Providing Veterans With Innovative Nursing Educational Opportunities.

This funded project assisted veteran students to obtain a baccalaureate in nursing by aligning the skills learned through military training with concepts and skills valued by professional nursing and taught to nursing students. Nine concepts or skills were identified by nursing faculty that validated for course credit. The identified concepts and skills were incorporated into four simulation scenarios. Veteran students' skills were validated through simulation experiences in place of taking a three-credit nursing course. Anecdotal data from the debriefing process were rich with students' experiences and gratitude for being recognized for their military knowledge and service.

Novel diagnosis for citrus stubborn disease by detection of a spiroplasma citri-secreted protein.

Citrus stubborn disease (CSD), first identified in California, is a widespread bacterial disease found in most arid citrus-producing regions in the United States and the Mediterranean Region. The disease is caused by Spiroplasma citri, an insect-transmitted and phloem-colonizing bacterium. CSD causes significant tree damage resulting in loss of fruit production and quality. Detection of CSD is challenging due to low and fluctuating titer and sporadic distribution of the pathogen in infected trees. In this study, we report the development of a novel diagnostic method for CSD using an S. citri-secreted protein as the detection marker. Microbial pathogens secrete a variety of proteins during infection that can potentially disperse systemically in infected plants with the vascular flow. Therefore, their distribution may not be restricted to the pathogen infection sites and could be used as a biological marker for infection. Using mass spectrometry analysis, we identified a unique secreted protein from S. citri that is highly expressed in the presence of citrus phloem extract. ScCCPP1, an antibody generated against this protein, was able to distinguish S. citri-infected citrus and periwinkle from healthy plants. In addition, the antiserum could be used to detect CSD using a simple direct tissue print assay without the need for sample processing or specialized lab equipment and may be suitable for field surveys. This study provides proof of a novel concept of using pathogen-secreted protein as a marker for diagnosis of a citrus bacterial disease and can probably be applied to other plant diseases.

Pseudomonas syringae type III effector HopZ1 targets a host enzyme to suppress isoflavone biosynthesis and promote infection in soybean.

Type III secreted effectors (T3SEs), such as Pseudomonas syringae HopZ1, are essential bacterial virulence proteins injected into the host cytosol to facilitate infection. However, few direct targets of T3SEs are known. Investigating the target(s) of HopZ1 in soybean, a natural P. syringae host, we find that HopZ1 physically interacts with the isoflavone biosynthesis enzyme, 2-hydroxyisoflavanone dehydratase (GmHID1). P. syringae infection induces gmhid1 expression and production of daidzein, a major soybean isoflavone. Silencing gmhid1 increases susceptibility to P. syringae infection, supporting a role for GmHID1 in innate immunity. P. syringae expressing active but not the catalytic mutant of HopZ1 inhibits daidzein induction and promotes bacterial multiplication in soybean. HopZ1-enhanced P. syringae multiplication is at least partially dependent on GmHID1. Thus, GmHID1 is a virulence target of HopZ1 to promote P. syringae infection of soybean. This work highlights the isoflavonoid biosynthesis pathway as an antibacterial defense mechanism and a direct T3SE target.

Catalytic domain of the diversified Pseudomonas syringae type III effector HopZ1 determines the allelic specificity in plant hosts.

The type III secretion systems (T3SS) and secreted effectors (T3SEs) are essential virulence factors in Gram-negative bacteria. During the arms race, plants have evolved resistance (R) genes to detect specific T3SEs and activate defence responses. However, this immunity can be efficiently defeated by the pathogens through effector evolution. HopZ1 of the plant pathogen Pseudomonas syringae is a member of the widely distributed YopJ T3SE family. Three alleles are known to be present in P. syringae, with HopZ1a most resembling the ancestral allelic form. In this study, molecular mechanisms underlying the sequence diversification-enabled HopZ1 allelic specificity is investigated. Using domain shuffling experiments, we present evidence showing that a central domain upstream of the conserved catalytic cysteine residue determines HopZ1 recognition specificity. Random and targeted mutagenesis identified three amino acids involved in HopZ1 allelic specificity. Particularly, the exchange of cysteine141 in HopZ1a with lysine137 at the corresponding position in HopZ1b abolished HopZ1a recognition in soybean. This position is under strong positive selection, suggesting that the cysteine/lysine mutation might be a key step driving the evolution of HopZ1. Our data support a model in which sequence diversification imposed by the plant R gene-associated immunity has driven HopZ1 evolution by allowing allele-specific substrate-binding.

