Dissertations / Theses on the topic 'Salmonella typhimurium LT 2'

O-Acetylserine Sulfhydrylase (OASS) is a pyridoxal phosphate enzyme that catalyzes the reaction of O-acetyl-Lserine with sulfide to give L-cysteine. OASS is present as two isoforms, designated -A and -B. The kinetic mechanism of OASS-A is well known and there is also much known concerning the acid-base chemistry of the enzyme. However, little is known concerning the location of the rate determining steps, the sequencing of chemical steps that occur at the active site, or the nature of the rate determining transition states. The studies performed to help elucidate these aspects of the OASS-A mechanism included determination of the thermodynamics of both half reactions, along with studies utilizing substrate analogs of OAS halting the reaction at specific points along the reaction pathway allowing the identification of reaction intermediates. The free energy change of the first half reaction was shown to be -5.7 Kcal/mole while the second half reaction was shown to be, for all intents and purposes, irreversible. Intermediates along the reaction pathway that have been previously identified include the internal Schiff base and the a-aminoacrylate. The external Schiff base was identified using the analogs cysteine, alanine, and glycine while the geminal diamine was identified using the analog serine. Formation of the external aldimine was shown to be pH dependent with a pK of 8.1 ± 0.3 most likely representing a general base that accepts a proton from the a-amine of cysteine to facilitate a nucleophilic attack on C4r of the PLP imine. Formation of the geminal diamine was also shown to be pH dependent with two pK values having an average value of 8.1. One of the groups most likely represents the general base which accepts a proton from the a-amine of cysteine while the second group likely interacts with the amino acid side chain to orientate the amino acid into the correct configuration.

Šiame tiriamajame darbe nagrinėjama bakterijos Salmonella enterica Serovar Typhimurium inkubacijos priklausomybė nuo inkubacinio periodo. Trumpai aptariami bakterijų inaktyvavimą aprašantys dažniausiai naudojami modeliai, skaitiškai išreiškiama lag fazė, randama jos ilgį aprašantį funkciją. Toliau vertinamos tiesinė ir liekamoji dalys bei išvedama inaktyvavimą aprašanti lygtis. Darbas baigiamas išvestinės formulės praktiniu panaudojimu ir rezultatų aptarimu.
Evaluation of Salmonella enterica Serovar Typhimurium inactivation by photosensitization and impact modeling The aim goal of this research is to evaluate the influence of irradiation of UV light and incubation period on Salmonella enterica Serovar Typhimurium bacteria. Shortly discussed most commonly used mathematical models of bacterial inactivation, expressed lag phase and its function. Next step is evaluation of line part and tail of inactivation (mortality) curve. At the end of the research the inactivation formula is deduced and the results are discussed.

Salmonella enterica serovar Typhimurium (S. Typhimurium), an intracellular pathogen, causes gastroenteritis in humans and a systemic disease in mice. The ability of Salmonella to replicate inside host cells requires translocation of effector proteins across the vacuolar membrane, mediated by the Salmonella pathogenicity island‐2 (SPI‐2) type three secretion system (T3SS). However, the repertoire of effectors involved in this process has not been defined. The first part of this PhD work focused on SrfJ, a putative effector of the SPI‐2 T3SS with similarity to human lysosomal glucosylceramidase. Expression of its gene was dependent on SsrA/B, a two‐component regulatory system required for expression of most SPI‐2 effector genes. Expression of srfJ was also shown to occur under SPI‐2 T3SS activation conditions. However, there was no detectable secretion or translocation of the protein, although a srfJ mutant strain had an intracellular replication defect in primary bone marrow‐derived macrophages. Using a dual‐fluorescence reporter system that allows direct measurement of intracellular replication, the contribution to replication of individual SPI‐2 T3SS effectors was investigated. The replication kinetics of S. Typhimurium deletion mutants for all known SPI‐2 effectors were measured and compared in mouse bone marrow‐derived macrophages. Several mutant strains with replication defects were identified, thereby revealing that intracellular replication is the result of the contribution of numerous effectors. Two S. Typhimurium polymutant strains were generated whose replication defects closely resemble that of a SPI‐2 T3SS null mutant and are severely attenuated in virulence in vivo. These strains retained an intact T3SS and delivered a CD8+ T cell epitope via the SPI‐2 T3SS into the cytoplasm of infected cells. Since an S. Typhi mutant strain lacking the SPI‐2 T3SS has been shown to be safe and immunogenic in humans, these polymutant strains could have applications in vaccine design, as carrier strains for the delivery of heterologous antigens.

Seminario de Título entregado a la Universidad de Chile en cumplimiento parcial de los requisitos para optar al Título de Ingeniero en Biotecnología Molecular.
