Isolation and characterization of powdery mildew-resistant Arabidopsis mutants

A compatible interaction between a plant and a pathogen is the result of a complex interplay between many factors of both plant and pathogen origin. Our objective was to identify host factors involved in this interaction. These factors may include susceptibility factors required for pathogen growth, factors manipulated by the pathogen to inactivate or avoid host defenses, or negative regulators of defense responses. To this end, we identified 20 recessive Arabidopsis mutants that do not support normal growth of the powdery mildew pathogen, Erysiphe cichoracearum. Complementation analyses indicated that four loci, designated powdery mildew resistant 1–4 (pmr1–4), are defined by this collection. These mutants do not constitutively accumulate elevated levels of PR1 or PDF1.2 mRNA, indicating that resistance is not simply due to constitutive activation of the salicylic acid- or ethylene- and jasmonic acid-dependent defense pathways. Further Northern blot analyses revealed that some mutants accumulate higher levels of PR1 mRNA than wild type in response to infection by powdery mildew. To test the specificity of the resistance, the pmr mutants were challenged with other pathogens including Pseudomonas syringae, Peronospora parasitica, and Erysiphe orontii. Surprisingly, one mutant, pmr1, was susceptible to E. orontii, a very closely related powdery mildew, suggesting that a very specific resistance mechanism is operating in this case. Another mutant, pmr4, was resistant to P. parasitica, indicating that this resistance is more generalized. Thus, we have identified a novel collection of mutants affecting genes required for a compatible interaction between a plant and a biotrophic pathogen.

Role of cysteines in Plasmodium falciparum circumsporozoite protein: Interactions with heparin can rejuvenate inactive protein mutants

Various pathogenic bacteria, viruses, and protozoan bind to glycosaminoglycan-based receptors on host cells and initiate an infection. Sporozoites of Plasmodium predominantly express circumsporozoite (CS) protein on their surface, which binds to heparan sulfate proteoglycans on liver cell surface that subsequently leads to malaria. Here we show that the interaction of free heparin with this parasite ligand has the potential to be a critical component of invasion. CS protein of P. falciparum contains four cysteines at positions 361, 365, 396, and 401. In this study, all four cysteine residues were mutagenized to alanine both individually and in different combinations. Conversion of cysteine 396 to alanine (protein CS3) led to a 10-fold increase in the binding activity of the protein to HepG2 cells. Replacement of cysteines at positions 361, 365, and 401 either alone or in different combinations led to a near total loss of binding. Surprisingly, activity in these inactive mutants could be effectively restored in the presence of submolar concentrations of heparin. Heparin also up-regulated binding of CS3 at submolar concentrations with respect to the protein but down-regulated binding when present in excess. Given the significantly different concentrations of heparin in different organs of the host and the in vitro results described here one can consider in vivo ramifications of this phenomenon for pathogen targeting of specific organs and for the functional effects of antigenic variation on receptor ligand interaction.

B7–CTLA4 interaction enhances both production of antitumor cytotoxic T lymphocytes and resistance to tumor challenge

Expression of B7-family costimulatory molecules CD80 (B7–1) and CD86 (B7–2) on tumor cells enhances host immunity. However, the role of the two B7 receptors, CD28 and CTLA4 (CD152), on T cells in antitumor immune response has not been clearly elucidated. Based on the effects of anti-CD28 and anti-CTLA4 mAbs on T cell response, it was proposed that CD28-B7 interaction promotes antitumor immunity, whereas B7-CTLA4 interaction down-regulates it. A critical test for the hypothesis is whether selective engagement of CTLA4 receptors by their natural ligands CD80 and CD86 enhances or reduces antitumor immunity. Here we used tumors expressing wild-type and mutant CD80, as well as mice with targeted mutation of CD28, to address this issue. We report that in syngeneic wild-type mice, B7W (W88>A), a CD80 mutant that has lost binding to CD28 but retained binding to CTLA4, can enhance the induction of antitumor cytotoxic T lymphocytes (CTL); B7Y (Y201>A), which binds neither CD28 nor CTLA4, fails to do so. Consistent with these observations, B7W-transfected J558 plasmocytoma and EL4 thymoma grow significantly more slowly than those transfected with either vector alone or with B7Y. Optimal tumor rejection requires wild-type CD80. Moreover, expression of a high level of CD80 on thymoma EL4 cells conveys immunity in mice with a targeted mutation of CD28 gene. Taken together, our results demonstrate that B7-CTLA4 interaction enhances production of antitumor CTL and resistance to tumor challenge and that optimal enhancement of antitumor immunity by CD80 requires its engagement of both CD28 and CTLA4.

