José L. Barra

Identification, Cloning, and Expression of Pseudomonas aeruginosa Phosphorylcholine Phosphatase Gene

Pseudomonas aeruginosa phosphorylcholine phosphatase (PChP) is a periplasmic enzyme produced simultaneously with the hemolytic phospholipase C (PLc-H) when the bacteria are grown in the presence of choline, betaine, dimethylglycine or carnitine. Molecular analysis of the P. aeruginosa mutant JUF8-00, after Tn5-751 mutagenesis, revealed that the PA5292 gene in the P. aeruginosa PAO1 genome was responsible for the synthesis of PChP. The enzyme expressed in E. coli, rPChP-Ec, purified by a chitin-binding column (IMPACT-CN system, New England BioLabs) was homogeneous after SDS-PAGE analysis. PChP was also expressed in P. aeruginosa PAO1-LAC, rPChP-Pa. Both recombinant enzymes exhibited a molecular mass of approximately 40 kDa, as expected for the size of the PA5292 gene, and catalyzed the hydrolysis of phosphorylcholine, phosphorylethanolamine, and p-nitrophenylphosphate. The saturation curve of rPChP-Ec and rPChP-Pa by phosphorylcholine revealed that these recombinant enzymes, like the purified native PChP, also contained the high- and low-affinity sites for phosphorylcholine and that the enzyme activity was inhibited by high substrate concentration.

Risk genes in head and neck cancer: a systematic review and meta-analysis of last 5 years

The aim of this work was to identify risk genes related to the development and progression of squamous cell carcinoma head and neck (SCCHN) and do a meta-analysis of available estimates. Eligible gene/polymorphism studies were identified by electronic searches. Individual participant data of 8540 patients with HNC and 9844 controls from 19 genetic studies were analyzed, yielding adjusted (tobacco, gender, age and alcohol) odds ratios (OR) and 95% confidence intervals (CIs) comparing cases with controls. A meta-analysis was done on the studies that applied fixed and random models. People have an increase of polymorphism expression related to inflammation (NFKB1-294-ATTG, TNFα308-A2A2/A2A1, and TNFβ252- B2B2/B2B1) or carcinogenic metabolism (GSTM1 null, and CYP1A1 m1/m1), representative of malignancy development. Furthermore, the increased expression of genes associated with the stabilization and repair of the cellular (OGG1-Asp267Asn, Ser279Gly Ile253Phe, 1578A>T, 1582C>T Ala399Glu (1542C>A) 1582insG 1543_1544delCT), and genes associated with the regulation of proliferation, apoptosis or tumor survival (miRNA499-CT/CC, CRYABC802G-CG/GG) are considered as risk factors. In this scheme, only the polymorphisms of ADH7A92G-GG and DEC1606-T/C genes are protective against malignancy transformation. The TP53, GSTM1 and CYPA1genes have been evaluated in more than one study and analyzed for homogeneity in each genotype. The meta-analysis showed no significant association between different allelic variants of Arg72Pro rs1042522 and SCCHN risk. In a model of tumorigenesis, an increased risk of SCCHN is associated with DNA repair and DNA stabilization genes. In addition, the polymorphisms involved in inflammation and carcinogenic metabolism processes represent an increased risk of SCCHN.

Glycine Betaine Transmethylase Mutant of Pseudomonas aeruginosa

The gene for glycine betaine transmethylase (gbt) was identified in Pseudomonas aeruginosa strain Fildes III by biochemical, physiological, and molecular approaches. Based on sequence analysis, the knockout gene corresponded to an open reading frame (ORF) named PA3082 in the genome of P. aeruginosa PAO1. The translated product of this ORF displayed similarity to transferases of different microorganisms. Mutation in gbt blocked the utilization of choline and glycine betaine as carbon and nitrogen sources.

Escherichia coli heat-labile enterotoxin preferentially interacts with blood group A-active glycolipids from pig intestinal mucosa and A- and B-active glycolipids from human red cells compared to H-active glycolipids

The capacity of cholera toxin (CT) and of the heat-labile enterotoxin produced by Escherichia coli isolated from humans (LTh) to interact with glycolipids bearing ABO(H) blood group determinants isolated from different sources and separated by thin layer chromatography was studied. Toxin binding to the ABO(H)-related glycolipids depends on the glycolipid source, the type of the blood group activity, and the toxin. LTh and CT were capable of interacting with several blood group-active glycolipids from pig intestinal mucosa and both toxins preferentially recognize glycolipids isolated from animals carrying A-blood group antigenic determinants compared to those isolated from animals lacking these antigens. In contrast, LTh but not CT was able to interact with ABO(H)-active glycolipids from human erythrocytes. LTh preferentially binds to glycolipids isolated from A, B, and AB compared to O red cells. Results from competition experiments between CT and LTh for binding to the blood group-active glycolipids suggest that the carbohydrate structure requirements for the interaction of each toxin are different. The present findings may help to understand the results of clinical studies indicating an association between ABO(H) blood groups and the severity of diarrheal diseases produced by some toxigenic enterobacteria.

