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.     

Mycolactone toxin induces an inflammatory response by targeting the IL-1β pathway: Mechanistic insight into Buruli ulcer pathophysiology

Mycolactone, a lipid-like toxin, is the major virulence factor of Mycobacterium ulcerans, the etiological agent of Buruli ulcer. Its involvement in lesion development has been widely described in early stages of the disease, through its cytotoxic and immunosuppressive activities, but less is known about later stages. Here, we revisit the role of mycolactone in disease outcome and provide the first demonstration of the pro-inflammatory potential of this toxin. We found that the mycolactone-containing mycobacterial extracellular vesicles produced by M. ulcerans induced the production of IL-1β, a potent pro-inflammatory cytokine, in a TLR2-dependent manner, targeting NLRP3/1 inflammasomes. We show our data to be relevant in a physiological context. The in vivo injection of these mycolactone-containing vesicles induced a strong local inflammatory response and tissue damage, which were prevented by corticosteroids. Finally, several soluble pro-inflammatory factors, including IL-1β, were detected in infected tissues from mice and Buruli ulcer patients. Our results revisit Buruli ulcer pathophysiology by providing new insight, thus paving the way for the development of new therapeutic strategies taking the pro-inflammatory potential of mycolactone into account.     

Subcutaneous injection of Mycobacterium ulcerans causes necrosis, chronic inflammatory response and fibrosis in skeletal muscle

Mycobacterium ulcerans (M. ulcerans) causes Buruli ulcer, a very debilitating disease that affects the skin and other tissues. The disease occurs mainly in children in sub-Sahara Africa. While contracture, fibrosis and functional limitation of range of motion are frequent complications of Buruli ulcer, no fundamental or clinical studies have investigated the impact of M. ulcerans infections on skeletal muscle. In the present study, we subcutaneously infected mice in the proximity of the right biceps muscle to evaluate the histological, biochemical and functional impact of M. ulcerans on skeletal muscles. The concentration of mast cells decreased but the number of neutrophils and macrophages increased steadily in proximate-infected biceps muscles. Pro- and anti-inflammatory cytokines as well as fibrogenic growth factor mRNA also increased. Significantly more membrane damage and fibrosis occurred in proximate-infected biceps muscles than in control and sham muscles. Passive biomechanical testing also revealed that the presence of M. ulcerans increased muscle stiffness. These findings show for the first time that M. ulcerans can induce local and chronic inflammatory responses in skeletal muscles that are associated with muscle fiber damage and fibrosis.     

Tumor necrosis factor-alpha promotes the accumulation of neutrophils and macrophages in skeletal muscle.

Tumor necrosis factor-alpha (TNF-alpha) has been associated with cachexia and is known to regulate multiple inflammatory cell (neutrophil and macrophage) responses. We tested the hypothesis that neutrophils and macrophages accumulate in the extensor digitorum longus (EDL) and soleus muscles of mice after chronic TNF-alpha administration. Murine recombinant TNF-alpha (approximately 100 microg x kg(-1) x day(-1)) in vehicle solution or vehicle solution alone (sham) was administered to C57BL/6 mice for 7 days via osmotic minipumps. In EDL muscles from TNF-alpha-treated mice, neutrophil and macrophage concentrations were elevated seven- and threefold, respectively, compared with sham mice. Neutrophil and macrophage concentrations were also elevated five- and twofold, respectively, in solei of TNF-alpha- relative to sham-treated mice. Treatment with TNF-alpha elevated ubiquitin content by approximately 25% relative to sham values for both the EDL and soleus muscles; however, these elevations were not statistically significant. No differences were observed between TNF-alpha- and sham-treated mice in body weight, food consumption, muscle mass, myofiber cross-sectional area, carbonyl groups, total protein content, or relative abundance of myosin heavy chain protein. Furthermore, no overt signs of muscle injury or regeneration were observed in muscles from TNF-alpha-treated mice in either the EDL or soleus muscles. These observations suggest that 7 days of TNF-alpha administration promote muscle inflammation as indicated by the accumulation of neutrophils and macrophages without overt signs of atrophy, injury, or regeneration.     

