- Madison, Wisconsin, United States
Adel Talaat
University of Wisconsin-Madison, Pathobiological Sciences, Faculty Member
Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), infects over two billion people, claiming around 1.5 million lives annually. The only vaccine approved for clinical use against this disease is the Bacillus... more
Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), infects over two billion people, claiming around 1.5 million lives annually. The only vaccine approved for clinical use against this disease is the Bacillus Calmette-Guérin (BCG) vaccine. Unfortunately, BCG has limited efficacy against the adult, pulmonary form of tuberculosis. This vaccine was developed from M. bovis with antigen expression and host specificity that differ from M. tuberculosis. To address these problems, we have designed two novel, live attenuated vaccine (LAV) candidates on an M. tuberculosis background: ΔmosR and ΔechA7. These targeted genes are important to M. tuberculosis pathogenicity during infection. To examine the efficacy of these strains, C57BL/6 mice were vaccinated subcutaneously with either LAV, BCG, or PBS. Both LAV strains persisted up to 16 weeks in the spleens or lungs of vaccinated mice, while eliciting minimal pathology prior to challenge. Following challenge with a selected, high virulence M. tuberculosis Beijing strain, protection was notably greater for both groups of LAV vaccinated animals as compared to BCG at both 30 and 60 days post-challenge. Additionally, vaccination with either ΔmosR or ΔechA7 elicited an immune response similar to BCG. Although these strains require further development to meet safety standards, this first evidence of protection by these two new, live attenuated vaccine candidates shows promise.
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Research article Optical mapping of the Mycobacterium avium subspecies paratuberculosis genome
Galactofuranose (Galf) is present in glycans critical for virulence and viability of several pathogenic microbes, including Mycobacterium tuberculosis, yet the monosaccharide is absent from mammalian glycans. Uridine... more
Galactofuranose (Galf) is present in glycans critical for virulence and viability of several pathogenic microbes, including Mycobacterium tuberculosis, yet the monosaccharide is absent from mammalian glycans. Uridine 5'-diphosphate-galactopyranose mutase (UGM) catalyzes the formation of UDP-Galf, which is required to produce Galf-containing glycoconjugates. Inhibitors of UGM have therefore been sought as antimicrobial leads and to delineate the roles of Galf in cells. Obtaining cell permeable UGM probes by either design or high throughput screens has been difficult, as has elucidating how UGM binds small molecule, non-carbohydrate inhibitors. To address these issues, we employed structure-based virtual screening to uncover new inhibitor chemotypes, including a triazolothiadiazine series. These compounds are among the most potent antimycobacterial UGM inhibitors described. They also facilitated determination of a UGM-small molecule inhibitor structure, which can guide optimizatio...
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Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) the causative agent of Johne's disease, is one of the most serious infectious diseases in dairy cattle worldwide. Due to the chronic nature of this disease and no... more
Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) the causative agent of Johne's disease, is one of the most serious infectious diseases in dairy cattle worldwide. Due to the chronic nature of this disease and no feasible control strategy, it is essential to have an efficient animal model which is representative of the natural route of infection as well as a viable treatment option. In this report, we evaluated the effect of different doses of M. paratuberculosis in their ability to colonize murine tissues following oral delivery and the ability of Lactobacillus casei ATCC 334, a nascent probiotic, to combat paratuberculosis. Oral inoculation of mice was able to establish paratuberculosis in a dose-dependent manner. Two consecutive doses of approximately 10(9) CFU per mouse resulted in a disseminated infection, whereas lower doses were not efficient to establish infection. All inoculated mice were colonized with M. paratuberculosis, maintained infection for up to...
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Mycobacterium avium subspecies paratuberculosis (M. ap), the causative agent of Johne's disease, infects many farmed ruminants, wild-life animals, and recently isolated from humans. To better understand the molecular pathogenesis of... more
Mycobacterium avium subspecies paratuberculosis (M. ap), the causative agent of Johne's disease, infects many farmed ruminants, wild-life animals, and recently isolated from humans. To better understand the molecular pathogenesis of these infections, we analyzed the whole-genome sequences of several M. ap and M. avium subspecies avium (M. avium) isolates to gain insights into genomic diversity associated with variable hosts and environments. Using Next-generation sequencing technology, all six M. ap isolates showed a high percentage of similarity (98%) to the reference genome sequence of M. ap K-10 isolated from cattle. However, two M. avium isolates (DT 78 and Env 77) showed significant sequence diversity (only 87 and 40% similarity, respectively) compared to the reference strain M. avium 104, a reflection of the wide environmental niches of this group of mycobacteria. Within the M. ap isolates, genomic rearrangements (insertions/deletions) were not detected, and only unique si...
