Mycobacterial Nucleoside Diphosphate Kinase Blocks Phagosome Maturation in Murine Raw 264.7 Macrophages

et al. (2010) Mycobacterial Nucleoside Diphosphate Kinase Blocks Phagosome Maturation in Murine Raw 264.7 Macrophages. PLoS ONE 5(1): e8769. doi:10.1371/journal.pone.0008769 Mycobacterial Nucleoside Diphosphate Kinase Blocks Phagosome Maturation in Murine Raw 264.7 Macrophages Jim Sun 0 Xuetao Wang 0 Alice Lau 0 Ting-Yu Angela Liao 0 Cecilia Bucci 0 Zakaria Hmama 0 Anil Kumar Tyagi, University of Delhi, India 0 1 Division of Infectious Diseases, Department of Medicine, University of British Columbia and Vancouver Coastal Health Research Institute , Vancouver, British Columbia , Canada , 2 Department of Biological and Environmental Sciences and Technologies, University of Salento , Lecce , Italy Background: Microorganisms capable of surviving within macrophages are rare, but represent very successful pathogens. One of them is Mycobacterium tuberculosis (Mtb) whose resistance to early mechanisms of macrophage killing and failure of its phagosomes to fuse with lysosomes causes tuberculosis (TB) disease in humans. Thus, defining the mechanisms of phagosome maturation arrest and identifying mycobacterial factors responsible for it are key to rational design of novel drugs for the treatment of TB. Previous studies have shown that Mtb and the related vaccine strain, M. bovis bacille Calmette-Gue rin (BCG), disrupt the normal function of host Rab5 and Rab7, two small GTPases that are instrumental in the control of phagosome fusion with early endosomes and late endosomes/lysosomes respectively. Methodology/Principal Findings: Here we show that recombinant Mtb nucleoside diphosphate kinase (Ndk) exhibits GTPase activating protein (GAP) activity towards Rab5 and Rab7. Then, using a model of latex bead phagosomes, we demonstrated that Ndk inhibits phagosome maturation and fusion with lysosomes in murine RAW 264.7 macrophages. Maturation arrest of phagosomes containing Ndk-beads was associated with the inactivation of both Rab5 and Rab7 as evidenced by the lack of recruitment of their respective effectors EEA1 (early endosome antigen 1) and RILP (Rab7interacting lysosomal protein). Consistent with these findings, macrophage infection with an Ndk knocked-down BCG strain resulted in increased fusion of its phagosome with lysosomes along with decreased survival of the mutant. Conclusion: Our findings provide evidence in support of the hypothesis that mycobacterial Ndk is a putative virulence factor that inhibits phagosome maturation and promotes survival of mycobacteria within the macrophage. - Funding: This work was supported by an operating grant from the Canadian Institutes of Health Research (MOP-84557) and British Columbia Lung Association. Z. Hmama was supported by scholar awards from the Michael Smith Foundation for Health Research and the TBVets Charitable Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Tuberculosis (TB) is a devastating disease caused by Mycobacterium tuberculosis (Mtb), which claims about 2 million lives every year [1]. Moreover, the emergence of drug resistant Mtb strains and their spread to the general population now pose unprecedented difficulties to the control of TB disease [2]. Given the persistent global burden of TB, it is crucial that research delineate the underlying mechanisms of Mtb pathogenesis in order to pave the road for developing better strategies to prevent and treat TB. The ability of Mtb to persist and replicate within the host macrophage is a central factor in the development of TB disease [3]. Intracellular survival of Mtb is aided by a combination of factors including a unique cell wall structure, which physically shields the bacterium from bactericidal and hydrolytic enzymes [4], and secretion of enzymes to combat host reactive oxygen and nitrogen radicals [5,6]. Although all these factors contribute to Mtb persistence within the macrophage, one recurring and highly important feature of this pathogen is inhibition of normal phagosome maturation process, thereby abrogating physical fusion of phagosome with lysosomes and ultimately protecting the bacterium from a bactericidal environment [7,8,9]. Phagosome biogenesis is characterized by a rapid and sequential fusion of vacuoles containing ingested pathogens with various endosomal compartments leading to acidification dependent on recruitment of the vacuolar proton ATPase subunits [8]. Thereafter, the acquisition of acidic lysosomal enzymes by the phagosome and their activation results in efficient killing and degradation of invading pathogens [10] from which the macrophage switch to the function of antigen presentation for proper detection by effectors of the adaptive immune response [10,11]. Rab GTPases play a major role in the control of normal phagosome biogenesis. Normally, phagosome biogenesis is initiated by fusion with endosomes coated with the small GTPase, Rab5. This step is essential for recruitment of the early endosome antigen 1 (EEA1), which drives the phagosome towards further maturation [12]. However, this early maturation event is disrupted by Mtb and the closely related vaccine strain M. bovis BCG, both of which exclude EEA1 from their phagosomes [13]. As the phagosome matures into more advanced stages, another prominent member of late phagosome markers, the GTPase Rab7 is recruited and serves as a docking site for RILP (Rab7-interacting lysosomal protein). RILP possesses two distinct domains: one that binds to the GTPbound form of Rab7 and another that recruits the dynein/ dynactin complex [14,15]. By simultaneously associating with both targets, RILP promotes the interaction of vesicles bearing active Rab7 with lysosomes [14]. Initially, one group demonstrated that Mtb phagosomes retained Rab7 on the phagosome despite arresting its maturation to phagolysosomes [16]. Our recent studies have furthered advanced these findings and demonstrated that Rab7 molecules on the membrane of mycobacterial phagosome are inactivated by secreted factor(s) from live pathogenic mycobacteria [17]. Therefore, mycobacteria disrupt phagolysosome fusion in a mechanism dependent on Rab7-RILP interaction. Rab cycling is a unique and essential characteristic of small GTPases including Rab5 and Rab7. These proteins bind GDP/ GTP and to be functionally active, they must be in the GTPbound state. Our previous studies demonstrated that mycobacteria interfere with this cycling through a GTPase activating protein (GAP)-like activity, which depletes the c-phosphate from GTPbound Rab7 molecules [17]. The finding that live mycobacteria export a variety of proteins and glycolipids intracellularly [18,19,20,21] and the demonstration that proteins with subunit size up to 70 kDa are able to cross the phagosomal membrane towards the cytosol supported the search for secreted mycobacterial products that might interact with a (...truncated)