was to demonstrate the biological significance of MgrA regulation of capsule and surface proteins. We found that strain Becker possessed one fibronectin-binding protein, FnbA, and that FnbA was the predominant protein involved in invasion of nonphagocytic HeLa cells. By genetic analysis of strains with different amounts of capsule, we demonstrated that capsule impeded invasion of HeLa cells by masking the bacterial cell wall-anchored protein FnbA. Using variants with different levels of mgrA transcription, we further demonstrated that MgrA negatively impacted invasion by activating the cap genes involved in capsule biosynthesis and repressing the fnbA gene. Thus, we conclude that MgrA negatively impacts cell invasion of S. aureus Becker by promoting capsule and repressing FnbA.

Uncariitannin, a polyphenolic polymer from Uncaria gambier, attenuates Staphylococcus aureus virulence through an MgrA-mediated regulation of α-hemolysin.

A global transcriptional regulator, MgrA, was previously identified as a key determinant of virulence in Staphylococcus aureus. An 80% EtOH extract of Uncaria gambier was found to attenuate the virulence of S. aureus via its effects on MgrA. Using bioassay-guided fractionation, a polyphenolic polymer, uncariitannin, was found to be the main bioactive constituent of the extract, and its structure was characterized using spectral and chemical analysis. The molecular weight and polydispersity of uncariitannin were determined by gel permeation chromatography-refractive index-light scattering analysis. An electrophoretic mobility shift assay showed that uncariitannin could effectively inhibit the interaction of MgrA with DNA in a dose-dependent manner. Treatment with uncariitannin could decrease the mRNA and protein levels of Hla in both the S. aureus Newman and USA300 LAC strains. Further analysis of Hla expression levels in the Newman ΔmgrA and Newman ΔmgrA/pYJ335-mgrA strains indicated that uncariitannin altered Hla expression primarily in an MgrA-dependent manner. A mouse model of infection indicated that uncariitannin could attenuate MRSA virulence. In conclusion, uncariitannin may be a potential candidate for further development as an antivirulence agent for the treatment of S. aureus infection.

MgrA Governs Adherence, Host Cell Interaction, and Virulence in a Murine Model of Bacteremia Due to Staphylococcus aureus.

BACKGROUND: MgrA is an important global virulence gene regulator in Staphylococcus aureus. In the present study, the role of mgrA in host-pathogen interactions related to virulence was explored in both methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) strains. METHODS: In vitro susceptibilities to human defense peptides (HDPs), adherence to fibronectin (Fn) and endothelial cells (ECs), EC damage, α-toxin production, expression of global regulator (eg, agr RNAIII) and its downstream effectors (eg, α-toxin [hla] and Fn binding protein A [fnbA]), MgrA binding to fnbA promoter, and the effect on HDP-induced mprF and dltA expression were analyzed. The impact of mgrA on virulence was evaluated using a mouse bacteremia model. RESULTS: mgrA mutants displayed significantly higher susceptibility to HDPs, which might be related to the decreased HDP-induced mprF and dltA expression but decreased Fn and EC adherence, EC damage, α-toxin production, agr RNAIII, hla and fnbA expression, and attenuated virulence in the bacteremia model as compared to their respective parental and mgrA-complemented strains. Importantly, direct binding of MgrA to the fnbA promoter was observed. CONCLUSIONS: These results suggest that mgrA mediates host-pathogen interactions and virulence and may provide a novel therapeutic target for invasive S. aureus infections.

The ArlR-MgrA regulatory cascade regulates PIA-dependent and protein-mediated biofilm formation in Rbf-dependent and Rbf-independent pathways.

The two-component system response regulator ArlR and the global regulator MgrA in Staphylococcus aureus participated in numerous biological processes including biofilm formation inhibition. Previous studies have shown that these two regulators could function as a regulatory cascade. Rbf is a positive regulator of biofilm formation enhancing the production of PIA (polysaccharide intercellular adhesin). Here we have demonstrated that both ArlR and MgrA can directly bind to the promoter of rbf and repress its expression. ArlR and MgrA can also directly bind to the promoter of ica operon and enhance the expression of icaA and PIA production, revealing that the ArlR-MgrA regulatory cascade controls PIA-dependent biofilm formation. In addition, we have found that Rbf can directly bind to the aur promoter and repress the expression of aur, which encodes a protease initiating a protease cascade to inhibit protein-mediated biofilm formation. Moreover, our data indicate that the ArlR-MgrA regulatory cascade can promote the expression of aur by directly binding to its promoter and inhibit protein-mediated biofilm formation. These findings shed light on the molecular mechanisms of both PIA-dependent and protein-mediated biofilm formation modulated by the ArlR-MgrA regulatory cascade and the new role of Rbf in protein-mediated biofilm formation, and broaden our understanding of the biofilm formation regulation in S. aureus.

