The PerR-Regulated P _1B-4 -Type ATPase (PmtA) Acts as a Ferrous Iron Efflux Pump in Streptococcus pyogenes

Abstract

Streptococcus pyogenes (group A Streptococcus [GAS]) is an obligate human pathogen responsible for a broad spectrum of human disease. GAS has a requirement for metal homeostasis within the human host and, as such, tightly modulates metal uptake and efflux during infection. Metal acquisition systems are required to combat metal sequestration by the host, while metal efflux systems are essential to protect against metal overload poisoning. Here, we investigated the function of PmtA (PerRregulated metal transporter A), a P 1B-4 -type ATPase efflux pump, in invasive GAS M1T1 strain 5448. We reveal that PmtA functions as a ferrous iron [Fe(II)] efflux system. In the presence of high Fe(II) concentrations, the 5448ΔpmtA deletion mutant exhibited diminished growth and accumulated 5-fold-higher levels of intracellular Fe(II) than did the wild type and the complemented mutant. The 5448ΔpmtA deletion mutant also showed enhanced susceptibility to killing by the Fe-dependent antibiotic streptonigrin as well as increased sensitivity to hydrogen peroxide and superoxide. We suggest that the PerRmediated control of Fe(II) efflux by PmtA is important for bacterial defense against oxidative stress. PmtA represents an exemplar for an Fe(II) efflux system in a host-adapted Gram-positive bacterial pathogen.KEYWORDS iron efflux, PmtA, oxidative stress response, PerR, group A Streptococcus, Streptococcus pyogenes D efense against peroxide is recognized as one of the most widespread stress responses in prokaryotes. A characteristic of the peroxide stress response is the increased expression of peroxidases that can directly remove hydrogen peroxide as well as the induction of systems that can repair damaged proteins and nucleic acids (1, 2). Streptococcus pyogenes (group A Streptococcus [GAS]) coordinates oxidative stress responses through the action of the regulator PerR, which functions as a repressor of oxidative stress defense genes under normal conditions through binding to DNA at per boxes upstream of these genes (3, 4). This stable complex typically exists with Fe as a prosthetic group; in the presence of hydrogen peroxide, Fe causes metal-catalyzed oxidation and damage of the complex, leading to its dissociation and the subsequent transcription of peroxide response genes (3-6). In GAS, the PerR-regulated oxidative stress response encompasses both direct mechanisms, involving the detoxification of reactive oxygen species (ROS), and indirect mechanisms, which involve the repair of biomolecules damaged by oxidative stress. Direct mechanisms involving the enzymatic detoxification of reactive oxygen species are achieved through alkyl hydroperoxidases and glutathione peroxidases (1, 2, 5) as well as a single superoxide dismutase, SodA, which is Mn dependent (7). An example of an indirect response mechanism is PolAI, a DNA polymerase that repairs oxidatively damaged DNA (8).

Ferrous iron efflux systems in bacteria

“…Two recent studies have confirmed the primary role of PmtA as an Fe 2+ -efflux pump 38, 39 . PmtA is important for resistance to iron intoxication, and a pmtA null mutant accumulates elevated levels of intracellular iron.…”

“…The P 1B4 -type ATPases were originally assigned a role in Co 2+ export, based on the properties of some of the first characterized members 53 . However, P 1B4 -type ATPases have recently been found to function instead, or in addition, as Fe 2+ efflux transporters including Bacillus subtilis PfeT 36 , Listeria monocytogenes FrvA 27 , Mycobacterium tuberculosis CtpD 37 , and group A Streptococcus PmtA 38, 39 .…”

“…In E. coli , H 2 O 2 induces an OxyR-activated Mn 2+ uptake system (MntH) 33, 34 , and in B. subtilis H 2 O 2 induces a PerR-regulated iron efflux system, PfeT 35, 36 . PfeT is a member of the P 1B4 -type ATPases, and recent results indicate that several close homologs also function as Fe 2+ efflux pumps 27, 37–39 . Fe 2+ efflux pumps have now been documented in a wide variety of bacteria, and include P 1B -type ATPases, cation diffusion facilitator (CDF) proteins, major facilitator superfamily (MFS) proteins, and membrane bound ferritin-like proteins (Table 1 & Fig.…”

Redox-Regulated Adaptation of Streptococcus oligofermentans to Hydrogen Peroxide Stress

“…Metal homeostasis plays a central role in oxidative stress resistance in Gram-positive bacteria (4,44,47,48). In addition to PerR, streptococci also employ MntR, a metalloregulator protein, to control cellular manganese and iron homeostasis (11,16,21,25). Oxidative inactivation of PerR and MntR derepresses the expression of the metal homeostasis-related genes dpr and mntABC (Table 3) (25).…”

PmtA functions as a ferrous iron and cobalt efflux pump in Streptococcus suis

“…Consequently, bacteria have evolved complex systems to maintain metal homeostasis, such as efflux or sequestration of metals [6]. Moreover, there is mounting evidence that metal homeostasis, maintained via metal efflux pumps, plays a critical role in bacterial physiology and pathogenesis [5][6][7][8][9][10][11].…”