Computational prediction of type III secreted proteins from gram-negative bacteria.

BACKGROUND: Type III secretion system (T3SS) is a specialized protein delivery system in gram-negative bacteria that injects proteins (called effectors) directly into the eukaryotic host cytosol and facilitates bacterial infection. For many plant and animal pathogens, T3SS is indispensable for disease development. Recently, T3SS has also been found in rhizobia and plays a crucial role in the nodulation process. Although a great deal of efforts have been done to understand type III secretion, the precise mechanism underlying the secretion and translocation process has not been fully understood. In particular, defined secretion and translocation signals enabling the secretion have not been identified from the type III secreted effectors (T3SEs), which makes the identification of these important virulence factors notoriously challenging. The availability of a large number of sequenced genomes for plant and animal-associated bacteria demands the development of efficient and effective prediction methods for the identification of T3SEs using bioinformatics approaches. RESULTS: We have developed a machine learning method based on the N-terminal amino acid sequences to predict novel type III effectors in the plant pathogen Pseudomonas syringae and the microsymbiont rhizobia. The extracted features used in the learning model (or classifier) include amino acid composition, secondary structure and solvent accessibility information. The method achieved a precision of over 90% on P. syringae in a cross validation study. In combination with a promoter screen for the type III specific promoters, this classifier trained on the P. syringae data was applied to predict novel T3SEs from the genomic sequences of four rhizobial strains. This application resulted in 57 candidate type III secreted proteins, 17 of which are confirmed effectors. CONCLUSION: Our experimental results demonstrate that the machine learning method based on N-terminal amino acid sequences combined with a promoter screen could prove to be a very effective computational approach for predicting novel type III effectors in gram-negative bacteria. Our method and data are available to the public upon request.

Allelic variants of the Pseudomonas syringae type III effector HopZ1 are differentially recognized by plant resistance systems.

The bacterial plant pathogen Pseudomonas syringae depends on the type III secretion system and type III-secreted effectors to cause disease in plants. HopZ is a diverse family of type III effectors widely distributed in P. syringae isolates. Among the HopZ homologs, HopZ1 is ancient to P. syringae and has been shown to be under strong positive selection driven by plant resistance-imposed selective pressure. Here, we characterized the virulence and avirulence functions of the three HopZ1 alleles in soybean and Nicotiana benthamiana. In soybean, HopZ1 alleles have distinct functions: HopZ1a triggers defense response, HopZ1b promotes bacterial growth, and HopZ1c has no observable effect. In N. benthamiana, HopZ1a and HopZ1b both induce plant defense responses. However, they appear to trigger different resistance pathways, evidenced by two major differences between HopZ1a- and HopZ1b-triggered hypersensitive response (HR): i) the putative N-acylation sites had no effect on HopZ1a-triggered cell death, whereas it greatly enhanced HopZ1b-triggered cell death; and ii) the HopZ1b-triggered HR, but not the HopZ1a-triggered HR, was suppressed by another HopZ homolog, HopZ3. We previously demonstrated that HopZ1a most resembled the ancestral allelic form of HopZ1; therefore, this new evidence suggested that differentiated resistance systems have evolved in plant hosts to adapt to HopZ1 diversification in P. syringae.

Convergent evolution of phytopathogenic pseudomonads onto hazelnut.

Pseudomonas syringae pv. avellanae (synonym: P. avellanae, Pav) is the causal agent of hazelnut decline in Greece and Italy. The population structure and evolutionary relationships of 22 strains from these two countries were examined by multilocus sequence typing (MLST) of four housekeeping genes (gapA, gltA, gyrB and rpoD). Neighbour-joining and maximum-likelihood phylogenetic analysis revealed that Greek strains isolated from the original 1976 outbreak of hazelnut decline through 1990 were very similar to Italian strains isolated from 2002 through 2004. Other Italian strains that were isolated during the 1990s were very homogeneous and clustered in a clade that was quite distinct from the Greek isolates and Italian isolates from the 2000s. A split decomposition analysis found evidence for recombination between these two highly divergent clades in two of the four MLST housekeeping genes. Incorporating these data into a broad MLST analysis of the P. syringae species complex showed that the Pav Greek and Italian strains from the 2000s clustered with P. syringae phylogroup 1, which is predominantly composed of pathogens of tomato and Brassicaceae hosts, while the Pav Italian strains from the 1990s clustered in P. syringae phylogroup 2 and are most closely related to pea (Pisum sativum L.) pathogens. These results clearly indicate that the ability to infect hazelnuts has arisen twice. This evolutionary process may be due to de novo adaptation to hazelnut by local P. syringae strains (such as the colonizers of Leguminosae crops), or the result of genetic exchange from the original Greek Pav clonal group into a phylogroup 2 strain. The latter explanation is intriguing since there is no exchange of hazelnut propagative material between Italy and Greece, which would be a likely vector for the movement of these pathogens.

