ycobacterium tuberculosis kills more humans than any other pathogen 1 . Whereas most bacterial pathogens cause acute disease, Mtb usually undergoes a years-long infection cycle. Mtb persists in humans in part through parasitism of macrophage phagosomes. Survival in this intracellular niche is accomplished by slowing phagosomal maturation and reducing intracellular killing mechanisms 2-4 , while offering partial cloaking from immune cells and access to lipids and other host nutrients 5,6 . As Mtb interactions with the host play out over years and at diverse anatomical sites, pinpointing specific events that determine tuberculosis (TB) disease outcome is challenging. However, a successful approach has been the comparative profiling of mycobacteria of varying virulence to discover factors selectively present in highly virulent species. Mycobacterium species naturally differ in their potential to infect, persist and cause TB, and transmit among hosts. With an estimated 1.7 billion infections worldwide 1 , only Mtb has broadly colonized the human species, and humans represent its only natural host. These observations highlight the need to identify factors selectively expressed in Mtb but not in other mycobacterial species.Comparative genomics and transcriptomics of Mtb and Bacille Calmette-Guèrin (BCG) have isolated factors selectively present in Mtb, such as the ESX-1 transporter 7 . Whereas genetic techniques are widely used, comparative chemical biology screens are uncommon in mycobacteria. An HPLC-mass spectrometry (MS)-based lipidomics platform was developed for analysis of all chloroform/methanol-extractable mycobacterial lipids 8,9 . Comparative lipidomics of Mtb and BCG identified a previously unknown, Mtb-specific lipid missed by genomics approaches: 1-tuberculosinyladenosine (1-TbAd, 1) 10 . Cyclization of geranylgeranyl pyrophosphate into tuberculosinyl pyrophosphate occurs via the enzyme, Rv3377c, and tuberculosinyl transferase (Rv3378c) generates 1-TbAd, which can chemically rearrange to N 6 -TbAd (2) [10][11][12] . So far 1-TbAd has been detected only in Mtb 12 , so its expression correlates with evolved virulence. However, 1-TbAd has been studied only in laboratoryadapted strains 12,13 , and the extent to which it is produced by patientderived Mtb strains remains unknown.Furthermore, 1-TbAd's function remains unknown. Transposon inactivation of Rv3377c or Rv3378c reduced Mtb uptake, phagosomal acidification and killing of Mtb in mouse macrophages 14 . Therefore, 1-TbAd might influence some aspects of these processes in host cells. However, any host receptor, receptor-independent mechanism or other target of 1-TbAd in host cells remains unknown. Commonly used bioinformatic predictors were not helpful for understanding 1-TbAd function, because it was not possible to identify orthologous biosynthetic genes or similar 1-linked purines in other species. Therefore, diverse candidate mechanisms

Mycobacterium kansasii can cause serious pulmonary disease. It belongs to a group of closely-related species of non-tuberculous mycobacteria known as the M. kansasii complex (MKC). Here, we report a population genomics analysis of 358 MKC isolates from worldwide water and clinical sources. We find that recombination, likely mediated by distributive conjugative transfer, has contributed to speciation and on-going diversification of the MKC. Our analyses support municipal water as a main source of MKC infections. Furthermore, nearly 80% of the MKC infections are due to closely-related M. kansasii strains, forming a main cluster that apparently originated in the 1900s and subsequently expanded globally. Bioinformatic analyses indicate that several genes involved in metabolism (e.g., maintenance of the methylcitrate cycle), ESX-I secretion, metal ion homeostasis and cell surface remodelling may have contributed to M. kansasii’s success and its ongoing adaptation to the human host.

Mycobacterium kansasii is an environmental nontuberculous mycobacterium that causes opportunistic tuberculosis-like disease. It is one of the most closely related species to the Mycobacterium tuberculosis complex. Using M. kansasii as a proxy for the M. kansasii-M. tuberculosis common ancestor, we asked whether introducing the M. tuberculosis-specific gene pair Rv3377c-Rv3378c into M. kansasii affects the course of experimental infection. Expression of these genes resulted in the production of an adenosine-linked lipid species, known as 1-tuberculosinyladenosine (1-TbAd), but did not alter growth in vitro under standard conditions. Production of 1-TbAd enhanced growth of M. kansasii under acidic conditions through a bacterial cell-intrinsic mechanism independent of controlling pH in the bulk extracellular and intracellular spaces. Production of 1-TbAd led to greater burden of M. kansasii in the lungs of C57BL/6 mice during the first 24 h after infection, and ex vivo infections of alveolar macrophages recapitulated this phenotype within the same time frame. However, in long-term infections, production of 1-TbAd resulted in impaired bacterial survival in both C57BL/6 mice and Ccr2−/− mice. We have demonstrated that M. kansasii is a valid surrogate of M. tuberculosis to study virulence factors acquired by the latter organism, yet shown the challenge inherent to studying the complex evolution of mycobacterial pathogenicity with isolated gene complementation. IMPORTANCE This work sheds light on the role of the lipid 1-tuberculosinyladenosine in the evolution of an environmental ancestor to M. tuberculosis. On a larger scale, it reinforces the importance of horizontal gene transfer in bacterial evolution and examines novel models and methods to provide a better understanding of the subtle effects of individual M. tuberculosis-specific virulence factors in infection settings that are relevant to the pathogen.