Importance of Host Cell Arginine Uptake in Francisella Phagosomal Escape and Ribosomal Protein Amounts*

Ramond, Elodie; Gesbert, Gaël; Guerrera, Ida Chiara; Chhuon, Cérina; Dupuis, Marion; Mélanie, Rigard; Henry, Thomas; Barel, Monique; Charbit, Alain

doi:10.1074/mcp.m114.044552

Cited by 24 publications

(26 citation statements)

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“…Functional complementation restored normal intracellular bacterial replication (in CpWT). The ability of the ∆ iglB mutants to induce phagosomal membrane rupture in BMMs was further tested by using a CCF4 assay, essentially as previously described (Ramond, Gesbert et al, 2015, Rigard et al, 2016). Wild-type F. novicida and CpWT strains showed similar amount of cleaved CCF4 at the three time-points tested (1 h, 3 h and 6 h) whereas there was no (or marginal) loss of FRET signal with ∆ iglB ( Figure supplement 5B ), Y139A, Y139F, and the two negative control strains (∆ FPI and ∆ bla ), indicating that the Y139A, Y139F mutant strains remained stuck in intact phagosomes during the time-period tested in BMMS.…”

Section: Resultsmentioning

confidence: 99%

“…Overlap PCRs were carried out using 100 ng of each purified PCR products and the resulting fragment of interest was purified from agarose gel. This fragment was then directly used to transform wild type F. novicida by chemical transformation (Gesbert, Ramond et al, 2015). Recombinant bacteria were isolated on Chocolate agar plates containing kanamycin (10 μg mL −1 ).…”

Section: Methodsmentioning

confidence: 99%

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A post-translational modification of the sheath modulatesFrancisellatype VI secretion system assembly and function

Ziveri

Cchuon

Jamet

et al. 2018

Preprint

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27Francisella tularensis is a facultative intracellular pathogen that causes the zoonotic 28 disease tularemia in human and animal hosts. This bacterium possesses a non-canonical 29 type VI secretion systems (T6SS) required for phagosomal escape and access to its 30 replicative niche in the cytosol of infected macrophages. KCl stimulation has been previously 31 used to trigger assembly and secretion of the Francisella T6SS in culture. We found that the 32 amounts of essentially all the TSS6 proteins remained unchanged upon KCl stimulation. We 33 therefore hypothesized that a post-translational modification might be involved in T6SS 34 assembly. A whole cell phosphoproteomic analysis allowed us to identify a unique 35 phosphorylation site on IglB, the TssC homologue and key component of the T6SS sheath. 36 Importantly, the phosphorylated form of IglB was not present in the contracted sheath and 3D 37 modeling indicated that the charge repulsion provoked by addition of a phosphogroup on 38 tyrosine 139 was likely to weaken the stability of the sheath structure. Substitutions of the 39 phosphorylatable residue of IglB (tyrosine 139) with alanine or with phosphomimetics 40 prevented T6SS formation and totally impaired phagosomal escape. In contrast, the 41 substitution with the non-phosphorylatable aromatic analog phenylalanine impaired but did 42 not prevent phagosomal escape and cytosolic bacterial multiplication in J774-1 43 macrophages. Altogether these data suggest that phosphorylation of the sheath participates 44 to T6SS disassembly. Post-translational modifications of the sheath may represent a 45 previously unrecognized mechanism to finely modulate the dynamics of T6SS assembly-46 disassembly. 47 48 Data are available via ProteomeXchange with identifier PXD012507. 49 50 3 Synopsis 51 52Francisella possesses a non-canonical T6SS that is essential for efficient phagosomal 53 escape and access to the cytosol of infected macrophages. KCl stimulation has been 54 previously used to trigger assembly and secretion of the Francisella T6SS in culture. We 55 found that KCl stimulation did not result in an increased production of TSS6 proteins. We 56 therefore hypothesized that a post-translational modification might be involved in T6SS 57 assembly. Using a global and site-specific phosphoproteomic analysis of Francisella we 58 identified a unique phosphorylation site on IglB, the TssC homologue and a key component 59 of the T6SS contractile sheath. We show that this site plays a critical role in T6SS biogenesis 60 and propose that phosphorylation may represent a new mechanism affecting the dynamics of 61 sheath formation. 62 63 64 65 66 67Francisella tularensis is the causative agent of the zoonotic disease tularemia (Luque-68

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A post-translational modification of the sheath modulatesFrancisellatype VI secretion system assembly and function

