Identification of vaccine candidate antigens of Staphylococcus aureus by serological proteome analysis

In this study we applied serological proteome analysis (Klade, C. S. et al. Proteomics 2001, 1, 890-898) for identification of bacterial vaccine candidate antigens. First, approximately one hundred sera from healthy individuals and patients suffering from Staphylococcus aureus infections were screened for antibodies against staphylococcal lysates and recombinant proteins representing surface antigens. Two pools (healthy donors, patients) each consisting of five sera with the highest antiproteinaceous IgG reactivity were selected. Second, S. aureus COL was grown under different conditions and the number of antigens expressed was monitored by Western blot analysis. Third, surface proteins were enriched by digesting the bacterial cell wall under isotonic conditions and subsequent removal of protoplasts. These protein preparations were resolved by two-dimensional electrophoresis (2-DE) (pI 4-7). 2-DE immunoblotting using the preselected serum pools at 1:10 000-1:100 000 dilutions revealed a number of highly immunogenic staphylococcal proteins. Twenty-one spots were isolated by preparative 2-DE, and analysed by matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry sequencing of tryptic peptides. This led to the identification of 15 proteins including known and novel vaccine candidates. Seroreactivity of several antigens including serine-aspartate repeat containing protein D, immuno-dominant staphylococcal antigen and a novel 309 amino acid lipoprotein was independently confirmed by enzyme-linked immunosorbent assay and Western blot analysis of purified recombinant proteins. In conclusion, serological proteome analysis proved to be a powerful tool for the identification of novel staphylococcal antigens, which provide a basis for rational vaccine design.     

Humoral response to Staphylococcus aureus antigens evaluated by the western blotting method]

Cellular antigens extracted from the cells of four Staphylococcus aureus strains from different kinds of infections (sepsis, osteomyelitis, furunculosis) were analysed by the western blotting technique. Antibiotic sensitivity pattern of the strains was compared. One isolate was found to be MRSA strain. Sera samples from patients of whom strains were isolated and four sera from blood donors (as a control) were used in the investigation. IgG levels for purified staphylococcal antigens (lipase, alpha-toxin and teichoic acid) were estimated. Interaction between extracted bacterial antigens and serum antibodies of IgG class were analysed in homologous and heterologous systems. The most strong immunological reaction of the investigated sera with staphylococcal antigens was observed in the case of homologous system. Serum from sepsis patient was found to be the most reactive serum with all staphylococcal antigens mixtures.     

Immunoblotting of a Staphylococcus aureus DNA library as a tool for isolating potential antigenic proteins

Abstract A Staphylococcus aureus DNA library was created in the lambda vector EMBL4 and expressed in Escherichia coli strain Y1090. Plaques were screened using serum from a S. aureus -infected patient. A number of positive clones that expressed proteins recognised by serum antibodies were further analysed using Western blots of total lysates from cultures infected with the lambda clones. Differences were found between patient and control sera in both the specificity and tite of anti- S. aureus antibodies. Expressed staphylococcal proteins appeared to be stable and were easily distinnguishable from E. coli proteins in the Western blot. This method may have applications in specifically selecting clones encoding antigens associated with infection and may be of potential use clinically.     

Dynamics of staphylococcal infection in guinea pigs with delayed hypersensitivity to Staphylococcus aureus surface antigens

The relationship between delayed hypersensitivity (DH) to S. aureus surface antigens and the intensity of the infectious process induced by the sublethal infection of guinea pigs with S. aureus was studied. The protective effect, manifested by a decrease in the staphylococcal contamination of the spleen tissue and by an increase in the level of the activation of lymphocytes, was shown to correlate with DH induced by inactivated staphylococcal cells. In infected guinea pigs having DH to different staphylococcal antigens the disease either took a more severe course (in cases of DH to cell wall or peptidoglycan) than in the animals subjected only to infection, or no aggravation of the disease was observed (in cases of DH to protein A).     

