Prevalence and risk factors associated with Haemophilus ducreyi cutaneous ulcers in Cameroon.

Epidemics of yaws-like cutaneous ulcers are regularly documented in children in the tropics. They occur mainly in poor and remote communities without access to health facilities. The integration of molecular tools into yaws control efforts has made it possible to describe Haemophilus ducreyi (HD) as a major cause of cutaneous ulcers. The objective of this study was to determine the prevalence of HD as cause of cutaneous ulcers, investigate its presence in asymptomatic individuals and identify associated risk factors. A cross-sectional study was conducted in yaws endemic districts of Cameroon. Participants included people presenting yaws-like ulcers and asymptomatic individuals. Swab samples were collected from each participant and tested for HD and Treponema pallidum (TP) using an established qPCR method. Additionally, demographic, habitat, proximity, and hygiene characteristics were collected using a structured questionnaire. A total of 443 individuals participated in the study, including 271 ulcer cases and 172 asymptomatic contacts. The prevalence of HD in ulcers was 30.3% (Confidence Interval (CI) 95% [24.8-35.7]) and the prevalence of asymptomatic HD carriage was 8.6% (CI95% [4.5-12.9]). TP was also detected in our sample among ulcer cases but in lower proportion (5.2% CI95% [2.5-7.8]) compared to HD. The adjusted logistic regression model showed that women were as much at risk of having HD cutaneous ulcer as men regardless of age. Physical proximity to a confirmed ulcer case was the major factor identified favouring HD transmission. HD ulcers were more likely to be present on Bantu individuals compared to Baka as well as HD colonization. These findings highlight HD as the most common cause of cutaneous ulcers in yaws-endemic communities in Cameroon. The exact implications of detecting HD on intact skin are not yet clear. Further studies are needed to understand the significance of this carriage in the spread dynamics of the disease.

Endemic infectious cutaneous ulcers syndrome in the Oti Region of Ghana: Study of cutaneous leishmaniasis, yaws and Haemophilus ducreyi cutaneous ulcers.

BACKGROUND: A recent study detected cutaneous leishmaniasis (CL) in 31.9% of persons with skin ulcers in the Oti Region of Ghana, resulting in a need to investigate other potential causes of the unexplained skin ulcers. METHODOLOGY/PRINCIPAL FINDINGS: A community based cross-sectional study was conducted in the Oti region to investigate skin ulcers of undetermined aetiologies. To confirm a diagnosis of cutaneous leishmaniasis, Buruli ulcer, Haemophilus ducreyi ulcers, or yaws, DNA obtained from each patient skin ulcer sample was systematically subjected to polymerase chain reaction (PCR) for Leishmania spp., Mycobacterium ulcerans, Haemophilus ducreyi, and Treponema pallidum sub species pertenue. A total of 101 skin ulcer samples were obtained from 101 persons. Co-infection of more than one organism was observed in 68.3% of the samples. Forty (39.6%) participants had a positive result for Leishmania spp., 68 (67.3%) for Treponema pallidum sub. Sp. pertenue, and 74 (73.3%) for H. ducreyi. Twenty (19.8%) of the patient ulcers were simultaneously infected with Leishmania spp., Treponema pallidum sub. Sp. pertenue, and H. ducreyi. None of the patients' lesions yielded a positive result for Mycobacterium ulcerans. CONCLUSIONS/SIGNIFICANCE: This study detected single and mixed occurrence of the causative organisms of CL, yaws, and H. ducreyi cutaneous ulcers in CL endemic communities of the Oti Region in Ghana. These findings emphasize the importance of integrating multiple skin diseases on a common research platform and calls for the development of a comprehensive guideline for diagnosing and treating tropical ulcers in the study areas.

Formate production is dispensable for Haemophilus ducreyi virulence in human volunteers.

Haemophilus ducreyi is a causative agent of cutaneous ulcers in children who live in the tropics and of the genital ulcer disease chancroid in sexually active persons. In the anaerobic environment of abscesses and ulcers, anaerobic respiration and mixed acid fermentation (MAF) can be used to provide cellular energy. In Escherichia coli, MAF produces formate, acetate, lactate, succinate, and ethanol; however, MAF has not been studied in H. ducreyi. In human challenge experiments with H. ducreyi 35000HP, transcripts of the formate transporter FocA and pyruvate formate lyase (PflB) were upregulated in pustules compared to the inocula. We made single and double mutants of focA and pflB in 35000HP. Growth of 35000HPΔfocA was similar to 35000HP, but 35000HPΔpflB and 35000HPΔfocA-pflB had growth defects during both aerobic and anaerobic growth. Mutants lacking pflB did not secrete formate into the media. However, formate was secreted into the media by 35000HPΔfocA, indicating that H. ducreyi has alternative formate transporters. The pH of the media during anaerobic growth decreased for 35000HP and 35000HPΔfocA, but not for 35000HPΔpflB or 35000HPΔfocA-pflB, indicating that pflB is the main contributor to media acidification during anaerobic growth. We tested whether formate production and transport were required for virulence in seven human volunteers in a mutant versus parent trial between 35000HPΔfocA-pflB and 35000HP. The pustule formation rate was similar for 35000HP (42.9%)- and 35000HPΔfocA-pflB (62%)-inoculated sites. Although formate production occurs during in vitro growth and focA-pflB transcripts are upregulated during human infection, focA and pflB are not required for virulence in humans.

