Peptidoglycan (PG) is an essential structural component of the bacterial cell wall and maintains the integrity and shape of the cell by forming a continuous layer around the cytoplasmic membrane. The ...thin PG layer of Escherichia coli resides in the periplasm, a unique compartment whose composition and pH can vary depending on the local environment of the cell. Hence, the growth of the PG layer must be sufficiently robust to allow cell growth and division under different conditions. We have analyzed the PG composition of 28 mutants lacking multiple PG enzymes (penicillin-binding proteins PBPs) after growth in acidic or near-neutral-pH media. Statistical analysis of the muropeptide profiles identified dd-carboxypeptidases (DD-CPases) that were more active in cells grown at acidic pH. In particular, the absence of the DD-CPase PBP6b caused a significant increase in the pentapeptide content of PG as well as morphological defects when the cells were grown at acidic pH. Other DD-CPases (PBP4, PBP4b, PBP5, PBP6a, PBP7, and AmpH) and the PG synthase PBP1B made a smaller or null contribution to the pentapeptide-trimming activity at acidic pH. We solved the crystal structure of PBP6b and also demonstrated that the enzyme is more stable and has a lower Km at acidic pH, explaining why PBP6b is more active at low pH. Hence, PBP6b is a specialized DD-CPase that contributes to cell shape maintenance at low pH, and E. coli appears to utilize redundant DD-CPases for normal growth under different conditions.
Escherichia coli requires peptidoglycan dd-carboxypeptidases to maintain cell shape by controlling the amount of pentapeptide substrates available to the peptidoglycan synthetic transpeptidases. Why E. coli has eight, seemingly redundant dd-carboxypeptidases has remained unknown. We now show that one of these dd-carboxypeptidases, PBP6b, is important for cell shape maintenance in acidic growth medium, consistent with the higher activity and stability of the enzyme at low pH. Hence, the presence of multiple dd-carboxypeptidases with different enzymatic properties may allow E. coli to maintain a normal cell shape under various growth conditions.
Teichoic acid (TA), a crucial cell wall constituent of the pathobiont Streptococcus pneumoniae, is bound to peptidoglycan (wall teichoic acid, WTA) or to membrane glycolipids (lipoteichoic acid, ...LTA). Both TA polymers share a common precursor synthesis pathway, but differ in the final transfer of the TA chain to either peptidoglycan or a glycolipid. Here, we show that LTA exhibits a different linkage conformation compared to WTA, and identify TacL (previously known as RafX) as a putative lipoteichoic acid ligase required for LTA assembly. Pneumococcal mutants deficient in TacL lack LTA and show attenuated virulence in mouse models of acute pneumonia and systemic infections, although they grow normally in culture. Hence, LTA is important for S. pneumoniae to establish systemic infections, and TacL represents a potential target for antimicrobial drug development.
Bacteria employ type VI secretion systems (T6SSs) to facilitate interactions with prokaryotic and eukaryotic cells. Despite the widespread identification of T6SSs among Gram-negative bacteria, the ...number of experimentally validated substrate effector proteins mediating these interactions remains small. Here, employing an informatics approach, we define novel families of T6S peptidoglycan glycoside hydrolase effectors. Consistent with the known intercellular self-intoxication exhibited by the T6S pathway, we observe that each effector gene is located adjacent to a hypothetical open reading frame encoding a putative periplasmically localized immunity determinant. To validate our sequence-based approach, we functionally investigate a representative family member from the soil-dwelling bacterium Pseudomonas protegens. We demonstrate that this protein is secreted in a T6SS-dependent manner and that it confers a fitness advantage in growth competition assays with Pseudomonas putida. In addition, we determined the 1.4 Å x-ray crystal structure of this effector in complex with its cognate immunity protein. The structure reveals the effector shares highest overall structural similarity to a glycoside hydrolase family associated with peptidoglycan N-acetylglucosaminidase activity, suggesting that T6S peptidoglycan glycoside hydrolase effector families may comprise significant enzymatic diversity. Our structural analyses also demonstrate that self-intoxication is prevented by the immunity protein through direct occlusion of the effector active site. This work significantly expands our current understanding of T6S effector diversity.
Background: The bacterial type VI secretion system (T6SS) translocates toxic effector proteins into target cells.
Results: Novel T6S peptidoglycan glycoside hydrolase effector-immunity families are identified and a representative pair is structurally and functionally characterized.
Conclusion: Peptidoglycan glycoside hydrolase effectors are an important component of the T6S effector arsenal.
