Synthesis of glycerol phosphate lipoteichoic acid in Staphylococcus aureus

Lipoteichoic acid (LTA), a glycerol phosphate surface polymer, is a component of the envelope of Gram-positive bacteria. However, the molecular basis for its synthesis or function is not known. Here we report that Staphylococcus aureus LtaS synthesizes glycerol phosphate LTA. Construction of a mutant S. aureus strain with inducible ltaS expression revealed that LTA synthesis is required for bacterial growth and cell division. An ltaS homologue of Bacillus subtilis restored LTA synthesis and the growth of ltaS mutant staphylococci. Thus, LtaS inhibition can be used as a target to treat human infections caused by antibiotic-resistant S. aureus or other bacterial pathogens.     

Crystal structure of Staphylococcus aureus transglycosylase in complex with a lipid II analog and elucidation of peptidoglycan synthesis mechanism

Bacterial transpeptidase and transglycosylase on the surface are essential for cell wall synthesis, and many antibiotics have been developed to target the transpeptidase; however, the problem of antibiotic resistance has arisen and caused a major threat in bacterial infection. The transglycosylase has been considered to be another excellent target, but no antibiotics have been developed to target this enzyme. Here, we determined the crystal structure of the Staphylococcus aureus membrane-bound transglycosylase, monofunctional glycosyltransferase, in complex with a lipid II analog to 2.3 Å resolution. Our results showed that the lipid II-contacting residues are not only conserved in WT and drug-resistant bacteria but also significant in enzymatic activity. Mechanistically, we proposed that K140 and R148 in the donor site, instead of the previously proposed E156, are used to stabilize the pyrophosphate-leaving group of lipid II, and E100 in the acceptor site acts as general base for the 4-OH of GlcNAc to facilitate the transglycosylation reaction. This mechanism, further supported by mutagenesis study and the structure of monofunctional glycosyltransferase in complex with moenomycin in the donor site, provides a direction for antibacterial drugs design.     

Protection against Staphylococcus aureus by antibody to the polyglycerolphosphate backbone of heterologous lipoteichoic acid

Type 1 lipoteichoic acid (LTA) is present in many clinically important gram-positive bacteria, including enterococci, streptococci, and staphylococci, and antibodies against LTA have been shown to opsonize nonencapsulated Enterococcus faecalis strains. In the present study, we show that antibodies against E. faecalis LTA also bind to type 1 LTA from other gram-positive species and opsonized Staphylocccus epidermidis and Staphylcoccus aureus strains as well as group B streptococci. Inhibition studies using teichoic acid oligomers indicated that cross-reactive opsonic antibodies bind to the teichoic acid backbone. Passive immunization with rabbit antibodies against E. faecalis LTA promoted the clearance of bacteremia by E. faecalis and S. epidermidis in mice. Furthermore, passive protection also reduced mortality in a murine S. aureus peritonitis model. The effectiveness of rabbit antibody against LTA suggests that this conserved bacterial structure could function as a single vaccine antigen that targets multiple gram-positive pathogens.     

Differential role of interleukin-6 in lung inflammation induced by lipoteichoic acid and peptidoglycan from Staphylococcus aureus

Lipoteichoic acids (LTA) and peptidoglycans (PepG) are major components of the cell walls of gram-positive bacteria that trigger inflammatory responses in vitro. To study the in vivo effects of LTA and PepG from Staphylococcus aureus in lungs and to determine the role of interleukin (IL)-6 herein, these compounds were intranasally administered to IL-6 gene deficient (IL-6(-/-)) and wild type (IL-6(+/+)) mice. In IL-6(+/+) mice, LTA and PepG induced acute pulmonary inflammation in a dose-dependent way, characterized by neutrophilic influx and IL-6 production in the bronchoalveolar lavage fluid. Endogenously produced IL-6 attenuated inflammation induced by 10 microg LTA, as reflected by enhanced neutrophil influx, and increased tumor necrosis factor-alpha, macrophage inflammatory protein-1-alpha, and cytokine-induced neutrophil chemoattractant (KC) release into bronchoalveolar lavage fluid of IL-6(-/-) mice, compared with IL-6(+/+) mice. By contrast, pulmonary inflammation induced by 100 microg LTA was similar (neutrophil influx) or even tended to be attenuated (cytokine and chemokine release) in IL-6(-/-) mice. Endogenous IL-6 increased inflammation induced by PepG, as reflected by decreased neutrophil influx into lungs of IL-6(-/-) mice, compared with IL-6(+/+) mice. These data suggest that IL-6 plays an anti-inflammatory role during LTA-induced pulmonary inflammation, which is dependent on the severity of the inflammatory challenge, and a proinflammatory role in peptidoglycan-induced acute lung inflammation. Thus, the contribution of IL-6 to lung inflammation may vary with the stimulus used.     

