Fusobacterium nucleatum. ¿un patógeno periodontal promotor de carcinogénesis colorrectal? / Fusobacterium nucleatum. is this periodontal pathogen a promoter of colorectal carcinogenesis?

El Fusobacterium nucleatum es una bacteria anaerobia Gram negativa,es un residente común en el biofi lm oral y se ha encontrado una estrechaasociación entre las fusobacterias y las periodontitis. El Fusobacteriumnucleatum se ha asociado con el cáncer colorrectal, pero la causalidad y el mecanismo subyacente aún no se han establecido. La microbiota intestinal humana tiene un papel reconocido en el cáncer colorrectal. Se ha encontrado que el Fn se adhiere, invade, e induce respuestas inflamatorias oncogénicas que estimulan el crecimiento de las células de cáncer colorrectal a través de un factor de la adhesina FadA.

The anaerobic, Gram-negative bacterial species Fusobacterium nucleatumis common in oral biofi lm and the association between it andperiodontitis is well-established. Fusobacterium nucleatum has beenassociated with colorectal cancer, though causality and the underlyingmechanism have yet to be determined. The role of the human gutmicrobiota in colorectal cancer has been acknowledged. Fusobacteriumnucleatum has been found to adhere to, invade, and induce oncogenicand infl ammatory responses that stimulate the growth of colorectalcancer cells through its unique FadA adhesin.

Leptospira spp.: Novel insights into host-pathogen interactions.

Leptospirosis is a widespread zoonosis caused by pathogenic Leptospira spp. It is an important infectious disease that affects humans and animals. The disease causes economic losses as it affects livestock, with decreased milk production and death. Our group is investigating the genome sequences of L. interrogans targeting surface-exposed proteins because, due to their location, these proteins are capable to interact with several host components that could allow establishment of the infection. These interactions may involve adhesion of the bacteria to extracellular matrix (ECM) components and, hence, help bacterial colonization. The bacteria could also react with the host fibrinolytic system and/or with the coagulation cascade components, such as, plasminogen (PLG) and fibrinogen (Fg), respectively. The binding with the first system generates plasmin (PLA), increasing the proteolytic power of the bacteria, while the second interferes with clotting in a thrombin-catalyzed reaction, which may promote hemorrhage foci and increase bacterial dissemination. Interaction with the complement system negative regulators may help bacteria to evade the host immune system, facilitating the invasion. This work compiles the main described leptospiral proteins that could act as adhesins, as PLG and fibrinogen receptors and as complement regulator binding proteins. We present models in which we suggest possible mechanisms of how leptospires might colonize and invade host tissues, causing the disease. Understanding leptospiral pathogenesis will help to identify antigen candidates that would contribute to the development of more effective vaccines and diagnostic tests.

Gut adhesive Bacillus subtilis spores as a platform for mucosal delivery of antigens.

Bacillus subtilis spores have been used as safe and heat-resistant antigen delivery vectors. Nonetheless, the oral administration of spores typically induces weak immune responses to the passenger antigens, which may be attributed to the fast transit through the gastrointestinal tract. To overcome this limitation, we have developed B. subtilis spores capable of binding to the gut epithelium by means of expressing bacterial adhesins on the spore surface. The resulting spores bound to in vitro intestinal cells, showed a longer transit through the mouse intestinal tract, and interacted with Peyer's patch cells. The adhesive spores increased the systemic and secreted antibody responses to the Streptococcus mutans P1 protein, used as a model antigen, following oral, intranasal, and sublingual administration. Additionally, P1-specific antibodies efficiently inhibited the adhesion of the oral pathogen Streptococcus mutans to abiotic surfaces. These results support the use of gut-colonizing B. subtilis spores as a new platform for the mucosal delivery of vaccine antigens.

Adhesin activity of Leptospira interrogans lipoprotein identified by in vivo and in vitro shotgun phage display.

