Genetic and chemical control of tuberculostearic acid production in Mycobacterium avium subspecies paratuberculosis.

Tuberculostearic acid (TBSA) is a fatty acid unique to mycobacteria and some corynebacteria and has been studied due to its diagnostic value, biofuel properties, and role in membrane dynamics. In this study, we demonstrate that TBSA production can be abrogated either by addition of pivalic acid to mycobacterial growth cultures or by a bfaA gene knockout encoding a flavin adenine dinucleotide (FAD)-binding oxidoreductase. Mycobacterium avium subspecies paratuberculosis (Map) growth and TBSA production were inhibited in 0.5-mg/mL pivalic acid-supplemented cultures, but higher concentrations were needed to have a similar effect in other mycobacteria, including Mycobacterium smegmatis. While Map C-type strains, isolated from cattle and other ruminants, will produce TBSA in the absence of pivalic acid, the S-type Map strains, typically isolated from sheep, do not produce TBSA in any condition. A SAM-dependent methyltransferase encoded by bfaB and FAD-binding oxidoreductase are both required in the two-step biosynthesis of TBSA. However, S-type strains contain a single-nucleotide polymorphism in the bfaA gene, rendering the oxidoreductase enzyme vestigial. This results in the production of an intermediate, termed 10-methylene stearate, which is detected only in S-type strains. Fatty acid methyl ester analysis of a C-type Map bfaA knockout revealed the loss of TBSA production, but the intermediate was present, similar to the S-type strains. Collectively, these results demonstrate the subtle biochemical differences between two primary genetic lineages of Map and other mycobacteria as well as explain the resulting phenotype at the genetic level. These data also suggest that TBSA should not be used as a diagnostic marker for Map.IMPORTANCEBranched-chain fatty acids are a predominant cell wall component among species belonging to the Mycobacterium genus. One of these is TBSA, which is a long-chain middle-branched fatty acid used as a diagnostic marker for Mycobacterium tuberculosis. This fatty acid is also an excellent biolubricant. Control of its production is important for industrial purposes as well as understanding the biology of mycobacteria. In this study, we discovered that a carboxylic acid compound termed pivalic acid inhibits TBSA production in mycobacteria. Furthermore, Map strains from two separate genetic lineages (C-type and S-type) showed differential production of TBSA. Cattle-type strains of Mycobacterium avium subspecies paratuberculosis produce TBSA, while the sheep-type strains do not. This important phenotypic difference is attributed to a single-nucleotide deletion in sheep-type strains of Map. This work sheds further light on the mechanism used by mycobacteria to produce tuberculostearic acid.

Molecular detection and characterization of the Mycobacterium tuberculosis complex subspecies responsible for bovine tuberculosis in Punjab, Pakistan.

Bovine tuberculosis (bTB), traditionally associated with Mycobacterium bovis, presents significant public health and economic challenges worldwide. This study investigated the causative agents of bTB in slaughtered cattle and buffalo in Lahore, Pakistan. Of the 3,581 animals screened, 34 were identified with gross TB-like lesions. The lesions were processed for culture, PCR, and Sanger sequencing to identify the causative agents of the disease. The results identified 10 Mycobacterium orygis and 8 Mycobacterium tuberculosis sensu stricto isolates. Whole-genome sequencing was performed on two M. orygis isolates, and the sequences were phylogenetically compared to 93 publicly available M. orygis sequences. The results also demonstrated that the JB21 and JB22 primers, which have been previously commonly applied to detect M. bovis in Pakistan, are unable to distinguish between M. tuberculosis complex subspecies. The identification of M. orygis and M. tuberculosis as causative agents of bTB in this slaughterhouse in Punjab may have important implications in identifying cases of zoonotic TB in humans and applying appropriate molecular tools to identify the prevalence of the disease. The data from this study align with recent findings suggesting M. orygis is the predominant cause of bTB in South Asia.IMPORTANCEThe study findings hold significant relevance to the Journal of Clinical Microbiology, as they directly impact the field. The first-time identification of Mycobacterium orygis and Mycobacterium tuberculosis as the predominant causative agents of bovine tuberculosis in Lahore, Pakistan underscores the urgent need for enhanced diagnostic methods. The study emphasizes the importance of improved assays for the accurate detection and differentiation of Mycobacterium subspecies. Additionally, the research addresses zoonotic risk assessment and public health implications, advocating for a multidisciplinary approach that integrates clinical microbiology with veterinary and human health sectors. These insights contribute to clinical microbiology knowledge, shaping effective strategies for disease prevention, surveillance, and control. The study's potential to advance the field makes it well suited for publication in the Microbiology Spectrum journal.

