Characterization of RD1 related secretory protein(s) from Mycobacterium tuberculosis H37Rv

Abstract

Tuberculosis (TB) has been the ancient curse to human and caused by the pathogenic gram positive bacteria Mycobacterium tuberculosis. Nearly 80% of global TB cases occur in developing countries. In 2009, about 55% of cases occurred in Asia followed by 30% in Africa. India accounts for about one fifth (21%) of TB cases worldwide. It caused over 9.4 million new case and two million deaths and about 30 times infection. This creates a huge reservoir of untreated latent TB infection (LTBI), which can reactivate later in life, and represents a major source of disease (WHO report, 2010). The fact that only 5–10% of recently exposed individuals develop clinically active TB in the first two years after exposure, together with the often casual nature of exposure, makes diagnosis of LTBI among recently exposed and potentially infected individuals extremely difficult. The reasons behind such large number of infection and mortality rate are lack of proper diagnosis, evolution of multi-drug resistant TB (MDR-TB), the appearance of extensively drug-resistance TB (XDRTB) and the destructive impact of TB / HIV co-infection. The global burden of MDR-TB, defined as resistance to isoniazid and rifampin, was estimated at 500,000 cases in 2006. XDR-TB, caused by MDR strains that are also resistant to a fluoroquinolone and at least one second-line injectable agent (amikacin, kanamycin and/or capreomycin), caught the world’s attention after an outbreak in KwaZulu-Natal, South Africa, where 52 of 53 infected patients died (Gandhi NR et al, 2006). HIV co-infection was a contributing factor in most of these deaths, and indeed, a deadly association between HIV and TB has been known almost since the start of the HIV-epidemic. Of the 1.7 million people who died from TB in 2006, an estimated 200,000 were co-infected with HIV. Another issue related with mycobacterium infection is its diagnosis. For many decades, different versions of the tuberculin skin test (TST) have been used. TST measures the delayed type hypersensitivity against M. tuberculosis infection at the intra-dermal inoculation with purified protein derivative (PPD), after 48-72 h. PPD is a mixture of proteins of low molecular weight secreted by M.tuberculosis in the liquid growth media. Historically, the foundation for TST was laid in 1891, when Robert Koch observed that subcutaneous inoculation of broth culture filtrate (old- tuberculin) of tubercle bacilli resulted in a characteristic febrile reaction in patients who had TB, but not in those who did not have TB. Although Koch’s use of tuberculin as a treatment failed, it did provide a diagnostic approach. Being a crude product, old tuberculin was replaced by Seibert and Glenn in 1934 by a standardized version of tuberculin, called PPD (Edwards PQ et al, 1960). Over the years, PPD has been used as the main test for the diagnosis of latent TB infection. However, the major drawbacks of PPD test are the poor specificity (as PPD contains various antigens widely shared among different species of mycobacteria) of this test reagent and the fact that 10–25% of culture-confirmed TB patients do not react to PPD, thereby decreasing test sensitivity in patients with advanced disease and for that matter even in immuno-compromising conditions (Huebner RE et al, 1993, Felten MK et al,1989). Further, it is unable to distinguish reliably individuals infected with Mycobacterium tuberculosis from those vaccinated with Bacillus Calmette-Guerin (BCG). Because of these limitations, effective approaches for early accurate diagnosis and alternatives to antibiotics are urgently needed for the control of TB. 1.1 RD regions of Mycobacterium tuberculosis M. tuberculosis, the causative agent of tuberculosis, is a gram positive intra-cellular pathogen that resides mainly within macrophages and is able to survive for many years in an intracellular habitat in a slow-replicating or non-replicating state that is induced by host immune responses and fibrotic encapsulation. The reasons for its survival within the hostile intracellular environment of immune host have been identified recently (Russell, DG 2001). M. tuberculosis responds to