MntP and YiiP contribute to manganese efflux in salmonella enterica Serovar Typhimurium under conditions of manganese overload and nitrosative stress

The divalent transition metal cation manganese is important for protein function, particularly under conditions of iron limitation, nitrosative stress, and oxidative stress, but can mediate substantial toxicity in excess. Salmonella enterica serovar Typhimurium possesses multiple manganese importers, but the pathways for manganese efflux remain poorly defined. The S. Typhimurium ATCC 14028s genome was analyzed for putative manganese export pathways, which identified a previously uncharacterized homologue of the Escherichia coli manganese exporter mntP, stm1834, and two cation diffusion facilitator family transporters, zitB (stm0758) and yiiP (stm4061). Manganese acquisition by S. Typhimurium has been shown to occur in response to nitric oxide, an important chemical mediator of the mammalian innate immune response. However, cellular manganese can rapidly return to prechallenge levels, strongly suggesting that one or more S. Typhimurium exporters may contribute to this process. Here, we report that mntP and yiiP contribute to manganese resistance and export in S. Typhimurium. YiiP, also known as FieF, has previously been associated with zinc and iron transport, although its physiological role remains ambiguous due to a lack of zinc-sensitive phenotypes in yiiP mutant strains of S. Typhimurium and E. coli. We report that S. Typhimurium ΔmntP ΔyiiP mutants are exquisitely sensitive to manganese and show that both YiiP and MntP contribute to manganese efflux following nitric oxide exposure. IMPORTANCE Transition metal cations are required for the function of many proteins but can mediate toxicity when present in excess. Identifying transporters that facilitate metal ion export, the conditions under which they are expressed, and the role they play in bacterial physiology is an evolving area of interest for environmental and pathogenic organisms. Determining the native targets of metal transporters has proved challenging since bioinformatic predictions, in vitro transport data, and mutant phenotypes do not always agree. This work identifies two transporters that mediate manganese efflux from the Gram-negative pathogen Salmonella enterica serovar Typhimurium in response to manganese overload and nitric oxide stress. While homologues of MntP have been characterized previously, this is the first observation of YiiP contributing to manganese export.Annie Ouyang, Kendall M. Gasner, Stephanie L. Neville, Christopher A. McDevitt, Elaine R. Frawle

The role of CopA in Streptococcus pyogenes copper homeostasis and virulence

Available online 6 January 2023Maintenance of intracellular metal homeostasis during interaction with host niches is critical to the success of bacterial pathogens. To prevent infection, the mammalian innate immune response employs metal-withholding and metal-intoxication mechanisms to limit bacterial propagation. The first-row transition metal ion copper serves critical roles at the host-pathogen interface and has been associated with antimicrobial activity since antiquity. Despite lacking any known copper-utilizing proteins, streptococci have been reported to accumulate significant levels of copper. Here, we report that loss of CopA, a copper-specific exporter, confers increased sensitivity to copper in Streptococcus pyogenes strain HSC5, with prolonged exposure to physiological levels of copper resulting in reduced viability during stationary phase cultivation. This defect in stationary phase survival was rescued by supplementation with exogeneous amino acids, indicating the pathogen had altered nutritional requirements during exposure to copper stress. Furthermore, S. pyogenes HSC5 ΔcopA was substantially attenuated during murine soft-tissue infection, demonstrating the importance of copper efflux at the host-pathogen interface. Collectively, these data indicate that copper can severely reduce the viability of stationary phase S. pyogenes and that active efflux mechanisms are required to survive copper stress in vitro and during infection.Tina H. Dao, Amy Iverson, Stephanie L. Neville, Michael D.L. Johnson, Christopher A. McDevitt, Jason W. Rosc

