Genome-wide phenotypic insights into mycobacterial virulence using Drosophila melanogaster

Drosophila melanogaster (Drosophila) is one of the most extensively studied animal models we have, with a broad, advanced, and organized research community. Yet, Drosophila has barely been exploited to understand the underlying mechanisms of mycobacterial infections, which cause some of the deadliest infectious diseases humans are currently battling. Here, we identified mycobacterial genes required for the pathogen's growth during Drosophila infection. Using Mycobacterium marinum (Mmar) to model mycobacterial pathogens, we first validated that an established mycobacterial virulence factor, EccB1 of the ESX-1 Type VII secretion system, is required for Mmar growth within the flies. Subsequently, we identified Mmar virulence genes in Drosophila in a high-throughput genome-wide phenotypic manner using transposon insertion sequencing. Of the 181 identified virulence genes, the vast majority (91%) had orthologs in the tuberculosis-causing M. tuberculosis (Mtb), suggesting that the encoded virulence mechanisms may be conserved across Mmar and Mtb species. By studying one of the identified genes in more depth, the putative ATP-binding protein ABC transporter encoded by mmar_1660, we found that both the Mmar gene and its Mtb ortholog (rv3041c) were required for virulence in human macrophages as well. We pinpointed the probable virulence mechanism of the genes to their requirements for growth during iron limitation, a condition met by mycobacteria during host infection. Together, our results bring forward Drosophila as a promising host model to study and identify mycobacterial virulence factors, providing insights that may transfer to Mtb human infection.