Immunogenicity Evaluation of Chimeric Subunit Vaccine Comprising Adhesion Coli Surface Antigens from Enterotoxigenic Escherichia coli

Enterotoxigenic <i>Escherichia coli</i> (ETEC) is the most common agent of diarrhea morbidity in developing countries. ETEC adheres to host intestinal epithelial cells via various colonization factors. The CooD and CotD proteins play a significant role in bacteria binding to the intestinal epithelial cells as adhesin tip subunits of CS1 and CS2 pili. The purpose here was to design a new construction containing <i>cooD</i> and <i>cotD</i> genes and use several types of bioinformatics software to predict the structural and immunological properties of the designed antigen. The fusion gene was synthesized with codon bias of <i>E. coli</i> in order to increase the expression level of the protein. The amino acid sequences, protein structure, and immunogenicity properties of potential antigens were analyzed in silico. The chimeric protein was expressed in <i>E. coli</i>BL21 (DE3). The antigenicity of the recombinant proteins was verified by Western blotting and ELISA. In order to assess the induced immunity, the immunized mice were challenged with wild-type ETEC by an intraperitoneal route. Immunological analyses showed the production of a high titer of IgG serum with no sign of serum-mucosal IgA antibody response. The result of the challenge assay showed that 30% of immunized mice survived. The results of this study showed that CooD-CotD recombinant protein can stimulate immunity against ETEC. The designed chimera could be a prototype for the subunit vaccine, which is worthy of further consideration.

Studies of the Listeria monocytogenes Cellobiose Transport Components and Their Impact on Virulence Gene Repression

<b><i>Background:</i></b> Many bacteria transport cellobiose via a phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). In <i>Listeria monocytogenes</i>, two pairs of soluble PTS components (EIIA^Cel1/EIIB^Cel1 and EIIA^Cel2/EIIB^Cel2) and the permease EIIC^Cel1 were suggested to contribute to cellobiose uptake. Interestingly, utilization of several carbohydrates, including cellobiose, strongly represses virulence gene expression by inhibiting PrfA, the virulence gene activator. <b><i>Results:</i></b> The LevR-like transcription regulator CelR activates expression of the cellobiose-induced PTS operons <i>celB1</i>-<i>celC1</i>-<i>celA1</i>, <i>celB2</i>-<i>celA2</i>, and the EIIC-encoding monocistronic <i>celC2</i>. Phosphorylation by P∼His-HPr at His550 activates CelR, whereas phosphorylation by P∼EIIB^Cel1 or P∼EIIB^Cel2 at His823 inhibits it. Replacement of His823 with Ala or deletion of both <i>celA</i> or <i>celB</i> genes caused constitutive CelR regulon expression. Mutants lacking EIIC^Cel1, CelR or both EIIA^Cel exhibited<i></i>slow cellobiose consumption. Deletion of <i>celC1</i> or <i>celR</i> prevented virulence gene repression by the disaccharide, but not by glucose and fructose. Surprisingly, deletion of both <i>celA</i> genes caused virulence gene repression even during growth on non-repressing carbohydrates. No cellobiose-related phenotype was found for the <i>celC2</i> mutant. <b><i>Conclusion:</i></b> The two EIIA/B^Cel pairs are similarly efficient as phosphoryl donors in EIIC^Cel1-catalyzed cellobiose transport and CelR regulation. The permanent virulence gene repression in the <i>celA</i> double mutant further supports a role of PTS^Cel components in PrfA regulation.

The Life Cycle of Dictyostelium discoideum Is Accelerated via MAP Kinase Cascade by a Culture Extract Produced by a Synthetic Microbial Consortium

A cellular slime mold, <i>Dictyostelium discoideum</i>, is an amoeboid organism that has a unique life cycle consisting of distinctly separated vegetative and developmental phases. Thus, this organism presents a rare opportunity in which to examine the effects of bioactive substances on separate cellular activities. In this research, we investigated the effect of a culture extract, termed EMXG, produced by a synthetic microbial consortium. EMXG promoted proliferative response of amoeba cells. It further accelerated the developmental phase, leading to the preferred fruiting body formation from fewer cells. Furthermore, EMXG modulated biological rhythm of this organism, that is, interval of oscillation of cAMP level observed in suspension starvation was significantly shortened. Concomitantly, the level of ERKB, a MAP kinase, was found to oscillate in a similar fashion to that of cAMP. Additionally, ErkB-deficient mutant amoeboid cells did not respond to proliferative stimulation by EMXG. These lines of evidence point to a likelihood that MAP kinase cascade is involved and further that ErkB could be the molecular target of EMXG.

