Defining the interaction of the Treponema pallidum adhesin Tp0751 with laminin

Various invasive pathogens attach to host tissues via the extracellular matrix component laminin, the major glycoprotein found within basement membranes. Previous investigations identified the laminin-binding adhesin Tp0751 within the spirochete bacterium Treponema pallidum. In the current study, Tp0751 was shown to attach to a variety of laminin isoforms that are widely distributed throughout the host, including laminins 1, 2, 4, 8, and 10. Such universal attachment is conducive for an adhesin present within a highly invasive pathogen that encounters a variety of tissue sites during the course of infection. Additional studies systematically identified the amino acid residues within Tp0751 that contribute to laminin binding using synthetic peptides designed from the mature protein sequence. The minimum laminin-binding region of the adhesin was localized to 10 amino acids; peptides containing these residues inhibited attachment of Tp0751 and T. pallidum to laminin. Further, Tp0751-specific antibodies inhibited attachment of T. pallidum to laminin. This study furthers our knowledge of the interaction of T. pallidum with laminin, an association that is proposed to facilitate bacterial traversal of basement membranes and subsequent entry into the circulation and tissue invasion. As such, these investigations will reveal new targets for possible prevention of bacterial dissemination and establishment of chronic infection.     

A newly identified leptospiral adhesin mediates attachment to laminin

Pathogenic leptospires have the ability to survive and disseminate to multiple organs after penetrating the host. Several pathogens, including spirochetes, have been shown to express surface proteins that interact with the extracellular matrix (ECM). This adhesin-mediated binding process seems to be a crucial step in the colonization of host tissues. This study examined the interaction of putative leptospiral outer membrane proteins with laminin, collagen type I, collagen type IV, cellular fibronectin, and plasma fibronectin. Six predicted coding sequences selected from the Leptospira interrogans serovar Copenhageni genome were cloned, and proteins were expressed, purified by metal affinity chromatography, and characterized by circular dichroism spectroscopy. Their capacity to mediate attachment to ECM components was evaluated by binding assays. We have identified a leptospiral protein encoded by LIC12906, named Lsa24 (leptospiral surface adhesin; 24 kDa) that binds strongly to laminin. Attachment of Lsa24 to laminin was specific, dose dependent, and saturable. Laminin oxidation by sodium metaperiodate reduced the protein-laminin interaction in a concentration-dependent manner, indicating that laminin sugar moieties are crucial for this interaction. Triton X-114-solubilized extract of L. interrogans and phase partitioning showed that Lsa24 was exclusively in the detergent phase, indicating that it is a component of the leptospiral membrane. Moreover, Lsa24 partially inhibited leptospiral adherence to immobilized laminin. This newly identified membrane protein may play a role in mediating adhesion of L. interrogans to the host. To our knowledge, this is the first leptospiral adhesin with laminin-binding properties reported to date.     

梅毒螺旋体Tp0751黏附蛋白的表达、纯化及免疫活性研究

目的 表达梅毒螺旋体黏附蛋白Tp0751,纯化表达产物并进行免疫活性分析,为探索Tp0751重组蛋白在梅毒致病过程中的作用奠定基础.方法 通过生物信息学分析,去除Tp0751信号肽序列,构建原核表达体进行诱导表达;Ni亲和层析柱纯化重组蛋白,Western blot检测其免疫反应性,用重组蛋白免疫新西兰家兔,评价其免疫原性.结果 成功构建了pET-28a(+)-0751原核表达载体,经表达、纯化后获得了相对分子质量约为26×103的融合蛋白;Western blot检测其能与梅毒患者阳性血清发生特异性反应;利用纯化的Tp0751重组蛋白免疫新西兰家兔,能诱导家兔产生特异性免疫应答,ELISA法测定免疫血清中特异性抗体滴度在1∶10 240以上.结论 重组表达的Tp0751黏附蛋白具有良好的免疫活性,为进一步研究其在梅毒致病过程中的作用和生物学功能奠定了基础.     

