A ciclodextrina glicosiltransferase (EC 2.4.1.19) é a única enzima capaz de converter amido e açúcares relacionados em ciclodextrinas através de reação de ciclização. Estes compostos podem formar complexos de inclusão com moléculas hidrofóbicas, tornando-se importante para aplicação nas indústrias alimentícias, farmacêuticas, agrícolas, químicas e de cosméticos. Um novo microorganismo produtor de CGTase foi encontrado entre bactérias isoladas do solo e foi identificado como Stenotrophomonas maltophilia. A enzima produzida por este microrganismo foi purificada em quatro etapas: precipitação por afinidade com amido, ultrafiltração, cromatografia de troca iônica e cromatografia de interação hidrofóbica. A CGTase assim purificada obteve, aproximadamente, atividade específica de 200.000 U/mg e fator de purificação de 2500. Com uma única banda, o peso molecular da enzima purificada foi estimado em 70kDa por SDS-PAGE. A temperatura ótima para atividade da enzima foi de 60 º C, enquanto que a atividade enzimática permaneceu praticamente estável entre pH 6 e 10, indicando natureza alcalotolerante. Km e Vmax para a enzima pura foram de 2,5 g/mL e 12,5 U/mg de proteína, respectivamente, usando amido solúvel como substrato. / Cyclodextrin glycosyltransferase (EC 2.4.1.19) is the unique enzyme able to convert starch and related sugars into cyclodextrins via cyclization reaction. These compounds can form inclusion complexes with hydrophobic molecules, becoming important for application in the food, pharmaceutical, agricultural, chemical and cosmetics industries. A new microorganism producing the CGTase was found among strains isolated from soil and identified as Stenotrophomonas maltophilia. The enzyme produced by this strain was purified by four steps: starch affinity precipitation, ultrafiltration, ion exchange chromatography and hydrophobic interaction chromatography. The CGTase thus obtaining specific activity 200,000 U.mg-1 and 2500-fold purification. With a single band, the molecular weight of the purified enzyme was estimated in 70kDa by SDS-PAGE. The optimum temperature for enzyme activity was 60ºC, whereas the enzyme activity remained almost stable between pH 6 to 10, indicating its alkalotolerant nature. Km e Vmax for the pure enzyme were 2.5 g/mL and 12.5 U/mg protein, respectively, using soluble starch as substrate.

A ciclodextrina glucosiltransferase (CGTase) é uma enzima industrialmente muito importante, capaz de converter o amido em ciclodextrinas (CDs). As CDs são capazes de formar complexos de inclusão com uma grande gama de compostos orgânicos e inorgânicos, podendo mudar suas propriedades químicas e físicas, propriedades estas que lhes confere extensiva aplicabilidade na indústria de alimentos, farmacêutica, química, cosmética e agrícola. Atualmente, diversas CGTases já foram isoladas e caracterizadas a partir de vários microrganismos, principalmente Bacillus, Paenibacillus, Pseudomonas, Klebsiella, Xanthomonas, Thermococcus, Vibrio, Geobacillus e Thermoanaerobacterium. Neste trabalho, demonstramos o primeiro relato envolvendo a clonagem e expressão heteróloga da CGTase de Stenotrophomonas maltophilia, microrganismo isolado do solo brasileiro. O gene codificador da CGTase de S. maltophilia, foi amplificado com êxito através da técnica de PCR, clonado no vetor pET-23a(+) e expresso em Escherichia coli BL21(DE3). As células recombinantes necessitaram de aproximadamente 4 horas de cultivo em meio Luria Bertani (LB) após a adição de 0,1 mM de IPTG para a expressão elevada da proteína alvo. Porém, a CGTase recombinante foi expressa sob forma de corpos de inclusão permanecendo na fração insolúvel, sendo necessário utilizar protocolos para solubilização, incluindo diferentes concentrações de uréia, mas a precipitação não foi eficaz. Embora tenha sido observada uma expressão elevada da proteína com cerca de 60 kDa em SDS-PAGE 12%, que correspondeu ao tamanho esperado da proteína, a forma ativa da enzima não foi obtida. Uma análise bioinformática foi realizada, onde foi observou-se uma proteína conhecida como uma importante possível facilitadora transmembrana (PMFTP – putative Major Facilitator Transmembrane Protein) que ancora o gene cgt. Proteína esta que pode ser utilizada em novos estudos a fim de desenvolver um novo e mais eficaz sistema para expressão da CGTase, podendo facilitar a sua expressão extracelular. Assim, mais estudos são necessários para desenvolver um sistema de co-expressão de rCGTase::PMFTP em E. coli e obter mais informações desta proteína de Stenotrophomonas maltophilia. / Cyclodextrin glucosyltransferase (CGTase) is an industrially important enzyme, capable to convert starch into cyclodextrins (CDs). The CDs are able to form inclusion complexes with a wide range of organic and inorganic compounds, which can change their chemical and physical properties, that gives them extensive applicability in the food, pharmaceutical, chemical, cosmetics and agricultural. Currently, many CGTases have been isolated and characterized from various microorganisms, particularly Bacillus, Paenibacillus, Pseudomonas, Klebsiella, Xanthomonas, Thermococcus, Vibrio, Geobacillus and Thermoanaerobacterium. This work demonstrates the first report involving cloning and expression of heterologous CGTase from Stenotrophomonas maltophilia, microorganism isolated from Brazilian soil. The gene encoding the S. maltophilia CGTase, was successfully amplified by PCR, cloned into the vector pET-23a (+) and expressed in Escherichia coli BL21(DE3). Recombinant cells required about 4 h of cultivation in Luria Bertani (LB) after addition of 0.1 mM IPTG for high expression of the target protein. However, the CGTase was expressed recombinant form of inclusion bodies remaining in the insoluble fraction, being necessary use protocols for solubilization, including different concentrations of urea, but the precipitation was not effective. Although it was observed a high expression of the protein about 60 kDa on SDS-PAGE 12%, corresponding to the expected size of the protein, but the active form of the enzyme was not obtained. Bioinformatic analysis was performed and observed a Putative Major Facilitator Transmembrane Protein (PMFTP) harboring the cgt gene. This protein can be used in further studies to develop a new and more effective system for expression of the CGTase, which may facilitate its extracellular expression. Thus, more studies are needed to develop a system of co-expression of rCGTase::PMFTP in E. coli and acquire more information about this protein of Stenotrophomonas maltophilia.

