D-alanylation Of Teichoic Acids Research Articles

Staphylococcus aureus surface attachment selectively influences tolerance against charged antibiotics

The threat of infection during implant placement surgery remains a considerable burden for millions of patients worldwide. To combat this threat, clinicians employ a range of anti-infective strategies and practices. One of the most common interventions is the use of prophylactic antibiotic treatment during implant placement surgery. However, these practices can be detrimental by promoting the resilience of biofilm-forming bacteria and enabling them to persist throughout treatment and re-emerge later, causing a life-threatening infection. Thus, it is of the utmost importance to elucidate the events occurring during the initial stages of bacterial surface attachment and determine whether any biological processes may be targeted to improve surgical outcomes. Using gene expression analysis, we identified a cellular mechanism of S. aureus which modifies its cell surface charge following attachment to a medical grade titanium surface. We determined the upregulation of two systems involved in the d-alanylation of teichoic acids and the lysylation of phosphatidylglycerol. We supported these molecular findings by utilizing synchrotron-sourced attenuated total reflection Fourier-transform infrared microspectroscopy to analyze the biomolecular properties of the S. aureus cell surface following attachment. As a direct consequence, S. aureus quickly becomes substantially more tolerant to the positively charged vancomycin, but not the negatively charged cefazolin. The present study can assist clinicians in rationally selecting the most potent antibiotic in prophylaxis treatments. Furthermore, it highlights a cellular process that could potentially be targeted by novel technologies and strategies to improve the outcome of antibiotic prophylaxis during implant placement surgery. Statement of significanceThe antibiotic tolerance of bacteria in biofilm is a well-established phenomenon. However, the physiological adaptations employed by Staphylococcus aureus to increase its antibiotic tolerance during the early stages of surface attachment are poorly understood. Using multiple techniques, including gene expression analysis and synchrotron-sourced Fourier-transform infrared microspectroscopy, we generated insights into the physiological response of S. aureus following attachment to a medical grade titanium surface. We showed that this phenotypic transition enables S. aureus to better tolerate the positively charged vancomycin, but not the negatively charged cefazolin. These findings shed light on the antibiotic tolerance mechanisms employed by S. aureus to survive prophylactically administered antibiotics and can help clinicians to protect patients from infections.

Resistance to host antimicrobial peptides mediates resilience of gut commensals during infection and aging in Drosophila.

Resilience to short-term perturbations, like inflammation, is a fundamental feature of microbiota, yet the underlying mechanisms of microbiota resilience are incompletely understood. Here, we show that Lactiplantibacillus plantarum, a major Drosophila commensal, stably colonizes the fruit fly gut during infection and is resistant to Drosophila antimicrobial peptides (AMPs). By transposon screening, we identified L. plantarum mutants sensitive to AMPs. These mutants were impaired in peptidoglycan O-acetylation or teichoic acid D-alanylation, resulting in increased negative cell surface charge and higher affinity to cationic AMPs. AMP-sensitive mutants were cleared from the gut after infection and aging-induced gut inflammation in wild-type, but not in AMP-deficient flies, suggesting that resistance to host AMPs is essential for commensal resilience in an inflamed gut environment. Thus, our work reveals that in addition to the host immune tolerance to the microbiota, commensal-encoded resilience mechanisms are necessary to maintain the stable association between host and microbiota during inflammation.

Metabolic reprogramming and altered cell envelope characteristics in a pentose phosphate pathway mutant increases MRSA resistance to β-lactam antibiotics.

Central metabolic pathways control virulence and antibiotic resistance, and constitute potential targets for antibacterial drugs. In Staphylococcus aureus the role of the pentose phosphate pathway (PPP) remains largely unexplored. Mutation of the 6-phosphogluconolactonase gene pgl, which encodes the only non-essential enzyme in the oxidative phase of the PPP, significantly increased MRSA resistance to β-lactam antibiotics, particularly in chemically defined media with physiologically-relevant concentrations of glucose, and reduced oxacillin (OX)-induced lysis. Expression of the methicillin-resistance penicillin binding protein 2a and peptidoglycan architecture were unaffected. Carbon tracing and metabolomics revealed extensive metabolic reprogramming in the pgl mutant including increased flux to glycolysis, the TCA cycle, and several cell envelope precursors, which was consistent with increased β-lactam resistance. Morphologically, pgl mutant cells were smaller than wild-type with a thicker cell wall and ruffled surface when grown in OX. The pgl mutation reduced resistance to Congo Red, sulfamethoxazole and oxidative stress, and increased resistance to targocil, fosfomycin and vancomycin. Levels of lipoteichoic acids (LTAs) were significantly reduced in pgl, which may limit cell lysis, while the surface charge of pgl cells was significantly more positive. A vraG mutation in pgl reversed the increased OX resistance phenotype, and partially restored wild-type surface charge, but not LTA levels. Mutations in vraF or graRS from the VraFG/GraRS complex that regulates DltABCD-mediated d-alanylation of teichoic acids (which in turn controls β-lactam resistance and surface charge), also restored wild-type OX susceptibility. Collectively these data show that reduced levels of LTAs and OX-induced lysis combined with a VraFG/GraRS-dependent increase in cell surface positive charge are accompanied by significantly increased OX resistance in an MRSA pgl mutant.

