Dlt Operon Research Articles

Characterization of the response to low pH ofLactobacillus caseiΔRR12, a mutant strain with low D-alanylation activity and sensitivity to low pH

To identify the differences that account for the acid sensitivity of Lactobacillus casei ΔRR12. RR12 controls the expression of the dlt operon, and its inactivation leads to a diminished teichoic acid D-alanylation activity. To this end, a comparison of its response of ΔRR12 to low pH with the parental strain Lact. casei BL23 was carried out. The ability to induce an acid tolerance response (ATR), fatty acid (FA) composition and proteome changes induced in both strains in response to acid were investigated. Results obtained showed that both strains induce a growth-phase-dependent ATR. However, significant differences in the content of FAs and membrane-associated proteins were detected. The greater abundance of cytoplasmic proteins in the membrane fraction of the mutant strain ΔRR12 suggests an increased permeability of the cell membrane in this strain. The analysis of the response to low pH of strain ΔRR12 indicated that the inactivation of TCS12 affected the content of FAs and proteins associated to the cell envelope. Increased abundance of cytoplasmic proteins suggested that low alanylation of teichoic acids affected the permeability of the cell membrane and possibly accounts for the acid sensitivity of strain ΔRR12.

Contributions of the σ(W) , σ(M) and σ(X) regulons to the lantibiotic resistome of Bacillus subtilis.

In Bacillus subtilis, the extracytoplasmic function (ECF) σ factors σ(M) , σ(W) and σ(X) all contribute to resistance against lantibiotics. Nisin, a model lantibiotic, has a dual mode of action: it inhibits cell wall synthesis by binding lipid II, and this complex also forms pores in the cytoplasmic membrane. These activities can be separated in a nisin hinge-region variant (N20P M21P) that binds lipid II, but no longer permeabilizes membranes. The major contribution of σ(M) to nisin resistance is expression of ltaSa, encoding a stress-activated lipoteichoic acid synthase, and σ(X) functions primarily by activation of the dlt operon controlling d-alanylation of teichoic acids. Together, σ(M) and σ(X) regulate cell envelope structure to decrease access of nisin to its lipid II target. In contrast, σ(W) is principally involved in protection against membrane permeabilization as it provides little protection against the nisin hinge region variant. σ(W) contributes to nisin resistance by regulation of a signal peptide peptidase (SppA), phage shock proteins (PspA and YvlC, a PspC homologue) and tellurite resistance related proteins (YceGHI). These defensive mechanisms are also effective against other lantibiotics such as mersacidin, gallidermin and subtilin and comprise an important subset of the intrinsic antibiotic resistome of B. subtilis.

Mechanisms of daptomycin resistance in Staphylococcus aureus: role of the cell membrane and cell wall.

The bactericidal, cell membrane-targeting lipopeptide antibiotic daptomycin (DAP) is an important agent in treating invasive Staphylococcus aureus infections. However, there have been numerous recent reports of development of daptomycin resistance (DAP-R) during therapy with this agent. The mechanisms of DAP-R in S. aureus appear to be quite diverse. DAP-R strains often exhibit progressive accumulation of single nucleotide polymorphisms in the multipeptide resistance factor gene (mprF) and the yycFG components of the yycFGHI operon. Both loci are involved in key cell membrane (CM) events, with mprF being responsible for the synthesis and outer CM translocation of the positively charged phospholipid, lysyl-phosphotidylglycerol (L-PG), while the yyc operon is involved in the generalized response to stressors such as antimicrobials. In addition, other perturbations of the CM have been identified in DAP-R strains, including extremes in CM order, resistance to CM depolarization and permeabilization, and reduced surface binding of DAP. Moreover, modifications of the cell wall (CW) appear to also contribute to DAP-R, including enhanced expression of the dlt operon (involved in d-alanylation of CW teichoic acids) and progressive CW thickening.

