Journal
ACS INFECTIOUS DISEASES
Volume 7, Issue 8, Pages 2285-2298Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acsinfecdis.0c00887
Keywords
Fusobacterium nucleatum; PBT2; anaerobe; metal homeostasis; zinc/iron; biofilm
Categories
Funding
- University of Otago Doctoral scholarship
- Maori Education Trust TiMaru Maori Trust scholarship
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Fusobacterium nucleatum is an opportunistic human pathogen commonly associated with periodontal disease and colorectal cancer. Current treatments using broad-spectrum antibiotics can harm normal microflora, making the development of narrow-spectrum antimicrobials essential. The antimicrobial Zn ionophore PBT2 shows strong inhibitory activity by disrupting metallostasis in F. nucleatum.
The Gram-negative anaerobe Fusobacterium nucleatum is an opportunistic human pathogen, most frequently associated with periodontal disease through dental biofilm formation and, increasingly, with colorectal cancer development and progression. F. nucleatum infections are routinely treated by broad-spectrum beta-lactam antibiotics and metronidazole. However, these antibiotics can negatively impact the normal microflora. Therefore, the development of novel narrow-spectrum antimicrobials active against anaerobic pathogens is of great interest. Here, we examined the antimicrobial Zn ionophore PBT2, an 8-hydroxyquinoline analogue with metal chelating properties, against a single type isolate F. nucleatum ATCC 25586. PBT2-Zn was a potent inhibitor of growth and exhibited synergistic bactericidal (>3-log(10) killing) activity at 5x MIC in planktonic cells, and at the MIC in biofilms grown in vitro. Physiological and transcriptional analyses uncovered a strong cellular response relating to Zn and Fe homeostasis in PBT2-Zn treated cells across subinhibitory and inhibitory concentrations. At IX MIC, PBT2 alone induced a 3.75-fold increase in intracellular Zn, whereas PBT2-Zn challenge induced a 19-fold accumulation of intracellular Zn after 2 h. A corresponding 2.1-fold loss of Fe was observed at 1X MIC. Transcriptional analyses after subinhibitory PBT2-Zn challenge (0.125 mu g/mL and 200 mu M ZnSO4) revealed significant differential expression of 15 genes at 0.5 h, and 12 genes at 1 h. Upregulated genes included those with roles in Zn homeostasis (e.g., a Zn-transporting ATPase and the Zn-sensing transcriptional regulator, smtB) and hemin transport (hmuTUV) to re-establish Fe homeostasis. A concentration-dependent protective effect was observed for cells pretreated with hemin (50 mu g/mL) prior to PBT2-Zn challenge. The data presented here supports our proposal that targeting the disruption of metallostasis by Zn-translocating ionophores is a strategy worth investigating further for the treatment of Gram-negative anaerobic pathogens.
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