Nucleotides and heteroduplex DNA preserve the active conformation of Pseudomonas aeruginosa MutS by preventing protein oligomerization.

MutS, a component of the mismatch repair system begins the DNA reparation process by recognizing base/base mismatches or small insertion/deletion loops. We have cloned the mutS gene from the human opportunistic pathogen Pseudomonas aeruginosa and analysed the biochemical properties of the encoded protein. Complementation of the hypermutator phenotype of a P. aeruginosa mutS mutant strain indicated that the isolated gene was functional. When purified MutS was incubated at 37 degrees C in the absence of ligands, a rapid inactivation of the oligonucleotide binding capability and ATPase activity occurred. However, the presence of ATP, ADP or heteroduplex oligonucleotides, but not homoduplex oligonucleotides, prevented the protein from being inactivated. The analysis of the protein by native PAGE indicated that the active conformation state correlates with the presence of MutS dimer. Analysis by gel-filtration chromatography showed that the inactive protein formed by incubation at 37 degrees C in the absence of ligands corresponds to the formation of a high molecular mass oligomer. The kinetic analysis of the oligomer formation showed that the extent of the reaction was markedly dependent on the temperature and the presence of MutS ligands. However, the protein inactivation apparently occurred before the maximum extent of MutS oligomerization. Further analysis of the MutS oligomers by electron microscopy showed the presence of regular structures consisting of four subunits, with each subunit probably representing a MutS homodimer. It is concluded that MutS possesses an intrinsic propensity to form oligomeric structures and that the presence of physiological ligands, such as nucleotides or heteroduplex DNA, but not homoduplex DNA, plays an important role in keeping the protein in an active conformation by preventing protein oligomerization.

Interaction of cholera toxin and Escherichia coli heat-labile enterotoxin with glycoconjugates from rabbit intestinal brush border membranes: Relationship with ABH blood group determinants

The capacity of cholera toxin (CT) and type I heat-labile enterotoxin produced by Escherichia coli isolated from human intestine (LTh) to interact with glycoconjugates bearing ABH blood group determinants from rabbit intestinal brush border membranes (BBM) was studied. On the basis of the type of intestinal compounds related to the human ABH blood group antigens, rabbits were classified as AB or H. Toxin binding to the intestinal glycolipids and glycoproteins depends on the blood group determinant borne by the glycoconjugate and on the analyzed toxin. LTh was capable of interacting preferentially with several blood group A- and B-active BBM glycolipids compared to those isolated from animals lacking these antigens (H rabbits). Also, LTh preferably bound to several BBM glycoproteins from AB rabbit intestines compared to those from H ones. One of these glycoproteins, the sucrase-isomaltase complex (EC 3.2.1.48-10) isolated from AB and H rabbits showed the same differential LTh binding. Conversely, CT practically did not recognize either blood group A-, B-, or H-active glycolipids and glycoproteins. These results may be relevant for carrying out in vivo experiments in rabbits in order to disclose the role of ABH active-glycoconjugates in the secretory response induced by LTh in rabbit intestine.

Pseudomonas aeruginosa MutL protein functions in Escherichia coli.

Escherichia coli MutS, MutL and MutH proteins act sequentially in the MMRS (mismatch repair system). MutH directs the repair system to the newly synthesized strand due to its transient lack of Dam (DNA-adenine methylase) methylation. Although Pseudomonas aeruginosa does not have the corresponding E. coli MutH and Dam homologues, and consequently the MMRS seems to work differently, we show that the mutL gene from P. aeruginosa is capable of complementing a MutL-deficient strain of E. coli. MutL from P. aeruginosa has conserved 21 out of the 22 amino acids known to affect functioning of E. coli MutL. We showed, using protein affinity chromatography, that the C-terminal regions of P. aeruginosa and E. coli MutL are capable of specifically interacting with E. coli MutH and retaining the E. coli MutH. Although, the amino acid sequences of the C-terminal regions of these two proteins are only 18% identical, they are 88% identical in the predicted secondary structure. Finally, by analysing (E. coli-P. aeruginosa) chimaeric MutL proteins, we show that the N-terminal regions of E. coli and P. aeruginosa MutL proteins function similarly, in vivo and in vitro. These new findings support the hypothesis that a large surface, rather than a single amino acid, constitutes the MutL surface for interaction with MutH, and that the N- and C-terminal regions of MutL are involved in such interactions.

