Fructose-Bisphophate Aldolase Exhibits Functional Roles between Carbon Metabolism and the hrp System in Rice Pathogen Xanthomonas oryzae pv. oryzicola

Fructose-bisphophate aldolase (FbaB), is an enzyme in glycolysis and gluconeogenesis in living organisms. The mutagenesis in a unique fbaB gene of Xanthomonas oryzae pv. oryzicola, the causal agent of rice bacterial leaf streak, led the pathogen not only unable to use pyruvate and malate for growth and delayed its growth when fructose was used as the sole carbon source, but also reduced extracellular polysaccharide (EPS) production and impaired bacterial virulence and growth in rice. Intriguingly, the fbaB promoter contains an imperfect PIP-box (plant-inducible promoter) (TTCGT-N9-TTCGT). The expression of fbaB was negatively regulated by a key hrp regulatory HrpG and HrpX cascade. Base substitution in the PIP-box altered the regulation of fbaB with the cascade. Furthermore, the expression of fbaB in X. oryzae pv. oryzicola RS105 strain was inducible in planta rather than in a nutrient-rich medium. Except other hrp-hrc-hpa genes, the expression of hrpG and hrpX was repressed and the transcripts of hrcC, hrpE and hpa3 were enhanced when fbaB was deleted. The mutation in hrcC, hrpE or hpa3 reduced the ability of the pathogen to acquire pyruvate and malate. In addition, bacterial virulence and growth in planta and EPS production in RΔfbaB mutant were completely restored to the wild-type level by the presence of fbaB in trans. This is the first report to demonstrate that carbohydrates, assimilated by X. oryzae pv. oryzicola, play critical roles in coordinating hrp gene expression through a yet unknown regulator

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

Detection and Functional Characterization of a 215 Amino Acid N-Terminal Extension in the Xanthomonas Type III Effector XopD

During evolution, pathogens have developed a variety of strategies to suppress plant-triggered immunity and promote successful infection. In Gram-negative phytopathogenic bacteria, the so-called type III protein secretion system works as a molecular syringe to inject type III effectors (T3Es) into plant cells. The XopD T3E from the strain 85-10 of Xanthomonas campestris pathovar vesicatoria (Xcv) delays the onset of symptom development and alters basal defence responses to promote pathogen growth in infected tomato leaves. XopD was previously described as a modular protein that contains (i) an N-terminal DNA-binding domain (DBD), (ii) two tandemly repeated EAR (ERF-associated amphiphillic repression) motifs involved in transcriptional repression, and (iii) a C-terminal cysteine protease domain, involved in release of SUMO (small ubiquitin-like modifier) from SUMO-modified proteins. Here, we show that the XopD protein that is produced and secreted by Xcv presents an additional N-terminal extension of 215 amino acids. Closer analysis of this newly identified N-terminal domain shows a low complexity region rich in lysine, alanine and glutamic acid residues (KAE-rich) with high propensity to form coiled-coil structures that confers to XopD the ability to form dimers when expressed in E. coli. The full length XopD protein identified in this study (XopD1-760) displays stronger repression of the XopD plant target promoter PR1, as compared to the XopD version annotated in the public databases (XopD216-760). Furthermore, the N-terminal extension of XopD, which is absent in XopD216-760, is essential for XopD type III-dependent secretion and, therefore, for complementation of an Xcv mutant strain deleted from XopD in its ability to delay symptom development in tomato susceptible cultivars. The identification of the complete sequence of XopD opens new perspectives for future studies on the XopD protein and its virulence-associated functions in planta

The Monofunctional Catalase KatE of Xanthomonas axonopodis pv. citri Is Required for Full Virulence in Citrus Plants

