Adel Talaat | University of Wisconsin-Madison (original) (raw)
Papers by Adel Talaat
Vaccine, 2015
Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), infects over two billion ... 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.
Research article Optical mapping of the Mycobacterium avium subspecies paratuberculosis genome
ACS chemical biology, Jan 27, 2015
Galactofuranose (Galf) is present in glycans critical for virulence and viability of several path... 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...
Drug metabolism and disposition: the biological fate of chemicals, 2015
Doxycycline is used in treating intracellular and extracellular infections. Liposomal (LE) antibi... more Doxycycline is used in treating intracellular and extracellular infections. Liposomal (LE) antibiotics allow for low frequency dosing and extended efficacy, compared to standard (STD) formulations. We developed a novel sulfuric acid loading method for doxycycline liposomes (LE-doxy). We hypothesized that a single s.c. injection of LE-doxy would be detectable in serum for at least two weeks at concentrations equal to or better than STD-doxy and would be bactericidal in an in vitro Mycobacterium smegmatis infection of J774A.1 macrophage cells. Liposomes were encapsulated by sulfuric acid gradient loading, and release kinetics were performed in vitro and in vivo. LE-doxy made using 8.25 mg/ mL doxycycline loaded for 24 hours achieved 97.77% capture in DPPC and 43.87% in sphingomyelin. Rats were injected s.c. with 50 mg/kg LE-doxy or 5 mg/kg STD-doxy, and serial blood samples were collected. Pharmacokinetics were analyzed using HPLC. Liver and injection site skin samples were collected ...
Frontiers in cellular and infection microbiology, 2015
Frontiers in cellular and infection microbiology, 2014
Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) the causative agent of Johne... 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...
Frontiers in microbiology, 2011
Mycobacterium avium subspecies paratuberculosis (M. ap), the causative agent of Johne's disea... 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...
Frontiers in Cellular and Infection Microbiology, 2012
More than 1.8 million people die annually from infection with Mycobacterium tuberculosis, the cau... more More than 1.8 million people die annually from infection with Mycobacterium tuberculosis, the causative agent of tuberculosis. The ability of M. tuberculosis to obtain and distribute micronutrients, including biometals, is known to play a role in its intracellular survival and virulence within a host. Techniques to detect elemental distributions within M. tuberculosis cells have previously been limited to bulk detection methods or low-resolution analyses. Here, we present a method for determining the elemental distribution within M. tuberculosis on a single-cell level, at high (individual nanometer) resolution, using scanning transmission electron microscopy (STEM) in concert with energy-dispersive X-ray spectroscopy (EDS). Results revealed the presence of large polyphosphate granules in all strains of Mycobacteria tested. These persisted even through starvation conditions, and might play a role connected to elemental homeostasis in M. tuberculosis. Associated with the polyphosphate granules were micronutrients such as calcium and magnesium. In addition, we expanded the technique beyond Mycobacteria to show that STEM and EDS could be used as a simple screen to detect the presence or absence of concentrated elements on a single-cell level within all six other bacterial types tested, with minimal processing to the bacteria. Overall, we believe that this technique represents a first step in developing a better understanding of the role that components of the intracellular milieu, including polyphosphates and biometals, play in the pathogenesis of M. tuberculosis, with potential future applications for in vivo analysis.
Molecular Microbiology, 2010
Copper is a required micronutrient that is also toxic at excess concentrations. Currently, little... more Copper is a required micronutrient that is also toxic at excess concentrations. Currently, little is known about the role of copper in interactions between bacterial pathogens and their human hosts. In this study, we elucidate a mechanism for copper homeostasis in the human pathogen Mycobacterium tuberculosis via characterization of a putative copper exporter, CtpV. CtpV was shown to be required by M. tuberculosis to maintain resistance to copper toxicity. Furthermore, the deletion of ctpV resulted in a 98-gene transcriptional response which elucidates the increased stress experienced by the bacteria in the absence of this detoxification mechanism. Interestingly, although the ΔctpV mutant survives close to the wild-type levels in both murine and guinea pig models of tuberculosis, animals infected with the ΔctpV mutant displayed decreased lung damage, and mutant-infected mice had a reduced immune response to the bacteria as well as a significant increase in survival time relative to mice infected with wild-type M. tuberculosis. Overall, our study provides the first evidence for a connection between bacterial copper response and the virulence of M. tuberculosis, supporting the hypothesis that copper response could be important to intracellular pathogens, in general.
Microbiology, 2015
Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is the causative agent of p... 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.
