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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 11  |  Issue : 1  |  Page : 10-15

Early secreted antigenic target of 6 kda-like proteins of mycobacterium tuberculosis: Diagnostic and vaccine relevance


Department of Microbiology, Faculty of Medicine, Kuwait University, Kuwait

Date of Submission06-Dec-2020
Date of Decision01-Jan-2022
Date of Acceptance20-Jan-2022
Date of Web Publication12-Mar-2022

Correspondence Address:
Abu Salim Mustafa
Department of Microbiology, Faculty of Medicine, Kuwait University, P.O. Box 24923, Safat 13110
Kuwait
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmy.ijmy_232_20

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  Abstract 


Background: Early secreted antigenic target of 6 kDa (ESAT6) is low-molecular-weight and immunodominant protein of Mycobacterium tuberculosis with relevance to diagnosis and vaccine development. Analysis of the M. tuberculosis genome has shown the existence of 23 ESAT6-like genes. This study was aimed to determine M. tuberculosis-specificity vis-à-vis crossreactivity of ESAT6-like genes and encoded proteins and their potential in the diagnosis and vaccine development. Methods: All ESAT6-like proteins were characterized using the webserver Tuberculist. The sequence identities were determined using basic local alignment search tool. Results: The genes for six ESAT6-like proteins were located in M. tuberculosis-specific genomic regions of differences (RDs), i.e., EsxA and EsxB in RD1, EsxO and EsxP in RD7, and EsxV and EsxW in RD9. The genes for other ESAT6-like proteins were located in the genomic regions shared with other mycobacteria. Based on sequence identities, the ESA6-like proteins were divided into four subfamilies of 15 proteins and no subfamily of 8 proteins. The members of subfamilies 1-4 shared extensive sequence identities among the members of each subfamily. Each member of subfamily 1 (EsxI, EsxL, EsxN, EsxO, EsxV) and subfamily 2 (EsxJ, EsxK, EsxM, EsxP, EsxW) were homologs. Hence, the gene sequences identical to EsxO and EsxP located in RD7, and EsxV and EsxW located in RD9 were also present in the regions of M. tuberculosis genome shared with other mycobacteria. Conclusion: Because of their specificity to M. tuberculosis, only EsxA (ESAT6) and EsxB (CFP10) will be useful in the specific diagnosis. However, other ESAT6-like proteins may be useful for vaccine development against tuberculosis.

Keywords: Diagnosis, early secreted antigenic target of 6 kDa-like proteins, sequence identity, subfamilies, vaccine


How to cite this article:
Mustafa AS. Early secreted antigenic target of 6 kda-like proteins of mycobacterium tuberculosis: Diagnostic and vaccine relevance. Int J Mycobacteriol 2022;11:10-5

How to cite this URL:
Mustafa AS. Early secreted antigenic target of 6 kda-like proteins of mycobacterium tuberculosis: Diagnostic and vaccine relevance. Int J Mycobacteriol [serial online] 2022 [cited 2022 May 21];11:10-5. Available from: https://www.ijmyco.org/text.asp?2022/11/1/10/339510




  Introduction Top


The early secreted antigenic target of 6 kDa (ESAT6) is low-molecular-weight and immunodominant protein of Mycobacterium tuberculosis with relevance to diagnosis and vaccine development.[1] Although ESAT6 is a secreted protein, it does not have the classical secretory signal sequences for its secretion and is secreted through alternative mechanisms.[2] ESAT6 was first isolated from the short-term culture filtrate of M. tuberculosis,[3] and was shown to be a major protein antigen recognized by T-cells from mice infected with M. tuberculosis.[4] Later, it was found to be a major antigen recognized by T-cells obtained from tuberculosis patients but not from Bacillus Calmette Guerin (BCG)-vaccinated healthy controls.[5],[6] The analysis of T-cell reactivity of ESAT6 using synthetic peptides showed that the full-length protein had several epitopes recognized by T-cells in association with frequently expressed human leukocyte antigen class 2 molecules.[7],[8],[9] Other studies have shown that immunization with ESAT6 either alone or in combination with another secreted protein antigen (Ag85B) protected animals against challenges with M. tuberculosis.[10],[11] The relevance of ESAT6 in diagnostic applications is also well established.[12]

