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.     

Transferrin binding in Staphylococcus aureus: involvement of a cell wall-anchored protein

The ability to gain access to iron is pivotal for bacterial pathogens during infection. Although much is known about iron acquisition systems in Gram-negative bacteria, comparatively little is known about how Gram-positive pathogens access iron from host iron sources. A previous study showed that, in the Gram-positive human pathogen Staphylococcus aureus, a cell surface-associated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzyme (Gap, or Tpn) is capable of binding human transferrin, representing a potential means by which this bacterium is able to access iron in vivo. We have investigated this property of S. aureus further and shown that, in S. aureus RN6390, GAPDH is expressed on the S. aureus cell surface independent of exogenous iron concentrations, and that overexpressed and purified Gap, although retaining GAPDH activity, has no affinity for human transferrin. Moreover, although a S. aureus gap mutant was devoid of surface-associated and cytoplasmic GAPDH activity, it retained the ability to bind human transferrin, equivalent to wild type. We concluded from these results that the Gap protein is not involved in S. aureus binding to human transferrin. We identified the transferrin-binding protein as a novel cell wall-anchored protein, designated StbA for staphylococcal transferrin-binding protein A, which shared no significant similarities with any other bacterial transferrin-binding proteins. StbA contained a C-terminal cell wall-anchoring motif (LPKTG), and expression of StbA in the cell wall was strictly controlled by exogenous iron concentrations. The stbA gene is found within a 7 kb region in the S. aureus chromosome that contains a total of six iron-regulated genes. Immediately downstream from stbA is an iron-regulated gene whose product was predicted to be another cell wall-anchored protein with no significant similarity to proteins with characterized functions. Transcribed in the opposite direction from stbA is a four-gene operon whose expression is also regulated by iron. While the deduced products of the first two genes lack similarity to known proteins, the last two genes encode, respectively, putative lipoprotein and permease components of an ABC transporter that shares significant similarities with several iron(III) ABC transporters in a variety of bacteria.     

沙门菌CWDMs乳酸脱氢酶同功酶的观察

采用聚丙烯酰胺凝胶电泳法检测伤寒杆菌和甲型副伤寒杆菌的ＣＷＤＭｓ及其宁代细菌型和伤寒杆菌粗糙型的乳酸脱氢酶（ＬＤＨ）同功酶,以了解沙门菌ＣＷＤＭｓ生物氧化的特点和机制,探讨ＣＷＤＭｓ变异的性质。结果表明,伤寒杆菌和甲型副伤寒杆菌的细菌型及伤寒杆粗糙型在聚丙烯酰胺凝胶电泳后显示出相同的４种具有不同泳动速率的ＬＤＨ同功酶,但ＣＷＤＭｓ仅显示２种ＬＤＨ。ＣＷＤＭｓ的２种ＬＤＨ同功酶与其亲代细菌型及伤寒杆     

Proteomics analysis of the causative agent of typhoid fever

Typhoid fever is a potentially fatal disease caused by the bacterial pathogen Salmonella enterica serotype Typhi ( S. typhi). S. typhi infection is a complex process that involves numerous bacterially encoded virulence determinants, and these are thought to confer both stringent human host specificity and a high mortality rate. In the present study, we used a liquid chromatography-mass spectrometry (LC-MS)-based proteomics strategy to investigate the proteome of logarithmic, stationary phase, and low pH/low magnesium (MgM) S. typhi cultures. This represents the first large-scale comprehensive characterization of the S. typhi proteome. Our analysis identified a total of 2066 S. typhi proteins. In an effort to identify putative S. typhi-specific virulence factors, we then compared our S. typhi results to those obtained in a previously published study of the S. typhimurium proteome under similar conditions ( Adkins, J. N. et al. Mol. Cell. Proteomics 2006, 5, 1450-1461 ). Comparative proteomics analysis of S. typhi strain Ty2 and S. typhimurium strain LT2 revealed a subset of highly expressed proteins unique to S. typhi that were exclusively detected under conditions that are thought to mimic the infective state in macrophage cells. These proteins included CdtB, HlyE, and gene products of t0142, t1108, t1109, t1476, and t1602. The differential expression of T1108, T1476, and HlyE was confirmed by Western blot analysis. When our observations are taken together with the current literature, they suggest that this subset of proteins may play a role in S. typhi pathogenesis and human host specificity.     