Diversifying selection drives the evolution of the type III secretion system pilus of Pseudomonas syringae.

The plant pathogenic bacterium Pseudomonas syringae uses a type III secretion system to inject virulence proteins directly into the cytoplasm of its hosts. The P. syringae type III secretion apparatus is encoded, in part, by the HrpZ operon, which carries the hrpA gene encoding the pilin subunit of the pilus, various components of the structural apparatus, and the HrpZ harpin protein that is believed to produce pores in the host cell membrane. The pilus of the type III system comes into direct contact with the host cell and is, therefore, a likely target of the host's pathogen surveillance systems. We sequenced and analyzed 22 HrpZ operons from P. syringae strains spanning the diversity of the species. Selection analyses, including K(a)/K(s) tests and Tajima's D, revealed strong diversifying selection acting on the hrpA gene. This form of selection enables pathogens to maintain genetic diversity within their populations and is often driven by selection imposed by host defense systems. The HrpZ operon also revealed a single significant recombination event that dramatically changed the evolutionary relationships among P. syringae strains from 2 quite distinct phylogroups. This recombination event appears to have introduced genetic diversity into a clade of strains that may now be undergoing positive selection. The identification of diversifying selection acting on the Hrp pilus across the whole population sample and positive selection within one P. syringae lineage supports a trench warfare coevolutionary model between P. syringae and its plant hosts.

Phylogenetic characterization of virulence and resistance phenotypes of Pseudomonas syringae.

Individual strains of the plant pathogenic bacterium Pseudomonas syringae vary in their ability to produce toxins, nucleate ice, and resist antimicrobial compounds. These phenotypes enhance virulence, but it is not clear whether they play a dominant role in specific pathogen-host interactions. To investigate the evolution of these virulence-associated phenotypes, we used functional assays to survey for the distribution of these phenotypes among a collection of 95 P. syringae strains. All of these strains were phylogenetically characterized via multilocus sequence typing (MLST). We surveyed for the production of coronatine, phaseolotoxin, syringomycin, and tabtoxin; for resistance to ampicillin, chloramphenicol, rifampin, streptomycin, tetracycline, kanamycin, and copper; and for the ability to nucleate ice at high temperatures via the ice-nucleating protein INA. We found that fewer than 50% of the strains produced toxins and significantly fewer strains than expected produced multiple toxins, leading to the speculation that there is a cost associated with the production of multiple toxins. None of these toxins was associated with host of isolation, and their distribution, relative to core genome phylogeny, indicated extensive horizontal genetic exchange. Most strains were resistant to ampicillin and copper and had the ability to nucleate ice, and yet very few strains were resistant to the other antibiotics. The distribution of the rare resistance phenotypes was also inconsistent with the clonal history of the species and did not associate with host of isolation. The present study provides a robust phylogenetic foundation for the study of these important virulence-associated phenotypes in P. syringae host colonization and pathogenesis.

Identification of four fimbria-encoding genomic islands that are highly specific for verocytotoxin-producing Escherichia coli serotype O157 strains.

Verocytotoxin-producing Escherichia coli causes zoonotic food- or waterborne infection that may be associated with massive outbreaks and with the serious complication of hemolytic uremic syndrome (HUS). Serotypes O157:H7 and O157:NM are more commonly associated with HUS and outbreaks than other serotypes, such as O26:H11. To determine whether a genetic basis exists for why serotype O157:H7/NM causes HUS and outbreaks more often than other serotypes, such as O26:H11, we conducted suppression subtractive hybridization (SSH) between the genomes of the sequenced O157:H7 strain EDL933 and CL1, a clinical serotype O26:H11 isolate. Genes from four EDL933 fimbria-encoding genomic O islands (OIs) (OI-1, -47, -141, and -154) were identified in the SSH library. OI-47 encodes several additional putative virulence factors, including secreted and signaling proteins, a hemolysin locus, a lipoprotein, an ABC transport system, and a lipid biosynthesis locus. The distribution of the OIs was investigated by PCR and Southern hybridization (when PCR was negative) with 69 VTEC strains belonging to 39 different serotypes corresponding to 5 seropathotypes that differ in their disease and epidemic potential. The four OIs described here were distributed almost exclusively in serotypes O157:H7 and O157:NM, which indicates that they may be associated with the ability of these strains to colonize human and/or animal intestinal tracts and to cause epidemic and serious disease more frequently than other serotypes. The occurrence of the four OIs in enteropathogenic E. coli O55:H7 strains is consistent with their vertical inheritance by VTEC O157:H7/NM from this clonally related ancestor.

