RESUMEN
To effectively analyze Mycoplasma gallisepticum for virulence-associated determinants, the ability to create stable genetic mutations is essential. Global M. gallisepticum mutagenesis is currently limited to the use of transposons. Using the gram-positive transposon Tn4001mod, a mutant library of 110 transformants was constructed and all insertion sites were mapped. To identify transposon insertion points, a unique primer directed outward from the end of Tn4001mod was used to sequence flanking genomic regions. By comparing sequences obtained in this manner to the annotated M. gallisepticum genome, the precise locations of transposon insertions were discerned. After determining the transposon insertion site for each mutant, unique reverse primers were synthesized based on the specific sequences, and PCR was performed. The resultant amplicons were used as unique Tn4001mod mutant identifiers. This procedure is referred to as signature sequence mutagenesis (SSM). SSM permits the comprehensive screening of the M. gallisepticum genome for the identification of novel virulence-associated determinants from a mixed mutant population. To this end, chickens were challenged with a pool of 27 unique Tn4001mod mutants. Two weeks postinfection, the birds were sacrificed, and organisms were recovered from respiratory tract tissues and screened for the presence or absence of various mutants. SSM is a negative-selection screening technique whereby those mutants possessing transposon insertions in genes essential for in vivo survival are not recovered from the host. We have identified a virulence-associated gene encoding dihydrolipoamide dehydrogenase (lpd). A transposon insertion in the middle of the coding sequence resulted in diminished biologic function and reduced virulence of the mutant designated Mg 7.
Asunto(s)
Elementos Transponibles de ADN , Dihidrolipoamida Deshidrogenasa/genética , Mutagénesis Insercional , Mycoplasma gallisepticum/patogenicidad , Enfermedades de las Aves de Corral/microbiología , Enfermedades de las Aves de Corral/patología , Animales , Proteínas Bacterianas , Pollos/microbiología , Dihidrolipoamida Deshidrogenasa/metabolismo , Infecciones por Mycoplasma/microbiología , Infecciones por Mycoplasma/patología , Infecciones por Mycoplasma/veterinaria , Mycoplasma gallisepticum/genética , Mycoplasma gallisepticum/metabolismo , Análisis de Secuencia de ADN , Organismos Libres de Patógenos Específicos , VirulenciaRESUMEN
It was previously demonstrated that avirulent Mycoplasma gallisepticum strain R(high) (passage 164) is lacking three proteins that are expressed in its virulent progenitor, strain R(low) (passage 15). These proteins were identified as the cytadhesin molecule GapA, the putative cytadhesin-related molecule CrmA, and a component of a high-affinity transporter system, HatA. Complementation of R(high) with wild-type gapA restored expression in the transformant (GT5) but did not restore the cytadherence phenotype and maintained avirulence in chickens. These results suggested that CrmA might play an essential role in the M. gallisepticum cytadherence process. CrmA is encoded by the second gene in the gapA operon and shares significant sequence homology to the ORF6 gene of Mycoplasma pneumoniae, which has been shown to play an accessory role in the cytadherence process. Complementation of R(high) with wild-type crmA resulted in the transformant (SDCA) that lacked the cytadherence and virulence phenotype comparable to that found in R(high) and GT5. In contrast, complementation of R(high) with the entire wild-type gapA operon resulted in the transformant (GCA1) that restored cytadherence to the level found in wild-type R(low). In vivo pathogenesis trials revealed that GCA1 had regained virulence, causing airsacculitis in chickens. These results demonstrate that both GapA and CrmA are required for M. gallisepticum cytadherence and pathogenesis.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Adhesión Bacteriana , Proteínas Bacterianas/metabolismo , Infecciones por Mycoplasma/veterinaria , Mycoplasma/patogenicidad , Enfermedades de las Aves de Corral/microbiología , Animales , Proteínas Bacterianas/genética , Pollos/microbiología , Mycoplasma/fisiología , Infecciones por Mycoplasma/microbiología , Infecciones por Mycoplasma/fisiopatología , Enfermedades de las Aves de Corral/fisiopatología , Análisis de Secuencia de ADN , Transformación Bacteriana , VirulenciaRESUMEN
The aim of this study was to assess the efficacy of a modified live Mycoplasma gallisepticum vaccine (GT5) for the protection of chickens against infection and respiratory disease. GT5 was constructed by the reconstitution of the avirulent high passage R (R(high)) strain with the gene encoding the major cytadhesin GapA. GT5 expressed GapA on its surface yet retained the phenotypic characteristics of the parental R(high) strain. Birds vaccinated with GT5 were protected upon challenge with the virulent low passage R (R(low)) strain as evidenced by a complete absence of tracheal lesions 2 and 4 weeks post-challenge, in contrast to sham immunized/challenged control birds. Modest amounts of IgG, and little, if any secretory IgA or IgM anti-M. gallisepticum were found in tracheal washings following vaccination. However, copious amounts of specific IgA were found following challenge, especially in sham immunized birds. This suggests that the tracheal IgG elicited by GT5 vaccination may have been responsible for blocking the initial colonization of R(low), thereby resulting in protection.