A mAb recognizing a surface antigen of Mycobacterium tuberculosis enhances host survival

Murine mAbs reactive with the surface of Mycobacterium tuberculosis were assayed for their ability to affect the course of infection in mice challenged with virulent organisms. An IgG3 mAb (9d8) specific for arabinomannan and reactive with purified antigen from a clinical isolate of M. tuberculosis conferred partial protection on mice after respiratory challenge (30–60% survival >75 days; P ≤ 0.05). Control mice pretreated with an irrelevant mAb of the same isotype succumbed to tuberculosis within 30 days. Mice with gene disruptions in interferon γ and major histocompatibility complex Class II also were partially protected from challenge. The protective mAb was neither bactericidal nor inhibitory of infection or bacterial replication. Nevertheless, it profoundly altered the nature of the granulomas in the infected lungs. Mice treated with mAb 9d8 and challenged with M. tuberculosis localized the pathogen within granuloma centers, suggesting that the mAb conferred protection by enhancing a cellular immune response.

Host microarray analysis reveals a role for the Salmonella response regulator phoP in human macrophage cell death

Bacterial pathogens manipulate host cells to promote pathogen survival and dissemination. We used a 22,571 human cDNA microarray to identify host pathways that are affected by the Salmonella enterica subspecies typhimurium phoP gene, a transcription factor required for virulence, by comparing the expression profiles of human monocytic tissue culture cells infected with either the wild-type bacteria or a phoP∷Tn10 mutant strain. Both wild-type and phoP∷Tn10 bacteria induced a common set of genes, many of which are proinflammatory. Differentially expressed genes included those that affect host cell death, suggesting that the phoP regulatory system controls bacterial genes that alter macrophage survival. Subsequent experiments showed that the phoP∷Tn10 mutant strain is defective for killing both cultured and primary human macrophages but is able to replicate intracellularly. These experiments indicate that phoP plays a role in Salmonella-induced human macrophage cell death.

Exploitation of host cells by enteropathogenic Escherichia coli

Microbial pathogens have evolved many ingenious ways to infect their hosts and cause disease, including the subversion and exploitation of target host cells. One such subversive microbe is enteropathogenic Escherichia coli (EPEC). A major cause of infantile diarrhea in developing countries, EPEC poses a significant health threat to children worldwide. Central to EPEC-mediated disease is its colonization of the intestinal epithelium. After initial adherence, EPEC causes the localized effacement of microvilli and intimately attaches to the host cell surface, forming characteristic attaching and effacing (A/E) lesions. Considered the prototype for a family of A/E lesion-causing bacteria, recent in vitro studies of EPEC have revolutionized our understanding of how these pathogens infect their hosts and cause disease. Intimate attachment requires the type III-mediated secretion of bacterial proteins, several of which are translocated directly into the infected cell, including the bacteria's own receptor (Tir). Binding to this membrane-bound, pathogen-derived protein permits EPEC to intimately attach to mammalian cells. The translocated EPEC proteins also activate signaling pathways within the underlying cell, causing the reorganization of the host actin cytoskeleton and the formation of pedestal-like structures beneath the adherent bacteria. This review explores what is known about EPEC's subversion of mammalian cell functions and how this knowledge has provided novel insights into bacterial pathogenesis and microbe-host interactions. Future studies of A/E pathogens in animal models should provide further insights into how EPEC exploits not only epithelial cells but other host cells, including those of the immune system, to cause diarrheal disease.