Deciphering the crosstalk between skin commensals and the immune system.

Abstract The mechanisms underlying the crosstalk between the microbiota and the immune system remains largely unknown. We previously established that skin colonization with Staphylococcus epidermidisresults in the induction of T cell responses that control tissue function in a way that both enhances cutaneous immunity and promotes tissue repair. To uncover the molecular determinants of S. epidermidisthat stimulate T cells, we engineered a library of S. epidermidismutants with defined cell envelope alterations. Under physiological cutaneous colonization, we found that S. epidermidiscell envelope mutants elicit marked changes in the composition and function of the T cell populations in the skin. The removal of cell envelope lipoproteins and D-alanylation of teichoic acids almost completely abolished CD8 +T cell induction without significant effect on CD4 +T cell stimulation. Deletion of Tlr2gene also strongly reduced the CD8 +T cell response after S. epidermidiscolonization, while the CD4 +T cell response remained unchanged, a phenotype similar to the elimination of cell envelope lipoproteins and D-alanylation of teichoic acid in S. epidermidis. Furthermore, suprabasal keratinocytes express the highest level of TLR2 among keratinocyte subsets. Together, these results support the notion that the ability of S. epidermidisto induce CD8 +T cells is partially dependent on the host sensing of S. epidermidiswall teichoic acids and lipoprotein through TLR2. Using complementary strategies including novel mice models and imaging, current studies are focusing on deciphering on which cell population(s) TLR2 sensing of S. epidermidismolecular determinants is required for the induction of CD8 +T cell responses.

DltC acts as an interaction hub for AcpS, DltA and DltB in the teichoic acid d-alanylation pathway of Lactiplantibacillus plantarum

Teichoic acids (TA) are crucial for the homeostasis of the bacterial cell wall as well as their developmental behavior and interplay with the environment. TA can be decorated by different modifications, modulating thus their biochemical properties. One major modification consists in the esterification of TA by d-alanine, a process known as d-alanylation. TA d-alanylation is performed by the Dlt pathway, which starts in the cytoplasm and continues extracellularly after d-Ala transportation through the membrane. In this study, we combined structural biology and in vivo approaches to dissect the cytoplasmic steps of this pathway in Lactiplantibacillus plantarum, a bacterial species conferring health benefits to its animal host. After establishing that AcpS, DltB, DltC1 and DltA are required for the promotion of Drosophila juvenile growth under chronic undernutrition, we solved their crystal structure and/or used NMR and molecular modeling to study their interactions. Our work demonstrates that the suite of interactions between these proteins is ordered with a conserved surface of DltC1 docking sequentially AcpS, DltA and eventually DltB. Altogether, we conclude that DltC1 acts as an interaction hub for all the successive cytoplasmic steps of the TA d-alanylation pathway.

Structural and functional analysis of the D-alanyl carrier protein ligase DltA from Staphylococcus aureus Mu50.

D-Alanylation of the teichoic acids of the Gram-positive bacterial cell wall plays crucial roles in bacterial physiology and virulence. Deprivation of D-alanine from the teichoic acids of Staphylococcus aureus impairs biofilm and colony formation, induces autolysis and ultimately renders methicillin-resistant S.aureus highly susceptible to antimicrobial agents and host defense peptides. Hence, the D-alanylation pathway has emerged as a promising antibacterial target against drug-resistant S. aureus. D-Alanylation of teichoic acids is mediated via the action of four proteins encoded by the dlt operon, DltABCD, all four of which are essential for the process. In order to develop novel antimicrobial agents against S. aureus, the D-alanyl carrier protein ligase DltA, which is the first protein in the D-alanylation pathway, was focused on. Here, the crystal structure of DltA from the methicillin-resistant S. aureus strain Mu50 is presented, which reveals the unique molecular details of the catalytic center and the role of the P-loop. Kinetic analysis shows that the enantioselectivity of S.aureus DltA is much higher than that of DltA from other species. In the presence of DltC, the enzymatic activity of DltA is increased by an order of magnitude, suggesting a new exploitable binding pocket. This discovery may pave the way for a new generation of treatments for drug-resistant S. aureus.

