Phenotypic Adaptation of Pseudomonas aeruginosa in the Presence of Siderophore-Antibiotic Conjugates during Epithelial Cell Infection

Abstract

Iron acquisition pathways have often been considered to be gateways for the uptake of antibiotics into bacteria. Bacteria excrete chelators, called siderophores, to access iron. Antibiotic molecules can be covalently attached to siderophores for their transport into pathogens during the iron-uptake process. P. aeruginosa produces two siderophores and is also able to use many siderophores produced by other bacteria. We investigated the phenotypic plasticity of iron-uptake pathway expression in an epithelial cell infection assay in the presence of two different siderophore–antibiotic conjugates, one with a hydroxamate siderophore and the second with a tris-catechol. Proteomic and RT-qPCR approaches showed that P. aeruginosa was able to sense the presence of both compounds in its environment and adapt the expression of its iron uptake pathways to access iron via them. Moreover, the catechol-type siderophore–antibiotic was clearly more efficient in inducing the expression of its corresponding transporter than the hydroxamate compound when both were simultaneously present. In parallel, the expression of the proteins of the two iron uptake pathways using siderophores produced by P. aeruginosa was significantly repressed in the presence of both conjugates. Altogether, the data indicate that catechol-type siderophores are more promising vectors for antibiotic vectorization using a Trojan-horse strategy.

The Esterase PfeE, the Achilles’ Heel in the Battle for Iron between Pseudomonas aeruginosa and Escherichia coli

“…In the presence of ENT, the activation of the transcription of the pfeA and pfeE genes via PfeS/PfeR goes hand in hand with the repression of the transcription of the genes involved in iron acquisition by the siderophore PCH, as we have already described previously ( Figure 2 and [ 32 , 33 ]). This repression is independent of PfeS/PfeR or the expression of PfeA, PfeE or PirA.…”

“…The experiment was repeated with 10 µM ENT in the growth media and we compared the level of transcription for each strain between cultures in the presence of ENT and those in its absence ( Figure 2 b). In PAO1, as previously described [ 24 , 29 , 32 , 33 ], the presence of 10 µM ENT in the growth medium induced the transcription of pfeA and pfeE , with a log 2 fold change of 5.5 and 5.2, respectively. As discussed above, the absence of the sensor PfeS of the two-component system (∆ pfeS strain) resulted in the induction of pfeA and pfeE gene transcription in the absence of ENT ( Figure 2 a).…”

“…We previously showed that 10 µM ENT in the growth medium of P. aeruginosa cells also represses transcription of the genes encoding for the proteins involved in iron acquisition by the siderophore PCH (PCH pathway) produced by P. aeruginosa [ 24 , 32 , 33 ]. Here, we observed the repression of fptA transcription (TBDT of PCH-Fe) by RT-qPCR, with a log 2 fold change between 2.3 and 3.5 for PAO1 and all mutants tested ( Figure 2 b), indicating that ENT-Fe does not need to enter P. aeruginosa cells by PfeA or PirA or interact somehow with the PfeS/PfeR two-component system to repress the endogenous PCH pathway.…”

“…Such repression occurred only when ENT was present in the growth media and was completely independent of the expression of PfeA, PfeE, PfeS, and PfeR. The observed decrease in transcription of fptA and of the genes of the PCH locus [ 24 , 32 , 33 ] is assuredly a consequence of the competition for iron between ENT and PCH in the bacterial environment, resulting in less PCH-Fe being imported by FptA to activate the positive transcriptional regulator, PchR [ 34 , 35 ]. There was no effect on the transcription of fpvA , the TBDT of PVD-Fe, the other siderophore produced by P. aeruginosa .…”

“…According to the literature, ENT-Fe complexes can be transported across the outer membrane by two TBDTs, PfeA and PirA [ 21 , 23 , 33 ]. Then, the siderophore-Fe complex is hydrolyzed by PfeE to facilitate iron release [ 25 ].…”