Search

ABSTRACT Escherichia coli has many periplasmic hydrolases to degrade and modify peptidoglycan (PG). However, the redundancy of eight PG endopeptidases makes it challenging to define specific roles to individual enzymes. Therefore, the cellular role of PBP7 (encoded by pbpG) is not clearly defined. In this work, we show that PBP7 localizes in the lateral cell envelope and at midcell. The C‐terminal α‐helix of PBP7 is crucial for midcell localization but not for its activity, which is dispensable for this localization. Additionally, midcell localization of PBP7 relies on the assembly of FtsZ up to FtsN in the divisome, and on the activity of PBP3. PBP7 was found to affect the assembly timing of FtsZ and FtsN in the divisome. The absence of PBP7 slows down the assembly of FtsN at midcell. The ΔpbpG mutant exhibited a weaker incorporation of the fluorescent D‐amino acid HADA, reporting on transpeptidase activity, compared to wild‐type cells. This could indicate reduced PG synthesis at the septum of the ΔpbpG strain, explaining the slower accumulation of FtsN and suggesting that endopeptidase‐mediated PG cleavage may be a rate‐limiting step for septal PG synthesis. [ABSTRACT FROM AUTHOR]

Orchestrated action of peptidoglycan (PG) synthetases and hydrolases is vital for bacterial growth and viability. Although the function of several PG synthetases and hydrolases is well understood, the function, regulation, and mechanism of action of PG hydrolases characterised as lysostaphin-like endopeptidases have remained elusive. Many of these M23 family members can hydrolyse glycyl-glycine peptide bonds and show lytic activity against Staphylococcus aureus whose PG contains a pentaglycine bridge, but their exact substrate specificity and hydrolysed bonds are still vaguely determined. In this work, we have employed NMR spectroscopy to study both the substrate specificity and the bond cleavage of the bactericide lysostaphin and the S. aureus PG hydrolase LytM. Yet, we provide substrate-level evidence for the functional role of these enzymes. Indeed, our results show that the substrate specificities of these structurally highly homologous enzymes are similar, but unlike observed earlier both LytM and lysostaphin prefer the D-Ala-Gly cross-linked part of mature peptidoglycan. However, we show that while lysostaphin is genuinely a glycyl-glycine hydrolase, LytM can also act as a D-alanyl-glycine endopeptidase.

During bacterial cell growth, hydrolases cleave peptide cross-links between strands of the peptidoglycan sacculus to allow new strand insertion. The Pseudomonas aeruginosa carboxyl-terminal processing protease (CTP) CtpA regulates some of these hydrolases by degrading them. CtpA assembles as an inactive hexamer composed of a trimer-of-dimers, but its lipoprotein binding partner LbcA activates CtpA by an unknown mechanism. Here, we report the cryo-EM structures of the CtpA–LbcA complex. LbcA has an N-terminal adaptor domain that binds to CtpA, and a C-terminal superhelical tetratricopeptide repeat domain. One LbcA molecule attaches to each of the three vertices of a CtpA hexamer. LbcA triggers relocation of the CtpA PDZ domain, remodeling of the substrate binding pocket, and realignment of the catalytic residues. Surprisingly, only one CtpA molecule in a CtpA dimer is activated upon LbcA binding. Also, a long loop from one CtpA dimer inserts into a neighboring dimer to facilitate the proteolytic activity. This work has revealed an activation mechanism for a bacterial CTP that is strikingly different from other CTPs that have been characterized structurally. Synopsis: The Pseudomonas aeruginosa cell envelope protease CtpA is activated by the LbcA lipoprotein to degrade cell-wall cross-link hydrolases. Here, structural, biochemical and in vivo approaches show how this activation occurs, revealing a unique mechanism compared to related proteases. CtpA alone is inactive and exists mainly as a dimer at physiological temperature. After LbcA binds to a CtpA dimer, the LbcA-CtpA complex oligomerizes into a CtpA hexamer with three bound LbcA proteins. Only one CtpA molecule is activated within each CtpA dimeric unit. The binding of LbcA triggers a series of conformational changes in the activated CtpA, including a shift of the PDZ domain, realignment of the catalytic site, and remodeling of substrate binding pocket. Structural, biochemical and in vivo approaches reveal a unique mechanism for cell envelope CtpA protease activation by its lipoprotein binding partner LbcA. [ABSTRACT FROM AUTHOR]

ABSTRACTGram-negative bacteria have a thin peptidoglycan layer between the cytoplasmic and outer membranes protecting the cell from osmotic challenges. Hydrolases of this structure are needed to cleave bonds to allow the newly synthesized peptidoglycan strands to be inserted by synthases. These enzymes need to be tightly regulated and their activities coordinated to prevent cell lysis. To better understand this process in Escherichia coli, we probed the genetic interactions of mrcA (encodes PBP1A) and mrcB (encodes PBP1B) with genes encoding peptidoglycan amidases and endopeptidases in envelope stress conditions. Our extensive genetic interaction network analysis revealed relatively few combinations of hydrolase gene deletions with reduced fitness in the absence of PBP1A or PBP1B, showing that none of the amidases or endopeptidases is strictly required for the functioning of one of the class A PBPs. This illustrates the robustness of the peptidoglycan growth mechanism. However, we discovered that the fitness of ∆mrcB cells is significantly reduced under high salt stress and in vitro activity assays suggest that this phenotype is caused by a reduced peptidoglycan synthesis activity of PBP1A at high salt concentration.IMPORTANCEEscherichia coli and many other bacteria have a surprisingly high number of peptidoglycan hydrolases. These enzymes function in concert with synthases to facilitate the expansion of the peptidoglycan sacculus under a range of growth and stress conditions. The synthases PBP1A and PBP1B both contribute to peptidoglycan expansion during cell division and growth. Our genetic interaction analysis revealed that these two penicillin-binding proteins (PBPs) do not need specific amidases, endopeptidases, or lytic transglycosylases for function. We show that PBP1A and PBP1B do not work equally well when cells encounter high salt stress and demonstrate that PBP1A alone cannot provide sufficient PG synthesis activity under this condition. These results show how the two class A PBPs and peptidoglycan hydrolases govern cell envelope integrity in E. coli in response to environmental challenges and particularly highlight the importance of PBP1B in maintaining cell fitness under high salt conditions.

