Listeria monocytogenes InlP interacts with afadin and facilitates basement membrane crossing.

During pregnancy, the placenta protects the fetus against the maternal immune response, as well as bacterial and viral pathogens. Bacterial pathogens that have evolved specific mechanisms of breaching this barrier, such as Listeria monocytogenes, present a unique opportunity for learning how the placenta carries out its protective function. We previously identified the L. monocytogenes protein Internalin P (InlP) as a secreted virulence factor critical for placental infection. Here, we show that InlP, but not the highly similar L. monocytogenes internalin Lmo2027, binds to human afadin (encoded by AF-6), a protein associated with cell-cell junctions. A crystal structure of InlP reveals several unique features, including an extended leucine-rich repeat (LRR) domain with a distinctive Ca2+-binding site. Despite afadin's involvement in the formation of cell-cell junctions, MDCK epithelial cells expressing InlP displayed a decrease in the magnitude of the traction stresses they could exert on deformable substrates, similar to the decrease in traction exhibited by AF-6 knock-out MDCK cells. L. monocytogenes ΔinlP mutants were deficient in their ability to form actin-rich protrusions from the basal face of polarized epithelial monolayers, a necessary step in the crossing of such monolayers (transcytosis). A similar phenotype was observed for bacteria expressing an internal in-frame deletion in inlP (inlP ΔLRR5) that specifically disrupts its interaction with afadin. However, afadin deletion in the host cells did not rescue the transcytosis defect. We conclude that secreted InlP targets cytosolic afadin to specifically promote L. monocytogenes transcytosis across the basal face of epithelial monolayers, which may contribute to the crossing of the basement membrane during placental infection.

Selective bacterial degradation of the extracellular matrix attaching the gingiva to the tooth.

The junctional epithelium (JE) is a specialized portion of the gingiva that seals off the tooth-supporting tissues from the oral environment. This relationship is achieved via a unique adhesive extracellular matrix that is, in fact, a specialized basal lamina (sBL). Three unique proteins - amelotin (AMTN), odontogenic ameloblast-associated (ODAM), and secretory calcium-binding phosphoprotein proline-glutamine rich 1 (SCPPPQ1) - together with laminin-332 structure the supramolecular organization of this sBL and determine its adhesive capacity. Despite the constant challenge of the JE by the oral microbiome, little is known of the susceptibility of the sBL to bacterial degradation. Assays with trypsin-like proteases, as well as incubation with Porphyromonas gingivalis, Prevotella intermedia, and Treponema denticola, revealed that all constituents, except SCPPPQ1, were rapidly degraded. Porphyromonas gingivalis was also shown to alter the supramolecular network of reconstituted and native sBLs. These results provide evidence that proteolytic enzymes and selected gram-negative periodontopathogenic bacteria can attack this adhesive extracellular matrix, intimating that its degradation could contribute to progression of periodontal diseases.

Role of prostatic epithelium basement membrane incompetence in the pathogenesis of chronic prostatitis.

Prostatic inflammation is associated with infections penetrating through the urethra. This inflammation is treated by long courses of wide-spectrum antibiotics. However, the most frequent cause of prostatitis is Escherichia coli and other enteric flora. Electron microscopy of biopsy specimens from the prostate detected gaps in the prostatic epithelium basement membrane, their size explaining the penetration of enteric flora into the prostate. These data suggest another view on the pathogenesis of prostatitis and approaches to improvement of therapy for this disease.

Influenza virus infection decreases tracheal mucociliary velocity and clearance of Streptococcus pneumoniae.

