Identification of the main glutamine and glutamate transporters in <i>Staphylococcus aureus</i> and their impact on c‐di‐AMP production

Zeden, Merve S; Kviatkovski, Igor; Schuster, Christopher; Thomas, Vinai Chittezham; Fey, Paul D.; Gründling, Angelika

doi:10.1111/mmi.14479

Cited by 30 publications

(28 citation statements)

References 67 publications

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“…Recent work in S. aureus and B. subtilis identified suppressor mutations in Gln and Glu transporters, respectively, which rescued the growth of mutants devoid of c-di-AMP (9,38). These results align well with those found in L. lactis and suggest that cells defective in c-di-AMP production are unable to regulate intracellular levels of major osmolyte amino acids.…”

supporting

confidence: 76%

Cyclic di-AMP Oversight of Counter-Ion Osmolyte Pools Impacts Intrinsic Cefuroxime Resistance in Lactococcus lactis

Pham

Shi

Xiang

et al. 2021

mBio

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The broadly conserved cyclic di-AMP (c-di-AMP) is a conditionally essential bacterial second messenger. The pool of c-di-AMP is fine-tuned through diadenylate cyclase and phosphodiesterase activities, and direct binding of c-di-AMP to proteins and riboswitches allows the regulation of a broad spectrum of cellular processes. c-di-AMP has a significant impact on intrinsic β-lactam antibiotic resistance in Gram-positive bacteria; however, the reason for this is currently unclear. In this work, genetic studies revealed that suppressor mutations that decrease the activity of the potassium (K+) importer KupB or the glutamine importer GlnPQ restore cefuroxime (CEF) resistance in diadenylate cyclase (cdaA) mutants of Lactococcus lactis. Metabolite analyses showed that glutamine is imported by GlnPQ and then rapidly converted to glutamate, and GlnPQ mutations or c-di-AMP negatively affects the pools of the most abundant free amino acids (glutamate and aspartate) during growth. In a high-c-di-AMP mutant, GlnPQ activity could be increased by raising the internal K+ level through the overexpression of a c-di-AMP-insensitive KupB variant. These results demonstrate that c-di-AMP reduces GlnPQ activity and, therefore, the level of the major free anions in L. lactis through its inhibition of K+ import. Excessive ion accumulation in cdaA mutants results in greater spontaneous cell lysis under hypotonic conditions, while CEF-resistant suppressors exhibit reduced cell lysis and lower osmoresistance. This work demonstrates that the overaccumulation of major counter-ion osmolyte pools in c-di-AMP-defective mutants of L. lactis causes cefuroxime sensitivity. IMPORTANCE The bacterial second messenger cyclic di-AMP (c-di-AMP) is a global regulator of potassium homeostasis and compatible solute uptake in many Gram-positive bacteria, making it essential for osmoregulation. The role that c-di-AMP plays in β-lactam resistance, however, is unclear despite being first identified a decade ago. Here, we demonstrate that the overaccumulation of potassium or free amino acids leads to cefuroxime sensitivity in Lactococcus lactis mutants partially defective in c-di-AMP synthesis. It was shown that c-di-AMP negatively affects the levels of the most abundant free amino acids (glutamate and aspartate) in L. lactis. Regulation of these major free anions was found to occur via the glutamine transporter GlnPQ, whose activity increased in response to intracellular potassium levels, which are under c-di-AMP control. Evidence is also presented showing that they are major osmolytes that enhance osmoresistance and cell lysis. The regulatory reach of c-di-AMP can be extended to include the main free anions in bacteria.

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supporting

confidence: 76%

Cyclic di-AMP Oversight of Counter-Ion Osmolyte Pools Impacts Intrinsic Cefuroxime Resistance in Lactococcus lactis

Pham

Shi

Xiang

et al. 2021

mBio

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“…aureus suppressor mutants resulted in the identification of the AlsT glutamine transporter, thus indicating that glutamine rather than glutamate is toxic for the S . aureus Δ dac mutant [ 39 , 42 ]. Again, these differences between the related organisms support the idea that c-di-AMP has global overarching functions in the different bacteria, but these can be put into practice differently.…”

