
==== Front
PLoS One
PLoS One
plos
PLOS ONE
1932-6203
Public Library of Science San Francisco, CA USA

10.1371/journal.pone.0310120
PONE-D-24-27526
Research Article
Medicine and Health Sciences
Pathology and Laboratory Medicine
Pathogens
Intracellular Pathogens
Biology and Life Sciences
Microbiology
Medical Microbiology
Microbial Pathogens
Bacterial Pathogens
Francisella Tularensis
Medicine and Health Sciences
Pathology and Laboratory Medicine
Pathogens
Microbial Pathogens
Bacterial Pathogens
Francisella Tularensis
Biology and Life Sciences
Organisms
Bacteria
Francisella
Francisella Tularensis
Biology and Life Sciences
Cell Biology
Cell Processes
Phagocytosis
Biology and Life Sciences
Biochemistry
Proteins
Contractile Proteins
Actins
Biology and Life Sciences
Biochemistry
Proteins
Cytoskeletal Proteins
Actins
Research and Analysis Methods
Animal Studies
Experimental Organism Systems
Model Organisms
Escherichia Coli
Research and Analysis Methods
Model Organisms
Escherichia Coli
Biology and Life Sciences
Microbiology
Medical Microbiology
Microbial Pathogens
Bacterial Pathogens
Escherichia Coli
Medicine and Health Sciences
Pathology and Laboratory Medicine
Pathogens
Microbial Pathogens
Bacterial Pathogens
Escherichia Coli
Biology and Life Sciences
Organisms
Bacteria
Enterobacteriaceae
Escherichia
Escherichia Coli
Biology and Life Sciences
Organisms
Bacteria
Gut Bacteria
Escherichia
Escherichia Coli
Research and Analysis Methods
Animal Studies
Experimental Organism Systems
Prokaryotic Models
Escherichia Coli
Biology and Life Sciences
Biochemistry
Proteins
Intracellular Receptors
Biology and Life Sciences
Cell Biology
Signal Transduction
Intracellular Receptors
Research and Analysis Methods
Microscopy
Light Microscopy
Confocal Microscopy
Medicine and Health Sciences
Medical Conditions
Infectious Diseases
Bacterial Diseases
Role of the JAK2/STAT3 pathway on infection of Francisella novicida
JAK2/STAT3 pathway on Francisella infection
Matsumoto Sonoko Conceptualization Data curation Formal analysis Funding acquisition Investigation Methodology Visualization Writing – original draft 1
https://orcid.org/0000-0003-1294-0799
Shimizu Takashi Conceptualization Data curation Formal analysis Funding acquisition Investigation Methodology Project administration Resources Supervision Validation Visualization Writing – original draft Writing – review & editing 2 *
Uda Akihiko Resources 3
Watanabe Kenta Formal analysis Investigation Writing – review & editing 1
Watarai Masahisa Conceptualization Formal analysis Funding acquisition Project administration Resources Writing – review & editing 1
1 Laboratory of Veterinary Public Health, Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi, Japan
2 One Welfare Education and Research Center, Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi, Japan
3 Department of Veterinary Science, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan
Shokoohi Ebrahim Editor
University of Limpopo, SOUTH AFRICA
Competing Interests: The authors have declared that no competing interests exist.

* E-mail: shimizut@yamaguchi-u.ac.jp
10 9 2024
2024
19 9 e03101207 7 2024
26 8 2024
© 2024 Matsumoto et al
2024
Matsumoto et al
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Francisella tularensis is a causative agent of the zoonotic disease tularemia, and is highly pathogenic to humans. The pathogenicity of this bacterium is largely attributed to intracellular growth in host cells. Although several bacterial factors important for the intracellular growth have been elucidated, including the type VI secretion system, the host factors involved in the intracellular growth of F. tularensis are largely unknown. To identify the host factors important for F. tularensis infection, 368 compounds were screened for the negative regulation of F. tularensis subsp. novicida (F. novicida) infection. Consequently, 56 inhibitors were isolated that decreased F. novicida infection. Among those inhibitors, we focused on cucurbitacin I, an inhibitor of the JAK2/ STAT3 pathway. Cucurbitacin I and another JAK2/STAT3 inhibitor, Stattic, decreased the intracellular bacterial number of F. novicida. However, these inhibitors failed to affect the cell attachment or the intrasaccular proliferation of F. novicida. In addition, treatment with these inhibitors destabilized actin filaments. These results suggest that the JAK2/STAT3 pathway plays an important role in internalization of F. novicida into host cells through mechanisms involving actin dynamics, such as phagocytosis.

