Browsing by Author "Sukkhu, Melisha."

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Autolytic characterization of selected Enterococcal strains, (previously Streptococcal)
(2007) Sukkhu, Melisha.; Beukes, Mervyn.
Autolysins are enzymes that cleave specific structural components within the bacterial cell wall. They contribute to numerous cellular activities such as cell growth, cell division, peptidoglycan recycling and turnover. In this study, twelve Enterococcal isolates (previously from the genus Streptococcus) were examined for susceptibility to the antibiotics Penicillin G and Vancomycin, using a Disk Diffusion and a Microtitre plate assay. In both methods, all twelve strains were resistant to Vancomycin. Six of these strains were susceptible to Penicillin G. The minimum inhibitory concentration (MIC) values were twice that of the disk diffusion assay values. In the presence of antibiotic, the growth rates for the six strains were halved. Autolysins were extracted from the respective cell cultures using a 4% SDS precipitation method. The protein concentrations were calculated and estimated to be within the range of 5.47- to 6.35 μg/μl. Profiles of the SDS precipitate were analyzed on SDS-PAGE. Autolytic proteins were identified and partially analyzed by renaturing SDS-PAGE (zymograms) using gels containing cell wall substrate. Seven lytic bands of molecular weights 25, 30, 50, 63, 75 95 and 145 kDa (designated Autolysin A to G, respectively) were selected for further analysis. The temporal distribution of the enzymes ranged from the mid exponential phase to the early death phase. The seven proteins were blotted onto polyvinylidene difluoride (PVDF) membranes and excised for N-terminal sequencing. Blast analysis of the respective N-terminal sequences showed autolysins A, C, D, E and F to have 100% similarity to the muramidase, amidase and peptidase from S. cremoris, S. suis, S. pneumonia, S. pyogenes and E. faecium, respectively. Biochemical characterization confirmed autolysins A, B, E and F to exhibit muramidase activity, and autolysin C and G to exhibit peptidase activity. Autolysin D displayed 100% similarity to the protein LytA, a peptidoglycan hydrolase that is known to exhibit amidase activity. Blast analysis could not determine any significant similarities for autolysins B and G to previously identified autolysins, thus indicating they may perhaps be novel autolysins.
Host induced microevolution of ESX secretion systems of M. Tuberculosis.
(2013) Sukkhu, Melisha.; Pym, Alexander S.
The ESX family of genes (esxA-W) in Mycobacterium tuberculosis (Mtb) encodes 23 effector molecules influencing immunogenicity and pathogenicity. This study was aimed at identifying and evaluating variations in ESX sequence and protein expression profiles in clinical isolates and examining how diversity might influence immune responses. 23 ESX genes from 55 clinical isolates (20 Beijing, 25 KZN and 10 Other) and 3 Laboratory strains (H37Rv, H37Ra and BCG) were sequenced. 482 single nucleotide polymorphisms (SNPs) were identified in 12 ESX genes relative to H37Rv. Majority of the identified 363 nsSNPs occured in Beijing isolates. No mutations were observed in esxA, B, C, E, G, H, J, R, S and T. Six unique nsSNPs were identified in the Beijing isolates: esxI (Q20L), esxO (E52G), 2 in esxP (T3S; N83D), esxU (P63S) and esxW (T2A). Three unique nsSNPs were identified in the KZN isolates: esxK (A58T), esxL (R33S). The esxL polymorphism resulted from a dinucleotide change. ESX gene transcription levels were evaluated using RT-qPCR. Varying expression levels were observed for esxA, B, C, F, M and Q across all clinical isolates with lowest levels seen amongst the Beijing isolates. This correlated with immunoblots with confirmed decreased esxAB protein expression relative to the other strains. The Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) spectral protein profiles were quantitatively compared within and between Mtb clinical and laboratory isolates. Protein spectral profiles within the mass range of the CFP-10 protein with variations in peak intensities were observed across all isolates. QILSS and Mtb9.9 peptides were tested individually for immune responses in TB infected patients. Healthy patients displayed no responses to QILSS and Mtb9.9, strong but variable immune responses were detected for specific regions of QILSS and Mtb9.9 in TB infected patients. These findings demonstrate that differences in sequence, transcriptional profiles and protein expression patterns in ESX secreted proteins exist between clinical isolates, and may translate into differences in human immune responses. Further research is needed to correlate human host immune responses to the phenotype and genotype of the infecting strain of Mtb to determine the consequences of specific variations of the other ESX members. These studies are important for the development of improved immune diagnostics and vaccines.