Structural asymmetry in a conserved signaling system that regulates division, replication, and virulence of an intracellular pathogen

Abstract

We have functionally and structurally defined an essential protein phosphorelay that regulates expression of genes required for growth, division, and intracellular survival of the global zoonotic pathogen Brucella abortus. Our study delineates phosphoryl transfer through this molecular pathway, which initiates from the sensor kinase CckA and proceeds through the ChpT phosphotransferase to two regulatory substrates: CtrA and CpdR. Genetic perturbation of this system results in defects in cell growth and division site selection, and a specific viability deficit inside human phagocytic cells. Thus, proper control of B. abortus division site polarity is necessary for survival in the intracellular niche. We further define the structural foundations of signaling from the central phosphotransferase, ChpT, to its response regulator substrate, CtrA, and provide evidence that there are at least two modes of interaction between ChpT and CtrA, only one of which is competent to catalyze phosphoryltransfer. The structure and dynamics of the active site on each side of the ChpT homodimer are distinct, supporting a model in which quaternary structure of the 2:2 ChpT-CtrA complex enforces an asymmetric mechanism of phosphoryl transfer between ChpT and CtrA. Our study provides mechanistic understanding, from the cellular to the atomic scale, of a conserved transcriptional regulatory system that controls the cellular and infection biology of B. abortus. More generally, our results provide insight into the structural basis of two-component signal transduction, which is broadly conserved in bacteria, plants, and fungi.rucellosis, caused by Brucella spp., is among the most common zoonotic diseases worldwide (1). These intracellular pathogens are estimated to cause at least 500,000 new human infections each year and, in areas of Africa, Asia, and South America, inflict significant agricultural losses due to decreased livestock production (2, 3). Survival of Brucella within mammals is linked to their ability to infect and survive inside professional phagocytic cells (2). If left untreated in human hosts, Brucella eventually spread to multiple tissue types, which can lead to a range of debilitating chronic sequelae including reticuloendothelial, cardiovascular, gastrointestinal, and neurological damage.Brucella are members of the α-proteobacteria, a diverse class of Gram-negative species adapted for growth across a range of environmental conditions including plant surfaces and roots, aquatic and soil ecosystems, and the interior of mammalian cells (4, 5). Among the central regulatory systems controlling the α-proteobacterial cell cycle is a multistep phosphorelay composed of four proteins: (i) the hybrid sensor histidine kinase (HK) CckA, (ii) the histidine phosphotransferase ChpT, (iii) the DNA-binding response regulator CtrA, and (iv) the phospho-receiver protein CpdR (Fig. 1). Our current understanding of this conserved regulatory system is based largely on studies of the related aquatic bacterium Caulobacter crescentus (6, 7). In Ca...

Regulation of signaling directionality revealed by 3D snapshots of a kinase:regulator complex in action

“…On the HK side, the main element engaged in the interface is helix α1 (HK) on the DHp domain from one DesK protomer, and minor contacts with helix α2’ (HK) from the second protomer. This interface broadly resembles the one found in other TCS complexes (Casino et al, 2009; Willett et al, 2015; Yamada et al, 2009; Zapf et al, 2000) (Figure 2A).

10.7554/eLife.21422.008Figure 2.The HK:RR interface and structural variability.( A ) Structural superposition of the DesK:DesR phosphatase complex (this study, PDB 5IUN) with HK853:RR468 from Thermotoga maritima (PDB 3DGE).

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“…Further supporting that this ensemble of conformations is functionally relevant, molecular dynamics simulations of the phosphotransferase complex (Figure 2—figure supplement 2) recapitulated the gliding shifts of one partner with respect to the other, reaching the different arrangements observed in the crystal structures at different moments of the simulations (Figure 2—figure supplement 2B). Loose HK:RR association architectures can actually be observed in other TCS-related complexes (Zapf et al, 2000; Casino et al, 2009; Willett et al, 2015), suggesting it is a universal feature. This explains the considerable promiscuity reported in different TCSs in in vitro assays, if long enough incubation times are allowed for a given HK to phosphotransfer to different RRs, including surrogate partners (Skerker et al, 2005).…”

“…In contrast, HK:RR TCSs with unidirectional P~His→Asp transfer, such as B. subtilis DesK:DesR (this study), Thermotoga maritima HK853:RR469 (Casino et al, 2009) and Rhodobacter sphaeroides CheA3:CheY6 (Bell et al, 2010), consistently show >6.6 Å HisNε2-AspOδ1 distances and correlated large reaction coordinate distances when these could be measured (Table 6). It will be interesting to test whether the phosphorelay pair ChpT:CtrA from Brucella abortus (Willett et al, 2015) displays reversible transfer, as anticipated on the basis of the reported 5.9 Å HisNε2-AspOδ1 distance.…”

A Kinase-Phosphatase Switch Transduces Environmental Information into a Bacterial Cell Cycle Circuit

“…Previous work in the nitrogen-fixing plant symbiont Sinorhizobium meliloti demonstrated that CtrA is strongly downregulated during the early steps of symbiosis [47]. In the pathogen Brucella abortus the CckA-ChpT-CtrA-CpdR pathway was shown to be required for intracellular survival in human macrophages [48]. Therefore, precise environmental regulation of CtrA abundance and activity likely plays an important role for the diverse functions that different alphaproteobacteria perform in the environment.…”

Brucella abortus Δ rpoE1 confers protective immunity against wild type challenge in a mouse model of brucellosis

“…Experiments were performed at B io s afety L evel 3 (BSL3) conditions as required by the Centers of Disease Control rules and regulations governing the use of Select Agents. Growth and maintenance of strains is as previously described (25). …”