Mycobacterium ulcerans Disease and Host Immune Responses

Abstract

Mycobacterium ulcerans is the causative agent of the subcutaneous necrotic condition known as Buruli ulcer (BU).BU is Neglected Tropical Disease. The bacillus is the third most common mycobacteria disease-causing agent after Mycobacterium tuberculosis and Mycobacterium leprae. M. ulcerans produces the toxin-Mycolactone, which plays a key role in the pathophysiological features of the disease. Buruli ulcer has been reported in 34 countries, mainly in the tropics and subtropics. Tropical countries include Benin, Cameroon, Ghana, Democratic Republic of Congo and Nigeria. BU is also prevalent in Queensland, a subtropical region, and in Victoria, a temperate area, all within Australia. The exact mode of the transmission remains unclear. However, M. ulcerans is believed to have an aquatic niche. Initial diagnosis of BU is based on the experience of the clinician, but PCR targeting the M. ulcerans DNA, IS2404, isolation and culture of the bacillus and histopathology are used for confirmation. The current, commonly used methods for confirmatory diagnosis have logistic and resource challenges. Novel cell mediated immunity (CMI) and serology-based tests would be beneficial to provide a more accurate assessment of population exposure.

1. Introduction

Mycobacterium ulcerans is the causative agent of the subcutaneous necrotic condition known as Buruli ulcer (Figure 1) [2, 3]. The bacillus is the third most common mycobacteria disease causing agent after Mycobacterium tuberculosis and Mycobacterium leprae [4].

Figure 1.

A severe Buruli ulcer on the upper limb. Image was taken from WHO website [1].

M. ulcerans produces the toxin-Mycolactone (Figure 2), which plays a key role in the pathophysiological features of the disease [6, 7, 8]. Buruli ulcer has been reported in 34 countries (Figure 3), mainly in the tropics and subtropics [10]. Tropical countries include Benin [11], Cameroon [12, 13, 14], Ghana [15], Democratic Republic of Congo [16] and Nigeria [17, 18]. BU is also prevalent in Queensland [19], a subtropical region, and in Victoria, a temperate area, all within Australia [20].

Figure 2.

Mycolactone structure [5].

Figure 3.

Distribution of Buruli ulcer, worldwide, 2020 [9].

The exact mode of the transmission remains unclear [10, 21]. However, M. ulcerans is believed to have an aquatic niche [22, 23, 24, 25, 26]. Aquatic insects, belonging to the Family: Belostomatidae and Naucoridae have been implicated as possible vectors in Africa [22, 27, 28]. In Australia, mosquitoes [20] and possum [29] have been suggested as vector and reservoir [20, 29] respectively. Initial diagnosis of BU is based on the experience of the clinician [30], but PCR targeting the M. ulcerans DNA, IS2404 [31], isolation and culture of the bacillus and histopathology are used for confirmation [32, 33, 34].

In Africa, the disease is common in the rural setting where access to medical facilities and other infrastructure is limited [35, 36]. The opposite is true in Australia [10]. More cases have been reported among people living near wetlands [21, 37]. Gender is not a risk factor [10, 38], however age has been identified as a non-environmental risk factor [10, 38]. BU is prevalence among children between 5 and 15 age group in Africa [38]. Whereas in Australia the >60 age group are most susceptible [39, 40, 41]. This has been attributed to the ageing population in Australia, as well as immunosenescence [41, 42, 43].

2. Mycobacterium ulcerans infection

The time of infection or exposure to M. ulcerans to showing clinical symptoms of BU ranges from 1 week to 9 months [44]. The median incubation period is 4.5 months [44]. Non-ulcerative and ulcerative are the two main forms of the disease. The non-ulcerative form manifests as either painless nodule, swelling, plaque or oedema [45].