La 4-amino-5-hidroximetil-2-metilpirimidina quinasa (HMPK, EC 2.7.1.49) es una enzima perteneciente a la superfamilia riboquinasa y participa en la biosíntesis de tiamina (vitamina B1) en bacterias. Se ha descrito que esta enzima es capaz de catalizar dos fosforilaciones consecutivas dependientes de ATP altamente específicas sobre el sustrato hidroximetil pirimidina (HMP), generando como producto hidroximetil pirimidina pirofosfato. Esto contrasta notablemente con lo que se ha observado en las piridoxal quinasas de bacterias Gram positivas (HMPK/PLK, EC 2.7.1.35), un grupo de enzimas homólogas cercanas capaces de fosforilar hidroximetil pirimidina, piridoxal, piridoxina y piridoxamina, pero incapaces de catalizar dos fosforilaciones consecutivas, por lo que sólo producen hidroximetil pirimidina fosfato. Las HMPKs no han sido estudiadas tan exhaustivamente como las HMPK/PLKs y sólo hay dos caracterizaciones breves disponibles en la literatura; la de la HMPK de Escherichia coli y la de Bacillus subtilis. Por lo tanto, aún no se conoce si las propiedades observadas en las enzimas descritas son ubicuas para linajes bacterianos distintos, especialmente aquellos filogenéticamente distantes y que han sido sometido a presiones selectivas fuertes, como los extremófilos. Por esta razón, en este trabajo se realizó la caracterización bioquímica de la HMPK de la enterobacteria Salmonella typhimurium (StHMPK) y de la bacteria termófila Thermus thermophilus (TtHMPK). A través de experimentos de estequiometría de reacción y análisis de generación de productos, se demostró que ambas enzimas son capaces de catalizar dos fosforilaciones consecutivas. Experimentos de especificidad de sustrato revelaron que ambas enzimas son altamente específicas por hidroximetil pirimidina. Análisis filogenéticos mostraron que estas enzimas están estrechamente relacionadas con las HMPKs/PLK de organismos gram positivos, y estas últimas parecen ser descendientes directos de las HMPKs. Por lo tanto, para estudiar cómo estos grupos de enzimas han divergido en términos de sus actividades catalíticas, se realizaron simulaciones de dinámica molecular del complejo ternario (Mg·ATP - HMP) de StHMPK, para analizar el sitio de unión a sustrato y compararlo con el de la HMPK/PLK de Staphylococcus aureus (SaPLK). Los resultados mostraron que existe un alto grado de conservación entre ambos sitios, existiendo sólo unas pocas diferencias que podrían explicar la divergencia funcional observada, principalmente la presencia de una treonina adyacente a la base catalítica en StHMPK, que es reemplazada por una alanina en SaPLK, y la presencia de una glutamina en StHMPK que forma puentes de hidrógeno con el HMP. La caracterización cinética de StHMPK y TtHMPK mostró que ambas enzimas poseen una KM similar para HMP (cercana a 30μM) y que la Vmax para TtHMPK es un orden de magnitud menor que para StHMPK a 37 °C. Sin embargo, estos parámetros fueron obtenidos para las curvas de saturación de HMP, las cuales mostraban un comportamiento del tipo Michaelis-Menten, mientras que las curvas de saturación para ATP mostraron una clara desviación de este modelo y por lo tanto, no se pudieron determinar parámetros cinéticos. Finalmente, se realizó una caracterización estructural y biofísica para evaluar diferencias de estabilidad. Ambas enzimas parecen ser monómeros en las condiciones estudiadas, a diferencia de lo reportado para la enzima de E. coli que forma un tetrámero. Experimentos de desplegamiento por temperatura y agentes químicos mostraron que TtHMPK es significativamente más estable que StHMPK. Las bases estructurales de estas diferencias fueron analizadas mediante simulaciones de dinámica molecular, las que revelaron que la proteína termoestable es más rígida, tiene un menor contenido de residuos polares en el núcleo y tiene mayor cantidad de interacciones electrostáticas que su homólogo mesoestable.
4-amino-5-hydroxymethyl-2-methylpyrimidine kinase (HMPK, EC 2.7.1.49) is a bacterial enzyme that belongs to the ribokinase superfamily and participates in the thiamine (vitamine B1) biosynthetic pathway. It has been described that this enzyme is capable to catalyze two consecutive highly specific ATP dependent phosphorylations on the substrate hydroxymethyl pyrimidine, yielding hydroxymethyl pyrimidine pyrophosphate. This contrast notoriously with what has been observed for the closely related homologous enzymes pyridoxal kinases from Gram positive bacteria (HMPK/PLK, EC 2.7.1.35), which can phosphorylate hydroxymethyl pyrimidine, pyridoxal, pyridoxine and pyridoxamine, but are unable to catalyze two consecutive phosphorylations, thus only produce hydroxymethyl pyrimidine phosphate. HMPKs have not been as extensively studied as HMPKs/PLK, and only two brief biochemical characterizations are available on the literature; the characterization of the HMPK from Escherichia coli and from Bacillus subtilis. Therefore, it is still unknown whether the properties observed in the described enzymes are ubiquitous among different bacterial lineages, especially those that come from a very distinct phylogenetic background and have been subject to strong selective pressures, as the enzymes from extremophilic organisms. For this reason, in this work we address the biochemical characterization of the HMPK from the enterobacteria Salmonella typhimurium (StHMPK) and the thermophilic bacteria Thermus thermophilus (TtHMPK). Through stoichiometric experiments and product generation analysis, it was established that both enzymes are able to perform two consecutive phosphorylations. Substrate specificity experiments revealed that both enzymes are highly specific for hydroxymethyl pyrimidine. Phylogenetic analysis of these enzymes showed that are closely related to HMPKs/PLK from Gram positive organisms, being the later a direct descendant from HMPKs. Therefore, to study how these two groups of enzymes have diverged so much in terms of their catalytic activities, we analysed the substrate binding site of StHMPK by molecular dynamics simulations of the ternary complex (Mg·ATP - HMP) and compared it to the binding site of the PLK from Staphylococcus aureus (SaPLK). The results showed that there is an overall great conservation among the active sites, with just a few differences that could be responsible for the functional divergences observed, mainly the presence of a threonine residue adjacent to the catalytic base in StHMPK which is replaced by an alanine in SaPLK, and the presence of a glutamine that forms hydrogen bonds with the HMP in StHMPK. Kinetic characterization of StHMPK and TtHMPK showed that both enzymes have a similar KM for HMP (around 30 μM) while the Vmax for TtHMPK is one order of magnitude lower than the Vmax for StHMPK. However, these parameters were obtained only for HMP saturation curves, which showed a Michaelis-Menten behaviour, whereas ATP saturation curves displayed a clear deviation from a Michaelis-Menten model and therefore, no kinetic parameters could be deduced from these experiments. Finally, a biophysical and structural characterization to assess stability differences was performed. Both enzymes seem to be in monomeric state under the conditions assayed, in contrast with what was reported for the enzyme from E. coli, which forms a tetramer. Thermal and chemical unfolding experiments showed that TtHMPK is significantly more stable than StHMPK. The structural basis for these differences were investigated through molecular dynamics simulations, which revealed that the thermostable protein is more rigid, has a reduced content of polar amino acids in its core, and has more electrostatic interactions than its mesostable homologous.