The role of ZntA in Klebsiella pneumoniae zinc homeostasis

Klebsiella pneumoniae is an opportunistic Gram-negative pathogen that is a leading cause of healthcare-associated infections, including pneumonia, urinary tract infections, and sepsis. Essential to the colonization and infection by K. pneumoniae is the acquisition of nutrients, such as the transition metal ion zinc. Zinc has crucial structural and catalytic roles in the proteome of all organisms. Nevertheless, in excess, it has the potential to mediate significant toxicity by dysregulating the homeostasis of other transition elements, disrupting enzymatic processes, and perturbing metalloprotein cofactor acquisition. Here, we sought to elucidate the zinc detoxification mechanisms of K. pneumoniae, which remain poorly defined. Using the representative K. pneumoniae AJ218 strain, we showed that the P-type ATPase, ZntA, which is upregulated in response to cellular zinc stress, was the primary zinc efflux pathway. Deletion of zntA rendered K. pneumoniae AJ218 highly susceptible to exogenous zinc stress and manifested as an impaired growth phenotype and increased cellular accumulation of the metal. Loss of zntA also increased sensitivity to cadmium stress, indicating a role for this efflux pathway in cadmium resistance. Disruption of zinc homeostasis in the K. pneumoniae AJ218 ΔzntA strain also impacted manganese and iron homeostasis and was associated with increased production of biofilm. Collectively, this work showed the critical role of ZntA in K. pneumoniae zinc tolerance and provided a foundation for further studies on zinc homeostasis and the future development of novel antimicrobials to target this pathway. IMPORTANCE: Klebsiella pneumoniae is a leading cause of healthcare-associated infections, including pneumonia, urinary tract infections, and sepsis. Treatment of K. pneumoniae infections is becoming increasingly challenging due to high levels of antibiotic resistance and the rising prevalence of carbapenem-resistant, extended-spectrum β-lactamases producing strains. Zinc is essential to the colonization and infection by many bacterial pathogens but toxic in excess. This work described the first dissection of the pathways associated with resisting extracellular zinc stress in K. pneumoniae. This study revealed that the P-type ATPase ZntA was highly upregulated in response to exogenous zinc stress and played a major role in maintaining bacterial metal homeostasis. Knowledge of how this major bacterial pathogen resists zinc stress provided a foundation for antimicrobial development studies to target and abrogate their essential function.Eve A. Maunders, Katherine Ganio, Andrew J. Hayes, Stephanie L. Neville, Mark R. Davies, Richard A. Strugnell, Christopher A. McDevitt, Aimee Ta

The structural basis of bacterial manganese import

Metal ions are essential for all forms of life. In prokaryotes, ATP-binding cassette (ABC) permeases serve as the primary import pathway for many micronutrients including the first-row transition metal manganese. However, the structural features of ionic metal transporting ABC permeases have remained undefined. Here, we present the crystal structure of the manganese transporter PsaBC from Streptococcus pneumoniae in an open-inward conformation. The type II transporter has a tightly closed transmembrane channel due to "extracellular gating" residues that prevent water permeation or ion reflux. Below these residues, the channel contains a hitherto unreported metal coordination site, which is essential for manganese translocation. Mutagenesis of the extracellular gate perturbs manganese uptake, while coordination site mutagenesis abolishes import. These structural features are highly conserved in metal-specific ABC transporters and are represented throughout the kingdoms of life. Collectively, our results define the structure of PsaBC and reveal the features required for divalent cation transport.Stephanie L. Neville, Jennie Sjöhamn, Jacinta A. Watts, Hugo MacDermott-Opeskin, Stephen J. Fairweather, Katherine Ganio, Alex Carey Hulyer, Aaron P. McGrath, Andrew J. Hayes, Tess R. Malcolm, Mark R. Davies, Norimichi Nomura, So Iwata, Megan L. O’Mara, Megan J. Maher, Christopher A. McDevit

Novel Loci for Adiponectin Levels and Their Influence on Type 2 Diabetes and Metabolic Traits : A Multi-Ethnic Meta-Analysis of 45,891 Individuals

J. Kaprio, S. Ripatti ja M.-L. Lokki työryhmien jäseniä.Peer reviewe

Manganese import protects Salmonella enterica serovar Typhimurium against nitrosative stress