ABSTRACT Most bacteria are surrounded by their cell wall, containing a highly cross-linked protective envelope of peptidoglycan. To grow, bacteria must continuously remodel their wall, inserting new material and breaking old bonds. Bond cleavage is performed by cell wall hydrolases, allowing the wall to expand. Understanding the functions of individual hydrolases has been impeded by their redundancy: single knockouts usually present no phenotype. We used an exhaustive multiple-knockout approach to determine the minimal set of hydrolases required for growth in Bacillus subtilis. We identified 42 candidate hydrolases. Strikingly, we were able to remove all but two of these genes in a single strain; this “∆40” strain shows only a mild reduction in growth rate, indicating that none of the 40 hydrolases are necessary for growth. The ∆40 strain does not detectably shed old wall, suggesting that turnover is not essential for growth. The remaining hydrolases in the ∆40 strain are LytE and CwlO, previously shown to be synthetically lethal. Either can be removed in ∆40, indicating that either hydrolase alone is sufficient for cell growth. Screening of environmental conditions and biochemistry revealed that LytE activity is inhibited by Mg2+ and that RlpA-like proteins may stimulate LytE activity. Together, these results suggest that the only essential function of cell wall hydrolases in B. subtilis is to enable cell growth by expanding the wall and that LytE or CwlO alone are sufficient for this function. These experiments introduce the ∆40 strain as a tool to study hydrolase activity and regulation in B. subtilis. IMPORTANCE In order to grow, bacterial cells must both create and break down their cell wall. The enzymes that are responsible for these processes are the target of some of our best antibiotics. Our understanding of the proteins that break down the wall— cell wall hydrolases—has been limited by redundancy among the large number of hydrolases many bacteria contain. To solve this problem, we identified 42 cell wall hydrolases in Bacillus subtilis and created a strain lacking 40 of them. We show that cells can survive using only a single cell wall hydrolase; this means that to understand the growth of B. subtilis in standard laboratory conditions, it is only necessary to study a very limited number of proteins, simplifying the problem substantially. We additionally show that the ∆40 strain is a research tool to characterize hydrolases, using it to identify three “helper” hydrolases that act in certain stress conditions.

Unregulated cell cycle progression may have lethal consequences and therefore, bacteria have various mechanisms in place for the precise spatiotemporal control of cell cycle events. We have uncovered a new link between chromosome replication/segregation and splitting of the division septum. We show that the DNA translocase domain‐containing divisome protein FtsK regulates cellular levels of a peptidoglycan hydrolase Sle1, which is involved in cell separation in the bacterial pathogen Staphylococcus aureus. FtsK interacts with a chaperone (trigger factor, TF) and establishes a FtsK‐dependent TF concentration gradient that is higher in the septal region. Trigger factor binds Sle1 and promotes its preferential export at the septal region, while also preventing Sle1 degradation by the ClpXP proteolytic machinery. Upon conditions that lead to paused septum synthesis, such as DNA damage or impaired DNA replication/segregation, TF gradient is dissipated and Sle1 levels are reduced, thus halting premature septum splitting. Synopsis: Bacteria complete cell division by splitting the division septum, a process that requires the regulated activity of peptidoglycan hydrolases. Here, Staphylococcus aureus divisome protein FtsK is shown to regulate daughter cell splitting by stabilizing the peptidoglycan hydrolase Sle1 and promoting its preferential septal export. The chaperone Trigger factor (TF) interacts with septal FtsK, establishing a TF concentration gradient towards the septal region.TF also interacts with newly synthesized Sle1 and promotes its preferential export at the septal region, where its hydrolytic activity is required to catalyze daughter cell splitting.In the absence of FtsK, the TF gradient is dissipated and Sle1 is degraded by ClpXP proteolytic machinery.Sle1 levels are reduced and septum splitting is delayed when chromosome replication and segregation are impaired. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a major causative agent of bovine mastitis, a disease considered one of the most economically devastating in the dairy sector. Considering the increasing prevalence of antibiotic-resistant strains, novel therapeutic approaches efficiently targeting extra- and intracellular bacteria and featuring high activity in the presence of raw milk components are needed. Here, we have screened a library of eighty peptidoglycan hydrolases (PGHs) for high activity against S. aureus in raw bovine milk, twelve of which were selected for further characterization and comparison in time-kill assays. The bacteriocins lysostaphin and ALE-1, and the chimeric PGH M23LST(L)_SH3b2638 reduced bacterial numbers in raw milk to the detection limit within 10 min. Three CHAP-based PGHs (CHAPGH15_SH3bAle1, CHAPK_SH3bLST_H, CHAPH5_LST_H) showed gradually improving activity with increasing dilution of the raw milk. Furthermore, we demonstrated synergistic activity of CHAPGH15_SH3bAle1 and LST when used in combination. Finally, modification of four PGHs (LST, M23LST(L)_SH3b2638, CHAPK_SH3bLST, CHAPGH15_SH3bAle1) with the cell-penetrating peptide TAT significantly enhanced the eradication of intracellular S. aureus in bovine mammary alveolar cells compared to the unmodified parentals in a concentration-dependent manner. [ABSTRACT FROM AUTHOR]

Orchestrated action of peptidoglycan (PG) synthetases and hydrolases is vital for bacterial growth and viability. Although the function of several PG synthetases and hydrolases is well understood, the function, regulation, and mechanism of action of PG hydrolases characterised as lysostaphin-like endopeptidases have remained elusive. Many of these M23 family members can hydrolyse glycyl-glycine peptide bonds and show lytic activity against Staphylococcus aureus whose PG contains a pentaglycine bridge, but their exact substrate specificity and hydrolysed bonds are still vaguely determined. In this work, we have employed NMR spectroscopy to study both the substrate specificity and the bond cleavage of the bactericide lysostaphin and the S. aureus PG hydrolase LytM. Yet, we provide substrate-level evidence for the functional role of these enzymes. Indeed, our results show that the substrate specificities of these structurally highly homologous enzymes are similar, but unlike observed earlier both LytM and lysostaphin prefer the D-Ala-Gly cross-linked part of mature peptidoglycan. However, we show that while lysostaphin is genuinely a glycyl-glycine hydrolase, LytM can also act as a D-alanyl-glycine endopeptidase., Competing Interests: LA, SV, CT, MJ, IP, HT, PP No competing interests declared, (© 2024, Antenucci et al.)

Bacterial cell walls are the guards of cell integrity. They are composed of peptidoglycan that provides rigidity to sustain internal turgor and ensures isolation from the external environment. In addition, they harbor the enzymatic machinery to secure cell wall modulations needed throughout the bacterial lifespan. The main players in this process are peptidoglycan hydrolases, a large group of enzymes with diverse specificities and different mechanisms of action. They are commonly, but not exclusively, found in prokaryotes. Although in most cases, these enzymes share the same molecular function, namely peptidoglycan hydrolysis, they are leveraged to perform a variety of physiological roles. A well-investigated family of peptidoglycan hydrolases is M23 peptidases, which display a very conserved fold, but their spectrum of lytic action is broad and includes both Gram- positive and Gram- negative bacteria. In this review, we summarize the structural, biochemical, and functional studies concerning the M23 family of peptidases based on literature and complement this knowledge by performing large-scale analyses of available protein sequences. This review has led us to gain new insight into the role of surface charge in the activity of this group of enzymes. We present relevant conclusions drawn from the analysis of available structures and indicate the main structural features that play a crucial role in specificity determination and mechanisms of latency. Our work systematizes the knowledge of the M23 family enzymes in the context of their unique antimicrobial potential against drug-resistant pathogens and presents possibilities to modulate and engineer their features to develop perfect antibacterial weapons.

