PtdIns(5)P activates the host cell PI3-kinase/Akt pathway during Shigella flexneri infection

The virulence factor IpgD, delivered into nonphagocytic cells by the type III secretion system of the pathogen Shigella flexneri, is a phosphoinositide 4-phosphatase generating phosphatidylinositol 5 monophosphate (PtdIns(5) P). We show that PtdIns(5) P is rapidly produced and concentrated at the entry foci of the bacteria, where it colocalises with phosphorylated Akt during the first steps of infection. Moreover, S. flexneri-induced phosphorylation of host cell Akt and its targets specifically requires IpgD. Ectopic expression of IpgD in various cell types, but not of its inactive mutant, or addition of short-chain penetrating PtdIns(5) P is sufficient to induce Akt phosphorylation. Conversely, sequestration of PtdIns(5) P or reduction of its level strongly decreases Akt phosphorylation in infected cells or in IpgD-expressing cells. Accordingly, IpgD and PtdIns(5) P production specifically activates a class IA PI 3-kinase via a mechanism involving tyrosine phosphorylations. Thus, S. flexneri parasitism is shedding light onto a new mechanism of PI 3-kinase/Akt activation via PtdIns(5) P production that plays an important role in host cell responses such as survival.

Genotypic and phenotypic diversity of a baculovirus population within an individual insect host

It is becoming increasingly apparent that many pathogen populations, including those of insects, show high levels of genotypic variation. Baculoviruses are known to be highly variable, with isolates collected from the same species in different geographical locations frequently showing genetic variation and differences in their biology. More recent Studies at smaller scales have also shown that virus DNA profiles from individual larvae can show polymorphisms within and between populations of the same species. Here, we investigate the genotypic and phenotypic variation of an insect baculovirus infection within a single insect host. Twenty four genotypically distinct nucleopolyhedrovirus (NPV) variants were isolated from an individual pine beauty moth, Panolis flammea, caterpillar by in vivo cloning techniques. No variant appeared to be dominant in the population. The Pafl NPV variants have been mapped using three restriction endonucleases and shown to contain three hypervariable regions containing insertions of 70-750 bp. Comparison of seven of these variants in an alternative host, Mamestra brassicae, demonstrated that the variants differed significantly in both pathogenicity and speed of kill. The generation and maintenance of pathogen heterogeneity are discussed. (c) 2005 Elsevier Inc. All rights reserved.

Restrictions to the adaptation of influenza A virus H5 hemagglutinin to the human host

The binding specificities of a panel of avian influenza virus subtype H5 hemagglutinin (RA) proteins bearing mutations at key residues in the receptor binding site were investigated. The results demonstrate that two simultaneous mutations in the receptor binding site resulted in H5 RA binding in a pattern similar to that shown by human viruses. Coexpression of the ion channel protein, M2, from most avian and human strains tested protected H5 RA conformation during trafficking, indicating that no genetic barrier to the reassortment of the H5 surface antigen gene with internal genes of human viruses existed at this level.

Phytohormones and plant-herbivore-pathogen interactions: integrating the molecular with the ecological

Current research into indirect phytopathogen–herbivore interactions (i.e., interactions mediated by the host plant) is carried out in two largely independent directions: ecological/mechanistic and molecular. We investigate the origin of these approaches and their strengths and weaknesses. Ecological studies have determined the effect of herbivores and phytopathogens on their host plants and are often correlative: the need for long-term manipulative experiments is pressing. Molecular/cellular studies have concentrated on the role of signaling pathways for systemic induced resistance, mainly involving salicylic acid and jasmonic acid, and more recently the cross-talk between these pathways. This cross-talk demonstrates how interactions between signaling mechanisms and phytohormones could mediate plant–herbivore–pathogen interactions. A bridge between these approaches may be provided by field studies using chemical induction of defense, or investigating whole-organism mechanisms of interactions among the three species. To determine the role of phytohormones in induced resistance in the field, researchers must combine ecological and molecular methods. We discuss how these methods can be integrated and present the concept of “kaleidoscopic defense.” Our recent molecular-level investigations of interactions between the herbivore Gastrophysa viridula and the rust fungus Uromyces rumicis on Rumex obtusifolius, which were well studied at the mechanistic and ecological levels, illustrate the difficulty in combining these different approaches. We suggest that the choice of the right study system (possibly wild relatives of model species) is important, and that molecular studies must consider the environmental conditions under which experiments are performed. The generalization of molecular predictions to ecologically realistic settings will be facilitated by “middle-ground studies” concentrating on the outcomes of the interactions.

