RESUMEN

Mycoplasma genitalium is the smallest self-replicating microorganism and is implicated in human diseases, including urogenital and respiratory infections and arthritides. M. genitalium colonizes host cells primarily through adherence mechanisms mediated by a network of surface-associated membrane proteins, including adhesins and cytadherence-related proteins. In this paper, we show that cytadherence in M. genitalium is affected by an unrelated protein known as peptide methionine sulfoxide reductase (MsrA), an antioxidant repair enzyme that catalyzes the reduction of methionine sulfoxide [Met(O)] residues in proteins to methionine. An msrA disruption mutant of M. genitalium, constructed through homologous recombination, displayed markedly reduced adherence to sheep erythrocytes. In addition, the msrA mutant was incapable of growing in hamsters and exhibited hypersensitivity to hydrogen peroxide when compared to wild-type virulent M. genitalium. These results indicate that MsrA plays an important role in M. genitalium pathogenicity, possibly by protecting mycoplasma protein structures from oxidative damage or through alternate virulence-related pathways.

Asunto(s)

Infecciones por Mycoplasma/microbiología , Infecciones por Mycoplasma/fisiopatología , Mycoplasma/enzimología , Mycoplasma/patogenicidad , Oxidorreductasas , Animales , Adhesión Bacteriana , Cricetinae , Eritrocitos/microbiología , Eliminación de Gen , Mesocricetus , Metionina Sulfóxido Reductasas , Mycoplasma/fisiología , Estrés Oxidativo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Ovinos , Virulencia/genética

RESUMEN

Although the complete genome of Mycoplasma genitalium has been sequenced, the functional identification of various genes, including those involved in virulence, has not been accomplished. Further compounding these difficulties has been the failure to develop genetic tools in mycoplasmas that permit the assessment of gene and operon function and regulation. To determine whether homologous recombination could be developed as a tool to analyze the function of genes in M. genitalium, a plasmid that replicates in Escherichia coli but not in M. genitalium was constructed to disrupt the cytadherence-related gene mg218 of M. genitalium. The electroporation of this disruption plasmid into wild-type hemadsorption-positive (HA+) M. genitalium cells permitted the isolation of HA- (strain JB1) and partial HA+ (strains JB2 and JB20) transformants. Analysis of the transformants by Southern hybridization indicated that homologous recombination occurred at the mg218 locus by single-crossover events in JB1 and JB2 and by a double-crossover event in JB20. While integration of the disruption construct abolished the expression MG218 in JB1, strains JB2 and JB20 exhibited a truncated MG218 protein (160 kDa), possibly because of in-frame fusion of the disrupted mg218 gene with sequences downstream of the gentamycin-resistance gene present in the disruption construct. Strain JB1, which lacked MG218, displayed a post-translational defect, being unable to maintain the structural integrity of the major adhesin P140 and its operon-related protein P110, in contrast to JB2 and JB20. It appears that MG218 influences the stability of other cytadherence-related proteins in vivo. Thus, targeted gene disruption through homologous recombination will be a powerful and promising tool for investigating the biology and pathogenesis of M. genitalium.

Asunto(s)

Adhesión Bacteriana/genética , Genes Bacterianos , Mycoplasma/citología , Mycoplasma/genética , Recombinación Genética , Farmacorresistencia Microbiana , Transformación Bacteriana

RESUMEN

A promoter probe vector, pGFPUV2, with Green Fluorescent Protein (GFP) as the reporter system was constructed to identify potential mycoplasmal promoter-containing regulatory sequences in E. coli. Libraries of M. pneumoniae and M. genitalium DNA constructed in pGFPUV2 and transformed into E. coli resulted in GFP-expressing clones. Primer extension analysis with E. coli RNA from five M. pneumoniae clones and two M. genitalium clones indicated that transcription originated from the insert DNA fragments of these promoter constructs. Primers based on the insert DNA sequences were used in primer extension reactions with total RNA isolated from M. pneumoniae or M. genitalium. Of the seven primers used, three generated products by primer extension with mycoplasmal RNA. However, only one of the DNAs had a 5' end similar to that obtained in a comparable reaction with E. coli RNA, and the start site of this transcript appeared to originate one base prior to the predicted open reading frame. These results indicate that E. coli can identify mycoplasmal promoters which have transcriptional elements resembling E. coli promoters.

