RESUMEN
Adhesion of pathogenic bacteria to target cells is a prerequisite for colonization and further infection. The main adhesins of the emerging sexually transmitted pathogen Mycoplasma genitalium, P140 and P110, interact to form a Nap complex anchored to the cell membrane. Herein, we present the crystal structures of the extracellular region of the virulence factor P110 (916 residues) unliganded and in complex with sialic acid oligosaccharides. P110 interacts only with the neuraminic acid moiety of the oligosaccharides and experiments with human cells demonstrate that these interactions are essential for mycoplasma cytadherence. Additionally, structural information provides a deep insight of the P110 antigenic regions undergoing programmed variation to evade the host immune response. These results enlighten the interplay of M. genitalium with human target cells, offering new strategies to control mycoplasma infections.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Infecciones por Mycoplasma/fisiopatología , Mycoplasma genitalium/fisiología , Receptores de Superficie Celular/metabolismo , Factores de Virulencia/metabolismo , Adhesinas Bacterianas/química , Adhesinas Bacterianas/genética , Sitios de Unión/genética , Membrana Celular/metabolismo , Eritrocitos/metabolismo , Eritrocitos/microbiología , Hemabsorción/genética , Humanos , Modelos Moleculares , Mutación , Infecciones por Mycoplasma/metabolismo , Mycoplasma genitalium/genética , Potasio/metabolismo , Unión Proteica , Conformación Proteica , Receptores de Superficie Celular/química , Factores de Virulencia/química , Factores de Virulencia/genéticaRESUMEN
A number of adherent mycoplasmas have developed highly complex polar structures that are involved in diverse aspects of the biology of these microorganisms and play a key role as virulence factors by promoting adhesion to host cells in the first stages of infection. Attachment activity of mycoplasma cells has been traditionally investigated by determining their hemadsorption ability to red blood cells and it is a distinctive trait widely examined when characterizing the different mycoplasma species. Despite the fact that protocols to qualitatively determine the hemadsorption or hemagglutination of mycoplasmas are straightforward, current methods when investigating hemadsorption at the quantitative level are expensive and poorly reproducible. By using flow cytometry, we have developed a procedure to quantify rapidly and accurately the hemadsorption activity of mycoplasmas in the presence of SYBR Green I, a vital fluorochrome that stains nucleic acids, allowing to resolve erythrocyte and mycoplasma cells by their different size and fluorescence. This method is very reproducible and permits the kinetic analysis of the obtained data and a precise hemadsorption quantification based on standard binding parameters such as the dissociation constant K d. The procedure we developed could be easily implemented in a standardized assay to test the hemadsorption activity of the growing number of clinical isolates and mutant strains of different mycoplasma species, providing valuable data about the virulence of these microorganisms.
Asunto(s)
Hemabsorción/fisiología , Mycoplasma/metabolismo , Mycoplasma/fisiología , Benzotiazoles , Biomasa , Diaminas , Eritrocitos/metabolismo , Eritrocitos/fisiología , Citometría de Flujo/métodos , Fluorescencia , Hemabsorción/genética , Hemaglutinación/genética , Hemaglutinación/fisiología , Mutación/genética , Mycoplasma/genética , Compuestos Orgánicos/metabolismo , Quinolinas , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Three recent isolates of measles virus Fu, IMA, and SMD obtained by using B95a cells did not exhibit hemadsorption with African green monkey red blood cells (AGM-RBC). After long-term passage in Vero cells, these Vero cell-adapted strains derived from three isolates obtained the activity to agglutinate AGM-RBC. The primary sequences of the hemagglutinin (H protein) and fusion glycoproteins (F protein) from these two types of viruses were compared and revealed that several important amino acid residues in the H protein do not converge. After adaptation, Fu strain has an Asn to Tyr substitution at position 481 and IMA strain has two substitutions--an Asp to Asn at position 14 and a Ser to Gly at position 546, SMD strain also has a Ser to Gly substitution at position 546. Since the sequences of the F protein were identical between both types of viruses, the hemadsorption alteration from negative to positive might be the result of these substitutions. Site-directed mutagenesis of the H genes were performed to confirm that the substitution of Ser --> Gly at position 546 and Asn --> Tyr at position 481 in the H protein were responsible for hemadsorption alteration. Anti-CD46 monoclonal antibody (M75 and M160) study made clear that these two substitutions also governed the MV H protein's interaction with CD46 receptor. Our results showed that two important amino acid residues in MV H protein govern the binding to CD46 receptor and hemadsorption. In this paper, we reported a novel amino acid residue at position 546 in MV H protein, which was critical for hemadsorption and CD46 binding.