[Isolation of Haemophilus no ducreyi in samples from the genital tract of men: Its clinical relationship]. / Aislamiento de Haemophilus no ducreyi en muestras del aparato genital de varones: relación con la clínica.

INTRODUCTION: With the advancement of microbiological methods, the isolation of less typical pathogens in cases of urethral and rectal infection is more frequent, apart from the classic etiological agents. One of them is formed by species of Haemophilus no ducreyi (HND). The objective of this work is to describe frequency, susceptibility to antibiotics, and clinical features of HDN urethritis and proctitis in adult males. PATIENTS AND METHODS: This is an observational retrospective descriptive study of the results obtained by the Microbiology laboratory of the Virgen de las Nieves University Hospital on the isolates of HND in genital and rectal samples from males between 2016 and 2019. RESULTS: HND was isolated in 135 (7%) of the genital infection episodes diagnosed in men. H. parainfluenzae was the most commonly isolated (34/45; 75.6%). The most frequent symptoms in men with proctitis were rectal tenesmus (31.6%) and lymphadenopathy (10.5%); in those with urethritis, dysuria (71.6%), urethral suppuration (46.7%) and gland lesions (27%), so differentiating it from infections caused by other genitopathogens is difficult. 43% of patients were HIV positive. Antibiotic resistance rates for H. parainfluenzae were high to quinolons, ampicillin, tetracycline and macrolides. CONCLUSION: HND species should be considered as possible etiologic agents in episodes of urethral and rectal infection in men, especially in cases with negative screening tests for agents that cause sexually transmitted infections (STIs). Its microbiological identification is essential for the establishment of an effective targeted treatment.

Chemoenzymatic Total Synthesis of Haemophilus ducreyi Lipooligosaccharide Core Octasaccharides Containing Natural and Unnatural Sialic Acids.

The first total synthesis of Haemophilus ducreyi lipooligosaccharide core octasaccharides containing natural and unnatural sialic acids has been achieved by an efficient chemoenzymatic approach. A highly convergent [3 + 3] coupling strategy was developed to chemically assemble a unique hexasaccharide bearing multiple rare higher-carbon sugars d-glycero-d-manno-heptose (d,d-Hep), l-glycero-d-manno-heptose (l,d-Hep), and 3-deoxy-α-d-manno-oct-2-ulosonic acid (Kdo). Key features include sequential one-pot glycosylations for oligosaccharide assembly and the construction of the challenging α-(1 → 5)-linked Hep-Kdo glycosidic bond by gold-catalyzed glycosylation with a glycosyl ortho-alkynylbenzoate donor. Furthermore, the sequential enzyme-catalyzed regio- and stereoselective introduction of a galactose residue using ß-1,4-galactosyltransferase and different sialic acids using a one-pot multienzyme sialylation system was efficiently accomplished to provide the target octasaccharides.

Dispensability of Ascorbic Acid Uptake and Utilization Encoded by ulaABCD for the Virulence of Haemophilus ducreyi in Humans.

Compared with wounded skin, ascorbic acid is enriched in pustules of humans experimentally infected with Haemophilus ducreyi. Compared with the broth-grown inocula, transcription of the H. ducreyi ulaABCD operon, which encodes genes for ascorbic acid uptake, is increased in pustules. We hypothesized that ascorbic acid uptake plays a role in H. ducreyi virulence. Five volunteers were infected with both H. ducreyi strain 35000HP and its isogenic ulaABCD deletion mutant at multiple sites; the papule and pustule formation rates of the mutant and parent strains were similar. Thus, ascorbic acid uptake is not essential for H. ducreyi virulence in humans.

Haemophilus ducreyi Infection Induces Oxidative Stress, Central Metabolic Changes, and a Mixed Pro- and Anti-inflammatory Environment in the Human Host.

