Non-specific immunoprecipitation of rotavirus Vp6 protein with Staphylococcus aureus

The specificity of Staphylococcus aureus and protein A-Sepharose (PA-S) were compared in the radioimmunoprecipitation assay for the characterization of monoclonal antibodies (mAbs) against rotavirus proteins. Five mAbs directed against bovine rotavirus Q17 proteins Vp6 and Vp7 and one mAb directed against human rotavirus protein Vp4 were used in this study. mAbs directed against other viruses, NS-1 culture supernatant and ascitic fluid, were used as control reagents. A non-specific immunoprecipitation of the viral protein Vp6 was always found with S. aureus, but not with PA-S. mAb 74 reacted with rotavirus antigens in ELISA and in indirect immunofluorescence assay but did not immunoprecipitate a viral protein with PA-S. This mAb immunoprecipitated the viral protein Vp6 when S. aureus reagent was used. This false positive reaction was always present and could lead to confusing results in the analysis and characterization of mAbs against rotavirus.     

Triton X-100 alters the resistance level of methicillin-resistant Staphylococcus aureus to oxacillin

Abstract We used population analysis to examine the effects of Triton X-100 on the level of resistance to oxacillin of 18 methicillin-resistant Staphylococcus aureus . In the presence of 0.02% Triton X-100, 17 formerly methicillin-resistiant strains exhibited enhanced sensitivity to oxacillin. One homogeneous isolate, KSAF1 was barely affected by the Triton X-100. Sensitivities of lysostaphin, 51 kDa N -acetylglucosaminidase and 62 kDa N -acetylmuramoyl-l-alanine amidase to heat-inactivated cells were not affected when the bacteria were grown in 0.02% Triton X-100. Our data, together with those of a previous study, suggested that Triton X-100 alters the resistance level of methicillin-resistant S. aureus by influencing a factor(s) other than PBPs, bacteriolytic enzymes, or femAB products.     

Successful selection of an infection‐protective anti‐Staphylococcus aureus monoclonal antibody and its protective activity in murine infection models

Recent clinical trials to develop anti‐methicillin‐resistant Staphylococcus aureus (MRSA) therapeutic antibodies have met unsuccessful sequels. To develop more effective antibodies against MRSA infection, a panel of mAbs against S. aureus cell wall was generated and then screened for the most protective mAb in mouse infection models. Twenty‐two anti‐S. aureus IgG mAbs were obtained from mice that had been immunized with alkali‐processed, deacetylated cell walls of S. aureus. One of these mAbs, ZBIA5H, exhibited life‐saving effects in mouse models of sepsis caused by community‐acquired MRSA strain MW2 and vancomycin‐resistant S. aureus strain VRS1. It also had a curative effect in a MW2‐caused pneumonia model. Curiously, the target of ZBIA5H was considered to be a conformational epitope of either the 1,4‐β‐linkage between N‐acetylmuramic acid and N‐acetyl‐D‐glucosamine or the peptidoglycan per se. Reactivity of ZBIA5H to S. aureus whole cells or purified peptidoglycan was weaker than that of most of the other mAbs generated in this study. However, the latter mAbs did not have the protective activities against S. aureus that ZBIA5H did. These data indicate that the epitopes that trigger production of high‐yield and/or high‐affinity antibodies may not be the most suitable epitopes for developing anti‐infective antibodies. ZBIA5H or its humanized form may find a future clinical application, and its target epitope may be used for the production of vaccines against S. aureus infection.     