PLS1, a gene encoding a tetraspanin-like protein, is required for penetration of rice leaf by the fungal pathogen Magnaporthe grisea

We describe in this study punchless, a nonpathogenic mutant from the rice blast fungus M. grisea, obtained by plasmid-mediated insertional mutagenesis. As do most fungal plant pathogens, M. grisea differentiates an infection structure specialized for host penetration called the appressorium. We show that punchless differentiates appressoria that fail to breach either the leaf epidermis or artificial membranes such as cellophane. Cytological analysis of punchless appressoria shows that they have a cellular structure, turgor, and glycogen content similar to those of wild type before penetration, but that they are unable to differentiate penetration pegs. The inactivated gene, PLS1, encodes a putative integral membrane protein of 225 aa (Pls1p). A functional Pls1p-green fluorescent protein fusion protein was detected only in appressoria and was localized in plasma membranes and vacuoles. Pls1p is structurally related to the tetraspanin family. In animals, these proteins are components of membrane signaling complexes controlling cell differentiation, motility, and adhesion. We conclude that PLS1 controls an appressorial function essential for the penetration of the fungus into host leaves.

Surface antigen cross-linking triggers forced exit of a protozoan parasite from its host.

We used the common fish pathogen Ichthyophthirius multifiliis as a model for studying interactions between parasitic ciliates and their vertebrate hosts. Although highly pathogenic, Ichthyophthirius can elicit a strong protective immune response in fish after exposure to controlled infections. To investigate the mechanisms underlying host resistance, a series of passive immunization experiments were carried out using mouse monoclonal antibodies against a class of surface membrane proteins, known as immobilization antigens (or i-antigens), thought to play a role in the protective response. Such antibodies bind to cilia and immobilize I. multifiliis in vitro. Surprisingly, we found that passive antibody transfer in vivo caused rapid exit of parasites from the host. The effect was highly specific for a given I. multifiliis serotype. F(ab)2 subfragments had the same effect as intact antibody, whereas monovalent Fab fragments failed to protect. The activity of Fab could, nevertheless, be restored after subsequent i.p. injection of bivalent goat anti-mouse IgG. Parasites that exit the host had detectable antibody on their surface and appeared viable in all respects. These findings represent a novel instance among protists in which protective immunity (and evasion of the host response) result from an effect of antibody on parasite behavior.

The replication initiator protein pi of the plasmid R6K specifically interacts with the host-encoded helicase DnaB.

The replication initiator protein pi of plasmid R6K is known to interact with the seven iterons of the gamma origin/enhancer and activate distant replication origins alpha and beta (ori alpha and ori beta) by pi-mediated DNA looping. Here we show that pi protein specifically interacts in vitro with the host-encoded helicase DnaB. The site of interaction of pi on DnaB has been localized to a 37-aa-long region located between amino acids 151 and 189 of DnaB. The surface of pi that interacts with DnaB has been mapped to the N-terminal region of the initiator protein between residues 1 and 116. The results suggest that during initiation of replication, the replicative helicase DnaB is first recruited to the gamma enhancer by the pi protein. In a subsequent step, the helicase probably gets delivered from ori gamma to ori alpha and ori beta by pi-mediated DNA looping.

Human protein Sam68 relocalization and interaction with poliovirus RNA polymerase in infected cells.

A HeLa cDNA expression library was screened for human polypeptides that interacted with the poliovirus RNA-dependent RNA polymerase, 3D, using the two-hybrid system in the yeast Saccharomyces cerevisiae. Sam68 (Src-associated in mitosis, 68 kDa) emerged as the human cDNA that, when fused to a transcriptional activation domain, gave the strongest 3D interaction signal with a LexA-3D hybrid protein. 3D polymerase and Sam68 coimmunoprecipitated from infected human cell lysates with antibodies that recognized either protein. Upon poliovirus infection, Sam68 relocalized from the nucleus to the cytoplasm, where poliovirus replication occurs. Sam68 was isolated from infected cell lysates with an antibody that recognizes poliovirus protein 2C, suggesting that it is found on poliovirus-induced membranes upon which viral RNA synthesis occurs. These data, in combination with the known RNA- and protein-binding properties of Sam68, make Sam68 a strong candidate for a host protein with a functional role in poliovirus replication.

