RESUMO
MAIN CONCLUSION: Tomato leaf curl New Delhi virus-derived AC4 protein interacts with host proteins involved in auxin biosynthesis and reprograms auxin biosynthesis/signaling to help in viral replication and manifestation of the disease-associated symptoms. Perturbations of phytohormone-mediated gene regulatory network cause growth and developmental defects. Furthermore, plant viral infections cause characteristic disease symptoms similar to hormone-deficient mutants. Tomato leaf curl New Delhi Virus (ToLCNDV)-encoded AC4 is a small protein that attenuates the host transcriptional gene silencing, and aggravated disease severity in tomato is correlated with transcript abundance of AC4. Hence, investigating the role of AC4 in pathogenesis divulged that ToLCNDV-AC4 interacted with host TAR1 (tryptophan amino transferase 1)-like protein, CYP450 monooxygenase-the key enzyme of indole acetic acid (IAA) biosynthesis pathway-and with a protein encoded by senescence-associated gene involved in jasmonic acid pathway. Also, ToLCNDV infection resulted in the upregulation of host miRNAs, viz., miR164, miR167, miR393 and miR319 involved in auxin signaling and leaf morphogenesis concomitant with the decline in endogenous IAA levels. Ectopic overexpression of ToLCNDV-derived AC4 in tomato recapitulated the transcriptomic and disruption of auxin biosynthesis/signaling features of the infected leaves. Furthermore, exogenous foliar application of IAA caused remission of the characteristic disease-related symptoms in tomato. The roles of ToLCNDV-AC4 in reprogramming auxin biosynthesis, signaling and cross-talk with JA pathway to help viral replication and manifest the disease-associated symptoms during ToLCNDV infection are discussed.
Assuntos
Geminiviridae , Ácidos Indolacéticos , Solanum lycopersicum , Geminiviridae/patogenicidade , Ácidos Indolacéticos/metabolismo , Solanum lycopersicum/genética , Solanum lycopersicum/virologia , Doenças das Plantas/virologia , Transdução de Sinais/genéticaRESUMO
Free-living amoebae of the genus Acanthamoeba have been associated with keratitis and encephalitis. Some factors related to their pathogenic potential have been described, including the release of hydrolytic enzymes, and the adhesion and phagocytosis processes. However, other factors such as their effect over the hemodynamics and microcirculation elements have not been fully investigated. This work determines the in vitro activity of potentially pathogenic environmental isolates of Acanthamoeba genotype T4 and T5 over erythrocytes and platelets. The hemolytic activity (dependent and independent of contact), as well as the production of ADP of ten environmental isolates of Acanthamoeba obtained from dental units, combined emergency showers, dust, and hospital water, were measured. Tests were carried out over erythrocytes in suspension and blood agar plates, incubated at 4 °C, room temperature and 37 °C. Erythrophagocytosis and platelet aggregation assays were also performed. Live trophozoites of all of the isolates tested showed a hemolytic activity that was temperature-dependent. Over erythrocytes in suspension, variable hemolysis percentages were obtained: a maximum of 41% and a minimum of 15%. Regarding hemolysis over agar plates, two patterns of hemolysis were observed: double and simple halos. Conditioned medium and crude extracts of trophozoites did not show hemolytic activity. Erythrophagocytosis by Acanthamoeba was also observed; however, no production of ADP was determined by the employed methodology.