Few studies have investigated host-bacterial interactions at sites of infection in humans using transcriptomics and metabolomics. Haemophilus ducreyi causes cutaneous ulcers in children and the genital ulcer disease chancroid in adults. We developed a human challenge model in which healthy adult volunteers are infected with H. ducreyi on the upper arm until they develop pustules. Here, we characterized host-pathogen interactions in pustules using transcriptomics and metabolomics and examined interactions between the host transcriptome and metabolome using integrated omics. In a previous pilot study, we determined the human and H. ducreyi transcriptomes and the metabolome of pustule and wounded sites of 4 volunteers (B. Griesenauer, T. M. Tran, K. R. Fortney, D. M. Janowicz, et al., mBio 10:e01193-19, 2019, https://doi.org/10.1128/mBio.01193-19). While we could form provisional transcriptional networks between the host and H. ducreyi, the study was underpowered to integrate the metabolome with the host transcriptome. To better define and integrate the transcriptomes and metabolome, we used samples from both the pilot study (n = 4) and new volunteers (n = 8) to identify 5,495 human differentially expressed genes (DEGs), 123 H. ducreyi DEGs, 205 differentially abundant positive ions, and 198 differentially abundant negative ions. We identified 42 positively correlated and 29 negatively correlated human-H. ducreyi transcriptome clusters. In addition, we defined human transcriptome-metabolome networks consisting of 9 total clusters, which highlighted changes in fatty acid metabolism and mitigation of oxidative damage. Taken together, the data suggest a mixed pro- and anti-inflammatory environment and rewired central metabolism in the host that provides a hostile, nutrient-limited environment for H. ducreyi. IMPORTANCE Interactions between the host and bacteria at sites of infection in humans are poorly understood. We inoculated human volunteers on the upper arm with the skin pathogen H. ducreyi or a buffer control and biopsied the resulting infected and sham-inoculated sites. We performed dual transcriptome sequencing (RNA-seq) and metabolic analysis on the biopsy samples. Network analyses between the host and bacterial transcriptomes and the host transcriptome-metabolome network were used to identify molecules that may be important for the virulence of H. ducreyi in the human host. Our results suggest that the pustule is highly oxidative, contains both pro- and anti-inflammatory components, and causes metabolic shifts in the host, to which H. ducreyi adapts to survive. To our knowledge, this is the first study to integrate transcriptomic and metabolomic responses to a single bacterial pathogen in the human host.

Two Streptococcus pyogenes emm types and several anaerobic bacterial species are associated with idiopathic cutaneous ulcers in children after community-based mass treatment with azithromycin.

BACKGROUND: In yaws-endemic areas, two-thirds of exudative cutaneous ulcers (CU) are associated with Treponema pallidum subsp. pertenue (TP) and Haemophilus ducreyi (HD); one-third are classified as idiopathic ulcers (IU). A yaws eradication campaign on Lihir Island in Papua New Guinea utilizing mass drug administration (MDA) of azithromycin initially reduced but failed to eradicate yaws; IU rates remained constant throughout the study. Using 16S rRNA gene sequencing, we previously determined that Streptococcus pyogenes was associated with some cases of IU. Here, we applied shotgun metagenomics to the same samples we analyzed previously by 16S rRNA sequencing to verify this result, identify additional IU-associated microorganisms, and determine why S. pyogenes-associated IU might have persisted after MDA of azithromycin. METHODOLOGY/PRINCIPAL FINDINGS: We sequenced DNA extracted from 244 CU specimens separated into four groups based upon microorganism-specific PCR results (HD+, TP+, TP+HD+, and TP-HD- or IU). S. pyogenes was enriched in IU (24.71% relative abundance [RA]) specimens compared to other ulcer sub-groups, confirming our prior results. We bioinformatically identified the emm (M protein gene) types found in the S. pyogenes IU specimens and found matches to emm156 and emm166. Only ~39% of IU specimens contained detectable S. pyogenes, suggesting that additional organisms could be associated with IU. In the sub-set of S. pyogenes-negative IU specimens, Criibacterium bergeronii, a member of the Peptostreptococcaceae, and Fusobacterium necrophorum (7.07% versus 0.00% RA and 2.18% versus 0.00% RA, respectively), were enriched compared to the S. pyogenes-positive sub-set. Although a broad range of viruses were detected in the CU specimens, none were specifically associated with IU. CONCLUSIONS/SIGNIFICANCE: Our observations confirm the association of S. pyogenes with IU in yaws-endemic areas, and suggest that additional anaerobic bacteria, but not other microorganisms, may be associated with this syndrome. Our results should aid in the design of diagnostic tests and selective therapies for CU.

