Biology subjects

Brignoli, T.

Publications and source records attributed to Brignoli, T..

7 recordsLinked to original sources

bioRxiv2025

Phosphate sensing by PhoPR regulates the cytotoxicity of Staphylococcus aureus

Staphylococcus aureus has evolved a complex regulatory network to coordinate expression of virulence factors, including cytolytic toxins, with host environmental signals. Central to this network are two-component systems, in which a histidine kinase senses an external signal and activates a response regulator via phosphorylation, leading to changes in gene expression. Using a comprehensive screen of transposon mutants in each of the non-essential histidine kinase and response regulatory genes in S. aureus, we demonstrate that 11 of these 16 systems regulate cytotoxicity. Further characterisation of a phoP mutant revealed that its impact on cytotoxicity is mediated through the Agr quorum-sensing system. Notably, we found that unphosphorylated PhoP is an activator of Agr activity, while phosphorylated PhoP also acts as a weak activator of Agr activity in high phosphate environments but as a repressor in low phosphate environments. Overall, we have demonstrated that phosphate sensing through PhoPR is a novel regulator of cytotoxicity in S. aureus. Moreover, our study challenges the canonical model of TCSs as simple on/off systems and highlights the importance of unphosphorylated response regulators in gene regulation. ImportanceThe production of cytolytic toxins is the major means by which bacterial pathogens damage host tissue and cause disease. Understanding the activity and regulation of these toxins is critical for the identification of means to block them and prevent the development of disease. In this study we focused on a specific regulatory mechanism, the two-component systems (TCSs), that enable bacteria to sense their environment and adapt accordingly. In the traditional model of a TCS, a response regulator (RR) is phosphorylated by a histidine kinase (HK), which enables it to activate or repress expression of target genes, which may include toxins or regulators of toxins. We found that 11 of S. aureus 16 TCSs affect toxin production, highlighting that S. aureus integrates a broad range of environmental cues to regulate toxicity. We focused on one of these TCSs, the PhoPR system and found that sensing of inorganic phosphate is a novel regulator of cytotoxicity in S. aureus. Furthermore, we found that the RR of this system acts as a strong activator of toxicity in its unphosphorylated form, challenging the traditional model of a TCS as only active upon signal activation.

microbiology↗

bioRxiv2023

Lipoteichoic acid biosynthesis by Staphylococcus aureus is controlled by the MspA protein through competitive interference.

Staphylococcus aureus produces a plethora of virulence factors critical to its ability to establish an infection and cause disease. We have previously characterised a small membrane protein, MspA, which has pleiotropic affects on virulence and contributes to S. aureus pathogenicity in vivo. Here we report that mspA inactivation triggers overaccumulation of the essential cell wall component lipoteichoic acid (LTA) which, in turn, decreases autolytic activity and leads to increased cell size due to a delay in cell separation. We show that MspA directly interacts with the LTA synthesis enzymes UgtP, LtaA and LtaS, and competitively interferes with the association between LtaA and LtaS. While complementation of the mspA mutant with wild-type mspA reduces the amount of LTA, expression of a mutated version of MspA that does not interfere with the interactions between LtaA and LtaS does not. We suggest that MspA contributes to maintaining a physiological level of LTA in the cell wall by interacting with the LTA synthetic enzymes. In conclusion, this study uncovers the critical role of the MspA protein in regulating cell envelope biosynthesis and pathogenicity. IMPORTANCEThe S. aureus cell envelope, comprising of the cytoplasmic membrane, a thick peptidoglycan layer and the anionic polymers lipoteichoic acid and wall teichoic acids, is fundamental for bacterial growth and division, as well as being the main interface between the pathogen and the host. It has become increasingly apparent that the synthesis and turnover of cell envelope components also affect the virulence of S. aureus. In this study, we show that MspA, a novel effector of S. aureus virulence, contributes to the maintenance of normal levels of lipoteichoic acid in the cell wall, with implications on cell cycle and size. These findings further our understanding of the connections between envelope synthesis and pathogenicity and suggests MspA as a novel a target for therapeutic intervention.

microbiology↗

bioRxiv2023

Extensive re-modelling of the cell wall during the development of Staphylococcus aureus bacteraemia

Introductory Paragraph / AbstractThe bloodstream represents a hostile environment that bacteria must overcome to cause bacteraemia. To understand how the major human pathogen Staphylococcus aureus manages this we have utilised a functional genomics approach to identify a number of new loci that affect the ability of the bacteria to survive exposure to serum, the critical first step in the development of bacteraemia. The expression of one of these genes, tcaA, was found to be induced upon exposure to serum, and we show that it is involved in the elaboration of a critical virulence factor, the wall teichoic acids (WTA), within the cell envelope. The activity of the TcaA protein alters the sensitivity of the bacteria to cell wall attacking agents, including antimicrobial peptides, human defence fatty acids, and several antibiotics. This protein also affects the autolytic activity and lysostaphin sensitivity of the bacteria, suggesting that in addition to changing WTA abundance in the cell envelope, it also plays a role in peptidoglycan crosslinking. With TcaA rendering the bacteria more susceptible to serum killing, while simultaneously increasing the abundance of WTA in the cell envelope, it was unclear what effect this protein may have during infection. To explore this, we examined human data and performed murine experimental infections. Collectively, our data suggests that whilst mutations in tcaA are selected for during bacteraemia, this protein positively contributes to the virulence of S. aureus through its involvement in altering the cell wall architecture of the bacteria, a process that appears to play a key role in the development of bacteraemia.

microbiology↗

bioRxiv2022

Wall teichoic acids facilitate the release of toxins from the surface of Staphylococcus aureus.