Opened necrotic lesion on the skin describes the ulcerative form and is categorised into 3 forms. Category 1 ulcers are single lesion on the skin and 5 cm in diameter [46]. Ulcers with size 5 to 15 cm is characterised as category 2 [46]. Multiples ulcers on different part of the body and/or ulcers greater than 15 cm is clinically described as category 3 [46]. African cohort usually report category 3 and 2 ulcers whereas category 1 are common in Australia [46]. This has been attributed to differences in geographical strains and virulence factor of M. ulcerans [47]. Access to adequate medical services in rural endemic regions in Africa and late presentation of condition to medical centres is another reason [30, 48].

Rook et al., described 3 phases of immune response to M. ulcerans in murine model in 1975 [49]. The first phase involves leucocyte migration and delayed hypersensitivity response at the site of infection [49]. This response is usually cell-mediated and involves proliferation of T-cells, monocytes and macrophages. In the next phase, as M. ulcerans cells multiple at the site of infection, there is subsequent reduction in the migration of inflammatory biomarkers at this site [49]. Though, there appears to be reactive cells in the lymph nodes [49]. The last phase is marked by depletion of T-cells [49]. This is attributed to excessive exposure to the bacillus toxins (mycolactone) [49]. Studies in tuberculosis and BU patients also confirms that individuals could have a negative skin-test while their lymph tissues is filled with enormous responsive cells [50, 51]. These 3 phases describes the phenomena of “sensitization” and “desensitisation” in mycobacteria infection. The latter being the non-appearances of cell-mediated response to a previously encountered antigen [49, 52].

3. Role of mycolactone in disease progression

Mycolactone is responsible for the necrotic nature of the disease [6, 7]. It is essentially a lipid-like toxin [6]. The exact role in the bacillus is unknown [6]. However, it is speculated to offer predatory protection from eukaryotic microorganisms [6]. Currently, 5 variants of mycolactone has been described and is grouped into A/B, C, D, E and F [53, 54]. Mycolactone A/B which is the most virulent variant is produced by M. ulcerans strains of African, Japan and Malaysia origin [55]. Ulcers are therefore, the most severe and difficult to treat [55]. Mycolactone C and D are localised to strains in the pacific regions, specifically Australia and China, respectively [55]. Mycobacterium liflandii, a West African frog pathogen produces the “E” variants [56, 57]. Mycolactone F is synthesised by Mycobacterium pseudoshottsii and Mycobacterium marinum [55].

Mycolactone has been observed to have cytopathic effect on host cells both in vitro and in vivo [6, 7, 58]. M. ulcerans upon entry into the body establishes a localised niche and multiplies along with mycolactone release [49]. The host mounts an initial cell-mediated response to clear the infection [49]. However, mycolactone functions as immune modulation and immunosuppressive agent [49, 59]. Thus preventing communication and recruitment of other important immune cells to the site of trauma [49, 60, 61]. Bacillus overload and dead inflammatory cells is usually seen as closed nodule-like or painless swellings (non-ulcerative). At this early stage, infection may clear or resolve naturally but persistent production of mycolactone eventually leads to necrotic and open ulcers [46, 49, 51].

4. Mycobacteria genus

Mycobacteria species are among well-studied genus of microorganism and M. tuberculosis has been an exemplar [62, 63]. The ubiquitous nature of organisms in this genus has huge medical implication [64]. Over 100 species have been identified and characterised from soil and water [64]. Mycobacteria could be divided into two main categories namely, tuberculous and non-tuberculous mycobacteria (NTM) or atypical [64]. Tuberculous mycobacteria causes diseases such as pulmonary tuberculous disease and leprosy [63]. Non-tuberculous mycobacterial species include M. ulcerans, M. avium, M. intracellularae, M. kansasii, M. xenopi, M. chelonae, M. marinum and M. fortuitum [63].