Release of protein A from the cell wall of Staphylococcus aureus

Staphylococcal protein A (SpA) is anchored to the cell wall envelope of Staphylococcus aureus by sortase A, which links the threonyl (T) of its C-terminal LPXTG motif to peptidoglycan cross-bridges (i.e., Gly₅). SpA binds the Fcγ domains of IgG and protects staphylococci from opsonophagocytic clearance. Moreover, SpA cross-links B-cell receptors to modify host adaptive immune responses. The mechanisms whereby SpA is released from the bacterial surface to access the host’s immune system are not known. Here we demonstrate that SpA is released with murein tetrapeptide-tetraglycyl [l-Ala-d-iGln-(SpA-Gly₅)l-Lys-d-Ala-Gly₄] linked to its C-terminal threonyl. LytN, a cross-wall murein hydrolase, contributes to the release of SpA by removing amino sugars [i.e., N-acetylmuramic acid-N-acetylglucosamine (MurNAc-GlcNAc)] from attached peptidoglycan, whereas LytM, a pentaglycyl-endopeptidase, triggers polypeptide release from the bacterial envelope. A model is proposed whereby murein hydrolases cleave the anchor structure of released SpA to modify host immune responses.The Gram-positive bacterium Staphylococcus aureus is a pathogen of humans (1). Cells of S. aureus are surrounded by a thick layer of highly cross-linked cell wall peptidoglycan (2). The peptidoglycan layer is formed from lipid II precursors, C₅₅-(PO₃)₂-N-acetylmuramic acid (MurNAc)-(l-Ala-d-iGln-(Gly₅)l-Lys-d-Ala-d-Ala)-GlcNAc (3), via the transpeptidation and transglycosylation reactions of cell wall synthesis to generate [MurNAc-(l-Ala-d-iGln-(Gly₅)l-Lys-d-Ala)-GlcNAc]_n polymer (4). Assembled peptidoglycan is a single large macromolecule that protects bacteria against osmotic lysis (5) and also functions as scaffold for the anchoring of wall teichoic acids (6) and proteins (7). These secondary cell wall polymers promote specific interactions between staphylococci and host tissues (8). Cell wall-anchored surface proteins are synthesized as precursors with N-terminal signal peptides and C-terminal LPXTG motif sorting signals (9). Following cleavage of the N-terminal signal peptide by signal peptidase, the C-terminal sorting signal is cleaved by sortase A between the threonyl (T) and the glycyl (G) of the LPXTG motif (10). Sortase A forms an acyl enzyme, capturing the C-terminal carboxyl group of cleaved surface proteins with its active site cysteine thiol (11). These acyl intermediates are relieved by the nucleophilic attack of the amino group of pentaglycyl within lipid II and incorporated into the cell wall via the transpeptidation and transglycosylation reactions (7, 12).The genomes of S. aureus isolates harbor 17 to 22 genes encoding LPXTG motif surface proteins, which can be further classified as precursors with canonical or YSIRK-G/S signal peptides (13). Surface proteins with canonical signal peptides are secreted and immobilized to peptidoglycan near the cell poles of dividing staphylococci (14). In contrast, precursors with YSIRK-G/S signal peptides are secreted into the cross-wall, a membrane enclosed compartment for the de novo synthesis of peptidoglycan that separates daughter cells during division (14). When precursors with YSIRK-G/S signal peptides and LPXTG motif sorting signals have been deposited at the cross-wall and its peptidoglycan has been split, surface proteins are displayed over the staphylococcal surface (14). YSIRK-G/S precursors include proteins with important virulence functions that are synthesized in large abundance, including clumping factor A (15), fibronectin binding proteins (16, 17), iron-regulated surface protein B (18), and staphylococcal protein A (SpA) (19).SpA binds human or animal Ig via its Ig-binding domains that capture the Fcγ domain of IgG or the Fab domain of V_H3-clan IgG and IgM antibodies (20, 21). SpA binding to the Fcγ domain blocks the ability of antibodies with specific binding activities for the staphylococcal surface to promote Fc receptor-mediated opsonophagocytosis and bacterial killing (22). SpA binding to the Fab domain of V_H3-clan IgM triggers B-cell receptor cross-linking and clonal expansion of B lymphocytes, which eventually undergo apoptotic collapse (23). During infection, this B-cell superantigen activity of SpA ablates host adaptive immune responses against many staphylococcal antigens (24). Although S. aureus disease predominantly manifests as localized skin or soft-tissue infection, its suppressive effects on the immune system appear to be general (25). If so, we wondered whether SpA, a key factor for staphylococcal immune evasion, is released from the bacterial surface.     

