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.

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.

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.

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.

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.

Cicatrising conjunctivitis with anti-basement membrane autoantibodies in ectodermal dysplasia.

AIMS: To report circulating and mucosa-deposited anti-basement membrane zone autoantibodies in a series of six ectodermal dysplasia patients with severe bilateral cicatrising conjunctivitis and blindness due to both corneal disease and intractable surface inflammation. We also report clinical improvement with steroid-sparing systemic immunosuppression combined with clearance of bacterial colonisation. METHODS: Conjunctival and buccal immunohistopathology, and serological analysis using a panel of epithelial basement membrane zone proteins including the bullous pemphigoid antigen 180 (BP180) were carried out as part of an ocular pemphigoid work-up in each patient. The degree of photophobia, conjunctival inflammation and visual acuity were monitored to evaluate the response to immunosuppression. The mean duration of follow-up was 31 (SD 6) months. RESULTS: Four of the six patients showed positive immunopathology: direct immunofluorescence testing of the conjunctiva in one patient demonstrated linear IgA deposition along the basement membrane zone, and IgG and IgM in the buccal mucosa of another patient. Circulating autoantibodies to BP180 were detected in two other patients. Treatment with systemic immunosuppression, combined with clearance of bacterial colonisation, reduced the severity of photophobia and degree of conjunctival inflammation in 5/6 (83%) patients. CONCLUSIONS: Systemic immunosuppression, used as steroid-sparing therapy, combined with clearance of bacterial colonisation can control inflammation and disabling photophobia, and allow improvement in vision, in a subgroup of ectodermal dysplasia patients who have severe cicatrising conjunctivitis which shares clinical and immunopathological features with ocular mucous membrane pemphigoid.

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.

Contribution of proteases and plasmin-acquired activity in migration of Peptostreptococcus micros through a reconstituted basement membrane.

BACKGROUND/AIMS: Peptostreptococcus micros is a gram-positive bacterium that has been associated with chronic periodontitis and endodontic infections. The aims of this study were to investigate the production of proteases and the acquisition of plasmin activity by rough and smooth morphotypes of P. micros. The contribution of these properties in the migration of bacteria through a reconstituted basement membrane was also evaluated. METHODS: Protease activities were determined using chromogenic and fluorogenic substrates as well as by zymography. Plasminogen binding activity was studied using an enzyme-linked immunosorbent assay. The role of proteases and plasmin-acquired activity in tissue penetration was investigated using Matrigel. RESULTS: The rough morphotype strains of P. micros, but not the smooth morphotype strains, were found to possess chymotrypsin-like and gelatinase activities, both of which were inhibited by a serine protease inhibitor. By zymography, three gelatinase bands (165, 129, and 115 kDa) were identified. Both morphotypes of P. micros can bind human plasminogen on their cell surface. Once bound to P. micros, plasminogen activators of bacterial (streptokinase) and human (urokinase) origins were found to activate plasminogen into plasmin. Our results also showed that plasmin activity can be acquired by P. micros following co-incubation with human brain microvascular endothelial cells in culture. When non-coated cells were used, the rough morphotype strain (HG1262), which possesses chymotrypsin-like and gelatinase activities, showed a better capacity to penetrate a reconstituted basement membrane (Matrigel) than the smooth morphotype strain (HG1251). Penetration of the Matrigel by P. micros HG1262 was inhibited by the presence of a serine protease inhibitor. In addition, cells of P. micros with plasmin activity showed a significantly greater tissue penetration capacity. CONCLUSION: Our study suggests that endogenous proteolytic activities of P. micros as well as plasmin-acquired activity, may facilitate dissemination of bacterial cells to surrounding periodontal tissues and blood vessels.

Binding of pro-matrix metalloproteinase 9 by Fusobacterium nucleatum subsp. nucleatum as a mechanism to promote the invasion of a reconstituted basement membrane.

In this study, we investigated the ability of Fusobacterium nucleatum subsp. nucleatum to increase its tissue-invasive potential by acquiring cell-associated human matrix metalloproteinase 9 (MMP-9) activity. Binding of pro-MMP-9 to fusobacteria was demonstrated by enzyme-linked immunosorbent assay. Zymography and a colorimetric assay showed that bound pro-MMP-9 can be converted into a proteolytically active form. The potential contribution of this acquired host activity in tissue invasion was demonstrated using a reconstituted basement membrane (Matrigel).

In vitro models of tissue penetration and destruction by Porphyromonas gingivalis.

