Short Variable Regions flaA Gene (SVR-flaA) Diversity and Virulence Profile of Multidrug-Resistant Campylobacter from Poultry and Poultry Meat in India.

Human gastrointestinal infections caused by Campylobacter species is the second most important foodborne illness after salmonellosis worldwide. Poultry represent one of the main sources of Campylobacter organisms. In the present study, the short variable region of flagellin gene (SVR-flaA) typing was carried out to determine the variation among the circulating strains of Campylobacter jejuni and Campylobacter coli. The C. jejuni and C. coli isolated from poultry and poultry meat were screened for the presence of virulence determinants like cadF, flaA, cdtB, and wlaN gene. The screening for wlaN gene is crucial in view of the fact that most patients with Guillian Barre's (GB) syndrome with a preceding history of diarrheal illness have been found to harbor wlaN gene-positive C jejuni strains. Out of the 200 samples comprising poultry meat and cloacal swabs, 21.5% of samples were found to harbor Campylobacter spp. of which 2.5% were Campylobacter jejuni, and 19% were confirmed as Campylobacter coli. The cadF, flaA, cdtB virulence genes were detected in all the Campylobacter spp. isolated in the present study. The presence of the wlaN gene in the Campylobacter jejuni isolated in the present study may pose a public health threat with long-term human health implications. The SVR-flaA typing of twelve Campylobacter isolates obtained in the present study revealed that Campylobacter coli flaA sequence OL471375 is a new strain with a novel allele type 1,675 and peptide sequence 5 which stands deposited in pubMLST database for Campylobacter. The other flaA-SVR gene sequences identified in this study were OL471369, OL471370, OL471371, OL471372, OL471373, and OL471374. Among twelve Campylobacter spp., three distinct DdeI-RFLP patterns were observed, each varying in size from 100 to 1,000 base pairs. Antimicrobial profiling of the Campylobacter spp. isolated in the present study revealed that 50% of the strains were multidrug resistant. All the Campylobacter spp. were resistant to ciprofloxacin (CIP), ampicillin (AMP), penicillin (PEN), and nalidixic acid (NAL) whereas 57.1% of strains were resistant to tetracycline (TET) and erythromycin (ERY) 28% to amoxicillin (AMX) and enrofloxacin (ENO), 85% to amikacin (AMK). The high degree of resistance to fluoroquinolones observed in the present study is crucial in view of fluoroquinolones being drugs of choice for the treatment of human Campylobacter infections.

Dual immunization with CdtB protein and flagellin epitope offers augmented protection against enteric fever in mice.

AIMS: Present study has explored the protective response of dual immunization using two different antigenic entities (i.e. flagellin epitope and cytolethal distending toxin subunit B (CdtB) protein) against lethal challenge of typhoidal serovars in a murine model. MAIN METHODS: In-vitro immunogenicity of flagellin epitope-BSA conjugate and CdtB protein was confirmed using Indirect ELISA of typhoid positive patients' sera. Further, both entities were administered intraperitoneally in mice individually or in combination, followed by lethal challenge of typhoidal Salmonellae. Various parameters were analysed such as bacterial burden, mice survival, histopathological analysis, cytokine analysis and immunophenotyping. Serum samples obtained from the immunized mice were used for passive immunization studies, wherein mice survival and mechanism of action of the generated antibodies was studied. KEY FINDINGS: Active immunization studies using the combination of both entities demonstrated improved mice survival after lethal challenge with typhoidal Salmonellae, reduced bacterial burden in organs, expression of immunophenotypic markers in splenocytes and restored tissue histoarchitecture. When used in combination, the effective doses of both the candidates reduced which may be attributed to multiprong approach used by the immune system to recognize Salmonella. Passive immunization studies further determined the protective efficacy of generated antibodies by different mechanisms such as complement mediated bactericidal action, swarming inhibition and increased phagocytic uptake. SIGNIFICANCE: Present study is the first phase of the proof-of-concept which may prove to be beneficial in developing an effective bi-functional vaccine candidate to render protection against both Vi-positive as well as Vi-negative Salmonella strains.

Presence of Helicobacter cinaedi in Atherosclerotic Abdominal Aortic Aneurysmal Wall.

