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Peptidoglycan hydrolases are promising alternatives for combating pathogens due to their specificity and potent bacteriolytic activity. In this study, a novel M23 peptidase from Streptococcus thermophilus NCTC10353, designated StM23, was discovered and characterized. It exhibited antibacterial activity against Listeria monocytogenes and other Gram-positive bacteria with meso -DAP-type peptidoglycan, including Bacillus subtilis and Bacillus cereus . To enhance StM23’s efficacy and specificity, a chimeric enzyme, StM23_CWT, was engineered by fusing its catalytic domain with a cell wall-targeting domain (CWT) from SpM23B, a peptidoglycan hydrolase found in  Staphylococcus pettenkoferi . The engineered chimera demonstrated expanded specificity, showing activity against Staphylococcus aureus and Enterococcus faecium . Its ability to disrupt L. monocytogenes cells was visualized by electron microscopy. The enzyme effectively disrupted biofilm structures and decontaminated surfaces like glass, stainless steel, and silicone, showcasing its industrial potential. Safety evaluations using zebrafish, moth larvae, and human cell models confirmed its non-toxic profile, supporting its broad applicability. Based on these findings, StM23_CWT is a novel and potent antimicrobial agent with significant potential to reduce the risk of listeriosis and control persistent pathogens.

Carboxy-terminal processing proteases (CTPs) occur in all three domains of life. In bacteria, some of them have been associated with virulence. However, the precise roles of bacterial CTPs are poorly understood, and few direct proteolytic substrates have been identified. One bacterial CTP is the CtpA protease of Pseudomonas aeruginosa , which is required for type III secretion system (T3SS) function and for virulence in a mouse model of acute pneumonia. Here, we have investigated the function of CtpA in P. aeruginosa and identified some of the proteins it cleaves. We discovered that CtpA forms a complex with a previously uncharacterized protein, which we have named LbcA ( l ipoprotein b inding partner of C tpA). LbcA is required for CtpA activity in vivo and promotes its activity in vitro . We have also identified four proteolytic substrates of CtpA, all of which are uncharacterized proteins predicted to cleave the peptide cross-links within peptidoglycan. Consistent with this, a ctpA null mutant was found to have fewer peptidoglycan cross-links than the wild type and grew slowly in salt-free medium. Intriguingly, the accumulation of just one of the CtpA substrates was required for some Δ ctpA mutant phenotypes, including the defective T3SS. We propose that LbcA-CtpA is a proteolytic complex in the P. aeruginosa cell envelope, which controls the activity of several peptidoglycan cross-link hydrolases by degrading them. Furthermore, based on these and other findings, we suggest that many bacterial CTPs might be similarly controlled by partner proteins as part of a widespread mechanism to control peptidoglycan hydrolase activity. IMPORTANCE Bacterial carboxy-terminal processing proteases (CTPs) are widely conserved and have been associated with the virulence of several species. However, their roles are poorly understood, and few direct substrates have been identified in any species. Pseudomonas aeruginosa is an important human pathogen in which one CTP, known as CtpA, is required for type III secretion system function and for virulence. This work provides an important advance by showing that CtpA works with a previously uncharacterized binding partner to degrade four substrates. These substrates are all predicted to hydrolyze peptidoglycan cross-links, suggesting that the CtpA complex is an important control mechanism for peptidoglycan hydrolysis. This is likely to emerge as a widespread mechanism used by diverse bacteria to control some of their peptidoglycan hydrolases. This is significant, given the links between CTPs and virulence in several pathogens and the importance of peptidoglycan remodeling to almost all bacterial cells. Bacterial carboxy-terminal processing proteases (CTPs) are widely conserved and have been associated with the virulence of several species. However, their roles are poorly understood, and few direct substrates have been identified in any species. Pseudomonas aeruginosa is an important human pathogen in which one CTP, known as CtpA, is required for type III secretion system function and for virulence. This work provides an important advance by showing that CtpA works with a previously uncharacterized binding partner to degrade four substrates. These substrates are all predicted to hydrolyze peptidoglycan cross-links, suggesting that the CtpA complex is an important control mechanism for peptidoglycan hydrolysis. This is likely to emerge as a widespread mechanism used by diverse bacteria to control some of their peptidoglycan hydrolases. This is significant, given the links between CTPs and virulence in several pathogens and the importance of peptidoglycan remodeling to almost all bacterial cells.

