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Title: Molecular analysis of pandemic and non-pandemic strains of vibrio parahaemolyticus and their interactions with caenorhabditis elegans
Other Titles: Liu xing xing yu fei liu xing xing fu rong xue xing hu jun de fen zi fen xi ji qi yu xiu li yin gan xian chong de xiang hu zuo yong
Authors: Jime, Jinath Sultana (0)
Department: Department of Biology and Chemistry
Degree: Doctor of Philosophy
Issue Date: 2010
Publisher: City University of Hong Kong
Subjects: Vibrio parahaemolyticus.
Caenorhabditis elegans.
Notes: CityU Call Number: QR82.V53 J55 2010
xxv, 226 leaves : ill. (some col.) 30 cm.
Thesis (Ph.D.)--City University of Hong Kong, 2010.
Includes bibliographical references (leaves 186-207)
Type: thesis
Abstract: Vibrio parahaemolyticus is a gram-negative halophilic bacterium that is recognized worldwide as a leading cause of sea-food related bacterial gastroenteritis in humans. Prior to 1995, V. parahaemolyticus infections were sporadic and associated with diverse serotypes which are regarded as non-pandemic strains. A sudden global upsurge of V. parahaemolyticus outbreaks was reported between 1996 and 1999, which was regarded as the 'first pandemic‘ in the history of V. parahaemolyticus. The pandemic was attributed to the emergence of a new clone of the O3:K6 serotype and its closely-related serovariants (O4:K68, O1:K25 and O1:KUT) which constitute the "predominant" group of pandemic strains. In recent years, other related serovariants of O3:K6 have emerged and are regarded as the "newly evolved" serovariants. The ancestor O3:K6 and its "predominant" and "newly evolved" serovariants are collectively regarded as the 'pandemic group' and are now recognized worldwide as a newly emerging group of pathogens. In this study, 85 strains (81 clinical and 4 environmental isolates) of V. parahaemolyticus from Bangladesh, Hong Kong and Taiwan isolated pre- and post-1995 were examined using a variety of molecular techniques. All strains were confirmed as V. parahaemolyticus by PCR that targeted a V. parahaemolyticus-specific region of the toxR gene. Serotyping was performed using commercial O and K antisera, which categorized the test strains into 15 different serotypes - O3:K6, O4:K68, O1:KUT, O1:K25, O5:KUT, O1:K56, O4:K22, O4:K10, O8:K22, O2:K3, O4:K11, O4:K55, O2:K8, O1:K38, O3:K29. Strains from the 15 serotypes were divided into the pandemic and non-pandemic group using pandemic strain-specific ORF8 and toxRS PCR. ORF8- and toxRS-PCR indicated that 51.8% (44/85) and 57.7% (49/85) of the strains belong to the pandemic group, and consisted of strains from both the "predominant" (O3:K6, O4:K68, O1:KUT, O1:K25) and "newly evolved" (O5:KUT) pandemic serovariants. The remaining serotypes (36 strains) belong to the non-pandemic group. To better understand the virulence and pathogenic potential of the serovariants from the pandemic and non-pandemic groups, various genotypic traits that are known to be associated with pathogenicity in V. parahaemolyticus were examined. Presence of tdh (that encode for thermostable direct hemolysin) and TDH production were examined by PCR and ELISA, respectively. All of the 49 pandemic and 26 out of 36 non-pandemic V. parahaemolyticus strains examined are toxigenic. PCR detection for the trh (tdh-related hemolysin), T3SS2 (Type Three Secretion System-2), T6SS (Type Six Secretion System) and Mtase (DNA methyl transferase) genes revealed that all tdh-positive pandemic (49/49) and 26 non-pandemic (26/36) strains harbored the T3SS2, Mtase and T6SS gene clusters. A few of the non-pandemic strains harbored the trh gene. The findings indicated that the presence of the T3SS2, T6SS and Mtase genes in V. parahaemolyticus is tightly correlated with the presence of the tdh gene regardless of their pandemic origin. The structure and distribution of four pathogenicity islands (VPaIs) -- VPaI-1, VPaI-2, VPaI-4 and VPaI-5 -- were investigated to better understand the association of these VPaIs to virulence in various pandemic strains. A novel 'Hybrid VPaI-2' was identified in pandemic V. parahaemolyticus strains, which comprises an intact 'VPaI-2 prototype I' (pandemic-strain specific) and an additional 6-kb DNA fragment from the 'VPaI-2 prototype II' (non-pandemic strain specific). 