Leptospira interrogans causes leptospirosis, one of the most common zoonotic diseases in the world. This pathogenic spirochete is able to bind to extracellular matrix, to express virulent factors and to cause host death. Until now, there is no effective human vaccine for the disease. Shotgun phage display genomic libraries of L. interrogans were constructed and used for in vivo biopanning in hamsters and screened for ligands able to bind to LLC-PK1 epithelial cells. In both panning procedures, clones coding for the putative lipoprotein LIC12976 were identified and, in order to confirm its adhesin activity, a recombinant protein was produced in Escherichia coli and showed to interact with A31 fibroblasts, LLC-PK1 and Vero epithelial cells in vitro. Moreover, rLIC12976 was shown to bind to laminin, indicating an adhesin function. This protein was also detected in extracts of L. interrogans from different serovars and it was found to be conserved among pathogenic leptospires. Further, the protein was tested as vaccine candidate and immunization of hamsters with LIC12976 did not confer protection against a lethal challenge with the homologous L. interrogans serovar Copenhageni. Nevertheless, LIC12976 seems to act as an adhesin, and may be important for the host-pathogen interaction, so that its study can contribute to the understanding of the virulence mechanisms in pathogenic leptospires.

Human sepsis-associated Escherichia coli (SEPEC) is able to adhere to and invade kidney epithelial cells in culture

The adhesins of extraintestinal pathogenic Escherichia coli are essential for mediating direct interactions between the microbes and the host cell surfaces that they infect. Using fluorescence microscopy and gentamycin protection assays, we observed that 49 sepsis-associated E. coli (SEPEC) strains isolated from human adults adhered to and invaded Vero cells in the presence of D-mannose (100%). In addition, bacteria concentrations of approximately 2 x 10(7) CFU/mL were recovered from Vero cells following an invasion assay. Furthermore, PCR analysis of adhesin genes showed that 98.0% of these SEPEC strains tested positive for fimH, 69.4% for flu, 53.1% for csgA, 38.8% for mat, and 32.7% for iha. Analysis of the invasin genes showed that 16.3% of the SEPEC strains were positive for tia, 12.3% for gimB, and 10.2% for ibeA. Therefore, these data suggest that SEPEC adhesion to cell surfaces occurs through non-fimH mechanisms. Scanning electron microscopy showed the formation of microcolonies on the Vero cell surface. SEPEC invasiveness was also confirmed by the presence of intracellular bacteria, and ultrastructural analysis using electron transmission microscopy revealed bacteria inside the Vero cells. Taken together, these results demonstrate that these SEPEC strains had the ability to adhere to and invade Vero cells. Moreover, these data support the theory that renal cells may be the predominant pathway through which SEPEC enters human blood vessels.

Human sepsis-associated Escherichia coli (SEPEC) is able to adhere to and invade kidney epithelial cells in culture.

The adhesins of extraintestinal pathogenic Escherichia coli are essential for mediating direct interactions between the microbes and the host cell surfaces that they infect. Using fluorescence microscopy and gentamycin protection assays, we observed that 49 sepsis-associated E. coli (SEPEC) strains isolated from human adults adhered to and invaded Vero cells in the presence of D-mannose (100%). In addition, bacteria concentrations of approximately 2 x 10(7) CFU/mL were recovered from Vero cells following an invasion assay. Furthermore, PCR analysis of adhesin genes showed that 98.0% of these SEPEC strains tested positive for fimH, 69.4% for flu, 53.1% for csgA, 38.8% for mat, and 32.7% for iha. Analysis of the invasin genes showed that 16.3% of the SEPEC strains were positive for tia, 12.3% for gimB, and 10.2% for ibeA. Therefore, these data suggest that SEPEC adhesion to cell surfaces occurs through non-fimH mechanisms. Scanning electron microscopy showed the formation of microcolonies on the Vero cell surface. SEPEC invasiveness was also confirmed by the presence of intracellular bacteria, and ultrastructural analysis using electron transmission microscopy revealed bacteria inside the Vero cells. Taken together, these results demonstrate that these SEPEC strains had the ability to adhere to and invade Vero cells. Moreover, these data support the theory that renal cells may be the predominant pathway through which SEPEC enters human blood vessels.