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Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is the causative agent of paratuberculosis, or Johne's disease, in cattle with potential involvement in cases of... more
Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is the causative agent of paratuberculosis, or Johne's disease, in cattle with potential involvement in cases of Crohn's disease in humans. Johne's disease is found worldwide and is economically important for both beef and dairy industries. In an effort to characterize this important infection in Egypt, we analyzed the ecological and genomic features of recent isolates of M. paratuberculosis. In this report, we examined 26 Holstein dairy herds distributed throughout Egypt from 2010 to 2013. Using PCR analysis of fecal samples, we estimated an average herd level prevalence of 65.4% with animal level infection that reached an average of 13.6% among animals suffering from diarrhea. Whole genome sequencing of field isolates identified numerous single nucleotide polymorphisms among field isolates relative to the standard M. paratuberculosis K-10 genome. Interestingly, the virulence of M. paratuberculosis isolates from Egypt revealed diverse virulence phenotypes in the murine model of paratuberculosis with significant differences in tissue colonization, particularly during the chronic stage of infection. Overall, our analysis confirmed that Johne's disease is a newly identified problem in Egypt and indicated that M. paratuberculosis has potentially diverse genotypes that impact its virulence. Further ecological mapping and genomic analysis of M. paratuberculosis will continue to enhance our understanding of the transmission and evolutionary dynamics of this pathogen under natural field conditions.
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Research Interests: Spleen, Liver, Mutation, Mice, Female, and 5 moreAnimals, Vaccination, Cattle, Time Factors, and Paratuberculosis
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Fourteen proteins of potential diagnostic value for bovine paratuberculosis were identified in the culture filtrate of Mycobacterium paratuberculosis JTC303 by immunoblot and mass spectrometry. The goals of the present study were to... more
Fourteen proteins of potential diagnostic value for bovine paratuberculosis were identified in the culture filtrate of Mycobacterium paratuberculosis JTC303 by immunoblot and mass spectrometry. The goals of the present study were to express these 14 ORFs in Escherichia coli and evaluate their antigenicity. All 14 proteins were expressed in E. coli BL21(DE3) after transformation with the pET-22b(+) vector. Yields of insoluble proteins were higher than those of the soluble proteins. Polyclonal rabbit antibodies directed against culture filtrate of JTC303 strain confirmed that five of the expressed and purified proteins are culture filtrate components: ModD, Antigen 85C, PepA, MAP1693c, and MAP2168c. Evaluation of ModD as an ELISA solid-phase antigen on a set of bovine sera from well-characterized paratuberculosis cases and infection-free controls revealed that there was strong serum antibody reactivity to rModD in many infected cattle. However, the overall rModD ELISA sensitivity and specificity for bovine paratuberculosis was not greater than those of ELISAs using crude antigens such as cellular extract or culture filtrate for plate coating, as judged by area under the curve (AUC) of Receiver-operating curve (ROC) analysis. However, an ELISA using natural ModD as the solid-phase antigen had a higher sensitivity and AUC than did rModD suggesting diminution of antigenicity in rModD. Taken together, our results showed that the natural forms of the identified proteins may be useful for diagnosis of bovine paratuberculosis.
