Type VI secretion delivers bacteriolytic effectors to target cells.

Peptidoglycan is the major structural constituent of the bacterial cell wall, forming a meshwork outside the cytoplasmic membrane that maintains cell shape and prevents lysis. In Gram-negative bacteria, peptidoglycan is located in the periplasm, where it is protected from exogenous lytic enzymes by the outer membrane. Here we show that the type VI secretion system of Pseudomonas aeruginosa breaches this barrier to deliver two effector proteins, Tse1 and Tse3, to the periplasm of recipient cells. In this compartment, the effectors hydrolyse peptidoglycan, thereby providing a fitness advantage for P. aeruginosa cells in competition with other bacteria. To protect itself from lysis by Tse1 and Tse3, P. aeruginosa uses specific periplasmically localized immunity proteins. The requirement for these immunity proteins depends on intercellular self-intoxication through an active type VI secretion system, indicating a mechanism for export whereby effectors do not access donor cell periplasm in transit.

Transfer of penicillin resistance from Streptococcus oralis to Streptococcus pneumoniae identifies murE as resistance determinant.

Beta-lactam resistant clinical isolates of Streptococcus pneumoniae contain altered penicillin-binding protein (PBP) genes and occasionally an altered murM, presumably products of interspecies gene transfer. MurM and MurN are responsible for the synthesis of branched lipid II, substrate for the PBP catalyzed transpeptidation reaction. Here we used the high-level beta-lactam resistant S. oralis Uo5 as donor in transformation experiments with the sensitive laboratory strain S. pneumoniae R6 as recipient. Surprisingly, piperacillin-resistant transformants contained no alterations in PBP genes but carried murEUo5 encoding the UDP-N-acetylmuramyl tripeptide synthetase. Codons 83-183 of murEUo5 were sufficient to confer the resistance phenotype. Moreover, the promoter of murEUo5 , which drives a twofold higher expression compared to that of S. pneumoniae R6, could also confer increased resistance. Multiple independent transformations produced S. pneumoniae R6 derivatives containing murEUo5 , pbp2xUo5 , pbp1aUo5 and pbp2bUo5 , but not murMUo5 sequences; however, the resistance level of the donor strain could not be reached. S. oralis Uo5 harbors an unusual murM, and murN is absent. Accordingly, the peptidoglycan of S. oralis Uo5 contained interpeptide bridges with one L-Ala residue only. The data suggest that resistance in S. oralis Uo5 is based on a complex interplay of distinct PBPs and other enzymes involved in peptidoglycan biosynthesis.

A widespread family of bacterial cell wall assembly proteins.

Teichoic acids and acidic capsular polysaccharides are major anionic cell wall polymers (APs) in many bacteria, with various critical cell functions, including maintenance of cell shape and structural integrity, charge and cation homeostasis, and multiple aspects of pathogenesis. We have identified the widespread LytR-Cps2A-Psr (LCP) protein family, of previously unknown function, as novel enzymes required for AP synthesis. Structural and biochemical analysis of several LCP proteins suggest that they carry out the final step of transferring APs from their lipid-linked precursor to cell wall peptidoglycan (PG). In Bacillus subtilis, LCP proteins are found in association with the MreB cytoskeleton, suggesting that MreB proteins coordinate the insertion of the major polymers, PG and AP, into the cell wall.

Specialized peptidoglycan hydrolases sculpt the intra-bacterial niche of predatory Bdellovibrio and increase population fitness.

Bdellovibrio are predatory bacteria that have evolved to invade virtually all gram-negative bacteria, including many prominent pathogens. Upon invasion, prey bacteria become rounded up into an osmotically stable niche for the Bdellovibrio, preventing further superinfection and allowing Bdellovibrio to replicate inside without competition, killing the prey bacterium and degrading its contents. Historically, prey rounding was hypothesized to be associated with peptidoglycan (PG) metabolism; we found two Bdellovibrio genes, bd0816 and bd3459, expressed at prey entry and encoding proteins with limited homologies to conventional dacB/PBP4 DD-endo/carboxypeptidases (responsible for peptidoglycan maintenance during growth and division). We tested possible links between Bd0816/3459 activity and predation. Bd3459, but not an active site serine mutant protein, bound ß-lactam, exhibited DD-endo/carboxypeptidase activity against purified peptidoglycan and, importantly, rounded up E. coli cells upon periplasmic expression. A ΔBd0816 ΔBd3459 double mutant invaded prey more slowly than the wild type (with negligible prey cell rounding) and double invasions of single prey by more than one Bdellovibrio became more frequent. We solved the crystal structure of Bd3459 to 1.45 Å and this revealed predation-associated domain differences to conventional PBP4 housekeeping enzymes (loss of the regulatory domain III, alteration of domain II and a more exposed active site). The Bd3459 active site (and by similarity the Bd0816 active site) can thus accommodate and remodel the various bacterial PGs that Bdellovibrio may encounter across its diverse prey range, compared to the more closed active site that "regular" PBP4s have for self cell wall maintenance. Therefore, during evolution, Bdellovibrio peptidoglycan endopeptidases have adapted into secreted predation-specific proteins, preventing wasteful double invasion, and allowing activity upon the diverse prey peptidoglycan structures to sculpt the prey cell into a stable intracellular niche for replication.

