M. avium, like M. tuberculosis, primarily infects the host mononuclear phagocytes. Targeting mononuclear phagocytes and being able to survive within the presence of efficient mechanisms of macrophage subversion, evolved by virulent.
In M. tuberculosis, PE-PGRS and PPE are two families of glycine-rich protein which constitute approximately 10% of the M. tuberculosis genome. Recent reports have suggested that these two gene families might be involved in antigen variation, eukaryotic cell binding, survival within macrophages and persistence in granulomas [19, 20]. Richardson and colleagues (2001) showed that a PPE protein (Rv1917) is expressed on the bacterial surface. Using signature-tagged mutagenesis, Camacho and colleagues identified a PPE gene (Rv3018c) associated with M. tuberculosis virulence in vivo . In addition, Ramakrishnan and colleagues observed that inactivation of PE-PGRS gene in Mycobacterium marinum resulted in attenuation of bacterial virulence in macrophages . In a recent report, Li and colleagues  demonstrated that an M. avium strain lacking a functional PPE protein, MAV_2928 (homologue to Rv1787), is attenuated in vivo and fails to inhibit both acidification of the vacuole, as well as phagosome-lysosome fusion. Mycobacterium avium MAV_2928 transposon mutant had comparable ability to enter the mononuclear phagocytes as the wild-type bacterium. The expression of MAV_2928 was noted following uptake of the wild-type bacterium by macrophages but not in culture media, suggesting a possible participation in the early events of the intracellular stage and possibly in phagosome formation .
The gene MAV_2928 is part of an M. avium chromosomal region with five PPE and PE genes, adjacent to the region homologous to the RD5 region in M. tuberculosis. The organization of this region suggests the existence of three promoters, one upstream of MAV_2928 inactivated in the 2D6 mutant, one between the second, and the third genes and another between the fourth and fifth genes in the downstream region . This specific region is also upstream of a region homologous to the RD1 region of M. tuberculosis. A PPE gene adjacent to the RD1 region in M. tuberculosis has been suggested to be associated with the transport of proteins . Because MAV_2928 is co-transcribed with MAV_2929, it is possible that some of the findings are due to the downstream gene. Complementation of the 2D6 mutant, however, has shown that most of the function lost with the inactivation of MAV_2928 is recovered . Interestingly, MAV_2925 has a high degree of homology with MAV_2928, but, based on the phenotype obtained with the inactivation of MAV_2928, we assume that the genes probably have unique functions.
Usually, upon bacterial uptake, a macrophage undergoes a series of events specifically designed to eliminate the engulfed microorganism. These include induction of reactive oxygen and nitrogen intermediates, gradual acidification of the phagosome, phagosome-lysosome fusion which loads the resulting compartment with acidic proteolytic enzymes, and antigen processing and presentation. The resulting lethal environment effectively kills the majority of the ingested bacteria. Pathogenic mycobacterial phagosomes, in contrast, show incomplete luminal acidification and absence of mature lysosomal hydrolases . Malik et al. [10, 23, 24] suggested that M. tuberculosis manipulation of calcium is in part responsible for the phagosome maturation arrest. The pathogenic mycobacterial phagosome has been shown to alter the trafficking of the plasma membrane markers, including MHC molecules , EEA-1 and LAMP-1 . M. tuberculosis-related blocking of phagosome maturation in macrophages appears to take place between the maturation stages controlled by early endocytic marker Rab5 and late endocytic marker Rab7 . The published data indicate that virulent mycobacterial phagosomes are selective in their fusion with various cytoplasmic organelles and do not mature into a phagosome-lysosome. Currently unknown is whether this ability to impact the docking and incorporation of proteins in the phagosome membrane is due completely, or partially, to the proteins that form the phagosome membrane is currently unknown. It is a plausible possibility. This interpretation could explain the differences between the vacuole proteomic between both bacterial strains.
Based on the results obtained in the macrophage transcriptome following infecting with M. avium or the 2D6 clone, it is clear that the mutant stimulates membrane-based signals and receptors that are bypassed by the wild-type bacterium.
Mass spectrometry analysis of the phagosomal proteins of 2D6 mutant and the wild-type bacterium yielded several differences in the protein expression in the vacuole membrane. For example, expression of EEA-1 and Rab5 effectors was seen on 2D6 phagosomes but not on the wild-type phagosomes, which is in agreement with the observation reported by Fratti et al. and Via et al. [6, 26]. The upregulation of Rab7 on the 2D6-infected macrophages indicates that the 2D6 mutant expresses late endosome markers and undergoes phagolysosome fusion .