Recently, the relationship between Helicobacter cinaedi (H. cinaedi) infection and several diseases, including cardiovascular and central nervous system disorders, bone and soft tissue disorders, and infectious abdominal aortic aneurysms (AAAs), has been reported. Moreover, H. cinaedi may be associated with arteriosclerosis. In the present study, we investigated the association between H. cinaedi infection and clinically uninfected AAAs. Genetic detection of H. cinaedi in the abdominal aneurysm wall was attempted in 39 patients with AAA undergoing elective open surgery between June 2019 and June 2020. DNA samples extracted from the arterial wall obtained during surgery were analyzed using nested polymerase chain reaction (PCR). The target gene region was the H. cinaedi-specific cytolethal distending toxin subunit B (cdtB). Nine (23.1%) of 39 patients showed positive bands corresponding to H. cinaedi, and further sequencing analyses demonstrated the presence of H. cinaedi DNAs in their aneurysm walls. In contrast, all the non-aneurysm arterial walls in our patients were negative for H. cinaedi. In conclusion, this is the first report of the detection of H. cinaedi in the walls of a clinically non-infectious AAA.

Detection of Salmonella Typhi nucleic acid by RT-PCR and anti-HlyE, -CdtB, -PilL, and -Vi IgM by ELISA at sites in Ghana, Madagascar and Ethiopia.

BACKGROUND: We aimed to assess the prevalence of Salmonella Typhi through DNA and IgM-antibody detection methods as a prelude to extended surveillance activities at sites in Ghana, Madagascar, and Ethiopia. METHODS: We performed species-specific real-time polymerase reaction (RT-PCR) to identify bacterial nucleic acid, and enzyme-linked immunosorbent assay (ELISA) for detecting HlyE/STY1498-, CdtB/STY1886-, pilL/STY4539- and Vi-antigens in blood and biopsy specimens of febrile and non-febrile subjects. We generated antigen-specific ELISA proxy cut-offs by change-point analyses, and utilized cumulative sum as detection method coupled with 1000 repetitive bootstrap analyses. We computed prevalence rates in addition to odds ratios to assess correlations between ELISA outcomes and participant characteristics. RESULTS: Definitive positive RT-PCR results were obtained from samples of febrile subjects originating from Adama Zuria/Ethiopia (1.9%, 2/104), Wolayita Sodo/Ethiopia (1.0%, 1/100), Diego/Madagascar (1.0%, 1/100), and Kintampo/Ghana (1.0%, 1/100), and from samples of non-febrile subjects from Wolayita Sodo/Ethiopia (1%, 2/201). While IgM antibodies against all antigens were identified across all sites, prevalence rates were highest at all Ethiopian sites, albeit in non-febrile populations. Significant correlations in febrile subjects aged < 15 years versus ≥ 15 years were detected for Vi (Odds Ratio (OR): 8.00, p = 0.034) in Adama Zuria/Ethiopia, STY1498 (OR: 3.21, p = 0.008), STY1886 (OR: 2.31, p = 0.054) and STY4539 (OR: 2.82, p = 0.022) in Diego/Madagascar, and STY1498 (OR: 2.45, p = 0.034) in Kintampo/Ghana. We found statistical significance in non-febrile male versus female subjects for STY1498 (OR: 1.96, p = 0.020) in Adama Zuria/Ethiopia, Vi (OR: 2.84, p = 0.048) in Diego/Madagascar, and STY4539 (OR: 0.46, p = 0.009) in Kintampo/Ghana. CONCLUSIONS: Findings indicate non-discriminatory stages of acute infections, though with site-specific differences. Immune responses among non-febrile, presumably healthy participants may mask recall and/or reporting bias leading to misclassification, or asymptomatic, subclinical infection signs induced by suppression of inflammatory responses. As most Ethiopian participants were ≥ 15 years of age and not at high-risk, the true S. Typhi burden was likely missed. Change-point analyses for generating ELISA proxy cut-offs appeared robust, though misclassification is possible. Our findings provided important information that may be useful to assess sites prior to implementing surveillance for febrile illness including Salmonella disease.

