Ismail, Che Muhammad Khairul Hisyam
(2022)
Transcriptional analysis and immunological characterization of iron uptake receptors of shigella flexneri 2a USM clinical isolates.
Masters thesis, Pusat Pengajian Sains Kesihatan.
Abstract
Shigellosis is a public health concern disease with high mortality and morbidity
rate, especially among children under the age of five. Without proper care, the disease
worsens from watery stool to acute bloody diarrheal. The rise of multi-drug resistance
strain in most gram-negative bacteria including Shigella species limits the current
clinical treatment as most of the antibiotic administrated is less effective. Besides the
unavailability of commercial or approved vaccines against shigellosis disease and the
report of the predominant shift from Shigella flexneri to Shigella sonnei, the
development of an effective and long-protection vaccine is urgently needed. Most
current developed vaccines are reported to cause side effects such as dysentery and the
protection efficacy varies among vaccinated individuals at different groups of ages. In
view of this, a comprehensive understanding of molecular pathogenesis among
infected individuals is of the utmost importance. Interestingly, iron molecules have
been known to play a critical role in bacterial survival and virulent determinant by
upregulation of multiple iron uptake receptor proteins within the infected individuals
to acquire enough iron sources. In this study, both of S. flexneri 2a Hospital USM
clinical isolates (SH057 and SH062) were grown under the presence of 200 μM of 2,2-
bipyridyl. The expression of gene encoding iron uptake receptors of S. flexneri 2a,
fepA, fhuA, IutA, efeU, and SitA were observed by quantitative polymerase chain
reaction (qPCR). All iron uptake receptors of S. flexneri 2a were further analyzed by
using the bioinformatic approach to further screening the potential target vaccine candidates against shigellosis disease. The selected iron uptake receptors then
proceeded for construction of recombinant protein and predict the antigenicity of the
recombinant protein corresponding to iron uptake receptors using dot-blot and ELISA
approach. The qPCR result showed the expression rate of majority iron uptake
receptors including fepA, fhuA, IutA, efeU, and sitA of both virulent (SH057) and mild
virulent (SH062) of S. flexneri 2a clinical isolates were high under iron-depleted
condition. Furthermore, the bioinformatics analysis showed that iron uptake receptors
of FepA, FhuA, and IutA contain promising antigenic regions to elicit both humoral
as well as cell-mediated immune responses. In addition, antigenicity analysis
demonstrated that the recombinant proteins corresponding to FepA, FhuA, and IutA
protein were antigenic against anti-Shigella group antigen-antibody and Shigellainfected
human serum. The findings could impact the development of an effective
vaccine against Shigella infection by the utilization of iron uptake receptors as part of
vaccine formulation. However, the protective efficacy and the effectiveness of these
proteins against shigellosis needs to be further evaluated through in vivo study.
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