LipL32 is an extracellular matrix-interacting protein of Leptospira spp. and Pseudoalteromonas tunicata

LipL32 is the major outer membrane protein in pathogenic Leptospira. It is highly conserved throughout pathogenic species and is expressed in vivo during human infection. While these data suggest a role in pathogenesis, a function for LipL32 has not been defined. Outer membrane proteins of gram-negative bacteria are the first line of molecular interaction with the host, and many have been shown to bind host extracellular matrix (ECM). A search for leptospiral ECM-interacting proteins identified the major outer membrane protein, LipL32. To verify this finding, recombinant LipL32 was expressed in Escherichia coli and was found to bind Matrigel ECM and individual components of ECM, including laminin, collagen I, and collagen V. Likewise, an orthologous protein found in the genome of Pseudoalteromonas tunicata strain D2 was expressed and found to be functionally similar and immunologically cross-reactive. Lastly, binding activity was mapped to the C-terminal 72 amino acids. These studies show that LipL32 and an orthologous protein in P. tunicata are immunologically cross-reactive and function as ECM-interacting proteins via a conserved C-terminal region.     

Recognition of extracellular matrix proteins by Paracoccidioides brasiliensis yeast cells.

The adhesion of microorganism to host cells or extracellular matrix (ECM) proteins is the first step in the establishment of an infectious process. Interaction between Paracoccidioides brasiliensis yeast cells and ECM proteins has been previously noted. In vivo, in the chronic phase of experimental paracoccidioidomycosis (PCM), laminin and fibronectin have been detected on the surface of yeast cells located inside granulomatous lesions. The aim of the present study was to examine the ability of P. brasiliensis yeast cells to interact with extracellular matrix proteins (laminin, fibrinogen and fibronectin) and to establish which molecules were involved in this interaction. Immunofluorescence microscopy and flow cytometry demonstrated that all three ECM proteins tested were able to bind to the surface of P. brasiliensis yeast cells. Treatment with trypsin, chymotrypsin, chitinase, proteinase K or different sugars resulted in no change in laminin binding. In addition, ligand affinity assays were performed using different yeast extracts (total homogenates, beta-mercaptoethanol, SDS extracts). These assays demonstrated the presence of 19 and 32-kDa proteins in the cell wall with the ability to bind to laminin, fibrinogen and fibronectin. This interaction could be important in mediating attachment of the fungus to host tissues and may consequently play a role in the pathogenesis of PCM.     

Enhanced levels of attachment of fibronectin-primed Treponema pallidum to extracellular matrix.

Freshly extracted Treponema pallidum organisms treated with exogenous human fibronectin (Fn) (Fn-primed treponemes) showed a 6- to 15-fold increased level of attachment to Fn-coated cover slips and to extracellular matrix (ECM) when compared with unprimed treponemes. Treponemes primed with collagen or laminin showed no similar enhanced binding to immobilized Fn or ECM. Preexposure of immobilized Fn and ECM to anti-Fn serum but not to anti-collagen or anti-laminin serum prevented treponemal adherence. Also, the presence of proteoglycanlike molecules such as dextran sulfate or heparan sulfate inhibited Fn-primed treponemal attachment to Fn or ECM. In contrast Fn-primed treponemes did not exhibit elevated levels of attachment to eucaryotic cell monolayers. To understand the increased tropism of Fn-primed T. pallidum organisms for Fn and ECM-like surfaces, we radiolabeled freshly extracted treponemes with [35S]methionine and examined them for the presence of surface immunoreactive Fn. Magnetic protAspheres and glass beads coated with monospecific anti-Fn serum bound only 20 to 30% of radiolabeled treponemes. Nonadherent treponemes failed to bind to gelatin-agarose, further confirming the absence of surface Fn or Fn-like material. Fn-free organisms, however, did attach to Fn-coated cover slips and to cell monolayers like treponemes of the original population. Incubation of Fn-free treponemes with human Fn resulted in almost total binding of organisms to anti-Fn antibody on glass beads and also produced increased attachment to Fn-coated cover slips and ECM. These results suggest that enhanced interactions between T. pallidum and the host are dependent on the presence of Fn on syphilis spirochetes and the specific location and orientation of Fn in vivo.     

