Biology subjects

Jeannot, K.

Publications and source records attributed to Jeannot, K..

5 recordsLinked to original sources

bioRxiv2026

Total Biosynthesis of Pseudomonas aeruginosa-Derived Azabicyclocarbamates Identifies Distinct Dehydrating Condensation Family Proteins

Bacterial azabicyclocarbamates and related pyrrolizidine alkaloids play important roles in microbial interactions, and are scaffolds of therapeutic potential. Their biosynthesis involves a bimodular non-ribosomal peptide synthetase (NRPS), as well as a Baeyer-Villiger monooxygenase and tailoring enzymes, the latter contributing to the structural diversification of these compounds. Azetidomonamide A, a core metabolite produced by the major human opportunistic pathogen Pseudomonas aeruginosa, is a rare 4,7-bicyclocarbamate involved in modulating bacterial virulence that belongs to a unique family of natural products targeting ClpP proteases. In this study, we elucidated the full set of reactions leading to the 7-membered cyclocarbamate warhead, and reconstituted in vitro the biosynthesis of azetidomonamide A. Notably, this approach allowed for detailed characterization of a condensation (C) domain-catalyzed online dehydration via chemical capture of NRPS-tethered intermediates. Furthermore, we identified the dehydratase AzeD as the founding member of a distinct group of standalone proteins of the C domain family. Via combined structural, docking and biochemical analyses, we provided evidence that AzeDs catalytic mechanism is distinct from that of dehydrating C domains, further expanding the known chemistry of these key biosynthetic enzymes.

biochemistry↗

bioRxiv2026

Mechanisms involved in cefiderocol resistance in French Pseudomonas aeruginosa clinical strains

Cefiderocol exhibits excellent in vitro activity against Pseudomonas aeruginosa; however, resistance can emerge. We investigated the molecular mechanisms underlying cefiderocol resistance (MIC >2 mg/L) in 103 clinical strains collected from 61 hospitals (2021-2024). MICs ranged from 4 to >128 mg/L, with 39.8% of strains showing MICs >8 mg/L. Although 37.8% were classified as difficult-to-treat resistant (DTR), acquired {beta}-lactamases were detected in 72.8% of strains, including carbapenemases (39.8%), mainly NDM-1 (29.1%), and Extended Spectrum {beta}-Lactamases (ESBLs) (38.8%). Cloning of 11 {beta}-lactamases into pUCP24, including the acquired cephalosporinase PAC-1 and ESBLs (VEB-1, and VEB-9), resulted in marked increases in cefiderocol MICs (up to 128-fold). Introduction of 6 mutations in the PDC enzyme into a PAO1{Delta}blaPDC-1 background increased MICs up to 4 mg/L and conferred cross-resistance to ceftolozane/tazobactam, notably F121L, G157D, T70I, and E219K. Alterations in siderophore transporters or regulators were identified in 38.8% of strains, most frequently a PirR frameshift (R132fs), consistent with PirR inactivation, which was confirmed in the PAO1 strain to contribute to cefiderocol resistance. Overall, cefiderocol resistance in clinical strains is multifactorial, mainly involving acquired {beta}-lactamases (ESBLs, carbapenemases) and impaired siderophore uptake (PiuA/PiuD, PirA, PiuC), leading to high-level resistance (>8 mg/L). The polyclonal distribution and diversity of mechanisms highlight the need for routine susceptibility testing and surveillance. Detection of NDM producers is critical, as cefiderocol should be used with caution in this context.

microbiology↗

bioRxiv2024

Serotype switching in Pseudomonas aeruginosa ST111 enhances adhesion and virulence

