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In JoVE (1)
Other Publications (7)
Articles by Isabel Murillo in JoVE
JoVE Immunology and Infection
Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human α-actinin by Confocal Imaging
Department of Cellular and Molecular Medicine, University of Bristol, UK
Neisseria meningitidis (Nm), a gram negative human-specific respiratory pathogen, can bind to human α-actinin. Here we present a protocol for visualisation of colocalisation of the bacterium with intracellular α-actinin after bacterial entry into human brain microvascular endothelial cells (HBMECs).
Other articles by Isabel Murillo on PubMed
Fungus- and Wound-induced Accumulation of MRNA Containing a Class II Chitinase of the Pathogenesis-related Protein 4 (PR-4) Family of Maize
Pathogenesis-related (PR) proteins are plant proteins that are induced in response to pathogen attack. PR proteins are grouped into independent families based on their sequences and properties. The PR-4 family comprises class I and class II chitinases. We have isolated a full-length cDNA encoding a chitinase from maize which shares a high degree of nucleotide and amino acid sequence homology with the class II chitinases of the PR-4 family of PR proteins. Our results indicate that fungal infection, and treatment either with fungal elicitors or with moniliformin, a mycotoxin produced by the fungus Fusarium moniliforme, increase the level of ZmPR4 mRNA. In situ mRNA hybridization analysis in sections obtained from fungus-infected germinating embryos revealed that ZmPR4 mRNA accumulation occurs in those cell types that first establish contact with the pathogen. ZmPR4 mRNA accumulation is also stimulated by treatment with silver nitrate whereas the application of the hormones gibberellic acid or acetylsalicylic acid has no effect. Wounding, or treatment with abscisic acid or methyl jasmonate, results in accumulation of ZmPR4 mRNA in maize leaves. Furthermore, the ZmPR4 protein was expressed in Escherichia coli, purified and used to obtain polyclonal antibodies that specifically recognized ZmPR4 in protein extracts from fungus-infected embryos. Accumulation of ZmPR4 mRNA in fungus-infected maize tissues was accompanied by a significant accumulation of the corresponding protein. The possible implications of these findings as part of the general defence response of maize plants against pathogens are discussed.
Engineering Photoassimilate Partitioning in Tobacco Plants Improves Growth and Productivity and Provides Pathogen Resistance
Expression of pathogenesis-related (PR) genes is part of the plant's natural defense response against pathogen attack. To study the in vivo role and function of the maize PRms protein, tobacco plants were transformed with the PRms cDNA under the control of the CaMV35S promoter. Transgenic tobacco plants grow faster and yield more leaf and seed biomass. By using immunoelectron microscopy, we found that PRms is associated with plasmodesmata in leaves of transgenic tobacco plants. Furthermore, we found that activation of sucrose efflux from photosynthetically active leaves and accumulation of higher levels of sucrose in leaf tissues are characteristic features of PRms tobacco plants. This, in turn, results in the constitutive expression of endogenous tobacco PR genes and resistance to phytopathogens. The expression of multiple plant defense genes can then be achieved by using a single transgene. These data provide a new approach for engineering disease-resistant plants while simultaneously improving plant yield and productivity through the modification of photoassimilate partitioning.
Bt Rice Harbouring Cry Genes Controlled by a Constitutive or Wound-inducible Promoter: Protection and Transgene Expression Under Mediterranean Field Conditions
Seven homozygous transgenic lines of two European commercial cultivars of rice (Ariete (A) and Senia (S)), harbouring the cry1B or cry1Aa Bacillus thuringiensis (Bt) delta-endotoxin genes, were field evaluated for protection from striped stem borer (SSB) (Chilo suppressalis) damage during the 2001 and 2002 summer crop seasons in the Delta de l'Ebre region, Spain. The plant codon-optimized toxin gene was placed under the control of the promoter of either the constitutive ubi1 gene or the wound-inducible mpi gene from maize. Stable, high-level, insecticidal protein accumulation was observed throughout root, leaf and seed tissues of field-grown plants harbouring the cry1B (lines A64.1, A33.1, A3.4 and S98.9) or cry1Aa (lines S05.1 and A19.14) genes under the control of the ubi1 promoter. Conversely, no toxin was detected in unwounded vegetative tissues of the A9.1 line harbouring the cry1B gene controlled by the mpi promoter, indicating that natural environmental stresses did not trigger the activity of the wound-inducible promoter. However, the toxin accumulated at 0.2% total soluble proteins in A9.1 sheath tissue exhibiting brown lesions resulting from SSB damage. The agronomical traits and performance of the transgenic lines were generally comparable with parental controls, except in the two lines accumulating Cry1Aa, which exhibited a high frequency of plants non-true to type. Natural infestation was assisted with manual infestations of L2/L3 SSB larvae in border control plants surrounding the experimental plots, which served as a reservoir for the second-cycle SSB population. The observation of damage (brown lesions and dead hearts) during the crop season and dissection of plants at harvest stage revealed a range of protection amongst the transgenic lines, which was highly consistent with the level of toxin accumulation and with previous experience in greenhouse assays. Lines A3.4 and S05.1 were found to exhibit stable and full protection against SSB attacks, mediated by the accumulation of Cry1B and Cry1Aa toxin, respectively, which was comparable with that afforded by the spraying of chemical insecticides on control plants. The wound-induced A9.1 line exhibited a satisfactory level of protection, with a notably low level of penetration of SSB larvae in the stems, but higher external symptoms than constitutive lines, probably due to the time lag to benefit from the protective effect of Cry1B.