Influenza virus infections increase susceptibility to secondary bacterial infections, such as pneumococcal pneumonia, resulting in increased morbidity and mortality. Influenza-induced tissue damage is hypothesized to increase susceptibility to Streptococcus pneumoniae infection by increasing adherence to the respiratory epithelium. Using a mouse model of influenza infection followed by S. pneumoniae infection, we found that an influenza infection does not increase the number of pneumococci initially present within the trachea, but does inhibit pneumococcal clearance by 2 hours after infection. To determine whether influenza damage increases pneumococcal adherence, we developed a novel murine tracheal explant system to determine influenza-induced tissue damage and subsequent pneumococcal adherence. Murine tracheas were kept viable ex vivo as shown by microscopic examination of ciliary beating and cellular morphology using continuous media flow for up to 8 days. Tracheas were infected with influenza virus for 0.5-5 days ex vivo, and influenza-induced tissue damage and the early stages of repair to the epithelium were assessed histologically. A prior influenza infection did not increase pneumococcal adherence, even when the basement membrane was maximally denuded or during the repopulation of the basement membrane with undifferentiated epithelial cells. We measured mucociliary clearance in vivo and found it was decreased in influenza-infected mice. Together, our results indicate that exposure of the tracheal basement membrane contributes minimally to pneumococcal adherence. Instead, an influenza infection results in decreased tracheal mucociliary velocity and initial clearance of pneumococci, leading to an increased pneumococcal burden as early as 2 hours after pneumococcal infection.

Identification of a novel protein promoting the colonization and survival of Finegoldia magna, a bacterial commensal and opportunistic pathogen.

Anaerobic bacteria dominate the human normal microbiota, but strikingly little is known about these commensals. Finegoldia magna is a Gram-positive anaerobe found in the skin and at other non-sterile body surfaces, but it is also an opportunistic pathogen. This study describes a novel protein designated FAF (F. magna adhesion factor) and expressed by more than 90% of F. magna isolates. The protein is present in substantial quantities at the F. magna surface but is also released from the surface. FAF forms large protein aggregates in solution and surface-associated FAF causes bacterial clumping. In skin F. magna bacteria were localized to the epidermis, where they adhere to basement membranes. FAF was found to mediate this adhesion via interactions with BM-40, a basement membrane protein. The biological significance of FAF is further underlined by the observation that it blocks the activity of LL-37, a major human antibacterial peptide. Altogether, the data demonstrate that FAF plays an important role in colonization and survival of F. magna in the human host.

Human laminin-332 degradation by Candida proteinases.

BACKGROUND: Human laminin-332 (Lm-332) degradation by 12 Candida strains and effects of synthetic proteinase inhibitors [Ilomastat (ILM), EDTA, chemically modified tetracycline-3(CMT-3), CMT-308, synthetic peptide CTT-2, and Pefabloc] were studied. MATERIALS AND METHODS: Laminin-332 was incubated with sonicated cell fractions and 10 times concentrated cell-free fractions of reference and clinical strains of C. albicans, C. dubliniensis, C. guilliermondii, C. glabrata, C. krusei, and C. tropicalis. Proteolysis, pH effects, and inhibitors were analyzed by fluorography and zymography. RESULTS: Cell fractions of all species except C. guilliermondii and cell-free fractions of C. albicans, and C. dubliniensis showed 20-70 kDa gelatinases at pH 5.0 and 6.0. At pH 7.6, C. glabrata, C. krusei, and C. tropicalis cell fractions and C. tropicalis cell-free fractions showed 55-70 kDa gelatinases. CMT-3, CMT-308, and CTT-2 inhibited Candida gelatinases slightly better than Pefabloc, ILM, and EDTA. No Candida fractions degraded Lm-332 at pH 7.6, but at pH 5.0, 100 kDa bands were generated by cell fractions of C. dubliniensis and C. tropicalis; C. albicans and C. glabrata clinical strains; and C. guilliermondii reference strain. C. krusei reference strain yielded three 100-130 kDa bands. C. albicans, C. dubliniensis, and C. tropicalis reference and clinical strain's cell-free fractions generated 100 kDa band. CONCLUSIONS: Laminin-332 degradation is pH-dependent and differences exist between studied Candida strains. Lm-332 degradation can exert functional disturbances on basement membrane integrity, possibly aiding Candida cell invasion into tissues. Certain synthetic matrix metalloproteinase inhibitors (CMTs, CTT) can inhibit Candida proteinases and may be therapeutically useful in future.

Interaction of Campylobacter jejuni with extracellular matrix components.

The adhesion of three strains of Campylobacter jejuni to coverslips and microwells coated with isolated extracellular matrix components, fibronectin, laminin and types I, III, IV and V collagens was studied. Fibronectin mediated the adherence of C. jejuni, but there were differences in the binding capacities of the strains. Type I, III and V collagens mediated very strongly the attachment of two strains of C. jejuni. All three strains attached weakly to basement membrane-specific type IV collagen. Laminin was capable of mediating the adhesion only when present at a higher concentration. The observations indicate that extracellular matrix components may serve as anchor molecules for C. jejuni adhesion and that several attachment mechanisms occur simultaneously.

