Effect of the deletion of lprG and p55 genes in the K10 strain of Mycobacterium avium subspecies paratuberculosis.

The lprG-p55 operon of Mycobacterium tuberculosis, M. bovis and M. avium strain D4ER has been identified as a virulence factor involved in the transport of toxic compounds. LprG is a lipoprotein that modulates the host immune response against mycobacteria, whereas P55 is an efflux pump that provides resistance to several drugs. In the present study we search for, and characterize, lprg and p55, putative virulence genes in Mycobacterium avium subsp. paratuberculosis (MAP) to generate a live-attenuated strain of MAP that may be useful in the future as live-attenuated vaccine. For this purpose, we generated and evaluated two mutants of MAP strain K10: one mutant lacking the lprG gene (ΔlprG) and the other lacking both genes lprG and p55 (ΔlprG-p55). None of the mutant strains showed altered susceptibility to first-line and second-line antituberculosis drugs or ethidium bromide, only the double mutant had two-fold increase in clarithromycin susceptibility compared with the wild-type strain. The deletion of lprG and of lprG-p55 reduced the replication of MAP in bovine macrophages; however, only the mutant in lprG-p55 grew faster in liquid media and showed reduced viability in macrophages and in a mouse model. Considering that the deletion of both genes lprG-p55, but not that of lprG alone, showed a reduced replication in vivo, we can speculate that p55 contributes to the survival of MAP in this animal model.

Efficient method for targeted gene disruption by homologous recombination in Mycobacterium avium subspecie paratuberculosis.

Targeted gene disruption by homologous recombination, has been widely used in mycobacterium species to understand the genetic basis of virulence and persistence in the host and to develop efficacious potential live vaccines. However, in slow growing pathogenic mycobacteria as Mycobacterium avium subsp paratuberculosis (MAP), these methods have been inefficient, in part due to the low frequency of legitimate homologous recombination. Another feature of mycobacteria is the low efficiency of transformation; therefore, some years ago, a phage-mediated transduction process was developed to introduce DNA into mycobacteria. This strategy is very efficient, due to the high rate of infection of the phage. This report describes a genetic method for the generation of targeted deletion mutations in MAP by allelic exchange using in vitro-generated specialized transducing mycobacteriophages, which does not require the critical packaging step and that could also be applied to other mycobacteria. We provide a detailed gene deletion methodology and demonstrate the use of this genetic system by deleting the mce4 operon of MAP. Finally, our results showed that the deletion of mce4 in MAP induces triacylglycerol accumulation; alter morphology and aggregation in liquid culture.

Protection efficacy of Argentinian isolates of Mycobacterium avium subsp. paratuberculosis with different genotypes and virulence in a murine model.

Paratuberculosis is a chronic disease caused by Mycobacterium avium subsp. paratuberculosis (Map). The disease causes economic losses and, therefore, it is imperative to follow proper control strategies, which should include an effective vaccine. Several strategies have assessed the virulence and immune response of Map strains that could be used as a vaccine. This study evaluates the degree of virulence, immune response, and protection of Argentinian strains of Map with different genotype in a murine model. Four local isolates (Cattle type) with different genotypes (analyzed by MIRU-VNTR and SSRs) were selected and evaluated in a virulence assay in BALB/c mice. This assay allowed us to differentiate virulent and low-virulence Map strains. The less virulent strains (1543/481 and A162) failed to induce a significant production of the proinflammatory cytokine IFNg, whereas the virulent strain 6611 established infection along with a proinflammatory immune response. On the other hand, the virulent strain 1347/498 was efficient in establishing a persistent infection, but failed to promote an important Th1 response compared with 6611 at the evaluated time. We selected the low-virulence strain 1543/498 as a live vaccine and the virulent strain 6611 as a live and inactivated vaccine in a protection assay in mice. Strain 1543/481 failed to protect the animals from challenge, whereas strain 6611, in its live and inactivated form, significantly reduced the CFUs count in the infected mice, although they had different immunological response profiles. The inactivated virulent strain 6611 is a potential vaccine candidate against paratuberculosis to be tested in cattle.

Diferencias proteómicas entre una cepa argentina de Mycobacterium bovis con la cepa de referencia AN5 / Proteomic differences between an argentine mycobacterium bovis strain and the reference AN5 strain

La tuberculosis bovina es en Argentina una enfermedad que provoca graves pérdidas económicas y que afecta a un 5 por ciento del ganado. En un trabajo previo de tipificación de cepas por RFLP mediante el uso de las sondas PGRS y DR se identificó una cepa altamente predominante que se llamó AA. A diferencia de la cepa salvaje AA, la cepa de referencia AN5, de origen europeo, que se utiliza para elaborar la tuberculina, es una cepa adaptada al crecimiento en laboratorio, que puede haber sufrido mutaciones en genes de antígenos o de virulencia. Para ello se analizó la producción de proteínas secretadas y del extracto celular, de la cepa salvaje AA comparada con la cepa de referencia AN5, con el propósito de identificar diferencias que puedan dar cuenta de la virulencia y para identificar nuevos antígenos. Se utilizaron técnicas como electroforesis en geles de policrilamida, geles de 2 dimensiones y Western blot utilizando antisueros específicos contra antígenos ya caracterizados y sueros de bovinos infectados con tuberculosis confirmada por aislamiento de M. bovis, empleando proteínas celulares y secretadas (a los 25 y 100 días de cultivo) de ambas cepas. Se pudieron identificar, una proteína secretada de aproximadamente 29 kDa y otra de 28 kDa del estracto celular que parecen ser exclusivas o producidas en mayor cantidad por la cepa AA. También, se identificaron otras pero cuyas bandas eran más débiles. En conclusión, algunas de las proteínas identificadas pueden servir para mejorar el diagnóstico de la tuberculosis bovina

Diferencias proteómicas entre una cepa argentina de Mycobacterium bovis con la cepa de referencia AN5 / Proteomic differences between an argentine mycobacterium bovis strain and the reference AN5 strain

La tuberculosis bovina es en Argentina una enfermedad que provoca graves pérdidas económicas y que afecta a un 5 por ciento del ganado. En un trabajo previo de tipificación de cepas por RFLP mediante el uso de las sondas PGRS y DR se identificó una cepa altamente predominante que se llamó AA. A diferencia de la cepa salvaje AA, la cepa de referencia AN5, de origen europeo, que se utiliza para elaborar la tuberculina, es una cepa adaptada al crecimiento en laboratorio, que puede haber sufrido mutaciones en genes de antígenos o de virulencia. Para ello se analizó la producción de proteínas secretadas y del extracto celular, de la cepa salvaje AA comparada con la cepa de referencia AN5, con el propósito de identificar diferencias que puedan dar cuenta de la virulencia y para identificar nuevos antígenos. Se utilizaron técnicas como electroforesis en geles de policrilamida, geles de 2 dimensiones y Western blot utilizando antisueros específicos contra antígenos ya caracterizados y sueros de bovinos infectados con tuberculosis confirmada por aislamiento de M. bovis, empleando proteínas celulares y secretadas (a los 25 y 100 días de cultivo) de ambas cepas. Se pudieron identificar, una proteína secretada de aproximadamente 29 kDa y otra de 28 kDa del estracto celular que parecen ser exclusivas o producidas en mayor cantidad por la cepa AA. También, se identificaron otras pero cuyas bandas eran más débiles. En conclusión, algunas de las proteínas identificadas pueden servir para mejorar el diagnóstico de la tuberculosis bovina (AU)