Natural antioxidants attenuate mycolactone toxicity to RAW 264.7 macrophages

Mycobacterium ulcerans produces a macrolide exotoxin, mycolactone which suppresses immune cells activity, is toxic to most cells and the key virulence factor in the pathogenesis of Buruli ulcer disease. Mycolactone is reported to mediate the production of reactive oxygen species in keratinocytes; cells that play critical role in wound healing. Increased levels of reactive oxygen species have been shown to disrupt the well-ordered process of wound repair; hence, the function of wound-healing cells such as macrophages, keratinocytes, and fibroblast could be impaired in the presence of the reactive oxygen species mediator, mycolactone. To ensure regeneration of tissues in chronic ulcers, with proper and timely healing of the wounds, natural antioxidants that can combat the effects of induced reactive oxygen species in wound-healing cells ought to be investigated. Reactive oxygen species activity was determined in mycolactone-treated RAW 264.7 macrophages and the scavenging ability of the antioxidants (ascorbic acid, gallic acid, and green tea kombucha) against mycolactone-induced reactive oxygen species (superoxide anions) was assessed using fluorescein probe (DCF-DA) and nitroblue tetrazolium dye. Cytotoxicity of the antioxidants, mycolactone, and the protective effect of the antioxidants on the cells upon treatment with mycolactone were determined using the Alamar blue assay. The expression levels of endogenous antioxidant enzyme genes (superoxide dismutase, catalase, and glutathione peroxidase) in response to mycolactone-mediated reactive oxygen species were determined using RT-qPCR. Mycolactone induced the production of reactive oxygen species in RAW 264.7 macrophages, and the resulting superoxide anions were scavenged by some of the antioxidants. The selected endogenous antioxidant enzyme genes in the macrophages were upregulated in the presence of the antioxidants and mycolactone. The exogenously supplied ascorbic acid and green tea kombucha offered moderate protection to the macrophages against the toxicity of mycolactone. We conclude that the results provide insights into alternate and adjunct therapeutic approaches in Buruli ulcer treatment, which could significantly attenuate the toxicity of the pathogenic factor; mycolactone.     

Spatiotemporal analysis of mycolactone distribution in vivo reveals partial diffusion in the central nervous system

Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU) disease, is unique amongst human pathogens in its capacity to produce a lipid toxin called mycolactone. While previous studies have demonstrated that bacterially-released mycolactone diffuses beyond infection foci, the spatiotemporal distribution of mycolactone remained largely unknown. Here, we used the zebrafish model to provide the first global kinetic analysis of mycolactone’s diffusion in vivo, and multicellular co-culture systems to address the critical question of the toxin’s access to the brain.Zebrafish larvae were injected with a fluorescent-derivative of mycolactone to visualize the in vivo diffusion of the toxin from the peripheral circulation. A rapid, body-wide distribution of mycolactone was observed, with selective accumulation in tissues near the injection site and brain, together with an important excretion through the gastro-intestinal tract. Our conclusion that mycolactone reached the central nervous system was reinforced by an in cellulo model of human blood brain barrier and a mouse model of M. ulcerans-infection.Here we show that mycolactone has a broad but heterogenous profile of distribution in vivo. Our investigations in vitro and in vivo support the view that a fraction of bacterially-produced mycolactone gains access to the central nervous system. The relative persistence of mycolactone in the bloodstream suggests that assays of circulating mycolactone are relevant for BU disease monitoring and treatment optimization.     

Antioxidants protect keratinocytes against M. ulcerans mycolactone cytotoxicity

Background

Mycobacterium ulcerans is the causative agent of necrotizing skin ulcerations in distinctive geographical areas. M. ulcerans produces a macrolide toxin, mycolactone, which has been identified as an important virulence factor in ulcer formation. Mycolactone is cytotoxic to fibroblasts and adipocytes in vitro and has modulating activity on immune cell functions. The effect of mycolactone on keratinocytes has not been reported previously and the mechanism of mycolactone toxicity is presently unknown. Many other macrolide substances have cytotoxic and immunosuppressive activities and mediate some of their effects via production of reactive oxygen species (ROS). We have studied the effect of mycolactone in vitro on human keratinocytes—key cells in wound healing—and tested the hypothesis that the cytotoxic effect of mycolactone is mediated by ROS.

