Short Communication SecA2 is not required for secretion of the surface autolysin IspC in Listeria monocytogenes serotype 4b Jennifer Ronholm 1,2 , Cathy X.Y. Zhang 1,2 , Xudong Cao 3 and Min Lin 1,2 1 Canadian Food Inspection Agency, Ottawa Laboratory Fallowfield, Ottawa, Ontario, Canada 2 Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada 3 Department of Chemical and Biological Engineering, University of Ottawa, Ottawa, Ontario, Canada Listeria monocytogenes is one of several Gram-positive bacteria known to contain an auxiliary ATPase (SecA2) involved in the Sec secretion of a subset of proteins important to bacterial pathogenesis, including autolysins. It is not known if IspC, a novel surface-associated autolysin essential for full virulence of L. monocytogenes serotype 4b, is SecA2-dependent for secretion. By creating a secA2 gene deletion (DsecA2) mutant from the wild type (WT) L. monocytogenes serotype 4b strain, in combination with the proteomic analysis of surface proteins and those secreted into the medium from both the mutant and the WT, we confirmed previous findings that two autolysins (p60 and NamA) are SecA2-dependent for secretion. However, this approach did not identify IspC as one of the surface proteins affected by the SecA2 deletion. Further experiments with immunofluores- cence microscopy and Western blotting indicated that IspC was well displayed on the surface of both the DsecA2 mutant and WT cells, while p60 was not, clearly indicating that the secretion of IspC is not attributed to the SecA2 pathway. This finding sets IspC apart from other autolysins involved in virulence, such as p60 and NamA, in that SecA2 is not required for IspC secretion. Keywords: Listeria monocytogenes / Secretion / SecA2 / IspC Received: January 7, 2013; accepted: April 13, 2013 DOI 10.1002/jobm.201300007 Introduction The widespread Gram-positive bacterium Listeria mono- cytogenes is the etiological agent of human listeriosis, a severe and life-threatening infection particularly in newborns, elderly persons, pregnant women, and immunocompromised individuals. Common clinical out- comes of L. monocytogenes infection include meningitis, fetal infection followed by abortion, septicaemia, and rarely gastroenteritis. L. monocytogenes has a complex lifecycle during which it alternates between living as an environmental saprophyte or an opportunistic intracel- lular parasite. Extracellular proteins participate in both lifestyles and help to signal when to switch from one to the other [1]. In the environment, L. monocytogenes cells secrete proteins, which participate in environmental sensing, degradation of substrates, and cell-to-cell communication [2]. As a parasite L. monocytogenes expresses surface proteins that mediate different stages of infection including: adhesion, internalization, and immune evasion, making some secreted proteins important virulence factors [3]. There- fore, an understanding of bacterial protein secretion is critical to understanding pathogenesis in L. monocytogenes. Most surface proteins in L. monocytogenes are secreted via the SecA pathway [4, 5]. An accessory SecA paralog (SecA2) has been discovered in several Gram-positive bacteria, including L. monocytogenes [6]. Unlike SecA, which is essential for L. monocytogenes survival [7], the SecA2 pathway is not required for bacterial viability; however, several of the proteins secreted by SecA2 are involved in pathogenesis [8]. SecA2-dependent proteins are necessary for persistent colonization of host cells by L. monocytogenes [8]. Proteins secreted by SecA2 are required for the development of protective memory CD8þ T cells in response to an infection [9]. Lenz et al. [8] suggest that autolysins are major products of Correspondence: Min Lin, Canadian Food Inspection Agency, 3851 Fallowfield Road, Ottawa, Ontario, Canada K2H 8P9 E-mail: Min.Lin@inspection.gc.ca Phone: þ613 228 6698 Fax: þ613 228 6667 Environment  Health  Techniques SecA2 is not required for IspC secretion 1 ß 2013 WILEYVCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com J. Basic Microbiol. 2013, 00,1–5