Significance: This work expands the repertoire of known T6S substrates and reports the first structure of a peptidoglycan glycoside hydrolase effector.
Summary
Staphylococcus species belong to one of the few bacterial genera that are completely lysozyme resistant, which greatly contributes to their persistence and success in colonizing the skin and ...mucosal areas of humans and animals. In an attempt to discover the cause of lysozyme resistance, we identified a gene, oatA, in Staphylococcus aureus. The corresponding oatA deletion mutant had an increased sensitivity to lysozyme. HPLC and electrospray ionization tandem mass spectrometry analyses of the cell wall revealed that the muramic acid of peptidoglycan of the wild‐type strain was O‐acetylated at C6‐OH, whereas the muramic acid of the oatA mutant lacked this modification. The complemented oatA mutant was lysozyme resistant. We identified the first bacterial peptidoglycan‐specific O‐acetyltransferase in S. aureus and showed that OatA, an integral membrane protein, is the molecular basis for the high lysozyme resistance in staphylococci.
Bacteria encase their cytoplasmic membrane with peptidoglycan (PG) to maintain the shape of the cell and protect it from bursting. The enlargement of the PG layer is facilitated by the coordinated ...activities of PG synthesising and -cleaving enzymes. In Escherichia coli, the cytoplasmic membrane-bound lytic transglycosylase MltG associates with PG synthases and was suggested to terminate the polymerisation of PG glycan strands. Using pull-down and surface plasmon resonance, we detected interactions between MltG from Bacillus subtilis and two PG synthases; the class A PBP1 and the class B PBP2B. Using in vitro PG synthesis assays with radio-labelled or fluorophore-labelled B. subtilis-type and/or E. coli-type lipid II, we showed that both, BsMltG and EcMltG, are lytic tranglycosylases and that their activity is higher during ongoing glycan strand polymerisation. MltG competed with the transpeptidase activity of class A PBPs, but had no effect on their glycosyltransferase activity, and produced glycan strands with a length of 7 disaccharide units from cleavage in the nascent strands. We hypothesize that MltG cleaves the nascent strands to produce short glycan strands that are used in the cell for a yet unknown process.
Summary
Acinetobacter baumannii has emerged as a leading nosocomial pathogen, infecting a wide range of anatomic sites including the respiratory tract and the bloodstream. In addition to being ...multi‐drug resistant, little is known about the molecular basis of A. baumannii pathogenesis. To better understand A. baumannii virulence, a combination of a transposon‐sequencing (TraDIS) screen and the neutropenic mouse model of bacteremia was used to identify the full set of fitness genes required during bloodstream infection. The lytic transglycosylase MltB was identified as a critical fitness factor. MltB cleaves the MurNAc‐GlcNAc bond of peptidoglycan, which leads to cell wall remodeling. Here we show that MltB is part of a complex network connecting resistance to stresses, membrane homeostasis, biogenesis of pili and in vivo fitness. Indeed, inactivation of mltB not only impaired resistance to serum complement, cationic antimicrobial peptides and oxygen species, but also altered the cell envelope integrity, activated the envelope stress response, drastically reduced the number of pili at the cell surface and finally, significantly decreased colonization of both the bloodstream and the respiratory tract.
Despite its clinical importance, relatively little is known about the molecular basis of Acinetobacter baumannii pathogenesis. Using a transposon‐sequencing (Tn‐Seq) screen and the mouse model of bloodstream infection, we identified the lytic transglycosylase MltB as being a critical fitness factor in A. baumannii. In this study, we showed that MltB is part of a complex network connecting resistance to stresses, cell envelope homeostasis, biogenesis of pili and in vivo fitness.
Streptococcus pneumoniae infections induce inflammatory responses that contribute toward both disease pathogenesis and immunity, but the host-pathogen interactions that mediate these effects are ...poorly defined. We used the surface lipoprotein-deficient ∆lgt pneumococcal mutant strain to test the hypothesis that lipoproteins are key determinants of TLR-mediated immune responses to S. pneumoniae. We show using reporter assays that TLR2 signaling is dependent on pneumococcal lipoproteins, and that macrophage NF-κB activation and TNF-α release were reduced in response to the ∆lgt strain. Differences in TNF-α responses between Δlgt and wild-type bacteria were abrogated for macrophages from TLR2- but not TLR4-deficient mice. Transcriptional profiling of human macrophages revealed attenuated TLR2-associated responses to ∆lgt S. pneumoniae, comprising many NF-κB-regulated proinflammatory cytokine and chemokine genes. Importantly, non-TLR2-associated responses were preserved. Experiments using leukocytes from IL-1R-associated kinase-4-deficient patients and a mouse pneumonia model confirmed that proinflammatory responses were lipoprotein dependent. Our data suggest that leukocyte responses to bacterial lipoproteins are required for TLR2- and IL-1R-associated kinase-4-mediated inflammatory responses to S. pneumoniae.
