ESAT-6 a Major Virulence Factor of Mycobacterium tuberculosis and a Target for New Therapeutic Interventions

1. Introduction

Human tuberculosis (TB) is one of the world’s deadliest infectious disease [1,2,3]. In 2022 the WHO reported approximately 1.6 million deaths, with 10 million new infections and an estimated one-quarter of the human population latently infected [3]. Infection control is hampered by the limited efficacy of a 100-year-old Bacille Calmette-Guérin (BCG) vaccine and by the emergence of multidrug-resistant strains to 70 year-old antibiotics, particularly those used as first line therapy [3]. There is also a need to develop better diagnostic tools, more sensitive, using simple instruments easy to handle in remote geographical areas, and cost-effective for rapid detection of Mtb. Moreover, although new methods are being implemented [4], currently available methods do not allow differentiation between different stages of infection [4,5,6,7,8].

Mycobacterium tuberculosis (Mtb) is considered the mainly causative agent of human TB [9]. Mtb infection primarily affects the lungs, a condition that contributes to the high transmissibility by the respiratory route[9]. Although not transmitted from person to person, extrapulmonary TB is a second form of disease manifestation that particularly affects less immunocompetent individuals, namely children or people co-infected with HIV [10,11,12]. In addition to Mtb, other pathogens can also infect humans and cause TB with similar clinical symptoms [13,14,15,16,17,18,19,20]. These, include M. africanum, which is restricted to humans in West Africa [21,22,23], where it causes nearly half of all pulmonary TB [18,19,20] and the animal-adapted species M. bovis, which is estimated to cause TB in about 2% of the world’s population [24,25]. M.caprae is responsible for a smaller burden of zoonotic TB [26]. They all belong to the Mycobacterium tuberculosis species complex (MTBC) which also includes the more distant M. canettii group and additional species that cause TB in animals such as M. microti [27], M. pinnipedii, M. orygis and M. mungi [20,23,28,29,30]. Although each of these animal MTBC variants causes TB in its host species, they may trigger slight or no disease outside of their adapted host, especially in immunocompetent hosts [13,30,31]. However, conditions such as geographic prevalence of infected animals, close humans contacts, route of transmission (milk, infected meet or airborne droplets containing pathogens), and conditions such as HIV infection or other immunosuppressive conditions should be taken in account to assess the true threat to public health [32].

The animal-adapted species are more recent pathogens that emerged as a crowd-associated diseases during the Neolithic demographic expansion along with the development of animal domestication [33]. Mtb ascended long before the establishment of crowdy populations, 70 thousand years ago and accompanied the exodus of Homo sapiens from Africa during the Neolithic expansion [34]. The initial virulence of Mtb is therefore strongly adapted to the occasional availability of the population to be infected and to an evolutionary ability to survive long in the host until the opportunity for transmission arises. Consequently, Mtb infection have evolved at low human population densities exhibiting a pattern of chronic development, accompanied by decades of latency before progressing to active disease [10,11,12]. It is a well-adapted human pathogen, requiring the induction of a strong inflammation and destruction of the lung tissue for transmission and evolutionary survival [9,34,35]. This feature is unusual in most pathogens where virulence is not associated with their spread to other hosts [36].

The pathogen does not seem to require virulence factors such as the usual virulence factors pili, toxins or capsules that are essential for the invasion of epithelial barriers [9]. Nevertheless, their potential involvement should not be ignored, as a capsule and pili have recently been identified in Mtb, although their roles during infection in vivo is still unclear [37,38,39]. Mtb virulence relies on its ability to manipulate host macrophages, where it establishes intracellular niches, to cross mucosal barriers and to avoid pathogen destruction. First, Mtb subverts the endocytic pathway preventing phagolysosome fusion and proteolytic digestion [40,41,42,43,44,45]. Second, it activates innate immune responses to induce their transmigration to the lung parenchyma [46,47]. There, infected macrophages attract more permissive cells, expanding intracellular niches [9,48,49,50]. Mtb induces the adaptive responses that stimulate their containment and long-live inside granulomas [51,52,53,54]. Finally, the pathogen induces necrotic cell death of macrophages, granuloma destruction and lung cavitation for transmission [47,49,55,56]. Common to all these events is the major virulence factor the “early secreted antigenic target of 6 kDa” (ESAT-6, also called EsxA). This review highlights the role of ESAT-6 in different phases of Mtb infection and its contribution to virulence. It also points ESAT-6 as a target for the development of better diagnostic tools and future vaccines for human tuberculosis.

