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 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.

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.

A combined effort of 11 laboratories in the WHO African region to improve quality of Buruli ulcer PCR diagnosis: The "BU-LABNET".

Buruli ulcer is one of the 20 neglected tropical diseases in the world. This necrotizing hypodermitis is a chronic debilitating disease caused by an environmental Mycobacterium ulcerans. At least 33 countries with tropical, subtropical and temperate climates have reported Buruli ulcer in African countries, South America and Western Pacific regions. Majority of cases are spread across West and Central Africa. The mode of transmission is unclear, hindering the implementation of adequate prevention for the population. Currently, early diagnosis and treatment are crucial to minimizing morbidity, costs and preventing long-term disability. Biological confirmation of clinical diagnosis of Buruli ulcer is essential before starting chemotherapy. Indeed, differential diagnosis are numerous and Buruli ulcer has varying clinical presentations. Up to now, the gold standard biological confirmation is the quantitative PCR, targeting the insertion sequence IS2404 of M. ulcerans performed on cutaneous samples. Due to the low PCR confirmation rate in endemic African countries (under 30% in 2018) for numerous identified reasons within this article, 11 laboratories decided to combine their efforts to create the network "BU-LABNET" in 2019. The first step of the network was to harmonize the procedures and ship specific reagents to each laboratory. With this system in place, implementation of these procedures for testing and follow-up was easy and the laboratories were able to carry out their first quality control with a very high success rate. It is now time to integrate other neglected tropical diseases to this platform, such as yaws or leprosy.

A combined field study of Buruli ulcer disease in southeast Benin proposing preventive strategies based on epidemiological, geographic, behavioural and environmental analyses.

Buruli ulcer is a neglected tropical disease caused by M. ulcerans, an environmental mycobacterium. This cutaneous infectious disease affects populations with poor access to sanitation, safe water and healthcare living in rural areas of West and Central Africa. Stagnant open bodies of surface water and slow-running streams are the only risk factor identified in Africa, and there is no human-to-human transmission. Appropriate and effective prevention strategies are required for populations living in endemic areas. Based on a multidisciplinary approach in an area in which Buruli ulcer is endemic in South Benin, we investigated the link between all human-environment interactions relating to unprotected water and behaviors associated with Buruli ulcer risk likely to affect incidence rates. We characterised the sources of water as well as water bodies and streams used by communities, by conducting a prospective case-control study directly coupled with geographic field observations, spatial analysis, and the detection of M. ulcerans in the environment. A full list of the free surface waters used for domestic activities was generated for a set of 34 villages, and several types of human behaviour associated with a higher risk of transmission were identified: (i) prolonged walking in water to reach cultivated fields, (ii) collecting water, (iii) and swimming. Combining the results of the different analyses identified the risk factor most strongly associated with Buruli ulcer was the frequency of contact with unprotected and natural water, particularly in regularly flooded or irrigated lowlands. We confirm that the use of clean water from drilled wells confers protection against Buruli ulcer. These specific and refined results provide a broader scope for the design of an appropriate preventive strategy including certain practices or infrastructures observed during our field investigations. This strategy could be improved by the addition of knowledge about irrigation practices and agricultural work in low-lying areas.

Small RNA mediated gradual control of lipopolysaccharide biosynthesis affects antibiotic resistance in Helicobacter pylori.

The small, regulatory RNA RepG (Regulator of polymeric G-repeats) regulates the expression of the chemotaxis receptor TlpB in Helicobacter pylori by targeting a variable G-repeat in the tlpB mRNA leader. Here, we show that RepG additionally controls lipopolysaccharide (LPS) phase variation by also modulating the expression of a gene (hp0102) that is co-transcribed with tlpB. The hp0102 gene encodes a glycosyltransferase required for LPS O-chain biosynthesis and in vivo colonization of the mouse stomach. The G-repeat length defines a gradual (rather than ON/OFF) control of LPS biosynthesis by RepG, and leads to gradual resistance to a membrane-targeting antibiotic. Thus, RepG-mediated modulation of LPS structure might impact host immune recognition and antibiotic sensitivity, thereby helping H. pylori to adapt and persist in the host.

Transcriptional adaptation of Mycobacterium ulcerans in an original mouse model: New insights into the regulation of mycolactone.