Associations between food-specific IgG antibodies and intestinal permeability biomarkers.

Increasing translational evidence suggests that intestinal permeability may be a contributing factor to systemic inflammatory events and numerous pathologies. While associations between IgE-mediated food allergies and increased intestinal permeability have been well-characterized, the relationship between IgG-mediated food sensitivities and intestinal permeability is not well-described in the literature. Thus, we tested for associations between intestinal permeability biomarkers and food-specific IgG antibodies in 111 adults, with and without gastrointestinal symptoms. All biomarkers and food-specific IgG antibodies were measured via ELISA. The intestinal permeability biomarkers anti-lipopolysaccharide (LPS) and anti-occludin IgG and IgA antibodies, but not anti-vinculin or anti-CdtB IgG antibodies, were significantly and positively associated with IgG-mediated food sensitivities. These significant relationships were attenuated by adjusting for the severity of wheat, dairy, and egg reactions. The results of this study support strong associations between titers of food-specific IgG antibodies and intestinal permeability biomarkers in adults, to the extent that the presence of multiple IgG antibodies to food, and increasing IgG food titers, can be considered indicative of increased antibodies to LPS and occludin. Notably, neither IgG titers to wheat, eggs, and dairy, nor permeability biomarkers, were increased in symptomatic participants compared to those without symptoms.

Contribution of typhoid toxin in the pathogenesis of Salmonella Typhi.

To persist and establish infection, Salmonella utilizes a battery of different virulence determinants at every stage of infection. Typhoid toxin, a newly identified toxin in Salmonella enterica serovar Typhi is recognized as one of the virulence factors that has been linked with Salmonella pathogenesis. In this study, we have further investigated the role of typhoid toxin in the symptomatology of typhoid fever through in-vivo and ex-vivo studies. In mice, administration of cloned and purified typhoid toxin induces similar symptoms observed during typhoid fever such as fever, weight loss with a decrease in peripheral leucocyte count along with an increase in levels of pro-inflammatory cytokines (Il-6, TNF-α). Results of DNA analysis, fluorescence microscopy and flow cytometry of typhoid toxin-treated macrophages (ex-vivo) altogether revealed the CdtB (subunit of typhoid toxin) mediated DNA damage that led to the apoptosis of cells. Furthermore, to validate CdtB's catalytic role, macrophages were treated with typhoid toxin preincubated with anti-CdtB antibodies (generated in mice). Re-assessment of macrophage DNA by gel electrophoresis and flow cytometry analysis indicated a significant decrease in DNA damage and cells undergoing apoptosis, respectively. Moreover, a significant reduction in in-vitro DNase activity of CdtB protein was also observed on preincubating holotoxin with anti-CdtB antibodies. In total, this study highlights the role of typhoid toxin in inducing typhoid fever-like symptomatology, which may be executed through the toxin's catalytic subunit CdtB.

Study of Antibodies to Cytolethal Distending Toxin B (CdtB) and Antibodies to Vinculin in Patients with Irritable Bowel Syndrome.

Background: Irritable bowel syndrome (IBS) is a common gastrointestinal disorder, categorized into various subtypes. Post-infection IBS may be attributed to the release of cytolethal distending toxin B (CdtB), which cross-reacts with the adhesion protein vinculin responsible for normal intestinal contractility. Objective: This study aims to identify anti-CdtB and anti-vinculin levels in IBS patients compared to healthy control. Subjects and methods: This retrospective case-control study was conducted on 100 patients with IBS, as determined by a questionnaire based on Rome IV criteria, recruited from the outpatient clinics of the Tropical Medicine at Mansoura University Hospital from January 2019 to January 2020. Results: Anti-vinculin and anti-CdtB levels were significantly elevated in patients with IBS (1.58±0.496ng/ml, 2.47±0.60ng/ml)  when compared to control subjects (1.13±0.249ng/ml, 2.1±0.24 ng/ml), respectively with P=0.001 for both.  Anti-vinculin level was significantly higher in the IBS-D subtype than the other subtypes (P=0.001) while, Anti-CdtB was significantly elevated in IBS-C, IBS-D subgroups compared to control subjects (P=0.001). Conclusion: Findings of the present study support the hypothesis that IBS results from post-infectious disorders initiated by bacterial enteritis. A hypothesis could be applied to all IBS subgroups. On the other hand. These biomarkers might reflect the post-infectious state's severity. These findings need further extensive longitudinal studies in patients with IBS.

