Adenylate Kinase Release as a High-Throughput-Screening-Compatible Reporter of Bacterial Lysis for Identification of Antibacterial Agents

Abstract

bAdenylate kinase (AK) is a ubiquitous intracellular enzyme that is released into the extracellular space upon cell lysis. We have shown that AK release serves as a useful reporter of bactericidal agent activity and can be exploited for antimicrobial screening purposes. The AK assay exhibits improved sensitivity over that of growth-based assays and can detect agents that are active against bacteria in clinically relevant growth states that are difficult to screen using conventional approaches, such as small colony variants (SCV) and bacteria within established biofilms. The usefulness of the AK assay was validated by screening a library of off-patent drugs for agents that exhibit antimicrobial properties toward a variety of bacterial species, including Escherichia coli and all members of the "ESKAPE" pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). The assay detected antibiotics within the library that were expected to be active against the organism screened. Moreover, 38 drugs with no previously reported antibacterial activity elicited AK release. Four of these were acquired, and all were verified to exhibit antimicrobial activity by standard susceptibility testing. Two of these molecules were further characterized. The antihistamine, terfenadine, was active against S. aureus planktonic, SCV population, and biofilm-associated cells. Tamoxifen, an estrogen receptor antagonist, was active toward E. faecium in vitro and also reduced E. faecium pathogenesis in a Galleria mellonella infection model. Our data demonstrate that the AK assay provides an attractive screening approach for identifying new antimicrobial agents. Further, terfenadine and tamoxifen may represent novel antimicrobial drug development scaffolds.

Synthetic Effect between Envelope Stress and Lack of Outer Membrane Vesicle Production in Escherichia coli

“…Adenylate kinase is a cytoplasmic enzyme that is released from lysed cells and can be detected with a luminescence assay (26). Following the protocol of Jacobs et al (26), we used heat-killed cells as our positive lysis control and to ensure that the content in our samples was not above the maximum detection limit of the assay (Fig.…”

Exo‐ and surface proteomes of the probiotic bacterium Lactobacillus acidophilus NCFM

“…Several exo‐ and surface proteome (Table ) proteins have been reported to be moonlighting proteins for other bacteria that may bind to ECM components (fibrinogen, collagen, plasminogen, mucin) and host epithelial cells . GAPDH is a universal moonlighting protein that has different roles in bacteria and eukaryotic cells .…”

“…Several exo-and surface proteome ( Table 1) proteins have been reported to be moonlighting proteins for other bacteria that may bind to ECM components (fibrinogen, collagen, plasminogen, mucin) and host epithelial cells [11,24,25]. GAPDH is a universal moonlighting protein that has dif- Signal Peptide, proteins that contain a signal peptide; Membrane, proteins predicted to be membrane proteins; Nonclassically secreted, proteins predicted to be secreted by nonclassical secretion systems [13,14] (B) Protein categories of exo-and surface proteomes of Lactobacillus acidophilus NCFM.…”

Adenylate kinase fromStreptococcus pneumoniaeis essential for growth through its catalytic activity

“…EC is the amount of energy readily accessible for cellular metabolism [4,5] that may affect other fundamental pathogenesis such as bacterial growth, virulence factors, and secretion pathways [6,7]. Adenylate kinase (AdK; ATP:AMP phosphotransferase; EC 2.7.4.3) catalyzes conversion between adenylate nucleotides [8]: Mg. ATP + AMP M Mg. ADP + ADP.…”