A ciclodextrina glucosiltransferase (CGTase) é uma enzima industrialmente muito importante, capaz de converter o amido em ciclodextrinas (CDs). As CDs são capazes de formar complexos de inclusão com uma grande gama de compostos orgânicos e inorgânicos, podendo mudar suas propriedades químicas e físicas, propriedades estas que lhes confere extensiva aplicabilidade na indústria de alimentos, farmacêutica, química, cosmética e agrícola. Atualmente, diversas CGTases já foram isoladas e caracterizadas a partir de vários microrganismos, principalmente Bacillus, Paenibacillus, Pseudomonas, Klebsiella, Xanthomonas, Thermococcus, Vibrio, Geobacillus e Thermoanaerobacterium. Neste trabalho, demonstramos o primeiro relato envolvendo a clonagem e expressão heteróloga da CGTase de Stenotrophomonas maltophilia, microrganismo isolado do solo brasileiro. O gene codificador da CGTase de S. maltophilia, foi amplificado com êxito através da técnica de PCR, clonado no vetor pET-23a(+) e expresso em Escherichia coli BL21(DE3). As células recombinantes necessitaram de aproximadamente 4 horas de cultivo em meio Luria Bertani (LB) após a adição de 0,1 mM de IPTG para a expressão elevada da proteína alvo. Porém, a CGTase recombinante foi expressa sob forma de corpos de inclusão permanecendo na fração insolúvel, sendo necessário utilizar protocolos para solubilização, incluindo diferentes concentrações de uréia, mas a precipitação não foi eficaz. Embora tenha sido observada uma expressão elevada da proteína com cerca de 60 kDa em SDS-PAGE 12%, que correspondeu ao tamanho esperado da proteína, a forma ativa da enzima não foi obtida. Uma análise bioinformática foi realizada, onde foi observou-se uma proteína conhecida como uma importante possível facilitadora transmembrana (PMFTP – putative Major Facilitator Transmembrane Protein) que ancora o gene cgt. Proteína esta que pode ser utilizada em novos estudos a fim de desenvolver um novo e mais eficaz sistema para expressão da CGTase, podendo facilitar a sua expressão extracelular. Assim, mais estudos são necessários para desenvolver um sistema de co-expressão de rCGTase::PMFTP em E. coli e obter mais informações desta proteína de Stenotrophomonas maltophilia. / Cyclodextrin glucosyltransferase (CGTase) is an industrially important enzyme, capable to convert starch into cyclodextrins (CDs). The CDs are able to form inclusion complexes with a wide range of organic and inorganic compounds, which can change their chemical and physical properties, that gives them extensive applicability in the food, pharmaceutical, chemical, cosmetics and agricultural. Currently, many CGTases have been isolated and characterized from various microorganisms, particularly Bacillus, Paenibacillus, Pseudomonas, Klebsiella, Xanthomonas, Thermococcus, Vibrio, Geobacillus and Thermoanaerobacterium. This work demonstrates the first report involving cloning and expression of heterologous CGTase from Stenotrophomonas maltophilia, microorganism isolated from Brazilian soil. The gene encoding the S. maltophilia CGTase, was successfully amplified by PCR, cloned into the vector pET-23a (+) and expressed in Escherichia coli BL21(DE3). Recombinant cells required about 4 h of cultivation in Luria Bertani (LB) after addition of 0.1 mM IPTG for high expression of the target protein. However, the CGTase was expressed recombinant form of inclusion bodies remaining in the insoluble fraction, being necessary use protocols for solubilization, including different concentrations of urea, but the precipitation was not effective. Although it was observed a high expression of the protein about 60 kDa on SDS-PAGE 12%, corresponding to the expected size of the protein, but the active form of the enzyme was not obtained. Bioinformatic analysis was performed and observed a Putative Major Facilitator Transmembrane Protein (PMFTP) harboring the cgt gene. This protein can be used in further studies to develop a new and more effective system for expression of the CGTase, which may facilitate its extracellular expression. Thus, more studies are needed to develop a system of co-expression of rCGTase::PMFTP in E. coli and acquire more information about this protein of Stenotrophomonas maltophilia.

