Disease resistance of pacu Piaractus mesopotamicus (Holmberg, 1887) fed with beta-glucan

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Dissecting Phaseolus vulgaris Innate Immune System against Colletotrichum lindemuthianum Infection

Background: The genus Colletotrichum is one of the most economically important plant pathogens, causing anthracnose on a wide range of crops including common beans (Phaseolus vulgaris L.). Crop yield can be dramatically decreased depending on the plant cultivar used and the environmental conditions. This study aimed to identify potential genetic components of the bean immune system to provide environmentally friendly control measures against this fungus. Methodology and Principal Findings: As the common bean is not amenable to reverse genetics to explore functionality and its genome is not fully curated, we used putative Arabidopsis orthologs of bean expressed sequence tag (EST) to perform bioinformatic analysis and experimental validation of gene expression to identify common bean genes regulated during the incompatible interaction with C. lindemuthianum. Similar to model pathosystems, Gene Ontology (GO) analysis indicated that hormone biosynthesis and signaling in common beans seem to be modulated by fungus infection. For instance, cytokinin and ethylene responses were up-regulated and jasmonic acid, gibberellin, and abscisic acid responses were down-regulated, indicating that these hormones may play a central role in this pathosystem. Importantly, we have identified putative bean gene orthologs of Arabidopsis genes involved in the plant immune system. Based on experimental validation of gene expression, we propose that hypersensitive reaction as part of effector-triggered immunity may operate, at least in part, by down-regulating genes, such as FLS2-like and MKK5-like, putative orthologs of the Arabidopsis genes involved in pathogen perception and downstream signaling. Conclusions/Significance: We have identified specific bean genes and uncovered metabolic processes and pathways that may be involved in the immune response against pathogens. Our transcriptome database is a rich resource for mining novel defense-related genes, which enabled us to develop a model of the molecular components of the bean innate immune system regulated upon pathogen attack.

Hemolytic activity of alternative complement pathway as an indicator of innate immunity in pacu (Piaractus mesopotamicus)

The objective of this study was to evaluate the methodology to establish the hemolytic activity of alternative complement pathway as an indicator of the innate immunity in Brazilian fish pacu (Piaractus mesopotamicus), in addition to verifying the influence of beta-glucan as an immunostimulant. Fish were fed with diets containing 0, 0.1 and 1% beta-glucan, during seven days, and then inoculated with Aeromonas hydrophila. Seven days after the challenge, they were bled for serum extraction. The methodology consisted of a kinetic assay that allows calculating the required time for serum proteins of the complement to promote 50% lysis of a rabbit red blood cell suspension. The method developed in mammals was successfully applied for pacu and determined that the hemolytic activity of the proteins of the complement system (alternative pathway) increased after the pathogen challenge, but was not influenced by the beta-glucan treatment.

Aspects of poriferan (Suberites domuncula) apoptosis and innate immune system and evolutionary implications

Survivin, a unique member of the family of inhibitors of apoptosis (IAP) proteins, orchestrates intracellular pathways during cell division and apoptosis. Its central regulatory function in vertebrate molecular pathways as mitotic regulator and inhibitor of apoptotic cell death has major implications for tumor cell proliferation and viability, and has inspired several approaches that target survivin for cancer therapy. Analyses in early-branching Metazoa so far propose an exclusive role of survivin as a chromosomal passenger protein, whereas only later during evolution the second, complementary antiapoptotic function might have arisen, concurrent with increased organismal complexity. To lift the veil on the ancestral function(s) of this key regulatory molecule, a survivin homologue of the phylogenetically oldest extant metazoan taxon (phylum Porifera) was identified and functionally characterized. SURVL of the demosponge Suberites domuncula shares significant similarities with its metazoan homologues, ranging from conserved exon/intron structures to the presence of localization signal and protein-interaction domains, characteristic of IAP proteins. Whereas sponge tissue displayed a very low steady-state level, SURVL expression was significantly up-regulated in rapidly proliferating primmorph cells. In addition, challenge of sponge tissue and primmorphs with cadmium and the lipopeptide Pam3Cys-Ser-(Lys)4 stimulated SURVL expression, concurrent with the expression of newly discovered poriferan caspases (CASL and CASL2). Complementary functional analyses in transfected HEK-293 revealed that heterologous expression of poriferan survivin in human cells not only promotes cell proliferation but also augments resistance to cadmium-induced cell death. Taken together, these results demonstrate both a deep evolutionary conserved and fundamental dual role of survivin, and an equally conserved central position of this key regulatory molecule in interconnected pathways of cell cycle and apoptosis. Additionally, SDCASL, SDCASL2, and SDTILRc (TIR-LRR containing protein) may represent new components of the innate defense sentinel in sponges. SDCASL and SDCASL2 are two new caspase-homolog proteins with a singular structure. In addition to their CASc domains, SDCASL and SDCASL2 feature a small prodomain NH2-terminal (effector caspases) and a remarkably long COOH-terminal domain containing one or several functional double stranded RNA binding domains (dsrm). This new caspase prototype can characterize a caspase specialization coupling pathogen sensing and apoptosis, and could represent a very efficient defense mechanism. SDTILRc encompasses also a unique combination of domains: several leucine rich repeats (LRR) and a Toll/IL-1 receptor (TIR) domain. This unusual domain association may correspond to a new family of intracellular sensing protein, forming a subclass of pattern recognition receptors (PRR).