Asunto(s)
Antígenos CD/metabolismo , Hemaglutininas Virales/metabolismo , Glicoproteínas de Membrana/metabolismo , Morbillivirus/metabolismo , Receptores Virales/metabolismo , Proteínas Virales de Fusión/metabolismo , Sustitución de Aminoácidos , Animales , Línea Celular , Chlorocebus aethiops , Hemabsorción/genética , Hemaglutininas Virales/química , Hemaglutininas Virales/genética , Humanos , Sarampión/virología , Proteína Cofactora de Membrana , Morbillivirus/genética , Mutagénesis Sitio-Dirigida , Unión Proteica , Células Vero , Proteínas Virales de Fusión/genéticaRESUMEN
The genes coding for the surface glycoproteins hemagglutinin-neuraminidase (HN) of the peste des petits ruminants virus (PPRV) and hemagglutinin (H) of rinderpest virus (RPV) were cloned in a cytomagalovirus promoter driven expression vector and expressed transiently in mammalian cells. The protein expression was apparent 24 h after transfection and the expressed proteins were detected at the cell surface. The transiently expressed PPRV HN protein was found to be biologically active in possessing hemadsorption and neuraminidase activities. On the other hand, RPV H protein exhibited neuraminidase activity but was deficient in hemadsorption activity. The substrate specificity of the neuraminidase activity of these two proteins differed distinctly. The presence of neuraminidase activity in both PPRV HN and RPV H proteins is unusual among members of the morbillivirus genus.
Asunto(s)
Proteína HN/biosíntesis , Proteína HN/genética , Virus de la Peste de los Pequeños Rumiantes/genética , Transfección , Animales , Línea Celular , Chlorocebus aethiops , Citomegalovirus/genética , Regulación Enzimológica de la Expresión Génica/genética , Regulación Viral de la Expresión Génica/genética , Genes Virales/genética , Hemabsorción/genética , Humanos , Neuraminidasa/genética , Virus de la Peste de los Pequeños Rumiantes/enzimología , Proteínas Recombinantes/biosíntesis , Células Vero , Proteínas Estructurales Virales/genéticaRESUMEN
The gene encoding the canine herpesvirus (CHV) glycoprotein C (gC) homologue has been identified by sequence homology analyses with other well studied herpesviruses. Previously, we have identified three CHV glycoproteins, gp145/112, gp80 and gp47 using a panel of monoclonal antibodies (MAbs). To determine which CHV glycoprotein corresponds to gC, a recombinant baculovirus which contains the putative CHV gC structural gene under the baculovirus polyhedrin promoter was constructed. The recombinant baculovirus expressed gC-related polypeptides (44-62 kDa), which reacted only with MAbs against CHV gp80, indicating that the previously identified CHV gp80 is the translation product of the gC gene. The baculovirus expressed gC was glycosylated and transported to the surface of infected cells. At least seven neutralizing epitopes were conserved on the gC produced in insect cells. It was found that the recombinant baculovirus infected cells adsorbed murine erythrocytes as is the case for CHV-infected cells. The hemadsorption activity was inhibited by heparin, indicating that the CHV gC binds to heparan sulfate on the surface of murine erythrocytes. Mice immunized with the recombinant gC produced strong neutralizing antibodies. Our results suggest that CHV gC produced in insect cells may be useful as a subunit vaccine to control CHV infections.