Assuntos
Acanthamoeba/fisiologia , Plaquetas/parasitologia , Meio Ambiente , Eritrócitos/parasitologia , Acanthamoeba/classificação , Acanthamoeba/genética , Acanthamoeba/patogenicidade , Difosfato de Adenosina/metabolismo , Doenças Transmissíveis Emergentes/parasitologia , Meios de Cultivo Condicionados , Eritrócitos/fisiologia , Genótipo , Hemólise , Humanos , Fagocitose , Agregação Plaquetária , Temperatura , Trofozoítos/classificação , Trofozoítos/genética , Trofozoítos/patogenicidade , Trofozoítos/fisiologiaRESUMO
Pasteurella multocida is one of the most important bacteria responsible for diseases of animals. Crude extracts from sonicated P. multocida strain Dainai-1, which is serotype A isolated from bovine pneumonia, were found to inhibit proliferation of mouse spleen cells stimulated with Con A. The crude extract was purified by cation and anion exchange chromatography and hydroxyapatite chromatography. Its molecular weight was 27 kDa by SDS-PAGE and it was named PM27. PM27 was found to inhibit proliferation of mouse spleen cells stimulated with Con A as effectively as did the crude extract; however, its activity was lost after heating to 100°C for 20 min. PM27 did not directly inhibit proliferation of HT-2 cells, which are an IL-2-dependent T cell line, nor did it modify IL-2 production by Con A-stimulated mouse spleen cells. The N-terminal amino acid sequence of PM27 was determined and BLAST analysis revealed its identity to uridine phosphorylase (UPase) from P. multocida. UPase gene from P. multocida Dainai-1 was cloned into expression vector pQE-60 in Escherichia coli XL-1 Blue. Recombinant UPase (rUPase) tagged with His at the C-terminal amino acid was purified with Ni affinity chromatography. rUPase was found to inhibit proliferation of mouse spleen cells stimulated with Con A; however, as was true for PM27, its activity was lost after heating to 100°C for 20 min. Thus, PM27/UPase purified from P. multocida has significant antiproliferative activity against Con A-stimulated mouse spleen cells and may be a virulence factor.
Assuntos
Proteínas da Membrana Bacteriana Externa/farmacologia , Proliferação de Células/efeitos dos fármacos , Pasteurella multocida/metabolismo , Uridina Fosforilase/isolamento & purificação , Uridina Fosforilase/farmacologia , Sequência de Aminoácidos , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Bovinos , Linhagem Celular/efeitos dos fármacos , Escherichia coli/genética , Humanos , Interleucina-2/metabolismo , Camundongos , Peso Molecular , Pasteurella multocida/genética , Fosforilases , Proteínas Recombinantes , Baço , Linfócitos T/efeitos dos fármacos , Uridina Fosforilase/genética , Uridina Fosforilase/metabolismoRESUMO
Garlic virus X (GarVX) encodes a 15 kDa cysteine-rich protein (CRP). To investigate the function(s) of p15, its subcellular localization, role as a symptom determinant and capacity to act as a viral suppressor of RNA silencing (VSR) were analysed. Results showed that GFP-tagged p15 was distributed in the cytoplasm, nucleus and nucleolus. Expression of p15 from PVX caused additional systemic foliar malformation and led to increased accumulation of PVX, showing that p15 is a virulence factor for reconstructed PVX-p15. Moreover, using a transient agro-infiltration patch assay and a Turnip crinkle virus (TCV) movement complementation assay, it was demonstrated that p15 possesses weak RNA silencing suppressor activity. Removal of an amino acid motif resembling a nuclear localization signal (NLS) prevented p15 from accumulating in the nucleus but did not abolish its silencing suppression activity. This study provides the first insights into the multiple functions of the GarVX p15 protein.
Assuntos
Flexiviridae/imunologia , Flexiviridae/patogenicidade , Interações Hospedeiro-Patógeno , Fatores Imunológicos/metabolismo , Doenças das Plantas/virologia , Proteínas Virais/metabolismo , Fatores de Virulência/metabolismo , Flexiviridae/genética , Fatores Imunológicos/genética , Interferência de RNA , Nicotiana/virologia , Proteínas Virais/genética , Fatores de Virulência/genéticaRESUMO
The pore-forming cytolysins of the RTX-toxin (Repeats in ToXin) family are a relatively small fraction of a steadily increasing family of proteins that contain several functionally important glycine-rich and aspartate containing nonapeptide repeats. These cytolysins produced by a variety of Gram-negative bacteria form ion-permeable channels in erythrocytes and other eukaryotic cells. Hemolytic and cytolytic RTX-toxins represent pathogenicity factors of the toxin-producing bacteria and are very often important key factors in pathogenesis of the bacteria. Channel formation by RTX-toxins lead to the dissipation of ionic gradients and membrane potential across the cytoplasmic membrane of target cells, which results in cell death. Here we discuss channel formation and channel properties of some of the best known RTX-toxins, such as α-hemolysin (HlyA) of Escherichia coli and the uropathogenic EHEC strains, the adenylate cyclase toxin (ACT, CyaA) of Bordetella pertussis and the RTX-toxins (ApxI, ApxII and ApxIII) produced by different strains of Actinobacillus pleuropneumoniae. The channels formed by these RTX-toxins in lipid bilayers share some common properties such as cation selectivity and voltage-dependence. Furthermore the channels are transient and show frequent switching between different ion-conducting states. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.
