RESUMO
We have previously reported the transcriptomic and lipidomic profile of the first-generation, hygromycin-resistant (HygR) version of the BCGΔBCG1419c vaccine candidate, under biofilm conditions. We recently constructed and characterized the efficacy, safety, whole genome sequence, and proteomic profile of a second-generation version of BCGΔBCG1419c, a strain lacking the BCG1419c gene and devoid of antibiotic markers. Here, we compared the antibiotic-less BCGΔBCG1419c with BCG. We assessed their colonial and ultrastructural morphology, biofilm, c-di-GMP production in vitro, as well as their transcriptomic and lipidomic profiles, including their capacity to activate macrophages via Mincle and Myd88. Our results show that BCGΔBCG1419c colonial and ultrastructural morphology, c-di-GMP, and biofilm production differed from parental BCG, whereas we found no significant changes in its lipidomic profile either in biofilm or planktonic growth conditions. Transcriptomic profiling suggests changes in BCGΔBCG1419c cell wall and showed reduced transcription of some members of the DosR, MtrA, and ArgR regulons. Finally, induction of TNF-α, IL-6 or G-CSF by bone-marrow derived macrophages infected with either BCGΔBCG1419c or BCG required Mincle and Myd88. Our results confirm that some differences already found to occur in HygR BCGΔBCG1419c compared with BCG are maintained in the antibiotic-less version of this vaccine candidate except changes in production of PDIM. Comparison with previous characterizations conducted by OMICs show that some differences observed in BCGΔBCG1419c compared with BCG are maintained whereas others are dependent on the growth condition employed to culture them.
Assuntos
Vacina BCG , Biofilmes , GMP Cíclico , Lipidômica , Macrófagos , Mycobacterium bovis , Fator 88 de Diferenciação Mieloide , Transcriptoma , Animais , Fator 88 de Diferenciação Mieloide/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Camundongos , Macrófagos/metabolismo , Macrófagos/imunologia , Vacina BCG/imunologia , GMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , Mycobacterium bovis/genética , Mycobacterium bovis/imunologia , Biofilmes/crescimento & desenvolvimento , Citocinas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Perfilação da Expressão Gênica , Lectinas Tipo CRESUMO
Mycobacterium tuberculosis and M. smegmatis form drug-tolerant biofilms through dedicated genetic programs. In support of a stepwise process regulating biofilm production in mycobacteria, it was shown elsewhere that lsr2 participates in intercellular aggregation, while groEL1 was required for biofilm maturation in M. smegmatis. Here, by means of RNA-Seq, we monitored the early steps of biofilm production in M. bovis BCG, to distinguish intercellular aggregation from attachment to a surface. Genes encoding for the transcriptional regulators dosR and BCG0114 (Rv0081) were significantly regulated and responded differently to intercellular aggregation and surface attachment. Moreover, a M. tuberculosis H37Rv deletion mutant in the Rv3134c-dosS-dosR regulon, formed less biofilm than wild type M. tuberculosis, a phenotype reverted upon reintroduction of this operon into the mutant. Combining RT-qPCR with microbiological assays (colony and surface pellicle morphologies, biofilm quantification, Ziehl-Neelsen staining, growth curve and replication of planktonic cells), we found that BCG0642c affected biofilm production and replication of planktonic BCG, whereas ethR affected only phenotypes linked to planktonic cells despite its downregulation at the intercellular aggregation step. Our results provide evidence for a stage-dependent expression of genes that contribute to biofilm production in slow-growing mycobacteria.
Assuntos
Proteínas de Bactérias/genética , Biofilmes , Mycobacterium bovis/genética , Mycobacterium tuberculosis/genética , Vacina BCG/genética , Vacina BCG/metabolismo , Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Mycobacterium bovis/metabolismo , Mycobacterium tuberculosis/fisiologia , Óperon , Regulon , Transcrição GênicaRESUMO
Mycobacterium tuberculosis (M. tuberculosis), the causative agent of human tuberculosis (TB), is estimated to be harbored by up to 2 billion people in a latent TB infection (LTBI) state. The only TB vaccine approved for use in humans, BCG, does not confer protection against establishment of or reactivation from LTBI, so new vaccine candidates are needed to specifically address this need. Following the hypothesis that mycobacterial biofilms resemble aspects of LTBI, we modified BCG by deleting the BCG1419c gene to create the BCGΔBCG1419c vaccine strain. In this study, we compared cytokine profiles, bacterial burden, and lung lesions after immunization with BCG or BCGΔBCG1419c before and after 6 months of aerosol infection with M. tuberculosis H37Rv in the resistant C57BL/6 mouse model. Our results show that in infected mice, BCGΔBCG1419c significantly reduced lung lesions and IL-6 in comparison to the unmodified BCG strain, and was the only vaccine that decreased production of TNF-α and IL-10 compared to non-vaccinated mice, while vaccination with BCG or BCGΔBCG1419c significantly reduced IFN-γ production. Moreover, transcriptome profiling of BCGΔBCG1419c suggests that compared to BCG, it has decreased expression of genes involved in mycolic acids (MAs) metabolism, and antigenic chaperones, which might be involved in reduced pathology compared to BCG-vaccinated mice.