PLoS ONE, 2014
The Gram-negative saprophytic bacterium Burkholderia pseudomallei is the causative agent of melio... more The Gram-negative saprophytic bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a severe infectious disease of both humans and animals. Severity of the disease is thought to be dependent on both the health status of the host, including diabetes mellitus and kidney disease, and bacterial-derived factors. To identify the bacterial factors important during an acute infection, gene expression profiles in the spleen, lung, and liver of BALB/c (Th2 prototype) and C57BL/6 mice (Th1 prototype) were determined using DNA microarrays. This analysis identified BPSS1521 (bprD), a predicted transcriptional regulator located in the type III secretion system (T3SS-3) operon, to be up regulated, specifically in C57BL/6 mice. BALB/c mice infected with a bprD mutant showed a shorter time to death and increased inflammation, as determined by histopathological analysis and enumeration of bacteria in the spleen. Elevated numbers of multinucleated giant cells (MNGCs), which is the hallmark of melioidosis, were detected in both the wild-type and the bprD mutants; a similar elevation occurs in melioidosis patients. One striking observation was the increased expression of BPSS1520 (bprC), located downstream of bprD, in the bprD mutant. BprC is a regulator of T6SS-1 that is required for the virulence of B. pseudomallei in murine infection models. Deletion of bprD led to the overexpression of bprC and a decreased time to death. bprD expression was elevated in C57BL/6 -as compared to BALB/c-mice, suggesting a role for BprD in the natural resistance of C57BL/6 mice to B. pseudomallei. Ultimately, this analysis using mice with different immune backgrounds may enhance our understanding of the outcomes of infection in a variety of models.
Frontiers in Cellular and Infection Microbiology, 2014
Johne's disease (JD) caused by Mycobacterium avium subspecies paratuberculosis (MAP) is a major t... more Johne's disease (JD) caused by Mycobacterium avium subspecies paratuberculosis (MAP) is a major threat to the dairy industry and possibly some cases of Crohn's disease in humans. A MAP vaccine that reduced of clinical disease and/or reduced fecal shedding would aid in the control of JD. The objectives of this study were (1) to evaluate the efficacy of 5 attenuated strains of MAP as vaccine candidates compared to a commercial control vaccine using the protocol proposed by the Johne's Disease Integrated Program (JDIP) Animal Model Standardization Committee (AMSC), and (2) to validate the AMSC Johne's disease goat challenge model. Eighty goat kids were vaccinated orally twice at 8 and 10 weeks of age with an experimental vaccine or once subcutaneously at 8 weeks with Silirum® (Zoetis), or a sham control oral vaccine at 8 and 10 weeks. Kids were challenged orally with a total of approximately 1.44 × 10 9 CFU divided in two consecutive daily doses using MAP ATCC-700535 (K10-like bovine isolate). All kids were necropsied at 13 months post-challenge. Results indicated that the AMSC goat challenge model is a highly efficient and valid model for JD challenge studies. None of the experimental or control vaccines evaluated prevented MAP infection or eliminated fecal shedding, although the 329 vaccine lowered the incidence of infection, fecal shedding, tissue colonization and reduced lesion scores, but less than the control vaccine. Based on our results the relative performance ranking of the experimental live-attenuated vaccines evaluated, the 329 vaccine was the best performer, followed by the 318 vaccine, then 316 vaccine, 315 vaccine and finally the 319 vaccine was the worst performer. The subcutaneously injected control vaccine outperformed the orally-delivered mutant vaccine candidates. Two vaccines (329 and 318) do reduce presence of JD gross and microscopic lesions, slow progression of disease, and one vaccine (329) reduced fecal shedding and tissue colonization.
Frontiers in Cellular and Infection Microbiology, 2014
Frontiers in Cellular and Infection Microbiology, 2014
Johne's disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), which results in ... more Johne's disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), which results in serious economic losses worldwide in farmed livestock such as cattle, sheep, and goats. To control this disease, an effective vaccine with minimal adverse effects is needed. In order to identify a live vaccine for Johne's disease, we evaluated eight attenuated mutant strains of MAP using a C57BL/6 mouse model. The persistence of the vaccine candidates was measured at 6, 12, and 18 weeks post vaccination. Only strains 320, 321, and 329 colonized both the liver and spleens up until the 12-week time point. The remaining five mutants showed no survival in those tissues, indicating their complete attenuation in the mouse model. The candidate vaccine strains demonstrated different levels of protection based on colonization of the challenge strain in liver and spleen tissues at 12 and 18 weeks post vaccination. Based on total MAP burden in both tissues at both time points, strain 315 (MAP1566::Tn5370) was the most protective whereas strain 318 (intergenic Tn5367 insertion between MAP0282c and MAP0283c) had the most colonization. Mice vaccinated with an undiluted commercial vaccine preparation displayed the highest bacterial burden as well as enlarged spleens indicative of a strong infection. Selected vaccine strains that showed promise in the mouse model were moved forward into a goat challenge model. The results suggest that the mouse trial, as conducted, may have a relatively poor predictive value for protection in a ruminant host such as goats.