Protein-protein interaction: a genetic selection for compensating mutations at the barnase-barstar interface.

Barnase and barstar are trivial names of the extracellular RNase and its intracellular inhibitor produced by Bacillus amyloliquefaciens. Inhibition involves the formation of a very tight one-to-one complex of the two proteins. With the crystallographic solution of the structure of the barnase-barstar complex and the development of methods for measuring the free energy of binding, the pair can be used to study protein-protein recognition in detail. In this report, we describe the isolation of suppressor mutations in barstar that compensate for the loss in interaction energy caused by a mutation in barnase. Our suppressor search is based on in vivo selection for barstar variants that are able to protect host cells against the RNAse activity of those barnase mutants not properly inhibited by wild-type barstar. This approach utilizes a plasmid system in which barnase expression is tightly controlled to keep the mutant barnase gene silent. When expression of barnase is turned on, failure to form a complex between the mutant barnase and barstar has a lethal effect on host cells unless overcome by substitution of the wild-type barstar by a functional suppressor derivative. A set of barstar suppressors has been identified for barnase mutants with substitutions in two amino acid positions (residues 102 and 59), which are critically involved in both RNase activity and barstar binding. The mutations selected as suppressors could not have been predicted on the basis of the known protein structures. The single barstar mutation with the highest information content for inhibition of barnase (H102K) has the substitution Y30W. The reduction in binding caused by the R59E mutation in barnase can be partly reversed by changing Glu-76 of barstar, which forms a salt bridge with the Arg-59 in the wild-type complex, to arginine, thus completing an interchange of the two charges.

Antigenic variation and the within-host dynamics of parasites.

Many parasites exhibit antigenic variation within their hosts. We use mathematical models to investigate the dynamical interaction between an antigenically varying parasite and the host's immune system. The models incorporate antigenic variation in the parasite population and the generation of immune responses directed against (i) antigens specific to individual parasite variants and (ii) antigens common to all the parasite variants. Analysis of the models allows us to evaluate the relative importance of variant-specific and cross-reactive immune responses in controlling the parasite. Early in the course of infection within the host, when parasite diversity is below a defined threshold value (the value is determined by the biological properties of the parasite and of the host's immune response), the variant-specific immune responses are predominant. Later, when the parasite diversity is high, the cross-reactive immune response is largely responsible for controlling the parasitemia. It is argued that increasing antigenic diversity leads to a switch from variant-specific to cross-reactive immune responses. These simple models mimic various features of observed infections recorded in the experimental literature, including an initial peak in parasitemia, a long and variable duration of infection with fluctuating parasitemia that ends with either the clearance of the parasite or persistent infection.

Fate of biological control introductions: monitoring an Australian fungal pathogen of grasshoppers in North America.

In North America there are two generally recognized pathotypes (pathotypes 1 and 2) of the fungus Entomophaga grylli which show host-preferential infection of grasshopper subfamilies. Pathotype 3, discovered in Australia, has a broader grasshopper host range and was considered to be a good biocontrol agent. Between 1989 and 1991 pathotype 3 was introduced at two field sites in North Dakota. Since resting spores are morphologically indistinguishable among pathotypes, we used pathotype-specific DNA probes to confirm pathotype identification in E. grylli-infected grasshoppers collected at the release sites in 1992, 1993, and 1994. In 1992, up to 23% of E. grylli-infected grasshoppers of the subfamilies Melanoplinae, Oedipodinae, and Gomphocerinae were infected by pathotype 3, with no infections > 1 km from the release sites. In 1993, pathotype 3 infections declined to 1.7%. In 1994 grasshopper populations were low and no pathotype 3 infections were found. The frequency of pathotype 3 infection has declined to levels where its long-term survival in North America is questionable. Analyses of biocontrol releases are critical to evaluating the environmental risks associated with these ecological manipulations, and molecular probes are powerful tools for monitoring biocontrol releases.

NDR1, a locus of Arabidopsis thaliana that is required for disease resistance to both a bacterial and a fungal pathogen.

