Mycobacterial toxin induces analgesia in buruli ulcer by targeting the angiotensin pathways.

Mycobacterium ulcerans, the etiological agent of Buruli ulcer, causes extensive skin lesions, which despite their severity are not accompanied by pain. It was previously thought that this remarkable analgesia is ensured by direct nerve cell destruction. We demonstrate here that M. ulcerans-induced hypoesthesia is instead achieved through a specific neurological pathway triggered by the secreted mycobacterial polyketide mycolactone. We decipher this pathway at the molecular level, showing that mycolactone elicits signaling through type 2 angiotensin II receptors (AT2Rs), leading to potassium-dependent hyperpolarization of neurons. We further validate the physiological relevance of this mechanism with in vivo studies of pain sensitivity in mice infected with M. ulcerans, following the disruption of the identified pathway. Our findings shed new light on molecular mechanisms evolved by natural systems for the induction of very effective analgesia, opening up the prospect of new families of analgesics derived from such systems.

Type-I interferons promote innate immune tolerance in macrophages exposed to Mycobacterium ulcerans vesicles.

Buruli ulcer is a chronic infectious disease caused by Mycobacterium ulcerans. The pathogen persistence in host skin is associated with the development of ulcerative and necrotic lesions leading to permanent disabilities in most patients. However, few of diagnosed cases are thought to resolve through an unknown self-healing process. Using in vitro and in vivo mouse models and M. ulcerans purified vesicles and mycolactone, we showed that the development of an innate immune tolerance was only specific to macrophages from mice able to heal spontaneously. This tolerance mechanism depends on a type I interferon response and can be induced by interferon beta. A type I interferon signature was further detected during in vivo infection in mice as well as in skin samples from patients under antibiotics regiment. Our results indicate that type I interferon-related genes expressed in macrophages may promote tolerance and healing during infection with skin damaging pathogen.

An Arginine-Rich Motif in the ORF2 capsid protein regulates the hepatitis E virus lifecycle and interactions with the host cell.

Hepatitis E virus (HEV) infection is the most common cause of acute viral hepatitis worldwide. Hepatitis E is usually asymptomatic and self-limiting but it can become chronic in immunocompromised patients and is associated with increased fulminant hepatic failure and mortality rates in pregnant women. HEV genome encodes three proteins including the ORF2 protein that is the viral capsid protein. Interestingly, HEV produces 3 isoforms of the ORF2 capsid protein which are partitioned in different subcellular compartments and perform distinct functions in the HEV lifecycle. Notably, the infectious ORF2 (ORF2i) protein is the structural component of virions, whereas the genome-free secreted and glycosylated ORF2 proteins likely act as a humoral immune decoy. Here, by using a series of ORF2 capsid protein mutants expressed in the infectious genotype 3 p6 HEV strain as well as chimeras between ORF2 and the CD4 glycoprotein, we demonstrated how an Arginine-Rich Motif (ARM) located in the ORF2 N-terminal region controls the fate and functions of ORF2 isoforms. We showed that the ARM controls ORF2 nuclear translocation likely to promote regulation of host antiviral responses. This motif also regulates the dual topology and functionality of ORF2 signal peptide, leading to the production of either cytosolic infectious ORF2i or reticular non-infectious glycosylated ORF2 forms. It serves as maturation site of glycosylated ORF2 by furin, and promotes ORF2-host cell membrane interactions. The identification of ORF2 ARM as a unique central regulator of the HEV lifecycle uncovers how viruses settle strategies to condense their genetic information and hijack cellular processes.

Changes in Inflammatory Markers in Patients Treated for Buruli Ulcer and Their Ability to Predict Paradoxical Reactions.

Mycobacterium ulcerans causes Buruli ulcer, the third most frequent mycobacterial disease after tuberculosis and leprosy. Transient clinical deteriorations, known as paradoxical reactions (PRs), occur in some patients during or after antibiotic treatment. We investigated the clinical and biological features of PRs in a prospective cohort of 41 patients with Buruli ulcer from Benin. Neutrophil counts decreased from baseline to day 90, and interleukin 6 (IL-6), granulocyte colony-stimulating factor, and vascular endothelial growth factor were the cytokines displaying a significant monthly decrease relative to baseline. PRs occurred in 10 (24%) patients. The baseline biological and clinical characteristics of the patients presenting with PRs did not differ significantly from those of the other patients. However, the patients with PRs had significantly higher IL-6 and tumor necrosis factor alpha (TNF-α) concentrations on days 30, 60, and 90 after the start of antibiotic treatment. The absence of a decrease in IL-6 and TNF-α levels during treatment should alert clinicians to the possibility of PR onset.