The genes for ESAT6 and another low molecular weight culture filtrate protein of molecular weight 10 kDa (CFP10) are located in the region of difference (RD) 1, which is present in the pathogenic strains of M. tuberculosis and Mycobacterium bovis but deleted/absent in all vaccine strains of BCG and most nontuberculous mycobacteria.[13],[14],[15],[16] The genes for ESAT6 and CFP10 are located in the ESX-1 locus of M. tuberculosis,[17] and the encoded proteins are known as EsxA (ESAT6) and EsxB (CFP10).[17] Recombinant BCG strains transformed with RD1 DNA and expressing ESAT6 and CFP10 proteins had increased pathogenesis in animals,[18] whereas deletion of RD1 from M. tuberculosis resulted in the loss of pathogenesis.[19] Hence, it has been suggested that EsxA and EsxB are virulence factors and important for the pathogenesis of M. tuberculosis/M. bovis.[20],[21] Since EsxA and EsxB are specific for M. tuberculosis/M. bovis and immunodominant,[22] they have been recommended in the specific diagnosis of infections caused by M. tuberculosis both in interferon-gamma (IFN-γ) release assays,[23],[24],[25],[26] and tuberculin-type skin tests.[27],[28],[29] Furthermore, immunizations with vaccine preparations containing EsxA and EsxB have shown to provide protection against challenge with M. tuberculosis in animal models of TB.[30],[31] In humans, the vaccine preparations containing EsxA and EsxB have shown to induce appropriate immune responses.[32],[33] However, EsxA and EsxB cannot be used for both vaccination and diagnosis, because their application in diagnosis among people vaccinated with preparations containing EsxA and EsxB will not differentiate between the effect of vaccination versus infection with M. tuberculosis. As the diagnostic use of EsxA and EsxB is well established and quite widespread,[23],[24],[25],[26] other appropriate antigens should be exploited for vaccine applications.

The analysis of M. tuberculosis genome has shown the existence of 21 additional ESAT6-like proteins, which may have roles in the diagnosis or vaccine development. However, their diagnostic efficacies will require that they are specific for M. tuberculosis. To predict their usefulness in the diagnosis and vaccine applications, this study was performed to determine the location of all genes of ESAT6-like proteins in M. tuberculosis genome and determine their specificity for M. tuberculosis and crossreactivity with the vaccine strains of BCG and nontuberculous mycobacteria.


  Materials and Methods Top


To characterize all ESAT6-like proteins, their genes were searched in M. tuberculosis H37Rv database using the webserver Tuberculist (http://genolist.pasteur.fr/TubercuList/). This webserver was also used to determine the location of each ESAT6-like gene in M. tuberculosis genome, size of the gene, the size and sequences of encoded proteins, and their description. The identified protein sequences belonging to ESAT6-like proteins were further analyzed for sequence identities by comparing the sequence of each protein with others using the bioinformatics tool basic local alignment search tool of the National Center for Biotechnology Information, USA. The proteins with significant identities were grouped into subfamilies. The ESAT6-like proteins were assigned to M. tuberculosis-specific genomic regions based on their location in the M. tuberculosis genome corresponding to the RDs that are present in M. tuberculosis but deleted/absent in the vaccine strains of BCG and other mycobacteria, as described by Behr et al.[14]


  Results Top


Early secreted antigenic target of 6 kDa-like proteins encoded by the genes in Mycobacterium tuberculosis genome

A search in the M. tuberculosis genome database using Tuberculist identified 23 genes (esxA to esxW) related to the esat6 operon, defining a novel gene family [Table 1]. The genes for ESAT6-like proteins were present at different locations in the M. tuberculosis genome [Table 1]. The genes' lengths varied from 285 to 387 base pairs (bps) and all of the encoded proteins were of low-molecular-weight containing 94 amino acids (aa) to 125 aa [Table 1].
Table 1: Gene name, gene length, location in Mycobacterium tuberculosis genome, and the length and description of early secreted antigenic target of 6 kDa like proteins

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Comparison of early secreted antigenic target of 6 kDa-like proteins for sequence identity and division into subfamilies