Establishment of persistent enteric mycobacterial infection following streptomycin pre-treatment.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis, a chronic gastrointestinal disease affecting ruminants. This disease remains widespread in part due to the limitations of available diagnostics and vaccines. A representative small animal model of disease could act as a valuable tool for studying its pathogenesis and to develop new methods for paratuberculosis control, but current models are lacking. Streptomycin pre-treatment can reduce colonization resistance and has previously been shown to improve enteric infection in a Salmonella model. Here, we investigated whether streptomycin pre-treatment of mice followed by MAP gavage could act as a model of paratuberculosis which mimics the natural route of infection and disease development in ruminants. The infection outcomes of MAP were compared to M. avium subsp. hominissuis (MAH), an environmental mycobacterium, and M. bovis and M. orygis, two tuberculous mycobacteria. Streptomycin pre-treatment was shown to consistently improve bacterial infection post-oral inoculation. This model led to chronic MAP infection of the intestines and mesenteric lymph nodes (MLNs) up to 24-weeks post-gavage, however there was no evidence of inflammation or disease. These infection outcomes were found to be specific to MAP. When the model was applied to a bacterium of lesser virulence MAH, the infection was comparatively transient. Mice infected with bacteria of greater virulence, M. bovis or M. orygis, developed chronic intestinal and MLN infection with pulmonary disease similar to zoonotic TB. Our findings suggest that a streptomycin pre-treatment mouse model could be applied to future studies to improve enteric infection with MAP and to investigate other modifications underlying MAP enteritis.

Development of a Multiplex Real-Time PCR Assay for Mycobacterium bovis BCG and Validation in a Clinical Laboratory.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is a live attenuated vaccine which can result in local or disseminated infection, most commonly in immunocompromised individuals. Differentiation of BCG from other members of the Mycobacterium tuberculosis complex (MTBC) is required to diagnose BCG disease, which requires specific management. Current methods for BCG diagnosis are based on mycobacterial culture and conventional PCR; the former is time-consuming and the latter often unavailable. Further, there are reports that certain BCG strains may be associated with a higher rate of adverse events. This study describes the development of a two-step multiplex real-time PCR assay which uses single nucleotide polymorphisms to detect BCG and identify early or late BCG strains. The assay has a limit of detection of 1 pg BCG boiled lysate DNA and was shown to detect BCG in both pure cultures and experimentally infected tissue. Its performance was assessed on 19 suspected BCG clinical isolates at Christian Medical College in Vellore, India, taken from January 2018 to August 2020. Of these 19 isolates, 10 were identified as BCG (6 early and 4 late strains), and 9 were identified as other MTBC members. Taken together, the results demonstrate the ability of this assay to identify and characterize BCG disease from cultures and infected tissue. The capacity to identify BCG may improve patient management, and the ability to discriminate between BCG strains may enable BCG vaccine pharmacovigilance. IMPORTANCE Vaccination against tuberculosis with bacillus Calmette-Guérin (BCG) can lead to adverse events, including a rare but life-threatening complication of disseminated BCG. This complication often occurs in young children with immunodeficiencies and is associated with an ∼60% mortality rate. A rapid method of reliably identifying BCG infection is important because BCG requires treatment unique to tuberculosis. BCG is resistant to the first-line antituberculosis drug pyrazinamide. Additionally, diagnosis of BCG disease would lead to further investigation of a possible underlying immune condition. We have developed a diagnostic assay to identify BCG which improves upon previously published methods and can reliably identify BCG from bacterial culture or directly from infected tissue. This assay can also differentiate between strains of BCG, which have been suggested to be associated with different rates of adverse events. This assay was validated on 19 clinical isolates collected at Christian Medical College in Vellore, India.

Reconsidering Mycobacterium bovis as a proxy for zoonotic tuberculosis: a molecular epidemiological surveillance study.

