Production of Poly-γ-Glutamic Acid (γ-PGA) by Clinical Isolates of Staphylococcus Epidermidis (original) (raw)
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Key role of poly-γ-dl-glutamic acid in immune evasion and virulence of Staphylococcus epidermidis
Journal of Clinical Investigation, 2005
Coagulase-negative staphylococci, with the leading species Staphylococcus epidermidis, are the predominant cause of hospital-acquired infections. Treatment is especially difficult owing to biofilm formation and frequent antibiotic resistance. However, virulence mechanisms of these important opportunistic pathogens have remained poorly characterized. Here we demonstrate that S. epidermidis secretes poly-γ-DL-glutamic acid (PGA) to facilitate growth and survival in the human host. Importantly, PGA efficiently sheltered S. epidermidis from key components of innate host defense, namely antimicrobial peptides and neutrophil phagocytosis, and was indispensable for persistence during device-related infection. Furthermore, PGA protected S. epidermidis from high salt concentration, a key feature of its natural environment, the human skin. Notably, PGA was synthesized by all tested strains of S. epidermidis and a series of closely related coagulase-negative staphylococci, most of which are opportunistic pathogens. Our study presents important novel biological functions for PGA and indicates that PGA represents an excellent target for therapeutic maneuvers aimed at treating disease caused by S. epidermidis and related staphylococci.
Immunochemical Properties of the Staphylococcal Poly-N-Acetylglucosamine Surface Polysaccharide
Infection and Immunity, 2002
Staphylococcus aureus and Staphylococcus epidermidis often elaborate adherent biofilms, which contain the capsular polysaccharide-adhesin (PS/A) that mediates the initial cell adherence to biomaterials. Biofilm cells produce another antigen, termed polysaccharide intercellular adhesin (PIA), which is composed of a ϳ28 kDa soluble linear (1-6)-linked N-acetylglucosamine. We developed a new method to purify PS/A from S. aureus MN8m, a strain hyperproducing PS/A. Using multiple analytical techniques, we determined that the chemical structure of PS/A is also (1-6)-N-acetylglucosamine (PNAG). We were unable to find N-succinylglucosamine residues in any of our preparations in contrast to previously reported findings (). PNAG was produced with a wide range of molecular masses that could be divided into three major fractions with average molecular masses of 460 kDa (PNAG-I), 100 kDa (PNAG-II), and 21 kDa (PNAG-III). The purified antigens were not soluble at neutral pH unless first dissolved in 5 M HCl and then neutralized with 5 M NaOH. PNAG-I was very immunogenic in rabbits, but the responses of individual animals were variable. Immunization of mice with various doses (100, 50, or 10 g) of PNAG-I, -II, and -III demonstrated that only PNAG-I was able to elicit an immunoglobulin G (IgG) immune response with the highest titers obtained with 100-g dose. When we purified a small fraction of PNAG with a molecular mass of ϳ780 kDa (PNAG-780) from PNAG-I, significantly higher IgG titers than those in mice immunized with the same doses of PNAG-I were obtained, suggesting the importance of the molecular mass of PNAG in the antibody response. These results further clarify the chemical structure of PS/A and help to differentiate it from PIA on the basis of immunogenicity, molecular size, and solubility.
Capsular polysaccharide serotyping scheme for Staphylococcus epidermidis
Journal of Clinical Microbiology, 1992
A scheme for the capsular typing of Staphylococcus epidermidis that is based on direct slide agglutination between proteinase-treated bacterial cells and specific antisera is described. Antisera were prepared from serum from rabbits immunized with two selected strains of encapsulated S. epidermidis isolated from bacteremic patients. Antisera were shown to be type specific and designated type 1 and type 2. Blood isolates of S. epidermidis from hospitals in different locations within the United States and Europe were serotyped, and it was found that over 90% of all strains were of type 1 or type 2. Type-specific antibodies mediated type-specific opsonophagocytosis and killing of S. epidermidis. The specificity was shown to be due to two distinct capsular polysaccharides. The data presented in this report may open a new window on the pathogenesis of S. epidermidis which could lead to the development of new vaccines and therapies.