Acid-induced disassembly of the Haemophilus ducreyi cytolethal distending toxin.

The cytolethal distending toxins (CDTs) produced by many Gram-negative pathogens are tripartite genotoxins with a single catalytic subunit (CdtB) and two cell-binding subunits (CdtA + CdtC). CDT moves by vesicle carriers from the cell surface to the endosomes and through the Golgi apparatus en route to the endoplasmic reticulum (ER). CdtA dissociates from the rest of the toxin before reaching the Golgi apparatus, and CdtB separates from CdtC in the ER. The free CdtB subunit, which is only active after holotoxin disassembly, then crosses the ER membrane and enters the nucleus where it generates DNA breaks. We hypothesized that the acidified lumen of the endosomes is responsible for separating CdtA from the CdtB/CdtC heterodimer. To test this prediction, possible acid-induced disruptions to the CDT holotoxin were monitored by size exclusion chromatography and surface plasmon resonance. We found that CDT could not efficiently assemble from its individual subunits at the early endosome pH of 6.3. Partial disassembly of the CDT holotoxin also occurred at pH 6.3, with complete separation of CdtA from an intact CdtB/CdtC heterodimer occurring at both pH 6.0 and the late endosome pH of 5.6. Acidification caused the precipitation of CdtA at pH 6.5 and below, but neither CdtB nor CdtC were affected by a pH as low as 5.2. Circular dichroism further showed that the individual CdtB subunit adopts a different secondary structure as compared to its structure in the holotoxin. We conclude the first stage of CDT disassembly occurs in the early endosomes, where an acid-induced alteration to CdtA releases it from the CdtB/CdtC heterodimer.

Genes Differentially Expressed by Haemophilus ducreyi during Anaerobic Growth Significantly Overlap Those Differentially Expressed during Experimental Infection of Human Volunteers.

Haemophilus ducreyi causes cutaneous ulcers in children and the genital ulcer disease chancroid in adults. In humans, H. ducreyi is found in the anaerobic environment of an abscess; previous studies comparing bacterial gene expression levels in pustules with the inocula (∼4-h aerobic mid-log-phase cultures) identified several upregulated differentially expressed genes (DEGs) that are associated with anaerobic metabolism. To determine how H. ducreyi alters its gene expression in response to anaerobiosis, we performed RNA sequencing (RNA-seq) on both aerobic and anaerobic broth cultures harvested after 4, 8, and 18 h of growth. Principal-coordinate analysis (PCoA) plots showed that anaerobic growth resulted in distinct transcriptional profiles compared to aerobic growth. During anaerobic growth, early-time-point comparisons (4 versus 8 h) identified few DEGs at a 2-fold change in expression and a false discovery rate (FDR) of <0.01. By 18 h, we observed 18 upregulated and 16 downregulated DEGs. DEGs involved in purine metabolism, the uptake and use of alternative carbon sources, toxin production, nitrate reduction, glycine metabolism, and tetrahydrofolate synthesis were upregulated; DEGs involved in electron transport, thiamine biosynthesis, DNA recombination, peptidoglycan synthesis, and riboflavin synthesis or modification were downregulated. To examine whether transcriptional changes that occur during anaerobiosis overlap those that occur during infection of human volunteers, we compared the overlap of DEGs obtained from 4 h of aerobic growth to 18 h of anaerobic growth to those found between the inocula and pustules in previous studies; the DEGs significantly overlapped. Thus, a major component of H. ducreyi gene regulation in vivo involves adaptation to anaerobiosis. IMPORTANCE In humans, H. ducreyi resides in the anaerobic environment of an abscess and appears to upregulate genes involved in anaerobic metabolism. How anaerobiosis alone affects gene transcription in H. ducreyi is unknown. Using RNA-seq, we investigated how anaerobiosis affects gene transcription over time compared to aerobic growth. Our results suggest that a substantial component of H. ducreyi gene regulation in vivo overlaps the organism's response to anaerobiosis in vitro. Our data identify potential therapeutic targets that could be inhibited during in vivo growth.

LAMP4yaws: Treponema pallidum, Haemophilus ducreyi loop mediated isothermal amplification - protocol for a cross-sectional, observational, diagnostic accuracy study.

