Whole cells of Vibrio cholerare serotype Inaba and serotype Ogawa (strains I389 and O395) were injected into rabbits to obtain antiserum. The antiserums were used for immune reaction with antigenic components of 25 strains of V.cholerae isolated from five provinces of Vietnam and the two standard strains I389 and O395 by Western-blot technique. Analysis of immune hybrid results showed that there were 11 antigenic components with molecular weights approximately 79kDa, 62kDa, 52kDa, 45kDa, 42kDa, 38kDa, 35kDa, 31kDa, 26kDa, 23kDa and 20kDa. In which the antigens of 45kDa, 42kDa, 31kDa and 20kDa were similar to OmpT, OmpS, Omp-31kDa and TcpA that have been considered as vaccine-candidate antigens. Among 25 V.cholerae strains, there were 6 antigenic components in common including 79kDa, 62kDa, 45kDa, 35kDa, 31kDa and 20kDa. 23/25 strains contained 42kDa antigen; 5/25 strains contained 38kDa and 23kDa antigens; 11/25 had 26kDa antigen. In addition, 7/25 strains contained antigens identical to V.cholerae I389 serotype Inaba; 6/25 strains contained antigens of I389 and O395; 12/25 strains had changes of antigenic components. These changes were actually the lack of antigens, not appearing new antigens. These results are considered as basis for researches about immune response and prevention of cholera disease. / Toàn bộ tế bào của các chủng Vibrio cholerare typ huyết thanh Inaba và typ huyết thanh Ogawa (chủng I389 và O395) được sử dụng để gây miễn dịch trên thỏ để thu kháng huyết thanh. Các kháng huyết thanh được dùng để thực hiện phản ứng miễn dịch với các thành phần kháng nguyên của 25 chủng V.cholerae phân lập từ 5 tỉnh thành của Việt Nam và hai chủng chuẩn I389 và O395 bằng kỹ thuật Western-blot. Phân tích kết quả lai miễn dịch cho thấy, có tổng số 11 thành phần kháng nguyên có kích thước khoảng 79kDa, 62kDa, 52kDa, 45kDa, 42kDa, 38kDa, 35kDa, 31kDa, 26kDa, 23kDa và 20kDa. Các kháng nguyên này chủ yếu là các protein màng ngoài (Omp) và kháng nguyên lông (TcpA). Trong đó các kháng nguyên 45kDa, 42kDa, 31kDa và 20kDa trùng với các kháng nguyên OmpS, OmpT, Omp-31kDa và TcpA được xem là những kháng nguyên dự tuyển vacxin tả. Có 6 kháng nguyên chung giữa 25 chủng với kích thước 79kDa, 62kDa, 45kDa, 35kDa, 31kDa và 20kDa. 7/25 chủng có các kháng nguyên giống với kháng nguyên của chủng V. cholerae I389 typ huyết thanh Inaba; 6/25 chủng có các kháng nguyên giống với kháng nguyên của cả hai chủng V.cholerae I389 và O395; 12/25 chủng có sự biến đổi thành phần kháng nguyên. Tuy nhiên, sự biến đổi này thực chất là sự thiếu hụt chứ không phải là sự xuất hiện các thành phần kháng nguyên mới. Các kết quả nghiên cứu này có thể được xem là nền tảng ban đầu cho các nghiên cứu về miễn dịch và dự phòng bệnh tả.

