Detection of virulence and pathogenicity genes in selected phytopathovars

Plant pathogenic organisms are known to infect host cell using various range of secretory proteins. Amongst all other secretion systems, type III secretion system (T3SS) is a key mechanism for bacterial pathogenesis for establishing and maintaining infection into the host. Expression levels of seven genes viz. avrXacE1, avrXacE2, hpaA and hrpG along with bacterial endogenous control lrp (leucine-responsive protein) were studied. The pathogenic organisms selected for the present study includes Enterobacter cloacae, Enterobacter spp., Pantoea ananatis, Xanthomonas campestris pv. Citri, Pantoea agglomerans, Ochrobactrum anthropi and Erwinia chrysanthemi. P. agglomerans and Enterobacter spp. gave high expression of above-mentioned virulence genes compared to Xanthomonas, while E. cloacae and P. ananatis showed similar expression with that of Xanthomonas. The detailed relationship of the expression profiles with respect to the selected organisms is discussed.     

Establishment of a gene expression system in Ochrobactrum anthropi

Genetic studies of Ochrobactrum anthropi are hindered by the lack of a suitable gene expression system. We constructed a set of vectors containing several promoters and a His tag fusion in the N terminus to facilitate protein detection and purification. The new vectors should significantly enhance the genetic manipulation and characterization of O. anthropi.     

Genetic and phenotypic microdiversity of Ochrobactrum spp

The diversity of Ochrobactrum anthropi, Ochrobactrum intermedium, Ochrobactrum tritici and Ochrobactrum grignonense in agricultural soil and on the wheat rhizoplane was investigated. O. anthropi was isolated both from soil and from the rhizoplane, O. intermedium and grignonense only from bulk soil, and O. tritici only from the wheat rhizoplane. On the genetic level, the immunotrapped isolates and a number of strains from culture collection mainly of clinical origin were compared with rep-PCR profiling using BOX primers, and a subset of these isolates and strains using REP primers. The isolates clustered according to their species affiliation. There was no correlation between rep clusters of O. anthropi isolates and habitat (place of isolation). The genetic diversity of Ochrobactrum at the species level as well as microdiversity of O. anthropi (number of BOX groups) was higher in soil than on the rhizoplane. Similarity values from genetic rep-PCR profiles correlated positively with DNA-DNA reassociation percentages. Isolates with >80.7% similarity in BOX profile and >86.4% in rep profile clustered within the same species. Similarity analysis of rep-PCR profiles is hence an alternative to DNA-DNA hybridization as a genomic criterion for species delineation within the genus Ochrobactrum. We used the substrate utilization system BIOLOG-GN to compare the immunotrapped isolates on the phenetic level. For the isolates from bulk soil, substrate utilization versatility (number of utilized substrates) and substrate utilization capacity (mean conversion rate of substrates) were slightly but significantly higher than for the isolates from the rhizoplane. This trend was also seen using API 20E and 20NE systems. Plate counts of total bacteria and the number of immunotrapped Ochrobactrum isolates per gram dry weight were higher for the rhizoplane than for the soil samples. The results of genetic and phenotypic analyses indicated a 'rhizosphere effect'; the diversity and metabolic capacity of Ochrobactrum isolates were higher in bulk soil, and the population density was higher on the wheat rhizoplane.     

Genome of Ochrobactrum anthropi ATCC 49188 T, a versatile opportunistic pathogen and symbiont of several eukaryotic hosts

Ochrobactrum anthropi is a common soil alphaproteobacterium that colonizes a wide spectrum of organisms and is being increasingly recognized as an opportunistic human pathogen. Potentially life-threatening infections, such as endocarditis, are included in the list of reported O. anthropi infections. These reports, together with the scant number of studies and the organism's phylogenetic proximity to the highly pathogenic brucellae, make O. anthropi an attractive model of bacterial pathogenicity. Here we report the genome sequence of the type strain O. anthropi ATCC 49188, which revealed the presence of two chromosomes and four plasmids.     

Insertion of the enteropathogenic Escherichia coli Tir virulence protein into membranes in vitro

Insertion of the enteropathogenic Escherichia coli Tir protein into the plasma membrane of intestinal epithelial cells is a crucial event in infection because it provides a receptor for intimate bacterial adherence. This interaction with the bacterial outer membrane protein intimin is also essential in generating a number of signaling activities associated with virulence. Tir can be modified at various sites by phosphorylation and functionally interacts with multiple host proteins. To investigate the mechanism of membrane insertion and to establish a model system in which the multiple interactions/functions of Tir can be uncoupled and independently characterized, we used intrinsic tryptophan fluorescence, surface plasmon resonance, and protease digestion assays to show that Tir can insert directly into phospholipid vesicles in a composition-dependent manner to generate the topology reported in vivo. This is the first time that Tir has been shown to insert into membranes in a simple model system in the absence of chemical modification or other factors. These data are consistent with the protein interacting with lipids through two sites. The major site is localized to the transmembrane/intimin-binding domain region and includes Trp235, which is shown to be an effective reporter of interaction. The minor site is located within the C-terminal domain. Together, these data support a model in which Tir is released into the cytoplasm by the type III translocon and then independently inserts into the plasma membrane from a cytoplasmic location. A thorough understanding of this mechanism will be crucial to understand the subtleties of enteropathogenic E. coli pathogenesis.     