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 p97 antigen is mapped to the q24-qter region of chromosome 3; the same region as the transferrin receptor.

Since the p97 antigen, a membrane-associated iron-binding protein, has extensive amino acid sequence with homology with transferrin, is functionally related to the transferrin receptor, and has been previously mapped to chromosome 3, we have performed additional studies for regional mapping of the gene expressing p97 antigen. In these experiments, Chinese hamster-human cell lines were chosen that contained a large spectrum of autosomal human chromosomes, but mainly consisted of clones expressing all or a part of chromosome 3. These cell lines included a clone that previously allowed for mapping of human transferrin receptor to q22-qter region. Human p97 expression was assessed by specific binding of [125I]monoclonal antibody 96.5, and human transferrin receptor expression was tested by specific [125I]human transferrin binding and [125I]monoclonal antibody OKT-9 specific for human transferrin receptor. Based on these analyses, both human p97 antigenic expression and human transferrin receptor are mapped concordantly to the q24-qter region. These data and previous reports, therefore, suggest that the related iron-transport proteins are closely linked and may be under coordinate regulation. However, studies of several cell lines that exhibit up-regulation of human transferrin receptor expression with cellular proliferation, and down-regulation of receptor with increased transferrin-iron in the media, showed no change in expression of p97 antigen. p97 antigenic expression increased when melanocyte-stimulating hormone was added to a human melanoma cell line in tissue culture. These latter studies suggest that in mammalian cells the two proteins do not show coordinate regulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of a molecular complex between ras proteins and transferrin receptor

Immunoprecipitation of extracts of human carcinoma cell lines with three different monoclonal antibodies generated against ras proteins revealed the coprecipitation of a 90,000 dalton protein. The coprecipitated protein was identified as the transferrin receptor by comigration in both reducing and nonreducing SDS-polyacrylamide gels, by absorption with a monoclonal antibody directed against transferrin receptor, and by analysis of partial proteolysis products. Coprecipitation of the transferrin receptor with three monoclonal antibodies with differing specificities to ras proteins, as well as the inability to coprecipitate the transferrin receptor from cell extracts from which ras proteins were depleted by preabsorption, indicates that ras proteins and the transferrin receptor form a molecular complex. This complex is disrupted by addition of transferrin to cell extracts. These findings suggest that ras proteins function in regulation of cell growth via interaction with the cell surface receptor for transferrin.     

Characterization of the human transferrin receptor produced in a baculovirus expression system

Recombinant human transferrin receptor has been produced in a baculovirus expression system. Magnetic particles coated with an anti-transferrin receptor monoclonal antibody were used to immunoselect virus-infected Sf9 insect cells expressing the human transferrin receptor on their cell surface. Recombinant virus containing the human transferrin receptor cDNA was then plaque-purified from these cells. Biosynthetic labeling studies of infected cells showed that the human transferrin receptor is one of the major proteins made 2-3 days postinfection. The recombinant receptor made in insect cells is glycosylated and is also posttranslationally modified by the addition of a fatty acid moiety. However, studies with tunicamycin and endoglycosidases H and F showed that the oligosaccharides displayed on the recombinant receptor differ from those found on the naturally occurring receptor in human cells. As a consequence, the human receptor produced in the baculovirus system has an Mr of 82,000 and is smaller in size than the authentic receptor. About 30% of human transferrin receptors made in insect cells do not form intermolecular disulfide bonds, but are recognized by the anti-transferrin receptor antibody, B3/25, and bind specifically to a human transferrin-Sepharose column. Binding studies using 125I-labeled human transferrin showed that insect cells infected with the recombinant virus expressed an average of 5.8 +/- 0.9 X 10(5) transferrin receptors (Kd = 63 +/- 9 nM) on their cell surface. Thus, the human transferrin receptor produced in insect cells is biologically active and appears suitable for structural and functional studies.     

