Serratia marcescens Is Able to Survive and Proliferate in Autophagic-Like Vacuoles inside Non-Phagocytic Cells Griselda V. Fedrigo1., Emanuel M. Campoy2., Gisela Di Venanzio1., Marı´a Isabel Colombo2, Eleonora Garcı´a Ve´ scovi1* 1 Instituto de Biolog´ıa Molecular y Celular de Rosario (IBR-CONICET), Departamento de Microbiolog´ıa, Facultad de Ciencias Bioqu´ımicas y Farmace´uticas, Universidad Nacional de Rosario, Rosario, Argentina, 2 Laboratorio de Biolog´ıa Celular y Molecular-Instituto de Histolog´ıa y Embriolog´ıa (IHEM), Facultad de Ciencias Me´dicas, Universidad de Cuyo, CONICET, Mendoza, Argentina Abstract Serratia marcescens is an opportunistic human pathogen that represents a growing problem for public health, particularly in hospitalized or immunocompromised patients. However, little is known about factors and mechanisms that contribute to S. marcescens pathogenesis within its host. In this work, we explore the invasion process of this opportunistic pathogen to epithelial cells. We demonstrate that once internalized, Serratia is able not only to persist but also to multiply inside a large membrane-bound compartment. This structure displays autophagic-like features, acquiring LC3 and Rab7, markers described to be recruited throughout the progression of antibacterial autophagy. The majority of the autophagic-like vacuoles in which Serratia resides and proliferates are non-acidic and have no degradative properties, indicating that the bacteria are capable to either delay or prevent fusion with lysosomal compartments, altering the expected progression of autophagosome maturation. In addition, our results demonstrate that Serratia triggers a non-canonical autophagic process before internalization. These findings reveal that S. marcescens is able to manipulate the autophagic traffic, generating a suitable niche for survival and proliferation inside the host cell. Citation: Fedrigo GV, Campoy EM, Di Venanzio G, Colombo MI, Ve´scovi EG (2011) Serratia marcescens Is Able to Survive and Proliferate in Autophagic-Like Vacuoles inside Non-Phagocytic Cells. PLoS ONE 6(8): e24054. doi:10.1371/journal.pone.0024054 Editor: Robin Charles May, University of Birmingham, United Kingdom Received February 10, 2011; Accepted August 1, 2011; Published August 25, 2011 Copyright: 2011 Fedrigo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: E. G. V. and M. I. C. are supported by grants from Agencia Nacional de Promocio´ n Cient´ıficay Tecnolo´ gica (ANPCyT- PICT 2007-02173) and from Consejo Nacional de Investigaciones Cient´ıficasy Tecnolo´ gicas (CONICET, PIP 112-200801-01063), Argentina. E. G. V. and M. I. C. are career investigators of CONICET, Argentina. G. V. F. has a fellowship from CONICET, and E. C. and G. D. V. have fellowships from ANPCyT, Argentina. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: garciavescovi@ibr.gov.ar . These authors contributed equally to this work. Introduction Serratia marcescens is a wide host range enterobacterium that can be isolated from plants, insects and nematodes, also being an opportunistic pathogen of mammals. In humans, S. marcescens is commonly isolated from urinary tract infections, nosocomial pneumonia, surgical wounds, and bloodstream infections, mostly in intensive care unit patients. The incidence of S. marcescens infections has increased over the last years. This emergence is mainly attributed to the acquisition of multiple antibiotic resistances and to the ability of the bacteria to adhere and persist on unanimated surfaces [1–3]. However, few reports have explored in depth the mechanisms that Serratia uses to invade, survive and proliferate in the host. Hertle and Schwartz [4] have previously shown that S. marcescens is able to invade epithelial cell lines. Supporting evidence that Serratia is able to invade non-phagocytic cells has been provided by Neheme and co-workers who found that Serratia invaded the Drosophila melanogaster mid-gut epithelial cells when it transverses the gut to reach the body cavity [5]. Besides, a multi-host screening performed in Caenorhabditis elegans, D. melanogaster and culture epithelial cells demonstrated the capacity of the bacteria to display pathogenic traits in a broad range of hosts [6]. Following their uptake into a host cell membrane-bound compartment, bacterial pathogens face the challenge posed by the intracellular innate defense responses. One of the most conspicuous removal mechanisms depends on the endocytic pathway. Shortly after entry, the bacterium is wrapped in a host-cell derived phagocytic compartment. This vesicle is expected to suffer a maturation process from early to late phagosome and fuse with lysosomes, turning into the degradative phagolysosome that finally gets rid of the microorganism [7]. In addition, along with a metabolic role in the recycling and degradation of cell components and nutrients, in recent years it has been demon- strated that autophagy actively collaborates with the delivery of the pathogen-containing vacuoles to lysosomes for degradation, in a process specifically known as xenophagy [8,9]. Once within the host cell, bacteria can take different fates. Pathogens such as Shigella, Listeria or Group A Streptococcus are able to disrupt the phagocytic vacuole and escape into the cytosol, where they manage to replicate and subsequently spread into neighbouring cells [10,11]. On the other hand, other bacteria, as is the case of Mycobacterium or Salmonella, can persist inside a phagocytic vacuole delaying or preventing the progression to a phagolysosome, or modifying the features of the lytic compart- ment. In addition, a variable fraction of the intracellular pathogen PLoS ONE | www.plosone.org 1 August 2011 | Volume 6 | Issue 8 | e24054