An Ochrobactrum anthropi gene conferring paraquat resistance to the heterologous host Escherichia coli

A new gene, pqrA, conferring paraquat resistance to the heterologous host Escherichia coli, from a chromosomal DNA library of Ochrobactrum anthropi JW2, was cloned and analyzed. Cells of E. coli transformed with a plasmid carrying the pqrA gene showed elevated resistance to paraquat, but not to hydrogen peroxide. The predicted amino acid sequence of the PqrA polypeptide showed 71% identity with mll7495 hypothetical membrane protein in Mesorhizobium loti, 49% identity with PA2269 protein in Pseudomonas aeruginosa, and significant identity with other previously reported drug transport proteins. The hydropathy pattern of the PqrA polypeptide showed a significant homology to those of 12-transmembrane-segment (TMS) family export proteins. Immunoblot analysis demonstrated that the PqrA protein found in the membrane protein fraction of O. anthropi JW2 has a molecular mass of 42 kDa. These results suggest that the PqrA protein is a membrane protein that plays an important role in protecting cells against paraquat toxicity.     

Expression of Vibrio cholerae virulence genes in response to environmental signals

Vibrio cholerae, the causative agent of Asiatic cholera, is a gram-negative motile bacterial species acquired via oral ingestion of contaminated food or water sources. The O1 serogroup of V. cholerae is responsible for pandemic cholera and is divided into two biotypes, classical and El Tor (Butterton and Calderwood, 1995; Mekalanos, 1985). The El Tor biotype is responsible for the current cholera pandemic. In the absence of disease, the vibrio life cycle consists of a free-swimming phase in marine and estuarine environments in association with zooplankton, crustaceans, insects, and water plants. Vibrios interact with various surfaces found in the environment to generate biofilms which may promote survival (Watnick etaL, 1999). Within the host the motile vibrios must evade the innate host defense mechanisms, penetrate the mucus layer covering the intestinal villi, adhere to and colonize the epithelial surface of the small intestine, assume a non-motile phase, replicate and cause disease by secreting numerous exoproteins at the site of infection (Oliver and Kaper, 1997). The voluminous diarrhea associated with cholera infection leads to the dissemination of the vibrios back into a watery environment and thus a continuation of the environmental phase of the life cycle. The host phase of the vibrio life cycle is only possible through the action of a group of virulence genes (ToxR-regulon) controlled by a complex and incompletely understood regulatory cascade. The ToxR regulon colonization and toxin genes are coordinately expressed in response to specific host signals that have yet to be completely defined (Skorupsky and Taylor 1997). Although little is known regarding the host signals that impact the ToxR regulatory cascade, it is clear that these intraintestinal signals play an important role in maximizing the ability of the vibrios to survive and multiply within the host. Key to understanding the complex events involved in the pathogenesis of V. cholerae will be elucidating the intraintestinal signaling molecules that trigger the expression of vibrio virulence genes. Understanding the molecular basis of this host-parasite interaction will provide important information with respect to how pathogenic bacteria establish infection and provide insights leading to novel methods for treating and/or preventing bacterial infections. This review will summarize what is known regarding host signaling and the complex ToxR regulatory system employed by V. cholerae to coordinate virulence gene expression within the host.     

Genotyping of Ochrobactrum anthropi by recA-based comparative sequence, PCR-RFLP, and 16S rRNA gene analysis

A recA-PCR restriction fragment length polymorphism assay was developed to study intraspecies variation among Ochrobactrum anthropi. Primers deduced from the known recA gene sequence of the genetically closely related genus Brucella allowed the specific amplification of a 1065 bp recA fragment from each of the 38 O. anthropi and the eight Brucella strains investigated. RecA was also amplified from the type strains of O. intermedium, O. tritici, and O. lupini but could not be generated from O. grignonense and O. gallinifaecis. Subsequent comparative recA sequence- and HaeIII-recA restriction fragment length polymorphism analysis identified nine different genospecies among the tested 38 O. anthropi isolates, whereas the recA sequences of the Brucella spp. were indistinguishable. Furthermore, Brucella spp., O. anthropi, O. intermedium, and O. tritici were clearly separated from each other by means of their recA sequences and HaeIII restriction patterns. Five strains of uncertain species status listed in the Culture Collection University of G?teborg bacterial culture collection as O. anthropi were characterized by recA analysis, and their phylogenetic position within the Brucella-Ochrobactrum group was determined. In summary, recA-sequence analysis provides a new reliable molecular subtyping tool to study the phylogeny of the Ochrobactrum taxon at both the inter- and intraspecies level.     