The biocontrol fungus pochonia chlamydosporia shows nematode host preference at the infraspecific level

A RAPD-PCR assay was developed and used to test For competitive variability in growth of the nematode biological control fungus Pochonia chlamydosporia. Saprophytic competence in soil with or without tomato plants was examined in three isolates of the fungus: RES 280 (J), originally isolated from potato cyst nematode (PCN) cysts; RES 200 (1) and RES 279 (S), both originally isolated from root knot nematode (RKN) eggs. Viable counts taken at 70 d indicated that I was the best saprophyte followed by S, with J the poorest. RAPD-PCR analysis of colonies from mixed treatments revealed that there was a cumulative effect of adding isolates to the system. This Suggested that the isolates did not interact and that they may occupy separate niches in soil and the rhizosphere. To investigate parasitic ability, soils were seeded with two isolates of the fungus: J and S, singly or in combination. Tomato or potato plants were grown in these soils; free of nematodes, or inoculated with PCN or RKN, and incubated for 77 d. The abundance of the PCN isolate J in PCN cysts was significantly greater than that of the RKN isolate S but in RKN egg masses, S was significantly more abundant than J. RAPD-PCR analysis of colonies from mixed treatments confirmed that J was more abundant than S ill PCN cysts whereas the converse was observed on RKN egg masses. This substantiates the phenomenon of nematode host preference at the infraspecific level of P. chlamydosporia and highlights its relevance for biological control of plant parasitic nematodes.

Changes in in vitro susceptibility of influenza A H3N2 viruses to a neuraminidase inhibitor drug during evolution in the human host

Objectives: Influenza A H3N2 viruses isolated recently have characteristic receptor binding properties that may decrease susceptibility to neuraminidase inhibitor drugs. A panel of clinical isolates and recombinant viruses generated by reverse genetics were characterized and tested for susceptibility to zanamivir. Methods: Plaque reduction assays and neuraminidase enzyme inhibition assays were used to assess susceptibility to zanamivir. Receptor binding properties of the viruses were characterized by differential agglutination of red blood cells (RBCs) from different species. Sequence analysis of the haemagglutinin (HA) and neuraminidase (NA) genes was carried out. Results: Characterization of a panel of H3N2 clinical isolates from 1968 to 2000 showed a gradual decrease in agglutination of chicken and guinea pig RBCs over time, although all isolates could agglutinate turkey RBCs equally. Sequence analysis of the HA and NA genes identified mutations in conserved residues of the HA1 receptor binding site, in particular Leu-226 --> Ile-226/Val-226, and modification of potential glycosylation site motifs. This may be indicative of changes in virus binding to sialic acid (SA) receptors in recent years. Although recent isolates had reduced susceptibility to zanamivir in MDCK cell based plaque reduction assays, no difference was found in an NA enzyme-inhibition assay. Assays with recombinant isogenic viruses showed that the recent HA, but not the NA, conferred reduced susceptibility to zanamivir. Conclusion: This study demonstrates that recent clinical isolates of influenza A H3N2 virus no longer agglutinate chicken RBCs, but despite significant receptor binding changes as a result of changes in HA, there was little variation in sensitivity of the NA to zanamivir.

