Curli fimbria: an Escherichia coli adhesin associated with human cystitis

Escherichia coli is the major causative agent of human cystitis. In this study, a preliminary molecular analysis carried out by PCR (polymerase chain reaction) demonstrated that 100% of 31 E. coli strains isolated from patients with recurrent UTIs (urinary tract infections) showed the presence of the curli fimbria gene (csgA). Curli fimbria is known to be associated with bacterial biofilm formation but not with the adhesion of human cystitis-associated E. coli. Therefore, this work aimed to study how curli fimbria is associated with uropathogenic E. coli (UPEC) as an adhesion factor. For this purpose, the csgA gene was deleted from strain UPEC-4, which carries three adhesion factor genes (csgA, fimH and ompA). The wild-type UPEC-4 strain and its mutant (ΔcsgA) were analyzed for their adhesion ability over HTB-9 (human bladder carcinoma), Vero (kidney cells of African green monkey) and HUVEC (human umbilical vein) cells in the presence of α-D-mannose. All the wild-type UPEC strains tested (100%) were able to adhere to all three cell types, while the UPEC-4 ΔcsgA mutant lost its adherence to HTB-9 but continued to adhere to the HUVEC and Vero cells. The results suggest that curli fimbria has an important role in the adhesion processes associated with human UPEC-induced cystitis.472414416CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQSem informaçã

Construction of attenuated strains of S. enterica Typhimurium producing antigen of Plasmodium

Orientador: Marcelo BrocchiDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A Salmonelose e a malária são duas doenças infecciosas negligenciadas de grande prevalência no mundo, mas acometendo particularmente populações humanas que vivem em regiões e países subdesenvolvidos ou em desenvolvimento. Apesar dos esforços ainda não existem formulações vacinais totalmente efetivas no controle destas enfermidades. Desta forma, nosso grupo tem buscado aprimorar a expressão do Domínio M2 do antígeno MAEBL de Plasmodium yoelii, que apresenta similaridade com a proteína de Plasmodium falciparum, em linhagens de Salmonella enterica Typhimurium, atenuadas para virulência. Em estudos prévios constatou-se que a expressão em níveis baixos não seria suficiente para a indução de uma resposta imune protetora. Com isso, ficou clara a necessidade de se aprimorar os níveis de expressão deste antígeno nas linhagens a serem testadas. Para tanto, o vetor de expressão anteriormente utilizado foi modificado, os codons da seqüência a ser expressa foram otimizados para expressão em salmonela e um sinal de secreção periplasmática foi adicionado no início da seqüência a ser expressa. Adicionalmente, modificamos novas linhagens atenuadas de S. enterica desenvolvidas por nosso grupo de tal forma a utiliza-las na expressão de antígenos heterólogos, empregando um sistema letal balanceado (Asd) de estabilização da expressão gênica. Apesar do sucesso na construção dos novos vetores, e da otimização de codons para a expressão, constatamos um possível efeito deletério do domínio M2 quando expresso em grande quantidade, o que impossibilitou a obtenção de algumas construções planejadas. Entretanto, os resultados obtidos na estabilização de plasmídios em novas linhagens vacinais significam um avanço no teste e uso de tais linhagens no desenvolvimento de vacinas multifatoriaisAbstract: Salmonellosis and malaria are two infectious diseases of high prevalence in the world, particularly affecting human populations living in underdeveloped regions and countries or developing countries. Despite several efforts, there is not a vaccinal formulation totally effective to control these diseases. This way, our group has sought to enhance the expression of M2 MAEBL's antigen Domain of Plasmodium yoelii, which is quite similar to same protein of P. falciparum, in attenuated strains of Salmonella enterica Typhimurium. In previous studies we found that the expression at low levels was not sufficient to induce a protective immune response. Thus, there was a clear need to improve M2 expression levels in the new strains to be tested. The expression vector previously used was modified, the m2 sequence was codon-optimized for expression in Salmonella, and a periplasmic secretion signal was added at the beginning of the sequence to be expressed. Additionally, new attenuated S. enterica strains developed by our group were modified in a way to use them in the expression of heterologous antigens, employing a balanced lethal system (ASD) for expression plasmid stabilization. Despite of the new vectors successfully built as well as codon optimization for m2 expression, we found a possible deleterious effect of the M2 domain when expressed in large quantities, making it impossible to obtain some planned constructs. However, the results obtained on stabilization of expression vectors in the new vaccine strains are an important advance for the use of such strains as multifactorial vaccinesMestradoBioquimicaMestre em Biologia Funcional e Molecula

Virulence evaluation and potential use as vaccine of Salmonella enterica mutants for nucleoid associated proteins