Innate and adaptive immunity in host-microbiota mutualism

Healthy individuals live in peaceful co-existence with an immense load of intestinal bacteria. This symbiosis is advantageous for both the host and the bacteria. For the host it provides access to otherwise undigestible nutrients and colonization resistance against pathogens. In return the bacteria receive an excellent nutrient habitat. The mucosal immune adaptations to the presence of this commensal intestinal microflora are manifold. Although bacterial colonization has clear systemic consequences, such as maturation of the immune system, it is striking that the mutualistic adaptive (T and B cells) and innate immune responses are precisely compartmentalized to the mucosal immune system. Here we summarize the mechanisms of mucosal immune compartmentalization and its importance for a healthy host-microbiota mutualism.

Stem cell-related "self-renewal" signature and high epidermal growth factor receptor expression associated with resistance to concomitant chemoradiotherapy in glioblastoma

PURPOSE: Glioblastomas are notorious for resistance to therapy, which has been attributed to DNA-repair proficiency, a multitude of deregulated molecular pathways, and, more recently, to the particular biologic behavior of tumor stem-like cells. Here, we aimed to identify molecular profiles specific for treatment resistance to the current standard of care of concomitant chemoradiotherapy with the alkylating agent temozolomide. PATIENTS AND METHODS: Gene expression profiles of 80 glioblastomas were interrogated for associations with resistance to therapy. Patients were treated within clinical trials testing the addition of concomitant and adjuvant temozolomide to radiotherapy. RESULTS: An expression signature dominated by HOX genes, which comprises Prominin-1 (CD133), emerged as a predictor for poor survival in patients treated with concomitant chemoradiotherapy (n = 42; hazard ratio = 2.69; 95% CI, 1.38 to 5.26; P = .004). This association could be validated in an independent data set. Provocatively, the HOX cluster was reminiscent of a "self-renewal" signature (P = .008; Gene Set Enrichment Analysis) recently characterized in a mouse leukemia model. The HOX signature and EGFR expression were independent prognostic factors in multivariate analysis, adjusted for the O-6-methylguanine-DNA methyltransferase (MGMT) methylation status, a known predictive factor for benefit from temozolomide, and age. Better outcome was associated with gene clusters characterizing features of tumor-host interaction including tumor vascularization and cell adhesion, and innate immune response. CONCLUSION: This study provides first clinical evidence for the implication of a "glioma stem cell" or "self-renewal" phenotype in treatment resistance of glioblastoma. Biologic mechanisms identified here to be relevant for resistance will guide future targeted therapies and respective marker development for individualized treatment and patient selection.

