Determination of PFOR gene expression in strains of G. intestinalis with different inhibitory concentrations of metronidazole.

INTRODUCTION: Giardia intestinalis is the most important and common diarrhea-causing parasitic protozoa worldwide with growing clinical relevance in public health. There are many documented cases of G. intestinalis resistance to metronidazole (MZ). Pyruvate: ferredoxin oxidoreductase (PFOR), the membrane-localized enzyme, plays a key role in the development of resistance to drugs. The aim of the present study was to evaluate the difference in the levels of PFOR gene expression between MZ-resistant and MZ-susceptible strains of G. intestinatlis. METHODOLOGY: From 159 samples with G. intestinalis cysts, 48 strains were successfully cultivated. Using specific pair primers, PFOR gene expressions were estimated in different groups of Giardia. The polymerase chain reaction (PCR) data were analyzed with Bayesian analysis of qRT-PCR data using MCMC.qpcr package, with relative expression software tool (REST) and quantitative PCR CopyCount web source. RESULTS: In the group of Giardia with minimum inhibitory concentration (MIC) of 6.3 µM, the level of PFOR gene expression was downregulated and compared with controls, differed by 1.5 to 2.8 times. At the same time, there was no significant difference in PFOR gene expression between the control (susceptible) group and the group with MIC of 3.2 µM. CONCLUSIONS: Though there is association between PFOR gene expression and metronidazole resistance of Giardia intestinalis, the level of PFOR gene expression cannot be a strong genetic marker to predict level of resistance to metronidazole based on MICs.

Improved production of tryptophan in genetically engineered Escherichia coli with TktA and PpsA overexpression.

Intracellular precursor supply is a critical factor for amino acid productivity. In the present study, ppsA and tktA genes were overexpressed in genetically engineered Escherichia coli to enhance the availability of two precursor substrates, phosphoenolpyruvate and erythrose-4-phosphate. The engineered strain, TRTH0709 carrying pSV709, produced 35.9 g/L tryptophan from glucose after 40 h in fed-batch cultivation. The two genes were inserted, independently or together, into a low-copy-number expression vector (pSTV28) and transferred to TRTH0709. Fed-batch fermentations at high cell densities of the recombination strains revealed that overexpression of the ppsA gene alone does not significantly increase tryptophan yield. On the other hand, overexpression of the tktA gene, alone or with the ppsA gene, could further improve tryptophan yield to a final tryptophan titer of 37.9 and 40.2 g/L, respectively. These results represent a 5.6% and 11.9% enhancement over the titer achieved by TRTH0709. No evident genetic modifications leading to growth impairment were observed.

Synthesis of pyruvate: ferredoxin oxidoreductase and alcohol dehydrogenase E enzymes during Giardia intestinalis excystation.

INTRODUCTION: Giardia intestinalis is a unicellular parasite of worldwide distribution. It causes an intestinal illness known as giardiasis, and it is probably the earliest diverging eukaryotic microorganism. Previously, changes have been reported in the expression of mRNAs at several stages of the life cycle; however specific enzymatic activity changes have not been explored. OBJECTIVE: The expression of pyruvate ferredoxin oxidoreductase (PFOR) and alcohol dehydrogenase E (ADHE) enzymes was measured in cyst and trophozoite stages, and during the excystation process. MATERIALS AND METHODS: Recombinant proteins were generated for PFOR and ADHE to be used as antigens in the production of polyclonal antibodies for the detection of native proteins by Western Blot. The enzymatic activity of ADHE and glutamate dehydrogenase (GDH) was evaluated by spectrophotometric assays. RESULTS: PFOR (139 kDa) and ADHE (97 kDa) proteins were detected in trophozoites, but not in cysts. During excystation, ADHE protein was detected after the first phase of induction, but the PFOR protein appeared only after the second phase. This indicated that both proteins were synthesized during excystation, although at different times. ADHE enzymatic activity was present only in trophozoites and not in cysts whereas GDH activity was detected in both stages. CONCLUSION: These results conclusively showed that PFOR and ADHE enzymes were translated during the excystation process and is strong evidence that active protein synthesis was occurring during excystation.

Immunization of broiler chickens against Clostridium perfringens-induced necrotic enteritis.

Necrotic enteritis (NE) in broiler chickens is caused by Clostridium perfringens. Currently, no vaccine against NE is available and immunity to NE is not well characterized. Our previous studies showed that immunity to NE followed oral infection by virulent rather than avirulent C. perfringens strains and identified immunogenic secreted proteins apparently uniquely produced by virulent C. perfringens isolates. These proteins were alpha-toxin, glyceraldehyde-3-phosphate dehydrogenase, pyruvate:ferredoxin oxidoreductase (PFOR), fructose 1,6-biphosphate aldolase, and a hypothetical protein (HP). The current study investigated the role of each of these proteins in conferring protection to broiler chickens against oral infection challenges of different severities with virulent C. perfringens. The genes encoding these proteins were cloned and purified as histidine-tagged recombinant proteins from Escherichia coli and were used to immunize broiler chickens intramuscularly. Serum and intestinal antibody responses were assessed by enzyme-linked immunosorbent assay. All proteins significantly protected broiler chickens against a relatively mild challenge. In addition, immunization with alpha-toxin, HP, and PFOR also offered significant protection against a more severe challenge. When the birds were primed with alpha-toxoid and boosted with active toxin, birds immunized with alpha-toxin were provided with the greatest protection against a severe challenge. The serum and intestinal washings from protected birds had high antigen-specific antibody titers. Thus, we conclude that there are certain secreted proteins, in addition to alpha-toxin, that are involved in immunity to NE in broiler chickens.

Heterologous expression of Escherichia coli ppsA (phosphoenolpyruvate synthetase) and galU (UDP-glucose pyrophosphorylase) genes in Corynebacterium glutamicum, and its impact on trehalose synthesis.

Trehalose is a disaccharide with a wide range of applications in the food industry. We recently proposed a strategy for trehalose production based on a Corynebacterium glutamicum strain expressing the Escherichia coli enzyme UDP-glucose pyrophosphorylase (GalU). Biochemical network analysis suggest a further bottleneck for trehalose synthesis resulting from the coupling of phosphotransferase (PTS) mediated glucose uptake, and glucose catabolism in C. glutamicum. To overcome this coupling, we propose the expression of E. coli phosphoenolpyruvate synthetase (PpsA), in addition to GalU expression, in C. glutamicum. Although GalU expression improved trehalose synthesis in C. glutamicum, the simultaneous expression of GalU and PpsA did not result in a further increase in trehalose yield, but resulted in an increased catabolic rate of glucose, which could be ascribed to the operation of a futile cycle between phosphoenolpyruvate and pyruvate. The impact of GalU and PpsA expression on polysaccharide content, side product excretion and metabolic fluxes is discussed, as well as alternative ways to decouple glucose uptake and catabolism, in order to increase trehalose yield.