Characterization of Mce4A protein of Mycobacterium tuberculosis: role in invasion and survival

Background The mce4 operon is one of the four homologues of mammalian cell entry (mce) operons of Mycobacterium tuberculosis. The mce4A (Rv3499c) gene within this operon is homologous to mce1A (Rv0169), that has a role in host cell invasion by M. tuberculosis. Our earlier reports show that mce4 operon is expressed during the stationary phase of growth of the bacillus in culture and during the course of infection in mammalian hosts. M. tuberculosis carrying mutation in mce4 operon shows growth defect and reduced survival in infected mice. However, the intracellular localization of Mce4A protein and its direct role in cell entry or survival of the bacillus has not been demonstrated so far. Results By transmission electron microscopy we have demonstrated that recombinant Mce4A protein facilitates the invasion of non-pathogenic strain of E. coli into non-phagocytic HeLa cells. We observe that mce4A gene has a role comparable to mce1A in the survival of recombinant E. coli in human macrophages. Using antibodies raised against Mce4A protein, we show that the protein is localized in the cell wall fraction of M. tuberculosis H37Rv stationary phase culture only. Conclusion Mce4A protein is expressed during the stationary phase of broth culture and localizes in the cell wall fraction of M. tuberculosis. Mce4A protein expressed in non-pathogenic E. coli enables it to enter and survive within HeLa cells and the macrophages. As Mce4A protein is expressed during later phase of mycobacterial growth, our results raise the possibility of it playing a role in maintenance of persistent tubercular infection.


Background
The World Health Organization has estimated that nearly one-third of the world's population is now latently infected with Mycobacterium tuberculosis and 8-10 million people develop active disease resulting in 2 million deaths each year [1,2]. The prevalence of this infection is largely due to the extended dormancy of M. tuberculosis in the host and its ability to cause disease even in the face of a highly orchestrated host immune response [3].
M. tuberculosis is believed to primarily invade and replicate within alveolar macrophages [4]. It has the ability to enter A549 cells of alveolar epithelial origin in culture [5] and also invade other non-phagocytic cells [6,7].
Role of Mce1A protein in the entry and survival of the pathogen is demonstrated using recombinant E. coli carrying cloned copy of the gene as well as latex microspheres coated with recombinant Mce1A protein, both of which were able to enter HeLa cells [8,9]. Additionally, it has been demonstrated that the recombinant E. coli expressing Mce1A can survive longer in these cells [8]. The continued analysis of the four mce operons of M. tuberculosis H37Rv genome has shown significant conservation of the protein sequences in the four operons [10]. The redundancy of these operons is explained by functional significance of the different mce operons of M. tuberculosis H37Rv by various studies including ours [11][12][13][14][15]. Earlier we reported the expression of mce4 and absence of mce1 transcripts in the specific tissues of both infected rabbits and guinea pigs during advanced disease conditions, which suggested that mce4 operon, may have a role in the long term survival of M. tuberculosis in host's tissue [11]. The survival mechanism of dormant tubercle bacilli is still under intense research [16][17][18]. It is suggested that mycobacterial persistence may rise as a response to environmental stress, such as reduced oxygen concentration within a host tissue [19]. It is demonstrated that deletion mutants of mce3 and mce4 operons of M. tuberculosis H37Rv are attenuated in mice [13]. By deleting eight genes and the first 250 bp of the ninth gene from mce4 operon it was shown that survival of M. tuberculosis was significantly reduced in mice [13]. However, direct involvement of individual proteins from mce4 operon in the survival or uptake of M. tuberculosis into the mammalian cells was not demonstrated.
In the present study we validate the role of Mce4A protein of mce4 operon not only in cell invasion but also in survival of the pathogen in human macrophages using recombinant E. coli expressing cloned mce4A gene. The cellular localization and in vitro expression of the Mce4A protein in M. tuberculosis was also analyzed. E. coli DH5α and BL-21 (DE3) cells were grown in Luria-Bertani (LB) broth or on LB agar in presence of kanamycin (25 μg/ml) where ever appropriate. The human epithelial cell line HeLa cells were grown in complete Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10 mM sodium pyruvate (Sigma), 10% FBS (fetal bovine serum, Gibco), HEPES (Sigma) and 10 μg of penicillin-streptomycin/ml (Sigma). THP-1 monocyte cell line was cultured in RPMI 1640 (Sigma) supplemented with 10% FCS (fetal calf serum, Gibco) and 2 mM Lglutamine (Sigma). Both the cell lines were grown at 37°C in presence of 5% CO 2 . PMA (phorbol myristate acetate, Sigma) was used for the adhesion of THP-1 cells.