Whole cells of Vibrio cholerare serotype Inaba and serotype Ogawa (strains I389 and O395) were injected into rabbits to obtain antiserum. The antiserums were used for immune reaction with antigenic components of 25 strains of V.cholerae isolated from five provinces of Vietnam and the two standard strains I389 and O395 by Western-blot technique. Analysis of immune hybrid results showed that there were 11 antigenic components with molecular weights approximately 79kDa, 62kDa, 52kDa, 45kDa, 42kDa, 38kDa, 35kDa, 31kDa, 26kDa, 23kDa and 20kDa. In which the antigens of 45kDa, 42kDa, 31kDa and 20kDa were similar to OmpT, OmpS, Omp-31kDa and TcpA that have been considered as vaccine-candidate antigens. Among 25 V.cholerae strains, there were 6 antigenic components in common including 79kDa, 62kDa, 45kDa, 35kDa, 31kDa and 20kDa. 23/25 strains contained 42kDa antigen; 5/25 strains contained 38kDa and 23kDa antigens; 11/25 had 26kDa antigen. In addition, 7/25 strains contained antigens identical to V.cholerae I389 serotype Inaba; 6/25 strains contained antigens of I389 and O395; 12/25 strains had changes of antigenic components. These changes were actually the lack of antigens, not appearing new antigens. These results are considered as basis for researches about immune response and prevention of cholera disease. / Toàn bộ tế bào của các chủng Vibrio cholerare typ huyết thanh Inaba và typ huyết thanh Ogawa (chủng I389 và O395) được sử dụng để gây miễn dịch trên thỏ để thu kháng huyết thanh. Các kháng huyết thanh được dùng để thực hiện phản ứng miễn dịch với các thành phần kháng nguyên của 25 chủng V.cholerae phân lập từ 5 tỉnh thành của Việt Nam và hai chủng chuẩn I389 và O395 bằng kỹ thuật Western-blot. Phân tích kết quả lai miễn dịch cho thấy, có tổng số 11 thành phần kháng nguyên có kích thước khoảng 79kDa, 62kDa, 52kDa, 45kDa, 42kDa, 38kDa, 35kDa, 31kDa, 26kDa, 23kDa và 20kDa. Các kháng nguyên này chủ yếu là các protein màng ngoài (Omp) và kháng nguyên lông (TcpA). Trong đó các kháng nguyên 45kDa, 42kDa, 31kDa và 20kDa trùng với các kháng nguyên OmpS, OmpT, Omp-31kDa và TcpA được xem là những kháng nguyên dự tuyển vacxin tả. Có 6 kháng nguyên chung giữa 25 chủng với kích thước 79kDa, 62kDa, 45kDa, 35kDa, 31kDa và 20kDa. 7/25 chủng có các kháng nguyên giống với kháng nguyên của chủng V. cholerae I389 typ huyết thanh Inaba; 6/25 chủng có các kháng nguyên giống với kháng nguyên của cả hai chủng V.cholerae I389 và O395; 12/25 chủng có sự biến đổi thành phần kháng nguyên. Tuy nhiên, sự biến đổi này thực chất là sự thiếu hụt chứ không phải là sự xuất hiện các thành phần kháng nguyên mới. Các kết quả nghiên cứu này có thể được xem là nền tảng ban đầu cho các nghiên cứu về miễn dịch và dự phòng bệnh tả.