All ESAT6-like proteins have similar size and gene organization and occur in pairs, except for Rv3017 [Table 1], and each pair is expected to be expressed from a single operon.[13] Thus, there are 13 operons involved in their expression, which are scattered in the genome of M. tuberculosis. Although these proteins are grouped as ESAT-6 like, because of their size and similar genomic organization, they do not share the significant level of sequence identity with either EsxA (<20% identity) or EsxB (<30% identity) [Table 2].
Table 2: Sequence identities between EsxA early secreted antigenic target of 6 kDa and EsxB culture filtrate protein of molecular weight 10 kDa with all ESAT6-like proteins

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However, sequence comparisons of all ESAT-like proteins with each other showed that several of them have more than 90% sequence identity with some of the other members. Thus, based on the level of sequence identities among the member proteins, the ESAT6-like proteins could be grouped into five groups, i.e., subfamilies 1–4 containing 15 proteins with high sequence identity and no subfamily of 8 proteins that lacked significant identities among themselves and with any other member of ESAT-like protein [Table 3].
Table 3: Sub-families of early secreted antigenic target of 6 kDa-like proteins, their sequence identities and gene location with respect to Mycobacterium tuberculosis-specific and shared regions

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The subfamilies 1 and 2 have five members in each group and account for 10 ESAT6-like proteins [Table 3]. The amino acid sequence identities among the members of subfamily 1 and subfamily 2 are the highest and range from 92%–97% to 97%–100%, respectively [Table 3]. Subfamily 3 and subfamily 4 have two and three members, respectively. The members of subfamily 3, i.e., EsxG and EsxS have 91% identity, and the members of subfamily 4, i.e., EsxH, EsxR, and EsxQ have 46%–84% identity with each other [Table 3]. The remaining 8 members of no subfamily of ESAT6-like proteins, including EsxA and EsxB, share <25% identity with each other [Table 3].

Further analysis with respect to the location of genes in M. tuberculosis genome showed that the genes for EsxA and EsxB were located in the genomic region RD1 specific for M. tuberculosis [Table 3], and there are no homologous genes in M. tuberculosis genome corresponding to these proteins. The genes for EsxO and EsxP are located in RD7 and the genes for EsxV and EsxW are located in RD9 [Table 3]. However, these proteins have homologs in M. tuberculosis genomic regions shared with BCG and nontuberculous mycobacteria [Table 3]. The genes for the remaining 17 ESAT6-like proteins are present in the genomic regions of M. tuberculosis shared with BCG and nontuberculous mycobacteria [Table 3].


  Discussion Top


There are genes for 23 ESAT6-like proteins in M. tuberculosis genome. These proteins have a small size and similar organization in the M. tuberculosis genome, but they do not share significant sequence identities with EsxA. None of them have signal sequences for secretion during the growth of M. tuberculosis. Their secretion out of the bacterial cells requires special secretion systems known as ESAT6 protein family secretion (ESX) systems, which are also known as type VII secretion systems.[34]

Based on sequence identities, this study has identified four subfamilies of ESAT6-like proteins. The individual members of subfamilies 1–4 have significant identities with each other. Six of the ESAT6-like proteins, two each, are present in RD1 (EsxA, EsxB), RD7 (EsxO, EsxP), and RD9 (EsxV, EsxW) genomic regions, which are M. tuberculosis-specific and deleted in all vaccine strains of BCG and other mycobacteria.[35] This suggested that these proteins may be useful in the diagnosis of tuberculosis. However, searches for sequence identities showed that EsxA and EsxB (both encoded by genes in RD1) also lack significant sequence identities with any other ESAT6-like protein. While four other proteins whose genes are located in M. tuberculosis-specific genomic regions RD7 (EsxO and EsxP) and RD9 (ExV and EsxW) have three additional homologs present in the genomic regions of M. tuberculosis shared with the vaccine strains of BCG and nontuberculous mycobacteria. Thus, none of the ESAT6-like proteins, other than EsxA and EsxB, will be useful in the specific diagnosis of tuberculosis. However, they may be useful in the development of subunit vaccines against tuberculosis. In this respect, immunizations with EsxV (Rv3619c) and/or EsxW (3620c) proteins have shown to induce antigen-specific humoral and cellular immune responses in mice.[36],[37],[38],[39],[40] Furthermore, immunizations of mice with EsxV (Rv3619c) were shown to provide protection against infection with M. tuberculosis.[41]

