<i>Francisella tularensis</i>type VI secretion system comes of age

Špidlová, Petra; Stulı́k, Jiřı́

doi:10.1080/21505594.2016.1278336

Cited by 13 publications

(11 citation statements)

References 30 publications

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“…5A). Confirming earlier reports (47), intracellular multiplication of the 'iglB mutant was essentially abolished and comparable to that of the ∆FPI mutant. The single amino acid substitution of Y139 by an alanine (Y139A) also abolished intracellular bacterial multiplication.…”

Section: Critical Role Of Residue Y139 In Francisella Pathogenesissupporting

confidence: 90%

Critical Role of a Sheath Phosphorylation Site On the Assembly and Function of an Atypical Type VI Secretion System

Ziveri

Chhuon

Jamet

et al. 2019

Molecular & Cellular Proteomics

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The bacterial pathogen Francisella tularensis possesses a non-canonical type VI secretion system (T6SS) that is required for phagosomal escape in infected macrophages. KCl stimulation has been previously used to trigger assembly and secretion of the T6SS in culture. By differential proteomics, we found here that the amounts of the T6SS proteins remained unchanged upon KCl stimulation, suggesting involvement of post-translational modifications in T6SS assembly. A phosphoproteomic analysis indeed identified a unique phosphorylation site on IglB, a key component of the T6SS sheath. Substitutions of Y139 with alanine or phosphomimetics prevented T6SS formation and abolished phagosomal escape whereas substitution with phenylalanine delayed but did not abolish phagosomal escape in J774-1 macrophages. Altogether our data demonstrated that the Y139 site of IglB plays a critical role in T6SS biogenesis, suggesting that sheath phosphorylation could participate to T6SS dynamics. Data are available via ProteomeXchange with identifier PXD013619; and on MS-viewer, key lkaqkllxwx. Proteomic and Phosphoproteomic analyses Reagents and chemicals. For protein digestion, dithiothreitol, iodoacetamide and ammonium bicarbonate were purchased from Sigma-Aldrich (St Louis, MO, USA). For phosphopeptide enrichment and LC-MS/MS analysis, trifluoroacetic acid (TFA), formic acid, acetonitrile and HPLC-grade water were purchased from Fisher Scientific (Pittsburgh, PA, USA) at the highest purity grade. Protein digestion. For proteomic analysis, F. novicida was analyzed in three independent biological replicates. Protein concentration was determined by DC assay (Biorad, CA, USA) according to the manufacturer's instructions. An estimated 1.2 mg of proteins for each

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Section: Critical Role Of Residue Y139 In Francisella Pathogenesissupporting

confidence: 90%

Critical Role of a Sheath Phosphorylation Site On the Assembly and Function of an Atypical Type VI Secretion System

Ziveri

Chhuon

Jamet

et al. 2019

Molecular & Cellular Proteomics

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“…The ability of wild-type F. novicida (WT), ∆ iglB and complemented strains (CpWT, Y139A and Y139F) to survive and multiply in murine macrophage-like J774.1 cells was monitored over a 24 h-period ( Figure 5A ). Confirming earlier reports (Spidlova & Stulik, 2017), intracellular multiplication of the Δ iglB mutant was essentially abolished and comparable to that of the ∆ FPI mutant. Remarkably, the single amino acid substitution of Tyr139 by Ala (Y139A) also abolished intracellular bacterial multiplication.…”

Section: Resultssupporting

confidence: 91%

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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“…For example, the proteins from the oxidative phosphorylation pathway or, even more relevant, the FPI proteins, represent a notable proof of this phenomenon. Some of the FPI proteins are structural and/or functional constituents of T6SS (Bröms et al, 2012; Clemens et al, 2015; Spidlova and Stulik, 2017); as such they are probably not released by the vesicular mechanism. Some of them are expressed and secreted (or mechanically shed) in quite high amounts so they can be found in the vesicular fractions as well (McCaig et al, 2013).…”

Section: Discussionmentioning

confidence: 99%

Francisella tularensis subsp. holarctica Releases Differentially Loaded Outer Membrane Vesicles Under Various Stress Conditions

et al. 2019

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Francisella tularensis is a Gram-negative, facultative intracellular bacterium, causing a severe disease called tularemia. It secretes unusually shaped nanotubular outer membrane vesicles (OMV) loaded with a number of virulence factors and immunoreactive proteins. In the present study, the vesicles were purified from a clinical isolate of subsp. holarctica strain FSC200. We here provide a comprehensive proteomic characterization of OMV using a novel approach in which a comparison of OMV and membrane fraction is performed in order to find proteins selectively enriched in OMV vs. membrane. Only these proteins were further considered to be really involved in the OMV function and/or their exceptional structure. OMV were also isolated from bacteria cultured under various cultivation conditions simulating the diverse environments of F. tularensis life cycle. These included conditions mimicking the milieu inside the mammalian host during inflammation: oxidative stress, low pH, and high temperature (42°C); and in contrast, low temperature (25°C). We observed several-fold increase in vesiculation rate and significant protein cargo changes for high temperature and low pH. Further proteomic characterization of stress-derived OMV gave us an insight how the bacterium responds to the hostile environment of a mammalian host through the release of differentially loaded OMV. Among the proteins preferentially and selectively packed into OMV during stressful cultivations, the previously described virulence factors connected to the unique intracellular trafficking of Francisella were detected. Considerable changes were also observed in a number of proteins involved in the biosynthesis and metabolism of the bacterial envelope components like O-antigen, lipid A, phospholipids, and fatty acids. Data are available via ProteomeXchange with identifier PXD013074.

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Francisella tularensistype VI secretion system comes of age

Cited by 13 publications

References 30 publications

Critical Role of a Sheath Phosphorylation Site On the Assembly and Function of an Atypical Type VI Secretion System

Critical Role of a Sheath Phosphorylation Site On the Assembly and Function of an Atypical Type VI Secretion System

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

Francisella tularensis subsp. holarctica Releases Differentially Loaded Outer Membrane Vesicles Under Various Stress Conditions

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