'Hybrid VPaI-2' was found to be present in 97.5% of pandemic strains (consisting of O3:K6, O4:K68, O1:KUT, O1:K25 and O5:KUT serovariants) isolated from diverse geographical locations such as Bangladesh, Hong Kong, Peru and India. Interestingly, none of the non-pandemic (32/32) or environmental (4/4) strains examined harbour the 'Hybrid VPaI-2'. Analysis by Long PCR of VPaI-1, VPaI-4 and VPaI-5 revealed the presence of intact forms of these three VPaIs in all 10 representative pandemic strains of diverse serotypes. None of the non-pandemic strains (10/10) examined harbour intact forms of VPaI-1, VPaI-4 or VPaI-5. Only portions of the VPaI-1 gene cluster were present in a few of the non-pandemic strains examined. Killing of C. elegans by E. coli recombinants containing VPaI-1, VPaI-4, VPaI-5 or Hybrid VPaI-2 revealed no killing effect on C. elegans as compared to the normal E. coli OP50 control. The evolutionary relatedness of various pandemic serovariants to the prototypical O3:K6 serotype, and among selected pandemic and non-pandemic strains were investigated using Arbitrarily Primed (AP)-PCR and Comparative Genomic Hybridization (CGH) analyses. AP-PCR analysis revealed that the genomes of all of the pandemic serovariants examined are identical to the prototypical O3:K6 serotype irrespective of their geographical origin, which strongly indicated that they are clonally related to O3:K6. In contrast, the genomes of the non-pandemic strains were shown not only to be divergent with each other, but are also distant to the pandemic O3:K6 clone. CGH analysis using DNA microarray revealed that both the "predominant" (O4:K68, O1:KUT, O1:K25) and "newly evolved" (O5:KUT) pandemic serovariants possess genetic backgrounds that are highly similar to that of the ancestral O3:K6 serotype, which suggest the former have likely evolved from the ancestral O3:K6 strain via alteration (by recombination) of the O and/or K-antigen gene clusters. In contrast, the non-pandemic strains (O4:K68 and O3:K29) were genetically highly dissimilar to the pandemic O3:K6 clone and likely originated from a different lineage. To investigate possible differences in virulence potential among the different pandemic and non-pandemic serotypes, V. parahaemolyticus-C. elegans interaction studies were carried out. In this project, C. elegans was used as a model host to assess the virulence potential of various V. parahaemolyticus strains based on TD50 values (time to kill 50% of C. elegans host). Life span of worms grown on pandemic V. parahaemolyticus strains were significantly shorter than on non-pandemic strains or E. coli OP50 (the normal food source of C. elegans). The intestines of C. elegans fed on pandemic V. parahaemolyticus were grossly distended. Killing of C. elegans required live bacteria and direct contact with V. parahaemolyticus, whereby accumulation of bacteria in the gut of C. elegans was the primary cause of death. During the infection process, C. elegans employed different strategies to resist and/or to mediate V. parahaemolyticus infection. Recognition of pathogen (tol-1, tir-1), physical barrier (srf-3) (cuticle) and resistance to lysosomal degradation were the defense mechanisms employed by C. elegans against V. parahaemolyticus infection. In contrast, ATP binding cassette transporter (pgp-3) and insulin/insulin-like growth factor (IGF) signaling pathways (daf-16, age-1) were the target of V. parahaemolyticus pathogenesis. Stabilization of the HIF-1 protein under normoxia was also involved in the pathogenesis of V. parahaemolyticus and therefore, killing of C. elegans. Overall, the findings of this study revealed that all serovariants belonging to the pandemic group possess a genetic background and pathogenic potential very similar to the O3:K6 ancestral clone, and suggest they likely originated from the O3:K6 clone. Altering the O and/or K antigenic structure (via genetic recombination) may have facilitated the emergence of new serovariants by circumventing the host-immune defense systems.
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