Host Cell Adhesion Research Articles

Immune responses to an encapsulated allogeneic islet β-cell line in diabetic NOD mice

Our goal is to develop effective islet grafts for treating type 1 diabetes. Since human islets are scarce, we evaluated the efficacy of a microencapsulated insulin-secreting conditionally transformed allogeneic β-cell line (βTC-tet) in non-obese diabetic mice treated with tetracycline to inhibit cell growth. Relatively low serum levels of tetracycline controlled proliferation of βTC-tet cells without inhibiting effective control of hyperglycemia in recipients. There was no significant host cellular reaction to the allografts or host cell adherence to microcapsules, and host cytokine levels were similar to those of sham-operated controls. We conclude that encapsulated allogeneic β-cell lines may be clinically relevant, because they effectively restore euglycemia and do not elicit a strong cellular immune response following transplantation. To our knowledge, this is the first extensive characterization of the kinetics of host cellular and cytokine responses to an encapsulated islet cell line in an animal model of type 1 diabetes.

Pathogenesis of Implant Infections by Enterococci

Enterococci are commensals of human and animal intestinal tract that have emerged in the last decades as a major cause of nosocomial infections of bloodstream, urinary tract and in infected surgical sites. Enterococcus faecalis is responsible for ca. 80% of all enterococcal infections while Enterococcus faecium accounts for most of the others; among the most relevant risk factors for development of enterococcal infections is the presence of implanted devices. The pathogenesis of such infections is poorly understood, but several virulence factors have been proposed. Among them, the ability to form biofilm has recently been shown to be one of the most prominent features of this microorganism, allowing colonization of inert and biological surfaces, while protecting against antimicrobial substances, and mediating adhesion and invasion of host cells and survival within professional phagocytes. Biofilm formation has been shown to be particularly important in the development of prosthetic valve enterococcal endocarditis and stent occlusion. Enterococci are also able to express other surface factors that may support colonization of both inert and biological surfaces, and that may be involved in the invasion of, and survival within, the host cell.

Calcineurin is required for Candida albicans to survive calcium stress in serum.

The calcium-activated protein phosphatase calcineurin plays a critical role in the virulence of Candida albicans. Previous studies demonstrated that calcineurin is not required for the yeast-hypha dimorphic transition, host cell adherence, or host cell injury, which are all established virulence attributes of this organism. Calcineurin is, however, essential for survival in serum and disseminated infection. Here we identify the component of serum that is toxic to calcineurin mutant cells. Proteins, peptides, lipids, and other hydrophobic components were all excluded as essential toxic elements. Upon testing of small molecules present in serum, we discovered that calcineurin protects cells from stress caused by the endogenous levels of calcium ions present in serum. These studies illustrate how calcineurin functions in a calcium homeostatic pathway that enables a common human commensal to survive passage through the hostile environment of the bloodstream to establish deep-seated infections and cause disease.

CEACAM engagement by human pathogens enhances cell adhesion and counteracts bacteria-induced detachment of epithelial cells

Exfoliation, which is the detachment of infected epithelial cells, is an innate defense mechanism to prevent bacterial colonization. Indeed, infection with Neisseria gonorrhoeae induced epithelial detachment from an extracellular matrix (ECM) substrate in vitro. Surprisingly, variants of N. gonorrhoeae that bind to human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) failed to induce detachment and, instead, promoted enhanced host cell adhesion to the ECM. Microarray analysis revealed that CEACAM engagement by several human pathogens triggers expression of CD105. Blockage of CD105 expression by antisense oligonucleotides abolished infection-induced cell adhesion. The expression of full-length CD105 promoted cell adhesion to the ECM and was sufficient to prevent infection-induced detachment. The CD105-mediated increase in cell adhesion was dependent on the presence and function of integrin β1. CD105 expression did not elevate cellular integrin levels but caused a dramatic increase in the ECM-binding capacity of the cells, suggesting that CD105 affects integrin activity. The exploitation of CEACAMs to trigger CD105 expression and to counteract infection-induced cell detachment represents an intriguing adaptation of pathogens that are specialized to colonize the human mucosa.