Section: Discussionmentioning

confidence: 99%

“…In S . aureus , c-di-AMP is dispensable for growth on complex medium under anaerobic conditions or when the bacteria acquire suppressor mutations that affect osmolyte or glutamine uptake [ 40 , 42 ]. Both types of studies support the idea that the control of potassium and osmolyte homeostasis is the central essential function of c-di-AMP in many bacteria that produce it [ 43 – 45 ].…”

Section: Introductionmentioning

confidence: 99%

Essentiality of c-di-AMP in Bacillus subtilis: Bypassing mutations converge in potassium and glutamate homeostasis

et al. 2021

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In order to adjust to changing environmental conditions, bacteria use nucleotide second messengers to transduce external signals and translate them into a specific cellular response. Cyclic di-adenosine monophosphate (c-di-AMP) is the only known essential nucleotide second messenger. In addition to the well-established role of this second messenger in the control of potassium homeostasis, we observed that glutamate is as toxic as potassium for a c-di-AMP-free strain of the Gram-positive model bacterium Bacillus subtilis. In this work, we isolated suppressor mutants that allow growth of a c-di-AMP-free strain under these toxic conditions. Characterization of glutamate resistant suppressors revealed that they contain pairs of mutations, in most cases affecting glutamate and potassium homeostasis. Among these mutations, several independent mutations affected a novel glutamate transporter, AimA (Amino acid importer A, formerly YbeC). This protein is the major transporter for glutamate and serine in B. subtilis. Unexpectedly, some of the isolated suppressor mutants could suppress glutamate toxicity by a combination of mutations that affect phospholipid biosynthesis and a specific gain-of-function mutation of a mechanosensitive channel of small conductance (YfkC) resulting in the acquisition of a device for glutamate export. Cultivation of the c-di-AMP-free strain on complex medium was an even greater challenge because the amounts of potassium, glutamate, and other osmolytes are substantially higher than in minimal medium. Suppressor mutants viable on complex medium could only be isolated under anaerobic conditions if one of the two c-di-AMP receptor proteins, DarA or DarB, was absent. Also on complex medium, potassium and osmolyte toxicity are the major bottlenecks for the growth of B. subtilis in the absence of c-di-AMP. Our results indicate that the essentiality of c-di-AMP in B. subtilis is caused by the global impact of the second messenger nucleotide on different aspects of cellular physiology.

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“…Mutation of the predicted glutamine transporter GlnPQ increased TCA cycle activity, decreased polysaccharide intercellular adhesin biosynthesis, and significantly reduced virulence in a rabbit endocarditis model [ 12 ]. Recently it was demonstrated that GlnPQ does not transport glutamine and that AlsT is instead the main glutamine transporter [ 9 ]. Moreover, AlsT-mediated glutamine uptake decreased c-di-AMP levels, which is known to effect cell envelope homeostasis, virulence and β-lactam resistance [ 9 , 25 ].…”

Section: Why Investigate Amino Acid and Peptide Transporters As Potenmentioning

confidence: 99%

“…Recently it was demonstrated that GlnPQ does not transport glutamine and that AlsT is instead the main glutamine transporter [ 9 ]. Moreover, AlsT-mediated glutamine uptake decreased c-di-AMP levels, which is known to effect cell envelope homeostasis, virulence and β-lactam resistance [ 9 , 25 ].…”

Section: Why Investigate Amino Acid and Peptide Transporters As Potenmentioning

confidence: 99%

Exploring amino acid and peptide transporters as therapeutic targets to attenuate virulence and antibiotic resistance in Staphylococcus aureus

et al. 2021

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Identification of the main glutamine and glutamate transporters in Staphylococcus aureus and their impact on c‐di‐AMP production

Cited by 30 publications

References 67 publications

Cyclic di-AMP Oversight of Counter-Ion Osmolyte Pools Impacts Intrinsic Cefuroxime Resistance in Lactococcus lactis

Cyclic di-AMP Oversight of Counter-Ion Osmolyte Pools Impacts Intrinsic Cefuroxime Resistance in Lactococcus lactis

Essentiality of c-di-AMP in Bacillus subtilis: Bypassing mutations converge in potassium and glutamate homeostasis

Exploring amino acid and peptide transporters as therapeutic targets to attenuate virulence and antibiotic resistance in Staphylococcus aureus

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