Functional and Evolutionary Characterization of a UDP-Xylose Synthase Gene from the Plant Pathogen <i>Xylella fastidiosa</i>, Involved in the Synthesis of Bacterial Lipopolysaccharide

<i>Xylella fastidiosa</i> is a plant-infecting bacillus, responsible for many important crop diseases, such as Pierce’s disease of vineyards, citrus variegated chlorosis, and coffee leaf scorch (CLS), among others. Recent genomic comparisons involving two CLS-related strains, belonging to <i>X. fastidiosa</i> subsp. <i>pauca</i>, revealed that one of them carries a frameshift mutation that inactivates a gene encoding an oxidoreductase of the short-chain dehydrogenase/reductase (SDR) superfamily, which may play important roles in determining structural variations in bacterial glycans and glycoconjugates. However, the exact nature of this SDR has been a matter of controversy, as different annotations of <i>X. fastidiosa</i> genomes have implicated it in distinct reactions. To confirm the nature of this mutated SDR, a comparative analysis was initially performed, suggesting that it belongs to a subgroup of SDR decarboxylases, representing a UDP-xylose synthase (Uxs). Functional assays, using a recombinant derivative of this enzyme, confirmed its nature as <i>Xf</i>Uxs, and carbohydrate composition analyses, performed with lipopolysaccharide (LPS) molecules obtained from different strains, indicate that inactivation of the <i>X. fastidiosa uxs</i> gene affects the LPS structure among CLS-related <i>X. fastidiosa</i> strains. Finally, a comparative sequence analysis suggests that this mutation is likely to result in a morphological and evolutionary hallmark that differentiates two subgroups of CLS-related strains, which may influence interactions between these bacteria and their plant and/or insect hosts

Functional and Evolutionary Characterization of a UDP-Xylose Synthase Gene from the Plant Pathogen <i>Xylella fastidiosa</i>, Involved in the Synthesis of Bacterial Lipopolysaccharide

<i>Xylella fastidiosa</i> is a plant-infecting bacillus, responsible for many important crop diseases, such as Pierce’s disease of vineyards, citrus variegated chlorosis, and coffee leaf scorch (CLS), among others. Recent genomic comparisons involving two CLS-related strains, belonging to <i>X. fastidiosa</i> subsp. <i>pauca</i>, revealed that one of them carries a frameshift mutation that inactivates a gene encoding an oxidoreductase of the short-chain dehydrogenase/reductase (SDR) superfamily, which may play important roles in determining structural variations in bacterial glycans and glycoconjugates. However, the exact nature of this SDR has been a matter of controversy, as different annotations of <i>X. fastidiosa</i> genomes have implicated it in distinct reactions. To confirm the nature of this mutated SDR, a comparative analysis was initially performed, suggesting that it belongs to a subgroup of SDR decarboxylases, representing a UDP-xylose synthase (Uxs). Functional assays, using a recombinant derivative of this enzyme, confirmed its nature as <i>Xf</i>Uxs, and carbohydrate composition analyses, performed with lipopolysaccharide (LPS) molecules obtained from different strains, indicate that inactivation of the <i>X. fastidiosa uxs</i> gene affects the LPS structure among CLS-related <i>X. fastidiosa</i> strains. Finally, a comparative sequence analysis suggests that this mutation is likely to result in a morphological and evolutionary hallmark that differentiates two subgroups of CLS-related strains, which may influence interactions between these bacteria and their plant and/or insect hosts

Aminotransferases four hours after liver reperfusion.

<p>1A –AST—aspartate aminotransferase; 1B –ALT—alanine aminotransferase CONTR—Control Group (n = 10); SAL—Saline Group (n = 15); TD—Trisulfated Disaccharide Group (n = 15). Data are expressed as mean values * (SAL vs TD and SAL vs CONTR) p<0.05.</p

Liver MDA and Lung microvascular permeability (EBD).

<p>3A –Liver MDA. Data are expressed as mean values * (SAL vs TD and SAL vs CONTR) p<0.05; 3B—Lung microvascular permeability (EBD) *(CONT vs SAL and CONTR vs TD) p<0.05. (TD vs SAL) NS. CONTR—Control Group; SAL (n = 10)–Saline Group (n = 15); TD—Trisulfated Disaccharide Group (n = 15). NS: No Significance.</p

Liver histology.

<p>Liver cell necrosis was significantly lower in the TD group when compared to the SAL group. 5A– Control Group (n = 10); 5B –Saline Group (n = 15); 5C –Trisulfated Disaccharide Group (n = 15).</p

Liver histologic analysis.

<p>Liver histologic analysis.</p

Liver mitochondrial function.

<p>2A –RCR—Respiratory control rate * (TD vs SAL) p<0.05; # (CONTR vs SAL) p<0,05; 2B—ADP/O—ADP/Oxygen ratio * (SAL vs TD and SAL vs CONTR) p<0.05; 2C –S3—State 3 respiration * (SAL vs TD and SAL vs CONTR) p<0.05; 2D –S4—State 4 respiration NS: No Significance. CONTR—Control Group (n = 10); SAL—Saline Group; TD (n = 15)—Trisulfated Disaccharide Group (n = 15). Data are expressed as mean values.</p

TD effect on thapsigargin-induced cytosolic calcium increase in liver cells (n = 3).

<p>Panel A—Images of 340/380 nm fluorescence ratio using a non-true color scale. Scale bars—50 μm; Time shown in white inside each frame; Panel B—Relative quantification of changes in calcium levels. Arrows indicate additions. THAP—4 μM thapsigargin; IONO—0.25 μM ionomycin; TD—30 μM trisulfated disaccharide.</p

Serum levels of inflammatory mediators.

<p>4A—TNF-α; 4B—IL-6; 4C—IL-10 * (TD vs SAL) p<0.05; # (CONTR vs SAL) p<0.05. CONTR—Control Group (n = 10); SAL—Saline Group (n = 15); TD—Trisulfated Disaccharide Group (n = 15). Data are expressed as mean values.</p