INTRODUCTION: Yaws, caused by the bacterium Treponema pallidum subsp. pertenue, is a neglected tropical disease targeted for eradication by 2030. Improved diagnostics will be essential to meet this goal. Diagnosis of yaws has relied heavily on clinical and serological tools. However, the presence of coendemic cutaneous skin ulcer diseases, such as lesions caused by Haemophilus ducreyi (HD), means these techniques do not provide a reliable diagnosis. Thus, new diagnostic tools are needed. Molecular tools such as PCR are ideal, but often expensive as they require trained technicians and laboratory facilities, which are often not available to national yaws programmes. METHODS AND ANALYSIS: The LAMP4yaws project is a cross-sectional, observational, diagnostic accuracy study of a combined Treponema pallidum (TP) and HD loop mediated isothermal amplification (TPHD-LAMP) test performed under real world conditions in three endemic countries in West Africa. Individuals with serologically confirmed yaws will be recruited in Cameroon, Côte d'Ivoire and Ghana. Each participant will provide paired swabs, one of which will be sent to the respective national reference laboratory for yaws quantitative PCR and the other will be tested for both TP and HD using the TPHD-LAMP test at local district laboratories. Sensitivity and specificity of the TPHD-LAMP test will be calculated against the reference standard qPCR. We will also assess the acceptability, feasibility and cost-effectiveness of the test. We anticipate that results from this study will support the adoption of the TPHD-LAMP test for use in global yaws eradication efforts. ETHICS AND DISSEMINATION: We have received ethical approval from all relevant institutional and national ethical committees. All participants, or their parents or guardians, must provide written informed consent prior to study enrolment. Study results will be published in an open access journal and disseminated with partners and the World Health Organization. TRIAL REGISTRATION NUMBER: NCT04753788.

Trial of Three Rounds of Mass Azithromycin Administration for Yaws Eradication.

BACKGROUND: Treponema pallidum subspecies pertenue causes yaws. Strategies to better control, eliminate, and eradicate yaws are needed. METHODS: In an open-label, cluster-randomized, community-based trial conducted in a yaws-endemic area of Papua New Guinea, we randomly assigned 38 wards (i.e., clusters) to receive one round of mass administration of azithromycin followed by two rounds of target treatment of active cases (control group) or three rounds of mass administration of azithromycin (experimental group); round 1 was administered at baseline, round 2 at 6 months, and round 3 at 12 months. The coprimary end points were the prevalence of active cases of yaws, confirmed by polymerase-chain-reaction assay, in the entire trial population and the prevalence of latent yaws, confirmed by serologic testing, in a subgroup of asymptomatic children 1 to 15 years of age; prevalences were measured at 18 months, and the between-group differences were calculated. RESULTS: Of the 38 wards, 19 were randomly assigned to the control group (30,438 persons) and 19 to the experimental group (26,238 persons). A total of 24,848 doses of azithromycin were administered in the control group (22,033 were given to the participants at round 1 and 207 and 2608 were given to the participants with yaws-like lesions and their contacts, respectively, at rounds 2 and 3 [combined]), and 59,852 doses were administered in the experimental group. At 18 months, the prevalence of active yaws had decreased from 0.46% (102 of 22,033 persons) at baseline to 0.16% (47 of 29,954 persons) in the control group and from 0.43% (87 of 20,331 persons) at baseline to 0.04% (10 of 25,987 persons) in the experimental group (relative risk adjusted for clustering, 4.08; 95% confidence interval [CI], 1.90 to 8.76). The prevalence of other infectious ulcers decreased to a similar extent in the two treatment groups. The prevalence of latent yaws at 18 months was 6.54% (95% CI, 5.00 to 8.08) among 994 children in the control group and 3.28% (95% CI, 2.14 to 4.42) among 945 children in the experimental group (relative risk adjusted for clustering and age, 2.03; 95% CI, 1.12 to 3.70). Three cases of yaws with resistance to macrolides were found in the experimental group. CONCLUSIONS: The reduction in the community prevalence of yaws was greater with three rounds of mass administration of azithromycin at 6-month intervals than with one round of mass administration of azithromycin followed by two rounds of targeted treatment. Monitoring for the emergence and spread of antimicrobial resistance is needed. (Funded by Fundació "la Caixa" and others; ClinicalTrials.gov number, NCT03490123.).

Evaluation of a laboratory-developed multiplex real-time PCR assay for diagnosis of syphilis, herpes and chancroid genital ulcers in four public health laboratories in the USA.

