Transcript Profiling of MRSA Biofilms Treated with a Halogenated Phenazine Eradicating Agent: A Platform for Defining Cellular Targets and Pathways Critical to Biofilm Survival

Abstract

Bacterial biofilms are surface-attached communities of non-replicating bacteria innately tolerant to antibiotics. Biofilms display differential gene expression profiles and physiologies as compared to their planktonic counterparts; however, their biology remains largely unknown. In this study, we used a halogenated phenazine (HP) biofilm eradicator in transcript profiling experiments (RNA-seq) to define cellular targets and pathways critical to biofilm viability. WoPPER analysis with time-course validation (RT-qPCR) revealed that HP-14 induces rapid iron starvation in MRSA biofilms, as evident by the activation of iron-acquisition gene clusters in 1 hour. Serine proteases and oligopeptide transporters were also found to be up-regulated, whereas glycolysis, arginine deiminase, and urease gene clusters were down-regulated. KEGG analysis revealed that HP-14 impacts metabolic and ABC transporter functional pathways. These findings suggest that MRSA biofilm viability relies on iron homeostasis.

Phenazine Antibiotic‐Inspired Discovery of Bacterial Biofilm‐Eradicating Agents

“…At ime-course assessment using RT-qPCR of MRSA biofilms treated with HP-14 (1/10 MBEC;0 .625 mm)r evealed that four iron acquisition gene clusters (isd, sbn, sfa, MW0695)w erea ctivated in 1hour. [130] In addition, HP-14-treated MRSA biofilms displayed higher levels of activation regarding these iron-acquisitiong ene clusters at 4a nd 8hours. Interestingly,m etalchelating agents N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) ande thylenediamine tetraacetic acid (ETDA)w ereu nable to activate iron-acquisition genes when tested alongside HP-14, aligning with our previousr esults demonstrating that these agents were unable to eradicate MRSA biofilms at high concentrations( EDTAa nd TPEN, MBEC > 2000 mm).…”

“…From >2700 MRSA‐1707 biofilm gene transcripts analyzed using RNA‐seq technology, 83 transcripts were down‐regulated and 134 transcripts were up‐regulated in response to HP‐14 . With >200 gene transcripts altered from HP‐14 treatment, we utilized the WoPPER analysis tool to focus attention on gene cluster activation or inhibition.…”

“…A time‐course assessment using RT‐qPCR of MRSA biofilms treated with HP‐14 (1/10 MBEC; 0.625 μ m ) revealed that four iron acquisition gene clusters ( isd, sbn, sfa, MW0695) were activated in 1 hour . In addition, HP‐14‐treated MRSA biofilms displayed higher levels of activation regarding these iron‐acquisition gene clusters at 4 and 8 hours.…”

“…Based on our collective data, we believe HP‐14 (CLogP=6.25) rapidly diffuses through bacterial cells and directly binds iron(II), following its release from a siderophore or heme. The binding of iron(II) by HP‐14 results in the rapid starvation of iron in MRSA biofilm cells, leading to the activation of iron‐uptake systems and eventual death (Figure ) . This mechanism is distinct from simple metal sequestration, as EDTA and TPEN are unable to eradicate biofilms and activate iron uptake systems when tested alongside HP‐14.…”

“…From > 2700 MRSA-1707 biofilm gene transcripts analyzed using RNA-seq technology,8 3t ranscripts were down-regulated and 134 transcripts were up-regulated in response to HP-14. [130] With > 200 gene transcripts altered from HP-14t reatment, we utilized the WoPPER analysist ool [131] to focus attention on gene cluster activation or inhibition. We found this approach to streamline our efforts, as 37 gene clusters were either up-o rd own-regulated from HP-14 treatment, including six activated gene clusters involved in iron uptake (Figure 13).…”