Julio del 2019

Analiza el efecto de la lactoferrina en la cinética de crecimiento de Salmonella typhimurium cepa SL 1344 ΔhilA, evalúa el efecto citotóxico del tratamiento con gentamicina a células HEp-2, determina el efecto in vitro de la lactoferrina sobre la adhesión de Salmonella typhimurium SL 1344 ΔhilA y especifica el tratamiento y concentración de lactoferrina que permita mayor disminución de la adherencia e invasión a las células HEp-2.
Tesis

Herbicides are a common tool in weed control. With the introduction of genetically modified herbicide-tolerant crops, there has been a dramatic increase in the use of particular herbicides. Herbicides contaminate the environment and food and feed and can come into contact with non-target organisms, especially bacteria. Salmonella enteric serovar Typhimurium, which is a human and animal pathogen, was chosen to investigate if the commercial formulations of three herbicides – Kamba, 2,4-D and Roundup are toxic to bacteria and whether sub-lethal concentrations cause a response to antibiotics. In addition, earlier work demonstrating an effect of salicylic acid on antibiotic response was reconfirmed in this study. The herbicides were toxic to S. typhimurium at concentrations above the manufacturers recommended application rates. A key finding of this study was that when S. typhimurium was grown in sub-lethal concentrations of the herbicides, it demonstrated a change in its susceptibility to various antibiotics. Kamba and 2,4-D caused increased tolerance of chloramphenicol, tetracycline, ampicillin and ciprofloxacin and increased sensitivity to kanamycin. Exposure to Roundup caused increased sensitivity to chloramphenicol and tetracycline and increased tolerance towards kanamycin and ciprofloxacin. Roundup had no measureable affect on ampicillin susceptibility. The minimum concentrations of herbicides that induced an antibiotic response were within the recommended application rates. Furthermore, the minimum 2,4-D concentration that induced tetracycline, chloramphenicol and ampicillin tolerance was at or below the maximum residue limits set for food and feed commodities. Simultaneous exposure to an herbicide and an antibiotic was necessary for the induction of antibiotic tolerance. In addition, the effect of the herbicide on the antibiotic response was faster than the lethal effect of the antibiotics. Kamba induced chloramphenicol, tetracycline, ampicillin and ciprofloxacin tolerance was maintained in the absence of Kamba once tolerance was induced by simultaneous exposure to Kamba and antibiotic. The emergence of antibiotic tolerance is an important health issue that may compromise treatment of serious bacterial infections. The widespread use of herbicides in agricultural, urban and domestic settings increases the number of bacteria that are exposed to herbicides. The tolerance induced by the herbicides may increase the frequency of antibiotic tolerant strains, increase the chance of co-exposure to antibiotics, and increase the potential for failure to treat bacterial infections as a result.

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Foi conduzido um experimento utilizando-se 630 pintos com um dia de idade com o objetivo de avaliar os efeitos de ácidos orgânicos, frente à inoculação experimental de Salmonella Typhimurium sobre a saúde intestinal, desempenho, bacteriologia de órgãos e função hepática. As aves foram distribuídas em delineamento inteiramente ao acaso com seis tratamentos e sete repetições com 15 pintos cada. O desafio experimental com a bactéria ocorreu por duas vias de administração: via inglúvio, ao primeiro dia após eclosão, e via ração durante o período de sete a 14 dias de idade. Estes grupos foram tratados ou não com ácidos orgânicos, definindo-se desta forma, um esquema fatorial de 3x2 (agente versus ácidos orgânicos). Os pintos dos tratamentos preconizados para inoculação no primeiro dia de vida receberam via inglúvio, a dose de 5,0 x 102 /0,5mL unidades formadoras de colônias (UFC) de Salmonella Typhimurium. Os tratamentos com contaminação via ração, receberam o desafio na dosagem de 5,0 x 102 UFC de Salmonella Typhimurium/ kg de ração. O teste de Tukey (5%) foi utilizado para análises das variáveis. Os grupos tratados com ácidos orgânicos apresentaram melhores de ganho de peso, peso médio e conversão alimentar (p<0,05) aos 14, 21 e de ganho de peso e peso médio (p<0,05) aos 28 dias de idade. Os grupos inoculados com Salmonella Typhimurium apresentaram piores índices de desempenho (p<0,05) aos sete, 14 e 28 dias. O peso do intestino delgado foi maior (p<0,05) para os grupos inoculados quando comparado ao grupo controle, entretanto com comprimento menor para o mesmo fragmento intestinal. O tratamento inoculado via inglúvio e tratado com ácidos orgânicos apresentou menores valores (p<0,05) de unidades formadoras de colônia/g de Escherichia coli em excretas do que os grupos que foi comparado. O pH do conteúdo do conteúdo cecal e do intestino delgado não foi afetado (p>0,05) pela adição de ácido, enquanto o pH do intestino delgado dos grupos inoculados foi menor (p<0,05) quando comparado ao grupo controle negativo durante todo o período experimental. Também foi verificado menor peso (p<0,05) de fígado para os grupos controle negativo aos 21 e 28 dias. Os grupos tratados com ácido, independente da via de administração com Salmonella Typhimurium, obtiveram menores freqüências de isolamento em todos os órgãos analisados. Foram também observadas alterações (p<0,05) na bioquímica sérica hepática e na análise histopatológica do fígado pela atuação da Salmonella. Pode-se concluir que o ácido utilizado na dosagem de 0,4% potencializou o desempenho, foi eficaz no controle de Salmonella Typhimurium e não promoveu lesões hepáticas, quando da inoculação experimental de Salmonella Typhimurium ácido nalidíxico resistente.