Mycobacterium ulcerans is the causal agent of Buruli ulcer, a chronic infectious disease and the third most common mycobacterial disease worldwide. Without early treatment, M. ulcerans provokes massive skin ulcers, caused by the mycolactone toxin, its main virulence factor. However, spontaneous healing may occur in Buruli ulcer patients several months or years after the disease onset. We have shown, in an original mouse model, that bacterial load remains high and viable in spontaneously healed tissues, with a switch of M. ulcerans to low levels of mycolactone production, adapting its strategy to survive in such a hostile environment. This original model offers the possibility to investigate the regulation of mycolactone production, by using an RNA-seq strategy to study bacterial adaptation during mouse infection. Pathway analysis and characterization of the tissue environment showed that the bacillus adapted to its new environment by modifying its metabolic activity and switching nutrient sources. Thus, M. ulcerans ensures its survival in healing tissues by reducing its secondary metabolism, leading to an inhibition of mycolactone synthesis. These findings shed new light on mycolactone regulation and pave the way for new therapeutic strategies.

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.

Skin-specific antibodies neutralizing mycolactone toxin during the spontaneous healing of Mycobacterium ulcerans infection.

Buruli ulcer, a neglected tropical infectious disease, is caused by Mycobacterium ulcerans. Without treatment, its lesions can progress to chronic skin ulcers, but spontaneous healing is observed in 5% of cases, suggesting the possible establishment of a host strategy counteracting the effects of M. ulcerans. We reveal here a skin-specific local humoral signature of the spontaneous healing process, associated with a rise in antibody-producing cells and specific recognition of mycolactone by the mouse IgG2a immunoglobulin subclass. We demonstrate the production of skin-specific antibodies neutralizing the immunomodulatory activity of the mycolactone toxin, and confirm the role of human host machinery in triggering effective local immune responses by the detection of anti-mycolactone antibodies in patients with Buruli ulcer. Our findings pave the way for substantial advances in both the diagnosis and treatment of Buruli ulcer in accordance with the most recent challenges issued by the World Health Organization.

Stable and Local Reservoirs of Mycobacterium ulcerans Inferred from the Nonrandom Distribution of Bacterial Genotypes, Benin.

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a neglected tropical disease found in rural areas of West and Central Africa. Despite the ongoing efforts to tackle Buruli ulcer epidemics, the environmental reservoir of its pathogen remains elusive, underscoring the need for new approaches to improving disease prevention and management. In our study, we implemented a local-scale spatial clustering model and deciphered the genetic diversity of the bacteria in a small area of Benin where Buruli ulcer is endemic. Using 179 strain samples from West Africa, we conducted a phylogeographic analysis combining whole-genome sequencing with spatial scan statistics. The 8 distinct genotypes we identified were by no means randomly spread over the studied area. Instead, they were divided into 3 different geographic clusters, associated with landscape characteristics. Our results highlight the ability of M. ulcerans to evolve independently and differentially depending on location in a specific ecologic reservoir.

An Optimized Method for Extracting Bacterial RNA from Mouse Skin Tissue Colonized by Mycobacterium ulcerans.

Bacterial transcriptome analyses during host colonization are essential to decipher the complexity of the relationship between the bacterium and its host. RNA sequencing (RNA-seq) is a promising approach providing valuable information about bacterial adaptation, the host response and, in some cases, mutual tolerance underlying crosstalk, as recently observed in the context of Mycobacterium ulcerans infection. Buruli ulcer is caused by M. ulcerans. This neglected disease is the third most common mycobacterial disease worldwide. Without treatment, M. ulcerans provokes massive skin ulcers. A healing process may be observed in 5% of Buruli ulcer patients several months after the initiation of disease. This spontaneous healing process suggests that some hosts can counteract the development of the lesions caused by M. ulcerans. Deciphering the mechanisms involved in this process should open up new treatment possibilities. To this end, we recently developed the first mouse model for studies of the spontaneous healing process. We have shown that the healing process is based on mutual tolerance between the bacterium and its host. In this context, RNA-seq seems to be the most appropriate method for deciphering bacterial adaptation. However, due to the low bacterial load in host tissues, the isolation of mycobacterial RNA from skin tissue for RNA-seq analysis remains challenging. We developed a method for extracting and purifying mycobacterial RNA whilst minimizing the amount of host RNA in the sample. This approach was based on the extraction of bacterial RNA by a differential lysis method. The challenge in the development of this method was the choice of a lysis system favoring the removal of host RNA without damage to the bacterial cells. We made use of the thick, resistant cell wall of M. ulcerans to achieve this end.