Transposon library screening for identification of genetic loci participating in intrinsic susceptibility and acquired resistance to antistaphylococcal agents

To establish an experimental platform in Staphylococcus aureus for identifying genetic loci that determine intrinsic antibiotic susceptibility and/or that have the potential to contribute to acquired antibiotic resistance. A near-saturation S. aureus transposon (Tn) library was screened for mutants exhibiting altered susceptibility to the antistaphylococcal agents daptomycin, vancomycin and nisin. S. aureus SH1000 was mutagenized with Tn InsTet(G+)2(Cm) by electroporation of transposomes. Approximately 20500 transposants were screened for increased or reduced susceptibility to the three antistaphylococcal agents and Tn insertion sites were mapped by DNA sequencing in mutants of interest. Transposants exhibiting hypersusceptibility or reduced susceptibility were identified for all three antibacterial agents; mapping of Tn insertion sites in these mutants identified genetic determinants of intrinsic susceptibility and potential contributors to acquired resistance, respectively. Tn insertions in the dlt operon caused cross-hypersusceptibility to vancomycin, daptomycin and nisin. Daptomycin hypersusceptibility was also associated with disruption of genes directing lipoteichoic acid and riboflavin biosynthesis, apparent inactivation of a putative membrane protein encoded by SAOUHSC_00957 and truncation of the cell-division gene ezrA. Tn-mediated disruption of the vraDE- and SAOUHSC_02953/4-encoded ABC transporters conferred hypersusceptibility to nisin. Reduced susceptibility to both daptomycin and vancomycin was associated with Tn insertions in rpsU and upstream of yycFG. Several loci were associated with reduced susceptibility to nisin, including two genes encoding putative glycosyltransferases. Tn library screening identified both known and novel modulators of antibacterial susceptibility in S. aureus and therefore represents a useful approach towards delineating the staphylococcal resistome.

Genome-wide transcriptional profiling of the cell envelope stress response and the role of LisRK and CesRK in Listeria monocytogenes

The Gram-positive bacterium Listeria monocytogenes is widely distributed in the environment and capable of causing food-borne infections in susceptible individuals. In this study, we investigated the cell envelope stress response in L. monocytogenes. Whole-genome transcriptional profiling was performed to investigate the response upon exposure to the cell wall antibiotic cefuroxime. Differential expression (at least twofold) of 558 genes was observed, corresponding to 20 % of the L. monocytogenes genome. The majority of genes that were strongly induced by cefuroxime exposure have cell-envelope-related functions, including the dlt operon and the gene encoding penicillin-binding protein PBPD2. A large overlap was observed between the cefuroxime stimulon and genes known to be induced in L. monocytogenes in blood and during intracellular infection, indicating that the cell envelope stress response is active at various stages of the infectious process. We analysed the roles of the two-component systems LisRK and CesRK in the cell envelope response, showing that activation of the most highly cefuroxime-induced genes was LisR- and CesR-dependent. In addition, multiple VirRS- and LiaSR-regulated genes were found to be induced in response to cefuroxime exposure. In total, 53 % of the genes upregulated at least fourfold by cefuroxime exposure are under positive control by one of the four two-component systems. Using genetic analyses, we showed that several genes of the cefuroxime stimulon contribute to the innate resistance of L. monocytogenes to cefuroxime and tolerance to other cell-envelope-perturbing conditions. Collectively, these findings demonstrate central roles for two-component systems in orchestrating the cell envelope stress response in L. monocytogenes.

Investigation of the Staphylococcus aureus GraSR Regulon Reveals Novel Links to Virulence, Stress Response and Cell Wall Signal Transduction Pathways

The GraS/GraR two-component system has been shown to control cationic antimicrobial peptide (CAMP) resistance in the major human pathogen Staphylococcus aureus. We demonstrated that graX, also involved in CAMP resistance and cotranscribed with graRS, encodes a regulatory cofactor of the GraSR signaling pathway, effectively constituting a three-component system. We identified a highly conserved ten base pair palindromic sequence (5′ ACAAA TTTGT 3′) located upstream from GraR-regulated genes (mprF and the dlt and vraFG operons), which we show to be essential for transcriptional regulation by GraR and induction in response to CAMPs, suggesting it is the likely GraR binding site. Genome-based predictions and transcriptome analysis revealed several novel GraR target genes. We also found that the GraSR TCS is required for growth of S. aureus at high temperatures and resistance to oxidative stress. The GraSR system has previously been shown to play a role in S. aureus pathogenesis and we have uncovered previously unsuspected links with the AgrCA peptide quorum-sensing system controlling virulence gene expression. We also show that the GraSR TCS controls stress reponse and cell wall metabolism signal transduction pathways, sharing an extensive overlap with the WalKR regulon. This is the first report showing a role for the GraSR TCS in high temperature and oxidative stress survival and linking this system to stress response, cell wall and pathogenesis control pathways.