Asunto(s)
Herpesvirus Cánido 1/genética , Nucleopoliedrovirus/genética , Proteínas Recombinantes/biosíntesis , Spodoptera/genética , Proteínas del Envoltorio Viral/química , Animales , Antígenos Virales/química , Perros , Hemabsorción/genética , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Spodoptera/citología , Spodoptera/metabolismo , Vacunas Sintéticas/inmunología , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Cytadherence and subsequent parasitism of host cells by the human pathogens, Mycoplasma pneumoniae and Mycoplasma genitalium, are mediated by adhesins and adherence-related accessory proteins. In this report we demonstrate the use of transposon Tn4001 to generate Tn-induced transformants displaying cytadherence-deficient characteristics. Mycoplasma pneumoniae Tn-generated transformant, designated 8R, lacked the high-molecular weight adherence-accessory proteins HMW1/4 and was deficient in hemadsorption and cytadherence capabilities. In transformant 8R, Tn4001 was not localized in or near the hmw1 gene or in the upstream adhesin (p30/hmw3) locus, suggesting an alternate site associated with the regulation of hmw1 gene expression. Sequence analysis identified the transposon insertion site at the crl locus previously reported, although the protein characteristics of transformant 8R differed from the earlier described transformants. The M. genitalium Tn-transformant, designated G26, was also defective in hemadsorption and cytadherence. However, transformant G26 synthesized adhesins P140 and P32 suggesting that Tn4001 transposed into a new gene or site previously unlinked to cytadherence, namely ORF MG032. This study demonstrates the utility of Tn4001 mutagenesis for both M. pneumoniae and M. genitalium which, in the latter case, has special relevance in light of the recent complete characterization of its continuous total genomic sequence.
Asunto(s)
Adhesinas Bacterianas/genética , Adhesión Bacteriana/genética , Moléculas de Adhesión Celular , Elementos Transponibles de ADN , Mycoplasma pneumoniae/genética , Mycoplasma/genética , Adhesinas Bacterianas/fisiología , Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Southern Blotting , Mapeo Cromosómico , Electroforesis en Gel de Poliacrilamida , Electroporación , Regulación Bacteriana de la Expresión Génica , Hemabsorción/genética , Immunoblotting , Proteínas de la Membrana/genética , Mutagénesis Insercional , Mycoplasma/patogenicidad , Mycoplasma pneumoniae/patogenicidad , Sistemas de Lectura Abierta , Operón , Plásmidos , Transformación BacterianaRESUMEN
The neuraminidase (NA) gene of influenza virus A/FPV/Rostock/34 virus (H7N1) was cloned and expressed in Sf9 cells using a baculovirus vector. The expression product had the expected molecular mass and showed neuraminidase activity. NA expressed in Sf9 cells also showed haemagglutinating activity as indicated by its ability to induce haemadsorption of chicken red blood cells. Haemadsorption depended on the presence of neuraminic acid on the erythrocytes, but was not blocked by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, a specific inhibitor of the enzymatic activity. These observations suggest that the N1 neuraminidase has two distinct reactive sites: a catalytic site and a receptor binding site. This concept was confirmed by site-directed mutagenesis which showed that the haemadsorbing activity of FPV NA was abolished when amino acids located on two surface loops at the putative binding site were exchanged. Substitutions on either of the two loops were sufficient for this effect. The mutated NAs retained full neuraminidase activity. In contrast, a mutated NA lacking neuraminidase activity still had haemadsorbing activity.
Asunto(s)
Hemabsorción , Virus de la Influenza A/enzimología , Neuraminidasa/sangre , Animales , Catálisis , Epítopos/genética , Hemabsorción/genética , Pruebas de Hemaglutinación , Virus de la Influenza A/genética , Virus de la Influenza A/metabolismo , Mutagénesis Sitio-Dirigida , Neuraminidasa/genética , Neuraminidasa/metabolismo , Receptores Virales/metabolismo , Especificidad de la Especie , Spodoptera/genéticaRESUMEN
An open reading frame, LMW8-DR, in the African swine fever virus (ASFV) genome possesses striking similarity to the lymphocyte membrane antigen CD2. All characterized CD2 domains, including the amino-terminal signal sequence, IgV, hinge, IgC2, stalk, transmembrane, and proline-rich carboxy cytoplasmic domains, are highly conserved in the ASFV gene. Critical residues for the binding of the lymphocyte function-associated antigen (LFA-3) and CD59 and for T-cell activation are also partially conserved. LMW8-DR is actively transcribed in ASFV-infected swine macrophages and Vero cells at late times in the infection cycle and Vero and COS cells transiently expressing the LMW8-DR open reading frame hemadsorbed swine red blood cells. The structural and functional similarities of LMW8-DR to CD2, a protein that is involved in cell-cell adhesion and immune response modulation, suggest a possible role in the pathogenesis of ASFV infection.