Methodology/Principal Findings

The effect of mycolactone on primary skin keratinocyte growth and cell numbers was investigated in serum free growth medium in the presence of different antioxidants. A concentration and time dependent reduction in keratinocyte cell numbers was observed after exposure to mycolactone. Several different antioxidants inhibited this effect partly. The ROS inhibiting substance deferoxamine, which acts via chelation of Fe²⁺, completely prevented mycolactone mediated cytotoxicity.

Conclusions/Significance

This study demonstrates that mycolactone mediated cytotoxicity can be inhibited by deferoxamine, suggesting a role of iron and ROS in mycolactone induced cytotoxicity of keratinocytes. The data provide a basis for the understanding of Buruli ulcer pathology and the development of improved therapies for this disease.     

Mycolactone diffuses into the peripheral blood of Buruli ulcer patients--implications for diagnosis and disease monitoring

Background

Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU), is unique among human pathogens in its capacity to produce a polyketide-derived macrolide called mycolactone, making this molecule an attractive candidate target for diagnosis and disease monitoring. Whether mycolactone diffuses from ulcerated lesions in clinically accessible samples and is modulated by antibiotic therapy remained to be established.

Methodology/Principal Finding

Peripheral blood and ulcer exudates were sampled from patients at various stages of antibiotic therapy in Ghana and Ivory Coast. Total lipids were extracted from serum, white cell pellets and ulcer exudates with organic solvents. The presence of mycolactone in these extracts was then analyzed by a recently published, field-friendly method using thin layer chromatography and fluorescence detection. This approach did not allow us to detect mycolactone accurately, because of a high background due to co-extracted human lipids. We thus used a previously established approach based on high performance liquid chromatography coupled to mass spectrometry. By this means, we could identify structurally intact mycolactone in ulcer exudates and serum of patients, and evaluate the impact of antibiotic treatment on the concentration of mycolactone.

Conclusions/Significance

Our study provides the proof of concept that assays based on mycolactone detection in serum and ulcer exudates can form the basis of BU diagnostic tests. However, the identification of mycolactone required a technology that is not compatible with field conditions and point-of-care assays for mycolactone detection remain to be worked out. Notably, we found mycolactone in ulcer exudates harvested at the end of antibiotic therapy, suggesting that the toxin is eliminated by BU patients at a slow rate. Our results also indicated that mycolactone titres in the serum may reflect a positive response to antibiotics, a possibility that it will be interesting to examine further through longitudinal studies.     

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.     

Subtractive hybridization reveals a type I polyketide synthase locus specific to Mycobacterium ulcerans

Mycobacterium ulcerans causes Buruli ulcer, the third most prevalent mycobacterial infection of immunocompetent humans after tuberculosis and leprosy. Recent work has shown that the production by M. ulcerans of mycolactone, a novel polyketide, may partly explain the pathogenesis of Buruli ulcer. To search for the genetic basis of virulence in M. ulcerans, we took advantage of the close genetic relationship between M. ulcerans and Mycobacterium marinum by performing genomic suppressive subtractive hybridization of M. ulcerans with M. marinum. We identified several DNA fragments specific to M. ulcerans, in particular, a type I polyketide synthase locus with a highly repetitive modular arrangement. We postulate that this locus is responsible for the synthesis of mycolactone in M. ulcerans.     

Mycolactone diffuses from Mycobacterium ulcerans-infected tissues and targets mononuclear cells in peripheral blood and lymphoid organs

Background

Buruli 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 Findings

Here 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.

Conclusion

In 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.     

Proteomic Analysis of the Action of the Mycobacterium ulcerans Toxin Mycolactone: Targeting Host Cells Cytoskeleton and Collagen

Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans. The tissue damage characteristic of BU lesions is known to be driven by the secretion of the potent lipidic exotoxin mycolactone. However, the molecular action of mycolactone on host cell biology mediating cytopathogenesis is not fully understood. Here we applied two-dimensional electrophoresis (2-DE) to identify the mechanisms of mycolactone''s cellular action in the L929 mouse fibroblast proteome. This revealed 20 changed spots corresponding to 18 proteins which were clustered mainly into cytoskeleton-related proteins (Dync1i2, Cfl1, Crmp2, Actg1, Stmn1) and collagen biosynthesis enzymes (Plod1, Plod3, P4ha1). In line with cytoskeleton conformational disarrangements that are observed by immunofluorescence, we found several regulators and constituents of both actin- and tubulin-cytoskeleton affected upon exposure to the toxin, providing a novel molecular basis for the effect of mycolactone. Consistent with these cytoskeleton-related alterations, accumulation of autophagosomes as well as an increased protein ubiquitination were observed in mycolactone-treated cells. In vivo analyses in a BU mouse model revealed mycolactone-dependent structural changes in collagen upon infection with M. ulcerans, associated with the reduction of dermal collagen content, which is in line with our proteomic finding of mycolactone-induced down-regulation of several collagen biosynthesis enzymes. Our results unveil the mechanisms of mycolactone-induced molecular cytopathogenesis on exposed host cells, with the toxin compromising cell structure and homeostasis by inducing cytoskeleton alterations, as well as disrupting tissue structure, by impairing the extracellular matrix biosynthesis.     

AMP-activated protein kinase activation prevents denervation-induced decline in gastrocnemius GLUT-4.

This study was designed to determine whether the reductions in GLUT-4 seen in 3-day-denervated muscles can be prevented through chemical activation of 5'-AMP-activated protein kinase (AMPK). Muscle AMPK can be chemically activated in rats using subcutaneous injections with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). In this study, the tibial nerve was sectioned on one side; the other was sham operated but without nerve section. Acute injections of AICAR resulted in significantly increased AMPK activity in denervated gastrocnemius but not soleus muscles. Acetyl-CoA carboxylase activity, a reporter of AMPK activation, declined in both gastrocnemius and soleus in both denervated and contralateral muscles. Three days after denervation, GLUT-4 levels were significantly decreased by approximately 40% in gastrocnemius muscles and by approximately 30% in soleus muscles. When rats were injected with AICAR (1 mg/g body wt) for 3 days, the decline in GLUT-4 levels was prevented in denervated gastrocnemius muscles but not in denervated soleus muscles. The extent of denervation-induced muscle atrophy was similar in AICAR-treated vs. saline-treated rats. These studies provide evidence that some effects of denervation may be prevented by chemical activation of the appropriate signaling pathways.     

Microbiological,Histological, Immunological,and Toxin Response to Antibiotic Treatment in the Mouse Model of Mycobacterium ulcerans Disease

Mycobacterium ulcerans infection causes a neglected tropical disease known as Buruli ulcer that is now found in poor rural areas of West Africa in numbers that sometimes exceed those reported for another significant mycobacterial disease, leprosy, caused by M. leprae. Unique among mycobacterial diseases, M. ulcerans produces a plasmid-encoded toxin called mycolactone (ML), which is the principal virulence factor and destroys fat cells in subcutaneous tissue. Disease is typically first manifested by the appearance of a nodule that eventually ulcerates and the lesions may continue to spread over limbs or occasionally the trunk. The current standard treatment is 8 weeks of daily rifampin and injections of streptomycin (RS). The treatment kills bacilli and wounds gradually heal. Whether RS treatment actually stops mycolactone production before killing bacilli has been suggested by histopathological analyses of patient lesions. Using a mouse footpad model of M. ulcerans infection where the time of infection and development of lesions can be followed in a controlled manner before and after antibiotic treatment, we have evaluated the progress of infection by assessing bacterial numbers, mycolactone production, the immune response, and lesion histopathology at regular intervals after infection and after antibiotic therapy. We found that RS treatment rapidly reduced gross lesions, bacterial numbers, and ML production as assessed by cytotoxicity assays and mass spectrometric analysis. Histopathological analysis revealed that RS treatment maintained the association of the bacilli with (or within) host cells where they were destroyed whereas lack of treatment resulted in extracellular infection, destruction of host cells, and ultimately lesion ulceration. We propose that RS treatment promotes healing in the host by blocking mycolactone production, which favors the survival of host cells, and by killing M. ulcerans bacilli.     

Estrogen Regulates Estrogen Receptors and Antioxidant Gene Expression in Mouse Skeletal Muscle

Background

Estrogens are associated with the loss of skeletal muscle strength in women with age. Ovarian hormone removal by ovariectomy in mice leads to a loss of muscle strength, which is reversed with 17β-estradiol replacement. Aging is also associated with an increase in antioxidant stress, and estrogens can improve antioxidant status via their interaction with estrogen receptors (ER) to regulate antioxidant gene expression. The purpose of this study was to determine if ER and antioxidant gene expression in skeletal muscle are responsive to changes in circulating estradiol, and if ERs regulate antioxidant gene expression in this tissue.