Analysis of key molecules of the innate immune system in mammary epithelial cells isolated from marker-assisted and conventionally selected cattle

Mastitis is the most prevalent infectious disease in dairy herds. Breeding programs considering mastitis susceptibility were adopted as approaches to improve udder health status. In recent decades, conventional selection criteria based on phenotypic characteristics such as somatic cell score in milk have been widely used to select animals. Recently, approaches to incorporate molecular information have become feasible because of the detection of quantitative trait loci (QTL) affecting mastitis resistance. The aims of the study were to explore molecular mechanisms underlying mastitis resistance and the genetic mechanisms underlying a QTL on Bos taurus chromosome 18 found to influence udder health. Primary cell cultures of mammary epithelial cells from heifers that were selected for high or low susceptibility to mastitis were established. Selection based on estimated pedigree breeding value or on the basis of marker-assisted selection using QTL information was implemented. The mRNA expression of 10 key molecules of the innate immune system was measured using quantitative real-time PCR after 1, 6, and 24 h of challenge with heat-inactivated mastitis pathogens (Escherichia coli and Staphylococcus aureus) and expression levels in the high and low susceptibility groups were compared according to selection criteria. In the marker-assisted selection groups, mRNA expression in cells isolated from less-susceptible animals was significantly elevated for toll-like receptor 2, tumor necrosis factor-alpha, IL-1beta, IL-6, IL-8, RANTES (regulated upon activation, normal t-cell expressed and secreted), complement factor C3, and lactoferrin. In the estimated pedigree breeding value groups, mRNA expression was significantly elevated only for V-rel reticuloendotheliosis viral oncogene homolog A, IL-1 beta, and RANTES. These observations provide first insights into genetically determined divergent reactions to pathogens in the bovine mammary gland and indicate that the application of QTL information could be a successful tool for the selection of animals resistant to mastitis.

Potent innate immune response to pathogenic leptospira in human whole blood.

BACKGROUND Leptospirosis is caused by pathogenic spirochetes of the genus Leptospira. The bacteria enter the human body via abraded skin or mucous membranes and may disseminate throughout. In general the clinical picture is mild but some patients develop rapidly progressive, severe disease with a high case fatality rate. Not much is known about the innate immune response to leptospires during haematogenous dissemination. Previous work showed that a human THP-1 cell line recognized heat-killed leptospires and leptospiral LPS through TLR2 instead of TLR4. The LPS of virulent leptospires displayed a lower potency to trigger TNF production by THP-1 cells compared to LPS of non-virulent leptospires. METHODOLOGY/PRINCIPAL FINDINGS We investigated the host response and killing of virulent and non-virulent Leptospira of different serovars by human THP-1 cells, human PBMC's and human whole blood. Virulence of each leptospiral strain was tested in a well accepted standard guinea pig model. Virulent leptospires displayed complement resistance in human serum and whole blood while in-vitro attenuated non-virulent leptospires were rapidly killed in a complement dependent manner. In vitro stimulation of THP-1 and PBMC's with heat-killed and living leptospires showed differential serovar and cell type dependence of cytokine induction. However, at low, physiological, leptospiral dose, living virulent complement resistant strains were consistently more potent in whole blood stimulations than the corresponding non-virulent complement sensitive strains. At higher dose living virulent and non-virulent leptospires were equipotent in whole blood. Inhibition of different TLRs indicated that both TLR2 and TLR4 as well as TLR5 play a role in the whole blood cytokine response to living leptospires. CONCLUSIONS/SIGNIFICANCE Thus, in a minimally altered system as human whole blood, highly virulent Leptospira are potent inducers of the cytokine response.

Benzoxazinoid Metabolites Regulate Innate Immunity against Aphids and Fungi in Maize