Staphylococcus spp. are Gram-positive bacteria that can be opportunistic pathogens capable of causing life-threatening infections. Staphylococcus aureus , the most harmful species among them produces heat-resistant endotoxins, which cause food intoxications. Staphylococci may form biofilms on food-processing surfaces and become more resistant to routinely used antimicrobials. The bacteriolytic enzymes Auresine and AuresinePlus, both derived from S. aureus LytM autolysin engineered catalytic domain, were tested for their specificity and activity against various staphylococcal species and other bacteria. Both enzymes exhibited lytic activity against all tested staphylococcal species except S. pasteuri. The specificity of staphylococcal strains to these enzymes depended on the composition of their peptidoglycan cross-bridges. The bacteriolytic effect of the enzymes depended on the growth phase, initial cell number of target bacteria and the enzyme concentration applied. Bacterial elimination and bactericidal effects of both Auresine and AuresinePlus against S. aureus biofilms were confirmed by live/dead staining, with AuresinePlus showing a more pronounced effect. Additionally, Auresine and AuresinePlus presented increased effectiveness in eliminating staphylococcal cells adhered to surfaces, when combined with a detergent or the enzymes - amylase and cellulase. Importantly, cells recovered from biofilms after enzyme treatment did not display decreased susceptibility to applied enzymes, indicating the sustained efficacy of Auresine and AuresinePlus. In conclusion, Auresine and AuresinePlus demonstrate promising bactericidal activity against various staphylococcal species, highlighting their potential as antibacterial agents. • Staphylococci form biofilms on food-processing surfaces and become more resistant to routinely used antimicrobials. • Auresine/AuresinePlus, engineered S. aureus autolysins specifically target Staphylococci , due to unique PG recognition site. • Enzybiotics eliminate bacteria from suspension, surfaces, biofilms. The effect depends on the growth phase and accessibility. • Biofilm matrix targeting agents applied simultaneously may improve performance of Auresine and AuresinePlus. [ABSTRACT FROM AUTHOR]

The synthesis of a peptidoglycan septum is a fundamental part of bacterial fission and is driven by a multiprotein dynamic complex called the divisome. FtsW and FtsI are essential proteins that synthesize the peptidoglycan septum and are controlled by the regulatory FtsBLQ subcomplex and the activator FtsN. However, their mode of regulation has not yet been uncovered in detail. Understanding this process in detail may enable the development of new compounds to combat the rise in antibiotic resistance. In this review, recent data on the regulation of septal peptidoglycan synthesis is summarized and discussed. Based on structural models and the collected data, multiple putative interactions within FtsWI and with regulators are uncovered. This elaborates on and supports an earlier proposed model that describes active and inactive conformations of the septal peptidoglycan synthesis complex that are stabilized by these interactions. Furthermore, a new model on the spatial organization of the newly synthesized peptidoglycan and the synthesis complex is presented. Overall, the updated model proposes a balance between several allosteric interactions that determine the state of septal peptidoglycan synthesis. [ABSTRACT FROM AUTHOR]

Peptidoglycan (PG) is an important architectural element that imparts physical toughness and rigidity to the bacterial envelope. It is also a dynamic structure that undergoes continuous turnover or autolysis. Escherichia coli possesses redundant PG degradation enzymes responsible for PG turnover; however, the advantage afforded by the existence of numerous PG degradation enzymes remains incompletely understood. In this study, we elucidated the physiological roles of MltE and MltC, members of the lytic transglycosylase (LTG) family that catalyze the cleavage of glycosidic bonds between disaccharide subunits within PG strands. MltE and MltC are acidic LTGs that exhibit increased enzymatic activity and protein levels under acidic pH conditions, respectively, and deletion of these two LTGs results in a pronounced growth defect at acidic pH. Furthermore, inactivation of these two LTGs induces increased susceptibility at acidic pH against various antibiotics, particularly vancomycin, which seems to be partially caused by elevated membrane permeability. Intriguingly, inactivation of these LTGs induces a chaining morphology, indicative of daughter cell separation defects, only under acidic pH conditions. Simultaneous deletion of PG amidases, known contributors to daughter cell separation, exacerbates the chaining phenotype at acidic pH. This suggests that the two LTGs may participate in the cleavage of glycan strands between daughter cells under acidic pH conditions. Collectively, our findings highlight the role of LTG repertoire diversity in facilitating bacterial survival and antibiotic resistance under stressful conditions., Competing Interests: The authors declare no conflict of interest.

Staphylococcus aureus is a major causative agent of bovine mastitis, a disease considered one of the most economically devastating in the dairy sector. Considering the increasing prevalence of antibiotic-resistant strains, novel therapeutic approaches efficiently targeting extra- and intracellular bacteria and featuring high activity in the presence of raw milk components are needed. Here, we have screened a library of eighty peptidoglycan hydrolases (PGHs) for high activity against S. aureus in raw bovine milk, twelve of which were selected for further characterization and comparison in time-kill assays. The bacteriocins lysostaphin and ALE-1, and the chimeric PGH M23LST(L)_SH3b2638 reduced bacterial numbers in raw milk to the detection limit within 10 min. Three CHAP-based PGHs (CHAPGH15_SH3bAle1, CHAPK_SH3bLST_H, CHAPH5_LST_H) showed gradually improving activity with increasing dilution of the raw milk. Furthermore, we demonstrated synergistic activity of CHAPGH15_SH3bAle1 and LST when used in combination. Finally, modification of four PGHs (LST, M23LST(L)_SH3b2638, CHAPK_SH3bLST, CHAPGH15_SH3bAle1) with the cell-penetrating peptide TAT significantly enhanced the eradication of intracellular S. aureus in bovine mammary alveolar cells compared to the unmodified parentals in a concentration-dependent manner.

Assembly of the bacterial flagellar rod, hook, and filament requires penetration through the peptidoglycan (PG) sacculus and outer membrane. In most β- and γ-proteobacteria, the protein FlgJ has two functional domains that enable PG hydrolyzing activity to create pores, facilitating proper assembly of the flagellar rod. However, two distinct proteins performing the same functions as the dual-domain FlgJ are proposed in δ- and ε-proteobacteria as well as spirochetes. The Lyme disease spirochete Borrelia burgdorferi genome possesses a FlgJ and a PG lytic SLT enzyme protein homolog (BB0259). FlgJ in B. burgdorferi is crucial for flagellar hook and filament assembly but not for the proper rod assembly reported in other bacteria. However, BB0259 has never been characterized. Here, we use cryo-electron tomography to visualize periplasmic flagella in different bb0259 mutant strains and provide evidence that the E580 residue of BB0259 is essential for PG-hydrolyzing activity. Without the enzyme activity, the flagellar hook fails to penetrate through the pores in the cell wall to complete assembly of an intact periplasmic flagellum. Given that FlgJ and BB0259 interact with each other, they likely coordinate the penetration through the PG sacculus and assembly of a functional flagellum in B. burgdorferi and other spirochetes. Because of its role, we renamed BB0259 as flagellar-specific lytic transglycosylase or LTaseBb. [ABSTRACT FROM AUTHOR]