BACKGROUND: Zoonotic tuberculosis is defined as human infection with Mycobacterium bovis. Although globally, India has the largest number of human tuberculosis cases and the largest cattle population, in which bovine tuberculosis is endemic, the burden of zoonotic tuberculosis is unknown. The aim of this study was to obtain estimates of the human prevalence of animal-associated members of the Mycobacterium tuberculosis complex (MTBC) at a large referral hospital in India. METHODS: We did a molecular epidemiological surveillance study of 940 positive mycobacteria growth indicator tube (MGIT) cultures, collected from patients visiting the outpatient department at Christian Medical College (Vellore, India) with suspected tuberculosis between Oct 1, 2018, and March 31, 2019. A PCR-based approach was applied to subspeciate cultures. Isolates identified as MTBC other than M tuberculosis or as inconclusive on PCR were subject to whole-genome sequencing (WGS), and phylogenetically compared with publicly available MTBC sequences from south Asia. Sequences from WGS were deposited in the National Center for Biotechnology Information Sequence Read Archive, accession number SRP226525 (BioProject database number PRJNA575883). FINDINGS: The 940 MGIT cultures were from 548 pulmonary and 392 extrapulmonary samples. A conclusive identification was obtained for all 940 isolates; wild-type M bovis was not identified. The isolates consisted of M tuberculosis (913 [97·1%] isolates), Mycobacterium orygis (seven [0·7%]), M bovis BCG (five [0·5%]), and non-tuberculous mycobacteria (15 [1·6%]). Subspecies were assigned for 25 isolates by WGS, which were analysed against 715 MTBC sequences from south Asia. Among the 715 genomes, no M bovis was identified. Four isolates of cattle origin were dispersed among human sequences within M tuberculosis lineage 1, and the seven M orygis isolates from human MGIT cultures were dispersed among sequences from cattle. INTERPRETATION: M bovis prevalence in humans is an inadequate proxy of zoonotic tuberculosis. The recovery of M orygis from humans highlights the need to use a broadened definition, including MTBC subspecies such as M orygis, to investigate zoonotic tuberculosis. The identification of M tuberculosis in cattle also reinforces the need for One Health investigations in countries with endemic bovine tuberculosis. FUNDING: Bill & Melinda Gates Foundation, Canadian Institutes for Health Research.

Broad-spectrum capture of clinical pathogens using engineered Fc-mannose-binding lectin enhanced by antibiotic treatment.

Background: Fc-mannose-binding lectin (FcMBL), an engineered version of the blood opsonin MBL that contains the carbohydrate recognition domain (CRD) and flexible neck regions of MBL fused to the Fc portion of human IgG1, has been shown to bind various microbes and pathogen-associated molecular patterns (PAMPs). FcMBL has also been used to create an enzyme-linked lectin sorbent assay (ELLecSA) for use as a rapid (<1 h) diagnostic of bloodstream infections. Methods: Here we extended this work by using the ELLecSA to test FcMBL's ability to bind to more than 190 different isolates from over 95 different pathogen species. Results: FcMBL bound to 85% of the isolates and 97 of the 112 (87%) different pathogen species tested, including bacteria, fungi, viral antigens and parasites. FcMBL also bound to PAMPs including, lipopolysaccharide endotoxin (LPS) and lipoteichoic acid (LTA) from Gram-negative and Gram-positive bacteria, as well as lipoarabinomannan (LAM) and phosphatidylinositol mannoside 6 (PIM 6) from Mycobacterium tuberculosis. Conclusions: The efficiency of pathogen detection and variation between binding of different strains of the same species could be improved by treating the bacteria with antibiotics, or mechanical disruption using a bead mill, prior to FcMBL capture to reveal previously concealed binding sites within the bacterial cell wall. As FcMBL can bind to pathogens and PAMPs in urine as well as blood, its broad-binding capability could be leveraged to develop a variety of clinically relevant technologies, including infectious disease diagnostics, therapeutics, and vaccines.

The Candy Smell Test in clinical routine.

BACKGROUND: The "Candy Smell Test" (CST) has been introduced as a new testing method for the evaluation of the human sense of smell. In contrast to other established orthonasal smell tests, the CST addresses the retronasal application of odors, typical for food aroma effects during mastication and swallowing. The aim of this study was to evaluate the CST in a clinical setting in patients with olfactory dysfunction and normal controls against the Sniffin' Sticks test. Furthermore, cutoff points for normal and pathological results in the CST should be determined. METHODS: The olfactory performance of 96 patients presenting with olfactory disorders and 71 healthy controls was evaluated with the CST-comprised of 23 different aromatized smell candies and the extended Sniffin' Sticks test (threshold, discrimination, and identification). The control group was gender matched but included also younger persons. RESULTS: The tested subjects could easily understand the procedures and were motivated to participate. The CST correlated well with the Sniffin' Sticks for all tested subjects and for patients (n = 96) and controls (n = 71). The proposed cutoff value to differentiate normosmia from hyposmia in the CST was a score of <16 (i.e., 16 correctly identified odors) of 23. A score below 13 in the CST was the cutoff value for anosmia. CONCLUSION: The CST is an easy-to-handle reliable tool to investigate retronasal olfaction suited for clinical determination of normosmia, hyposmia, and ansomia. In addition, it can be used for investigation where self-application is necessary such as in large survey studies.