Horizontal Transfer of Microbial Toxin Genes to Gall Midge Genomes.

A growing body of evidence has underscored the role of horizontal gene transfer (HGT) in animal evolution. Previously, we discovered the horizontal transfer of the gene encoding the eukaryotic genotoxin cytolethal distending toxin B (cdtB) from the pea aphid Acyrthosiphon pisum secondary endosymbiont (APSE) phages to drosophilid and aphid nuclear genomes. Here, we report cdtB in the nuclear genome of the gall-forming "swede midge" Contarinia nasturtii (Diptera: Cecidomyiidae) via HGT. We searched all available gall midge genome sequences for evidence of APSE-to-insect HGT events and found five toxin genes (aip56, cdtB, lysozyme, rhs, and sltxB) transferred horizontally to cecidomyiid nuclear genomes. Surprisingly, phylogenetic analyses of HGT candidates indicated APSE phages were often not the ancestral donor lineage of the toxin gene to cecidomyiids. We used a phylogenetic signal statistic to test a transfer-by-proximity hypothesis for animal HGT, which suggested that microbe-to-insect HGT was more likely between taxa that share environments than those from different environments. Many of the toxins we found in midge genomes target eukaryotic cells, and catalytic residues important for toxin function are conserved in insect copies. This class of horizontally transferred, eukaryotic cell-targeting genes is potentially important in insect adaptation.

The Importance of Therapeutically Targeting the Binary Toxin from Clostridioides difficile.

Novel therapeutics are needed to treat pathologies associated with the Clostridioides difficile binary toxin (CDT), particularly when C. difficile infection (CDI) occurs in the elderly or in hospitalized patients having illnesses, in addition to CDI, such as cancer. While therapies are available to block toxicities associated with the large clostridial toxins (TcdA and TcdB) in this nosocomial disease, nothing is available yet to treat toxicities arising from strains of CDI having the binary toxin. Like other binary toxins, the active CDTa catalytic subunit of CDT is delivered into host cells together with an oligomeric assembly of CDTb subunits via host cell receptor-mediated endocytosis. Once CDT arrives in the host cell's cytoplasm, CDTa catalyzes the ADP-ribosylation of G-actin leading to degradation of the cytoskeleton and rapid cell death. Although a detailed molecular mechanism for CDT entry and host cell toxicity is not yet fully established, structural and functional resemblances to other binary toxins are described. Additionally, unique conformational assemblies of individual CDT components are highlighted herein to refine our mechanistic understanding of this deadly toxin as is needed to develop effective new therapeutic strategies for treating some of the most hypervirulent and lethal strains of CDT-containing strains of CDI.

Helicobacter hepaticus Induce Colitis in Male IL-10-/- Mice Dependent by Cytolethal Distending Toxin B and via the Activation of Jak/Stat Signaling Pathway.

It has been well documented that cytolethal distending toxin (CDT) from Helicobacter hepaticus (H. hepaticus), Campylobacter jejuni (C. jejuni) and other Gram-negative intestinal pathogens is linked to the inflammatory bowel disease (IBD). However, the mechanisms underlying the progression of H. hepaticus induced colitis remains unclear. In this study, male B6.129P2-IL10tm1Cgn /J mice were infected by H. hepaticus and ΔCdtB H. hepaticus for 6, 12, 18, and 24 weeks. Histopathology, H. hepaticus colonization levels, expression of inflammatory cytokines, signaling pathways, and content of NO in proximal colon were examined. We found that Cytolethal distending toxin subunit B (CdtB) deletion had no influence on colonization ability of H. hepaticus in colon of B6.129P2-IL10tm1cgn/J mice, and there was no significant difference in abundance of colonic H. hepaticus over infection duration. H. hepaticus aggravated rectocele and proximal colonic inflammation, especially at 24 WPI, while ΔCdtB H. hepaticus could not cause significant symptom. Furthermore, mRNA levels of Il-6, Tnf-α, Il-1ß, and iNOS significantly increased in the proximal colon of H. hepaticus-infected mice compared to ΔCdtB H. hepaticus infected group from 12 WPI to 24 WPI. In addition, the elevated content of NO and activated Stat3 and Jak2 in colon were observed in H. hepaticus infected mice. These data demonstrated that CdtB promote colitis development in male B6.129P2-IL10tm1Cgn /J mice by induction of inflammatory response and activation of Jak-Stat signaling pathway.