Bacterial peptidoglycan hydrolases play an essential role in cell wall metabolism during bacterial growth, division, and elongation (autolysins) or in the elimination of closely related species from the same ecological niche (bacteriocins). Most studies concerning the peptidoglycan hydrolases present in Gram-positive bacteria have focused on clinically relevant Staphylococcus aureus or the model organism Bacillus subtilis , while knowledge relating to other species remains limited. Here, we report two new peptidoglycan hydrolases from the M23 family of metallopeptidases derived from the same staphylococcal species, Staphylococcus pettenkoferi. They share modular architecture, significant sequence identity (60%), catalytic and binding residue conservation, and similar modes of activation, but differ in gene distribution, putative biological role, and, strikingly, in their isoelectric points (pIs). One of the peptides has a high pI, similar to that reported for all M23 peptidases evaluated to date, whereas the other displays a low pI, a unique feature among M23 peptidases. Consequently, we named them SpM23_B (Staphylococcus pettenkoferi M23 "Basic") and SpM23_A (Staphylococcus pettenkoferi M23 "Acidic"). Using genetic and biochemical approaches, we have characterized these two novel lytic enzymes, both in vitro and in their physiological context. Our study presents a detailed characterization of two novel and clearly distinct peptidoglycan hydrolases to understand their role in bacterial physiology. [ABSTRACT FROM AUTHOR]

Assembly of the bacterial flagellar rod, hook, and filament requires penetration through the peptidoglycan (PG) sacculus and outer membrane. In most β- and γ-proteobacteria, the protein FlgJ has two functional domains that enable PG hydrolyzing activity to create pores, facilitating proper assembly of the flagellar rod. However, two distinct proteins performing the same functions as the dual-domain FlgJ are proposed in δ- and ε-proteobacteria as well as spirochetes. The Lyme disease spirochete Borrelia burgdorferi genome possesses a FlgJ and a PG lytic SLT enzyme protein homolog (BB0259). FlgJ in B. burgdorferi is crucial for flagellar hook and filament assembly but not for the proper rod assembly reported in other bacteria. However, BB0259 has never been characterized. Here, we use cryo-electron tomography to visualize periplasmic flagella in different bb0259 mutant strains and provide evidence that the E580 residue of BB0259 is essential for PG-hydrolyzing activity. Without the enzyme activity, the flagellar hook fails to penetrate through the pores in the cell wall to complete assembly of an intact periplasmic flagellum. Given that FlgJ and BB0259 interact with each other, they likely coordinate the penetration through the PG sacculus and assembly of a functional flagellum in B. burgdorferi and other spirochetes. Because of its role, we renamed BB0259 as flagellar-specific lytic transglycosylase or LTaseBb.

Bacterial peptidoglycan hydrolases play an essential role in cell wall metabolism during bacterial growth, division, and elongation (autolysins) or in the elimination of closely related species from the same ecological niche (bacteriocins). Most studies concerning the peptidoglycan hydrolases present in Gram-positive bacteria have focused on clinically relevant Staphylococcus aureus or the model organism Bacillus subtilis, while knowledge relating to other species remains limited. Here, we report two new peptidoglycan hydrolases from the M23 family of metallopeptidases derived from the same staphylococcal species, Staphylococcus pettenkoferi. They share modular architecture, significant sequence identity (60%), catalytic and binding residue conservation, and similar modes of activation, but differ in gene distribution, putative biological role, and, strikingly, in their isoelectric points (pIs). One of the peptides has a high pI, similar to that reported for all M23 peptidases evaluated to date, whereas the other displays a low pI, a unique feature among M23 peptidases. Consequently, we named them SpM23_B (Staphylococcus pettenkoferi M23 “Basic”) and SpM23_A (Staphylococcus pettenkoferi M23 “Acidic”). Using genetic and biochemical approaches, we have characterized these two novel lytic enzymes, both in vitro and in their physiological context. Our study presents a detailed characterization of two novel and clearly distinct peptidoglycan hydrolases to understand their role in bacterial physiology.

Gram-negative bacteria have a thin peptidoglycan layer between the cytoplasmic and outer membranes protecting the cell from osmotic challenges. Hydrolases of this structure are needed to cleave bonds to allow the newly synthesized peptidoglycan strands to be inserted by synthases. These enzymes need to be tightly regulated and their activities coordinated to prevent cell lysis. To better understand this process in Escherichia coli , we probed the genetic interactions of mrcA (encodes PBP1A) and mrcB (encodes PBP1B) with genes encoding peptidoglycan amidases and endopeptidases in envelope stress conditions. Our extensive genetic interaction network analysis revealed relatively few combinations of hydrolase gene deletions with reduced fitness in the absence of PBP1A or PBP1B, showing that none of the amidases or endopeptidases is strictly required for the functioning of one of the class A PBPs. This illustrates the robustness of the peptidoglycan growth mechanism. However, we discovered that the fitness of ∆ mrcB cells is significantly reduced under high salt stress and in vitro activity assays suggest that this phenotype is caused by a reduced peptidoglycan synthesis activity of PBP1A at high salt concentration.IMPORTANCE Escherichia coli and many other bacteria have a surprisingly high number of peptidoglycan hydrolases. These enzymes function in concert with synthases to facilitate the expansion of the peptidoglycan sacculus under a range of growth and stress conditions. The synthases PBP1A and PBP1B both contribute to peptidoglycan expansion during cell division and growth. Our genetic interaction analysis revealed that these two penicillin-binding proteins (PBPs) do not need specific amidases, endopeptidases, or lytic transglycosylases for function. We show that PBP1A and PBP1B do not work equally well when cells encounter high salt stress and demonstrate that PBP1A alone cannot provide sufficient PG synthesis activity under this condition. These results show how the two class A PBPs and peptidoglycan hydrolases govern cell envelope integrity in E. coli in response to environmental challenges and particularly highlight the importance of PBP1B in maintaining cell fitness under high salt conditions., Competing Interests: The authors declare no conflict of interest.

Pediococcus acidilactici NCDC 252 is a Gram-positive lactic acid bacteria (LAB) that is being studied to develop it as probiotic and starter culture for dairy and meat industry. Peptidoglycan hydrolases (PGHs) are also gaining interest because of their antibacterial activity. NCDC 252 genome was searched for PGH encoded genes and product of these genes were in silico characterized for different parameters. Extracellular PGHs of NCDC 252 were precipitated and characterized. PGHs were screened turbidometrically with Bacillus cereus and Staphylococcus albus as substrates. Spectrum of PGH(s) was studied in renaturing SDS-PAGE by varying substrates and renaturing conditions. PGH(s) band intensity decreased in the presence of NaCl whereas some additional secondary lytic bands appeared in the presence of EDTA. Autolysis of LAB in cheese and other fermented food products is associated with release of oligopeptides by proteolysis and other enzyme activities. Autolytic phenotype of P. acidilactici was evaluated and characterized. Lytic activity studies revealed 45–60% activity against Micrococcus lysodeikticus and B. cereus greater than for P. acidilactici. PGH of P. acidilactici exhibited broad growth inhibiting spectrum of antibacterial activity against studied Gram positive and Gram negative bacteria and thus can be promising in controlling pathogenic bacteria. This also confers probiotic attribute to the studied strain. [ABSTRACT FROM AUTHOR]

ABSTRACT 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 (PscBCC). 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.

