Genomics and X-ray microanalysis indicate that Ca2+ and thiols mediate the aggregation and adhesion of Xylella fastidiosa

The availability of the genome sequence of the bacterial plant pathogen Xylella fastidiosa, the causal agent of citrus variegated chlorosis, is accelerating important investigations concerning its pathogenicity. Plant vessel occlusion is critical for symptom development. The objective of the present study was to search for information that would help to explain the adhesion of X. fastidiosa cells to the xylem. Scanning electron microscopy revealed that adhesion may occur without the fastidium gum, an exopolysaccharide produced by X. fastidiosa, and X-ray microanalysis demonstrated the presence of elemental sulfur both in cells grown in vitro and in cells found inside plant vessels, indicating that the sulfur signal is generated by the pathogen surface. Calcium and magnesium peaks were detected in association with sulfur in occluded vessels. We propose an explanation for the adhesion and aggregation process. Thiol groups, maintained by the enzyme peptide methionine sulfoxide reductase, could be active on the surface of the bacteria and appear to promote cell-cell aggregation by forming disulfide bonds with thiol groups on the surface of adjacent cells. The enzyme methionine sulfoxide reductase has been shown to be an auxiliary component in the adhesiveness of some human pathogens. The negative charge conferred by the ionized thiol group could of itself constitute a mechanism of adhesion by allowing the formation of divalent cation bridges between the negatively charged bacteria and predominantly negatively charged xylem walls.

Characterization of ß-trypsin at acid pH by differential scanning calorimetry

Trypsin is a serino-protease with a polypeptide chain of 223 amino acid residues and contains six disulfide bridges. It is a globular protein with a predominance of antiparallel ß-sheet and helix in its secondary structure and has two domains with similar structures. We assessed the stability of ß-trypsin in the acid pH range using microcalorimetric (differential scanning calorimetry) techniques. Protein concentrations varied in the range of 0.05 to 2.30 mg/ml. Buffer solutions of 50.0 mM ß-alanine and 20.0 mM CaCl2 at different pH values (from 2.0 to 4.2) and concentrations of sorbitol (1.0 and 2.0 M), urea (0.5 M) or guanidinium hydrochloride (0.5 and 1.0 M) were used. The data suggest that we are studying the same conformational transition of the protein in all experimental situations using pH, sorbitol, urea and guanidinium hydrochloride as perturbing agents. The observed van't Hoff ratios (deltaHcal/deltaHvH) of 1.0 to 0.5 in the pH range of 3.2 to 4.2 suggest protein aggregation. In contrast, deltaHcal/deltaHvH ratios equal to one in the pH range of 2.0 to 3.2 suggest that the protein unfolds as a monomer. At pH 3.00, ß-trypsin unfolded with Tm = 54ºC and deltaH = 101.8 kcal/mol, and the change in heat capacity between the native and unfolded forms of the protein (deltaCp) was estimated to be 2.50 ± 0.07 kcal mol-1 K-1. The stability of ß-trypsin calculated at 298 K was deltaG D = 5.7 kcal/mol at pH 3.00 and deltaG D = 15.2 kcal/mol at pH 7.00, values in the range expected for a small globular protein.

Secretion of Streptomyces tendae antifungal protein 1 by Lactococcus lactis

Lactococcus lactis, the model lactic acid bacterium, is a good candidate for heterologous protein production in both foodstuffs and the digestive tract. We attempted to produce Streptomyces tendae antifungal protein 1 (Afp1) in L. lactis with the objective of constructing a strain able to limit fungal growth. Since Afp1 activity requires disulfide bond (DSB) formation and since intracellular redox conditions are reportedly unfavorable for DSB formation in prokaryotes, Afp1 was produced as a secreted form. An inducible expression-secretion system was used to drive Afp1 secretion by L. lactis; Afp1 was fused or not with LEISSTCDA, a synthetic propeptide (LEISS) that has been described to be a secretion enhancer. Production of Afp1 alone was not achieved, but production of LEISS-Afp1 was confirmed by Western blot and immunodetection with anti-Afp1 antibodies. This protein (molecular mass: 9.8 kDa) is the smallest non-bacteriocin heterologous protein ever reported to be secreted in L. lactis via the Sec-dependent pathway. However, no anti-fungal activity was detected, even in concentrated samples of induced supernatant. This could be due to a too low secretion yield of Afp1 in L. lactis, to the absence of DSB formation, or to an improper DSB formation involving the additional cysteine residue included in LEISS propeptide. This raises questions about size limits, conformation problems, and protein secretion yields in L. lactis.