A novel Treponema pallidum antigen, TP0136, is an outer membrane protein that binds human fibronectin

The antigenicity, structural location, and function of the predicted lipoprotein TP0136 of Treponema pallidum subsp. pallidum were investigated based on previous screening studies indicating that anti-TP0136 antibodies are present in the sera of syphilis patients and experimentally infected rabbits. Recombinant TP0136 (rTP0136) protein was purified and shown to be strongly antigenic during human and experimental rabbit infection. The TP0136 protein was exposed on the surface of the bacterial outer membrane and bound to the host extracellular matrix glycoproteins fibronectin and laminin. In addition, the TP0136 open reading frame was shown to be highly polymorphic among T. pallidum subspecies and strains at the nucleotide and amino acid levels. Finally, the ability of rTP0136 protein to act as a protective antigen to subsequent challenge with infectious T. pallidum in the rabbit model of infection was assessed. Immunization with rTP0136 delayed ulceration but did not prevent infection or the formation of lesions. These results demonstrate that TP0136 is expressed on the outer membrane of the treponeme during infection and may be involved in attachment to host extracellular matrix components.     

The role of extracellular matrix proteins in the control of phagocytosis

The phagocytic function of human-peripheral-blood-derived mononuclear and polymorphonuclear leukocytes can be regulated by external stimuli. In particular, the extracellular matrix (ECM) proteins fibronectin and laminin and serum amyloid P component can increase the phagocytic activity of these cells. Phagocytosis enhancement by the ECM requires two distinct signals to the ingesting cell. First, a ligand for interaction of the target particle with phagocytic cells is required. Generally this occurs because of opsonins such as antibody or complement on the phagocytic target and is independent of the ECM proteins. The phagocytosis-enhancing effect of the connective tissue proteins also requires direct interaction of these proteins with phagocytic cells and occurs through cell surface receptors for these molecules. In the case of neutrophils, sensitivity to the phagocytosis-enhancing effects of connective tissue proteins requires prior stimulation with the chemotactic peptides C5a or f-met-leu-phe. The present state or knowledge of the mechanism of phagocytosis enhancement by ECM proteins and the implications of the phenomenon for host defense are discussed.     

Serodiagnosis of syphilis: antibodies to recombinant Tp0453, Tp92, and Gpd proteins are sensitive and specific indicators of infection by Treponema pallidum

Syphilis serodiagnosis relies on a combination of nonspecific screening tests (antilipoidal antibodies) and Treponema pallidum-specific tests (anti-T. pallidum antibodies). We studied a group of six recombinant T. pallidum antigens for their sensitivities and specificities with sera from individuals with syphilis (n = 43), relapsing fever (n = 8), Lyme disease (n = 8), and leptospirosis (n = 9) and from uninfected individuals (n = 15). Three recombinant proteins, Tp0155, Tp0483, and Tp0751, demonstrated sensitivity values that ranged from 28 to 42%. In contrast, three other recombinant proteins exhibited the following sensitivity and specificity values: Tp0453, 100% sensitivity and 100% specificity; Tp92 (Tp0326), 98% sensitivity and 97% specificity; and Gpd (Tp0257), 91% sensitivity and 93% specificity. Tp0453, Tp92, and Gpd also were recognized by sera from individuals with early primary syphilis that were nonreactive with the antilipoidal Venereal Disease Research Laboratory test. The reactivities of syphilis patient sera with Tp0453, Tp92, and Gpd were proportional to the titers of these sera with the treponemal test MHA-TP (microhemagglutination assay for T. pallidum). Thus, the recombinant T. pallidum antigens Tp0453, Tp92, and Gpd show promise as diagnostic antigens in the enzyme-linked immunosorbent assay-based assay.     

Attachment characteristics of bovine bronchial epithelial cells to extracellular matrix components