OBJECTIVE: To evaluate the field performance of a multiplex PCR (M-PCR) assay for detection of herpes simplex virus (HSV)-1 and HSV-2, Treponema pallidum (T. pallidum) and Haemophilus ducreyi (H. ducreyi) in genital ulcer disease (GUD) specimens. METHODS: GUD M-PCR was performed on 186 remnant specimens, previously collected for HSV testing, by four public health laboratories (PHLs) and the Laboratory Reference and Research Branch (LRRB) at the Centers for Disease Control and Prevention. The results from the PHLs were compared with those of LRRB, which served as the reference testing method, and percentage agreement was calculated. RESULTS: HSV was detected in 31 of 52 (59.6%), 20 of 40 (50%), 43 of 44 (97.7%) and 19 of 50 (38.0%) specimens from PHL1, PHL2, PHL3 and PHL4, respectively. There were seven discrepant results for HSV, and the overall percent agreement between the PHLs and the LRRB was 94%-100%, with a kappa value of 0.922, which demonstrates high agreement. T. pallidum was identified in 7 of 51 (13.7%) specimens from PHL1 with 94.1% agreement and in 2 of 40 (5.0%) specimens from PHL2 with 100% agreement. The LRRB identified three additional T. pallidum-positive specimens from PHL1. The kappa value (0.849) for T. pallidum testing suggests good agreement. Consistent with the LRRB results, no T. pallidum was detected in specimens from PHL3 and PHL4, and H. ducreyi was not detected at any of the study sites. CONCLUSIONS: The GUD M-PCR assay performed well in four independent PHLs and 12 suspected syphilis cases were identified in this study. The M-PCR assay could provide improved diagnostic options for GUD infections in state and local PHLs.

Engineering of Cytolethal Distending Toxin B by Its Reducing Immunogenicity and Maintaining Stability as a New Drug Candidate for Tumor Therapy; an In Silico Study.

The cytolethal distending toxin (CDT), Haemophilus ducreyi, is one of the bacterial toxins that have recently been considered for targeted therapies, especially in cancer therapies. CDT is an A-B2 exotoxin. Its catalytic subunit (CdtB) is capable of inducing DNA double strand breaks, cell cycle arrest and apoptosis in host eukaryotic cells. The sequence alignment indicates that the CdtB is structurally homologyr to phosphatases and deoxyribonucleases I (DNase I). Recently, it has been found that CdtB toxicity is mainly related to its nuclease activity. The immunogenicity of CDT can reduce its effectiveness in targeted therapies. However, the toxin can be very useful if its immunogenicity is significantly reduced. Detecting hotspot ectopic residues by computational servers and then mutating them to eliminate B-cell epitopes is a promising approach to reduce the immunogenicity of foreign protein-based therapeutics. By the mentioned method, in this study, we try to reduce the immunogenicity of the CdtB- protein sequence. This study initially screened residue of the CdtB is B-cell epitopes both linearly and conformationally. By overlapping the B-cell epitopes with the excluded conserve residues, and active and enzymatic sites, four residues were allowed to be mutated. There were two mutein options that show reduced antigenicity probability. Option one was N19F, G74I, and S161F with a VaxiJen score of 0.45 and the immune epitope database (IEDB) score of 1.80, and option two was N19F, G74I, and S161W with a VaxiJen score of 0.45 and IEDB score of 1.88. The 3D structure of the proposed sequences was evaluated and refined. The structural stability of native and mutant proteins was accessed through molecular dynamic simulation. The results showed that the mutations in the mutants caused no considerable changes in their structural stability. However, mutant 1 reveals more thermodynamic stability during the simulation. The applied approaches in this study can be used as rough guidelines for finding hot spot immunogen regions in the therapeutic proteins. Our results provide a new version of CdtB that, due to reduced immunogenicity and increased stability, can be used in toxin-based drugs such as immunotoxins.

Endocytosis of the CdtA subunit from the Haemophilus ducreyi cytolethal distending toxin.

Many Gram-negative pathogens produce a cytolethal distending toxin (CDT) with two cell-binding subunits (CdtA + CdtC) and a catalytic CdtB subunit. After adhesion to the plasma membrane of a target cell, CDT moves by retrograde transport to endoplasmic reticulum. CdtB then enters the nucleus where it generates DNA breaks that lead to cell cycle arrest and apoptosis or senescence. CdtA anchors the CDT holotoxin to the plasma membrane and is thought to remain on the cell surface after endocytosis of the CdtB/CdtC heterodimer. Here, we re-examined the potential endocytosis and intracellular transport of CdtA from the Haemophilus ducreyi CDT. We recorded the endocytosis of holotoxin-associated CdtA with a cell-based enzyme-linked immunoabsorbent assay (CELISA) and visualised its presence in the early endosomes by confocal microscopy 10 min after CDT binding to the cell surface. Western blot analysis documented the rapid degradation of internalised CdtA. Most of internalised CdtB and CdtC were degraded as well. The rapid rate of CDT internalisation and turnover, which could explain why CdtA endocytosis was not detected in previous studies, suggests only a minor pool of cell-associated CdtB reaches the nucleus. Our work demonstrates that CDT is internalised as an intact holotoxin and identifies the endosomes as the site of CdtA dissociation from CdtB/CdtC. TAKE AWAYS: During the endocytosis of CDT, CdtA is thought to remain at the cell surface. A cell-based ELISA documented the rapid endocytosis of CdtA. CdtA was visualised in the early endosomes by confocal microscopy. Intracellular CdtA was rapidly degraded, along with most of CdtB and CdtC.

