Ail Proteins of Yersinia pestis and Y. pseudotuberculosis Have Different Cell Binding and Invasion Activities

The Yersinia pestis adhesin Ail mediates host cell binding and facilitates delivery of cytotoxic Yop proteins. Ail from Y. pestis and Y. pseudotuberculosis is identical except for one or two amino acids at positions 43 and 126 depending on the Y. pseudotuberculosis strain. Ail from Y. pseudotuberculosis strain YPIII has been reported to lack host cell binding ability, thus we sought to determine which amino acid difference(s) are responsible for the difference in cell adhesion. Y. pseudotuberculosis YPIII Ail expressed in Escherichia coli bound host cells, albeit at ∼50% the capacity of Y. pestis Ail. Y. pestis Ail single mutants, Ail-E43D and Ail-F126V, both have decreased adhesion and invasion in E. coli when compared to wild-type Y. pestis Ail. Y. pseudotuberculosis YPIII Ail also had decreased binding to the Ail substrate fibronectin, relative to Y. pestis Ail in E. coli. When expressed in Y. pestis, there was a 30–50% decrease in adhesion and invasion depending on the substitution. Ail-mediated Yop delivery by both Y. pestis Ail and Y. pseudotuberculosis Ail were similar when expressed in Y. pestis, with only Ail-F126V giving a statistically significant reduction in Yop delivery of 25%. In contrast to results in E. coli and Y. pestis, expression of Ail in Y. pseudotuberculosis led to no measurable adhesion or invasion, suggesting the longer LPS of Y. pseudotuberculosis interferes with Ail cell-binding activity. Thus, host context affects the binding activities of Ail and both Y. pestis and Y. pseudotuberculosis Ail can mediate cell binding, cell invasion and facilitate Yop delivery.     

Swabbing Often Fails to Detect Amphibian Chytridiomycosis under Conditions of Low Infection Load

The pathogenic chytrid fungus, Batrachochytrium dendrobatidis (denoted Bd), causes large-scale epizootics in naïve amphibian populations. Intervention strategies to rapidly respond to Bd incursions require sensitive and accurate diagnostic methods. Chytridiomycosis usually is assessed by quantitative polymerase chain reaction (qPCR) amplification of amphibian skin swabs. Results based on this method, however, sometimes yield inconsistent results on infection status and inaccurate scores of infection intensity. In Asia and other regions where amphibians typically bear low Bd loads, swab results are least reliable. We developed a Bd-sampling method that collects zoospores released by infected subjects into an aquatic medium. Bd DNA is extracted by filters and amplified by nested PCR. Using laboratory colonies and field populations of Bombina orientalis, we compare results with those obtained on the same subjects by qPCR of DNA extracted from swabs. Many subjects, despite being diagnosed as Bd-negative by conventional methods, released Bd zoospores into collection containers and thus must be considered infected. Infection loads determined from filtered water were at least 1000 times higher than those estimated from swabs. Subjects significantly varied in infection load, as they intermittently released zoospores, over a 5-day period. Thus, the method might be used to compare the infectivity of individuals and study the periodicity of zoospore release. Sampling methods based on water filtration can dramatically increase the capacity to accurately diagnose chytridiomycosis and contribute to a better understanding of the interactions between Bd and its hosts.     

Toxigenic fungi associated with stored corn

A total of 246 fungal isolates were obtained from 25 moldy corn samples collected in central Iowa. Either water or ether extracts of all corn samples except one exhibited some degree of toxicity in mice and/or ducklings. At least one toxigenic isolate was obtained from each corn sample. Extracts of cultures of 99 of the fungal isolates, involving 13 genera, produced death in one or more of the assay animals. The majority of the toxigenic isolates belonged to the generaAspergillus andPenicillium. Three isolates ofTrichothecium roseum were highly toxic to ducklings and mice. The ducklings exhibited a flaccid paralysis shortly after receiving an oral dose of extracts of these three isolates. Death frequently occurred following a second oral dose given 24 hr later. Mice given intraperitoneal injections of extracts ofT. roseum first exhibited lethargy and intermittent tremors, then hyperemotivity, roughening of the hair coat, abdominal respirations, incoordination, dyspnea and clonic convulsions with death occurring usually within 10 to 20 min.

