Interaction of the sensor module of Mycobacterium tuberculosis H37Rv KdpD with members of the Lpr family

Abstract

The genetic and biochemical mechanisms by which Mycobacterium tuberculosis senses and responds to the complex environment that it encounters during infection and persistence within the host remain unknown. In a number of bacterial species, the Kdp signal transduction pathway appears to be the primary response to environmental osmotic stress, which is primarily mediated by K+ concentration in bacteria. We show that kdp encodes for components of a mycobacterial signalling pathway by demonstrating the K+ dependence of kdpFABC expression in both M. tuberculosis H37Rv and Mycobacterium smegmatis. To identify proteins of M. tuberculosis that participate in this signalling pathway, we used the N-terminal sensing module of the histidine kinase KdpD as bait in a yeast two-hybrid screen. We show that the sensing domain of KdpD interacts specifically with two membrane lipoproteins, LprJ (Rv1690) and LprF (Rv1368). Overexpression of lprF and lprJ alleles in mycobacterial kdpF-lacZ reporter strains enabled us to identify alleles that modulate kdpFABC expression. By exploiting the yeast three-hybrid system, we have found that the histidine kinase domain of KdpD forms ternary complexes with LprF and LprJ and the sensing module of KdpD. Our results establish a role for membrane proteins in the Kdp signalling pathway and suggest that LprF and LprJ function as accessory or ligand-binding proteins that communicate directly with the sensing domain of KdpD to modulate kdp expression.

Dissecting virulence pathways of Mycobacterium tuberculosis through protein–protein association

“…Cultivation and transformation of Mtb H37Rv and Msm mc 2 155 were performed as described (7). When necessary, Middlebrook medium was supplemented with KAN (25 g͞ml), HYG (50 g͞ml), or TRIM (40-50 g͞ml).…”

“…Previously, the mDHFR fragments were engineered such that the interacting protein pairs and F [1,2] and F [3] were separated by a flexible glycine linker peptide of 10 amino acids (9). In proof-of-concept experiments designed to demonstrate the feasibility of M-PFC to detect a diverse range of proteinprotein associations in Msm, we selected several well characterized interacting partners, namely Saccharomyces cerevisiae GCN4 (9), Mtb two-component proteins KdpD (Rv1028c)͞ KdpE (Rv1027c) (7) and Mtb secreted antigens Esat-6 (Rv3875)͞Cfp-10 (Rv3874) (13). We generated these bait and prey plasmids as C-terminal fusions with the complementary fragments of mDHFR to generate the interacting protein pairs GCN4 [F1,2] …”

“…An exception is the protein network of Helicobacter pylori (5) that yielded Ͼ1,000 Y2H interactions, connecting close to half of the proteome. Previously, we successfully exploited the Y2H system to study Mtb virulence (6) and signal transduction (7). Nevertheless, yeast does have certain limitations: (i) interactions occur in the nucleus, (ii) membrane proteins represent a problem, (iii) bacterial proteins do not undergo appropriate posttranslational modification, (iv) self activation can be a significant problem, and finally (v) high GϩC DNA is sometimes not well tolerated.…”

The N-terminal Input Domain of the Sensor Kinase KdpD of Escherichia coli Stabilizes the Interaction between the Cognate Response Regulator KdpE and the Corresponding DNA-binding Site

“…In this regard it is interesting to note that H-NS (hns) and thioredoxin reductase (trxB) somehow affect kdpFABC expression in E. coli (47). Furthermore, a very recent report (48) proposes the interaction of the input domain of KdpD and lipoproteins, namely LprJ and LprF, in Mycobacterium tuberculosis H37Rv. All these data indicate a more complex regulatory network for the regulation of kdpFABC expression than originally assumed for the KdpD/ KdpE two-component system.…”

Mycobacterium tuberculosis Rv1395 Is a Class III Transcriptional Regulator of the AraC Family Involved in Cytochrome P450 Regulation

“…Compared with other bacterial genera, little functional data about gene regulation in M. tuberculosis are currently available, due to the late development of mycobacterial genetics (14,15) and the slow growing nature of M. tuberculosis. However, many studies have recently appeared, focused especially on sigma factors (16 -20) and two-component systems such as mtrA-mtrB (21,22), trcS-trcR (23)(24)(25), mprA (26), prrA-prrB (25), devS-devR (27)(28)(29)(30), phoP-phoQ (31,32), senX3-regX3 (25,33) and more recently kdpE-kdpD (34). Some regulators such as IdeR, an iron-responsive DNA-binding protein from the DtxR family (35)(36)(37), LexA, involved in DNA repair (38,39), FurA (40,41), and WhiB3 (42) have also been partially characterized.…”