An Overview of 10 Years of Activity of a Molecular Laboratory for Buruli Ulcer Diagnosis at a Field Hospital in Benin.

Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans. Early diagnosis is crucial to prevent morbidity. In November 2012, a field laboratory fully equipped for the rapid on-site quantitative PCR (qPCR) diagnosis of M. ulcerans was established at the Buruli ulcer treatment center (CDTLUB) center in Pobè Benin, a region where BU is endemic. We describe its first 10 years of activity and its gradual evolution into an expert laboratory for BU diagnosis. From 2012 to 2022, the laboratory analyzed 3,018 samples from patients attending consultations for suspected BU at the CDTLUB in Pobè. Ziehl-Neelsen staining and qPCR targeting the IS2404 sequence were performed. Since 2019, the laboratory has also received and analyzed 570 samples from other centers. The laboratory confirmed the diagnosis of BU by qPCR for 39.7% samples: M. ulcerans DNA was detected in 34.7% of swabs, 47.2% of all fine needle aspiration samples (FNA) and 44.6% of all skin biopsy specimens. Positive Ziehl-Neelsen staining results were obtained for 19.0% samples. Bacterial load, estimated by qPCR, was significantly greater for the Ziehl-Neelsen-positive samples than for Ziehl-Neelsen-negative samples, and detection rates were highest for FNA samples. Overall, 26.3% of the samples received from other centers were positive for BU. Most of these samples were sent by the CDTLUBs of Lalo, Allada, and Zagnanado, Benin. The establishment of the laboratory in the CDTLUB of Pobè has been a huge success. Optimal patient care depends on the close proximity of a molecular biology structure to BU treatment centers. Finally, FNA should be promoted among caregivers. IMPORTANCE Here, we describe the first 10 years of activity at a field laboratory established at the Buruli ulcer treatment center (CDTLUB) in Pobè, Benin, a country in which Mycobacterium ulcerans is endemic. Between 2012 and 2022, the laboratory analyzed 3,018 samples from patients consulting the CDTLUB of Pobè with a suspected clinical BU. Ziehl-Neelsen staining and qPCR targeting the IS2404 sequence were performed. In total, 39.7% of samples tested positive by qPCR and 19.0% tested positive by Ziehl-Neelsen staining. Detection rates were highest for FNA samples, and the bacterial loads estimated by qPCR were significantly higher for Ziehl-Neelsen-positive samples than for Ziehl-Neelsen-negative samples. Since 2019, the laboratory has also analyzed 570 samples received from outside the CDTLUB of Pobè, 26.3% of which were positive for BU. Most of these samples were sent by the CDTLUBs of Lalo, Allada, and Zagnanado in Benin. The establishment of the laboratory in the CDTLUB of Pobè has been a huge success, with major benefits for both the medical staff and patients. Our findings illustrate that the usefulness and feasibility of having a diagnostic center in rural Africa, where the disease is endemic, is a key part of optimal patient care, and that FNA should be promoted to increase detection rates.

Ketogenic Diet Impairment of Mycobacterium ulcerans Growth and Toxin Production and Enhancement of Host Response to Infection in an Experimental Mouse Model.

Ketogenic diets have been used to treat diverse conditions, and there is growing evidence of their benefits for tissue repair and in inflammatory disease treatment. However, their role in infectious diseases has been little studied. Buruli ulcer (Mycobacterium ulcerans infection) is a chronic infectious disease characterized by large skin ulcerations caused by mycolactone, the major virulence factor of the bacillus. In the current study, we investigated the impact of ketogenic diet on this cutaneous disease in an experimental mouse model. This diet prevented ulceration, by modulating bacterial growth and host inflammatory response. ß-hydroxybutyrate, the major ketone body produced during ketogenic diet and diffusing in tissues, impeded M. ulcerans growth and mycolactone production in vitro underlying its potential key role in infection. These results pave the way for the development of new patient management strategies involving shorter courses of treatment and improving wound healing, in line with the major objectives of the World Health Organization.

