Isolation ofMycobacterium tuberculosismutants defective in the arrest of phagosome maturation

Abstract

Mycobacterium tuberculosis resides within the phagocytes of its host. It ensures its continued survival through arresting the normal maturation of its phagosome, which is retained within the early endosomal system of the macrophage. Although individual bacterial components have been shown to modulate phagosome biogenesis, the mechanism(s) active in live, intact bacteria remain elusive. We have developed a genetic screen that facilitates the isolation of mutants defective in arresting the maturation of their phagosomes. Macrophages were incubated with iron-dextran that was chased into lysosomes. The cells were subsequently infected with M. tuberculosis from a library of transposon-mutagenized bacteria. After four rounds of enrichment, the majority of mutants isolated were unable to prevent acidification of their phagosomes and were attenuated for intracellular survival. The genes affected range in function from those with no known homologues to putative transporters and lipid synthesis enzymes. Further characterization of these bacteria is needed. In addition to clarifying the processes active in modulation of phagosome biogenesis by M. tuberculosis, this screen may be applicable to other pathogens that restrict the maturation of their phagosome. P athogenic Mycobacterium spp. are known to reside in vacuoles that fail to exhibit the normal progression of phagosomes to phagolysosomes. Studies from several groups (1-3) have demonstrated that the phagosomes containing these bacilli retain many of the characteristics of early endosomes and remain accessible to material internalized by means of the rapid recycling endosomal system.The normal progression of phagosomes after internalization of inert particles by phagocytes involves the transient acquisition of the GTPase rab5, phosphorylation of phosphatidylinositol (PI) to generate PI-3-phosphate (PI3P) by the PI kinase VPS34, and accumulation of the PI3P-binding protein early endosome autoantigen 1 (EEA1) (4-7). As the association with EEA1 diminishes, phagosomes show increased accumulation of rab7 and increased fusion with lysosomes. Within the lumen of phagosomes, one observes increased acidification and the accumulation of lysosomal hydrolases that are processed into their active forms as the environment within phagosomes becomes increasingly hydrolytic. In contrast, the phagosomes containing pathogenic Mycobacterium spp. fail to acidify below pH 6.2 (8, 9), remain positive for the early endosomal GTPase rab5 (10-12), and do not acquire EEA1 (13,14). Despite the extensive documentation of the aberrant retention of a range of host proteins, these studies do not provide an explanation of how the bacterium achieves this process of arrest.Several mechanisms have been proposed for this phenomenon, including the effects of ammonia production (15), the close apposition between the bacterium and its vacuole membrane (16), the ability of surface lipids such as lipoarabinomannan (LAM) and cord factor to inhibit vesicular fusion (13,14,17), and the activity of a bacterial ser...

Dormant Mycobacterium tuberculosis Fails To Block Phagosome Maturation and Shows Unexpected Capacity To Stimulate Specific Human T Lymphocytes

“…R-Mtb is highly adapted to life within macrophages, and it can modulate macrophage defenses and inhibit the processing of its Ags for the MHC class II pathway to promote its intracellular survival (32,33). In particular, metabolically active M. tuberculosis is endowed with the capacity to arrest phagosomal maturation (34)(35)(36)(37).…”

Molecular profiling ofMycobacterium tuberculosisidentifies tuberculosinyl nucleoside products of the virulence-associated enzyme Rv3378c

“…The Rv3377c-Rv3378c locus is required for optimal phagosome maturation arrest (13). The discovery of extracellular 1-TbAd provides specific insight into mechanisms by which an enzyme that is thought to be localized in the cytosol affects events outside the bacterium (13). To our knowledge, neither Rv3378c nor free tuberculosinol has been detected in culture filtrates (18).…”

“…More direct evidence for a role of this locus in virulence comes from transposon studies showing that Rv3377c and Rv3378c play nonredundant roles in phagosome-lysosome fusion and survival in macrophages (13). This key finding initiated an intensive search for the actual functions of these virulence-associated genes.…”

“…Within minutes of phagocytosis, M. tuberculosis inhibits host defenses, including phagosome acidification and phagolysosome fusion (34,35). The Rv3377c-Rv3378c locus is required for optimal phagosome maturation arrest (13). The discovery of extracellular 1-TbAd provides specific insight into mechanisms by which an enzyme that is thought to be localized in the cytosol affects events outside the bacterium (13).…”

“…Previously, Rv3378c was thought to generate free tuberculosinol and isotuberculosinol (10)(11)(12). This discovery revises the enzymatic function of Rv3378c, which acts as a virulence factor to inhibit phagolysosome fusion (13). Whereas current models of prenyl transferase function emphasize iterative lengthening of prenyl pyrophosphates using one binding pocket, the crystal structure of Rv3378c identifies two pockets in the catalytic site, establishing a mechanism for heterologous prenyl transfer to nonprenyl metabolites.…”