Cloning, expression and purification of Mce1A and Mce4A
Genomic DNA from M. tuberculosis H37Rv was extracted according to the CTAB (cetyltrimethylammonium bromide) method [20]. Full length mce1A (Rv0169, 1365 bp) and mce4A (Rv3499c, 1203 bp) genes were amplified from M. tuberculosis genomic DNA [21] by polymerase chain reaction (PCR) using the primers mce1F and mce1R for mce1A gene, mce4F and mce4R for mce4A gene as listed in Table 1. Each forward primer contained SacI restriction enzyme site whereas the reverse primers contained a HindIII restriction site.
The amplicons were cloned in pET28a (Novagen) to generate pET28a/mce1A and pET28a/mce4A with hexa histidine tags. Both DH5α and BL-21 (DE3) strains of E. coli were transformed with the recombinant plasmids for expression studies. The presence of the inserts was confirmed by restriction enzyme digestion and sequencing.
Overnight grown culture of E. coli BL-21 (DE3) containing the recombinant expression plasmids was diluted 1:10 in fresh LB broth containing kanamycin and grown with vigorous shaking at 37°C to an optical density (OD) of 0.6 at 600 nm. For transcription induction, IPTG (isopropyl thio-b-D-galactoside, Sigma) to a final concentration of 0.5 mM was added to the culture and incubation was continued for 2 h. The N-terminal His-tagged recombinant proteins were purified under denaturing condition on nitrilotriacetic acid (Ni-NTA) agarose columns (Qiagen) according to the manufacturer's instructions.

Production of polyclonal antibodies against Mce4A protein
The antibodies against the Mce4A protein were raised in 5-month-old female NZW rabbit. For each immunization, 300 μg of protein in 0.5 ml PBS was mixed with 0.6 ml of incomplete Freund's adjuvant (Sigma) and administered subcutaneously at five sites in rabbit, followed by four booster injections of 300 μg each 2, 3, 4 and 6 weeks after the first injection. Serum was prepared from blood collected 2 weeks after the last booster dose. The animal handling was according to the guidelines of the Institutional Ethical Committee and Indian Council of Medical Research, India.

Preparation of subcellular fractions
M. tuberculosis H37Rv was grown in one liter of 7H9 Middlebrook medium to mid-log phase (day 6) at 37°C on shaking (200 rpm) up to an OD of 0.6 at 600 nm. Cells were harvested by centrifugation at 10,000 g for 30 min and the culture supernatant was concentrated to obtain the culture filtrate protein (CFP). Pelleted cells were washed, resuspended in PBS and subcellular fractions were prepared as previously described [22,23]. Similarly, cellular fractions were also prepared from the stationary phase culture (day 10, OD of 1.0 at 600 nm) of M. tuberculosis H37Rv.

Detection of protein expression
The protocol followed for Western blotting of purified Mce1A and Mce4A proteins was as described previously [24]. Anti-His monoclonal antibody (1:2000 dilution) and peroxidase-conjugated goat anti-mouse IgG (1:2000) were used as primary and secondary antibody respectively. Western blot was carried out to detect purified

Invasion assay and Electron microscopy
Invasion of HeLa cells seeded at 2 × 10 6 cells per well into 6-well plates by BL-21 cells transformed with pET28a/ mce1A, pET28a/mce4A and empty vector pET28a was assayed. Bacterial cultures grown overnight were diluted (1:10) in fresh medium containing kanamycin and incubated further with vigorous shaking to reach an OD of 0.6 at 600 nm and induced with 0.5 mM IPTG for 2 h at 37°C. Following the induction, the cells were washed once with PBS and resuspended in 5 ml of PBS. Prior to the infection, monolayer of HeLa cells were charged with fresh medium (DMEM, supplemented with 10% FCS) and incubated at 37°C for 30 min. Recombinant E. coli cells were added to the monolayer at a multiplicity of infection (MOI) of 10:1 and incubated at 37°C for 4 h, washed three times with PBS and processed for electron microscopy as described previously [9] and examined under CM-10 electron microscope. Since the E. coli strain used is kanamycin resistant, the cells were not treated with kanamycin or gentamycin but were extensively washed postinfection to remove the extra-cellular bacteria that failed to enter the mammalian cells.

