Transcriptional Profiling of Mycobacterium tuberculosis Replicating Ex vivo in Blood from HIV- and HIV+ Subjects Michelle B. Ryndak 1. , Krishna K. Singh 1.¤ , Zhengyu Peng 2 , Susan Zolla-Pazner 3,1 , Hualin Li 4 , Lu Meng 2 , Suman Laal 3,1 * 1 Department of Pathology, New York University Langone Medical Center, New York, New York, United States of America, 2 Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China, 3 Veterans Affairs New York Harbor Healthcare System, New York, New York, United States of America, 4 Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America Abstract Hematogenous dissemination of Mycobacterium tuberculosis (M. tb) occurs during both primary and reactivated tuberculosis (TB). Although hematogenous dissemination occurs in non-HIV TB patients, in ,80% of these patients, TB manifests exclusively as pulmonary disease. In contrast, extrapulmonary, disseminated, and/or miliary TB is seen in 60–70% of HIV- infected TB patients, suggesting that hematogenous dissemination is likely more common in HIV+ patients. To understand M. tb adaptation to the blood environment during bacteremia, we have studied the transcriptome of M. tb replicating in human whole blood. To investigate if M. tb discriminates between the hematogenous environments of immunocompetent and immunodeficient individuals, we compared the M. tb transcriptional profiles during replication in blood from HIV- and HIV+ donors. Our results demonstrate that M. tb survives and replicates in blood from both HIV- and HIV+ donors and enhances its virulence/pathogenic potential in the hematogenous environment. The M. tb blood-specific transcriptome reflects suppression of dormancy, induction of cell-wall remodeling, alteration in mode of iron acquisition, potential evasion of immune surveillance, and enhanced expression of important virulence factors that drive active M. tb infection and dissemination. These changes are accentuated during bacterial replication in blood from HIV+ patients. Furthermore, the expression of ESAT-6, which participates in dissemination of M. tb from the lungs, is upregulated in M. tb growing in blood, especially during growth in blood from HIV+ patients. Preliminary experiments also demonstrate that ESAT-6 promotes HIV replication in U1 cells. These studies provide evidence, for the first time, that during bacteremia, M. tb can adapt to the blood environment by modifying its transcriptome in a manner indicative of an enhanced-virulence phenotype that favors active infection. Additionally, transcriptional modifications in HIV+ blood may further accentuate M. tb virulence and drive both M. tb and HIV infection. Citation: Ryndak MB, Singh KK, Peng Z, Zolla-Pazner S, Li H, et al. (2014) Transcriptional Profiling of Mycobacterium tuberculosis Replicating Ex vivo in Blood from HIV- and HIV+ Subjects. PLoS ONE 9(4): e94939. doi:10.1371/journal.pone.0094939 Editor: Anil Kumar Tyagi, University of Delhi, India Received November 7, 2013; Accepted March 21, 2014; Published April 22, 2014 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: The work was supported by the Center for AIDS Research (CFAR) grant (P30 AI027742) awarded to K.K.S., and the Veterans Affairs Research Career Scientist Award and the National Institutes of Health (NIH)/ Fogarty International Center training grant (D43 TW001409) both awarded to S.L. This material is also based upon work supported in part by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development with salary support to Research Microbiologist and Research Career Scientist, S.L. The funders had no role in the 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: Suman.Laal@nyumc.org ¤ Current address: Siemens Healthcare Diagnostics, Tarrytown, New York, United States of America . These authors contributed equally to this work. Introduction Infection with M. tuberculosis (M. tb) is initiated by the few bacilli in a droplet that are inhaled into the alveolus, which results either in establishment of infection and progression to primary TB or, by 4–5 weeks post-infection, elicitation of immune responses that control bacterial replication and result in latent TB. Studies in animal models have demonstrated hematogenous dissemination of M. tb from the lungs to various organs, including lymph nodes, spleen, liver, pancreas, adrenal, and heart, as well as reseeding of the lung, during primary infection [1,2,3]. Studies performed in the pre-ATT (anti TB treatment) era demonstrated hematogenous dissemination of M. tb in a majority of pediatric cases of primary TB [4]. Viable M. tb have been demonstrated in lungs, liver, spleen and kidneys of healthy individuals who died of causes unrelated to TB, in a TB endemic setting [5]. This again provides evidence of hematogenous dissemination of M. tb during primary infection resulting in latent TB [5]. Bacteremia in TB patients with pulmonary TB has been demonstrated for several decades [4,6,7]. Evidence for disseminated M. tb in patients who reactivated their latent infection also exists [8]. Thus, bacteremia and hematoge- nous dissemination of M. tb is an important component of the natural course of establishment of infection and progression to both primary and reactivated TB. TB is one of the most common infections in HIV-infected (HIV+) individuals in TB endemic countries, where 60–70% of the HIV+ patients develop TB [9]. Co-infected individuals are at a ,20–40 fold higher risk for TB [10], and manifestation of TB is independent of CD4+ T cell status and peripheral viral load [11,12,13,14,15]. Extrapulmonary TB (EPTB), disseminated TB PLOS ONE | www.plosone.org 1 April 2014 | Volume 9 | Issue 4 | e94939