BmaC, a novel autotransporter of Brucella suis, is involved in bacterial adhesion to host cells.

Brucella is an intracellular pathogen responsible of a zoonotic disease called brucellosis. Brucella survives and proliferates within several types of phagocytic and non-phagocytic cells. Like in other pathogens, adhesion of brucellae to host surfaces was proposed to be an important step in the infection process. Indeed, Brucella has the capacity to bind to culture human cells and key components of the extracellular matrix, such as fibronectin. However, little is known about the molecular bases of Brucella adherence. In an attempt to identify bacterial genes encoding adhesins, a phage display library of Brucella suis was panned against fibronectin. Three fibronectin-binding proteins of B. suis were identified using this approach. One of the candidates, designated BmaC was a very large protein of 340 kDa that is predicted to belong to the type I (monomeric) autotransporter family. Microscopy studies showed that BmaC is located at one pole on the bacterial surface. The phage displaying the fibronectin-binding peptide of BmaC inhibited the attachment of brucellae to both, HeLa cells and immobilized fibronectin in vitro. In addition, a bmaC deletion mutant was impaired in the ability of B. suis to attach to immobilized fibronectin and to the surface of HeLa and A549 cells and was out-competed by the wild-type strain in co-infection experiments. Finally, anti-fibronectin or anti-BmaC antibodies significantly inhibited the binding of wild-type bacteria to HeLa cells. Our results highlight the role of a novel monomeric autotransporter protein in the adhesion of B. suis to the extracellular matrix and non-phagocytic cells via fibronectin binding.

FHA-mediated cell-substrate and cell-cell adhesions are critical for Bordetella pertussis biofilm formation on abiotic surfaces and in the mouse nose and the trachea.

Bordetella spp. form biofilms in the mouse nasopharynx, thereby providing a potential mechanism for establishing chronic infections in humans and animals. Filamentous hemagglutinin (FHA) is a major virulence factor of B. pertussis, the causative agent of the highly transmissible and infectious disease, pertussis. In this study, we dissected the role of FHA in the distinct biofilm developmental stages of B. pertussis on abiotic substrates and in the respiratory tract by employing a murine model of respiratory biofilms. Our results show that the lack of FHA reduced attachment and decreased accumulation of biofilm biomass on artificial surfaces. FHA contributes to biofilm development by promoting the formation of microcolonies. Absence of FHA from B. pertussis or antibody-mediated blockade of surface-associated FHA impaired the attachment of bacteria to the biofilm community. Exogenous addition of FHA resulted in a dose-dependent inhibitory effect on bacterial association with the biofilms. Furthermore, we show that FHA is important for the structural integrity of biofilms formed on the mouse nose and trachea. Together, these results strongly support the hypothesis that FHA promotes the formation and maintenance of biofilms by mediating cell-substrate and inter-bacterial adhesions. These discoveries highlight FHA as a key factor in establishing structured biofilm communities in the respiratory tract.

The localized adherence pattern of an atypical enteropathogenic Escherichia coli is mediated by intimin omicron and unexpectedly promotes HeLa cell invasion.

Enteropathogenic Escherichia coli (EPEC) forms attaching and effacing lesions in the intestinal mucosa characterized by intimate attachment to the epithelium by means of intimin (an outer membrane adhesin encoded by eae). EPEC is subgrouped into typical (tEPEC) and atypical (aEPEC); only tEPEC carries the EAF (EPEC adherence factor) plasmid that encodes the bundle-forming pilus (BFP). Characteristically, after 3 h of incubation, tEPEC produces localized adherence (LA) (with compact microcolonies) in HeLa/HEp-2 cells by means of BFP, whereas most aEPEC form looser microcolonies. We have previously identified nine aEPEC strains displaying LA in extended (6 h) assays (LA6). In this study, we analysed the kinetics of LA6 pattern development and the role of intimin in the process. Transmission electron microscopy and confocal laser microscopy showed that the invasive process of strain 1551-2 displays a LA phenotype. An eae-defective mutant of strain 1551-2 prevented the invasion although preserving intense diffused adherence. Sequencing of eae revealed that strain 1551-2 expresses the omicron subtype of intimin. We propose that the LA phenotype of aEPEC strain 1551-2 is mediated by intimin omicron and hypothesize that this strain expresses an additional novel adhesive structure. The present study is the first to report the association of compact microcolony formation and an intense invasive ability in aEPEC.

