INTRODUCTION
The Lyme disease agent Borrelia (Borreliella )burgdorferi is maintained in an enzootic cycle traversing betweenIxodes ticks and vertebrates (1,2). The spirochete regulates gene expression in a phase-specific fashion as it cycles between its tick vector and its vertebrate hosts (3,4). Ixodes larvae feed on an infected host and acquire the spirochetes, which persist in the midgut. Following the molt, nymphs feed on a vertebrate and the spirochetes migrate from the midgut through the hemocoel to the salivary glands and transmit to the naïve host (5,6). Gene expression is globally regulated by the alternative sigma factors RpoS (σS) and RpoN (σN) (7-9), the stringent response via guanosine tetraphosphate and pentaphosphate [(p)ppGpp] (10,11), and cyclic dimeric GMP (c-di-GMP) (12-15). While transcriptomics studies of these signaling systems have revealed gene products important for infectivity and persistence, our understanding of post-transcriptional regulation remains sparse.
Many mechanisms of post-transcriptional gene regulation rely on RNA chaperones, a heterogeneous group of proteins that modulate RNA structures by disrupting RNA secondary and tertiary structure (unwinding, unfolding, and strand-displacing) or accelerating base pairing of RNAs (annealing) (16-18). Bacterial RNA chaperones include Hfq, StpA, FinO/ProQ, CsrA, cold shock proteins (CSPs), and the ribosomal proteins S1 and S12; mutants have pleiotropic phenotypes including reduced virulence, impaired growth, and reduced ability to respond to and survive environmental stresses (19,20). RNA chaperones can display sequence-independent, transient annealing and strand displacement activity (17,18,21). Hfq and, likely, ProQ are considered “matchmaker” chaperones that facilitate base pairing oftrans -acting small regulatory RNAs and their RNA targets (19,22-24). B. burgdorferi harbors several RNA-binding proteins that regulate post-transcriptional gene expression, but their RNA chaperone activity and mechanisms of regulation have not been extensively studied (4). In 2010, we identified BB0268 as a unique RNA chaperone (25). BB0268 partially complements an Escherichia colihfq mutant and the hfq gene of E. coli partially complements several defects of the pleiotropic bb0268 mutant ofB. burgdorferi ; however, BB0268 has negligible homology with Hfq orthologs from well-studied model bacteria (25).
Small RNAs (sRNAs) regulate gene expression, which depends on their three-dimensional structure as well as RNA chaperones (26,27). Many pathogenic bacteria utilize sRNAs to regulate virulence (20,28-30). Only three sRNAs to date have been functionally characterized in B. burgdorferi : DsrABb, a trans -acting sRNA, regulates translation of an rpoS mRNA species (31); Bb6S RNA, a protein-interacting sRNA, globally regulates gene expression by affecting RNA polymerase σ factor selectivity (32); and ittA , another sRNA, affects expression of several genes and is required for dissemination in the vertebrate host by an unknown molecular mechanism (33). However, the catalog of non-coding sRNAs in B. burgdorferiincreased dramatically following high-throughput sequencing studies (4,34-38).
c-di-GMP is a global secondary messenger responsible for numerous physiological adaptations of bacteria, including biofilm formation, virulence, motility, phage resistance, and cell morphology (39-43). Diguanylate cyclases synthesize c-di-GMP from two GTP molecules and phosphodiesterases degrade c-di-GMP into linear 5′-phosphoguanylyl-(3′-5′)-guanosine (pGpG) and/or GMP. In B. burgdorferi , c-di-GMP is produced by a sole diguanylate cyclase, Rrp1, and degraded by two phosphodiesterases, PdeA and PdeB (44-47). Rrp1 forms a prototypical two-component signal transduction system with histidine kinase 1 (Hk1), although the activating ligand remains unknown (12,48). rrp1 and hk1 mutants have wild-type transmission to and colonization of vertebrates via needle inoculation; however, both mutants are quickly destroyed in the tick midgut during larvae and nymph blood meal feeding (12,13,49,50). In other bacteria, c-di-GMP regulates gene expression at the transcriptional, post-transcriptional, and post-translational levels by binding to different effectors (riboswitches, transcriptional regulators, enzymes, and proteins) (51). In most B. burgdorferi , PlzA is the only c-di-GMP effector identified to date (52-55). Mouse infectivity is markedly impaired in aplzA mutant (52,56,57), unlike the hk1 and rrp1mutants. Groshong et al. (14) recently demonstrated that a plzApoint mutant unable to bind c-di-GMP (R145D) is fully infectious in mice confirming that the function of PlzA in mammals is c-di-GMP-independent. The c-di-GMP-dependent role of PlzA in the tick is controversial. Kostick-Dunn et al. (56) found that PlzA is not required to establish infection in ticks, while Pitzer et al. (55) report that plzAmutants do not survive during acquisition.
The binding of c-di-GMP induces a large conformational change in PlzA (14,53). The crystal structure of holo -PlzA revealed a two-domain structure separated by a short linker (58). Both the N - andC -terminal domains contain seven-stranded β-barrels that are nearly superimposable. Differences between the two domains are found in the loops connecting the β-strands. The N -terminal domain has α helices joining the β-strands, while the C -terminal domain has disordered linkers. Notably, apo -PlzA did not yield crystals, suggesting more structural flexibility than the liganded form.
In this study, we demonstrate that PlzA has RNA chaperone activity that includes RNA annealing, strand displacement and unfolding. Our data show that c-di-GMP inhibits PlzA strand displacement and RNA unfolding activities in vitro and in a heterologous system in vivo . We hypothesize that unliganded apo -PlzA facilitates conformational changes in RNA that alter their expression, remodeling the RNA structure to stimulate or inhibit transcription or translation, thus enabling spirochete transmission and survival in the vertebrate. In contrast, c-di-GMP does not affect the RNA annealing activity of PlzAin vitro , as apo - and holo -PlzA have similar RNA annealing properties. We hypothesize that holo -­ andapo -PlzA are “matchmaker” RNA chaperones that facilitate sRNA and target RNA annealing resulting in the regulation of gene expression throughout the B. burgdorferi enzootic cycle. To our knowledge, this is the first protein with RNA chaperone activity that is modulated by a second messenger.