A human transferrin-binding protein of Staphylococcus aureus is immunogenic in vivo and has an epitope in common with human transferrin receptor

To understand human immune responses against the human transferrin-binding protein of Staphylococcus aureus (SA-tbp), we examined cell wall proteins from S. aureus ATCC 6538 using human convalescent sera, and a monoclonal antibody specific for human transferrin receptor (McAb-HTR). The SA-tbp, detected by immunoblot assay, was iron-repressible, reacted with the convalescent sera, and cross-reacted with McAb-HTR. Immunoelectron microscopy probed with McAb-HTR showed a reaction zone around the test strain from the deferrated BHI. After being preincubated with an S. aureus -bacteremic serum, the electroblot of the SA-tbp still reacted with McAb-HTR, but not with human transferrin-horseradish peroxidase conjugate. We conclude, there are at least two kinds of epitopes in the SA-tbp; one able to bind to human transferrin is immunogenic in humans, but the other sharing epitopes common with human transferrin receptor is not immunogenic in humans.     

Characterization and comparative bactericidal activity of monoclonal antibodies to Bordetella pertussis lipo-oligosaccharide A.

Spleen cells from mice immunized with a Bordetella pertussis N-lauroyl sarcosine membrane extract (SME) were used to generate hybridoma cells lines producing monoclonal antibodies (mAbs). Seven mAbs were shown to be specific to B. pertussis lipo-oligosaccharide (LOS) by immunoblotting of SME or purified LOS following SDS-PAGE. All mAbs reacted with the B. pertussis Tohama I strain of the LOS AB phenotype, and did not react with the atypical variant strain 134 of the LOS B phenotype. The immune reactivity of the mAbs was retained after treatment of SME with proteinase K and was lost after sodium periodate treatment. No cross-reactivity was observed with the mAbs when tested against B. parapertussis and other Gram-negative bacteria. However, all mAbs reacted with B. bronchiseptica. Binding assays with live B. pertussis cells demonstrated that mAbs strongly reacted with cell surface exposed antigenic determinants. High bacterial cell lytic capability was observed for five of these mAbs. Concentrations between 0.22 and 2.2 micrograms mAb ml-1 (0.1 and 1 microgram per 450 microliter assay) purified by protein A were required to kill at least 50% of the bacteria. Competition immunoassays with biotinylated antibodies showed that the bacteriolytic and non-bacteriolytic mAbs were directed to different epitopes of the B. pertussis LOS A.     

Suppression of penicillin-induced lysis of Staphylococcus aureus by cibacron blue 3G-A

The effect of cibacron blue 3G-A (CB) on bacteriolysis induced by penicillin G was investigated using Staphylococcus aureus FDA 209P. Penicillin-induced lysis was completely inhibited by 30 microM CB. However, the bactericidal effect of penicillin G was not influenced by CB. These results indicate that a bacteriolytic process is not essential for penicillin to kill S. aureus.     

Effect of combined physico-chemical preservatives on enterocin AS-48 activity against the enterotoxigenic Staphylococcus aureus CECT 976 strain

AIMS: Control of the enterotoxigenic Staphylococcus aureus CECT 976 strain by enterocin AS-48 in laboratory cultures, and behaviour of the AS-48 activity in the presence of food preservatives. METHODS AND RESULTS: Enterocin AS-48 shows inhibitory activity on the majority of the Staphylococcus species tested. This enterocin has a bactericidal and bacteriolytic mode of action on S. aureus CECT 976, a strain selected for this study by its enterotoxigenic character (SEA production). The inhibitory effect of AS-48 was pH and temperature dependent, and enterocin activity was higher at pH 5. The minimum bactericidal concentration (MBC) of AS-48, decreased from 15 microg ml(-1) at 37 degrees C to 10 microg ml(-1) at 15 degrees C. Sublethally injured cells showed an increased sensitivity with a MBC of 5 microg ml(-1). In this way, the highest effectiveness of Ent AS-48 against S. aureus CECT 976 was obtained at 4 degrees C in combination with high concentrations of NaCl (6 and 7%). Interestingly, enterotoxin SEA production by strain CECT 976 was markedly inhibited by subinhibitory concentrations of Ent AS-48. These low concentrations also provoked a delay of bacterial growth. CONCLUSION: The results presented indicated that Ent AS-48 has a potential for application as a protective agent against S. aureus in foods. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we have established the conditions for an efficient inhibition of growth and enterotoxin production by S. aureus CECT 976 in culture media by a combination of environmental factors and Ent AS-48.     