Vaccine-Specific Immune Responses against Mycobacterium ulcerans Infection in a Low-Dose Murine Challenge Model.

The neglected tropical disease Buruli ulcer (BU) is an infection of subcutaneous tissue with Mycobacterium ulcerans There is no effective vaccine. Here, we assessed an experimental prime-boost vaccine in a low-dose murine tail infection model. We used the enoyl reductase (ER) domain of the M. ulcerans mycolactone polyketide synthases electrostatically coupled with a previously described Toll-like receptor 2 (TLR-2) agonist-based lipopeptide adjuvant, R4Pam2Cys. Mice were vaccinated and then challenged via tail inoculation with 14 to 20 CFU of a bioluminescent strain of M. ulcerans Mice receiving either the experimental ER vaccine or Mycobacterium bovis bacillus Calmette-Guérin (BCG) were equally protected, with both groups faring significantly better than nonvaccinated animals (P < 0.05). To explore potential correlates of protection, a suite of 29 immune parameters were assessed in the mice at the end of the experimental period. Multivariate statistical approaches were used to interrogate the immune response data to develop disease-prognostic models. High levels of interleukin 2 (IL-2) and low gamma interferon (IFN-γ) produced in the spleen best predicted control of infection across all vaccine groups. Univariate logistic regression revealed vaccine-specific profiles of protection. High titers of ER-specific IgG serum antibodies together with IL-2 and IL-4 in the draining lymph node (DLN) were associated with protection induced by the ER vaccine. In contrast, high titers of IL-6, tumor necrosis factor alpha (TNF-α), IFN-γ, and IL-10 in the DLN and low IFN-γ titers in the spleen were associated with protection following BCG vaccination. This study suggests that an effective BU vaccine must induce localized, tissue-specific immune profiles with controlled inflammatory responses at the site of infection.

The potent effect of mycolactone on lipid membranes.

Mycolactone is a lipid-like endotoxin synthesized by an environmental human pathogen, Mycobacterium ulcerans, the causal agent of Buruli ulcer disease. Mycolactone has pleiotropic effects on fundamental cellular processes (cell adhesion, cell death and inflammation). Various cellular targets of mycolactone have been identified and a literature survey revealed that most of these targets are membrane receptors residing in ordered plasma membrane nanodomains, within which their functionalities can be modulated. We investigated the capacity of mycolactone to interact with membranes, to evaluate its effects on membrane lipid organization following its diffusion across the cell membrane. We used Langmuir monolayers as a cell membrane model. Experiments were carried out with a lipid composition chosen to be as similar as possible to that of the plasma membrane. Mycolactone, which has surfactant properties, with an apparent saturation concentration of 1 µM, interacted with the membrane at very low concentrations (60 nM). The interaction of mycolactone with the membrane was mediated by the presence of cholesterol and, like detergents, mycolactone reshaped the membrane. In its monomeric form, this toxin modifies lipid segregation in the monolayer, strongly affecting the formation of ordered microdomains. These findings suggest that mycolactone disturbs lipid organization in the biological membranes it crosses, with potential effects on cell functions and signaling pathways. Microdomain remodeling may therefore underlie molecular events, accounting for the ability of mycolactone to attack multiple targets and providing new insight into a single unifying mechanism underlying the pleiotropic effects of this molecule. This membrane remodeling may act in synergy with the other known effects of mycolactone on its intracellular targets, potentiating these effects.

ArfGAP1 restricts Mycobacterium tuberculosis entry by controlling the actin cytoskeleton.