MENEZES, Francisca Gleire Rodrigues de. Caracterização fenotípica e genotípica de bactérias do gênero Vibrio isoladas em alguns estuários do Estado do Ceará. 2011. 94 f. : Tese (doutorado) - Universidade Federal do Ceará, Centro de Ciências Agrárias, Departamento de Engenharia de Pesca, Fortaleza-CE, 2011 / Submitted by Nádja Goes (nmoraissoares@gmail.com) on 2016-07-26T13:27:46Z No. of bitstreams: 1 2011_tese_fgrmenezes.pdf: 2453594 bytes, checksum: 0370cb8f6e2c333d91e3ccc575df6f2b (MD5) / Approved for entry into archive by Nádja Goes (nmoraissoares@gmail.com) on 2016-07-26T13:28:01Z (GMT) No. of bitstreams: 1 2011_tese_fgrmenezes.pdf: 2453594 bytes, checksum: 0370cb8f6e2c333d91e3ccc575df6f2b (MD5) / Made available in DSpace on 2016-07-26T13:28:01Z (GMT). No. of bitstreams: 1 2011_tese_fgrmenezes.pdf: 2453594 bytes, checksum: 0370cb8f6e2c333d91e3ccc575df6f2b (MD5) Previous issue date: 2011 / The frequent association of environmental aquatic contamination with vibriosis in humans suggests the need for systematic monitoring and study of environmental vibrio strains and their pathogenic potential and clinical significance. The objective of this study was to evaluate the diversity of vibrio species in four estuaries (Pacoti, Choró, Pirangi and Jaguaribe) in Ceará, Northeastern Brazil. Nineteen vibrio species were identified in 32 water samples and 32 sediment samples collected between January and April 2009. Overall, V. parahaemolyticus and V. alginolyticus were the most abundant (the former in Choró, the latter in Pacoti). The isolated strains were submitted to antibiogram testing with 15 antibiotics. All strains (n=197) were susceptible to sulfametoxazol-trimetoprim, ciprofloxacin, nalidixic acid and chloramphenicol. Resistance was observed to penicillin G (n=163; 82%), ampicillin (n=108; 54%), cephalothin (n=15; 7%), aztreonam (n=3; 1%), gentamicin, cefotaxime, ceftriaxone (1 each; 0.5%). Partial resistance was observed to cefalotin (n=52; 25%), ampicillin (n=28; 14%), aztreonam (n=10; 5%), tetracycline (n=8; 4%), oxytetracycline (n=2; 1%), and florfenicol, cefotaxime, ceftriaxone, streptomycin and gentamicin (1 each; 0.5%). Five species known to be pathogenic to humans were chosen for analysis of factors of pathogenicity. Strains belonging to the species V. parahaemolyticus (n=64) and V. cholerae (n=9) were submitted to molecular analysis using genes to confirm the species and indicate virulence. Sixty-three strains of V. parahaemolyticus were positive for species-specific tl, 57 were positive for tdh and 20 for trh. Five strains of V. cholerae were positive for species-specific ompW, but no strains presented the genes ctxA, zot, tcp or rfbO1. In conclusion, the estuaries surveyed presented a great diversity of vibrio species, the most abundant of which were V. parahaemolyticus and V. alginolyticus. Resistance to penicillin and ampicillin was elevated and positivity for virulence factors was considerable among strains of species pathogenic to humans. V. parahaemolyticus strains presented virulence genes indicating risk to public health. V. cholerae was identified in samples of both water and sediment / Muitas pesquisas têm associado contaminação aquática ambiental com infecções de Vibrio em humanos, sugerindo que a importância do monitoramento sistemático das cepas ambientais se faz necessário para definir seu possível potencial patogênico e sua significância clínica. O objetivo dessa pesquisa foi estudar a diversidade do gênero Vibrio isolado de quatro regiões estuarinas no Estado do Ceará, (Pacoti, Choró, Pirangi e Jaguaribe). As coletas realizadas resultaram num total de 32 amostras de água e 32 de sedimento, durante os meses de janeiro a abril de 2009. Foram catalogadas 19 espécies de bactérias pertencentes ao gênero Vibrio, das quais Vibrio parahaemolyticus e Vibrio alginolyticus foram as mais abundantes nos quatro estuários: V. parahaemolyticus no Rio Choró e V. alginolyticus no Rio Pacoti. As cepas identificadas foram submetidas a testes de susceptibilidade a quinze antimicrobianos. Todas as cepas analisadas (197) apresentaram susceptibilidade a sulfazotrim, ciprofloxacin, ácido nalidíxico e cloranfenicol, sendo que cento e sessenta e três (82%) apresentaram resistência a penicilina G, cento e oito (54%) a ampicilina, quinze (7%) a cefalotina, três (1%) a aztreonam, uma (0,5%) a gentamicina, a cefotaxima e a ceftriaxona. Cinquenta e uma cepas (25%) apresentaram comportamento intermediário frente à cefalotina, vinte e oito cepas (14%) a ampicilina, dez (5%) a aztreonam, oito (4%) a tetracilina, duas (1%) a oxitetraciclina e uma (0,5%) a florfenicol, a cefotaxima, a ceftriaxona, a estreptomicina e a gentamicina. Foram escolhidas cinco espécies patógenas ao homem para verificação de seus fatores de patogenicidade. As cepas identificadas como V. parahaemolyticus (64) e V. cholerae (9) foram analisadas através de técnicas de biologia molecular, usando genes que confirmam as espécies e genes que indicam virulência. Das 64 amostras de V. parahaemolyticus analisadas, 63 foram positivas para o gene tl, específico para espécie, 57 para o gene tdh e 20 para o trh, genes que indicam patogenicidade. Das nove cepas de V. cholerae, cinco foram positivas para o gene ompW, gene específico para espécie, porém, nenhuma amostra apresentou os genes de virulência ctxA, zot, tcp e rfbO1. Com isso conclui-se que os estuários dos rios analisados apresentam uma elevada abundância de espécies, tendo V. parahaemolyticus e V. alginolyticus como as mais abundantes. O antibiograma das cepas isoladas mostrou uma elevada resistência à penicilina e a ampicilina. Foram encontradas elevada positividade para a presença dos fatores de virulência nas cepas pertencentes às espécies de Vibrio patógenas a humanos. As cepas de V. parahaemolyticus apresentaram genes de virulência indicando que as cepas podem acarretar danos à saúde pública. A presença do V. cholerae foi confirmada nas águas e sedimento dos estuários