1HN, 13C, and 15N resonance assignments of the Clostridioides difficile receptor binding domain 2 (CDTb, residues 757-876).

Clostridioides difficile is a bacterial pathogen responsible for the majority of nosocomial infections in the developed world. C. difficile infection (CDI) is difficult to treat in many cases because hypervirulent strains have evolved that contain a third toxin, termed the C. difficile toxin (CDT), in addition to the two enterotoxins TcdA and TcdB. CDT is a binary toxin comprised of an enzymatic, ADP-ribosyltransferase (ART) toxin component, CDTa, and a pore-forming or delivery subunit, CDTb. In the absence of CDTa, CDTb assembles into two distinct di-heptameric states, a symmetric and an asymmetric form with both states having two surface-accessible host cell receptor-binding domains, termed RBD1 and RBD2. RBD1 has a unique amino acid sequence, when aligned to other well-studied binary toxins (i.e., anthrax), and it contains a novel Ca2+-binding site important for CDTb stability. The other receptor binding domain, RBD2, is critically important for CDT toxicity, and a domain such as this is missing altogether in other binary toxins and shows further that CDT is unique when compared to other binary toxins. In this study, the 1H, 13C, and 15N backbone and sidechain resonances of the 120 amino acid RBD2 domain of CDTb (residues 757-876) were assigned sequence-specifically and provide a framework for future NMR-based drug discovery studies directed towards targeting the most virulent strains of CDI.

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.

The Nuclear Remodeling Induced by Helicobacter Cytolethal Distending Toxin Involves MAFB Oncoprotein.

Enterohepatic Helicobacters, such as Helicobacter hepaticus and Helicobacter pullorum, are associated with several intestinal and hepatic diseases. Their main virulence factor is the cytolethal distending toxin (CDT). In the present study, whole genome microarray-based identification of differentially expressed genes was performed in vitro in HT-29 intestinal cells while following the ectopic expression of the active CdtB subunit of H. hepaticus CDT. A CdtB-dependent upregulation of the V-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) gene encoding the MAFB oncoprotein was found, as well as the CdtB-dependent regulation of several MAFB target genes. The transduction and coculture experiments confirmed MAFB mRNA and protein induction in response to CDT and its CdtB subunit in intestinal and hepatic cell lines. An analysis of MAFB protein subcellular localization revealed a strong nuclear and perinuclear localization in the CdtB-distended nuclei in intestinal and hepatic cells. MAFB was also detected at the cell periphery of the CdtB-induced lamellipodia in some cells. The silencing of MAFB changed the cellular response to CDT with the formation of narrower lamellipodia, a reduction of the increase in nucleus size, and the formation of less γH2AX foci, the biomarker for DNA double-strand breaks. Taken together, these data show that the CDT of enterohepatic Helicobacters modulates the expression of the MAFB oncoprotein, which is translocated in the nucleus and is associated with the remodeling of the nuclei and actin cytoskeleton.

Cytolethal Distending Toxin Subunit B: A Review of Structure-Function Relationship.

The Cytolethal Distending Toxin (CDT) is a bacterial virulence factor produced by several Gram-negative pathogenic bacteria. These bacteria, found in distinct niches, cause diverse infectious diseases and produce CDTs differing in sequence and structure. CDTs have been involved in the pathogenicity of the associated bacteria by promoting persistent infection. At the host-cell level, CDTs cause cell distension, cell cycle block and DNA damage, eventually leading to cell death. All these effects are attributable to the catalytic CdtB subunit, but its exact mode of action is only beginning to be unraveled. Sequence and 3D structure analyses revealed similarities with better characterized proteins, such as nucleases or phosphatases, and it has been hypothesized that CdtB exerts a biochemical activity close to those enzymes. Here, we review the relationships that have been established between CdtB structure and function, particularly by mutation experiments on predicted key residues in different experimental systems. We discuss the relevance of these approaches and underline the importance of further study in the molecular mechanisms of CDT toxicity, particularly in the context of different pathological conditions.

