Iron depletion enhances production of antimicrobials by Pseudomonas

Transcription

1 Iron depletion enhances production of antimicrobials by Pseudomonas aeruginosa. Angela T. Nguyen 1, Jace W. Jones 1, Max A. Ruge 1, Maureen A. Kane 1, and Amanda G. Oglesby-Sherrouse 1,2 * University of Maryland 1 School of Pharmacy, Department of Pharmaceutical Sciences and 2 School of Medicine, Department of Microbiology and Immunology, Baltimore, Maryland, Supplemental Materials

2 Figure S1. Iron-regulated antimicrobial activity against S. aureus require coculture with P. aeruginosa. S. aureus cell density was measured spectroscopically at an OD630 after 18 hours growth with culture supernatants from the indicated P. aeruginosa strains in transwell cell culture plates. Error bars indicate standard deviation of three biological replicates.

3 Figure S2: Static growth of PAO1 produces significantly less PQS in high iron than shaking growth. PAO1 was grown with shaking at 37 C for 18 hours overnight or diluted to an OD of.05 and inoculated into a cell culture plate and left static in a 37 C incubator for 18 hours. AQs were extracted and run on TLC as previously described. PQS/C9-PQS densitometry was quantified and normalized to OD 600. Error bars indicate standard deviation of three biological replicates. * p <.05 indicates significance when comparing shaking and static cultures or between high and low iron conditions as indicated *p <. 05; **p <. 005.

4 Figure S3. Tandem mass spectra of AQ standards HHQ, HQNO, and PQS. Experimental m/z values were given and measured with mass accuracies less than 5 ppm. The precursor ions were designated [M+H] + and the representative structure, molecular formula, and exact mass were displayed. An associated molecular formula corresponding to the neutral loss and a tentative structure of the product ion were displayed next to the m/z values. A.) Tandem mass spectrum for HHQ. The precursor ion of m/z was isolated and fragmented via collision-induced dissociation (CID). The product ions at m/z values 172.1, 159.1, and were identified by their neutral loss and assigned a tentative structure starting from the base HHQ structure. B.) Tandem mass spectrum for HQNO. The precursor ion of m/z was isolated and fragmented via CID. The product ions at m/z values 186.1, 172.1, 159.1, and were identified by their neutral loss and assigned a tentative structure starting from the base HQNO structure. Note, m/z values 172.1, 159.1, and have the same tentative structure as HHQ (A.). C.) Tandem mass spectrum for PQS. The precursor ion of m/z was isolated and fragmented via CID. The product ions at m/z values 188.1, 175.1, and were identified by their neutral loss and assigned a tentative structure starting from the base PQS structure. Note, m/z value had the same tentative structure as HHQ (A.) and HQNO (B.).

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6 Figure S4. Extracted ion chromatogram (XIC) for m/z and associated tandem mass spectra for highlighted peaks in the XIC. A.) The XIC of m/z with four highlighted chromatographic peaks corresponding to C9-QNO* (rt = 5.0 min), C9-PQS (rt = 6.3 min), C9:1-QNO (rt = 7.0 min), and C9-QNO (rt = 7.5 min). Note, C9:1 QNO had a point of unsaturation and corresponded to a precursor ion at m/z It was extracted in the m/z XIC as a result of isotopic overlap. B.) The tandem mass spectrum for the precursor ion at m/z (rt = 6.3 min) and identified as C9-PQS. The accurate mass measurement of the precursor ion (< 5 ppm) and the highlighted m/z values and (diagnostic for the PQS ring structure) provided confident structure assignment. C.) The tandem mass spectrum for the precursor ion at m/z (rt = 7.5 min) and identified as C9-QNO. The accurate mass measurement of the precursor ion (< 5 ppm) and the highlighted m/z values 186.1, 172.1, 159.1, and (diagnostic for the QNO ring structure) provided confident structure assignment. D.) The tandem mass spectrum for the precursor ion at m/z (rt = 5.0 min) and identified as C9-QNO. The accurate mass measurement of the precursor ion (< 5 ppm) and the highlighted m/z values and (diagnostic for the QNO ring structure) provided confident structure assignment. E.) The tandem mass spectrum for the precursor ion at m/z (rt = 7.0 min) and identified as C9:1-QNO. The accurate mass measurement of the precursor ion (< 5 ppm) and the highlighted m/z values 184.1, 172.1, and (diagnostic for the QNO ring structure) provided confident structure assignment. The product ion at m/z localized the double bond location to the proximal C1 position.

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