Characterization in Helicobacter pylori of a Nickel Transporter Essential for Colonization That Was Acquired during Evolution by Gastric Helicobacter Species.

Metal acquisition is crucial for all cells and for the virulence of many bacterial pathogens. In particular, nickel is a virulence determinant for the human gastric pathogen Helicobacter pylori as it is the cofactor of two enzymes essential for in vivo colonization, urease and a [NiFe] hydrogenase. To import nickel despite its scarcity in the human body, H. pylori requires efficient uptake mechanisms that are only partially defined. Indeed, alternative ways of nickel entry were predicted to exist in addition to the well-described NixA permease. Using a genetic screen, we identified an ABC transporter, that we designated NiuBDE, as a novel H. pylori nickel transport system. Unmarked mutants carrying deletions of nixA, niuD and/or niuB, were constructed and used to measure (i) tolerance to toxic nickel exposure, (ii) intracellular nickel content by ICP-OES, (iii) transport of radioactive nickel and (iv) expression of a reporter gene controlled by nickel concentration. We demonstrated that NiuBDE and NixA function separately and are the sole nickel transporters in H. pylori. NiuBDE, but not NixA, also transports cobalt and bismuth, a metal currently used in H. pylori eradication therapy. Both NiuBDE and NixA participate in nickel-dependent urease activation at pH 5 and survival under acidic conditions mimicking those encountered in the stomach. However, only NiuBDE is able to carry out this activity at neutral pH and is essential for colonization of the mouse stomach. Phylogenomic analyses indicated that both nixA and niuBDE genes have been acquired via horizontal gene transfer by the last common ancestor of the gastric Helicobacter species. Our work highlights the importance of this evolutionary event for the emergence of Helicobacter gastric species that are adapted to the hostile environment of the stomach where the capacity of Helicobacter to import nickel and thereby activate urease needs to be optimized.

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.

Evolution of Helicobacter: Acquisition by Gastric Species of Two Histidine-Rich Proteins Essential for Colonization.

Metal acquisition and intracellular trafficking are crucial for all cells and metal ions have been recognized as virulence determinants in bacterial pathogens. Virulence of the human gastric pathogen Helicobacter pylori is dependent on nickel, cofactor of two enzymes essential for in vivo colonization, urease and [NiFe] hydrogenase. We found that two small paralogous nickel-binding proteins with high content in Histidine (Hpn and Hpn-2) play a central role in maintaining non-toxic intracellular nickel content and in controlling its intracellular trafficking. Measurements of metal resistance, intracellular nickel contents, urease activities and interactomic analysis were performed. We observed that Hpn acts as a nickel-sequestration protein, while Hpn-2 is not. In vivo, Hpn and Hpn-2 form homo-multimers, interact with each other, Hpn interacts with the UreA urease subunit while Hpn and Hpn-2 interact with the HypAB hydrogenase maturation proteins. In addition, Hpn-2 is directly or indirectly restricting urease activity while Hpn is required for full urease activation. Based on these data, we present a model where Hpn and Hpn-2 participate in a common pathway of controlled nickel transfer to urease. Using bioinformatics and top-down proteomics to identify the predicted proteins, we established that Hpn-2 is only expressed by H. pylori and its closely related species Helicobacter acinonychis. Hpn was detected in every gastric Helicobacter species tested and is absent from the enterohepatic Helicobacter species. Our phylogenomic analysis revealed that Hpn acquisition was concomitant with the specialization of Helicobacter to colonization of the gastric environment and the duplication at the origin of hpn-2 occurred in the common ancestor of H. pylori and H. acinonychis. Finally, Hpn and Hpn-2 were found to be required for colonization of the mouse model by H. pylori. Our data show that during evolution of the Helicobacter genus, acquisition of Hpn and Hpn-2 by gastric Helicobacter species constituted a decisive evolutionary event to allow Helicobacter to colonize the hostile gastric environment, in which no other bacteria persistently thrives. This acquisition was key for the emergence of one of the most successful bacterial pathogens, H. pylori.