Neospora caninum protein disulfide isomerase is involved in tachyzoite-host cell interaction

We have previously shown that treatment of Neospora caninum tachyzoites with the aspartyl protease inhibitor pepstatin A reduces host cell invasion [Naguleswaran, A., Muller, N., Hemphill, A., 2003. Neospora caninum and Toxoplasma gondii: a novel adhesion/invasion assay reveals distinct differences in tachyzoite-host cell interactions. Exp. Parasitol. 104, 149–158]. Pepstatin A-affinity-chromatography led to the isolation of a major band of ∼52 kDa which was identified as a homologue of a previously described Toxoplasma gondii putative protein disulfide isomerase (TgPDI) through tandem mass spectrometry. A BLAST search against N. caninum expressed sequence tags (ESTs) on the ApiDots server using TgPDI cDNA as query sequence revealed a 2251 bp PDI-like consensus (NcPDI), which shows 94% identity to the T. gondii homologue. In N. caninum tachyzoites, NcPDI was found mainly in the soluble hydrophilic fraction. Immunofluorescence showed that expression of NcPDI was dramatically down-regulated in the bradyzoite stage, and immunogold-EM on tachyzoites localised the protein to the cytoplasm, mostly in close vicinity to the nuclear membrane, to the micronemes, and to the parasite cell surface. However, NcPDI was absent in rhoptries and dense granules. Preincubation of tachyzoites with the sulfhydryl blocker 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), p-chloromercuribenzoic acid (pCMBA), and with the PDI inhibitor bacitracin reduced adhesion of parasites to host cells. In addition, incubation of N. caninum tachyzoites with affinity-purified anti-NcPDI antibodies reduced host cell adhesion. PDIs catalyse the formation, reduction or isomerisation of disulfide bonds. Many major components of the adhesion and invasion machinery of apicomplexan parasites are cysteine-rich and dependent on correct folding via disulfide bond formation. Thus, our data points towards an important role for surface-associated NcPDI in Neospora-host cell interaction.

Solving a sticky problem: new genetic approaches to host cell adhesion by the Lyme disease spirochete

The Lyme disease spirochetes, comprised of at least three closely related species, Borrelia burgdorferi, Borrelia garinii and Borrelia afzelii, are fascinating and enigmatic bacterial pathogens. They are maintained by tick-mediated transmission between mammalian hosts, usually small rodents. The ability of these bacteria, which have relatively small genomes, to survive and disseminate in both an immunocompetent mammal and in an arthropod vector suggests that they have evolved elegant and indispensable strategies for interacting with their hosts. Recognition of specific mammalian and tick tissues is likely to be essential for successful completion of the enzootic life cycle but, given the historical difficulties in genetic manipulation of these organisms, characterization of factors promoting cell adhesion has until recently largely been confined to either the manipulation of host cells or the analysis of potential bacterial ligands in the form of recombinant proteins. These studies have led to the identification of several mammalian receptors for Lyme disease spirochetes, including glycosaminoglycans, decorin, fibronectin and integrins, as well as a tick receptor for the bacterium, and also candidate cognate bacterial ligands. Recent advances in our ability to genetically manipulate Lyme disease spirochetes, particularly B. burgdorferi, are now providing us with firm evidence that these ligands indeed do promote bacterial adherence to host cells, and with new insights into the roles of these multifacted Borrelia-host cell interactions during mammalian and arthropod infection.

Cellular and molecular mechanisms that underlie Entamoeba histolytica pathogenesis: prospects for intervention

The protozoan parasite Entamoeba histolytica is the causative agent of amoebic dysentery. It is prevalent in developing countries that cannot prevent its fecal-oral spread and ranks second in worldwide causes of morbidity by parasitic infection. Improvements in sanitation would help curb disease spread. However, a lack of significant progress in this area has resulted in the need for a better understanding of the molecular and cellular biology of pathogenesis in order to design novel methods of disease treatment and prevention. Recent insight into the cellular mechanisms regulating virulence of E. histolytica has indicated that processes such as endocytosis, secretion, host cell adhesion and encystation play major roles in the infectious process. This review focuses on components of the molecular machinery that govern these cellular processes and their role in virulence, and discusses how an understanding of this might reveal opportunities to interfere with E. histolytica infection.

