Nitric oxide donor sodium nitroprusside serves as a source of iron supporting Pseudomonas aeruginosa growth and biofilm formation

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Fairfull-Smith , View ORCID Profile Jilong Qin , View ORCID Profile Makrina Totsika doi: https://doi.org/10.1101/2025.03.12.642859 Xavier Bertran i Forga 1 Centre for Immunity and Infection Control, School of Biomedical Sciences, Queensland University of Technology , Brisbane, QLD, Australia 2 Max Planck Queensland Centre, Queensland University of Technology , Brisbane, QLD, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Xavier Bertran i Forga Yaoqin Hong 2 Max Planck Queensland Centre, Queensland University of Technology , Brisbane, QLD, Australia 3 Biomedical Sciences and Molecular Biology, College of Medicine and Dentistry, James Cook University , Douglas, QLD, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yaoqin Hong Kathryn E. Fairfull-Smith 4 School of Chemistry and Physics, Queensland University of Technology , Brisbane, QLD, Australia 5 Centre for Materials Science, Queensland University of Technology , Brisbane, QLD, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jilong Qin 1 Centre for Immunity and Infection Control, School of Biomedical Sciences, Queensland University of Technology , Brisbane, QLD, Australia 2 Max Planck Queensland Centre, Queensland University of Technology , Brisbane, QLD, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jilong Qin For correspondence: Jilong.qin{at}qut.edu.au Makrina.totsika{at}qut.edu.au Makrina Totsika 1 Centre for Immunity and Infection Control, School of Biomedical Sciences, Queensland University of Technology , Brisbane, QLD, Australia 2 Max Planck Queensland Centre, Queensland University of Technology , Brisbane, QLD, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Makrina Totsika For correspondence: Jilong.qin{at}qut.edu.au Makrina.totsika{at}qut.edu.au Abstract Full Text Info/History Metrics Preview PDF Abstract Biofilm dispersal agents, like nitric oxide (NO), restore antimicrobial effectiveness against biofilm infections by inducing bacteria to shift from a biofilm to a planktonic state, thereby overcoming the antimicrobial tolerance typically associated with biofilms. Sodium nitroprusside (SNP) is a widely used NO donor for investigating the molecular mechanisms underlying NO-mediated biofilm dispersal in the nosocomial pathogen Pseudomonas aeruginosa . However, the biofilm effects of SNP are variable depending on the in vitro experimental conditions, with some studies reporting enhanced growth in both planktonic and biofilm forms instead of dispersal. These discrepancies suggest that SNP affects P. aeruginosa biofilm-residing cells beyond the release of NO. In this study, we compared SNP with another NO donor, Spermine NONOate, to systematically contrast their effects on biofilm and planktonic cultures of P. aeruginosa . We found that SNP, but not Spermine NONOate, increased the biomass of P. aeruginosa biofilms in microplate cultures. This effect was also observed when biofilms were supplemented with iron. Additionally, supplementation with SNP rescued the planktonic growth of P. aeruginosa in iron-depleted media, similar to FeSO 4 supplementation, suggesting that SNP may serve as an iron source. Our findings suggest that SNP’s potential as an NO agent used for biofilm dispersal may be confounded by its role in promoting both biofilm and planktonic growth through its iron centre. Our study cautions investigators using SNP for studying NO-mediated biofilm dispersal. Importance Research into biofilm dispersal agent nitric oxide (NO) holds promise for treating biofilm-associated infections. Sodium nitroprusside (SNP), an NO donor widely used in antibiofilm research, has been shown in this study to enhance cell growth and biofilm formation in P. aeruginosa by acting as a source of iron. Our results suggest that SNP functions both as NO and iron donor, with its iron-releasing properties playing a more dominant role in promoting biofilm growth in closed culture systems. This study underscores the dual but conflicting roles of SNP in biofilm growth, which caution its future development as a NO-based therapeutic strategy for biofilm-associated infections. Observation Biofilms are microbial communities encapsulated in an extracellular polymeric matrix that are ubiquitous in both natural and clinical environments. They cause significant damage through biofouling of equipment and serve as a reservoir for recurrent chronic infections, as well as for food and water contamination 