Orientador: Yuji Takahata
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica
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Mestrado

Bacteria produce and respond to external stimuli using molecules termed autoinducers. Poultry meat contains inhibitors which interfere with AI-2 signaling. The primary objective of this work was to understand the effects of AI-2 on the virulence and growth of Salmonella Typhimurium, and if the introduction of AI-2 inhibiting compounds would influence these effects. Using DNA microarray analysis, expression of 1136 virulence-related genes in a Salmonella Typhimurium wild type and a luxS mutant strain, PJ002 (unable to produce AI-2), was monitored after exposure to treatments containing in vitro synthesized AI-2 (AI-2) and poultry meat (PM) inhibitors. Responding gene expression was unique in the presence of AI-2, with 23 genes differentially expressed at least 1.5-fold (p < 0.05). The combined AI-2 + PM treatment resulted in 22 genes being differentially expressed. Identification of inhibitory compounds was attempted using GC analysis on a hexane solvent extract obtained from a PM wash. From this analysis, chemical standards of linoleic, oleic, palmitic, and stearic acid were tested for inhibition using V. harveyi BB170. Combined fatty acids (FA) demonstrated inhibition against AI-2 at 60 % while 10-fold and 100-fold concentrations had inhibition of 84 % and 70 %, respectively. Growth of PJoo2, was studied using M-9 minimal medium with FA of varying concentrations, supplemented with either AI-2, or 1X phosphate buffered saline (PBS). Comparative analysis was done calculating the growth constants based on OD 600 values for each treatment. No significant difference in the combined FA + AI-2 treatments was observed against the AI-2 treatment. A significant increase in the growth rate constants of the AI-2 treatments was observed, however, compared to the PBS control (P = 0.01). Bacterial invasiveness, using a murine macrophage cell line, RAW 264.7, was also studied. AI-2 decreased cell invasiveness (P = 0.02), while the addition of combined FA improved invasiveness to normal levels. The results of these studies indicate that AI-2 does have an effect on the growth and virulence of Salmonella, but this is not uniformly modulated by the introduction of fatty acids, that inhibit AI-2 activity, suggesting that inhibition may be based on species specific transport systems.

碩士
國防醫學院
微生物及免疫學研究所
92
The objects of this study contains three fold; firstly, to investigate the expression of NS1 protein of Dengue virus via a Caf1 cherapone/usher secretion pathway; secondly, to study the immune response to NS1:Caf1 fusion protein delivered by attenuated Salmonella typhimurium SL1344 strain in a murine infection model; and thirdly, to determine whether protein priming-oral Salmonella boosting immunization against the Dengue NS1 antigen could induce a protective immune response against Dengue virus challenge. Our results demonstrated that NS1 antigen of Dengue virus could be successfully expressed as a NS1:Caf1 fusion protein and secreted on the surface of the cells in both E.coli and Salmonella via Caf1 secretion system. In particular overexpression of Caf1A resulted in higher level of soluble NS1:Caf1 protein present in the extracellular compartment as compared to that of the non-induced one. This results indicated that Caf1A may also play a critical role in promotion of the assembly and folding of sCaf1 (signal sequence) targeting polypeptide, such as NS1:Caf1 fusion protein. However, mice administrated with single oral dose of S.typhimurium SL1344/pLT105 was not appeared to be eliciting high level of specific anti-NS1 serum titer. But it was significantly increased by 2-3 fold in the anti-NS1 serum titer when co-dosed with amphotericin B suggesting that amphotericin B was likely to be as a potential adjuvant for Salmonella oral vaccine. Furthermore, co-dosing with AmB and S. typhimurium SL1344/pLT105 mice obtained 70% protection against the Dengue virus challenge in comparison with that of 30 % for S.typhimurium SL1344/pLT105 alone. In addition mice immunized with a parenteral NS1 protein vaccine followed by an oral boosting with S. typhimurium SL1344/pLT105 could obtain better protection than by single immunized regime. Taken together the results clearly demonstrated that Dengue NS1 protein antigen could be expressed as a soluble NS1:Caf1 fusion protein and highly displayed on the cell surface in E. coli and Salmonella. Single oral dose with S. typhimurium SL1344/pLT105 could confer partial protection for mice against lethal dose of Dengue virus challenge. Protein priming-oral Salmonella boosting regime could provide an effective way to increasely elicit protective immune response to against Dengue virus challenge. As an adjuvant amphotericin B is able to augment the immune response for Salmonella oral vaccine.

