Use of a marker plasmid to examine differential rates of growth and death between clinical and environmental strains of Vibrio vulnificus in experimentally infected mice

Vibrio vulnificus is Gram-negative bacterium that contaminates oysters, causing highly lethal sepsis after consumption of raw oysters and wound infection. We previously described two sets of V. vulnificus strains with different levels of virulence in subcutaneously inoculated iron dextran-treated mice. Both virulent, clinical strains and attenuated, environmental strains could be recovered in high numbers from skin lesions and livers; however, the attenuated environmental strains required significantly higher numbers of colony-forming units (cfu) in the inoculum to produce lethal infection. Using some of these strains and an additional clinical strain, we presently asked if the different abilities to cause infection between the clinical and environmental strains were due to differences in rates of growth or death of the bacteria in the mouse host. We therefore constructed a marker plasmid, pGTR902, that functions as a replicon only in the presence of arabinose, which is not present in significant levels in animal tissues. V. vulnificus strains containing pGTR902 were inoculated into iron dextran-treated and untreated mice. Measuring the proportion of bacteria that had maintained the marker plasmid recovered from mice enabled us to monitor the number of in vivo divisions, hence growth rate; whereas measuring the number of marker plasmid-containing bacteria recovered enabled the measurement of death of the vibrios in the mice. The numbers of bacterial divisions in vivo for all of the strains over a 12-15 h infection period were not significantly different in iron dextran-treated mice; however, the rate of death of one environmental strain was significantly higher compared with the clinical strains. Infection of non-iron dextran-treated mice with clinical strains demonstrated that the greatest effect of iron dextran-treatment was increased growth rate, while one clinical strain also experienced increased death in untreated mice. V. vulnificus inoculated into iron dextran-treated mice replicated extremely rapidly over the first 4 h of infection with doubling times of approximately 15-28 min. In contrast, one of the environmental strains exhibited a reduced early growth rate. These results demonstrate that differences in virulence among naturally occurring V. vulnificus can be explained by diverse abilities to replicate rapidly in or resist defences of the host. The marker plasmid pGTR902 should be useful for examining virulence of bacteria in terms of differentiating growth verses death in animal hosts for most Gram-negative bacteria.     

Yeast functional genomic screens lead to identification of a role for a bacterial effector in innate immunity regulation

Numerous bacterial pathogens manipulate host cell processes to promote infection and ultimately cause disease through the action of proteins that they directly inject into host cells. Identification of the targets and molecular mechanisms of action used by these bacterial effector proteins is critical to understanding pathogenesis. We have developed a systems biological approach using the yeast Saccharomyces cerevisiae that can expedite the identification of cellular processes targeted by bacterial effector proteins. We systematically screened the viable yeast haploid deletion strain collection for mutants hypersensitive to expression of the Shigella type III effector OspF. Statistical data mining of the results identified several cellular processes, including cell wall biogenesis, which when impaired by a deletion caused yeast to be hypersensitive to OspF expression. Microarray experiments revealed that OspF expression resulted in reversed regulation of genes regulated by the yeast cell wall integrity pathway. The yeast cell wall integrity pathway is a highly conserved mitogen-activated protein kinase (MAPK) signaling pathway, normally activated in response to cell wall perturbations. Together these results led us to hypothesize and subsequently demonstrate that OspF inhibited both yeast and mammalian MAPK signaling cascades. Furthermore, inhibition of MAPK signaling by OspF is associated with attenuation of the host innate immune response to Shigella infection in a mouse model. These studies demonstrate how yeast systems biology can facilitate functional characterization of pathogenic bacterial effector proteins.     

Role of sphingosine-1 phosphate in the enhancement of endothelial barrier integrity by platelet-released products

In vitro and in vivo evidence indicates that circulating platelets affect both vascular integrity and hemostasis. How platelets enhance the permeability barrier of the vascular endothelium is not well understood. We measured the effect of isolated human platelets on human pulmonary artery endothelial cell (EC) barrier integrity by monitoring transmonolayer electrical resistance. EC barrier function was significantly increased by the addition of platelets ( approximately 40% maximum, 2.5 x 106 platelets/ml). Platelet supernatants, derived from 2.5 x 106 platelets/ml, reproduced the barrier enhancement and reversed the barrier dysfunction produced by the edemagenic agonist thrombin, which implicates a soluble barrier-promoting factor. The barrier-enhancing effect of platelet supernatants was heat stable but was attenuated by either charcoal delipidation (suggesting a vasoactive lipid mediator) or pertussis toxin, implying involvement of a Gialpha-coupled receptor signal transduction pathway. Sphingosine-1-phosphate (S1P), a sphingolipid that is released from activated platelets, is known to ligate G protein-coupled EC differentiation gene (EDG) receptors, increase EC electrical resistance, and reorganize the actin cytoskeleton (Garcia JG, Liu F, Verin AD, Birukova A, Dechert MA, Gerthoffer WT, Bamberg JR, and English D. J Clin Invest 108: 689-701, 2001). Infection of EC with an adenoviral vector expressing an antisense oligonucleotide directed against EDG-1 but not infection with control vector attenuated the barrier-enhancing effect of both platelet supernatants and S1P. These results indicate that a major physiologically relevant vascular barrier-protective mediator produced by human platelets is S1P.     

