Importance of Metabolic Adaptations in Francisella Pathogenesis

Ziveri, Jason; Barel, Monique; Charbit, Alain

doi:10.3389/fcimb.2017.00096

Cited by 29 publications

(21 citation statements)

References 61 publications

(82 reference statements)

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“…Replication and survival in the cytosol are essential to the virulence of L. monocytogenes and other cytosol-adapted pathogens. Metabolic adaptations to the cytosolic environment are essential for not only replication but also tolerance of cytosolic stresses (45,46). Our work indicates that DHNA, independent of the ETC, is central to L. monocytogenes intracellular survival.…”

Section: Discussionmentioning

confidence: 73%

Mutation of the Transcriptional Regulator YtoI Rescues Listeria monocytogenes Mutants Deficient in the Essential Shared Metabolite 1,4-Dihydroxy-2-Naphthoate (DHNA)

et al. 2019

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Listeria monocytogenes, a Gram-positive, facultative intracellular pathogen, survives and replicates in the cytosol of host cells. Synthesis of 1,4-dihydroxy-2-naphthoate (DHNA), an intermediate of menaquinone biosynthesis, is essential for cytosolic survival of L. monocytogenes independent from its role in respiration. Here, we demonstrate that DHNA is essential for virulence in a murine model of listeriosis due to both respiration-dependent and -independent functions. In addition, DHNA can be both secreted and utilized as an extracellular shared metabolite to promote cytosolic survival inside host macrophages. To understand the role(s) of DHNA in L. monocytogenes intracellular survival and virulence, we isolated DHNA-deficient (ΔmenD strain) suppressor mutants that formed plaques in monolayers of fibroblasts. Five ΔmenD suppressor (mds) mutants additionally rescued at least 50% of the cytosolic survival defect of the parent ΔmenD mutant. Whole-genome sequencing revealed that four of the five suppressor mutants had independent missense mutations in a putative transcriptional regulator, ytoI (lmo1576). Clean deletion and complementation in trans confirmed that loss of ytoI could restore plaquing and cytosolic survival of DHNA-deficient L. monocytogenes. RNA-seq transcriptome analysis revealed five genes (lmo0944, lmo1575, lmo1577, lmo2005, and lmo2006) expressed at a higher level in the ΔytoI strain than in the wild-type strain, whereas two genes (lmo1917 and lmo2103) demonstrated lower expression in the ΔytoI mutant. Intriguingly, the majority of these genes are involved in controlling pyruvate flux. Metabolic analysis confirmed that acetoin, acetate, and lactate flux were altered in a ΔytoI mutant, suggesting a critical role for regulating these metabolic programs. In conclusion, we have demonstrated that, similar to findings in select other bacteria, DHNA can act as a shared resource, and it is essential for cytosolic survival and virulence of L. monocytogenes. Furthermore, we have identified a novel transcriptional regulator in L. monocytogenes and determined that its metabolic regulation is implicated in cytosolic survival of L. monocytogenes.

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Section: Discussionmentioning

confidence: 73%

Mutation of the Transcriptional Regulator YtoI Rescues Listeria monocytogenes Mutants Deficient in the Essential Shared Metabolite 1,4-Dihydroxy-2-Naphthoate (DHNA)

et al. 2019

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“…Our results identify a convergence between central carbon metabolism, amino acid starvation and OMV/T formation. Central metabolism plays a critical role in Francisella virulence, and in the virulence of bacterial pathogens in general (Santic and Abu Kwaik, ; Wyatt et al ., ; Ziveri et al ., ). Our findings provide a framework for understanding the genetic and environmental cues that drive biogenesis of bacterial membrane‐derived structures, and suggest that nutrient cues may be sensed by Francisella to regulate OMV/T production during infection.…”

Section: Discussionmentioning

confidence: 97%

Amino acid deprivation and central carbon metabolism regulate the production of outer membrane vesicles and tubes by Francisella

