Buruli Ulcer Lesions Research Articles

A Mycobacterium ulcerans toxin, mycolactone, induces apoptosis in primary human keratinocytes and in HaCaT cells

The pathogenicity of Mycobacterium ulcerans (Buruli ulcer) depends on cytotoxic effect of its exotoxin mycolactone. Since epidermis represents a barrier against infectious agents and balanced apoptosis is essential in epidermal homeostasis, we explored if mycolactone A/B induces apoptosis on two human keratinocyte populations, stem cells (KSC) and transit amplifying cells (TAC), and on human keratinocyte line, HaCaT. Treatment of TAC with 1 and 10 ng/ml mycolactone-induced 60 and 90% apoptosis. KSC were more resistant than TAC: 50 and 75% of cells underwent apoptosis after 10 and 100 ng/ml toxin-treatment. Higher doses (1000 ng/ml) induced about 30% apoptosis on HaCaT. In contrast, mycolactone A/B was devoid of toxicity neither on human hepatoma HuH7 nor on human embryonic kidney HEK 293 T cell lines. In conclusion, mycolactone induces apoptosis in human keratinocytes, thus contributing to Buruli ulcer lesions development.

Fine-Needle Aspiration, an Efficient Sampling Technique for Bacteriological Diagnosis of Nonulcerative Buruli Ulcer

Invasive punch or incisional skin biopsy specimens are currently employed for the bacteriological confirmation of the clinical diagnosis of Buruli ulcer (BU), a cutaneous infectious disease caused by Mycobacterium ulcerans. The efficacy of fine-needle aspirates (FNA) using fine-gauge needles (23G by 25 mm) for the laboratory confirmation of BU was compared with that of skin tissue fragments obtained in parallel by excision or punch biopsy. In three BU treatment centers in Benin, both types of diagnostic material were obtained from 33 clinically suspected cases of BU and subjected to the same laboratory analyses: i.e., direct smear examination, IS2404 PCR, and in vitro culture. Twenty-three patients, demonstrating 17 ulcerative and 6 nonulcerative lesions, were positive by at least two tests and were therefore confirmed to have active BU. A total of 68 aspirates and 68 parallel tissue specimens were available from these confirmed patients. When comparing the sensitivities of the three confirmation tests between FNA and tissue specimens, the latter yielded more positive results, but only for PCR was this significant. When only nonulcerative BU lesions were considered, however, the sensitivities of the confirmation tests using FNA and tissue specimens were not significantly different. Our results show that the minimally invasive FNA technique offers enough sensitivity to be used for the diagnosis of BU in nonulcerative lesions.

Mycolactone Inhibits Monocyte Cytokine Production by a Posttranscriptional Mechanism

The virulence and immunosuppressive activity of Mycobacterium ulcerans is attributed to mycolactone, a macrolide toxin synthesized by the bacteria. We have explored the consequence and mechanism of mycolactone pretreatment of primary human monocytes activated by a wide range of TLR ligands. The production of cytokines (TNF, IL-1beta, IL-6, IL-10, and IFN-gamma-inducible protein-10), chemokines (IL-8), and intracellular effector molecules (exemplified by cyclooxygenase-2) was found to be powerfully and dose dependently inhibited by mycolactone, irrespective of the stimulating ligand. However, mycolactone had no effect on the activation of signaling pathways that are known to be important in inducing these genes, including the MAPK and NF-kappaB pathways. Unexpectedly, LPS-dependent transcription of TNF, IL-6, and cyclooxygenase-2 mRNA was found not to be inhibited, implying that mycolactone has a novel mechanism of action and must function posttranscriptionally. We propose that mycolactone mediates its effects by inhibiting the translation of a specific subset of proteins in primary human monocytes. This mechanism is distinct from rapamycin, another naturally occurring immunosuppressive lactone. The current findings also suggest that monocyte-derived cytokine transcript and protein levels may not correlate in Buruli ulcer lesions, and urge caution in the interpretation of RT-PCR data obtained from patient biopsy samples.

Mycolactone Diffuses from Mycobacterium ulcerans–Infected Tissues and Targets Mononuclear Cells in Peripheral Blood and Lymphoid Organs

