D-Alanine substitution of teichoic acids as a modulator of protein folding and stability at the cytoplasmic membrane/cell wall interface of Bacillus subtilis

The extracytoplasmic folding of secreted proteins in Gram-positive bacteria is influenced by the microenvironment of the compartment into which they are translocated, namely the negatively charged matrix of the cell wall polymers. In this compartment, the PrsA lipoprotein facilitates correct post-translocational folding or prevents misfolding of secreted proteins. In this study, a secretion mutant of B. subtilis (prsA3) encoding a defective PrsA protein was mutagenized and screened for restored secretion of the AmyQ alpha-amylase. One mini-Tn10 insertion, which partially suppressed the secretion deficiency, was found to interrupt dlt, the operon involved in the d-alanylation of teichoic acids. The inactivation of dlt rescued the mutant PrsA3 protein from degradation, and the increased amount of PrsA3 was shown to enhance the secretion of PrsA-dependent proteins. Heterologous or abnormal secreted proteins, which are prone to degradation after translocation, were also stabilized and secreted in increased quantities from a dlt prsA(+) strain. Furthermore, the dlt mutation partially suppressed the lethal effect of PrsA depletion, suggesting that the dlt deficiency also leads to stabilization of an essential cell wall protein(s). Our results suggest that main influence of the increased net negative charge of the wall caused by the absence of d-alanine is to increase the rate of post-translocational folding of exported proteins.     

Optimization of the cell wall microenvironment allows increased production of recombinant Bacillus anthracis protective antigen from B. subtilis.

The stability of heterologous proteins secreted by gram-positive bacteria is greatly influenced by the microenvironment on the trans side of the cytoplasmic membrane, and secreted heterologous proteins are susceptible to rapid degradation by host cell proteases. In Bacillus subtilis, degradation occurs either as the proteins emerge from the presecretory translocase and prior to folding into their native conformation or after the native conformation has been reached. The former process generally involves membrane- and/or cell wall-bound proteases, while the latter involves proteases that are released into the culture medium. The identification and manipulation of factors that influence the folding of heterologous proteins has the potential to improve the yield of secreted heterologous proteins. Recombinant anthrax protective antigen (rPA) has been used as a model secreted heterologous protein because it is sensitive to proteolytic degradation both before and after folding into its native conformation. This paper describes the influence of the microenvironment on the trans side of the cytoplasmic membrane on the stability of rPA. Specifically, we have determined the influence of net cell wall charge and its modulation by the extent to which the anionic polymer teichoic acid is D-alanylated on the secretion and stability of rPA. The potential role of the dlt operon, responsible for D-alanylation, was investigated using a Bacillus subtilis strain encoding an inducible dlt operon. We show that, in the absence of D-alanylation, the yield of secreted rPA is increased 2.5-fold. The function of D-alanylation and the use of rPA as a model protein are evaluated with respect to the optimization of B. subtilis for the secretion of heterologous proteins.     

Expression of a pertussis toxin S1 fragment by inducible promoters in oral Streptococcus and the induction of immune responses during oral colonization in mice

Previous work aimed at developing a live oral vaccine expressing pertussis toxin S1 fragment on the surface of the bacterium Streptococcus gordonii elicited a lower than expected antibody response, perhaps because of low antigen expression. In this study, in-frame promoter fusions were constructed to investigate whether an increase in antigen production by the streptococcal vaccine strain results in a better antibody response. The promoters tested were (i) the Streptococcus mutans sucrose-inducible fructosyltransferase (ftf) promoter and (ii) the Bacillus subtilis/Escherichia coli chimeric tetracycline-inducible xyl/tetO promoter. Each of these two promoters was placed upstream of the spaP/s1 fusion gene to drive its expression. The constructs were introduced into S. gordonii DL1 and S. mutans 834. The inducibility of the promoters was confirmed through the determination of SpaP/S1 production via Western blottings. Induced production of SpaP/S1 was observed in S. gordonii and S. mutans with each of the promoters, but the level of expression was the highest in S. mutans, using the xyl/tetO promoter. Thus, S. mutans carrying the xyl/tetO/spaP/s1 construct (S. mutans PM14) was used in oral colonization studies in BALB/c mice. Streptococccus mutans PM14 was able to colonize the animals for the 14-week duration of experimentation. A mucosal IgA response was observed in all the treatment groups but was highest in mice receiving tetracycline induction. In the mouse model of Bordetella pertussis respiratory infection, animals colonized with S. mutans PM14 showed a decreased in B. pertussis lung colony count (P = 0.03) on day 3 compared with control mice colonized by the parent S. mutans 834.     