Phenotypic, Proteomic, and Genomic Characterization of a Putative ABC-Transporter Permease Involved in Listeria monocytogenes Biofilm Formation

The foodborne pathogen Listeria monocytogenes is able to form biofilms in food processing environments. Previously, we have reported that an lm.G_1771 gene (encoding a putative ABC-transporter permease) was involved in negative regulation of L. monocytogenes biofilm formation using LM-49, a biofilm-enhanced mutant isolated on Tn917 mutagenesis (AEM 2008 p.7675-7683). Here, the possible action of this ABC-transporter permease in L. monocytogenes biofilm formation was characterized by phenotypic, proteomic, and genomic analyses using an lm.G_1771 gene deletant (Δ1771). The Δ1771 mutant exhibited the same enhanced ability for biofilm formation as the LM-49 strain using a crystal violet staining assay. DNA microarrays and two-dimensional gel electrophoresis revealed 49 and 11 differentially expressed (twofold or more) genes or proteins in Δ1771, respectively. The transcriptomics study indicated that lm.G_1771 could play a vital role in regulating candidate genes involved in biofilm formation such as genes encoding cell surface proteins (Dlt), cell surface anchor proteins (SrtA), and transcriptional regulators (GntR) contributing to negative regulation of biofilm formation by L. monocytogenes. The mutant Δ1771 was more sensitive to Triton X-100 and less resistant to cationic antibiotics, which might be explained by the down-regulation of dlt operon in this deletant and the fact that dlt involves the incorporation of D-alanine residues into lipoteichoic acids, resulting in a positive net charge on the teichoic acids. Therefore, lm.G_1771 is considered to be involved in negative regulation of biofilm formation, and the results from this work provide a possible molecular mechanism of biofilm formation regulated by lm.G_1771 in L. monocytogenes.

ThedltOperon ofBacillus cereusIs Required for Resistance to Cationic Antimicrobial Peptides and for Virulence in Insects

The dlt operon encodes proteins that alanylate teichoic acids, the major components of cell walls of gram-positive bacteria. This generates a net positive charge on bacterial cell walls, repulsing positively charged molecules and conferring resistance to animal and human cationic antimicrobial peptides (AMPs) in gram-positive pathogenic bacteria. AMPs damage the bacterial membrane and are the most effective components of the humoral immune response against bacteria. We investigated the role of the dlt operon in insect virulence by inactivating this operon in Bacillus cereus, which is both an opportunistic human pathogen and an insect pathogen. The Delta dlt(Bc) mutant displayed several morphological alterations but grew at a rate similar to that for the wild-type strain. This mutant was less resistant to protamine and several bacterial cationic AMPs, such as nisin, polymyxin B, and colistin, in vitro. It was also less resistant to molecules from the insect humoral immune system, lysozyme, and cationic AMP cecropin B from Spodoptera frugiperda. Delta dlt(Bc) was as pathogenic as the wild-type strain in oral infections of Galleria mellonella but much less virulent when injected into the hemocoels of G. mellonella and Spodoptera littoralis. We detected the dlt operon in three gram-negative genera: Erwinia (Erwinia carotovora), Bordetella (Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), and Photorhabdus (the entomopathogenic bacterium Photorhabdus luminescens TT01, the dlt operon of which did not restore cationic AMP resistance in Delta dlt(Bc)). We suggest that the dlt operon protects B. cereus against insect humoral immune mediators, including hemolymph cationic AMPs, and may be critical for the establishment of lethal septicemia in insects and in nosocomial infections in humans.

Neutrophil antimicrobial defense against Staphylococcus aureus is mediated by phagolysosomal but not extracellular trap-associated cathelicidin.