http://dx.doi.org/10.13039/501100002241 Japan Science and Technology Agency JPMJSP2111 Matsumoto Sonoko JSPS KAKENHI 22K07054 https://orcid.org/0000-0003-1294-0799
Shimizu Takashi JSPS KAKENHI 21H02360 Watarai Masahisa "SM: JST SPRING Grant Number JPMJSP2111, TS: JSPS KAKENHI Grant Number 22K07054, MW: JSPS KAKENHI Grant Number 21H02360" The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data AvailabilityAll relevant data are within the paper and its Supporting Information files.
Data Availability

All relevant data are within the paper and its Supporting Information files.
==== Body
pmcIntroduction

Francisella tularensis are gram-negative bacteria that can be distinguished into four subspecies; F. tularensis subsp. tularensis (type A), F. tularensis subsp. holarctica (type B), F. tularensis subsp. mediasiatica, and F. tularensis subsp. novicida (F. novicida). Among them type A and type B F. tularensis have been reported to be pathogenic to humans [1]. F. tularensis is the zoonotic causative agent of tularemia, which occurs in Northern Hemisphere countries [1]. As few as 10 bacteria of type A F. tularensis inhaled through aerosols develop tularemia in humans [2]. Consequently, because of the high infectivity of F. tularensis, the Centers for Disease Control and Prevention (USA) is concerned for misuse of F. tularensis in terrorism and has classified this bacterium as a category A bioterrorism agent [3]. F. novicida is highly related to type A F. tularensis genetically but exhibits low pathogenicity in humans [1]. However, F. novicida is pathogenic to mice and is a commensal intracellular pathogen that replicates in both human and mouse macrophages in the same way as seen with type A F. tularensis [4]. Thus F. novicida is thought to have considerable homology with type A F. tularensis and is often used as a practical surrogate of type A F. tularensis [5]. Although there are many reports on virulence factors of Francisella, including the presence of a type VI secretion system [6], the host factors important for the infection of the bacteria are largely unknown.

Francisella are ingested through the pseudopodial loop of macrophages and incorporated into spacious vacuoles that have endosomal markers [7, 8]. The bacterium then escapes from the phagosomal membrane and replicates in the cytoplasm [9]. In the late stages of infection, Francisella bacteria re-enter the autophagosome [10] and acquire amino acids from degraded proteins and replicate [10]. Cytoplasmic bacteria with defective or damaged replication are trapped in Francisella-containing vacuoles, which are lysosome-associated membrane protein 1 (LAMP-1)-positive autophagosomes and are degraded via the ubiquitin-SQSTM1-LC3 pathway [11, 12].

The JAK2/STAT3 pathway is involved in various biological processes such as immunity, cell division, cell death, and tumor formation [13]. Activation of the JAK2/STAT3 pathway occurs via the binding of the extracellular domain of specific intracellular receptors (RTKs) to hormones (e.g., prolactin), growth factors (e.g., epidermal growth factor, EGF), and cytokines (e.g., the interleukin-6, IL-6, family). JAK2 mediates signaling through several cytokine receptors, including IL-6 and IFN-γ. The interaction between ligands and receptors induces dimerization of the receptor subunits. The close proximity of JAK2 noncovalently bound to the intracellular domain of the receptor causes autophosphorylation of JAK2 and stimulates the kinase activity [14, 15]. When the JAK2 protein is phosphorylated, tyrosine residues in the intracellular domain of the receptor are phosphorylated by the activated JAK2 kinase domain, creating a docking site for STAT3 within the SH2 domains of the receptor. This allows cytoplasmic STAT3 protein to bind to the phosphorylated tyrosine residues on the receptor. STAT3 is then phosphorylated by JAK2, dimerizes, dissociates from the receptor, and moves to the nucleus. The phosphorylated STAT3 dimer binds to specific DNA sequences, inducing transcription of target genes, including Cyclin D1, cMyc, Bclx1, bcl-2, MCL-1, and P53, leading to cell proliferation, differentiation, apoptosis, and immune regulation [15]. Furthermore, JAK2/STAT3 signaling can interact with other pathways, including the MAPK/ERK and PI3K/ACT/mTOR signaling pathways, to activate specific cellular responses [16].

