Anupama Ghosh

Metabolic priming by a secreted fungal effector

Maize smut caused by the fungus Ustilago maydis is a widespread disease characterized by the development of large plant tumours. U. maydis is a biotrophic pathogen that requires living plant tissue for its development and establishes an intimate interaction zone between fungal hyphae and the plant plasma membrane. U. maydis actively suppresses plant defence responses by secreted protein effectors. Its effector repertoire comprises at least 386 genes mostly encoding proteins of unknown function and expressed exclusively during the biotrophic stage. The U. maydis secretome also contains about 150 proteins with probable roles in fungal nutrition, fungal cell wall modification and host penetration as well as proteins unlikely to act in the fungal-host interface like a chorismate mutase. Chorismate mutases are key enzymes of the shikimate pathway and catalyse the conversion of chorismate to prephenate, the precursor for tyrosine and phenylalanine synthesis. Root-knot nematodes inject a secreted chorismate mutase into plant cells likely to affect development. Here we show that the chorismate mutase Cmu1 secreted by U. maydis is a virulence factor. The enzyme is taken up by plant cells, can spread to neighbouring cells and changes the metabolic status of these cells through metabolic priming. Secreted chorismate mutases are found in many plant-associated microbes and might serve as general tools for host manipulation.

Hydrogen peroxide-induced apoptosis-like cell death in Entamoeba histolytica

The microaerophilic intestinal parasitic protozoan Entamoeba histolytica has been previously shown to be highly susceptible to oxidative stress induced by hydrogen peroxide. However the mechanism of cell death was not investigated. Studies presented in this paper demonstrate several morphological features in the parasite when exposed to H(2)O(2) which are identical to metazoan apoptotic phenotype indicating a possible apoptosis-like cell death exhibited by E. histolytica in response to H(2)O(2) treatment. Trophozoite cell shrinkage, DNA fragmentation, phosphatidyl serine externalization and increased endogenous reactive oxygen species level have been observed in the protozoan parasite when exposed to 2.0mM H(2)O(2) for different time periods. Although the parasite genome is completely devoid of any of the homologues of mammalian caspases it still codes for a huge number of cysteine proteases which may take over the apoptotic function of the caspases. But the present study indicates the existence of a cysteine protease independent programmed cell death in the parasite since E-64 the specific cysteine protease inhibitor could not rescue the cells from H(2)O(2) induced apoptosis-like cell death.

Small heat shock proteins (HSP12, HSP20 and HSP30) play a role in Ustilago maydis pathogenesis

Small heat shock proteins (HSP) have multiple functions within a cell. These functions primarily include regulation of growth and survival in response to different stresses. However in some cases small HSPs have been shown to play crucial roles in microbial pathogenesis. Ustilago maydis genome also codes for a number of small HSPs. In the present study we elucidate the role of U. maydis small HSPs in the pathogenicity as well as general stress response of the fungus. Through quantitative real time PCR analysis the expression levels of small HSP genes in comparison with other HSPs were assessed both during infection of the host plant Zea mays and when the pathogen was subjected to an abiotic stress such as oxidative stress. This study revealed that contrary to other HSPs, small HSPs showed an increased level of differential expression under both the tested conditions, indicating a possible role of small HSPs in the pathogenicity and stress response of U. maydis This has been further confirmed by generation of deletion and complementation strains of three putative small HSPs.

EhMAPK, the Mitogen-Activated Protein Kinase from Entamoeba histolytica Is Associated with Cell Survival

Mitogen Activated Protein Kinases (MAPKs) are a class of serine/threonine kinases that regulate a number of different cellular activities including cell proliferation, differentiation, survival and even death. The pathogen Entamoeba histolytica possess a single homologue of a typical MAPK gene (EhMAPK) whose identification was previously reported by us but its functional implications remained unexplored. EhMAPK, the only mitogen-activated protein kinase from the parasitic protist Entamoeba histolytica with Threonine-X-Tyrosine (TXY) phosphorylation motif was cloned, expressed in E. coli and functionally characterized under different stress conditions. The expression profile of EhMAPK at the protein and mRNA level remained similar among untreated, heat shocked and hydrogen peroxide-treated samples in all cases of dose and time. But a significant difference was obtained in the phosphorylation status of the protein in response to different stresses. Heat shock at 43°C or 0.5 mM H2O2 treatment enhanced the phosphorylation status of EhMAPK and augmented the kinase activity of the protein whereas 2.0 mM H2O2 treatment induced dephosphorylation of EhMAPK and loss of kinase activity. 2.0 mM H2O2 treatment reduced parasite viability significantly but heat shock and 0.5 mM H2O2 treatment failed to adversely affect E. histolytica viability. Therefore, a distinct possibility that activation of EhMAPK is associated with stress survival in E. histolytica is seen. Our study also gives a glimpse of the regulatory mechanism of the protein under in vivo conditions. Since the parasite genome lacks any typical homologue of mammalian MEK, the dual specificity kinases which are the upstream activators of MAPK, indications of the existence of some alternate regulatory mechanisms of the EhMAPK activity is perceived. These may include the autophosphorylation activity of the protein itself in combination with some upstream phosphatases which are not yet identified.

Molecular and functional characterisation of a stress responsive cysteine protease, EhCP6 from Entamoeba histolytica

Entamoeba histolytica cysteine protease 6 (EhCP6) is a stress responsive cysteine protease that is upregulated in response to heat shock and during pathogen invasion of the host tissue. In the present study an attempt has been made to express and purify recombinant EhCP6 in order to gain insights into its biochemical properties. The recombinant and refolded protein has been shown to undergo autoproteolysis in the presence of DTT and SDS to give rise to ∼25kDa mature form. The mature form of the protein was found to exhibit a protease activity that is sensitive to E-64, a specific cysteine protease inhibitor. In silico homology modelling of EhCP6 revealed that the protein exhibits conservation of almost all the major structural features of cathepsin-L like cysteine proteases. Further in vivo studies are needed to decipher the function of the protein in response to different stressed conditions.