金黄葡萄球菌fnbB基因的克隆及在大肠杆菌中的表达

金黄色葡萄球菌（Staphylococcus aureus）是引起奶牛乳房炎主要致病菌之一,主要通过其菌体表面的黏附素侵入寄主细胞引起疾病,为奶牛业造成巨大损失。金黄色葡萄球菌表面蛋白纤连蛋白结合蛋白(fibronectin-binding protein,FnBP)是其关键的黏附因子,在研制抗金黄色葡萄球菌的新型疫苗中占有重要地位.本文根据GenBank中纤连蛋白结合蛋白B基因（fnbB）序列设计特异性引物,以金黄葡萄球菌基因组DNA为模板,进行PCR扩增,获得3 458 bp 的DNA片段。使用T-A克隆技术,将PCR产物克隆至pGEM T easy Vector中,成功构建出了克隆质粒pGEM-fnbB。以 BamHI和XhoI 双酶切pGEM-fnbB和pET28a(+),并将纯化的基因fnbB 亚克隆至pET28a(+)中,构建出原核表达质粒pET28a-fnbB,并将其转化至E.coli BL21(DE3)感受态细胞中,经1 mmol/L的IPTG诱导和SDS-PAGE分析,在约165 ku 处出现了与预期目的蛋白相一致的外源蛋白带,Western blot分析结果表明该蛋白具有金黄葡萄球菌的抗原性。金黄葡萄球菌pET28a-fnbB成功表达为金黄葡萄球菌引起的奶牛乳房炎的诊断和研究新型疫苗奠定基础。     

TRAIL expression is induced in both osteoblasts containing intracellular Staphylococcus aureus and uninfected osteoblasts in infected cultures

Staphylococcus aureus is the principal etiological agent of osteomyelitis (bone infection), which is characterized by a progressive inflammatory response resulting in extensive damage to bone tissue. Recent studies have demonstrated the ability of S. aureus to invade and persist inside osteoblasts (bone matrix-forming cells) and other eukaryotic cells. The presence of intracellular S. aureus in bone tissue may be relevant to the pathology of osteomyelitis, a disease often refractory to antibiotic treatment and subject to recurrence months and even years after apparently successful therapy. The present study examined the production of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) following S. aureus infection, and whether expression of the molecule was induced by those osteoblasts containing intracellular S. aureus. Results from this study suggest that osteoblasts containing intracellular S. aureus induce TRAIL expression in uninfected osteoblasts present in infected cultures.     

Inhibitory antibodies to human angiotensin-converting enzyme: fine epitope mapping and mechanism of action

Angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, consists of two homologous domains (N and C), each bearing a Zn-dependent active site. We modeled the 3D-structure of the ACE N-domain using known structures of the C-domain of human ACE and the ACE homologue, ACE2, as templates. Two monoclonal antibodies (mAb), 3A5 and i2H5, developed against the human N-domain of ACE, demonstrated anticatalytic activity. N-domain modeling and mutagenesis of 21 amino acid residues allowed us to define the epitopes for these mAbs. Their epitopes partially overlap: amino acid residues K407, E403, Y521, E522, G523, P524, D529 are present in both epitopes. Mutation of 4 amino acid residues within the 3A5 epitope, N203E, R550A, D558L, and K557Q, increased the apparent binding of mAb 3A5 with the mutated N-domain 3-fold in plate precipitation assay, but abolished the inhibitory potency of this mAb. Moreover, mutation D558L dramatically decreased 3A5-induced ACE shedding from the surface of CHO cells expressing human somatic ACE. The inhibition of N-domain activity by mAbs 3A5 and i2H5 obeys similar kinetics. Both mAbs can bind to the free enzyme and enzyme-substrate complex, forming E.mAb and E.S.mAb complexes, respectively; however, only complex E.S can form a product. Kinetic analysis indicates that both mAbs bind better with the ACE N-domain in the presence of a substrate, which, in turn, implies that binding of a substrate causes conformational adjustments in the N-domain structure. Independent experiments with ELISA demonstrated better binding of mAbs 3A5 and i2H5 in the presence of the inhibitor lisinopril as well. This effect can be attributed to better binding of both mAbs with the "closed" conformation of ACE, therefore, disturbing the hinge-bending movement of the enzyme, which is necessary for catalysis.     

