Research Article: Zinc-dependent substrate-level phosphorylation powers Salmonella growth under nitrosative stress of the innate host response

Date Published: October 26, 2018

Publisher: Public Library of Science

Author(s): Liam Fitzsimmons, Lin Liu, Steffen Porwollik, Sangeeta Chakraborty, Prerak Desai, Timothy Tapscott, Calvin Henard, Michael McClelland, Andres Vazquez-Torres, Eric P. Skaar.

http://doi.org/10.1371/journal.ppat.1007388

Abstract

The metabolic processes that enable the replication of intracellular Salmonella under nitrosative stress conditions engendered in the innate response of macrophages are poorly understood. A screen of Salmonella transposon mutants identified the ABC-type high-affinity zinc uptake system ZnuABC as a critical determinant of the adaptation of Salmonella to the nitrosative stress generated by the enzymatic activity of inducible nitric oxide (NO) synthase of mononuclear phagocytic cells. NO limits the virulence of a znuB mutant in an acute murine model of salmonellosis. The ZnuABC transporter is crucial for the glycolytic function of fructose bisphosphate aldolase, thereby fueling growth of Salmonella during nitrosative stress produced in the innate response of macrophages. Our investigations demonstrate that glycolysis mediates resistance of Salmonella to the antimicrobial activity of NO produced in an acute model of infection. The ATP synthesized by substrate-level phosphorylation at the payoff phase of glycolysis and acetate fermentation powers the replication of Salmonella experiencing high levels of nitrosative stress. In contrast, despite its high potential for ATP synthesis, oxidative phosphorylation is a major target of inhibition by NO and contributes little to the antinitrosative defenses of intracellular Salmonella. Our investigations have uncovered a previously unsuspected conjunction between zinc homeostasis, glucose metabolism and cellular energetics in the adaptation of intracellular Salmonella to the reactive nitrogen species synthesized in the innate host response.

Partial Text

Many of the more than 2,500 serovars of Salmonella enterica cause gastrointestinal or disseminated infections in millions of people and livestock every year [1, 2]. Reactive nitrogen species synthesized abiotically in the gastric lumen and the extreme acidity of the stomach constitute a formidable barrier to most microorganisms. However, Salmonella and other enteropathogens can endure these innate host defenses [3]. In the gastrointestinal tract, Salmonella competes for nutrients and space with members of the resident microbiota and, aided by the cytoskeletal remodeling induced by effectors of the Salmonella pathogenicity island-1 (SPI-1) type-III secretion system, forces its way into enterocytes and M cells of Peyer’s patches. The Salmonella SPI-1 effector SopB activates the transcription of Nos2-encoded inducible nitric oxide synthase (iNOS) long after invasion [4, 5]. Transcription of Nos2 is independently activated in mononuclear phagocytic cells in response to lipopolysaccharide, fimbriae or porins imbedded in Salmonella’s cell envelope [6]. The iNOS flavohemoprotein synthesizes nitric oxide (NO) from the guanidino group of L-arginine and molecular oxygen (O2) [6–8]. The diatomic gas NO combines with O2, superoxide anion, iron and low-molecular weight thiols, generating a plethora of reactive nitrogen species that are endowed with vigorous antimicrobial activity [6].

Zn2+, the second most abundant metal cofactor, provides structural, regulatory, antioxidant, and catalytic properties to diverse metalloproteins [39, 40]. Given its critical importance in bacterial cell physiology, mammalian hosts actively limit the bioavailability of Zn2+ to bacterial pathogens, thereby contributing to what is now known as nutritional immunity [41]. As a countermeasure, bacterial pathogens use high affinity Zn2+ transporters, such as ZnuABC or the Gram-positive AdcABC orthologue, to compete with the host for zinc [41]. Interestingly, Salmonella utilizes ZnuABC to compete for zinc with indigenous microbiota of the gut, and exploits this high-affinity uptake system to gain advantages in systemic sites and macrophages [33, 42]. The mechanisms underlying poor growth of ΔznuB Salmonella within macrophages have not been identified yet. Herein, we show that ZnuABC potentiates Salmonella pathogenesis by in part antagonizing the nitrosative stress generated in the innate response of macrophages and mice. Despite its widespread utilization in metabolism and multiple regulatory processes, the scarcity of zinc in ΔznuB Salmonella is particularly detrimental to zinc-dependent fructose bisphosphate aldolase in glycolysis as suggested by the complementation of the intracellular growth defects of ΔznuB Salmonella with the fbaB gene encoding zinc-independent fructose bisphosphate aldolase. Thus, not only does high affinity zinc uptake defend microbes against the metabolic stress associated with either nutritional immunity or competition with microbiota, but it also arms bacteria with the glycolytic flexibility needed to overcome the deficiencies in energetics that are triggered by the inhibition of cytochromes by NO of the innate response of professional phagocytes.

 

Source:

http://doi.org/10.1371/journal.ppat.1007388

 

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