Frontiers in Cellular and Infection Microbiology, 2014
Since the early 1980s, several investigations have focused on developing a vaccine against Mycoba... more Since the early 1980s, several investigations have focused on developing a vaccine against Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease in cattle and sheep. These studies used whole-cell inactivated vaccines that have proven useful in limiting disease progression, but have not prevented infection. In contrast, modified live vaccines that invoke a Th1 type immune response, may improve protection against infection. Spurred by recent advances in the ability to create defined knockouts in MAP, several independent laboratories have developed modified live vaccine candidates by transpositional mutation of virulence and metabolic genes in MAP. In order to accelerate the process of identification and comparative evaluation of the most promising modified live MAP vaccine candidates, members of a multi-institutional USDA-funded research consortium, the Johne's disease integrated program (JDIP), met to establish a standardized testing platform using agreed upon protocols. A total of 22 candidates vaccine strains developed in five independent laboratories in the United States and New Zealand voluntarily entered into a double blind stage gated trial pipeline. In Phase I, the survival characteristics of each candidate were determined in bovine macrophages. Attenuated strains moved to Phase II, where tissue colonization of C57/BL6 mice were evaluated in a challenge model. In Phase III, five promising candidates from Phase I and II were evaluated for their ability to reduce fecal shedding, tissue colonization and pathology in a baby goat challenge model. Formation of a multi-institutional consortium for vaccine strain evaluation has revealed insights for the implementation of vaccine trials for Johne's disease and other animal pathogens. We conclude by suggesting the best way forward based on this 3-phase trial experience and challenge the rationale for use of a macrophage-to-mouse-to native host pipeline for MAP vaccine development.
Veterinary Microbiology, 2007
An international committee of Johne's disease (JD) researchers was convened to develop guidelines... more An international committee of Johne's disease (JD) researchers was convened to develop guidelines for JD challenge studies in multiple animal species. The intent was to develop and propose international standard guidelines for models based on animal www.elsevier.com/locate/vetmic Veterinary Microbiology 122 (2007) 197-222 species that would gain acceptance worldwide. Parameters essential for the development of long-term and short-term infection models were outlined and harmonized to provide a ''best fit'' JD challenge model for cattle, goats, sheep, cervids, and mice. These models will be useful to study host-pathogen interactions, host immunity at the local and systemic level, and for evaluating vaccine candidates and therapeutics. The consensus guidelines herein list by animal species strains of Mycobacterium avium subsp. paratuberculosis used, challenge dose, dose frequency, age of challenge, route of challenge, preparation of inoculum, experimental animal selection, quality control, minimal experimental endpoints and other parameters. #
Veterinary Microbiology, 1999
Despite the ubiquitous presence of atypical mycobacteria in the environment and the potential ris... more Despite the ubiquitous presence of atypical mycobacteria in the environment and the potential risk of infection in humans and animals, the pathogenesis of diseases caused by infection with atypical mycobacteria has been poorly characterized. In this study, goldfish, Carassius auratus were infected either with the rapidly growing fish pathogen, Mycobacterium fortuitum or with another rapidly growing mycobacteria, Mycobacterium smegmatis. Bacterial persistence and pathological host response to mycobacterial infection in the goldfish are described. Mycobacteria were recovered from a high percentage of inoculated fish that developed a characteristic chronic granulomatous response similar to that associated with natural mycobacterial infection. Both M. fortuitum and M. smegmatis were pathogenic to fish. Fish infected with M. smegmatis ATCC 19420 showed the highest level of giant cell recruitment compared to fish inoculated with M. smegmatis mc 2 155 and M. fortuitum. Of the three strains of mycobacteria examined, M. smegmatis ATCC 19420 was the most virulent strain to goldfish followed by M. fortuitum and M. smegmatis mc 2 155, respectively. #
Protein Expression and Purification, 2007
Fourteen proteins of potential diagnostic value for bovine paratuberculosis were identified in th... 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.
