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: 3.73). 06/2011; 79(8):3273-83. DOI: 10.1128/IAI.05153-11
Source: PubMed


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 Km 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 · 3.99 Impact Factor
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    • "However, Western blot analysis showed that both proteins are expressed at wild-type levels in the pdeB mutant, eliminating the possibility of a receptor-c-di-GMP complex directly regulating the expression of chemotaxis proteins (Sultan et al., 2011). However, Sultan et al. demonstrated that a pdeBplzA double mutant in B. burgdorferi constantly flexed, which is similar to the phenotype of the cheX mutant (Motaleb et al., 2005; Sultan et al., 2011), implying that PlzA may control the chemotaxis system either through modulating the activity of CheX or CheY3 (Sultan et al., 2011). Alternatively, the hydrolyzed products of the phosphodiesterases may be responsible for the phenotypic differences observed in the pdeA and pdeB mutants. "
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    ABSTRACT: In nature, the Lyme disease spirochete Borrelia burgdorferi cycles between the unrelated environments of the Ixodes tick vector and mammalian host. In order to survive transmission between hosts, B. burgdorferi must be able to not only detect changes in its environment, but also rapidly and appropriately respond to these changes. One manner in which this obligate parasite regulates and adapts to its changing environment is through cyclic-di-GMP (c-di-GMP) signaling. c-di-GMP has been shown to be instrumental in orchestrating the adaptation of B. burgdorferi to the tick environment. B. burgdorferi possesses only one set of c-di-GMP-metabolizing genes (one diguanylate cyclase and two distinct phosphodiesterases) and one c-di-GMP-binding PilZ-domain protein designated as PlzA. While studies in the realm of c-di-GMP signaling in B. burgdorferi have exploded in the last few years, there are still many more questions than answers. Elucidation of the importance of c-di-GMP signaling to B. burgdorferi may lead to the identification of mechanisms that are critical for the survival of B. burgdorferi in the tick phase of the enzootic cycle as well as potentially delineate a role (if any) c-di-GMP may play in the transmission and virulence of B. burgdorferi during the enzootic cycle, thereby enabling the development of effective drugs for the prevention and/or treatment of Lyme disease.
    Frontiers in Cellular and Infection Microbiology 05/2014; 4:56. DOI:10.3389/fcimb.2014.00056 · 3.72 Impact Factor
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    • "The action of the HD-GYP domain as a PDE active against c-di-GMP has been demonstrated in proteins from several bacteria including Xcc, Pseudomonas aeruginosa and Borrelia burgdorferi [8], [9], [35]. To test whether the regulatory influence on virulence and virulence factor synthesis in Xoc depended upon the c-di-GMP PDE activity, our approach was to examine the effects of introducing alanine substitutions in the presumed HD catalytic diad [8], [36], [37], [38] on both the enzymatic and regulatory activities of the protein. "
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    ABSTRACT: Bacterial leaf streak caused by Xanthomonas oryzae pv. oryzicola (Xoc) is one of the most important diseases in rice. However, little is known about the pathogenicity mechanisms of Xoc. Here we have investigated the function of three HD-GYP domain regulatory proteins in biofilm formation, the synthesis of virulence factors and virulence of Xoc. Deletion of rpfG resulted in altered production of extracellular polysaccharides (EPS), abolished virulence on rice and enhanced biofilm formation, but had little effect on the secretion of proteases and motility. In contrast, mutational analysis showed that the other two HD-GYP domain proteins had no effect on virulence factor synthesis and tested phenotypes. Mutation of rpfG led to up-regulation of the type III secretion system and altered expression of three putative glycosyltransferase genes gumD, pgaC and xagB, which are part of operons directing the synthesis of different extracellular polysaccharides. The pgaABCD and xagABCD operons were greatly up-regulated in the Xoc ΔrpfG mutant, whereas the expression of the gum genes was unaltered or slightly enhanced. The elevated biofilm formation of the Xoc ΔrpfG mutant was dramatically reduced upon deletion of gumD, xagA and xagB, but not when pgaA and pgaC were deleted. Interestingly, only the ΔgumD mutant, among these single gene mutants, exhibits multiple phenotype alterations including reduced biofilm and EPS production and attenuated virulence on rice. These data indicate that RpfG is a global regulator that controls biofilm formation, EPS production and bacterial virulence in Xoc and that both gumD- and xagB-dependent EPS contribute to biofilm formation under different conditions.
    PLoS ONE 03/2013; 8(3):e59428. DOI:10.1371/journal.pone.0059428 · 3.23 Impact Factor
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