Some amino acids of the Pseudomonas aeruginosa MutL D(Q/M)HA(X)(2)E(X)(4)E conserved motif are essential for the in vivo function of the protein but not for the in vitro endonuclease activity.

Human and Saccharomyces cerevisiae MutLα, and some bacterial MutL proteins, possess a metal ion-dependent endonuclease activity which is important for the in vivo function of these proteins. Conserved amino acids of the C-terminal region of human PMS2, S. cerevisiae PMS1 and of some bacterial MutL proteins have been implicated in the metal-binding/endonuclease activity. However, the contribution of individual amino acids to these activities has not yet been fully elucidated. In this work we show that Pseudomonas aeruginosa MutL protein possess an in vitro metal ion-dependent endonuclease activity. In agreement with previous published results, we observed that mutation of the aspartic acid, the first histidine or the first glutamic acid of the conserved C-terminal DMHAAHERITYE region results in nonfunctional in vivo proteins. We also determined that the arginine residue is essential for the in vivo function of this protein. However, we unexpectedly observed that although the first glutamic acid mutant derivative is not functional in vivo, its in vitro endonuclease activity is even higher than that of the wild-type protein.

Expression of a bioactive fusion protein of Escherichia coli heat-labile toxin B subunit to a synapsin peptide

The B subunit of Escherichia coli heat-labile toxin (LTB) may function as an efficient carrier molecule for the delivery of genetically coupled antigens across the mucosal barrier. We constructed vectors for the expression of LTB and LTBSC proteins. LTBSC is a fusion protein that comprises the amino acid sequence from the C-domain of rat synapsin fused to the C-terminal end of LTB. Both constructions have a coding sequence for a 6His-tag fused in-frame. LTBSC was expressed in E. coli as inclusion bodies. The inclusion bodies were isolated and purified by Ni2+-chelating affinity chromatography under denaturing condition. Purified LTBSC was diluted in several refolding buffers to gain a soluble and biologically active protein. Refolded LTBSC assembled as an active oligomer which binds to the GM1 receptor in an enzyme-linked immunosorbent assay (ELISA). Soluble LTB in the E. coli lysate was also purified by Ni2+-chelating affinity chromatography and the assembled pentamer was able to bind with high affinity to GM1 in vitro. LTBSC and LTB were fed to rats and the ability to induce antigen-specific tolerance was tested. LTBSC inhibited the specific delayed-type hypersensitivity (DTH) response and induced decreased antigen-specific in vivo and in vitro cell proliferation more efficiently than LTB. Thus, the novel hybrid molecule LTBSC when orally delivered was able to elicit a systemic immune response. These results suggest that LTBSC could be suitable for exploring further therapeutic treatment of autoimmune inflammatory diseases involving antigens from central nervous system.

Escherichia coli Frameshift Mutation Rate Depends on the Chromosomal Context but Not on the GATC Content Near the Mutation Site

Different studies have suggested that mutation rate varies at different positions in the genome. In this work we analyzed if the chromosomal context and/or the presence of GATC sites can affect the frameshift mutation rate in the Escherichia coli genome. We show that in a mismatch repair deficient background, a condition where the mutation rate reflects the fidelity of the DNA polymerization process, the frameshift mutation rate could vary up to four times among different chromosomal contexts. Furthermore, the mismatch repair efficiency could vary up to eight times when compared at different chromosomal locations, indicating that detection and/or repair of frameshift events also depends on the chromosomal context. Also, GATC sequences have been proved to be essential for the correct functioning of the E. coli mismatch repair system. Using bacteriophage heteroduplexes molecules it has been shown that GATC influence the mismatch repair efficiency in a distance- and number-dependent manner, being almost nonfunctional when GATC sequences are located at 1 kb or more from the mutation site. Interestingly, we found that in E. coli genomic DNA the mismatch repair system can efficiently function even if the nearest GATC sequence is located more than 2 kb away from the mutation site. The results presented in this work show that even though frameshift mutations can be efficiently generated and/or repaired anywhere in the genome, these processes can be modulated by the chromosomal context that surrounds the mutation site.