Vaccine, 2015
Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), infects over two billion ... 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.
Research article Optical mapping of the Mycobacterium avium subspecies paratuberculosis genome
ACS chemical biology, Jan 27, 2015
Galactofuranose (Galf) is present in glycans critical for virulence and viability of several path... 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...
Drug metabolism and disposition: the biological fate of chemicals, 2015
Doxycycline is used in treating intracellular and extracellular infections. Liposomal (LE) antibi... more Doxycycline is used in treating intracellular and extracellular infections. Liposomal (LE) antibiotics allow for low frequency dosing and extended efficacy, compared to standard (STD) formulations. We developed a novel sulfuric acid loading method for doxycycline liposomes (LE-doxy). We hypothesized that a single s.c. injection of LE-doxy would be detectable in serum for at least two weeks at concentrations equal to or better than STD-doxy and would be bactericidal in an in vitro Mycobacterium smegmatis infection of J774A.1 macrophage cells. Liposomes were encapsulated by sulfuric acid gradient loading, and release kinetics were performed in vitro and in vivo. LE-doxy made using 8.25 mg/ mL doxycycline loaded for 24 hours achieved 97.77% capture in DPPC and 43.87% in sphingomyelin. Rats were injected s.c. with 50 mg/kg LE-doxy or 5 mg/kg STD-doxy, and serial blood samples were collected. Pharmacokinetics were analyzed using HPLC. Liver and injection site skin samples were collected ...
Frontiers in cellular and infection microbiology, 2015
Frontiers in cellular and infection microbiology, 2014
Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) the causative agent of Johne... 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...
Frontiers in microbiology, 2011
Mycobacterium avium subspecies paratuberculosis (M. ap), the causative agent of Johne's disea... 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...
Frontiers in Cellular and Infection Microbiology, 2012
More than 1.8 million people die annually from infection with Mycobacterium tuberculosis, the cau... more More than 1.8 million people die annually from infection with Mycobacterium tuberculosis, the causative agent of tuberculosis. The ability of M. tuberculosis to obtain and distribute micronutrients, including biometals, is known to play a role in its intracellular survival and virulence within a host. Techniques to detect elemental distributions within M. tuberculosis cells have previously been limited to bulk detection methods or low-resolution analyses. Here, we present a method for determining the elemental distribution within M. tuberculosis on a single-cell level, at high (individual nanometer) resolution, using scanning transmission electron microscopy (STEM) in concert with energy-dispersive X-ray spectroscopy (EDS). Results revealed the presence of large polyphosphate granules in all strains of Mycobacteria tested. These persisted even through starvation conditions, and might play a role connected to elemental homeostasis in M. tuberculosis. Associated with the polyphosphate granules were micronutrients such as calcium and magnesium. In addition, we expanded the technique beyond Mycobacteria to show that STEM and EDS could be used as a simple screen to detect the presence or absence of concentrated elements on a single-cell level within all six other bacterial types tested, with minimal processing to the bacteria. Overall, we believe that this technique represents a first step in developing a better understanding of the role that components of the intracellular milieu, including polyphosphates and biometals, play in the pathogenesis of M. tuberculosis, with potential future applications for in vivo analysis.
Molecular Microbiology, 2010
Copper is a required micronutrient that is also toxic at excess concentrations. Currently, little... more Copper is a required micronutrient that is also toxic at excess concentrations. Currently, little is known about the role of copper in interactions between bacterial pathogens and their human hosts. In this study, we elucidate a mechanism for copper homeostasis in the human pathogen Mycobacterium tuberculosis via characterization of a putative copper exporter, CtpV. CtpV was shown to be required by M. tuberculosis to maintain resistance to copper toxicity. Furthermore, the deletion of ctpV resulted in a 98-gene transcriptional response which elucidates the increased stress experienced by the bacteria in the absence of this detoxification mechanism. Interestingly, although the ΔctpV mutant survives close to the wild-type levels in both murine and guinea pig models of tuberculosis, animals infected with the ΔctpV mutant displayed decreased lung damage, and mutant-infected mice had a reduced immune response to the bacteria as well as a significant increase in survival time relative to mice infected with wild-type M. tuberculosis. Overall, our study provides the first evidence for a connection between bacterial copper response and the virulence of M. tuberculosis, supporting the hypothesis that copper response could be important to intracellular pathogens, in general.
Microbiology, 2015
Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is the causative agent of p... 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.
