Abstract: Rv3873 is a relatively large size protein (371 amino acids in length) and its gene is located in the immunodominant genomic region of difference (RD)1 that is present in the genome of Mycobacterium tuberculosis but deleted from the genomes of all the vaccine strains of Bacillus Calmette Guerin (BCG) and most other mycobacteria. However, when tested for cellular immune responses using peripheral blood mononuclear cells from tuberculosis patients and BCG-vaccinated healthy subjects, this protein was found to be a major stimulator of cell mediated immune responses in both groups of subjects. In order to further identify the sequence of immunodominant epitopes and explore their Human Leukocyte Antigen (HLA)-restriction for epitope recognition, 24 peptides (25-mers overlapping with the neighboring peptides by 10 residues) covering the sequence of Rv3873 were synthesized chemically using fluorenylmethyloxycarbonyl chemistry and tested in cell mediated immune responses. The results of these experiments helped in the identification of an immunodominant peptide P9 that was recognized by people expressing varying HLA-DR types. Furthermore, it was also predicted to be a promiscuous binder with multiple epitopes for binding to HLA-DR, HLA-DP and HLA-DQ alleles of HLA-class II molecules that present antigens to T helper cells, and to HLA-class I molecules that present antigens to T cytotoxic cells. In addition, the evaluation of peptide P9 using an immunogenicity predictor server yielded a high score (0.94), which indicated a greater probability of this peptide to elicit a protective cellular immune response. In conclusion, P9, a peptide with multiple epitopes and ability to bind several HLA class I and class II molecules for presentation to cells of the cellular immune response, may be useful as a peptide-based vaccine against tuberculosis.
Abstract: The comparisons of mycobacterial genomes have identified several Mycobacterium tuberculosis-specific genomic regions that are absent in other mycobacteria and are known as regions of differences. Due to M. tuberculosis-specificity, the peptides encoded by these regions could be useful in the specific diagnosis of tuberculosis. To explore this possibility, overlapping synthetic peptides corresponding to 39 proteins predicted to be encoded by genes present in regions of differences were tested for antibody-reactivity with sera from tuberculosis patients and healthy subjects. The results identified four immunodominant peptides corresponding to four different proteins, with three of the peptides showing significantly stronger antibody reactivity and rate of positivity with sera from tuberculosis patients than healthy subjects. The fourth peptide was recognized equally well by the sera of tuberculosis patients as well as healthy subjects. Predication of antibody epitopes by bioinformatics analyses using ABCpred server predicted multiple linear epitopes in each peptide. Furthermore, peptide sequence analysis for sequence identity using BLAST suggested M. tuberculosis-specificity for the three peptides that had preferential reactivity with sera from tuberculosis patients, but the peptide with equal reactivity with sera of TB patients and healthy subjects showed significant identity with sequences present in nob-tuberculous mycobacteria. The three identified M. tuberculosis-specific immunodominant peptides may be useful in the serological diagnosis of tuberculosis.
Abstract: Performing culture and characterization of mycobacteria in low resource settings like Nigeria is a very difficult task to undertake because of the very few and limited laboratories carrying out such an experiment; this is a largely due to stringent and laborious nature of the tests. Hence, a rapid, simple and accurate test for characterization is needed. The “SD BIOLINE TB Ag MPT 64 Rapid ®” is a simple and rapid immunochromatographic test used in differentiating Mycobacteria into Mycobacterium tuberculosis (NTM). The 100 sputa were obtained from patients suspected to be infected with tuberculosis and presented themselves to hospitals for check-up and treatment were involved in the study. The samples were cultured in a class III Biosafety cabinet and level III biosafety practices were followed. Forty isolates were obtained from the cultured sputa, and there were identified as Acid-fast bacilli (AFB) using Zeihl-Neelsen acid-fast stain. All the isolates (AFB positive) were then subjected to the SD BIOLINE Analyses. A total of 31 (77.5%) were characterized as MTBC, while nine (22.5%) were NTM. The total turnaround time for the rapid assay was just 30 minutes as compared to a few days of phenotypic and genotypic method. It was simple, rapid and reliable test to differentiate MTBC from NTM.
