Pathway-Based Toxicity: History, Current Approaches and Liver Fibrosis and Steatosis as Prototypes

The Human Toxicology Project Consortium (HTPC) was created to accelerate implementation of the science and policies required to achieve a pathway-based foundation for toxicology as articulated in the 2007 National Research Council report, Toxicity Testing in the 21st Century: a Vision and a Strategy. The HTPC held a workshop, “Building Shared Experience to Advance Practical Application of Pathway-Based Toxicology: Liver Toxicity Mode-of-Action,” in January, 2013, in Baltimore, MD, to further the science of pathway-based approaches to liver toxicity. This review was initiated as a thought-starter for this workshop and has since been updated to include insights from the workshop and other activities occurring in 2013. The report of the workshop has been published elsewhere in this journal (Willett, 2014).JRC.I.5-Systems Toxicolog

Multiple Recombination Events Drive the Current Genetic Structure of Xanthomonas perforans in Florida

Prior to the identification of Xanthomonas perforans associated with bacterial spot of tomato in 1991, X. euvesicatoria was the only known species in Florida. Currently, X. perforans is the Xanthomonas sp. associated with tomato in Florida. Changes in pathogenic race and sequence alleles over time signify shifts in the dominant X. perforans genotype in Florida. We previously reported recombination of X. perforans strains with closely related Xanthomonas species as a potential driving factor for X. perforans evolution. However, the extent of recombination across the X. perforans genomes was unknown. We used a core genome multilocus sequence analysis approach to identify conserved genes and evaluated recombination-associated evolution of these genes in X. perforans. A total of 1,356 genes were determined to be “core” genes conserved among the 58 X. perforans genomes used in the study. Our approach identified three genetic groups of X. perforans in Florida based on the principal component analysis (PCA) using core genes. Nucleotide variation in 241 genes defined these groups, that are referred as Phylogenetic-group Defining (PgD) genes. Furthermore, alleles of many of these PgD genes showed 100% sequence identity with X. euvesicatoria, suggesting that variation likely has been introduced by recombination at multiple locations throughout the bacterial chromosome. Site-specific recombinase genes along with plasmid mobilization and phage associated genes were observed at different frequencies in the three phylogenetic groups and were associated with clusters of recombinant genes. Our analysis of core genes revealed the extent, source, and mechanisms of recombination events that shaped the current population and genomic structure of X. perforans in Florida

Building Shared Experience to Advance Practical Application of Pathway-Based Toxicology: Liver Toxicity Mode-of-Action

A workshop sponsored by the Human Toxicology Project Consortium (HTPC), “Building Shared Experience to Advance Practical Application of Pathway-Based Toxicology: Liver Toxicity Mode-of-Action” brought together experts from a wide range of perspectives to inform the process of pathway development and to advance two prototype pathways initially developed by the European Commission Joint Research Center (JRC): liver-specific fibrosis and steatosis. The first half of the workshop focused on the theory and practice of pathway development; the second on liver disease and the two prototype pathways. Participants agreed pathway development is extremely useful for organizing information and found that focusing the theoretical discussion on a specific AOP is helpful. It is important to include several perspectives during pathway development, including information specialists, pathologists, human health and environmental risk assessors, and chemical and product manufacturers, to ensure the biology is well captured and end use is considered

Aconitase B Is Required for Optimal Growth of Xanthomonas campestris pv. vesicatoria in Pepper Plants

The aerobic plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) colonizes the intercellular spaces of pepper and tomato. One enzyme that might contribute to the successful proliferation of Xcv in the host is the iron-sulfur protein aconitase, which catalyzes the conversion of citrate to isocitrate in the tricarboxylic acid (TCA) cycle and might also sense reactive oxygen species (ROS) and changes in cellular iron levels. Xcv contains three putative aconitases, two of which, acnA and acnB, are encoded by a single chromosomal locus. The focus of this study is aconitase B (AcnB). acnB is co-transcribed with two genes, XCV1925 and XCV1926, encoding putative nucleic acid-binding proteins. In vitro growth of acnB mutants was like wild type, whereas in planta growth and symptom formation in pepper plants were impaired. While acnA, XCV1925 or XCV1926 mutants showed a wild-type phenotype with respect to bacterial growth and in planta symptom formation, proliferation of the acnB mutant in susceptible pepper plants was significantly impaired. Furthermore, the deletion of acnB led to reduced HR induction in resistant pepper plants and an increased susceptibility to the superoxide-generating compound menadione. As AcnB complemented the growth deficiency of an Escherichia coli aconitase mutant, it is likely to be an active aconitase. We therefore propose that optimal growth and survival of Xcv in pepper plants depends on AcnB, which might be required for the utilization of citrate as carbon source and could also help protect the bacterium against oxidative stress