Assuntos
Bactérias/metabolismo , Infecções Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Membrana Celular/metabolismo , Canais Iônicos/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Animais , Humanos , Ativação do Canal Iônico , Bicamadas Lipídicas/metabolismoRESUMO
The pathogenicity of intracellular plant pathogenic bacteria is associated with the action of pathogenicity factors/effectors, but their physiological roles for most phytoplasma species, including 'Candidiatus Phytoplasma solani' are unknown. Six putative pathogenicity factors/effectors from six different strains of 'Ca. P. solani' were selected by bioinformatic analysis. The way in which they manipulate the host cellular machinery was elucidated by analyzing Nicotiana benthamiana leaves after Agrobacterium-mediated transient transformation with the pathogenicity factor/effector constructs using confocal microscopy, pull-down, and co-immunoprecipitation, and enzyme assays. Candidate pathogenicity factors/effectors were shown to modulate plant carbohydrate metabolism and the ascorbate-glutathione cycle and to induce autophagosomes. PoStoSP06, PoStoSP13, and PoStoSP28 were localized in the nucleus and cytosol. The most active effector in the processes studied was PoStoSP06. PoStoSP18 was associated with an increase in phosphoglucomutase activity, whereas PoStoSP28, previously annotated as an antigenic membrane protein StAMP, specifically interacted with phosphoglucomutase. PoStoSP04 induced only the ascorbate-glutathione cycle along with other pathogenicity factors/effectors. Candidate pathogenicity factors/effectors were involved in reprogramming host carbohydrate metabolism in favor of phytoplasma own growth and infection. They were specifically associated with three distinct metabolic pathways leading to fructose-6-phosphate as an input substrate for glycolysis. The possible significance of autophagosome induction by PoStoSP28 is discussed.
RESUMO
Verticillium dahliae is a hemibiotrophic pathogen responsible for great losses in dicot crop production. An ExoPG gene (VDAG_03463,) identified using subtractive hybridization/cDNA-AFLP, showed higher expression levels in highly aggressive than in weakly aggressive V. dahliae isolates. We used a vector-free split-marker recombination method with PEG-mediated protoplast to delete the ExoPG gene in V. dahliae. This is the first instance of using this method for V. dahliae transformation. Only two PCR steps and one transformation step were required, markedly reducing the necessary time for gene deletion. Six mutants were identified. ExoPG expressed more in the highly aggressive than in the weakly aggressive isolate in response to potato leaf and stem extracts. Its expression increased in both isolates during infection, with higher levels in the highly aggressive isolate at early infection stages. The disruption of ExoPG did not influence virulence, nor did it affect total exopolygalacturonase activity in V. dahliae. Full genome analysis showed 8 more genes related to polygalacturonase/pectinase activity in V. dahliae. Transcripts of PGA increased in the â³exopg mutant in response to potato leaf extracts, compared to the wild type. The expression pattern of those eight genes showed similar trends in the â³exopg mutant and in the weakly aggressive isolate in response to potato extracts, but without the increase of PGA in the weakly aggressive isolate to leaf extracts. This indicated that the â³exopg mutant of V. dahliae compensated by the suppression of ExoPG by activating other related gene.