Serodiagnostic evaluation of recombinant CdtB of S. Typhi as a potential candidate for acute typhoid.

Typhoid fever caused by human restricted Salmonella typhi presents a considerable health burden on developing South-Asian nations like India. The suboptimal sensitivity and specificity associated with culture-based isolation of etiological agent and the extensively used surface antigen-based serological assays often lead to misdiagnosis and inappropriate antimicrobial treatment. The increasing reports of the emergence of resistant strains and undefined disease burden signify the critical need for an inexpensive, reliable, easy-to-use, and highly sensitive diagnostic test for typhoid fever. Utilizing S. typhi-specific and immunogenic antigens in sero-diagnostic assays could lead to precise diagnosis of acute typhoid and prompt treatment. In this study, we report cloning, expression, and purification of recombinant Cytolethal distending toxin subunit B (CdtB) of S. typhi, which is reported to be highly specific, immunogenic, and expressed only upon S. typhi infection. We further evaluated the purified recombinant CdtB for its diagnostic potential in an IgM-based indirect ELISA format using 33 human samples. Twenty-one serum samples from blood culture confirmed cases (n = 21) of typhoid and 12 samples from healthy controls (n = 12) were tested. The assay showed sensitivity of 100% and specificity of 83.3% respectively with positive and negative predictive values of 91.3 and 100% respectively. Efficient detection of specific IgM antibodies indicates that CdtB could be highly valuable in sero-diagnosis of acute typhoid and rapid screening of clinical samples.

Prevalence of binary-toxin genes (cdtA and cdtB) among clinical strains of Clostridium difficile isolated from diarrheal patients in Iran.

AIM: In this study we investigated the prevalence of binary toxin genes, cdtA and cdtB, in clinical isolates of C. difficile from hospitalized patients with diarrhea. BACKGROUND: C. difficile binary toxin (CDT) is an action-specific ADP-ribosyltransferase that is produced by some strains of C. difficile. Co-expression of this toxin with tcdA and tcdB can lead to more severe disease in CDI patients. METHODS: Totally, 930 patients suspected of having CDI was included in this study. All samples were treated with methanol and cultured on selective C. difficile agar plates. The C. difficile isolates were further identified by PCR. Presence of tcdA, tcdB, cdtA, and cdtB genes among the strains were examined by PCR. RESULTS: Analysis of the PCR results showed a prevalence of 85.2% (144/169) for toxigenic C. diffidile. Toxin genotyping of the strains for tcdA and tcdB genes revealed the toxin profiles of A+B+, A+B-, A-B+ accounting for 86.1% (124/144), 7.6% (11/144), 6.2% (9/144) among the strains, respectively. Totally, 12.4% (21/169) of the C. difficile strains were binary toxin-positive. cdtA-B+, cdtA+ B + and cdtA+B- were detected in 43% (9/21), 38% (8/21) and 19% (4/21) of the strains, respectively. Interestingly, 12% (3/25) of nontoxigenic C. difficile strains (tcdA-B-) had either cdtA+ B + or cdtA-B+ profiles. CONCLUSION: This is the first report for the prevalence of binary toxin genes in C. difficile strains isolated from Iran. Further studies are required to investigate the exact role of binary toxins in the pathogenesis of C. difficile particularly in patients with chronic diarrhea among Iranian populations.

Clinical experience with the use of anti-CdtB and anti-vinculin antibodies in patients with diarrhea in Mexico. / Experiencia clínica con el uso de los anticuerpos anti-CdtB y anti-vinculina en pacientes con diarrea en México.

