Expression profiling of Mycobacterium tuberculosis H37Rv and Mycobacterium smegmatis in acid-nitrosative multi-stress displays defined regulatory networks Andrea Cossu a, * , Leonardo Antonio Sechi b , Ennio Bandino c , Stefania Zanetti b , Valentina Rosu c a Department of Food Science, Division of Food Microbiology, University of Massachusetts, Amherst, MA 01003, USA b Department of Biomedical Sciences, Division of Experimental and Clinical Microbiology, University of Sassari, Sassari 07100, Italy c Experimental Zooprophylactic Institute of Sardinia, Department of Nuoro, Nuoro 08100, Italy article info Article history: Received 14 January 2013 Received in revised form 17 October 2013 Accepted 23 October 2013 Available online 1 November 2013 Keywords: Mycobacterium tuberculosis Mycobacterium smegmatis Functional genomics DNA-Microarray Acid-nitrosative multi-stress Virulence factors abstract Several studies regarding the transcriptome of Mycobacterium tuberculosis following the exposure to various in vitro simulated phagosomal stressors, have already tried to elucidate the bacterium behavior during the intracellular infection. An in vitro acid-nitrosative multi-stress was carried out for M. tuber- culosis H37Rv and Mycobacterium smegmatis MC 2 155 in order to analyze by DNA-microarray the gene expression changes associated respectively to pathogenic and non-pathogenic mycobacterial species. During acid-nitrosative multi-stress both mycobacteria shift their transcriptome to allow the anaerobic respiratory state and energy pathways characteristic of starvation. M. tuberculosis counteracts the combined acid-nitrosative stress more efficiently than M. smegmatis as also shown by the up-regulation of glbN and hmp genes, that are specifically directed to NO detoxification. Moreover, the down-regulation of some virulence factors involved in phthiocerol dimycocerosates synthesis strengthens the hypothesis that these major virulence determinants may be attenuated by M. tuberculosis in the presence of reactive nitrogen species. In fact, it down-regulates other genes implicated in the synthesis of membrane structural lipids but in contrast to M. smegmatis, M. tuberculosis up-regulates many genes annotated for the synthesis of peptidoglycan. Results suggest a gene regulation of M. tuberculosis which reveals a distinctive expression pattern under stressful environment. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Mycobacterium tuberculosis (MTB) remains one of the most important human pathogens being spread in about 1/3 of the whole world population [1]. One of the main features of this suc- cessful infectious agent is its capacity of resistance to several drug treatments which has led to the selection of many multi-drug resistance (MDR) strains [2] and its ability to persist in a dormant form within the host by eluding the human immune response [3]. The development of lung tuberculosis (TB) is most likely due to the specificity and adaptation of MTB to mammalian macrophages [4] and to some effective and not fully understood mechanisms concerning the interaction with the host immune response. The majority of infected patients do not develop overt symptoms of TB, but undergo a lag phase [5] where MTB enters an energy-metabolic quiescent phase awaiting reactivation when host is immune- compromised [6]. The host niche of MTB is the macrophage within which the bacteria are able to resist various physical and chemical insults. Several works on in vitro simulated phagosomal conditions have been very useful in highlighting the gene regulation of MTB during different environmental stresses including acid stress [7], heat [8], oxidative stress [9], nutrient starvation [10], anoxia [11], anti- bacterial compounds [12] and inhibitors of metabolism [13]. However, many of these experiments were performed by treating mycobacteria with a single stressful agent each time [14] that does not effectively mimic what happens in vivo where the bacteria may simultaneously face more than one stress. Therefore, in this study we reproduced an in vitro multi-stress condition in order to identify differentially regulated genes peculiar of pathogenic mycobacterial * Corresponding author. Department of Food Science, Division of Food Microbi- ology, University of Massachusetts, Chenoweth Laboratory, 102 Holdsworth Way, Amherst, MA 01003, USA. Tel.: þ1 413 545 1008; fax: þ1 413 545 1262. E-mail address: andrcossu@yahoo.it (A. Cossu). Contents lists available at ScienceDirect Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath 0882-4010/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.micpath.2013.10.004 Microbial Pathogenesis 65 (2013) 89e96