Dogmas and controversies in the handling of nitrogenous wastes: expression of arginase Type I and II genes in rainbow trout: influence of fasting on liver enzyme activity and mRNA levels in juveniles.

Through analysis of a cDNA library and third-party annotation of available database sequences, we characterized the full-length coding regions of rainbow trout (Oncorhynchus mykiss) Type I, Onmy-ARG01, and Type II, Onmy-ARG02, arginase genes. Two partial related arginase sequences, Onmy-ARG01b and Onmy-ARG02b, and a full-length zebrafish arginase coding region (Danio rerio), Dare-ARG02, are also reported. Comparison of vertebrate arginase sequences shows that both Type I and Type II genes in bony fishes contain a mitochondrial targeting N-terminal domain. This suggests that the cytosolic Type I arginase found in ureotelic vertebrates arose in the common ancestor of amphibia and mammals. Onmy-ARG01 and Onmy-ARG02 mRNA was detected in liver, kidney, gill, intestine, red muscle and heart tissues. Onmy-ARG01 was expressed at a significantly higher level relative to Onmy-ARG02 in liver and red muscle tissue. To investigate whether there was differential regulation of Onmy-ARG01 and Onmy-ARG02, juvenile trout were fasted for 6 weeks and hepatic enzyme activities and mRNA levels were compared with those of fed control fish. There was a 3-fold increase in liver arginase activity and a 2-fold increase in Onmy-ARG02 mRNA levels but no change in Onmy-ARG01 mRNA levels in fasted fish relative to fed fish. These findings indicate that both types of arginase genes are present and expressed in rainbow trout and that the pattern of expression varies between tissues. The increase in liver arginase activity after a 6-week fast is due, in part, to an increase in the expression of Onmy-ARG02 mRNA levels.

Regulation of a renal urea transporter with reduced salinity in a marine elasmobranch, Raja erinacea.

Marine elasmobranchs retain urea and other osmolytes, e.g. trimethylamine oxide (TMAO), to counterbalance the osmotic pressure of seawater. We investigated whether a renal urea transporter(s) would be regulated in response to dilution of the external environment. A 779 bp cDNA for a putative skate kidney urea transporter (SkUT) was cloned, sequenced and found to display relatively high identity with facilitated urea transporters from other vertebrates. Northern analysis using SkUT as a probe revealed three signals in the kidney at 3.1, 2.8 and 1.6 kb. Upon exposure to 50% seawater, the levels of all three SkUT transcripts were significantly diminished in the kidney (by 1.8- to 3.5-fold). In response to environmental dilution, renal tissue osmolality and urea concentration decreased, whereas water content increased. There were no significant differences in osmolyte and mRNA levels between the dorsal-lateral bundle and ventral sections of the kidney. Taken together, these findings provide evidence that the downregulation of SkUT may play a key role in lowering tissue urea levels in response to external osmolality.

Urea transport in kidney brush-border membrane vesicles from an elasmobranch, Raja erinacea.

Marine elasmobranch fishes maintain high urea concentrations and therefore must minimize urea loss to the environment in order to reduce the energetic costs of urea production. Previous studies have identified a facilitated urea transporter in the kidney of the dogfish. We examined mechanisms of urea transport in the kidney of the little skate Raja erinacea using an isolated brush-border membrane vesicle preparation. Urea uptake by brush-border membrane vesicles is by a phloretin-sensitive, non-saturable uniporter in the dorsal section and a phloretin-sensitive, sodium-linked urea transporter (Km = 0.70 mmol l(-1), Vmax = 1.18 micromol h(-1) mg(-1) protein) in the ventral section of the kidney. This provides evidence for two separate urea transporters in the dorsal versus ventral sections of the kidney. We propose that these two mechanisms of urea transport are critical for renal urea reabsorption in the little skate.