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Cloning and expression of diadenosine 5',5'''-P1,P4-tetraphosphate hydrolase from Lupinus angustifolius L.

ABSTRACT: The first isolation, cloning and expression of cDNA encoding an asymmetric diadenosine 5',5'''P1,P4-tetraphosphate pyrophosphohydrolase (Ap4A hydrolase) from a higher plant is described. Ap4A hydrolase protein was purified from seeds of both Lupinus luteus and Lupinus angustifolius and partially sequenced. The Ap4A hydrolase cDNA was cloned from L. angustifolius cotyledonary polyadenylated RNA using reverse transcription and PCR with primers based on the amino acid sequence. The cDNA encoded a protein of 199 amino acids, molecular mass 22982Da. When expressed in Escherichia coli fused to a maltose-binding protein, the enzyme catalysed asymmetric cleavage of Ap4A to AMP and ATP which was inhibited at concentrations of F- as low as 3 microM. These are properties characteristic of Ap4A hydrolase (asymmetrical) (EC 3.6.1. 17). Comparison of the Ap4A hydrolase sequences derived from the four known cDNAs from pig, human, lupin and fission yeast showed that, like the mammalian hydrolase, the lupin enzyme possesses a Mut T motif but no other significant similarities. No sequence similarity to the human fragile histidine triad protein, as found in the Ap4A hydrolase from Schizosaccharomyces pombe, was detected in the Ap4A hydrolase from lupin.

SUBMITTER: Maksel D

PROVIDER: S-EPMC1219046 | biostudies-other | 1998 Jan

REPOSITORIES: biostudies-other

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Project description:The opportunistic bacterial pathogen Pseudomonas aeruginosa causes a wide range of infections that are difficult to treat, largely because of the spread of antibiotic-resistant isolates. Antivirulence therapy, í.e. the use of drugs that inhibit the expression or activity of virulence factors, is currently considered an attractive strategy to reduce P. aeruginosa pathogenicity and complement antibiotic treatments. Because of the multifactorial nature of P. aeruginosa virulence and the broad arsenal of virulence factors this bacterium can produce, the regulatory networks that control the expression of multiple virulence traits have been extensively explored as potential targets for antivirulence drug development. The intracellular signaling molecule diadenosine tetraphosphate (Ap4A) has been reported to control stress resistance and virulence-related traits in some bacteria, but its role has not been investigated in P. aeruginosa so far. To fill this gap, we generated a mutant of the reference strain P. aeruginosa PAO1 that lacks the Ap4A-hydrolysing enzyme ApaH and, consequently, accumulates high intracellular levels of Ap4A. Phenotypic and transcriptomic analyses revealed that the lack of ApaH causes a drastic reduction in the expression of several virulence factors, including extracellular proteases, elastases, siderophores, and quorum sensing signal molecules. Accordingly, infection assays in plant and animal models demonstrated that ApaH-deficient cells are significantly impaired in infectivity and persistence in different hosts, including mice. Finally, deletion of apaH in P. aeruginosa clinical isolates demonstrated that the positive effect of ApaH on the production of virulence-related traits and on infectivity is conserved in P. aeruginosa. This study provides the first evidence that the Ap4A-hydrolysing enzyme ApaH is important for P. aeruginosa virulence, highlighting this protein as a novel potential target for antivirulence therapies against P. aeruginosa.

Project description:Diadenosine tetraphosphate (Ap4A) is a dinucleotide found in both prokaryotes and eukaryotes. In bacteria, its cellular levels increase following exposure to various stress signals and stimuli, and its accumulation is generally correlated with increased sensitivity to a stressor(s), decreased pathogenicity, and enhanced antibiotic susceptibility. Ap4A is produced as a by-product of tRNA aminoacylation, and is cleaved to ADP molecules by hydrolases of the ApaH and Nudix families and/or by specific phosphorylases. Here, considering evidence that the recombinant protein YqeK from Staphylococcus aureus copurified with ADP, and aided by thermal shift and kinetic analyses, we identified the YqeK family of proteins (COG1713) as an unprecedented class of symmetrically cleaving Ap4A hydrolases. We validated the functional assignment by confirming the ability of YqeK to affect in vivo levels of Ap4A in B. subtilis YqeK shows a catalytic efficiency toward Ap4A similar to that of the symmetrically cleaving Ap4A hydrolases of the known ApaH family, although it displays a distinct fold that is typical of proteins of the HD domain superfamily harboring a diiron cluster. Analysis of the available 3D structures of three members of the YqeK family provided hints to the mode of substrate binding. Phylogenetic analysis revealed the occurrence of YqeK proteins in a consistent group of Gram-positive bacteria that lack ApaH enzymes. Comparative genomics highlighted that yqeK and apaH genes share a similar genomic context, where they are frequently found in operons involved in integrated responses to stress signals.IMPORTANCE Elevation of Ap4A level in bacteria is associated with increased sensitivity to heat and oxidative stress, reduced antibiotic tolerance, and decreased pathogenicity. ApaH is the major Ap4A hydrolase in gamma- and betaproteobacteria and has been recently proposed as a novel target to weaken the bacterial resistance to antibiotics. Here, we identified the orphan YqeK protein family (COG1713) as a highly efficient Ap4A hydrolase family, with members distributed in a consistent group of bacterial species that lack the ApaH enzyme. Among them are the pathogens Staphylococcus aureus, Streptococcus pneumoniae, and Mycoplasma pneumoniae By identifying the player contributing to Ap4A homeostasis in these bacteria, we disclose a novel target to develop innovative antibacterial strategies.

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Cloning and expression of diadenosine 5',5'''-P1,P4-tetraphosphate hydrolase from Lupinus angustifolius L.

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