Importance: Neisseria gonorrhoeae is unusual in that the bacteria release larger amounts of cell wall material as they grow as compared to related bacteria, and the released cell wall fragments induce inflammation that leads to tissue damage in infected people. The study of MltG revealed the importance of this enzyme for controlling cell wall growth, cell wall fragment production, and bacterial cell size and suggests a role for MltG in a cell wall synthesis and degradation complex. The increased antibiotic sensitivities of mltG mutants suggest that an antimicrobial drug inhibiting MltG would be useful in combination therapy to restore the sensitivity of the bacteria to cell wall targeting antibiotics to which the bacteria are currently resistant., Competing Interests: The authors declare no conflict of interest.

ABSTRACT The human eukaryotic pathogen Trichomonas vaginalis causes trichomoniasis, a prevalent sexually transmitted infection. This extracellular protozoan is intimately associated with the human vaginal mucosa and microbiota, but key aspects of the complex interactions between the parasite and the vaginal bacteria remain elusive. We report that T. vaginalis has acquired, by lateral gene transfer from bacteria, genes encoding peptidoglycan hydrolases of the NlpC/P60 family. Two of the T. vaginalis enzymes were active against bacterial peptidoglycan, retaining the active-site fold and specificity as dl-endopeptidases. The endogenous NlpC/P60 genes are transcriptionally upregulated in T. vaginalis in the presence of bacteria. The overexpression of an exogenous copy enables the parasite to outcompete bacteria from mixed cultures, consistent with the biochemical activity of the enzyme. Our study results highlight the relevance of the interactions of this eukaryotic pathogen with bacteria, a poorly understood aspect of the biology of this important human parasite. IMPORTANCE Trichomonas vaginalis is a parasitic protozoan of the human urogenital tract that causes trichomoniasis, a very common sexually transmitted disease. Despite residing extracellularly and in close association with the vaginal bacteria (i.e., the microbiota), very little is known about the nature of the parasite-bacterium interactions. Our study showed that this parasite had acquired genes from bacteria which retained their original function. They produce active enzymes capable of degrading peptidoglycan, a unique polymer of the bacterial cell envelope, helping the parasite to outcompete bacteria in mixed cultures. This study was the first to show that a laterally acquired group of genes enables a eukaryotic mucosal pathogen to control bacterial population. We highlight the importance of understanding the interactions between pathogens and microbiota, as the outcomes of these interactions are increasingly understood to have important implications on health and disease.

The peptidoglycan of the bacterial cell wall undergoes a permanent turnover during cell growth and differentiation. In the Gram-positive pathogen Staphylococcus aureus, the major peptidoglycan hydrolase Atl is required for accurate cell division, daughter cell separation and autolysis. Atl is a bifunctional N-acetylmuramoyl-L-alanine amidase/endo-β-N-acetylglucosaminidase that releases peptides and the disaccharide N-acetylmuramic acid-β-1,4-N-acetylglucosamine (MurNAc-GlcNAc) from the peptido-glycan. Here we revealed the recycling pathway of the cell wall turnover product MurNAc-GlcNAc in S. aureus. The latter disaccharide is internalized and concomitantly phosphorylated by the phosphotransferase system (PTS) transporter MurP, which had been implicated previously in the uptake and phosphorylation of MurNAc. Since MurP mutant cells accumulate MurNAc-GlcNAc and not MurNAc in the culture medium during growth, the disaccharide represents the physiological substrate of the PTS transporter. We further identified and characterized a novel 6-phospho-N-acetylmuramidase, named MupG, which intracellularly hydrolyses MurNAc 6-phosphate-GlcNAc, the product of MurP-uptake and phosphorylation, yielding MurNAc 6-phosphate and GlcNAc. MupG is the first characterized representative of a novel family of glycosidases containing domain of unknown function 871 (DUF871). The corresponding gene mupG (SAUSA300_0192) of S. aureus strain USA300 is the first gene within a putative operon that also includes genes encoding the MurNAc 6-phosphate etherase MurQ, MurP, and the putative transcriptional regulator MurR. Using mass spectrometry, we observed cytoplasmic accumulation of MurNAc 6-phosphate-GlcNAc in ΔmupG and ΔmupGmurQ markerless non-polar deletion mutants, but not in the wild type or in the complemented ΔmupG strain. MurNAc 6-phosphate-GlcNAc levels in the mutants increased during stationary phase, in accordance with previous observations regarding peptidoglycan recycling in S. aureus.

ABSTRACT 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 (lipoprotein binding partner of CtpA). 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 ΔctpA 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.

Importance: In order to grow, bacterial cells must both create and break down their cell wall. The enzymes that are responsible for these processes are the target of some of our best antibiotics. Our understanding of the proteins that break down the wall- cell wall hydrolases-has been limited by redundancy among the large number of hydrolases many bacteria contain. To solve this problem, we identified 42 cell wall hydrolases in Bacillus subtilis and created a strain lacking 40 of them. We show that cells can survive using only a single cell wall hydrolase; this means that to understand the growth of B. subtilis in standard laboratory conditions, it is only necessary to study a very limited number of proteins, simplifying the problem substantially. We additionally show that the ∆40 strain is a research tool to characterize hydrolases, using it to identify three "helper" hydrolases that act in certain stress conditions., Competing Interests: The authors declare no conflict of interest.