Teichoic acids are temporal and spatial regulators of peptidoglycan cross-linking in Staphylococcus aureus

The cell wall of Staphylococcus aureus is characterized by an extremely high degree of cross-linking within its peptidoglycan (PGN). Penicillin-binding protein 4 (PBP4) is required for the synthesis of this highly cross-linked peptidoglycan. We found that wall teichoic acids, glycopolymers attached to the peptidoglycan and important for virulence in Gram-positive bacteria, act as temporal and spatial regulators of PGN metabolism, controlling the level of cross-linking by regulating PBP4 localization. PBP4 normally localizes at the division septum, but in the absence of wall teichoic acids synthesis, it becomes dispersed throughout the entire cell membrane and is unable to function normally. As a consequence, the peptidoglycan of TagO null mutants, impaired in wall teichoic acid biosynthesis, has a decreased degree of cross-linking, which renders it more susceptible to the action of lysozyme, an enzyme produced by different host organisms as an initial defense against bacterial infection.     

Autocrine regulation of toxin synthesis by Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen causing diseases which range from minor skin infection to endocarditis and toxic shock syndrome. The pathogenesis of S. aureus is due primarily to the production of toxic exoproteins, whose synthesis is controlled by a global regulatory system, agr. We show here that agr is autoinduced by a proteinaceous factor produced and secreted by the bacteria and that it is inhibited by a peptide produced by an exoprotein-deficient S. aureus mutant strain. The inhibitor, RIP, competes with the activator, RAP, and may be a mutational derivative. Our results suggest two possible approaches, independent of antibiotics, to the control of S. aureus infections. RIP may prove useful as a direct inhibitor of virulence and RAP as a vaccine against the expression of agr-induced virulence factors; either could interfere with the ability of the bacteria to establish and maintain an infection.     

Demonstration of the role of cell wall homeostasis in Staphylococcus aureus growth and the action of bactericidal antibiotics

Bacterial cell wall peptidoglycan is essential, maintaining both cellular integrity and morphology, in the face of internal turgor pressure. Peptidoglycan synthesis is important, as it is targeted by cell wall antibiotics, including methicillin and vancomycin. Here, we have used the major human pathogen Staphylococcus aureus to elucidate both the cell wall dynamic processes essential for growth (life) and the bactericidal effects of cell wall antibiotics (death) based on the principle of coordinated peptidoglycan synthesis and hydrolysis. The death of S. aureus due to depletion of the essential, two-component and positive regulatory system for peptidoglycan hydrolase activity (WalKR) is prevented by addition of otherwise bactericidal cell wall antibiotics, resulting in stasis. In contrast, cell wall antibiotics kill via the activity of peptidoglycan hydrolases in the absence of concomitant synthesis. Both methicillin and vancomycin treatment lead to the appearance of perforating holes throughout the cell wall due to peptidoglycan hydrolases. Methicillin alone also results in plasmolysis and misshapen septa with the involvement of the major peptidoglycan hydrolase Atl, a process that is inhibited by vancomycin. The bactericidal effect of vancomycin involves the peptidoglycan hydrolase SagB. In the presence of cell wall antibiotics, the inhibition of peptidoglycan hydrolase activity using the inhibitor complestatin results in reduced killing, while, conversely, the deregulation of hydrolase activity via loss of wall teichoic acids increases the death rate. For S. aureus, the independent regulation of cell wall synthesis and hydrolysis can lead to cell growth, death, or stasis, with implications for the development of new control regimes for this important pathogen.

