Manganese Homeostasis in Group A Streptococcus Is Critical for Resistance to Oxidative Stress and Virulence

Abstract

Streptococcus pyogenes (group A Streptococcus [GAS]) is an obligate human pathogen responsible for a spectrum of human disease states. Metallobiology of human pathogens is revealing the fundamental role of metals in both nutritional immunity leading to pathogen starvation and metal poisoning of pathogens by innate immune cells. Spy0980 (MntE) is a paralog of the GAS zinc efflux pump CzcD. Through use of an isogenic mntE deletion mutant in the GAS serotype M1T1 strain 5448, we have elucidated that MntE is a manganese-specific efflux pump required for GAS virulence. The 5448ΔmntE mutant had significantly lower survival following infection of human neutrophils than did the 5448 wild type and the complemented mutant (5448ΔmntE::mntE). Manganese homeostasis may provide protection against oxidative stress, explaining the observed ex vivo reduction in virulence. In the presence of manganese and hydrogen peroxide, 5448ΔmntE mutant exhibits significantly lower survival than wild-type 5448 and the complemented mutant. We hypothesize that MntE, by maintaining homeostatic control of cytoplasmic manganese, ensures that the peroxide response repressor PerR is optimally poised to respond to hydrogen peroxide stress. Creation of a 5448ΔmntE-ΔperR double mutant rescued the oxidative stress resistance of the double mutant to wild-type levels in the presence of manganese and hydrogen peroxide. This work elucidates the mechanism for manganese toxicity within GAS and the crucial role of manganese homeostasis in maintaining GAS virulence.

The PerR-Regulated P 1B-4 -Type ATPase (PmtA) Acts as a Ferrous Iron Efflux Pump in Streptococcus pyogenes

“…The necessity of maintaining the Fe/Mn ratio in Gram-positive bacteria such as Streptococcus pneumoniae and GAS is particularly important for defense against oxidative stress and pathogenesis (48)(49)(50). During oxidative stress, mismetallation of proteins can occur when the Fe/Mn ratio is disrupted (44).…”

“…All GAS mutants were constructed according to standard operating procedures (50). Briefly, the construction of 5448ΔpmtA was achieved by the amplification of 5448 WT genomic DNA with primers pmtA-KO-1 (incorporating an XhoI site at the 5= end) and pmtA-KO-2 (with homology to Km r at the 5= end) to create the 5=-flanking region of pmtA.…”

Functional Determinants of Metal Ion Transport and Selectivity in Paralogous Cation Diffusion Facilitator Transporters CzcD and MntE in Streptococcus pneumoniae

“…CDFs are a large family of divalent metal/H ϩ antiporters that collectively possess broad substrate (metal) specificity and play important roles in global intracellular metal homeostasis in bacteria and in the pathogenicity of bacteria (18,(20)(21)(22). CDF transporters are dimeric, with each protomer containing an N-terminal six-helical transmembrane domain (TMD; transmembrane [TM] helices TM1 to TM6) and a C-terminal mixed ␣/␤ cytoplasmic domain (CTD) that varies in length ( Fig.…”

“…Previous studies have consistently shown that pneumococcal cells lacking czcD are specifically sensitive to Zn toxicity, while mntE mutants are sensitive to Mn overload (21,22,34,35). During metal stress, czcD and mntE mutants accumulate Zn and Mn, respectively.…”

“…Regardless of the mechanism of metal toxicity, bacteria have evolved ways to minimize the deleterious impact of metal ion excess. One well-characterized strategy involves the use of metal efflux transporters, such as cation diffusion facilitator (CDFs) (18) and P-type ATPase proteins (19), to remove excess metal ions from the cytoplasm.CDFs are a large family of divalent metal/H ϩ antiporters that collectively possess broad substrate (metal) specificity and play important roles in global intracellular metal homeostasis in bacteria and in the pathogenicity of bacteria (18,(20)(21)(22). CDF transporters are dimeric, with each protomer containing an N-terminal six-helical transmembrane domain (TMD; transmembrane [TM] helices TM1 to TM6) and a C-terminal mixed ␣/␤ cytoplasmic domain (CTD) that varies in length ( Fig.…”