Neutrophils kill invading pathogens by AMPs, including cathelicidins, ROS, and NETs. The human pathogen Staphylococcus aureus exhibits enhanced resistance to neutrophil AMPs, including the murine cathelicidin CRAMP, in part, as a result of alanylation of teichoic acids by the dlt operon. In this study, we took advantage of the hypersusceptible phenotype of S. aureus DeltadltA against cationic AMPs to study the impact of the murine cathelicidin CRAMP on staphylococcal killing and to identify its key site of action in murine neutrophils. We demonstrate that CRAMP remained intracellular during PMN exudation from blood and was secreted upon PMA stimulation. We show first evidence that CRAMP was recruited to phagolysosomes in infected neutrophils and exhibited intracellular activity against S. aureus. Later in infection, neutrophils produced NETs, and immunofluorescence revealed association of CRAMP with S. aureus in NETs, which similarly killed S. aureus wt and DeltadltA, indicating that CRAMP activity was reduced when associated with NETs. Indeed, the presence of DNA reduced the antimicrobial activity of CRAMP, and CRAMP localization in response to S. aureus was independent of the NADPH oxidase, whereas killing was partially dependent on a functional NADPH oxidase. Our study indicates that neutrophils use CRAMP in a timed and locally coordinated manner in defense against S. aureus.

Inactivation of DltA Modulates Virulence Factor Expression in Streptococcus pyogenes

BackgroundD-alanylated lipoteichoic acid is a virtually ubiquitous component of Gram-positive cell walls. Mutations in the dltABCD operon of numerous species exhibit pleiotropic effects, including reduced virulence, which has been attributed to increased binding of cationic antimicrobial peptides to the more negatively charged cell surface. In this study, we have further investigated the effects that mutating dltA has on virulence factor expression in Streptococcus pyogenes.Methodology/Principal FindingsIsogenic ΔdltA mutants had previously been created in two distinct M1T1 isolates of S. pyogenes. Immunoblots, flow cytometry, and immunofluorescence were used to quantitate M protein levels in these strains, as well as to assess their ability to bind complement. Bacteria were tested for their ability to interact with human PMN and to grow in whole human blood. Message levels for emm, sic, and various regulatory elements were assessed by quantitative RT-PCR. Cell walls of ΔdltA mutants contained much less M protein than cell walls of parent strains and this correlated with reduced levels of emm transcripts, increased deposition of complement, increased association of bacteria with polymorphonuclear leukocytes, and reduced bacterial growth in whole human blood. Transcription of at least one other gene of the mga regulon, sic, which encodes a protein that inactivates antimicrobial peptides, was also dramatically reduced in ΔdltA mutants. Concomitantly, ccpA and rofA were unaffected, while rgg and arcA were up-regulated.Conclusions/SignificanceThis study has identified a novel mechanism for the reduced virulence of dltA mutants of Streptococcus pyogenes in which gene regulatory networks somehow sense and respond to the loss of DltA and lack of D-alanine esterification of lipoteichoic acid. The mechanism remains to be determined, but the data indicate that the status of D-alanine-lipoteichoic acid can significantly influence the expression of at least some streptococcal virulence factors and provide further impetus to targeting the dlt operon of Gram-positive pathogens in the search for novel antimicrobial compounds.

Crystal Structure of Bacillus cereusd-Alanyl Carrier Protein Ligase (DltA) in Complex with ATP

d-Alanylation of lipoteichoic acids modulates the surface charge and ligand binding of the Gram-positive cell wall. Disruption of the bacterial dlt operon involved in teichoic acid alanylation, as well as inhibition of the DltA ( d-alanyl carrier protein ligase) protein, has been shown to render the bacterium more susceptible to conventional antibiotics and host defense responses. The DltA catalyzes the adenylation and thiolation reactions of d-alanine. This enzyme belongs to a superfamily of AMP-forming domains such as the ubiquitous acetyl-coenzyme A synthetase. We have determined the 1.9-Å-resolution crystal structure of a DltA protein from Bacillus cereus in complex with ATP. This structure sheds light on the geometry of the bound ATP. The invariant catalytic residue Lys492 appears to be mobile, suggesting a molecular mechanism of catalysis for this superfamily of enzymes. Specific roles are also revealed for two other invariant residues: the divalent cation-stabilizing Glu298 and the β-phosphate-interacting Arg397. Mutant proteins with a glutamine substitution at position 298 or 397 are inactive.