Methodology/Principal Findings

Adult C57BL/6 mice underwent ovariectomies or sham surgeries to remove circulating estrogens. These mice were implanted with placebo or 17β-estradiol pellets acutely or chronically. A separate experiment examined mice that received weekly injections of Faslodex to chronically block ERs. Skeletal muscles were analyzed for expression of ER genes and proteins and antioxidant genes. ERα was the most abundant, followed by Gper and ERβ in both soleus and EDL muscles. The loss of estrogens through ovariectomy induced ERα gene and protein expression in the soleus, EDL, and TA muscles at both the acute and chronic time points. Gpx3 mRNA was also induced both acutely and chronically in all 3 muscles in mice receiving 17β-estradiol. When ERs were blocked using Faslodex, Gpx3 mRNA was downregulated in the soleus muscle, but not the EDL and TA muscles.

Conclusions/Significance

These data suggest that Gpx3 and ERα gene expression are sensitive to circulating estrogens in skeletal muscle. ERs may regulate Gpx3 gene expression in the soleus muscle, but skeletal muscle regulation of Gpx3 via ERs is dependent upon muscle type. Further work is needed to determine the indirect effects of estrogen and ERα on Gpx3 expression in skeletal muscle, and their importance in the aging process.     

beta-Adrenoceptor signaling in regenerating skeletal muscle after beta-agonist administration

Stimulating the beta-adrenoceptor (beta-AR) signaling pathway can enhance the functional repair of skeletal muscle after injury, but long-term use of beta-AR agonists causes beta-AR downregulation, which may limit their therapeutic effectiveness. The aim was to examine beta-AR signaling during early regeneration in rat fast-twitch [extensor digitorum longus (EDL)] and slow-twitch (soleus) muscles after bupivacaine injury and test the hypothesis that, during regeneration, beta-agonist administration does not cause beta-AR desensitization. Rats received either the beta-AR agonist fenoterol (1.4 mgxkg(-1)xday(-1) ip) or saline for 7 days postinjury. Fenoterol reduced beta-AR density in regenerating soleus muscles by 42%. Regenerating EDL muscles showed a threefold increase in beta-AR density, and, again, these values were 43% lower with fenoterol treatment. An amplified adenylate cyclase (AC) response to isoproterenol was observed in cell membrane fragments from EDL and soleus muscles 7 days postinjury. Fenoterol attenuated this increase in regenerating EDL muscles but not soleus muscles. beta-AR signaling mechanisms were assessed using AC stimulants (NaF, forskolin, and Mn(2+)). Although beta-agonist treatment reduces beta-AR density in regenerating muscles, these muscles can produce large cAMP responses relative to healthy (uninjured) muscles. Desensitization of beta-AR signaling in regenerating muscles is prevented by altered rates of beta-AR synthesis and/or degradation, changes in G protein populations and coupling efficiency, and altered AC activity. These mechanisms have important therapeutic implications for modulating beta-AR signaling to enhance muscle repair after injury.     

Analysis of the Vaccine Potential of Plasmid DNA Encoding Nine Mycolactone Polyketide Synthase Domains in Mycobacterium ulcerans Infected Mice

