Abstract
The small, regulatory RNA RepG (
Re
gulator of
p
olymeric
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.
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.
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.
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.
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.
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.
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
infection. Buruli ulcer is caused by
. This neglected disease is the third most common mycobacterial disease worldwide. Without treatment,
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
. 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
to achieve this end.
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.
Abstract
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.
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.
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