INTRODUCTION: Circulating anti-CdtB/anti-vinculin antibodies have been validated as biomarkers to distinguish IBS-D from IBD, but there is no experience with them in Latin America. MATERIALS AND METHODS: The analysis was carried out on patients seen at a FGIDs/motility clinic over the last 7 months for diarrhea with abdominal pain and/or bloating who were tested for these antibodies. The patients were diagnosed according to the Rome III criteria or with organic disorders, and those presenting with IBS were further classified as post-infectious (PI) or non-PI-IBS. RESULTS: Thirty patients were studied. Positive biomarkers were found in IBS-D and IBS-D Overlap (58.8%) and IBS-M (33.3%), with no differences between PI-IBS (71.4%) vs. non-PI-IBS (41.7%) subjects (P=.21). There was no positivity in patients with other FGIDs or organic diarrhea, except for one with small intestinal bacterial overgrowth (SIBO). CONCLUSIONS: Our findings support the use of this test as a first-line diagnostic tool to confirm the presence of IBS-D/IBS-M according to the Rome III criteria.

(1)H(N), (13)C, and (15)N resonance assignments of the CDTb-interacting domain (CDTaBID) from the Clostridium difficile binary toxin catalytic component (CDTa, residues 1-221).

Once considered a relatively harmless bacterium, Clostridium difficile has become a major concern for healthcare facilities, now the most commonly reported hospital-acquired pathogen. C. difficile infection (CDI) is usually contracted when the normal gut microbiome is compromised by antibiotic therapy, allowing the opportunistic pathogen to grow and produce its toxins. The severity of infection ranges from watery diarrhea and abdominal cramping to pseudomembranous colitis, sepsis, or death. The past decade has seen a marked increase in the frequency and severity of CDI among industrialized nations owing directly to the emergence of a highly virulent C. difficile strain, NAP1. Along with the large Clostridial toxins expressed by non-epidemic strains, C. difficile NAP1 produces a binary toxin, C. difficile transferase (CDT). As the name suggests, CDT is a two-component toxin comprised of an ADP-ribosyltransferase (ART) component (CDTa) and a cell-binding/translocation component (CDTb) that function to destabilize the host cytoskeleton by covalent modification of actin monomers. Central to the mechanism of binary toxin-induced pathogenicity is the formation of CDTa/CDTb complexes at the cell surface. From the perspective of CDTa, this interaction is mediated by the N-terminal domain (residues 1-215) and is spatially and functionally independent of ART activity, which is located in the C-terminal domain (residues 216-420). Here we report the (1)H(N), (13)C, and (15)N backbone resonance assignments of a 221 amino acid, ~26 kDa N-terminal CDTb-interacting domain (CDTaBID) construct by heteronuclear NMR spectroscopy. These NMR assignments represent the first component coordination domain for a family of Clostridium or Bacillus species harboring ART activity. Our assignments lay the foundation for detailed solution state characterization of structure-function relationships, toxin complex formation, and NMR-based drug discovery efforts.

Complete genome sequence of the Streptomyces sp. strain CdTB01, a bacterium tolerant to cadmium.

Streptomyces sp. Strain CdTB01, which is tolerant to high concentrations of heavy metals, particularly cadmium, was isolated from soil contaminated with heavy metals. Two contigs with total genome size of 10.19Mb were identified in the whole genome sequencing and assembly, and numerous homologous genes known to be involved in heavy metal resistance were found in the genome.

Functional significance of active site residues in the enzymatic component of the Clostridium difficile binary toxin.

Clostridium difficile binary toxin (CDT) is an ADP-ribosyltransferase which is linked to enhanced pathogenesis of C. difficile strains. CDT has dual function: domain a (CDTa) catalyses the ADP-ribosylation of actin (enzymatic component), whereas domain b (CDTb) transports CDTa into the cytosol (transport component). Understanding the molecular mechanism of CDT is necessary to assess its role in C. difficile infection. Identifying amino acids that are essential to CDTa function may aid drug inhibitor design to control the severity of C. difficile infections. Here we report mutations of key catalytic residues within CDTa and their effect on CDT cytotoxicity. Rather than an all-or-nothing response, activity of CDTa mutants vary with the type of amino acid substitution; S345A retains cytotoxicity whereas S345Y was sufficient to render CDT non-cytotoxic. Thus CDTa cytotoxicity levels are directly linked to ADP-ribosyltransferase activity.