How bacteria grow and divide is a fundamental question in microbiology, where many of the essential processes involved are the targets of clinically important antibiotics. The cell wall is crucial for bacterial survival, forming the interface between the external and internal environments and maintaining internal turgor pressure (1, 2). The major cell wall structural component is peptidoglycan (PG), a polymer of glycan strands and peptide cross-links (3–5), the synthesis of which is the target of antibiotics including β-lactams and glycopeptides (6). These cell wall antibiotics inhibit the final stages of PG synthesis where building blocks are incorporated into the existing structure via the action of penicillin-binding proteins (PBPs) (6). Several mechanisms linking the action of antibiotics to the inhibition of essential processes in cell wall growth and division have been suggested, including lytic and nonlytic death, oxidative stress, and futile PG synthesis (7–12).As a single macromolecule that surrounds the cell, PG can increase in surface area to permit growth and division while maintaining cellular integrity. It has been proposed that areal PG growth occurs as a consequence of both synthesis and hydrolysis (4, 13, 14), with new material being covalently bound to the existing macrostructure and hydrolysis of existing bonds allowing expansion. This leads to a simple set of hypotheses for growth but also makes predictions as to the effects of inhibition of PG homeostasis activities, including cell wall antibiotics (Fig. 1A). The lack of either PG synthesis or hydrolysis will result in cell death because of the continued activity of the other, but the loss of both will lead to stasis.Open in a separate windowFig. 1.The role of regulation of PG hydrolases (PGHs) by WalKR in life and death. (A) Predictive model for how cell wall homeostasis governs bacterial life and death. Both cell wall synthesis and hydrolysis are required for growth, loss of either results in death, or both, cell stasis. (B–H) Effect of 10 × minimum inhibitory concentration (MIC) vancomycin for 3 h on conditional lethal strain S. aureus P_spac-walKR (without inducer; WalKR OFF) compared to the control (with inducer; WalKR ON). (B) CFU relative to T = 0; after t test with Welch''s correction: P (WalKR OFF − WalKR OFF + vancomycin, **) = 6.9 × 10⁻³. (C and D) PG synthesis and transpeptidase activity measured by ¹⁴C-GlcNAc and Atto 488 dipeptide (53) incorporation, normalized against WalKR ON. (E) Transmission electron microscopy (TEM) (scale bars, 300 nm). (F) Quantification of bacterial phenotypes (SI Appendix, Fig. S2; dark green: no septum, mid-green: incomplete septum, light green: complete septum, and yellow: growth defects). For samples shown, the number of individual cells quantified was n > 300. (G) AFM topographic images of sacculi (scale bars, 150, 300, and 300 nm; data scales [DS], 85, 200, and 85 nm, respectively, from Left to Right). (i) Insets show sacculus external architecture from Left to Right, (WalKR ON) from dashed box in panel G, (WalKR OFF) from SI Appendix, Fig. S2E, (WalKR OFF+Van) from SI Appendix, Fig. S2D, respectively (scale bars, 50 nm; DS, 30, 52, and 32 nm, respectively, from Left to Right; images were analyzed with NanoscopeAnalysis from Bruker using the default color scale). (H) Thickness distribution values for sacculi with SD (n = 5). For sample size and data reproducibility, see Materials and Methods.Staphylococcus aureus is a major human antimicrobial-resistant pathogen. As a spheroid cell with a simple growth and division cycle, it forms an excellent subject to demonstrate the basic principles underlying growth, division, and the action of antibiotics. Many organisms have multiple PBPs, but S. aureus has only four, of which PBP1 and PBP2 are essential for growth and division (15–19). S. aureus also has many PG hydrolases (PGHs), including SagB, which is involved in cell growth (20, 21). The bifunctional Atl is involved in generalized cell lysis and cell separation after septation and contains both amidase and glucosaminidase domains (22, 23). PGHs often show functional redundancy with several enzymes involved in the same process (20, 24). In S. aureus, no individual PGH alone has been shown to be required for either growth or division, but multiple PGHs are positively regulated by an essential two-component system, WalKR (25–27), further suggesting that their collective activity is required.Recently, using atomic force microscopy (AFM), we have revealed that the molecular architecture of the PG is that of an expanded hydrogel whose mature external surface is a porous open network but with an interior surface characterized by a much smoother and denser mesh of PG material (28). This provides an architectural framework from which to begin to elucidate the roles of PG synthesis and hydrolysis. Here, we have taken an integrated approach to determine the role of PG homeostasis in S. aureus growth, division, and the bactericidal action of cell wall antibiotics.     

