Expression of <i>Francisella</i> pathogenicity island protein intracellular growth locus E (IglE) in mammalian cells is involved in intracellular trafficking, possibly through microtubule organizing center

Shimizu, Takashi; Otonari, Shiho; Suzuki, Jin; Uda, Akihiko; Watanabe, Kenta; Watarai, Masahisa

doi:10.1002/mbo3.684

Cited by 8 publications

(12 citation statements)

References 55 publications

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“…Clemens et al showed that in THP-1 macrophage infection assays IglA and IglB tagged with split GFP could be seen as bright foci in F. novicida cells as soon as 15 min postinfection, and by 22 h up to 70% of intracellular F. novicida had bright IglA-GFP foci (77). A recent study by Shimizu et al reported that in, 293T cell infection assays, while IglH, IglI, and IglC tagged with GFP localized diffusely in host cells, PdPA and IglE had a punctate localization (56). For THP-1 cells incubated with F. tularensis LVS strains containing opiA-emgfp, increased fluorescence was observed under the microscope and increased fluorescence was observed in cell lysates, a finding consistent with the secretion of OpiA-EmGFP.…”

Section: Discussionmentioning

confidence: 95%

“…Of the known secreted effector proteins of the T6SS ii in Francisella (PdpC, PdpD, OpiA, OpiB, IglD, and IglE), all have so far been shown be involved in virulence in host cells (37,38,(54)(55)(56)(57)(58). In F. tularensis LVS, there was a slight, but statistically significant, attenuation of virulence in an in vivo chicken embryo infection model.…”

Section: Discussionmentioning

confidence: 99%

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OpiA, a Type Six Secretion System Substrate, Localizes to the Cell Pole and Plays a Role in Bacterial Growth and Viability inFrancisella tularensisLVS

2020

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Francisella tularensis is an intracellular pathogen and the causative agent of tularemia. The F. tularensis type six secretion system (T6SS) is required for a number of host-pathogen interactions, including phagolysosomal escape and invasion of erythrocytes. One known effector of the T6SS, OpiA, has recently been shown to be a phosphatidylinositol-3 kinase. To investigate the role of OpiA in erythrocyte invasion, we constructed an opiA-null mutant in the live vaccine strain, F. tularensis LVS. OpiA was not required for erythrocyte invasion; however, deletion of opiA affected growth of F. tularensis LVS in broth cultures in a medium-dependent manner. We also found that opiA influenced cell size, gentamicin sensitivity, bacterial viability, and the lipid content of F. tularensis. A fluorescently tagged OpiA (OpiA–emerald-green fluorescent protein [EmGFP]) accumulated at the cell poles of F. tularensis, which is consistent with the location of the T6SS. However, OpiA-EmGFP also exhibited a highly dynamic localization, and this fusion protein was detected in erythrocytes and THP-1 cells in vitro, further supporting that OpiA is secreted. Similar to previous reports with F. novicida, our data demonstrated that opiA had a minimal effect on intracellular replication of F. tularensis in host immune cells in vitro. However, THP-1 cells infected with the opiA mutant produced modestly (but significantly) higher levels of the proinflammatory cytokine tumor necrosis factor alpha compared to these host cells infected with wild-type bacteria. We conclude that, in addition to its role in host-pathogen interactions, our results reveal that the function of opiA is central to the biology of F. tularensis bacteria. IMPORTANCE F. tularensis is a pathogenic intracellular pathogen that is of importance for public health and strategic defense. This study characterizes the opiA gene of F. tularensis LVS, an attenuated strain that has been used as a live vaccine but that also shares significant genetic similarity to related Francisella strains that cause human disease. The data presented here provide the first evidence of a T6SS effector protein that affects the physiology of F. tularensis, namely, the growth, cell size, viability, and aminoglycoside resistance of F. tularensis LVS. This study also adds insight into our understanding of OpiA as a determinant of virulence. Finally, the fluorescence fusion constructs presented here will be useful tools for dissecting the role of OpiA in infection.