Generation and characterization of EphA1 receptor tyrosine kinase reporter knockout mice.

Eph receptor tyrosine kinases (RTKs) are a highly conserved family of signaling proteins with functions in cellular migration, adhesion, apoptosis, and proliferation during both adult and embryonic life. Here, we describe a knock-in mouse in which EphA1 expression is disrupted via the insertion of an internal ribosome entry site (IRES)-human placental alkaline phosphatase (ALPP) reporter cassette into exon II of the EphA1 gene. This was shown to successfully knockout expression of endogenous EphA1 and enforce expression of the ALPP reporter by the EphA1 promoter. Staining for the ALPP reporter protein demonstrated an epithelially restricted expression pattern in mouse tissues. In EphA1 null mice, two separate phenotypes were identified: abnormal tail development manifesting as a kinky tail was found in approximately 80% of homozygous adults. A second, distinct abnormality present in approximately 18% of females was characterized by imperforate uterovaginal development with hydrometrocolpos and caused by a resistance of cells to apoptosis during reproductive tract canalization. These results indicate a possible role for EphA1 in tissue patterning and hormone-induced apoptotic processes.

Epithelial morphogenesis and intestinal cancer: new insights in signaling mechanisms.

In this review, the major signal transduction pathways that have been shown to play an important role in intestinal homeostasis are highlighted. Each of them, the Wnt, Notch, Hedgehog, and Bone Morphogenetic Protein, as well as growth-factor regulated Receptor Tyrosine Kinases are depicted with a special emphasis through their involvement in stem cell maintenance and their role in intestinal tumorigenesis. Finally, we discuss recent data on the final steps of tumor progression, notably the formation of distant metastases. This multistep process is highly complex and still far from being understood while being of major importance for the survival of patients with digestive cancer.

Maxillary transverse discrepancies and potentially impacted maxillary canines in mixed-dentition patients.

OBJECTIVE: To investigate the correlation between maxillary transverse discrepancy and the occurrence of impacted canines in patients during the mixed-dentition stage. MATERIALS AND METHODS: Panoramic radiographs and dental casts were evaluated of randomly selected patients in the mixed dentition. The experimental group consisted of 84 orthodontic patients with a maxillary transverse discrepancy. The control group included 100 orthodontic patients without a maxillary transverse discrepancy. Intermolar widths of the experimental group were measured and recorded. The permanent canines of both groups were placed into a sector classification by using a panoramic radiograph. The experimental group was then analyzed to identify whether these patients had an impacted maxillary canine associated with the transverse discrepancy. The results were further evaluated based on type of impaction (unilateral or bilateral). RESULTS: Results of this study showed that patients with a transverse discrepancy are more likely to have an impacted canine than those patients without a transverse discrepancy, with the impaction more likely being unilateral. However, patients with a transverse discrepancy do not have a greater likelihood of having a bilateral impaction compared with patients without a transverse discrepancy. CONCLUSIONS: There appears to be an association between potentially impacted canines and transverse discrepancies. Identification can be made early based on proper panoramic evaluation and clinical detection. If a possibly impacted canine is detected early, appropriate treatment should be taken to minimize complications and avoid definitive impaction.

Over-expression of Eph and ephrin genes in advanced ovarian cancer: ephrin gene expression correlates with shortened survival.