PLoS ONE, 2014
The Gram-negative saprophytic bacterium Burkholderia pseudomallei is the causative agent of melio... more The Gram-negative saprophytic bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a severe infectious disease of both humans and animals. Severity of the disease is thought to be dependent on both the health status of the host, including diabetes mellitus and kidney disease, and bacterial-derived factors. To identify the bacterial factors important during an acute infection, gene expression profiles in the spleen, lung, and liver of BALB/c (Th2 prototype) and C57BL/6 mice (Th1 prototype) were determined using DNA microarrays. This analysis identified BPSS1521 (bprD), a predicted transcriptional regulator located in the type III secretion system (T3SS-3) operon, to be up regulated, specifically in C57BL/6 mice. BALB/c mice infected with a bprD mutant showed a shorter time to death and increased inflammation, as determined by histopathological analysis and enumeration of bacteria in the spleen. Elevated numbers of multinucleated giant cells (MNGCs), which is the hallmark of melioidosis, were detected in both the wild-type and the bprD mutants; a similar elevation occurs in melioidosis patients. One striking observation was the increased expression of BPSS1520 (bprC), located downstream of bprD, in the bprD mutant. BprC is a regulator of T6SS-1 that is required for the virulence of B. pseudomallei in murine infection models. Deletion of bprD led to the overexpression of bprC and a decreased time to death. bprD expression was elevated in C57BL/6 -as compared to BALB/c-mice, suggesting a role for BprD in the natural resistance of C57BL/6 mice to B. pseudomallei. Ultimately, this analysis using mice with different immune backgrounds may enhance our understanding of the outcomes of infection in a variety of models.
Frontiers in Cellular and Infection Microbiology, 2014
Johne's disease (JD) caused by Mycobacterium avium subspecies paratuberculosis (MAP) is a major t... more Johne's disease (JD) caused by Mycobacterium avium subspecies paratuberculosis (MAP) is a major threat to the dairy industry and possibly some cases of Crohn's disease in humans. A MAP vaccine that reduced of clinical disease and/or reduced fecal shedding would aid in the control of JD. The objectives of this study were (1) to evaluate the efficacy of 5 attenuated strains of MAP as vaccine candidates compared to a commercial control vaccine using the protocol proposed by the Johne's Disease Integrated Program (JDIP) Animal Model Standardization Committee (AMSC), and (2) to validate the AMSC Johne's disease goat challenge model. Eighty goat kids were vaccinated orally twice at 8 and 10 weeks of age with an experimental vaccine or once subcutaneously at 8 weeks with Silirum® (Zoetis), or a sham control oral vaccine at 8 and 10 weeks. Kids were challenged orally with a total of approximately 1.44 × 10 9 CFU divided in two consecutive daily doses using MAP ATCC-700535 (K10-like bovine isolate). All kids were necropsied at 13 months post-challenge. Results indicated that the AMSC goat challenge model is a highly efficient and valid model for JD challenge studies. None of the experimental or control vaccines evaluated prevented MAP infection or eliminated fecal shedding, although the 329 vaccine lowered the incidence of infection, fecal shedding, tissue colonization and reduced lesion scores, but less than the control vaccine. Based on our results the relative performance ranking of the experimental live-attenuated vaccines evaluated, the 329 vaccine was the best performer, followed by the 318 vaccine, then 316 vaccine, 315 vaccine and finally the 319 vaccine was the worst performer. The subcutaneously injected control vaccine outperformed the orally-delivered mutant vaccine candidates. Two vaccines (329 and 318) do reduce presence of JD gross and microscopic lesions, slow progression of disease, and one vaccine (329) reduced fecal shedding and tissue colonization.
Frontiers in Cellular and Infection Microbiology, 2014
Frontiers in Cellular and Infection Microbiology, 2014
Johne's disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), which results in ... more Johne's disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), which results in serious economic losses worldwide in farmed livestock such as cattle, sheep, and goats. To control this disease, an effective vaccine with minimal adverse effects is needed. In order to identify a live vaccine for Johne's disease, we evaluated eight attenuated mutant strains of MAP using a C57BL/6 mouse model. The persistence of the vaccine candidates was measured at 6, 12, and 18 weeks post vaccination. Only strains 320, 321, and 329 colonized both the liver and spleens up until the 12-week time point. The remaining five mutants showed no survival in those tissues, indicating their complete attenuation in the mouse model. The candidate vaccine strains demonstrated different levels of protection based on colonization of the challenge strain in liver and spleen tissues at 12 and 18 weeks post vaccination. Based on total MAP burden in both tissues at both time points, strain 315 (MAP1566::Tn5370) was the most protective whereas strain 318 (intergenic Tn5367 insertion between MAP0282c and MAP0283c) had the most colonization. Mice vaccinated with an undiluted commercial vaccine preparation displayed the highest bacterial burden as well as enlarged spleens indicative of a strong infection. Selected vaccine strains that showed promise in the mouse model were moved forward into a goat challenge model. The results suggest that the mouse trial, as conducted, may have a relatively poor predictive value for protection in a ruminant host such as goats.