Processivity of peptidoglycan synthesis provides a built-in mechanism for the robustness of straight-rod cell morphology

The propagation of cell shape across generations is remarkably robust in most bacteria. Even when deformations are acquired, growing cells progressively recover their original shape once the deforming factors are eliminated. For instance, straight-rod-shaped bacteria grow curved when confined to circular microchambers, but straighten in a growth-dependent fashion when released. Bacterial cell shape is maintained by the peptidoglycan (PG) cell wall, a giant macromolecule of glycan strands that are synthesized by processive enzymes and cross-linked by peptide chains. Changes in cell geometry require modifying the PG and therefore depend directly on the molecular-scale properties of PG structure and synthesis. Using a mathematical model we quantify the straightening of curved Caulobacter crescentus cells after disruption of the cell-curving crescentin structure. We observe that cells straighten at a rate that is about half (57%) the cell growth rate. Next we show that in the absence of other effects there exists a mathematical relationship between the rate of cell straightening and the processivity of PG synthesis—the number of subunits incorporated before termination of synthesis. From the measured rate of cell straightening this relationship predicts processivity values that are in good agreement with our estimates from published data. Finally, we consider the possible role of three other mechanisms in cell straightening. We conclude that regardless of the involvement of other factors, intrinsic properties of PG processivity provide a robust mechanism for cell straightening that is hardwired to the cell wall synthesis machinery.     

The cell wall components peptidoglycan and lipoteichoic acid from Staphylococcus aureus act in synergy to cause shock and multiple organ failure.

Although the incidence of Gram-positive sepsis has risen strongly, it is unclear how Gram-positive organisms (without endotoxin) initiate septic shock. We investigated whether two cell wall components from Staphylococcus aureus, peptidoglycan (PepG) and lipoteichoic acid (LTA), can induce the inflammatory response and multiple organ dysfunction syndrome (MODS) associated with septic shock caused by Gram-positive organisms. In cultured macrophages, LTA (10 micrograms/ml), but not PepG (100 micrograms/ml), induces the release of nitric oxide measured as nitrite. PepG, however, caused a 4-fold increase in the production of nitrite elicited by LTA. Furthermore, PepG antibodies inhibited the release of nitrite elicited by killed S. aureus. Administration of both PepG (10 mg/kg; i.v.) and LTA (3 mg/kg; i.v.) in anesthetized rats resulted in the release of tumor necrosis factor alpha and interferon gamma and MODS, as indicated by a decrease in arterial oxygen pressure (lung) and an increase in plasma concentrations of bilirubin and alanine aminotransferase (liver), creatinine and urea (kidney), lipase (pancreas), and creatine kinase (heart or skeletal muscle). There was also the expression of inducible nitric oxide synthase in these organs, circulatory failure, and 50% mortality. These effects were not observed after administration of PepG or LTA alone. Even a high dose of LTA (10 mg/kg) causes only circulatory failure but no MODS. Thus, our results demonstrate that the two bacterial wall components, PepG and LTA, work together to cause systemic inflammation and multiple systems failure associated with Gram-positive organisms.     

EsxA and EsxB are secreted by an ESAT-6-like system that is required for the pathogenesis of Staphylococcus aureus infections

Mycobacterium tuberculosis secretes ESAT-6, a virulence factor that triggers cell-mediated immune responses and IFN-gamma production during tuberculosis. ESAT-6 is transported across the bacterial envelope by a specialized secretion system with a FSD (FtsK-SpoIIIE domain) membrane protein. Although the presence of ESAT-6-like genes has been identified in the genomes of other microbes, the possibility that they may encode general virulence functions has hitherto not been addressed. Herein we show that the human pathogen Staphylococcus aureus secretes EsxA and EsxB, ESAT-6-like proteins, across the bacterial envelope. Staphylococcal esxA and esxB are clustered with six other genes and some of these are required for synthesis or secretion of EsxA and EsxB. Mutants that failed to secrete EsxA and EsxB displayed defects in the pathogenesis of S. aureus murine abscesses, suggesting that this specialized secretion system may be a general strategy of human bacterial pathogenesis.     