伤寒沙门菌野生型与粗糙型菌体蛋白质的双向电泳分析与研究

了解伤寒沙门菌野生型与粗糙型菌株的菌体蛋白质组成特点,探讨伤寒沙门菌粗糙型变异的遗传学基础。分离提取伤寒沙门菌野生型与粗糙型菌株的菌体蛋白质,聚丙烯酰胺凝胶双向电泳(2D-PAGE)和考马斯亮蓝染色,计算机分析与比较两菌株的蛋白质组成特点及其相关性。伤寒沙门菌野生型与粗糙型具有相似的蛋白质电泳图谱,相似系数为78%。多数蛋白质斑点分布于pH 3.0~6.4之间,并且分子量小于30kDa。两菌株的蛋白质电泳图谱之间的主要差异共计36处,多数差异蛋白质的分子量小于20kDa。伤寒沙门菌粗糙型与野生型菌体蛋白质组成的差异显示,粗糙型变异绝不仅仅是O抗原多糖的缺失,也可发生菌体蛋白质组成的改变与缺失。伤寒沙门菌粗糙型保留了同其亲代野生型菌株一致的绝大多数菌体蛋白质组成,在2D-PAGE中形成伤寒沙门菌特征性的基本蛋白质图谱,有助于对粗糙型菌株进行蛋白质分子同源性与变异性的分析与鉴别。     

Role of Genomic Rearrangements in Producing New Ribotypes of Salmonella typhi

Salmonella typhi is the only species of Salmonella which grows exclusively in humans, in whom it causes enteric typhoid fever. Strains of S. typhi show very little variation in electrophoretic types, restriction fragment length polymorphisms, cell envelope proteins, and intervening sequences, but the same strains are very heterogeneous for ribotypes which are detected with the restriction endonuclease PstI. In addition, the genome of S. typhi has been proven to undergo genomic rearrangement due to homologous recombination between the seven copies of rrn genes. The relationship between ribotype heterogeneity and genomic rearrangement was investigated. Strains of S. typhi which belong to 23 different genome types were analyzed by ribotyping. A limited number of ribotypes were found within the same genome type group; e. g., most strains of genome type 3 belonged to only two different ribotypes, which result from recombination between rrnH and rrnG operons. Different genome type groups normally have different ribotypes. The size and identity of the PstI fragment containing each of the seven different rrn operons from S. typhi Ty2 were determined, and from these data, one can infer how genomic rearrangement forms new ribotypes. It is postulated that genomic rearrangement, rather than mutation, is largely responsible for producing the ribotype heterogeneity in S. typhi.     

Salmonella typhi does not inhibit phagosome-lysosome fusion in human monocyte-derived macrophages

Abstract We examined phagosome-lysosome fusion in Salmonella typhi -infected human monocyte-derived macrophages and its relevance to the intracellular survival of this bacterium in vitro. S. typhi was found to survive and multiply in human monocyte-derived macrophages, whereas S. typhimurium was killed easily, indicating that the survival of Salmonella serovars is host-specific. Neither S. typhi nor S. typhimurium inhibited phagosome-lysosome fusion in human monocyte-derived macrophages. No difference between the phagosome-lysosome fusibilities of freshly prepared human monocytes and monocyte-derived macrophages was observed. These results suggest that S. typhi may survive by adapting to the conditions within fused phagolysosomes of human monocyte-derived macrophages.     

The primary immune response of brown trout (Salmo trutta) to injection with cellular antigens

The primary immune response of brown trout ( Salmo trutta L.) was studied after injections of two cellular antigens– Salmonella typhi H, flagellar antigen d, and human group 'O'Rh+ red blood cells. Both intraperitoneal and intramuscular injections were employed. Agglutinins and complement–fixing antibodies were produced to S. typhi and haem–agglutinins to human 'O' red blood cells. Maximum titres to S. typhi were reached after 49 days in the case of both agglutinins and complement-fixing antibody. Haem-agglutinins reached a maximum value of 1 : 512 between 35 and 42 days. Haemagglutinins to human 'O' red blood cells were detected as early as 7 days after injection. Antibodies against S. typhi were found after 14 days. Natural haemolysins were present against horse, sheep and human groups 'A', 'B' and 'AB' but not with group 'O'. No natural haemagglutinins were present to the six types of red blood cells tested. No precipitins were detected to either S. typhi or human 'O' red blood cells by immunodiffusion.     