The Active Subunit of the Cytolethal Distending Toxin, CdtB, Derived From Both Haemophilus ducreyi and Campylobacter jejuni Exhibits Potent Phosphatidylinositol-3,4,5-Triphosphate Phosphatase Activity.

Human lymphocytes exposed to Aggregatibacter actinomycetemcomitans (Aa) cytolethal distending toxin (Cdt) undergo cell cycle arrest and apoptosis. In previous studies, we demonstrated that the active Cdt subunit, CdtB, is a potent phosphatidylinositol (PI) 3,4,5-triphosphate phosphatase. Moreover, AaCdt-treated cells exhibit evidence of PI-3-kinase (PI-3K) signaling blockade characterized by reduced levels of PIP3, pAkt, and pGSK3ß. We have also demonstrated that PI-3K blockade is a requisite of AaCdt-induced toxicity in lymphocytes. In this study, we extended our observations to include assessment of Cdts from Haemophilus ducreyi (HdCdt) and Campylobacter jejuni (CjCdt). We now report that the CdtB subunit from HdCdt and CjCdt, similar to that of AaCdt, exhibit potent PIP3 phosphatase activity and that Jurkat cells treated with these Cdts exhibit PI-3K signaling blockade: reduced levels of pAkt and pGSK3ß. Since non-phosphorylated GSK3ß is the active form of this kinase, we compared Cdts for dependence on GSK3ß activity. Two GSK3ß inhibitors were employed, LY2090314 and CHIR99021; both inhibitors blocked the ability of Cdts to induce cell cycle arrest. We have previously demonstrated that AaCdt induces increases in the CDK inhibitor, p21CIP1/WAF1, and, further, that this was a requisite for toxin-induced cell death via apoptosis. We now demonstrate that HdCdt and CjCdt also share this requirement. It is also noteworthy that p21CIP1/WAF1 was not involved in the ability of the three Cdts to induce cell cycle arrest. Finally, we demonstrate that, like AaCdt, HdCdt is dependent upon the host cell protein, cellugyrin, for its toxicity (and presumably internalization of CdtB); CjCdt was not dependent upon this protein. The implications of these findings as they relate to Cdt's molecular mode of action are discussed.

Ulcerative skin lesions among children in Cameroon: It is not always Yaws.

Outbreaks of yaws-like ulcerative skin lesions in children are frequently reported in tropical and sub-tropical countries. The origin of these lesions might be primarily traumatic or infectious; in the latter case, Treponema pallidum subspecies pertenue, the yaws agent, and Haemophilus ducreyi, the agent of chancroid, are two of the pathogens commonly associated with the aetiology of skin ulcers. In this work, we investigated the presence of T. p. pertenue and H. ducreyi DNA in skin ulcers in children living in yaws-endemic regions in Cameroon. Skin lesion swabs were collected from children presenting with yaws-suspected skin lesions during three outbreaks, two of which occurred in 2017 and one in 2019. DNA extracted from the swabs was used to amplify three target genes: the human ß2-microglobulin gene to confirm proper sample collection and DNA extraction, the polA gene, highly conserved among all subspecies of T. pallidum, and the hddA gene of H. ducreyi. A fourth target, the tprL gene was used to differentiate T. p. pertenue from the other agents of human treponematoses in polA-positive samples. A total of 112 samples were analysed in this study. One sample, negative for ß2-microglobulin, was excluded from further analysis. T. p. pertenue was only detected in the samples collected during the first 2017 outbreak (12/74, 16.2%). In contrast, H. ducreyi DNA could be amplified from samples from all three outbreaks (outbreak 1: 27/74, 36.5%; outbreak 2: 17/24, 70.8%; outbreak 3: 11/13, 84.6%). Our results show that H. ducreyi was more frequently associated to skin lesions in the examined children than T. p. pertenue, but also that yaws is still present in Cameroon. These findings strongly advocate for a continuous effort to determine the aetiology of ulcerative skin lesions during these recurring outbreaks, and to inform the planned mass treatment campaigns to eliminate yaws in Cameroon.