Abstract: This research work presents the surface
thermodynamics approach to M-TB/HIV-Human sputum
interactions. This involved the use of the Hamaker coefficient
concept as a surface energetics tool in determining the interaction
processes, with the surface interfacial energies explained using van
der Waals concept of particle interactions. The Lifshitz derivation for
van der Waals forces was applied as an alternative to the contact
angle approach which has been widely used in other biological
systems. The methodology involved taking sputum samples from
twenty infected persons and from twenty uninfected persons for
absorbance measurement using a digital Ultraviolet visible
Spectrophotometer. The variables required for the computations with
the Lifshitz formula were derived from the absorbance data. The
Matlab software tools were used in the mathematical analysis of the
data produced from the experiments (absorbance values). The
Hamaker constants and the combined Hamaker coefficients were
obtained using the values of the dielectric constant together with the
Lifshitz Equation. The absolute combined Hamaker coefficients
A132abs and A131abs on both infected and uninfected sputum samples
gave the values of A132abs = 0.21631x10-21Joule for M-TB infected
sputum and Ã132abs = 0.18825x10-21Joule for M-TB/HIV infected
sputum. The significance of this result is the positive value of the
absolute combined Hamaker coefficient which suggests the existence
of net positive van der waals forces demonstrating an attraction
between the bacteria and the macrophage. This however, implies that
infection can occur. It was also shown that in the presence of HIV,
the interaction energy is reduced by 13% conforming adverse effects
observed in HIV patients suffering from tuberculosis.
Abstract: Altered drug binding may be an important factor in isoniazid (INH) resistance, rather than major changes in the enzyme’s activity as a catalase or peroxidase (KatG). The identification of structural or functional defects in the mutant KatGs responsible for INH resistance remains as an area to be explored. In this connection, the differences in the binding affinity between wild-type (WT) and mutants of KatG were investigated, through the generation of three mutants of KatG, Ser315Thr [S315T], Ser315Asn [S315N], Ser315Arg [S315R] and a WT [S315]) with the help of software-MODELLER. The mutants were docked with INH using the software-GOLD. The affinity is lower for WT than mutant, suggesting the tight binding of INH with the mutant protein compared to WT type. These models provide the in silico evidence for the binding interaction of KatG with INH and implicate the basis for rationalization of INH resistance in naturally occurring KatG mutant strains of Mycobacterium tuberculosis.
Abstract: Pyrazinamide (PZA) is among the first-line pro-drugs in the tuberculosis (TB) combination chemotherapy used to treat Mycobacterium tuberculosis. Numerous reports have suggested that hepatotoxicity due to pyrazinamide in patients is due to inappropriate dosing. It is, therefore necessary to develop sensitive and reliable techniques for determining the PZA metabolic profile of diagnosed patients promptly and at point-of-care. This study reports the determination of PZA based on nanobiosensor systems developed from disuccinimidyl octanedioate modified Cytochrome P450-2E1 (CYP2E1) electrodeposited on gold substrates derivatised with (poly(8-anilino-1-napthalene sulphonic acid) PANSA/PVP-AgNPs nanocomposites. The rapid and sensitive amperometric PZA detection gave a dynamic linear range of 2µM to 16µM revealing a
limit of detection of 0.044µM and a sensitivity of 1.38µA/µM. The Michaelis-Menten parameters; KM, KM app and IMAX were calculated to be 6.0µM, 1.41µM and 1.51x10-6 A, respectively, indicating a nanobiosensor suitable for use in serum.
Abstract: Tuberculosis (TB) is a bacterial infectious disease caused by the obligate human pathogen, Mycobacterium tuberculosis. Multidrug-resistant tuberculosis (MDR-TB) is a global reality that threatens tuberculosis control. Resistance to antibiotic Rifampicin, occurs in 95% of cases through nucleotide substitutions in an 81-bp core region of the rpoB i.e; beta subunit of DNA dependant RNA polymerase. In this paper, we studied the Rifampicin-rpoB receptor interactions In silico. First, homology modeling was performed to obtain the three dimensional structure of Mycobacterium rpoB. Sixty analogs of Rifampicin were prepared using Marvin sketch software. Both original Rifampicin and the analogs were docked with rpoB and energy values were obtained. Out of sixty analogs, 43 analogs had lesser energy values than conventional Rifampicin and hence are predicted to have greater binding affinity to rpoB. Thus, this study offers a route for the development of Rifampicin analogs against multi drug resistant Mycobacterium rpoB.