Characterization of DalS, an ATP-binding cassette transporter for D-alanine, and its role in pathogenesis in Salmonella enterica.

Expansion into new host niches requires bacterial pathogens to adapt to changes in nutrient availability and to evade an arsenal of host defenses. Horizontal acquisition of Salmonella Pathogenicity Island (SPI)-2 permitted the expansion of Salmonella enterica serovar Typhimurium into the intracellular environment of host cells by allowing it to deliver bacterial effector proteins across the phagosome membrane. This is facilitated by the SsrA-SsrB two-component regulatory system and a type III secretion system encoded within SPI-2. SPI-2 acquisition was followed by evolution of existing regulatory DNA, creating an expanded SsrB regulon involved in intracellular fitness and host infection. Here, we identified an SsrB-regulated operon comprising an ABC transporter in Salmonella. Biochemical and structural studies determined that the periplasmic solute-binding component, STM1633/DalS, transports D-alanine and that DalS is required for intracellular survival of the bacteria and for fitness in an animal host. This work exemplifies the role of nutrient exchange at the host-pathogen interface as a critical determinant of disease outcome.

Structural basis for type VI secreted peptidoglycan DL-endopeptidase function, specificity and neutralization in Serratia marcescens.

Some Gram-negative bacteria target their competitors by exploiting the type VI secretion system to extrude toxic effector proteins. To prevent self-harm, these bacteria also produce highly specific immunity proteins that neutralize these antagonistic effectors. Here, the peptidoglycan endopeptidase specificity of two type VI secretion-system-associated effectors from Serratia marcescens is characterized. These small secreted proteins, Ssp1 and Ssp2, cleave between γ-D-glutamic acid and L-meso-diaminopimelic acid with different specificities. Ssp2 degrades the acceptor part of cross-linked tetratetrapeptides. Ssp1 displays greater promiscuity and cleaves monomeric tripeptides, tetrapeptides and pentapeptides and dimeric tetratetra and tetrapenta muropeptides on both the acceptor and donor strands. Functional assays confirm the identity of a catalytic cysteine in these endopeptidases and crystal structures provide information on the structure-activity relationships of Ssp1 and, by comparison, of related effectors. Functional assays also reveal that neutralization of these effectors by their cognate immunity proteins, which are called resistance-associated proteins (Raps), contributes an essential role to cell fitness. The structures of two immunity proteins, Rap1a and Rap2a, responsible for the neutralization of Ssp1 and Ssp2-like endopeptidases, respectively, revealed two distinct folds, with that of Rap1a not having previously been observed. The structure of the Ssp1-Rap1a complex revealed a tightly bound heteromeric assembly with two effector molecules flanking a Rap1a dimer. A highly effective steric block of the Ssp1 active site forms the basis of effector neutralization. Comparisons with Ssp2-Rap2a orthologues suggest that the specificity of these immunity proteins for neutralizing effectors is fold-dependent and that in cases where the fold is conserved sequence differences contribute to the specificity of effector-immunity protein interactions.

Acquisition of VanB-type vancomycin resistance by Bacillus subtilis: the impact on gene expression, cell wall composition and morphology.

The vancomycin resistance operons from Enterococci, Staphylococci and Actinomycetes encode a VanRS two-component signal transduction system (TCS) and a suite of enzymes to modify the peptidoglycan biosynthetic precursor lipid II and to eliminate the D-Ala-D-Ala from the cell. Commingling of these regulatory and enzymatic activities with host functions has the potential to significantly impact host gene expression and cell wall metabolism. Here we report the effects of individually expressing the VanR(B) S(B) TCS and the VanY(B) WH(B) BX(B) resistance proteins in Bacillus subtilis. VanY(B) WH(B) BX(B) expression confers resistance to 2 µg ml(-1) of vancomycin with concomitant reduced Van-FL staining and leads to a cell division defect. In contrast to E. faecalis and S. aureus, VanS(B) is active in B. subtilis without vancomycin addition. Individual expression of the VanR(B) S(B) TCS and the VanY(B) WH(B) BX(B) resistance proteins repress and increase, respectively, expression of PhoPR regulon genes in the phosphate-limited state. When vancomycin-resistant cells are exposed to elevated vancomycin levels, mutant strains with increased resistance to vancomycin and a growth dependency on vanY(B) WH(B) BX(B) expression frequently arise. Mutation of the endogenous Ddl ligase is the necessary and sufficient cause of both phenotypes. We discuss how these effects may influence establishment of van operons in new host bacteria.

