Proteomics 2013, 13, 89–107 89 DOI 10.1002/pmic.201200227 RESEARCH ARTICLE Host response to intravenous injection of epsilon toxin in mouse model: A proteomic view Bhoj Kumar 1 , Syed Imteyaz Alam 1 and Om Kumar 2 1 Biotechnology Division, Defence Research & Development Establishment, Gwalior, India 2 Pharmacology and Toxicology Division, Defence Research & Development Establishment, Gwalior, India Epsilon toxin (ETX) is an extremely potent pore-forming toxin and a category B biological agent. ETX is a major virulence determinant of Clostridium perfringens toxinotypes B and D, and is implicated in pathogenesis of rapidly fatal economically important pulpy kidney disease in lambs caused by toxinotype D. Despite being a toxin, ETX can be utilized as a tool to target glutamatergic neurons and for drug delivery into the CNS. 2DE-MS approach was employed to elucidate the host response to ETX following intravenous injection in mouse model. In total, 136 proteins were identified either differentially expressed in brain (18) and kidney (33); showing specific interaction with ETX from lysates of brain (4), kidney (21), or from plasma (42); and urine markers (18) of intoxication. Differentially expressed proteins in kidney included those involved in calcium homeostasis and cytoskeletal organization. Proteins involved in ER and oxidative stress and energy metabolism also showed differential levels in the target tissue after ETX treatment. The known functions of the proteins differentially expressed and those interacting with ETX indicate involvement of interlinked pathways. This study provides first proteomic account of host response to ETX exposure providing clues to mechanism of toxicity and potential therapeutic targets. Keywords: Clostridium perfringens / Differential proteomics / Epsilon toxin / Intoxication / Microbiology Received: June 8, 2012 Revised: October 12, 2012 Accepted: October 25, 2012  Additional supporting information may be found in the online version of this article at the publisher’s web-site 1 Introduction Clostridium perfringens is an important clostridial pathogen and causative agent of several diseases in humans and ani- mals including gas gangrene, food poisoning, necrotizing en- terocolitis of infants, and enteritis necroticans [1, 2]. Clostrid- ium perfringens causes diseases owing to its ability to produce several extracellular toxins including the lethal toxins, alpha, beta, epsilon, and iota [3]. Among the six toxinotypes known, Type B or type D strains produce epsilon toxin (ETX). Toxino- type B causes lamb dysenteriae, whereas toxinotype D is the Correspondence: Dr. Syed Imteyaz Alam, Biotechnology Divi- sion, Defence Research & Development Establishment, Gwalior 474002, India E-mail: syimteyaz@gmail.com Fax: 91-751-2341148 Abbreviation: ETX, epsilon toxin causative agent for an economically important and fatal en- terotoxemia of sheep found world over. ETX along with beta toxin is the major virulence determinant for the pathogenesis of toxinotypes B and D. The enterotoxemia or pulpy kidney disease in lambs caused by C. perfringens type D strains is rapidly fatal. In the peracute clinical form, the disease leads to sudden death without any premonitory signs. The rapid acute form lasts for a few minutes to several hours (no more than 12 h) exhibiting excitatory type neurological symptoms including violent convulsions, hyperthermia, and coma [2]. ETX is a member of the family of aerolysin pore-forming toxins and its exact mode of action, explaining the high tox- icity, remains elusive [4]. It is intriguing that ETX, which has a pore-forming activity similar to that of aerolysin and C. septicum alpha toxin, is significantly more active. ETX is a single protein (32 981 Da) containing a signal pep- tide (32 N-terminal amino acids) and secreted as an inac- tive prototoxin [5]. The prototoxin is activated by proteases such as trypsin, -chymotrypsin, and -protease produced by C  2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com