Bacterial cell division is a central process that requires exquisite orchestration of both the cell wall biosynthetic and lytic machineries. The essential membrane complex FtsEX, widely conserved across bacteria, plays a central role by recruiting proteins to the divisome apparatus and by regulating periplasmic muralytic activity from the cytosol. FtsEX is a member of the type VII family of the ABC-superfamily, but instead of being a transporter, it couples the ATP hydrolysis catalyzed by FtsE to mechanically transduce a conformational signal that provokes the activation of peptidoglycan (PG) hydrolases. So far, no structural information is available for FtsE. Here, we provide the structural characterization of FtsE, confirming its ATPase nature and revealing regions with high structural plasticity which are key for FtsE binding to FtsX. The complementary binding region in FtsX has also been identified and validated in vivo . Our results provide evidence on how the difference between the ATP/ADP-bound states in FtsE would dramatically alter the interaction of FtsEX with the PG hydrolase PcsB in pneumococcal division. FtsEX is a membrane complex widely conserved across diverse bacterial genera and involved in critical processes such as recruitment of division proteins and in spatial and temporal regulation of muralytic activity during cell division or sporulation. FtsEX is a member of the ABC transporter superfamily. The component FtsX is an integral membrane protein, whereas FtsE is an ATPase and is required for the transmission of a conformational signal from the cytosol through the membrane to regulate the activity of cell wall hydrolases in the periplasm. Both proteins are essential in the major human respiratory pathogenic bacterium Streptococcus pneumoniae , and FtsX interacts with the modular peptidoglycan hydrolase PcsB at the septum. Here, we report high-resolution structures of pneumococcal FtsE bound to different nucleotides. Structural analysis revealed that FtsE contains all the conserved structural motifs associated with ATPase activity and afforded interpretation of the in vivo dimeric arrangement in both the ADP and ATP states. Interestingly, three specific FtsE regions with high structural plasticity were identified that shape the cavity in which the cytosolic region of FtsX would be inserted. The residues corresponding to the FtsX coupling helix, responsible for contacting FtsE, were identified and validated by in vivo mutagenesis studies showing that this interaction is essential for cell growth and proper morphology. IMPORTANCE Bacterial cell division is a central process that requires exquisite orchestration of both the cell wall biosynthetic and lytic machineries. The essential membrane complex FtsEX, widely conserved across bacteria, plays a central role by recruiting proteins to the divisome apparatus and by regulating periplasmic muralytic activity from the cytosol. FtsEX is a member of the type VII family of the ABC-superfamily, but instead of being a transporter, it couples the ATP hydrolysis catalyzed by FtsE to mechanically transduce a conformational signal that provokes the activation of peptidoglycan (PG) hydrolases. So far, no structural information is available for FtsE. Here, we provide the structural characterization of FtsE, confirming its ATPase nature and revealing regions with high structural plasticity which are key for FtsE binding to FtsX. The complementary binding region in FtsX has also been identified and validated in vivo . Our results provide evidence on how the difference between the ATP/ADP-bound states in FtsE would dramatically alter the interaction of FtsEX with the PG hydrolase PcsB in pneumococcal division.

Staphylococcus aureus is a major threat due to its ability to generate antibiotic-resistant strains. Understanding S. aureus division is therefore of great importance, but we do not know how septum formation is coordinated with cell scission. Previous works have shown that both mechanical stress and autolysin activity play key roles in scission, but it is unclear how mechanical and biochemical cues work together. Here, we propose a “mechanical trigger” model for the interplay between mechanical stress and autolysin activation. We use mathematical modeling to show that stress decreases in the S. aureus cell wall close to the division site as the septum is formed, and we propose that this could trigger autolysin activity. Our model explains reports of diverse division outcomes in the presence of mutations and antibiotics and points to a general link between cell geometry and antibiotic resistance.