BACKGROUND: Xanthomonas axonopodis pv. citri (Xac) is an obligate aerobic phytopathogen constantly exposed to hydrogen peroxide produced by normal aerobic respiration and by the plant defense response during plant-pathogen interactions. Four putative catalase genes have been identified in silico in the Xac genome, designated as katE, catB, srpA (monofunctional catalases) and katG (bifunctional catalase). METHODOLOGY/PRINCIPAL FINDINGS: Xac catalase activity was analyzed using native gel electrophoresis and semi-quantitative RT-PCR. We demonstrated that the catalase activity pattern was regulated in different growth stages displaying the highest levels during the stationary phase. KatE was the most active catalase in this phase of growth. At this stage cells were more resistant to hydrogen peroxide as was determined by the analysis of CFU after the exposition to different H(2)O(2) concentrations. In addition, Xac exhibited an adaptive response to hydrogen peroxide, displaying higher levels of catalase activity and H(2)O(2) resistance after treatment with sub-lethal concentrations of the oxidant. In the plant-like medium XVM2 the expression of KatE was strongly induced and in this medium Xac was more resistant to H(2)O(2). A XackatE mutant strain was constructed by insertional mutagenesis. We observed that catalase induction in stationary phase was lost meanwhile the adaptive response to peroxide was maintained in this mutant. Finally, the XackatE strain was assayed in planta during host plant interaction rendering a less aggressive phenotype with a minor canker formation. CONCLUSIONS: Our results confirmed that in contrast to other Xanthomonas species, Xac catalase-specific activity is induced during the stationary phase of growth in parallel with the bacterial resistance to peroxide challenge. Moreover, Xac catalases expression pattern is modified in response to any stimuli associated with the plant or the microenvironment it provides. The catalase KatE has been shown to have an important function for the colonization and survival of the bacterium in the citrus plant during the pathogenic process. Our work provides the first genetic evidence to support a monofunctional catalase as a virulence factor in Xac

Temperature Shift and Host Cell Contact Up-Regulate Sporozoite Expression of Plasmodium falciparum Genes Involved in Hepatocyte Infection

Plasmodium sporozoites are deposited in the skin by Anopheles mosquitoes. They then find their way to the liver, where they specifically invade hepatocytes in which they develop to yield merozoites infective to red blood cells. Relatively little is known of the molecular interactions during these initial obligatory phases of the infection. Recent data suggested that many of the inoculated sporozoites invade hepatocytes an hour or more after the infective bite. We hypothesised that this pre-invasive period in the mammalian host prepares sporozoites for successful hepatocyte infection. Therefore, the genes whose expression becomes modified prior to hepatocyte invasion would be those likely to code for proteins implicated in the subsequent events of invasion and development. We have used P. falciparum sporozoites and their natural host cells, primary human hepatocytes, in in vitro co-culture system as a model for the pre-invasive period. We first established that under co-culture conditions, sporozoites maintain infectivity for an hour or more, in contrast to a drastic loss in infectivity when hepatocytes were not included. Thus, a differential transcriptome of salivary gland sporozoites versus sporozoites co-cultured with hepatocytes was established using a pan-genomic P. falciparum microarray. The expression of 532 genes was found to have been up-regulated following co-culture. A fifth of these genes had no orthologues in the genomes of Plasmodium species used in rodent models of malaria. Quantitative RT-PCR analysis of a selection of 21 genes confirmed the reliability of the microarray data. Time-course analysis further indicated two patterns of up-regulation following sporozoite co-culture, one transient and the other sustained, suggesting roles in hepatocyte invasion and liver stage development, respectively. This was supported by functional studies of four hitherto uncharacterized proteins of which two were shown to be sporozoite surface proteins involved in hepatocyte invasion, while the other two were predominantly expressed during hepatic parasite development. The genome-wide up-regulation of expression observed supports the hypothesis that the shift from the mosquito to the mammalian host contributes to activate quiescent salivary gland sporozoites into a state of readiness for the hepatic stages. Functional studies on four of the up-regulated genes validated our approach as one means to determine the repertoire of proteins implicated during the early events of the Plasmodium infection, and in this case that of P. falciparum, the species responsible for the severest forms of malaria

Malaria Parasite Invasion of the Mosquito Salivary Gland Requires Interaction between the Plasmodium TRAP and the Anopheles Saglin Proteins