2. Virulence evolution among MTBC

Comparative genomic analysis among MTBC species reveals more than 99,95% sequence homology [57], but they differ mainly by large sequence polymorphisms [18] relative to Mtb reflected by the so-called regions of difference (RD) and translated into deletions [57,58,59,60,61] or punctual insertional sequences [62]. These observations reinforce the ancestral origin of Mtb reflecting a loss of genes during transmission to animals that allowed fitness gain in the new host and loss of robustness in humans. In the case of M. africanum lineage 5 (L5) is more associated with Mtb-like lineages while lineages L6 and 9 displays RD more associated with M. bovis-like animal adapted species [19,20].

Important cumulative findings from these studies have identified more pathogenic MTBC disease-causing species from less fit ones with deletions/insertions/mutations in RD regions affecting the PhoPR two-component virulence system, or the genes under the control of this signal transduction regulator [63,64]. This includes the regions called region of difference 1 (RD1) and the region of difference 8 (RD8), which are responsible for the production and secretion of the virulence factor ESAT-6 [29,62].

Particularly the RD1 region is deleted in the animal-adapted species M. microtii and was first described in BCG, where it is absent and associated with attenuation of this live vaccine during its propagation in vitro [23,65,66]. The RD8 deletion is associated with animal-adapted species and affects the regulatory PhoPR-dependent region of the espACD operon involved in ESAT-6 secretion (Figure 1). Genetic transfer of mutations affecting the PhoP binding region in M. bovis and in the closely related M. africanum L6 into Mtb sensu stricto human species, resulted in reduced ESAT-6 secretion, and lower virulence [29]. Remarkably, the deleterious effects of these mutations were partially compensated by RD8 deletions, in both species, allowing ESAT-6 secretion to some extent by creating alternative regulatory sequences [62]. The observed attenuated ESAT-6 responses contribute for the observed slower clinical progression from infection to disease when compared to Mtb [18,67]. Conversely, the insertion of the IS6110 element upstream of the PhoP binding locus resulted in the upregulation of the operon in one multidrug-resistant M. bovis strain, responsible for an unusually high human transmissibility, partially reverting the phoPR-bovis-associated fitness loss [68,69].

The ESAT-6 gene (esxA) is part of the the esx-1 locus, a group of genes encoding a the type VII secretion system that allows the secretion of the virulence factor ESAT-6 from the pathogen, known as ESAT-6 secretion system 1 (ESX-1) [70,71] (Figure 1).

3. ESAT-6 is required for virulence of Mtb

ESAT-6 was first identified in 1995 by the discovery of a potent T-cell antigen in the short-term culture filtrate of Mtb [72,73]. The initial focus was on its potential use as a target for a TB vaccine to replace BCG [74]. Moreover, since patients and animals infected with MTBC respond strongly to ESAT-6 antigens, parallel emphasis was given to the development of a better diagnostic tool for TB [75].

The export of ESAT-6 from the bacilli, requires a secretory apparatus consisting of proteins that are assembled on the inner surface of the cell membrane and are strongly recognized by T cells [70,71,76]. The encoded genes are all part of the esx-1 operon (Figure 1), and there is increasing evidence that they are under selective pressure imposed by the host immune system [76]. Concomitant with these studies, comparative functional genomics among virulent, attenuated, or saprophytic mycobacterial species have contributed to the inclusion of ESAT-6 in Mtb virulence [77]. Curiously, the saprophyte species M. smegmatis (Ms) also encodes for an ESX-1 apparatus [78], however it does not appear to confer Ms virulence capabilities as demonstrated by its inability to survive in human macrophages [79] or in amoeba in the environment [9]. The emergence of the ESX-1-associated virulence in Mtb stems from the original role of ESX-1 in Ms and other GC-rich soil species in inducing horizontal gene transfer between bacteria [80,81,82]. Predatory amoeba may have contributed to the evolutionary pressure that selected mycobacterial pathogens for intracellular survival. ESAT-6 requires an extended esx-1 locus to be secreted, a putative genomic island containing the espACD locus which is adjacent to the region of difference 8 (RD8) (Figure 1). It has been hypothesized that this phenotype may be associated with independent horizontal gene transfer from pre-pathogenic mycobacteria in the contact with soil bacteria and positively selected while in amoeba phagosomes [83,84].