Benzoxazinoids (BXs), such as 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), are secondary metabolites in grasses. The first step in BX biosynthesis converts indole-3-glycerol phosphate into indole. In maize (Zea mays), this reaction is catalyzed by either BENZOXAZINELESS1 (BX1) or INDOLE GLYCEROL PHOSPHATE LYASE (IGL). The Bx1 gene is under developmental control and is mainly responsible for BX production, whereas the Igl gene is inducible by stress signals, such as wounding, herbivory, or jasmonates. To determine the role of BXs in defense against aphids and fungi, we compared basal resistance between Bx1 wild-type and bx1 mutant lines in the igl mutant background, thereby preventing BX production from IGL. Compared to Bx1 wild-type plants, BX-deficient bx1 mutant plants allowed better development of the cereal aphid Rhopalosiphum padi, and were affected in penetration resistance against the fungus Setosphaeria turtica. At stages preceding major tissue disruption, R. padi and S. turtica elicited increased accumulation of DIMBOA-glucoside, DIMBOA, and 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one-glucoside (HDMBOA-glc), which was most pronounced in apoplastic leaf extracts. Treatment with the defense elicitor chitosan similarly enhanced apoplastic accumulation of DIMBOA and HDMBOA-glc, but repressed transcription of genes controlling BX biosynthesis downstream of BX1. This repression was also obtained after treatment with the BX precursor indole and DIMBOA, but not with HDMBOA-glc. Furthermore, BX-deficient bx1 mutant lines deposited less chitosan-induced callose than Bx1 wild-type lines, whereas apoplast infiltration with DIMBOA, but not HDMBOA-glc, mimicked chitosan-induced callose. Hence, DIMBOA functions as a defense regulatory signal in maize innate immunity, which acts in addition to its well-characterized activity as a biocidal defense metabolite.

Signal signature of aboveground-induced resistance upon belowground herbivory in maize

Plants activate local and systemic defence mechanisms upon exposure to stress. This innate immune response is partially regulated by plant hormones, and involves the accumulation of defensive metabolites. Although local defence reactions to herbivores are well studied, less is known about the impact of root herbivory on shoot defence. Here, we examined the effects of belowground infestation by the western corn rootworm Diabrotica virgifera virgifera on aboveground resistance in maize. Belowground herbivory by D. v. virgifera induced aboveground resistance against the generalist herbivore Spodoptera littoralis, and the necrotrophic pathogen Setosphaeria turcica. Furthermore, D. v. virgifera increased shoot levels of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), and primed the induction of chlorogenic acid upon subsequent infestation by S. littoralis. To gain insight into the signalling network behind this below- and aboveground defence interaction, we compiled a set of 32 defence-related genes, which can be used as transcriptional marker systems to detect activities of different hormone-response pathways. Belowground attack by D. v. virgifera triggered an ABA-inducible transcription pattern in the shoot. The quantification of defence hormones showed a local increase in the production of oxylipins after root and shoot infestation by D. v. virgifera and S. littoralis, respectively. On the other hand, ABA accumulated locally and systemically upon belowground attack by D. v. virgifera. Furthermore, D. v. virgifera reduced the aboveground water content, whereas the removal of similar quantities of root biomass had no effect. Our study shows that root herbivory by D. v. virgifera specifically alters the aboveground defence status of a maize, and suggests that ABA plays a role in the signalling network mediating this interaction.

Functional characterization of YODA, a mitogen-activated protein kinase kinase kinase (MAP3K) that regulates a novel innate immunity pathway in Arabidopsis thaliana