Orientador: Marcelo BrocchiTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Salmonella enterica é um bacilo Gram-negativo pertencente à família Enterobacteriaceae responsável por grandes perdas econômicas e de saúde pública, podendo causar doenças ao homem, que variam de gastroenterites a infecções sistêmicas, e em animais, provocando grandes perdas econômicas. As sorovariedade Enteritidis e Typhimurium são as sorovariedade mais comumente isoladas em surtos de salmonelose no Brasil, Europa e Estados Unidos, tendo como principal fonte da infecção ovos e outros produtos derivados do frango. Uma das formas de se controlar infecções por S. enterica é através do uso de vacinas, as quais já vem sendo utilizadas em diversos países, apesar de ainda haver necessidade de melhora destas. Neste trabalho enfatizamos o uso de linhagens selvagens de S. enterica, realizando a caracterização genotípica e fenotípica destas linhagens, e comparando com mutantes obtidos a partir das mesmas, para testar a viabilidade em utiliza-los como vetores vacinais. Essas linhagens tiveram seus genomas sequenciados. Foram obtidos mutantes para os genes fis, hupA e hupB à partir dessas linhagens com o intuito de verificar o potencial destes mutantes no uso como vacinas vivas atenuadas. As proteínas codificadas por esses genes se associam ao nucleóide bacteriano e influenciam direta ou indiretamente na expressão de genes de virulência e quando deletadas podem levar a diferentes níveis de atenuação. Os mutantes de S. enterica Typhimurium para o gene fis demonstraram serem suficientemente atenuados e, portanto, passíveis de serem testados como vacinas vivas atenuadas. Mutantes para os genes hupA e hupB de S. enterica Enteritidis também foram obtidos e, embora os mutantes com apenas uma mutação (hupA ou hupB) não fossem atenuados, o mutante duplo hupAB mostrou-se suficientemente atenuado, permitindo doses superiores a 109 UFC por animal. Além disso, os mutantes atenuados obtidos neste estudo foram capazes de provocar uma resposta imune capaz de proteger camundongos Balb/c contra um desafio com a linhagem selvagem. Esses resultados demonstram claramente que as linhagens aqui desenvolvidas têm potencial para utilização como vacinas vivas atenuadasAbstract: Salmonella enterica is a gram-negative bacillus belonging to the family Enterobacteriaceae responsible for major economic and public health losses, may cause diseases to humans ranging from gastroenteritis to systemic infections and in animals, causing great economic losses. The most commonly isolated serovar in salmonellosis outbreaks in Brazil, Europe and the United States, are Enteritidis and Typhimurium, being eggs and other products derived from chicken the main source of infection. One way to control infections caused by Salmonella is by vaccination, a strategy that is already being used in several countries, although there is still need to improve those vaccines. Here we emphasize the use of wild strains of S. enterica, performing genotypic and phenotypic characterization of these strains, and compared with mutants for nucleoid associated proteins obtained from them, to test the feasibility of using these mutants as vaccine vectors. The wild-type strains had their genome sequenced. Starting from this wild-type strains we obtained mutants for the genes fis, hupA e hupB, intendind to verify their potential use as live-attenuated vaccines. The proteins encoded by these genes are associated to the bacterial nucleoid and directly or indirectly influences expression of virulence genes and when deleted may lead to different levels of attenuation. The S. enterica Typhimurium mutants for the fis gene showed to be sufficiently attenuated and therefore capable of being tested as live attenuated vaccines. S. enterica Enteritidis mutants for hupA and hupB genes were also obtained, although the single mutants (hupA or hupB) were not attenuated, the double mutant hupAB showed to be very attenuated, allowing doses greater than 109 CFU. Additionally, the attenuated mutants obtained in this study were able to elicit an immune response capable of protecting BALB / c mice against a challenge with wild type strain. These results clearly demonstrate that the strains developed herein have potential for use as live-attenuated vaccinesDoutoradoBioquimicaDoutor em Biologia Funcional e Molecular2012/05382-6FAPES

Whole-Genome Sequence of Salmonella enterica Serovar Enteritidis Phage Type 4, Isolated from a Brazilian Poultry Farm

Submitted by Sandra Infurna ([email protected]) on 2017-01-01T17:07:50Z No. of bitstreams: 1 dalia3_rodrigues_etal_IOC_2016.pdf: 160781 bytes, checksum: 360b608d178d2a484e75b781402a4dd7 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2017-01-01T17:25:12Z (GMT) No. of bitstreams: 1 dalia3_rodrigues_etal_IOC_2016.pdf: 160781 bytes, checksum: 360b608d178d2a484e75b781402a4dd7 (MD5)Made available in DSpace on 2017-01-01T17:25:12Z (GMT). No. of bitstreams: 1 dalia3_rodrigues_etal_IOC_2016.pdf: 160781 bytes, checksum: 360b608d178d2a484e75b781402a4dd7 (MD5) Previous issue date: 2016Universidade de Campinas. Instituto de Biologia. Departamento de Genética, Evolução e Bioagentes. Campinas, SP, Brasil.Universidade de Campinas. Instituto de Biologia. Departamento de Genética, Evolução e Bioagentes. Laboratório de Genômica e Expressão (LGE). Campinas, SP, Brasil / Universidade de Campinas. Laboratório Central de Tecnologias de Alto Desempenho. Campinas, SP, Brasil.Biovet Laboratório. Vargem Grande Paulista, SP. Brasil.Biovet Laboratório. Vargem Grande Paulista, SP. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Bacteriologia. Rio de Janeiro, RJ, Brasil.Universidade de Campinas. Instituto de Biologia. Departamento de Genética, Evolução e Bioagentes. Laboratório de Genômica e Expressão (LGE). Campinas, SP, Brasil.Universidade de Campinas. Instituto de Biologia. Departamento de Genética, Evolução e Bioagentes. Campinas, SP, Brasil.The draft genome of Salmonella enterica serovar Enteritidis phage type 4 (PT4) strain IOC4647/2004, isolated from a poultry farm in São Paulo state, was obtained with high-throughput Illumina sequencing platform, generating 4,173,826 paired-end reads with 251 bp. The assembly of 4,804,382 bp in 27 scaffolds shows strong similarity to other S Enteritidis strains