Bacterial cell division is a complex process brought about by the coordinated action of multiple proteins. Separation of daughter cells during the final stages of division involves cleavage of new cell wall laid down at the division septum. In E. coli, this process is governed by the action of N-acetylmuramoyl-L-alanine amidases AmiA/B/C, which are regulated by their LytM activators EnvC and NlpD. While much is known about the regulation of septum cleavage in E. coli, the mechanism of daughter cell separation is not clear in Caulobacter crescentus, a dimorphic crescent-shaped bacterium. In this work, we characterized the role of AmiC, the only annotated amidase in C. crescentus. AmiC from C. crescentus is functional in E. coli and restores cell separation defects seen in E. coli amidase mutants, suggesting that AmiC has septum splitting activity. The medial localization of AmiC was independent of DipM, an LytM domain-containing endopeptidase. Our results indicate that enzymatic activity is essential for medial recruitment of AmiC. Overexpression of AmiC causes cell separation defects and formation of chains. Finally, overexpression of AmiC in cells inhibited for cell division leads to lysis. Collectively, our findings reveal that regulation of daughter cell separation in C. crescentus differs from that of E. coli and can serve as a model system to study bacterial cytokinesis. [ABSTRACT FROM AUTHOR]

The screening of three strains of lactic acid bacteria identified as Lactobacillus rhamnosus, Lactobacillus reuteri, and Lactobacillus helveticus showed significant antagonistic activity against Klebsiella pneumoniae strains characterized by multiple antibiotic resistance. Lactobacilli cocultivated with the Klebsiella strains inhibited their growth 20 to 86% on the first and second days, respectively. Exoproteome analysis of L. rhamnosus cocultivated with K. pneumoniae revealed the induction of peptidoglycan hydrolases, including extracellular lytic transglycosylases, family II (MltA), and endopeptidases capable of disrupting the peptidoglycan bacterial cell wall. [ABSTRACT FROM AUTHOR]

ABSTRACT Neisseria gonorrhoeae gonococcus (GC) is a Gram-negative betaproteobacterium and causative agent of the sexually transmitted infection gonorrhea. During growth, GC releases lipooligosaccharide (LOS) and peptidoglycan (PG) fragments, which contribute significantly to the inflammatory damage observed during human infection. In ascending infection of human Fallopian tubes, inflammation leads to increased risk of ectopic pregnancy, pelvic inflammatory disease, and sterility. Of the PG fragments released by GC, most are disaccharide peptide monomers, and of those, 80% have tripeptide stems despite the observation that tetrapeptide stems make up 80% of the assembled cell wall. We identified a serine-protease l,d-carboxypeptidase, NGO1274 (LdcA), as the enzyme responsible for converting cell wall tetrapeptide-stem PG to released tripeptide-stem PG. Unlike characterized cytoplasmic LdcA homologs in gammaproteobacteria, LdcA in GC is exported to the periplasm, and its localization is critical for its activity in modifying PG fragments for release. Distinct among other characterized l,d-carboxypeptidases, LdcA from GC is also capable of catalyzing the cleavage of specific peptide cross-bridges (endopeptidase activity). To define the role of ldcA in pathogenesis, we demonstrate that ldcA disruption results in both loss of NOD1-dependent NF-κB activation and decreased NOD2-dependent NF-κB activation while not affecting Toll-like receptor (TLR) agonist release. Since the human intracellular peptidoglycan receptor NOD1 (hNOD1) specifically recognizes PG fragments with a terminal meso-DAP rather than d-alanine, we conclude that LdcA is required for GC to provoke NOD1-dependent responses in cells of the human host. IMPORTANCE The macromolecular meshwork of peptidoglycan serves essential functions in determining bacterial cell shape, protecting against osmotic lysis, and defending cells from external assaults. The conserved peptidoglycan structure, however, is also recognized by eukaryotic pattern recognition receptors, which can trigger immune responses against bacteria. Many bacteria can induce an inflammatory response through the intracellular peptidoglycan receptor NOD1, but Neisseria gonorrhoeae serves as an extreme example, releasing fragments of peptidoglycan into the environment during growth that specifically antagonize human NOD1. Understanding the peptidoglycan breakdown mechanisms that allow Neisseria to promote NOD1 activation, rather than avoiding or suppressing immune detection, is critical to understanding the pathogenesis of this increasingly drug-resistant organism. We identify a peptidoglycan l,d-carboxypeptidase responsible for converting liberated peptidoglycan fragments into the human NOD1 agonist and find that the same enzyme has endopeptidase activity on certain peptidoglycan cross-links, the first described combination of those two activities in a single enzyme.

Peptidoglycan hydrolases (PGHs) have been suggested as novel therapeutics for the treatment of bovine mastitis. However, activity in the presence of cow's milk is an important requirement for drugs administered into the bovine udder. We have screened a library of >170 recombinant PGHs, including engineered bacteriophage endolysins, for enzymes with activity against Staphylococcus aureus in milk, using a microtiter plate-based protocol. Nine suitable PGH constructs were identified by this approach and further compared in time-kill assays for their efficacy against S. aureus in heat-treated milk. The three most active enzymes (lysostaphin, Ami2638A, and CHAPK_CWT-LST) reduced S. aureus in milk to undetectable numbers within minutes at nanomolar concentrations. Due to their different peptidoglycan cleavage sites, these PGH constructs revealed synergistic activity in most combinations, as demonstrated by checkerboard assays, spot assays, and time-kill experiments. Furthermore, they proved active against a selection of staphylococcal mastitis isolates from different geographical regions when applied individually or in synergistic combination. The most effective PGH combination completely eradicated S. aureus from milk, with no more bacteria being detected within 24 h after addition of the enzymes, corresponding to a reduction of >9 log units compared to the control. Efficacy was also retained at different inoculum levels (3 versus 6 log CFU/ml) and when S. aureus was grown in milk as opposed to broth prior to the experiments. In raw cow's milk, CHAPK_CWT-LST showed reduced efficacy, whereas both Ami2638A and lysostaphin retained their activity, reducing bacterial numbers by >3.5 log units within 3 h. IMPORTANCE Staphylococci and S. aureus in particular are a major cause of bovine mastitis, an inflammation of the mammary gland in cows associated with high costs and risks for consumers of milk products. S. aureus-induced mastitis, commonly treated by intramammary infusion of antibiotics, is characterized by low cure rates and increasing antibiotic resistance in bacteria. Therefore, alternative treatment options are highly desirable. PGHs, including bacteriophage endolysins, rapidly and specifically kill selected pathogens by degrading their cell wall and are refractory to resistance development, therefore holding promise as novel antibacterial agents. This study employed a screening approach to identify PGH constructs with high staphylolytic activity in cow's milk within a large collection of enzymes. Our results suggest that the most promising enzymes identified by this strategy hold potential as novel mastitis therapeutics and support their further characterization in animal models. [ABSTRACT FROM AUTHOR]

Predatory microbes like Bdellovibrio feed on other bacteria by invading their periplasm, replicating within the bacterial shell that is now a feeding trough, and ultimately lysing the prey and disseminating. A new study by E. J. Banks, C. Lambert, S. Mason, J. Tyson, et al. (J Bacteriol 205:e00475-22, 2023, https://doi.org/10.1128/jb.00475-22) highlights the great lengths to which Bdellovibrio must go to affect host cell remodeling: A secreted cell wall lytic enzyme with specificity for the host septal cell wall maximizes the size of the attacker's meal and the size of the restaurant in which it can spread out. This study provides novel insights into bacterial predator-prey dynamics and showcases elegant co-option of an endogenous cell wall turnover enzyme refurbished as a warhead to enhance prey consumption.

