Genome-wide screening identified SEC61A1 as an essential factor for mycolactone-dependent apoptosis in human premonocytic THP-1 cells.

Buruli ulcer is a chronic skin disease caused by a toxic lipid mycolactone produced by Mycobacterium ulcerans, which induces local skin tissue destruction and analgesia. However, the cytotoxicity pathway induced by mycolactone remains largely unknown. Here we investigated the mycolactone-induced cell death pathway by screening host factors using a genome-scale lenti-CRISPR mutagenesis assay in human premonocytic THP-1 cells. As a result, 884 genes were identified as candidates causing mycolactone-induced cell death, among which SEC61A1, the α-subunit of the Sec61 translocon complex, was the highest scoring. CRISPR/Cas9 genome editing of SEC61A1 in THP-1 cells suppressed mycolactone-induced endoplasmic reticulum stress, especially eIF2α phosphorylation, and caspase-dependent apoptosis. Although previous studies have reported that mycolactone targets SEC61A1 based on mutation screening and structural analysis in several cell lines, we have reconfirmed that SEC61A1 is a mycolactone target by genome-wide screening in THP-1 cells. These results shed light on the cytotoxicity of mycolactone and suggest that the inhibition of mycolactone activity or SEC61A1 downstream cascades will be a novel therapeutic modality to eliminate the harmful effects of mycolactone in addition to the 8-week antibiotic regimen of rifampicin and clarithromycin.

Mycolactone cytotoxicity in Schwann cells could explain nerve damage in Buruli ulcer.

Buruli ulcer is a chronic painless skin disease caused by Mycobacterium ulcerans. The local nerve damage induced by M. ulcerans invasion is similar to the nerve damage evoked by the injection of mycolactone in a Buruli ulcer mouse model. In order to elucidate the mechanism of this nerve damage, we tested and compared the cytotoxic effect of synthetic mycolactone A/B on cultured Schwann cells, fibroblasts and macrophages. Mycolactone induced much higher cell death and apoptosis in Schwann cell line SW10 than in fibroblast line L929. These results suggest that mycolactone is a key substance in the production of nerve damage of Buruli ulcer.

[Review of WHO Expert Committee on Leprosy 8th report, --comparison to 7th report].

In 2012 the WHO Expert Committee on Leprosy published its 8th report, 14 years after the publication of its 7th report in 1998. This report, the first since the leprosy reduction goal was met in 2000, highlights key points such as improvements in the quality of various services available to patients and the efforts of individuals and societies, in addition to medical progress in diagnosis and treatment. This review will mainly describe the changes made since the 7th report. Some of the main modifications are the deletion of single lesion paucibacillary type, elongated treatment of patients with high bacterial indices, the introduction of promising new drugs, and a shift from reducing the statistical number of patients to a new target for disability prevention.

[Role of mycolactone in the nerve damage of Buruli ulcer (Mycobacterium ulcerans infection)].

Buruli ulcer is a skin disease caused by Mycobacterium ulcerans (M. ulcerans). In this review, we introduce our recent studies and other important works. Lesions of Buruli ulcer are usually painless, despite the extensive tissue necrosis. We have reported that mice inoculated with M ulcerans show nerve degeneration and absence of pain, but the mechanism evoking the nerve damage have not been clarified. In order to define whether mycolactone, a toxic lipid produced by M. ulcerans, can induce nerve damages, we have injected mycolactone A/B to BALB/c mouse footpads. Mycolactone induced footpad swelling, and sensory test showed hyperesthesia on day 7 and 14, recovery on day 21, and hypoesthesia on days 28 and 42. Histologically, nerve bundles showed hemorrhage, neutrophilic infiltration, and loss of Schwann cell nuclei on days 7 and 14. Semithin section studies revealed vacuolar change of Schwann cells started on day 14, which subsided by day 42, but myelinated fiber density remained low. This study suggests that mycolactone directly damages nerves and is responsible for the absence of pain characteristic of Buruli ulcer. In the human lesions, presence of neuritis is reported (Rondini S, 2006), and murine studies showed "autoamputation" (Addo P, 2005). In order to prevent the serious deformities evoked by Buruli ulcer, further studies are necessary.

[Power of music that moves mind and body--music therapy in the Hansen's disease sanatorium in Japan].

