New Treatment Strategies
There are very few effective antifungal drugs, and there is increasing concern that fungi are developing resistance to the few drugs that are available. We are therefore working to develop new antifungal therapies. The first is based on drug synergy, to enable our current drugs to be more effective and less likely to induce resistance. The second is based on antimicrobial honey, an ancient medicine that is gaining new traction as a broad-spectrum medicine for treating superficial infections.
Drug synergy for the enhancement of antifungal agents
The lactoferrin molecule. Reproduced from https://www.hindawi.com/journals/bri/2013/271641/
Proteome analysis identified FPPS as a differentially regulated protein in response to fluconazole. Reproduced from https://doi.org/10.1371/journal.pone.0042835
Drug synergy is used to enhance an antifungal drug with a second agent or drug, such that working together they are substantially more potent than either is when used alone. We used proteomics and transcriptomics to examine how fungi responded to antifungal treatments and from this identified potential synergising targets that were subsequently validated and refined.
1. Amphotericin B and lactoferrin/lactofungin
This work began by testing various iron chelators for their ability to synergise with antifungal drugs. Unfortunately the only synergistic pair was amphotericin B (AMB) and lactofungin. The latter is a protein found in milk and tears, and has known antimicrobial properties. In fact, it wasn't the iron chelating properties that were synergistic but a peptide present in lactoferrin, which we purified and named lactofungin. The following papers detail our work in this area:
Fernandes, K.E., Payne, R.J and Carter, D.A. (2020) Lactoferrin-derived peptide lactofungin is potently synergistic with amphotericin B. Antimicrobial Agents and Chemotherapy. 64:e00842-20. https://doi.org/10.1128/AAC.00842-20.
Fernandes, K.E., Weeks, K. and Carter, D.A. (2020) Lactoferrin is broadly active against yeasts and highly synergistic with amphotericin B. Antimicrobial Agents and Chemotherapy. 64:e02284-19. https://doi.org/10.1128/AAC.02284-19.
Lai, Y-W., Pang, C.N.I., Campbell, L.T., Chen, S C.–A., Wilkins, M.R. Carter, D.A. (2019) Different pathways mediate amphotericin-lactoferrin drug synergy in Cryptococcus and Saccharomyces. Frontiers in Microbiology 10: 2195. | https://doi.org/10.3389/fmicb.2019.02195
Pang, C.N.I., Lai, Y-W., Campbell, L.T., Chen, S.C.A., Carter, D.A* and Wilkins, M.R*(2017). Transcriptome and network analyses in Saccharomyces cerevisiae reveal that amphotericin B and lactoferrin synergy disrupt metal homeostasis and stress response. Scientific Reports 7, 40232. DOI: 10.1038/srep40232
Lai, Y-W., Campbell, L.T., Wilkins, M.R., Pang, C.N.I., Chen, C. and Carter, D.A. (2016) Synergy and antagonism between iron chelators and antifungals in Cryptococcus. International Journal of Antimicrobial Agents 48(4): 388-394. https://doi.org/10.1016/j.ijantimicag.2016.06.012
2: Azole drugs and bisphosphonates
In a proteomic analysis of the response of Cryptococcus to the azole drug fluconazole we found upregulation of farnesyl pyrophosphate synthetase (FPPS), an enzyme that catalases the production of squalene, which feeds into the ergosterol biosynthesis pathway that is the target of azole drugs. FPPS is targeted by bisphosphonate drugs such as alendronate and zolendronate. When tested these produced strong synergy with fluconazole, providing proof-of-concept that FPPS inhibition will enhance azole action. These papers detail this work:
Kane, A. and Carter, D.A. (2022) Augmenting azoles with drug synergy to expand the antifungal toolbox. Pharmaceuticals 15 (4):482 https://doi.org/10.3390/ph15040482
Kane, A., Campbell, L., Ky, D., Hibbs, D. and Carter, D. The antifungal and synergistic effect of bisphosphonates in Cryptococcus. Antimicrobial Agents and Chemotherapy. 65(2), e01753-20. https://doi.org/10.1128/AAC.01753-20
Chong, H.S., Campbell, L.T., Padula, M., Hill, C.P., Harry, E., Li, S., Wilkins, M.R, Herbert, B., and Carter, D.A. (2012) Time-course proteome analysis reveals the dynamic response of Cryptococcus gattii cells to fluconazole. PLOS ONE 7 (8) e42835. https://doi.org/10.1371/journal.pone.0042835
Chong, H.S., Dagg, R., Malik, R. Chen, S. and Carter, D.A. (2010) In vitro susceptibility of the yeast pathogen Cryptococcus to fluconazole and other azoles varies with molecular genotype. Journal of Clinical Microbiology 48: 4115–4120. DOI: 10.1128/JCM.01271-10
Significant synergy between AMB and lactoferrin. Reproduced from https://doi.org/10.1016/j.ijantimicag.2016.06.012
Antimicrobial honey for the treatment of bacterial and fungal infections