Calcium binding activity of the epidermal growth factor-like domains of the apicomplexan microneme protein EtMIC4

Microneme proteins are secreted from apicomplexan parasites during invasion of host cells and they play crucial roles in parasite–host cell adhesion. EtMIC4 is a 240 kDa transmembrane protein from Eimeria tenella that contains 31 tandemly arranged epidermal growth factor (EGF), like repeats within its extracellular domain. The majority of these repeats have calcium binding (cb) consensus sequences. Little is known about cbEGFs in apicomplexan parasites but their presence in microneme proteins suggests that they may contribute to parasite–host interactions. To investigate the potential role of cbEGFs we have expressed and correctly refolded a cbEGF triplet from EtMIC4 (cbEGF 7–9) and demonstrated that this triplet binds calcium. Circular dichroism spectroscopic analysis of cbEGF 7–9 demonstrates that the molecule undergoes a gradual change in conformation with increasing levels of calcium. In the presence of calcium, the triplet becomes resistant to proteolytic degradation by a variety of proteases, a characteristic feature of cbEGF repeats from higher eukaryotic proteins, such as fibrillin, suggesting that calcium binding induces the formation of a rigid conformation. Moreover, mass spectrometric mapping of the cleavage sites that are protected by calcium shows that these sites are located both close to and distant from the calcium binding sites, indicating that protection is not due to steric hindrance by calcium ions, but rather due to the overall conformation adopted by the triplet in the presence of calcium. Thus, the tandemly-arranged cbEGF repeats within EtMIC4 provide a mechanism whereby, in the calcium-rich extracellular environment, the molecule could adopt a protease-resistant, rigid structure that could favour its interaction with host cell ligands.

Adherence to and Invasion of Human Brain Microvascular Endothelial Cells Are Promoted by Fibrinogen-Binding Protein FbsA ofStreptococcus agalactiae

Streptococcus agalactiae is a frequent cause of bacterial sepsis and meningitis in neonates. During the course of infection, S. agalactiae colonizes and invades a number of host compartments, thereby interacting with different host tissues. Deletion of the fbsA gene, encoding the fibrinogen protein FbsA, significantly impaired the adherence and invasion of human brain microvascular endothelial cells (HBMEC) by S. agalactiae. The adherence and invasiveness of an fbsA deletion mutant were restored by reintroducing the fbsA gene on an expression vector. Heterologous expression of fbsA in Lactococcus lactis enabled this bacterium to adhere to but not to invade HBMEC, suggesting that FbsA is a streptococcal adhesin. Finally, host cell adherence and invasion were significantly blocked in competition experiments with either purified FbsA fusion protein or a monoclonal antibody directed against the fibrinogen-binding epitope of FbsA. The S. agalactiae fbsA mutant induced a release of the neutrophil chemoattractant interleukin-8 (IL-8) equal to that induced by the wild type. Taken together, our studies demonstrate that FbsA promotes the adherence of S. agalactiae to HBMEC but that FbsA neither mediates the bacterial invasion into host cells nor plays a role in IL-8 release for HBMEC.

Global epithelial cell transcriptional responses reveal Streptococcus pyogenes Fas regulator activity association with bacterial aggressiveness

