SpoT Induces Intracellular <italic toggle="yes">Salmonella</italic> Virulence Programs in the Phagosome

ABSTRACT Guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), together named (p)ppGpp, regulate diverse aspects of Salmonella pathogenesis, including synthesis of nutrients, resistance to inflammatory mediators, and expression of secretion systems. In Salmonella, these nucleotide...

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Autores principales:	Liam F. Fitzsimmons, Lin Liu, Sashi Kant, Ju-Sim Kim, James K. Till, Jessica Jones-Carson, Steffen Porwollik, Michael McClelland, Andres Vazquez-Torres
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	Salmonella bacterial pathogenesis genetics innate immunity macrophages stringent response Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/c6f901b0ad8541e8ac3c523e7681884f
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spelling	oai:doaj.org-article:c6f901b0ad8541e8ac3c523e7681884f2021-11-15T15:56:58ZSpoT Induces Intracellular <italic toggle="yes">Salmonella</italic> Virulence Programs in the Phagosome10.1128/mBio.03397-192150-7511https://doaj.org/article/c6f901b0ad8541e8ac3c523e7681884f2020-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.03397-19https://doaj.org/toc/2150-7511ABSTRACT Guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), together named (p)ppGpp, regulate diverse aspects of Salmonella pathogenesis, including synthesis of nutrients, resistance to inflammatory mediators, and expression of secretion systems. In Salmonella, these nucleotide alarmones are generated by the synthetase activities of RelA and SpoT proteins. In addition, the (p)ppGpp hydrolase activity of the bifunctional SpoT protein is essential to preserve cell viability. The contribution of SpoT to physiology and pathogenesis has proven elusive in organisms such as Salmonella, because the hydrolytic activity of this RelA and SpoT homologue (RSH) is vital to prevent inhibitory effects of (p)ppGpp produced by a functional RelA. Here, we describe the biochemical and functional characterization of a spoT-Δctd mutant Salmonella strain encoding a SpoT protein that lacks the C-terminal regulatory elements collectively referred to as “ctd.” Salmonella expressing the spoT-Δctd variant hydrolyzes (p)ppGpp with similar kinetics to those of wild-type bacteria, but it is defective at synthesizing (p)ppGpp in response to acidic pH. Salmonella spoT-Δctd mutants have virtually normal adaptations to nutritional, nitrosative, and oxidative stresses, but poorly induce metal cation uptake systems and Salmonella pathogenicity island 2 (SPI-2) genes in response to the acidic pH of the phagosome. Importantly, spoT-Δctd mutant Salmonella replicates poorly intracellularly and is attenuated in a murine model of acute salmonellosis. Collectively, these investigations indicate that (p)ppGpp synthesized by SpoT serves a unique function in the adaptation of Salmonella to the intracellular environment of host phagocytes that cannot be compensated by the presence of a functional RelA. IMPORTANCE Pathogenic bacteria experience nutritional challenges during colonization and infection of mammalian hosts. Binding of the alarmone nucleotide guanosine tetraphosphate (ppGpp) to RNA polymerase coordinates metabolic adaptations and virulence gene transcription, increasing the fitness of diverse Gram-positive and Gram-negative bacteria as well as that of actinomycetes. Gammaproteobacteria such as Salmonella synthesize ppGpp by the combined activities of the closely related RelA and SpoT synthetases. Due to its profound inhibitory effects on growth, ppGpp must be removed; in Salmonella, this process is catalyzed by the vital hydrolytic activity of the bifunctional SpoT protein. Because SpoT hydrolase activity is essential in cells expressing a functional RelA, we have a very limited understanding of unique roles these two synthetases may assume during interactions of bacterial pathogens with their hosts. We describe here a SpoT truncation mutant that lacks ppGpp synthetase activity and all C-terminal regulatory domains but retains excellent hydrolase activity. Our studies of this mutant reveal that SpoT uniquely senses the acidification of phagosomes, inducing virulence programs that increase Salmonella fitness in an acute model of infection. Our investigations indicate that the coexistence of RelA/SpoT homologues in a bacterial cell is driven by the need to mount a stringent response to a myriad of physiological and host-specific signatures.Liam F. FitzsimmonsLin LiuSashi KantJu-Sim KimJames K. TillJessica Jones-CarsonSteffen PorwollikMichael McClellandAndres Vazquez-TorresAmerican Society for MicrobiologyarticleSalmonellabacterial pathogenesisgeneticsinnate immunitymacrophagesstringent responseMicrobiologyQR1-502ENmBio, Vol 11, Iss 1 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Salmonella bacterial pathogenesis genetics innate immunity macrophages stringent response Microbiology QR1-502