Proteins with a catalytically inactive LytM-type endopeptidase domain are important regulators of cell wall-degrading enzymes in bacteria. Here, we study their representative DipM, a factor promoting ...cell division in Caulobacter crescentus. We show that the LytM domain of DipM interacts with multiple autolysins, including the soluble lytic transglycosylases SdpA and SdpB, the amidase AmiC and the putative carboxypeptidase CrbA, and stimulates the activities of SdpA and AmiC. Its crystal structure reveals a conserved groove, which is predicted to represent the docking site for autolysins by modeling studies. Mutations in this groove indeed abolish the function of DipM in vivo and its interaction with AmiC and SdpA in vitro. Notably, DipM and its targets SdpA and SdpB stimulate each other's recruitment to midcell, establishing a self-reinforcing cycle that gradually increases autolytic activity as cytokinesis progresses. DipM thus coordinates different peptidoglycan-remodeling pathways to ensure proper cell constriction and daughter cell separation.
Summary
Clostridium difficile remains the leading cause of antibiotic‐associated diarrhoea in hospitals worldwide, linked to significant morbidity and mortality. As a strict anaerobe, it produces ...dormant cell forms – spores – which allow it to survive in the aerobic environment. Importantly, spores are the transmission agent of C. difficile infections. A key aspect of sporulation is the engulfment of the future spore by the mother cell and several proteins have been proposed to be involved. Here, we investigated the role of the SpoIID, SpoIIM and SpoIIP (DMP) machinery and its interplay with the SpoIIQ:SpoIIIAH (Q:AH) complex in C. difficile. We show that, surprisingly, SpoIIM, the proposed machinery anchor, is not required for efficient engulfment and sporulation. We demonstrate the requirement of DP for engulfment due to their sequential peptidoglycan degradation activity, both in vitro and in vivo. Finally, new interactions within DMP and between DMP and Q:AH suggest that both systems form a single engulfment machinery to keep the mother cell and forespore membranes together throughout engulfment. This work sheds new light upon the engulfment process and on how different sporeformers might use the same components in different ways to drive spore formation.
Clostridium difficile is a major cause of antibiotic related diarrhoea. C. difficile infections spread via spores, a highly resistant dormant cell form that involves a small cell being engulfed by a larger cell. We show evidence of a novel organisation of the protein machinery driving this process: two protein complexes (DP and Q:AH) act concertedly to keep the membranes tightly together as sequential enzymatic activity of SpoIIP and SpoIID drives engulfment.
Peptidoglycan is the major structural component of the Staphylococcus aureus cell wall, in which it maintains cellular integrity, is the interface with the host, and its synthesis is targeted by some ...of the most crucial antibiotics developed. Despite this importance, and the wealth of data from in vitro studies, we do not understand the structure and dynamics of peptidoglycan during infection. In this study we have developed methods to harvest bacteria from an active infection in order to purify cell walls for biochemical analysis ex vivo. Isolated ex vivo bacterial cells are smaller than those actively growing in vitro, with thickened cell walls and reduced peptidoglycan crosslinking, similar to that of stationary phase cells. These features suggested a role for specific peptidoglycan homeostatic mechanisms in disease. As S. aureus missing penicillin binding protein 4 (PBP4) has reduced peptidoglycan crosslinking in vitro its role during infection was established. Loss of PBP4 resulted in an increased recovery of S. aureus from the livers of infected mice, which coincided with enhanced fitness within murine and human macrophages. Thicker cell walls correlate with reduced activity of peptidoglycan hydrolases. S. aureus has a family of 4 putative glucosaminidases, that are collectively crucial for growth. Loss of the major enzyme SagB, led to attenuation during murine infection and reduced survival in human macrophages. However, loss of the other three enzymes Atl, SagA and ScaH resulted in clustering dependent attenuation, in a zebrafish embryo, but not a murine, model of infection. A combination of pbp4 and sagB deficiencies resulted in a restoration of parental virulence. Our results, demonstrate the importance of appropriate cell wall structure and dynamics during pathogenesis, providing new insight to the mechanisms of disease.
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