BackgroundBuruli ulcer (BU) is a progressive disease of subcutaneous tissues caused by Mycobacterium ulcerans. The pathology of BU lesions is associated with the local production of a diffusible substance, mycolactone, with cytocidal and immunosuppressive properties. The defective inflammatory responses in BU lesions reflect these biological properties of the toxin. However, whether mycolactone diffuses from infected tissues and suppresses IFN-γ responses in BU patients remains unclear.Methodology/Principal FindingsHere we have investigated the pharmacodistribution of mycolactone following injection in animal models by tracing a radiolabeled form of the toxin, and by directly quantifying mycolactone in lipid extracts from internal organs and cell subpopulations. We show that subcutaneously delivered mycolactone diffused into mouse peripheral blood and accumulated in internal organs with a particular tropism for the spleen. When mice were infected subcutaneously with M. ulcerans, this led to a comparable pattern of distribution of mycolactone. No evidence that mycolactone circulated in blood serum during infection could be demonstrated. However, structurally intact toxin was identified in the mononuclear cells of blood, lymph nodes and spleen several weeks before ulcerative lesions appear. Importantly, diffusion of mycolactone into the blood of M. ulcerans–infected mice coincided with alterations in the functions of circulating lymphocytes.ConclusionIn addition to providing the first evidence that mycolactone diffuses beyond the site of M. ulcerans infection, our results support the hypothesis that the toxin exerts immunosuppressive effects at the systemic level. Furthermore, they suggest that assays based on mycolactone detection in circulating blood cells may be considered for diagnostic tests of early disease.

Phagocytosis ofMycobacterium ulceransin the course of rifampicin and streptomycin chemotherapy in Buruli ulcer lesions

Infection with Mycobacterium ulcerans involves a devastating skin disease called Buruli ulcer (BU). Currently, dual therapy with rifampicin and streptomycin (R/S) for 8 weeks as well as surgery are the standard treatments. To elucidate the processes taking place in BU lesions in the course of chemotherapy we performed an in-depth histological analysis of lesions after 4 weeks of R/S treatment and compared results with findings in untreated lesions and lesions treated for 8 weeks. Tissue specimens were collected from patients who had no treatment and from patients after 4 and 8 weeks of R/S treatment. The main features evaluated were local immune responses, histopathological alterations and bacterial distribution. After 4 weeks of R/S treatment we observed a large proportion of mycobacteria inside macrophages, occasionally forming globus-like aggregations. While distinct bands of inflammatory leucocytes surrounded the necrotic core in an ulcer and early granuloma formation was apparent in the healthy-appearing margins, acute cellular infiltration covering the whole lesion had developed in a nodular lesion. In contrast, ulcerative lesions after 8 weeks of chemotherapy showed intra- and extracellular bacterial debris as well as the presence of extensive chronic infiltrates forming huge granulomas. R/S treatment of BU results in a rapid onset of local cellular immune responses associated with phagocytosis of the extracellular M. ulcerans. This may be related to declining levels of the macrolide toxin mycolactone in the tissue, thus leading to an enhanced chemotherapy-induced clearance of the infection.

Development of highly organized lymphoid structures in Buruli ulcer lesions after treatment with rifampicin and streptomycin.

BackgroundBuruli ulcer caused by Mycobacterium ulcerans is an infection of the subcutaneous tissue leading to chronic necrotising skin ulcers. The pathogenesis is associated with the cytocidal and immunosuppressive activities of a macrolide toxin. Histopathological hallmark of progressing disease is a poor inflammatory response despite of clusters of extracellular bacilli. While traditionally wide excision of the infected tissue was the standard treatment, provisional WHO guidelines now recommend an eight week pre-treatment with streptomycin and rifampicin.Methodology/Principal FindingsWe conducted a detailed immunohistochemical analysis of tissue samples from Buruli patients who received antibiotic treatment. Cellular immune response along with bacterial load and distribution were monitored. We demonstrate that this treatment leads to the development of highly organized cellular infiltration surrounding areas of coagulative necrosis. Diffuse infiltrates, granulomas and dense lymphocyte aggregation close to vessels were observed. Mycobacterial material was primarily located inside mononuclear phagocytes and microcolonies consisting of extracellular rod-shaped mycobacteria were no longer found. In observational studies some patients showed no clinical response to antibiotic treatment. Corresponding to that, one of five lesions analysed presented with huge clusters of rod-shaped bacilli but no signs of infiltration.Conclusions/SignificanceResults signify that eight weeks of antibiotic treatment reverses local immunosuppression and leads to an active inflammatory process in different compartments of the skin. Structured leukocyte infiltrates with unique signatures indicative for healing processes developed at the margins of the lesions. It remains to be analysed whether antibiotic resistance of certain strains of M. ulcerans, lacking patient compliance or poor drug quality are responsible for the absent clinical responses in some patients. In future, analysis of local immune responses could serve as a suitable surrogate marker for the efficacy of alternative treatment strategies.