spellingShingle	Salmonella bacterial pathogenesis genetics innate immunity macrophages stringent response Microbiology QR1-502 Liam F. Fitzsimmons Lin Liu Sashi Kant Ju-Sim Kim James K. Till Jessica Jones-Carson Steffen Porwollik Michael McClelland Andres Vazquez-Torres SpoT Induces Intracellular <italic toggle="yes">Salmonella</italic> Virulence Programs in the Phagosome
description	ABSTRACT Guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), together named (p)ppGpp, regulate diverse aspects of Salmonella pathogenesis, including synthesis of nutrients, resistance to inflammatory mediators, and expression of secretion systems. In Salmonella, these nucleotide alarmones are generated by the synthetase activities of RelA and SpoT proteins. In addition, the (p)ppGpp hydrolase activity of the bifunctional SpoT protein is essential to preserve cell viability. The contribution of SpoT to physiology and pathogenesis has proven elusive in organisms such as Salmonella, because the hydrolytic activity of this RelA and SpoT homologue (RSH) is vital to prevent inhibitory effects of (p)ppGpp produced by a functional RelA. Here, we describe the biochemical and functional characterization of a spoT-Δctd mutant Salmonella strain encoding a SpoT protein that lacks the C-terminal regulatory elements collectively referred to as “ctd.” Salmonella expressing the spoT-Δctd variant hydrolyzes (p)ppGpp with similar kinetics to those of wild-type bacteria, but it is defective at synthesizing (p)ppGpp in response to acidic pH. Salmonella spoT-Δctd mutants have virtually normal adaptations to nutritional, nitrosative, and oxidative stresses, but poorly induce metal cation uptake systems and Salmonella pathogenicity island 2 (SPI-2) genes in response to the acidic pH of the phagosome. Importantly, spoT-Δctd mutant Salmonella replicates poorly intracellularly and is attenuated in a murine model of acute salmonellosis. Collectively, these investigations indicate that (p)ppGpp synthesized by SpoT serves a unique function in the adaptation of Salmonella to the intracellular environment of host phagocytes that cannot be compensated by the presence of a functional RelA. IMPORTANCE Pathogenic bacteria experience nutritional challenges during colonization and infection of mammalian hosts. Binding of the alarmone nucleotide guanosine tetraphosphate (ppGpp) to RNA polymerase coordinates metabolic adaptations and virulence gene transcription, increasing the fitness of diverse Gram-positive and Gram-negative bacteria as well as that of actinomycetes. Gammaproteobacteria such as Salmonella synthesize ppGpp by the combined activities of the closely related RelA and SpoT synthetases. Due to its profound inhibitory effects on growth, ppGpp must be removed; in Salmonella, this process is catalyzed by the vital hydrolytic activity of the bifunctional SpoT protein. Because SpoT hydrolase activity is essential in cells expressing a functional RelA, we have a very limited understanding of unique roles these two synthetases may assume during interactions of bacterial pathogens with their hosts. We describe here a SpoT truncation mutant that lacks ppGpp synthetase activity and all C-terminal regulatory domains but retains excellent hydrolase activity. Our studies of this mutant reveal that SpoT uniquely senses the acidification of phagosomes, inducing virulence programs that increase Salmonella fitness in an acute model of infection. Our investigations indicate that the coexistence of RelA/SpoT homologues in a bacterial cell is driven by the need to mount a stringent response to a myriad of physiological and host-specific signatures.
format	article
author	Liam F. Fitzsimmons Lin Liu Sashi Kant Ju-Sim Kim James K. Till Jessica Jones-Carson Steffen Porwollik Michael McClelland Andres Vazquez-Torres
author_facet	Liam F. Fitzsimmons Lin Liu Sashi Kant Ju-Sim Kim James K. Till Jessica Jones-Carson Steffen Porwollik Michael McClelland Andres Vazquez-Torres
author_sort	Liam F. Fitzsimmons
title	SpoT Induces Intracellular <italic toggle="yes">Salmonella</italic> Virulence Programs in the Phagosome
title_short	SpoT Induces Intracellular <italic toggle="yes">Salmonella</italic> Virulence Programs in the Phagosome
title_full	SpoT Induces Intracellular <italic toggle="yes">Salmonella</italic> Virulence Programs in the Phagosome
title_fullStr	SpoT Induces Intracellular <italic toggle="yes">Salmonella</italic> Virulence Programs in the Phagosome
title_full_unstemmed	SpoT Induces Intracellular <italic toggle="yes">Salmonella</italic> Virulence Programs in the Phagosome
title_sort	spot induces intracellular <italic toggle="yes">salmonella</italic> virulence programs in the phagosome
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/c6f901b0ad8541e8ac3c523e7681884f
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SpoT Induces Intracellular <italic toggle="yes">Salmonella</italic> Virulence Programs in the Phagosome

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