Research Article: Revealing how an adenylate cyclase toxin uses bait and switch tactics in its activation

Date Published: February 27, 2018

Publisher: Public Library of Science

Author(s): Natosha L. Finley

Abstract: Dissecting how bacterial pathogens escape immune destruction and cause respiratory infections in humans is a work in progress. One tactic employed by microbes is to use bacterial adenylate cyclase toxins (ACTs) to disarm immune cells and disrupt cellular signaling in host cells, which facilitates the infection process. Several clinically significant pathogens, such as Bacillus anthracis and Bordetella pertussis, have ACTs that are stimulated by an activator protein in human cells. Research has shown that these bacterial ACTs have evolved distinct ways of controlling their activities, but our understanding of how the B. pertussis ACT does this is limited. In a recent study, O’Brien and colleagues provide new and exciting evidence demonstrating that the regulation of B. pertussis ACT involves conformational switching between flexible and rigid states, which is triggered upon binding the host activator protein. This study increases our knowledge of how bacterial ACTs are unique enzymes, representing a potentially novel class of drug targets that may open new pathways to combat reemerging infectious diseases.

Partial Text: Whooping cough (also known as pertussis) is an infectious respiratory disease found in adults and adolescents, but which is most harmful and even fatal to infants and young children. The respiratory pathogen B. pertussis uses a number of damaging enzymes, including an adenylate cyclase toxin (ACT) known as CyaA, to facilitate causing whooping cough in humans. Historically, whooping cough infections killed infants around the globe, but modern medicine reduces the impact of this deadly disease using a combination of antibiotics and immunization. At present, pertussis infections are increasing, despite being a vaccine-preventable disease and the emergence of antibiotic resistant strains is a global public health concern [1].

The bacterial ACTs convert adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP), allowing for its high intracellular accumulation to pathogenic levels. There are different bacterial ACTs—edema factor (EF) released by Bacillus anthracis, CyaA made by B. pertussis, and ExoY, which is produced by Pseudomonas aeruginosa. Both EF and CyaA are calmodulin (CaM)-dependent, while ExoY is stimulated by actin, the significance of which is reviewed elsewhere [2]. EF entry into the host cell requires endocytosis and translocation through a pore formed by an accessory protein known as protective antigen. However, CyaA is composed of a C-terminal pore-forming hemolysin domain that transports the N-terminal adenylate cyclase domain of CyaA (CyaA-ACD) into the host cell for CaM-dependent activation. The early establishment of B. pertussis infections is facilitated by CyaA, which disrupts the action of immune cells, allowing the microbe to escape destruction [3,4]. CyaA-deficient B. pertussis strains are less virulent [5], and antibody-mediated neutralization of CyaA protects against infection [6]. In order to facilitate bacterial colonization during infection, ACTs must compete with other effector proteins such as host cell adenylate cyclases (ACs) for CaM association. A persistent question in the study of B. pertussis pathogenesis has been: how does CyaA selectively target host cell CaM?

In this primer, we focus on how CaM-dependent bacterial ACTs work by highlighting the unique mechanism by which CyaA activity is stimulated by the host activator CaM. This ubiquitous eukaryotic protein is a globular metal-sensing protein, which is important in a variety biological functions such as cellular signaling, metabolism, memory formation, and channel activation in host cells [7]. CaM is composed on N-terminal and C-terminal domains that bind intracellular Ca2+ and Mg2+, with one of its primary physiological roles being Ca2+-regulated enzyme stimulation. One normal physiological function of CaM is to activate host ACs through moderate affinity interactions, leading to the increased production of intracellular cAMP, a critical secondary messenger molecule in the cell. Interestingly, bacterial ACTs such as CyaA remain largely inactivated until they gain access to the host cell, where they wreak havoc by disrupting cAMP levels through CaM-dependent activation [8]. Much speculation exists as to how bacterial ACTs exert their selective effects during host infection. There is compelling evidence that CyaA exploits its high-affinity interaction with CaM to commandeer this metal-sensing protein, and the physiological consequences are likely to be disruption of cellular responses to Ca2+ fluctuations [9]. To this end, other urgent questions emerge, such as how does the activation of CyaA-ACD differ from other CaM-dependent bacterial ACTs and what is the role of metal binding in these processes?

There is little conservation at the amino acid level between bacterial ACTs; therefore, EF and CyaA are not structural homologs. However, they do share functional similarities. A detailed picture of EF structural transitions exists due in part to the availability of experimentally determined structures and biophysical characterization of CaM/EF complexes [10,11]. The activation of EF is physically blocked by the presence of the helical domain (Fig 1A and 1B). Upon CaM interaction and in the presence of high Ca2+concentration, the helical domain is rotated, exposing the ATP-binding pocket, which ultimately results in EF stimulation.

Unfortunately, the absence of high-resolution structures of CyaA (both free and bound to intact CaM) limits the understanding of how CaM-binding induces activation. The conformation of free CyaA is known to be heterogeneous, and structural studies performed using isolated CyaA-ACD do not mitigate this intrinsic property of the toxin, as it too remains recalcitrant to structure determination. Aggregation of CyaA in the absence of CaM is modulated by sample conditions and posttranslational modifications allowing it to be folded into monodisperse, functional conformers [13,14]. In the presence of CaM, isolated CyaA-ACD forms stable complexes that are monodisperse and functionally active [15,16], which is advantageous in the structural study of this system.

While there are examples of CaM-binding inducing structural order in other enzymes, such as calcineurin [25], the findings reported in PLOS Biology point toward the likelihood that B. pertussis ACT exhibits a previously uncharacterized mechanism for CaM-induced enzymatic stimulation that functions on a global scale. As such, the work of O’Brien and colleagues provides a springboard for continued research and presents a number of thought-provoking questions to explore. For example, what is the role of induced structural changes that occur in regions outside of the primary CaM-binding determinant? Other studies have shown that mutation in these regions of CyaA-ACD impacts conformation and activation, suggesting sites of importance in CaM-dependent stimulation of this enzyme [16,18]. O’Brien reports that the solvent accessibility of CaM is impacted by both intact CyaA and CyaA-ACD with some minor differences. It is tempting to speculate on the significance of any differences in induced conformational changes in CaM outside of the primary C-terminal binding sites. CyaA-ACD association does not mediate large conformational changes in N-terminal CaM, which is in good agreement with other studies. Perhaps we can take another step toward elucidating the role of N-terminal CaM in CyaA-ACD activation by exploring the range of detection that the solvent accessibility experiments have in capturing transient interactions. Future experiments should test the “crowding” hypothesis by continuing to explore the contribution of N-terminal CaM in CyaA-ACD stimulation using distance measurements. Furthermore, elucidating the novel mechanism by which intact CaM activates CyaA might permit the design of highly specific compounds targeting this interaction. The possibility of designer drugs targeting only CyaA-ACD that do not disrupt mammalian AC interaction with CaM offers promise in the development of antitoxin therapies that may prevent or treat pertussis in the age of evolving antibiotic resistance.