Albumin inhibition of transferrin low-affinity binding to K562 cells

Several reports have suggested that variations of albumin concentration in the incubation medium can modulate the magnitude of transferrin binding to the cells. We have investigated this problem further using K562 cells. In the absence of human serum albumin, transferrin binding demonstrated a non-saturable curve which, upon Scatchard analysis, showed two components with high and low affinities. In the presence of 0.5% human serum albumin, the low-affinity but not the high-affinity component was totally inhibited and, thus, the binding showed a saturation plateau at transferrin concentration of 6 micrograms/ml. Increasing concentrations of human serum albumin in the incubation medium led to progressive inhibition of transferrin binding, reaching a plateau at 0.2% human serum albumin. At this concentration transferrin binding was about 12 ng/10(6) cells, corresponding to the saturation plateau for high-affinity binding. Low-affinity transferrin binding in the absence of human serum albumin could readily be displaced by subsequent addition of albumin. Similar inhibition was obtained by another serum protein, ceruloplasmin, suggesting that this inhibition is not unique to albumin and may be a common property of all proteins. Incubation at 37 degrees C with 59Fe-labeled transferrin indicated that all iron uptake occurs through high-affinity binding. We conclude that the reported variations in magnitude of transferrin binding by the cell due to variations in albumin concentration are the result of inhibition of low-affinity binding of transferrin by albumin.     

Polysaccharides and their common proteinic carrier in the Vi antigens of Citrobacter ballerup and Salmonella typhi Ty2

Immunochemical analysis of Citrobacter ballerup and Salmonella typhi Ty2 showed that the strains share native and heat-resistant proteins that are, apparently, the carriers of a common polysaccharidic determinant present in their respective somatic antigens. After the classic acetic acid hydrolysis, the somatic antigen of C. ballerup reacted, in agar gel, against the homologous antiserum by two precipitation lines, one of which also precipitated against the anti S, typhi Ty2 serum; the hydrolysis of the S. typhi Ty2 somatic antigen demonstrated that, in addition to the 'O' polysaccharide, reacting against all the S. typhi antisera, it contains a polysaccharide that precipitated against the anti-C. ballerup serum. The elusiveness in the agglutinability of only freshly isolated bacterial authorizes some doubt concerning the responsibility of the antipolysaccharide antibodies in the agglutinating Vi sera; in order to induce anitpolysaccharides hyperimmunizations are needed while antiproteins are easily induced by short immunizations.     

Newly synthesized proteins in seminiferous intertubular and intratubular compartments of the rat testis

Two-dimensional gel electrophoresis combined with autoradiography and Western blot procedures have been used to characterize newly synthesized proteins in testicular intertubular fluid (TIF) and seminiferous tubular fluid (SNF). Fluids were collected following in vivo and in vitro intratesticular injection of [35S]methionine into control and hypophysectomized adult rats. A discrete number of [35S]methionine-labeled proteins were detected within TIF and SNF. Their presence and relative abundance varied according to in vivo and in vitro labeling conditions. While two major blood plasma proteins, albumin and transferrin, were radioactively labeled after in vivo labeling, these two proteins were insignificantly labeled in samples collected after in vitro labeling. Three acidic proteins, possibly secreted by Sertoli cells (Mr = 72,000, 45,000 and 35,000), were more abundant in TIF samples collected after in vitro [35S]methionine labeling than after in vivo labeling. Incubated seminiferous tubules and TIF of hypophysectomized rats showed a decrease in [35S]methionine-labeling intensity of the Mr = 72,000 acidic protein, possibly reflecting changes in the seminiferous epithelium caused by pituitary hormonal deprivation. Autoradiographs of TIF and most remarkably, of SNF, showed many protein spots that suggested cell breakage and leakage during sample collection. Results of this study suggest that most albumin and transferrin found in TIF and SNF have an extratesticular origin and that proteins secreted by the Sertoli cell can gain access to both TIF and SNF.     