碩士
國防醫學院
微生物及免疫學研究所
90
The Caf1 capsule protein is encoded by the Caf1 gene cluster of Yersinia pestis. It is secreted via the Caf1 M chaperone/Caf1 A usher pathway. We investigated the ability of this secretion system to facilitate secretion of dengue virus type 2 NS1 full-length protein fused to the Caf1 subunit in Escherichia coli and Salmonella enterica serovar Typhimurium aroA. The processed product consisting of mature NS1 and Caf1 remained insoluble in spite of correct processing of this chimeric protein. However, co-expression of this chimera with a functional Caf1 M chaperone led to the accumulation of soluble NS1:Caf1 in the periplasm, which can be released by osmotic shock. Souble NS1:Caf1 reacted with monoclonal antibody directed against structural epitopes of NS1 protein. The result suggest that Caf1 M-induced release of NS1:Caf1 from the plasma membrane promotes folding of the NS1 domain. In this system, gene encoding chimeric protein was created in which the NS1 was sandwiched between the Caf1 signal peptide and the mature Caf1 subunit, leaving the C terminus of the nature of the N-terminal heterologous protein, the Caf1 domain of the chimera remained free to interact with Caf1 M and that this interaction enhanced the solubility of the periplasmic NS1 protein. Following co-expressing of NS1:Caf1 with both Caf1 M chaperone and Caf1 A outer membrane protein. NS1:Caf1 fusion protein could be detected on the cell surface and in the culture broth of E. coli and Salmonella enterica serovar Typhimurium aroA . These results indicate the potential application of the Caf1 protein secretion system in the transport of NS1:Caf1 fusion protein.

I formally joined Prof. M. R. N. Murthy’s laboratory at the Molecular Biophysics Unit, Indian institute of Science, on 1st August 2001. During that time, the interest in the laboratory was mainly focused on structural studies on a number of capsid mutants of two plant viruses, sesbania mosaic virus and physalis mottle virus, to gain an insight into the virus structure and its assembly. Besides these two projects, there were a few other collaborative projects running in the lab at that time such as NIa protease from pepper vein banding virus and diaminopropionate ammonia lyase from Escherichia coli with Prof. H. S. Savithri, triosephosphate isomerase from Plasmodium falciparum with Prof. P. Balaram and Prof. H. Balaram and a DNA binding protein (TP2) with Prof. M. R. S. Rao. During my first semester, along with my course work, I was assigned to make an attempt to purify and crystallize recombinant NIa protease and TP2 protein. I started with NIa protease which could be purified using one step Ni-NTA affinity column chromatography. Although the expression and protein yield were reasonably good, protein precipitated with in a couple of hours after purification. Attempts were made to prevent the precipitation of the purified enzyme and towards this end we were successful to some extent. However, during crystallization trials most of the crystallization drops precipitated completely even at low protein oncentration. TP2 protein was purified using three-step chromatographic techniques by one of the project assistant in Prof. M. R. S. Rao’s laboratory. Because of low expression level and three step purification protocol, protein yield was not good enough for complete crystallization screening. Hits obtained from our initial screening could not be confirmed because of low protein yield as well as batch to batch variation. My attempts to crystallize these two proteins remained unsuccessful but in due course I had learnt a great deal about the tips and tricks of expression, purification and mainly crystallization. To overcome the problems faced with these two proteins, we decided to make some changes in the gene construct and try different expression systems. By this time (beginning of 2002), I had finished my first semester and a major part of the course work, so we decided to start a new project focusing on some of the unknown enzymes from a metabolic pathway. Dr. Parthasarathy, who had finished his Ph. D. from the lab, helped me in literature work and in finding targets for structural studies. Finally, we decided to target enzymes involved in the propionate etabolism. The pathways for propionate metabolism in Escherichia coli as well as Salmonella typhimurium were just established and there were no structural information available for most of the enzymes involved in these pathways. Since, propionate metabolic pathways were well described in the case of Salmonella typhimurium, we decided to use this as the model organism. We first started with the enzymes present in the propionate catabolic pathway “2-methylcitrate pathway”, which converts propionate into pyruvate and succinate. 2-methylcitrate pathway resembles the well-studied glyoxylate and TCA cycle. Most of the enzymes involved in 2-methylcitrate pathway were not characterized biochemically as well as structurally. First, we cloned all the four enzymes PrpB, PrpC, PrpD and PrpE present in the prpBCDE operon along with PrpR, a transcription factor, with the help of Dr. P.S. Satheshkumar from Prof. H. S. Savithri’s laboratory. Since these five proteins were cloned with either N- or C-terminal hexa-histidine tag, they could be purified easily using one-step Ni-NTA affinity column chromatography. PrpB, PrpC and PrpD had good expression levels but with PrpE and PrpR, more than 50% of the expressed protein went into insoluble fraction, probably due to the presence of membrane spanning domains in these two enzymes. Around this time, crystallization report for the PrpD from Salmonella was published by Ivan Rayment’s group, so after that we focused only on the remaining four proteins leaving out PrpD. Our initial attempts to crystallize these proteins became successful in case of PrpB, 2-methylisocitrate lyase. We collected a complete diffraction data to a resolution of 2.5 Å which was later on extended to a resolution of 2.1 Å using another crystal. Repeated crystallization trials with PrpC also gave small protein crystals but they were not easy to reproduce and size and diffraction quality always remained a problem. Using one good crystal obtained for PrpC, data to a resolution of 3.5 Å could be collected. Unfortunately, during data collection due to failure of the cryo-system, a complete dataset could not be collected. Further attempts to crystallize this protein made by Nandashree, one of my colleagues in the lab at that time, was also without much success. Attempts to