1 . In addition, bacteria in biofilms exhibit increased tolerance to antimicrobials and disinfectants compared to their planktonic counterparts, which undermines therapeutic efficacy 2 . One approach to address the substantial economic and health challenges posed by bacterial biofilms is to induce a transition to the planktonic state using biofilm dispersal agents, thereby reducing their antimicrobial resistance. Nitric oxide (NO), a well-known biofilm dispersal agent, has been exploited as an antibiofilm agent against several clinically and industrially relevant biofilm-forming species 3 , 4 . In Pseudomonas aeruginosa , NO functions as a signalling molecule, promoting the degradation of the exopolysaccharide matrix and the assembly of flagella and therefore reverting biofilm cells to the planktonic (free-living) state 5 , 6 . As a result, NO has been shown to enhance the effectiveness of antibiotics by resensitising biofilm bacteria to antimicrobial treatment, aiding in biofilm eradication through this shift to the planktonic form 4 , 7 . In P. aeruginosa, Escherichia coli and Staphylococcus aureus , NO has been reported to disrupt Fe-S clusters through S-nitrosylation or heme-containing proteins, which are implicated in cell signalling and metabolic processes 8 – 11 . The reaction of oxygen radicals with NO leads to the formation of peroxynitrite, a highly reactive nitrosative species that may damage lipids and DNA 12 . The precise delivery of gaseous NO at desired concentrations has remained a challenge in both clinical and laboratory settings due to its gaseous and highly reactive nature (14). This has prompted the development of NO-releasing drugs, which are generally soluble compounds that have aided in the controlled delivery of NO and have thus been instrumental to investigate the mechanisms involved in associated biofilm dispersal responses. The metal-nitrosyl complex sodium nitroprusside (SNP), an FDA-approved vasodilator, has been a model compound in studying NO-induced biofilm dispersal mechanisms in P. aeruginosa 5 – 7 , 13 , 14 . SNP consists of a ferrous (Fe 2+ ) ion coordinating five cyanide groups and a nitrosonium group (NO + ), which is released as NO together with cyanide upon reduction in aqueous solution ( Fig 1A ) 15 . Due to its reported success in dispersing biofilms in vitro , SNP is currently being explored in combination with nanoparticles as an in-situ NO-delivery method with antibiofilm properties using a mouse skin infection model 16 . However, biofilms grown in closed culture systems (i.e., microtiter plates) showed confounding results following 12-24h of SNP treatment, with effects ranging from growth promotion of planktonic and biofilm bacteria to biofilm biomass reduction 17 – 19 . In contrast, other NO donors, such as N- diazenium diolates (NONOates), are consistently reported to reduce the biomass of biofilms in vitro 11 , 17 . A primary distinction between these two NO donors is the ferrous iron present in SNP that is lacking from NONOates. Iron has been consistently reported to aid in the formation of P. aeruginosa biofilms 20 , 21 . Therefore, we hypothesised that the variable effects reported for SNP on biofilms in vitro may be substantially influenced by the other components in its chemical structure. Download figure Open in new tab Fig 1. SNP promotes biomass increase of P. aeruginosa PAO1 biofilms. (A) Chemical structure of Sodium Nitroprusside and Spermine-NONOate. (B) Changes in biofilm biomass over 24h. 10 7 CFU/ml bacterial solutions were prepared from overnight cultures using M9 media, transferred to 24-well plates and incubated at 37 ºC shaking. Biofilm biomass was quantified every 2h by crystal violet staining for up to 24h. (C) 4h biofilms were treated with SP-NONOate for 15-min, or FeSO 4 or SNP for 30-min, at the indicated concentrations. Data represent at least four independent replicates. (D) 4h biofilms of either PAO1 WT or fhp ::Tn were treated with either SP-NONOate or SNP for 15-min or 30-min respectively at the indicated concentrations. Data represent two independent replicates. (E) Biofilm cultures were grown in M9 media supplemented with 31.25µM of SNP or FeSO 4 . Two independent cultures were included. The means ± SD are represented in the graph. SNP increases biofilm biomass of P. aeruginosa PAO1 in vitro To explore the effects of SNP on microplate biofilms of P. aeruginosa (a model biofilm-forming pathogen), we first measured biofilm growth kinetics in minimal media by tracking the biomass of P. aeruginosa PAO1 biofilms over 24 hours in microtiter plates ( Fig 1A , Text S1). In this system, P. aeruginosa biofilms reached a peak biomass at 4 hours (biomass max ), followed by a gradual decline, with <10% biofilm biomass remaining by 14 hours. As the biomass max achieved in microplates occurs at 4 h of incubation, 4h-old biofilms were subsequently used to evaluate dispersal effects by two NO-donors, SNP and Spermine NONOate (SP-NONOate), measured as significant reduction in remaining biofilm biomass. SP-NONOate is an N- diazeniumdiolate that spontaneously releases NO in aqueous solution 11 , 17 , 22 . Therefore, SP-NONOate was used to compare the effects of SNP on biofilm biomass. Consistent with previous reports, SP-NONOate induced a reduction of P. aeruginosa biofilm biomass within 15 minutes ( Fig 1C , Fig S1A and S1B) 22 . Instead, SNP induced a dose-dependent biofilm biomass increase after 30 minutes, with a maximum 1.25-fold increase observed at 500 µM SNP ( Fig 1C , Fig S1B). As a treatment strategy, NO is proposed to reduce biofilm-associated antimicrobial tolerance by reverting P. aeruginosa biofilm cells to their planktonic state. This hypothesis has been routinely tested using SNP to induce NO-mediated dispersal of P aeruginosa biofilms 5 , 6 , 14 , 17 . However, our findings contradict this expectation, as we observed that SNP instead promotes a rapid increase in biofilm biomass. A previous study similarly reported that short exposure to low concentrations of NO correlated with increased P. aeruginosa biofilm biomass 11 , suggesting that SNP might be releasing NO at concentrations insufficient for dispersal, thereby promoting biofilm growth instead. To investigate this possibility, we treated PAO1 biofilms with a range of sub-dispersing concentrations of SP-NONOate (Fig. S2), yet observed no significant biomass increase. These results suggest that SNP may enhance biofilm growth through a mechanism independent of NO release. A major difference in chemical structures between SNP and SP-NONOate is the presence of an iron centre in SNP ( Fig 1A ). Iron has been described as an essential micronutrient for the development of biofilms in P. aeruginosa 20 . Therefore, we reasoned that the observed increase in biomass by SNP ( Fig 1C ) could be derived from the surplus in bioavailable iron released from the SNP molecule. This hypothesis is supported by previous reports showing that P. aeruginosa biofilms grown in iron-supplemented media reached a higher biomass, and that the addition of iron to established biofilms induced rapid surface attachment of planktonic cells 21 , 22 . Therefore, we supplemented established P. aeruginosa biofilms with matching concentrations of FeSO 4 , which induced a similar increase in biofilm biomass to that of SNP treatment ( Fig 1C , Fig S1B). Unlike SP-NONOate, which significantly reduced biofilm biomass accumulation (Fig S3), media supplementation with SNP or FeSO 4 prolonged the biofilm formation phase to 8 h compared to the control media group (4 h), allowing for increased accumulation of biomass and a higher biomass max ( Fig 1E ). Therefore, these data suggest SNP as a potential iron donor to support biofilm growth. SNP rescues growth in iron-depleted conditions To further test this hypothesis, we next examined whether supplementing iron-chelated M9 medium with SNP could rescue the growth of P. aeruginosa planktonic cultures. While M9 supported the growth of P. aeruginosa , we found that growth in M9 was inhibited when iron was depleted from culture media by the iron chelator 2,2’-bipyridyl ( Fig 2 , Text S1). However, the addition of SNP reversed the growth inhibition caused by iron depletion, and that this rescue of otherwise defective growth of PAO1 in iron-chelated media, by the addition of SNP, was comparable to the effect of FeSO 4 supplementation in alleviating growth arrest triggered by iron deficiency ( Fig 2 , Fig S4). Together, these data suggest that SNP acts as an iron donor, supporting both biofilm and planktonic growth of P. aeruginosa . Download figure Open in new tab Fig 2. Growth defects of P. aeruginosa PAO1 in iron-depleted media can be rescued by SNP or FeSO 4 . Initial bacterial cultures were inoculated at an OD 600 of 0.05 and grown in M9 media pre-treated with 100µM of the iron scavenger 2,2’-bipyridyl. Cultures were supplemented with SNP or FeSO 4 under shaking conditions in 96-well microtiter plates. Culture optical density was periodically recorded in a plate reader by measuring the absorbance at 600nm. Data represents two biological replicates. The means ± SD are represented in the graph. While the mechanism by