the host immune system with dynamic transcriptional changes of a subset of its 4000 genes. Mimicking growth conditions in vivo by O2 depletion, nutrient starvation or nitric oxide (NO) addition has led to the identification of several M. tuberculosis genes, the expression of which is rapidly altered to enable intracellular survival (e.g. the dormancy (Dos R) regulon, which consists of 48 genes) (Sherman, DR. et al, 2001). Despite the existence of effective treatment regimens, control of tuberculosis is complicated by the chronic nature of the infection. Bacille Calmette-Guérin (BCG), an attenuated strain of M. bovis, is currently the only available vaccine against TB. Since 1974, BCG vaccination has been included in the WHO Expanded Program on Immunization. It is estimated that more than 3 billion individuals have been immunized with BCG and over 100 million doses of BCG are administered annually, making it the most widely used vaccine in humans. Metaanalysis studies have confirmed that BCG protects children, providing >80% efficacy against severe forms of TB, including TB meningitis and miliary TB (Colditz GA et al, 1995, Trunz BB et al, 2006). In contrast, evidence for protection against pulmonary TB in adolescents and adults remains contentious as efficacy estimates from clinical trials, observational case control studies and contact studies range from 0 to 80% (Brewer TF, 2000 Colditz GA 1994). The reasons for the variable protective efficacy are unknown but several hypotheses have been proposed, including differences among the vaccine strains used in clinical studies, exposure of trial populations to environmental mycobacteria, nutritional or genetic differences in human populations, differences in trial methods, and variations among clinical M. tuberculosis strains. (Brandt L et al, 2002, Demangel C et al, 2005, Fine PE et al,1995, Behr MA. 2002). These explanations are not mutually exclusive and all may contribute to the heterogeneity in vaccine efficacy. The only available vaccine for tuberculosis is BCG which has been developed by passaging the M. bovis strain 230 times between 1908 and 1921. BCG was a balance of a strain with low virulence and high immunogenicity. But due to inability to preserve the live bacteria, the vaccine required continuous passaging. Thus, BCG has evolved over a period of time. To understand the genetic differences developed in vaccine strain relative to pathogenic strain comparative genomic studies were performed. Initially, Mahairas GG et al (1996), compared the genomes of virulent M. tuberculosis and M. bovis with avirulent M. bovis BCG using the subtractive genomic hybridization. During this effort three genomic regions of differences (named RD1 to RD3) representing approximately 30 kb of DNA were found to be deleted from the BCG genome. Subsequently, existence of these RD was confirmed by comparing the physical maps of BCG and M. tuberculosis chromosomes (Philipp WJ et al, 1996). Another study used microarray based comparisons followed by PCR sequencing across deleted regions. The study identified 16 regions of differences (RD’s) in BCG relative to M. tuberculosis H37Rv. These were named as RD1 to RD16 which are deleted in BCG vaccine strain and encompass 129 open reading frames (ORF’s). Of these 16 RD’s nine are missing from BCG and all virulent M. bovis strains, two are missing from BCG and some of M. bovis strains, one is missing from all BCG strains and four are missing from certain BCG strains only (Behr MA et al, 1999). Genomic analysis by Gordon SV, et al, 1999, study using BAC array identified 10 genomic loci that were absent in M. bovis BCG relative to M. tuberculosis. Of these seven deletions were also present in M. bovis relative to M. tuberculosis. The detailed analysis revealed that M. bovis BCG specific three regions were identical to RD1-RD3 regions defined by Mahairas GG et al, 1996. The detailed analysis of the deleted regions is given below. 