Evolutionary History of the Live-Bearing Endemic Allotoca diazi Species Complex (Actinopterygii,Goodeinae): Evidence of Founder Effect Events in the Mexican Pre-Hispanic Period

The evolutionary history of Mexican ichthyofauna has been strongly linked to natural events, and the impact of pre-Hispanic cultures is little known. The live-bearing fish species Allotoca diazi, Allotoca meeki and Allotoca catarinae occur in areas of biological, cultural and economic importance in central Mexico: Pátzcuaro basin, Zirahuén basin, and the Cupatitzio River, respectively. The species are closely related genetically and morphologically, and hypotheses have attempted to explain their systematics and biogeography. Mitochondrial DNA and microsatellite markers were used to investigate the evolutionary history of the complex. The species complex shows minimal genetic differentiation. The separation of A. diazi and A. meeki was dated to 400–7000 years ago, explained by geological and climate events. A bottleneck and reduction of genetic diversity in Allotoca diazi was detected, attributed to recent climate fluctuations and anthropogenic activity. The isolation of A. catarinae occurred ~1900 years ago. No geological events are documented in the area during this period, but the date is contemporary with P’urhépecha culture settlements. This founder effect represents the first evidence of fish species translocation by a pre-Hispanic culture of Mexico. The response of the complex to climate fluctuation, geological changes and human activity in the past and the future according to the ecological niches predictions indicates areas of vulnerability and important information for conservation. The new genetic information showed that the Allotoca diazi complex consist of two genetic groups with an incomplete lineage sorting pattern: Pátzcuaro and Zirahuén lakes, and an introduced population in the Cupatitzio River.     

Eimeria acervulina: DNA cloning and characterization of recombinant sporozoite and merozoite antigens

Genes encoding antigens of Eimeria acervulina were cloned from cDNA expression libraries prepared from the sporozoite and merozoite stages in order to examine humoral and cellular immune responses to this protozoan parasite. Two clones expressing surface antigens were characterized by DNA hybridization studies to identify homologous genomic DNA fragments. The proteins they encode were identified by 125I-labeling, immunoblotting, immunofluorescence, and T-cell activation experiments. One, designated cSZ-1, encodes a 130-kDa beta-galactosidase fusion protein which represents a portion of a p240/p160 immunodominant sporozoite surface antigen. Immunofluorescence studies using anti-cSZ-1 sera and live or 1% paraformaldehyde-fixed E. acervulina sporozoites have confirmed this surface locale. Purified cSZ-1 fusion protein, which is not recognized by sera from E. acervulina-infected chickens, induced the activation of immune T lymphocytes in vitro. Another cDNA clone, designated cMZ-8, gives rise to a 150-kDa fusion protein and encodes a portion of a p250 immunodominant merozoite surface antigen. This was established by immunoblotting of 125I-labeled merozoite proteins with anti-cMZ-8 sera and immunofluorescence staining of live and 1% paraformaldehyde-fixed E. acervulina merozoites. Purified cMZ-8 is recognized by sera from E. acervulina-infected chickens and induces a significant activation of immune T lymphocytes in vitro.     

Inhibition of STAT3 promotes the efficacy of adoptive transfer therapy using type-1 CTLs by modulation of the immunological microenvironment in a murine intracranial glioma

