Biology subjects

Barbuti, M. D.

Publications and source records attributed to Barbuti, M. D..

4 recordsLinked to original sources

bioRxiv2025

Cell splitting in Staphylococcus aureus is controlled by an adaptor protein facilitating degradation of a peptidoglycan hydrolase

Regulated protein degradation by Clp proteases is a highly conserved post-translational control mechanism in bacteria. In Staphylococcus aureus, the ClpXP complex targets the peptidoglycan hydrolase Sle1, maintaining a tightly regulated balance between peptidoglycan biosynthesis and hydrolysis, which is required to ensure proper cell splitting without compromising cell integrity. {beta}-lactams antibiotics disturb this balance, leading to their bactericidal effects. The mechanism underlying the specific targeting of Sle1 by the conserved ClpXP complex remains unknown. From a genome-wide screen for determinants of penicillin G susceptibility in S. aureus, we here identify the uncharacterized protein CxaR (for ClpXP-associated autolytic regulator). Growth defects, premature cell splitting, and increased cell lysis were observed in the absence of CxaR. Interestingly, these defects were mitigated by sublethal concentrations of {beta}-lactams. Through sequencing cxaR suppressor mutants, followed by immunoblotting, we show that the cxaR phenotypes are caused by excessive Sle1 accumulation. Indeed, exposure to {beta}-lactams reduces Sle1 levels, thereby rescuing the cells lacking CxaR. Furthermore, in vivo protein-protein interaction assays demonstrated that CxaR directly interacts with both ClpXP and Sle1, whereas no direct interaction was detected between Sle1 and ClpX. In line with this, CxaR was found to co-localize with ClpX adjacent to the septum. Taken together, these findings reveal that CxaR is a new regulatory factor controlling staphylococcal cell splitting by acting as an adaptor protein for controlled ClpXP-mediated degradation of Sle1.

microbiology↗

bioRxiv2025

Temperature-dependent regulation of bacterial cell division hydrolases by the coordinated action of a regulatory RNA and the ClpXP protease

A defining feature of bacteria is the peptidoglycan cell wall which provides structural integrity and prevents osmotic lysis. While peptidoglycan hydrolases are required for daughter cell separation, dysregulated cell wall degradation may result in cell lysis. The mechanisms allowing bacteria to control these deadly enzymes in response to environmental changes remain incompletely understood. Here, we find that in Staphylococcus aureus, temperature-dependent regulation of such hydrolases occurs by the coordinated action of a CHAP domain-specific regulatory RNA and the ClpXP protease. Using a proteomics approach, we identify a hitherto uncharacterized ClpXP controlled autolysin, CxcA, with a catalytic CHAP domain and show that it contributes to separation of daughter cells. CxcA is positively controlled by a non-coding RNA, named Rbc1 (for RNA binding to CHAP domain) transcribed from the antisense strand of cxcA. Notably, Rbc1 is capable of base pairing with RNAs encoding the CHAP domains of numerous cell wall hydrolases and we show that Rbc1 works in trans to upregulate the cell division hydrolase Sle1. Specifically, Rbc1 functions as a thermosensor allowing for upregulation of CxcA and Sle1 at low temperature where daughter cell separation is impeded. Interestingly, the Rbc1-mediated up-regulation of CxcA and Sle1 does not involve mRNA stabilization or increased translation; instead, Rbc1 depletion increases ClpXP-mediated degradation. In conclusion, we identify a novel cell division hydrolase that is highly conserved in Staphylococci and show that it is co-regulated with enzymes containing the catalytic CHAP domain via transcriptional regulation, an RNA-RNA temperature sensory mechanism and the ClpXP protease.

microbiology↗

bioRxiv2024

The Spx stress regulator confers high-level β-lactam resistance and decreases susceptibility to last-line antibiotics in methicillin resistant Staphylococcus aureus

Infections caused by methicillin resistant Staphylococcus aureus (MRSA) are a leading cause of mortality worldwide. MRSA have acquired resistance to next generation {beta}-lactam antibiotics through the horizontal acquisition of the mecA resistance gene. Development of high resistance is, however, often associated with additional mutations in a set of chromosomal core genes, known as potentiators which through poorly described mechanisms enhance resistance. The yjbH gene was recently identified as a hot spot for adaptive mutations during severe infections. Here, we show that inactivation of yjbH increased {beta}-lactam MICs up to 16-folds and transformed MRSA cells with low level of resistance to being homogenously highly resistant to {beta}-lactams. The yjbH gene encodes an adaptor protein that targets the transcriptional stress regulator Spx for degradation by the ClpXP protease. Using CRISPRi to knock down spx transcription, we unambiguously linked hyper-resistance to accumulation of Spx. Spx was previously proposed to be essential, however, our data indicate that Spx is dispensable for growth at 37{degrees}C but becomes essential in the presence of antibiotics with various targets. On the other hand, high Spx levels bypassed the role of PBP4 in {beta}-lactam resistance and broadly decreased MRSA susceptibility to compounds targeting the cell wall or the cell membrane including vancomycin, daptomycin, and nisin. Strikingly, Spx potentiated resistance independently of its redox sensing switch. Collectively, our study identifies a general stress pathway that, in addition to promoting the development of high-level, broad-spectrum {beta}-lactam resistance, also decreases MRSA susceptibility to critical antibiotics of last resort.

microbiology↗

bioRxiv2023

The function of CozE proteins is linked to lipoteichoic acid biosynthesis in Staphylococcus aureus

To maintain cell integrity and facilitate cell division in Staphylococcus aureus, a well-coordinated interplay between membrane biogenesis, peptidoglycan formation, and teichoic acid synthesis is crucial. However, the molecular mechanisms and regulatory pathways that underpin their coordination are still poorly understood. CozE constitute a conserved family of membrane proteins implicated in cell division via regulation of penicillin binding proteins. It has been shown that the two staphylococcal cozE genes (cozEa and cozEb) constitute a synthetic lethal gene pair. Depletion of CozEa and CozEb simultaneously in S. aureus resulted in severely defective cell division phenotypes, reminiscent of cell lacking lipoteichoic acid (LTA). Indeed, we demonstrate that there is an intricate interplay between CozE, biosynthesis of LTA, and membrane homeostasis in S. aureus. By screening for potential genetic links, we establish that there is synthetic lethal relationship between CozE and UgtP, the enzyme synthesizing the LTA glycolipid anchor Glc2DAG. On the contrary, in cells lacking LtaA, the flippase of Glc2DAG, the essentiality of CozEa and CozEb was alleviated. Furthermore, by immunoblotting, we found that CozEb plays a unique role in controlling LTA polymer length and stability. Using reconstituted proteoliposomes, we also demonstrated that CozE proteins modulate the glycolipid flipping activity of LtaA in vitro. Together, the results demonstrate a new function of CozE proteins, facilitating proper membrane homeostasis and LTA biosynthesis in S. aureus.

microbiology↗