Asunto(s)

Escherichia coli/genética , Genes Reporteros/genética , Vectores Genéticos/genética , Proteínas Luminiscentes/genética , Mycoplasma pneumoniae/genética , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Bacterianos/genética , Células Clonales/química , Células Clonales/citología , Células Clonales/metabolismo , Clonación Molecular , Cartilla de ADN/genética , Cartilla de ADN/metabolismo , Sondas de ADN/genética , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Bases de Datos Factuales , Escherichia coli/química , Genes Bacterianos/genética , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/análisis , Biología Molecular , Mycoplasma/química , Mycoplasma/genética , Mycoplasma pneumoniae/química , Regiones Promotoras Genéticas/genética , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Proteínas Recombinantes/genética , Alineación de Secuencia , Transcripción Genética/genética

RESUMEN

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 Bacteriana

RESUMEN

Adhesins and adhesin-related accessory proteins of the bacterial pathogen, Mycoplasma pneumoniae, are proline-rich in composition and mediate successful parasitism of host target cells. A specific class of peptidyl-proyl cis-trans isomerases (PPIs), called cyclophilins (Cyps), activate proline-rich proteins, and this enzymatic activity is inhibited by the drug, cyclosporin A (CsA). This study builds upon the connection between the structural/functional properties of the proline-rich proteins of M. pneumoniae and the mode of action of CsA to demonstrate that CsA reduces cytadherence capabilities of mycoplasmas, affects colony morphology and can be mycoplasmacidal. As a consequence of CsA treatment early passage mycoplasmas lacked the major adhesin, P1, explaining their cytadherence-negative phenotype. Three mycoplasma proteins with molecular masses of 160, 84 and 80 kDa were identified by CsA-affinity chromatography. A PCR cloned partial cyp gene of M. pneumoniae, which exhibited sequence homologies with prokaryotic and eukaryotic cyclophilins, was present in multiple copies. These results implicate the role of PPIs as important regulators of cytadherence, virulence and growth cycle events in mycoplasmas.

Asunto(s)

Isomerasas de Aminoácido/efectos de los fármacos , Adhesión Bacteriana , Proteínas Portadoras/efectos de los fármacos , Ciclosporinas/metabolismo , Mycoplasma pneumoniae , Isomerasas de Aminoácido/genética , Isomerasas de Aminoácido/fisiología , Secuencia de Bases , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Clonación Molecular , Farmacorresistencia Microbiana/genética , Hemabsorción , Immunoblotting , Datos de Secuencia Molecular , Mycoplasma pneumoniae/química , Mycoplasma pneumoniae/crecimiento & desarrollo , Mycoplasma pneumoniae/fisiología , Isomerasa de Peptidilprolil , Reacción en Cadena de la Polimerasa , Virulencia

RESUMEN

Adhesins and adhesin-related accessory proteins of pathogenic mycoplasmas are required for cytadherence and the subsequent development of disease pathology. The classic example has been Mycoplasma pneumoniae, which causes primary atypical pneumonia in humans. Mutants of M. pneumoniae defective in adhesins (P1 and P30) or in adherence-accessory proteins (HMW1 through HMW4) are unable to colonize host tissues and are avirulent. Mycoplasma genitalium, implicated in nongonococcal, nonchlamydial urethritis, pneumonia, arthritis, and AIDS progression, was found to encode a 140-kDa adhesin that shared both DNA and protein sequence similarities with P1, a major adhesin of M. pneumoniae. In this report, we show that M. genitalium possesses additional homolog sequences to well-characterized adherence-related genes and proteins of M. pneumoniae. The M. genitalium homologs are designated P32 and P69 and correspond to P30 and HMW3 of M. pneumoniae, respectively (J. B. Baseman, p. 243-259, in S. Rottem and I. Kahane, ed., Subcellular biochemistry, vol. 20. Mycoplasma cell membranes, 1993, and D. C. Krause, D. K. Leith, R. M. Wilson, and J. B. Baseman, Infect. Immun. 35:809-817, 1982). Interestingly, the operon-like organizations of P32 and P69 in the M. genitalium genome are similar to the organizations of P30 and HMW3 genes of M. pneumoniae, suggesting that the conservation of these adherence-related genes and proteins might have occurred through horizontal gene transfer events originating from an ancestral gene family.

Asunto(s)

Adhesión Bacteriana/genética , Moléculas de Adhesión Celular/genética , Genes Bacterianos , Mycoplasma/genética , Adhesinas Bacterianas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Clonación Molecular , Immunoblotting , Datos de Secuencia Molecular , Familia de Multigenes , Mycoplasma pneumoniae/genética , Sistemas de Lectura Abierta , Estructura Secundaria de Proteína , Mapeo Restrictivo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Especificidad de la Especie