Fimbriae of uropathogenic Proteus mirabilis.

Proteus mirabilis is a common causative agent of cystitis and pyelonephritis in patients with urinary catheters or structural abnormalities of the urinary tract. Several types of fimbriae, which are potentially involved in adhesion to the uroepithelium, can be expressed simultaneously by P. mirabilis: mannose-resistant/Proteus-like (MR/P) fimbriae, P. mirabilis fimbriae (PMF), uroepithelial cell adhesin (UCA), renamed by some authors nonagglutinating fimbriae (NAF), and ambient-temperature fimbriae (ATF). Proteus mirabilis is a common cause of biofilm formation on catheter material and MR/P fimbriae are involved in this process. The considerable serious pathology caused by P. mirabilis in the urinary tract warrants the development of a prophylactic vaccine, and several studies have pointed to MR/P fimbriae as a potential target for immunization. This article reviews P. mirabilis fimbriae with regard to their participation in uropathogenesis, biofilm formation and as vaccine targets.

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.

Adenylate cyclase influences filamentous haemagglutinin-mediated attachment of Bordetella pertussis to epithelial alveolar cells.

Attachment to epithelial cells in the respiratory tract is a key event in Bordetella pertussis colonization. Filamentous haemagglutinin (FHA) is an important virulence factor mediating adhesion to host cells. In this study, the relevance of the interaction between FHA and adenylate cyclase toxin (ACT) during bacterial attachment was investigated. Mutants lacking either FHA or ACT showed significantly decreased adherence to epithelial respiratory cells. The use of several ACT-specific monoclonal antibodies and antiserum showed that the decrease in attachment of strains lacking ACT expression could not be explained by the adhesin-like activity of ACT, or a change of any of the biological activities of ACT. Immunoblot analysis showed that the lack of ACT expression did not interfere with FHA localization. An heparin-inhibitable carbohydrate-binding site is crucial in the process of FHA-mediated bacterial binding to epithelial cells. In the presence of heparin attachment of wild-type B. pertussis, but not of the isogenic ACT defective mutant, to epithelial cells was significantly decreased. These results suggest that ACT enhances the adhesive functions of FHA, and modifies the performance of the FHA heparin-inhibitable carbohydrate binding site. We propose that the presence of ACT in the outer membrane of B. pertussis to play a role in the functionality of FHA.

Role of EspA and intimin in expression of proinflammatory cytokines from enterocytes and lymphocytes by rabbit enteropathogenic Escherichia coli-infected rabbits.