Multidimensional proteomic analysis of the soluble subproteome of the emerging nosocomial pathogen Ochrobactrum anthropi

We report the first large-scale gel-free proteomic analysis of the soluble subproteome of the emerging pathogen Ochrobactrum anthropi. Utilizing our robust offline multidimensional protein identification protocol, a total of 57 280 peptides were initially identified utilizing automated MS/MS analysis software. We describe our investigation of the heuristic protein validation tool PROVALT and demonstrate its ability to increase the speed and accuracy of the curation process of large-scale proteomic datasets. PROVALT reduced our peptide list to 8517 identified peptides and further manual curation of these peptides led to a final list of 984 uniquely identified peptides that resulted in the positive identification of 249 proteins. These identified proteins were functionally classified and physiochemically characterized. A variety of typical "housekeeping" functions identified within the proteome included nucleic acid, amino and fatty acid anabolism and catabolism, glycolysis, TCA cycle, and pyruvate and selenoamino acid metabolism. In addition, a number of potential virulence factors of relevance to both plant and human disease were identified.     

Specificity inversion of Ochrobactrum anthropi D-aminopeptidase to a D,D-carboxypeptidase with new penicillin binding activity by directed mutagenesis

The serine penicillin-recognizing proteins have been extensively studied. They show a wide range of substrate specificities accompanied by multidomain features. Their adaptation capacity has resulted in the emergence of pathogenic bacteria resistant to beta-lactam antibiotics. The most divergent enzymatic activities in this protein family are those of the Ochrobactrum anthropi D-aminopeptidase and of the Streptomyces R61 D,D-carboxypeptidase/transpeptidase. With the help of structural data, we have attempted to identify the factors responsible for this opposite specificity. A loop deletion mutant of the Ochrobactrum anthropi D-aminopeptidase lost its original activity in favor of a new penicillin-binding activity. D-aminopeptidase activity of the deletion mutant can be restored by complementation with another deletion mutant corresponding to the noncatalytic domain of the wild-type enzyme. By a second step site-directed mutagenesis, the specificity of the Ochrobactrum anthropi D-aminopeptidase was inverted to a D,D-carboxypeptidase specificity. These results imply a core enzyme with high diversity potential surrounded by specificity modulators. It is the first example of drastic specificity change in the serine penicillin-recognizing proteins. These results open new perspectives in the conception of new enzymes with nonnatural specificities. The structure/specificity relationship in the serine penicillin-recognizing proteins are discussed.     

Genetics and evolution of parasites and hosts: some comments

We provide a brief commentary on aspects of the analysis of the genetics and evolution of malaria parasites. Any attempt to understand the nature and manifestations of an infectious disease requires an understanding of the genetics of both pathogen and host. The outcome of a malaria infection, i.e. whether it is asymptomatic, mild, severe or causes cerebral malaria, is due to a complex interaction between the products of parasite and host genes. In general terms, genes in the parasite determine its ability to infect the host, its virulence, etc., while host genes will determine resistance or susceptibility to infection. More than this, however, genetics is about the spread of genes in populations, how they mutate and recombine to produce novel genotypes, and how the parasite and its hosts co-evolve with changing environments. This is a complex subject, and we present some discussion of a few aspects of its analysis.     

Specificity of six gene sequences for the detection of the genus Brucella by DNA amplification

M. DA COSTA, J.-P. GUILLOU, B. GARIN-BASTUJI, M. THIÉBAUD AND G. DUBRAY. 1996. DNA extracted from all Brucella species, reference and vaccine strains were amplified by PCR using primers specific for the genes encoding a 31-kDa Brucella protein, the heat shock proteins (DnaJ, DnaK, HtrA and GroEL) and 16S RNA. No difference was found between Brucella species and biovars with all primer pairs used, even after restriction enzyme analysis of the amplified fragments. The specificity of the amplified products was confirmed by hybridization with a digoxigenin 3'-labelled specific probe and by PCR using 98 non- Brucella micro-organisms' DNA. Only Ochrobactrum anthropi and Phyllobacterium spp. yielded a PCR product by using 31-kDa DnaK, DnaJ, GroEL and 16S RNA primers. After hybridization and restriction analysis, 16S RNA fragments of 3301 and 3331 O. anthropi strains showed a total similarity to those from Brucella. A similar result was shown with DnaJ fragments obtained with 3301 strain of O. anthropi after Eco RI digestion.     