Ziveri

Cchuon

Jamet

et al. 2018

Preprint

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“…We also showed that the permease ArgP of Francisella was a high affinity arginine transporter (Ramond et al, 2015). ArgP-mediated arginine uptake appeared to be crucial for efficient phagosomal escape.…”

Section: Amino Acids Constitute a Major Carbon Source For Intracellulmentioning

confidence: 90%

“…Indeed, in bacteria grown under arginine limiting conditions, the majority of the ribosomal proteins identified (app. 80%) were present in lower amount in the Δ argP mutant compared to wild-type, suggesting possible links between ribosomal proteins amounts and phagosomal escape (Ramond et al, 2015). Of note, ArgP is the closest paralogue of GadC within the APC family of transporter (Meibom and Charbit, 2010).…”

Section: Amino Acids Constitute a Major Carbon Source For Intracellulmentioning

confidence: 99%

“…Macrophages are able to synthesize their own arginine and to import it via constitutive and inducible cationic amino acid transporters CAT-1 (or SLC7A1) and CAT-2 (or SLC7A2), respectively (Ramond et al, 2015). Arginine serves either to produce nitrogen reactive species (NO, via NO synthases) or convert it to ornithine and urea (via type 1 arginase).…”

Section: Could Amino Acids Serve As Sensors Of the Intracellular Milieu?mentioning

confidence: 99%

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Importance of Metabolic Adaptations in Francisella Pathogenesis

Ziveri

Barel

Charbit

2017

Front. Cell. Infect. Microbiol.

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Francisella tularensis is a highly infectious Gram-negative bacterium and the causative agent of the zoonotic disease tularemia. This bacterial pathogen can infect a broad variety of animal species and can be transmitted to humans in numerous ways with various clinical outcomes. Although, Francisella possesses the capacity to infect numerous mammalian cell types, the macrophage constitutes the main intracellular niche, used for in vivo bacterial dissemination. To survive and multiply within infected macrophages, Francisella must imperatively escape from the phagosomal compartment. In the cytosol, the bacterium needs to control the host innate immune response and adapt its metabolism to this nutrient-restricted niche. Our laboratory has shown that intracellular Francisella mainly relied on host amino acid as major gluconeogenic substrates and provided evidence that the host metabolism was also modified upon Francisella infection. We will review here our current understanding of how Francisella copes with the available nutrient sources provided by the host cell during the course of infection.

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A study of innate immune kinetics reveals a role for a chloride transporter in a virulent Francisella tularensis type B strain

Matz

Petrosino

2021

MicrobiologyOpen

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Tularemia is a zoonotic disease of global proportions. Francisella tularensis subspecies tularensis (type A) and holarctica (type B) cause disease in healthy humans, with type A infections resulting in higher mortality. Repeated passage of a type B strain in the mid‐20th century generated the Live Vaccine Strain (LVS). LVS remains unlicensed, does not protect against high inhalational doses of type A, and its exact mechanisms of attenuation are poorly understood. Recent data suggest that live attenuated vaccines derived from type B may cross‐protect against type A. However, there is a dearth of knowledge regarding virulent type B pathogenesis and its capacity to stimulate the host's innate immune response. We therefore sought to increase our understanding of virulent type B in vitro characteristics using strain OR96‐0246 as a model. Adding to our knowledge of innate immune kinetics in macrophages following infection with virulent type B, we observed robust replication of strain OR96‐0246 in murine and human macrophages, reduced expression of pro‐inflammatory cytokine genes from “wild type” type B‐infected macrophages compared to LVS, and delayed macrophage cell death suggesting that virulent type B may suppress macrophage activation. One disruption in LVS is in the gene encoding the chloride transporter ClcA. We investigated the role of ClcA in macrophage infection and observed a replication delay in a clcA mutant. Here, we propose its role in acid tolerance. A greater understanding of LVS attenuation may reveal new mechanisms of pathogenesis and inform strategies toward the development of an improved vaccine against tularemia.

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Importance of Host Cell Arginine Uptake in Francisella Phagosomal Escape and Ribosomal Protein Amounts*

Cited by 24 publications

References 54 publications

A post-translational modification of the sheath modulatesFrancisellatype VI secretion system assembly and function

A post-translational modification of the sheath modulatesFrancisellatype VI secretion system assembly and function

Importance of Metabolic Adaptations in Francisella Pathogenesis

A study of innate immune kinetics reveals a role for a chloride transporter in a virulent Francisella tularensis type B strain

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