A relatively large body of published data suggests the role of complement receptors CR1, CR3 and CR4  and a mannose receptor  in the uptake of M. tuberculosis by macrophages. It has been shown that CR3 is one of the main receptors involved in phagocytosis of M. avium by macrophages and monocytes [28, 29]. The CR2 was identified among various receptors on M. avium phagosomes. Studies have suggested an important role of CR1/2, CR3 and CR4 in host defense against Streptococcus pneumoniae infections . Functional studies have demonstrated that CR2 mediates tyrosine phosphorylation of 95 kDa nucleolin and its interaction with phosphatidylinositol 3 kinase .
Surfactant-associated proteins A and D (SP-D) are pulmonary collectins that bind to bacterial, fungal and viral pathogens and have multiple classes of receptors on both pneumocytes and macrophages . In addition, they act as chemoattractant to phagocytes. Surfactant proteins A and D (SP-A and -D) participate in the innate response to inhaled microorganisms and organic antigens and contribute to immune and inflammatory regulation within the lung . Ferguson and colleagues showed that SP-D binds to M. tuberculosis, resulting in decreased uptake and inhibition of bacterial growth . The presence of SP-D in phagosomes MAC 109 suggests a host attempt to eliminate the pathogen. Surfactant protein A (SP-A) expressed on M. tuberculosis vacuoles has been shown to be involved in enhancing the uptake of bacteria by macrophages [35–37].
The lack of MHC class II molecule expression in M. avium phagosomes, and its presence in the attenuated 2D6 mutant phagosomes in our data, is in agreement with the above findings that MHC class II molecules are down-regulated upon mycobacterial infection [38–40]. The MHC class I molecules are involved in presentation of the antigens located in the cytoplasm. The fact that MHC class I molecules were found on 2D6 mutant phagosomes, at 24 h time point, may reflect altered trafficking by the bacteria. In addition, MHC class I expression at early time points on the phagosome would suggest that the protein being present on the cell surface, during phagocytosis, would have been ingested upon during vacuole formation. The presence of MHC class I molecules on the 2D6 phagosomes could also be due to the fact that mycobacterial antigens are processed by MHC class I . The MHC class I has been reported to be present on latex bead phagosomes .
Several proteins not previously shown to be associated with the mycobacterial phagosomes were identified in the phagosomal preparations. Because we could not completely rule out the possibility of contamination of the phagosome preparations with other organelles, which indeed is a limiting factor of most subcellular fractionation techniques, we confirmed the findings by identifying proteins by fluorescence microscopy and Western blot. Recent studies on Legionella and Brucella have shown that these organisms reside in compartments displaying features of endoplasmic reticulum (ER) . In addition, there is evidence of recruitment of endoplasmic reticulum (ER) to nascent phagosomes containing inert particles or Leishmania and having a major contribution to the phagosomal membrane . This explains how antigens of vacuolar pathogens are presented to T lymphocytes via MHC class I machinery located on ER. Considering this information, it would be plausible to find ER particles on mycobacterial phagosomes. Some of the mitochondrial proteins, such as ATP synthase and HSP60 found in our preparations, have also been shown to be present in latex bead containing phagosomes .
A recent report on the elemental analysis of M. avium phagosomes in Balb/c mouse macrophages revealed high concentrations of potassium and chlorine at 24 h time point and correlated it to the microbiocidal killing similar to that observed in neutrophils . The increase in expression of CHP (potassium channel regulator) in the 2D6-infected macrophages, added to the finding that K-Cl co-transporter is also increased (proteomic results) on the 2D6 mutant phagosomes at 24 h time point, could support, at least in part, the above published report, since the 2D6 mutant is unable to survive within the macrophages . Therefore, there is a possibility that K-Cl transporter and CHP could be involved in the augmentation of the potassium and chlorine concentrations in the phagosome, leading to mutant killing, but this will have to be tested in future work. Because of the observed difference in vacuole membrane between the two tested bacterial strains, it was hypothesized that the difference might impact the content of the metals in the vacuole environment.
Measurement of the intravacuolar concentration of single elements demonstrates that the 2D6 mutant's vacuole is depleted of several important elements at 24 h after infection. The decrease in the intravacuolar concentrations of Ca++ and Zn++ suggests that the wild-type bacteria are capable of retaining the elements, but the PPE mutant is not, probably indicating that the mutant cannot suppress the transport mechanisms or cannot continue to induce uptake of the metals.
We studied protein expression of the mycobacterial phagosome and compared it to a isogenic mutant. We identified several proteins, either previously described or not reported to be present on the phagosomes. The modifications appear to have a significant effect on the intravacuolar environments. Nonetheless, the use of the MAV_2928 mutant established the possibility that one protein may have key function in modulating the formation of the phagosome, perhaps by altering initial events. Alternatively, the PPE-PE operon may be part of a complex system influencing or impacting the expression of other bacterial genes or involved in the transport of bacterial proteins. Change in single element concentrations in the bacterial environment can have significant effect on gene regulation . Future studies will address some of the differences found and will possibly provide insights into the mechanisms of pathogenesis and survival of mycobacteria inside the host.