In planta proteomics and proteogenomics of the biotrophic barley fungal pathogen blumeria graminis f. sp hordei

To further our understanding of powdery mildew biology during infection, we undertook a systematic shotgun proteomics analysis of the obligate biotroph Blumeria graminis f. sp. hordei at different stages of development in the host. Moreover we used a proteogenomics approach to feed information into the annotation of the newly sequenced genome. We analyzed and compared the proteomes from three stages of development representing different functions during the plant-dependent vegetative life cycle of this fungus. We identified 441 proteins in ungerminated spores, 775 proteins in epiphytic sporulating hyphae, and 47 proteins from haustoria inside barley leaf epidermal cells and used the data to aid annotation of the B. graminis f. sp. hordei genome. We also compared the differences in the protein complement of these key stages. Although confirming some of the previously reported findings and models derived from the analysis of transcriptome dynamics, our results also suggest that the intracellular haustoria are subject to stress possibly as a result of the plant defense strategy, including the production of reactive oxygen species. In addition, a number of small haustorial proteins with a predicted N-terminal signal peptide for secretion were identified in infected tissues: these represent candidate effector proteins that may play a role in controlling host metabolism and immunity. Molecular & Cellular Proteomics 8: 2368-2381, 2009.

Stabilization of a helical water chain in a metal-organic host of a trinuclear Schiff base complex

Three heterometallic trinuclear Schiff base complexes, [{GuL(1)(H2O)}(2)Ni(CN)(4)]center dot 4H(2)O (1), [{CuL2(H2O)}(2)Ni(CN)(4)] (2), and [{CuL3(H2O)}(2)Ni(CN)(4)] (3) (HL1 = 7-amino-4-methyl-5-azahept-3-en-2-one, HL2 = 7-methylamino-4-methyl-5-azahept-3-en-2-one, and HL3 = 7-dimethylamino-4-methyl-5-azahept-3-en-2-one), were synthesized. All three complexes were characterized by elemental analysis, IR and UV spectroscopies, and thermal analysis. Two of them (1 and 3) were also characterized by single crystal X-ray crystallography. Complex 1 forms a hydrogen-bonded one-dimensional metal-organic framework that stabilizes a helical water chain into its cavity, but when any of the amine hydrogen atoms of the Schiff base are replaced by methyl groups, as in L 2 and L 3, the water chain, vanishes, showing explicitly the importance of the host-guest H-bonding interactions for the stabilization of a water cluster.

Nanosized polymetallic resorcinarene-based host assemblies that strongly bind fullerenes

Polymetallic nanodimensional assemblies have been prepared via metal directed assembly of dithiocarbamate functionalized cavitand structural frameworks with late transition metals (Ni, Pd, Cu, Au, Zn, and Cd). The coordination geometry about the metal centers is shown to dictate the architecture adopted. X-ray crystallographic studies confirm that square planar coordination geometries result in "cagelike" octanuclear complexes, whereas square-based pyramidal metal geometries favor hexanuclear "molecular loop" structures. Both classes of complex are sterically and electronically complementary to the fullerenes (C-60 and C-70). The strong binding of these guests occurred via favorable interactions with the sulfur atoms of multiple dithiocarbamate moieties of the hosts. In the case of the tetrameric copper(II) complexes, the lability of the copper(II)-dithiocarbamate bond enabled the fullerene guests to be encapsulated in the electron-rich cavity of the host, over time. The examination of the binding of fullerenes has been undertaken using spectroscopic and electrochemical methods, electrospray mass spectrometry, and molecular modeling.

Incorporation of a sodium ion guest in the host of copper(II)-Schiff-base complexes: Structural characterization and magnetic study

