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

Abstract

Zinc is an essential trace metal to all forms of life 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 factor, which we respectively named ZccE and ZccR, 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 Zn:Mn ratios leading to heightened peroxide sensitivity that was alleviated by excess 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.

Metal Homeostasis in Pathogenic Streptococci

“…The most well characterized Zn efflux system is CzcD, which has been described in a few pathogenic species including S. agalactiae , S. pneumoniae , and S. pyogenes [ 16 , 58 , 88 ]. Recently, a unique P-type ATPase exporter named ZccE was also discovered in S. mutans , which, notably, does not have a CzcD homolog [ 30 ]. ZccE is shown to contribute to Zn tolerance and virulence, which is discussed in a later section.…”

“…Zn, Zn transport systems, and Pht proteins are also known to contribute to Streptococcal adherence, invasion, and biofilm formation [ 26 , 30 , 88 , 100 , 105 , 106 , 107 ]. For example, S. agalactiae and S. pyogenes AdcAII/Lmb/Lsp homologues can bind to the extracellular matrix protein laminin, which is important for adhesion and invasion into epithelial cells [ 107 , 108 , 109 ].…”

“…This has been shown in S. pneumoniae , S. pyogenes , S. mutans , and S. suis [ 28 , 106 , 111 , 112 ]. The Zn efflux systems in Streptococci are, therefore, necessary to overcome Zn toxicity by ridding the cell of extra Zn and contributing to Streptococcal infections [ 16 , 30 , 58 , 96 , 113 , 114 , 115 ].…”

“…Overall, Streptococci have multiple mechanisms to maintain Zn homeostasis in different environments, including import and export systems, Zn binding proteins, and Zn-dependent regulatory factors. However, there are still ranges of tolerance for Zn starvation and intoxication depending on the species and strain variability, which has been exemplified in recent publications [ 30 , 116 ]. Therefore, the role of Zn in Streptococcus spp.…”