The GraRS regulatory system controls Staphylococcus aureus susceptibility to antimicrobial host defenses

BackgroundModification of teichoic acids with D-alanine by the products of the dlt operon protects Gram-positive bacteria against major antimicrobial host defense molecules such as defensins, cathelicidins, myeloperoxidase or phospholipase. The graRS regulatory genes have recently been implicated in the control of D-alanylation in Staphylococcus aureus.ResultsTo determine the impact of the GraRS regulatory system on resistance to antimicrobial host defense mechanisms and virulence of S. aureus, we compared inactivation of S. aureus SA113 wild type and its isogenic graRS deletion mutant by the human cathelicidin LL-37 or human neutrophil granulocytes in vitro, and the ability to cause infection in vivo. We show here that graRS deletion considerably alters bacterial surface charge, increases susceptibility to killing by human neutrophils or the defense peptide LL-37, and attenuates virulence of S. aureus in a mouse infection model.ConclusionOur results indicate that S. aureus can regulate its surface properties in order to overcome innate host defenses.

Molecular Basis of Resistance to Muramidase and Cationic Antimicrobial Peptide Activity of Lysozyme in Staphylococci

It has been shown recently that modification of peptidoglycan by O-acetylation renders pathogenic staphylococci resistant to the muramidase activity of lysozyme. Here, we show that a Staphylococcus aureus double mutant defective in O-acetyltransferase A (OatA), and the glycopeptide resistance-associated two-component system, GraRS, is much more sensitive to lysozyme than S. aureus with the oatA mutation alone. The graRS single mutant was resistant to the muramidase activity of lysozyme, but was sensitive to cationic antimicrobial peptides (CAMPs) such as the human lysozyme-derived peptide 107R-A-W-V-A-W-R-N-R115 (LP9), polymyxin B, or gallidermin. A comparative transcriptome analysis of wild type and the graRS mutant revealed that GraRS controls 248 genes. It up-regulates global regulators (rot, sarS, or mgrA), various colonization factors, and exotoxin-encoding genes, as well as the ica and dlt operons. A pronounced decrease in the expression of the latter two operons explains why the graRS mutant is also biofilm-negative. The decrease of the dlt transcript in the graRS mutant correlates with a 46.7% decrease in the content of esterified d-alanyl groups in teichoic acids. The oatA/dltA double mutant showed the highest sensitivity to lysozyme; this mutant completely lacks teichoic acid–bound d-alanine esters, which are responsible for the increased susceptibility to CAMPs and peptidoglycan O-acetylation. Our results demonstrate that resistance to lysozyme can be dissected into genes mediating resistance to its muramidase activity (oatA) and genes mediating resistance to CAMPs (graRS and dlt). The two lysozyme activities act synergistically, as the oatA/dltA or oatA/graRS double mutants are much more susceptible to lysozyme than each of the single mutants.

D‐Alanyl ester depletion of teichoic acids in Lactobacillus reuteri 100‐23 results in impaired colonization of the mouse gastrointestinal tract

The dlt operon of Gram-positive bacteria encodes proteins required for the incorporation of D-alanine esters into cell wall-associated teichoic acids (TA). D-alanylation of TA has been shown to be important for acid tolerance, resistance to antimicrobial peptides, adhesion, biofilm formation, and virulence of a variety of pathogenic organisms. The aim of this study was to determine the importance of D-alanylation for colonization of the gastrointestinal tract by Lactobacillus reuteri 100-23. Insertional inactivation of the dltA gene resulted in complete depletion of D-alanine substitution of lipoteichoic acids. The dlt mutant had similar growth characteristics as the wild type under standard in vitro conditions, but formed lower population sizes in the gastrointestinal tract of ex-Lactobacillus-free mice, and was almost eliminated from the habitat in competition experiments with the parental strain. In contrast to the wild type, the dlt mutant was unable to form a biofilm on the forestomach epithelium during gut colonization. Transmission electron microscope observations showed evidence of cell wall damage of mutant bacteria present in the forestomach. The dlt mutant had impaired growth under acidic culture conditions and increased susceptibility to the cationic peptide nisin relative to the wild type. Ex vivo adherence of the dlt mutant to the forestomach epithelium was not impaired. This study showed that D-alanylation is an important cell function of L. reuteri that seems to protect this commensal organism against the hostile conditions prevailing in the murine forestomach.

