Transient Requirement of the PrrA-PrrB Two-Component System for Early Intracellular Multiplication of Mycobacterium tuberculosis

Adaptive regulation of gene expression in response to environmental changes is a general property of bacterial pathogens. By screening an ordered transposon mutagenesis library of Mycobacterium tuberculosis, we have identified three mutants containing a transposon in the coding sequence or in the 5′ regions of genes coding for two-component signal transduction systems (trcS, regX3, prrA). The intracellular multiplication capacity of the three mutants was investigated in mouse bone marrow-derived macrophages. Only the prrA mutant showed a defect in intracellular growth during the early phase of infection, and this defect was fully reverted when the mutant was complemented with prrA-prrB wild-type copies. The mutant phenotype was transient, as after 1 week this strain recovered full growth capacity to reach levels similar to that of the wild type at day 9. Moreover, a transient induction of prrA promoter activity was observed during the initial phase of macrophage infection, as shown by a prrA promoter-gfp fusion in M. bovis BCG infecting the mouse macrophages. The concordant transience of the prrA mutant phenotype and prrA promoter activity indicates that the PrrA-PrrB two-component system is involved in the environmental adaptation of M. tuberculosis, specifically in an early phase of the intracellular growth, and that, similar to other facultative intracellular parasites, M. tuberculosis can use genes temporarily required at different stages in the course of macrophage infection

Persistence and Protective Efficacy of a Mycobacterium tuberculosis Auxotroph Vaccine

New vaccines against tuberculosis are urgently required because of the impressive incidence of this disease worldwide and the highly variable protective efficacy of the current vaccine. The possibility of creating new live vaccines by the rational attenuation of strains from the Mycobacterium tuberculosis complex was investigated. Two auxotrophic mutants of M. tuberculosis and M. bovis BCG were constructed by disruption of one of their purine biosynthetic genes. These mutants appeared unable to multiply in vitro within mouse bone-marrow derived macrophages. They were also attenuated in vivo in the mouse and guinea pig animal models. In guinea pigs, the two mutants induced strong delayed-type hypersensitivity response to purified protein derivative. In a preliminary experiment, the two mutants were compared to the BCG vaccine for their protective efficacy in a challenge against aerosolized virulent M. tuberculosis in the guinea pig model. Both mutants conferred some level of protection. These experiments demonstrate that the rational attenuation of M. tuberculosis could lead to the design of new candidate live vaccines against tuberculosis

Neutrophils rapidly migrate via lymphatics after Mycobacterium bovis BCG intradermal vaccination and shuttle live bacilli to the draining lymph nodes

International audienceThe early innate response after Mycobacterium bovis BCG vaccination is poorly characterized, but probably decisive for subsequent protective immunity against tuberculosis. Therefore, we vaccinated mice with fluorescent BCG strains in the ear dorsum, as a surrogate of intradermal vaccination in humans. During the first three days, we tracked BCG host cells migrating out of the dermis to the auricular draining lymph nodes (ADLN). Resident skin dendritic cells (DCs) or macrophages did not play a predominant role in early BCG capture and transport to ADLN. The main BCG host cells rapidly recruited both in the dermis and ADLN were neutrophils. Fluorescent green or red BCG strains injected into non-overlapping sites were essentially sheltered by distinct neutrophils in the ADLN capsule indicating that neutrophils had captured bacilli in peripheral tissue and transported them to the lymphoid organ. Strikingly, we observed BCG-infected neutrophils in the lumen of lymphatic vessels by confocal microscopy on ear dermis. Fluorescent-labeled neutrophils injected into the ears, accumulated exclusively into the ipsilateral ADLN capsule after BCG vaccination. Thus, we provide in vivo evidence that neutrophils, like DCs or inflammatory monocytes, migrate via afferent lymphatics to lymphoid tissue and can shuttle live microorganisms

Attenuation of virulence by disruption of the Mycobacterium tuberculosis erp gene

International audienceThe virulence of the mycobacteria that cause tuberculosis depends on their ability to multiply in mammalian hosts. Disruption of the bacterial erp gene, which encodes the exported repetitive protein, impaired multiplication of M. tuberculosis and M. bovis Bacille Calmette-Guérin in cultured macrophages and mice. Reintroduction of erp into the mutants restored their ability to multiply. These results indicate that erp contributes to the virulence of M. tuberculosis