Las cascadas de señalización mediadas por proteína quinasas activadas por mitógeno (MAP quinasas) son capaces de integrar y transducir señales ambientales en respuestas celulares. Entre estas señales se encuentran los PAMPs/MAMPs (Pathogen/Microbe-Associated Molecular Patterns), que son moléculas de patógenos o microorganismos, o los DAMPs (Damaged-Associated Molecular Patterns), que son moléculas derivadas de las plantas producidas en respuesta a daño celular. Tras el reconocimiento de los PAMPs/DAMPs por receptores de membrana denominados PRRs (Pattern Recognition Receptors), como los receptores con dominio quinasa (RLKs) o los receptores sin dominio quinasa (RLPs), se activan respuestas moleculares, incluidas cascadas de MAP quinasas, que regulan la puesta en marcha de la inmunidad activada por PAMPs (PTI). Esta Tesis describe la caracterización funcional de la MAP quinasa quinasa quinasa (MAP3K) YODA (YDA), que actúa como un regulador clave de la PTI en Arabidopsis. Se ha descrito previamente que YDA controla varios procesos de desarrollo, como la regulación del patrón estomático, la elongación del zigoto y la arquitectura floral. Hemos caracterizado un alelo mutante hipomórfico de YDA (elk2 o yda11) que presenta una elevada susceptibilidad a patógenos biótrofos y necrótrofos. Notablemente, plantas que expresan una forma constitutivamente activa de YDA (CA-YDA), con una deleción en el dominio N-terminal, presentan una resistencia de amplio espectro frente a diferentes tipos de patógenos, incluyendo hongos, oomicetos y bacterias, lo que indica que YDA juega un papel importante en la regulación de la resistencia de las plantas a patógenos. Nuestros datos indican que esta función es independiente de las respuestas inmunes mediadas por los receptores previamente caracterizados FLS2 y CERK1, que reconocen los PAMPs flg22 y quitina, respectivamente, y que están implicados en la resistencia de Arabidopsis frente a bacterias y hongos. Hemos demostrado que YDA controla la resistencia frente al hongo necrótrofo Plectosphaerella cucumerina y el patrón estomático mediante su interacción genética con la RLK ERECTA (ER), un PRR implicado en la regulación de estos procesos. Por el contrario, la interacción genética entre ER y YDA en la regulación de otros procesos de desarrollo es aditiva en lugar de epistática. Análisis genéticos indicaron que MPK3, una MAP quinasa que funciona aguas abajo de YDA en el desarrollo estomático, es un componente de la ruta de señalización mediada por YDA para la resistencia frente a P. cucumerina, lo que sugiere que el desarrollo de las plantas y la PTI comparten el módulo de transducción de MAP quinasas asociado a YDA. Nuestros experimentos han revelado que la resistencia mediada por YDA es independiente de las rutas de señalización reguladas por las hormonas de defensa ácido salicílico, ácido jasmónico, ácido abscísico o etileno, y también es independiente de la ruta de metabolitos secundarios derivados del triptófano, que están implicados en inmunidad vegetal. Además, hemos demostrado que respuestas asociadas a PTI, como el aumento en la concentración de calcio citoplásmico, la producción de especies reactivas de oxígeno, la fosforilación de MAP quinasas y la expresión de genes de defensa, no están afectadas en el mutante yda11. La expresión constitutiva de la proteína CA-YDA en plantas de Arabidopsis no provoca un aumento de las respuestas PTI, lo que sugiere la existencia de mecanismos de resistencia adicionales regulados por YDA que son diferentes de los regulados por FLS2 y CERK1. En línea con estos resultados, nuestros datos transcriptómicos revelan una sobre-representación en plantas CA-YDA de genes de defensa que codifican, por ejemplo, péptidos antimicrobianos o reguladores de muerte celular, o proteínas implicadas en la biogénesis de la pared celular, lo que sugiere una conexión potencial entre la composición e integridad de la pared celular y la resistencia de amplio espectro mediada por YDA. Además, análisis de fosfoproteómica indican la fosforilación diferencial de proteínas relacionadas con la pared celular en plantas CA-YDA en comparación con plantas silvestres. El posible papel de la ruta ER-YDA en la regulación de la integridad de la pared celular está apoyado por análisis bioquímicos y glicómicos de las paredes celulares de plantas er, yda11 y CA-YDA, que revelaron cambios significativos en la composición de la pared celular de estos genotipos en comparación con la de plantas silvestres. En resumen, nuestros datos indican que ER y YDA forman parte de una nueva ruta de inmunidad que regula la integridad de la pared celular y respuestas defensivas, confiriendo una resistencia de amplio espectro frente a patógenos. ABSTRACT Plant mitogen-activated protein kinase (MAPK) cascades transduce environmental signals and developmental cues into cellular responses. Among these signals are the pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs) and the damage-associated molecular patterns (DAMPs). These PAMPs/DAMPs, upon recognition by plant pattern recognition receptors (PRRs), such as Receptor-Like Kinases (RLKs) and Receptor-Like Proteins (RLPs), activate molecular responses, including MAPK cascades, which regulate the onset of PAMP-triggered immunity (PTI). This Thesis describes the functional characterization of the MAPK kinase kinase (MAP3K) YODA (YDA) as a key regulator of Arabidopsis PTI. YDA has been previously described to control several developmental processes, such as stomatal patterning, zygote elongation and inflorescence architecture. We characterized a hypomorphic, non-embryo lethal mutant allele of YDA (elk2 or yda11) that was found to be highly susceptible to biotrophic and necrotrophic pathogens. Remarkably, plants expressing a constitutive active form of YDA (CA-YDA), with a deletion in the N-terminal domain, showed broad-spectrum resistance to different types of pathogens, including fungi, oomycetes and bacteria, indicating that YDA plays a relevant function in plant resistance to pathogens. Our data indicated that this function is independent of the immune responses regulated by the well characterized FLS2 and CERK1 RLKs, which are the PRRs recognizing flg22 and chitin PAMPs, respectively, and are required for Arabidopsis resistance to bacteria and fungi. We demonstrate that YDA controls resistance to the necrotrophic fungus Plectosphaerella cucumerina and stomatal patterning by genetically interacting with ERECTA (ER) RLK, a PRR involved in regulating these processes. In contrast, the genetic interaction between ER and YDA in the regulation of other ER-associated developmental processes was additive, rather than epistatic. Genetic analyses indicated that MPK3, a MAP kinase that functions downstream of YDA in stomatal development, also regulates plant resistance to P. cucumerina in a YDA-dependent manner, suggesting that the YDA-associated MAPK transduction module is shared in plant development and PTI. Our experiments revealed that YDA-mediated resistance was independent of signalling pathways regulated by defensive hormones like salicylic acid, jasmonic acid, abscisic acid or ethylene, and of the tryptophan-derived metabolites pathway, which are involved in plant immunity. In addition, we showed that PAMP-mediated PTI responses, such as the increase of cytoplasmic Ca2+ concentration, reactive oxygen species (ROS) burst, MAPK phosphorylation, and expression of defense-related genes are not impaired in the yda11 mutant. Furthermore, the expression of CA-YDA protein does not result in enhanced PTI responses, further suggesting the existence of additional mechanisms of resistance regulated by YDA that differ from those regulated by the PTI receptors FLS2 and CERK1. In line with these observations, our transcriptomic data revealed the over-representation in CA-YDA plants of defensive genes, such as those encoding antimicrobial peptides and cell death regulators, and genes encoding cell wall-related proteins, suggesting a potential link between plant cell wall composition and integrity and broad spectrum resistance mediated by YDA. In addition, phosphoproteomic data revealed an over-representation of genes encoding wall-related proteins in CA-YDA plants in comparison with wild-type plants. The putative role of the ER-YDA pathway in regulating cell wall integrity was further supported by biochemical and glycomics analyses of er, yda11 and CA-YDA cell walls, which revealed significant changes in the cell wall composition of these genotypes compared with that of wild-type plants. In summary, our data indicate that ER and YDA are components of a novel immune pathway that regulates cell wall integrity and defensive responses, which confer broad-spectrum resistance to pathogens.

