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

Abstract

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.

Figure 1

SDS-polyacrylamide gel electrophoresis and Immunoblot analysis. (A) SDS-polyacrylamide gel electrophoresis of whole-cell lysates of uninduced and induced recombinant E. coli cells. Lane 1, 4- Molecular mass marker (116 kDa, 66.2, 45, 35, 25, 18.4, 14.4, Fermentas), the molecular mass standards are indicated in kDa on left side. Lane 2, 3- Lysate of uninduced and induced E. coli (pET28a/mce1A) cells respectively. Lane 5, 6- Lysate of uninduced and induced E. coli (pET28a/mce4A) cells respectively. Mce1A and Mce4A are indicated by arrows. (B) Immunoblot of purified Mce1A and Mce4A proteins. Anti-His-tag antibody was used to demonstrate the fusion proteins. Lane1-Purified Mce1A protein (2 μg). Lane 2- Pre-stained molecular mass marker (188 kDa, 62, 49, 38, 28, 18, 14, 6, 3, Invitrogen), the molecular mass standards in kDa are indicated on left side. Lane 3-Purified Mce4A protein (2 μg). Mce1A and Mce4A are indicated by arrows.

Figure 2

Immunoblot analysis of Mce4A protein. (A) Purified Mce4A protein separated by SDS-PAGE and electrotransferred to nitrocellulose membrane. Rabbit polyclonal antibody to Mce4A protein used at a dilution of 1:2000, detected signal of purified Mce4A protein. Lane 1-Pre-stained molecular mass marker (188 kDa, 62, 49, 38, 28, 18, 14, 6, 3, Invitrogen), the molecular mass standards are indicated in kDa on left side. Lane 2: Purified Mce4A protein (1 μg). Mce4A protein is indicated by arrow. (B) Subcellular fractions of M. tuberculosis H37Rv, stationary phase culture were fractionated on SDS-PAGE and Western blotting was performed using polyclonal anti-Mce4A antibodies for localization of Mce4A in various fractions. Concentration of protein loaded per well is indicated in parenthesis. Lane CL-Whole cell lysate (50 μg), Lane C-Cytoplasm (40 μg), Lane CFP-Culture filtrate proteins (30 μg), Lane CM-Cytoplasmic membrane (30 μg), Lane M-Pre-stained molecular mass marker (188 kDa, 62, 49, 38, 28, 18, 14, 6, 3, Invitrogen), the molecular mass standards are indicated in kDa on right side, Lane CW-Cell wall fraction (30 μg). Mce4A protein is detected only in the whole cell lysate and cell wall fraction from stationary phase culture.

Figure 3

Invasion of HeLa cells by recombinant E. coli. The invasion of HeLa cells by E. coli expressing Mce4A and Mce1A proteins was monitored by transmission electron microscopy. The stages of invasion by recombinant E. coli (pET28a/mce4A) (fig. 3A), E. coli (pET28a/mce1A) (fig. 3B) and E. coli (pET28a) (fig. 3C: j) are shown. A: a and B: f infection with uninduced recombinant E. coli. A: b, c, d, e and B: g, h, i infection with induced recombinant E. coli expressing Mce4A and Mce1A respectively. Unfilled arrow (bacteria attached to cell membrane), thick arrow (membrane ruffling), thin arrow (internalized bacteria). Bars = 1 μm in all the cases, except in A: c, d and B: g, h, where it is 0.1 μm.