Frontiers in Cellular and Infection Microbiology, 2014
Since the early 1980s, several investigations have focused on developing a vaccine against Mycoba... more Since the early 1980s, several investigations have focused on developing a vaccine against Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease in cattle and sheep. These studies used whole-cell inactivated vaccines that have proven useful in limiting disease progression, but have not prevented infection. In contrast, modified live vaccines that invoke a Th1 type immune response, may improve protection against infection. Spurred by recent advances in the ability to create defined knockouts in MAP, several independent laboratories have developed modified live vaccine candidates by transpositional mutation of virulence and metabolic genes in MAP. In order to accelerate the process of identification and comparative evaluation of the most promising modified live MAP vaccine candidates, members of a multi-institutional USDA-funded research consortium, the Johne's disease integrated program (JDIP), met to establish a standardized testing platform using agreed upon protocols. A total of 22 candidates vaccine strains developed in five independent laboratories in the United States and New Zealand voluntarily entered into a double blind stage gated trial pipeline. In Phase I, the survival characteristics of each candidate were determined in bovine macrophages. Attenuated strains moved to Phase II, where tissue colonization of C57/BL6 mice were evaluated in a challenge model. In Phase III, five promising candidates from Phase I and II were evaluated for their ability to reduce fecal shedding, tissue colonization and pathology in a baby goat challenge model. Formation of a multi-institutional consortium for vaccine strain evaluation has revealed insights for the implementation of vaccine trials for Johne's disease and other animal pathogens. We conclude by suggesting the best way forward based on this 3-phase trial experience and challenge the rationale for use of a macrophage-to-mouse-to native host pipeline for MAP vaccine development.
Veterinary Microbiology, 2007
An international committee of Johne's disease (JD) researchers was convened to develop guidelines... more An international committee of Johne's disease (JD) researchers was convened to develop guidelines for JD challenge studies in multiple animal species. The intent was to develop and propose international standard guidelines for models based on animal www.elsevier.com/locate/vetmic Veterinary Microbiology 122 (2007) 197-222 species that would gain acceptance worldwide. Parameters essential for the development of long-term and short-term infection models were outlined and harmonized to provide a ''best fit'' JD challenge model for cattle, goats, sheep, cervids, and mice. These models will be useful to study host-pathogen interactions, host immunity at the local and systemic level, and for evaluating vaccine candidates and therapeutics. The consensus guidelines herein list by animal species strains of Mycobacterium avium subsp. paratuberculosis used, challenge dose, dose frequency, age of challenge, route of challenge, preparation of inoculum, experimental animal selection, quality control, minimal experimental endpoints and other parameters. #
Veterinary Microbiology, 1999
Despite the ubiquitous presence of atypical mycobacteria in the environment and the potential ris... more Despite the ubiquitous presence of atypical mycobacteria in the environment and the potential risk of infection in humans and animals, the pathogenesis of diseases caused by infection with atypical mycobacteria has been poorly characterized. In this study, goldfish, Carassius auratus were infected either with the rapidly growing fish pathogen, Mycobacterium fortuitum or with another rapidly growing mycobacteria, Mycobacterium smegmatis. Bacterial persistence and pathological host response to mycobacterial infection in the goldfish are described. Mycobacteria were recovered from a high percentage of inoculated fish that developed a characteristic chronic granulomatous response similar to that associated with natural mycobacterial infection. Both M. fortuitum and M. smegmatis were pathogenic to fish. Fish infected with M. smegmatis ATCC 19420 showed the highest level of giant cell recruitment compared to fish inoculated with M. smegmatis mc 2 155 and M. fortuitum. Of the three strains of mycobacteria examined, M. smegmatis ATCC 19420 was the most virulent strain to goldfish followed by M. fortuitum and M. smegmatis mc 2 155, respectively. #
Protein Expression and Purification, 2007
Fourteen proteins of potential diagnostic value for bovine paratuberculosis were identified in th... 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.