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Deciphering the Che2 chemosensory pathway and the roles of individual Che2 proteins from Pseudomonas aeruginosa.

ABSTRACT: Pseudomonas aeruginosa is an opportunistic pathogen that senses and responds to its environment via four chemosensory systems. Oxygen activates the Che2 chemosensory system by binding to the PAS-heme domain of the Aer2 receptor. Ostensibly, the output of Che2 occurs via its response regulator CheY2, but controversy persists over CheY2's exact role. In this study, we show that CheY2 does not interact with the flagellar motor and that the Che2 system does not transfer phosphoryl groups to the chemotaxis (Che) system. We show that CheY2 instead provides feedback control of Aer2 adaptation. In the presence of O₂ , Aer2 signaling increases the autophosphorylation of the histidine kinase CheA2, followed by CheY2-mediated dephosphorylation. CheY2 does not stably retain phosphate and may not signal the output of the Che2 system. Rather, CheY2 activity enhances the direct interaction of CheY2 with the adaptation protein CheD (a role often facilitated by CheC, which P. aeruginosa lacks). In the absence of O₂ , Aer2 does not signal, and CheY2/CheD interactions attenuate. This frees CheD to augment CheR2-mediated methylation of Aer2, which enhances Aer2 signaling. CheD does not interact with CheR2, but most likely interacts with Aer2 via conserved CheD-binding motifs to make Aer2 a better methylation substrate.

SUBMITTER: Orillard E

PROVIDER: S-EPMC7897226 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Pseudomonas aeruginosa produces a class C ?-lactamase (e.g., PDC-3) that robustly hydrolyzes early generation cephalosporins often at the diffusion limit; therefore, bacteria possessing these ?-lactamases are resistant to many ?-lactam antibiotics. In response to this significant clinical threat, ceftolozane, a 3' aminopyrazolium cephalosporin, was developed. Combined with tazobactam, ceftolozane promised to be effective against multidrug-resistant P. aeruginosa Alarmingly, ?-loop variants of the PDC ?-lactamase (V213A, G216R, E221K, E221G, and Y223H) were identified in ceftolozane/tazobactam-resistant P. aeruginosa clinical isolates. Herein, we demonstrate that the Escherichia coli strain expressing the E221K variant of PDC-3 had the highest minimum inhibitory concentrations (MICs) against a panel of ?-lactam antibiotics, including ceftolozane and ceftazidime, a cephalosporin that differs in structure largely in the R2 side chain. The k cat values of the E221K variant for both substrates were equivalent, whereas the Km for ceftolozane (341?±?64?µM) was higher than that for ceftazidime (174?±?20?µM). Timed mass spectrometry, thermal stability, and equilibrium unfolding studies revealed key mechanistic insights. Enhanced sampling molecular dynamics simulations identified conformational changes in the E221K variant ?-loop, where a hidden pocket adjacent to the catalytic site opens and stabilizes ceftolozane for efficient hydrolysis. Encouragingly, the diazabicyclooctane ?-lactamase inhibitor avibactam restored susceptibility to ceftolozane and ceftazidime in cells producing the E221K variant. In addition, a boronic acid transition state inhibitor, LP-06, lowered the ceftolozane and ceftazidime MICs by 8-fold for the E221K-expressing strain. Understanding these structural changes in evolutionarily selected variants is critical toward designing effective ?-lactam/?-lactamase inhibitor therapies for P. aeruginosa infections.IMPORTANCE The presence of ?-lactamases (e.g., PDC-3) that have naturally evolved and acquired the ability to break down ?-lactam antibiotics (e.g., ceftazidime and ceftolozane) leads to highly resistant and potentially lethal Pseudomonas aeruginosa infections. We show that wild-type PDC-3 ?-lactamase forms an acyl enzyme complex with ceftazidime, but it cannot accommodate the structurally similar ceftolozane that has a longer R2 side chain with increased basicity. A single amino acid substitution from a glutamate to a lysine at position 221 in PDC-3 (E221K) causes the tyrosine residue at 223 to adopt a new position poised for efficient hydrolysis of both cephalosporins. The importance of the mechanism of action of the E221K variant, in particular, is underscored by its evolutionary recurrences in multiple bacterial species. Understanding the biochemical and molecular basis for resistance is key to designing effective therapies and developing new ?-lactam/?-lactamase inhibitor combinations.

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Deciphering the Che2 chemosensory pathway and the roles of individual Che2 proteins from Pseudomonas aeruginosa.

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