Expression and immunogenicity of a recombinant diphtheria toxin fragment A in Streptococcus gordonii

A nontoxic mutant diphtheria toxin fragment A (DTA) was genetically fused in single, double, or triple copy to the major surface protein antigen P1 (SpaP) and surface expressed in Streptococcus gordonii DL-1. The expression was verified by Western immunoblotting. Mouse antisera raised against the recombinant S. gordonii recognized the native diphtheria toxinm suggesting the recombinant DTA was immunogenic. When given intranasally to mice with cholera toxin subunit B as the adjuvant, the recombinant S. gordonii expressing double copies of DTA (SpaP-DTA(2)) induced a mucosal immunoglobulin A response and a weak systemic immunoglobulin G response. S. gordonii SpaP-DTA(2) was able to orally colonize BALB/c mice for a 15-week period and elicited a mucosal response, but a serum immunoglobulin G response was not apparent. The antisera failed to neutralize diphtheria toxin cytotoxicity in a Vero cell assay.     

Mutants of Streptococcus gordonii Challis over-producing glucosyltransferase

Two mutants of Streptococcus gordonii which over-produced extracellular polysaccharide when grown on sucrose-containing medium were isolated after mutagenesis of strain Challis with ethyl methanesulphonate. The mutants, designated strains OB20 and OB30, expressed 2.6-fold and 4.7-fold respectively more glucosyltransferase (GTF) activities than the wild-type strain. Transformation experiments suggested that the two mutants carried different mutations, denoted gtf-20 and gtf-30. A double mutant (gtf-20 gtf-30) was constructed and this strain produced 6.4-fold more GTF. Enzymes from wild-type and mutant strains were biochemically indistinguishable and they synthesized structurally identical glucans. Increasing the Na+ concentration of the bacterial growth medium reduced GTF production in all strains by about 60%. Tween 80 also inhibited enzyme production and more specifically reduced GTF synthesis by the mutants. The mutations gtf-20 and gtf-30 appear to define separate genetic loci involved in regulating expression of GTF activity in S. gordonii.     

高SO2产量啤酒酵母工业菌株的构建

二氧化硫在啤酒中具有抗氧化的重要功能,而在其形成过程中APS激酶(MET14编码)起着非常重要的作用。以二氧化硫产量较高的青岛啤酒酵母(Saccharomyces cerevisiae)YSF-5的总DNA为模板,用PCR方法克隆得到MET14基因。为使目的基因在酿酒酵母中表达,以大肠杆菌-酿酒酵母穿梭质粒YEp352为载体,以PGK1强启动子为调控元件,构建了重组表达质粒pPM,并转化酿酒酵母YS58。转化子在YNB添加亮氨酸、组氨酸和色氨酸的选择性培养基上筛选鉴定,盐酸副玫瑰苯胺法测得转化子的SO2产量是受体菌的2倍左右。在重组表达质粒pPM的基础上添加铜抗性标记基因构建了重组表达质粒pCPM,并转化青岛啤酒工业酵母菌株YSF-38,转化子在YEPD 4mmol/L CuSO4的选择性培养基上筛选鉴定,实验室条件下培养后,测得转化子YSF-38(pCPM)的SO2产量是受体菌的3.2倍。用该转化子在青岛啤酒厂进行小型发酵实验,结果表明在发酵结束时,YSF-38(pCPM)转化子的SO2产量是受体菌的1.4倍。因此,MET14基因的有效表达可以提高啤酒工业酵母的SO2产量。     

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.     

