ZccE is a Novel P-type ATPase That Protects Streptococcus mutans Against Zinc Intoxication

Abstract

Zinc is a trace metal that is essential to all forms of life, but that becomes toxic at high concentrations. Because it has both antimicrobial and anti-inflammatory properties and low toxicity to mammalian cells, zinc has been used as a therapeutic agent for centuries to treat a variety of infectious and non-infectious conditions. While the usefulness of zinc-based therapies in caries prevention is controversial, zinc is incorporated into toothpaste and mouthwash formulations to prevent gingivitis and halitosis. Despite this widespread use of zinc in oral healthcare, the mechanisms that allow Streptococcus mutans, a keystone pathogen in dental caries and prevalent etiological agent of infective endocarditis, to overcome zinc toxicity are largely unknown. Here, we discovered that S. mutans is inherently more tolerant to high zinc stress than all other species of streptococci tested, including commensal streptococci associated with oral health. Using a transcriptome approach, we uncovered several potential strategies utilized by S. mutans to overcome zinc toxicity. Among them, we identified a previously uncharacterized P-type ATPase transporter and cognate transcriptional regulator, which we named ZccE and ZccR respectively, as responsible for the remarkable high zinc tolerance of S. mutans. In addition to zinc, we found that ZccE, which was found to be unique to S. mutans strains, mediates tolerance to at least three additional metal ions, namely cadmium, cobalt, and copper. Loss of the ability to maintain zinc homeostasis when exposed to high zinc stress severely disturbed zinc:manganese ratios, leading to heightened peroxide sensitivity that was alleviated by manganese supplementation. Finally, we showed that the ability of the ΔzccE strain to stably colonize the rat tooth surface after topical zinc treatment was significantly impaired, providing proof of concept that ZccE and ZccR are suitable targets for the development of antimicrobial therapies specifically tailored to kill S. mutans.

Fig 1. Disc diffusion assay indicating that S. mutans is inherently more tolerant to Zn than other streptococci.

Cultures were grown to mid-exponential phase, spread onto BHI plates, and topped with filter paper discs saturated with 1 mM ZnSO₄. Growth inhibition zones (in mm) around the discs were measured after 24 h incubation at 37°C in 5% CO₂. Data represent means and standard deviations of results from 3 independent experiments. One-way ANOVA was used to compare the zones of growth inhibition of each strain to that of S. mutans UA159. A p value of <0.05 was considered significant (*).

Fig 2. Summary of RNA-Seq analysis of S. mutans UA159 treated with 4 mM ZnSO₄ for 15 (A) or 90 (B) minutes compared to untreated control.

The y-axis indicates the log₂ fold change in expression compared to the control, while the x-axis separates genes according to their average expression levels as compared to all other genes. The identities of selected genes of interest are indicated. The differentially expressed genes from both time points were grouped according to Clusters of Orthologous Groups (COG) functional categories [39].

Fig 3. Phylogenetic tree of P-type ATPases including ZccE and 38 other P-type ATPases identified from the literature and from BLASTP searches for comparison.

The tree distance is the calculated distance from the multiple sequence alignment in Clustal Omega. ZccE is highlighted in red.

Fig 4. ZccE is a multi-metal exporter responsible for the high Zn tolerance of S. mutans.

(A-B) Growth curves of S. mutans UA159, ΔzccE and ΔzccE^comp strains in FMC (A) or BHI (B) with or without 1 mM Zn supplementation. Data represent means from 3 independent experiments and the error bars represent standard deviations of the results. A nonlinear curve fit analysis and measuring the doubling time indicated that the growth defect of the ΔzccE strain was statistically significant when compared to either UA159 or ΔzccE^comp strains. (C) Plate titration (spot test) of S. mutans UA159, ΔzccE and ΔzccE^comp strains on BHI agar with or without 1 mM Zn supplementation. (D) Plate titration of S. mutans UA159 and ΔzccE strains on BHI agar supplemented with 4 mM Mn, 4 mM Fe, 1 mM Cu, 1 mM Ni or 5 μM Cd. (C-D) Images were taken after 24 h incubation at 37°C in 5% CO₂ and are representative of at least 3 independent experiments.

Fig 5. CopA plays a minor role in Zn tolerance while ZccE and CopA work interchangeably to protect against Cu intoxication in oxidizing environments.

(A-B) Plate titration (spot test) of S. mutans UA159, ΔzccE, ΔcopYAZ, and ΔzccEΔcopYAZ strains on BHI agar with varying concentrations of Zn (A) or Cu (B). Images were taken after 24 h incubation at 37°C in 5% CO₂ and are representative of at least 3 independent experiments.

Fig 6. The MerR-type transcription factor ZccR is a positive regulator of zccE.