Activation of human monocytes induces differential resistance to apoptosis with rapid down regulation of caspase-8/FLICE

Cells of the monocyte/macrophage lineage play a central role in both innate and acquired immunity of the host. However, the acquisition of functional competence and the ability to respond to a variety of activating or modulating signals require maturation and differentiation of circulating monocytes and entail alterations in both biochemical and phenotypic profiles of the cells. The process of activation also confers survival signals essential for the functional integrity of monocytes enabling the cells to remain viable in microenvironments of immune or inflammatory lesions that are rich in cytotoxic inflammatory mediators and reactive free-radical species. However, the molecular mechanisms of activation-induced survival signals in monocytes remain obscure. To define the mechanistic basis of activation-induced resistance to apoptosis in human monocytes at the molecular level, we evaluated the modulation of expression profiles of genes associated with the cellular apoptotic pathways upon activation and demonstrate the following: (i) activation results in selective resistance to apoptosis particularly to that induced by signaling via death receptors and DNA damage; (ii) concurrent with activation, the most apical protease in the death receptor pathway, caspase-8/FLICE is rapidly down-regulated at the mRNA level representing a novel regulatory mechanism; and (iii) activation of monocytes also leads to dramatic induction of the Bfl-1 gene, an anti apoptotic member of the Bcl-2 family. Our findings thus provide a potential mechanistic basis for the activation-induced resistance to apoptosis in human monocytes.