The interaction of Mycobacterium tuberculosis (Mtb) with pulmonary epithelial cells is critical for early stages of bacillus colonization and during the progression of tuberculosis. Entry of Mtb into epithelial cells has been shown to depend on F-actin polymerization, though the molecular mechanisms are still unclear. Here, we demonstrate that mycobacterial uptake into epithelial cells requires rearrangements of the actin cytoskeleton, which are regulated by ADP-ribosylation factor 1 (Arf1) and phospholipase D1 (PLD1), and is dependent on the M3 muscarinic receptor (M3R). We show that this pathway is controlled by Arf GTPase-activating protein 1 (ArfGAP1), as its silencing has an impact on actin cytoskeleton reorganization leading to uncontrolled uptake and replication of Mtb. Furthermore, we provide evidence that this pathway is critical for mycobacterial entry, while the cellular infection with other pathogens, such as Shigella flexneri and Yersinia pseudotuberculosis, is not affected. Altogether, these results reveal how cortical actin plays the role of a barrier to prevent mycobacterial entry into epithelial cells and indicate a novel role for ArfGAP1 as a restriction factor of host-pathogen interactions.

FVB/N Mice Spontaneously Heal Ulcerative Lesions Induced by Mycobacterium ulcerans and Switch M. ulcerans into a Low Mycolactone Producer.

Buruli ulcer, a debilitating disease, is caused by Mycobacterium ulcerans. The incidence of this neglected tropical disease is steadily increasing. As a rule, without treatment, skin ulcers occur and a lengthy healing process may be observed associated with severe functional disabilities. Mouse models are already available to study establishment of lesions or evaluation of therapy but a lack of a suitable animal model, mimicking all clinical stages, in particular the healing process, remains an obstacle to understand the pathophysiology of M. ulcerans infection. M. ulcerans was s.c. inoculated in three consanguine mouse strains, that is, BALB/c and C57BL/6, classically used to study mycobacterial infection, and FVB/N. Strikingly, FVB/N mice, although as sensitive as all other mouse strains with respect to M. ulcerans infection, presented a spontaneous healing after the ulcerative phase despite stable bacterial load, and mycolactone toxin was not detected in the healed tissues. The spontaneous healing process was accompanied by an activation of the innate immune system. The adaptive response initiated by FVB/N mice was not involved in the healing process and did not confer protection against M. ulcerans. Our work highlights the importance of innate immune responses to control M. ulcerans infection. This in vivo model of M. ulcerans infection now paves the way for new avenues of research toward the elucidation of critical stages of this disease, such as the characterization of the regulation of mycolactone production, a better understanding of the pathophysiology of M. ulcerans infection, and the development of new therapeutic strategies.

Seasonal Patterns of Buruli Ulcer Incidence, Central Africa, 2002-2012.

To determine when risk for Buruli ulcer is highest, we examined seasonal patterns in a highly disease-endemic area of Cameroon during 2002-2012. Cases peaked in March, suggesting that risk is highest during the high rainy season. During and after this season, populations should increase protective behaviors, and case detection efforts should be intensified.

Extrahuman epidemiology of Acinetobacter baumannii in Lebanon.

The presence of Acinetobacter baumannii outside hospitals is still a controversial issue. The objective of our study was to explore the extrahospital epidemiology of A. baumannii in Lebanon. From February 2012 to October 2013, a total of 73 water samples, 51 soil samples, 37 raw cow milk samples, 50 cow meat samples, 7 raw cheese samples, and 379 animal samples were analyzed by cultural methods for the presence of A. baumannii. Species identification was performed by rpoB gene sequencing. Antibiotic susceptibility was investigated, and the A. baumannii population was studied by two genotyping approaches: multilocus sequence typing (MLST) and blaOXA-51 sequence-based typing (SBT). A. baumannii was detected in 6.9% of water samples, 2.7% of milk samples, 8.0% of meat samples, 14.3% of cheese samples, and 7.7% of animal samples. All isolates showed a susceptible phenotype against most of the antibiotics tested and lacked carbapenemase-encoding genes, except one that harbored a blaOXA-143 gene. MLST analysis revealed the presence of 36 sequence types (STs), among which 24 were novel STs reported for the first time in this study. blaOXA-51 SBT showed the presence of 34 variants, among which 21 were novel and all were isolated from animal origins. Finally, 30 isolates had new partial rpoB sequences and were considered putative new Acinetobacter species. In conclusion, animals can be a potential reservoir for A. baumannii and the dissemination of new emerging carbapenemases. The roles of the novel animal clones identified in community-acquired infections should be investigated.