产SEB金黄色葡萄球菌α-溶血毒素基因缺失菌株的构建

构建产肠毒素B(Staphylococcal enterotoxin B ,SEB)的金黄色葡萄球菌α-溶血毒素（α-hemolysin, α-HL)缺失菌株。首先构建用于α-HL基因敲除的同源重组质粒pMHL-α,经金黄色葡萄球菌RN4220修饰后再通过原生质体转入金黄色葡萄球菌SM-01。含重组质粒pMHL-α的金黄色葡萄球菌SM-01在42℃诱导条件下培养多代,最终筛选出α-溶血毒素基因缺失菌株。经序列分析和血平板溶血实验结果证明最终获得产SEB金黄色葡萄球菌α-HL缺失菌株。为野生型金黄色葡萄球菌的体内遗传操作及构建产超抗原药物金黄色葡萄球菌基因工程菌株提供了一定的理论基础和方法。     

Production of Monoclonal Antibodies to Barley Mild Mosaic Virus and Their Use for Strain Differentiation

A panel of five stable hybridoma cell lines secreting mono- clonal antibodies (mAbs) were produced using a French mechanically transmitted isolate of barley mild mosaic virus (BaMMV-MF) as antigen. All mAbs reacted with BaMMV-MF in two enzyme-linked immunosorbent assay (ELISA) formats: triple antibody sandwich (TAS)-ELISA and antigen-coated plate (ACP)-ELISA. These mAbs recognized epitopes, present on both degraded virions and intact particles. Four mAbs (5C8, 1D5, 1B12, 1A12) belong to the immunoglobulin (Ig)G class and one mAb (3A9) represents an IgM. The five mAbs were compared in TAS- and ACP-ELISA for reactivity with numerous French isolates. These isolates were detected in TAS- and ACP-ELISA with four mAbs (5C8, 1D5, 1B12, 3A9). In both ELISA systems the mAb 1A12 recognized only an epitope specific for BaMMV-MF. All mAbs, except 1A12 recognized also the German (BaMMV-MG), Italian (BaMMV-I) and Japanese (BaMMV-Ka1) isolates in both TAS- and ACP-ELISA. The Japanese isolate (BaMMV-Na1) only reacted with two mAbs (1D5, 5C8) in TAS-ELISA. Only one mAb (3A9) reacted with BaMMV-MF, BaMMV-PF, BaMMV-I,BaMMV-MG and BaMMV-Ka1 in Western blot. These mAbs make it possible to distingish between the three BaMMV serotypes.     

Fluorescence correlation spectroscopy as a sensitive and useful tool for revealing potential overlaps between the epitopes of monoclonal antibodies on viral particles

