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EXPRESSION AND CHARACTERIZATION OF MYCOBACTERIUM PARATUBERCULOSIS 19KDA WITH POSTTRANSLATIONAL MODIFICATION
Title
EXPRESSION AND CHARACTERIZATION OF MYCOBACTERIUM PARATUBERCULOSIS 19KDA WITH POSTTRANSLATIONAL MODIFICATION.
Creator
Safavi-Khasraghi, Mitra, Naser, Saleh, University of Central Florida
Abstract / Description
Despite the fact that E. coli supports limited posttranslational modification, this bacterium has been universally used as the expression system of choice. Expression of modified proteins in E. coli may lead to expression of recombinant proteins that lack essential immunomodulatory or catalytic components essentials for infectious processes. Previously in our laboratory, pMptb#28 plasmid containing a 4.8 kb insert from M. paratuberculosis has been identified which expressed 16 kDa recombinant...
Show moreDespite the fact that E. coli supports limited posttranslational modification, this bacterium has been universally used as the expression system of choice. Expression of modified proteins in E. coli may lead to expression of recombinant proteins that lack essential immunomodulatory or catalytic components essentials for infectious processes. Previously in our laboratory, pMptb#28 plasmid containing a 4.8 kb insert from M. paratuberculosis has been identified which expressed 16 kDa recombinant protein in E. coli and 19 kDa recombinant protein in Mycobacterium smegmatis. The objective of this study is to identify the ORF sequence, investigate possible posttranslational modification and characterize the protein forms in the two hosts. Earlier in the study, the genome sequence for M. paratuberculosis was not available and therefore sequencing both the 5' and 3' ends of the 4.8 kb insert did not help in the identification of the ORF. However, unidirectional Exonuclease deletion resulted in identification of subclones containing possible ORF sequence. Later on, the publication of the M. paratuberculosis genome sequence along with BLAST analysis of sequences from the subclones resulted in the identification of 486 bp ORF with significant identity to that from M. tuberculosis and M. leprae. Cloning of the 486 ORF coding sequence in E. coli resulted in the expression of 16 kDa protein similar to the calculated predicted size of translated peptide. Cloning of the 486 bp ORF coding sequence in M. smegmatis resulted in the expression of 19 kDa protein similar to that from M. paratuberculosis. The 16/19 kDa forms of the same protein were verified using rabbit anti-M. paratuberculosis antibodies adsorbed in E. coli and M. smegmatis lysates. The size of the 19 kDa proteins was not reduced following treatment with deglycosylation enzymes in absence of any enzyme inhibitors. The 19 kDa product was confirmed not be a glycoprotein when failed to react with ConA stain. The 16/19 kDa forms of the protein were evaluated against T-lymphocytes from Crohn's disease patients and normal controls. T- proliferation assay included controls such as PHA and PPD from M. paratuberculosis. There was not a significant difference between the two forms of the protein (16/19 kDa) against T-cell response from both populations. Overall, the study identified the ORF of the 19 kDa non-glycoprotein from M. paratuberculosis. Moreover, this is the first study which reports that the zoonotic M. paratuberculosis supports posttranslational modification similar to M. tuberculosis and M. leprae pathogens. Although the posttranslational modification component in this 19 kDa nonglycoprotein did not affect T- cell response, the finding is significant toward glycoproteins from M. paratuberculosis and their role in the pathogenesis of this bacterial infection in animals and humans.
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Date Issued
2006
Identifier
CFE0001170, ucf:52851
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0001170
Evaluation of Intestinal Microbial Diversity and a New Antibiotic Regimen in Crohn's Disease Patients
Title
Evaluation of Intestinal Microbial Diversity and a New Antibiotic Regimen in Crohn's Disease Patients.
Creator
Alcedo, Karel, Naser, Saleh, Cheng, Zixi, Siddiqi, Shadab, University of Central Florida
Abstract / Description
Crohn's disease (CD) is a chronic granulomatous inflammatory bowel disease involving Mycobacterium avium subspecies paratuberculosis (MAP). Other microorganisms such as adherent-invasive Escherichia coli (AIEC) have also been proposed in CD association. To date, only one study investigated both MAP and AIEC simultaneously using peripheral blood but not in affected intestinal tissues. A standardized and effective antibiotic therapy against MAP and/or AIEC is needed for better treatment. Three...
