Investigating the therapeutic potential of the antibacterial drug nitroxoline in combating cryptococcal infection

Cryptococcosis is a major health threat among immunocompromised individuals, resulting in life-threatening consequences secondary to brain invasion. The limited treatment options and the increased incidence of treatment failure and relapses urge the need for more effective therapies. In this study, we investigated the therapeutic potential of nitroxoline (NTX) in treating cryptococcal infection. NTX exhibited potent fungicidal activity against Cryptococcus neoformans/gattii clinical isolates, with an MIC90 of 1 µg/mL. In addition, NTX substantially inhibits the main virulence traits of cryptococcal cells, including the polysaccharide capsule, melanin production, and the urease enzyme, which are crucial for the infection progression. In exploring the potential mechanism of action, our results revealed the relationship between the metal-chelating properties of the NTX scaffold and its antifungal activity. Interfering metal chelation, either through structural modification or exogenous supplementation with varied divalent cations, significantly reduced the antifungal and anti-virulence activities of NTX. Given its potential therapeutic role, we evaluated the interaction between NTX and the clinically used antifungals amphotericin B, flucytosine and fluconazole. Additionally, we investigated the tendency of cryptococcal cells to develop resistance following repeated exposure to NTX, as well as its efficacy in the Caenorhabditis elegans nematode model of cryptococcal infection. Notably, cryptococcal cells exhibited consistent susceptibility to NTX upon repeated exposure to a subinhibitory concentration. In the C. elegans model, NTX significantly reduced the fungal burden of the infected worms, achieving a 2-log10 reduction, and consistently enhanced their survival compared to untreated group. These findings highlight the potential role of the NTX scaffold in combating cryptococcal infection that warrants further investigation.Author summary: Nitroxoline (NTX) antibiotic has been prescribed for decades to treat acute and recurrent bacterial urinary tract infections. Its extensive urinary excretion has limited its therapeutic utility for invasive bacterial infections. However, its favorable safety profile, excellent oral bioavailability, and the low potential of resistance, have encouraged its repurposing against a diverse range of pathogens. In this study, we investigated NTX activity against Cryptococcus fungal pathogen that can disseminate to the central nervous system, leading to fatal meningoencephalitis in immunocompromised patients. The mortality rate of this opportunistic infection can exceed 60% especially in resource-limited regions. NTX exhibited potent fungicidal activity against proliferated cryptococcal cells, with an inhibitory activity on the main virulence factors involved in the initial colonization, including capsule formation, melanin pigmentation and urease enzyme. This anti-virulence activity is directly related to the metal chelation nature of the NTX scaffold, enabling the depletion of the intracellular content of essential cations. Furthermore, cryptococcal cells exhibited a negligible resistance tendency during continuous exposure to NTX. Overall, these findings demonstrate that NTX exerts both direct antifungal and anti-virulence activities on Cryptococcus through a metal chelation-dependent mechanism and highlight its potential role as a repurposed therapeutic for the treatment of cryptococcosis.