Identification of a Genetic Locus in Pseudomonas aureofaciens Involved in Fungal Inhibition

In iron-rich conditions, Pseudomonas aureofaciens PA147-2 produces an antibiotic-like compound that inhibits the growth of a plant fungal pathogen, Aphanomyces euteiches. To contribute to the potential use of PA147-2 as a biocontrol organism, we report the identification of a genetic locus important for antibiotic biosynthesis. Mutants defective for fungal inhibition (Af^-) were generated by Tn5 mutagenesis. Southern hybridization of total DNAs from three Af^- mutants indicated that loss of fungal inhibition was due to a single Tn5 insertion in each mutant. Restriction mapping of the mutation points showed that in two mutants the Tn5 insertions were in the same 16.0-kb EcoRI fragment and were separated by 2.1 kb. A genomic library of PA147-2 was constructed and screened by using a region of DNA flanking the Tn5 insertion in one mutant (PA109) as a probe to recover complementing cosmids. Three cosmids containing a 16.0-kb EcoRI fragment complementary to the two mutants were recovered. Allele replacement by homologous recombination with putative complementing cosmids restored one mutant to antifungal activity against A. euteiches. Southern analysis of the complemented mutants confirmed that allele replacement had occurred between cosmid DNA and Tn5. The wild-type 16.0-kb EcoRI fragment was cloned from the cosmid and complemented the two mutants to antifungal activity. An antifungal compound was isolated from PA147-2 grown on solid medium. Antifungal activity correlated to a peak on high-pressure liquid chromatography analysis. Under the same growth and extraction conditions, the antifungal activity seen in PA147-2 was absent in two Af^- mutants. Furthermore, absence of an antifungal compound in each mutant correlated to the absence of the wild-type “antifungal” peak on high-pressure liquid chromatography analysis.     

Characterization of Serratia entomophila Bacteriophages and the Phage-Resistant Mutant Strain BC4B

Successful large-scale fermentations of the bacterium Serratia entomophila for use in biological control of the soil-dwelling insect Costelytra zealandica has required the development of a phage-resistant mutant, BC4B. We report our investigations into S. entomophila phages and the nature of the phage resistance mechanism of strain BC4B. The parental strain of BC4B, A1MO2, was found to contain two previously unidentified prophages, (phi)9A and (phi)9B, which were UV inducible and also released spontaneously in large numbers. BC4B was shown to be completely cured of (phi)9A. Single lysogens of (phi)9A and (phi)9B were not homoimmune to any other S. entomophila phages. However, on the basis of DNA-DNA homology, all S. entomophila phages except (phi)CW3 were shown to have significant regions of homology and also packaged their DNA via pac-like mechanisms. The failure of phage particles to adsorb was identified as the basis of phage resistance in BC4B. In addition, it was demonstrated that all known S. entomophila phages are naturally temperature sensitive.     

Parallel regulation of IL-4 and IL-5 in human helminth infections.

To investigate the relationship between cytokine production and the increased levels of serum IgE and peripheral eosinophilia commonly accompanying human helminth infections, we studied the ability of PBMC of normal (N1) (n = 18) and eosinophilic individuals with helminth infections (H1) (n = 9) to produce IL-3, IL-4, IL-5, granulocyte-macrophage-CSF, and IFN-gamma in vitro after stimulation with PMA (50 ng/ml) and ionomycin (1 microgram/ml). The two groups differed in both the levels of serum IgE and eosinophilia. For mitogen-induced production of granulocyte-macrophage-CSF and IFN-gamma, there was no difference in cytokine production between the two groups. In marked contrast, supernatants from PBMC of infected individuals had significantly higher levels of IL-4 (mean = 213 pg/ml for N1 and 944 pg/ml for H1, p less than 0.02), IL-5 (mean = 180 pg/ml for N1 and 1118 pg/ml for HL, p less than 0.001), and IL-3 (mean = 13900 pg/ml for N1, 28029 pg/ml for H1, p less than 0.05). In addition, helminth-infected patients had approximately 5-fold greater numbers of T cells capable of producing IL-5 and 2.5-fold greater frequency of IL-4-secreting cells than did normal individuals; GM-CSF- and IFN-gamma-producing T cell numbers were not significantly different in the two groups. IL-3-producing cell frequencies could not be evaluated by this method. There was a direct correlation between IL-4 production and IL-5 production at the level of both protein production and frequency of T cells capable of producing these cytokines. These data indicate that individuals with reactive eosinophilia and elevated serum IgE have an expanded population of lymphocytes producing IL-4 and IL-5 and the association of the two suggests that the regulation of IL-4 and IL-5 may be linked.     