Lytic transglycosylases cut peptidoglycan backbones, facilitating a variety of functions within bacteria, including cell division, pathogenesis, and insertion of macromolecular machinery into the cell envelope. Here, we identify a novel role of a secreted lytic transglycosylase associated with the predatory lifestyle of Bdellovibrio bacteriovorus strain HD100. During wild-type B. bacteriovorus prey invasion, the predator rounds up rod-shaped prey into spherical prey bdelloplasts, forming a spacious niche within which the predator grows. Deleting the MltA-like lytic transglycosylase Bd3285 still permitted predation but resulted in three different, invaded prey cell shapes: spheres, rods, and "dumbbells." Amino acid D321 within the catalytic C-terminal 3D domain of Bd3285 was essential for wild-type complementation. Microscopic analyses revealed that dumbbell-shaped bdelloplasts are derived from Escherichia coli prey undergoing cell division at the moment of Δ bd3285 predator invasion. Prelabeling of E. coli prey peptidoglycan prior to predation with the fluorescent D-amino acid HADA showed that the dumbbell bdelloplasts invaded by B. bacteriovorus Δ bd3285 contained a septum. Fluorescently tagged Bd3285, expressed in E. coli, localized to the septum of dividing cells. Our data indicate that B. bacteriovorus secretes the lytic transglycosylase Bd3285 into the E. coli periplasm during prey invasion to cleave the septum of dividing prey, facilitating prey cell occupation. IMPORTANCE Antimicrobial resistance is a serious and rapidly growing threat to global health. Bdellovibrio bacteriovorus can prey upon an extensive range of Gram-negative bacterial pathogens and thus has promising potential as a novel antibacterial therapeutic and is a source of antibacterial enzymes. Here, we elucidate the role of a unique secreted lytic transglycosylase from B. bacteriovorus which acts on the septal peptidoglycan of its prey. This improves our understanding of mechanisms that underpin bacterial predation.

The dynamic composition of the peptidoglycan cell wall has been the subject of intense research for decades, yet how bacteria coordinate the synthesis of new peptidoglycan with the turnover and remodeling of existing peptidoglycan remains elusive. Diversity and redundancy within peptidoglycan synthases and peptidoglycan autolysins, enzymes that degrade peptidoglycan, have often made it challenging to assign physiological roles to individual enzymes and determine how those activities are regulated. For these reasons, peptidoglycan glycosidases, which cleave within the glycan strands of peptidoglycan, have proven veritable masters of misdirection over the years. Unlike many of the broadly conserved peptidoglycan synthetic complexes, diverse bacteria can employ unrelated glycosidases to achieve the same physiological outcome. Additionally, although the mechanisms of action for many individual enzymes have been characterized, apparent conserved homologs in other organisms can exhibit an entirely different biochemistry. This flexibility has been recently demonstrated in the context of three functions critical to vegetative growth: (i) release of newly synthesized peptidoglycan strands from their membrane anchors, (ii) processing of peptidoglycan turned over during cell wall expansion, and (iii) removal of peptidoglycan fragments that interfere with daughter cell separation during cell division. Finally, the regulation of glycosidase activity during these cell processes may be a cumulation of many factors, including protein-protein interactions, intrinsic substrate preferences, substrate availability, and subcellular localization. Understanding the true scope of peptidoglycan glycosidase activity will require the exploration of enzymes from diverse organisms with equally diverse growth and division strategies.

Bacteria have to avoid recognition by the host immune system in order to establish a successful infection. Peptidoglycan, the principal constituent of virtually all bacterial surfaces, is a specific molecular signature recognized by dedicated host receptors, present in animals and plants, which trigger an immune response. Here we report that autolysins from Gram-positive pathogenic bacteria, enzymes capable of hydrolyzing peptidoglycan, have a major role in concealing this inflammatory molecule from Drosophila peptidoglycan recognition proteins (PGRPs). We show that autolysins trim the outermost peptidoglycan fragments and that in their absence bacterial virulence is impaired, as PGRPs can directly recognize leftover peptidoglycan extending beyond the external layers of bacterial proteins and polysaccharides. The activity of autolysins is not restricted to the producer cells but can also alter the surface of neighboring bacteria, facilitating the survival of the entire population in the infected host.

Bacterial cell walls are the guards of cell integrity. They are composed of peptidoglycan that provides rigidity to sustain internal turgor and ensures isolation from the external environment. In addition, they harbor the enzymatic machinery to secure cell wall modulations needed throughout the bacterial lifespan. The main players in this process are peptidoglycan hydrolases, a large group of enzymes with diverse specificities and different mechanisms of action. They are commonly, but not exclusively, found in prokaryotes. Although in most cases, these enzymes share the same molecular function, namely peptidoglycan hydrolysis, they are leveraged to perform a variety of physiological roles. A well-investigated family of peptidoglycan hydrolases is M23 peptidases, which display a very conserved fold, but their spectrum of lytic action is broad and includes both Gram- positive and Gram- negative bacteria. In this review, we summarize the structural, biochemical, and functional studies concerning the M23 family of peptidases based on literature and complement this knowledge by performing large-scale analyses of available protein sequences. This review has led us to gain new insight into the role of surface charge in the activity of this group of enzymes. We present relevant conclusions drawn from the analysis of available structures and indicate the main structural features that play a crucial role in specificity determination and mechanisms of latency. Our work systematizes the knowledge of the M23 family enzymes in the context of their unique antimicrobial potential against drug-resistant pathogens and presents possibilities to modulate and engineer their features to develop perfect antibacterial weapons., Competing Interests: IS holds the patent EP2699254 “The method of proteolysis, peptidase, the composition to be used as a bacteriostatic and bactericidal agent, the mixture and the applications of the active form of LytM from S. aureus or derivatives thereof.” The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Razew, Schwarz, Mitkowski, Sabala and Kaus-Drobek.)

Bacteria of the genus Staphylococcus are common pathogens responsible for a broad spectrum of human and animal infections and belong to the most important etiological factors causing food poisoning. Because of rapid increase in the prevalence of isolation of staphylococci resistant to many antibiotics, there is an urgent need for the development of new alternative chemotherapeutics. A number of studies have recently demonstrated the strong potential of peptidoglycan hydrolases (PHs) to control and treat infections caused by this group of bacteria. PHs cause rapid lysis and death of bacterial cells. The review concentrates on enzymes hydrolyzing peptidoglycan of staphylococci. Usually, they are characterized by high specificity to only Staphylococcus aureus cell wall components; however, some of them are also able to lyse cells of other staphylococci, e.g., Staphylococcus epidermidis-human pathogen of growing importance and also other groups of bacteria. Some PHs strengthen the bactericidal or bacteriostatic activity of common antibiotics, and as a result, they should be considered as component of combined therapy which could definitely reduced the development of bacterial resistance to both enzymes and antibiotics. The preliminary research revealed that most of these enzymes can be produced using heterologous, especially Escherichia coli expression systems; however, still much effort is required to develop more efficient and large-scale production technologies. This review discusses current state on knowledge with emphasis on the possibilities of application of PHs in the context of therapeutics for infections caused by staphylococci. [ABSTRACT FROM AUTHOR]