A variety of cancers, including malignant gliomas, show aberrant activation of STAT3, which plays a pivotal role in negative regulation of antitumor immunity. We hypothesized that inhibition of STAT3 signals would improve the efficacy of T cell adoptive transfer therapy by reversal of STAT3-induced immunosuppression in a murine GL261 intracranial glioma model. In vitro treatment of GL261 cells with JSI-124, a STAT3 inhibitor, reversed highly phosphorylated status of STAT3. Systemic i.p. administration of JSI-124 in glioma-bearing immunocompetent mice, but not athymic mice, resulted in prolonged survival, suggesting a role of adaptive immunity in the antitumor effect. Furthermore, JSI-124 promoted maturation of tumor-infiltrating CD11c(+) dendritic cells and activation of tumor-conditioned cytotoxic T cells, enhanced dendritic cells and GL261 production of CXCL-10, a critical chemokine for attraction of Tc1 cells. When i.p. JSI-124 administration was combined with i.v. transfer of Pmel-I mouse-derived type-1 CTLs (Tc1), glioma-bearing mice exhibited prolonged survival compared with i.p. JSI-124 or i.v. Tc1 therapy alone. Flow cytometric analyses of brain infiltrating lymphocytes revealed that JSI-124-treatment enhanced the tumor-homing of i.v. transferred Tc1 cells in a CXCL-10-dependent fashion. Systemic JSI-124 administration also up-regulated serum IL-15 levels, and promoted the persistence of transferred Tc1 in the host. These data suggest that systemic inhibition of STAT3 signaling can reverse the suppressive immunological environment of intracranial tumor bearing mice both systemically and locally, thereby promoting the efficacy of adoptive transfer therapy with Tc1.     

Shifting white pox aetiologies affecting Acropora palmata in the Florida Keys, 1994–2014

We propose ‘the moving target hypothesis’ to describe the aetiology of a contemporary coral disease that differs from that of its historical disease state. Hitting the target with coral disease aetiology is a complex pursuit that requires understanding of host and environment, and may lack a single pathogen solution. White pox disease (WPX) affects the Caribbean coral Acropora palmata. Acroporid serratiosis is a form of WPX for which the bacterial pathogen (Serratia marcescens) has been established. We used long-term (1994–2014) photographic monitoring to evaluate historical and contemporary epizootiology and aetiology of WPX affecting A. palmata at eight reefs in the Florida Keys. Ranges of WPX prevalence over time (0–71.4%) were comparable for the duration of the 20-year study. Whole colony mortality and disease severity were high in historical (1994–2004), and low in contemporary (2008–2014), outbreaks of WPX. Acroporid serratiosis was diagnosed for some historical (1999, 2003) and contemporary (2012, 2013) outbreaks, but this form of WPX was not confirmed for all WPX cases. Our results serve as a context for considering aetiology as a moving target for WPX and other coral diseases for which pathogens are established and/or candidate pathogens are identified. Coral aetiology investigations completed to date suggest that changes in pathogen, host and/or environment alter the disease state and complicate diagnosis.     

A missense mutation in the 20S proteasome β2 subunit of Great Danes having harlequin coat patterning

Harlequin is a pigmentary trait of the domestic dog that is controlled by two autosomal loci: the melanosomal gene, SILV, and a modifier gene, harlequin (H), previously localized to chromosome 9. Heterozygosity for a retrotransposon insertion in SILV and a mutation in H causes a pattern of black patches on a white background. Homozygosity for H is embryonic lethal. Fine mapping of the harlequin locus revealed a 25 kb interval wherein all harlequin Great Danes are heterozygous for a common haplotype. This region contains one gene, PSMB7, which encodes the β2 catalytic subunit of the proteasome. Sequence analysis identified a coding variant in exon 2 that segregates with harlequin patterning. The substitution predicts the replacement of a highly conserved valine with a glycine. Described herein is the identification of a naturally-occurring mutation of the ubiquitin proteasome system that is associated with a discernable phenotype of dogs.     

A novel insertion mutation in atlastin 1 is associated with spastic quadriplegia,increased membrane tethering,and aberrant conformational switching

Hereditary spastic paraplegia (HSP) comprises a heterogeneous group of neuropathies affecting upper motor neurons and causing progressive gait disorder. Mutations in the gene SPG3A/atlastin-1 (ATL1), encoding a dynamin superfamily member, which utilizes the energy from GTP hydrolysis for membrane tethering and fusion to promote the formation of a highly branched, smooth endoplasmic reticulum (ER), account for approximately 10% of all HSP cases. The continued discovery and characterization of novel disease mutations are crucial for our understanding of HSP pathogenesis and potential treatments. Here, we report a novel disease-causing, in-frame insertion in the ATL1 gene, leading to inclusion of an additional asparagine residue at position 417 (N417ins). This mutation correlates with complex, early-onset spastic quadriplegia affecting all four extremities, generalized dystonia, and a thinning of the corpus callosum. We show using limited proteolysis and FRET-based studies that this novel insertion affects a region in the protein central to intramolecular interactions and GTPase-driven conformational change, and that this insertion mutation is associated with an aberrant prehydrolysis state. While GTPase activity remains unaffected by the insertion, membrane tethering is increased, indicative of a gain-of-function disease mechanism uncommon for ATL1-associated pathologies. In conclusion, our results identify a novel insertion mutation with altered membrane tethering activity that is associated with spastic quadriplegia, potentially uncovering a broad spectrum of molecular mechanisms that may affect neuronal function.