The bacterial human pathogen Streptococcus pyogenes (group A streptococci, GAS) is able to adhere to, internalize into and cross-talk on multiple levels with its host cells. To gain insight into the Fas function in pathogenesis we used Affymetrix human genome DNA-arrays to measure temporal and global transcriptional responses of HEp-2 cells infected with M49 S. pyogenes wild-type bacteria and DeltafasX, an isogenic S. pyogenes two-component-signal-transduction system mutant. A modified stringent statistical analysis method identified a total of 86 HEp-2 cell genes as differentially transcribed upon infection over the investigated time course. Increased expression of genes encoding proteins involved in GAS host cell adherence and internalization (fibronectin, integrin-alpha5) was found as a common response. In contrast to earlier reports investigating other GAS serotype strains, Ras superfamily and RhoA pathways are exploited by M49 GAS, suggesting serotype specific interactions with the host cell cytoskeleton. Despite transcriptional induction, secreted IL-8 levels of deltafasX mutant infected cells were below those of non-infected cells, indicating an absence of Fas expression could be important for GAS tissue colonization and long-term intracellular persistence. Oppositely, activity of the S. pyogenes Fas-system apparently promotes high adherence and internalization rates, massive cytokine gene transcription and cytokine release, host cell apoptosis via a caspase-2 activation pathway, and cytotoxicity. Thus, the S. pyogenes Fas two-component signal transduction system could be involved in local tissue destruction and general bacterial aggressiveness towards host cells.

REDUCED INFECTION AND PROTECTION FROM CLINICAL SIGNS OF CEREBRAL NEOSPOROSIS IN C57BL/6 MICE VACCINATED WITH RECOMBINANT MICRONEME ANTIGEN NCMIC1

NcMIC1 is a 460 amino acid Neospora caninum microneme protein implicated in host cell adhesion and invasion processes. In this study, we assessed the potential protectivity of NcMIC1-based vaccination against experimental N. caninum infection in mice, employing both recombinant antigen vaccines and DNA vaccines. Recombinant NcMIC1 (recNcMIC1) was expressed in Escherichia coli as gluthatione-S-transferase-fusion protein. The corresponding NcMIC1 cDNA was cloned into the pcDNA3.1 expression plasmid (pcDNA-MIC1), and expression was checked in transfected Vero cells. Mice (10 animals/group) were vaccinated either with recNcMIC1 antigen suspended in Ribi-adjuvant (3 intraperitoneal injections), pcDNA-NcMIC1 (3 intramuscular injections), or pcDNA-NcMIC1 (twice intramuscularly), followed by 1 intraperitoneal recNcMIC1 antigen boost. Control groups included corresponding treatments with adjuvant, pcDNA3.1 without insert, and PBS (= infection control). All vaccinated and control groups were then challenged intraperitoneally with 2 x 10(6) N. caninum tachyzoites. Animals were inspected daily for a period of 3 wk postinfection (PI). At day 21, all animals were killed and assessed for infection. Before day 21 PI, clinical signs such as walking disorders, rounded back, apathy, and paralysis occurred in infection controls (50% of the mice), pcDNA and adjuvant controls (20% each), and the combined pcDNA-NcMIC1/recNcMIC1-treated group (30%). No clinical symptoms were observed in the recNcMIC1 and pcDNA-NcMIC1 vaccinated groups. All mice were positive for cerebral N. caninum infection as assessed by PCR of brain tissue. However, quantitative real-time PCR revealed that the infection intensity was significantly reduced in the group vaccinated with recNcMIC1 antigen. Immunohistochemistry confirmed these findings. In contrast, the infection intensity was highest in the group vaccinated with the pcDNA-NcMIC1/recNcMIC1 combination, indicating that the sequential application of the DNA vaccine and recombinant antigen had a deleterious effect. Serological analysis showed that only recNcMIC1-immunized animals generated detectable antibody levels recognizing native NcMIC1. Thus, of all protocols applied here, only recNcMIC1 vaccination appears to be suited to reduce cerebral infection in mice challenged with N. caninum tachyzoites.

Trichomonas vaginalis Polyamine Metabolism Is Linked to Host Cell Adherence and Cytotoxicity