Honey is an ancient medicine that is gaining popularity as a topical agent. Working with colleagues Liz Harry, Nural Cokcetin and Shona Blair at the University of Technology Sydney, we have undertaken research to provide a mechanistic basis for the activity of honey and thereby increase acceptance in modern medicine. Papers from our work in this area are listed below:
Guttentag, A., Krishnakumar, K., Cokcetin, N., Hainsworth, S., Elizabeth Harry, E., Carter, D. (2021) Inhibition of dermatophyte fungi by Australian jarrah honey. Pathogens 10, 194. https://doi.org/10.3390/pathogens10020194
Guttentag, A., Krishnakumar, K., Cokcetin, N., Elizabeth Harry, E., Carter, D. (2021) Factors affecting the production and measurement of hydrogen peroxide in bioactive honey samples. Access Microbiology 3(3) https://doi.org/10.1099/acmi.0.000198
Lu, J., Cokcetin, N., Burke, C.M., Turnbull, L., Liu, M., Carter, D.A., Whitchurch, C.B. and Harry, E.J. (2019) Honey can inhibit and eliminate biofilms produced by Pseudomonas aeruginosa wound isolates. Scientific Reports 9, 18160. https://doi.org/10.1038/s41598-019-54576-2
Lehmann, D. M., K. Krishnakumar, K., Batres, M.A., Hakola-Parry, A., Cokcetin, N., Harry, E. and Carter, D. A. (2019) A cost-effective colorimetric assay for quantifying hydrogen peroxide in honey. Access Microbiology, 1 (10) e000065. doi: 10.1099/acmi.0.000065
Liu, M,Y., Cokcetin, N., Lu, J., Turnbull, L., Carter, D.A., Whitchurch, C.B, & Harry, E.J. (2018). Rifampicin-manuka honey combinations are superior to other antibiotic-manuka honey combinations in eradicating Staphylococcus aureus biofilms. Frontiers in Microbiology 8, 2653. DOI: 10.3389/fmicb.2017.02653
Cokcetin, N.N., Pappalardo, M., Campbell, L.T., Brooks, P., Carter, D.A., Blair, S.E. and Harry, E.J. (2016) The antibacterial activity of Australian Leptospermum honey correlates with methylglyoxal levels. PLOS ONE 11(12): e0167780. DOI: 10.1371/journal.pone.0167780
Carter, D.A., Blair, S.E, Cokcetin, N., Bouzo, D., Brooks, P., Schothauer, R. and Harry, E.J. (2016) Therapeutic manuka honey: no longer so alternative. Frontiers in Microbiology. 7: 569. https://doi.org/10.3389/fmicb.2016.00569
Liu, M., Lu, J., Müller, P., Turnbull, L., Burke, C., Schlothauer, R., Carter, D., Whitchurch, C. B., Harry, E. J. (2015). Antibiotic-specific differences in the response of Staphylococcus aureus to treatment with antimicrobials combined with manuka honey. Frontiers in Microbiology. 5:779. DOI: 10.3389/fmicb.2014.00779
Lu, J., Turnbull, L., Burke, C.M., Liu, M., Carter, D.A., Schlothauer, R. Whitchurch, C.B. and Harry, E.J. (2014) Manuka-type honeys can eradicate biofilms produced by Staphylococcus aureus strains with different biofilm-forming abilities. PeerJ 2:e326. http://dx.doi.org/10.7717/peerj.326
Müller, P., Alber, D.G., Turnbull, L., Schlothauer, R.C., Carter, D.A., Whitchurch, C.B. and Harry, E.J. (2013) Synergism between Medihoney and rifampicin against methicillin-resistant Staphylococcus aureus (MRSA). PLOS ONE 8(2): e57679. DOI: 10.1371/journal.pone.0057679
Lu, J., Carter, D.A., Turnbull, L., Rosendale, D., Hedderley, D., Stephens, J., Gannabathula, S., Steinhorn, G., Schlothauer, R.C., Whitchurch, C.B., & Harry, E. J. (2013) The effect of New Zealand kanuka, manuka and clover honeys on bacterial growth dynamics and cellular morphology varies according to the species. PLOS ONE 8(2):e55898. doi: 10.1371/journal.pone.0055898
Chen, C., Campbell, L.T., Blair, S.E. and Carter, DA. (2012) The effect of heat treatment on the antimicrobial properties of honey. Frontiers in Microbiology 3: Article 265. doi: 10.3389/ fmicb.2012.00265. https://doi.org/10.3389/fmicb.2012.00265
Packer, J.M., Irish, J., Herbert, B., Hill, C., Padula, M., Blair, S. Carter, D.A. and Harry, E.J. (2012) Specific non-peroxide antibacterial effect of manuka honey on the Staphylococcus aureus proteome. International Journal of Antimicrobial Agents 40:43–50. https://doi.org/10.1016/j.ijantimicag.2012.03.012
Irish, J., Blair, S. and Carter, D.A. (2011) The antibacterial activity of honey derived from Australian flora. PLoS ONE 6(3): e18229. https://doi.org/10.1371/journal.pone.0018229
Blair S.E., Cokcetin, N.N., Harry, E. J. and Carter, D.A. (2009) The unusual antibacterial activity of medical-grade Leptospermum honey: antibacterial spectrum, resistance and transcriptome analysis. European Journal of Clinical Medicine and Infectious Diseases 28: 1199–1208. DOI: 10.1007/s10096-009-0763-z
Flowers of certain Leptospermum species produce New Zealand manuka nnd Australian Jellybush honey with potent antimicrobial activity
Hot-spots for highly active honey (pink diamonds) from eastern Australia. Reproduced from https://doi.org/10.1371/journal.pone.0018229
Antibacterial Medihoney produced by Comvita NZ was used in many of our studies that were funded by an ARC Linkage grant.