Porphyromonas gingivalis is a gram-negative anaerobic bacterium that is considered the key etiologic agent of chronic periodontitis. Arg- and Lys-gingipain cysteine proteinases produced by P. gingivalis are key virulence factors and are believed to be essential for significant tissue component degradation, leading to host tissue invasion by periodontopathogens. Two in vitro models were used to determine the extent to which P. gingivalis can reach connective tissue. The tissue penetration potential of P. gingivalis was first investigated by using an engineered human oral mucosa model composed of normal human epithelial cells and fibroblasts. Internalized bacteria were assessed by transmission electron microscopy. Bacteria were observed within multilayered gingival epithelial cells and in the space between the stratified epithelium and the lamina propria. A gingipain-null mutant strain of P. gingivalis was found to be less potent in penetrating tissue than the wild-type strain. Proinflammatory responses to P. gingivalis infection were evaluated. P. gingivalis increased the secretion of interleukin-1 beta, interleukin-6, interleukin-8, and tumor necrosis factor alpha. In the second part of the study, the contribution of P. gingivalis gingipains to tissue penetration was investigated by using a reconstituted basement membrane model (Matrigel). The penetration of (14)C-labeled P. gingivalis cells through Matrigel was significantly reduced when leupeptin, a specific inhibitor of Arg-gingipain activity, was added or when a gingipain-null mutant was used. The results obtained with these two relevant models support the capacities of P. gingivalis to infiltrate periodontal tissue and to modulate the proinflammatory response and suggest a critical role of gingipains in tissue destruction.

Binding of plasminogen to Pseudomonas aeruginosa results in formation of surface-associated plasmin and enhanced bacterial invasiveness.

The interaction of Pseudomonas aeruginosa with plasminogen (Plg) is herein reported. Plg bound similarly to laboratory and clinical P. aeruginosa isolates from blood of septicemic patients and stools of asymptomatic carriers. No difference in Plg capture was detected between the piliated PAK strain and its isogenic nonpiliated mutant. Western immunoblotting results suggested that low molecular weight nonpilus adhesins from the bacterial outer membranes accounted for the Plg capture. Bacteria-bound Plg was converted to bioactive plasmin in the presence of exogenous urokinase-type Plg activator. The presence of surface-bound plasmin enhanced significantly the P. aeruginosa capability to invade fibrin gels and a reconstituted basement membrane matrix. These findings support the concept that Plg capture by P. aeruginosa may represent a mechanism which offers advantages to bacterial invasiveness through tissue barriers.

Prevention of fungal infection in transplantation.

Despite significant advances in the diagnosis, prevention, and treatment of fungal infection in transplant recipients, this infection complication remains a major cause of morbidity and mortality. Our understanding of the pathogenesis of the different fungal microorganisms has enabled us to identify patients at risk for such infections. While Candida infection remains a major complication in patients with intra-abdominal solid organ transplantations in which the bowel is surgically manipulated, Aspergillus infection remains the main fungal complication in lung transplantation recipients. The incidence of all types of fungal infection remains around 5-10%, while mortality following Aspergillus infection remains around 70%. Suppression of Candida growth at the time of surgical manipulation of the bowel should be the mainstay of prevention of this infection in intra-abdominal organ transplantation. Fluconazole is effective and relatively safe at 100-400 mg daily for the first 1-3 months post-liver transplantation. Prevention strategies toward Aspergillus infections remain elusive, but a number of manipulations, such as inhaled liposomal preparations post-organ transplantation or the preemptive use or universal prophylaxis of itraconazole are being validated. The next step is to determine the clinical value of molecular diagnostic techniques for the identification and preemptive therapy of patients at risk for the variety fungal infections.

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.

Streptococcus anginosus adheres to vascular endothelium basement membrane and purified extracellular matrix proteins.

The mechanisms of bacterial adherence in the initial stages of native valve endocarditis are unclear, especially in patients without valve disease or the presence of a platelet-fibrin thrombus. Extracellular matrix may act as a ligand in areas of exposed basement membrane on the endothelial monolayer. In this study, adherence of 55 clinical blood and 21 oral viridans streptococcal isolates was examined using purified extracellular matrix compounds. The majority of blood and oral isolates exhibited adherence to purified laminin, fibronectin, and fibrinogen, with lesser adherence to type I and IV collagens. Adherence to laminin and fibronectin was concentration dependent, saturable, and competitively inhibited with soluble ligand. A Streptococcus anginosus isolate and other viridans strains exhibiting a strong laminin adherence phenotype bound extensively to the endothelial aspect of human and porcine valve tissue sections and were inhibited by soluble laminin and anti-laminin antibody fragments. Using a novel native porcine valve explant adherence model, we localized binding to areas of exposed basement membrane by confocal and scanning electron microscopy. These studies support the hypothesis that bacterial adherence to exposed basement membrane plays a role in the initial phase of native valve endocarditis.