We have employed Arabidopsis thaliana as a model host plant to genetically dissect the molecular pathways leading to disease resistance. A. thaliana accession Col-0 is susceptible to the bacterial pathogen Pseudomonas syringae pv. tomato strain DC3000 but resistant in a race-specific manner to DC3000 carrying any one of the cloned avirulence genes avrB, avrRpm1, avrRpt2, and avrPph3. Fast-neutron-mutagenized Col-0 M2 seed was screened to identify mutants susceptible to DC3000(avrB). Disease assays and analysis of in planta bacterial growth identified one mutant, ndr1-1 (nonrace-specific disease resistance), that was susceptible to DC3000 expressing any one of the four avirulence genes tested. Interestingly, a hypersensitive-like response was still induced by several of the strains. The ndr1-1 mutation also rendered the plant susceptible to several avirulent isolates of the fungal pathogen Peronospora parasitica. Genetic analysis of ndr1-1 demonstrated that the mutation segregated as a single recessive locus, located on chromosome III. Characterization of the ndr1-1 mutation suggests that a common step exists in pathways of resistance to two unrelated pathogens.

Leishmaniose visceral americana: avaliação dos parâmetros da capacidade vetorial de Lutzomyia longipalpis em área urbana do muncípio de Panorama, São Paulo, Brasil

Introdução: A leishmaniose visceral (LV) é um importante problema de saúde pública no Brasil, com cerca 3000 mil casos notificados anualmente. Nos últimos anos, a LV tem ampliado sua distribuição em vários estados do país, associada principalmente aos processos socioambientais, antrópicos e migratórios. A LV é causada pela infecção com Leishmania infantum chagasi, transmitida, principalmente, por Lutzomyia longipalpis (Diptera: Psychodidae). Este flebotomíneo apresenta ampla distribuição nas Américas, todavia, evidências sugerem que se constitui em um complexo de espécies crípticas. A dinâmica de transmissão da LV é modulada por fatores ecológicos locais que influenciam a interação entre populações do patógeno, do vetor e dos hospedeiros vertebrados. Portanto, o estudo das variáveis associadas a esta interação pode contribuir para elucidar aspectos dos elos epidemiológicos e contribuir para a tomada de decisões em saúde pública. Objetivo: Avaliar parâmetros relacionados à capacidade vetorial da população de Lu. longipalpis presente em área urbana do município de Panorama, estado de São Paulo. Métodos: Foram realizadas capturas mensais durante 48 meses para avaliar a distribuição espaço-temporal de Lu. longipalpis e investigar a circulação de Le. i. chagasi. Também foram realizados os seguintes experimentos com o vetor: captura-marcação-soltura-recaptura para estimar a sobrevida da população e a duração do seu ciclo gonotrófico, a atratividade dos hospedeiros mais frequentes em áreas urbanas, a proporção de repasto em cão, infecção experimental e competência vetorial. Resultados: Observou-se que no município de Panorama, Lu. longipalpis apresentou as frequências mais elevadas na estação chuvosa (entre outubro e março), maior densidade em áreas com presença de vegetação e criação de animais domésticos, locais aonde também foi demonstrada a circulação natural de espécimes de Lu. longipalpis infectados com Le. i. chagasi. Além disto, foi corroborado que a população de Lu. longipalpis apresentou hábito hematofágico eclético, altas taxas de sobrevivência e que foi competente para transmitir o agente da LV. Nos experimentos de laboratório foi evidenciada a heterogeneidade na infecção de fêmeas de Lu. longipalpis desafiadas a se alimentarem em cães comprovadamente infectados por L. i. chagasi e o rápido desenvolvimento do parasita neste vetor natural. Conclusões. As observações do presente estudo corroboram a capacidade vetora de Lu. longipalpis para transmitir a Le. i. chagasi e ressaltam a importância da espécie na transmissão do agente etiológico da LV. Ações de manejo ambiental, educação e promoção à saúde são recomendadas às autoridades municipais para diminuir o risco potencial de infecção na população humana e canina, considerando-se o elevado potencial vetor de Lu. longipalpis e a presença de condições que favorecem a interação dos componentes da tríade epidemiológica da LV.