Isolation and analysis of cell wall components from Streptococcus pneumoniae.

The complex and heterogeneous cell wall of the pathogenic bacterium Streptococcus pneumoniae is composed of peptidoglycan and a covalently attached wall teichoic acid. The net-like peptidoglycan is formed by glycan chains that are crosslinked by short peptides. We have developed a method to purify the glycan chains, and we show that they are longer than approximately 25 disaccharide units. From purified peptidoglycan, we released 50 muropeptides that differ in the length of their peptides (tri-, tetra-, or pentapeptides with or without mono- or dipeptide branch), the degree of peptide crosslinking (monomer, dimer, or trimer), and the presence of modifications in the glycan chains (N-deacetylation, O-acetylation, or lack of GlcNAc or GlcNAc-MurNAc) or peptides (glutamic acid instead of glutamine). We also established a method to isolate wall teichoic acid chains and show that the most abundant chains have 6 or 7 repeating units. Finally, we obtained solid-state nuclear magnetic resonance spectra of whole insoluble cell walls. These novel tools will help to characterize mutant strains, cell wall-modifying enzymes, and protein-cell wall interactions.

Dynamics characterization of fully hydrated bacterial cell walls by solid-state NMR: evidence for cooperative binding of metal ions.

The bacterial cell wall maintains a cell's integrity while allowing growth and division. It is made up of peptidoglycan (PG), a biopolymer forming a multigigadalton bag-like structure, and, additionally in gram-positive bacteria, of covalently linked anionic polymers collectively called teichoic acids. These anionic polymers are thought to play important roles in host-cell adhesion, inflammation, and immune activation. In this Article, we compare the flexibility and the organization of peptidoglycans from gram-negative bacteria (E. coli) with its counterpart from different gram-positive bacteria using solid-state nuclear magnetic resonance spectroscopy (NMR) under magic-angle sample spinning (MAS). The NMR fingerprints suggest an identical local conformation of the PG in all of these bacterial species. Dynamics in the peptidoglycan network decreases from E. coli to B. subtilis and from B. subtilis to S. aureus and correlates mainly with the degree of peptide cross-linkage. For intact bacterial cells and isolated cell walls, we show that (31)P solid-state NMR is particularly well adapted to characterize and differentiate wall teichoic acids of different species. We have further observed complexation with divalent ions, highlighting an important structural aspect of gram-positive cell wall architecture. We propose a new model for the interaction of divalent cations with both wall teichoic acids and carbonyl groups of peptidoglycan.

The peptidoglycan sacculus of Myxococcus xanthus has unusual structural features and is degraded during glycerol-induced myxospore development.

Upon nutrient limitation cells of the swarming soil bacterium Myxococcus xanthus form a multicellular fruiting body in which a fraction of the cells develop into myxospores. Spore development includes the transition from a rod-shaped vegetative cell to a spherical myxospore and so is expected to be accompanied by changes in the bacterial cell envelope. Peptidoglycan is the shape-determining structure in the cell envelope of most bacteria, including myxobacteria. We analyzed the composition of peptidoglycan isolated from M. xanthus. While the basic structural elements of peptidoglycan in myxobacteria were identical to those in other gram-negative bacteria, the peptidoglycan of M. xanthus had unique structural features. meso- or LL-diaminopimelic acid was present in the stem peptides, and a new modification of N-acetylmuramic acid was detected in a fraction of the muropeptides. Peptidoglycan formed a continuous, bag-shaped sacculus in vegetative cells. The sacculus was degraded during the transition from vegetative cells to glycerol-induced myxospores. The spherical, bag-shaped coats isolated from glycerol-induced spores contained no detectable muropeptides, but they contained small amounts of N-acetylmuramic acid and meso-diaminopimelic acid.

Significant contribution of the pgdA gene to the virulence of Streptococcus suis.