Findings in patients from Benin with osteomyelitis and polymerase chain reaction-confirmed Mycobacterium ulcerans infection.

BACKGROUND: Mycobacterium ulcerans is known to cause Buruli ulcer (BU), a necrotizing skin disease leading to extensive cutaneous and subcutaneous destruction and functional limitations. However, M. ulcerans infections are not limited to skin, and osteomyelitis, still poorly described in the literature, occurs in numerous young patients in Africa. METHODS: In a retrospective matched case-control study conducted in a highly endemic area in Benin, we analyzed demographic, clinical, biological, and radiological features in all patients with M. ulcerans infections with bone involvement, identified from a cohort of 1257 patients with polymerase chain reaction-proved M. ulcerans infections. RESULTS: The 81 patients studied had a median age of 11 years (interquartile range, 7-16 years) and were predominantly male (male-female ratio, 2:1). Osteomyelitis was observed beneath active BU lesions (60.5%) or at a distance from active or apparently healed BU lesions (14.8%) but also in patients without a history of BU skin lesions (24.7%). These lesions had an insidious course, with nonspecific clinical findings leading to delayed diagnosis. A comparison with findings in 243 age- and sex-matched patients with BU without osteomyelitis showed that case patients were less likely to have received BCG immunization than controls (33.3% vs 52.7%; P = .01). They were also at higher risk of longer hospital stay (118 vs 69 days; P = .001), surgery (92.6% vs 63.0%; P = .001), and long-term crippling sequelae (55.6% vs 15.2%; P < .001). CONCLUSIONS: This study highlighted the difficulties associated with diagnosis of M. ulcerans osteomyelitis, with one-fourth of patients having no apparent history of BU skin lesions, including during the current course of illness. Delays in treatment contributed to the high proportion (55.6%) of patients with crippling sequelae.

Ecological niche modelling of Hemipteran insects in Cameroon; the paradox of a vector-borne transmission for Mycobacterium ulcerans, the causative agent of Buruli ulcer.

BACKGROUND: The mode of transmission of the emerging neglected disease Buruli ulcer is unknown. Several potential transmission pathways have been proposed, such as amoebae, or transmission through food webs. Several lines of evidence have suggested that biting aquatic insects, Naucoridae and Belostomatidae, may act as vectors, however this proposal remains controversial. MATERIALS AND METHODS: Herein, based on sampling in Cameroon, we construct an ecological niche model of these insects to describe their spatial distribution. We predict their distribution across West Africa, describe important environmental drivers of their abundance, and examine the correlation between their abundance and Buruli ulcer prevalence in the context of the Bradford-Hill guidelines. RESULTS: We find a significant positive correlation between the abundance of the insects and the prevalence of Buruli ulcer. This correlation changes in space and time, it is significant in one Camerounese study region in (Akonolinga) and not other (Bankim). We discuss notable environmental differences between these regions. CONCLUSION: We interpret the presence of, and change in, this correlation as evidence (though not proof) that these insects may be locally important in the environmental persistence, or transmission, of Mycobacterium. ulcerans. This is consistent with the idea of M. ulcerans as a pathogen transmitted by multiple modes of infection, the importance of any one pathway changing from region to region, depending on the local environmental conditions.

Extensive buruli ulcer in a patient returning from Mali and Senegal.

no abstract requested for correspondance items.

Emergence and spread of Mycobacterium ulcerans at different geographic scales.

The classical lineage of Mycobacterium ulcerans is the most prevalent clonal group associated with Buruli ulcer in humans. Its reservoir is strongly associated with the environment. We analyzed together 1,045 isolates collected from 13 countries on two continents to define the evolutionary history and population dynamics of this lineage. We confirm that this lineage spread over 7,000 years from Australia to Africa with the emergence of outbreaks in distinct waves in the 18th and 19th centuries. In sharp contrast with its global spread over the last century, transmission chains are now mostly local, with little or no dissemination between endemic areas. This study provides new insights into the phylogeography and population dynamics of M. ulcerans, highlighting the importance of comparative genomic analyses to improve our understanding of pathogen transmission. IMPORTANCE: Mycobacterium ulcerans is an environmental mycobacterial pathogen that can cause Buruli ulcer, a severe cutaneous infection, mostly spread in Africa and Australia. We conducted a large genomic study of M. ulcerans, combining genomic and evolutionary approaches to decipher its evolutionary history and pattern of spread at different geographic scales. At the scale of villages in an endemic area of Benin, the circulating genotypes have been introduced in recent decades and are not randomly distributed along the river. On a global scale, M. ulcerans has been spreading for much longer, resulting in distinct and compartmentalized endemic foci across Africa and Australia.