RESUMO
Colletotrichum scovillei, an ascomycete phytopathogenic fungus, is the main causal agent of serious yield losses of economic crops worldwide. The fungus causes anthracnose disease on several fruits, including peppers. However, little is known regarding the underlying molecular mechanisms involved in the development of anthracnose caused by this fungus. In an initial step toward understanding the development of anthracnose on pepper fruits, we retrieved 624 transcription factors (TFs) from the whole genome of C. scovillei and comparatively analyzed the entire repertoire of TFs among phytopathogenic fungi. Evolution and proliferation of members of the homeobox-like superfamily, including homeobox (HOX) TFs that regulate the development of eukaryotic organisms, were demonstrated in the genus Colletotrichum. C. scovillei was found to contain 10 HOX TF genes (CsHOX1 to CsHOX10), which were functionally characterized using deletion mutants of each CsHOX gene. Notably, CsHOX1 was identified as a pathogenicity factor required for the suppression of host defense mechanisms, which represents a new role for HOX TFs in pathogenic fungi. CsHOX2 and CsHOX7 were found to play essential roles in conidiation and appressorium development, respectively, in a stage-specific manner in C. scovillei. Our study provides a molecular basis for understanding the mechanisms associated with the development of anthracnose on fruits caused by C. scovillei, which will aid in the development of novel approaches for disease management. IMPORTANCE The ascomycete phytopathogenic fungus, Colletotrichum scovillei, causes serious yield loss on peppers. However, little is known about molecular mechanisms involved in the development of anthracnose caused by this fungus. We analyzed whole-genome sequences of C. scovillei and isolated 624 putative TFs, revealing the existence of 10 homeobox (HOX) transcription factor (TF) genes. We found that CsHOX1 is a pathogenicity factor required for the suppression of host defense mechanism, which represents a new role for HOX TFs in pathogenic fungi. We also found that CsHOX2 and CsHOX7 play essential roles in conidiation and appressorium development, respectively, in a stage-specific manner in C. scovillei. Our study contributes to understanding the mechanisms associated with the development of anthracnose on fruits caused by C. scovillei, which will aid for initiating novel approaches for disease management.
Assuntos
Capsicum/microbiologia , Colletotrichum/crescimento & desenvolvimento , Colletotrichum/genética , Genes Homeobox , Interações Hospedeiro-Patógeno/genética , Doenças das Plantas/genética , Fatores de Transcrição/genética , Colletotrichum/patogenicidade , Mecanismos de Defesa , Genoma Fúngico , Doenças das Plantas/microbiologia , Esporos Fúngicos/crescimento & desenvolvimentoRESUMO
Lipopolysaccharide (LPS), localized in the outer leaflet of the outer membrane, serves as the major surface component of the Gram-negative bacterial cell envelope responsible for the activation of the host's innate immune system. Variations of the LPS structure utilized by Gram-negative bacteria promote survival by providing resistance to components of the innate immune system and preventing recognition by TLR4. This review summarizes studies of the biosynthesis of Yersinia pseudotuberculosis complex LPSs, and the roles of their structural components in molecular mechanisms of yersiniae pathogenesis and immunogenesis.
Assuntos
Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata/genética , Lipopolissacarídeos/química , Yersinia pseudotuberculosis/química , Interações Hospedeiro-Patógeno/genética , Humanos , Lipídeo A/genética , Lipídeo A/imunologia , Lipopolissacarídeos/genética , Lipopolissacarídeos/imunologia , Estrutura Molecular , Relação Estrutura-Atividade , Yersinia pseudotuberculosis/imunologia , Yersinia pseudotuberculosis/patogenicidadeRESUMO
The Gram-negative bacterium Yersinia pestis causes plague, a fatal flea-borne anthropozoonosis, which can progress to aerosol-transmitted pneumonia. Y. pestis overcomes the innate immunity of its host thanks to many pathogenicity factors, including plasminogen activator, Pla. This factor is a broad-spectrum outer membrane protease also acting as adhesin and invasin. Y. pestis uses Pla adhesion and proteolytic capacity to manipulate the fibrinolytic cascade and immune system to produce bacteremia necessary for pathogen transmission via fleabite or aerosols. Because of microevolution, Y. pestis invasiveness has increased significantly after a single amino-acid substitution (I259T) in Pla of one of the oldest Y. pestis phylogenetic groups. This mutation caused a better ability to activate plasminogen. In paradox with its fibrinolytic activity, Pla cleaves and inactivates the tissue factor pathway inhibitor (TFPI), a key inhibitor of the coagulation cascade. This function in the plague remains enigmatic. Pla (or pla) had been used as a specific marker of Y. pestis, but its solitary detection is no longer valid as this gene is present in other species of Enterobacteriaceae. Though recovering hosts generate anti-Pla antibodies, Pla is not a good subunit vaccine. However, its deletion increases the safety of attenuated Y. pestis strains, providing a means to generate a safe live plague vaccine.
