Phenotypic characterization of the Francisella tularensis ΔpdpC and ΔiglG mutants

Ožanič, Mateja; Marečić, Valentina; Lindgren, Marie; Sjöstedt, Anders; Šantić, Marina

doi:10.1016/j.micinf.2016.07.006

Cited by 9 publications

(10 citation statements)

References 45 publications

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“…has shown that F. novicida PdpC and PdpD are released by the T6SS in an in vitro secretion assay, supporting the hypothesis that these proteins function as secreted effectors in the target cell. Moreover, F. tularensis and F. holarctica lacking pdpC are unable to escape from the phagosome, induce cytotoxicity and replicate intracellularly, and they are avirulent in a mouse model of tularaemia4344464748. These observations support our conclusions that PdpC contribute to Francisella virulence, independent of the Francisella tularensis subspecies.…”

Section: Discussionsupporting

confidence: 85%

“…On the other hand, the FPI cluster contains many genes of unknown function, such as iglF , iglI , iglJ , pdpA , pdpC , pdpE , pdpD and anmK . PdpA, PdpC and PdpD were identified by mass-spectrometry as secreted by Francisella T6SS and PdpC/PdpD were proposed to be effectors required for phagosomal escape, intracellular growth and virulence42434445464748. Interestingly, the FPI cluster lacks a homologue of an unfoldase ClpV, which is present in all canonical T6SS clusters and recycles contracted sheaths14244950.…”

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confidence: 99%

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Francisella requires dynamic type VI secretion system and ClpB to deliver effectors for phagosomal escape

Brodmann¹,

Dreier²,

Brož³

et al. 2017

Nat Commun

110

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Francisella tularensis is an intracellular pathogen that causes the fatal zoonotic disease tularaemia. Critical for its pathogenesis is the ability of the phagocytosed bacteria to escape into the cell cytosol. For this, the bacteria use a non-canonical type VI secretion system (T6SS) encoded on the Francisella pathogenicity island (FPI). Here we show that in F. novicida T6SS assembly initiates at the bacterial poles both in vitro and within infected macrophages. T6SS dynamics and function depends on the general purpose ClpB unfoldase, which specifically colocalizes with contracted sheaths and is required for their disassembly. T6SS assembly depends on iglF, iglG, iglI and iglJ, whereas pdpC, pdpD, pdpE and anmK are dispensable. Importantly, strains lacking pdpC and pdpD are unable to escape from phagosome, activate AIM2 inflammasome or cause disease in mice. This suggests that PdpC and PdpD are T6SS effectors involved in phagosome rupture.

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Section: Discussionsupporting

confidence: 85%

mentioning

confidence: 99%

Francisella requires dynamic type VI secretion system and ClpB to deliver effectors for phagosomal escape

Brodmann¹,

Dreier²,

Brož³

et al. 2017

Nat Commun

110

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“…Given the intracellular nature of F. tularensis and the success of LVS as a vaccine, a live attenuated type A strain with a defined mutation would logically be expected to be the most efficacious to induce protective immunity against infection. Targeted mutations that have been successful in attenuating F. tularensis have included surface components such as the lipopolysaccharide (LPS) and some outer membrane proteins (OMPs) (Thomas et al, 2007 ; Meibom et al, 2009 ; Qin et al, 2009 ; Reed et al, 2014 ), and the Francisella pathogenicity island (FPI) (Ozanic et al, 2016 ).…”

Section: Introductionmentioning

confidence: 99%

Further Characterization of the Capsule-Like Complex (CLC) Produced by Francisella tularensis Subspecies tularensis: Protective Efficacy and Similarity to Outer Membrane Vesicles

Champion

Bandara

Mohapatra

et al. 2018

Front. Cell. Infect. Microbiol.