High-Throughput Screening Strategies for the Development of Anti-Virulence Inhibitors Against Staphylococcus aureus.

BACKGROUND: The increasing threats of antibiotic resistance urge the need for developing new approaches to combat bacterial infections including those caused by Staphylococcus aureus (S. aureus). Unlike conventional antibiotics that aim to kill bacteria or inhibit their growth, targeting bacterial virulence may be a promising alternative approach, which imposes less selective pressure for antibiotic resistance in future generations. OBJECTIVE: Our goal is to provide a systematic review about developing high-throughput screening (HTS) strategies for the identification of inhibitors targeting virulence of S. aureus. We also describe an overview of virulence regulatory pathways for potential antivirulence targets. METHODS: We focus on five potential targets or target families, including agr quorum sensing system, SarA/MgrA protein family, sortase A, Clp protease and eukaryotic-like Ser/Thr phosphatase (Stp1). For each target, we introduce its role in virulence regulation, summarize the HTS approaches that are used to identify novel anti-virulence inhibitors, and discuss the advantages and disadvantages of these strategies. CONCLUSION: The discovery of anti-virulence inhibitors via HTS underlines the promising potential of anti-virulence therapy for S. aureus. The development of HTS strategies can facilitate the identification of novel anti-virulence inhibitors for combating S. aureus infection, and may also advance our understanding on virulence regulation in S. aureus.

Structure-activity relationships of pyrazole-4-carbodithioates as antibacterials against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of serious hospital-acquired infections and is responsible for significant morbidity and mortality in residential care facilities. New agents against MRSA are needed to combat rising resistance to current antibiotics. We recently reported 5-hydroxy-3-methyl-1-phenyl-1H-pyrazole-4-carbodithioate (HMPC) as a new bacteriostatic agent against MRSA that appears to act via a novel mechanism. Here, twenty nine analogs of HMPC were synthesized, their anti-MRSA structure-activity relationships evaluated and selectivity versus human HKC-8 cells determined. Minimum inhibitory concentrations (MIC) ranged from 0.5 to 64 µg/mL and up to 16-fold selectivity was achieved. The 4-carbodithioate function was found to be essential for activity but non-specific reactivity was ruled out as a contributor to antibacterial action. The study supports further work aimed at elucidating the molecular targets of this interesting new class of anti-MRSA agents.

MgrA Negatively Regulates Biofilm Formation and Detachment by Repressing the Expression of psm Operons in Staphylococcus aureus.

Phenol-soluble modulins (PSMs) are amphipathic peptides that are produced by staphylococci and play important roles in Staphylococcus aureus biofilm formation and dissemination. Although the multiple functions of PSMs have been recognized, the regulatory mechanisms controlling the expression of psm operons remain largely unknown. In this study, we identified MgrA in a DNA pulldown assay and further demonstrated, by electrophoretic mobility shift assays and DNase I footprinting assays, that MgrA could bind specifically to the promoter regions of psm operons. We then constructed an isogenic mgrA deletion strain and compared biofilm formation and detachment in the wild-type and isogenic mgrA deletion strains. Our results indicated that biofilm formation and detachment were significantly increased in the mgrA mutant strain. Real-time quantitative reverse transcription-PCR data indicated that MgrA repressed the transcription of psm operons in cultures and biofilms, suggesting that MgrA is a negative regulator of psm expression. Furthermore, we analyzed biofilm formation by the psm mutant strains, and we found that PSMs promoted biofilm structuring and development in the mgrA mutant strain. These findings reveal that MgrA negatively regulates biofilm formation and detachment by repressing the expression of psm operons through direct binding to the psm promoter regions.IMPORTANCEStaphylococcus aureus is a human and animal pathogen that can cause biofilm-associated infections. PSMs have multiple functions in biofilm development and virulence in staphylococcal pathogenesis. This study has revealed that MgrA can negatively regulate psm expression by binding directly to the promoter regions of psm operons. Furthermore, our results show that MgrA can modulate biofilm structuring and development by repressing the production of PSMs in S. aureus Our findings provide novel insights into the regulatory mechanisms of S. aureus psm gene expression, biofilm development, and pathogenesis.