Role of the cystic fibrosis transmembrane conductance regulator in innate immunity to Pseudomonas aeruginosa infections

Chronic Pseudomonas aeruginosa infection occurs in 75–90% of patients with cystic fibrosis (CF). It is the foremost factor in pulmonary function decline and early mortality. A connection has been made between mutant or missing CF transmembrane conductance regulator (CFTR) in lung epithelial cell membranes and a failure in innate immunity leading to initiation of P. aeruginosa infection. Epithelial cells use CFTR as a receptor for internalization of P. aeruginosa via endocytosis and subsequent removal of bacteria from the airway. In the absence of functional CFTR, this interaction does not occur, allowing for increased bacterial loads in the lungs. Binding occurs between the outer core of the bacterial lipopolysaccharide and amino acids 108–117 in the first predicted extracellular domain of CFTR. In experimentally infected mice, inhibiting CFTR-mediated endocytosis of P. aeruginosa by inclusion in the bacterial inoculum of either free bacterial lipopolysaccharide or CFTR peptide 108–117 resulted in increased bacterial counts in the lungs. CFTR is also a receptor on gastrointestinal epithelial cells for Salmonella enterica serovar Typhi, the etiologic agent of typhoid fever. There was a significant decrease in translocation of this organism to the gastrointestinal submucosa in transgenic mice that are heterozygous carriers of a mutant ΔF508 CFTR allele, suggesting heterozygous CFTR carriers may have increased resistance to typhoid fever. The identification of CFTR as a receptor for bacterial pathogens could underlie the biology of CF lung disease and be the basis for the heterozygote advantage for carriers of mutant alleles of CFTR.

Mice deficient in interleukin-4 (IL-4) or IL-4 receptor alpha have higher resistance to sporozoite infection with Plasmodium berghei (ANKA) than do naive wild-type mice

BALB/c interleukin-4 (IL-4(-/-)) or IL-4 receptor-alpha (IL-4ralpha(-/-)) knockout (KO) mice were used to assess the roles of the IL-4 and IL-13 pathways during infections with the blood or liver stages of plasmodium in murine malaria. Intraperitoneal infection with the blood-stage erythrocytes of Plasmodium berghei (ANKA) resulted in 100% mortality within 24 days in BALB/c mice, as well as in the mutant mouse strains. However, when infected intravenously with the sporozoite liver stage, 60 to 80% of IL-4(-/-) and IL-4ralpha(-/-) mice survived, whereas all BALB/c mice succumbed with high parasitemia. Compared to infected BALB/c controls, the surviving KO mice showed increased NK cell numbers and expression of inducible nitric oxide synthase (iNOS) in the liver and were able to eliminate parasites early during infection. In vivo blockade of NO resulted in 100% mortality of sporozoite-infected KO mice. In vivo depletion of NK cells also resulted in 80 to 100% mortality, with a significant reduction in gamma interferon (IFN-gamma) production in the liver. These results suggest that IFN-gamma-producing NK cells are critical in host resistance against the sporozoite liver stage by inducing NO production, an effective killing effector molecule against Plasmodium. The absence of IL-4-mediated functions increases the protective innate immune mechanism identified above, which results in immunity against P. berghei infection in these mice, with no major role for IL-13.

Mechanisms of inducible resistance against Bacillus thuringiensis endotoxins in invertebrates

Abstract Resistance in insect pests against the endotoxin of Bacillus thuringiensis (Berliner) (Bt) is a major threat to the usefulness of this biopesticide, both used as traditional formulations and in transgenic crops. A crucial requirement for the development of successful resistance management strategies is a molecular understanding of the nature and inheritance of resistance mechanisms. This information can be used to design management strategies that will delay or counteract Bt resistance. The best known Bt resistance mechanism is inactivation of brush border membrane receptors. This type of resistance has a largely recessive mode of inheritance, which has enabled the design of resistance management approaches involving high dose and refuge strategies. Recent observations suggest that other resistance mechanisms are possible, including a mechanism that sequesters the toxin in the gut lumen through inducible immune reactions. The elevated immune status associated with tolerance to the toxin can be transmitted to subsequent generations by a maternal effect, which has implications for resistance management in the field. The high dose/refuge strategy may not be appropriate for the management of these alternative resistance mechanisms and other strategies have to be developed if inducible dominant resistance or tolerance mechanisms occur frequently in the field.