Abstract: Peptidoglycan hydrolases (PGHs) are bacterial enzymes that can hydrolyze the peptidoglycan in bacterial cell wall leading to autolysis. By releasing intracellular enzymes, autolysis of Lactobacillus helveticus has important applications in cheese ripening as its extent varied from strain to strain. Nine genes coding PGHs were previously annotated in the genome of the high autolytic strain L. helveticus DPC 4571. This study was conducted to evaluate the clone diversity of the nine PGHs genes within a collection of 24 L. helveticus strains, highly diverse in terms of origin, biotope and autolytic activity. Pulsed field gel electrophoresis was applied to assess the genomic diversity of the 24 strains. The presence or absence of nine PGHs genes was verified for all L. helveticus strains. Nucleotide and deduced amino acid sequence were compared for six relevant strains. Finally, gene expression was monitored by reverse transcription during growth and by zymogram for 12 strains. The nine PGHs genes are ubiquitous and transcripted early during growth. Zymograms were similar in terms of molecular size of the bands, but exhibited strain to strain variations in the number of bands revealing from 2 to 5 lytic bands per strain. [Copyright &y& Elsevier]

The secreted Micrococcus luteus protein, Rpf, is required for successful resuscitation of dormant “non-culturable” M. luteus cells and for growth stimulation in poor media. The biochemical mechanism of Rpf action remained unknown. Theoretical predictions of Rpf domain architecture and organization, together with a recent NMR analysis of the protein structure, indicate that the conserved Rpf domain has a lysozyme-like fold. In the present study, we found that both the secreted native protein and the recombinant protein lyse crude preparations of M. luteus cell walls. They also hydrolyze 4-methylumbelliferyl-β-D-N,N′,N″-triacetylchitotrioside, a synthetic substrate for peptidoglycan muramidases, with optimum activity at pH 6. The Rpf protein also has weak proteolytic activity against N-CBZ-Gly-Gly-Arg-β-naphthylamide, a substrate for trypsin-like enzymes. Rpf activity towards 4-methylumbelliferyl-β-D-N,N′,N″-triacetylchitotrioside was reduced when the glutamate residue at position 54, invariant for all Rpf family proteins and presumably involved in catalysis, was altered. The same amino acid substitution resulted in impaired resuscitation activity of Rpf. The data indicate that Rpf is a peptidoglycan-hydrolyzing enzyme, and strongly suggest that this specific activity is responsible for its growth promotion and resuscitation activity. A possible mechanism of Rpf-mediated resuscitation is discussed. [ABSTRACT FROM AUTHOR]

Aim: To determine the autolytic phenotype of five species in the Bacillus cereus group. Methods and Results: The autolytic rate of 96 strains belonging to five species in the B. cereus group was examined under starvation conditions at pH 6, 6·5 and 8·5 in different buffers. The autolytic rate was strain-dependent with a wide variability at pH 6, but higher and more uniform at pH 6·5. At pH 8·5, and respect to the extent of autolysis at pH 6·5, it was relatively low for most of the strains with the lowest values between 13 and 52% in Bacillus mycoides and Bacillus pseudomycoides. Peptidoglycan hydrolase patterns evaluated by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis using cells of Bacillus thuringiensis ssp. tolworthi HD125 as an indicator, revealed complex profiles with lytic bands of about 90, 63, 46, 41, 38, 32, 28 and 25 kDa in B. cereus, B. thuringiensis and Bacillus weihenstephanensis. Bacillus mycoides and B. pseudomycoides had simpler profiles with lytic bands of 63, 46 and 38 kDa. Changes in the autolytic pattern were observed for cells harvested at the stationary phase of growth (72 h) showing an increase in the intensity of the 25 kDa band in the case of B. cereus, B. thuringiensis and B. weihenstephanensis, while no changes were observed for B. mycoides. Using Micrococcus lysodeicticus and Listeria monocytogenes as indicators lytic activity was retained by proteins of 63, 46, 38, 32 and 25 kDa and a new one of about 20 kDa in B. mycoides. Growth in the different media did not affect the autolytic pattern. NaCl abolished the activity of all the peptidoglycan hydrolases except for those of B. mycoides and B. weihenstephanensis. Lytic activity was retained in the presence of MgCl2, MnCl2 and EDTA and increased at basic pH. Conclusions: Bacillus cereus/ B. thuringiensis/ B. weihenstephanensis showed a high extent of autolysis around neutral pH, even though they presented relatively complex autolysin profiles at alkaline pH. Bacillus mycoides/ B. pseudomycoides had a higher extent of autolysis at acidic pH and a simpler autolysin pattern. Significance and Impact of the Study: Information on the autolytic phenotype expand the phenotypic characterization of the different species in the B. cereus group. [ABSTRACT FROM AUTHOR]

Abstract: Lysis of dairy starters is a prerequisite for optimum cheese maturation, since intracellular starter enzymes, particularly peptidases, can then play their role. Here we describe the different methods used to detect starter lysis in situ and current knowledge concerning the impact of lysis on cheese ripening, particularly the increase of free amino acids due to early lysis and the reduction of bitterness by hydrolysis of large hydrophobic peptides. Recent results obtained on the impact of lysis on lipolysis and amino acid catabolism are also described. Then, we present current knowledge regarding the mechanisms involved, focussing mainly on the model most investigated: Lactococcus lactis. Recent advances concerning the molecular characterization of peptidoglycan hydrolases are summarized (sequence, structure, regulation) together with current knowledge of the relationship between lysogeny and lysis. Lastly, we review the different approaches proposed to control or induce lysis in situ. In conclusion, we point out unaddressed questions. [Copyright &y& Elsevier]

The human-restricted pathogen Neisseria meningitidis, which is best known for causing invasive meningococcal disease, has a nonpathogenic lifestyle as an asymptomatic colonizer of the human naso- and oropharyngeal space. N. meningitidis releases small peptidoglycan (PG) fragments during growth. It was demonstrated previously that N. meningitidis releases low levels of tripeptide PG monomer, which is an inflammatory molecule recognized by the human intracellular innate immune receptor NOD1. In the present study, we demonstrated that N. meningitidis released more PG-derived peptides than PG monomers. Using a reporter cell line overexpressing human NOD1, we showed that N. meningitidis activates NOD1 using PG-derived peptides. The generation of such peptides required the presence of the periplasmic N- acetylmuramyl-l-alanine amidase AmiC and the outer membrane lipoprotein NlpD. AmiC and NlpD were found to function in cell separation, and mutation of either amiC or nlpD resulted in large clumps of unseparated N. meningitidis cells instead of the characteristic diplococci. Using stochastic optical reconstruction microscopy, we demonstrated that FLAG epitope-tagged NlpD localized to the septum, while similarly tagged AmiC was found at the septum in some diplococci but was distributed around the cell in most cases. In a human whole-blood infection assay, an nlpD mutant was severely attenuated and showed particular sensitivity to complement. Thus, in N. meningitidis, the cell separation proteins AmiC and NlpD are necessary for NOD1 stimulation and survival during infection of human blood.