Abstract: Kwashiorkor is one of nutritional problem in
Indonesia, which lead to decrease immune system. This condition
causes susceptibility to infectious disease, especially tuberculosis.
Development of new tuberculosis vaccine will be an important
strategy to eliminate tuberculosis in kwashiorkor. Previous research
showed that 38-kDa Mycobacterium tuberculosis protein is one of the
potent immunogen. However, the role of oral immunization with 38-
kDa Mycobacterium tuberculosis protein to the number of
lymphocytes in the rat model of kwashiorkor is still unknown. We
used kwashiorkor rat model groups with 4% and 2% low protein diet.
Oral immunization with 38-kDa Mycobacterium tuberculosis protein
given with 2 booster every week. The lymphocytes number were
measured by flowcytometry. There was no significant difference
between the number of lymphocytes in the normal rat group and the
kwashiorkor rat groups. It may reveal the role of 38-kDa
Mycobacterium tuberculosis protein as a potent immunogen that can
increase the lymphocytes number from kwashiorkor rat model same
as normal rat.
Abstract: A series of 1-(1H-benzimidazol-2-yl)-3-(substituted phenyl)-2-propen-1-one were allowed to react with hydrazine hydrate and phenyl hydrazine in submitted reactions to get pyrazoline and phenyl pyrazoline derivatives. All the compounds entered for screening at the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF) for their in vitro antibacterial activity against Mycobacterium tuberculosis H37Rv strain (ATCC 27294) using Microplate Alamar Blue Assay (MABA) susceptibility test. The results expressed as MIC (minimum inhibitory concentration) in μg/mL. Among the fifteen compounds, eight compounds were found to have MIC values less than 10 μg/mL. These were subjected for cytotoxicity assay in VERO cells to determine CC50 (cytotoxic concentration 50%) values and finally SI (Selectivity Index) were calculated. Compound (XV) 2-[5-(4- fluorophenyl)-1-phenyl-4,5-dihydro-1H-3-pyrazolyl]-1Hbenzimidazole was considered the best candidate of the series that could be a good starting point to develop new lead compounds in the fight against tuberculosis.
Abstract: There is an urgent need to develop novel
Mycobacterium tuberculosis (Mtb) drugs that are active against drug
resistant bacteria but, more importantly, kill persistent bacteria. Our
study structured based on integrated analysis of metabolic pathways,
small molecule screening and similarity Search in PubChem
Database. Metabolic analysis approaches based on Unified weighted
used for potent target selection. Our results suggest that pantothenate
synthetase (panC) and and 3-methyl-2-oxobutanoate hydroxymethyl
transferase (panB) as a appropriate drug targets. In our study, we
used pantothenate synthetase because of existence inhibitors. We
have reported the discovery of new antitubercular compounds
through ligand based approaches using computational tools.
Abstract: Aptamers are useful tools in microorganism
researches, diagnoses, and treatment. Aptamers are specific target
molecules formed by oligonucleic acid molecules, and are not
decomposed by alcohol. Aptamers used to detect Mycobacterium
tuberculosis (MTB) have been proved to have specific affinity to the
outer membrane proteins of MTB. This article presents a biosensor
chip set with aptamers for early detection of MTB with high specificity
and sensitivity, even in very low concentration. Meanwhile, we have
already made a modified hydrophobic facial mask module with
internal rendering hydrophobic for effectively collecting M.
Abstract: An alarming emergence of multidrug-resistant strains
of the tuberculosis pathogen Mycobacterium tuberculosis and
continuing high worldwide incidence of tuberculosis has invigorated
the search for novel drug targets. The enzyme glutamate racemase
(MurI) in bacteria catalyzes the stereoconversion of L-glutamate to
D-glutamate which is a component of the peptidoglycan cell wall of
the bacterium. The inhibitors targeted against MurI from several
bacterial species have been patented and are advocated as promising
antibacterial agents. However there are none available against MurI
from Mycobacterium tuberculosis, due to the lack of its threedimensional
structure. This work accomplished two major objectives.
First, the tertiary structure of MtMurI was deduced computationally
through homology modeling using the templates from bacterial
homologues. It is speculated that like in other Gram-positive bacteria,
MtMurI exists as a dimer and many of the protein interactions at the
dimer interface are also conserved. Second, potent candidate
inhibitors against MtMurI were identified through docking against
already known inhibitors in other organisms.