Asunto(s)
Vacunas Bacterianas/uso terapéutico , Infecciones por Mycoplasma/prevención & control , Infecciones por Mycoplasma/veterinaria , Mycoplasma/inmunología , Enfermedades de las Aves de Corral/microbiología , Enfermedades de las Aves de Corral/prevención & control , Infecciones del Sistema Respiratorio/prevención & control , Infecciones del Sistema Respiratorio/veterinaria , Vacunación/veterinaria , Animales , Vacunas Bacterianas/administración & dosificación , Pollos , Vías de Administración de Medicamentos/veterinaria , Inmunidad Mucosa , Inmunoglobulina A Secretora/biosíntesis , Inmunoglobulina A Secretora/sangre , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/sangre , Isotipos de Inmunoglobulinas/biosíntesis , Isotipos de Inmunoglobulinas/sangre , Inmunoglobulina M/biosíntesis , Inmunoglobulina M/sangre , Mycoplasma/aislamiento & purificación , Infecciones del Sistema Respiratorio/microbiología , Tráquea/inmunología , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/uso terapéuticoRESUMEN
Comparison of the phenotypic expression of Mycoplasma gallisepticum strain R low (passage 15) to that of strain R high (passage 164) revealed that three proteins, i.e., the cytadhesin molecule GapA, a 116-kDa protein (p116), and a 45-kDa protein (p45), are missing in strain R high. Sequence analysis confirmed that the insertion of an adenine 105 bp downstream of the gapA translational start codon resulted in premature termination of translation in R high. A second adenine insertion had also occurred at position 907. Restoration of expression of wild-type gapA in R high (clone designated GT5) allowed us to evaluate the extent to which the diminished cytadherence capacity could be attributed to GapA alone. The results indicated that GT5 attached to the same limited extent as the parental R high, from which it was derived. The cytadherence capability of the parental R high was not restored solely by gapA complementation alone, indicating that either p116 or p45 or both may play a role in the overall cytadherence process. The gene encoding p116 was found to be immediately downstream of gapA in the same operon and was designated crmA. This gene exhibited striking homology to genes encoding molecules with cytadhesin-related functions in both Mycoplasma pneumoniae and Mycoplasma genitalium. Transcriptional analysis revealed that crmA is not transcribed in R high. We are currently constructing a shuttle vector containing both the wild-type gapA and crmA for transformation into R high to assess the role of CrmA in the cytadherence process.
Asunto(s)
Adhesinas Bacterianas/fisiología , Adhesión Bacteriana , Mycoplasma/fisiología , Adhesinas Bacterianas/química , Adhesinas Bacterianas/genética , Secuencia de Aminoácidos , Células Cultivadas , Humanos , Datos de Secuencia Molecular , Mycoplasma/genética , Sistemas de Lectura AbiertaRESUMEN
A family of abundant surface proteins (Vpmas [variable proteins of Mycoplasma agalactiae]) undergoing phase variation in M. agalactiae has been characterized using monoclonal antibodies and specific polyclonal sera. Two expressed members of 39 kDa (Vpma39) and 34 kDa (Vpma34), which varied in expression between clones of a lineage, shared a common amino-terminal sequence but were immunologically distinct. An amino-terminal oligonucleotide probe identified multiple vpma genes which were clustered within a 14-kb ClaI genomic fragment. Rearrangements were found to have occurred within the vpma locus between clones which correlated with changes in their Vpma phenotype. Two neighboring vpma genes were cloned and sequenced from one M. agalactiae clonal variant expressing Vpma39. The two genes, vpmaX and vpmaY, were orientated divergently and shared highly homologous 5' untranslated regions, 25-amino-acid (aa) lipoprotein leader sequences, and amino-terminal sequences. The vpmaY gene coded for 346 aa and 84% of the open reading frame, comprised of 1.5 units of a large repeat of 186 aa. Although the sequence for an entire second vpmaY repeat was present, it was prematurely terminated by insertion of two nucleotides. The vpmaX gene encoded 221 aa and possessed 102 aa of the 186-aa repeat of vpmaY. Many of the features in common between the vpma genes were also found to be shared by the vsp genes of M. bovis, which also undergo DNA rearrangements concomitant with phenotypic changes. Since M. bovis is the closest phylogenetic relative to M. agalactiae, the vpma and vsp gene families most probably represent homologous systems.