Bacterial Nucleotidyl Cyclases Activated by Calmodulin or Actin in Host Cells: Enzyme Specificities and Cytotoxicity Mechanisms Identified to Date

Abstract

Many pathogens manipulate host cell cAMP signaling pathways to promote their survival and proliferation. Bacterial Exoenzyme Y (ExoY) toxins belong to a family of invasive, structurally-related bacterial nucleotidyl cyclases (NC). Inactive in bacteria, they use proteins that are uniquely and abundantly present in eukaryotic cells to become potent, unregulated NC enzymes in host cells. Other well-known members of the family include Bacillus anthracis Edema Factor (EF) and Bordetella pertussis CyaA. Once bound to their eukaryotic protein cofactor, they can catalyze supra-physiological levels of various cyclic nucleotide monophosphates in infected cells. Originally identified in Pseudomonas aeruginosa, ExoY-related NC toxins appear now to be more widely distributed among various γ- and β-proteobacteria. ExoY-like toxins represent atypical, poorly characterized members within the NC toxin family. While the NC catalytic domains of EF and CyaA toxins use both calmodulin as cofactor, their counterparts in ExoY-like members from pathogens of the genus Pseudomonas or Vibrio use actin as a potent cofactor, in either its monomeric or polymerized form. This is an original subversion of actin for cytoskeleton-targeting toxins. Here, we review recent advances on the different members of the NC toxin family to highlight their common and distinct functional characteristics at the molecular, cytotoxic and enzymatic levels, and important aspects that need further characterizations.

Functional and structural insights into the multi-step activation and catalytic mechanism of bacterial ExoY nucleotidyl cyclase toxins bound to actin-profilin

“…Bacterial NC toxins belong to the class II adenylate cyclases (ACs). Their catalytic domain may represent a potential drug target against bacterial infections (1, 2) because it is not structurally related to widespread class III mammalian adenylyl or guanylyl cyclases (GCs) that use a dimeric catalytic core (18–23). The monomeric catalytic domain of NC toxins can be divided structurally into subdomains C A and C B with the catalytic site located in a groove at their interface (20).…”

A Robust and Sensitive Spectrophotometric Assay for the Enzymatic Activity of Bacterial Adenylate Cyclase Toxins

“…Many pathogens have evolved sophisticated strategies to disrupt cNMP metabolism in the hosts they attempt to colonize [ 2 ]. In particular, several bacterial pathogens produce toxins that are endowed with endogenous nucleotidyl cyclase activity [ 3 ]. These toxins are able to invade eukaryotic cells where they are stimulated by endogenous co-factors to produce large amounts of cNMP, thus disrupting cell signaling and altering cell physiology.…”

“…Both CyaA and EF are potently activated by the eukaryotic protein, calmodulin (CaM), and have very high catalytic efficiency (with kcat > 1000 s −1 ). Their catalytic moieties share remarkable structural similarity, while these two toxins differ markedly in their sequences, their secretion mechanisms, and their modes of invasion of eukaryotic target cells [ 3 ]. A distinct sub-family of virulent factors with nucleotidyl cyclase activity [ 10 , 11 ] has been recently characterized as being activated by eukaryotic actin [ 12 ].…”

“…A distinct sub-family of virulent factors with nucleotidyl cyclase activity [ 10 , 11 ] has been recently characterized as being activated by eukaryotic actin [ 12 ]. It includes the ExoY toxin that is a type-3 virulent effector produced by P. aeruginosa and various ExoY-like modules present in different MARTX (Multifunctional-Autoprocessing Repeats-in-Toxin) toxins produced by certain Vibrio species as well as by other Gram-negative bacteria [ 3 , 13 ].…”

“…The characterization of these virulent factors frequently requires the determination of their enzymatic activities, which is the conversion of adenosine triphosphate (ATP) into cyclic AMP (cAMP) and pyrophosphate (PPi). Indeed, numerous studies have been devoted to the identification of critical residues in the catalytic reaction and/or the binding of the toxins to their eukaryotic co-factors [ 2 , 3 ]. Simple and rapid assays for determining enzymatic activities of recombinant enzymes harboring specific mutations would greatly facilitate such investigations.…”