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Zusammenfassung Insgesamt sind 246 Pilzisolierungen von 25 verschimmelten Getreideproben, die in Zentral-Iowa gesammelt worden sind, erhalten worden. Sowohol Wasser- wie Aether-Extrakte von allen Getreideproben zeigten eine gewisse Toxizität in Mäusen als auch in Entchen außer einer einzigen Probe. Kulturextrakte von 99 der Pilzisolierungen, die 13 Gattungen umfaßten, verursachten den Tod in einem oder in mehreren Versuchstieren. Die Mehrheit der toxischen Isolierungen gehörte den GattungenAspergillus undPenicillium an. Drei Isolierungen vonTrichothecium roseum waren hoch toxisch für Entchen und Mäuse. Die Entchen zeigten eine schlaffe Paralyse kurz nachdem sie eine orale Dosis der Auszüge dieser drei Isolierungen erhalten hatten. Tod erfolgte häufig nach der zweiten oralen Dosis innerhalb 24 Stunden. Mäuse, die eine intraperitoneale Injektion der Auszüge vonT. roseum erhielten, zeigten erst eine Lethargie und einen intermittierenden Tremor, dann eine Hyperemotivität, aufgerauhte Haarbedeckung, abdominale Atmung, Inkoordination, Dyspnoe, klonische Zuckungen und Tod gewöhnlich innerhalb 10 bis 20 Minuten.

     

Biological effects of toxic products from Trichothecium roseum link

Extracts of rice cultures of 8 of 16 isolates ofTrichothecium roseum killed one or more ducklings or mice or both. One of the isolates (MC-156) obtained from shelled corn was the most toxigenic; extracts of this isolate killed all treated ducklings and mice.Doses of purified toxic fraction TR-1 of 166 mg/kg body weight given intraperitoneally killed all test mice but none of the mice given 100 mg/kg doses died. However, a partially purified fraction (Fraction VII), from which toxic fraction TR-1 was derived, killed two of three mice given 78 mg/kg doses.Crude ether extracts of rice cultures ofT. roseum (MC-156) produced death when injected intraperitoneally in rabbits and a 19-day-old pig and also produced dermal necrosis when applied to the skin of rabbits. The latter phenomenon was not observed when 5 mg of toxic fraction TR-1 was applied to the skin of rabbits.The greatest production of toxin occurred in rice cultures when incubated at 28° C and with 20 % (ml/g) water added to the rice. Incubation of rice cultures ofT. roseum (MC-156) under CO₂ tension suppressed toxin production.

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Zusammenfassung Auszüge von Reiskulturen von acht der 16 Stämme vonTrichothecium roseum haben mehrere Entchen und/oder Mäuse getötet. Einer der Auszüge (MC-156) von abgeschälten Mais war das toxischste; Auszüge dieses Isolates hat alle Entchen und Mäuse getötet. Dosen der gereinigten toxischen Fraktion TR-1, 166 mg/Kg Körpergewicht, intraperitonial verabreicht, haben alle Mäuse getötet, aber keine der Mäuse starb mit der Dose von 100 mg/Kg. Jedoch hat eine teilweise gereinigte Fraktion (Fraktion VII), von welcher die toxische Fraktion TR-1 gewonnen war, zwei von drei Mäusen mit 78 mg/Kg Dose getötet. Ungereinigte Ätherauszüge vonT. roseum (MC-156) waren tödlich, wenn sie in Kaninchen, in ein 19 Tage altes Schweinchen intraperitonel injiziert worden sind. Sie haben auch eine Hautnekrose hervorgerufen, wenn sie auf die Haut von Kaninchen gebracht worden sind. Das letztere Phänomen war nicht beobachtet, wenn 5 mg der toxischen Fraktion TR-1 an Kaninchenhaut gebracht worden ist. Die größte Produktion des Toxins fand in Reiskulturen statt, wenn sie bei 28° C und mit 20 Perzent (ml/g) Wasser bebrütet worden sind. Inkubation von Reiskulturen vonT. roseum (MC-156) unter CO₂-Druck hat die Toxinproduktion unterdrückt.

     

Deterioration of High-Moisture Corn

Two small, leaky silos were filled with normal high-moisture corn (HMC), and two with HMC severely infested by Helminthosporium maydis. Counts of mesophilic bacteria, lactobacilli, coliforms, yeasts, and molds were made on corn samples as received and periodically thereafter during 220 days of storage. Temperature and gas levels also were monitored. Sequential changes in the populations of lactobacilli, yeasts, and molds were determined during spoilage of HMC. These population changes were compared on the basis of the variables encountered in the present study as well as with the results of previous studies conducted on normal HMC stored under adequate conditions. Heavy infestation by H. maydis had no appreciable effect on HMC preservation.     