双歧杆菌脂磷壁酸延缓细胞衰老的作用机制

目的 探讨双歧杆菌脂磷壁酸(Lipoteichoic acid,LTA)在延缓H2O2诱导细胞衰老中的作用.方法 H2O2诱导WI-38细胞衰老,β-半乳糖苷酶细胞化学染色计算衰老细胞百分率变化,RT-PCR和Western印迹检测衰老细胞p21、细胞周期蛋白E(cyclin E)和周期蛋白依赖性蛋白激酶2(CDK2)表达水平的变化.结果 双歧杆菌LTA处理后,β-半乳糖苷酶细胞化学染色阳性细胞百分率较衰老模型组降低(P＜0.01).与年轻对照组相比,衰老模型组细胞中p21的表达增高,cyclin E和CDK2表达降低,而双歧杆菌LTA能够逆转上述变化(P＜0.01).结论 双歧杆菌能延缓H2O2诱导的细胞衰老,机制可能与改变p21,cyclin E和CDK2表达水平有关.     

Inhibition of intracellular macromolecular synthesis in Staphylococcus aureus by thrombin-induced platelet microbicidal proteins.

Thrombin-induced platelet microbicidal proteins (tPMP-1 and tPMP-2) are believed to initiate their staphylocidal effects via cytoplasmic membrane perturbation. The aim of the present study was to investigate the role of subsequent inhibition of macromolecular synthesis in the staphylocidal mechanisms of tPMP-1 and tPMP-2 in an isogenic tPMP-susceptible and -resistant strain pair (ISP479C and ISP479R, respectively). In ISP479C, tPMP-1 and tPMP-2 (2 microg/mL) exerted significant bactericidal effects and significantly reduced DNA and RNA synthesis (P <.05 vs. control). In contrast, tPMP-1 and tPMP-2 exerted reduced staphylocidal effects and significantly reduced inhibition of DNA and RNA synthesis against ISP479R, as compared with ISP479C (P <.05). However, tPMP-1 and tPMP-2 (2 microg/mL) caused equivalent degrees of inhibition of protein synthesis in both ISP479C and ISP479R. Collectively, these observations are consistent with the hypothesis that inhibition of specific macromolecular synthesis pathways is integral to the overall staphylocidal mechanism(s) of tPMPs.     

Small molecule inhibitor of lipoteichoic acid synthesis is an antibiotic for Gram-positive bacteria

