TY - JOUR

T1 - Uptake Mechanisms and Regulatory Responses to MECAM- and DOTAM-Based Artificial Siderophores and Their Antibiotic Conjugates in Pseudomonas aeruginosa

AU - Fritsch, Sarah

AU - Gasser, Véronique

AU - Peukert, Carsten

AU - Pinkert, Lukas

AU - Kuhn, Lauriane

AU - Perraud, Quentin

AU - Normant, Vincent

AU - Brönstrup, Mark

AU - Schalk, Isabelle J.

N1 - Funding Information: This work was partially funded by the Centre National de la Recherche Scientifique, a grant from the Joint Programming Initiative on Antimicrobial Resistance (JPI AMR, grant number: 01KI1825) and the Interdisciplinary Thematic Institute (ITI) InnoVec (Innovative Vectorization of Biomolecules, IdEx, ANR-10-IDEX-0002) and SFRI (ANR-20-SFRI-0012). The mass spectrometry instrumentation at the IBMC was funded by the University of Strasbourg, IdEx “Equipement mi-lourd” 2015. The equipment at the IPHC was partly funded by the French Proteomics Infrastructure (ProFI; ANR-10-INSB-08-03). Q.P. had a fellowship from the associations Vaincre la Mucoviscidose and Gregory Lemarchal. C.P. had a Kekulé stipend from the ‘Verband der chemischen Industrie,’ and L.P. was funded by the DFG project number BR 3572/4-1.

PY - 2022/6/10

Y1 - 2022/6/10

N2 - The development of new antibiotics against Gram-negative bacteria has to deal with the low permeability of the outer membrane. This obstacle can be overcome by utilizing siderophore-dependent iron uptake pathways as entrance routes for antibiotic uptake. Iron-chelating siderophores are actively imported by bacteria, and their conjugation to antibiotics allows smuggling the latter into bacterial cells. Synthetic siderophore mimetics based on MECAM (1,3,5-N,N′,N″-tris-(2,3-dihydroxybenzoyl)-triaminomethylbenzene) and DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane) cores, both chelating iron via catechol groups, have been recently applied as versatile carriers of functional cargo. In the present study, we show that MECAM and the MECAM-ampicillin conjugate 3 transport iron into Pseudomonas aeruginosa cells via the catechol-type outer membrane transporters PfeA and PirA and DOTAM solely via PirA. Differential proteomics and quantitative real-time polymerase chain reaction (qRT-PCR) showed that MECAM import induced the expression of pfeA, whereas 3 led to an increase in the expression of pfeA and ampc, a gene conferring ampicillin resistance. The presence of DOTAM did not induce the expression of pirA but upregulated the expression of two zinc transporters (cntO and PA0781), pointing out that bacteria become zinc starved in the presence of this compound. Iron uptake experiments with radioactive 55Fe demonstrated that import of this nutrient by MECAM and DOTAM was as efficient as with the natural siderophore enterobactin. The study provides a functional validation for DOTAM- and MECAM-based artificial siderophore mimetics as vehicles for the delivery of cargo into Gram-negative bacteria.

AB - The development of new antibiotics against Gram-negative bacteria has to deal with the low permeability of the outer membrane. This obstacle can be overcome by utilizing siderophore-dependent iron uptake pathways as entrance routes for antibiotic uptake. Iron-chelating siderophores are actively imported by bacteria, and their conjugation to antibiotics allows smuggling the latter into bacterial cells. Synthetic siderophore mimetics based on MECAM (1,3,5-N,N′,N″-tris-(2,3-dihydroxybenzoyl)-triaminomethylbenzene) and DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane) cores, both chelating iron via catechol groups, have been recently applied as versatile carriers of functional cargo. In the present study, we show that MECAM and the MECAM-ampicillin conjugate 3 transport iron into Pseudomonas aeruginosa cells via the catechol-type outer membrane transporters PfeA and PirA and DOTAM solely via PirA. Differential proteomics and quantitative real-time polymerase chain reaction (qRT-PCR) showed that MECAM import induced the expression of pfeA, whereas 3 led to an increase in the expression of pfeA and ampc, a gene conferring ampicillin resistance. The presence of DOTAM did not induce the expression of pirA but upregulated the expression of two zinc transporters (cntO and PA0781), pointing out that bacteria become zinc starved in the presence of this compound. Iron uptake experiments with radioactive 55Fe demonstrated that import of this nutrient by MECAM and DOTAM was as efficient as with the natural siderophore enterobactin. The study provides a functional validation for DOTAM- and MECAM-based artificial siderophore mimetics as vehicles for the delivery of cargo into Gram-negative bacteria.

KW - antibiotic vectorization

KW - iron uptake

KW - outer membrane transporters

KW - Pseudomonas aeruginosa

KW - siderophores

KW - Trojan horse

UR - http://www.scopus.com/inward/record.url?scp=85130030556&partnerID=8YFLogxK

U2 - 10.1021/acsinfecdis.2c00049

DO - 10.1021/acsinfecdis.2c00049

M3 - Article

C2 - 35500104

AN - SCOPUS:85130030556

VL - 8

SP - 1134

EP - 1146

JO - ACS infectious diseases

JF - ACS infectious diseases

SN - 2373-8227

IS - 6

ER -