Please cite this article in press as: Arif, M., et al., Quantification of cell infection caused by Listeria monocytogenes invasion. J. Biotechnol. (2011), doi:10.1016/j.jbiotec.2011.03.008 ARTICLE IN PRESS G Model BIOTEC-5700; No. of Pages 8 Journal of Biotechnology xxx (2011) xxx–xxx Contents lists available at ScienceDirect Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec Quantification of cell infection caused by Listeria monocytogenes invasion Muhammad Arif a,* , Nasir M. Rajpoot b,** , Tim W. Nattkemper c , Ulrike Technow d , Trinad Chakraborty d , Nicole Fisch e , Nickels A. Jensen e , Karsten Niehaus e a Department of Electrical Engineering, Pakistan Institute of Engineering & Applied Sciences (PIEAS), P.O. Nilore, Islamabad 45650, Pakistan b Department of Computer Science, University of Warwick, Coventry CV4 7AL, United Kingdom c Biodata Mining Group, Faculty of Technology, Bielefeld University, Germany d Giessen University, Research Center for Infectious Disease, Germany e Faculty of Biology, Department for Proteome and Metabolome Research, Bielefeld University, Germany article info Article history: Received 14 April 2010 Received in revised form 8 March 2011 Accepted 10 March 2011 Available online xxx Keywords: High content screen Listeria monocytogenes Bioimage informatics Bacterial infection Cell segmentation Pattern recognition abstract Listeria monocytogenes causes a life-threatening food-borne disease known as Listeriosis. Elderly, immunocompromised, and pregnant women are primarily the victims of this facultative intracellular Gram-positive pathogen. Since the bacteria survive intracellularly within the human host cells they are protected against the immune system and poorly accessed by many antibiotics. In order to screen pharmaceutical substances for their ability to interfere with the infection, persistence and release of L. monocytogenes a high content assay is required. We established a high content screen (HCS) using the RAW 264.7 mouse macrophage cell line seeded into 96-well glass bottom microplates. Cells were infected with GFP-expressing L. monocytogenes and stained thereafter with Hoechst 33342. Automated image acquisition was carried out by the Scan R screening station. We have developed an algorithm that automatically grades cells in microscopy images of fluorescent-tagged Listeria for the severity of infec- tion. The grading accuracy of this newly developed algorithm is 97.1% as compared to a 74.3% grading accuracy we obtained using the commercial Olympus Scan R software. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Listeria monocytogenes is a Gram-positive pathogenic bacterium that causes a food-borne disease called Listeriosis in both humans and animals. Listeriosis is a rare but serious disease with a high overall mortality rate of 30%, most common in pregnant women or immunocompromised individuals (Ramaswamy et al., 2007). The bacteria cause severe gastroenteritis and central nervous system infections and are able to cross the intestinal, materno-fetal and blood–brain barrier (Barbuddhe and Chakraborty, 2009; Drevets and Bronze, 2008). L. monocytogenes is a facultatively intracellular pathogen, which has evolved several mechanisms for exploiting the hosts’ cellular machinery for infection and proliferation (Portnoy et al., 2002). Because of its ability to transit from extracellular environments to the intracellular milieu of infected eukaryotic cells, it is an important model organism for infection, intracellular proliferation and host–pathogen interactions and presently represents one of * Corresponding author. Tel.: +92 51 2208357; fax: +92 51 2208070. ** Corresponding author. Tel: +44 24 7657 3795; fax: +44 24 7657 3024. E-mail addresses: fac097@pieas.edu.pk, gilgiti@yahoo.com (M. Arif), nasir@dcs.warwick.ac.uk (N.M. Rajpoot). the most well studied pathogens (Cossart, 2007). Several Listeria strains with varying virulence and pathogenicity are known (Liu et al., 2003; Roche et al., 2003). The pathogen can provoke its internalisation in normally non- phagocytic cells such as fibroblasts and can survive the engulfment by macrophages (Birmingham et al., 2008). The cellular invasion and proliferation of cells occurs in consequential stages (Portnoy et al., 2002). First the pathogen is internalised or provokes its inter- nalisation in host cells with virulence factors called internalins (Seveau et al., 2007). Inside the cell, the bacterium lyses the vacuole and escapes to the cytoplasm, where it proliferates. By recruitment of the hosts’ actin cytoskeleton, the bacteria move in the cytoplasm and spread from one cell to another (Vázquez-Boland et al., 2001). Those intracellular bacteria are sheltered from the host immune system and are poorly accessible for treatment with antibiotics. Therefore, the invasion of the host cells is an important and crucial step in Listeria pathogenesis and virulence (Ireton, 2007). This pathogenesis and infection can be elucidated using in vivo or in vitro cell culture infection assays (Liu et al., 2007). In conven- tional in vitro cell assays, a defined number of Listeria is incubated with a confluent mammalian cell layer (Roche et al., 2001). After a defined incubation time extracellular bacteria are washed away or killed by gentamycine treatment. The remaining intracellular bacteria are enumerated on solid agar medium after lysis of the 0168-1656/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2011.03.008