Paez JIG. Fatores associados à mortalidade em infecções nosocomiais por Stenotrophomonas maltophilia [dissertação]. São Paulo: Faculdade de Medicina, Universidade de São Paulo; 2007. 154p. Infecção de corrente sanguínea (ICS) e pneumonia por S. maltophilia são associadas á alta mortalidade. A identificação de fatores relacionados à mortalidade em pacientes com infecções por esse agente pode permitir intervenções no sentido de diminuir a sua mortalidade. Realizamos um estudo retrospectivo com 60 pacientes com ICS ou pneumonia de origem hospitalar no Instituto Central do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (ICHC-FMUSP) durante o período de 30 de julho de 1999 a 30 de julho de 2005. Analisamos os fatores de risco relacionados à mortalidade global e a mortalidade nos primeiros 14 dias da infecção por meio de um estudo de coorte retrospectivo comparando os pacientes que apresentaram óbito com os que não apresentaram óbito.As seguintes características foram encontradas na população estudada no momento da infecção, 57 (85%) dos pacientes receberam antibióticos prévios, 88% tinham cateter venoso central, 57% estavam em uso de ventilação mecânica, 35% em uso de quimioterapia e 75% estavam internados em unidade de cuidado intensivo. Neoplasia foi a principal doença de base presente em 45%. Choque séptico foi descrito em 30% dos casos, a média de pontuação na escala de APACHE II foi de 17 pontos e a média de pontuação da escala SOFA foi de 7 pontos. Foram diagnosticadas 8 pneumonias e 52 ICS, 47% foram ICS primárias, entre estas 13% foram ICS relacionadas ao cateter. Foram diagnosticadas também 40% ICS secundárias, sendo o principal foco pulmonar (18%). 27% das infecções foram polimicrobianas. Os fatores de risco independentes associados à mortalidade nos primeiros 14 dias identificados na análise multivariada foram, pontuação maior que 6 no índice SOFA (RR=18,9. IC95%=2,4-146,2) e presença de choque séptico (RR=11,6. IC95%=1,3-105,9). O fator de risco associado à mortalidade global na análise multivariada foi, pontuação maior que 6 no índice SOFA (RR=37,1. IC95%=2,8-494,3). A instituição de terapia antimicrobiana inadequada para o tratamento das infecções por S. maltophilia foi freqüente, sendo observada em 40 (85%) pacientes, principalmente por atraso no inicio e por tempo curto de tratamento. Não houve diferença estatística quando comparado o tratamento adequado do tratamento inadequado. A curva de sobrevida de Kaplan-Meier mostrou que pacientes com APACHE II >20 e SOFA > 10 tinham respectivamente uma chance de sobrevida menor que 8% e menor que 10% (P=<0.001) até 21 dias após a primeira cultura positiva. A mortalidade global foi de 75% e a mortalidade nos primeiros 14 dias de infecção foi de 38%. Estes resultados mostram que infecções por S. maltophilia acontecem em pacientes gravemente doentes com múltiplos fatores de risco e que os fatores associados à mortalidade são principalmente relacionados ao estado da condição clínica de base e gravidade do paciente. / Paez JIG. Factors associated with mortality in Nosocomial infections with Stenotrophomonas maltophilia [dissertation]. São Paulo: ?Faculdade de Medicina, Universidade de São Paulo?; 2007. 154p. Bloodstream infections (BSI) and pneumonia caused by Stenotrophomonas maltophilia are associated with high mortality. A retrospective study with 60 patients with nosocomial BSI and pneumonia was done at the Instituto Central do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (ICHC-FMUSP). Cases were selected from July 30, 1999 through July 30, 2005. Risk factors associated with overall mortality and 14-day mortality after the onset of infection were accessed. 57 (85%) patients had received previous antimicrobial therapy, 88% had CVC, 57% mechanical ventilation and 75% stay in intensive care unit in the onset of infection. Malignancy (45%) was the most frequent underlying diseases. From 60 patients, 30% had septic shock, the mean of APACHE II score was 17 points and the mean of SOFA index was 7 points. A total of 60 infections were identified. Among these, 8 were pneumonias and 52 BSI, that for this turn, 33% were primary BSI, 13% were CVC-related and 40% secondary. 35% of the infections were polymicrobial. Risk factors associated with 14-day mortality after the onset of infections in the multivariate analysis were, SOFA index > 6 points (RR=18.9. 95%CI=2.4-146.2) and septic shock (RR=11.6. 95%IC=1.3-105.9). Risk factor associated with overall mortality was SOFA index > 6 points, (RR=37.1. 95%IC=2.8-494.3). Used of inappropriate antimicrobial therapy was observed in 40 (85%) patients, of whom received therapy with more 72 hours and received therapy for an insufficient length of time and there was no difference when compared appropriated and non-appropriated therapy. Kaplan-Meier estimations curves showed that patients with APACHE II >20 and SOFA > 10 had respectively a survival chance less than 8% and less than 10% (P=<0.001) at 21 days after the first positive S. maltophilia culture. The overall mortality and 14-day mortality after the onset of infections rates were 75% and 38% respectively. Our results showed that infections caused by S. maltophilia occur in critically ill patients with multiple risk factors and that the most important risk factors associated with mortality are the initial clinical condition and severity of diseases.