Although the enzyme-linked immunosorbent assay (ELISA) is well established for quantitating epitopes on inactivated virions used as vaccines, it is less suited for detecting potential overlaps between the epitopes recognized by different antibodies raised against the virions. We used fluorescent correlation spectroscopy (FCS) to detect the potential overlaps between 3 monoclonal antibodies (mAbs 4B7-1H8-2E10, 1E3-3G4, 4H8-3A12-2D3) selected for their ability to specifically recognize poliovirus type 3. Competition of the Alexa488-labeled mAbs with non-labeled mAbs revealed that mAbs 4B7-1H8-2E10 and 4H8-3A12-2D3 compete strongly for their binding sites on the virions, suggesting an important overlap of their epitopes. This was confirmed by the cryo-electron microscopy (cryo EM) structure of the poliovirus type 3 complexed with the corresponding antigen-binding fragments (Fabs) of the mAbs, which revealed that Fabs 4B7-1H8-2E10 and 4H8-3A12-2D3 epitopes share common amino acids. In contrast, a less efficient competition between mAb 1E3-3G4 and mAb 4H8-3A12-2D3 was observed by FCS, and there was no competition between mAbs 1E3-3G4 and 4B7-1H8-2E10. The Fab 1E3-3G4 epitope was found by cryoEM to be close to but distinct from the epitopes of both Fabs 4H8-3A12-2D3 and 4B7-1H8-2E10. Therefore, the FCS data additionally suggest that mAbs 4H8-3A12-2D3 and 4B7-1H8-2E10 bind in a different orientation to their epitopes, so that only the former sterically clashes with the mAb 1E3-3G4 bound to its epitope. Our results demonstrate that FCS can be a highly sensitive and useful tool for assessing the potential overlap of mAbs on viral particles.     

Specific association of megalin and the Na+/H+ exchanger isoform NHE3 in the proximal tubule.

We investigated whether the renal brush border Na+/H+ exchanger NHE3 exists in assemblies with other proteins in native kidney membranes. To this end we generated monoclonal antibodies (mAbs) against affinity purified NHE3 protein complexes. Hybridomas were selected based on ability to immunoprecipitate NHE3. One of the resulting mAbs (10A3) labeled a high molecular mass (>200 kDa) protein and stained primarily the coated pit region of the proximal tubule in a manner similar to that described for megalin (gp330). We then confirmed that both mAb 10A3 and a known anti-megalin mAb immunoprecipitated and immunoblotted the same protein, namely megalin. mAb 10A3 specifically co-precipitated NHE3 but not villin or NaPi-2 from solubilized renal membranes, indicating specificity of the NHE3-megalin interaction. When immunoprecipitations were performed using either 10A3 or anti-NHE3 mAb 2B9 after separation of solubilized renal proteins by sucrose velocity gradient centrifugation, we found that NHE3 exists in two states with distinct sedimentation coefficients, a 9.6 S megalin-free form and a 21 S megalin-bound form, and that when NHE3 assembles with megalin, epitopes within the carboxyl-terminal 131 amino acids of NHE3 are blocked. Taken together, these findings indicate that a significant pool of NHE3 exists as a multimeric complex with megalin in the brush border of the proximal tubule.     

Alteration of Bacteriolytic Enzyme Profile of Staphylococcus aureus during Growth

Profiles of cell-associated bacteriolytic activities and those in the culture supernatant of Staphylococcus aureus FDA209P at various stages of growth were analyzed using sodium dodecyl sulfate-polyacrylamide gels containing Micrococcus luteus or S. aureus. In the logarithmic growth phase, the cell-associated bacteriolytic activities extracted with Triton X-100 contained a number of bacteriolytic proteins, the profiles of which were similar to those we reported elsewhere (Sugai, M., Akiyama, T., Komatsuzawa, H., Miyake, Y., and Suginaka, H.(1990) J. Bacteriol., 172, 6494-6498). The proteins include P1, P2, P7, P9, PX, P13, P18 and other minor components. At the stationary growth phase, the bacteriolytic band-profile of the Triton X-100 extract changed dramatically. P1, P7 and P9 disappeared, and the other minor bands had markedly decreased band intensities. On the other hand, P2, PX, P13, and P18 retained their band intensities during the stationary growth phase. The band intensities of P7, P13, PX, and P18 increased in the supernatant during the logarithmic growth phase. These results indicated that the bacteriolytic band-profile changes during growth.     