Show moreCrohn's disease (CD) is a chronic granulomatous inflammatory bowel disease involving Mycobacterium avium subspecies paratuberculosis (MAP). Other microorganisms such as adherent-invasive Escherichia coli (AIEC) have also been proposed in CD association. To date, only one study investigated both MAP and AIEC simultaneously using peripheral blood but not in affected intestinal tissues. A standardized and effective antibiotic therapy against MAP and/or AIEC is needed for better treatment. Three antibiotic drugs (-) Clarithromycin (CLA), Rifabutin (RIF), and Clofazimine (CLO) have been used to treat CD patients suspected with MAP infection. However, the outcome has been controversial. The treatment dosage is high, the duration is long, and the reported drug side effects resulted in patient non-compliance; therefore, a lower and effective drug dosage is needed. In this study, we developed two aims 1) to evaluate RHB 104, a drug formula comprised of low dosages of CLA, RIF, and CLO, against clinical MAP strains in-vitro using fluorescence quenching method, and 2) to develop a fluorescence in-situ hybridization method to detect both MAP and AIEC simultaneously in intestinal tissues of CD patients. A total of 16 clinical MAP strains and 19 non-MAP strains were tested against varied concentrations of RHB 104, CLA, RIF, and CLO. Although the MIC for all drugs ranged between 0.5-20 ?g/ml, the MIC for RHB 104 was significantly lower against most MAP strains. The effect of RHB 104 against MAP was bactericidal. Unlike RHB-104 formula, CLA, CLO, and RIF dosage similar to those in RHB-104 did not inhibit MAP growth when trialed individually and in dual-drug combinations. The data illustrated the presence of synergistic anti-MAP activity of low dosage of the three antibiotics in RHB-104. We also developed a rapid and sensitive multicolor in-situ hybridization technique that can detect MAP and AIEC using tagged-oligonucleotide probes. Non-pathogenic Escherichia coli (npEC) was used as a control for the study. Specifically, cultured MAP and npEC were fixed and hybridized with MAP488 and EC647 probes, respectively. Confocal laser scanning microscope (CLSM) revealed specific signals at 488nm for MAP and 647nm for npEC, indicating probe binding to each bacteria. This was confirmed with hybridization of MAP with EC647 and npEC with MAP488 resulting in absence of signals. Intestinal tissue samples from 9 CD patients were then analyzed using our technique. Preliminary data indicated positive results in 6/6 samples for MAP, 6/6 for npEC, 3/3 for AIEC, and 2/2 for both MAP and AIEC with MAP being more dominant. This protocol shortened the FISH procedure from multiple days to short-hours. The protocol allows the investigation of more than one pathogen simultaneously in the same clinical sample. A quantitative measurement of the signals is needed.
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Date Issued
2015
Identifier
CFE0005917, ucf:50831
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005917
Dubious role of Mycobacterium paratuberculosis in pathogenesis of Type I diabetes
Title
Dubious role of Mycobacterium paratuberculosis in pathogenesis of Type I diabetes.
Creator
Thanigachalam, Saisathya, Naser, Saleh, Singla, Dinender, Siddiqi, Shadab, Jewett, Travis, University of Central Florida
Abstract / Description
INTRODUCTION: Type 1 Diabetes Mellitus (T1DM) is a chronic disorder with unknown etiology and associated with insulin deficiency. Mycobacterium avium subsp. paratuberculosis (MAP), the etiologic agent of paratuberculosis in cattle, has been implicated in many autoimmune diseases including Crohn's disease, TIDM and others. We hypothesize that the molecular mimicry including epitope homology between MAP-Hsp65 and pancreatic Glutamic Acid Decarboxylase65 (GAD65) may play a role in the auto...