Inhibition of d-alanylation of teichoic acids overcomes resistance of methicillin-resistant Staphylococcus aureus.

BackgroundMRSA are high-priority multidrug-resistant pathogens. Although there are still some antibiotics active against MRSA, continuous efforts to discover new antibiotics and treatment strategies are needed because resistance to these new drugs has already been reported.ObjectivesHere we explore if d-alanylation of teichoic acids (TAs) mediated by the dlt operon gene products might be a druggable target to overcome β-lactam-resistance of MRSA.MethodsMICs and bactericidal effects of several β-lactam antibiotics were monitored in a panel of clinical MRSA strains with genetic or chemically induced deficiency in d-alanylation of TAs. Efficiency of the chemical inhibitor to rescue MRSA-infected larvae of Galleria mellonella as well as its ability to prevent or eradicate biofilms of S. aureus were analysed.ResultsGenetic inactivation of the Dlt system or its chemical inhibition re-sensitizes MRSA to β-lactams. Among the 13 strains, the most pronounced effect was obtained using the inhibitor with imipenem, reducing the median MIC from 16 to 0.25 mg/L. This combination was also bactericidal in some strains and significantly protected G. mellonella larvae from lethal MRSA infections. Finally, inactivation of d-alanylation potentiated the effect of imipenem on inhibition and/or eradication of biofilm.ConclusionsOur combined results show that highly efficient inhibitors of d-alanylation of TAs targeting enzymes of the Dlt system should be promising therapeutic adjuvants, especially in combination with carbapenems, for restoring the therapeutic efficacy of this class of antibiotics against MRSA.

Genetic and pharmacological inactivation of d-alanylation of teichoic acids sensitizes pathogenic enterococci to β-lactams.

Enterococci intrinsically resistant to cephalosporins represent a major cause of healthcare-associated infections, and the emergence of MDR makes therapeutic approaches particularly challenging. Teichoic acids are cell wall glycopolymers present in Gram-positive bacteria. Teichoic acids can be modified by d-alanylation, which requires four proteins encoded by the dltABCD operon. Our objective was to evaluate the Dlt system as a druggable target to treat enterococcal infections. The susceptibility of a d-alanylation-deficient strain of Enterococcus faecalis to β-lactam antibiotics individually and/or in combination was analysed. Moreover, a DltA inhibitor was synthesized to test pharmacological inhibition of d-alanylation in vivo and in host using the animal model Galleria mellonella with different clinical isolates of E. faecalis and Enterococcus faecium. Most cephalosporins used as mono treatment had no impact on survival of the parental strain, but were slightly lethal for the dltA mutant of E. faecalis. Addition of a very low concentration of amoxicillin significantly increased killing of the dltA mutant under these conditions. The most spectacular effect was obtained with a combination of cefotaxime (1 mg/L) and amoxicillin (0.03 mg/L). In the presence of the inhibitor, the WT strain was as susceptible to this combination treatment as the dltA mutant. This molecule associated with the antibiotics was also effective in killing other E. faecalis clinical isolates and successfully prevented death of Galleria infected with either E. faecalis or E. faecium. The combined results support the potential usefulness of the Dlt system as a target to potentiate antibiotic combination therapies for the treatment of drug-resistant enterococci.

A partial reconstitution implicates DltD in catalyzing lipoteichoic acid d-alanylation