Enteropathogenic Escherichia coli (EPEC) produces attaching and effacing (A/E) lesions and watery diarrhea, both of which are intimin and EspA dependent. In this work, we explored the mucosal immune response by detecting cytokine induction in rabbits with diarrhea caused by rabbit EPEC (REPEC). Orally inoculated rabbits exhibited weight loss and mucosal inflammation, developed watery diarrhea, and died (day 7). At day 6 postinoculation, animals were analyzed for the induction of proinflammatory cytokines in enterocytes. The role of lymphocyte-dependent immunity was determined through the expression of proinflammatory cytokines by lymphocytes from Peyer's patches (PP) and the spleen. EspA and intimin mutants were used to explore the role of A/E lesions in the expression of these cytokines. REPEC-infected rabbit enterocytes showed increased interleukin 1beta (IL-1beta), IL-6, IL-8, and tumor necrosis factor alpha (TNF-alpha) mRNA expression, but that of anti-inflammatory IL-10 was increased only slightly. In contrast, intimin mutant-infected rabbits were unable to produce this proinflammatory cytokine profile but did produce a remarkable increase in IL-10 expression. Bacteria lacking EspA increased the expression of IL-8 and TNF-alpha, but that of IL-10 was increased only slightly. PP lymphocytes also produced proinflammatory cytokines, which were dependent on EspA (except for TNF-alpha) and intimin, while IL-10 was induced by EspA and intimin mutants. In contrast, spleen lymphocytes (systemic compartment) were unable to produce IL-1beta and TNF-alpha. These data show the importance of the proinflammatory cytokines secreted by enterocytes and those expressed locally by PP lymphocytes, which can activate effector mechanisms at the epithelium. Furthermore, this cytokine profile, including IL-6 and IL-1beta, which may be involved in the diarrhea produced by EPEC, depends on intimin.

DNA microarray-based genome comparison of a pathogenic and a nonpathogenic strain of Xylella fastidiosa delineates genes important for bacterial virulence.

Xylella fastidiosa is a phytopathogenic bacterium that causes serious diseases in a wide range of economically important crops. Despite extensive comparative analyses of genome sequences of Xylella pathogenic strains from different plant hosts, nonpathogenic strains have not been studied. In this report, we show that X. fastidiosa strain J1a12, associated with citrus variegated chlorosis (CVC), is nonpathogenic when injected into citrus and tobacco plants. Furthermore, a DNA microarray-based comparison of J1a12 with 9a5c, a CVC strain that is highly pathogenic and had its genome completely sequenced, revealed that 14 coding sequences of strain 9a5c are absent or highly divergent in strain J1a12. Among them, we found an arginase and a fimbrial adhesin precursor of type III pilus, which were confirmed to be absent in the nonpathogenic strain by PCR and DNA sequencing. The absence of arginase can be correlated to the inability of J1a12 to multiply in host plants. This enzyme has been recently shown to act as a bacterial survival mechanism by down-regulating host nitric oxide production. The lack of the adhesin precursor gene is in accordance with the less aggregated phenotype observed for J1a12 cells growing in vitro. Thus, the absence of both genes can be associated with the failure of the J1a12 strain to establish and spread in citrus and tobacco plants. These results provide the first detailed comparison between a nonpathogenic strain and a pathogenic strain of X. fastidiosa, constituting an important step towards understanding the molecular basis of the disease.

Characterization of an outer membrane protein associated with haemagglutination and adhesive properties of enteroaggregative Escherichia coli O111:H12.

Diarrhoeagenic Escherichia coli strains of serotype O111:H12 are characterized by their aggregative pattern of adherence on cultured epithelial cells and thus are considered enteroaggregative E. coli (EAEC). We have previously shown that these EAEC strains lack the genes encoding the aggregative fimbriae I and II described in other heterologous EAEC strains. In this paper, we show compelling data suggesting that a plasmid-encoded outer membrane 58 kDa protein termed aggregative protein 58 (Ap58) produced by EAEC O111:H12 strains, is associated with the adherence capabilities and haemagglutination of animal red blood cells. This conclusion is supported by several lines of evidence: (i) adherent O111:H12 strains are able to produce Ap58; (ii) non-adherent O111:H12 strains are unable to produce Ap58; (iii) antibodies raised against Ap58 inhibited adherence and haemagglutination of epithelial and bovine red blood cells, respectively; (iv) a non-adherent E. coli K-12 host strain containing the ap58 gene determinant on plasmid pVM15 displayed abundant adherence to cultured HEp-2 cells; and (v) the purified Ap58 bound specifically to HEp-2 and bovine red blood cells. Our findings indicate that the aggregative adherence in the O111:H12 strains may be also mediated by non-fimbrial adhesins. We believe our data contribute to the understanding of the adherence mechanisms of these organisms.

Systemic dissemination in tuberculosis and leprosy: do mycobacterial adhesins play a role?