In this study, we sought to identify the host factors important for Francisella infection and screened 368 compounds for those that inhibited F. novicida infection. Consequently, we focused on cucurbitacin I, an inhibitor of JAK2/STAT3 [17], and investigated the effect of JAK2/STAT3 pathway on F. novicida infection.

Materials and methods

All experiments were conducted in accordance with the institutional biosecurity guidelines and were approved by Yamaguchi University.

Inhibitor library and inhibitors

The inhibitor compound library was obtained from Molecular Profiling Committee, Grant-in-Aid for Transformative Research Areas “Advanced Animal Model Support (AdAMS)” from The Ministry of Education, Culture, Sports, Science, and Technology, Japan (JSPS KAKENHI Grant Number JP 22H04922). Cucurbitacin I (Merck, Darmstadt, Germany) and Stattic (Merck) were dissolved in dimethyl sulfoxide (DMSO) at 2 mM and then diluted to 200, 20, and 2 μM. The same amount of the inhibitors and DMSO control were added to culture medium at a final concentration of 10, 1, 0.1, and 0.01 μM.

Cell culture

The mouse monocyte-macrophage J774.1 cell line was cultured at 37°C under 5% CO2 in Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher, Waltham, MA) supplemented with 10% fetal bovine serum (Thermo Fisher).

Bacteria strains and culture conditions

F. novicida ATCC 15482 strain was cultured aerobically at 37°C with brain heart infusion broth (Becton, Dickinson and Company, Franklin Lakes, NJ) supplemented with cysteine (BHIc), BHIc plates containing 1.5% Agar (Wako Laboratory Chemicals, Osaka, Japan) [18], or chemically defined medium (CDM) [19]. Green-Fluorescent protein (GFP)-expressing F. novicida was cultured with BHIc containing 2.5 μg/mL chloramphenicol [20]. Escherichia coli ATCC15482 strain was cultured aerobically at 37°C in Luria–Bertani medium (LB) (Nacalai Tesque, Kyoto, Japan) or LB plates containing 1.5% agar. Bacterial concentrations were adjusted based on their optical density at 595 nm.

Screening of inhibitors

J774.1 cells (25 × 104 cells/mL) were seeded at 100 μL/well in a 96-well plate or 500 μL/well in a 24-well plate and cultured overnight. Cells were treated with 10 or 1 μM of inhibitors for 2 h; DMSO was used as the control. After treatment, cells were infected with GFP-expressing F. novicida at multiplicity of infection (MOI) of 1 and incubated for 24 h. Cells were then washed three times with phosphate-buffered saline (PBS) and fluorescence intensity was measured using plate reader 2030 ARVO X4 (Perlin Elmer, Waltham, MA). An intensity at 4,000 lower than that of the DMSO control showed inhibition of infection, whereas an intensity at 5000 higher than that of the control showed enhancement of infection (S1 Table).

Inhibitor treatment before infection

J774.1 cells (25 × 104 cells/mL) were seeded at 100 μL/well in a 96-well plate or 500 μL/well in a 24-well plate and cultured overnight. Cells were treated with indicated concentration of cucurbitacin I or Stattic for 2 h; DMSO was used as the control. After treatment, cells were infected with F. novicida at an MOI of 1. Plates were centrifuged for 10 min at 300 × g and incubated for 1 h at 37°C. Cells were cultured in culture medium containing inhibitors, and 50 μg/mL of gentamycin added for 1 h to kill extracellular bacteria. Cells were then washed three times with PBS and cultured in medium containing inhibitors at 37°C.