Trichomonas vaginalis secretes putrescine that is readily detected in vaginal secretions. We wanted to examine the effect of decreased putrescine synthesis by inhibition of ornithine decarboxylase (ODC) on T. vaginalis. One reason is because inhibition of Tritrichomonas foetus ODC results in growth arrest, destruction of hydrogenosomes, and decreased amounts of hydrogenosomal enzymes. Treatment of T. vaginalis T016 with >/=20 mM 1,4-diamino-2-butanone (DAB) to inhibit ODC resulted in growth arrest, which was reversed by addition of exogenous putrescine. No similar reversal of growth arrest was achieved with the polyamines spermine or spermidine or with iron. Electron microscopic examination of control versus DAB-treated trichomonads did not reveal any adverse effects on the number and integrity of hydrogenosomes. Further, the adhesins AP65, AP51, and AP33 mediating binding to immortalized vaginal epithelial cells (VECs) share identity to enzymes of the hydrogenosome organelle, and there was no difference in amounts of adhesins between control versus DAB-treated T. vaginalis parasites. Likewise, similar patterns and extent of fluorescence were evident for the prominent AP65 adhesin. Surprisingly, DAB treatment increased by 4- to 20-fold above untreated trichomonads handled identically the level of adherence mediated by adhesins. Interestingly, the enhanced attachment to VECs was reversed by exogenous putrescine added to DAB-treated trichomonads. Equally noteworthy was that DAB-treated T. vaginalis with enhanced adherence did not possess the previously reported ability to kill host cells in a contact-dependent fashion mediated by cysteine proteinases, and total cysteine proteinase activity patterns were identical between control and DAB-treated trichomonads. Overall, these data suggest that polyamine metabolism and secreted putrescine are linked to host cell adherence and cytotoxicity.

The Use of Carbohydrate Microarrays to Study Carbohydrate-Cell Interactions and to Detect Pathogens

The use of carbohydrate microarrays to investigate the carbohydrate binding specificities of bacteria, to detect pathogens, and to screen antiadhesion therapeutics is reported. This system is ideal for whole-cell applications because microarrays present carbohydrate ligands in a manner that mimics interactions at cell-cell interfaces. Other advantages include assay miniaturization, since minimal amounts (approximately picomoles) of a ligand are required to observe binding, and high throughput, since thousands of compounds can be placed on an array and analyzed in parallel. Pathogen detection experiments can be completed in complex mixtures of cells or protein using the known carbohydrate binding epitopes of the pathogens in question. The nondestructive nature of the arrays allows the pathogen to be harvested and tested for antibacterial susceptibility. These investigations allow microarray-based screening of biological samples for contaminants and combinatorial libraries for antiadhesion therapeutics.

The Fibrinogen Receptor FbsA Promotes Adherence of Streptococcus agalactiae to Human Epithelial Cells

Streptococcus agalactiae is a major cause of bacterial pneumonia, sepsis, and meningitis in human neonates. During the course of infection, S. agalactiae adheres to a variety of epithelial cells but the underlying mechanisms are only poorly understood. The present report demonstrates the importance of the fibrinogen receptor FbsA for the streptococcal adherence and invasion of epithelial cells. Deletion of the fbsA gene in various S. agalactiae strains substantially reduced their binding of soluble fibrinogen and their adherence to and invasion of epithelial cells, indicating a role of FbsA in these different processes. The adherence and invasiveness of an fbsA deletion mutant were partially restored by reintroducing the fbsA gene on an expression vector. Heterologous expression of fbsA in Lactococcus lactis enabled this bacterium to adhere to but not to invade epithelial cells, suggesting that FbsA is a streptococcal adhesin. Flow cytometry experiments revealed a dose-dependent binding of FbsA to the surface of epithelial cells. Furthermore, tissue culture experiments exhibited an intimate contact of FbsA-coated latex beads with the surfaces of human epithelial cells. Finally, host cell adherence and invasion were significantly blocked in competition experiments with either purified FbsA protein or a monoclonal antibody directed against the fibrinogen-binding epitope of FbsA. Taken together, our studies demonstrate that FbsA promotes the adherence of S. agalactiae to epithelial cells but that FbsA does not mediate the bacterial invasion into host cells. Our results also indicate that fibrinogen-binding epitopes within FbsA are involved in the adherence of S. agalactiae to epithelial cells.