Posttranslocation chaperone PrsA2 regulates the maturation and secretion of Listeria monocytogenes proprotein virulence factors

PrsA2 is a conserved posttranslocation chaperone and a peptidyl prolyl cis-trans isomerase (PPIase) that contributes to the virulence of the Gram-positive intracellular pathogen Listeria monocytogenes. One of the phenotypes associated with a prsA2 mutant is decreased activity of the broad-range phospholipase C (PC-PLC). PC-PLC is made as a proenzyme whose maturation is mediated by a metalloprotease (Mpl). The proforms of PC-PLC and Mpl accumulate at the membrane-cell wall interface until a decrease in pH triggers their maturation and rapid secretion into the host cell. In this study, we examined the mechanism by which PrsA2 regulates the activity of PC-PLC. We observed that in the absence of PrsA2, the proenzymes are secreted at physiological pH and do not mature upon a decrease in pH. The sensitivity of the prsA2 mutant to cell wall hydrolases was modified. However, no apparent changes in cell wall porosity were detected. Interestingly, synthesis of PC-PLC in the absence of its propeptide lead to the secretion of a fully active enzyme in the cytosol of host cells independent of PrsA2, indicating that neither the propeptide of PC-PLC nor PrsA2 is required for native folding of the catalytic domain, although both influence secretion of the enzyme. Taken together, these results suggest that PrsA2 regulates compartmentalization of Mpl and PC-PLC, possibly by influencing cell wall properties and interacting with the PC-PLC propeptide. Moreover, the ability of these proproteins to respond to a decrease in pH during intracellular growth depends on their localization at the membrane-cell wall interface.     

Development of PLEX, a plasmid-based expression system for production of heterologous gene products by the gram-positive bacteria Streptococcus gordonii

While Escherichia coli expression systems have been widely utilized for the production of heterologous proteins, these systems have limitations with regard to the production of particular protein products, including poor expression, expression of insoluble proteins into inclusion bodies, and/or expression of a truncated product. Using the surface protein expression (SPEX) system, chromosomally integrated heterologous genes are expressed and secreted into media by the naturally competent gram-positive organism Streptococcus gordonii. After E. coli turned out to be an inappropriate expression system to produce sufficient quantities of intact product, we successfully utilized SPEX to produce the heterologous antigen BH4XCRR that is designed from sequences homologous to the S. pyogenes M-protein C-repeat region. To further enhance production of this product by S. gordonii, we sought to develop a novel system for the production and secretion of heterologous proteins. We observed that under various growth conditions, S. gordonii secreted high levels of a 172 kDa protein, which was identified by N-terminal sequence analysis as the glucosyltransferase GTF. Here we report on the development of a plasmid-based expression system, designated as PLEX, which we used to enhance production of BH4XCRR by S. gordonii. A region from the S. gordonii chromosome that contains the positive regulatory gene rgg, putative gtfG promoter, and gtfG secretion-signal sequence was cloned into the E. coli/Streptococcus shuttle plasmid pVA838. Additionally, the bh4xcrr structural gene was cloned into the same plasmid downstream and in-frame with rgg and gtfG. This plasmid construct was transformed into S. gordonii and BH4XCRR was detected in culture supernatants from transformants at greater concentrations than in supernatants from a SPEX strain expressing the same product. BH4XCRR was easily purified from culture supernatant using a scalable two-step purification process involving hydrophobic-interaction and gel-filtration chromatography.     