Streptococcus suis is a major swine pathogen and emerging zoonotic agent. In this study we have determined the muropeptide composition of S. suis peptidoglycan (PG) and found, among other modifications, N-deacetylated compounds. Comparison with an isogenic mutant showed that the product of the pgdA gene is responsible for this specific modification which occurred in very low amounts. Low level of PG N-deacetylation correlated with absence of significant lysozyme resistance when wild-type S. suis was grown in vitro. On the other hand, expression of the pgdA gene was increased upon interaction of the bacterium with neutrophils in vitro as well as in vivo in experimentally inoculated mice, suggesting that S. suis may enhance PG N-deacetylation under these conditions. Evaluation of the DeltapgdA mutant in both the CD1 murine and the porcine models of infection revealed a significant contribution of the pgdA gene to the virulence traits of S. suis. Reflecting a severe impairment in its ability to persist in blood and decreased ability to escape immune clearance mechanisms mediated by neutrophils, the DeltapgdA mutant was highly attenuated in both models. The results of this study suggest that modification of PG by N-deacetylation is an important factor in S. suis virulence.

Interrupting peptidoglycan deacetylation during Bdellovibrio predator-prey interaction prevents ultimate destruction of prey wall, liberating bacterial-ghosts.

The peptidoglycan wall, located in the periplasm between the inner and outer membranes of the cell envelope in Gram-negative bacteria, maintains cell shape and endows osmotic robustness. Predatory Bdellovibrio bacteria invade the periplasm of other bacterial prey cells, usually crossing the peptidoglycan layer, forming transient structures called bdelloplasts within which the predators replicate. Prey peptidoglycan remains intact for several hours, but is modified and then degraded by escaping predators. Here we show predation is altered by deleting two Bdellovibrio N-acetylglucosamine (GlcNAc) deacetylases, one of which we show to have a unique two domain structure with a novel regulatory"plug". Deleting the deacetylases limits peptidoglycan degradation and rounded prey cell "ghosts" persist after mutant-predator exit. Mutant predators can replicate unusually in the periplasmic region between the peptidoglycan wall and the outer membrane rather than between wall and inner-membrane, yet still obtain nutrients from the prey cytoplasm. Deleting two further genes encoding DacB/PBP4 family proteins, known to decrosslink and round prey peptidoglycan, results in a quadruple mutant Bdellovibrio which leaves prey-shaped ghosts upon predation. The resultant bacterial ghosts contain cytoplasmic membrane within bacteria-shaped peptidoglycan surrounded by outer membrane material which could have promise as "bacterial skeletons" for housing artificial chromosomes.

Ankyrin-mediated self-protection during cell invasion by the bacterial predator Bdellovibrio bacteriovorus.

Predatory Bdellovibrio bacteriovorus are natural antimicrobial organisms, killing other bacteria by whole-cell invasion. Self-protection against prey-metabolizing enzymes is important for the evolution of predation. Initial prey entry involves the predator's peptidoglycan DD-endopeptidases, which decrosslink cell walls and prevent wasteful entry by a second predator. Here we identify and characterize a self-protection protein from B. bacteriovorus, Bd3460, which displays an ankyrin-based fold common to intracellular pathogens of eukaryotes. Co-crystal structures reveal Bd3460 complexation of dual targets, binding a conserved epitope of each of the Bd3459 and Bd0816 endopeptidases. Complexation inhibits endopeptidase activity and cell wall decrosslinking in vitro. Self-protection is vital - ΔBd3460 Bdellovibrio deleteriously decrosslink self-peptidoglycan upon invasion, adopt a round morphology, and lose predatory capacity and cellular integrity. Our analysis provides the first mechanistic examination of self-protection in Bdellovibrio, documents protection-multiplicity for products of two different genomic loci, and reveals an important evolutionary adaptation to an invasive predatory bacterial lifestyle.

Structure and function of a spectrin-like regulator of bacterial cytokinesis.

Bacterial cell division is facilitated by a molecular machine--the divisome--that assembles at mid-cell in dividing cells. The formation of the cytokinetic Z-ring by the tubulin homologue FtsZ is regulated by several factors, including the divisome component EzrA. Here we describe the structure of the 60-kDa cytoplasmic domain of EzrA, which comprises five linear repeats of an unusual triple helical bundle. The EzrA structure is bent into a semicircle, providing the protein with the potential to interact at both N- and C-termini with adjacent membrane-bound divisome components. We also identify at least two binding sites for FtsZ on EzrA and map regions of EzrA that are responsible for regulating FtsZ assembly. The individual repeats, and their linear organization, are homologous to the spectrin proteins that connect actin filaments to the membrane in eukaryotes, and we thus propose that EzrA is the founding member of the bacterial spectrin family.

In vitro peptidoglycan synthesis assay with lipid II substrate.