Early host cell-Molluscum contagiosum virus interactions.

Basal and suprabasal layers of human epidermis infected with the poxvirus Molluscum contagiosum have been examined with the technique of serial sectioning. Phagocytic vacuoles, formerly not observed in human epidermis, were found exclusively in the basal region. They did not fuse with other virus-containing vacuoles or with lysosomes to form digestive vacuoles. Various stages of uncoating, preceding ejection of the virus core into the cytoplasm, were observed in the virus-containing vacuole. Clusters of cores were commonly found close to or even associated with centriolar structures. Their possible interference with mitosis is discussed in relation to alterations observed in the plasma membrane. It is assumed that excision of gap junction elements precedes the induction of mitosis.

Heparan sulfate as a mediator of herpes simplex virus binding to basement membrane.

Explants of human lip and oral mucosa were infected with herpes simplex virus (HSV) in vitro and the expression of viral antigen was investigated by immunofluorescent antibody staining. Viral antigen was demonstrated in the cells of basal cell layer and lower prickle cell layers. Moreover, an accumulation of viral antigen in the epithelial-mesenchymal junction was observed. To examine the possibility that the basement membrane has an affinity for HSV, the interaction between HSV and major basement membrane components including type IV collagen, laminin, fibronectin, and heparan sulfate was investigated. When tested by a plaque-reduction assay, only heparan sulfate inhibited HSV plaque formation by competing for the virus adsorption to HEp-2 cells. The inhibitory effects of heparan sulfate and heparin were not affected by pre-incubation of these glycosaminoglycans with antithrombin III, whereas de-N-sulfation resulted in a significant reduction of their inhibitory activity. These findings suggest that heparan sulfate is involved in the binding of HSV to the basement membrane and that N-sulfated glucosamine residues of heparan sulfate are essential for HSV binding. The basement membrane may act as a reservoir of HSV in muco-cutaneous tissues.

Normal Breast Milk Limits the Development of Colitis in IL-10-Deficient Mice.

BACKGROUND AND AIMS: This study examined the role of breast milk in neonatal bacterial colonization of the colon and disease progression in IL-10-deficient mice. METHODS: IL-10-deficient mice were cross-fostered at birth and raised until weaning with a normal mother. Results were compared with normal pups cross-fostered to an IL-10-deficient mother. Mice were examined at various ages for histologic disease, levels of colonic bacteria, and proinflammatory cytokine secretion. RESULTS: IL-10-deficient mice that had been cross-fostered to a normal mother demonstrated normal levels of colonic adherent bacteria and reduced TNFalpha and IFN gamma secretion at 2 to 12 weeks of age. Histologic disease was significantly reduced up to 12 weeks of age. Normal mice cross-fostered to an IL-10-deficient mother had increased levels of adherent bacteria at 2 and 4 weeks and increased IFN gamma secretion. This group also demonstrated slight inflammation up until 12 weeks of age. CONCLUSION: Breast milk has a role in neonatal bacterial colonization. Changing the luminal environment of IL-10-deficient mice during the neonatal period alters the natural disease course.

Characterization of the lactoferrin-dependent inhibition of the adhesion of Actinobacillus actinomycetemcomitans, Prevotella intermedia and Prevotella nigrescens to fibroblasts and to a reconstituted basement membrane.