Show moreINTRODUCTION: Type 1 Diabetes Mellitus (T1DM) is a chronic disorder with unknown etiology and associated with insulin deficiency. Mycobacterium avium subsp. paratuberculosis (MAP), the etiologic agent of paratuberculosis in cattle, has been implicated in many autoimmune diseases including Crohn's disease, TIDM and others. We hypothesize that the molecular mimicry including epitope homology between MAP-Hsp65 and pancreatic Glutamic Acid Decarboxylase65 (GAD65) may play a role in the auto destruction of pancreatic beta cells leading to insufficient insulin production and the development of TIDM, following exposure to MAP. METHODOLOGY: Peptide sequences of MAP-Hsp65 and GAD65 were analyzed using BLAST and PyMOL bioinformatics tools. Cross reactivity between the two proteins were evaluated using immunoblot and ELISA. Furthermore, coded EDTA blood samples were collected from 18 subjects (12 DM and 6 controls) and investigated for the presence or exposure to MAP. Peripheral leukocytes were investigated for harboring viable MAP using long-term culture followed by nested PCR. Clinical plasma samples were used for measurement of anti-MAP IgG titer as well as glucose and Insulin concentrations. Moreover, coded bovine sera from 100 cattle (50 MAP infected and 50 healthy) were investigated for possible correlation between MAP infection and plasma levels of glucose and insulin. RESULT: Peptide BLAST analysis revealed a 44% identity between MAP Hsp65 and GAD65 proteins with 75% positive identities in a 16 amino acid region. PyMOL 3-D structural analyses identified a shared epitope region within the 16 amino acid motif which is known to be an antigenic site on GAD65 antigen. MAP DNA and anti-MAP IgG were detected in the blood of TD8, a TIDM subject. Strong cross reactivity was observed between plasma from TD8 and MAP Hsp65 in proteins samples from M. tuberculosis, and E. coli recombinant clone expressing MAP Hsp65. A weak cross reactivity was also observed between rat pancreatic tissue homogenate and rabbit anti-MAP IgG. Long term culture of leukocytes from 18 blood samples resulted in the detection of MAP in 3/10 (30%) TIDM and 4/8 (50%) control subjects whereas anti-MAP IgG were detected in 5/10 (50%) TIDM samples compared to 3/8 (37.5 %) controls. In MAP infected cattle, insulin level ranged from below 0.1ng/ml to 2.456 ng/ml with an average of 0.36 +/- 0.57ng/ml compared to 0.1ng/ml to 13.47ng/ml with an average of 2.86 +/- 3.00ng/ml in healthy cattle (P(<)0.0001). CONCLUSION: We identified and confirmed a shared epitope region between MAP Hsp65 and human pancreatic GAD65. The shared epitope is a known antigenic binding site. Although MAP DNA was detected in both TIDM and control subjects, a strong correlation was found between anti-MAP IgG titer and MAP-positive culture in clinical samples, regardless of diagnosis. The correlation between MAP infection and insulin level in cattle is significant. Overall the result is intriguing and requires further investigation of MAP in well-characterized clinical samples.
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Date Issued
2012
Identifier
CFE0004608, ucf:49924
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0004608
MOLECULAR TYPING OF MYCOBACTERIAL ISOLATES CULTURED FROM THE TISSUE OF INFLAMMATORY BOWEL DISEASE (CROHN'S DISEASE) PATIENTS
Title
MOLECULAR TYPING OF MYCOBACTERIAL ISOLATES CULTURED FROM THE TISSUE OF INFLAMMATORY BOWEL DISEASE (CROHN'S DISEASE) PATIENTS.
Creator
Adams, Leanne M, Naser, Saleh, University of Central Florida
Abstract / Description
The role of Mycobacterium avium subsp paratuberculosis (MAP) in the etiology and pathogenesis of inflammatory bowel disease (IBD) including Crohn's Disease (CD), has been investigated. The fastidious characteristics and cross reactivity of MAP with other members in Mycobacteria have produced significant challenges in their detection and identification. In this two year pilot study, an array of three PCR molecular assays based on the detection of sequences from the16S rRNA, IS1245, and IS900...