Protein 3-nitrotyrosine formation during Trypanosoma cruzi infection in mice

Nitric oxide (·NO) is a diffusible messenger implicated in Trypanosoma cruzi resistance. Excess production of ·NO and oxidants leads to the generation of nitrogen dioxide (·NO2), a strong nitrating agent. Tyrosine nitration is a post-translational modification resulting from the addition of a nitro (-NO2) group to the ortho-position of tyrosine residues. Detection of protein 3-nitrotyrosine is regarded as a marker of nitro-oxidative stress and is observed in inflammatory processes. The formation and role of nitrating species in the control and myocardiopathy of T. cruzi infection remain to be studied. We investigated the levels of ·NO and protein 3-nitrotyrosine in the plasma of C3H and BALB/c mice and pharmacologically modulated their production during the acute phase of T. cruzi infection. We also looked for protein 3-nitrotyrosine in the hearts of infected animals. Our results demonstrated that C3H animals produced higher amounts of ·NO than BALB/c mice, but their generation of peroxynitrite was not proportionally enhanced and they had higher parasitemias. While N G-nitro-arginine methyl ester treatment abolished ·NO production and drastically augmented the parasitism, mercaptoethylguanidine and guanido-ethyl disulfide, at doses that moderately reduced the ·NO and 3-nitrotyrosine levels, paradoxically diminished the parasitemia in both strains. Nitrated proteins were also demonstrated in myocardial cells of infected mice. These data suggest that the control of T. cruzi infection depends not only on the capacity to produce ·NO, but also on its metabolic fate, including the generation of nitrating species that may constitute an important element in parasite resistance and collateral myocardial damage.

Participation of the endoplasmic reticulum protein chaperone thio-oxidoreductase in gonadotropin-releasing hormone receptor expression at the plasma membrane

Chaperone members of the protein disulfide isomerase family can catalyze the thiol-disulfide exchange reaction with pairs of cysteines. There are 14 protein disulfide isomerase family members, but the ability to catalyze a thiol disulfide exchange reaction has not been demonstrated for all of them. Human endoplasmic reticulum protein chaperone thio-oxidoreductase (ERp18) shows partial oxidative activity as a protein disulfide isomerase. The aim of the present study was to evaluate the participation of ERp18 in gonadotropin-releasing hormone receptor (GnRHR) expression at the plasma membrane. Cos-7 cells were cultured, plated, and transfected with 25 ng (unless indicated) wild-type human GnRHR (hGnRHR) or mutant GnRHR (Cys14Ala and Cys200Ala) and pcDNA3.1 without insert (empty vector) or ERp18 cDNA (75 ng/well), pre-loaded for 18 h with 1 µCi myo-[2-3H(N)]-inositol in 0.25 mL DMEM and treated for 2 h with buserelin. We observed a decrease in maximal inositol phosphate (IP) production in response to buserelin in the cells co-transfected with hGnRHR, and a decrease from 20 to 75 ng of ERp18 compared with cells co-transfected with hGnRHR and empty vector. The decrease in maximal IP was proportional to the amount of ERp18 DNA over the range examined. Mutants (Cys14Ala and Cys200Ala) that could not form the Cys14-Cys200 bridge essential for plasma membrane routing of the hGnRHR did not modify maximal IP production when they were co-transfected with ERp18. These results suggest that ERp18 has a reduction role on disulfide bonds in wild-type hGnRHR folding.

cDNA and deduced primary structure of basic phospholipase A2 with neurotoxic activity from the venom secretion of the Crotalus durissus collilineatus rattlesnake

To illustrate the construction of precursor complementary DNAs, we isolated mRNAs from whole venom samples. After reverse transcription polymerase chain reaction (RT-PCR), we amplified the cDNA coding for a neurotoxic protein, phospholipase A2 D49 (PLA2 D49), from the venom of Crotalus durissus collilineatus (Cdc PLA2). The cDNA encoding Cdc PLA2 from whole venom was sequenced. The deduced amino acid sequence of this cDNA has high overall sequence identity with the group II PLA2 protein family. Cdc PLA2 has 14 cysteine residues capable of forming seven disulfide bonds that characterize this group of PLA2 enzymes. Cdc PLA2 was isolated using conventional Sephadex G75 column chromatography and reverse-phase high performance liquid chromatography (RP-HPLC). The molecular mass was estimated using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. We tested the neuromuscular blocking activities on chick biventer cervicis neuromuscular tissue. Phylogenetic analysis of Cdc PLA2 showed the existence of two lines of N6-PLA2, denominated F24 and S24. Apparently, the sequences of the New World’s N6-F24-PLA2 are similar to those of the agkistrodotoxin from the Asian genus Gloydius. The sequences of N6-S24-PLA2 are similar to the sequence of trimucrotoxin from the genus Protobothrops, found in the Old World.