Research Interests: Mass Spectrometry, Gene expression, Escherichia coli, Animals, Cattle, and 10 moreMolecular cloning, Protein expression and purification, Rabbits, Base Sequence, Sensitivity and Specificity, Area Under the Curve, Enzyme Linked Immunosorbent Assay, Biochemistry and cell biology, Paratuberculosis, and Immunoblotting
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Research Interests: Bacteriology, Data Analysis, Time Use, Oxidative Stress, Biological Sciences, and 15 moreMycobacterium tuberculosis, Infectious Disease, United States, Stress response, Animals, Polymerase Chain Reaction, Cattle, Dairy Cattle, Heat Shock, Dairy industry, Murine Model, High Sensitivity, Hydrogen-Ion Concentration, *Hot Temperature, and Paratuberculosis
Research Interests: Genetics, Bacteriology, Sequence Analysis, Biological Sciences, Virulence, and 16 moreTuberculosis, Humans, Regulation of Gene Expression, Animals, Genome Analysis, Cattle, Health Problems, Captive Wild Animals, Genetic variation, Economic Loss, Genome Rearrangement, Species Specificity, Dairy industry, Crohn Disease, Mycobacterium avium, and Paratuberculosis
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Mycobacterium avium subsp. paratuberculosis causes Johne's disease, an enteric infection in cattle and other ruminants, greatly afflicting the dairy industry worldwide. Once inside the cell, M. avium subsp.... more
Mycobacterium avium subsp. paratuberculosis causes Johne's disease, an enteric infection in cattle and other ruminants, greatly afflicting the dairy industry worldwide. Once inside the cell, M. avium subsp. paratuberculosis is known to survive harsh microenvironments, especially those inside activated macrophages. To improve our understanding of M. avium subsp. paratuberculosis pathogenesis, we examined phagosome maturation associated with transcriptional responses of M. avium subsp. paratuberculosis during macrophage infection. Monitoring cellular markers, only live M. avium subsp. paratuberculosis bacilli were able to prevent phagosome maturation and reduce its acidification. On the transcriptional level, over 300 M. avium subsp. paratuberculosis genes were significantly and differentially regulated in both naive and IFN-γ-activated macrophages. These genes include the sigma factor H (sigH) that was shown to be important for M. avium subsp. paratuberculosis survival inside gamma interferon (IFN-γ)-activated bovine macrophages. Interestingly, an sigH-knockout mutant showed increased sensitivity to a sustained level of thiol-specific oxidative stress. Large-scale RNA sequence analysis revealed that a large number of genes belong to the sigH regulon, especially following diamide stress. Genes involved in oxidative stress and virulence were among the induced genes in the sigH regulon with a putative consensus sequence for SigH binding that was recognized in a subset of these genes (n = 30), suggesting direct regulation by SigH. Finally, mice infections showed a significant attenuation of the ΔsigH mutant compared to its parental strain, suggesting a role for sigH in M. avium subsp. paratuberculosis virulence. Such analysis could identify potential targets for further testing as vaccine candidates against Johne's disease.
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To develop and evaluate protocols for genetic manipulations (transformation and transposition) of the fish pathogen, Mycobacterium marinum. Isolates of M. marinum obtained from fish and humans. Electrocompetent cells were prepared from... more
To develop and evaluate protocols for genetic manipulations (transformation and transposition) of the fish pathogen, Mycobacterium marinum. Isolates of M. marinum obtained from fish and humans. Electrocompetent cells were prepared from isolates of M. marinum grown to various growth phases at several temperatures and with or without the addition of ethionamide or cycloheximide. Mycobacterial cells were transformed by electroporation with a replicative Escherichia coli-mycobacteria shuttle vector (pYUB18) as well as suicide vectors (pYUB285 and pUS252) that carried transposable elements (IS1096 and IS6110, respectively). Mutants from both isolates of M. marinum were recovered on 7H10 agar plates supplemented with kanamycin. Transformation and transposition efficiencies for various protocols were compared. Southern hybridization analysis was performed on mycobacterial mutants to confirm transposition events. Competent cells prepared at room temperature (23-25 C) from organisms in late-exponential growth phase yielded higher transposition efficiency, compared with cells prepared at 4 C or from organisms in early- or mid-exponential growth phase. Naturally developing kanamycin-resistant colonies of M. marinum were not detected. Only the IS1096-derived transposition was able to efficiently mutate M. marinum. Southern hybridization of M. marinum mutants revealed random integration of IS 1096 into the M. marinum genome. Transposition and transformation efficiencies were comparable, suggesting that the limiting factor in transposition is the transformation step. Most of the experiments resulted in transposition of IS1096; however, better approaches are needed to improve transposition efficiency.