A major feature of the pathogenicity of Staphylococcus aureus is its ability to secrete cytolytic toxins. This process involves the translocation of the toxins from the cytoplasm, through the bacterial membrane and the cell wall to the external environment. The process of their movement through the membrane is relatively well defined, involving both general and toxin-specific secretory systems. Movement of the toxins through the cell wall was considered to involve the passive diffusion of the proteins through the porous cell wall structures, however, recent work suggests that this is more complex, and here we demonstrate a role for the wall teichoic acids (WTA) in this process. Utilising a genome-wide association approach we identified a polymorphism in the locus encoding the WTA biosynthetic machinery as associated with the cytolytic activity of the bacteria. We verified this association using an isogenic mutant set and found that WTA is required for the release of several cytolytic toxins from the bacterial cells. We show this effect is mediated by a change in the electrostatic charge across the cell envelope that results from the loss of WTA. As a major target for the development of novel therapeutics, it is important that we fully understand the entire process of cytolytic toxin production and release. These findings open up a new aspect to this process that requires in-depth investigation, while also demonstrating that clinical isolates can utilise WTA production to vary their cytotoxicity, thereby altering their pathogenic capabilities. ImportanceThe production and release of cytolytic toxins is a critical aspect to the pathogenicity of many bacterial pathogens. In this study we demonstrate a role for wall teichoic acids, molecules that are anchored to the peptidoglycan of the bacterial cell wall, in the release of toxins from S. aueus cells into the extracellular environment. Our findings suggest this effect is mediated by a gradient of electrostatic charge the presence of the negatively charged WTA molecules create across the cell envelope. This work brings an entirely new aspect to our understanding of the cytotoxicity of S. aureus and demonstrates a further means by which this major human pathogen can adapt its pathogenic capabilities.

microbiology↗

bioRxiv2021

Self-sensitisation of Staphylococcus aureus to the antimicrobial factors found in human blood.

Introductory ParagraphFor opportunistic pathogens, the switch from a commensal to an invasive lifestyle is often considered an accidental event. But with plentiful opportunity, what leads one accidental event to result in an invasive infection, and another not to? And how much of this apparent stochasticity is driven by bacterial factors? To answer these questions, here we focussed on the major human pathogen Staphylococcus aureus, which can both reside asymptomatically as a member of our respiratory microbiome, or become invasive and cause infections as severe as bacteraemia. Survival upon exposure to the antibacterial factors found in serum is a critical aspect of their ability to cause bacteraemia, and across a collection of 300 clinical isolates we found there to be significant variability in this capability. Utilising a GWAS approach we have uncovered the genetic basis of much of this variability through the identification and functional verification of a number of new polymorphic loci that affect serum survival: tcaA, tarK, gntR, ilvC, arsB, yfhO, and pdhD. The expression of one of these genes, tcaA, was found to be induced upon exposure to serum, while simultaneously enhancing the sensitivity of S. aureus to serum through a process involving the ligation of wall teichoic acids into the cell wall. As blood-stage infections are a transmission dead-end for the bacteria, that S. aureus actively responds to serum to produce a protein which specifically limits their ability to survive in this environment demonstrates that the switch from the commensal to the invasive lifestyle is complex, and that TcaA may contribute to the long-term success of S. aureus by restricting the bacteria to their more readily transmissible commensal state.

microbiology↗

bioRxiv2021

The MpsB protein contributes to both the toxicity and immune evasion capacity of Staphylococcus aureus.

Understanding the role specific bacterial factors play in the development of severe disease in humans is critical if new approaches to tackle such infections are to be developed. In this study we focus on genes we have found to be associated with patient outcome following bacteraemia caused by the major human pathogen Staphylococcus aureus. By examining the contribution these genes make to the ability of the bacteria to survive exposure to the antibacterial factors found in serum, we identify three novel serum resistance associated genes, mdeA, mpsB and yycH. Detailed analysis of an MpsB mutant supports its previous association with the slow growing SCV phenotype of S. aureus, and we demonstrate that the effect this mutation has on membrane potential prevents the activation of the Agr quorum sensing system, and as a consequence the mutant bacteria do not produce cytolytic toxins. Given the importance of both toxin production and immune evasion to the ability of S. aureus to cause disease, we believe these findings explain the role of the mpsB gene as a mortality-associate locus during human disease.

microbiology↗

bioRxiv2021

A Functional Menadione Biosynthesis Pathway is Required for Capsule Production by Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen that utilises a wide array of pathogenic and immune evasion strategies to cause disease. One immune evasion strategy, common to many bacterial pathogens, is the ability of S. aureus to produce a capsule that protects the bacteria from several aspects of the human immune system. To identify novel regulators of capsule production by S. aureus we applied a genome wide association study (GWAS) to a collection of 300 bacteraemia isolates that represent the two major MRSA clones in UK and Irish hospitals: CC22 and CC30. One of the loci associated with capsule production, the menD gene, encodes an enzyme critical to the biosynthesis of menadione. Mutations in this gene that result in menadione auxotrophy induce the slow growing small-colony variant (SCV) form of S. aureus often associated with chronic infections due to their increased resistance to antibiotics and ability to survive inside phagocytes. Utilising such an SCV we functionally verified this association between menD and capsule production. Although the clinical isolates with polymorphisms in the menD gene in our collections had no apparent growth defects, they were more resistant to gentamicin when compared to those with the wild-type menD gene. Our work suggests that menadione plays a critical role in the production of the S. aureus capsule, and that amongst clinical isolates polymorphisms exist in the menD gene that confer the characteristic increased gentamicin resistance, but not the major growth defect associated with SCV phenotype.

microbiology↗