<p dir="ltr"><i>Stenotrophomonas maltophilia</i> is an emerging multidrug-resistant, Gram-negative opportunistic pathogen. It causes many hospital-acquired infections such as sepsis, endocarditis, meningitis, and catheter-related urinary tract infections. It also affects individuals with cystic fibrosis, exacerbating their lung condition. <i>S. maltophilia</i> often causes pathogenesis through the formation of biofilms. However, the molecular mechanisms <i>S. maltophilia</i> uses to carry out these pathogenic steps are unclear. The SMF-1 chaperone/usher pilus has been thought to mediate <i>S. maltophilia</i> attachment. To confirm this role, we created an isogenic deletion of the <i>smf-1</i> pilin gene and observed a defect in biofilm compared to wild type. We also discovered 2 additional chaperone/usher pilus operons, mutation of which also caused attenuation in biofilm levels. Analysis of <i>S. maltophilia</i> clinical strains and <i>S. maltophili</i><i>a</i> complete genomes listed in NCBI showed that these three pili are prevalent and highly conserved, suggesting a vital role in infection. Intriguingly, through TEM studies, we found that the mutation of one pilus is not phenotypically compensated by another. Infection of <i>Galleria mellonella</i> larvae revealed increased virulence of the pilus mutants. Additionally, we also demonstrated a relationship between pilus and flagella contributing to the overall biofilm development of <i>S. maltophilia</i>. Understanding their activity may help identify therapeutic targets for this pathogen.</p>

Thesis (Ph. D.)--Miami University, Dept. of Chemistry and Biochemistry, 2004. / Title from second page of PDF document. Includes bibliographical references.

Thesis (Ph. D.)--Miami University, Dept. of Chemistry and Biochemistry, 2004. / Title from second page of PDF document. Includes bibliographical references.