Characterization of Fus3 localization: active Fus3 localizes in complexes of varying size and specific activity

The MAP kinase Fus3 regulates many different signal transduction outputs that govern the ability of Saccharomyces cerevisiae haploid cells to mate. Here we characterize Fus3 localization and association with other proteins. By indirect immunofluorescence, Fus3 localizes in punctate spots throughout the cytoplasm and nucleus, with slightly enhanced nuclear localization after pheromone stimulation. This broad distribution is consistent with the critical role Fus3 plays in mating and contrasts that of Kss1, which concentrates in the nucleus and is not required for mating. The majority of Fus3 is soluble and not bound to any one protein; however, a fraction is stably bound to two proteins of approximately 60 and approximately 70 kDa. Based on fractionation and gradient density centrifugation properties, Fus3 exists in a number of complexes, with its activity critically dependent upon association with other proteins. In the presence of alpha factor, nearly all of the active Fus3 localizes in complexes of varying size and specific activity, whereas monomeric Fus3 has little activity. Fus3 has highest specific activity within a 350- to 500-kDa complex previously shown to contain Ste5, Ste11, and Ste7. Ste5 is required for Fus3 to exist in this complex. Upon alpha factor withdrawal, a pool of Fus3 retains activity for more than one cell cycle. Collectively, these results support Ste5's role as a tether and suggest that association of Fus3 in complexes in the presence of pheromone may prevent inactivation in addition to enhancing activation.     

The influence of antibiotic production and pre-emptive colonization on the population dynamics of Pantoea agglomerans (Erwinia herbicola) Eh1087 and Erwinia amylovora in planta

Stigma colonization by Erwinia amylovora is the crucial first step in the development of most fire blight infections in apple and pear trees. Suppression at this point of the disease process by antagonists of E. amylovora, such as Pantoea agglomerans (Erwinia herbicola) strain Eh1087, is a rational approach to control fire blight. We tested the hypothesis that the ability of E. amylovora to compete with Eh1087 for colonization of a stigma is reduced by the potential for Eh1087 to produce the phenazine antibiotic, d-alanylgriseoluteic acid (AGA). In competition experiments on the stigmas of apple flowers, E. amylovora was significantly less successful against Eh1087 (AGA+) than against EhDeltaAGA (AGA-). Further experiments to test the importance of pre-emptive colonization of the stigma by either the pathogen or the antagonist suggested that AGA production significantly enhanced the competitiveness of Eh1087 when it was applied at the same time or 24 h before the pathogen. We also found that pre-emptive stigma colonization by either the pathogen or the antagonist resulted in a population that was resilient to subsequent invasion by a second species suggesting that niche exclusion has a dominant influence on the dynamics of bacterial populations on stigmas.     

Expression of the antifeeding gene anfA1 in Serratia entomophila requires rpoS

The rpoS gene of Serratia entomophila BC4B was cloned and used to create rpoS-mutant strain BC4BRS. Larvae of the New Zealand grass grub Costelytra zealandica infected with BC4BRS became amber colored but continued to feed, albeit to a lesser extent than infected larvae. Subsequently, we found that expression of the antifeeding gene anfA1 in trans was substantially reduced in BC4BRS relative to that in the parental strain BC4B. Our data show that a functional rpoS gene is vital for full expression of anfA1 and for development of the antifeeding component of amber disease.     

Comparative Genomic Analysis of Buffalo (Bubalus bubalis) NOD1 and NOD2 Receptors and Their Functional Role in In-Vitro Cellular Immune Response

Nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs) are innate immune receptors that recognize bacterial cell wall components and initiate host immune response. Structure and function of NLRs have been well studied in human and mice, but little information exists on genetic composition and role of these receptors in innate immune system of water buffalo—a species known for its exceptional disease resistance. Here, a comparative study on the functional domains of NOD1 and NOD2 was performed across different species. The NOD mediated in-vitro cellular responses were studied in buffalo peripheral blood mononuclear cells, resident macrophages, mammary epithelial, and fibroblast cells. Buffalo NOD1 (buNOD1) and buNOD2 showed conserved domain architectures as found in other mammals. The domains of buNOD1 and buNOD2 showed analogy in secondary and tertiary conformations. Constitutive expressions of NODs were ubiquitous in different tissues. Following treatment with NOD agonists, peripheral lymphocytes showed an IFN-γ response along-with production of pro-inflammatory cytokines. Alveolar macrophages and mammary epithelial cells showed NOD mediated in-vitro immune response through NF-κB dependent pathway. Fibroblasts showed pro-inflammatory cytokine response following agonist treatment. Our study demonstrates that both immune and non-immune cells could generate NOD-mediated responses to pathogens though the type and magnitude of response depend on the cell types. The structural basis of ligand recognition by buffalo NODs and knowledge of immune response by different cell types could be useful for development of non-infective innate immune modulators and next generation anti-inflammatory compounds.     

A Comparative Study of Peripheral Immune Responses to Taenia solium in Individuals with Parenchymal and Subarachnoid Neurocysticercosis

Background

The ability of Taenia solium to modulate the immune system likely contributes to their longevity in the human host. We tested the hypothesis that the nature of the immune response is related to the location of parasite and clinical manifestations of infection.