Capsule and d-alanylated lipoteichoic acids protect Streptococcus pneumoniae against neutrophil extracellular traps

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. Pneumococci can counteract the action of neutrophils with an antiphagocytic capsule and through electrochemical repulsion of antimicrobial peptides via addition of positive charge to the surface. Pneumococci are captured, but not killed in neutrophil extracellular traps (NETs). Here, we study the role of the polysaccharide capsule and lipoteichoic acid (LTA) modification on pneumococcal interaction with NETs. Expression of capsule (serotypes 1, 2, 4 and 9V) significantly reduced trapping by NETs, but was not required for resistance to NET-mediated killing. Pneumococci contain a dlt operon that mediates the incorporation of d-alanine residues into LTAs, thereby introducing positive charge. Genetic inactivation of dltA in non-encapsulated pneumococci rendered the organism sensitive to killing by antimicrobial components present in NETs. However, the encapsulated dltA mutant remained resistant to NET-mediated killing in vitro. Nevertheless, in a murine model of pneumococcal pneumonia, the encapsulated dltA-mutant strain was outcompeted by the wild-type upon invasion into the lungs and bloodstream. This suggests a non-redundant role for LTA alanylation in pneumococcal virulence at the early stage of invasive disease when capsule expression has been shown to be low.

A functional dlt operon, encoding proteins required for incorporation of d-alanine in teichoic acids in gram-positive bacteria, confers resistance to cationic antimicrobial peptides in Streptococcus pneumoniae.

Streptococcus pneumoniae is one of the few species within the group of low-G +C gram-positive bacteria reported to contain no d-alanine in teichoic acids, although the dltABCD operon encoding proteins responsible for d-alanylation is present in the genomes of two S. pneumoniae strains, the laboratory strain R6 and the clinical isolate TIGR4. The annotation of dltA in R6 predicts a protein, d-alanine-d-alanyl carrier protein ligase (Dcl), that is shorter at the amino terminus than all other Dcl proteins. Translation of dltA could also start upstream of the annotated TTG start codon at a GTG, resulting in the premature termination of dltA translation at a stop codon. Applying a novel integrative translation probe plasmid with Escherichia coli 'lacZ as a reporter, we could demonstrate that dltA translation starts at the upstream GTG. Consequently, S. pneumoniae R6 is a dltA mutant, whereas S. pneumoniae D39, the parental strain of R6, and Rx, another derivative of D39, contained intact dltA genes. Repair of the stop codon in dltA of R6 and insertional inactivation of dltA in D39 and Rx yielded pairs of dltA-deficient and dltA-proficient strains. Subsequent phenotypic analysis showed that dltA inactivation resulted in enhanced sensitivity to the cationic antimicrobial peptides nisin and gallidermin, a phenotype fully consistent with those of dltA mutants of other gram-positive bacteria. In addition, mild alkaline hydrolysis of heat-inactivated whole cells released d-alanine from dltA-proficient strains, but not from dltA mutants. The results of our study suggest that, as in many other low-G+C gram-positive bacteria, teichoic acids of S. pneumoniae contain d-alanine residues in order to protect this human pathogen against the actions of cationic antimicrobial peptides.

D-alanyl ester depletion of teichoic acids in Lactobacillus plantarum results in a major modification of lipoteichoic acid composition and cell wall perforations at the septum mediated by the Acm2 autolysin.

The insertional inactivation of the dlt operon from Lactobacillus plantarum NCIMB8826 had a strong impact on lipoteichoic acid (LTA) composition, resulting in a major reduction in D-alanyl ester content. Unexpectedly, mutant LTA showed high levels of glucosylation and were threefold longer than wild-type LTA. The dlt mutation resulted in a reduced growth rate and increased cell lysis during the exponential and stationary growth phases. Microscopy analysis revealed increased cell length, damaged dividing cells, and perforations of the envelope in the septal region. The observed defects in the separation process, cell envelope perforation, and autolysis of the dlt mutant could be partially attributed to the L. plantarum Acm2 peptidoglycan hydrolase.