Master of Science / Division of Biology / Michael A. Herman / Stenotrophomonas maltophilia is an opportunistic bacterial pathogen found ubiquitously in the environment. Although S. maltophilia is an emerging pathogen associated with hospital-acquired infections in patients with respiratory diseases, particularly cystic fibrosis, very little is known about its mechanism of pathogenesis in any system. In addition, S. maltophilia isolates vary in pathogenicity to several hosts and are genetically diverse, including variation in virulence factors. In this thesis, I address the genetic basis of S. maltophilia pathogenesis from both host and bacterial perspectives. Our lab has previously developed Caenorhabditis elegans as a model for S. maltophilia infection. Stenotrophomonas is found in relatively high abundance in the microbiome of C. elegans, making it a suitable platform for studying S. maltophilia-host interactions. I performed a transcriptomic analysis to determine C. elegans responses to several S. maltophilia strains of varying pathogenicity. Treatments included K279a, an avirulent clinical isolate, JCMS, a virulent environmental strain isolated in association with nematodes near Manhattan, KS, and JV3, an even more virulent environmental isolate. Overall, I found that most genes (89%) that are differentially expressed in response to pathogenic S. maltophilia strains are upregulated, with many even further upregulated in response to the more virulent strain, JV3. Using information from a variety of transcriptomic datasets, I found that most of these genes are also commonly differentially expressed in C. elegans in response to other pathogens. Many more genes were differentially expressed specifically in response to JV3 when compared to all other strains (221 genes) than JCMS as compared to all other strains (14 genes), suggesting JV3 has unique virulence mechanisms that could explain its observed increased virulence. Candidate genes were chosen from the above differentially expressed gene sets (differentially expressed in response to both pathogenic S. maltophilia strains or in a strain-specific manner) for functional analysis. Mutational analysis of these candidate genes revealed that several mutants caused increased susceptibility of C. elegans to pathogenic S. maltophilia, regardless of the strain(s) that caused differential expression of that gene. Furthermore, many of these mutants also caused increased susceptibility to K279a, suggesting that K279a may also employ virulence mechanisms that wild-type C. elegans are able to defend against. To address the pathogen side of the interaction, we analyzed draft assemblies of the S. maltophilia strains, with the addition of another slightly pathogenic environmental strain, R551-3. We hypothesized that differences in observed pathogenicity and host responses to strains of S. maltophilia could be explained by differences in their genomes. When comparing draft assemblies to their respective reference genomes, few differences were observed. However, several genomic features were present in some strains and absent in others, including components of the CmeABC efflux pump and the Type IV secretion system, that might play a role in different virulence mechanisms. Genome-wide comparison of shared and unique genetic features across many S. maltophilia strains revealed that most S. maltophilia genes are strain-specific, suggesting that many potential virulence factors are unique and have yet to be functionally analyzed. Overall, variation in observed pathogenicity, differences in host transcriptional responses, and comparative genomics of S. maltophilia strains reveal that strain-specific mechanisms play important roles in S. maltophilia pathogenesis.

Doctor of Philosophy / Biology / Michael A. Herman / Nematodes play an important role in various habitats where numerous factors serve to shape their communities. One such factor is the potentially pathogenic nematode-prey interaction. This project is focused on the elucidation of the genes that the bacterivorous nematode Caenorhabditis elegans employs to respond to the emerging nosocomial bacterial pathogen Stenotrophomonas maltophilia. A virulent S. maltophilia strain JCMS requires the action of several C. elegans conserved innate immune pathways that serve to protect the nematode from other pathogenic bacteria. However, insulin-like DAF-2/16 signaling pathway mutants that are typically pathogen resistant are susceptible to JCMS, and several DAF-2/16 regulated genes are not significantly differentially expressed between JCMS and avirulent E. coli OP50. We have determined the complete set of mRNA transcripts under different bacterial treatments to identify genes that might explain this JCMS specific DAF-2/16 pathway evasion. The identified set included 438 differentially expressed transcripts among pairwise comparisons of wild-type nematodes fed OP50, JCMS or avirulent S. maltophilia K279a. Candidate genes were nominated from this list of differentially expressed genes using a probabilistic functional connection model. Six of seven genes that were highly connected within a gene network generated from this model showed a significant effect on nematode survival by mutation. Of these genes, C48B4.1, mpk-2, cpr-4, clec-67 and lys-6 are needed for combating JCMS, while dod-22 was solely involved in K279a response. Only dod-22 had a documented role in innate immunity, which merits our approach in the identification of gene candidates. To a lesser extent, we have also focused on the identification of virulence factors and the mode of action employed by S. maltophilia. JCMS virulence requires rpfF, xps and involves living bacteria that accumulate in the intestinal lumen. Additionally, the bacterial secretion encoding genes cs, p773, p1176, pi1y1 and xdi are involved in JCMS evasion of daf-2. In summary, we have discovered a novel host-pathogen interaction between C. elegans and S. maltophilia JCMS, revealed genes that are involved in each partner of the interaction, and established a new animal model for the study of S. maltophilia mode of action.

No description available.