Gene Regulation by the LiaSR Two-Component System in Streptococcus mutans

The LiaSR two-component signal transduction system regulates cellular responses to several environmental stresses, including those that induce cell envelope damages. Downstream regulons of the LiaSR system have been implicated in tolerance to acid, antibiotics and detergents. In the dental pathogen Streptococcus mutans, the LiaSR system is necessary for tolerance against acid, antibiotics, and cell wall damaging stresses during growth in the oral cavity. To understand the molecular mechanisms by which LiaSR regulates gene expression, we created a mutant LiaR in which the conserved aspartic acid residue (the phosphorylation site), was changed to alanine residue (D58A). As expected, the LiaR-D58A variant was unable to acquire the phosphate group and bind to target promoters. We also noted that the predicted LiaR-binding motif upstream of the lia operon does not appear to be well conserved. Consistent with this observation, we found that LiaR was unable to bind to the promoter region of lia; however, we showed that LiaR was able to bind to the promoters of SMU.753, SMU.2084 and SMU.1727. Based on sequence analysis and DNA binding studies we proposed a new 25-bp conserved motif essential for LiaR binding. Introducing alterations at fully conserved positions in the 25-bp motif affected LiaR binding, and the binding was dependent on the combination of positions that were altered. By scanning the S. mutans genome for the occurrence of the newly defined LiaR binding motif, we identified the promoter of hrcA (encoding a key regulator of the heat shock response) that contains a LiaR binding motif, and we showed that hrcA is negatively regulated by the LiaSR system. Taken together our results suggest a putative role of the LiaSR system in heat shock responses of S. mutans.     

Penicillin‐binding protein folding is dependent on the PrsA peptidyl‐prolyl cis‐trans isomerase in Bacillus subtilis

The PrsA protein is a membrane‐anchored peptidyl‐prolyl cis‐trans isomerase in Bacillus subtilis and most other Gram‐positive bacteria. It catalyses the post‐translocational folding of exported proteins and is essential for normal growth of B. subtilis. We studied the mechanism behind this indispensability. We could construct a viable prsA null mutant in the presence of a high concentration of magnesium. Various changes in cell morphology in the absence of PrsA suggested that PrsA is involved in the biosynthesis of the cylindrical lateral wall. Consistently, four penicillin‐binding proteins (PBP2a, PBP2b, PBP3 and PBP4) were unstable in the absence of PrsA, while muropeptide analysis revealed a 2% decrease in the peptidoglycan cross‐linkage index. Misfolded PBP2a was detected in PrsA‐depleted cells, indicating that PrsA is required for the folding of this PBP either directly or indirectly. Furthermore, strongly increased uniform staining of cell wall with a fluorescent vancomycin was observed in the absence of PrsA. We also demonstrated that PrsA is a dimeric or oligomeric protein which is localized at distinct spots organized in a helical pattern along the cell membrane. These results suggest that PrsA is essential for normal growth most probably as PBP folding is dependent on this PPIase.     

Formation of D-alanyl-lipoteichoic acid is required for adhesion and virulence of Listeria monocytogenes

The dlt operon of Gram-positive bacteria comprises four genes (dltA, dltB, dltC and dltD) that catalyse the incorporation of D-alanine residues into the cell wall-associated lipoteichoic acids (LTAs). In this work, we characterized the dlt operon of Listeria monocytogenes and constructed a D-Ala-deficient LTA mutant by inactivating the first gene (dltA) of this operon. The DltA- mutant did not show any morphological alterations and its growth rate was similar to that of the wild-type strain. However, it exhibited an increased susceptibility to the cationic peptides colistin, nisin and polymyxin B. The virulence of the DltA- mutant was severely impaired in a mouse infection model (4 log increase in the LD50) and, in vitro, the adherence of the mutant to various cell lines (murine bone marrow-derived macrophages and hepatocytes and a human epithelial cell line) was strongly restricted, although the amounts of surface proteins implicated in virulence (ActA, InlA and InlB) remains unaffected. We suggest that the decreased adherence of the DltA- mutant to non-phagocytic and phagocytic cells might be as a result of the increased electronegativity of its charge surface and/or the presence at the bacterial surface of adhesins possessing altered binding activities. These results show that the D-alanylation of the LTAs contributes to the virulence of the intracellular pathogen L. monocytogenes.     

