RESUMO
Cyclic AMP (cAMP) receptor protein (CRP), encoded by crp, is a global regulator that is activated by cAMP, a second messenger synthesized by a class I adenylate cyclase (AC-I) encoded by cyaA in Escherichia coli. cAMP-CRP is required for growth on nonpreferred carbon sources and is a global regulator. We constructed in-frame nonpolar deletions of the crp and cyaA homologs in Vibrio parahaemolyticus and found that the Δcrp mutant did not grow in minimal media supplemented with nonpreferred carbon sources, but the ΔcyaA mutant grew similarly to the wild type. Bioinformatics analysis of the V. parahaemolyticus genome identified a 181-amino-acid protein annotated as a class IV adenylate cyclase (AC-IV) named CyaB, a member of the CYTH protein superfamily. AC-IV phylogeny showed that CyaB was present in Gammaproteobacteria and Alphaproteobacteria as well as Planctomycetes and Archaea. Only the bacterial CyaB proteins contained an N-terminal motif, HFxxxxExExK, indicative of adenylyl cyclase activity. Both V. parahaemolyticus cyaA and cyaB genes functionally complemented an E. coli ΔcyaA mutant. The Δcrp and ΔcyaB ΔcyaA mutants showed defects in growth on nonpreferred carbon sources and in swimming and swarming motility, indicating that cAMP-CRP is an activator. The ΔcyaA and ΔcyaB single mutants had no defects in these phenotypes, indicating that AC-IV complements AC-I. Capsule polysaccharide and biofilm production assays showed significant defects in the Δcrp, ΔcyaBΔcyaA, and ΔcyaB mutants, whereas the ΔcyaA strain behaved similarly to the wild type. This is consistent with a role of cAMP-CRP as an activator of these phenotypes and establishes a cellular role for AC-IV in capsule and biofilm formation, which to date has been unestablished. IMPORTANCE Here, we characterized the roles of CRP and CyaA in V. parahaemolyticus, showing that cAMP-CRP is an activator of metabolism, motility, capsule production, and biofilm formation. These results are in contrast to cAMP-CRP in V. cholerae, which represses capsule and biofilm formation. Previously, only an AC-I CyaA had been identified in Vibrio species. Our data showed that an AC-IV CyaB homolog is present in V. parahaemolyticus and is required for optimal growth. The data demonstrated that CyaB is essential for capsule production and biofilm formation, uncovering a physiological role of AC-IV in bacteria. The data showed that the cyaB gene was widespread among Vibrionaceae species and several other Gammaproteobacteria, but in general, its phylogenetic distribution was limited. Our phylogenetic analysis also demonstrated that in some species the cyaB gene was acquired by horizontal gene transfer.
Assuntos
Adenilil Ciclases , Vibrio parahaemolyticus , Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Filogenia , AMP Cíclico/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteína Receptora de AMP Cíclico/genética , Proteína Receptora de AMP Cíclico/metabolismo , Biofilmes , PolissacarídeosRESUMO
Borrelia burgdorferi, the causative agent of Lyme disease, traverses through vastly distinct environments between the tick vector and the multiple phases of the mammalian infection that requires genetic adaptation for the progression of pathogenesis. Borrelial gene expression is highly responsive to changes in specific environmental signals that initiate the RpoS regulon for mammalian adaptation, but the mechanism(s) for direct detection of environmental cues has yet to be identified. Secondary messenger cyclic adenosine monophosphate (cAMP) produced by adenylate cyclase is responsive to environmental signals, such as carbon source and pH, in many bacterial pathogens to promote virulence by altering gene regulation. B. burgdorferi encodes a single non-toxin class IV adenylate cyclase (bb0723, cyaB). This study investigates cyaB expression along with its influence on borrelial virulence regulation and mammalian infectivity. Expression of cyaB was specifically induced with co-incubation of mammalian host cells that was not observed with cultivated tick cells suggesting that cyaB expression is influenced by cellular factor(s) unique to mammalian cell lines. The 3' end of cyaB also encodes a small RNA, SR0623, in the same orientation that overlaps with bb0722. The differential processing of cyaB and SR0623 transcripts may alter the ability to influence function in the form of virulence determinant regulation and infectivity. Two independent cyaB deletion B31 strains were generated in 5A4-NP1 and ML23 backgrounds and complemented with the cyaB ORF alone that truncates SR0623, cyaB with intact SR0623, or cyaB with a mutagenized full-length SR0623 to evaluate the influence on transcriptional and posttranscriptional regulation of borrelial virulence factors and infectivity. In the absence of cyaB, the expression and production of ospC was significantly reduced, while the protein levels for BosR and DbpA were substantially lower than parental strains. Infectivity studies with both independent cyaB mutants demonstrated an attenuated phenotype with reduced colonization of tissues during early disseminated infection. This work suggests that B. burgdorferi utilizes cyaB and potentially cAMP as a regulatory pathway to modulate borrelial gene expression and protein production to promote borrelial virulence and dissemination in the mammalian host.
