Downloaded from www.microbiologyresearch.org by IP: 52.87.207.59 On: Tue, 25 Jul 2017 11:07:39 Intracellular autoregulation of the Mycobacterium tuberculosis PrrA response regulator Fanny Ewann, Camille Locht and Philip Supply Correspondence Philip Supply philip.supply@pasteur-lille.fr INSERM U447, Institut Pasteur de Lille, 1 rue du Professeur Calmette, F-59019 Lille Cedex, France Received 27 May 2003 Revised 17 September 2003 Accepted 19 September 2003 Two-component systems are major regulatory systems for bacterial adaptation to environmental changes. During the infectious cycle of Mycobacterium tuberculosis, adaptation to an intracellular environment is critical for multiplication and survival of the micro-organism within the host. The M. tuberculosis prrA gene, encoding the regulator of the two-component system PrrA–PrrB, has been shown to be induced upon macrophage phagocytosis and to be transiently required for the early stages of macrophage infection. In order to study the mechanisms of regulation of the PrrA–PrrB two-component system, PrrA and the cytoplasmic part of the PrrB histidine kinase were produced and purified as hexahistidine-tagged recombinant proteins. Electrophoretic mobility shift assays indicated that PrrA specifically binds to the promoter of its own operon, with increased affinity upon phosphorylation. Moreover, induction of fluorescence was observed after phagocytosis of a wild-type M. tuberculosis strain containing the gfp reporter gene under the control of the prrA–prrB promoter, while this induction was not seen in a prrA/B mutant strain containing the same construct. These results indicate that the early intracellular induction of prrA depends on the autoregulation of this two-component system. INTRODUCTION Two-component systems are major elements in bacterial adaptation to environmental changes. These systems are implicated in a large variety of adaptive responses, such as quorum sensing, chemotaxis and metabolic changes [for reviews, see Hoch (2000) and Stock et al. (2000)]. In many pathogenic bacteria, two-component systems are central regulatory elements for the production of virulence factors. Two-component systems basically involve a histidine kinase and a response regulator, which communicate through a phosphorylation cascade. In addition to a so-called receiver domain, which includes a conserved aspartyl residue as the final target of the phosphorelay, most response regulators contain an effector domain able to bind specific DNA sequences, and to thereby act as transcriptional regulators. Mycobacterium tuberculosis possesses a complex infectious cycle, which includes intra- and extra-cellular phases, both within and outside the lungs, as well as a latency phase, suggesting that the expression of many of its genes must be subjected to regulation. However, the regulatory mechan- isms governing the adaptive responses of M. tuberculosis, especially during phagocytosis, are still poorly understood. The M. tuberculosis genome contains 11 pairs of genes encoding two-component systems, in addition to a few isolated genes encoding orphan histidine kinases or response regulators (Cole et al., 1998). Several of these genes have been characterized at least partially (Dasgupta et al., 2000; Ewann et al., 2002; Graham & Clark-Curtiss, 1999; Haydel et al., 1999; Himpens et al., 2000; Perez et al., 2001; Sherman et al., 2001; Supply et al., 1997; Via et al., 1996; Zahrt & Deretic, 2000, 2001). For example, an M. tuberculosis strain with a mutation in the phoP gene was found to be impaired in intra-cellular growth within macrophages, one of the major target cells of M. tuberculosis, and its virulence was found to be attenuated in mice (Perez et al., 2001). MprA–MprB is required for persistence in murine infection (Zahrt & Deretic, 2001) and DevR–DevS is induced in response to hypoxia and required to survive it (Boon & Dick, 2002; Park et al., 2003; Sherman et al., 2001). MtrA–MtrB was found to be essential for survival, as so far it has not been possible to obtain mtrA knockout strains of M. tuberculosis (Zahrt & Deretic, 2000). In addition, mtrA has been shown to be upregulated upon phagocytosis in Mycobacterium bovis BCG but not in M. tuberculosis (Via et al., 1996). The upregulating mechanism in M. bovis BCG has not yet been identified. The PrrA–PrrB system has been found to be induced after macrophage phagocytosis and to be transiently required during the early stages of the macrophage infection (Ewann et al., 2002; Graham & Clark- Curtiss, 1999). The PrrA–PrrB system belongs to a wide subfamily of two-component systems, of which OmpR– EnvZ is the prototype. Many members of this subfamily have been demonstrated to be autoregulated. However, autoregulation is not a general rule, as illustrated by the hilA gene in Salmonella typhimurium (Bajaj et al., 1996; Lucas Abbreviation: His 6 , hexahistidine. 0002-6516 G 2004 SGM Printed in Great Britain 241 Microbiology (2004), 150, 241–246 DOI 10.1099/mic.0.26516-0