Show moreThe role of Mycobacterium avium subsp paratuberculosis (MAP) in the etiology and pathogenesis of inflammatory bowel disease (IBD) including Crohn's Disease (CD), has been investigated. The fastidious characteristics and cross reactivity of MAP with other members in Mycobacteria have produced significant challenges in their detection and identification. In this two year pilot study, an array of three PCR molecular assays based on the detection of sequences from the16S rRNA, IS1245, and IS900 genes, belonging to members of the MAC, have been developed and optimized into a common protocol to be used as a rapid and accurate diagnostic tool regarding M. avium complex (MAC) infection. The PCR protocol time was reduced by half, and the sensitivity and specificity of the molecular assays has been significantly improved barring the need for southern hybridization. This improved methodology was employed for the molecular typing of MAC in 100 resected, full-thickness tissue samples removed from IBD patients. The tissue samples were homogenized, decontaminated, and inoculated into two mycobacterial culture media systems. A total of 328 Bactec and Mycobacteria Growth Indicator Tube (MIGT) cultures were evaluated for positive MAC growth. Harvested cells were then subjected to genomic DNA extraction and subsequent PCR typing. The I6 S rRNA-based PCR resulted in detection of 26/28 (93%) MAC in Bactec cultures. Specifically, 25/28 (89%) of positive MAC indicated the presence of IS1245 specific to M. avium subsp avium (MAV), and 6/28 (21%) produced results consistent with the presence of IS900 following nested PCR. Moreover, 20/100 (20%) of MGIT cultures were positive for MAP. Sequence analysis was performed on amplified regions of the IS900 element from seven isolates. A nucleotide alignment revealed that 2/7 isolates demonstrated 100% homology to Bovine MAP and 5/7 isolates showed 96-99% homology to sequenced Bovine MAP published in GenBank. The detection of at least two Bovine derived MAP in IBD tissue will have great impact on the epidemiology and reclassification of IBD. The significant homology of the other five isolates to Bovine derived MAP suggests a diversity in the geographical distribution of MAP regarding Johne's disease and CD. Ultimately, the etiology, diagnosis, and the treatment of IBD as well as control and prevention measures may be enhanced with better tools for investigating emerging infectious diseases.
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Date Issued
2004
Identifier
CFE0000031, ucf:46125
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0000031
Role of Single Nucleotide Polymorphisms (SNPs) in PTPN2/22 and Mycobacterium avium subspecies paratuberculosis (MAP) in Rheumatoid Arthritis and Crohn's Disease
Title
Role of Single Nucleotide Polymorphisms (SNPs) in PTPN2/22 and Mycobacterium avium subspecies paratuberculosis (MAP) in Rheumatoid Arthritis and Crohn's Disease.
Creator
Sharp, Robert, Naser, Saleh, Parks, Griffith, Roy, Herve, Singla, Dinender, University of Central Florida
Abstract / Description
Both genetic pre-disposition and potential environmental triggers are shared between Rheumatoid arthritis (RA) and Crohn's disease (CD). We hypothesized that single nucleotide polymorphisms (SNPs) in the negative T-cell regulators Protein Tyrosine Phosphatase Non-receptor type 2 and 22 (PTPN2/22) lead to a dysregulated immune response as seen in RA and CD. To test the hypothesis, peripheral leukocytes samples from 204 consented subjects were TaqMan genotyped for 9 SNPs in PTPN2/22. The SNPs...
Show moreBoth genetic pre-disposition and potential environmental triggers are shared between Rheumatoid arthritis (RA) and Crohn's disease (CD). We hypothesized that single nucleotide polymorphisms (SNPs) in the negative T-cell regulators Protein Tyrosine Phosphatase Non-receptor type 2 and 22 (PTPN2/22) lead to a dysregulated immune response as seen in RA and CD. To test the hypothesis, peripheral leukocytes samples from 204 consented subjects were TaqMan genotyped for 9 SNPs in PTPN2/22. The SNPs effect on PTPN2/22 and IFN-y expression was determined using RT-PCR. Blood samples were analyzed for the Mycobacterium avium subspecies paratuberculosis (MAP) IS900 gene by nPCR. T-cell proliferation and response to phytohematoagglutonin (PHA) mitogen and MAP cell lysate were determined by BrdU proliferation assay. Out of 9 SNPs, SNP alleles of PTPN2:rs478582 occurred in 79% RA compared to 60% control (p-values ? 0.05). SNP alleles of PTPN22:rs2476601 occurred in 29% RA compared to 6% control (p-values ? 0.05). For the haplotype combination of PTPN2:rs478582/PTPN22rs2476601, 21.4% RA had both SNPs (C-A) compared to 2.4% control (p-values ? 0.05). PTPN2/22 expression in RA was decreased by an average of 1.2 fold. PTPN2:rs478582 upregulated IFN-y in RA by an average of 1.5 fold. Combined PTPN2:rs478582/PTPN22:rs2476601 increased T-cell proliferation by an average of 2.7 fold when treated with PHA. MAP DNA was detected in 34% RA compared to 8% controls (p-values ? 0.05), where samples with PTPN2:rs478582 and/or PTPN22:rs2476601 were more MAP positive. PTPN2:rs478582/PTPN22:rs2476601 together with MAP infection significantly increased T-cell response and IFN-y expression in RA samples. The same experimental approach was followed on blood samples from CD patients. Both PTPN2:rs478582/PTPN22:rs2476601 affected PTPN2/22 and IFN-y expression along with T-cell proliferation significantly more than in RA. MAP DNA was detected in 64% of CD. This is the first study to report the correlation between SNPs in PTPN2/22, IFN-y expression and MAP in autoimmune disease.