The current epidemic of infections caused by antibiotic-resistant Gram-positive bacteria requires the discovery of new drug targets and the development of new therapeutics. Lipoteichoic acid (LTA), a cell wall polymer of Gram-positive bacteria, consists of 1,3-polyglycerol-phosphate linked to glycolipid. LTA synthase (LtaS) polymerizes polyglycerol-phosphate from phosphatidylglycerol, a reaction that is essential for the growth of Gram-positive bacteria. We screened small molecule libraries for compounds inhibiting growth of Staphylococcus aureus but not of Gram-negative bacteria. Compound 1771 [2-oxo-2-(5-phenyl-1,3,4-oxadiazol-2-ylamino)ethyl 2-naphtho[2,1-b]furan-1-ylacetate] blocked phosphatidylglycerol binding to LtaS and inhibited LTA synthesis in S. aureus and in Escherichia coli expressing ltaS. Compound 1771 inhibited the growth of antibiotic-resistant Gram-positive bacteria and prolonged the survival of mice with lethal S. aureus challenge, validating LtaS as a target for the development of antibiotics.Methicillin-resistant Staphylococcus aureus (MRSA), glycopeptide-resistant enterococci (GRE), and antibiotic-resistant Clostridium difficile are examples for newly emerging or reemerging drug-resistant Gram-positive pathogens with significant human morbidity (1). As commensals of the human skin or intestine, these microbes are continuously exposed to antibiotics and have evolved resistance traits against commonly used therapeutics (2). To develop novel antibiotics, a suitable molecular target must be identified (3). Ideal targets are found only in bacteria, not in humans, and are positioned in the bacterial envelope, accessible for small molecule inhibitors yet out of reach of multi–drug-resistance transporters protecting cytoplasmic targets (3, 4). Such attributes are found in penicillin-binding proteins and their inhibitors, β-lactam or glycopeptide antibiotics; however, efforts to identify other extracellular targets in Gram-positive bacteria have met with little success (5).Gram-positive bacteria incorporate lipoteichoic acids (LTAs) into their envelope to scavenge magnesium ions, direct autolysins to subcellular cell wall locations, and enable bacterial cell division (6, 7). LTA from S. aureus and other bacteria is composed of 1,3-polyglycerol-phosphate linked to glycolipid, which provides for LTA anchoring in membranes (8). The glycolipid moiety is composed of β-gentiobiosyldiacylglycerol [glucosyl-(1→6)-glucosyl-(1→3)-diacylglycerol (Glc₂-DAG)] (8, 9). PgcA (α-phosphoglucomutase), GtaB (UTP:α-glucose-1-phosphate uridyl transferase), and YpfP (glycosyl-transferase) catalyze the three steps of glycolipid synthesis (10–12), whereas LtaA transports Glc₂-DAG across the lipid bilayer (10). S. aureus ltaA, ypfP, pgcA, or gtaB mutants cannot assemble Glc₂-DAG but continue to synthesize polyglycerol-phosphate (10, 12). Glycolipid synthesis mutants are viable; however, the variants display an increase in size and aberrant cell shapes (10, 12).LTA synthesis involves the polymerization of polyglycerol-phosphate and its transfer to Glc₂-DAG (13). This reaction is catalyzed by LTA synthase (LtaS), a protein with five transmembrane domains in its N-terminal domain and an extracellular sulfatase-like domain (pfam00884) at the C-terminal end (14). Genetic depletion or loss of the ltaS gene in S. aureus results in severe cell division defects, a phenotype that is exacerbated when staphylococci are grown at >30 °C (14, 15). Characterization of LtaS in Bacillus anthracis, Bacillus subtilis, and Listeria monocytogenes confirmed that membrane proteins with pfam00884 domains are indeed responsible for the synthesis of polyglycerol-phosphate LTA (7). Where examined, ltaS mutants exhibited diminished viability, increased cell size, and altered morphology (16–19). These results suggested that LtaS of Gram-positive bacteria may represent an extracellular target for the development of antibiotics against drug-resistant Gram-positive bacteria. We here provide proof for this hypothesis by isolating a small molecule inhibitor of LTA synthesis.     

金黄色葡萄球菌对万古霉素耐药机制的初步研究

目的了解金黄色葡萄球菌对万古霉素耐药机制。方法按照美国临床实验室标准化委员会(NCCLS)标准,应用琼脂平板稀释法及MRSA特异性基因mecA的扩增鉴定MRSA,诱导产生万古霉素耐药株,然后以超声破碎法提取外膜蛋白(OMP),经聚丙烯酰胺凝胶电泳(SDS-PAGE)分析OMP的成分,分光光度法扫描仪测定相关膜蛋白的相对含量。结果耐万古霉素金葡菌菌株中,分子量为45KD和14KD的膜蛋白的相对含量较金葡菌ATCC25923株和对万古霉素敏感的MRSA少。结论提示45KD和14KD膜蛋白减少或缺失与金葡菌对万古霉素耐药可能有密切关系。     

甲氧西林耐药金黄色葡萄球菌对氨基糖苷抗生素耐药机制的研究

为研究甲氧要耐药金黄色球菌（ＭＲＳＡ）对氨基糖苷类抗生素的耐药机制，应用氨基醭 抗生素耐药谱推测法、核素标记分析法、Ｓｏｕｔｈｅｍ印迹和斑点杂交试验对１００株ＭＲＳＡ进行了耐药研究。结果：根据细菌对氨基糖苷抗生素的耐药谱，１００株ＭＲＳＡ可以分成４类，６５株细菌产生ＡＡＣ（６）－ＡＰＨ（２）钝化酶，２４株产生ＡＡＣ（６）－ＡＰＨ（２）＋ＡＰＨ（３），１０株产生ＡＡＣ（６），还有１株产生ＡＡＣ（６）