There is no effective vaccine against Buruli ulcer. In experimental footpad infection of C57BL/6 mice with M. ulcerans, a prime-boost vaccination protocol using plasmid DNA encoding mycolyltransferase Ag85A of M. ulcerans and a homologous protein boost has shown significant, albeit transient protection, comparable to the one induced by M. bovis BCG. The mycolactone toxin is an obvious candidate for a vaccine, but by virtue of its chemical structure, this toxin is not immunogenic in itself. However, antibodies against some of the polyketide synthase domains involved in mycolactone synthesis, were found in Buruli ulcer patients and healthy controls from the same endemic region, suggesting that these domains are indeed immunogenic. Here we have analyzed the vaccine potential of nine polyketide synthase domains using a DNA prime/protein boost strategy. C57BL/6 mice were vaccinated against the following domains: acyl carrier protein 1, 2, and 3, acyltransferase (acetate) 1 and 2, acyltransferase (propionate), enoylreductase, ketoreductase A, and ketosynthase load module. As positive controls, mice were vaccinated with DNA encoding Ag85A or with M. bovis BCG. Strongest antigen specific antibodies could be detected in response to acyltransferase (propionate) and enoylreductase. Antigen-specific Th1 type cytokine responses (IL-2 or IFN-γ) were induced by vaccination against all antigens, and were strongest against acyltransferase (propionate). Finally, vaccination against acyltransferase (propionate) and enoylreductase conferred some protection against challenge with virulent M. ulcerans 1615. However, protection was weaker than the one conferred by vaccination with Ag85A or M. bovis BCG. Combinations of these polyketide synthase domains with the vaccine targeting Ag85A, of which the latter is involved in the integrity of the cell wall of the pathogen, and/or with live attenuated M. bovis BCG or mycolactone negative M. ulcerans may eventually lead to the development of an efficacious BU vaccine.     

Mycobacterium ulcerans infections cause progressive muscle atrophy and dysfunction, and mycolactone impairs satellite cell proliferation

Clinical observations from Buruli ulcer (BU) patients in West Africa suggest that severe Mycobacterium ulcerans infections can cause skeletal muscle contracture and atrophy leading to significant impairment in function. In the present study, male mice C57BL/6 were subcutaneously injected with M. ulcerans in proximity to the right biceps muscle, avoiding direct physical contact between the infectious agent and the skeletal muscle. The histological, morphological, and functional properties of the muscles were assessed at different times after the injection. On day 42 postinjection, the isometric tetanic force and the cross-sectional area of the myofibers were reduced by 31% and 29%, respectively, in the proximate-infected muscles relative to the control muscles. The necrotic areas of the proximate-infected muscles had spread to 7% of the total area by day 42 postinjection. However, the number of central nucleated fibers and myogenic regulatory factors (MyoD and myogenin) remained stable and low. Furthermore, Pax-7 expression did not increase significantly in mycolactone-injected muscles, indicating that the satellite cell proliferation is abrogated by the toxin. In addition, the fibrotic area increased progressively during the infection. Lastly, muscle-specific RING finger protein 1 (MuRF-1) and atrogin-1/muscle atrophy F-box protein (atrogin-1/MAFbx), two muscle-specific E3 ubiquitin ligases, were upregulated in the presence of M. ulcerans. These findings confirmed that skeletal muscle is affected in our model of subcutaneous infection with M. ulcerans and that a better understanding of muscle contractures and weakness is essential to develop a therapy to minimize loss of function and promote the autonomy of BU patients.     

Oxidative stress of myosin contributes to skeletal muscle dysfunction in rats with chronic heart failure

Intrinsic muscle abnormalities affecting skeletal muscle are often reported during chronic heart failure (CHF). Because myosin is the molecular motor of force generation, we sought to determine whether its dysfunction contributes to skeletal muscle weakness in CHF and, if so, to identify the underlying causative factors. Severe CHF was induced in rats by aortic stenosis. In diaphragm and soleus muscles, we investigated in vitro mechanical performance, myosin-based actin filament motility, myosin heavy (MHC) and light (MLC) chain isoform compositions, MLC integrity, caspase-3 activation, and oxidative damage. Diaphragm and soleus muscles from CHF exhibited depressed mechanical performance. Myosin sliding velocities were 16 and 20% slower in CHF than in sham in diaphragm (1.9 +/- 0.1 vs. 1.6 +/- 0.1 microm/s) and soleus (0.6 +/- 0.1 vs. 0.5 +/- 0.1 microm/s), respectively (each P < 0.05). The ratio of slow-to-fast myosin isoform did not differ between sham and CHF. Immunoblots with anti-MLC antibodies did not detect the presence of protein fragments, and no activation of caspase-3 was evidenced. Immunolabeling revealed oxidative damage in CHF muscles, and MHC was the main oxidized protein. Lipid peroxidation and expression of oxidized MHC were significantly higher in CHF than in shams. In vitro myosin exposure to increasing ONOO(-) concentrations was associated with an increasing amount of oxidized MHC and a reduced myosin velocity. These data provide experimental evidence that intrinsic myosin dysfunction occurs in CHF and may be related to oxidative damage to myosin.