Nitric oxide (NO•) is a radical molecule produced by mammalian phagocytic cells as part of the innate immune response to bacterial pathogens. It exerts its antimicrobial activity in part by impairing the function of metalloproteins, particularly those containing iron and zinc cofactors. The pathogenic Gram-negative bacterium Salmonella enterica serovar typhimurium undergoes dynamic changes in its cellular content of the four most common metal cofactors following exposure to NO• stress. Zinc, iron and magnesium all decrease in response to NO• while cellular manganese increases significantly. Manganese acquisition is driven primarily by increased expression of the mntH and sitABCD transporters following derepression of MntR and Fur. ZupT also contributes to manganese acquisition in response to nitrosative stress. S. Typhimurium mutants lacking manganese importers are more sensitive to NO•, indicating that manganese is important for resistance to nitrosative stress.Shehla Yousuf, Joyce E. Karlinsey, Stephanie L. Neville, Christopher A. McDevitt, Stephen J. Libby, Ferric C. Fang and Elaine R. Frawle

Cadmium stress dictates central carbon flux and alters membrane composition in Streptococcus pneumoniae

Metal ion homeostasis is essential for all forms of life. However, the breadth of intracellular impacts that arise upon dysregulation of metal ion homeostasis remain to be elucidated. Here, we used cadmium, a non-physiological metal ion, to investigate how the bacterial pathogen, Streptococcus pneumoniae, resists metal ion stress and dyshomeostasis. By combining transcriptomics, metabolomics and metalloproteomics, we reveal that cadmium stress dysregulates numerous essential cellular pathways including central carbon metabolism, lipid membrane biogenesis and homeostasis, and capsule production at the transcriptional and/or functional level. Despite the breadth of cellular pathways susceptible to metal intoxication, we show that S. pneumoniae is able to maintain viability by utilizing cellular pathways that are predominately metal-independent, such as the pentose phosphate pathway to maintain energy production. Collectively, this work provides insight into the cellular processes impacted by cadmium and how resistance to metal ion toxicity is achieved in S. pneumoniae.Stephanie L. Neville, Bart A. Eijkelkamp, Amber Lothian, James C. Paton, Blaine R. Roberts, Jason W. Rosch and Christopher A. McDevit

Disruption of phosphate homeostasis sensitizes Staphylococcus aureus to nutritional immunity

To control infection, mammals actively withhold essential nutrients, including the transition metal manganese, by a process termed nutritional immunity. A critical component of this host response is the manganese-chelating protein calprotectin. While many bacterial mechanisms for overcoming nutritional immunity have been identified, the intersection between metal starvation and other essential inorganic nutrients has not been investigated. Here, we report that overexpression of an operon encoding a highly conserved inorganic phosphate importer, PstSCAB, increases the sensitivity of Staphylococcus aureus to calprotectin-mediated manganese sequestration. Further analysis revealed that overexpression of pstSCAB does not disrupt manganese acquisition or result in over-accumulation of phosphate by S. aureus However, it does reduce the ability of S. aureus to grow in phosphate-replete defined medium. Overexpression of pstSCAB does not aberrantly activate the phosphate-responsive two-component system PhoPR, nor was this two-component system required for sensitivity to manganese starvation. In a mouse model of systemic staphylococcal disease, a pstSCAB-overexpressing strain is significantly attenuated compared to wild-type S. aureus This defect is partially reversed in a calprotectin-deficient mouse, in which manganese is more readily available. Given that expression of pstSCAB is regulated by PhoPR, these findings suggest that over-activation of PhoPR would diminish the ability of S. aureus to resist nutritional immunity and cause infection. As PhoPR is also necessary for bacterial virulence, these findings imply that phosphate homeostasis represents a critical regulatory node whose activity must be precisely controlled in order for S. aureus and other pathogens to cause infection.Jessica L. Kelliher, Erin B. Brazel, Jana N. Radin, Eliot S. Joya, Paola K. Párraga Solórzano ... Christopher A. McDevitt ... et al