Average age of residents living in National sanatorium Hoshizuka-Keiaien where people have past history of Hansen disease is around 80 years old at present, and many of them spend their whole days in watching TV or sleeping almost alone in their rooms. Therefore music therapy was introduced in order to improve their daily activities in our sanatorium. Singing, listening to music, playing the musical instruments, and dancing were performed, either in a group or individually. Reactivation of their brain function such as recollection, sense of unity and relaxation were expected. Improvement of cardiopulmonary function was also expected. Solidarity and relaxed state were observed by being with the other participants in the group therapy. For example, when using musical instruments, some participants with hesitation tried to use their instruments, and had good performance. They seemed to be satisfied and became confident with the musical instruments. Then their confidence and satisfaction activated the group. After the sessions, mutual conversation increased. These processes obtained a synergy effect, which means that a group affects of individuals at first and next alteration of individual behavior influences the group. We could observe a better effect in their motivation and activity in their daily life in the individual therapy. The music therapy was applied to the senior participants by the music therapist in this study. The participants could easily reinforce their mind and body through this therapy. Music therapy will be continued for the improvement of quality of life of residents in the sanatorium.

Mycolactone is responsible for the painlessness of Mycobacterium ulcerans infection (buruli ulcer) in a murine study.

Buruli ulcer is a chronic skin disease caused by Mycobacterium ulcerans, which produces a toxic lipid mycolactone. Despite the extensive necrosis and tissue damage, the lesions are painless. This absence of pain prevents patients from seeking early treatment and, as a result, many patients experience severe sequelae, including limb amputation. We have reported that mice inoculated with M. ulcerans show loss of pain sensation and nerve degeneration. However, the molecules responsible for the nerve damage have not been identified. In order to clarify whether mycolactone alone can induce nerve damage, mycolactone A/B was injected to footpads of BALB/c mice. A total of 100 microg of mycolactone induced footpad swelling, redness, and erosion. The von Frey sensory test showed hyperesthesia on day 7, recovery on day 21, and hypoesthesia on day 28. Histologically, the footpads showed epidermal erosion, moderate stromal edema, and moderate neutrophilic infiltration up to day 14, which gradually resolved. Nerve bundles showed intraneural hemorrhage, neutrophilic infiltration, and loss of Schwann cell nuclei on days 7 and 14. Ultrastructurally, vacuolar change of myelin started on day 14 and gradually subsided by day 42, but the density of myelinated fibers remained low. This study demonstrated that initial hyperesthesia is followed by sensory recovery and final hypoesthesia. Our present study suggests that mycolactone directly damages nerves and is responsible for the absence of pain characteristic of Buruli ulcer. Furthermore, mice injected with 200 microg of mycolactone showed pulmonary hemorrhage. This is the first study to demonstrate the systemic effects of mycolactone.

Mycobacterium leprae in neurons of the medulla oblongata and spinal cord in leprosy.

Peripheral neuropathy has been extensively studied in leprosy, a chronic disease caused by Mycobacterium leprae, but the central nervous system (CNS) is thought to be free from bacilli. Involvement of the CNS was explored in autopsy cases of clinically cured lepromatous leprosy (n = 67) and in non-leprosy cases (n = 15). Paraffin sections of the medulla oblongata and spinal cord were subjected to hematoxylin and eosin staining, Fite acid-fast staining, and anti-phenolic glycolipid-I (PGL-I) immunostaining. PGL-I-positive areas were microdissected from selected cases and nested polymerase chain reaction (PCR) targeting the M. leprae-specific repetitive sequence was performed. Of the 67 cases of leprosy, 44 (67%) had vacuolar changes of motor neurons either in medulla oblongata (nucleus ambiguous or hypoglossal nucleus) or spinal cord. Fite staining was negative, but PGL-I was positive in vacuolated areas. PCR revealed M. leprae-specific genomic DNA in 18 of 19 cases (95%) with vacuolated changes and 5 of 8 (63%) without vacuolated changes. All of above findings were negative in control cases. Terminal deoxynucleotidyl transferase dUTP nick-end labeling staining did not show a significant increase of apoptosis in the neurons. The PCR positivity had a significant correlation with PGL-I immunostaining (p < 0.05). The presence of vacuolar changes in the spinal cord was correlated with hand and feet deformity grades (p = 0.04). This study provides significant additional evidence to indicate that M. leprae is present in the CNS in a subset of patients. Further investigation is required to correlate this finding to motor dysfunction and silent neuropathy in leprosy.