Assuntos
Ativadores de Plasminogênio/metabolismo , Mapas de Interação de Proteínas/fisiologia , Yersinia pestis/metabolismo , Animais , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Humanos , Peste/genética , Peste/metabolismo , Peste/prevenção & controle , Vacina contra a Peste/administração & dosagem , Vacina contra a Peste/genética , Vacina contra a Peste/metabolismo , Ativadores de Plasminogênio/química , Ativadores de Plasminogênio/genética , Mutação Puntual/fisiologia , Estrutura Secundária de Proteína , Yersinia pestis/classificação , Yersinia pestis/genéticaRESUMO
Virus-derived siRNAs (vsiRNAs) generated by the host RNA silencing mechanism are effectors of plant's defense response and act by targeting the viral RNA and DNA in post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) pathways, respectively. Contrarily, viral suppressors of RNA silencing (VSRs) compromise the host RNA silencing pathways and also cause disease-associated symptoms. In this backdrop, reports describing the modulation of plant gene(s) expression by vsiRNAs via sequence complementarity between viral small RNAs (sRNAs) and host mRNAs have emerged. In some cases, silencing of host mRNAs by vsiRNAs has been implicated to cause characteristic symptoms of the viral diseases. Similarly, viroid infection results in generation of sRNAs, originating from viroid genomic RNAs, that potentially target host mRNAs causing typical disease-associated symptoms. Pathogen-derived sRNAs have been demonstrated to have the propensity to target wide range of genes including host defense-related genes, genes involved in flowering and reproductive pathways. Recent evidence indicates that vsiRNAs inhibit host RNA silencing to promote viral infection by acting as decoy sRNAs. Nevertheless, it remains unclear if the silencing of host transcripts by viral genome-derived sRNAs are inadvertent effects due to fortuitous pairing between vsiRNA and host mRNA or the result of genuine counter-defense strategy employed by viruses to enhance its survival inside the plant cell. In this review, we analyze the instances of such cross reaction between pathogen-derived vsiRNAs and host mRNAs and discuss the molecular insights regarding the process of pathogenesis.
RESUMO
Plant viruses rely on both host plant and vectors for a successful infection. Essentially to simplify studies, transmission has been considered for decades as an interaction between two partners, virus and vector. This interaction has gained a third partner, the host plant, to establish a tripartite pathosystem in which the players can react with each other directly or indirectly through changes induced in/by the third partner. For instance, viruses can alter the plant metabolism or plant immune defence pathways to modify vector's attraction, settling or feeding, in a way that can be conducive for virus propagation. Such changes in the plant physiology can also become favourable to the vector, establishing a mutualistic relationship. This review focuses on the recent molecular data on the interplay between viral and plant factors that provide some important clues to understand how viruses manipulate both the host plants and vectors in order to improve transmission conditions and thus ensuring their survival.