More than one century after the discovery of their etiological agents, tuberculosis and leprosy remain as major health threats for humans, and the molecular mechanisms that lead to the development of both diseases are poorly understood. The elucidation of these mechanisms, and especially those allowing for the mycobacteria to systemically disseminate, should facilitate the development of new prophylactic and/or therapeutic strategies. This review is focused on the routes that Mycobacterium tuberculosis and Mycobacterium leprae may use to disseminate within the human body, and the potential roles played by recently characterized adhesins in this process.

Helicobacter pylori: Factores de virulencia, patología y diagnóstico / Helicobacter pylori: virulence factors and their relationship with the gastroduodenal pathology

En este artículo revisamos la evidencia concerniente a los factores de virulencia de Helicobacter pylori, la principal causa de gastritis, úlceras pépticas y algunos tipos de neoplasias. También se hace un resumen de los hallazgos acerca de los posibles modos de transmisión, incluyendo las siguientes rutas: oral-oral, gastro-oral y fecal-oral. La principal evidencia para cada una de esas rutas consisten el aislamiento e identificación del DNA de H pylori en saliva, placa dental, heces y en el agua. También se describen algunos factores de virulencia tales como a) la actividad de ureasa que promueve la liberación de amonia la cual puede inducir daño en el epitelio gástrico; b) adhesinas bacterianas que son fundamentales para el proceso de colonización; c) hemaglutininas las cuales inducen auto-anticuerpos debido a su similaridad bioquímica con antígenos presentes en los grupos sanguíneos; d) presencia del gen asociado a la vacuolización (vacA) y del gen asociado a la citotoxicidad (cagA) que correlaciona con cepas virulentas que exhiben actividad de citotóxica. El perfil de la distribución epidemiológica a nivel mundial de H. pylori está correlacionado con la distribución del cáncer gástrico. También describimos en este artículo la metodología para la diagnosis de H. pylori mediante el cultivo, incluyendo un método económico que genera la atmósfera microaerofílica requerida, un método de ureasa rápido y de bajo costo, así como la visualización de bacterias curvas en frotis de biopsias gástricas.

[Mechanisms of pathogenicity and adhesion in Pasteurella haemolytica]. / Mecanismos de patogenicidad y adherencia de Pasteurella haemolytica.

Pasteurella haemolytica is one of the bacteria most commonly isolated from pneumonic cases in ruminants. Some of the mechanisms and factors involved in the pathogenesis of the disease are partially documented; and the early stages of bacterial colonization have not been totally clarified. Therefore a review is presented in this paper, particularly related with the mechanisms of bacterial pathogenicity responsible of pulmonary damage to ruminants, as well as a detailed analysis of the adherence process.

Hemagglutination properties of Enterococcus.

In total, 86 enterococcal strains including representatives of most of the described species were tested for the ability to agglutinate human, sheep, and rabbit erythrocytes. Five strains did not react with any of the erythrocytes tested, and 81 (94.2%) strains agglutinated only rabbit erythrocytes. The hemagglutination titers ranged from 2 to 64. Loss of the hemagglutination activity was observed when rabbit erythrocytes were treated with trypsin or neuraminidase. Trypsin treatment of the bacterial suspensions also caused loss of the agglutination ability. On the other hand, heat treatment of bacterial suspensions increased the efficiency of the interactions, and higher titers were obtained. Assays for inhibition of hemagglutination were performed with alpha-D-fucose, alpha-D-galactose, beta-D-galactose, D-glucose, N-acetyl-galactosamine, N-acetyl-glucosamine, N-acetylneuraminic acid, N-acetylneuraminic acid-lactose, and fetuin. Only fetuin was able to inhibit the hemagglutination reactions. The results showed that hemagglutination properties are common to the different enterococcal species tested. They also suggest that enterococci possess hemagglutinins of proteic and non-proteic nature that are involved in the attachment to sialic acid-containing receptors on the surface of rabbit erythrocytes.