(A) Genetic organization of zccE (smu2057c) and zccR (smu2058) in the S. mutans UA159 chromosome (Created with BioRender.com). (B) qRT-PCR analysis of zccE mRNA expression in the ΔzccR strain relative to the parent UA159 strain before and after exposure to 4 mM Zn. Data represent means and standard deviations of results from 3 independent experiments. One-way ANOVA was used to determine significance (*, p <0.001; ns, not significant). (C) Plate titration (spot test) of UA159, ΔzccE, ΔzccR and ΔzccR^comp strains on BHI with or without 1 mM Zn supplementation. Images were taken after 24 h incubation at 37°C in 5% CO₂ and are representative of at least 3 independent experiments. (D) EMSA showing direct interaction of ZccR with the 160-bp zccE-zccR intergenic region (IGR). To determine ZccR binding specificity, addition of 100X (molar excess) of either non-biotin labeled (cold) zccE-zccR IGR or non-specific competitor DNA (mntH promoter region) was added to the reaction. To explore the role of Zn on ZccR binding activity, 3 mM TPEN (Zn specific chelator) was added to the reaction alone or in the presence of 2.5 mM ZnSO₄.

Fig 7. ICP-MS quantifications of intracellular Zn, Mn, Cu and Fe in UA159, ΔzccE, ΔzccR and respective complemented strains.

Strains were grown in BHI to mid-exponential phase at which point the experimental groups were exposed to 1 mM Zn for 90 minutes. The graphs indicate intracellular parts per billion Zn (A), Mn (B), Cu (C), and Fe (D) normalized by milligram of total protein. Data represent average and standard deviation of values from four independent biological replicates. Two-way ANOVA was used to determine significance between metal content of either the same strain before and after Zn exposure or among different strains after Zn exposure. Error bars represent standard deviations of results from at least 3 independent experiments. A p value of <0.05 was considered significant (*), and only comparisons that were statistically significant are indicated in the figure.

Fig 8. ICP-MS quantifications of intracellular Zn, Cu, Co, and Cd in UA159, ΔzccE, and complemented strains.

Strains were grown in BHI to mid-exponential phase at which point the experimental groups were exposed to a mixture of sub-inhibitory concentrations of the 4 metals (0.5 mM Zn, 0.5 mM Cu, 0.5 mM Co and 2.5 μM Cd) for 90 minutes. The graphs indicate intracellular parts per billion of Zn (A), Cu (B), Co (C) and Cd (D) normalized by milligram of total protein. Two-way ANOVA was used to determine significant differences in intracellular metal content among strains after metal exposure. A p value of <0.05 was considered significant (*). Average and error bars represent standard deviations of results from at least 3 independent experiments.

Fig 9. Disruption of Zn homeostasis increases H₂O₂ sensitivity in the zccE strain.

(A) Growth inhibition zones for S. mutans UA159, ΔzccE, and ΔzccE^Comp strains grown on BHI agar with or without Zn supplementation and exposed to filter paper discs saturated with 0.25% H₂O₂. Two-way ANOVA was used to determine significance (*, p <0.05). (B) Growth inhibition zones of S. mutans UA159 and ΔzccE strains by peroxigenic S. gordonii DL-1 spotted on BHI agar with or without 50 μM Zn supplementation. The S. mutans-S. gordonii competition assay was repeated with catalase overlaid onto the S. gordonii spot to inactivate H₂O₂. Images are representative of results from three independent experiments.

Fig 10. Colonization efficiency of S. mutans UA159 or ΔzccE on the teeth of rats after topical treatment with Zn.

(A-B) Bacterial CFU recovered from rat jaws by plating on (A) MS agar for S. mutans and (B) blood agar for total flora. (C) Percentage of S. mutans-like colonies over total flora were calculated by dividing the values shown in (A) by those shown in (B). The dashed line indicates the limit of detection. One-way ANOVA was used to determine significance, and only statistically significant comparisons with p value <0.05 (*) are indicated in the figure.

Fig 11. Model figure showing that ZccE (a P-type ATPase transporter) and CzcD (a CDF-type transporter) mediate high Zn tolerance in streptococci.

Both czcD and zccE encode for Zn efflux systems and each system is positively regulated by cognate transcriptional regulators (SzcA for czcD and ZccR for zccE) that are activated under high Zn conditions. The CzcD/SzcA pair is found in most streptococci while ZccE/ZccR is unique to S. mutans and, based on Zn sensitivity assays and mutational analysis, is directly responsible for the exceptionally high Zn tolerance of S. mutans when compared to other streptococcal species. AdcABC is an ABC-type transporter conserved among streptococci and primarily responsible for Zn import. This figure was created with Biorender.