Assuntos
Vetores de Doenças , Interações Hospedeiro-Patógeno , Doenças das Plantas/virologia , Vírus de Plantas/fisiologia , Animais , Resistência à Doença , Regulação Viral da Expressão Gênica , Transporte Proteico , Transdução de Sinais , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Geminiviruses are a significant group of emergent plant DNA viruses causing devastating diseases in food crops worldwide, including the Southern United States, Central America and the Caribbean. Crop failure due to geminivirus-related disease can be as high as 100%. Improved global transportation has enhanced the spread of geminiviruses and their vectors, supporting the emergence of new, more virulent recombinant strains. With limited coding capacity, geminiviruses encode multifunctional proteins, including the AC2/C2 gene that plays a central role in the viral replication-cycle through suppression of host defenses and transcriptional regulation of the late viral genes. The AC2/C2 proteins encoded by mono- and bipartite geminiviruses and the curtovirus C2 can be considered virulence factors, and are known to interact with both basal and inducible systems. This review highlights the role of AC2/C2 in affecting the jasmonic acid and salicylic acid (JA and SA) pathways, the ubiquitin/proteasome system (UPS), and RNA silencing pathways. In addition to suppressing host defenses, AC2/C2 play a critical role in regulating expression of the coat protein during the viral life cycle. It is important that the timing of CP expression is regulated to ensure that ssDNA is converted to dsDNA early during an infection and is sequestered late in the infection. How AC2 interacts with host transcription factors to regulate CP expression is discussed along with how computational approaches can help identify critical host networks targeted by geminivirus AC2 proteins. Thus, the role of AC2/C2 in the viral life-cycle is to prevent the host from mounting an efficient defense response to geminivirus infection and to ensure maximal amplification and encapsidation of the viral genome.
RESUMO
Verticillium wilt, caused by the ascomycete fungus Verticillium dahliae (Vd), is a devastating disease of numerous plant species. However, the pathogenicity/virulence-related genes in this fungus, which may be potential targets for improving plant resistance, remain poorly elucidated. For the study of these genes in Vd, we used a well-established host-induced gene silencing (HIGS) approach and identified 16 candidate genes, including a putative adenylate kinase gene (VdAK). Transiently VdAK-silenced plants developed milder wilt symptoms than control plants did. VdAK-knockout mutants were more sensitive to abiotic stresses and had reduced germination and virulence on host plants. Transgenic Nicotiana benthamiana and Arabidopsis thaliana plants that overexpressed VdAK dsRNAs had improved Vd resistance than the wild-type. RT-qPCR results showed that VdAK was also crucial for energy metabolism. Importantly, in an analysis of total small RNAs from Vd strains isolated from the transgenic plants, a small interfering RNA (siRNA) targeting VdAK was identified in transgenic N. benthamiana. Our results demonstrate that HIGS is a promising strategy for efficiently screening pathogenicity/virulence-related genes of Vd and that VdAK is a potential target to control this fungus.
Assuntos
Adenilato Quinase/genética , Ascomicetos/genética , Metabolismo Energético , Proteínas Fúngicas/genética , Inativação Gênica , Doenças das Plantas/microbiologia , Adenilato Quinase/metabolismo , Arabidopsis/genética , Arabidopsis/microbiologia , Ascomicetos/patogenicidade , Ascomicetos/fisiologia , Resistência à Doença , Proteínas Fúngicas/metabolismo , Interações Hospedeiro-Patógeno , Doenças das Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/microbiologia , Nicotiana/genética , Nicotiana/microbiologiaRESUMO
Human adenoviruses cause different organ infections of varying severity, from asymptomatic to severe cases with lethal outcome, that are registered everywhere. Detailed and focused study of factors predisposing to a severe course of infection is required. The literature contains information indicating the association of severe adenoviral respiratory diseases with certain types of adenovirus, primarily type 7. This review highlights the possible causes of increased pathogenicity of some types of adenovirus and their association with severe forms of infection. Pathogenicity factors include the ability of adenovirus to bind the specific cellular receptors, the formation of subviral particles, the interaction with blood proteins, in particular the coagulation factor X, as well as the features of the early genes E1A, E1B, E3, E4. In addition, the severity of the disease may be affected by the presence or absence of pre-existing antibodies specific to certain types of adenoviruses. Chronic diseases or immunosuppression also increase the risk of severe adenovirus infection. The information presented in this review may elucidate the pathogenesis of adenovirus infection, and help to develop new features for prevention and treatment.