Bibliography: leaves 160-205. / 205, [128] leaves, [27] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Aims to characterise the region of the chromosome associated with the gene encoding the MFRHA (Mannose-Fucose Resistant Haemagglutinin) / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1994

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 135-141).

Although the importance of cation/proton antiporters in cellular physiology is well recognized and widely studied, many antiport systems remain underinvestigated. In this work, I report the phenotypic and biochemical effects of deletion of the cytoplasmic C-terminal tail of the NhaP2 antiporter from Vibrio cholerae (Vc-NhaP2). Namely, deletion of the C-terminal tail results in diminished K+/H+ and Na+/H+ antiport activity, as well as a 5-fold decrease in affinity for its major substrate, K+ (measured as the apparent Km at pH 7.5). Furthermore, reconstitution of antiport activity in the truncation mutant upon addition of exogenous C-terminal tail is demonstrated. Currently, the only known mechanism of antiport is for NhaA, which lacks a cytoplasmic tail. Therefore, these results suggest that NhaP2 may employ a novel mechanism of antiport in which the cytoplasmic tail is directly or indirectly involved.

Cholera, an acute diarrheal diseases caused by the intestinal infection of the pathogenic bacterium Vibrio cholerae, continues to be a global killer in the world today. PrtV, a secreted zinc metalloprotease, is a potent cytotoxic virulence factor of V. cholerae. The 102 kDa full length multi-domain PrtV protein undergoes several N and C terminal modifications before being secreted as a 81 kDa pro-protein. The activation of the pro-protein is calcium dependent. The removal of calcium triggers auto-proteolysis to give a stable active protease with the catalytic zinc binding domain. The aim of the thesis was to study the structure and function of the PrtV protein. The results from paper I, identified the end product of the maturation of PrtV as the stable 37 kDa M6 active domain, and not a 55 kDa complex as reported earlier. Results also showed the this 37 kDa active M6 domain alone was sufficient for catalytic activity. A revised model for the maturation of PrtV was proposed. Individual domains were isolated from the PrtV protein by domain phasing methods. This included the N-terminal domain (residues 23-103), the PKD1 domain (residues 755-839), and a 25 kDa fragment (residues 589-839). The isolated domains were recombinantly over expressed as fusion proteins to increase expression and solubility. The PKD1 domain was purified to homogeneity and crystallized. The structure of the PKD1 domain reported in paper II, was solved by X-ray crystallography at an atomic resolution of 1.1 Å. From the structure, a previously unknown calcium binding site was identified at the N-terminal of the PKD1 domain. The structure also revealed two conformations for the PKD1 domain depending on free or bound calcium. From the structure, a function of the PKD1 domain as a protector of the cleavage site in the linker region between the M6 domain and the PKD1 domain in the presence of calcium was elucidated. A new model for the activation of PrtV was given. In paper III, the structure of the N-terminal domain solved by NMR spectroscopy was reported. The structure revealed two well defined helices but a third predicted helix was found to be unstructured.