Attachment of cells to extracellular matrix (ECM) plays an important role in the regulation of cell growth and differentiated function. We hypothesized that bronchial epithelial cells preferentially attach to ECM proteins and utilize specific receptors for ECM proteins. Bronchial epithelial cells were obtained from bovine lung by protease digestion. Both freshly isolated and cultured bronchial epithelial cells were plated onto plastic petri dishes coated with bovine serum albumin, type I collagen, type IV collagen, fibronectin, laminin, ECM synthesized by cultured bronchial epithelial cells, or uncoated. Freshly isolated cells demonstrated significant attachment to ECM but weak attachment to other matrix proteins. Cultured bronchial epithelial cells attached well to ECM; however, they had relatively increased attachment to type I collagen, type IV collagen, fibronectin, and laminin compared to freshly isolated cells. To determine whether the attachment of bronchial epithelial cells is arginine-glycine-aspartic acid (RGD)-mediated, an RGD-containing peptide known to block attachment mediated by many integrin receptors was added to the media (400 micrograms/ml). There was no inhibition of attachment of freshly isolated cells; however, there was significant but not complete inhibition of the attachment of the cultured cells to type IV collagen, laminin, and fibronectin, but not to type I collagen or ECM. Thus, freshly isolated bronchial epithelial cells readily adhere to ECM, and the attachment does not appear to be mediated by RGD-dependent receptors. Cultured bronchial epithelial cells demonstrate increased attachment to component proteins of ECM, and this attachment is, in part, to RGD-dependent receptors.     

Identification of a beta 1 integrin on Mycobacterium avium-Mycobacterium intracellulare.

Mycobacterium avium-Mycobacterium intracellulare (MAI) is an opportunistic intracellular pathogen responsible for the highest incidence of disseminated bacterial infection in patients with AIDS. Treatment of the infection is extremely difficult and has shown limited efficacy. A critical event in the initiation of a variety of bacterial infections involves the adherence of bacteria to host cell surfaces. In the present study, we have shown that MAI organisms bind avidly to extracellular matrix proteins such as laminin, collagen I, and fibronectin in an in vitro attachment assay. Immunoblot analysis of a sonicate of MAI with polyclonal antibodies against different integrin receptors indicated that the sonicate cross-reacts with polyclonal antibodies against a human laminin-binding integrin, alpha 3 beta 1, and a human fibronectin-binding integrin, alpha 5 beta 1, although it is reactive with only the beta 1 subunit in the case of both antisera. Antibodies against the alpha 3 beta 1 and alpha 5 beta 1 integrins specifically inhibited the binding of MAI to laminin, collagen I, and fibronectin by 70 to 97%, depending on the ligand, suggesting that the attachment of MAI to these extracellular matrix proteins may be mediated by a beta 1 integrin. Furthermore, the attachment of MAI to laminin, collagen I, and fibronectin was found to be cation dependent. MAI may use this and other beta 1-containing integrins to adhere and penetrate through basement membrane structures that underlie host cell linings. An understanding of the mechanism of attachment and a definition of the adhesive molecules on the surface of MAI may open up new approaches to the prevention of serious infection caused by this organism.     

梅毒螺旋体黏附素Tp0751 核酸菌影的构建及免疫原性研究

目的:构建梅毒螺旋体(Tp)黏附素Tp0751 的重组真核大肠埃希菌菌影(EBG)并检测其在免疫鼠中的免疫原性,为探讨新型梅毒疫苗奠定基础。方法:构建pcDNA3.1(+) / Tp0751 真核表达载体,将其装载入已构建的空EBG 中,形成重组核酸菌影pcD/ Tp0751-BG,计算装载率;将核酸菌影转染鼠源性巨噬细胞RAW264.7,Western blot(WB)鉴定目的蛋白表达。将雌性BALB/ c 鼠随机分为A(PBS)、B(空EBG)、C(空pcDNA3.1)三个对照组和D(pcD/ Tp0751)、E(pcD/ Tp0751-BG)、F(pcD/ Tp0751-BG+rTp0751)三个实验组,各组间隔两周肌注免疫共三次,检测特异性血清IgG 及生殖道黏膜SIgA、小鼠脾细胞增殖水平和分泌IFN-γ水平。结果:重组真核质粒对菌影的装载率为76.1%;WB 显示此转染细胞能有效表达重组目的蛋白。D、E、F 实验组小鼠特异性血清IgG 与生殖道SIgA 效价均随免疫次数增加而增加,各时间点均显著高于三个对照组(P<0.01),于末次加免后第8 周达到峰值,此时F 组IgG 与SIgA 效价分别为1 :102 400 与1 :12 800;首次加免2 周后,E、F 组均显著高于D 组(P<0.01);末次加免2 周后,F 组显著高于E 组(P<0.01)。末次加免后第8 周,D、E、F 组的刺激指数(SI)值与IFN-γ水平均分别显著高于三个对照组(P<0.01);E、F 组均分别显著高于D 组(P<0.01);F 组分别均高于E 组(P<0.05)。结论:Tp0751 真核质粒菌影具有良好的免疫原性,在小鼠体内诱生了有效的系统和黏膜的体液应答以及系统细胞免疫应答;异源加免较同源加免免疫效果更好。     

Laminin enhances binding of Toxoplasma gondii tachyzoites to J774 murine macrophage cells.