Assuntos
Infecções por Adenovirus Humanos , Adenovírus Humanos , Regulação Viral da Expressão Gênica , Infecções Respiratórias , Infecções por Adenovirus Humanos/genética , Infecções por Adenovirus Humanos/metabolismo , Infecções por Adenovirus Humanos/patologia , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , Adenovírus Humanos/patogenicidade , Fator X/metabolismo , Humanos , Infecções Respiratórias/genética , Infecções Respiratórias/metabolismo , Infecções Respiratórias/patologia , Infecções Respiratórias/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
In a number of compatible plant-bacterium interactions, a rise in apoplastic Ca2+ levels is observed, suggesting that Ca2+ represents an important environmental clue, as reported for bacteria infecting mammalians. We demonstrate that Ca2+ entry in Pseudomonas savastanoi pv. savastanoi (Psav) strain DAPP-PG 722 is mediated by a Na+ /Ca2+ exchanger critical for virulence. Using the fluorescent Ca2+ probe Fura 2-AM, we demonstrate that Ca2+ enters Psav cells foremost when they experience low levels of energy, a situation mimicking the apoplastic fluid. In fact, Ca2+ entry was suppressed in the presence of high concentrations of glucose, fructose, sucrose or adenosine triphosphate (ATP). Since Ca2+ entry was inhibited by nifedipine and LiCl, we conclude that the channel for Ca2+ entry is a Na+ /Ca2+ exchanger. In silico analysis of the Psav DAPP-PG 722 genome revealed the presence of a single gene coding for a Na+ /Ca2+ exchanger (cneA), which is a widely conserved and ancestral gene within the P. syringae complex based on gene phylogeny. Mutation of cneA compromised not only Ca2+ entry, but also compromised the Hypersensitive response (HR) in tobacco leaves and blocked the ability to induce knots in olive stems. The expression of both pathogenicity (hrpL, hrpA and iaaM) and virulence (ptz) genes was reduced in this Psav-cneA mutant. Complementation of the Psav-cneA mutation restored both Ca2+ entry and pathogenicity in olive plants, but failed to restore the HR in tobacco leaves. In conclusion, Ca2+ entry acts as a 'host signal' that allows and promotes Psav pathogenicity on olive plants.
Assuntos
Proteínas de Bactérias/metabolismo , Olea/microbiologia , Pseudomonas/patogenicidade , Trocador de Sódio e Cálcio/metabolismo , Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Cálcio/metabolismo , Cromossomos Bacterianos/genética , Citosol/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Ácidos Indolacéticos/farmacologia , Mutação/genética , Olea/efeitos dos fármacos , Fenótipo , Doenças das Plantas/microbiologia , Reguladores de Crescimento de Plantas/metabolismo , Pseudomonas/efeitos dos fármacos , Nicotiana/microbiologia , Virulência/efeitos dos fármacosRESUMO
OBJECTIVES: Antibiotic use and immunocompromised status in haematology patients have been shown to determine the constituents of commensal microbiota with highly increased resistance, including vancomycin resistant enterococci. We compared the carriage of virulence factor genes and the capacity for biofilm formation in vancomycin resistant enterococci (VRE) originating from the oropharyngeal and stool cultures of haematology patients. DESIGN: PCR tests were used to investigate the presence of genes encoding pathogenicity factors (esp and hyl) in VRE isolates. The genotype of vancomycin resistance was investigated by multiplex PCR tests for vanA and vanB genes. PFGE typing was conducted to exclude the duplicate isolates. RESULTS: Of 3310 pharyngeal swabs taken from inpatients at a clinic for haematology, Enterococcus species were recovered in 6.46%. All VRE investigated were identified as Enterococcus faecium and were highly vancomycin resistant. VanA genotype was confirmed in all. In the group of oropharyngeal carriers (nâ¯=â¯8 patients), 15 strains were recovered from oropharyngeal specimens and PFGE typing revealed 5 types and 3 subtypes. Identical types of VRE in the oropharynx and stool cultures were found in three patients from this group. In the group of stool carriers (nâ¯=â¯24 patients) VRE were obtained from stools only and placed in 21 macro-restriction patterns. The esp gene was more common in VRE isolated from the oropharynx than in isolates from stools (pâ¯=â¯0.014). Results were not significant when we compared the presence of hyl genes in oropharyngeal isolates with those from stool cultures (pâ¯=â¯0.66) or when we investigated the association between esp and hyl gene carriage and capability of biofilm formation in non-repeated VRE. CONCLUSIONS: In the present study, isolation of VRE from the oropharynx in haematology patients was associated with esp gene carriage. Further research is needed to investigate the clinical and long-term effects of this finding.