Inhibitor treatment after infection

J774.1 cells (25 × 104 cells/mL) were seeded at 100 μL/well in a 96-well plate or 500 μL/well in a 24-well plate and cultured overnight. Cells were infected with F. novicida at an MOI of 1. Plates were centrifuged for 10 min at 300 × g and incubated for 1 h at 37°C. Cells were treated with 50 μg/mL of gentamycin to kill extracellular bacteria. Cells were washed three times with culture medium and treated with indicated concentrations of cucurbitacin I or Stattic.

Colony forming units (CFU)

J774.1 cells (25 × 104 cells/mL) were seeded at 100 μL/well in a 96-well plate and cultured overnight. After inhibitor treatment and infection described above, cells were washed three times with PB and then disrupted with 0.1% Triton-X in CDM for 1 min and 900 μL of CDM was immediately added. Samples were diluted with CDM and cultured on a BHIc plate overnight, and the number of colonies were counted.

Laser scanning confocal microscopy

J774.1 cells (25 × 104 cells/mL) were seeded at 500 μL/well in a 24-well plate with 120-mm glass coverslips (Matsunami, Osaka, Japan) and cultured overnight. After treatment with inhibitors and infection with GFP-expressing F. novicida as described above, cells were washed three times with PBS and fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. Images of the cells were obtained using FluoView FV100 confocal laser scanning microscope (Olympus, Tokyo, Japan).

Measurement of phagocytotic activity

The activity of phagocytosis against E. coli was measured as previously described with slight modification [21, 22]. J774.1 cells (25 × 104 cells/mL) were seeded at 100 μL/well in a 96-well plate or 500 μL/well in a 24-well plate and cultured overnight. Cells were treated with the indicated concentration of cucurbitacin I, Stattic, or DMSO control for 2 h. After treatment, cells were infected with E. coli for 3 h and treated with gentamycin to kill extracellular bacteria. Cells were then washed three times with PBS and disrupted with 0.1% Triton-X in PBS for 1 min followed by immediate addition of 900 μL PBS. CFU was counted as described above.

Visualization of actin filaments

J774.1 cells (25 × 104 cells/mL) were seeded at 500 μL/well in a 24-well plate with 12-mm glass coverslips and cultured overnight. After treatment of inhibitors and infection described above, cells were washed three times with PBS and fixed with 4% paraformaldehyde in PBS. After being washed three times with PBS, cells were permeabilized with 0.1% Triton-X in PBS for 5 min and washed three times with PBS. Cells were then blocked with 2% of bovine serum albumin in PBS for 1 h. and stained with 0.1 μM of phalloidin-rhodamine B isothiocyanate (P1951, Thermo Fisher) for 2 h. Cells were washed three times with PBS and observed with laser scanning confocal microscopy.

Statistical analysis

Significant differences are determined by P < 0.05 or P < 0.01 using Student’s t-test or Dunnett’s test, or Tukey–Kramer method, and indicated with * and ** respectively.

Results

Screening of inhibitors

To identify host factors important for the infection of F. novicida, 368 inhibitor compounds were screened to identify those that inhibited the growth of F. novicida. J774.1 cells treated with inhibitors for 2 h were infected with GFP-expressing F. novicida, and cells with a lower fluorescence compared with that of untreated cells were selected (S1 Table). Consequently, 56 inhibitors were selected to inhibit the growth of F. novicida in J774.1 cells (Table 1). A number of the identified inhibitors have been demonstrated to possess antibiotic properties. Among the non-antibiotic inhibitors, three were found to be involved in the Jak-2/Stat3 pathway. Consequently, we considered that the Jak-2/Stat3 pathway was a key factor in F. novicida infection and focused on cucurbitacin I as a representative inhibitor of the JAK2/STAT3 pathway.

10.1371/journal.pone.0310120.t001 Table 1 Inhibitors negatively regulated the infection of F. novicida.