Secretome analysis of the phytopathogen Macrophomina phaseolina cultivated in liquid medium supplemented with and without soybean leaf infusion

Macrophomina phaseolina (Tassi) Goid. is a fungal pathogen that causes root and stem rot in several economically important crops. However, most of disease control strategies have shown limited effectiveness. Despite its impact on agriculture, molecular mechanisms involved in the interaction with host plant remains poorly understood. Nevertheless, it has been proven that fungal pathogens secrete a variety of proteins and metabolites to successfully infect their host plants. In this study, a proteomic analysis of proteins secreted by M. phaseolina in culture media supplemented with soybean leaf infusion was performed. A total of 250 proteins were identified with a predominance of hydrolytic enzymes. Plant cell wall degrading enzymes together peptidases were found, probably involved in the infection process. Predicted effector proteins were also found that could induce plant cell death or suppress plant immune response. Some of the putative effectors presented similarities to known fungal virulence factors. Expression analysis of ten selected protein-coding genes showed that these genes are induced during host tissue infection and suggested their participation in the infection process. The identification of secreted proteins of M. phaseolina could be used to improve the understanding of the biology and pathogenesis of this fungus. Although leaf infusion was able to induce changes at the proteome level, it is necessary to study the changes induced under conditions that mimic the natural infection process of the soil-borne pathogen M. phaseolina to identify virulence factors.     

Trade-offs and the evolution of virulence of microparasites: do details matter?

Models of the within-host dynamics of parasites have been used to consider the evolution of microparasites causing acute infections in vertebrate hosts. In this paper, we use these models to examine how the level of virulence to which a parasite evolves, depends on factors such as the relationship between parasite density and its rate of transmission from infected hosts, and the mechanism of parasite-induced pathogenesis. We show that changes in the terms describing transmissibility and pathogenesis may lead to dramatic differences in the level of virulence to which a parasite evolves. This suggests that no single factor is likely to be responsible for the differences in virulence of different parasites, and that understanding of the evolution of virulence of parasites will require a detailed quantitative understanding of the interaction between the parasite and its host.     

Commingling regulatory systems following acquisition of virulence plasmids by Bacillus anthracis

The conversion of a bacterium from a non-pathogenic to a pathogenic existence is usually associated with the acquisition of virulence factors, the genes of which gain entry through bacteriophage infection, transposable elements or plasmid transfer. Pathogenesis research is mostly focused on how these factors enable the bacterium to infect the host or evade the repertoire of host defenses. Less effort is expended on understanding how the invading genes are affected by the complex regulatory circuits of the bacterium and how virulence is the result of converting these regulatory circuits to make them complicit with pathogenesis. An example of such a conversion is seen in Bacillus anthracis, and how acquired plasmid regulatory functions affect the activity of the regulatory processes of the bacterium, and vice versa, is now being revealed.     

A Computational Study Identifies HIV Progression-Related Genes Using mRMR and Shortest Path Tracing

Since statistical relationships between HIV load and CD4+ T cell loss have been demonstrated to be weak, searching for host factors contributing to the pathogenesis of HIV infection becomes a key point for both understanding the disease pathology and developing treatments. We applied Maximum Relevance Minimum Redundancy (mRMR) algorithm to a set of microarray data generated from the CD4+ T cells of viremic non-progressors (VNPs) and rapid progressors (RPs) to identify host factors associated with the different responses to HIV infection. Using mRMR algorithm, 147 gene had been identified. Furthermore, we constructed a weighted molecular interaction network with the existing protein-protein interaction data from STRING database and identified 1331 genes on the shortest-paths among the genes identified with mRMR. Functional analysis shows that the functions relating to apoptosis play important roles during the pathogenesis of HIV infection. These results bring new insights of understanding HIV progression.     

Bacterial Serine/Threonine Protein Kinases in Host-Pathogen Interactions

In bacterial pathogenesis, monitoring and adapting to the dynamically changing environment in the host and an ability to disrupt host immune responses are critical. The virulence determinants of pathogenic bacteria include the sensor/signaling proteins of the serine/threonine protein kinase (STPK) family that have a dual role of sensing the environment and subverting specific host defense processes. STPKs can sense a wide range of signals and coordinate multiple cellular processes to mount an appropriate response. Here, we review some of the well studied bacterial STPKs that are essential virulence factors and that modify global host responses during infection.