5. General overview of immune response in mammals

The natural and initial response of mammals to invading pathogens, including mycobacteria is the innate immune response [65, 66]. This involves recruitment of inflammatory cells (granulocytes) to the site of infection and subsequent ingestion and disruption of the harmful agent, which then is engulfed by macrophages [67, 68]. These are necessitated by innate immune cells, hence the name cell-mediated immunity [66]. Cell-mediate response is transient (acute) and last 1 day to 2 weeks [66]. However, humoral (antibodies) response or immunity which forms after initial expose to a pathogen is long lasting [66]. In adaptive immunity, two cells play key role [66]. These are the T (T-cell) and B (B-cell) lymphocytes [66]. T-cells develop in the bone marrow and matures in the thymus, they recognises and fights viral particulates. B-cells on the other hand matures in the bone marrow and work to eliminate bacteria and viruses from the body [66].

5.1 T-cells

T-cell is divided into “helper T cell (T_h)”, “cytotoxic T cell (T_c)”, “natural killer T cell (NKT)” and “memory T cell (T_m)” [66]. T_h cells are responsible for activation and proliferation of cytokines. They also assist in the maturation of B cells and activation of T_c and macrophages [66]. Helper T cell is divided into Type 1 (T_h1) and Type 2 (T_h2) T-helper cells [66]. Cell-mediated response is usually aided by T_h1. Resulting in the recruitment of macrophages to an infected site for action [66]. Interferon gamma (IFNγ) and macrophages are the main effector biomarkers of T_h1 immune response [66]. T_h1 response is usually mounted against intracellular bacteria and protozoa [66]. T_h2 on the other hand aids in the activation and proliferation of B cells, and is a component of humoral immunity [66]. T_h2 cells functions to clear extracellular bacteria from the body [66]. Eosinophils basophils, and mast cells are important effector agents of T_h2 immune response [66]. Cytotoxic T cell (T_c) is responsible for the disruption and cytolysis of host cells infected by virus [66]. They also play key role in tumour and cancer immunity [66]. Natural killer T cell (NKT) also assist in B cell activation and microbial defence [66].

5.2 B-cells

B cells is a component of the white blood cells and humoral immunity [66]. B lymphocytes secrete antibodies which are critical in pathogen specific recognition and elimination [66]. These cells are essential in the production of antigen-specific immunoglobulin (Ig) which are the agents that binds to pathogens [66]. Major sites of B cells in human include the blood and the lymph nodes [66]. Spleen and tonsil are other areas B cell are be found [66]. B cells and T cells work in concert to eliminate pathogens from the body [66]. After production and maturation in the bone marrow, B cells circulate through the blood and lymphoid system. Upon encounter with a specific pathogen, binding occurs. Subsequent assistance from the T cells lead to the elimination of the invading cell from the host [66].

6. Immune profile of active BU patients, exposed and unexposed controls

Screening for cytokines and antibodies specific responses in BU patients is still a matter of intense research [59, 60, 69, 70, 71, 72, 73, 74, 75]. This is because individuals with active disease are incapable of mounting an effective specific immune response to M. ulcerans [63]. It is known that patients seem to have reduced expression of T_h1 cytokines [49, 69, 70]. In comparison, T_h2 response is common among active ulcerative patients and those with healed ulcers [69, 70]. The T_h2 cytokine cells include interleukin (IL) 4, IL-5, IL-6, and IL-10. Exposed household contact with no clinical symptoms also have T_h2 response [69, 70]. However, BU patients are more likely to express IL-4 and IL-10 [69, 70, 76]. T_h1 immune response is characterised by expression of IFNγ and IL-12, critical mediators for macrophages recruitments [66, 69, 70, 76]. Interestingly, previous studies have shown that histopathology of ulcerative tissue biopsy shows macrophages with acid fast bacteria overload (likely to be M. ulcerans) [73, 77]. Mycolactone functions to either supress or deviate immune cells that could lead to the effective clearance of M. ulcerans from the host [61, 78]. Therefore, first responder cytokines such as tumour necrotic factors (TNF) is downregulated. TNF is responsible for septic shock and it works in concert with IL-17 in the release of inflammation infiltrates to infected areas and induces fever [60, 79].