RNAIII of the Staphylococcus aureus agr system activates global regulator MgrA by stabilizing mRNA.

RNAIII, the effector of the agr quorum-sensing system, plays a key role in virulence gene regulation in Staphylococcus aureus, but how RNAIII transcriptionally regulates its downstream genes is not completely understood. Here, we show that RNAIII stabilizes mgrA mRNA, thereby increasing the production of MgrA, a global transcriptional regulator that affects the expression of many genes. The mgrA gene is transcribed from two promoters, P1 and P2, to produce two mRNA transcripts with long 5' UTR. Two adjacent regions of the mgrA mRNA UTR transcribed from the upstream P2 promoter, but not the P1 promoter, form a stable complex with two regions of RNAIII near the 5' and 3' ends. We further demonstrate that the interaction has several biological effects. We propose that MgrA can serve as an intermediary regulator through which agr exerts its regulatory function.

Scaffold assembly based on genome rearrangement analysis.

Advances in DNA sequencing technology over the past decade have increased the volume of raw sequenced genomic data available for further assembly and analysis. While there exist many algorithms for assembly of sequenced genomic material, they often experience difficulties in constructing complete genomic sequences. Instead, they produce long genomic subsequences (scaffolds), which then become a subject to scaffold assembly aimed at reconstruction of their order along genome chromosomes. The balance between reliability and cost for scaffold assembly is not there just yet, which inspires one to seek for new approaches to address this problem. We present a new method for scaffold assembly based on the analysis of gene orders and genome rearrangements in multiple related genomes (some or even all of which may be fragmented). Evaluation of the proposed method on artificially fragmented mammalian genomes demonstrates its high reliability. We also apply our method for incomplete anophelinae genomes, which expose high fragmentation, and further validate the assembly results with referenced-based scaffolding. While the two methods demonstrate consistent results, the proposed method is able to identify more assembly points than the reference-based scaffolding.

The impact of mgrA on progression of Staphylococcus aureus sepsis.

Sepsis induced by Staphylococcus aureus has worse outcome with the appearance of methicillin-resistant Staphylococcus aureus (MRSA) because of multi-resistance to a large group of antibiotics, which may lead to death from septic shock. Pathogenesis of S. aureus infections are involved in the production of a wide variety of virulence factors. MgrA, a noval global regulator, is a member of the MarR (multiple antibiotic resistance regulator)/SarA (staphylococcal accessory regulator A) family proteins, which plays a key role in regulating the expression of major virulence factors in S. aureus. In the present study, by using a murine model of sepsis, we investigated the role of mgrA in onset and progression of S. aureus induced sepsis. We found that mice inoculated with wild-type strain Newman had significantly higher mortality (p = 0.029), more weight lost, more bacterial load in blood, spleen and kidney, more intense inflammation response, and worse histopathology than mice inoculated with mgrA knockout strain. Our results has provided evidence that mgrA is a global regulator in S. aureus, and play an important role in S. aureus sepsis, could increase mortality and accelerate the onset and development of sepsis.

MgrA activates expression of capsule genes, but not the α-toxin gene in experimental Staphylococcus aureus endocarditis.

BACKGROUND: Staphylococcus aureus produces numerous virulence factors but little is known about their in vivo regulation during an infection. METHODS: The production of capsule and α-toxin, and the expression of their respective genes, cap5 and hla, were analyzed by comparing CYL11481 (derivative of Newman) and its isogenic regulatory mutants in vitro. The temporal expression of cap5 and hla and the regulatory genes in vivo was carried out using a rat infective endocarditis model. RESULTS: In vitro analyses showed that capsule was positively regulated by MgrA, Agr, Sae, ArlR, and ClpC, and negatively by CodY and SbcDC. The α-toxin was positively regulated by MgrA, Agr, Sae, ArlR, and SbcDC but negatively by ClpC and CodY. In vivo analyses showed that cap5 expression correlated best with mgrA expression, whereas hla expression correlated best with sae expression. Mutation in mgrA drastically reduced cap5 expression in vivo. CONCLUSIONS: Our results suggest that, in vitro, Agr is the most important regulator for capsule and α-toxin production, as well as for cap5 transcription, but SaeR is the most critical for hla transcription. However, in vivo, MgrA is the major transcriptional regulator of capsule, but not α-toxin, whereas saeR expression correlates best with hla expression.