Streptococcus pyogenes Is Associated with Idiopathic Cutaneous Ulcers in Children on a Yaws-Endemic Island.

Exudative cutaneous ulcers (CU) in yaws-endemic areas are associated with Treponema pallidum subsp. pertenue (TP) and Haemophilus ducreyi (HD), but one-third of CU cases are idiopathic (IU). Using mass drug administration (MDA) of azithromycin, a yaws eradication campaign on Lihir Island in Papua New Guinea reduced but failed to eradicate yaws; IU rates remained constant throughout the campaign. To identify potential etiologies of IU, we obtained swabs of CU lesions (n = 279) and of the skin of asymptomatic controls (AC; n = 233) from the Lihir Island cohort and characterized their microbiomes using a metagenomics approach. CU bacterial communities were less diverse than those of the AC. Using real-time multiplex PCR with pathogen-specific primers, we separated CU specimens into HD-positive (HD+), TP+, HD+TP+, and IU groups. Each CU subgroup formed a distinct bacterial community, defined by the species detected and/or the relative abundances of species within each group. Streptococcus pyogenes was the most abundant organism in IU (22.65%) and was enriched in IU compared to other ulcer groups. Follow-up samples (n = 31) were obtained from nonhealed ulcers; the average relative abundance of S. pyogenes was 30.11% in not improved ulcers and 0.88% in improved ulcers, suggesting that S. pyogenes in the not improved ulcers may be azithromycin resistant. Catonella morbi was enriched in IU that lacked S. pyogenes As some S. pyogenes and TP strains are macrolide resistant, penicillin may be the drug of choice for CU azithromycin treatment failures. Our study will aid in the design of diagnostic tests and selective therapies for CU.IMPORTANCE Cutaneous ulcers (CU) affect approximately 100,000 children in the tropics each year. While two-thirds of CU are caused by Treponema pallidum subspecies pertenue and Haemophilus ducreyi, the cause(s) of the remaining one-third is unknown. Given the failure of mass drug administration of azithromycin to eradicate CU, the World Health Organization recently proposed an integrated disease management strategy to control CU. Success of this strategy requires determining the unknown cause(s) of CU. By using 16S rRNA gene sequencing of swabs obtained from CU and the skin of asymptomatic children, we identified another possible cause of skin ulcers, Streptococcus pyogenes Although S. pyogenes is known to cause impetigo and cellulitis, this is the first report implicating the organism as a causal agent of CU. Inclusion of S. pyogenes into the integrated disease management plan will improve diagnostic testing and treatment of this painful and debilitating disease of children and strengthen elimination efforts.

Yaws, Haemophilus ducreyi, and Other Bacterial Causes of Cutaneous Ulcer Disease in the South Pacific Islands.

Cutaneous ulcers in the tropics are a painful and debilitating condition that anchors people into poverty. In rural regions of the South Pacific, infectious cutaneous ulcers are caused mainly by bacteria, including Treponema pallidum pertenue (yaws), Haemophilus ducreyi, and polymicrobial ulcers. For this group of infections the term cutaneous ulcer disease (CUD) is proposed. Some infections can cause malformations on the bone that have a permanent impact on lives in endemic communities. Better characterization of CUD may help design diagnostic tools and more effective antimicrobial therapies. This review updates the knowledge of CUD and discusses optimized terminology and syndromic management.

Functional Study of Haemophilus ducreyi Cytolethal Distending Toxin Subunit B.

The Cytolethal Distending Toxin (CDT) is produced by many Gram-negative pathogenic bacteria responsible for major foodborne diseases worldwide. CDT induces DNA damage and cell cycle arrest in host-cells, eventually leading to senescence or apoptosis. According to structural and sequence comparison, the catalytic subunit CdtB is suggested to possess both nuclease and phosphatase activities, carried by a single catalytic site. However, the impact of each activity on cell-host toxicity is yet to be characterized. Here, we analyze the consequences of cell exposure to different CDT mutated on key CdtB residues, focusing on cell viability, cell cycle defects, and DNA damage induction. A first class of mutant, devoid of any activity, targets putative catalytic (H160A), metal binding (D273R), and DNA binding residues (R117A-R144A-N201A). The second class of mutants (A163R, F156-T158, and the newly identified G114T), which gathers mutations on residues potentially involved in lipid substrate binding, has only partially lost its toxic effects. However, their defects are alleviated when CdtB is artificially introduced inside cells, except for the F156-T158 double mutant that is defective in nuclear addressing. Therefore, our data reveal that CDT toxicity is mainly correlated to CdtB nuclease activity, whereas phosphatase activity may probably be involved in CdtB intracellular trafficking.