Intracellular Accumulation of Staphylopine Can Sensitize Staphylococcus aureus to Host-Imposed Zinc Starvation by Chelation-Independent Toxicity

Abstract

The host restricts the availability of zinc to prevent infection. To overcome this defense, Staphylococcus aureus and Pseudomonas aeruginosa rely on zincophore-dependent zinc importers. Synthesis of the zincophore staphylopine by S. aureus and its import are both necessary for the bacterium to cause infection. In this study, we sought to elucidate how loss of zincophore efflux impacts bacterial resistance to host-imposed zinc starvation. In culture and during infection, mutants lacking CntE, the staphylopine efflux pump, were more sensitive to zinc starvation imposed by the metal-binding immune effector calprotectin than those lacking the ability to import staphylopine. However, disruption of staphylopine synthesis reversed the enhanced sensitivity phenotype of the ΔcntE mutant to calprotectin, indicating that intracellular toxicity of staphylopine is more detrimental than the impaired ability to acquire zinc. Unexpectedly, intracellular accumulation of staphylopine does not increase the expression of metal importers or alter cellular metal concentrations, suggesting that, contrary to prevailing models, the toxicity associated with staphylopine is not strictly due to intracellular chelation of metals. As P. aeruginosa and other pathogens produce zincophores with similar chemistry, our observations on the crucial importance of zincophore efflux are likely to be broadly relevant. IMPORTANCE Staphylococcus aureus and many other bacterial pathogens rely on metal-binding small molecules to obtain the essential metal zinc during infection. In this study, we reveal that export of these small molecules is critical for overcoming host-imposed metal starvation during infection and prevents toxicity due to accumulation of the metal-binding molecule within the cell. Surprisingly, we found that intracellular toxicity of the molecule is not due to chelation of cellular metals.

Bioinformatic Mapping of Opine-Like Zincophore Biosynthesis in Bacteria

“…If this is true, zincophore systems may be excellent potential targets for antimicrobial therapies. One therapeutic avenue could be to inhibit zincophore efflux, since deletion of CntE in S. aureus causes increased sensitivity to calprotectin in vitro and a dramatic reduction in virulence ( 41 , 42 ). Similarly, in P. aeruginosa , deletion of the efflux system CntI causes a growth defect that is rescued by preventing pseudopaline synthesis, as well as causing a reduction in virulence in burn wound and lung infection models ( 26 , 29 ).…”

Zinc: Multidimensional Effects on Living Organisms

“…The intracellular concentration of zinc must be tightly controlled to ensure that it is high enough to sustain life but low enough not to cause toxic effects that can lead to cell death [ 6 , 12 , 85 , 86 , 87 , 88 ]. Through the molecular interactions with biomolecules, zinc participates in a wide variety of metabolic processes as well as repair and maintenance of cell structures and biomolecules that are essential for bacterial cell growth [ 7 , 13 , 81 , 89 , 90 ]. However, higher than needed concentrations of zinc are highly detrimental to the life of the bacterial cell.…”

“…While nutritional immunity produces deprivation of the metal ions, intracellular and other niches have the opposite effect [ 50 , 56 ]. As a consequence of the strict requirements for intracellular zinc concentrations, bacteria evolved several tightly regulated uptake, efflux, binding, and sensing mechanisms critical for pathogenicity [ 6 , 12 , 23 , 24 , 25 , 33 , 34 , 35 , 36 , 37 , 76 , 86 , 89 , 90 , 91 , 92 , 93 , 94 ]. This section will discuss the major and better-known mechanisms of zinc import and export.…”

“…Although other zincophores were subsequently discovered, research on these elements is in its infancy. Many more will most probably be detected and characterized in the future [ 90 , 114 , 115 , 116 ].…”

Zinc: Multidimensional Effects on Living Organisms

“…Although other zincophores were subsequently discovered, research on these elements is in its infancy. Many more will most probably be detected and characterized in the future [100,[122][123][124].…”