The fibronectin-binding capacity and host cell adherence of Streptococcus pyogenes strains are discordant with each other

Surface exposed fibronectin-binding proteins (FBPs) play an important role in the adherence of S treptococcus pyogenes (group A streptococcus, GAS) to host cells. This pathogen expresses numerous FBPs, of which SfbI, SfbII and PrtF2 are major surface exposed FBPs. However, GAS strains differ in the genetic potential to express these proteins. To test whether this difference reflects in differences in fibronectin (Fn) binding, a set of circulating strains previously examined for adherence to host cells was used. The 68 distinct strains were isolated from throat, skin and blood. They were analyzed for (a) the presence of genes for SfbI, SfbII and PrtF2 and (b) the extent of Fn binding. The results suggest that strains possessing two or more of the genes for these FBPs bound Fn significantly more than strains possessing none or one of the genes. No correlation between the extent of Fn binding and the tissue site of isolation was found. Furthermore, together with our previous studies on adherence capacity of these GAS strains, we found no correlation between Fn binding ability and the avidity of the strains to adhere to epithelial cells. We suggest that while Fn binding is important for adhesion, for many GAS strains the extent of Fn binding is not the critical determinant of adherence.

Cell-surface-associated tissue transglutaminase is a target of MMP-2 proteolysis.

MT1-MMP, a prototypic member of a membrane-type metalloproteinase subfamily, is an invasion promoting protease and an activator of MMP-2. In addition, MT1-MMP proteolysis regulates the functionality of cell-surface adhesion/signaling receptors including tissue transglutaminase (tTG). tTG is known to serve as an adhesion coreceptor for beta1/beta3 integrins and as an enzyme that catalyzes the cross-linking of proteins and the conjugation of polyamines to proteins. Here, we report that MMP-2, functioning in concert with MT1-MMP, hydrolyzes cell-surface-associated tTG, thereby further promoting the effect initiated by the activator of MMP-2. tTG, in return, preferentially associates with the activation intermediate of MMP-2. This event decreases the rate of MMP-2 maturation and protects tTG against proteolysis by MMP-2. Our cell culture, in vitro experiments, and in silico modeling indicate that the catalytic domain of MMP-2 directly associates with the core enzymatic domain II of tTG (the K(d) = 380 nM). The follow-up cleavage of the domain II eliminates both the receptor and the enzymatic activity of tTG. Our data illuminate the coordinated interplay involving the MT1-MMP/MMP-2 protease tandem in the regulation of the cell receptors and explain the underlying biochemical mechanisms of the extensive tTG proteolysis that exists at the normal tissue/tumor boundary. Our findings also suggest that neoplasms, which express functionally active MT1-MMP and, therefore, activate soluble MMP-2, can contribute to the degradation of tTG expressed in neighboring host cells. The loss of adhesive and enzymatic activities of tTG at the interface between tumor and normal tissue will decrease cell-matrix interactions and inhibit matrix cross-linking, causing multiple pathological alterations in host cell adhesion and locomotion.

Identification and characterization of a Neospora caninum microneme-associated protein (NcMIC4) that exhibits unique lactose-binding properties.

Microneme proteins have been shown to play an important role in the early phase of host cell adhesion, by mediating the contact between the parasite and host cell surface receptors. In this study we have identified and characterized a lectin-like protein of Neospora caninum tachyzoites which was purified by alpha-lactose-agarose affinity chromatography. Upon separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this lactose-binding protein migrated at 70 and 55 kDa under reducing and nonreducing conditions, respectively. Immunofluorescence and immunogold electron microscopy with affinity-purified antibodies showed that the protein was associated with the tachyzoite micronemes. Mass spectrometry analyses and expressed sequence tag database mining revealed that this protein is a member of the Neospora microneme protein family; the protein was named NcMIC4 (N. caninum microneme protein 4). Upon two-dimensional gel electrophoresis, NcMIC4 separated into seven distinct isoforms. Incubation of extracellular parasites at 37 degrees C resulted in the secretion of NcMIC4 into the medium as a soluble protein, and the secreted protein exhibited a slightly reduced M(r) but retained its lactose-binding properties. Immunofluorescence was used to investigate the temporal and spatial distribution of NcMIC4 in tachyzoites entering their host cells and showed that reexpression of NcMIC4 took place 30 min after entry into the host cell. Incubation of secreted fractions and purified NcMIC4 with Vero cells demonstrated binding of NcMIC4 to Vero cells as well as binding to chondroitin sulfate A glycosaminoglycans.