Modifications to the Gram-positive bacterial cell wall play important roles in antibiotic resistance and pathogenesis, but the pathway for the d-alanylation of teichoic acids (DLT pathway), a ubiquitous modification, is poorly understood. The d-alanylation machinery includes two membrane proteins of unclear function, DltB and DltD, which are somehow involved in transfer of d-alanine from a carrier protein inside the cell to teichoic acids on the cell surface. Here, we probed the role of DltD in the human pathogen Staphylococcus aureus using both cell-based and biochemical assays. We first exploited a known synthetic lethal interaction to establish the essentiality of each gene in the DLT pathway for d-alanylation of lipoteichoic acid (LTA) and confirmed this by directly detecting radiolabeled d-Ala-LTA both in cells and in vesicles prepared from mutant strains of S. aureus We developed a partial reconstitution of the pathway by using cell-derived vesicles containing DltB, but no other components of the d-alanylation pathway, and showed that d-alanylation of previously formed lipoteichoic acid in the DltB vesicles requires the presence of purified and reconstituted DltA, DltC, and DltD, but not of the LTA synthase LtaS. Finally, based on the activity of DltD mutants in cells and in our reconstituted system, we determined that Ser-70 and His-361 are essential for d-alanylation activity, and we propose that DltD uses a catalytic dyad to transfer d-alanine to LTA. In summary, we have developed a suite of assays for investigating the bacterial DLT pathway and uncovered a role for DltD in LTA d-alanylation.

Monitoring metabolic reactions in Staphylococcus epidermidis exposed to silicon nitride using in situ time-lapse Raman spectroscopy.

Staphylococcus epidermidis (S. epidermidis) is one of the leading nosocomial pathogens, particularly associated with periprosthetic infections of biomedical implants. Silicon nitride (Si3N4), a nonoxide biomaterial widely used in spinal implants, has shown bacteriostatic effects against both gram-positive and gram-negative bacteria; however, the physicochemical interactions between Si3N4 and bacteria yet remain conspicuously unexplored. In situ time-lapse Raman spectroscopic experiments were conducted by exposing S. epidermidis for 12, 24, and 48h to Si3N4 substrates to understand the evolution of bacterial metabolism and to elucidate the ceramics antimicrobial behavior. The Raman probe captured an initial metabolic response of the bacteria to the adverse chemistry of the Si3N4 surface, which included peroxidation of membrane phospholipids and protein structural modifications to adjust for survivorship. However, beyond 24h of exposure, the Raman signals representing DNA, lipids, proteins, and carbohydrates showed clear fingerprints of bacterial lysis. Bands related to biofilm formation completely disappeared or underwent drastically reduced intensity. Bacterial lysis was confirmed by conventional fluorescence microscopy methods. Spectroscopic experiments suggested that a pH change at the Si3N4's surface induced variations in the membrane structure and D-alanylation of teichoic acids in its peptidoglycan layer. Concurrent stimulation of peptidoglycan hydrolase (i.e., an enzyme involved with autolysis) ultimately led to membrane degradation and cellular death. An additional finding was that modulating the Si3N4 surface by increasing the population of amine groups improved the efficiency of the substrate against S. epidermidis, thus suggesting that optimization of the near-surface (alkaline) conditions may be a viable approach to bacterial reduction.

D-Alanylation of teichoic acids contributes to Lactobacillus plantarum-mediated Drosophila growth during chronic undernutrition.

SummaryThe microbial environment influence animal physiology. However, the underlying molecular mechanisms of such functional interactions are largely undefined. Previously, we showed that upon chronic undernutrition, strains of Lactobacillus plantarum, a major commensal partner of Drosophila, promote host juvenile growth and maturation partly via enhanced expression of intestinal peptidases. By screening a transposon insertion library of Lactobacillus plantarum in gnotobiotic Drosophila larvae, we identify a bacterial cell wall modifying machinery encoded by the pbpX2-dlt operon that is critical to enhance host digestive capabilities and promote animal growth and maturation. Deletion of this operon leads to bacterial cell wall alteration with a complete loss of teichoic acids D-alanylation. We show that L. plantarum cell walls bearing D-alanylated teichoic acids are directly sensed by Drosophila enterocytes to ensure optimal intestinal peptidase expression and activity, juvenile growth and maturation upon chronic undernutrition. We thus conclude that besides peptidoglycan, teichoic acids modifications participate in the host-commensal bacteria molecular dialogue occurring in the intestine.

DltX of Bacillus thuringiensis Is Essential for D-Alanylation of Teichoic Acids and Resistance to Antimicrobial Response in Insects.