人转铁蛋白受体及其特异性抗体的制备

目的:从胎盘中提取转铁蛋白受体并获得抗转铁蛋白受体的抗体。方法:人新鲜胎盘组织被破碎后,用去污剂TritonX-100裂解细胞膜,释放膜蛋白。利用膜蛋白中的转铁蛋白受体能与铁-转铁蛋白复合物特异性结合的特性对其进行亲和纯化。对纯化得到的目的蛋白,经脱盐后进行ELISA及肽质量图谱分析,证明为所需的转铁蛋白受体后,以其包被免疫管,从全合成人源噬菌体抗体库中筛选抗体。结果:从人源噬菌体抗体库中筛选到5个能够与转铁蛋白受体特异性结合的噬菌体单链抗体。结论:以人源转铁蛋白受体为抗体,可从全人源噬菌体抗体库中筛选到其特异性的抗体。     

伤寒沙门氏菌SOD的提取及抗原性研究

经硫酸铵分级沉淀,ＤＥＡＥ纤维素柱层析提取了伤寒沙门氏菌ＳＯＤ。提取后酶的比活性为３２７０Ｕ／ｍｇ,经聚丙烯酰胺凝胶电泳,蛋白质染色及酶活性染色显示提取的ＳＯＤ达到了电泳纯。酶活性染色法和原子吸收分光光度法测定结果表明提取的ＳＯＤ为Ｆｅ－ＳＯＤ。双向琼脂扩散试验结果显示抗伤寒沙门氏菌Ｆｅ－ＳＯＤ血清与牛红细胞ＳＯＤ不形成沉淀线,提示伤寒沙门氏菌Ｆｅ－ＳＯＤ与牛红细胞ＳＯＤ无交叉反应,抗体对酶活性抑制试验结果显示抗Ｆｅ－ＳＯＤ血清可抑制伤寒沙门氏菌及鼠伤寒沙门氏菌的Ｆｅ－ＳＯＤ和Ｆｅ／Ｍｎ－ＳＯＤ活性,对Ｍｎ－ＳＯＤ活性无抑制作用,说明伤寒沙门氏菌和鼠伤寒沙门氏菌的ＳＯＤ之间有共同抗原,也提示ＳＯＤ的辅基似乎决定了其抗原特异性。     

Apoptosis of human keratinocytes after bacterial invasion

In this study, we examined the invasive capacity of Staphylococcus aureus and Salmonella typhi in human keratinocytes and monitored the number of viable intracellular bacteria at different post-infection times. The strains tested entered keratinocytes; both S. typhi and S. aureus were internalized within 30 min to 2 h after infection. No intracellular multiplication was observed, but S. typhi and S. aureus remained viable 72 h after infection. We also demonstrated that keratinocyte death following S. typhi and S. aureus invasion occurs by apoptosis as shown by DNA fragmentation. After 24 h of infection with S. typhi, the number of cells undergoing apoptosis were higher compared to infection with S. aureus. For prolonged infection times (48 h, 72 h) with both bacteria, there was no significant change in the number of cells undergoing apoptosis. The results demonstrated that viable intracellular S. typhi and S. aureus induced apoptosis in keratinocyte cells.     

伤寒沙门菌CT18和新疆XJ19体外培养条件下的蛋白质组差异分析

目的：分析我国新疆伤寒沙门菌分离株XJ19与全基因组测序的国际标准菌株CT18的蛋白表达差异,并推算基因差异。方法：运用二维蛋白电泳,对CT18和XJ19在体外培养基中的全菌蛋白进行分离,使用PDQuest软件找到其差异蛋白,进行质谱鉴定;对CT18差异蛋白编码基因设计引物,以XJ19DNA为模板进行PCR扩增,检测CT18差异蛋白编码基因在XJ19的存在情况。结果：菌株XJ19中存在53个特异蛋白点,鉴定出47个,但这些蛋白的编码基因在CT18中均存在,其中36个蛋白点在CT18的蛋白谱中不存在,11个蛋白在CT18中处于其他修饰状态;CT18中找到13个特异蛋白点,质谱鉴定出7个,其中6个所对应基因在XJ19中均能扩增出目的片段,但点C9蛋白的编码基因在XJ19中不存在。菌株XJ19中的多个差异蛋白参与磷酸戊糖途径的代谢及信号感应调控,此外超氧化物歧化酶、外膜蛋白OmpA呈现与CT18不同的修饰状态。结论：我们认为不同伤寒沙门菌分离株的遗传差异不仅仅是基因的有或无,还包括蛋白的不同表达和修饰所造成的不同调控机制和代谢的差异。