Compound	Category	
5,15-DPP	STAT3	
ABT-702	AK	
Actinonin	aminopeptidase M	
AG957	Bcr-abl	
Amastatin	aminopeptidase A	
AMD3100 octahydrochloride	CXCR4	
Aminoglutethimide	aromatase	
anisomycin	stress inducer	
Bafilomycin A1	V-ATPase	
BH3I-1	Bcl-XL	
bortezomib	Proteasome	
brefeldin A	golgi inhibitor	
CA-074	cathepsin B	
Cdk4 inhibitor	CDK	
Chetomin	HIF	
Cucurbitacin I	Jak-2	
cyclopamine	Hedgehog	
Cytochalasin D	actin filament	
E-64d	calpain	
Formestane	aromatase	
gefitinib	EGFR	
Glibenclamide	K channel	
HA 14–1	Bcl-2	
imatinib mesylate	Bcr-Abl/Kit	
Jervine	Hedgehog	
KT5823	PKG	
Leptomycin B	CRM1	
LFM-A13	Burton’s tyrosine kinase(BTK)	
MG-132	proteasome	
Mifepristone	progesterone receptor	
Monensin	Na ionophore	
Nifedipine	Ca channel	
Nigericin	K ionophore	
nilotinib	Bcr-Abl	
NSC625987	CDK	
NSC95397	Cdc25	
Ouabain	Na/K ATPase	
Pepstatin A	cathepsin D	
Pifithrin-a (cyclic)	p53	
PIM1 Inhibitor II	PIM	
PKR inhibitor	PKR	
PRIMA-1	p53 activator	
radicicol	Hsp90	
Rotenone	mitochondrial complex I	
RS 102895	CCR2	
Sanguinarine	Na/K/Mg ATPase	
SB 328437	CCR3	
sorafenib	Multi-kinases	
sunitinib malate	Multi-kinases	
temsirolimus	mTOR	
Thapsigargin	Ca-ATPase	
TOFA	acetyl-CoA carboxylase (ACC)	
vorinostat	HDAC	
WP1066	STAT3	
YM155	Survivin	
Z-GLF-CMK	cathepsin G	

Effect of inhibitors on F. novicida infection

To assess whether cucurbitacin I suppressed F. novicida infection, J774.1 cells treated with cucurbitacin I were infected them with GFP-expressing F. novicida, and intracellular bacteria were observed using confocal microscopy. Cucurbitacin I-treated J774.1 cells were also infected with F. novicida, and the number of intracellular bacteria was measured by colony counting. Cucurbitacin I treatment decreased the number of intracellular F. novicida (Fig 1A, 1C), indicating that the JAK2/STAT3 pathway is important for F. novicida infection. To confirm this, the effect of another JAK2/STAT3 pathway inhibitor Stattic was evaluated. Stattic treatment decreased the number of intracellular F. novicida as assessed by both microscopic observation and colony counting (Fig 1B, 1D). A concentration of 1 μM cucurbitacin and 10 μM of Stattic were significantly effective and were therefore used in subsequent experiments.

10.1371/journal.pone.0310120.g001 Fig 1 Suppression of F. novicida infection by JAK2/STAT3 inhibitors.

J774.1 cells treated with 0.01 to 10 μM of cucurbitacin I (A, C) or Stattic (B, D) were infected with F. novicida (C, D) or GFP-expressing F. novicida (A, B). Cells were treated with gentamicin and incubated for 12 h and then observed with confocal microscopy (A, B), or the number of intracellular bacterial was counted (C, D). Data represent the average and standard deviation of three identical experiments. Differences were analyzed with multiple comparison (Dunnett’s test) and indicated by asterisks, **P < 0.01, *P < 0.05. Scale bar: 10μm.

Growth of F. novicida in BHIc

To check the direct effect of inhibitors on the growth of F. novicida, cucurbitacin I or Stattic was added into the growth medium, and the growth was measured by optical density and colony count. No significant differences in counts were observed between inhibitors and DMSO control (Fig 2A–2D). GFP-expressing F. novicida cultured with inhibitors were washed and infected into J774.1 cells, and intracellular bacteria were observed using confocal microscopy. The same levels of intracellular F. novicida was observed in bacteria cultured with cucurbitacin I or Stattic compared with that of DMSO control (Fig 3). Therefore, cucurbitacin I and Stattic had no direct effect on the growth and infectivity of F. novicida.

10.1371/journal.pone.0310120.g002 Fig 2 Growth of F. novicida in culture medium with inhibitors.