Heterogenetic antigens of gram-positive bacteria

Chorpenning, Frank W. (The Ohio State University, Columbus), and Matthew C. Dodd. Heterogenetic antigens of gram-positive bacteria. J. Bacteriol. 91:1440-1445. 1966.-Soluble antigens obtained by various methods from gram-positive bacteria were used to modify erythrocytes whose hemagglutinating reactions with immune rabbit sera and normal human sera were then studied. Antigens from all gram-positive organisms studied except corynbacteria altered red cells, causing them to react with specific bacterial antisera and with normal human sera; however, cross-absorption and inhibition tests indicated that at least three different specificites were involved. One of these antigens seemed to be similar to Rantz's streptococcal NSS, which is shared with Staphylococcus aureus and Bacillus spp., and is therefore heterogenetic. Another was found in streptococci but was apparently not present in S. aureus and Bacillus spp. A third antigen, also heterogenetic, appeared to be shared by several species of Bacillus and by S. aureus, but not by streptococci or any gram-negative bacteria. The third antigen was heat-stable at pH 8.0, and appeared to be essentially polysaccharide in nature. Normal human sera varied in their content of antibodies which reacted with erythrocytes modified by extracts from gram-positive bacteria. Whereas some sera reacted very broadly with red cells modified by extracts of practically any gram-positive organism, other sera agglutinated only cells which had been modified by streptococcal antigen.     

Effects of murine endotoxemia on lymphocyte subsets and clearance of staphylococcal pulmonary infection

In a model of staphylococcal pneumonia initiated during systemic endotoxemia in BALB/c mice, a significant reduction of the number of circulating CD4+ and CD8+ T-lymphocytes, B-lymphocytes, and NK cells, as well as lung-resident total T- and CD4+ T-lymphocytes was demonstrated. Staphylococcus aureus exposure only induced a similar decrease of lymphocyte subsets in the blood. However, the number of lung-resident total T- and CD4+ T-lymphocytes was increased. More viable bacteria were recovered from the lungs of S. aureus-infected mice than from those animals previously treated with lipopolysaccharide (LPS) followed by a staphylococcal challenge. These results indicate that LPS-induced reduction in the number of circulating lymphocyte subsets and lung-resident total T- and CD4+ T-lymphocytes do not increase susceptibility to staphylococcal respiratory infection. Moreover, LPS challenge prior to S. aureus exposure significantly improves clearance of the bacteria in the lung.     

The serological specificities of Salmonella typhi antigens.

Sera prepared with two different strains of Salmonella typhi were analysed against all the soluble antigens isolated from S. typhi 0901, S. typhi Ty2 and S. typhi Vi. Agar-gel diffusion against individual sera showed that, in all the sera, antibodies were induced against somatic antigens and free proteins. Absorptions of the sera with polysaccharides, split from the somatic antigens, removed the antibodies induced against the polysaccharide and its proteinic carrier in most of the somatic antigens of S. typhi 0901. The antibodies left in the absorbed sera reacted against the proteinic moieties of more complex somatic antigens of S. typhi and against free proteins from all the analysed strains. Only the absorption with proteins removed all the precipitating antibodies from the sera. Moreover, in incomplete absorptions with proteins, the first antibodies removed are the antipolysaccharides, since antibodies are never induced against the haptenic polysaccharide but against somatic conjugates; in these the proteinic moiety eventually varies with every batch of bacteria. The sera exhausted of precipitins still agglutinate the bacteria, thus confirming the assumption that agglutinins and precipitins may be different antibodies.     

The adhesion of protein A-bearing Staphylococcus aureus organisms to soluble-staphylococcal-antigens-coated HeLa cells mediated by specific antibodies

The adhesion of staphylococcal protein A (SpA)-bearing Staphylococcus aureus Cowan I organisms to HeLa cells was enhanced by pretreatment of HeLa cells with staphylococcal extracellular antigens and antibodies to them. The adhesion of HLj, an SpA-poor mutant derived from Cowan I, to HeLa cells was not enhanced by the same pretreatment of HeLa cells. Furthermore, the enhanced staphylococcal adhesion was inhibited by soluble SpA. The antigen(s) responsible for the enhanced staphylococcal adhesion was(were) heat stable. Pretreatment of HeLa cells with the mixture of staphylococcal extracellular antigens and antibodies to them also enhanced the adhesion of Cowan I. Similarly the adhesion of Cowan I was enhanced by pretreatment of HeLa cells with extracellular antigens of Pseudomonas aeruginosa and antibodies to them. These results indicated that cell-bound SpA mediated the binding of S. aureus to immune complexes composed of extracellular bacterial products and antibodies to them bound to the surface of HeLa cells, and suggested another role of cell-bound SpA as a co-adhesin with other factors in infections due to S. aureus.     