BACKGROUND: Increased expression of Eph receptor tyrosine kinases and their ephrin ligands has been implicated in tumor progression in a number of malignancies. This report describes aberrant expression of these genes in ovarian cancer, the commonest cause of death amongst gynaecological malignancies. METHODS: Eph and ephrin expression was determined using quantitative real time RT-PCR. Correlation of gene expression was measured using Spearman's rho statistic. Survival was analysed using log-rank analysis and (was visualised by) Kaplan-Meier survival curves. RESULTS: Greater than 10 fold over-expression of EphA1 and a more modest over-expression of EphA2 were observed in partially overlapping subsets of tumors. Over-expression of EphA1 strongly correlated (r = 0.801; p < 0.01) with the high affinity ligand ephrin A1. A similar trend was observed between EphA2 and ephrin A1 (r = 0.387; p = 0.06). A striking correlation of both ephrin A1 and ephrin A5 expression with poor survival (r = -0.470; p = 0.02 and r = -0.562; p < 0.01) was observed. Intriguingly, there was no correlation between survival and other clinical parameters or Eph expression. CONCLUSION: These data imply that increased levels of ephrins A1 and A5 in the presence of high expression of Ephs A1 and A2 lead to a more aggressive tumor phenotype. The known functions of Eph/ephrin signalling in cell de-adhesion and movement may explain the observed correlation of ephrin expression with poor prognosis.

Expression analysis of the Epha1 receptor tyrosine kinase and its high-affinity ligands Efna1 and Efna3 during early mouse development.

Interaction of Eph receptor tyrosine kinases with their membrane bound ephrin ligands initiates bidirectional signaling events that regulate cell migratory and adhesive behavior. Whole-mount in situ hybridization revealed overlapping expression of the Epha1 receptor and its high-affinity ligands ephrin A1 (Efna1) and ephrin A3 (Efna3) in the primitive streak and the posterior paraxial mesoderm during early mouse development. These results show complex and dynamic expression for all three genes with expression domains that are successively complementary, overlapping, and divergent.

Inhibition of stress-inducible kinase pathways by tumorigenic mutant p53.

More than 50% of human cancers contain p53 gene mutations and as a result accumulate altered forms of the full-length p53 protein. Although certain tumor types expressing mutant p53 protein have a poor prognostic process, the precise role of mutant p53 protein in highly malignant tumor cells is not well defined. Some p53 mutants, but not wild-type p53, are shown here to interact with Daxx, a Fas-binding protein that activates stress-inducible kinase pathways. Interaction of Daxx with p53 is highly dependent upon the specific mutation of p53. Tumorigenic mutants of p53 bind to Daxx and inhibit Daxx-dependent activation of the apoptosis signal-regulating kinase 1 stress-inducible kinases and Jun NH(2)-terminal kinase. Mutant p53 forms complexes with Daxx in cells, and consequently, mutant p53 is able to rescue cells from Daxx-dependent inhibition of proliferation. Thus, the accumulation of mutant p53 in tumor cells may contribute to tumorigenesis by inhibiting stress-inducible kinase pathways.

A novel p53-inducible apoptogenic gene, PRG3, encodes a homologue of the apoptosis-inducing factor (AIF).

The p53 tumor suppressor protein induces cell cycle arrest or apoptosis in response to cellular stresses. We have identified PRG3 (p53-responsive gene 3), which is induced specifically under p53-dependent apoptotic conditions in human colon cancer cells, and encodes a novel polypeptide of 373 amino acids with a predicted molecular mass of 40.5 kDa. PRG3 has significant homology to bacterial oxidoreductases and the apoptosis-inducing factor, AIF, and the gene was assigned to chromosome 10q21.3-q22.1. Expression of PRG3 was induced by the activation of endogenous p53 and it contains a p53-responsive element. Unlike AIF, PRG3 localizes in the cytoplasm and its ectopic expression induces apoptosis. An amino-terminal deletion mutant of PRG3 that lacks a putative oxidoreductase activity retains its apoptotic activity, suggesting that the oxidoreductase activity is dispensable for the apoptotic function of PRG3. The PRG3 gene is thus a novel p53 target gene in a p53-dependent apoptosis pathway.

The role of the Eph-ephrin signalling system in the regulation of developmental patterning.

The Eph and ephrin system, consisting of fourteen Eph receptor tyrosine kinase proteins and nine ephrin membrane proteins in vertebrates, has been implicated in the regulation of many critical events during development. Binding of cell surface Eph and ephrin proteins results in bi-directional signals, which regulate the cytoskeletal, adhesive and motile properties of the interacting cells. Through these signals Eph and ephrin proteins are involved in early embryonic cell movements, which establish the germ layers, cell movements involved in formation of tissue boundaries and the pathfinding of axons. This review focuses on two vertebrate models, the zebrafish and mouse, in which experimental perturbation of Eph and/or ephrin expression in vivo have provided important insights into the role and functioning of the Eph/ephrin system.