F. novicida was cultured in BHIc with cucurbitacin I (A, C) or Stattic (B, D) and optical density (λ = 595 nm) was measured at the indicated time point (A, B). The number of CFU at 0 and 12 h was counted (C, D). Data represent the average and standard deviation of three identical experiments. Differences were analyzed with multiple comparison (Tukey–Kramer method) (A, C) or Student t-test (B, D) and indicated by asterisks, *P < 0.05.

10.1371/journal.pone.0310120.g003 Fig 3 Intracellular growth of F. novicida cultured with inhibitors.

F. novicida was cultured in BHIc with cucurbitacin I or Stattic. J774.1 cells were infected with inhibitor-treated F. novicida and observed at 12 h post infection. Scale bar: 10μm.

Cell adhesion, invasion, and intracellular proliferation of F. novicida

Infection of F. novicida is established via three infections steps: attachment, internalization, and proliferation. To investigate which step of infection is affected by the identified inhibitors, the attachment of F. novicida was initially tested. J774.1 cells were treated with cucurbitacin or Stattic and then infected with F. novicida. The number of bacteria attached to cells just after infection (10 and 30 min) was measured by colony counting. No significant difference was observed in colony counts between control DMSO and inhibitor treatments (Fig 4). Next, to test the effect of inhibitors of internalization and proliferation, J774.1 cells were treated with inhibitors and infected with F. novicida. Cells were incubated for 1 h to allow internalization of bacteria while attached bacterial cells were removed by gentamicin treatment. After 1.5 and 12 h incubation, the number of internalized and proliferated F. novicida were measured. The number of bacteria was decreased in cells treated with each inhibitor compared with that treated with DMSO, indicating that either internalization or proliferation were affected by the inhibitors (Fig 5). To determine which steps of internalization and proliferation was affected by inhibitors, the proliferation of F. novicida was examined. J774.1 cells were infected with F. novicida and incubated for 1 h to allow internalization of bacteria. Attached bacteria were removed by gentamicin treatment and infected cells were treated with inhibitors. The number of bacteria in inhibitor-treated cells were unaffected by inhibitors (Fig 6). Thus, cucurbitacin I and Stattic affect internalization but not proliferation in F. novicida infection.

10.1371/journal.pone.0310120.g004 Fig 4 Attachment of F. novicida to cell surface.

J774.1 cells treated with 1 μM of cucurbitacin I (A, C) or 10 μM Stattic (B, D) were infected with F. novicida (C, D) or GFP-expressing F. novicida (A, B). Cells were observed with confocal microscopy (A, B), or the intracellular bacterial number was counted (C, D) at 10 or 30 min post infection. The data represent the averages and standard deviations of three identical experiments. Differences were analyzed with Student t-test and indicated by asterisks, *P < 0.05. Scale bar: 10μm.

10.1371/journal.pone.0310120.g005 Fig 5 Internalization and proliferation of F. novicida.

J774.1 cells treated with 1 μM of cucurbitacin I (A, C) or 10 μM of Stattic (B, D) were infected with F. novicida (C, D) or GFP-expressing F. novicida (A, B). Cells were treated with gentamicin and incubated for 1.5 or 12 h, Cells were then observed with confocal microscopy (A, B), or the intracellular bacterial number was counted (C, D). Data represent the average and standard deviation of three identical experiments. Differences were analyzed with Student t-test and indicated by asterisks, **P < 0.01, *P < 0.05. Scale bar: 10μm.

10.1371/journal.pone.0310120.g006 Fig 6 Intracellular proliferation of F. novicida.

J774.1 cells were infected with F. novicida (B, C) or GFP-expressing F. novicida (A). Cells were treated with gentamicin and cultured with 1 μM of cucurbitacin I (A, B) or 10 μM of Stattic (A, C) for 12 h. Cells were observed with confocal microscopy(A), or the intracellular bacterial number was counted (B, C). Data represent the average and standard deviation of three identical experiments. Differences were analyzed with multiple comparison (Dunnett’s test) and indicated by asterisks, **P < 0.01. Scale bar: 10μm.

