Resumen de Role of pknh in mycobacterium tuberculosis complex virulence. Involvement of lung myeloid cells in vaccine-induced protection of pulmonary delivered bcg
Tuberculosis (TB) is nowadays the most devastating infectious disease associated with a single pathogen. Despite the existence of the widely administered BCG vaccine, its variable protection against pulmonary TB along with the rising appearance of drug resistant strains makes this disease an alarming global health problem, causing an estimated 1.5 million deaths in 2018.
TB is caused in humans and animals by organisms from the Mycobacterium tuberculosis Complex (MTBC), that show more than 99% genetic identity but exhibit distinct host preference and virulence. Unlike the human restriction of M. tuberculosis, Mycobacterium bovis, the causative agent of bovine TB, has one of the broadest host ranges of all known pathogens and exacts a global tremendous economic burden in both developed and developing countries. Although it mainly infects cattle, M. bovis exhibits transmissibility across many other mammalian species and it has been found to be more virulent than M. tuberculosis in different animal models. However, the molecular genetic changes that underly host specificity and infection phenotype within MTBC members have not been fully elucidated.
In this work, we performed a comparison study between M. tuberculosis, M. bovis and M. africanum strains regarding their virulence and dissemination ability in vivo. In addition, we analysed RD900 genomic region across MTBC members using whole genome sequences from different MTBC strains so as to determine its role in the context of MTBC evolutionary history. The RD900 region comprises two homologous genes encoding the serine/threonine protein kinase PknH flanking the tbd2 gene. PknH is involved in the regulation of several bacterial processes and has been associated with virulence in M. tuberculosis. This analysis revealed that RD900 has been independently lost in different MTBC lineages and different strains, resulting in the generation of a single pknH gene. Importantly, all the analysed M. bovis and M. caprae strains carry a conserved deletion within a proline rich-region of pknH genes independent of RD900 presence or absence. We hypothesized that conservation of this deletion in M. bovis may affect PknH function, having a potential role in its virulence and evolutionary adaptation. To explore this hypothesis, we constructed and characterized M. bovis and M. tuberculosis pknH mutant strains. M. bovis knock-in strains containing the M. tuberculosis complete pknH gene revealed a reduced virulence compared to the wild type in the mouse model, suggesting that PknH plays an important role in the differential virulence phenotype of M. bovis vs M. tuberculosis.
The failure of BCG to protect against pulmonary TB results in an urgent need for the development of new effective TB vaccines strategies. The use of alternative routes of administration for the delivery of BCG or new vaccine candidates has emerged a renewed interest during the last years. One of the most studied non-canonical routes of administration is the pulmonary delivery, with the rationale of mimicking the natural route of M. tuberculosis infection. In this regard, preclinical studies in different animal models have shown that pulmonary BCG confers significantly improved protection compared to subcutaneous or intradermal vaccination. However, current immunogenicity studies about mucosal immune responses elicited by pulmonary BCG are focused on adaptive responses, with few data of local innate immunity and BCG interaction with lung myeloid cells.
In the present study, we pulmonary delivered GFP-expressing virulent and attenuated mycobacteria to characterize infected lung myeloid populations and differences in cellular infection dynamics occurring in vivo in the mouse model. We found profound differences in dissemination patterns between M. tuberculosis and BCG. Whereas BCG mainly remained in the macrophage compartment, M. tuberculosis actively spread from macrophages to neutrophils in a mechanism dependent on RD1-containg genes. BCG presence in lungs leads to the activation of lung macrophages. As a consequence, M. tuberculosis replication and neutrophil dissemination is impaired when M. tuberculosis is engulfed by BCG pre-activated macrophages in pulmonary, but not subcutaneous, vaccinated mice, which correlates with a strong vaccine-induced protection. Furthermore, we show that lung macrophages are activated by pulmonary BCG in a mechanism that required the contribution of CD4 T cells for the induction, but not maintenance, of their activation status. Noteworthy, pulmonary BCG induced long-term activation of alveolar macrophages, which maintain their activation status and early bactericidal capacity long after vaccine clearance, suggesting the generation of a memory-like response in lung macrophages. These results highlight a crucial importance of bacterial cell-to-cell infection dynamics for early M. tuberculosis infection success, and indicate that lung macrophages might crucially contribute to the early protection provided by pulmonary live TB vaccines.
