Deletion of Braun lipoprotein gene (lpp) attenuates Yersinia pestis KIM/D27 strain: Role of Lpp in modulating host immune response, NF-kB activation and cell death Tie Liu 1 , Stacy L. Agar 1 , Jian Sha, Ashok K. Chopra * Department of Microbiology & Immunology, Medical Research Building, University of Texas Medical Branch, Galveston, TX 77555-1070, USA article info Article history: Received 2 May 2009 Received in revised form 25 August 2009 Accepted 1 September 2009 Available online 6 September 2009 Keywords: Yersinia pestis Braun lipoprotein Mouse model of infection NF-kB Apoptosis Host immune response abstract The pathogenic species of yersiniae potently blocks immune responses in host cells by using the type III secretion apparatus and its effector proteins. In this study, we characterized potential mechanisms associated with the Braun lipoprotein (Lpp) that contributed to a further attenuation of a pigmentation locus-minus Yersinia pestis KIM/D27 mutant strain and its ability to generate immune responses in mice. The lpp gene encodes one of the major outer membrane lipoproteins that is involved in inflammatory responses and septic shock. We found that sera and splenocytes from Dlpp mutant-immunized mice, when transferred to naı¨ve animals, provided protection to the latter against challenge with a lethal dose of the Y. pestis parental strain. Further, the Dlpp mutant promoted ex vivo a significantly higher inter- leukin (IL)-2 and interferon-gamma production from T cells of immunized mice, when compared to those from animals infected with the sub-lethal dose of the parental Y. pestis KIM/D27 strain. Likewise, murine primary macrophages infected with the mutant, when compared to those infected with the parental strain in vitro, produced significantly higher IL-12 levels. Importantly, increased nuclear factor-kappa B activation and decreased apoptosis were noted in splenocytes and primary macrophages of mice chal- lenged with the Dlpp mutant, when compared to those in animals infected with the parental Y. pestis KIM/D27 strain. Finally, significantly higher levels of antibodies specific for the parental Y. pestis antigens were developed in mice first immunized with the Dlpp mutant and then challenged with the parental strain, compared to the antibody levels in animals that were immunized and then infected with the parental KIM/D27 strain. To our knowledge, this is the first report of a mechanistic basis for attenuation and immunological responses associated with deletion of the lpp gene from the Y. pestis KIM/D27 strain. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction The genus Yersinia includes three human pathogenic species, Yersinia pestis, Yersinia enterocolitica, and Yersinia pseudotubercu- losis. Y. pestis is the causative agent of bubonic and pneumonic plague, both of which remain serious public health threats in some regions of the world, accounting for the deaths of approximately 200 million people throughout recorded history [1]. Y. pestis is generally transmitted to humans via the bite of an infected rodent flea and is endemic to Africa, India, and the southwestern United States [2–4]. Because plague is highly infectious and can readily spread by aerosolization, it poses a bioterrorism threat [2,5]. Yersiniae type III secretion system (T3SS) expression is induced by contact with host cells in a temperature-dependent fashion resulting in secretion and/or translocation of Yersinia outer membrane proteins (Yops), including the low calcium response V antigen (LcrV) [6–8]. The T3SS and its effectors Yops are encoded on a 70-kb plasmid present in all of the pathogenic species of yersiniae [1]. In addition, Y. pestis synthesizes an anti-phagocytic capsule, the genes for which are encoded on a 110-kb plasmid [1]. Yersiniae that can successfully translocate Yops into the host cytosol are capable of modulating the host’s response to infection, thereby increasing their own survival [9]. Lipoproteins are outer membrane modified proteins with an N-terminal diacyl cysteine and are present in many bacterial pathogens [10,11]. Some of these lipoproteins are important viru- lence factors during the transmission, colonization and persistence of bacterial pathogens in the host [12–15]. For example, Braun lipoprotein (Lpp), specific for gram-negative bacteria of Enter- obacteriaceae family, has been shown to trigger the innate immune response and to induce apoptosis of the host cell through the toll- like receptor (TLR)-2 [16]. * Corresponding author. Tel.: þ1 409 747 0578; fax: þ1 409 747 6869. E-mail address: achopra@utmb.edu (A.K. Chopra). 1 Contributed equally to this manuscript. Contents lists available at ScienceDirect Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath 0882-4010/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2009.09.002 Microbial Pathogenesis 48 (2010) 42–52