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Date Issued
2018
Identifier
CFE0007371, ucf:52094
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0007371
Downregulation in IFNGR1 increases suspectiblity to Mycobacterium avium subspecies paratuberculosis infection in Crohn's disease
Title
Downregulation in IFNGR1 increases suspectiblity to Mycobacterium avium subspecies paratuberculosis infection in Crohn's disease.
Creator
Htun, Zin Mar, Naser, Saleh, Andl, Claudia, Tigno-Aranjuez, Justine, University of Central Florida
Abstract / Description
BACKGROUND: Crohn's disease (CD) is an inflammatory bowel disease (IBD) and has been associated with Mycobacterium avium subspecies paratuberculosis (MAP). MAP has been detected in stool, tissue and blood samples from patients with CD. Gamma interferon (?-IFN) is an inflammatory cytokine that plays a crucial role in killing intracellular pathogens like MAP, and its receptor (IFNGR1) mutations cause immunodeficiency and severe disseminated mycobacterial infections. The role of MAP in...
Show moreBACKGROUND: Crohn's disease (CD) is an inflammatory bowel disease (IBD) and has been associated with Mycobacterium avium subspecies paratuberculosis (MAP). MAP has been detected in stool, tissue and blood samples from patients with CD. Gamma interferon (?-IFN) is an inflammatory cytokine that plays a crucial role in killing intracellular pathogens like MAP, and its receptor (IFNGR1) mutations cause immunodeficiency and severe disseminated mycobacterial infections. The role of MAP in association with IFNGR1 mutation in CD patients have not been investigated.METHODS: In this study, we investigated blood samples of 79 human subjects for MAP infection in association with IFNGR1 gene dysfunction. Samples were divided into 22 CD, 6 Ulcerative colitis (UC), 32 normal healthy and 19 non-inflammatory bowel disease (NIBD). Five variants of IFNGR1 single nucleotide polymorphisms (SNP) were investigated using Taqman Genotyping assay, then IFNGR1 expression measured by RT-PCR and serum IFNGR1 and ?-IFN levels were measured using ELISA. MAP infection was detected using nested PCR. RESULTS: Among 28 IBD patients, 4/6 (66.67%) of UC and 18/22 (81.82%) of CD are tested positive for at least one SNP homozygous minor form compared to 21.88% and 47.37%% in 32 healthy and 19 NIBD (P (<)0.05). IFNGR1 gene expression was downregulated 1.4-fold in IBD patients (P =0.07) and 1.7-fold downregulated in MAP positive IBD patients compared to MAP negative IBD patients (P=0.06). Serum IFNGR1 protein levels were downregulated 1.53-fold in IBD patients compared to normal, and 1.4-fold downregulated in MAP positive IBD patients compared to MAP negative IBD patients. MAP infection is more common in rs2234711 SNP positive patients (5/7 =71.42%) (P(<)0.05). Serum ?-IFN levels were not elevated in both groups.CONCLUSION: IFNGR1 SNP's, MAP infection and IFNGR1 downregulation were found in higher incidence in IBD, suggesting role of IFNGR1 in susceptibility of MAP infection in IBD patients.
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Date Issued
2017
Identifier
CFE0007121, ucf:51951
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0007121