Local Activation of the Innate Immune System in Buruli Ulcer Lesions

Buruli ulcer (BU) caused by Mycobacterium ulcerans is a chronic necrotizing disease of the skin and the underlying soft tissue. Fat tissue necrosis accompanied by minimal inflammation is considered the most reliable histopathologic feature of BU. There may be a constant influx of inflammatory cells to the sites of active infection but these are thought to be killed by mycolactone, a polyketide toxin produced by M. ulcerans, through apoptosis and necrosis. Here we describe the spatial correlations between mycobacterial load and the expression of dendritic cell (DC) surface markers (cluster of differentiation (CD)83, CD11c, and CD123), the Toll-like receptor (TLR) 9 and pro- and anti-inflammatory cytokines (IL-8, IL-6, tumor necrosis factor-alpha (TNF-alpha), IFN-alpha, IL-12p40, IL-10, and IFN-gamma) within BU lesions. Although IL-8, IL-6, and TNF-alpha messenger RNA (mRNA) was detectable by real-time PCR in all lesions, the expression of the other cytokines was only found as small foci in some lesions. Correlations of the distribution of mRNA encoding the activation marker CD83 and the DC subset markers CD123 and CD11c indicate that both activated plasmacytoid and myeloid dendritic cells were present in the lesions. Results suggest that M. ulcerans specific immune responses may develop once therapeutic interventions have limited the production of mycolactone.

Contiguous spread of Mycobacterium ulcerans in Buruli ulcer lesions analysed by histopathology and real‐time PCR quantification of mycobacterial DNA

The distribution of M. ulcerans in Buruli ulcer lesions was analysed by IS2404 real-time PCR quantification of M. ulcerans DNA and by semi-quantitative microscopic assessment of the number of acid-fast bacilli (AFB). Mycobacterial burden was compared with histopathological changes. Focal distribution of tissue destruction extending into areas with high and low mycobacterial burden was a feature in all lesions analysed. Even where most of the mycobacteria were washed out of ulcerative lesions, peaks of mycobacterial DNA and AFB in the necrotic base of the ulcers still marked the position of the primary infection focus. Significant amounts of mycobacterial DNA and microcolonies were also present in samples from more peripheral regions and occasionally in excised margins of macroscopically and histologically healthy-appearing excised tissue margins. Additional peaks of mycobacterial DNA clearly marked sites where satellite lesions were developing. Even when granulomas provided evidence for the development of cell-mediated immunity, development of satellite lesions by contiguous spreading was not completely prevented. Areas free of mycobacterial DNA were found between primary and secondary infection foci and around scarring tissue of healing lesions. These results demonstrate that IS2404 real-time PCR analysis is a better tool than the less sensitive and only semi-quantitative microscopic enumeration of AFB for studying the dynamics of M. ulcerans infection in situ.

HIGH RATES OF APOPTOSIS IN HUMAN MYCOBACTERIUM ULCERANS CULTURE-POSITIVE BURULI ULCER SKIN LESIONS

Buruli ulcer, a disease caused by Mycobacterium ulcerans, causes ulcerative skin disease likely generated by a toxin that mediates apoptosis. We analyzed paraffin-embedded sections of surgically excised Buruli ulcer lesions (two ulcers and one edematous plaque) and adjacent non-lesional skin samples (n = 9) for apoptosis by an indirect immunofluorescent terminal deoxynucleotide transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay. All samples were stained for acid-fast bacilli (AFB) and cultured for mycobacteria, and most were analyzed with an M. ulcerans-specific diagnostic polymerase chain reaction (PCR). TUNEL (+) bodies were numerous in both ulcers and the plaque, and sparse or absent in adjacent non-lesional skin. The AFB tissue stains and cultures for M. ulcerans were positive only in the three lesions. The result of the PCR for M. ulcerans was positive in all three lesions and in four of six non-lesional tissue samples; three contained sparse TUNEL (+) bodies. An abundance of TUNEL (+) bodies in the three AFB stain (+), culture (+), and PCR (+) Buruli ulcer lesional samples, but not in nearby AFB stain (-), culture (-), and PCR (+) non-lesional skin samples, strengthen the evidence that apoptosis is an important tissue destruction mechanism in human lesions closely associated with viable M. ulcerans.

Distribution of Buruli ulcer lesions over body surface area in a large case series in Ghana: uncovering clues for mode of transmission

We studied hospital records of 750 consecutive Buruli ulcer patients in a highly endemic area in Amansie West, Ghana. Although more Buruli ulcer lesions were found on the right side of the body, comparison of lesions on arms and legs showed a bilaterally symmetrical distribution. Upper and lower extremities were affected equally by Buruli ulcers, if correction was made for differences in body surface area. Patients from outside the Amansie West district presented significantly more often with ulcerated lesions, which were more often located on a joint, than patients who lived in Amansie West, suggesting that longer travel distance might have caused delay. Our observations of a bilaterally symmetrical distribution of lesions on extremities and equal upper and lower extremity involvement are compatible with a mode of transmission that involves passive exposure of exposed body parts. An asymmetrical distribution of lesions was found in an earlier study, suggesting transmission by vegetation near the ground, through activities like farming or play. Perhaps, transmission in or near water, e.g. by bites of infected aquatic insects, might favour the pattern of distribution of lesions that we found.