ESAT-6 secretion from the bacilli requires both the expression of the esx-1 locus for the type VII secretion apparatus and for the transcription of both the ESAT-6 gene (esxA) and the culture filtrate protein 10 (CFP-10) gene (esx-B) contained in the RD1 region. In addition, it requires the protein EspA which is not encoded in the esx-1 locus but in the extended espACD operon adjacent to RD8 [85] (Figure 1), which is located 260 kb upstream of esx-1 [71].

All species and strains deleted in the esx-1 locus or in the internal RD1 region, or in the the esx-1 extended locus espACD exhibits an attenuated phenotype [86]. Mutants with deletion on ESX-1 of Mtb are attenuated in virulence translated into reduced survival of mycobacteria in cultured macrophages or in experimental animal models of TB [87,88,89,90,91,92], which is consistent with the attenuation of BCG vaccine, or the species M. microti due to the deletion of the region of difference RD1 [65,66]. Likewise, the introduction of RD1 into BCG restores the virulence [90,93]. The reference laboratory strain Mtb H37Ra has a mutation in the phoP regulatory region responsible for the attenuated phenotype compared to its virulent counterpart Mtb H37Rv resulting in impaired ESAT-6 secretion [63,94]. Clinical Mtb isolates were shown to secrete higher levels of ESAT-6 than the reference laboratory strain Mtb H37Rv. Comparative analysis of genetic polymorphisms between clinical and laboratory strains revealed whiB6 (rv3862c), a gene upstream of the ESX-1 genetic locus. It encodes for a regulatory protein that activates promoters under the esx-1 and extended espACD locus responsible for ESAT-6 production and secretion, respectively [95] (Figure 1).

Clearly, the loss or gain of mycobacterial virulence is closely linked to the ability of mycobacteria to produce and secrete ESAT-6 and the extension of virulence is correlated with the amount of protein secreted.

3.1. During the early phases of infection: the innate phase

Upon inhalation of aerosols containing the pathogen, bacilli that manage to reach the alveoli are phagocytosed by alveolar macrophages [10]. These professional phagocytic cells are permissive to Mtb and provide a critical niche for the survival of the intracellular pathogen [9,44,96,97]. In the endocytic pathway the pathogen inhibits phagosome fusion with lysosomes, prevents vesicle acidification and its proteolytic destruction by lysosomal enzymes allowing the pathogen to replicate in early phagosomes [40,41,42,43,44,45]. Recent studies showed that ESAT-6 inhibits IL-18-mediated phagolysosome fusion by regulating microRNA-30a in mycobacteria-infected macrophages [98,99]. ESAT-6 was indeed associated with the blockade of phagosomal maturation because mutants in ESAT-6 of M. marinum, a pathogen that causes a TB-like disease in fish, were found to be mainly located in lysosomes in contrast to the wild-type strain, which is located in early phagosomes [100].

However, luminal studies have challenged the dogma of the exclusive intracellular localization on phagosomes of both Mtb and M. marinum [101,102,103,104,105]. Studies using ultrastructural observations generated by electron microscopy and, more recently, the development of a Fluorescent Resonance Energy Transfer (FRET) method, demonstrate the translocation and escape of both pathogens from phagolysosomal compartments into the cytosol [101,102].

It has been shown that ESAT-6 induces phagosomal membrane rupture allowing pathogens to gain access to the cytosol, in contrast to the corresponding mutants, which instead accumulate in phagosomes. Moreover, following phagolysosomal escape, a necrotic form of cell death of infected macrophages was observed 3 to 4 days post-infection in ex-vivo studies [101].