Three copper(II) complexes, [CuL1], [CuL2] and [CuL3] where L-1, L-2 and L-3 are the tetradentate di-Schiff-base ligands prepared by the condensation of acetylacetone and appropriate diamines (e.g. 1,2-diaminoethane, 1,2-diaminopropane and 1,3-diaminopropane, respectively) in 2:1 ratios, have been prepared. These complexes act as host molecules and include a guest sodium ion by coordinating through the oxygen atoms to result in corresponding new trinuclear complexes, [(CuL1)(2)Na(ClO4)(H2O)][CuL1], [(CuL2)(2)Na(ClO4)(H2O)] (2) and [(CuL3)(2)Na(ClO4)(H2O)] (3) when crystallized from methanol solution containing sodium perchlorate. All three complexes have been characterized by single crystal X-ray crystallography. In all the complexes, the sodium cation has a six-coordinate distorted octahedral environment being bonded to four oxygen atoms from two Schiff-base complexes of Cu(II) in addition to a perchlorate anion and a water molecule. The copper atoms are four coordinate in a square planar environment being bonded to two oxygen atoms and two nitrogen atoms of the Schiff-base ligand. The variable temperature susceptibilities for complexes 1-3 were measured over the range 2-300 K. The isotropic Hamiltonian, H = g(1)beta HS1 + g(2)beta HS2 + J(12)S(1)S(2) + g(3)beta HS3 for complex 1 and H = g(1)beta HS1 + g2 beta HS2 +J(12)S(1)S(2) for complexes 2 and 3 has been used to interpret the magnetic data. The best fit parameters obtained are: g(1) = g(2) = 2.07(0), J = - 1.09(1) cm(-1) for complex 1, g(1) = g(2) = 2.06(0), J = -0.55(1) cm(-1) for complex 2 and g1 = g2 = 2.07(0).J = -0.80(1) cm(-1) for 3. Electrochemical studies displayed an irreversible Cu(II)/Cu(I) one-electron reduction process. (C) 2008 Elsevier Ltd. All rights reserved.

Top-down systems biology modeling of host metabotype-microbiome associations in obese rodents

Covariation in the structural composition of the gut microbiome and the spectroscopically derived metabolic phenotype (metabotype) of a rodent model for obesity were investigated using a range of multivariate statistical tools. Urine and plasma samples from three strains of 10-week-old male Zucker rats (obese (fa/fa, n = 8), lean (fal-, n = 8) and lean (-/-, n = 8)) were characterized via high-resolution H-1 NMR spectroscopy, and in parallel, the fecal microbial composition was investigated using fluorescence in situ hydridization (FISH) and denaturing gradient gel electrophoresis (DGGE) methods. All three Zucker strains had different relative abundances of the dominant members of their intestinal microbiota (FISH), with the novel observation of a Halomonas and a Sphingomonas species being present in the (fa/fa) obese strain on the basis of DGGE data. The two functionally and phenotypically normal Zucker strains (fal- and -/-) were readily distinguished from the (fa/fa) obese rats on the basis of their metabotypes with relatively lower urinary hippurate and creatinine, relatively higher levels of urinary isoleucine, leucine and acetate and higher plasma LDL and VLDL levels typifying the (fa/fa) obese strain. Collectively, these data suggest a conditional host genetic involvement in selection of the microbial species in each host strain, and that both lean and obese animals could have specific metabolic phenotypes that are linked to their individual microbiomes.

The impact of genome defense on mobile elements in Microbotryum

Repeat induced point mutation (RIP), a mechanism causing hypermutation of repetitive DNA sequences in fungi, has been described as a ‘genome defense’ which functions to inactivate mobile elements and inhibit their deleterious effects on genome stability. Here we address the interactions between RIP and transposable elements in the Microbotryum violaceum species complex. Ten strains of M. violaceum, most of which belong to different species of the fungus, were all found to contain intragenomic populations of copia-like retrotransposons. Intragenomic DNA sequence variation among the copia-like elements was analyzed for evidence of RIP. Among species with RIP, there was no significant correlation between the frequency of RIP-induced mutations and inferred transposition rate based on diversity. Two strains of M. violaceum, from two different plant species but belonging to the same fungal lineage, contained copia-like elements with very low diversity, as would result from a high transposition rate, and these were also unique in showing no evidence of the hypermutation patterns indicative of the RIP genome defense. In this species, evidence of RIP was also absent from a Class II helitron-like transposable element. However, unexpectedly the absolute repetitive element load was lower than in other strains.

Coping with multiple enemies - the evolution of resistance and host-parasitoid community structure

Recent work has shown that the evolution of Drosophila melanogaster resistance to attack by the parasitoid Asobara tabida is constrained by a trade-off with larval competitive ability. However, there are two very important questions that need to be answered. First, is this a general cost, or is it parasitoid specific? Second, does a selected increase in immune response against one parasitoid species result in a correlated change in resistance to other parasitoid species? The answers to both questions will influence the coevolutionary dynamics of these species, and also may have a previously unconsidered, yet important, influence on community structure.