Glycobiology, 2009
Secondary cell wall polysaccharides of Bacillus anthracis are antigens that contain specific epitopes which cross-react with three pathogenic Bacillus cereus strains that caused severe disease, and other epitopes common to all the Bacillus cereus strains tested The immunoreactivities of hydrogen fluoride (HF)-released cell wall polysaccharides (HF-PSs) from selected Bacillus anthracis and Bacillus cereus strains were compared using antisera against live and killed B. anthracis spores. These antisera bound to the HF-PSs from B. anthracis and from three clinical B. cereus isolates (G9241, 03BB87, and 03BB102) obtained from cases of severe or fatal human pneumonia but did not bind to the HF-PSs from the closely related B. cereus ATCC 10987 or from B. cereus type strain ATCC 14579. Antiserum against a keyhole limpet hemocyanin conjugate of the B. anthracis HF-PS (HF-PS-KLH) also bound to HF-PSs and cell walls from B. anthracis and the three clinical B. cereus isolates, and B. anthracis spores. These results indicate that the B. anthracis HF-PS is an antigen in both B. anthracis cell walls and spores, and that it shares cross-reactive, and possibly pathogenicity-related, epitopes with three clinical B. cereus isolates that caused severe disease. The anti-HF-PS-KLH antiserum cross-reacted with the bovine serum albumin (BSA)-conjugates of all B. anthracis and all B. cereus HF-PSs tested, including those from nonclinical B. cereus ATCC 10987 and ATCC 14579 strains. Finally, the serum of vaccinated (anthrax vaccine adsorbed (AVA)) Rhesus macaques that survived inhalation anthrax contained IgG antibodies that bound the B. anthracis HF-PS-KLH conjugate. These data indicate that HF-PSs from the cell walls of the bacilli tested here are (i) antigens that contain (ii) a potentially virulence-associated carbohydrate antigen motif, and (iii) another antigenic determinant that is common to B. cereus strains.
Comparative evaluation of protective antigen produced from Bacillus anthracis & Escherichia coli
The Indian journal of medical research, 2003
Anthrax has been reported from almost every country and India is endemic for this disease. There is considerable under reporting of the disease because of lack of microbiological facilities and diagnostic reagents. In India only conventional methods which have limitations, are being used to diagnose the disease. Hence the aim of this study was to isolate and purify protective antigen (PA) using different protocols and to use this PA for detection of anti-PA antibodies from sera samples. Protective antigen was isolated and purified from the Sterne strain of Bacillus anthracis. B. anthracis lacking pXO1 and pXO2 transformed with pYS5 (B. anthracis pYS5) and recombinant Escherichia coli transformed with pQE30 containing PA gene using hydroxyapatite (HA), Q-sepharose fast protein liquid chromatography (FPLC) and nickel-nitrilotriacetic acid (Ni-NTA) chromatographic methods, respectively. A mixture of PA and edema factor (EF) was injected subcutaneously into rabbits to test the biologica...
Infection and immunity, 2016
Poly-N-acetylglucosamine (PNAG) is a major component of the Staphylococcus epidermidis biofilm extracellular matrix. However, it is not yet clear how this polysaccharide impacts the host immune response and infection-associated pathology. Faster neutrophil recruitment and bacterial clearance were observed in mice challenged intraperitoneally with S. epidermidis biofilm cells of the PNAG-producing 9142 strain than in mice similarly challenged with the isogenic PNAG-defective M10 mutant. Moreover, intraperitoneal priming with 9142 cells exacerbated liver inflammatory pathology induced by a subsequent intravenous S. epidermidis challenge, compared to priming with M10 cells. The 9142-primed mice had elevated splenic CD4(+) T cells producing gamma interferon and interleukin-17A, indicating that PNAG promoted cell-mediated immunity. Curiously, despite having more marked liver tissue pathology, 9142-primed mice also had splenic T regulatory cells with greater suppressive activity than thos...
In Vitro Study of the Toxicity Effects of Bacillus anthracis Protective Antigen
Journal of Applied Biotechnology Reports, 2017
Anthrax, a common disease of human and cattle, is caused by Bacillus anthracis infection. Protective antigen (PA) from Bacillus anthracis is a potent immunogen, which has been of interest in the development of new candidate vaccines against the disease. In this study, the toxicity effects of this antigen on prokaryotic (Escherichia coli and Staphylococcus aureus) and eukaryotic (MCF-7) cells were investigated. Antibacterial effects of the recombinant PA were analyzed using MTT and MIC (Minimum Inhibitory Concentration) assays. Cytotoxicity effect of the recombinant protein (in concentrations ranging from 0.5 to 2 µg/ml) on MCF-7 cell line was analyzed using MTT, Neutral red uptake, and comet assays. MCF-7 cells' oxidative stress following the treatment with PA (0.5-2 μg/ml) was analyzed by NO assay, reduced glutathione assay (GSH), and catalase activity assay. MTT and MIC assays showed that PA has a low inhibitory effect on Escherichia coli and no inhibitory effect on Staphyloco...
