Pertussis Adenylyl Cyclase Research Articles

Structural Investigation into Calmodulin's Role in Activating Bordetella Pertussis Adenylyl Cyclase Toxin CyaA

Whooping cough is a respiratory disease resulting from Bordetella pertussis infection that remains a globally significant cause of vaccine-preventable deaths. As a major virulence factor, the adenylyl cyclase toxin (ACT) of Bordetella pertussis (CyaA) has evolved a unique mode of activation by host cell calmodulin (CaM), thereby making it a potential target for the design of novel therapeutics. Currently, no high-resolution structure of intact CaM bound to CyaA exists, so the molecular pathways controlling enzymatic activation remain unclear. Previously, we have identified unique sites of intermolecular association between CyaA and CaM that directly impact CaM's structure, metal-binding properties, and conformational stability. In this study, we utilized site-directed mutagenesis, biophysical, and biochemical techniques to investigate the roles of metal-binding and CaM-dependent interactions in regulating CyaA structure and function. Site-specific mutations mapping to metal-binding site I or II of CaM reduced CyaA binding affinity and cAMP production without significantly altering CaM conformation. Moreover, inactivation of sites I or II in CaM modulated the global conformation of the CaM/CyaA complex in a metal-dependent manner. In the absence of CaM, CyaA exhibited multiple thermal transitions during unfolding experiments, but domain-specific interactions with wild-type or mutant CaM increased the thermal stability of the complex. Together, these data support that CaM-dependent CyaA activation occurs by novel structural mechanisms. This study sheds light on the molecular details of CaM-regulated ACTs, knowledge which may be exploited in the rationale design of inhibitory compounds.

Interactions of Bordetella pertussis adenylyl cyclase toxin CyaA with calmodulin mutants and calmodulin antagonists: Comparison with membranous adenylyl cyclase I

The adenylyl cyclase (AC) toxin CyaA from Bordetella pertussis constitutes an important virulence factor for the pathogenesis of whooping cough. CyaA is activated by calmodulin (CaM) and compromises host defense by excessive cAMP production. Hence, pharmacological modulation of the CyaA/CaM interaction could constitute a promising approach to treat whooping cough, provided that interactions of endogenous effector proteins with CaM are not affected. As a first step toward this ambitious goal we examined the interactions of CyaA with wild-type CaM and four CaM mutants in which most methionine residues were replaced by leucine residues and studied the effects of the CaM antagonists calmidazolium, trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). CyaA/CaM interaction was monitored by CaM-dependent fluorescence resonance energy transfer (FRET) between tryptophan residues in CyaA and 2′-(N-methylanthraniloyl)-3′-deoxy-adenosine 5′-triphosphate and catalytic activity. Comparison of the concentration/response curves of CaM and CaM mutants for FRET and catalysis revealed differences, suggesting a two-step activation mechanism of CyaA by CaM. Even in the absence of CaM, calmidazolium inhibited catalysis, and it did so according to a biphasic function. Trifluoperazine and W-7 did not inhibit FRET or catalysis. In contrast to CyaA, some CaM mutants were more efficacious than CaM at activating membranous AC isoform 1. The slope of CyaA activation by CaM was much steeper than of AC1 activation. Collectively, the two-step activation mechanism of CyaA by CaM offers opportunities for pharmacological intervention. The failure of classic CaM inhibitors to interfere with CyaA/CaM interactions and the different interactions of CaM mutants with CyaA and AC1 point to unique CyaA/CaM interactions.

Quantitative Mass Spectrometry for Bacterial Protein Toxins — A Sensitive, Specific, High-Throughput Tool for Detection and Diagnosis

Matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometry (MS) is a valuable high-throughput tool for peptide analysis. Liquid chromatography electrospray ionization (LC-ESI) tandem-MS provides sensitive and specific quantification of small molecules and peptides. The high analytic power of MS coupled with high-specificity substrates is ideally suited for detection and quantification of bacterial enzymatic activities. As specific examples of the MS applications in disease diagnosis and select agent detection, we describe recent advances in the analyses of two high profile protein toxin groups, the Bacillus anthracis toxins and the Clostridium botulinum neurotoxins. The two binary toxins produced by B. anthracis consist of protective antigen (PA) which combines with lethal factor (LF) and edema factor (EF), forming lethal toxin and edema toxin respectively. LF is a zinc-dependent endoprotease which hydrolyzes specific proteins involved in inflammation and immunity. EF is an adenylyl cyclase which converts ATP to cyclic-AMP. Toxin-specific enzyme activity for a strategically designed substrate, amplifies reaction products which are detected by MALDI-TOF-MS and LC-ESI-MS/MS. Pre-concentration/purification with toxin specific monoclonal antibodies provides additional specificity. These combined technologies have achieved high specificity, ultrasensitive detection and quantification of the anthrax toxins. We also describe potential applications to diseases of high public health impact, including Clostridium difficile glucosylating toxins and the Bordetella pertussis adenylyl cyclase.

Structural basis for the interaction of Bordetella pertussis adenylyl cyclase toxin with calmodulin

CyaA is crucial for colonization by Bordetella pertussis, the etiologic agent of whooping cough. Here we report crystal structures of the adenylyl cyclase domain (ACD) of CyaA with the C-terminal domain of calmodulin. Four discrete regions of CyaA bind calcium-loaded calmodulin with a large buried contact surface. Of those, a tryptophan residue (W242) at an alpha-helix of CyaA makes extensive contacts with the calcium-induced, hydrophobic pocket of calmodulin. Mutagenic analyses show that all four regions of CyaA contribute to calmodulin binding and the calmodulin-induced conformational change of CyaA is crucial for catalytic activation. A crystal structure of CyaA-calmodulin with adefovir diphosphate, the metabolite of an approved antiviral drug, reveals the location of catalytic site of CyaA and how adefovir diphosphate tightly binds CyaA. The ACD of CyaA shares a similar structure and mechanism of activation with anthrax edema factor (EF). However, the interactions of CyaA with calmodulin completely diverge from those of EF. This provides molecular details of how two structurally homologous bacterial toxins evolved divergently to bind calmodulin, an evolutionarily conserved calcium sensor.

The cox-2-specific inhibitor celecoxib inhibits adenylyl cyclase.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are well-known causes of acute renal insufficiency and gastropathy in patients with chronic inflammatory diseases. This action is presumed to result from nonselective inhibition of both constitutive and inducible forms of prostaglandin H synthases, also known as the cyclooxygenase enzymes (i.e., COX-1 amd COX-2). Celecoxib (Celebrex) is a COX-2 enzyme inhibitor and has emerged as a preferred therapeutic agent for the treatment of rheumatoid arthritis as compared to other NSAIDs. Celecoxib has recently been the subject of criticism for its side effects, mainly arterial thrombosis and renal hemorrhage, although it is considered a superior drug in protecting the gastrointestinal tract. In the present study, we report that celecoxib not only inhibited COX-2, but also exhibited the property of inhibiting adenylyl cyclase, an important enzyme forming the intracellular second messenger 3',5'-adenosine monophosphate (cAMP) from adenosine triphosphate (ATP). Celecoxib also inhibited cholera toxin-stimulated cAMP formation, which indicated its ability to permeate cell membranes in order to reach intracellular adenylyl cyclase. It inhibited in vitro adenylyl cyclase activity in both human colonic epithelial cells and purified adenylyl cyclase from Bordetella pertussis. The IC50 of celecoxib for B. pertussis adenylyl cyclase was calculated to be 0.375 mM. Lineweaver-Burk analysis showed that the type of enzyme inhibition was competitive. The apparent Km and Vm of adenylyl cyclase was calculated as 25.0 nM and 7.14 nmol/min/mg, respectively. Celecoxib changed the Km value to 66.6 nM without affecting the Vmax. The current study suggests that apart from inflammation, celecoxib therapy could be further extended to diseases involving cAMP upregulation either by endogenous reactions or exogenous agents. These new data showing inhibition of adenylyl cyclase should be considered in light of the drug's pathological effects or in patients specifically excluded from treatment (e.g., asthmatics).