Expression of the Maize Proteinase Inhibitor (mpi) Gene in Rice Plants Enhances Resistance Against the Striped Stem Borer (Chilo Suppressalis): Effects on Larval Growth and Insect Gut Proteinases
The maize proteinase inhibitor (mpi) gene was introduced into two elite japonica rice varieties. Both constitutive expression of the mpi gene driven by the maize ubiquitin 1 promoter and wound-inducible expression of the mpi gene driven by its own promoter resulted in the accumulation of MPI protein in the transgenic plants. No effect on plant phenotype was observed in mpi-expressing lines. The stability of transgene expression through successive generations of mpi rice lines (up to the T(4) generation) and the production of functional MPI protein were confirmed. Expression of the mpi gene in rice enhanced resistance to the striped stem borer (Chilo suppressalis), one of the most important pests of rice. In addition, transgenic mpi plants were evaluated in terms of their effects on the growth of C. suppressalis larvae and the insect digestive proteolytic system. An important dose-dependent reduction of larval weight of C. suppressalis larvae fed on mpi rice, compared with larvae fed on untransformed rice plants, was observed. Analysis of the digestive proteolytic activity from the gut of C. suppressalis demonstrated that larvae adapted to mpi transgene expression by increasing the complement of digestive proteolytic activity: the serine and cysteine endoproteinases as well as the exopeptidases leucine aminopeptidase and carboxypeptidases A and B. However, the induction of such proteolytic activity did not prevent the deleterious effects of MPI on larval growth. The introduction of the mpi gene into rice plants can thus be considered as a promising strategy to protect rice plants against striped stem borer.
Expression of Gp91phox/Nox2 in COS-7 Cells: Cellular Localization of the Protein and the Detection of Outward Proton Currents
We have reported previously that gp91phox, expressed in CHO (Chinese hamster ovary) cells, functions as a voltage-dependent proton channel. However, others have reported that COS-7 cells expressing gp91phox failed to exhibit outward proton currents, and concluded that gp91phox does not function as a proton channel. To investigate this clear difference in findings, we have examined the expression and cellular localization of the fusion protein EGFP-C-91, in which gp91phox is fused to the C-terminus of enhanced green fluorescent protein. EGFP-C-91 was observed in the plasma membrane and intracellular membranes of 30% of the transfected COS-7 cells. In the remaining COS-7 cells, EGFP-C-91 was detected in the intracellular membranes only. In CHO cells EGFP-C-91 was present in both the plasma membrane and the intracellular membranes of all transfected cells. Under the whole-cell configuration, outward currents were recorded from COS-7 cells expressing gp91phox. These increased in magnitude and lost their 'droop' over time as the pipette solution equilibrated with the cell cytoplasm (50 min). The threshold activation voltage for the currents was shifted by approximately 60 mV for a 1 unit difference in bath pH. Zn2+ inhibited the outward currents observed in COS-7 cells expressing gp91phox. The tail current reversal potential was -64 mV at a pH(o) (external pH) of 8.0, -40 mV at pH(o) 7.4 and -8 mV at pH(o) 7.0, indicating that the current arises from the movement of protons. Outward currents were exhibited by 37.5% of the COS-7 cells expressing gp91phox. Proton currents were recorded following the excision of inside-out patches from cells transfected with gp91phox. The presence of outward proton currents in COS-7 cells expressing gp91phox provides further support for our proposed role for gp91phox as the NADPH oxidase-associated proton channel.
Neisseria Meningitidis Opc Invasin Binds to the Cytoskeletal Protein Alpha-actinin
Neisseria meningitidis Opc protein is an effective invasin for human endothelial cells. We have investigated novel human endothelial receptors targeted by Opc and observed that Opc-expressing bacteria interacted with a 100 kDa protein in whole-cell lysates of human endothelial and epithelial cells. The identity of the protein was established as alpha-actinin by mass spectrometry. Opc expression was essential for the recognition of alpha-actinin whether provided in a purified form or in cell extracts. The interaction of the two proteins did not involve intermediate molecules. As there was no demonstrable expression of alpha-actinin on the surfaces of any of the eight cell lines studied, the likelihood of the interactions after meningococcal internalization was examined. Confocal imaging demonstrated considerable colocalization of N. meningitidis with alpha-actinin especially after a prolonged period of internalization. This may imply that bacteria and alpha-actinin initially occur in separate compartments and co-compartmentalization occurs progressively over the 8 h infection period used. In conclusion, these studies have identified a novel and an intracellular target for the N. meningitidis Opc invasin. Since alpha-actinin is a modulator of a variety of signalling pathways and of cytoskeletal functions, its targeting by Opc may enable bacteria to survive/translocate across endothelial barriers.
Meningococcal Interactions with the Host
Neisseria meningitidis interacts with host tissues through hierarchical, concerted and co-ordinated actions of a number of adhesins; many of which undergo antigenic and phase variation, a strategy that helps immune evasion. Three major structures, pili, Opa and Opc predominantly influence bacterial adhesion to host cells. Pili and Opa proteins also determine host and tissue specificity while Opa and Opc facilitate efficient cellular invasion. Recent studies have also implied a role of certain adhesin-receptor pairs in determining increased host susceptibility to infection. This chapter examines our current knowledge of meningococcal adhesion and invasion mechanisms particularly related to human epithelial and endothelial cells which are of primary importance in the disease process.