Bacterial Mutation During Seasonal Epidemics

Rapidly evolving bacterial pathogens pose a unique challenge for long-term plant disease management. In this study, we investigated the types and rate of mutations in bacterial populations during seasonal disease epidemics. Two phylogenetically distinct strains of the bacterial spot pathogen, Xanthomonas perforans, were marked, released in tomato fields, and recaptured at several time points during the growing season. Genomic variations in recaptured isolates were identified by comparative analysis of their whole-genome sequences. In total, 180 unique variations (116 substitutions, 57 insertions/deletions, and 7 structural variations) were identified from 300 genomes, resulting in the overall host-associated mutation rate of ∼0.3 to 0.9/genome/week. This result serves as a benchmark for bacterial mutation during epidemics in similar pathosystems. [Graphic: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license

Whole-Genome Sequences of Xanthomonas euvesicatoria Strains Clarify Taxonomy and Reveal a Stepwise Erosion of Type 3 Effectors

International audienceMultiple species of Xanthomonas cause bacterial spot of tomato (BST) and pepper. We sequenced five Xanthomonas euvesicatoria strains isolated from three continents (Africa, Asia, and South America) to provide a set of representative genomes with temporal and geographic diversity. LMG strains 667, 905, 909, and 933 were pathogenic on tomato and pepper, except LMG 918 elicited a hypersensitive reaction (HR) on tomato. Furthermore, LMG 667, 909, and 918 elicited a HR on Early Cal Wonder 30R containing Bs3. We examined pectolytic activity and starch hydrolysis, two tests which are useful in differentiating X. euvesicatoria from X. perforans, both causal agents of BST. LMG strains 905, 909, 918, and 933 were nonpectolytic while only LMG 918 was amylolytic. These results suggest that LMG 918 is atypical of X. euvesicatoria. Sequence analysis of all the publicly available X. euvesicatoria and X. perforans strains comparing seven housekeeping genes identified seven haplotypes with few polymorphisms. Whole genome comparison by average nucleotide identity (ANI) resulted in values of >99% among the LMG strains 667, 905, 909, 918, and 933 and X. euvesicatoria strains and >99.6% among the LMG strains and a subset of X. perforans strains. These results suggest that X. euvesicatoria and X. perforans should be considered a single species. ANI values between strains of X. euvesicatoria, X. perforans, X. allii, X. alfalfa subsp. citrumelonis, X. dieffenbachiae, and a recently described pathogen of rose were >97.8% suggesting these pathogens should be a single species and recognized as X. euvesicatoria. Analysis of the newly sequenced X. euvesicatoria strains revealed interesting findings among the type 3 (T3) effectors, relatively ancient stepwise erosion of some T3 effectors, additional X. euvesicatoria-specific T3 effectors among the causal agents of BST, orthologs of avrBs3 and avrBs4, and T3 effectors shared among xanthomonads pathogenic against various hosts. The results from this study supports the finding that T3 effector repertoire and host range are fundamental for the study of host-microbe interaction but of little relevance to bacterial speciation

Transgenic Expression of EFR and Bs2 Genes for Field Management of Bacterial Wilt and Bacterial Spot of Tomato

Field trials were conducted at two locations in Florida to evaluate transgenic tomato expressing the ELONGATION FACTOR TU RECEPTOR (EFR) gene from Arabidopsis thaliana, the Bs2 gene from pepper, or both Bs2 and EFR (Bs2/EFR) for managing bacterial wilt caused by Ralstonia solanacearum and bacterial spot caused by Xanthomonas perforans. Expression of EFR or Bs2/EFR in the susceptible genotype Fla. 8000 significantly reduced bacterial wilt incidence (50 to 100%) and increased total yield (57 to 114%) relative to lines expressing only Bs2 or the nontransformed Fla. 8000 control, although the marketable yield was not significantly affected. Following harvest, surviving symptomatic and nonsymptomatic plants were assessed for colonization by R. solanacearum. There were no significant differences in the population at the lower stem. Interestingly, in the middle stem, no bacteria could be recovered from EFR or Bs2/EFR lines but viable bacterial populations were recovered from Bs2 and nontransformed control lines at 102 to 105 CFU/g of stem tissue. In growth-chamber experiments, the EFR transgenic tomato lines were found to be effective against seven different R. solanacearum strains isolated from the southeastern United States, indicating utility across the southeastern United States. In all of the bacterial spot trials, EFR and Bs2/EFR lines had significantly reduced disease severity (22 to 98%) compared with the Fla. 8000 control. The marketable and total yield of Bs2/EFR were significantly higher (43 to 170%) than Fla. 8000 control in three of four field trials. These results demonstrate for the first time the potential of using the EFR gene for field management of bacterial wilt and bacterial spot diseases of tomato