It is unlikely that in all contexts of infection in vivo, ESAT-6 will induce complete rupture of the phagosome membrane and total escape of the bacteria into the cytosol, ending with necrotic cell death. Necrotic cell death occurring in vivo will induce a strong inflammatory response and tissue destruction. There is a possibility that punctual membrane perturbations may create local conditions for the transfer of Mtb proteins with less induced stress in the cytosol. ESAT- 6 is a pathogen-associated molecular pattern (PAMP) sensed by innate cytosolic receptors and is the major Mtb PAMP that activates the NLRP3 inflammasome [47]. This platform is required for caspase-1-mediated processing of the cytokines IL-1β and IL-18. The combination of purified ESAT-6 protein with other PAMPS such as Ag85 adds a significant impact on NLRP3 activation and IL-1β secretion demonstrating that ESAT-6 helps other Mtb PAMPS to reach the cytosol [47]. IL-1β drives neutrophil recruitment by several mechanisms and, if not controlled, neutrophils are major instructors for tissue destruction [55,106]. The mechanism is controlled by nitric oxide (NO) released by IFN𝛾-activated macrophages [55,106]. NO in turn controls the inhibition of the NLRP3 inflammasome, which regulates the amounts of cytokines secreted.

Independent studies have shown that NLRP3 activation does not always results in necrotic cell death of infected cells and tissue destruction. Transmigration of infected alveolar macrophages (AM) into the lung parenchyma is dependent on Mtb ESX-1 inducing IL-1β via the NLRP3 inflammasome. As a result, IL-1R signalling on alveolar pneumocytes affects alveolar permeability and lung tissue access without tissue destruction [46,47].

Once inside the lung, infected macrophages, activate pneumocytes surrounding the nascent granuloma to secrete matrix metalloproteinase 9 (MMP9) in an ESAT-6-dependent manner [107,108,109]. Consequently, an influx of more permissive macrophages following the MMP9 signals reach the nascent granuloma and efficiently find and perform efferocytosis of dying apoptotic infected cells [110,111]. Continuous cycles of this process allow the expansion of Mtb intracellular niches [107,108,112]. The intracellular replication and bacterial load are controlled by distinct mechanisms, some of which depend on perturbations of the ESAT-6 phagosomal membrane with concomitant cytosolic PAMPS that activate different antimicrobial mechanisms including apoptosis [48,110] and autophagy [113]. The ability to induce apoptosis is a feature of virulent strains of M. tuberculosis, in a process that involves ESAT-6 [50,114]. Moreover has also been shown that inhibition of apoptosis by non-virulent mutants of M. marinum impairs the spread of infection and bacterial expansion on nascent granulomas [52]. The apoptotic form of controlling intracellular bacterial loads counteracts the necrotic forms of death usually observed during high load infection of macrophages in vitro [115], and contributes to cell-to-cell spread of Mtb.

Autophagy is a relevant innate response to control intracellular pathogens including Mtb [116]. ESAT-6 contributes to this pathway [113,117,118]. Its functions in perturbing the phagosomal membrane will expose PAMPS including mycobacterial DNA, a signature that can be sensed by host innate cytosolic receptors. These include at least 3 cytosolic sensors, two involving the inflammasomes NOD-, LRR-, and pyrin domain-containing 3 (NLRP3) and absent in melanoma 2 (AIM2), and the third the cyclic GMP-AMP synthase (cGAS) [117]. The latter leads to the synthesis of the second messenger cyclic GMP-AMP (cGAMP), which activates the endoplasmic reticulum-associated stimulator of interferon genes (STING) and the downstream serine/ threonine-protein kinase (TBK1)-interferon regulatory factor 3 (IRF3)–IFN-I signaling pathway (Figure 2) [113,119]. TBK1 provides a bridge for Mtb destruction by targeting intracellular bacteria to the ubiquitin-mediated autophagic pathway in macrophages [113].