Identification of a domain in Bordetella pertussis adenylyl cyclase important for subunit interactions and cell invasion activity

Bordetella pertussis produces a calmodulin-stimulated adenylyl cyclase that invades animal cells and raises intracellular cAMP levels. The enzyme does not enter animal cells by receptormediated endocytosis, but the mechanism for invasion of animal cells has not been defined. We have proposed that the 177 kDa adenylyl cyclase is proteolyzed to a 45 kDa catalytic subunit and one or more polypeptides (invasive factor) that facilitate entry of the catalytic subunit into animal cells. in this study, we report the identification of a sequence of amino acids within the adenylyl cyclase catalytic subunit that is important for entry of the enzyme into eukaryotic cells. A synthetic peptide corresponding to amino acids 313-339 within the catalytic subunit was shown to inhibit invasion of neuroblastoma cells by the adenylyl cyclase. In addition, this peptide inhibited the association of the catalytic subunit with invasive factor. We propose that this domain is a site for interaction between the catalytic subunit and invasive factor.

Virulence of a Bordetella pertussis strain expressing a mutant adenylyl cyclase with decreased calmodulin affinity

Bordetella pertussis, the pathogen responsible for whooping cough, produces a toxic calmodulin-sensitive adenylyl cyclase which enters animal cells and increases intracellular cAMP. A point mutant of B. pertussis with abolished adenylyl cyclase catalytic activity was over 1000-fold less pathogenic to newborn mice than wild-type bacteria, demonstrating the importance of the adenylyl cyclase for B. pertussis virulence (Gross et al. ). The B. pertussis adenylyl cyclase is highly sensitive to calmodulin with an apparent Kd for calmodulin of approximately 1 nM. The importance of this high-affinity calmodulin binding for virulence in vivo was examined by the creation of a B. pertussis point mutant (Trp-242 to Glu-242) with 200-fold lower calmodulin affinity than the native enzyme. This mutant B. pertussis strain retained its virulence in a newborn mouse model of pertussis, but the time course for establishment of a lethal infection in vivo was significantly delayed for the mutant strain. These data illustrate that high-affinity calmodulin binding is not obligatory for the activity of this toxin but is important for the rate for establishment of a lethal infection.

High-affinity calmodulin binding is required for the rapid entry of Bordetella pertussis adenylyl cyclase into neuroblastoma cells.

Bordetella pertussis produces a calmodulin-stimulated adenylyl cyclase that invades animal cells and raises intracellular cAMP levels [Confer, D. L., & Eaton, J. W. (1982) Science 217, 948-950; Shattuck, R. L., & Storm, D. R. (1985) Biochemistry 24, 6323-6328]. The mechanism for invasion of animal cells by this enzyme has not been defined, but there is considerable evidence that it does not enter by receptor-mediated endocytosis [Gordon, V. M., Leppla, S. H., & Hewlett, E. L. (1988) Infect. Immun. 56, 1066-1069; Donovan, M. G., & Storm, D. R. (1990) J. Cell. Physiol. 145, 444-449]. In this study, the importance of high-affinity calmodulin (CaM) binding for entry of the enzyme into neuroblastoma cells was evaluated using a mutant enzyme that has significantly lower affinity for calmodulin than the wild-type enzyme. Oligonucleotide-directed site-specific mutagenesis was used to create a point mutant at a critical tryptophan residue (Trp-242) within the proposed CaM binding domain of the B. pertussis adenylyl cyclase. Substitution of Trp-242 with Glu lowered the apparent affinity of the enzyme for calmodulin by 250-fold; however, the maximal enzyme activity in the presence of saturating calmodulin was equivalent to the wild-type enzyme. The Glu-242 mutant adenylyl cyclase was returned to B. pertussis by homologous recombination, and the enzyme produced by this strain was examined for invasion of neuroblastoma cells. Although the mutant enzyme stimulated the production of intracellular cAMP in neuroblastoma cells, the rate of cAMP accumulation was at least 10-fold lower than that caused by the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

A monoclonal antibody directed against the catalytic site of Bacillus anthracis adenylyl cyclase identifies a novel mammalian brain catalytic subunit.

