PATHOGENESIS MICROBIAL Microbial Pathogenesis 42 (2007) 2–10 Role of the universal stress protein UspA of Salmonella in growth arrest, stress and virulence Wen-Tssann Liu 1 , Michail H. Karavolos, David M. Bulmer, Abdelmounaaı¨m Allaoui 2 , Raquel Demarco Carlos E. Hormaeche, Jeong Jin Lee 3 , C.M. Anjam Khan à Institute for Cell and Molecular Biosciences and School of Biomedical Sciences, The Medical School, University of Newcastle, Newcastle upon Tyne NE2 4HH, UK Received 24 May 2006; received in revised form 15 September 2006; accepted 15 September 2006 Available online 1 November 2006 Abstract Pathogenic bacteria employ a variety of mechanisms to resist a barrage of stresses they encounter during active growth in or outside the host as well as during growth stasis. An in silico screen of the Salmonella genome sequence revealed that Salmonella typhimurium LT2 possesses a homologue belonging to the universal stress protein A (UspA) family. We assessed the transcriptional profile of uspA in S. typhimurium C5 by constructing a lacZ fusion revealing that uspA is induced by metabolic, oxidative, and temperature stresses. The highest transcriptional levels occurred in cells entering stationary phase, an observation consistent with expression patterns in Escherichia coli. The protein was purified as a fusion with GST (UspA F ) and antibodies raised against UspA F revealed elevated protein levels in stressed and growth-arrested cells. Inactivation of uspA in S. typhimurium C5, lead to increased susceptibility to stress conditions. Furthermore, UspA makes an important contribution to the in vivo virulence of Salmonella in mice thus highlighting the importance of stress resistance regulation in pathogenicity and survival within the host. Crown Copyright r 2006 Published by Elsevier Ltd. All rights reserved. Keywords: Microbial pathogenesis; Salmonella; Universal stress; Virulence 1. Introduction The universal stress protein (UspA) superfamily repre- sents a growing set of small cytoplasmic proteins whose expression is affected by a wide variety of internal or external stresses [1,2]. UspA proteins or conserved Usp domains are also present in eukaryotes, and therefore appear to be of immense importance in these organisms as well as in bacteria and archae [1]. Small Usp proteins have one domain whereas the larger Usp versions consist of two domains arranged in tandem [1,2]. The precise physiologi- cal role of the Usp proteins remains to be elucidated [2]. E. coli has six usp family genes that are divided into two sub-families on the basis of sequence similarity of the Usp domain and one tandem-type gene UspE [3]. It has recently been shown that usp paralogues respond to elevated levels of cytoplasmic polyphosphates (ppGpp) and are implicated in protecting DNA from UV damage [3]. The best characterised orthologue is the E. coli UspA [4]. The protein is synthesised in response to growth inhibition caused by starvation for carbon, nitrogen, sulphate or phosphate, by osmotic shock, high pH, heat, or by heavy metals, oxidants, acids and antibiotics [4–6]. UspA is phosphorylated at both serine and threonine phosphoryla- tion sites upon entry to the stationary phase [7,8]. Mutants devoid of UspA die prematurely during stasis whereas overproduction induces a continuous growth-arrest state [9–11]. Furthermore, it is suggested that UspA is a member of the RecA-dependant DNA protection and repair system since lack of UspA enhances sensitivity to UV irradiation and mitomycin C exposure [12]. Transcription of uspA is activated by ppGpp [3,13] and RecA [3,12] and repressed ARTICLE IN PRESS www.elsevier.com/locate/micpath 0882-4010/$ - see front matter Crown Copyright r 2006 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2006.09.002 à Corresponding author. Tel.: +44 191 2227066; fax: +44 191 2227424. E-mail address: anjam.khan@ncl.ac.uk (C.M. Anjam Khan). 1 Present address: Institute of Preventive Medicine, National Defense Medical Center, Taipei, Taiwan. 2 Present address: Molecular Bacteriology Laboratory, Faculty of Medicine, Free University of Brussels, Brussels, Belgium. 3 Present address: LG Life Sciences, Ltd., R&D Park 104-1, Moonji- dong, Yuseong-gu, Daejeon, South Korea.