Comparative genomic characterization of citrus-associated <it>Xylella fastidiosa </it>strains

Abstract Background The xylem-inhabiting bacterium Xylella fastidiosa (Xf) is the causal agent of Pierce's disease (PD) in vineyards and citrus variegated chlorosis (CVC) in orange trees. Both of these economically-devastating diseases are caused by distinct strains of this complex group of microorganisms, which has motivated researchers to conduct extensive genomic sequencing projects with Xf strains. This sequence information, along with other molecular tools, have been used to estimate the evolutionary history of the group and provide clues to understand the capacity of Xf to infect different hosts, causing a variety of symptoms. Nonetheless, although significant amounts of information have been generated from Xf strains, a large proportion of these efforts has concentrated on the study of North American strains, limiting our understanding about the genomic composition of South American strains – which is particularly important for CVC-associated strains. Results This paper describes the first genome-wide comparison among South American Xf strains, involving 6 distinct citrus-associated bacteria. Comparative analyses performed through a microarray-based approach allowed identification and characterization of large mobile genetic elements that seem to be exclusive to South American strains. Moreover, a large-scale sequencing effort, based on Suppressive Subtraction Hybridization (SSH), identified 290 new ORFs, distributed in 135 Groups of Orthologous Elements, throughout the genomes of these bacteria. Conclusion Results from microarray-based comparisons provide further evidence concerning activity of horizontally transferred elements, reinforcing their importance as major mediators in the evolution of Xf. Moreover, the microarray-based genomic profiles showed similarity between Xf strains 9a5c and Fb7, which is unexpected, given the geographical and chronological differences associated with the isolation of these microorganisms. The newly identified ORFs, obtained by SSH, represent an approximately 10% increase in our current knowledge of the South American Xf gene pool and include new putative virulence factors, as well as novel potential markers for strain identification. Surprisingly, this list of novel elements include sequences previously believed to be unique to North American strains, pointing to the necessity of revising the list of specific markers that may be used for identification of distinct Xf strains.</p

Heparin modulates the endopeptidase activity of Leishmania mexicana cysteine protease cathepsin L-Like rCPB2.8

Cysteine protease B is considered crucial for the survival and infectivity of the Leishmania in its human host. Several microorganism pathogens bind to the heparin-like glycosaminoglycans chains of proteoglycans at host-cell surface to promote their attachment and internalization. Here, we have investigated the influence of heparin upon Leishmania mexicana cysteine protease rCPB2.8 activity. The data analysis revealed that the presence of heparin affects all steps of the enzyme reaction: (i) it decreases 3.5-fold the k1 and 4.0-fold the k−1, (ii) it affects the acyl-enzyme accumulation with pronounced decrease in k2 (2.7-fold), and also decrease in k3 (3.5-fold). The large values of ΔG = 12 kJ/mol for the association and dissociation steps indicate substantial structural strains linked to the formation/dissociation of the ES complex in the presence of heparin, which underscore a conformational change that prevents the diffusion of substrate in the rCPB2.8 active site. Binding to heparin also significantly decreases the α-helix content of the rCPB2.8 and perturbs the intrinsic fluorescence emission of the enzyme. The data strongly suggest that heparin is altering the ionization of catalytic (Cys25)-S−/(His163)-Im+ H ion pair of the rCPB2.8. Moreover, the interaction of heparin with the N-terminal pro-region of rCPB2.8 significantly decreased its inhibitory activity against the mature enzyme. Taken together, depending on their concentration, heparin-like glycosaminoglycans can either stimulate or antagonize the activity of cysteine protease B enzymes during parasite infection, suggesting that this glycoconjugate can anchor parasite cysteine protease at host cell surface

Arrhenius and Eyring plots of the <i>k_cat/K_M</i> and <i>k_cat</i> for the Z-FR-MCA hydrolisys by rCPB2.8.