FtsX is a ubiquitous bacterial integral membrane protein involved in cell division that regulates the activity of peptidoglycan (PG) hydrolases. FtsX is representative of a large group of ABC3 superfamily proteins that function as “mechanotransmitters,” proteins that relay signals from the inside to the outside of the cell. Here, we present a structural characterization of the large extracellular loop, ECL1, of FtsX from the opportunistic human pathogen S. pneumoniae . We show the molecular nature of the direct interaction between the peptidoglycan hydrolase PcsB and FtsX and demonstrate that this interaction is essential for cell viability. As such, FtsX represents an attractive, conserved target for the development of new classes of antibiotics. Streptococcus pneumoniae is a leading killer of infants and immunocompromised adults and has become increasingly resistant to major antibiotics. Therefore, the development of new antibiotic strategies is desperately needed. Targeting bacterial cell division is one such strategy, specifically by targeting proteins that are essential for the synthesis and breakdown of peptidoglycan. One complex important to this process is FtsEX. FtsEX comprises a cell division-regulating integral membrane protein (FtsX) and a cytoplasmic ATPase (FtsE) that resembles an ATP-binding cassette (ABC) transporter. Here, we present nuclear magnetic resonance (NMR) solution structural and crystallographic models of the large extracellular domain of FtsX, denoted extracellular loop 1 (ECL1). The structure of ECL1 reveals an upper extended β-hairpin and a lower α-helical lobe, each extending from a mixed α-β core. The helical lobe mediates a physical interaction with the peptidoglycan hydrolase PcsB via the coiled-coil domain of PcsB (PscB CC ). Characterization of S. pneumoniae strain D39-derived strains harboring mutations in the α-helical lobe shows that this subdomain is essential for cell viability and required for proper cell division of S. pneumoniae . IMPORTANCE FtsX is a ubiquitous bacterial integral membrane protein involved in cell division that regulates the activity of peptidoglycan (PG) hydrolases. FtsX is representative of a large group of ABC3 superfamily proteins that function as “mechanotransmitters,” proteins that relay signals from the inside to the outside of the cell. Here, we present a structural characterization of the large extracellular loop, ECL1, of FtsX from the opportunistic human pathogen S. pneumoniae . We show the molecular nature of the direct interaction between the peptidoglycan hydrolase PcsB and FtsX and demonstrate that this interaction is essential for cell viability. As such, FtsX represents an attractive, conserved target for the development of new classes of antibiotics.

Comparative transcriptome analysis and de novo short-read assembly of S. aureus Newman strains revealed significant transcriptional changes in response to the exposure to triple-acting staphylolytic peptidoglycan hydrolase (PGH) 1801. Most altered transcriptions were associated with the membrane, cell wall, and related genes, including amidase, peptidase, holin, and phospholipase D/transphosphatidylase. The differential expression of genes obtained from RNA-seq was confirmed by reverse transcription quantitative PCR. Moreover, some of these gene expression changes were consistent with the observed structural perturbations at the DNA and RNA levels. These structural changes in the genes encoding membrane/cell surface proteins and altered gene expressions are the candidates for resistance to these novel antimicrobials. The findings in this study could provide insight into the design of new antimicrobial agents.

Staphylococcus aureus is an important human and livestock pathogen that is well-protected against environmental insults by a thick cell wall. Accordingly, the wall is a major target of present-day antimicrobial therapy. Unfortunately, S. aureus has mastered the art of antimicrobial resistance, as underscored by the global spread of methicillin-resistant S. aureus (MRSA). The major cell wall component is peptidoglycan. Importantly, the peptidoglycan network is not only vital for cell wall function, but it also represents a bacterial Achilles’ heel. In particular, this network is continuously opened by no less than 18 different peptidoglycan hydrolases (PGHs) encoded by the S. aureus core genome, which facilitate bacterial growth and division. This focuses attention on the specific functions executed by these enzymes, their subcellular localization, their control at the transcriptional and post-transcriptional levels, their contributions to staphylococcal virulence and their overall importance in bacterial homeostasis. As highlighted in the present review, our understanding of the different aspects of PGH function in S. aureus has been substantially increased over recent years. This is important because it opens up new possibilities to exploit PGHs as innovative targets for next-generation antimicrobials, passive or active immunization strategies, or even to engineer them into effective antimicrobial agents.