Brachyspira (Serpulina) pilosicoli of human origin interfere with the haemolytic activity and the growth of Staphylococcus aureus beta-toxin producer

Brachyspira (Serpulina) pilosicoli related to intestinal spirochaetosis were found to interfere in vitro with the haemolytic activity and the growth of Staphylococcus aureus beta-toxin producer. This interference was clearly appreciated because a reduction of the zone of the staphylococcal beta-toxin activity, the reduction and/or absence of cooperative haemolysis between bacteria, and the growth reduction of S. aureus were observed when B. (S.) pilosicoli were grown 72-96 hours sooner than S. aureus and after the inoculum of the latter the plates were anaerobically incubated for additional 48-72 hours. The phenomenon was more clearly observed when B. (S.) pilosicoli had a concentration of 8x10(6)-8x10(7) CFU/ml and S. aureus at a concentration ranging from 10(7) to 10(1) CFU/ml was inoculated at a distance from the streaks of B. (S.) pilosicoli ranging from 0-10 mm. When B. (S.) pilosicoli and S. aureus were inoculated at the same time and when B. (S.) pilosicoli grew 24-48 hours sooner than S. aureus only a cooperative haemolysis was observed.     

IgY antibodies of chicken do not bind staphylococcal binder of immunoglobulin (Sbi) from Staphylococcus aureus

The immunoglobulin (Ig) binding proteins of Staphylococcus aureus namely staphylococcal protein A (SpA) and staphylococcal binder of immunoglobulin (Sbi) are responsible for false positives during immunoassays. Avian IgY antibodies were reported to have no affinity to SpA and thus are safe for use in immunoassays. However, the behaviour of Sbi with IgY was not reported. The purpose of the present study is to evaluate the interactions between IgY antibodies and Sbi protein from different S. aureus strains. Initially, heterologous cloning and expression of complete sbi gene in Escherichia coli was undertaken. Recombinant Sbi protein was utilized to generate polyclonal anti-Sbi IgY and anti-Sbi antibodies in chicken and BALB/c mice respectively. Indirect ELISA and Western blotting were performed to evaluate the reactivity of anti-Sbi antibodies. Non-reducing PAGE followed by Western blotting and double-antibody sandwich dot-ELISA were performed to analyze the reactivity of IgY antibodies with recombinant Sbi and native Sbi from S. aureus strains. To avoid the possible interference of enzyme-conjugated secondary antibodies from mammalian sources, mouse anti-Sbi revealing antibodies were labeled with biotin so that streptavidin-HRP was used as developing reagent for chromogenic reaction. Sbi was highly immunogenic in chicken and mouse with antibody titers of 1:128,000 and 1:64,000 dilutions respectively. We observed that unimmunized IgY antibodies showed no affinity to either recombinant Sbi or native Sbi from S. aureus strains in Western blotting and double antibody sandwich ELISA. In view of these observations, we recommend that IgY antibodies are safe and free from false positives due to SpA and Sbi in immunoassays involving detection of S. aureus antigens/exotoxins.     

Zero order kinetics of cell wall turnover inStaphylococcus aureus

Cells exponentially grown from four strains ofS. aureus (SG 511, H, 52A5G, and248 PN-1) and uniformly labeled in their walls with³H-N-acetylglucosamine, were found to turn over their old walls at constant rates of up to 25% per generation. Wall turnover was not observed to follow first order kinetics, thus ruling out the implication that maintenance of normal wall thickness was achieved by a random distribution of new wall components in the old wall. Instead, wall turnover in all cases strictly followed zero order kinetics, indicating that newly synthesized wall material was placed layer by layer beneath the inner surface of the old cell wall. This finding correlates with evidence obtained from earlier electron microscopic investigations into the regeneration of the staphylococcal cell wall after chloramphenicol treatment. Based on the experimental data presented, a simplified model for wall turnover of the growing staphylococcal cell was proposed. The model also takes into account the finding, derived from additional experiments with strainSG 511, that the total cell wall turned over at a somewhat higher rate than the old portions of the wall. The rates of cell wall turnover found inS. aureus SG 511 are the highest reported to date for pathogenic bacteria. The medical implications of this finding were discussed.     