Lactoferrin functions: current status and perspectives.

Lactoferrin, an iron-binding glycoprotein synthesized by neutrophils and exocrine glands, plays an important role in human innate defense mechanisms against bacteria, fungi, and viruses. First, a bacteriostatic activity of lactoferrin, depending on iron withholding to bacteria, and successively a bactericidal iron-independent effect, related to its binding on bacterial surfaces, was recognized. Many other functions have been ascribed to this cationic protein, including the inhibiting action toward bacterial adhesion and invasion of target host cells. Recent research also reported the lactoferrin influence on bacterial aggregation and biofilm development of Pseudomonas aeruginosa and Streptococcus mutans. The different lactoferrin functions can be justified by different physicochemical properties of the molecule, which include the iron-binding capability, the binding to anionic cell surfaces and molecules, and serine protease activity.

The influence of surface carbohydrates during in vitro infection of mammalian cells by the dermatophyte Trichophyton rubrum

In order to better understand the role played by surface glycoconjugates during host cell adhesion and endocytosis of Trichophyton rubrum, we looked for the presence of carbohydrate-binding adhesins on the microconidia surface and their role on cellular interaction with epithelial and macrophages cells. The interaction of T. rubrum with chinese hamster ovary epithelial cells and their glycosylation-deficient mutants demonstrated a higher adhesion index in Lec1 and Lec2 mutants, that express mannose and galactose, respectively. Endocytosed fungi were shown preferentially in Lec2 cells. Addition of the carbohydrates to the interaction medium, pretreatment with lectins and with sodium periodate decreased the adhesion and endocytic index for all mutants. The ability of the fungus to penetrate into mammalian cells was confirmed in experiments using macrophages treated with cytochalasin D. Flow cytometric analysis showed that this fungus recognizes mannose and galactose. The binding was inhibited by the addition of methyl α- d-mannopyranoside and methyl α- d-galactopyranoside, and showed higher fluorescence intensity at 37 than at 28 °C. Trypsin treatment and heating of the cells reduced the binding, suggesting a (glyco) protein nature for the microconidia adhesins. The presence of lectin-like molecules in fungus cell could be observed by scanning electron microscopy of the fungus incubated with colloidal-gold labeled neoglycoproteins. Our results suggest that T. rubrum has the ability to invade mammalian cells and expresses carbohydrate-specific adhesins on microconidia surface that recognize mannose and galactose. These adhesins may play an important role on the adhesion and invasion of the fungus during the infectious process of dermatophytosis.

From the inside out--processing of the Chlamydial autotransporter PmpD and its role in bacterial adhesion and activation of human host cells.

Polymorphic membrane protein (Pmp)21 otherwise known as PmpD is the longest of 21 Pmps expressed by Chlamydophila pneumoniae. Recent bioinformatical analyses annotated PmpD as belonging to a family of exported Gram-negative bacterial proteins designated autotransporters. This prediction, however, was never experimentally supported, nor was the function of PmpD known. Here, using 1D and 2D PAGE we demonstrate that PmpD is processed into two parts, N-terminal (N-pmpD), middle (M-pmpD) and presumably third, C-terminal part (C-pmpD). Based on localization of the external part on the outer membrane as shown by immunofluorescence, immuno-electron microscopy and immunoblotting combined with trypsinization, we demonstrate that N-pmpD translocates to the surface of bacteria where it non-covalently binds other components of the outer membrane. We propose that N-pmpD functions as an adhesin, as antibodies raised against N-pmpD blocked chlamydial infectivity in the epithelial cells. In addition, recombinant N-pmpD activated human monocytes in vitro by upregulating their metabolic activity and by stimulating IL-8 release in a dose-dependent manner. These results demonstrate that N-PmpD is an autotransporter component of chlamydial outer membrane, important for bacterial invasion and host inflammation.