Transposon mutagenesis identifies novel Mycobacterium bovis BCG genes involved in a dynamic interactions between BCG vaccine and bovine host

BCG is the most widely used vaccine of all time and remains the only licensed vaccine for use against tuberculosis in humans. BCG also protects other species such as cattle against tuberculosis, but due to its incompatibility with current tuberculin testing regimens it is not currently licensed. BCG’s efficacy relates to its ability to persist in the host for weeks, months or even years. However, it is unclear if this ability to persist after vaccination is an active processes maintained by a dynamic interaction between the vaccine strain and its host or whether the bacilli are passive. To investigate this question, we constructed transposon mutant libraries in both BCG Pasteur and BCG Danish strains and inoculated them into bovine lymph nodes. Cattle are well suited to such an assay, as they are naturally susceptible to tuberculosis and are one of the few animal species for which BCG has been proposed to be used. After three weeks, the BCG were recovered and the input and output libraries compared to identify mutants with in vivo fitness defects. Less than 10% of the mutants were identified as affecting in vivo fitness. Phenotypic groups predicted to be important for survival included mycobactin synthesis, sugar transport, reductive sulphate assimilation, PDIM synthesis and cholesterol metabolism, functional groups that are similar to those previously identified as virulence factors in pathogenic mycobacteria. To verify our conclusions we tested the in vivo phenotypes of three genes identified from the transposon library as having a fitness phenotype that had not previously been recognised as involved in virulence, namely pyruvate carboxylase, a hypothetical protein, and a putative cyclopropane-fatty-acyl-phospholipid synthase. BCG strain lacking these genes were able to survive as well as wild type in bovine macrophages yet demonstrated a marked attenuation after inoculation into bovine lymph nodes confirming that they were indeed involved in persistence of BCG in the host. These data show that BCG is far from passive during its interaction with the host, rather it continues to employ its remaining virulence factors, to interact with the host’s innate immune system to persist, a property that is important for its protective efficacy.