Evolution of the highly successful and multidrug resistant clone ST111 in Pseudomonas aeruginosa involves serotype switching from O-antigen O4 to O12. How expression of a different O-antigen serotype alters pathogen physiology to enable global dissemination of this high-risk clone-type is not understood. Here, we engineered isogenic laboratory and clinical P. aeruginosa strains that express the different O-antigen gene clusters to assess the correlation of structural differences of O4 and O12 O-antigens to pathogen-relevant phenotypic traits. We show that serotype O12 is associated with enhanced adhesion, type IV pili dependent twitching motility, and tolerance to host defense molecules and serum. Moreover, we find that serotype O4 is less virulent compared to O12 in an acute murine pneumonia infection in terms of both colonization and survival rate. Finally, we find that these O-antigen effects may be explained by specific biophysical properties of the serotype repeat unit found in O4 and O12, and by differences in membrane stability between O4 and O12 expressing cells. The results demonstrate that differences in O-antigen sugar composition can directly affect P. aeruginosa pathogenicity traits, and provide a better understanding of the potential selective advantages that underlie serotype switching and emergence of serotype O12 ST111.

microbiology↗

bioRxiv2023

Mutations in genes lpxL1, bamA and pmrB impair the susceptibility of cystic fibrosis strains of Pseudomonas aeruginosa to murepavadin

Murepavadin is a peptidomimetic exhibiting specific inhibitory activity against Pseudomonas species. In the present study, its in vitro activity was assessed on 230 cystic fibrosis (CF) strains of P. aeruginosa isolated from twelve French hospitals, in comparison with twelve other antipseudomonal antibiotics. Although murepavadin is still in pre-clinical stage of development, 9.1% (n=21) of the strains displayed a resistance superior to 4 mg/L, a level at least 128-fold higher than the modal MIC value of the whole collection ([≤] 0.06 mg/L). Whole-genome sequencing of these 21 strains along with more susceptible isogenic counterparts coexisting in the same patients revealed diverse mutations in genes involved in the synthesis (lpxL1 and lpxL2) or transport of lipopolysaccharides (bamA, lptD, and msbA), or encoding histidine kinases of two-component systems (pmrB and cbrA). Allelic replacement experiments with wild-type reference strain PAO1 confirmed that alteration of genes lpxL1, bamA and/or pmrB can increase murepavadin resistance from 8- to 32-fold. Furthermore, we found that specific amino-acid substitutions in histidine kinase PmrB (G188D, Q105P, and D45E) reduce the susceptibility of P. aeruginosa to murepavadin, colistin and tobramycin, three antibiotics used or intended to be used (murepavadin) in aerosols to treat colonized CF patients. Whether colistin or tobramycin may select mutants resistant to murepavadin or the opposite needs to be addressed by clinical studies.

microbiology↗

bioRxiv2023

Pseudomonas aeruginosa MipA-MipB envelope proteins act as new sensors of polymyxin

Due to the rising incidence of antibiotic resistant infections, the last-line antibiotics polymyxins have resurged in the clinics in parallel with new bacterial strategies of escape. The Gram-negative opportunistic pathogen Pseudomonas aeruginosa develops resistance to colistin/polymyxin by distinct molecular mechanisms, mostly through modification of the lipid A component of the LPS by proteins encoded within the arnBCDATEF-ugd (arn) operon. In this work, we characterized a polymyxin-induced operon, named mipBA, notably present in P. aeruginosa strains devoid of the arn operon. We showed that mipBA is activated by ParR/ParS two-component regulatory system in response to polymyxin. MipA and MipB localize to bacterial outer membrane and form a complex in vitro. Structural modeling revealed that the lipoprotein MipB adopts a {beta}-lactamase fold with two additional C-terminal domains,while MipA folds as an outer-membrane {beta}-barrel, harboring an internal negatively charged channel, able to host a polymyxin molecule. Nano differential scanning fluorimetry (DSF) showed that polymyxin stabilized MipA protein in vitro. Mass spectrometry-based quantitative proteomics on whole bacterial membranes demonstrated that the {Delta}mipBA mutant synthesized less MexXY-OprA proteins in response to polymyxin compared to the wild-type strain, as a consequence of impaired transcriptional activation of the mex operon. We propose MipA/MipB to act as membrane (co)sensors working in concert to activate ParS histidine kinase and help the bacterium to cope with polymyxin-mediated envelope stress through synthesis of the efflux pomp, MexXY-OprA.

microbiology↗