乳杆菌抑制金黄色葡萄球菌对HeLa细胞的粘附

对弯曲乳杆菌Lactobacillus crispatus T79-3和T90-1、詹氏乳杆菌Lactobacillus jensenii T118-3和T231-1四株乳杆菌对金黄色葡萄球菌生长的抑制效果以及抑菌成分进行了分析,比较乳杆菌排除、竞争、置换3种不同作用方式对金黄色葡萄球菌粘附HeLa细胞的抑制作用。结果表明4株乳杆菌皆能抑制金黄色葡萄球菌的生长及其粘附HeLa细胞的能力,分析发现4株乳杆菌发挥抑制作用的主要成分是有机酸,同时比较分析乳杆菌3种不同作用方式发现它们对金黄色葡萄球菌粘附HeLa细胞的抑制效果不同,其中,排除作用方式效果最好。另外,乳杆菌对金黄色葡萄球菌粘附HeLa细胞的抑制作用具有浓度依赖性,随着乳杆菌浓度增大,抑制作用增强并逐渐达到饱和。4株乳杆菌中,T79-3粘附能力最强,对金黄色葡萄球菌的抑制作用最强,排除作用方式抑制金黄色葡萄球菌粘附HeLa细胞作用效果较好,提示乳杆菌T79-3有可能作为益生菌防治妇女泌尿生殖道感染。     

Monoclonal antibodies to CNA, a collagen-binding microbial surface component recognizing adhesive matrix molecules, detach Staphylococcus aureus from a collagen substrate

Previous studies showed that Staphylococcus aureus expresses a collagen-binding MSCRAMM (Microbial Surface Component Recognizing Adhesive Matrix Molecules), CNA, that is necessary and sufficient for S. aureus cells to adhere to cartilage and is a virulence factor in experimental septic arthritis. We have now used a monoclonal antibody (mAb) approach to further analyze the structure and function of CNA. 22 mAbs raised against the minimal ligand binding domain, CNA-(151-318), were shown to bind to the MSCRAMM with similar affinity. All mAbs appear to recognize conformation-dependent epitopes that were mapped throughout the CNA-(151-318) domain using a chimeric strategy where segments of CNA are grafted on ACE, a structurally related MSCRAMM from Enterococcus faecalis. These mAbs were able to inhibit (125)I-collagen binding to CNA-(151-318) as well as to intact S. aureus cells. They also interfered with the attachment of bacteria to collagen substrates. Furthermore, some of the mAbs could effectively displace (125)I-collagen bound to the bacteria. These displacing mAbs were also able to detach bacteria that had adhered to a collagen substrate in a preincubation, raising the possibility that some of the mAbs may be used as therapeutic agents.     

Peptides of human thyroglobulin reactive with sera of patients with autoimmune thyroid disease

Autoantibodies to thyroglobulin (Tg) are a prominent feature of the two autoimmune thyroid diseases, chronic lymphocytic (Hashimoto's) thyroiditis and Graves' disease. Similar autoantibodies are found in the serum of many normal individuals without evidence of thyroid disease. Previous studies have indicated that patients with autoimmune thyroid disease recognize epitopes of Tg which are not usually recognized by normal individuals. The goal of this investigation was to identify peptide fragments of Tg bearing these disease-associated epitopes. For this purpose, we utilized a panel of mAbs that bind to different epitopes of the Tg molecule. One of these mAbs (137C1) reacted with an epitope that was also recognized by the sera of patients with autoimmune thyroiditis. In the present study, we show that two peptides (15 and 23 kDa) that reacted with mAb 137C1 are located in different parts of the Tg molecule. Each peptide inhibited the binding of mAb 137C1 to the other peptide and to the intact Tg, indicating that the same epitope was represented on the two peptides. Loops and helices of the secondary structure of the two peptides might be involved in the conformational epitope recognized by mAb 137C1. A striking finding of this study is that two apparently unrelated fragments of the Tg molecule bind to the same mAb. These findings may have important ramifications with regard to epitope spread and the progression of the autoimmune response to disease.