In addition to the ESAT6-like proteins whose genes are located in the M. tuberculosis-specific genomic regions, other ESAT6-like proteins have also been tested for their vaccine potentials. In a study by Villarreal et al., the authors prepared five plasmid DNA constructs containing 15 ESAT6-like proteins.[42] Vaccination with all the constructs induced robust antigen-specific IFN-γ responses in mice to all antigens with multifunctional CD4 Th1 and CD8 T-cell responses.[42] When all constructs were combined into a cocktail of 15 ESAT6-like antigens, the T-cell responses induced were substantially broad to all antigens and superior to BCG.[42] Immunization of mice with Rv1198 (EsxL) induced a pro-inflammatory response with elevated levels of tumor necrosis factor (TNF) and interleukin (IL)-6, along with low induction of IFN-γ, IL-2, and IL-10, but no induction of IL-4.[43] Immunization of mice with Rv3444c (EsxT) or Rv3445c (EsxU) resulted in the induction of antigen-specific long-term humoral and cellular immune responses.[44] A fusion protein containing five ESAT6-like proteins, i.e., EsxB (CFP10), EsxD (Rv3891), EsxG (Rv0287), EsxU (Rv3445), and EsxM (Rv1792) induced the production of Th1 cytokines (IFN-γ, TNF, and IL-2) and multifunctional CD4+ T-cells, and provided protection challenge with M. bovis BCG in mice.[45] Another fusion protein-based vaccine containing HspX/EsxS, when injected into mice with appropriate carrier and adjuvant, induced mucosal and systemic immune responses, including Th1, Th17, IgA, IgG1, and IgG2a immune responses.[46]

A multiprotein vaccine candidate, ID93, containing four M. tuberculosis proteins, two of which were ESAT-6 like proteins, i.e., Rv3619c (EsxV) and Rv3620c (EsxW), has been developed.[47] Immunization with this subunit vaccine did not induce sensitivity to purified protein derivative (PPD) of M. tuberculosis, suggesting that its use as a vaccine will not interfere with the use of PPD as a skin test reagent for the diagnosis of tuberculosis.[48] Immunization of mice with ID93 in combination with glucopyranosyl lipid adjuvant (GLA) in a stable nano-emulsion (SE) known as ID93/GLA-SE protected them when infected with a clinical isolate of M. tuberculosis and a hyper-virulent Korean Beijing strain K of M. tuberculosis.[49],[50],[51],[52] The ID93/GLA-SE vaccine has also been tested in the Phase 1 clinical trial in BCG-vaccinated adults.[53] The vaccine was well-tolerated, and the vaccinees did not develop severe or serious vaccine-related adverse reactions.[54] Moreover, the vaccination with ID93/GLA-SE induced long-lasting antibody and Th1-type cellular immune responses.[53],[54] The ID93/GLA-SE vaccine is currently undergoing a Phase 2 clinical trial.[55]


  Conclusion Top


The genome of M. tuberculosis contains genes for encoding 23 ESAT6-like proteins. These proteins have a small size and similar organization in the M. tuberculosis genome, but they do not share significant sequence identities with ESAT6 (EsxA). The genes of six of these proteins are located in M. tuberculosis-specific genomic segments RD1, RD7, and RD9. Furthermore, only RD1 encoded EsxA and EsxB are M. tuberculosis-specific as they do not have any homologs in the M. tuberculosis genome. The remaining 17 ESAT6-like proteins are present in the M. tuberculosis genomic regions shared with BCG and nontuberculous mycobacteria. Furthermore, the ESAT6-like proteins encoded by genes in M. tuberculosis-specific genomic regions RD7 (EsxP and EsxO) and RD9 (EsxV and EsxW) have three homologs present in the genomic regions shared by BCG and nontuberculous mycobacteria. Hence, only, EsxA and EsxB will be of use in the specific diagnosis of infection with M. tuberculosis, but other proteins may be exploited as subunit vaccines against tuberculosis. In this respect, a vaccine candidate containing two ESAT6-like proteins encoded by genes in RD9 (EsxV and EsxW) is undergoing clinical trials in humans.

Financial support and sponsorship

The study was supported by Kuwait University Research Sector grant MI02/12.

Conflicts of interest

There are no conflicts of interest.



 
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