Survey and properties of Staphylococcus aureus isolated from Japanese-style desserts

We surveyed the contamination of 315 Japanese- and western-style desserts and 247 human hands by Staphylococcus aureus and other staphylococcal bacteria. The most frequently isolated staphylococcal bacterium was S. warneri, followed by S. aureus. Only 1.9% of western-style desserts were contaminated by S. aureus strains, while 19.4% and 13.0% of Japanese-style desserts and human hands respectively were contaminated. Ninety-four isolates of S. aureus were characterized as to their biological properties and enterotoxigenicity. Although staphylococcal enterotoxins (SEs) were detected by enzyme-linked immunosorbent assay in the cultured broth of all S. aureus isolates, the reversed passive latex agglutination method and the polymerase chain reaction showed only 39 (41.5%) and 40 (42.6%) samples respectively as SE-positive. The predominant type of SE was SEB (67.5%), and eight strains produced SEA. None of the S. aureus strains had penicillin-binding protein 2', showing that methicillin-resistant S. aureus was not present in the samples.     

Target cell specificity of a bacteriocin molecule: a C-terminal signal directs lysostaphin to the cell wall of Staphylococcus aureus.

Microbial organisms secrete antibiotics that cause the selective destruction of specific target cells. Although the mode of action is known for many antibiotics, the mechanisms by which these molecules are directed specifically to their target cells hitherto have not been described. Staphylococcus simulans secretes lysostaphin, a bacteriolytic enzyme that cleaves staphylococcal peptidoglycans in general but that is directed specifically to Staphylococcus aureus target cells. The sequence element sufficient for the binding of the bacteriocin as well as of hybrid indicator proteins to the cell wall of S.aureus consisted of 92 C-terminal lysostaphin residues. Targeting to the cell wall of S.aureus occurred either when the hybrid indicator molecules were added externally to the bacteria or when they were synthesized and exported from their cytoplasm by an N-terminal leader peptide. A lysostaphin molecule lacking the C-terminal targeting signal was enzymatically active but had lost its ability to distinguish between S.aureus and S.simulans cells, indicating that this domain functions to confer target cell specificity to the bacteriolytic molecule.     

The characteristics of Staphylococcus aureus peptidoglycan in the spectrum of the reactivity of human lymphocytes to bacterial peptidoglycans]

The sensitivity of lymphocytes of healthy persons to S. aureus peptidoglycan as compared with that to the polyclonal stimulator zymosan C3b and peptidoglycans of other bacteria (Streptococcus faecalis, Escherichia coli, Bacterium bifidum) was analyzed with a test system permitting the determination of specific reactivity to peptidoglycans. The analysis showed that at the peak of luminol-dependent chemiluminescence (25-30 minutes) individual reactivity to S. aureus peptidoglycan varied within wide limits (the coefficient of lymphocyte stimulation was 1.4-9.6, 3.5 +/- 0.6), exceeding sensitivity to other bacteria, as well as the values obtained in the negative control. The conclusion of the wide spread of sensitization to S. aureus peptidoglycan and the possibility of using this preparation for the study of cell-mediated immunity reactions was made.     

Mitogenic activity of the antigens isolated from different Staphylococcus aureus strains in relation to mouse and guinea pig splenocytes

The mitogenic effect of corpuscular antigens with respect to the splenocytes of animals was found to depend on the strain of Staphylococcus aureus. The maximum synthesis of DNA in the cells was induced by corpuscular antigen Smith and the minimum synthesis, by Wood-46. The synthesis of DNA was activated in both B- and T-splenocytes in response to corpuscular antigens Wood-46, Cowan-1 and Smith, as well as to the cell wall and protein A. Peptidoglycan produced a mitogenic effect only in B-lymphocytes, and teichoic acid showed no mitogenic activity in mouse splenocytes. The mitogenic effect of staphylococcal antigens on splenocytes depended on the dose of the antigen and the time of cultivation. After 48-hour cultivation the incorporation of 3H-thymidine into the DNA of mouse cells was 5 times higher than into the DNA of guinea-pig cells. The optimum mitogenic dose in thymectomized BALB/c mice with respect to splenocytes was higher than in normal BALB/c mice practically by one order.     

Evaluation and Standardization of an Agglutination Test for Human Listeriosis

Human sera from patients with culturally confirmed listeriosis were tested for immunoglobulin M (IgM) and immunoglobulin G (IgG) agglutinating antibodies with trypsinized antigens of Listeria monocytogenes, Streptococcus faecalis, and Staphylococcus aureus. The response of humans to listeria infections is mainly IgM rather than IgG as found in animals. The antigens prepared from L. monocytogenes serotypes 1a, 1b, 2, 4b, and 4d were evaluated for specificity with normal sera, sera from patients with various other diseases, and sera from patients with listeriosis. The trypsinized antigens appeared to be specific for listeria antibodies with a cross-reaction rate of from 5.4 to 6%. Cross-reaction with S. aureus can be eliminated by absorption of the serum with S. aureus. This agglutination technique appears to be applicable for diagnostic testing, but, as with all serological procedures, both acute and convalescent sera should be tested.     