Non-Caloric Artificial Sweeteners Modulate the Expression of Key Metabolic Genes in the Omnipresent Gut Microbe Escherichia coli

The human gut is inhabited by several hundred different bacterial species. These bacteria are closely associated with our health and well-being. The composition of these diverse commensals is influenced by our dietary intakes. Non-caloric artificial sweeteners (NAS) have gained global popularity, particularly among diabetic patients, due to their perceived health benefits, such as reduction of body weight and maintenance of blood glucose level compared to caloric sugars. Recent studies have reported that these artificial sweeteners can alter the composition of gut microbiota and, thus, affect our normal physiological state. Here, we investigated the effect of aspartame and acesulfame potassium (ace-K), two popular NAS, in a commercial formulation on the growth and metabolic pathways of omnipresent gut commensal <i>Escherichia coli</i>by analyzing the relative expression levels of the key genes, which control over twenty important metabolic pathways. Treatment with NAS preparation (aspartame and ace-K) modulates the growth of <i>E. coli</i>as well as inducing the expression of important metabolic genes associated with glucose (<i>pfkA, sucA, aceE, pfkB, lpdA</i>), nucleotide (<i>tmk, adk, tdk, thyA</i>), and fatty acid (<i>fabI</i>) metabolisms, among others. Several of the affected genes<b><i></i></b>were previously reported to be important for the colonization of the microbes in the gut. These findings may shed light on the mechanism of alteration of gut microbes and their metabolism by NAS.

Production of Mycobacterium bovis Antigens Included in Recombinant Occlusion Bodies of Baculovirus

Bovine tuberculosis (bTB) is a disease produced by <i>Mycobacterium bovis</i> that affects livestock, wild animals, and humans. The classical diagnostic method to detect bTB is measuring the response induced with the intradermal injection of purified protein derivative of <i>M. bovis</i> (PPDb). Another ancillary bTB test detects IFN-γ produced in whole blood upon stimulation with PPDb, protein/peptide cocktails, or individual antigens. Among the most used <i>M. bovis</i> antigens in IFN-γ assays are the secreted proteins ESAT-6 and CFP-10, which together with antigen Rv3615c improve the sensitivity of the test in comparison to PPDb. Protein reagents for immune stimulation are generally obtained from <i>Escherichia coli</i>, because this bacterium produces a high level of recombinant proteins. However, <i>E. coli</i> recombinant antigens are in general contaminated with lipopolysaccharides and other components that produce non-specific IFN-γ secretion in in vitro assays. In this work, we produced the relevant ESAT-6, CFP-10, and Rv3615c <i>M. bovis</i> antigens as fusions to the polyhedrin protein from the baculovirus AcMNPV. We obtained chimeric proteins effectively incorporated to the occlusion bodies and easily purified the recombinant polyhedra with no reactive contaminants. In an IFN-γ assay, these fusion proteins showed equivalent sensibility but better specificity than the same <i>M. bovis</i> proteins produced in <i>E. coli</i>.

Enhanced Fermentable Sugar Production from Enteromorpha Polysaccharides by the Crude Enzymes of Vibrio sp. H11

In recent years, large-scale outbreaks of the green alga <i>Enteromorpha prolifera</i> in China’s offshore waters have posed a serious threat. This study aimed to improve <i>Enteromorpha</i> polysaccharide (EP) enzymatic sugar production using the hydrolase system of <i>Vibrio</i> sp. H11, an EP-utilizing microbial strain. Strain H11 was found to contain 711 carbohydrate-related genes, and 259 genes belong to glycoside hydrolases that have the potential to hydrolyze EP. To maximize the capability of strain H11 to hydrolyze EP, both the culture medium and the composition were optimized. Response surface methodology analysis showed that maximal enzymatic production from strain H11 was 8.43 U/mL after 26-h incubation. When 50 g/L of EP were treated with crude H11 enzyme, the concentration of fermentation sugars increased by 36.12%. Under these conditions, the hydrolysates were capable of generating 3,217 mL/L of biogas and 6.74 g/L of biosolvents, with increases of 28.17 and 7.29%, respectively, compared to controls. The combined application of the H11 enzymatic system and anaerobic fermentation has the potential to improve the comprehensive application of EP.