Plant Stress Response: Hsp70 in the Spotlight

Heat Shock Protein 70 (Hsp70) is an evolutionarily conserved family of proteins which carry out multiple cellular functions such as protein biogenesis, protection during stress, prevention of formation of protein aggregates, assistance in protein translocation and many others. Hsp70, being the major cytoprotective molecular chaperone, plays a crucial role in protecting against a stunning array of stresses and in the re-establishment of cellular homeostasis. This book chapter gives an overview of the multifaceted Hsp70s in plants, with special emphasis on their association with plant response to various stress conditions and eventually, stress acclimation. The contribution of plant stress-responsive proteomics studies towards putting Hsp in the spotlight has also been brought forth. The road ahead is to decipher the underlying mechanisms of Hsp70-mediated multiple cross tolerance, that is likely to lead to new strategies to enhance crop tolerance to environmental stress.

Deciphering the mode of action of a mutant Allium sativum Leaf Agglutinin (mASAL), a potent antifungal protein on Rhizoctonia solani

BackgroundMutant Allium sativum leaf agglutinin (mASAL) is a potent, biosafe, antifungal protein that exhibits fungicidal activity against different phytopathogenic fungi, including Rhizoctonia solani.MethodsThe effect of mASAL on the morphology of R.solani was monitored primarily by scanning electron and light microscopic techniques. Besides different fluorescent probes were used for monitoring various intracellular changes associated with mASAL treatment like change in mitochondrial membrane potential (MMP), intracellular accumulation of reactive oxygen species (ROS) and induction of programmed cell death (PCD). In addition ligand blot followed by LC-MS/MS analyses were performed to detect the putative interactors of mASAL.ResultsKnowledge on the mode of function for any new protein is a prerequisite for its biotechnological application. Detailed morphological analysis of mASAL treated R. solani hyphae using different microscopic techniques revealed a detrimental effect of mASAL on both the cell wall and the plasma membrane. Moreover, exposure to mASAL caused the loss of mitochondrial membrane potential (MMP) and the subsequent intracellular accumulation of reactive oxygen species (ROS) in the target organism. In conjunction with this observation, evidence of the induction of programmed cell death (PCD) was also noted in the mASAL treated R. solani hyphae.Furthermore, we investigated its interacting partners from R. solani. Using ligand blots followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses, we identified different binding partners including Actin, HSP70, ATPase and 14-3-3 protein.ConclusionsTaken together, the present study provides insight into the probable mode of action of the antifungal protein, mASAL on R. solani which could be exploited in future biotechnological applications.

Induction of apoptosis-like cell death and clearance of stress-induced intracellular protein aggregates: dual roles for Ustilago maydis metacaspase Mca1

Metacaspases primarily associate with induction and execution of programmed cell death in protozoa, fungi and plants. In the recent past, several studies have also demonstrated cellular functions of metacaspases other than cell death in different organisms including yeast and protozoa. This study shows similar dual function for the only metacaspase of a biotrophic phytopathogen, Ustilago maydis. In addition to a conventional role in the induction of cell death, Mca1 has been demonstrated to play a key role in maintaining the quality of the cellular proteome. On one hand, Mca1 could be shown to bring about apoptosis‐like phenotypic changes in U. maydis on exposure to oxidative stress, on the other hand, the protein was found to regulate cellular protein quality control. U. maydis metacaspase has been found to remain closely associated with the insoluble intracellular protein aggregates, generated during an event of stress exposure to the fungus. The study, therefore, provides direct evidence for a role of U. maydis metacaspase in the clearance of the stress‐induced intracellular insoluble protein aggregates. Furthermore, host infection assays with mca1 deletion strain also revealed a role of the protein in the virulence of the fungus.

Involvement of Heat Shock Protein 70 (Hsp70) in Gastrointestinal Cancers

Intracellular protein homeostasis is largely controlled by Heat shock proteins (Hsp). Heat shock proteins (Hsp) impart an age-old defense mechanism for all forms of life on earth. Misfolded proteins are refolded with the aid of Hsp and proteins which are damaged beyond repair are eliminated with assistance from Hsp. Hsp are known as molecular chaperones for their cytoprotective roles. In cancer cells the Hsp are frequently overexpressed and are assumed to be associated with tumor formation. Hsp demonstrate specific affinity to particular classes of oncogenic peptides and client proteins in cancer cells, and are able to stabilize mutated oncogene proteins. They play a key regulatory role in prevention of apoptotic cell death during tumorigenesis and thereby enhance cell growth and proliferation. They may also promote chemoresistance in cancer cells. Here we present the current knowledge on the role of molecular chaperones in particular heat shock protein 70 (Hsp70) in human gastrointestinal cancers along with their therapeutic targeting. This review will focus on the role of Hsp 70 and related chaperones in several gastrointestinal cancers such as pancreatic, gastric, and liver cancers.

Proteases from Protozoa and Their Role in Infection

One of the major classes of virulence factors acting in different host-pathogen interaction systems is comprised of proteases. Pathogen-secreted or membrane-associated proteases could be found to participate in different stages of establishment of infection. They are explored as candidate drug targets due to their key participation in the disease development process carried out by the pathogen. In this chapter we present an extensive review of the proteases of different protozoan parasites. Throughout the article we have made an effort to provide a comprehensive list of different proteases from various parasitic protozoa that have been demonstrated to execute major functions in the respective infection processes. Attempts have also been made to present their mode of action with respect to host invasion and disease development.