SignificancePeptidoglycan, the main structural component of the bacterial cell wall, is made of glycan strands cross-linked by short peptides. It has long been assumed that class A penicillin-binding proteins (PBPs) are the only enzymes capable of synthesizing glycan strands from lipid II. Recently, however, it was discovered that two non-PBP proteins, FtsW and RodA, constitute the core peptidoglycan polymerizing enzymes of the divisome and elongasome, respectively. What, then, is the role of class A PBPs in the construction of the bacterial cell wall? In contrast to previous assumptions, our results strongly suggest that class A PBPs are not an intrinsic part of the divisome and elongasome but have important autonomous roles in construction of the fully mature bacterial cell wall. In oval-shaped Streptococcus pneumoniae, septal and longitudinal peptidoglycan syntheses are performed by independent functional complexes: the divisome and the elongasome. Penicillin-binding proteins (PBPs) were long considered the key peptidoglycan-synthesizing enzymes in these complexes. Among these were the bifunctional class A PBPs, which are both glycosyltransferases and transpeptidases, and monofunctional class B PBPs with only transpeptidase activity. Recently, however, it was established that the monofunctional class B PBPs work together with transmembrane glycosyltransferases (FtsW and RodA) from the shape, elongation, division, and sporulation (SEDS) family to make up the core peptidoglycan-synthesizing machineries within the pneumococcal divisome (FtsW/PBP2x) and elongasome (RodA/PBP2b). The function of class A PBPs is therefore now an open question. Here we utilize the peptidoglycan hydrolase CbpD that targets the septum of S. pneumoniae cells to show that class A PBPs have an autonomous role during pneumococcal cell wall synthesis. Using assays to specifically inhibit the function of PBP2x and FtsW, we demonstrate that CbpD attacks nascent peptidoglycan synthesized by the divisome. Notably, class A PBPs could process this nascent peptidoglycan from a CbpD-sensitive to a CbpD-resistant form. The class A PBP-mediated processing was independent of divisome and elongasome activities. Class A PBPs thus constitute an autonomous functional entity which processes recently formed peptidoglycan synthesized by FtsW/PBP2×. Our results support a model in which mature pneumococcal peptidoglycan is synthesized by three functional entities, the divisome, the elongasome, and bifunctional PBPs. The latter modify existing peptidoglycan but are probably not involved in primary peptidoglycan synthesis.

Bacterial cell wall peptidoglycan is essential, maintaining both cellular integrity and morphology, in the face of internal turgor pressure. Peptidoglycan synthesis is important, as it is targeted by cell wall antibiotics, including methicillin and vancomycin. Here, we have used the major human pathogen Staphylococcus aureus to elucidate both the cell wall dynamic processes essential for growth (life) and the bactericidal effects of cell wall antibiotics (death) based on the principle of coordinated peptidoglycan synthesis and hydrolysis. The death of S. aureus due to depletion of the essential, two-component and positive regulatory system for peptidoglycan hydrolase activity (WalKR) is prevented by addition of otherwise bactericidal cell wall antibiotics, resulting in stasis. In contrast, cell wall antibiotics kill via the activity of peptidoglycan hydrolases in the absence of concomitant synthesis. Both methicillin and vancomycin treatment lead to the appearance of perforating holes throughout the cell wall due to peptidoglycan hydrolases. Methicillin alone also results in plasmolysis and misshapen septa with the involvement of the major peptidoglycan hydrolase Atl, a process that is inhibited by vancomycin. The bactericidal effect of vancomycin involves the peptidoglycan hydrolase SagB. In the presence of cell wall antibiotics, the inhibition of peptidoglycan hydrolase activity using the inhibitor complestatin results in reduced killing, while, conversely, the deregulation of hydrolase activity via loss of wall teichoic acids increases the death rate. For S. aureus, the independent regulation of cell wall synthesis and hydrolysis can lead to cell growth, death, or stasis, with implications for the development of new control regimes for this important pathogen.

The cell wall of Gram-positive bacteria is a complex assemblage of glycopolymers and proteins. It consists of a thick peptidoglycan sacculus that surrounds the cytoplasmic membrane and that is decorated with teichoic acids, polysaccharides, and proteins. It plays a major role in bacterial physiology since it maintains cell shape and integrity during growth and division; in addition, it acts as the interface between the bacterium and its environment. Lactic acid bacteria (LAB) are traditionally and widely used to ferment food, and they are also the subject of more and more research because of their potential health-related benefits. It is now recognized that understanding the composition, structure, and properties of LAB cell walls is a crucial part of developing technological and health applications using these bacteria. In this review, we examine the different components of the Gram-positive cell wall: peptidoglycan, teichoic acids, polysaccharides, and proteins. We present recent findings regarding the structure and function of these complex compounds, results that have emerged thanks to the tandem development of structural analysis and whole genome sequencing. Although general structures and biosynthesis pathways are conserved among Gram-positive bacteria, studies have revealed that LAB cell walls demonstrate unique properties; these studies have yielded some notable, fundamental, and novel findings. Given the potential of this research to contribute to future applied strategies, in our discussion of the role played by cell wall components in LAB physiology, we pay special attention to the mechanisms controlling bacterial autolysis, bacterial sensitivity to bacteriophages and the mechanisms underlying interactions between probiotic bacteria and their hosts.