We investigated the effects of the extracellular matrix proteins laminin and fibronectin on the attachment of tachyzoites of Toxoplasma gondii to the murine macrophage cell line J774. Laminin but not fibronectin increased parasite attachment to J774 cells in a dose-dependent fashion. Cyclic YIGSR, a laminin-derived peptide which inhibits laminin binding to the 32/67-kDa laminin-binding protein on host cells, blocked the laminin-mediated enhancement of parasite attachment. An antiserum to the 32/67-kDa laminin-binding protein also inhibited binding of parasites to J774 cells. These results, in conjunction with our previous observations (G. C. Furtado, F. L. Collins, and K. A. Joiner, submitted for publication), demonstrate that tachyzoites bearing surface laminin bind to multiple laminin receptors in attaching to different target cells.     

Products of cells cultured from gliomas. VII. Extracellular matrix proteins of gliomas which contain glial fibrillary acidic protein

Extracellular matrix (ECM) components of two glial fibrillary acidic protein positive (GFAP+) glioma lines U251 and UM6 were studied by silver stain, morphometry, immunofluorescence, enzyme-linked immunosorbent assay, and biosynthetic labeling. Both GFAP+ lines expressed the following qualitative features in common with previously studied GFAP-negative gliomas: (a) laminin, (b) type IV collagen, (c) extracellular fibrils of silver-reducing collagen (d) pattern of reactivity with lectins. Quantitative differences in GFAP+ glioma proteins included less collagen and more laminin than GFAP-negative gliomas. Sparse collagen of GFAP+ gliomas aggregated as extracellular masses. Individual cells of UM6 simultaneously expressed GFAP and mesenchymal ECM components. Results show qualitative similarities of ECM expression among GFAP+ and negative gliomas suggesting a common lineage of these two glioma cell types and universal expression of two epithelial components of ECM, laminin and type IV collagen, among cultured gliomas. Moreover, there is a diversity of quantity and type of ECM proteins of GFAP+ gliomas with the U251 line most restricted in its expression of ECM components and with UM6 manifesting markers of epithelial and mesenchymal lineage. This diversity suggests a capacity for regulation of phenotypic expression of ECM beyond that explained simply by the presence of two cell types of different lineage.     

Effect of matrix metalloproteinases inhibition on the proliferation and differentiation of HUCB-NSCs cultured in the presence of adhesive substrates

Cell adhesion to extracellular matrix (ECM) generates intracellular signals that modulate cell survival, proliferation, migration and differentiation. Because of its heterogeneous nature, ECM has the potential to induce unique responses that are composition-dependent. One approach to study the effect of ECM signals on cell development, independently on signals from other extracellular sources, has been to deprive cells of serum and to analyze the influence of specific ligands. In the current work we determine the potential of different ECM proteins (fibronectin, laminin, collagen) on the proliferation and differentiation of human umbilical cord blood-derived neural stem cells (HUCB-NSCs) cultured in serum-free conditions. The effect of tested ECM components on the above processes might be associated with the particular pattern of their proteolysis. In this context enzymes that are responsible for the modification of ECM proteins are of particular pertinence. Matrix metalloproteinases (MMPs) represent a family of enzymes known to play role in the modification of ECM and by this can change the cell-ECM substrate interaction, required for cell development. In an effort to elucidate the participation of MMPs in the proliferation and differentiation HUCB-NSCs were cultured in the presence or absence of MMPs inhibitors - GM6001 and doxycycline. Our results show that addition of the above inhibitors interfered with both the proliferation and differentiation of progenitor cells toward the neuronal lineage. This effect depends on the adhesive ECM substrate and is most pronounced in the presence of fibronectin and laminin. In conclusion, our results suggest that MMPs modulate interaction between HUCB-NSCs and their environment and therefore might be an important component in neurogenesis-associated processes.     