Lactoferrin was previously shown to inhibit the adhesion of A. actinomycetemcomitans, P. intermedia and P. nigrescens to human cells. Lactoferrin was also shown to competitively inhibit the binding of these bacteria to the basement membrane protein laminin. The present study aimed to determine the type of interactions inhibited by lactoferrin. Lactoferrin binds to fibroblast monolayers and Matrigel, a reconstituted basement membrane, through ionic interactions. The adhesion of A. actinomycetemcomitans to these substrata was mainly dependent on the ionic strength of the environment. P. intermedia and P. nigrescens also adhere to fibroblasts mainly by ionic interactions, while their adhesion to Matrigel seems to be mediated by specific mechanisms. Lectin-type interactions were not found to be involved in the binding of these bacteria to the substrata. Treatment of either A. actinomycetemcomitans or fibroblasts with lactoferrin decreased the adhesion in a dose-dependent manner, while lactoferrin treatment of Matrigel alone had no adhesion-counteracting effect. Adhesion of P. intermedia and P. nigrescens to Matrigel was not significantly affected by the ionic strength, but the presence of lactoferrin inhibited the adhesion. Lactoferrin bound to Matrigel, P. intermedia and P. nigrescens was rapidly released, while lactoferrin bound to A. actinomycetemcomitans and fibroblasts was retained. These findings indicate that lactoferrin-dependent inhibition of the adhesion of A. actinomycetemcomitans, P. intermedia and P. nigrescens to fibroblasts and Matrigel can involve binding of lactoferrin to both the bacteria and substrata. The decreased adhesion may be due to blocking of both specific adhesin-ligand as well as non-specific charge-dependent interactions.

Penetration of fimbriate enteric bacteria through basement membranes: a hypothesis.

A mechanism for penetration of basement membranes by Escherichia coli is presented. The mechanism is based on the ability of the S fimbriae of meningitis-associated E. coli to bind to vascular endothelium and choroid plexuses in brain and to basement membranes. On the other hand, the S and the type 1 fimbriae of E. coli immobilize plasminogen and tissue-type plasminogen activator; this process generates proteolytic plasmin activity on the surface of fimbriate cells. Our hypothesis is that bacterium-bound plasma activity, directed to basement membranes through fimbrial binding, promotes bacterial penetration through basement membranes.

Degradation of human subendothelial extracellular matrix by proteinase-secreting Candida albicans.

Candida albicans infections in severely immunocompromized patients are not confined to mucosal surfaces; instead the fungus can invade through epithelial and endothelial layers into the bloodstream and spread to other organs, causing disseminated infections with often fatal outcome. We investigated whether secretion of the C. albicans acid proteinase facilitates invasion into deeper tissues by degrading the subendothelial basement membrane. After cultivation under conditions that induce the secretion of the acid proteinase, C. albicans degraded radioactively metabolically labeled extracellular matrix proteins from a human endothelial cell line. The degradation was inhibited in the presence of pepstatin A, an inhibitor of acid proteinases. Pepstatin A-sensitive degradation of the soluble and immobilized extracellular matrix proteins fibronectin and laminin by proteinase-producing C. albicans was also detected, whereas no degradation was observed when the expression of the acid proteinase was repressed. Our results demonstrate that the C. albicans acid proteinase degrades human subendothelial extracellular matrix; this may be of importance in the penetration of C. albicans into circulation and deep organs.

Growth of Mycoplasma bovis in organ cultures of bovine foetal trachea and comparison with Mycoplasma dispar.

Inoculation of tracheal organ cultures from bovine foetuses with Mycoplasma bovis resulted in a loss of cellular structure of the lamina propria, followed 20-22 days later by lifting and detachment of overlying epithelium. The effect was associated with large numbers of M. bovis, identified by immunoperoxidase labelling and electromicroscopy, infiltrating between the epithelial cells and amassing in the lamina propria, especially in the region of the basement membrane of the epithelium. Ciliary activity was undiminished for up to 18 days following inoculation and little or no cytopathic effect on the ciliated epithelium was seen in spite of the close proximity of large numbers of organisms. In contrast, M. dispar was restricted to the margin of the ciliated epithelium where, as previously reported, it caused pyknosis, sloughing and flattening of the epithelium with consequent loss of ciliary activity. The cytopathology observed for each mycoplasma bore a close similarity to the behaviour of the two mycoplasmas in vivo and it is suggested that the organ culture system may be a useful and relevant system to elucidate the pathogenic mechanisms for each mycoplasma.

Middle ear effusions and structure of the tympanic membrane.

