Host induced microevolution of ESX secretion systems of M. Tuberculosis.
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Date
2013
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Abstract
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.
Description
Thesis (Ph.D.)-University of University of Natal, Durban, 2013.
Keywords
Mycobacterium tuberculosis., Immune response., Vaccines., Theses--Medical microbiology.