The Gram-negative pathogen Vibrio cholerae uses variety of regulatory molecules to modulate expression of virulence factors. One important regulatory element of microorganisms is small non-coding RNAs (sRNAs), which control various cell functions such as expression of cell membrane proteins, mRNA decay and riboswitches. In this thesis studies, we demonstrated the roles of the sRNAs VrrA in regulation of outer membrane protein expression, biofilm formation and expression of ribosome binding proteins. In addition, we showed that VrrB, a newly discovered sRNA, played a role in amino acid dependent starvation survival of V. cholerae and might functioned as a riboswitch. VrrA, a 140-nt sRNAs in V. cholerae, was controlled by the alternative sigma factor σE. The outer membrane protein, OmpT is known to be regulated by environmental signals such as pH and temperature via the ToxR regulon and carbon source signals via the cAMP–CRP complex. Our studies provide new insight into the regulation of OmpT by signals received via the σE regulon through VrrA. We demonstrated that VrrA down-regulate ompT translation by base-pairing with the 5′ region of the ompT mRNA in a Hfq (RNA chaperone protein) dependent manner. V. cholerae biofilms contain three matrix proteins—RbmA, RbmC and Bap1—and exopolysaccharide. While much is known about exopolysaccharide regulation, little is known about the mechanisms by which the matrix protein components of biofilms are regulated. In our studies, we demonstrated that VrrA negatively regulated rbmC translation by pairing to the 5' untranslated region of the rbmC transcript and that this regulation was not stringently dependent on Hfq. In V. cholerae, VC0706 (Vrp) and VC2530 proteins are homologous to ribosome-associated inhibitor A (RaiA) and hibernation promoting factor (HPF) of Escherichia coli, respectively. HPF facilitates stationary phase survival through ribosome hibernation. We showed that VrrA repressed Vrp protein expression by base-pairing to the 5´ region of vrp mRNA and that this regulation required Hfq. We also showed that Vrp was highly expressed during stationary phase growth and associated with the ribosomes of V. cholerae. We further demonstrated that Vrp and VC2530 were important for V. cholerae starvation survival under nutrient-deficient conditions. While VC2530 was down-regulated in bacterial cells lacking vrrA, mutation of vrp resulted in increased expression of VC2530. Riboswitches are an important class of regulators in bacteria, which are most often located in the 5' untranslated region (5´ UTR) of bacterial mRNA. In this study, we discovered the novel non-coding sRNA, VrrB located at the 5´ UTR of a downstream gene encoding Vibrio auxotropic factor A (VafA) for phenylalanine. In V. cholerae, reduced production of VafA was observed in the presence of phenylalanine and phenylpyruvate in the culture media. Some analogs of phenylalanine and phenylpyruvate could also modulate the expression of VafA. Furthermore, bacterial cells lacking the vrrB gene exhibited high production of VafA, suggesting that VrrB might function as a riboswitch that controls VafA expression.

Bibliography: leaves 169-208 / ix, 208 leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1988

Corrigenda pasted onto front fly-leaf. / Bibliography: leaves 247-286. / xv, 286 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis examines an aspect of the pathogenesis of a model extracellular enteric pathogen, the causative agent of human cholera. The export of TcpA (Toxin-Coregulated Pilus) and assembly of the TCP is explored as a paradigm of macromolecular export in Gram negative bacteria. TcpA is examined in detail in an attempt to define strictly conserved regions between species. The TCP of the emergent 0139 (Bengal) serotype is demonstrated to be of El Tor type. The possibily that proteases such as the soluble haemagglutinin (SHA) may have a detachase role centring on TCP dispersal/TcpA degradation is also discussed. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology, 1997