SM1 is a twelve-amino-acid peptide that binds tightly to the Anopheles salivary gland and inhibits its invasion by Plasmodium sporozoites. By use of UV-crosslinking experiments between the peptide and its salivary gland target protein, we have identified the Anopheles salivary protein, saglin, as the receptor for SM1. Furthermore, by use of an anti-SM1 antibody, we have determined that the peptide is a mimotope of the Plasmodium sporozoite Thrombospondin Related Anonymous Protein (TRAP). TRAP binds to saglin with high specificity. Point mutations in TRAP's binding domain A abrogate binding, and binding is competed for by the SM1 peptide. Importantly, in vivo down-regulation of saglin expression results in strong inhibition of salivary gland invasion. Together, the results suggest that saglin/TRAP interaction is crucial for salivary gland invasion by Plasmodium sporozoites

Roles of the Amino Terminal Region and Repeat Region of the Plasmodium berghei Circumsporozoite Protein in Parasite Infectivity

The circumsporozoite protein (CSP) plays a key role in malaria sporozoite infection of both mosquito salivary glands and the vertebrate host. The conserved Regions I and II have been well studied but little is known about the immunogenic central repeat region and the N-terminal region of the protein. Rodent malaria Plasmodium berghei parasites, in which the endogenous CS gene has been replaced with the avian Plasmodium gallinaceum CS (PgCS) sequence, develop normally in the A. stephensi mosquito midgut but the sporozoites are not infectious. We therefore generated P. berghei transgenic parasites carrying the PgCS gene, in which the repeat region was replaced with the homologous region of P. berghei CS (PbCS). A further line, in which both the N-terminal region and repeat region were replaced with the homologous regions of PbCS, was also generated. Introduction of the PbCS repeat region alone, into the PgCS gene, did not rescue sporozoite species-specific infectivity. However, the introduction of both the PbCS repeat region and the N-terminal region into the PgCS gene completely rescued infectivity, in both the mosquito vector and the mammalian host. Immunofluorescence experiments and western blot analysis revealed correct localization and proteolytic processing of CSP in the chimeric parasites. The results demonstrate, in vivo, that the repeat region of P. berghei CSP, alone, is unable to mediate sporozoite infectivity in either the mosquito or the mammalian host, but suggest an important role for the N-terminal region in sporozoite host cell invasion

Evolutionary Patterning: A Novel Approach to the Identification of Potential Drug Target Sites in Plasmodium falciparum

Malaria continues to be the most lethal protozoan disease of humans. Drug development programs exhibit a high attrition rate and parasite resistance to chemotherapeutic drugs exacerbates the problem. Strategies that limit the development of resistance and minimize host side-effects are therefore of major importance. In this study, a novel approach, termed evolutionary patterning (EP), was used to identify suitable drug target sites that would minimize the emergence of parasite resistance. EP uses the ratio of non-synonymous to synonymous substitutions (ω) to assess the patterns of evolutionary change at individual codons in a gene and to identify codons under the most intense purifying selection (ω≤0.1). The extreme evolutionary pressure to maintain these residues implies that resistance mutations are highly unlikely to develop, which makes them attractive chemotherapeutic targets. Method validation included a demonstration that none of the residues providing pyrimethamine resistance in the Plasmodium falciparum dihydrofolate reductase enzyme were under extreme purifying selection. To illustrate the EP approach, the putative P. falciparum glycerol kinase (PfGK) was used as an example. The gene was cloned and the recombinant protein was active in vitro, verifying the database annotation. Parasite and human GK gene sequences were analyzed separately as part of protozoan and metazoan clades, respectively, and key differences in the evolutionary patterns of the two molecules were identified. Potential drug target sites containing residues under extreme evolutionary constraints were selected. Structural modeling was used to evaluate the functional importance and drug accessibility of these sites, which narrowed down the number of candidates. The strategy of evolutionary patterning and refinement with structural modeling addresses the problem of targeting sites to minimize the development of drug resistance. This represents a significant advance for drug discovery programs in malaria and other infectious diseases