Highly conserved surface proteins of oral spirochetes as adhesins and potent inducers of proinflammatory and osteoclastogenic factors

Oral spirochetes include enormously heterogeneous Treponema species, and some have been implicated in the etiology of periodontitis. In this study, we characterized highly conserved surface proteins in four representative oral spirochetes (Treponema denticola, T. lecithinolyticum, T. maltophilum, and T. socranskii subsp. socranskii) that are homologs of T. pallidum Tp92, with opsonophagocytic potential and protective capacity against syphilis. Tp92 homologs of oral spirochetes had predicted signal peptides (20 to 31 amino acids) and molecular masses of 88 to 92 kDa for mature proteins. They showed amino acid sequence identities of 37.9 to 49.3% and similarities of 54.5 to 66.9% to Tp92. The sequence identities and similarities of Tp92 homologs of oral treponemes to one another were 41.6 to 71.6% and 59.9 to 85.6%, respectively. The tp92 gene homologs were successfully expressed in Escherichia coli, and the recombinant proteins were capable of binding to KB cells, an epithelial cell line, and inhibited the binding of the whole bacteria to the cells. Antiserum (the immunoglobulin G fraction) raised against a recombinant form of the T. denticola Tp92 homolog cross-reacted with homologs from three other species of treponemes. The Tp92 homologs stimulated various factors involved in inflammation and osteoclastogenesis, like interleukin-1beta (IL-1beta), tumor necrosis factor alpha, IL-6, prostaglandin E(2), and matrix metalloproteinase 9, in host cells like monocytes and fibroblasts. Our results demonstrate that Tp92 homologs of oral spirochetes are highly conserved and may play an important role in cell attachment, inflammation, and tissue destruction. The coexistence of various Treponema species in a single periodontal pocket and, therefore, the accumulation of multiple Tp92 homologs may amplify the pathological effect in periodontitis.     

Extracellular matrix proteins and integrin receptors in reactive and non-reactive lymph nodes.

The extracellular matrix (ECM) proteins collagen I, III and IV, laminin, fibronectin, vitronectin, thrombospondin, tenascin and their integrin receptors of the beta 1 and beta 3 subfamilies showed characteristic patterns of distribution in different compartments of non-reactive and reactive lymph nodes (human and monkey). This was particularly evident during development of germinal centres. Thus, ECM proteins (collagens, laminin, fibronectin and tenascin) were abundant in the interfollicular (T-cell rich) compartments of non-reactive as well as reactive lymph nodes. In primary follicles, collagen I, III and fibronectin were expressed but displaced by the expanding germinal centre during the formation of secondary follicles in reactive lymphoid tissues. The integrin subunits were mainly associated with endothelial cells and lymphoid cells in interfollicular areas, but were absent or only poorly expressed in primary as well as secondary follicles. Evidently the expression of ECM components and their integrin receptors is markedly down-regulated in the reactive, highly proliferative germinal centres.     

Identification of the surface component of Streptococcus defectivus that mediates extracellular matrix adherence.

Bacterial attachment to host tissue is considered to be a crucial primary step in pathogen infection. Previous studies have shown that Streptococcus defectivus adheres specifically to cell-secreted extracellular matrix (ECM). Though generally not exposed in vivo, this host tissue is exposed at endothelial cell junctions and sites of tissue injury. In this report, we identify a ca. 200-kDa surface protein of S. defectivus involved in ECM adherence. Nitrous acid-derived mutant strains that were unable to bind ECM and which failed to adsorb adhesin-specific antibody from polyclonal inhibitory sera were isolated. A surface protein (ca. 200 kDa) was absent from ECM-nonadherent mutants, indicating its involvement in ECM attachment. Additionally, affinity-purified antibody to the ca. 200-kDa protein inhibited whole-cell S. defectivus ECM attachment, whereas antibody to the same region of the nonadherent mutant cell wall-associated protein profile did not. Furthermore, solubilized cell wall-associated protein extracts of parent but not mutant strains bound ECM, confirming the significance of this protein in ECM adherence. Therefore, we propose that the ca. 200-kDa protein is the major S. defectivus surface component that mediates the ECM attachment of these organisms.