Assuntos
Proteínas de Bactérias/genética , Enterococcus faecium/genética , Fezes/microbiologia , Hematologia , Proteínas de Membrana/genética , Microbiota/genética , Orofaringe/microbiologia , Fatores de Virulência/genética , Antibacterianos/farmacologia , Carbono-Oxigênio Ligases/genética , DNA Bacteriano , Enterococcus/efeitos dos fármacos , Enterococcus/genética , Enterococcus/isolamento & purificação , Enterococcus faecium/efeitos dos fármacos , Enterococcus faecium/isolamento & purificação , Genes Bacterianos , Genótipo , Humanos , Microbiota/efeitos dos fármacos , Reação em Cadeia da Polimerase Multiplex , Enterococos Resistentes à Vancomicina/efeitos dos fármacos , Enterococos Resistentes à Vancomicina/genéticaRESUMO
In this study the distribution of species and antimicrobial resistance among vancomycin resistant enterococci (VRE) recovered from clinical specimens obtained from five hospitals in Belgrade was analyzed. Strains were further characterized by pulsed-field gel electrophoresis (PFGE). Polymerase chain reaction (PCR) was used to investigate the presence of vanA and vanB genes and pathogenicity factor genes. Identification of 194 VRE isolates revealed 154 Enterococcus faecium, 21 Enterococcus faecalis, 10 Enterococcus raffinosus and 9 Enterococcus gallinarum. This study revealed existence of 8 major clones of VRE. PCR determined vanA gene to be present in all of the VRE studied. Esp and hyl genes were present in 29.22% and 27.92% of E. faecium, respectively, and in 76.19% and 0 of E. faecalis, respectively. Esp and hyl genes were not found more frequently in members of predominant clones of E. faecium than in single isolates; nor was their presence connected to invasiveness.
Assuntos
Antibacterianos/farmacologia , Infecções por Bactérias Gram-Positivas/microbiologia , Resistência a Vancomicina/genética , Enterococos Resistentes à Vancomicina/isolamento & purificação , Análise por Conglomerados , Eletroforese em Gel de Campo Pulsado , Genótipo , Infecções por Bactérias Gram-Positivas/epidemiologia , Humanos , Testes de Sensibilidade Microbiana , Tipagem Molecular , Reação em Cadeia da Polimerase , Estudos Prospectivos , Sérvia/epidemiologia , Especificidade da Espécie , Vancomicina/farmacologia , Enterococos Resistentes à Vancomicina/efeitos dos fármacos , Enterococos Resistentes à Vancomicina/genética , Enterococos Resistentes à Vancomicina/patogenicidade , Fatores de Virulência/genéticaRESUMO
Saprolegniosis, the disease caused by Saprolegnia sp., results in considerable economic losses in aquaculture. Current control methods are inadequate, as they are either largely ineffective or present environmental and fish health concerns. Vaccination of fish presents an attractive alternative to these control methods. Therefore we set out to identify suitable antigens that could help generate a fish vaccine against Saprolegnia parasitica. Unexpectedly, antibodies against S. parasitica were found in serum from healthy rainbow trout, Oncorhynchus mykiss. The antibodies detected a single band in secreted proteins that were run on a one-dimensional SDS-polyacrylamide gel, which corresponded to two protein spots on a two-dimensional gel. The proteins were analysed by liquid chromatography tandem mass spectrometry. Mascot and bioinformatic analysis resulted in the identification of a single secreted protein, SpSsp1, of 481 amino acid residues, containing a subtilisin domain. Expression analysis demonstrated that SpSsp1 is highly expressed in all tested mycelial stages of S. parasitica. Investigation of other non-infected trout from several fish farms in the United Kingdom showed similar activity in their sera towards SpSsp1. Several fish that had no visible saprolegniosis showed an antibody response towards SpSsp1 suggesting that SpSsp1 might be a useful candidate for future vaccination trial experiments.