Cytochemical evaluation of localization and secretion of a heterologous enzyme displayed on yeast cell surface

A starch-utilizing Saccharomyces cerevisiae strain was constructed by cell surface engineering. Distribution of the heterologous glucoamylase-alpha-agglutinin fusion protein on the yeast cell was analyzed by indirect fluorescence microscopy using an anti-glucoamylase antibody. Most of the intense fluorescence was first localized in the small bud, then observed on the entire cell wall of the daughter and mother cells. Fluorescence also accumulated at the neck region. These observations suggest that the display of the heterologous protein on the cell surface is carried with other cell wall components to the areas in which the cell wall is newly synthesized; the distribution is controlled by the cell cycle. Then, the heterologous protein-encoding gene was expressed in a sec1 mutant, in which secretory vesicles accumulate under restrictive temperature, and the produced protein was detected by immunoelectron microscopy. Most of the gold particles that reacted with the fusion protein were not localized in vesicles but in expanding endoplasmic reticulum. This phenomenon may be due to overproduction of the heterologous protein which was designed to be displayed on the cell wall. Artificial production of heterologous protein may have caused a relative shortage of glycosyl phosphatidylinositol anchors.     

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.     

Inactivation of the dlt operon in Staphylococcus aureus confers sensitivity to defensins, protegrins, and other antimicrobial peptides

Positively charged antimicrobial peptides with membrane-damaging activity are produced by animals and humans as components of their innate immunity against bacterial infections and also by many bacteria to inhibit competing microorganisms. Staphylococcus aureus and Staphylococcus xylosus, which tolerate high concentrations of several antimicrobial peptides, were mutagenized to identify genes responsible for this insensitivity. Several mutants with increased sensitivity were obtained, which exhibited an altered structure of teichoic acids, major components of the Gram-positive cell wall. The mutant teichoic acids lacked D-alanine, as a result of which the cells carried an increased negative surface charge. The mutant cells bound fewer anionic, but more positively charged proteins. They were sensitive to human defensin HNP1-3, animal-derived protegrins, tachyplesins, and magainin II, and to the bacteria-derived peptides gallidermin and nisin. The mutated genes shared sequence similarity with the dlt genes involved in the transfer of D-alanine into teichoic acids from other Gram-positive bacteria. Wild-type strains bearing additional copies of the dlt operon produced teichoic acids with higher amounts of D-alanine esters, bound cationic proteins less effectively and were less sensitive to antimicrobial peptides. We propose a role of the D-alanine-esterified teichoic acids which occur in many pathogenic bacteria in the protection against human and animal defense systems.     

The major autolysin of Streptococcus gordonii is subject to complex regulation and modulates stress tolerance, biofilm formation, and extracellular-DNA release

A gene, designated atlS, encoding a major autolysin from Streptococcus gordonii, was identified and characterized. The predicted AtlS protein is 1,160 amino acids and 127 kDa and has a conserved β1,4-N-acetylmuramidase domain. Zymographic analysis of wild-type S. gordonii revealed peptidoglycan hydrolase activities with molecular masses of 130 and 90 kDa that were absent in an atlS deletion mutant. Western blotting revealed that the 90-kDa band was derived from the 130-kDa protein. Inactivation of atlS resulted in formation of long chains by the cells, markedly decreased autolytic capacity, poor biofilm formation, diminished tolerance of acid and oxidative stress, and decreased production of extracellular DNA (eDNA). The biofilm-forming capacity of the atlS mutant could be almost completely restored to that of the wild-type strain by adding purified recombinant AtlA autolysin of S. mutans but was only partially restored by addition of eDNA. Autolysis, eDNA release, and atlS expression increased sharply when cells entered stationary phase and were greatly enhanced in cells growing with aeration. The LytST and VicRK two-component systems were both required for the induction of atlS by aeration, and purified LytT was able to bind to the promoter region of atlS in vitro. Thus, AtlS and its associated regulatory cascade dominantly control phenotypes of S. gordonii that are critical to colonization, persistence, and competition with other commensal and pathogenic oral bacteria in response to the redox environment and growth domain.