Antimelanoma effect of Salmonella Typhimurium integration host factor mutant in murine model

CNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOThis study aimed to evaluate an attenuated Salmonella ihfA-null mutant strain as therapeutic agent to control tumor growth. After bacterial toxicity evaluation, C57BL/6JUnib mice were inoculated with B16F10 cells and treated with two Salmonella strains (122023672378CNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO308955/2012-92014/13412-8; 2012/25426-8; 2012/05382-6141629/2012-

A lipidomics approach in the characterization of zika-infected mosquito cells: potential targets for breaking the transmission cycle

Recent outbreaks of Zika virus in Oceania and Latin America, accompanied by unexpected clinical complications, made this infection a global public health concern. This virus has tropism to neural tissue, leading to microcephaly in newborns in a significant proportion of infected mothers. The clinical relevance of this infection, the difficulty to perform accurate diagnosis and the small amount of data in literature indicate the necessity of studies on Zika infection in order to characterize new biomarkers of this infection and to establish new targets for viral control in vertebrates and invertebrate vectors. Thus, this study aims at establishing a lipidomics profile of infected mosquito cells compared to a control group to define potential targets for viral control in mosquitoes. Thirteen lipids were elected as specific markers for Zika virus infection (Brazilian strain), which were identified as putatively linked to the intracellular mechanism of viral replication and/or cell recognition. Our findings bring biochemical information that may translate into useful targets for breaking the transmission cycle1110CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPSem informaçãoSem informação2011/50400-0; 2015/06809-1; 2014/00084-2; 2014/00302-0; 2013/11343-

ZIKV-specific NS1 epitopes as serological markers of acute zika virus infection

Differentiate ZIKV from other cocirculating flaviviruses for accurate diagnosis remains a challenge. We investigated antibody responses in 51 acute ZIKV-infected adult patients from Campinas, Brazil, including 7 pregnant women who later delivered during the study. Using enzyme-linked immunosorbent assays, levels of antibody response were measured and specific epitopes identified. Several antibody-binding hot spots were identified in ZIKV immunogenic antigens, including membrane, envelope (E) and nonstructural protein 1 (NS1). Interestingly, specific epitopes (2 from E and 2 from NS1) strongly recognized by ZIKV-infected patients' antibodies were identified and were not cross-recognized by dengue virus (DENV)-infected patients' antibodies. Corresponding DENV peptides were not strongly recognized by ZIKV-infected patients' antibodies. Notably, ZIKV-infected pregnant women had specific epitope recognition for ZIKV NS1 (amino acid residues 17-34), which could be a potential serological marker for early ZIKV detection.This study identified 6 linear ZIKV-specific epitopes for early detection of ZIKV infections. We observed differential epitope recognition between ZIKV-infected and DENV-infected patients. This information will be useful for developing diagnostic methods that differentiate between closely related flaviviruses2202203212CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPnão tem2016/00194-8; 2013/25807-4This work is supported by the Biomedical Research Council (BMRC) core research grants to the Singapore Immunology Network and the Zika Virus Consortium Fund, led by BMRC A*Star (project number 15/1/82/27/001); Agency for Science, Technology and Research, Singapore; Fundação de Amparo à Pesquisa do Estado de São Paulo (grant numbers 2016/00194-8 and 2013/25807-4 fellowship to J. A. L.); and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (research fellowships to G. P. M. and F. T. M. C.

Efficient detection of Zika virus RNA in patients' blood from the 2016 outbreak in Campinas, Brazil

Abstract Infection with Zika virus (ZIKV), a mosquito-borne flavivirus has been casually linked with increased congenital microcephaly in Brazil from 2015 through 2016. Sensitive and specific diagnosis of patients with Zika fever (ZIKF) remains critical for patient management. We developed a ZIKV NS5 qRT-PCR assay by combining primers described by Balm et al. and a new Taqman probe. The assay was evaluated and compared with another assay described by Lanciotti et al. (ZIKV 1107) using 51 blood and 42 urine samples from 54 suspected ZIKV patients. ZIKV NS5 performed better in terms of sensitivity with more samples detected as ZIKV-positive (n = 37) than ZIKV 1107 (n = 34) for urine, and ZIKV-positive (n = 29) than ZIKV 1107 (n = 26) for blood. Both assays displayed good overall agreement for urine (κappa = 0.770) and blood (κappa = 0.825) samples. Improved availability of validated diagnostic tests, such ZIKV NS5 qRT-PCR, will be critical to ensure adequate and accurate ZIKV diagnosis