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Francisella tularensis is the etiologic agent of tularemia, and subspecies tularensis (type A) is the most virulent subspecies. The live vaccine strain (LVS) of subspecies holarctica produces a capsule-like complex (CLC) that consists of a large variety of glycoproteins. Expression of the CLC is greatly enhanced when the bacteria are subcultured in and grown on chemically defined medium. Deletion of two genes responsible for CLC glycosylation in LVS results in an attenuated mutant that is protective against respiratory tularemia in a mouse model. We sought to further characterize the CLC composition and to determine if a type A CLC glycosylation mutant would be attenuated in mice. The CLCs isolated from LVS extracted with 0.5% phenol or 1 M urea were similar, as determined by gel electrophoresis and Western blotting, but the CLC extracted with urea was more water-soluble. The CLC extracted with either 0.5% phenol or 1 M urea from type A strains was also similar to the CLC of LVS in antigenic properties, electrophoretic profile, and by transmission electron microscopy (TEM). The solubility of the CLC could be further enhanced by fractionation with Triton X-114 followed by N-Lauroylsarcosine detergents; the largest (>250 kDa) molecular size component appeared to be an aggregate of smaller components. Outer membrane vesicles/tubules (OMV/T) isolated by differential centrifugation and micro-filtration appeared similar to the CLC by TEM, and many of the proteins present in the OMV/T were also identified in soluble and insoluble fractions of the CLC. Further investigation is warranted to assess the relationship between OMV/T and the CLC. The CLC conjugated to keyhole limpet hemocyanin or flagellin was highly protective against high-dose LVS intradermal challenge and partially protective against intranasal challenge. A protective response was associated with a significant rise in cytokines IL-12, IL-10, and IFN-γ. However, a type A CLC glycosylation mutant remained virulent in BALB/c mice, and immunization with the CLC did not protect mice against high dose respiratory challenge with type A strain SCHU S4.

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“…Positive doubling rates were interpreted as an indication that replication was outpacing escape, and negative doubling rates were interpreted as an indication that escape was outpacing replication. Supernatants were assayed for the release of lactate dehydrogenase (LDH), an intracellular enzyme whose release is indicative of cell death ( 39 , 40 ). Supporting our previous findings with THP-1 cells, F. tularensis SchuS4Δ fptB exhibited a significant growth defect that was quantified as reduced intracellular bacterial numbers, evident by 15 hpi ( P < 0.05) ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Deletion of the Major Facilitator Superfamily Transporter fptB Alters Host Cell Interactions and Attenuates Virulence of Type A Francisella tularensis

et al. 2018

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Francisella tularensis is a Gram-negative, facultative, intracellular coccobacillus that can infect a wide variety of hosts. In humans, F. tularensis causes the zoonosis tularemia following insect bites, ingestion, inhalation, and the handling of infected animals. The fact that a very small inoculum delivered by the aerosol route can cause severe disease, coupled with the possibility of its use as an aerosolized bioweapon, has led to the classification of Francisella tularensis as a category A select agent and has renewed interest in the formulation of a vaccine. To this end, we engineered a type A strain SchuS4 derivative containing a targeted deletion of the major facilitator superfamily (MFS) transporter fptB. Based on the attenuating capacity of this deletion in the F. tularensis LVS background, we hypothesized that the deletion of this transporter would alter the intracellular replication and cytokine induction of the type A strain and attenuate virulence in the stringent C57BL/6J mouse model. Here we demonstrate that the deletion of fptB significantly alters the intracellular life cycle of F. tularensis, attenuating intracellular replication in both cell line-derived and primary macrophages and inducing a novel cytosolic escape delay. Additionally, we observed prominent differences in the in vitro cytokine profiles in human macrophage-like cells. The mutant was highly attenuated in the C57BL/6J mouse model and provided partial protection against virulent type A F. tularensis challenge. These results indicate a fundamental necessity for this nutrient transporter in the timely progression of F. tularensis through its replication cycle and in pathogenesis.

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Phenotypic characterization of the Francisella tularensis ΔpdpC and ΔiglG mutants

Cited by 9 publications

References 45 publications

Francisella requires dynamic type VI secretion system and ClpB to deliver effectors for phagosomal escape

Francisella requires dynamic type VI secretion system and ClpB to deliver effectors for phagosomal escape

Further Characterization of the Capsule-Like Complex (CLC) Produced by Francisella tularensis Subspecies tularensis: Protective Efficacy and Similarity to Outer Membrane Vesicles

Deletion of the Major Facilitator Superfamily Transporter fptB Alters Host Cell Interactions and Attenuates Virulence of Type A Francisella tularensis

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