Inhibition of the adherence of Escherichia coli strains to basement membrane by Lactobacillus crispatus expressing an S-layer.

AIMS: This study aimed to evaluate the efficiency with which Lactobacillus crispatus JCM 5810 inhibited the adhesion of enteric pathogens to a synthetic basement membrane and to elucidate the mechanism underlying the inhibition. METHODS AND RESULTS: Lactobacillus crispatus JCM 5810 inhibited the adhesion of three diarrhoeagenic Escherichia coli strains to a reconstituted basement membrane preparation called Matrigel, used as a model of a damaged intestinal tissue site. Inhibition was also observed with the use of immobilized laminin, a major component of Matrigel, but diminished after the removal of S-layer protein (CbsA) from JCM 5810 cells. The isolated CbsA inhibited the adhesion of E. coli to both Matrigel and immobilized laminin. Lactobacillus crispatus JCM 5810 and CbsA seem to inhibit pathogenic E. coli from adhering to basement membrane via competition with laminin molecules for binding sites. CONCLUSIONS: These results suggested that not only Lact. crispatus JCM 5810 cells but CbsA alone might prevent pathogens from colonizing damaged intestinal tissues. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to show the applied aspect of Lactobacillus S-layer protein.

Peritonitis increases MMP-9 activity in peritoneal effluent from CAPD patients.

Patients undergoing long-term continuous ambulatory peritoneal dialysis (CAPD) sometimes experience ultrafiltration failure. Mesothelial basement membrane thickening and the accumulation of submesothelial fibrotic tissue are common features of the diseased peritoneum. Peritonitis can lead to ultrafiltration failure, but the precise mechanism is not clear. The key enzymes in extracellular matrix (ECM) remodeling, namely matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), are produced by human peritoneal mesothelial cells. Using peritoneal effluent from 13 CAPD patients with peritonitis and 7 noninfected CAPD control individuals, we examined MMP and TIMP activities by gelatin and reverse zymography. Latent and activated types of MMP-2 and -9, and TIMP-1 and -2 were identified in peritoneal effluent (from all CAPD patients). Levels of latent and activated type MMP-9, as well as of TIMP-1 activities were higher at the onset of peritonitis than either during the recovery phase of peritonitis and/ or in control individuals. Activated MMP-9 activity positively correlated with leukocyte numbers and IL-6 levels in peritoneal effluent. Activities of MMP-2 and TIMP-2 in peritoneal effluent did not change between the onset of peritonitis and recovery. We concluded that increased MMP-9 and TIMP-1 levels might be associated with peritoneal ECM remodeling during peritonitis.

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.

Role of the cell wall phenolic glycolipid-1 in the peripheral nerve predilection of Mycobacterium leprae.

The cell wall of pathogenic mycobacteria is abundant with complex glycolipids whose roles in disease pathogenesis are mostly unknown. Here, we provide evidence for the involvement of the specific trisaccharide unit of the phenolic glycolipid-1 (PGL-1) of Mycobacterium leprae in determining the bacterial predilection to the peripheral nerve. PGL-1 binds specifically to the native laminin-2 in the basal lamina of Schwann cell-axon units. This binding is mediated by the alpha(2LG1, alpha2LG4, and alpha2LG5 modules present in the naturally cleaved fragments of the peripheral nerve laminin alpha2 chain, and is inhibited by the synthetic terminal trisaccharide of PGL-1. PGL-1 is involved in the M. leprae invasion of Schwann cells through the basal lamina in a laminin-2-dependent pathway. The results indicate a novel role of a bacterial glycolipid in determining the nerve predilection of a human pathogen.

Plasminogen activation in degradation and penetration of extracellular matrices and basement membranes by invasive bacteria.

Methods to assess in vitro the role of plasminogen activation in enterobacterial degradation of extracellular matrices and their protein components as well as in penetration through basement membrane are described. Development of these methods was initiated after the findings that enterobacterial surface structures (fimbriae and the Pla surface protease) function in plasminogen activation as well as in laminin- and/or fibronectin-specific adhesion. Enterobacteria with these properties degrade radiolabeled laminin as well as metabolically labeled extracellular matrix from cultured endothelial or epithelial cells. Plasmin-coated bacteria also penetrate through the reconstituted basement membrane preparation Matrigel. The processes are dependent on plasminogen activation by the invasive bacteria. The results suggest a pathogenic similarity between enterobacteria and tumor cells in cellular metastasis through tissue barriers.