Iron dependent membrane vesicle production in M. tuberculosis

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Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is one of the oldest known human maladies and a major cause of mortality worldwide, killing 1.5 million people each year. Despite the widespread use of an attenuated live vaccine and several antibiotics, there is currently more TB than ever before, with one third of the world population infected with Mtb. This dire situation is compounded by increasing prevalence of antibiotic-resistant Mtb, whose emergence is facilitated by the lengthy course of antibiotic treatment and the ability of Mtb to persist in the host. During infection, the host scavenges essential metal ions, particularly iron, as part of an antimicrobial strategy known as “nutritional immunity”. In response to iron limitation, pathogens, including Mtb, synthesize not only high affinity iron sequestering molecules, but also virulence determinants and other factors that allow the pathogen to withstand the immune attack. Accumulating evidence shows that pathogenic bacteria concentrate and pack virulence factors into small membrane vesicles (MVs) that are released into the extracellular milieu and have the capacity to influence pathogen-host interplay. In particular, it was shown recently that Mtb also produces and releases MVs, which contain immunologically active molecules. Thus, MVs might be a tool used by Mtb to overcome host defenses and, as such, are potential targets of therapeutic interference. Furthermore, immunization with isolated MVs elicits a protective immune response against TB in mice. Although these findings indicate that MVs play an important role in host- pathogen interactions, very little is known regarding the biogenesis, regulation and functions of Mtb MVs. Our findings indicate that in response to iron limitation Mtb significantly enhances production of MVs and modifies their content. To identify factors involved in MV biogenesis in Mtb, we have designed a genetic screen based on the properties of MVs produced under iron limitation to identify mutants with altered MVs production. In addition to identifying genes required for normal MV formation, the availability of these mutants will facilitate effective efforts to elucidate the relevance of MV production for Mtb pathogenesis. In a complementary approach, we will test the hypothesis that under conditions of iron limitation, MV production is controlled by the machinery that regulates iron homeostasis in Mtb. Specifically, we will test whether the master transcriptional regulator of iron uptake, iron transporters, and siderophores are required for normal MV biogenesis during iron limitation. The results of these studies will help elucidate a still poorly understood mechanism used by Mtb to interact with the host and identify new points of intervention for development of new therapeutic or preventive therapies.