1.1.1 Region of Deletion 1 RD1 is absent from all the vaccine strains of BCG consistently but it is present in pathogenic strain of M. bovis, M. tuberculosis, M. africanum and four non-tuberculosis mycobacteria (M. kansasii, M. szulgai, M. flavescens, and M. marinum). The deletion of RD1 genes results into a phenotype cable of growth in THP-1 cell line but unable to spread to uninfected macrophages (Guinn KM, 2004). The first experimental evidence in this line came with the complementation of M. bovis BCG and M . microti with RD1 region. Complete restoration of RD1 region and its flanking region resulted in pathogenecity though not upto the level of M. tuberculosis (Pym AS et al, 2002, Pym AS et al, 2003). The in-silico and microarray analysis found this region of particular interest due the conservation of gene content and gene order even in distantly related species such as M. marinum, M. leprae and M. smegmatis. The region comprises of nine ORF’s within 9455 bp region located from Rv3871 to Rv3879c. The proteins encoded by them are of interest due to several reasons including theirinvolvement in virulence, use as potential vaccine candidates and use in diagnostic purposes. The members of RD1 region have different functions but together they form a secretory apparatus for secretion of virulent factors. The virulent factors are encoded by ORF’s Rv3874 and Rv3875 which encode proteins CFP-10 (esx B) and ESAT-6 (esx A) respectively. The region is also termed as ESAT-6 secretion system-1 or ESX-1 since the reintroduction of this region in M. bovis BCG and M. microti leads to the secretion of virulent factors and restoration of virulence. ESAT-6 and CFP-10 are well studied members of the region (Pym AS et al, 1999). They elicit strong immune response in experimental animals and humans as well. They will be discussed in detail in later sections. The ORF Rv3871codes for a dimeric membrane bound ATPase belonging to the FtsK/SpoIIIE family. These kinds of ATPases are present in gram negative bacteria as part of type IV protein secretion system. They are supposed to generate the energy for secretion of virulent factors (Pallen MJ, 2002). Rv3872 and Rv3873 are the PE (PE35) and PPE (ppe 68) family proteins rich in Pro-Glu and Pro-Pro- Glu motif. Deletion of Rv3872 also results into lack of expression of ESAT-6 and CFP-10. Functionally, Rv3872 is strong candidate for serodiagnosis of tuberculosis. Mukherjee P et al, 2007, found Rv3872 and the peptides derived from it are suitable candidates for pulmonary and extra-pulmonary tuberculosis diagnosis. Another gene of RD1 region, Rv3876, is proline rich polypeptide, which is weakly related to putative chaperone and its function predicted to be in cell division and chromosome partitioning. Rv3877 codes for trans-membrane protein which contains 12 trans-membrane helices. Rv3878 encodes a conserved hypothetical Ala rich protein and Rv3879 encodes an Ala and Pro rich protein (Pallen MJ, 2002). Besides RD1, an extended region has also been reported to be a part of ESX-1 secretory apparatus. This extended region includes Rv3866, Rv3868, Rv3870, Rv3882c and Rv3883c. Rv3868 belongs to CbbX family of proteins with ATPase activity (Luthra A et al, 2008). Rv3868 through Rv3871and Rv3877 is required for secretion of ESAT-6 and CFP-10 (Figure 1). Rv3883c encodes for serine protease named as serine protease mycosin 1 (myc p1). MycP1 is predicted to be located in periplasmic space, which may function to cleave signal peptide from ESAT-6 or CFP-10. But, this does not happen actually, because there is no reduction in size of ESAT-6 or CFP-10 after secretion from the mycobaceteria in host cell (Converse SE et al, 2005). The predicted function of Rv3870 is similar to Rv3871 as it belongs to FtsK/SpoIIIE family (Pallen MJ, 2002). Stanley SA et al, 2003, found that Rv3870, Rv3871 and Rv3877 are required for secretion of ESAT-6 and CFP-10 renamed these proteins as Snm1, Snm2 and Snm4. The deletion of these genes results into failure of Snm mutants to replicate in the cultured macrophages and to inhibit macrophage inflammatory responses (Stanley SA et al, 2003). The Snm2 is cytosolic component of ESX- 1 secretory system which interacts with Snm1 and it also recognizes the unstructured Cterminal of CFP-10. The mutation at the end of CFP-10 results into loss of binding with snm2 and consequently lack of secretion of ESAT-6 and CFP-10. The deletion of last seven residues is sufficient to abolish the interaction between CFP-10 and snm2 (Champion PAD et al, 2006).