RESUMO
The type III secretion system (T3SS) plays an important role in the pathogenesis of Pseudomonas aeruginosa. Expression of the T3SS is controlled under a complicate regulatory network. In this study, we demonstrate that NrtR (PA4916) is involved in the T3SS expression and pathogenesis of P. aeruginosa in a mouse acute pneumonia model. Overexpression of the T3SS central activator ExsA or exogenous supplementation of cAMP restored the expression of T3SS in the ΔnrtR mutant, suggesting that NrtR might regulate T3SS through the cAMP-Vfr signaling pathway. Further experiments demonstrated that the decrease of cAMP content is not due to the expression change of adenylate cyclases or phosphodiesterase in the ΔnrtR mutant. As it has been shown that nadD2 is upregulated in the ΔnrtR mutant, we overexpressed nadD2 in wild type PAK, which reduced the intracellular cAMP level and the expression of the T3SS genes. Meanwhile, deletion of nadD2 in the ΔnrtR mutant restored the expression and secretion of the T3SS. Co-immunoprecipitation assay revealed an interaction between NadD2 and the catalytic domain of the adenylate cyclase CyaB. Further in vitro assay indicated that NadD2 repressed the enzymatic activity of CyaB. Therefore, we have identified a novel regulatory mechanism of T3SS in P. aeruginosa.
RESUMO
BACKGROUND: Thiamine triphosphate (ThTP) is present in most organisms and might be involved in intracellular signaling. In mammalian cells, the cytosolic ThTP level is controlled by a specific thiamine triphosphatase (ThTPase), belonging to the CYTH superfamily of proteins. CYTH proteins are present in all superkingdoms of life and act on various triphosphorylated substrates. METHODS: Using crystallography, mass spectrometry and mutational analysis, we identified the key structural determinants of the high specificity and catalytic efficiency of mammalian ThTPase. RESULTS: Triphosphate binding requires three conserved arginines while the catalytic mechanism relies on an unusual lysine-tyrosine dyad. By docking of the ThTP molecule in the active site, we found that Trp-53 should interact with the thiazole part of the substrate molecule, thus playing a key role in substrate recognition and specificity. Sea anemone and zebrafish CYTH proteins, which retain the corresponding Trp residue, are also specific ThTPases. Surprisingly, the whole chromosome region containing the ThTPase gene is lost in birds. CONCLUSIONS: The specificity for ThTP is linked to a stacking interaction between the thiazole heterocycle of thiamine and a tryptophan residue. The latter likely plays a key role in the secondary acquisition of ThTPase activity in early metazoan CYTH enzymes, in the lineage leading from cnidarians to mammals. GENERAL SIGNIFICANCE: We show that ThTPase activity is not restricted to mammals as previously thought but is an acquisition of early metazoans. This, and the identification of critically important residues, allows us to draw an evolutionary perspective of the CYTH family of proteins.