which SNP releases iron remains unclear and is beyond the scope of this work, our data suggest that physiologically relevant iron concentrations become readily available upon SNP addition, potentially overshadowing its NO-related effects. This is evidenced by the rapid increase in biofilm biomass within 30 minutes of SNP exposure, similar to that induced by FeSO 4 , possibly due to the upregulation of extracellular matrix polysaccharide Psl synthesis and/or enhanced attachment of planktonic bacteria 22 . Importantly, our findings highlight the iron-releasing properties of SNP, a factor that may not have been fully considered in previous studies investigating its therapeutic potential, likely introducing bias to our understanding of SNP as a NO-donor. While studies contrasting the phenotypic and genotypic behaviours of biofilms grown in each culture system are lacking, existing data suggest that biofilms grown in open flow systems exhibit distinct phenotypic profiles compared to those in closed microplate environments. This is evident from the opposing responses of P. aeruginosa biofilms to iron chelators and tobramycin, depending on the culture system used 23 . These findings highlight the value of microplates as a low-cost, rapid, and high-throughput system usable to control for potentially masked effects by treatments tested in open cultures. In terms of potentiating antibiofilm treatments, SNP was reported previously to synergise with tobramycin against P. aeruginosa biofilms, an antibiotic commonly used to treat biofilm infections in cystic fibrosis patients 7 , 24 and more effective in metabolically active bacteria 27,28 . Here we showed that SNP supplementation resulted in a higher growth rate for P. aeruginosa due to its iron donation, which could lead to a more active metabolism, potentially resulting in sensitisation of biofilm-embedded bacteria to tobramycin. In addition, iron supplementation has also been shown to increase the effectiveness of antibiotics by enhancing the production of reactive oxygen species. This would also explain the reported increases in effectiveness of nanoparticles when tethered to SNP, as these depend on light-activated ROS generation 25 . Considering that SNP acts as a source of readily available iron, ROS-induced damage to the biofilms might have been enhanced by the presence of ferrous iron in SNP, which may trigger localised production of superoxide radicals through the Fenton reaction leading to a severe disruption of iron homeostasis 25 . Conclusion Altogether, our results demonstrate that SNP provides a readily available source of iron to growing biofilms of P. aeruginosa . Considering most assays studying NO dispersal have been conducted with SNP as the NO donor, our findings should elicit caution when solely attributing reported SNP biofilm responses to NO, as masked secondary effects might have been overlooked. Author contributions XB and JQ conceptualised the project. XB, JQ and YH contributed to experimental design. XB conducted all experiments, and contributed to data collection, analysis and visualisation. XB, JQ, YH and MT contributed to data interpretation. JQ and MT supervised the project. KFS and MT obtained the funding. XB wrote the original draft, and all authors edited the manuscript. Competing interests MT is an employee of the GSK group of companies. All remaining authors declare no competing interests. This research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest. Data availability All data generated or analysed during this study were included in this article and supplementary files. Acknowledgements This work is funded in part by an Australian Research Council project grant (DP210101317), the Max Planck Queensland Centre on the Materials Science of Extracellular Matrices to MT, and the QUT Amplify Scholarship provided by the Queensland University of Technology (Australia) to XB. The Ian Potter Foundation sponsored the CLARIOStar high-performance microplate reader (BMG, Australia). 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Iron Homeostasis Affects Antibiotic-mediated Cell Death in Pseudomonas Species . Journal of Biological Chemistry 2010 ; 285 : 22689 – 22695 . OpenUrl Abstract / FREE Full Text View the discussion thread. Back to top Previous Next Posted March 12, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Nitric oxide donor sodium nitroprusside serves as a source of iron supporting Pseudomonas aeruginosa growth and biofilm formation Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. 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