The dlt operon of Gram-positive bacteria is required for the incorporation of D-alanine esters into cell wall-associated teichoic acids (TAs). Addition of D-alanine to TAs reduces the negative charge of the cell envelope thereby preventing cationic antimicrobial peptides (CAMPs) from reaching their target of action on the bacterial surface. In most gram-positive bacteria, this operon consists of five genes dltXABCD but the involvement of the first ORF (dltX) encoding a small protein of unknown function, has never been investigated. The aim of this study was to establish whether this protein is involved in the D-alanylation process in Bacillus thuringiensis. We, therefore constructed an in frame deletion mutant of dltX, without affecting the expression of the other genes of the operon. The growth characteristics of the dltX mutant and those of the wild type strain were similar under standard in vitro conditions. However, disruption of dltX drastically impaired the resistance of B. thuringiensis to CAMPs and significantly attenuated its virulence in two insect species. Moreover, high-performance liquid chromatography studies showed that the dltX mutant was devoid of D-alanine, and electrophoretic mobility measurements indicated that the cells carried a higher negative surface charge. Scanning electron microscopy experiments showed morphological alterations of these mutant bacteria, suggesting that depletion of D-alanine from TAs affects cell wall structure. Our findings suggest that DltX is essential for the incorporation of D-alanyl esters into TAs. Therefore, DltX plays a direct role in the resistance to CAMPs, thus contributing to the survival of B. thuringiensis in insects. To our knowledge, this work is the first report examining the involvement of dltX in the D-alanylation of TAs.

ANTIMICROBIAL PEPTIDE P34 INFLUENCES GENE EXPRESSION OF LISTERIA MONOCYTOGENES GROWING IN SOFT CHEESE

Objective: To evaluate whether antimicrobial substances produced by autochthonous lactic acid bacteria (LAB) from Minas Frescal cheese are able to enhance the activity of bacteriocin P34 against Listeria monocytogenes and investigate the influence of P34 in specific gene expression of this bacterium after the inoculation in Minas Frescal cheese.Methods: Bacillus sp. P34 and L. monocytogenes ATCC 7644 were used in this study. The antimicrobial peptide P34 was purified and applied (0, 800 or 6400 AU/ml) to cheese surface before inoculation with L. monocytogenes. Antimicrobial activity and synergism were detected using the agar diffusion technique. Expression levels of D-Alanine-D-alanyl carrier protein ligase (dltA), Putative phospholipid lysinylation (Imo 1695) and EIIABMan of mannose-specific PTS (mptA) mRNAs in bacteriocin-treated L. monocytogenes growing in Minas Frescal cheese were determined using real-time PCR.Results: The peptide P34 showed increased antilisterial activity when combined with culture supernatants of some selected LAB isolated from Minas Frescal cheese. The addition of peptide P34 to cheese caused a decrease of up to 3 log cycles in viable counts of artificially inoculated L. monocytogenes. The influence of peptide P34 on the expression of genes associated with components of the cell surface of L. monocytogenes was investigated by real-time PCR. A significant increase in the expression of the genes dltA, Imo 1695 and mptA was observed after 96 h in the presence of peptide P34.Conclusion: These results suggest that the peptide P34 influences the expression of genes involved in D-alanylation of teichoic acids and lipoteichoic acids and lysination of the cell membrane of phospholipids.

Daptomycin Tolerance in the Staphylococcus aureus pitA6 Mutant Is Due to Upregulation of the dlt Operon.

Understanding the mechanisms of how bacteria become tolerant toward antibiotics during clinical therapy is a very important object. In a previous study, we showed that increased daptomycin (DAP) tolerance of Staphylococcus aureus was due to a point mutation in pitA (inorganic phosphate transporter) that led to intracellular accumulation of both inorganic phosphate (Pi) and polyphosphate (polyP). DAP tolerance in the pitA6 mutant differs from classical resistance mechanisms since there is no increase in the MIC. In this follow-up study, we demonstrate that DAP tolerance in the pitA6 mutant is not triggered by the accumulation of polyP. Transcriptome analysis revealed that 234 genes were at least 2.0-fold differentially expressed in the mutant. Particularly, genes involved in protein biosynthesis, carbohydrate and lipid metabolism, and replication and maintenance of DNA were downregulated. However, the most important change was the upregulation of the dlt operon, which is induced by the accumulation of intracellular Pi The GraXRS system, known as an activator of the dlt operon (d-alanylation of teichoic acids) and of the mprF gene (multiple peptide resistance factor), is not involved in DAP tolerance of the pitA6 mutant. In conclusion, DAP tolerance of the pitA6 mutant is due to an upregulation of the dlt operon, triggered directly or indirectly by the accumulation of Pi.

D-Alanylation of Teichoic Acids and Loss of Poly-N-Acetyl Glucosamine in Staphylococcus aureus during Exponential Growth Phase Enhance IL-12 Production in Murine Dendritic Cells.