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Section: Discussionmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 99%

OpiA, a Type Six Secretion System Substrate, Localizes to the Cell Pole and Plays a Role in Bacterial Growth and Viability inFrancisella tularensisLVS

2020

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“…novicida were generated through group-II intron insertion using the TargeTron Gene Knockout System (Sigma-Aldrich) modified for Francisella species [20], as previously described [21]. The slt (FTN_0496) deletion mutant (Δ slt ) was generated via homologous recombination using the Francisella suicide vector pFRSU [21]. The upstream and downstream regions of slt (1.5 kbp each) were cloned into the BamHI site of pFRSU to generate pFRSU-slt.…”

Section: Methodsmentioning

confidence: 99%

“…The GFP- and mCherry-expressing plasmids pOM5-GFP and pOM5-mCherry were constructed according to published procedures [21]. The chromosomal slt gene with its native promoter region (200 bp upstream) from the F .…”

Section: Methodsmentioning

confidence: 99%

Soluble lytic transglycosylase SLT of Francisella novicida is involved in intracellular growth and immune suppression

et al. 2019

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Francisella tularensis, a category-A bioterrorism agent causes tularemia. F. tularensis suppresses the immune response of host cells and intracellularly proliferates. However, the detailed mechanisms of immune suppression and intracellular growth are largely unknown. Here we developed a transposon mutant library to identify novel pathogenic factors of F. tularensis. Among 750 transposon mutants of F. tularensis subsp. novicida (F. novicida), 11 were isolated as less cytotoxic strains, and the genes responsible for cytotoxicity were identified. Among them, the function of slt, which encodes soluble lytic transglycosylase (SLT) was investigated in detail. An slt deletion mutant (Δslt) was less toxic to the human monocyte cell line THP-1 vs the wild-type strain. Although the wild-type strain proliferated in THP-1 cells, the number of intracellular Δslt mutant decreased in comparison. The Δslt mutant escaped from phagosomes during the early stages of infection, but the mutant was detected within the autophagosome, followed by degradation in lysosomes. Moreover, the Δslt mutant induced host cells to produce high levels of cytokines such as tumor necrosis factor-α, interleukin (IL)-6, and IL-1β, compared with the wild-type strain. These results suggest that the SLT of F. novicida is required for immune suppression and escape from autophagy to allow its survival in host cells.

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“…A deletion mutant of dotU (DdotU) was previously constructed (Shimizu et al, 2019) through group II intron insertion using a TargeTron Gene Knockout System (Sigma-Aldrich), which was modified for Francisella species (Rodriguez et al, 2008). A deletion mutant of mltA (DmltA) was generated via homologous recombination using the Francisella suicide vector pFRSU (Shimizu et al, 2019). The upstream and downstream regions of mltA (1.5 kbp each) were cloned into the BamHI site of pFRSU to generate pFRSU-mltA.…”

Section: Construction Of F Novicida Mutantsmentioning

confidence: 99%

Identification of Membrane-Bound Lytic Murein Transglycosylase A (MltA) as a Growth Factor for Francisella novicida in a Silkworm Infection Model

Nakamura

Shimizu

Inagaki

et al. 2021

Front. Cell. Infect. Microbiol.

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Francisella tularensis, the causative agent of tularemia, is transmitted by arthropod vectors within mammalian hosts. The detailed mechanisms contributing to growth and survival of Francisella within arthropod remain poorly understood. To identify novel factors supporting growth and survival of Francisella within arthropods, a transposon mutant library of F. tularensis subsp. novicida (F. novicida) was screened using an F. novicida–silkworm infection model. Among 750 transposon mutants screened, the mltA-encoding membrane-bound lytic murein transglycosylase A (MltA) was identified as a novel growth factor of F. novicida in silkworms. Silkworms infection with an mltA deletion mutant (ΔmltA) resulted in a reduction in the number of bacteria and prolonged survival. The ΔmltA strain exhibited limited intracellular growth and cytotoxicity in BmN4 silkworm ovary cells. Moreover, the ΔmltA strain induced higher expression of the antimicrobial peptide in silkworms compared to the wild-type strain. These results suggest that F. novicida MltA contributes to the survival of F. novicida in silkworms via immune suppression-related mechanisms. Intracellular growth of the ΔmltA strain was also reduced in human monocyte THP-1 cells. These results also suggest the contribution of MltA to pathogenicity in humans and utility of the F. novicida–silkworm infection model to explore Francisella infection.

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Expression of Francisella pathogenicity island protein intracellular growth locus E (IglE) in mammalian cells is involved in intracellular trafficking, possibly through microtubule organizing center

Cited by 8 publications

References 55 publications

OpiA, a Type Six Secretion System Substrate, Localizes to the Cell Pole and Plays a Role in Bacterial Growth and Viability inFrancisella tularensisLVS

OpiA, a Type Six Secretion System Substrate, Localizes to the Cell Pole and Plays a Role in Bacterial Growth and Viability inFrancisella tularensisLVS

Soluble lytic transglycosylase SLT of Francisella novicida is involved in intracellular growth and immune suppression

Identification of Membrane-Bound Lytic Murein Transglycosylase A (MltA) as a Growth Factor for Francisella novicida in a Silkworm Infection Model

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