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bioRxiv · 10.1101/2025.07.10.664208

Zinc-Enhanced Activity of an Antimicrobial Halogenated Phenazine Against Streptococcus mutans and Other Gram-positive Bacteria

Lemos, J. A.·Kajfasz, J. K.·Huigens, R. W.·Hosay, H. B.·Gao, Q.
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Abstract

Halogenated phenazine (HP) compounds have shown promise as antimicrobial agents, particularly against biofilm-associated Gram-positive pathogens. Among these compounds, HP-29 demonstrates potent activity against methicillin-resistant Staphylococcus aureus by inducing rapid iron starvation. As maintenance of trace metals homeostasis is critical for the survival of Streptococcus mutans, this study investigated the antimicrobial efficacy of HP-29 and the impact of metal supplementation on this major oral and occasional systemic pathogen. As anticipated, HP-29 inhibited S. mutans growth in a dose-dependent manner, with iron supplementation alleviating the antimicrobial effect. Cobalt, manganese, or nickel supplementation also mitigated the inhibitory activity of HP-29 but, unexpectedly, the addition of zinc greatly enhanced HP-29 antimicrobial activity. This zinc-driven potentiation of HP-29 extended to other Gram-positive pathogens, including Enterococcus faecalis and S. aureus. Inductively coupled plasma mass spectrometry analysis revealed that intracellular iron content decreased significantly following exposure to HP-29. At the same time, exposure to HP-29 led to a slight increase in intracellular zinc, mirroring the increase observed in cells exposed to excess zinc. When combined with zinc, HP-29 triggered a 5-fold increase in intracellular zinc and reduced manganese levels by [~]50%. Transcriptome analysis showed that HP-29, with or without zinc, altered expression of genes linked to iron and manganese uptake as well as zinc efflux, suggesting broad disruption of metal ion regulation. These findings highlight HP-29 as a potent antimicrobial that broadly impairs metal homeostasis. The unexpected synergy of HP-29 with zinc points toward a promising dual-agent therapeutic strategy against Gram-positive pathogens. IMPORTANCEWidespread development of antibiotic resistance has created a constantly moving target when combating infectious microbes. Here, we further explore an antimicrobial halogenated phenazine, HP-29, which is effective against Gram-positive bacteria through disruption of intracellular trace metal equilibrium. We showed that HP-29 inhibits growth of the oral and systemic pathogen Streptococcus mutans and that its antimicrobial effect is greatly potentiated by the addition of zinc. The zinc-mediated enhancement of HP-29s efficacy was also observed in other Gram-positive pathogens, including Enterococcus faecalis and Staphylococcus aureus. Intracellular trace metal quantifications and transcriptome analysis confirmed that HP-29 treatment impairs trace metal homeostasis, an outcome that is exacerbated when S. mutans is treated with both HP-29 and zinc. The observed synergy of HP-29 with zinc supports the development of a dual-agent therapeutic strategy against Gram-positive pathogens.

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Lemos, J. A., Kajfasz, J. K., Huigens, R. W., Hosay, H. B., Gao, Q.. 2025-07-10. Zinc-Enhanced Activity of an Antimicrobial Halogenated Phenazine Against Streptococcus mutans and Other Gram-positive Bacteria. https://doi.org/10.1101/2025.07.10.664208

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