7. Immune profile of BU patients undergoing antibiotic treatment

BU is curable [80], but treatment could take months if not diagnosed early [80]. Like many bacterial infection, appropriate antimicrobial medications is required in killing M. ulcerans to wane off mycolactone production [81, 82]. Currently, 8 weeks of oral rifampicin, is the preferred antibiotic regimen in many medical centres [46, 83]. Like mycolactone, rifampicin is a polyketide, and functions to inhibit the ribonucleic acid (RNA) polymerase activity of bacteria [84]. Thus leading to the shutdown of important amino acids, protein synthesis and inhibiting the growth of the bacillus [84]. Other medications/treatment tested or used in combination with rifampicin include Streptomycin, Oral Fluoroquinolones [85], Amikacin [86], Clofazimine [87], Co-trimoxazole [87], Ofloxacin [87], Cephalosporin [87], Penicillin [87], Metronidazol [87], Minocyclin [87], dapsone [87], Phenytoin powder [88], Topical nitrogen oxides, mycobacteriophage D29 [89], clay and local heat [87]. Cytokine profile of patients undergoing antibiotic treatment have shown that there is gradual re-establishment of T_h1 immune response during the healing process [90]. These inflammatory biomarkers include IL-4, IL-7 and IL-8 [90].

8. Paradoxical reactions in BU treatment

Paradoxical reaction in the healing process has also been reported [91]. This describes a stage where instead of the ulcers healing during antibiotic treatment, it rather becomes worst [91]. The exact mechanism underlining paradoxical reaction in BU healing is poorly understood. It has been speculated that immediate restoration of cytokine response to M. ulcerans antigens after mycolactone wash out could be the plausible explanation.

9. BU vaccines

There is currently no specific vaccine for BU [92]. But Bacillus Calmette–Guérin (BCG) vaccine has been used to check for cross-protection in M. ulcerans infection in Uganda [50]. BCG is a purified protein derivative (PPD) from Mycobacterium bovis [63]. It is the main vaccination regimen for TB and some non-tuberculous mycobacteria (NTM) [63, 64]. It stimulates a strong T_h1 immune response. This include IFNγ-secreting CD4+ T cell response in recipient [64]. It was observed that BCG offered a protective effect between 47 to 50% among those tested in Uganda [50, 93]. However, this protection was short-lived, the reason is unclear [50, 94]. The main protective agent in BCG vaccine is the peptide Ag85A [92]. It is a highly conserved motif and plays a critical role in cell wall synthesis [95, 96]. Ag85A homologue from M. ulcerans have also been screened for cross-protection in BU disease and have shown prospect [96]. M. ulcerans surface protein −18 kDa small heat shock protein (MUL_2232) [97] and MUL_3720 [98] have also been used in vaccine formulation against M. ulcerans [99]. In a murine experiment, a recombinant (r) vaccine formulation (rMUL2232 and rMUL3720) failed to protect against BU even though antibodies were present [99]. It appears that M. ulcerans specific antibodies fail to protect against developing overt disease [100]. Although mycolactone is the culprit in this debilitating disease [6], vaccine based on it has been thought off as a “goose chase”. The reason being, mycolatone is a polyketide, a lipid-like derivative and not necessary immunogenic [92]. Animal experiment by Robert et al 1997 did not find mycolactone an ideal candidate for diagnosis or vaccine [58].

10. BU and host susceptibility

M. ulcerans is an environmental pathogen [64]. Several studies have reported M. ulcerans DNA target both in BU endemic and non-endemic regions [21]. In addition, Sero-epidemiological studies have also shown that antibodies for the immuno-dominant protein, 18 kDa shsp is present in healthy controls [100]. This has raised numerous questions and hypotheses on host genetics and susceptibility [36]. From previous reports, it appears not all those exposed to M. ulcerans develop overt disease [36]. Some genetic marks such as solute carrier family 11, member 1 (SLC11A1) [36], autophagy-related genes E3 ubiquitin-protein ligase (PARK2) and autophagy-related protein 16–1 (ATG16L1) [36] nucleotide-binding oligomerization domain-containing 2 (NOD2) have been suggested as key players in BU susceptibility [36]. If host susceptibility is at play, it could change our perception on the mode of transmission and treatment of BU.