A brain adenylyl cyclase was shown to contain an epitope closely related to that specified by a conserved sequence containing a nucleotide-binding consensus sequence GXXXXGKS and located in the catalytic sites of bacterial, calmodulin-dependent adenylyl cyclases [Goyard, S., Orlando, C., Sabatier, J.-M., Labruyere, E., d'Alayer, J., Fontan, G., van Rietschoten, J., Mock, M., Danchin, A., Ullmann, A., & Monneron, A. (1989) Biochemistry 28, 1964-1967]. A monoclonal antibody, mab 164, produced against a peptide corresponding to this conserved sequence specifically inhibited the Bordetella pertussis adenylyl cyclase. It also specifically inhibited rat and rabbit brain synaptosomal adenylyl cyclases. The extent of inhibition depended upon the type of enzyme purification, reaching 90% for the calmodulin-sensitive species of enzyme and 20-35% for the forskolin-agarose-retained species. The extent of inhibition in a given fraction also depended upon the effector present. mab 164 reacted on Western blots of forskolin-agarose-retained fractions with a 175-kDa component and did not recognize the Gs alpha stimulatory subunit. Consequently, the 175-kDa protein was considered as a good candidate for an adenylyl cyclase catalyst. The adenylyl cyclase activity contained in forskolin-agarose-retained fractions was further purified on calmodulin-Sepharose. On Western blots of such fractions, mab 164 reacted with a 140-kDa protein, a component that appeared to derive from the 175-kDa protein enriched in the previous step. The kcat of this 140-kDa presumptive adenylyl cyclase was estimated to be of the order of 600 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunological relatedness between Bordetella pertussis and rat brain adenylyl cyclases

A prokaryotic adenylyl cyclase, secreted by Bordetella pertussis, shares a common functional property with eukaryotic adenylyl cyclases, i.e., regulation by the eukaryotic protein calmodulin. Making use of polyclonal antibodies raised against the bacterial adenylyl cyclase and the rat brain adenylyl cyclase catalytic component, respectively, we showed an immunological cross-reactivity between the two enzymes. Furthermore, B. pertussis adenylyl cyclase was inhibited and immunoprecipitated by the homologous and one of the heterologous immune sera. These results suggest an evolutionary relationship between the B. pertussis enzyme and its eukaryotic counterpart.

Gentamicin nucleotidyltransferase. Stereochemical inversion at phosphorus in enzymatic 2'-deoxyadenylyl transfer to tobramycin.

Gentamicin nucleotidyltransferase-catalyzed reaction of (Sp)-[alpha-17O]dATP with tobramycin produced 2"-(2'-deoxyadenosine 5'-[17O]phosphoryl)tobramycin. The configuration at phosphorus in this product was shown to be Rp by chemical degradation to chiral [17O, 18O]dAMP using a stereochemically defined procedure, and determination of the configuration at phosphorus in this product. Periodate-base treatment of 2"-(2'-deoxyadenosine 5'-[17O]phosphoryl)tobramycin followed by NaBH4 reduction produced (2-glyceryl)-[17O]dAMP, which upon snake venom phosphodiesterase-catalyzed hydrolysis in H(2)18O produced [17O,18O] dAMP. The configuration at phosphorus in this product was shown to be S by enzymatic phosphorylation to [17O,18O]dATP, adenylylcyclase (Bordetella pertussis)-catalyzed cyclization to 3',5'-cyclic [17O,18O]dAMP, and 31P NMR analysis of the ethyl esters. Since snake venom phosphodiesterase-catalyzed hydrolyses proceed with retention of configuration at phosphorus, (Sp)-[17O,18O]dAMP must have been produced from (Rp)-(2-glyceryl)-[17O]dAMP; and since the chemical degradation to the latter compound did not involve cleavage of any bonds to phosphorus, the initial enzymatic product must have been (Rp)-2"-(2'-deoxyadenosine 5'-[17O]phosphoryl)tobramycin. Therefore, nucleotidyl transfer catalyzed by gentamicin nucleotidyl-transferase proceeds with inversion of configuration at phosphorus, and the reaction mechanism involves an uneven number of phosphotransfer steps. Inasmuch as this is an uncomplicated two-substrate group transfer reaction, the mechanism probably involves direct nucleotidyl transfer from the nucleoside triphosphate to the aminoglycoside. The B. pertussis adenylylcyclase reaction was shown to proceed with inversion at phosphorus, as has been established for other adenylylcyclases.