Iron Efflux by PmtA Is Critical for Oxidative Stress Resistance and Contributes Significantly to Group A Streptococcus Virulence

Abstract

Group A Streptococcus (GAS) is a human-only pathogen that causes a spectrum of disease conditions. Given its survival in inflamed lesions, the ability to sense and overcome oxidative stress is critical for GAS pathogenesis. PerR senses oxidative stress and coordinates the regulation of genes involved in GAS antioxidant defenses. In this study, we investigated the role of PerR-controlled metal transporter A (PmtA) in GAS pathogenesis. Previously, PmtA was implicated in GAS antioxidant defenses and suggested to protect against zinc toxicity. Here, we report that PmtA is a P 1B4 -type ATPase that functions as an Fe(II) exporter and aids GAS defenses against iron intoxication and oxidative stress. The expression of pmtA is specifically induced by excess iron, and this induction requires PerR. Furthermore, a pmtA mutant exhibited increased sensitivity to iron toxicity and oxidative stress due to an elevated intracellular accumulation of iron. RNA-sequencing analysis revealed that GAS undergoes significant alterations in gene expression to adapt to iron toxicity. Finally, using two mouse models of invasive infection, we demonstrated that iron efflux by PmtA is critical for bacterial survival during infection and GAS virulence. Together, these data demonstrate that PmtA is a key component of GAS antioxidant defenses and contributes significantly to GAS virulence.KEYWORDS bacterial pathogenesis, gene regulation, iron efflux, metal homeostasis, oxidative stress I ron is a critical micronutrient required for bacterial survival and proliferation due to its role as a cofactor in cellular macromolecules involved in metabolism and electron transport. Given the bacterial requirement for iron, hosts limit iron availability to pathogens by using a variety of extracellular and intracellular mechanisms (1, 2). The eukaryotic host recruits an array of extracellular antimicrobial factors to chelate iron present at the microbial colonization surfaces to retard bacterial growth (2-4). As a countermeasure, pathogens employ high-affinity iron importers and iron-scavenging siderophores to acquire metal ions from nutrient-sparse infection sites (5). Consistent with this, the inactivation of iron importers resulted in an attenuated virulence of several pathogens, underscoring their importance to bacterial pathogenesis (5-7). Emerging evidence indicates that the host also employs metal toxicity at microbial colonization surfaces, predominantly within phagosomes, to mediate microbial killing and control bacterial infection (8,9). Host innate immune cells, such as neutrophils and macrophages, accumulate copper and zinc around phagocytosed bacteria and impose metal toxicity (8-12). Conversely, bacteria employ metal efflux pumps to reduce the intracellular metal concentration and negate host-induced metal toxicity (8,11,12). In

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 .…”

“…Since GAS is catalase negative, PmtA might be a frontline defense against peroxide stress. PmtA is also a critical virulence factor and is required for survival during infection in both intramuscular and subcutaneous mouse models 38 , which again links iron efflux and peroxide resistance to pathogen virulence.…”

Zinc’ing it out: zinc homeostasis mechanisms and their impact on the pathogenesis of human pathogen group A streptococcus

“…79 Given that pmtA expression was specifically induced by iron excess, and inactivation of pmtA resulted in increased bacterial sensitivity to iron stress and elevated levels of intracellular iron, it was further confirmed that iron, not zinc, is the physiological ligand for PmtA. 79 However, similar studies with CzcD indicated that zinc is its cognate ligand (Fig. 1).…”

“…Although PmtA was originally thought to be a zinc efflux pump, 69 recent studies showed that PmtA aids GAS antioxidant defense by exporting excess iron out of the bacterial cytosol. 79 Given that pmtA expression was specifically induced by iron excess, and inactivation of pmtA resulted in increased bacterial sensitivity to iron stress and elevated levels of intracellular iron, it was further confirmed that iron, not zinc, is the physiological ligand for PmtA. 79 However, similar studies with CzcD indicated that zinc is its cognate ligand (Fig.…”