LipC (Rv0220) Is an Immunogenic Cell Surface Esterase of Mycobacterium tuberculosis

Abstract

We have reported previously the identification of novel proteins of Mycobacterium tuberculosis by the immunoscreening of an expression library of M. tuberculosis genomic DNA with sera obtained from M. tuberculosis-infected rabbits at 5 weeks postinfection. In this study, we report the further characterization of one of these antigens, LipC (Rv0220). LipC is annotated as a member of the Lip family based on the presence of the consensus motif "GXSXG" characteristic of esterases. Although predicted to be a cytoplasmic enzyme, we provide evidence that LipC is a cell surface protein that is present in both the cell wall and the capsule of M. tuberculosis. Consistent with this localization, LipC elicits strong humoral immune responses in both HIV-negative (HIV ؊ ) and HIV-positive (HIV ؉ ) tuberculosis (TB) patients. The absence of anti-LipC antibodies in sera from purified protein derivative-positive (PPD ؉ ) healthy subjects confirms its expression only during active M. tuberculosis infection. Epitope mapping of LipC identified 6 immunodominant epitopes, 5 of which map to the exposed surface of the modeled LipC protein. The recombinant LipC (rLipC) protein also elicits proinflammatory cytokine and chemokine responses from macrophages and pulmonary epithelial cells. rLipC can hydrolyze short-chain esters with the carbon chain containing 2 to 10 carbon atoms. Together, these studies demonstrate that LipC is a novel cell surface-associated esterase of M. tuberculosis that is highly immunogenic and elicits both antibodies and cytokines/chemokines. There are an estimated 9 ϫ 10 6 new cases of tuberculosis (TB) and 2 ϫ10 6 TB-related deaths every year (55). Infection with Mycobacterium tuberculosis is initiated with the inhalation of a droplet bearing bacteria, and it takes months or years to progress to clinical TB. During this progression from initial infection to clinical disease, the in vivo bacteria adapt to continuously changing environments by altering their gene expressions (31,34,48,50). Previous studies to delineate culture filtrate (CF) proteins of M. tuberculosis that are recognized by antibodies during the natural course of disease progression demonstrated that the repertoire of antigens enlarges with the progression of infection (34)(35)(36)41). Interestingly, several of the M. tuberculosis antigens (malate synthase, MPT51, and ESAT6, for example) that elicit immune responses during the early stages of active infection have also been demonstrated to play important roles in the host-pathogen interaction (19-21, 57).To identify additional antigenic proteins of M. tuberculosis that are expressed during the early stages of active infection, we used sera obtained from M. tuberculosis aerosol-infected rabbits that were bled at 4 to 5 weeks postinfection to screen an expression library of M. tuberculosis genomic DNA (44). Antibodies in these sera identified several proteins known to contribute to the infection and survival of M. tuberculosis (exported repetitive protein [ERP], KatG, and MtrA) as well as novel prote...

Characterization of a novel esterase Rv1497 of Mycobacterium tuberculosis H37Rv demonstrating β-lactamase activity

“…Bioinformatically no signal sequence was identified in Rv1497 protein [3,25] while BlaC was shown to posses signal sequence [26]. Few Lip gene family members (LipC, LipF), have been predicted as cytoplasmic, while later on these were confirmed as extracellular or membrane bound [27,28]. Therefore, the localization of Rv1497 needed to be explored further in detail.…”

Targeting Lipid Esterases in Mycobacteria Grown Under Different Physiological Conditions Using Activity-based Profiling with Tetrahydrolipstatin (THL)

“…A family of 24 carboxyl ester hydrolases called "lip" genes (lipC to Z, except K and S) has been predicted to play a role in lipid catabolism (14). Among these, only a few have been functionally characterized and related to mycobacterial dormancy and resuscitation (15)(16)(17)(18).…”

Development of a POC Test for TB Based on Multiple Immunodominant Epitopes of M. tuberculosis Specific Cell-Wall Proteins

“…Our earlier results with the recombinant proteins demonstrated that antibodies to PTRP and LipC were present in sera from 80–90% of the ∼50 of HIV+TB+ patients tested [7], [8]. While the number of HIV+TB+ patients whose sera have been included in the evaluation of reactivity with the BSA-peptide conjugates on the lateral flow format is small, and sera from larger cohorts will need to be examined, it is reassuring that the high sensitivity of anti-peptide antibody detection was obtained on the rapid format also.…”

“…Our earlier studies in the ELISA format had demonstrated that antibodies to these individual immunodominant epitopes are detected in sera from at least 40% of the TB patients tested, but not in subjects with latent M. tb infection [7], [8]. Moreover, since a vast majority of the specimens included were from India, where BCG vaccination is given at birth, antibodies to these peptides are also absent in the vaccinated individuals.…”

“…The identification of immunodominant epitopes of 2 cell-wall proteins of M. tb , PTRP (Rv0538) and LipC (Rv0220) by epitope mapping by ELISA was previously reported [7], [8]. Briefly, overlapping peptides, each with a biotin residue attached to the N-terminal, and covering the sequence of the two proteins were synthesized commercially.…”