Vulnerabilities in Yersinia pestis caf Operon Are Unveiled by a Salmonella Vector Ling Cao1, Timothy Lim1, SangMu Jun1, Theresa Thornburg1, Recep Avci2, Xinghong Yang 1 * 1 Immunology and Infectious Diseases, Montana State University, Bozeman, Montana, United States of America, 2 Imaging and Chemical Analysis Laboratory, Department of Physics, Montana State University, Bozeman, Montana, United States of America Abstract During infection, Yersinia pestis uses its F1 capsule to enhance survival and cause virulence to mammalian host. Since F1 is produced in large quantities and secreted into the host tissues, it also serves as a major immune target. To hold this detrimental effect under proper control, Y. pestis expresses the caf operon (encoding the F1 capsule) in a temperature- dependent manner. However, additional properties of the caf operon limit its expression. By overexpressing the caf operon in wild-type Salmonella enterica serovar Typhimurium under a potent promoter, virulence of Salmonella was greatly attenuated both in vitro and in vivo. In contrast, expression of the caf operon under the regulation of its native promoter exhibited negligible impairment of Salmonellae virulence. In-depth investigation revealed all individual genes in the caf operon attenuated Salmonella when overexpressed. The deleterious effects of caf operon and the caf individual genes were further confirmed when they were overexpressed in Y. pestis KIM6+. This study suggests that by using a weak inducible promoter, the detrimental effects of the caf operon are minimally manifested in Y. pestis. Thus, through tight regulation of the caf operon, Y. pestis precisely balances its capsular anti-phagocytic properties with the detrimental effects of caf during interaction with mammalian host. Citation: Cao L, Lim T, Jun S, Thornburg T, Avci R, et al. (2012) Vulnerabilities in Yersinia pestis caf Operon Are Unveiled by a Salmonella Vector. PLoS ONE 7(4): e36283. doi:10.1371/journal.pone.0036283 Editor: Dipshikha Chakravortty, Indian Institute of Science, India Received October 24, 2011; Accepted March 28, 2012; Published April 30, 2012 Copyright: 2012 Cao 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: This work is supported by grants from National Institute of Allergy and Infectious Diseases (R21 AI-080960), National Center for Research Resources (RR020185-09), and the National Institute of General Medical Sciences (GM103500-09) from the National Institutes of Health. It is supported in part by an equipment grant from the M.J. Murdock Charitable Trust, Montana Agricultural Station, and U.S. Department of Agriculture Formula Funds. It is also supported in part by Office of Navy Research Award (N00014-10-1-0946). 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: yangxh@montana.edu Introduction Plague is an ancient disease, responsible for 200 million deaths worldwide and still exists in parts of the world today [1,2]. Plague is caused by the gram-negative bacterium Yersinia pestis carried by rodents and spread by a flea vector [2]. Plague transmitted to humans by the bite of infected fleas leads to the bubonic form of the disease [3], which can result in 50% mortality if left untreated [4]. Some individuals bitten by infected fleas will not develop bubonic plague, but rather develop septicemic plague, a more lethal form [5]. Although neither bubonic nor septicemic plague is contagious, a small minority of patients develop secondary pneumonic plague. Pneumonic plague is highly contagious since the distance required for effective aerosol transmission is 2 meters [2]. Pneumonic plague can cause 100% mortality if not treated and 50% mortality when antimicrobial treatment begins within 20 hours of the onset of symptoms [5]. To date, endemic areas include China, central and southern Africa, vast areas of Asia and South America, and the southwest portion of the United States. In addition, plague has been classified as a re-emerging disease by the World Health Organization [1]. Two types of clinical isolates of Y. pestis have been disclosed: the F1 capsular positive and negative strains. Although the F1 capsule has been shown to be required for successful transmission from the flea to the mammalian host [6], contradictory findings regarding its requirement for mammalian infection have been reported [7,8]. A more recent elaborate study comparing the caf operon deletion mutant with its parent wild-type (wt) strain Y. pestis CO92 showed that caf is required for maintaining virulence in mice although it depends on the mouse background [9], suggesting the F1 capsule is an important virulence factor. However, in order to utilize the F1 capsule to assist in infection, the pathogen must reduce exposure of F1 capsular protein to the mammalian immune system since it is also a protective antigen [10]. To minimize the exposure of F1 proteins, the pathogen has adopted a strategy of placing caf operon expression under tight control of a temperature-sensitive promoter [11], such that, at mammalian body temperature, the caf operon becomes activated to produce virulence effects, i.e., anti- phagocysis [12]. It can be inferred that the advantage gained from the anti-phagocytic properties of F1 outweighs the potential disadvantage of immunogenicity, since F1 capsular positive strains are more frequently isolated in nature than F1 capsular negative strains [13]. Additionally, bubonic plague symptoms usually appear after 2–8 days of exposure to the bacteria, while pneumonic plague has an incubation period of 1–6 days [14]. Such a short time from incubation to disease does not allow for protective immune responses to develop against the F1 capsule. Since the F1 capsule provides an overall beneficial effect for Y. pestis, we questioned why it does not utilize a potent constitutive promoter rather than an inducible one. We therefore hypothesized PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e36283