purify and crystallize PrpE and PrpR were made by me as well as one of my colleagues, Anupama. In this case, besides crystallization, low expression and precipitation of the protein after purification were major problems. Our attempt to phase the PrpB data using the closest search model (phosphoenolpyruvate mutase) by molecular replacement technique was unsuccessful,probably because of low sequence identity between them (24%). Further attempts were made to obtain heavy atom derivatives of PrpB crystal. We could obtain a mercury derivative using PCMBS. However, an electron density map based on this single derivative was not nterpretable. Around this time, the structure of 2-methylisocitrate lyase (PrpB) from E. coli was published by Grimm et. al. The structure of Salmonella PrpB could easily be determined using the E. coli PrpB enzyme as the starting model. We also solved the structure of PrpB in complex with pyruvate and Mg2+. Our attempts to crystallize PrpB with other ligands were not successful. Using the structures of PrpB and its complex with pyruvate and Mg2+, we carried out comparative studies with the well-studied structural and functional homologue, isocitrate lyase. These studies provided the plausible rationale for different substrate specificities of these two enzymes. Due to unavailability of PrpB substrate commercially and the extensive biochemical and mutational studies carried out by two different groups made us turn our attention to other enzymes in this metabolic pathway. Since our repeated attempts to obtain good diffraction quality crystals of PrpC, PrpE and PrpR continued to be unsuccessful, we decided to target other enzymes involved in propionate metabolism. We looked into the literature for the metabolic pathways by which propionate is synthesized in the Salmonella typhimurium and finally decided to target enzymes present in the metabolic pathway which converts L-threonine to propionate. Formation of propionate from L-threonine is the most direct route in many organisms. During February 2003, we initiated these studies with the last enzyme of this pathway, propionate kinase (TdcD), and within a couple of months we could obtain a well-diffracting crystal in complex with ADP and with a non-hydrolysable ATP analog, AMPPNP. TdcD structure was solved by molecular replacement using acetate kinase as a search model. Propionate kinase, like acetate kinase, contains a fold with the topology βββαβαβα, identical with that of glycerol kinase, hexokinase, heat shock cognate 70 (Hsc70) and actin, the superfamily of phosphotransferases. Examination of the active site pocket in propionate kinase revealed a plausible structural rationale for the greater specificity of the enzyme towards propionate than acetate. One of the datasets of TdcD obtained in the presence of ATP showed extra continuous density beyond the γ-phosphate. Careful examination of this extra electron density finally allowed us to build diadenosine tetraphosphate (Ap4A) into the active site pocket, which fitted the density very well. Since the data was collected at a synchrotron source to a resolution of 1.98 Å, we could identify the ligand in the active site pocket solely on the basis of difference Fourier map. Later on, co-crystallization trials of TdcD with commercially available Ap4A confirmed its binding to the enzyme. These studies suggested the presence of a novel Ap4A synthetic activity in TdcD, which is further being examined by biochemical experiments using mass-spectrometry as well as thin-layer chromatography experiments. By the end of 2004, we shifted our focus to the first enzyme involved in the anaerobic degradation of L-threonine to propionate, a biodegradative threonine deaminase (TdcB). Sagar Chittori, who had joined the lab as an integrated Ph. D student, helped me in cloning this enzyme. My attempt to crystallize this protein became finally successful and datasets in three different crystal forms were collected. Dataset for TdcB in complex with CMP was collected during a synchrotron trip to SPring8, Japan by my colleague P. Gayathri and Prof. Murthy. TdcB structure was solved by molecular replacement using the N-terminal domain of biosynthetic threonine deaminase as a search model. Structure of TdcB in the native form and in complex with CMP helped us to understand several unanswered questions related to ligand mediated oligomerization and enzyme activation observed in this enzyme. The structural studies carried out on these three enzymes have provided structural as well as functional insights into the catalytic process and revealed many unique features of these metabolic enzymes. All these have been possible mainly due to proper guidance and encouragement from Prof. Murthy and Prof. Savithri. Prof. Murthy’s teaching as well as discussions during the course of investigation has helped me in a great deal to learn and understand crystallography. Collaboration with Prof. Savithri kept me close to biochemistry and molecular biology, the background with which I entered the world of structural biology. The freedom to choose the project and carry forward some of my own ideas has given me enough confidence to enjoy doing research in future.

Three-dimensional structures of proteins provide insights into the mechanisms of macromolecular assembly, enzyme catalysis and mode of activation, substrate-specificity, ligand-binding properties, stability and dynamical features. X-ray crystallography has become the method of choice in structural biology due to the remarkable methodological advances made in the generation of intense X-ray beams with very low divergence, cryocooling methods to prolong useful life of irradiated crystals, sensitive methods of Xray diffraction data collection, automated and fast methods for data processing, advances and automation in methods of computational crystallography, comparative analysis of macromolecular structures along with parallel advances in biochemical and molecular biology methods that allow production of the desired biomolecule in quantities sufficient for X-ray diffraction studies. Advances in molecular biology techniques and genomic data have helped in identifying metabolic pathways responsible for metabolism of short-chain fatty acids (SCFAs). The primary objective of this thesis is application of crystallographic techniques for understanding the structure and function of enzymes involved in the metabolism of SCFAs in S. typhimurium. Pathways chosen for the present study are (i) propionate degradation to pyruvate and succinate by 2-methylcitrate