Analysis of the HD-GYP domain cyclic dimeric GMP phosphodiesterase reveals a role in motility and the enzootic life cycle of Borrelia burgdorferi

Department of Microbiology and Immunology, Brody School of Medicine, East Carolina University, Greenville, NC 27834, USA.
Infection and immunity (Impact Factor: 4.16). 06/2011; 79(8):3273-83. DOI: 10.1128/IAI.05153-11
Source: PubMed

ABSTRACT HD-GYP domain cyclic dimeric GMP (c-di-GMP) phosphodiesterases are implicated in motility and virulence in bacteria. Borrelia burgdorferi possesses a single set of c-di-GMP-metabolizing enzymes, including a putative HD-GYP domain protein, BB0374. Recently, we characterized the EAL domain phosphodiesterase PdeA. A mutation in pdeA resulted in cells that were defective in motility and virulence. Here we demonstrate that BB0374/PdeB specifically hydrolyzed c-di-GMP with a K(m) of 2.9 nM, confirming that it is a functional phosphodiesterase. Furthermore, by measuring phosphodiesterase enzyme activity in extracts from cells containing the pdeA pdeB double mutant, we demonstrate that no additional phosphodiesterases are present in B. burgdorferi. pdeB single mutant cells exhibit significantly increased flexing, indicating a role for c-di-GMP in motility. Constructing and analyzing a pilZ pdeB double mutant suggests that PilZ likely interacts with chemotaxis signaling. While virulence in needle-inoculated C3H/HeN mice did not appear to be altered significantly in pdeB mutant cells, these cells exhibited a reduced ability to survive in Ixodes scapularis ticks. Consequently, those ticks were unable to transmit the infection to naïve mice. All of these phenotypes were restored when the mutant was complemented. Identification of this role of pdeB increases our understanding of the c-di-GMP signaling network in motility regulation and the life cycle of B. burgdorferi.

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    • "RpfG from Xanthomonas campestris promotes synthesis of virulence factors and affects biofilm formation and motility (Ryan et al., 2010, 2012). The Borrelia burgdorferi PdeB controls motility and promotes virulence in ticks (Sultan et al., 2011). Two HD-GYP domain proteins from Pseudomonas aeruginosa control swarming motility and production of virulence factors (Ryan et al., 2009). "
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    ABSTRACT: We surveyed the eight putative cyclic-di-GMP-modulating response regulators (RRs) in Desulfovibrio vulgaris Hildenborough that are predicted to function via two-component signaling. Using purified proteins, we examined cyclic-di-GMP (c-di-GMP) production or turnover in vitro of all eight proteins. The two RRs containing only GGDEF domains (DVU2067, DVU0636) demonstrated c-di-GMP production activity in vitro. Of the remaining proteins, three RRs with HD-GYP domains (DVU0722, DVUA0086, and DVU2933) were confirmed to be Mn(2+)-dependent phosphodiesterases (PDEs) in vitro and converted c-di-GMP to its linear form, pGpG. DVU0408, containing both c-di-GMP production (GGDEF) and degradation domains (EAL), showed c-di-GMP turnover activity in vitro also with production of pGpG. No c-di-GMP related activity could be assigned to the RR DVU0330, containing a metal-dependent phosphohydrolase HD-OD domain, or to the HD-GYP domain RR, DVU1181. Studies included examining the impact of overexpressed cyclic-di-GMP-modulating RRs in the heterologous host E. coli and led to the identification of one RR, DVU0636, with increased cellulose production. Evaluation of a transposon mutant in DVU0636 indicated that the strain was impaired in biofilm formation and demonstrated an altered carbohydrate:protein ratio relative to the D. vulgaris wild type biofilms. However, grown in liquid lactate/sulfate medium, the DVU0636 transposon mutant showed no growth impairment relative to the wild-type strain. Among the eight candidates, only the transposon disruption mutant in the DVU2067 RR presented a growth defect in liquid culture. Our results indicate that, of the two diguanylate cyclases (DGCs) that function as part of two-component signaling, DVU0636 plays an important role in biofilm formation while the function of DVU2067 has pertinence in planktonic growth.
    Frontiers in Microbiology 07/2014; 5:382. DOI:10.3389/fmicb.2014.00382 · 3.94 Impact Factor
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    • "Cyclic di-GMP signalling is not required for virulence of Y. pestis in a plague mouse model (Bobrov et al., 2011) and also the Lyme disease spirochaete Borrelia burgdorferi can successfully cause infection in mice without using its cyclic di-GMP signalling system (He et al., 2011). Even more, uncontrolled high cyclic di-GMP levels achieved by deletion of the single phosphodiesterase inhibit acute infection through the expression of extracellular biofilm matrix components (Bobrov et al., 2011; Sultan et al., 2011). On the other hand, biofilm formation, which is stimulated by cyclic di-GMP signalling, is a virulence factor in chronic infections (Tamayo et al., 2007). "
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    ABSTRACT: The secondary messenger cyclic di-GMP coordinately regulates the transition between motility/sessility/virulence in bacterial populations and upon adaptation to novel habitats. Thereby, multiple independent regulatory circuits regulate a diversity of targets. This specific output response is surprising considering the diverse physiological processes regulated by this signalling molecule, which range from transcription to proteolysis and clearly demonstrates the presence of sophisticated developmental programmes in these so-called simple organisms.
    Environmental Microbiology 10/2011; 14(8):1817-29. DOI:10.1111/j.1462-2920.2011.02617.x · 6.24 Impact Factor
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    ABSTRACT: Cyclic-di-GMP is a near-ubiquitous bacterial second messenger that is important in localized signal transmission during the control of various processes, including virulence and switching between planktonic and biofilm-based lifestyles. Cyclic-di-GMP is synthesized by GGDEF diguanylate cyclases and hydrolyzed by EAL or HD-GYP phosphodiesterases, with each functional domain often appended to distinct sensory modules. HD-GYP domain proteins have resisted structural analysis, but here we present the first structural representative of this family (1.28 Å), obtained using the unusual Bd1817 HD-GYP protein from the predatory bacterium Bdellovibrio bacteriovorus. Bd1817 lacks the active-site tyrosine present in most HD-GYP family members yet remains an excellent model of their features, sharing 48% sequence similarity with the archetype RpfG. The protein structure is highly modular and thus provides a basis for delineating domain boundaries in other stimulus-dependent homologues. Conserved residues in the HD-GYP family cluster around a binuclear metal center, which is observed complexed to a molecule of phosphate, providing information on the mode of hydroxide ion attack on substrate. The fold and active site of the HD-GYP domain are different from those of EAL proteins, and restricted access to the active-site cleft is indicative of a different mode of activity regulation. The region encompassing the GYP motif has a novel conformation and is surface exposed and available for complexation with binding partners, including GGDEF proteins.
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