Wound colonization with methicillin-resistant Staphylococcus aureus and hypotheses about acquisition routes in rural health care settings in Sub-Saharan Africa: Perspective from a center devoted to the treatment of cutaneous neglected tropical diseases.

We identified a high prevalence (46.4%) of wound colonization with methicillin-resistant Staphylococcus aureus (MRSA) in patients hospitalized in a center devoted to the treatment of cutaneous tropical diseases in Benin. The proportion of MRSA among S aureus isolates was 54.3%. Thirty percent of these MRSA were identified in outpatients. The analysis of pulsed-field gel electrophoresis demonstrated an important diversity of strains but also identified 8 small clusters containing between 2 and 4 isolates suggesting cross-transmission.

[Buruli ulcer: a dynamic transversal research model performed through the international network of Pasteur Institutes]. / L'ulcère de Buruli: un modèle de recherche transversale dynamique au sein du réseau des Instituts Pasteur.

Buruli ulcer is an endemic severe human skin disease caused by Mycobacterium ulcerans, which prevails in western Africa in swampy areas and primarily hits children. Its gravity comes from the extent of tissue destruction, created by the toxin mycolactone. We describe here how the Centre Pasteur of Cameroon, with the help of the ministry of Health, gathered a network of multidisciplinary partners to fight against Buruli ulcer starting in the years 2000. The Centre Pasteur develops three missions : patient care, training of health care workers and research on the insect vector. Ten years of efforts resulted in significant medical advances such as the design of an early diagnostic test using PCR, or the observation that bed net use significantly decreased the risk of Buruli ulcer, offering useful prevention ; on the research side, entomological studies on aquatic bugs, coupled with epidemiological data, point to the role of these insects in the transmission of the disease. This study examplifies how an efficient network can contribute to the prevention and treatment of debilitating infectious diseases.

A photoswitchable inhibitor of TREK channels controls pain in wild-type intact freely moving animals.

By endowing light control of neuronal activity, optogenetics and photopharmacology are powerful methods notably used to probe the transmission of pain signals. However, costs, animal handling and ethical issues have reduced their dissemination and routine use. Here we report LAKI (Light Activated K+ channel Inhibitor), a specific photoswitchable inhibitor of the pain-related two-pore-domain potassium TREK and TRESK channels. In the dark or ambient light, LAKI is inactive. However, alternating transdermal illumination at 365 nm and 480 nm reversibly blocks and unblocks TREK/TRESK current in nociceptors, enabling rapid control of pain and nociception in intact and freely moving mice and nematode. These results demonstrate, in vivo, the subcellular localization of TREK/TRESK at the nociceptor free nerve endings in which their acute inhibition is sufficient to induce pain, showing LAKI potential as a valuable tool for TREK/TRESK channel studies. More importantly, LAKI gives the ability to reversibly remote-control pain in a non-invasive and physiological manner in naive animals, which has utility in basic and translational pain research but also in in vivo analgesic drug screening and validation, without the need of genetic manipulations or viral infection.

Purification and Characterization of Extracellular Vesicles from Mycobacterium ulcerans Culture.

Extracellular vesicles (EVs) from both eukaryotic and prokaryotic cells have been characterized over decades and present many biological properties. Since it has been shown that mycobacterial extracellular vesicles (MEVs) of M. ulcerans contain the macrolide toxin mycolactone, MEVs are known to be associated with the pathogenesis of mycobacteria. This chapter describes a method for purifying and characterizing vesicles from in vitro cultures of M. ulcerans. We also describe how purified vesicles can be used in cellular tests, to determine their role in the pathophysiology of M. ulcerans infection.