3.2. During latency

The latency period can last for decades after the initial infection. It is defined as a state of immune activation in the absence of disease symptoms [11]. During the innate granuloma expansion, the adaptive responses are activated, and the latency phase begins with the arrival of effector T-lymphocytes to the granuloma [10]. The adaptive granuloma is a structure that contains Mtb in the lung, but it is also the result of a strategic manipulation by the pathogen to ensure a long life in the host. A fine balance of proinflammatory microbicidal mechanisms with immunosuppressive events drives the process. Bacterial loads are kept at bay in macrophages by effector CD4⁺ T-cells, such as Th1 secreting the cytokines IFN𝛾 and TNFα, Th17 secreting IL-17 and, in much smaller numbers, Th2 and regulatory T-cells that counteract the inflammatory effects of the former [10]. The adaptive T-cells are constantly arriving at the granuloma, forming a surface layer, and a few infiltrating cells will license infected macrophages to exert their activity. In the case of Th1, this licensing induces macrophages to secrete IL-12 allowing efficient IFN𝛾 and TNFα release from effector T-cells. Mtb ESAT6 has been shown to induce immunosuppressive regulatory T cell populations that delay effector T cells migration into the granuloma, a process that is reversed by IL-12 [120]. In turn, IFN𝛾 and TNFα activate infected macrophages to become more microbicidal. However, Mtb resists these effects by reducing macrophage responsiveness to signaling by IFN𝛾 [121]. IFN𝛾 also contributes to NO release from activated macrophages, a mechanism required to control IL-1β and tissue destruction, helping to preserve the structure of the granuloma [55].

In the case of CD8⁺ T-lymphocytes, ESAT-6 in infected macrophages interacts with beta-2-microglobulin (β2M) in the endoplasmic reticulum, affecting antigen presentation to effector cytotoxic T-cells and impairing their microbicidal activity [122,123].

While macrophages become more microbicidal, the pathogen activates the two-component PhoPR system, which allows the pathogen to adapt to a stressful environment such as hypoxia and low pH, while activating the dormancy regulon that puts the bacilli in a low metabolic state [124].

Activated macrophages secrete TNFα and IL-1β which permeabilize the endothelia to feed the granuloma with newly arrived macrophages and neutrophils to be infected. All of this cell arrival dynamic is fuelled by new blood vessel formation induced by an ESAT-6 driven release of the angiogenesis factor VEGF from infected cells in the granuloma. [125].

4. ESAT-6 from a virulence factor to diagnostic tools and vaccines for TB

Research on ESAT-6 and its involvement in several steps of Mtb pathogenesis, together with the strong antigenic recognition in TB patients, reveals its potential for therapeutic and diagnostic applications.

ESAT-6 through its duality of virulence and antigenicity, is a target for the design of more effective vaccines than BCG. The strategic design of new live attenuated vaccines should preserve Mtb antigens while removing virulence factors to prevent host damage. MTBVAC, a vaccine in development that has just entered phase 3 clinical trials (see [137]), is the only vaccine based on an attenuated Mtb strain [138]. It is conceivable that MTBVAC, by targeting epitopes from the RD1 that are missing from the BCG vaccine, could provide better protection against TB.

The attenuated virulence phenotype is based on PhoP mutants that are unable to secrete ESAT-6. The protein is synthesized but remains inside the pathogen with its antigenic potential. PhoP mutations in the attenuated vaccine strain prevent EspA translation from the espACD locus. A second deletion affects the gene required for the biosynthesis and export of phthiocerol dimycocerosates (PDIM), the major virulence-associated cell-wall lipids of Mtb. Both EspA and PDIM act together in the phagosomal secretion of ESAT-6 [105,139].

Subunit vaccine candidates are designed to boost BCG-primed responses or to induce specific immune responses as examples of the therapeutic vaccines under development to be used in conjugation with antibiotic treatment [140,141,142,143]. ESAT- 6-based subunit vaccines, already in clinical trials, include TB/FLU-04L and use a live-attenuated influenza A virus vector. Other subunit vaccines in clinical evaluation such as H6, H56:IC31 and GamTBvac are provided in non-viral delivery systems and are based on fusion immunogenic proteins, including ESAT-6 together with adjuvants [142,144,145,146].