Sampath

McCaig

Thanassi

2018

Molecular Microbiology

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Francisella tularensis is a highly virulent Gram-negative bacterial pathogen that causes the zoonotic disease tularemia. F. novicida, a model tularemia strain, produces spherical outer membrane vesicles (OMV), as well as novel tubular vesicles and extensions of the cell surface. These OMV and tubes (OMV/T) are produced in a regulated manner and contain known virulence factors. Mechanisms by which bacterial vesicles are produced and regulated are not well understood. We performed a genetic screen in F. novicida to decipher the molecular basis for regulated OMV/T formation, and identified both hypo- and hyper-vesiculating mutants. Mutations in fumA and tktA, involved in central carbon metabolism, and in FTN_0908 and FTN_1037, of unknown function, resulted in severe defects in OMV/T production. Cysteine deprivation was identified as the signal that triggers OMV/T formation in F. novicida during growth in rich medium. We also found that fully virulent F. tularensis produces OMV/T in a similarly regulated manner. Further analysis revealed that OMV/T production is responsive to deprivation of essential amino acids in addition to cysteine, and that the hypo-vesiculating mutants are defective in responding to this signal. Thus, amino acid starvation, such as encountered by Francisella during host cell invasion, regulates the production of membrane-derived structures.

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“…Genetic screens resulted in the identification of the gluconeogenic enzyme class II fructose-1,6-bisphosphatase (FtFBPaseII), the product of the glpX gene, as one of the most important contributors to the virulence of F. tularensis strains SchuS4 and LVS (Kadzhaev et al, 2009). Further work has demonstrated that Francisella relies on the supply of amino acids from the host as major gluconeogenic substrates for intracellular growth (Ziveri et al, 2017), highlighting the importance of the FtFBPaseII enzyme; no other FBPases have been characterized in F. tularensis. Thus, targeted inactivation of FtFBPaseII should prevent the bacterium from producing fructose 6-phosphate (F6P) during intracellular growth, regardless of the specific gluconeogenic host-derived carbon source that is available to the pathogen (Radlinski et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Structural and biochemical characterization of the class II fructose-1,6-bisphosphatase from Francisella tularensis

et al. 2020

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The crystal structure of the class II fructose-1,6-bisphosphatase (FBPaseII) from the important pathogen Francisella tularensis is presented at 2.4 Å resolution. Its structural and functional relationships to the closely related phosphatases from Mycobacterium tuberculosis (MtFBPaseII) and Escherichia coli (EcFBPaseII) and to the dual phosphatase from Synechocystis strain 6803 are discussed. FBPaseII from F. tularensis (FtFBPaseII) was crystallized in a monoclinic crystal form (space group P21, unit-cell parameters a = 76.30, b = 100.17, c = 92.02 Å, β = 90.003°) with four chains in the asymmetric unit. Chain A had two coordinated Mg2+ ions in its active center, which is distinct from previous findings, and is presumably deactivated by their presence. The structure revealed an approximate 222 (D 2) symmetry homotetramer analogous to that previously described for MtFBPaseII, which is formed by a crystallographic dyad and which differs from the exact tetramer found in EcFBPaseII at a 222 symmetry site in the crystal. Instead, the approximate homotetramer is very similar to that found in the dual phosphatase from Synechocystis, even though no allosteric effector was found in FtFBPase. The amino-acid sequence and folding of the active site of FtFBPaseII result in structural characteristics that are more similar to those of the previously published EcFBPaseII than to those of MtFBPaseII. The kinetic parameters of native FtFBPaseII were found to be in agreement with published studies. Kinetic analyses of the Thr89Ser and Thr89Ala mutations in the active site of the enzyme are consistent with the previously proposed mechanism for other class II bisphosphatases. The Thr89Ala variant enzyme was inactive but the Thr89Ser variant was partially active, with an approximately fourfold lower K m and V max than the native enzyme. The structural and functional insights derived from the structure of FtFBPaseII will provide valuable information for the design of specific inhibitors.

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Importance of Metabolic Adaptations in Francisella Pathogenesis

Cited by 29 publications

References 61 publications

Mutation of the Transcriptional Regulator YtoI Rescues Listeria monocytogenes Mutants Deficient in the Essential Shared Metabolite 1,4-Dihydroxy-2-Naphthoate (DHNA)

Mutation of the Transcriptional Regulator YtoI Rescues Listeria monocytogenes Mutants Deficient in the Essential Shared Metabolite 1,4-Dihydroxy-2-Naphthoate (DHNA)

Amino acid deprivation and central carbon metabolism regulate the production of outer membrane vesicles and tubes by Francisella

Structural and biochemical characterization of the class II fructose-1,6-bisphosphatase from Francisella tularensis

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