<p><i>A</i>, Arrhenius plot of the <i>ln</i> (<i>k_cat/K</i>_M<i>)</i> versus 1000/T. <i>B</i>, Arrhenius plot of the <i>ln k</i>_cat versus 1000/T. <i>C</i>, Eyring plot of the <i>ln (k_cat/K</i>_M.T<i>)</i> versus 1000/T. <i>D</i>, Eyring plot of the <i>ln (k_cat/T)</i> versus 1000/T. The experimental conditions were: the values of the kinetic parameters <i>k_cat</i> and <i>K</i>_M for rCPB2.8 was determined using 100 mM sodium acetate buffer, 20% glycerol, 5 mM DTT, pH 5.5 activating the enzyme for 10 min in the temperature range 10°C to 55°C as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080153#s2" target="_blank">Methods</a>.” The data were obtained in the absence (•–•) or in the presence (○–○) of 40 µM heparin. The continuous lines (<i>slops</i>) were drawn according the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080153#pone.0080153.e006" target="_blank">Equations 6</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080153#pone.0080153.e007" target="_blank">7</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080153#pone.0080153.e009" target="_blank">9</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080153#pone.0080153.e010" target="_blank">10</a>, with the best-fit parameter values listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080153#pone-0080153-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080153#pone-0080153-t002" target="_blank">2</a>.</p

Kinetic rate constants of hydrolysis of Z-FR-MCA by rCPB2.8 in absence or in the presence of 40 µM heparin.

<p>Kinetic rate constants of hydrolysis of Z-FR-MCA by rCPB2.8 in absence or in the presence of 40 µM heparin.</p

Effect of Heparin upon the rCPB2.8 Endopeptidase Activity.

<p>The influence of heparin upon rCPB2.8 endopeptidase activity was monitored fluorometrically using the fluorogenic substrate Z-FR-MCA. The steady-state kinetic assays with fluorogenic substrate were performed in 100 mM sodium acetate buffer (pH 5.5) containing 20% glycerol and 5 mM DTT at 35°C. The enzyme was activated by its pre-incubation in the assay buffer for 5 min at 35°C before the substrate addition. <i>A</i>, the rate of substrate Z-FR-MCA hydrolysis as a function of substrate concentration. The kinetic parameters were determined by measuring the initial rate of hydrolysis at various substrate concentrations in the absence or in the presence of heparin, control (▵–▵); 8 µM (•–•); 16 µM (•–•); 32 µM (□–□); 42 µM (▴–▴); 48 µM (○–○); 62 µM (*–*) of heparin. <i>B</i><b>,</b> the reciprocal plot 1/<i>V</i> versus 1/[S] in the presence of different concentrations of heparin (0 — 62 µM). <i>C</i>, replots of <i>slope</i>_1/[S] versus [heparin]. <i>D</i>, replot of 1/<i>V</i>_−axis intercept versus [heparin]. The data of replots were taken from the reciprocal plot 1/<i>V</i> versus 1/[S].</p

Heparin binding-induced rCPB2.8 conformational change.

<p><i>A</i>, the intrinsic fluorescence of tryptophan residues of the rCPB2.8 was monitored in 50 mM sodium phosphate buffer containing 20% glycerol at 35 °C by measuring the emission of fluorescence between at 300 — 450 nm after excitation at λ_ex  =  290 nm in the absence or in the presence of different concentrations of heparin (0 — 167 µM). The insert in <i>A</i> shows the variation of the intrinsic fluoresce emission ΔF (F-F₀) as a function of heparin concentration. <i>B</i>, effects of heparin on rCPB2.8 circular dichroism spectra. About 2 µM rCPB2.8 were determined in 5 mM sodium phosphate buffer pH 5.8 in the absence (•–•) or in the presence (□–□) of 40 µM heparin.</p

Activation energies, entropies, enthalpies and Gibbs free energies of hydrolysis of Z-FR-MCA by rCPB2.8 in absence or in the presence of 40 µM heparin.

<p>Activation energies, entropies, enthalpies and Gibbs free energies of hydrolysis of Z-FR-MCA by rCPB2.8 in absence or in the presence of 40 µM heparin.</p

Effects of heparin on the <i>N</i>-terminal pro-region of rCPB2.8.

<p><i>A</i>, the influence of heparin concentration upon <i>N</i>-terminal pro-region segment intrinsic fluorescence emission. The insert in <i>A</i> shows the variation of the intrinsic fluoresce emission ΔF (F-F₀) of the <i>N</i>-terminal pro-region as a function of heparin concentration (0 — 167 µM) as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080153#s2" target="_blank">Methods</a>.” <i>B</i>, heparin prevents the inhibitory activity of <i>N</i>-terminal pro-region upon rCPB2.8. The remaining activity of the enzyme was plotted as a function of <i>N</i>-terminal pro-region concentration in the absence (○–○) or in the presence (•–•) of 40 µM heparin.</p