Proceedings of the National Academy of Sciences, 2003
Both the protective antigen (PA) and the poly(␥-D-glutamic acid) capsule (␥DPGA) are essential for the virulence of Bacillus anthracis. A critical level of vaccine-induced IgG anti-PA confers immunity to anthrax, but there is no information about the protective action of IgG anti-␥DPGA. Because the number of spores presented by bioterrorists might be greater than encountered in nature, we sought to induce capsular antibodies to expand the immunity conferred by available anthrax vaccines. The nonimmunogenic ␥DPGA or corresponding synthetic peptides were bound to BSA, recombinant B. anthracis PA (rPA), or recombinant Pseudomonas aeruginosa exotoxin A (rEPA). To identify the optimal construct, conjugates of B. anthracis ␥DPGA, Bacillus pumilus ␥DLPGA, and peptides of varying lengths (5-, 10-, or 20-mers), of the D or L configuration with active groups at the N or C termini, were bound at 5-32 mol per protein. The conjugates were characterized by physico-chemical and immunological assays, including GLC-MS and matrix-assisted laser desorption ionization time-of-flight spectrometry, and immunogenicity in 5-to 6-week-old mice. IgG anti-␥DPGA and antiprotein were measured by ELISA. The highest levels of IgG anti-␥DPGA were elicited by decamers of ␥DPGA at 10-20 mol per protein bound to the Nor C-terminal end. High IgG anti-␥DPGA levels were elicited by two injections of 2.5 g of ␥DPGA per mouse, whereas three injections were needed to achieve high levels of protein antibodies. rPA was the most effective carrier. Anti-␥DPGA induced opsonophagocytic killing of B. anthracis tox؊, cap؉. ␥DPGA conjugates may enhance the protection conferred by PA alone. ␥DPGA-rPA conjugates induced both anti-PA and anti-␥DPGA. A nthrax probably caused the ''festering boils'' of the people and cattle of Egypt described in the sixth plague of the Old Testament. After the discovery of Bacillus anthracis by Robert Koch in 1880 (1), Pasteur (2) developed a vaccine for sheep composed of chemically treated attenuated strains. Routine use of a noncapsulated strain has virtually eliminated anthrax among domesticated animals (3). In the only controlled study of an anthrax vaccine in humans, culture-supernatant from a capϪ nonproteolytic strain that produced protective antigen (PA), conferred 92% efficacy among woolsorters (4). The Centers for Disease Control monitored the anthrax vaccine adsorbed (AVA) in industrial settings between 1962 and 1974: none of 34 cases occurred in fully vaccinated individuals. A similar vaccine is used in the U.K. (5). This and other evidence indicate that serum IgG anti-PA confers immunity to cutaneous and inhalational anthrax in humans (6, 7). The structure and expression of the essential virulence factors of B. anthracis are controlled by two plasmids. pX01 encodes anthrax toxin (AT) composed of the PA (binding subunit of AT), and two enzymes known as lethal factor and edema factor (8, 9). Administration of AT to primates mimics the symptoms of anthrax (9). pX02 encodes the poly(␥-D-glutamic acid) (␥DPGA) capsule of B. anthracis (10, 11). Other bacilli produce poly(␥glutamic acid) (␥PGA) but only B. anthracis synthesizes it entirely in the D conformation (12). ␥DPGA is a surface structure (13), inhibits in vitro phagocytosis and, when injected, is a poor immunogen even as a bacterial component (14-18); the protective effect of anti-␥DPGA has not been reported. The capsule shields the vegetative form of B. anthracis from agglutination by monoclonal antibodies to its cell wall polysaccharide (19). Systemic infection with B. anthracis induces ␥DPGA antibodies (20). Antibodies to D-amino acid polymers may be induced in animals by injection of ␥DPGA methylated BSA complexes along with Freund's adjuvant, i.v. injections of a formalin-treated capsulated B. anthracis, or by peptidyl proteins (16, 21). We report the synthesis and evaluation of conjugates that induce ␥DPGA antibodies under conditions suitable for clinical use. Experimental Procedures Bacterial Strains. Bacillus pumilus strain Sh18 and B. anthracis strain A34, a pX01Ϫ, pX0 2ϩ variant derived from the Ames strain by repeated passage at 43°C, have been described (10, 22). Analytic. Amino acid analyses were done by GLC-MS after hydrolysis with 6 M HCl, 150°C, 1 h, derivatization to heptafluorobutyryl R-(Ϫ)isobutyl esters, and assayed with a Hewlett-Packard apparatus (model HP 6890) with a HP-5 0.32 ϫ 30 mm glass capillary column, temperature programming at 8°C per min, from 125°C to 250°C in the electron ionization (106 eV) mode (24). Under these conditions, we could separate Dglutamic acid from the L-enantiomer. The amount of each was calculated based on the ratio of D-glutamic acid relative to L-glutamic acid residues in the protein (Fig. 1). The number of peptide chains in L-peptide conjugates was calculated by the increase of total L-glutamic acid relative to aspartic acid. Protein concentration was measured by the method of Lowry (25), free amino groups were measured by Fields' assay (26), thiolation was measured by release of 2-pyridylthio groups (A 343) (27), and hydrazide was measured as reported (28). SDS͞PAGE used 14% gels according to the manufacturer's instructions. Double immunodiffusion was performed in 1.0% agarose gel in PBS. Matrix-Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF). Mass spectra were obtained with a PerSeptive BioSystems Voyager Elite DE-STR MALDI-TOF instrument (Applied Biosystems) operated in the linear mode, 25-kV accelerating voltage, and a 300-nsec ion extraction delay time. Samples for analysis were prepared by a ''sandwich'' of matrix and analyte. First, 1 l of matrix (saturated solution of sinnapinic acid made