Effect of d-Alanylation of (Lipo)Teichoic Acids of Staphylococcus aureus on Host Secretory Phospholipase A2 Action before and after Phagocytosis by Human Neutrophils

Invading bacteria such as Staphylococcus aureus induce mobilization of professional phagocytes (e.g., neutrophils) and extracellular antibacterial proteins (e.g., group IIA phospholipase A2 (gIIA PLA2)). Accumulation of gIIA PLA2 in inflammatory fluids confers potent extracellular antistaphylococcal activity and at lower concentrations promotes bacterial phospholipid degradation during phagocytosis of S. aureus by human neutrophils. D-alanylation of (lipo) teichoic acids of S. aureus increases bacterial resistance to gIIA PLA2 approximately 100-fold, raising the possibility that the resistance of ingested S. aureus to related gV and gX secretory PLA2 present in human neutrophil granules depends on D-alanylation mediated by the dlt operon. However, we show that isogenic wild-type and dltA S. aureus are equally resistant to gV/X PLA2 during phagocytosis and when exposed to the purified enzymes. The fates of wild-type and dltA S. aureus exposed to serum and human neutrophils differed significantly only when extracellular gIIA PLA2 was also present before phagocytosis. The extreme potency of the gIIA PLA2 toward dltA S. aureus suggests that even small amounts of this extracellular enzyme mobilized early in inflammation could contribute substantially to the overall cytotoxicity of acute inflammatory exudates toward S. aureus when D-alanylation of (lipo)teichoic acids is limiting.

DNA micro-array-based identification of bile-responsive genes in Lactobacillus plantarum

The purpose of this study was to determine the global transcriptional response in a food-associated lactic acid bacterium during bile stress. Clone-based DNA micro-arrays were employed to describe the global transcriptional response of Lactobacillus plantarum WCFS1 towards 0.1% porcine bile. Comparison of differential transcript profiles obtained during growth of Lact. plantarum on plates with and without bile revealed 28 and 62 putative genes, of which the expression was at least 2.5-fold up- or down-regulated by bile, respectively. Approximately, 50% of these genes appeared genetically linked, and 12 bile-responsive gene clusters were identified. Seven of the identified bile-responsive genes and gene clusters encode typical stress-related functions, including glutathione reductase and glutamate decarboxylase, involved in oxidative and acid stress, respectively. Moreover, 14 bile-responsive genes and gene clusters were identified that encode proteins that are located in the cell envelope, including the dlt operon and the F1F0 ATPase. The identification of a relatively high number of genes encoding cell envelope functions indicates a major impact of bile acids on the integrity and/or functionality of the cytoplasmic membrane and cell wall. The data presented here provide valuable clues towards the defence mechanisms that play a role during bile stress in Lact. plantarum.

A continuum of anionic charge: structures and functions of D-alanyl-teichoic acids in gram-positive bacteria.

Teichoic acids (TAs) are major wall and membrane components of most gram-positive bacteria. With few exceptions, they are polymers of glycerol-phosphate or ribitol-phosphate to which are attached glycosyl and D-alanyl ester residues. Wall TA is attached to peptidoglycan via a linkage unit, whereas lipoteichoic acid is attached to glycolipid intercalated in the membrane. Together with peptidoglycan, these polymers make up a polyanionic matrix that functions in (i) cation homeostasis; (ii) trafficking of ions, nutrients, proteins, and antibiotics; (iii) regulation of autolysins; and (iv) presentation of envelope proteins. The esterification of TAs with D-alanyl esters provides a means of modulating the net anionic charge, determining the cationic binding capacity, and displaying cations in the wall. This review addresses the structures and functions of D-alanyl-TAs, the D-alanylation system encoded by the dlt operon, and the roles of TAs in cell growth. The importance of dlt in the physiology of many organisms is illustrated by the variety of mutant phenotypes. In addition, advances in our understanding of D-alanyl ester function in virulence and host-mediated responses have been made possible through targeted mutagenesis of dlt. Studies of the mechanism of D-alanylation have identified two potential targets of antibacterial action and provided possible screening reactions for designing novel agents targeted to D-alanyl-TA synthesis.