An intranasally administered subunit vaccine combining ESAT-6 and cyclic dimeric adenosine monophosphate (c-di-AMP) was designed to promote macrophage autophagy via the STING pathway with impact on pathogen killing and humoral and cellular immune responses [147]. Other fusion proteins, such as dodicin-ESAT-6, result in the increased expression of the costimulatory molecules CD80/CD86 and the antigen presenting machinery MHC-II in a mouse model of the infection [148]. In the improvement of these combinations with ESAT-6 in subunit vaccine candidates the chaperone-like protein HtpG_Mtb of Mtb has been studied and structure-based design studies points for more effective antigen properties for immunization [149,150,151].

Some challenges in the development of ESAT-6-based subunit vaccines is that while CD4 T cells are maintained in the lung parenchyma due to continuous antigenic stimulation, the protective immunity is limited by functional exhaustion [152].

Another relevant application on ESAT-6 has been the development of TB diagnostics such as the IFNγ release assays (IGRAs) [153]. It was initially designed to guide preventive treatment of infected individuals at risk of developing active TB. Additionally, the ESAT-6-based IGRA allows differentiation between BCG vaccinated and unvaccinated individuals as BCG does not possess or secrete these proteins.

However, the challenges associated with subunit ESAT-6 based subunit vaccines is that they will virtually override all modern available immunodiagnostics (IGRAs and skin tests) thereby limiting the ability to distinguish immunized from infected people. To overcome this ESAT-6 free IGRA tests are under development aimed at differentiating ESAT-6 subunit responses in vaccinated individuals. These assays are based on the combination of a cocktail of proteins that are part of ESX-1 operon including the CFP-10 chaperon but excluding ESAT-6. Preliminary results show promising effects on the induction IFNγ and the chemokine biomarker IP-10 allowing distinguish subunit-vaccinated from non-vaccinated individuals [75].

An ESAT-6/CFP-10-based skin test, C-Tb, has revealed similar sensitivity for active TB compared with tuberculin skin test (TST) and QuantiFERON-TB-Gold-In-Tube (QFT), but with limited sensitivity in children and HIV-infected individuals [154]. The diagnosis of TB in children as in HIV-infected individuals is often difficult due to several factors, including the frequent extrapulmonary TB, a recurrent associated sputum smear-negative and the fact that that they have a low humoral response to mycobacterial antigens using conventional enzyme-linked immunosorbent assays (ELISA) for TB immunodiagnosis [51,155]. To overcome this, immunodiagnostic methods using antigen combinations are being developed, using “cocktails” of PhoP, ESAT-6, CFP-10 and the latency-associated antigen Acr-1. This combination of antigens includes proteins that are associated with the different stages of disease progression [156] and has been shown to significantly improve the sensitivity. Recently, another cocktail using biosynthetically derived peptides of ESAT-6 and Ag-85 allowed the detection of irrespective specific-IgG in patients’ sera, providing a reliable diagnosis of active TB in children [157].

Other situations were extrapulmonary TB is frequent is in bovine TB and the corresponding zoonotic form in humans. The available methods to distinguish M.bovis from Mtb are based in polymerase chain reaction and genomic sequencing. This imposes limitations requiring bacilli isolation difficult to obtain in this context and the lack of technical capacity in high burden countries [32].

Future directions on diagnostic tools should allow to determine differences between latent TB infection, BCG/sub-unit vaccination, and active TB infection, extrapulmonary TB with enough sensitivity to detect all cases of TB including children and HIV-infected people and MTBC species. This could revolutionize TB diagnostics and treatment strategies, allowing the development of differentiating biomarkers so relevant to evaluate the status of immune activation and/or the stage of the infection.

5. Conclusions

The studies on ESAT-6 allowed to define relevant steps in Mtb pathogenesis and to distinguish virulence from attenuated phenotypes. The lessons from this knowledge will allow us to foster our understanding of this proteinaceous army that makes Mtb such a successful human pathogen. The likelihood that this could enable us to target ESAT-6 and the host hijacking pathways involved to halt the spread of disease is a possibility for the near future. Perhaps this will open new avenues leading to the development of novel immunotherapeutic strategies to stop of TB in the 21st century.