A Riboflavin Transporter in Bdellovibrio exovorous JSS

The ImpX transporters of the drug/metabolite transporter superfamily were first proposed to transport riboflavin (RF; vitamin B2) based on findings of a <i>cis</i>-regulatory RNA element responding to flavin mononucleotide (an FMN riboswitch). <i>Bdellovibrio exovorous</i> JSS has a homolog belonging to this superfamily. It has 10 TMSs and shows 30% identity to the previously characterized ImpX transporter from <i>Fusobacterium nucleatum</i>. However, the ImpX homolog is not regulated by an FMN-riboswitch. In order to test the putative function of the ImpX homolog from <i>B. exovorous</i> (BexImpX), we cloned and heterologously expressed its gene. We used functional complementation, growth inhibition experiments, direct uptake experiments and inhibition studies, suggesting a high degree of specificity for RF uptake. The EC50 for growth with RF was estimated to be in the range 0.5–1 µM, estimated from the half-maximal RF concentration supporting the growth of a RF auxotrophic <i>Escherichia coli</i> strain, but the K_half for RF uptake was 20 µM. Transport experiments suggested that the energy source is the proton motive force but that NaCl stimulates uptake. Thus, members of the ImpX family members are capable of RF uptake, not only in RF prototrophic species such as <i>F. nucleatum</i>, but also in the B2 auxotrophic species, <i>B. exovorous</i>.

Study of the Enzymatic Capacity of Kluyveromyces marxianus for the Synthesis of Esters

Recently, biotechnological opportunities have been found in non-<i>Saccharomyces</i> yeasts because they possess metabolic characteristics that lead to the production of compounds of interest. It has been observed that <i>Kluyveromyces marxianus</i> has a great potential in the production of esters, which are aromatic compounds of industrial importance. The genetic bases that govern the synthesis of esters include a large group of enzymes, among which the most important are alcohol acetyl transferases (AATases) and esterases (AEATases), and it is known that some are present in <i>K. marxianus</i>, because it has genetic characteristics like <i>S. cerevisiae</i>. It also has a physiology suitable for biotechnological use since it is the eukaryotic microorganism with the fastest growth rate and has a wide range of thermotolerance with respect to other yeasts. In this work, the enzymatic background of <i>K. marxianus</i> involved in the synthesis of esters is analyzed, based on the sequences reported in the NCBI database.

Characterization of Alanine Dehydrogenase and Its Effect on Streptomyces coelicolorA3(2) Development in Liquid Culture

<i>Streptomyces</i>, the most important group of industrial microorganisms, is harvested in liquid cultures for the production of two-thirds of all clinically relevant secondary metabolites. It is demonstrated here that the growth of <i>Streptomyces coelicolor</i> A3(2) is impacted by the deletion of the alanine dehydrogenase (ALD), an essential enzyme that plays a central role in the carbon and nitrogen metabolism. A long lag-phase growth followed by a slow exponential growth of <i>S. coelicolor</i> due to ALD gene deletion was observed in liquid yeast extract mineral salt culture. The slow lag-phase growth was replaced by the normal wild-type like growth by ALD complementation engineering. The ALD enzyme from <i>S. coelicolor</i> was also heterologously cloned and expressed in <i>Escherichia coli</i> for characterization. The optimum enzyme activity for the oxidative deamination reaction was found at 30°C, pH 9.5 with a catalytic efficiency, k_cat/K_M, of 2.0 ± 0.1 mM^–1 s^–1. The optimum enzyme activity for the reductive amination reaction was found at 30°C, pH 9.0 with a catalytic efficiency, k_cat/K_M, of 1.9 ± 0.1 mM^–1 s^–1.