OBJECTIVE: To study the effect of various middle ear effusions on the structure of the lamina propria of the tympanic membrane. METHODS: Sterile and infective middle ear effusions were induced by obstruction of the eustachian tube in specific pathogen-free (SPF) rats and in rats with upper airway infections (URI), respectively. The condition of the tympanic membrane was monitored at regular intervals. After varying survival times, the animals were killed and the tympanic membranes processed for light and electron microscopy. RESULTS: Sterile effusions always resulted in tympanosclerotic lesions. These lesions did not develop in the presence of primary-infected effusions. These effusions had a severe destructive effect on the lamina propria, followed by fibrosis. Generally, secondary infection did not markedly affect preexisting tympanosclerotic lesions. Moreover, calcification disappeared when re-aeration of the middle ear occurred, but the abnormal collagen depositions persisted. CONCLUSIONS: Both sterile and infective effusions result in comprehensive irreversible changes in the lamina propria of the pars tensa. The development of tympanosclerosis is confined to sterile effusions. Mechanical injury and compromised vascularization of the lamina propria are likely to be important etiological factors in the development of tympanosclerosis.

Perlecan in the basement membrane of corneal epithelium serves as a site for P. aeruginosa binding.

PURPOSE: To determine whether binding of Pseudomonas aeruginosa (P. aeruginosa) to the scarified mouse cornea depends on interaction with proteoglycans (PGs). METHODS: Scarified corneas were treated with anti-proteoglycan monoclonal antibodies (MAbs), glycosaminoglycans (GAGs), heparinase III or chondroitin ABC lyase before inoculation with P. aeruginosa strain ATCC 19660 or PAO1. Scanning electron microscopy (SEM) was used to quantitate adherent bacteria. Frozen sections of unwounded and wounded mouse cornea, the latter treated or not treated with heparinase III were stained to spatially localize perlecan [core protein or heparan sulfate (HS) side chains]. Anti-perlecan MAb against the heparan sulfate proteoglycan core protein and succinyl wheat germ agglutinin (sWGA), a lectin which recognizes N-acetyl-glucosamine in heparan sulfate, respectively were used. RESULTS: Anti-perlecan MAb, as well as heparan sulfate, heparin and heparinase III decreased the binding of both bacterial strains to cornea, and the decrease was concentration-dependent. Fluorescence microscopic analysis of sections of mouse cornea immunostained with anti-perlecan MAb showed that perlecan was localized to the epithelial basement membrane. Scarification of the mouse cornea exposed perlecan in the basement membrane and increased bacterial binding to this site was consistent with this exposure. Lectin staining revealed that heparinase treatment removed heparan sulfate side chains of perlecan from the exposed basement membrane, and this removal was consistent with a decrease in bacterial binding. CONCLUSIONS: These studies provide evidence that perlecan, core protein and its heparan sulfate side chains serve as a binding site for Pseudomonas aeruginosa when the basement membrane of the cornea is exposed.

Bacterial adherence to the surface and isolated cell epithelium of the palatine tonsils.

Bacterial adherence to the oropharyngeal epithelium is a significant factor in normal microecology, etiopathogenesis of diseases (tonsillitis, gingivitis) and possibly also induction of immune response. Bacterial adhesion to human tonsillar epithelium of whole tonsils and swabs was studied by fluorescence, scanning, and transmission electron microscopy. The epithelial cell borders were well demarcated. On the apical surface of the cells there were irregular microridges. All forms of microscopy visualized epithelial cells with attached bacteria, often forming microcolonies on the free surface. Some bacteria formed excavations on the cell surface. Most attached bacteria were coccoid, but variously sized rods were also visible. In transmission electron microscopy, epithelial cells with intracellular bacteria were regularly observed.

Diverse microbial interactions with the basement membrane barrier.

During primary contact with susceptible hosts, microorganisms face an array of barriers that thwart their invasion process. Passage through the basement membrane (BM), a 50-100-nm-thick crucial barrier underlying epithelia and endothelia, is a prerequisite for successful host invasion. Such passage allows pathogens to reach nerve endings or blood vessels in the stroma and to facilitate spread to internal organs. During evolution, several pathogens have developed different mechanisms to cross this dense matrix of sheet-like proteins. To breach the BM, some microorganisms have developed independent mechanisms, others hijack host cells that are able to transverse the BM (e.g. leukocytes and dendritic cells) and oncogenic microorganisms might even trigger metastatic processes in epithelial cells to penetrate the underlying BM.