Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Wed, 07 Nov 2018 06:43:59 Molecular pathogenesis of Listeria monocytogenes in the alternative model host Galleria mellonella Susan A. Joyce 1,2 and Cormac G. M. Gahan 1,2,3 Correspondence Cormac G. M. Gahan c.gahan@ucc.ie Received 21 April 2010 Revised 9 July 2010 Accepted 3 August 2010 1 Department of Microbiology, University College Cork, Cork, Ireland 2 Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland 3 School of Pharmacy, University College Cork, Cork, Ireland Larvae of Galleria mellonella, the greater wax moth, provide an alternative infection model for many human pathogens as they are amenable to use at elevated incubation temperatures (37 6C). This study and a parallel study by Mukherjee et al. [Mukherjee, K., Altincicek, B., Hain, T., Domann, E., Vilcinskas, A. & Chakraborty, T. (2010). Appl Environ Microbiol 76, 310–317] establish this insect host as an appropriate model to investigate the pathogenesis of Listeria species. In this study we show that inoculation with Listeria monocytogenes initiates a dynamic infection in G. mellonella and that production of the cytolysin listeriolysin O (LLO) is necessary for toxicity and bacterial growth. Production of LLO by the non-pathogenic species Lactococcus lactis is sufficient to induce mortality in the insect model. We employed real-time bioluminescence imaging to examine the dynamics of listerial growth and virulence gene expression in the G. mellonella model. Analysis of lux promoter fusions demonstrated significant induction of virulence gene expression upon introduction of the pathogen into insects at both 30 and 37 6C. The host response to listerial infection was examined which demonstrated that haemocyte destruction accompanies L. monocytogenes pathogenesis and is preceded by activation of the phenoloxidase system. Furthermore, we demonstrate that Listeria innocua is pathogenic to G. mellonella through a persistence mechanism that implicates an alternative mechanism for pathogenicity in this model. INTRODUCTION Listeria monocytogenes is an invasive foodborne Gram- positive pathogen that causes sporadic and epidemic disease (listeriosis) that is often fatal (20–30 % case fatality rates). The pathogen has a facultative intracellular life cycle with the capacity for cellular invasion, intracellular replication and movement from cell to cell without an extracellular phase. The intracellular pathogenesis of L. monocytogenes is mainly attributed to the presence of a multi-gene virulence island that is regulated by the CRP/ FNR-type protein PrfA (Freitag et al., 2009; Greene & Freitag, 2003; Sheehan et al., 1995). PrfA also regulates expression of factors required for cellular invasion (InlA and InlB) and intracellular growth (Hpt) that are located elsewhere on the chromosome (Hamon et al., 2006). Virulence factors encoded on the virulence gene island include listeriolysin O (LLO, encoded by hly), a protein that is absolutely required for phagosomal escape and pathogenesis of L. monocytogenes in mammalian models of disease (Freitag et al., 2009; Hamon et al., 2006). Also present at this locus is a gene encoding ActA, a protein required for actin polymerization and intracytoplasmic movement (Hamon et al., 2006). Clearly PrfA is essential in order to direct virulence gene expression in L. mono- cytogenes during the invasive and systemic phases of infection and PrfA deletion mutants are significantly attenuated in the murine model of disease (Chakraborty et al., 1992). In addition, the stress response factor SigB plays a significant role within the gastrointestinal tract and also positively regulates PrfA and other factors during adaptation to stress (Garner et al., 2006; Nadon et al., 2002). The non-pathogenic species Listeria innocua appears to have evolved from L. monocytogenes and lacks the virulence locus as well as specific genes (inlA, inlB, bsh and hpt) that encode virulence factors (Buchrieser et al., 2003). Many studies have established the murine model as a useful model for analysis of the systemic phase of listerial pathogenesis (Lecuit, 2007). However, the murine model has a number of limitations including poor interaction between bacterial InlA and the cognate host receptor (E- cadherin), relatively high cost and ethical considerations associated with the analysis of mammalian hosts (Disson et al., 2008, 2009; Lecuit, 2007). Alternative model systems Abbreviations: AMP, antimicrobial peptide; BHI, brain heart infusion; CFSE, carboxylfluorescein succinimydyl ester; IVIS, in vitro imaging system; LLO, listeriolysin O; PGRP-LE, peptidoglycan recognition protein; PO, phenoloxidase; PPO, pro-phenoloxidase. Two supplementary tables and two supplementary figures are available with the online version of this paper. Microbiology (2010), 156, 3456–3468 DOI 10.1099/mic.0.040782-0 3456 040782 G 2010 SGM Printed in Great Britain