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Tuberculosis is a treatable but severe disease caused by Mycobacterium tuberculosis (Mtb). Recent statistics by international health organizations estimate the Mtb exposure to have reached over two billion individuals. Delay in disease... more
Tuberculosis is a treatable but severe disease caused by Mycobacterium tuberculosis (Mtb). Recent statistics by international health organizations estimate the Mtb exposure to have reached over two billion individuals. Delay in disease diagnosis could be fatal, especially to the population at risk, such as individuals with compromised immune systems. Intelligent decision systems (IDS) provide a promising tool to expedite discovery of biomarkers, and to boost their impact on earlier prediction of the likelihood of the disease onset. A novel IDS (iTB) is designed that integrates results from molecular medicine and systems biology of Mtb infection to estimate model parameters for prediction of the dynamics of the gene networks in Mtb-infected laboratory animals. The mouse model identifies a number of genes whose expressions could be significantly altered during the TB activation. Among them, a much smaller number of the most informative genes for prediction of the onset of TB are selected using a modified version of Empirical Risk Minimization as in Vapnik’s statistical learning theory. A hybrid intelligent system is designed to take as input the mRNA abundance at a near genome-size from the individual-to-be-tested, measured 3-4 times. The algorithms determine if that individual is at risk of the onset of the disease based on our current analysis of mRNA data, and to predict the values of the biomarkers for a future period (of up to 60 days for mice; this may differ for humans). An early warning sign allows conducting gene expression analysis during the activation which aims to find key genes that are expressed. With rapid advances in low-cost genome-based diagnosis, this IDS architecture provides a promising platform to advance Personalized Health Care based on sequencing the genome and microarray analysis of samples obtained from individuals at risk. The novelty of the design of iTB lies in the integration of the IDS design principles and the solution of the biological problems hand-in-hand, so as to provide an AI framework for biologically better-targeted personalized prevention/treatment for the high-risk groups. The iTB design applies in more generality, and provides the potential for extension of our AI-approach to personalized-medicine to prevent other public health pandemics.
Research Interests: Health Care, Personalized Medicine, Public Health, System Biology, Genome Size, and 13 moreStatistical Learning Theory, Early Warning, Tuberculosis, Mycobacterium tuberculosis, Laboratory Animals, Intelligent System, Gene Network, Mouse Model, Immune system, Microarray Analysis, International health, International Health, and Gene Expression Analysis
Infection with Mycobacterium tuberculosis causes the illness tuberculosis with an annual mortality of 2 million. Understanding the nature of the host-pathogen interactions at different stages of tuberculosis is central to new strategies... more
Infection with Mycobacterium tuberculosis causes the illness tuberculosis with an annual mortality of 2 million. Understanding the nature of the host-pathogen interactions at different stages of tuberculosis is central to new strategies for developing chemotherapies and vaccines. Toward this end, we adapted microarray technology to analyze the change in gene expression profiles of M. tuberculosis during infection in mice. This protocol provides the transcription profile of genes expressed during the course of early tuberculosis in immune-competent (BALB/c) and severe combined immune-deficient (SCID) hosts in comparison with growth in medium. The microarray analysis revealed clusters of genes that changed their transcription levels exclusively in the lungs of BALB/c, SCID mice, or medium over time. We identified a set of genes (n = 67) activated only in BALB/c and not in SCID mice at 21 days after infection, a key point in the progression of tuberculosis. A subset of the lung-activated genes was previously identified as induced during mycobacterial survival in a macrophage cell line. Another group of in vivo-expressed genes may also define a previously unreported genomic island. In addition, our analysis suggests the similarity between mycobacterial transcriptional machinery during growth in SCID and in broth, which questions the validity of using the SCID model for assessing mycobacterial virulence. The in vivo expression-profiling technology presented should be applicable to any microbial model of infection.
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Research Interests: Microbiology, Immunology, Electron Microscopy, Biofilms, Medical Microbiology, and 22 moreScanning Electron Microscopy, Veterinary, Virulence, Biofilm, Animals, Male, Bacteria, Microbial, Microbial Pathogenesis, Risk factors, Genes, Cattle, Risk Factors, Lipid Profile, Cell Wall, Mutants, Virulence Factors, Pathogenicity, Antigens, Pathogenic Bacteria, Lipopeptides, and Paratuberculosis
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An assay is described utilizing PCR technology for a rapid diagnostic test to identify fish infection with Mycobacterium marinum, M. fortuitum and M. chelonae. A 924 bp DNA fragment from a highly conserved area of the mycobacterial 16S... more
An assay is described utilizing PCR technology for a rapid diagnostic test to identify fish infection with Mycobacterium marinum, M. fortuitum and M. chelonae. A 924 bp DNA fragment from a highly conserved area of the mycobacterial 16S rRNA gene was amplified using mycobacteria genus-specific primers and digested with restriction enzymes (BanI and ApaI). This examination yielded unique restriction patterns for each mycobacterial specie enabling identification of mycobacteria infecting fish to the species level. The protocol can be applied to purified DNA, a simple colony preparation or infected fish tissue. This protocol can be completed in 1-2 days.