Bacteriophage conversion as a factor modifying the intensity of phagocytosis of Staphylococcus aureus by human leukocytes

Staphylococcus aureus NCTC 8325-4 and its eight variants lysogenized with phages responsible for the synthesis of staphylococcal staphylokinase were used for the study. Influence of phage conversion of S. aureus on its interaction with human leucocytes and influence of prophage on strain susceptibility to intracellular killing by human granulocytes without opsonins were evaluated. It was found that lysogenization of the strain with the bacteriophages decreased in each case reactivity of human leucocytes for staphylococcal strain what was expressed by lower bioluminescence values and by lower percentage of intracellular killing of bacterial cells carrying prophage.     

Intracellular metalloporphyrin metabolism in Staphylococcus aureus

The bacterial pathogen Staphylococcus aureus is responsible for a significant amount of human morbidity and mortality, and the ability of S. aureus to cause disease is absolutely dependent on the acquisition of iron from the host. The most abundant iron source to invading staphylococci is in the form of the porphyrin heme. S. aureus is capable of acquiring nutrient iron from heme and hemoproteins via two heme-acquisition systems, the iron-regulated surface determinant system (Isd) and the heme transport system (Hts). Heme acquisition through these systems is involved in staphylococcal pathogenesis suggesting that the intracellular fate of heme plays a significant role in the infectious process. The valuable heme molecule presents a paradox to invading bacteria because although heme is an abundant source of nutrient iron, the extreme reactivity of heme makes it toxic at high concentrations. Therefore, bacteria must regulate the levels of intracellular heme to avoid toxicity. Although the molecular mechanisms responsible for staphylococcal heme acquisition are beginning to emerge, the mechanisms by which S. aureus regulate intracellular heme homeostasis are largely unknown. In this review we describe three potential fates of host-derived heme acquired by S. aureus during infection: (i) degradation for use as a nutrient iron source, (ii) incorporation into bacterial heme-binding proteins for use as an enzyme cofactor, or (iii) efflux through a dedicated ABC-type transport system. We hypothesize that the ultimate fate of exogenously acquired heme in S. aureus is dependent upon the intracellular and extracellular availability of both iron and heme.     

Enhanced staphylolytic activity of the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88 HydH5 virion-associated peptidoglycan hydrolase: fusions, deletions, and synergy with LysH5

Virion-associated peptidoglycan hydrolases have potential as antimicrobial agents due to their ability to lyse Gram-positive bacteria on contact. In this work, our aim was to improve the lytic activity of HydH5, a virion-associated peptidoglycan hydrolase from the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88. Full-length HydH5 and two truncated derivatives containing only the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain exhibited high lytic activity against live S. aureus cells. In addition, three different fusion proteins were created between lysostaphin and HydH5, each of which showed higher staphylolytic activity than the parental enzyme or its deletion construct. Both parental and fusion proteins lysed S. aureus cells in zymograms and plate lysis and turbidity reduction assays. In plate lysis assays, HydH5 and its derivative fusions lysed bovine and human S. aureus strains, the methicillin-resistant S. aureus (MRSA) strain N315, and human Staphylococcus epidermidis strains. Several nonstaphylococcal bacteria were not affected. HydH5 and its derivative fusion proteins displayed antimicrobial synergy with the endolysin LysH5 in vitro, suggesting that the two enzymes have distinct cut sites and, thus, may be more efficient in combination for the elimination of staphylococcal infections.     

The effect of immunotherapy with cell-free vaccines made from the antigens of opportunistic microorganisms on the dynamics of DNA antibody formation]

The sera of patients subjected to immunotherapy with staphylococcal vaccine and with multicomponent vaccine (i.e. the mixture of the antigenic preparations of Staphylococcus aureus, Klebsiella pneumoniae, Proteus vulgaris, Escherichia coli) were studied by the method of the enzyme immunoassay on the basis of cattle spleen DNA. Immunotherapy with staphylococcal vaccine was given to patients with dermal diseases, chronic obstructive bronchitis and pulmonary abscess. Multicomponent vaccine was introduced to patients with the infectious allergic form of bronchial asthma, moderate or severe. Immunotherapy with both preparations under study was shown to produce no accumulation of antibodies to native and denatured DNA.