Methodology

Peripheral blood mononuclear cells (PBMC) were obtained from untreated patients with neurocysticercosis (NCC), categorized as having parenchymal or subarachnoid infection by the presence of cysts exclusively within the parenchyma or in subarachnoid spaces of the brain, and from uninfected (control) individuals matched by age and gender to each patient. Using multiplex detection technology, sera from NCC patients and controls and cytokine production by PBMC after T. solium antigen (TsAg) stimulation were assayed for levels of inflammatory and regulatory cytokines. PBMC were phenotyped by flow cytometry ex vivo and following in vitro stimulation with TsAg.

Principal Findings

Sera from patients with parenchymal NCC demonstrated significantly higher Th1 (IFN-γ/IL-12) and Th2 (IL-4/IL-13) cytokine responses and trends towards higher levels of IL-1β/IL-8/IL-5 than those obtained from patients with subarachnoid NCC. Also higher in vitro antigen-driven TNF-β secretion was detected in PBMC supernatants from parenchymal than in subarachnoid NCC. In contrast, there was a significantly higher IL-10 response to TsAg stimulation in patients with subarachnoid NCC compared to parenchymal NCC. Although no differences in regulatory T cells (Tregs) frequencies were found ex vivo, there was a trend towards greater expansion of Tregs upon TsAg stimulation in subarachnoid than in parenchymal NCC when data were normalized for the corresponding controls.

Conclusions/Significance

T. solium infection of the subarachnoid space is associated with an enhanced regulatory immune response compared to infection in the parenchyma. The resulting anti-inflammatory milieu may represent a parasite strategy to maintain a permissive environment in the host or diminish inflammatory damage from the host immune response in the central nervous system.     

CD8-mediated protection against Ebola virus infection is perforin dependent

CD8 T cells have been shown to play an important role in the clearance and protection against fatal Ebola virus infection. In this study, we examined the mechanisms by which CD8 T cells mediate this protection. Our data demonstrate that all normal mice infected s.c. with a mouse-adapted Ebola virus survived the infection, as did 100% of mice deficient in Fas and 90% of those deficient in IFN-gamma. In contrast, perforin-deficient mice uniformly died after s.c. challenge. Perforin-deficient mice failed to clear viral infection even though they developed normal levels of neutralizing anti-Ebola Abs and 5- to 10-fold higher levels of IFN-gamma than control mice. Using MHC class I tetramers, we have also shown that perforin-deficient mice have 2- to 4-fold higher numbers of Ebola-specific CD8s than control mice. These findings suggest that the clearance of Ebola virus is perforin-dependent and provide an additional example showing that this basic immunologic mechanism is not limited to the clearance of noncytopathic viruses.     

Biochemical and molecular analyses of copper-zinc superoxide dismutase from a C4 plant Pennisetum glaucum reveals an adaptive role in response to oxidative stress

Cadherin 23 (CDH23) is an important constituent of the hair cell tip link in the organ of Corti. Mutations in cdh23 are associated with age-related hearing loss (AHL). In this study, we proposed that the Cdh23(nmf308/nmf308) mice with progressive hair cell loss had specific morphological changes and suffered a base to apex gradient and age-related hearing loss, and that mutations in cdh23 were linked to AHL. The Cdh23(nmf308/nmf308) mice produced by the N-nitrosourea (ENU) mutagenesis program were used as an animal model to study AHL and progressive hair cell loss. RT-PCR was performed to confirm the cdh23 mutation in Cdh23(nmf308/nmf308) mice and genetic analysis was used to map the specific mutation site. Distortion product otoacoustic emission (DPOAE) assay and acoustic brainstem evoked response (ABR) threshold analysis were carried out to evaluate the AHL. Cochlear histology was examined with scanning electron microscope (SEM) and transmission electron microscope (TEM), as well as the nuclear labeling by propidium iodide staining; terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay and caspase-3 activities were examined to evaluate cell apoptosis. Genetic mapping identified the candidate gene linking AHL in Cdh23(nmf308/nmf308) mice as cdh23. A mutation in exon3 (63 T>C) was screened as compared with the sequence of the same position of the gene from B6 (+/+) mice. The cochleae outer hair cells were reduced from 5-10% at one month to 100% at three months in the basal region. DPOAE and ABR exhibited an increasing threshold at high frequencies (≥16kHz) from one month of age. Morphological and cellular analysis showed that Cdh23(nmf308/nmf308) mice exhibited a time course of histological alterations and cell apoptosis of outer hair cells. Our results suggest that the cdh23 mutation may be harmful to the stereociliary tip link and cause the hair cell apoptosis. Due to the same cdh23 mutations in human subjects with presbycusis (Petit et al., 2001; Zheng et al., 2005), the Cdh23(nmf308/nmf308) mouse is an excellent animal model for investigating the mechanisms involved in human AHL.