Statistical analysis
The CFU of recombinant E. coli and the percentage survival of recombinant E. coli cells are represented as mean ± standard deviation. The difference in percentage survival of recombinant E. coli cells (pET28a/mce1A and pET28a/ mce4A) as compared to E. coli (pET28a) was calculated using student's t test using graph pad prism Tm software.

Cloning, expression and purification of Mce1A and Mce4A proteins
The expression of the fusion proteins was studied in E. coli BL-21 (DE3) strain. The His-tagged Mce1A and Mce4A were expressed at high level on induction with IPTG and the purified proteins were detected using anti-His-tag monoclonal antibody (Fig. 1B). The fusion proteins were of the expected size of 45 kDa and 43 kDa respectively (Fig. 1A). Further we confirmed the identity of purified Mce4A protein with rabbit anti-Mce4A polyclonal antibodies raised in our laboratory ( Fig. 2A). Mce4A was purified as a single band, while Mce1A was detected as a doublet. Since anti-His-tag antibodies detected the lower molecular weight band, it is probably due to partial degradation. Similar profile for Mce1A protein was reported earlier also [25].

Growth-phase dependent expression and subcellular localization of Mce4A protein of M. tuberculosis
Immunochemical detection of Mce4A protein in whole cell lysate from different phase of M. tuberculosis cultures shows that it is expressed only in stationary phase culture (data not shown) and the subcellular fractions from stationary phase culture show that Mce4A is localized in cell wall fraction only (Fig. 2B). In a similar experiment subcellular fraction from mid-log phase culture of M. tuberculosis H37Rv, antibodies against Mce4A did not detect any protein either in whole cell lysate or any subcellular fraction (data not shown), which indicates absence of expression of Mce4A during early phase of growth.

Invasion of HeLa cells by recombinant E. coli expressing Mce4A protein
The invasion of HeLa cells by recombinant E. coli was monitored by transmission electron microscopy. The results show that uninduced E. coli (pET28a/mce4A and SDS-polyacrylamide gel electrophoresis and Immunoblot analysis

Discussion
The initial step in the pathogenesis by intracellular pathogens is the invasion of host cells. In case of M. tuberculosis, Mce1A protein of mce1 operon was the first protein of the pathogen to be implicated in invasion and survival inside macrophages [8].  [12].
The recently emerging reports on the role of mce4 operon in survival of M. tuberculosis in host tissue indicates that Mce4 proteins may be involved in maintaining the M. tuberculosis in a nutrient deficient environment for long term survival [14]. It has been suggested that the proteins of the mce4 operon may operate as a major cholesterol import system of M. tuberculosis because strains lacking mce4 operon exhibit drastically reduced ability to take up and metabolize cholesterol in vitro and hence grow poorly when cholesterol is the primary source of carbon [14]. It was reported earlier that mutation in mce1 operon produces growth defect in early phase of infection in mice and mutation in mce4 operon produces growth defect after 3-4 weeks post infection, indicating thereby that mce4 operon is required in later phase of infection [28].
The localization study showed that the Mce4A protein was present in the cell wall fraction of the stationary phase cul-ture of M. tuberculosis H37Rv further strengthening our results of transmission electron microscopy and cell invasion studies.
We conclude that the results of the present study strongly support the role of Mce4A protein in invasion and the intracellular survival of M. tuberculosis and suggest that it may have a role in persistence of mycobacterial infection in the host tissue.

Conclusion
There are many studies reported in the literature to elucidate dormancy in M. tuberculosis and the survival mechanism of dormant tubercle bacilli is not fully understood. It is suggested that mycobacterial persistence might arise as a response to environmental stress, such as reduced oxygen concentration within host tissue. Our earlier observation of preferential expression of mce4 operon in the internal organs of rabbit and guinea pig and also during the stationary phase in culture in conjunction with the results presented here strongly support a role for Mce4A protein in the long term survival of M. tuberculosis inside its host.

Authors' contributions
MB conceived the study. MB, VB and NKS designed the experiments, interpreted the results and worked on the manuscript. NKS carried out major experimental work. MS was associated with tissues culture studies. AC and RP participated in the designing of the experiments and the interpretation of the results. All authors read and approved the final manuscript.