pathway involving gene products of the prp operon, (ii) acetate activation to acetyl-CoA by AckA-Pta pathway involving gene products of the ack-pta operon, (iii) threonine degradation to propionate involving gene products of the tdc operon, (iv) 1,2-propanediol (1,2-PD) degradation to propionate involving gene products of the pdu operon. These metabolic pathways utilize a large number of enzymes with diverse catalytic mechanisms. The main objectives of the work include structural and functional studies on 2-methycitrate synthase (PrpC), acetate kinase (AckA), propionate kinase isoforms (PduW and TdcD) and propanol dehydrogenase (PduQ) from S. typhimurium. In the present work, these proteins were cloned, expressed, purified and characterized. The purified proteins were crystallized using standard methods. The crystals were placed in an X-ray beam and diffraction data were collected and used for the elucidation of structure of the proteins. The structures were subjected to rigorous comparative analysis and the results were complemented with suitable biochemical and biophysical experiments. The thesis begins with a review of the current literature on SCFAs metabolism in bacteria, emphasizing studies carried out on S. typhimurium and the closely related E. coli as well as organisms for which the structure of a homologue has been determined (Chapter 1). Metabolic pathways involving acetate utilization by activation to acetyl- CoA, propionate degradation to pyruvate and succinate, anaerobic degradation of Lthreonine to propionate and, 1,2-PD degradation to propionate are described in this chapter. Common experimental and computational methods used during the course of investigations are described in Chapter 2, as most of these are applicable to all structure determinations and analyses. Experimental procedures described here include cloning, overexpression, purification, crystallization and intensity data collection. Computational methods covered include details of various programs used during data processing, structure solution, refinement, model building, validation and structural analysis. In Chapter 3, X-ray crystal structure of S. typhimurium 2-methylcitrate synthase (StPrpC; EC 2.3.3.5) determined at 2.4 Å resolution and its functional characterization is reported. StPrpC catalyzes aldol-condensation of oxaloacetate and propionyl-CoA to 2- methylcitrate and CoA in the second step of 2-methylcitrate pathway. StPrpC forms a dimer in solution and utilizes propionyl-CoA more efficiently than acetyl-CoA or butyryl- CoA. The polypeptide fold and the catalytic residues of StPrpC are conserved in citrate synthases (CSs) suggesting similarities in their functional mechanisms. Tyr197 and Leu324 of StPrpC are structurally equivalent to the ligand binding residues His and Val, respectively, of CSs. These substitutions might be responsible for the specificities for acyl-CoAs of these enzymes. Structural comparison with the ligand free (open) and bound (closed) states of CSs showed that StPrpC represents the first apo structure among xvi CS homologs in a nearly closed conformation. StPrpC molecules were organized as decamers, composed of five identical dimer units, in the P1 crystal cell. Higher order oligomerization of StPrpC is likely to be due to high pH (9.0) of the crystallization condition. In gram-negative bacteria, a hexameric form, believed to be important for regulation of activity by NADH, is also observed. Structural comparisons with hexameric E. coli CS suggested that the key residues involved in NADH binding are not conserved in StPrpC. Structural and functional studies on S. typhimurium acetate kinase (StAckA; EC 2.7.2.1) are described in Chapter 4. Acetate kinase, an enzyme widely distributed in the bacteria and archaea domains, catalyzes the reversible phosphoryl transfer from ATP to acetate in the presence of a metal ion during acetate metabolism. StAckA catalyzes Mg2+ dependent phosphate transfer from ATP to acetate 10 times more efficiently when compared to propionate. Butyrate was found to inhibit the activity of the enzyme. Kinetic analysis showed that ATP and Mg2+ could be effectively substituted by other nucleoside 5′-triphosphates (GTP, UTP and CTP) and divalent cations (Mn2+ and Co2+), respectively. The X-ray crystal structure of StAckA was determined in two different forms at 2.70 Å (Form-I) and 1.90 Å (Form-II) resolutions, respectively. StAckA contains a fold with the topology βββαβαβα, similar to those of glycerol kinase, hexokinase, heat shock cognate 70 (Hsc70) and actin. StAckA consists of two domains with an active site cleft at the domain interface. Comparison of StAckA structure with those of ligand complexes of other acetokinase family proteins permitted the identification of residues essential for substrate binding and catalysis. Conservation of most of these residues points to both structural and mechanistic similarities between enzymes of this family. Examination of the active site pocket revealed a plausible structural rationale for the greater specificity of the enzyme towards acetate than propionate. Intriguingly, a major conformational reorganization and partial disorder in a large segment consisting of residues 230-297 of the polypeptide was observed in Form-II. Electron density corresponding to a plausible xvii citrate was observed at a novel binding pocket present at the dimeric interface. Citrate bound at this site might be responsible for the observed disorder in the Form-II structure. A similar ligand binding pocket and residues lining the pocket were also found to be conserved in other structurally known enzymes of acetokinase family. These observations and examination of enzymatic reaction in the presence of citrate and succinate (tricarboxylic acid cycle intermediates) suggested that binding of ligands at this pocket might be important for allosteric regulation in this family of enzymes. Propionate kinase (EC 2.7.2.15) catalyzes reversible conversion of propionylphosphate and ADP to propionate and ATP. S. typhimurium possess two isoforms of propionate kinase, PduW and TdcD, involved in 1,2-propanediol degradation to propionate and in L-threonine degradation to propionate, respectively. In Chapter 5, structural and functional analyses of PduW and TdcD, carried out to gain insights into the substrate-binding pocket and catalytic mechanism of these enzymes, are described. Both isoforms showed broad specificity for utilization of SCFAs (propionate > acetate), nucleotides (ATP ≈ GTP > UTP > CTP) and metal ions (Mg2+ ≈ Mn2+). Molecular modeling of StPduW indicated that the enzyme is likely to adopt a fold similar to other members of acetokinase family. The residues at the active site are well conserved. Differences in the size of hydrophobic pocket where the substrate binds, particularly the replacement of a valine residue in acetate kinases (StAckA: Val93) by an alanine in propionate kinases (StPduW: Ala92; StTdcD: Ala88), could account for the observed greater affinity towards their cognate SCFAs. Crystal structures of TdcD from S. typhimurium in complex with various nucleotides were determined using native StTdcD as the phasing model. Nucleotide complexes of StTdcD provide a structural rationale for the broad specificity of the enzyme for its cofactor. Binding of ethylene glycol close to the γ-phosphate of GTP might suggest a direct in-line transfer mechanism. The thesis concludes with a brief discussion on the future prospects of the work. xviii Projects carried out as part of Master of Science projects and as additional activity during the course of the thesis work are described in three appendices. Analysis of the genomic sequences of E. coli and S. typhimurium has revealed the presence of hpa operon essential for 4-hydroxyphenylacetate (4-HPA) catabolism. S. typhimurium hpaE gene encodes for a 55 kDa polypeptide (StHpaE; EC 1.2.1.60) which catalyzes conversion of 5-carboxymethyl-2-hydroxymuconic semialdehyde (CHMS) to 5-carboxymethyl-2-hydroxymuconic aldehyde (CHMA) in 4-HPA metabolism. Sequence analysis of StHpaE showed that it belongs to aldehyde dehydrogenase (ALDH) superfamily and possesses residues equivalent to the catalytic glutamate and cysteine residues of homologous enzymes (Appendix A). The gene was cloned in pRSET C expression vector and the recombinant protein was purified using Ni-NTA affinity chromatography. The enzyme forms a tetramer in solution and shows catalytic activity toward the substrate analog adipic semialdehyde. Crystal structure of StHpaE revealed that it contains three domains; two dinucleotide-binding domains, a Rossmann-fold type domain, and a small three-stranded β-sheet domain, which is involved in tetrameric interactions. NAD+-bound crystal of StHpaE permitted identification of active site pocket and residues important for ligand anchoring and catalysis. Mutarotases or aldose 1-epimerases (EC 5.1.3.3) play a key role in carbohydrate metabolism by catalyzing the interconversion of α- and β-anomers of sugars. S. typhimurium YeaD (StYeaD), annotated as aldose 1-epimerase, has very low sequence identity with other well characterized mutarotases. In Appendix B, the crystal structure of StYeaD determined in orthorhombic and monoclinic crystal forms at 1.9 Å and 2.5 Å resolutions, respectively are reported. StYeaD possesses a fold similar to those of galactose mutarotases (GalMs). Structural comparison of StYeaD with GalMs has permitted identification of residues involved in catalysis and substrate anchoring. In spite xix of the similar fold and conservation of catalytic residues, minor but significant differences in the substrate binding pocket were observed compared to GalMs. Therefore, the substrate specificity of YeaD like proteins seems to be distinct from those of GalMs. Pepper Vein Banding Virus (PVBV) is a member of the genus potyvirus and infects Solanaceae plants. PVBV is a single-stranded positive-sense RNA virus with a genome-linked viral protein (VPg) covalently attached at the 5'-terminus. In order to establish the role of VPg in the initiation of replication of the virus, recombinant PVBV VPg was over-expressed in E. coli and purified using Ni-NTA affinity chromatography (Appendix C). PVBV NIb was found to uridylylate Tyr66 of VPg in a templateindependent manner. Studies on N- and C-terminal deletion mutants of VPg revealed that N-terminal 21 and C-terminal 92 residues of PVBV VPg are dispensable for in vitro uridylylation by PVBV NIb.

Small proline-rich protein-2 (SPRR2) functions as a determinant of flexibility and permeability in the mature cornified envelope of the skin. SPRR2 is strongly upregulated by the commensal flora and may mediate signaling to differentiated epithelia of the small intestine and colon. Yet, SPRR2 function in the GI tract is largely unexplored. Using the Caco-2 model of intestinal epithelial differentiation along the crypt-villus axis, we hypothesized that SPRR2 would be preferentially expressed in post-confluent differentiated Caco-2 cells and examined SPRR2 regulation by the protein kinase A pathway (PKA) and short chain fatty acids (SCFAs). Differentiation-dependent SPRR2 expression was examined in cytoskeletal-, membrane-, and nuclear-enriched fractions by immunoblotting and confocal immunofluorescence. We studied the effect of SCFAs, known inducers of differentiation, on SPRR2 expression in pre-confluent undifferentiated Caco-2 cells and explored potential mechanisms involved in this induction using MAP kinase inhibitors. SPRR2 expression was also compared between HIEC crypt cells and 16 to 20 week primary fetal villus cells as well as in different segments in mouse small intestine and colon. We determined if SPRR2 is increased by gram negative bacteria such as S. typhimurium. SPRR2 expression increased in a differentiation-dependent manner in Caco-2 cells and was present in human fetal epithelial villus cells but absent in HIEC crypt cells. Differentiation-induced SPRR2 was down-regulated by 8-Br-cAMP as well as by forskolin/IBMX co-treatment. SPRR2 was predominantly cytoplasmic and did not accumulate in Triton X-100-insoluble cytoskeletal fractions. SPRR2 was present in the membrane- and nuclear-enriched fractions and demonstrated co-localization with F-actin at the apical actin ring. No induction was seen with the specific HDAC inhibitor trichostatin A, while SCFAs and the HDAC inhibitor SBHA all induced SPRR2. SCFA responses were inhibited by MAP kinase inhibitors SB203580 and U0126, thus suggesting that the SCFA effect may be mediated by orphan G-protein receptors GPR41 and GPR43. S. typhimurium induced SPRR2 in undifferentiated cells. We conclude that SPRR2 protein expression is associated with differentiated epithelia and is regulated by PKA signaling and by by-products of the bowel flora. This is the first report to establish an in vitro model to study the physiology and regulation of SPRR2.
Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2008-04-25 12:39:06.427
This work was funded by the CIHR GIDRU Training Grant and Aid in Research from Crohn's and Colitis Foundation of Canada