Domain V peptides inhibit beta2-glycoprotein I-mediated mesenteric ischemia/reperfusion-induced tissue damage and inflammation

Reperfusion of ischemic tissue induces significant tissue damage in multiple conditions, including myocardial infarctions, stroke, and transplantation. Although not as common, the mortality rate of mesenteric ischemia/reperfusion (IR) remains >70%. Although complement and naturally occurring Abs are known to mediate significant damage during IR, the target Ags are intracellular molecules. We investigated the role of the serum protein, β2-glycoprotein I as an initiating Ag for Ab recognition and β2-glycoprotein I (β2-GPI) peptides as a therapeutic for mesenteric IR. The time course of β2-GPI binding to the tissue indicated binding and complement activation within 15 min postreperfusion. Treatment of wild-type mice with peptides corresponding to the lipid binding domain V of β2-GPI blocked intestinal injury and inflammation, including cellular influx and cytokine and eicosanoid production. The optimal therapeutic peptide (peptide 296) contained the lysine-rich region of domain V. In addition, damage and most inflammation were also blocked by peptide 305, which overlaps with peptide 296 but does not contain the lysine-rich, phospholipid-binding region. Importantly, peptide 296 retained efficacy after replacement of cysteine residues with serine. In addition, infusion of wild-type serum containing reduced levels of anti-β2-GPI Abs into Rag-1(-/-) mice prevented IR-induced intestinal damage and inflammation. Taken together, these data suggest that the serum protein β2-GPI initiates the IR-induced intestinal damage and inflammatory response and as such is a critical therapeutic target for IR-induced damage and inflammation.     

Ail protein binds ninth type III fibronectin repeat (9FNIII) within central 120-kDa region of fibronectin to facilitate cell binding by Yersinia pestis

The Yersinia pestis adhesin molecule Ail interacts with the extracellular matrix protein fibronectin (Fn) on host cells to facilitate efficient delivery of cytotoxic Yop proteins, a process essential for plague virulence. A number of bacterial pathogens are known to bind to the N-terminal region of Fn, comprising type I Fn (FNI) repeats. Using proteolytically generated Fn fragments and purified recombinant Fn fragments, we demonstrated that Ail binds the centrally located 120-kDa fragment containing type III Fn (FNIII) repeats. A panel of monoclonal antibodies (mAbs) that recognize specific epitopes within the 120-kDa fragment demonstrated that mAb binding to (9)FNIII blocks Ail-mediated bacterial binding to Fn. Epitopes of three mAbs that blocked Ail binding to Fn were mapped to a similar face of (9)FNIII. Antibodies directed against (9)FNIII also inhibited Ail-dependent cell binding activity, thus demonstrating the biological relevance of this Ail binding region on Fn. Bacteria expressing Ail on their surface could also bind a minimal fragment of Fn containing repeats (9-10)FNIII, and this binding was blocked by a mAb specific for (9)FNIII. These data demonstrate that Ail binds to (9)FNIII of Fn and presents Fn to host cells to facilitate cell binding and delivery of Yops (cytotoxins of Y. pestis), a novel interaction, distinct from other bacterial Fn-binding proteins.     

Exome sequencing in multiplex families with left-sided cardiac defects has high yield for disease gene discovery

Congenital heart disease (CHD) is a common group of birth defects with a strong genetic contribution to their etiology, but historically the diagnostic yield from exome studies of isolated CHD has been low. Pleiotropy, variable expressivity, and the difficulty of accurately phenotyping newborns contribute to this problem. We hypothesized that performing exome sequencing on selected individuals in families with multiple members affected by left-sided CHD, then filtering variants by population frequency, in silico predictive algorithms, and phenotypic annotations from publicly available databases would increase this yield and generate a list of candidate disease-causing variants that would show a high validation rate. In eight of the nineteen families in our study (42%), we established a well-known gene/phenotype link for a candidate variant or performed confirmation of a candidate variant’s effect on protein function, including variants in genes not previously described or firmly established as disease genes in the body of CHD literature: BMP10, CASZ1, ROCK1 and SMYD1. Two plausible variants in different genes were found to segregate in the same family in two instances suggesting oligogenic inheritance. These results highlight the need for functional validation and demonstrate that in the era of next-generation sequencing, multiplex families with isolated CHD can still bring high yield to the discovery of novel disease genes.