Staphylococcus aureus is a major human pathogen that has evolved very efficient immune evading strategies leading to persistent colonization. During different stages of growth, S. aureus express various surface molecules, which may affect the immune stimulating properties, but very little is known about their role in immune stimulation and evasion. Depending on the growth phase, S. aureus may affect antigen presenting cells differently. Here, the impact of growth phases and the surface molecules lipoteichoic acid, peptidoglycan and poly-N-acetyl glucosamine on the induction of IL-12 imperative for an efficient clearance of S. aureus was studied in dendritic cells (DCs). Exponential phase (EP) S. aureus was superior to stationary phase (SP) bacteria in induction of IL-12, which required actin-mediated endocytosis and endosomal acidification. Moreover, addition of staphylococcal cell wall derived peptidoglycan to EP S. aureus stimulated cells increased bacterial uptake but abrogated IL-12 induction, while addition of lipoteichoic acid increased IL-12 production but had no effect on the bacterial uptake. Depletion of the capability to produce poly-N-acetyl glucosamine increased the IL-12 inducing activity of EP bacteria. Furthermore, the mutant dltA unable to produce D-alanylated teichoic acids failed to induce IL-12 but like peptidoglycan and the toll-like receptor (TLR) ligands LPS and Pam3CSK4 the mutant stimulated increased macropinocytosis. In conclusion, the IL-12 response by DCs against S. aureus is highly growth phase dependent, relies on cell wall D-alanylation, endocytosis and subsequent endosomal degradation, and is abrogated by receptor induced macropinocytosis.

Disentangling gene networks

Cell-wall biogenesis in bacteria involves multiple intersecting gene networks. A powerful approach that allies synthetic lethality with small-molecule discovery has now been used to probe these networks and has revealed that the pathway for D-alanylation of teichoic acids in Staphylococcus aureus is a viable target for new antibacterials.

A synthetic lethal approach for compound and target identification in Staphylococcus aureus.

The majority of bacterial proteins are dispensable for growth in the laboratory, but nevertheless play important physiological roles. There are no systematic approaches to identify cell-permeable small molecule inhibitors of these proteins. We demonstrate a strategy to identify such inhibitors that exploits synthetic lethal relationships both for small molecule discovery and for target identification. Applying this strategy in Staphylococcus aureus, we have identified a compound that inhibits DltB, a component of the teichoic acid D-alanylation machinery, which has been implicated in virulence. This D-alanylation inhibitor sensitizes S. aureus to aminoglycosides and cationic peptides and is lethal in combination with a wall teichoic acid inhibitor. We conclude that DltB is a druggable target in the D-alanylation pathway. More broadly, the work described demonstrates a systematic method to identify biologically active inhibitors of important bacterial processes that can be adapted to numerous organisms.

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.

The Impact of Lactobacillus plantarum WCFS1 Teichoic Acid D-Alanylation on the Generation of Effector and Regulatory T-cells in Healthy Mice

To date it remains unclear how probiotics affect the immune system. Bacterial envelope components may play an essential role, as these are the first to establish bacterial-host cell interactions. Teichoic acids (TAs), and especially lipoteichoic acids, are the most pro-inflammatory components of the gram-positive bacterial envelope. This effect is dependent on D-alanyl substitution of the TA backbone and interactions with TLR2 on host cells. Although the pro-inflammatory properties of TAs have been established in vitro, it remains unclear how TAs affect immunomodulation in vivo. In this study, we investigated the role of TA D-alanylation on L. plantarum–induced intestinal and systemic immunomodulation in vivo. For this, we compared the effect of L. plantarum WCFS1 and its TA D-Alanylation negative derivative (dltX-D) on the distribution of dendritic cell and T cell populations and responses in healthy mice. We demonstrated that the majority of the L. plantarum-induced in vivo immunomodulatory effects were dependent on D-alanylation (D-Ala), as some L. plantarum WCFS1-induced immune changes were not observed in the dltX-D-treated group and some were only observed after treatment with dltX-D. Strikingly, not only pro-inflammatory immune responses were abolished in the absence of D-Ala substitution, but also anti-inflammatory responses, such as the L. plantarum-induced generation of regulatory T cells in the spleen. With this study we provide insight in host-microbe interactions, by demonstrating the involvement of D-alanylation of TAs on the bacterial membrane in intestinal and systemic immunomodulation in healthy mice.