Bacterial cell wall peptidoglycan is synthesized from lipid II precursor by two reactions. Glycosyltransferases polymerize the glycan chains and transpeptidases form the peptide cross-links. The bifunctional class A penicillin-binding proteins catalyze both of these reactions. Here, we describe an in vitro peptidoglycan synthesis assay utilizing radiolabeled lipid II substrate to monitor simultaneously peptidoglycan glycosyltransferase and transpeptidase activities. The products of the reaction are separated by high-pressure liquid chromatography and quantified by flow-through scintillation counting.

Growth medium-dependent glycine incorporation into the peptidoglycan of Caulobacter crescentus.

The peptidoglycan (PG) is a macromolecular component of the bacterial cell wall that maintains the shape and integrity of the cell. The PG of Caulobacter crescentus, unlike that of many other Gram-negative bacteria, has repeatedly been shown to contain significant amounts of glycine. This compositional peculiarity has been deemed an intrinsic characteristic of this species. By performing a comprehensive qualitative and quantitative analysis of the C. crescentus PG by high-performance liquid chromatography (HPLC) and mass spectrometry (MS), we show here that glycine incorporation into the C. crescentus PG depends on the presence of exogenous glycine in the growth medium. High levels of glycine were detected at the fifth position of the peptide side chains of PG isolated from C. crescentus cells grown in the complex laboratory medium PYE or in defined medium (M2G) supplemented with casamino acids or glycine alone. In contrast, glycine incorporation was undetectable when cells were grown in M2G medium lacking glycine. Remarkably, glycine incorporation into C. crescentus peptidoglycan occurred even in the presence of low millimolar to sub-millimolar concentrations of free glycine. High glycine content in the PG had no obvious effects on growth rates, mode of PG incorporation or cell morphology. Hence, the C. crescentus PG is able to retain its physiological functions in cell growth and morphogenesis despite significant alterations in its composition, in what we deem to be unprecedented plasticity.

Structure of a peptidoglycan amidase effector targeted to Gram-negative bacteria by the type VI secretion system.

The target range of a bacterial secretion system can be defined by effector substrate specificity or by the efficacy of effector delivery. Here, we report the crystal structure of Tse1, a type VI secretion (T6S) bacteriolytic amidase effector from Pseudomonas aeruginosa. Consistent with its role as a toxin, Tse1 has a more accessible active site than related housekeeping enzymes. The activity of Tse1 against isolated peptidoglycan shows its capacity to act broadly against Gram-negative bacteria and even certain Gram-positive species. Studies with intact cells indicate that Gram-positive bacteria can remain vulnerable to Tse1 despite cell wall modifications. However, interbacterial competition studies demonstrate that Tse1-dependent lysis is restricted to Gram-negative targets. We propose that the previously observed specificity for T6S against Gram-negative bacteria is a consequence of high local effector concentration achieved by T6S-dependent targeting to its site of action rather than inherent effector substrate specificity.

A widespread bacterial type VI secretion effector superfamily identified using a heuristic approach.

Sophisticated mechanisms are employed to facilitate information exchange between interfacing bacteria. A type VI secretion system (T6SS) of Pseudomonas aeruginosa was shown to deliver cell wall-targeting effectors to neighboring cells. However, the generality of bacteriolytic effectors and, moreover, of antibacterial T6S remained unknown. Using parameters derived from experimentally validated bacterial T6SS effectors we identified a phylogenetically disperse superfamily of T6SS-associated peptidoglycan-degrading effectors. The effectors separate into four families composed of peptidoglycan amidase enzymes of differing specificities. Effectors strictly co-occur with cognate immunity proteins, indicating that self-intoxication is a general property of antibacterial T6SSs and effector delivery by the system exerts a strong selective pressure in nature. The presence of antibacterial effectors in a plethora of organisms, including many that inhabit or infect polymicrobial niches in the human body, suggests that the system could mediate interbacterial interactions of both environmental and clinical significance.

Development of screening assays and discovery of initial inhibitors of pneumococcal peptidoglycan deacetylase PgdA.

The essential cell wall peptidoglycan is the target of several components of the innate immune system and its disruption results in lysis of invading bacteria. The pathogen Streptococcus pneumoniae produces a peptidoglycan N-acetylglucosamine deacetylase, PgdA, to modify the peptidoglycan structure. The activity of PgdA contributes to the bacteria's resistance to lysozyme, which is an important antimicrobial factor of the human innate immune system. In this study we report on the activity of PgdA against natural and artificial substrates. We have also established a virtual high-throughput screening and a new enzyme assay to search for compounds inhibiting PgdA. Two compounds with IC(50) values in the micromolar range have been identified and they could serve as leads for the search of inhibitors of PgdA, an important pneumococcal virulence factor.