Phagocytotic activity

As cucurbitacin I and Stattic affected the internalization of F. novicida, the effect of inhibitors on phagocytosis was investigated using the nonintracellular bacteria E. coli. J774.1 cells were treated with cucurbitacin I or Stattic and infected with E. coli. Cells were incubated for 3 h to allow phagocytosis and attached cells were removed by gentamicin treatment for 30 min. At 3.5 h post infection, the internal bacterial number was measured by colony counting. Addition of inhibitors decreased the number of internalized E. coli (Fig 7), indicating cucurbitacin I and Stattic affect host cell phagocytosis.

10.1371/journal.pone.0310120.g007 Fig 7 Effects of inhibitors on phagocytosis.

J774.1 cells treated with 1 μM of cucurbitacin I (A, B) or 10 μM of Stattic (A, B) were infected with E. coli (B) or GFP-expressing E.coli (A). Cells were treated with gentamicin and incubated for 3 h. Cells were observed with confocal microscopy (A), or the intracellular bacterial number was counted (B). Data represent the average and standard deviation of three identical experiments. Differences were analyzed with multiple comparison (Dunnett’s test) and indicated by asterisks, **P < 0.01. Scale bar: 10μm.

Actin filaments

Phagocytosis results from polymerization, depolymerization, and rearrangement of actin [23]. To investigate the effect of inhibitors on actin polymerization, J774.1 cells were treated with cucurbitacin I or Stattic for 2 h and infected with F. novicida, and then actin was visualized using immunofluorescence microscopy. Abnormal arrangements of actin were observed in inhibitor-treated cells compared with those in cells treated with DMSO control (Fig 8). These results suggest that the correct arrangement of actin is crucial for the infection of F. novicida.

10.1371/journal.pone.0310120.g008 Fig 8 Effects of inhibitors on actin filaments.

J774.1 cells treated with 1 μM of cucurbitacin I or 10 μM of Stattic were infected with GFP-expressing F. novicida for the indicated time. Cells were stained with phalloidin-rhodamine and observed by confocal microscopy. Scale bar: 10μm.

Discussion

To identify the host factors important for Francisella infection, 368 inhibitors were screened, and those that affected F. novicida infection were selected. Consequently, 56 compounds inhibited the infection of F. novicida while eight enhanced the infection. In this study we focused on 56 inhibitors that negatively affected infection to identify the host factors important for infection. Most of the 56 inhibitors possessed antibiotic property, whereas three inhibitors were related to the JAK2/STAT3 pathway. Therefore, we focused on the inhibitors related to the JAK2/STAT3 pathway without antibiotic properties, and cucurbitacin I was selected for farther study.

Cucurbitacin I is a triterpenoid compound derived from the fruit extract of plants, such as cucumber, in the Cucurbitaceae family [24]. Cucurbitacin I inhibits JAK2 phosphorylation and thereby suppresses the levels of tyrosine-phosphorylated STAT3 [17]. In this study, cucurbitacin I inhibited the infection of F. novicida but failed to affect the growth of F. novicida in culture medium. To confirm the involvement of the JAK2/STAT3 pathway in F. novicida infection, another inhibitor of the JAK2/STAT3 pathway, Stattic was tested. Stattic is a nonpeptide small molecule that inhibits the dimerization of STAT3 through the SH2 domain [25]. Similar to cucurbitacin I, Stattic did not affect growth in the culture medium but did inhibit F. novicida infection. In addition, F. novicida infection tended to enhance activation of STAT3 just after infection. These results indicate that the JAK2/STAT3 pathway plays an important role in F. novicida infection.

To examine which of the three steps of adhesion, invasion, and intracellular proliferation is inhibited by cucurbitacin I or Stattic, cells were treated with inhibitors and the subsequent effects observed at different time points. Treatment of inhibitors failed to affect the attachment of F. novicida to the cells at 10 or 30 min post infection, indicating that cucurbitacin I and Stattic affects the internalization or intracellular proliferation. Cucurbitacin I or Stattic treatment after infection failed to decrease the number of intracellular F. novicida, indicating that intracellular proliferation was not affected by the inhibitors, indicating that the JAK2/STAT3 pathway is important for the internalization step of F. novicida. These results are consistent with the various reports concerning intracellular infection by other bacteria. In infection by Brucella abortus, the AK2/STAT3 pathway is important for the intracellular survival of the bacteria [26]. In Helicobacter pylori infection, inhibition of the JAK2/STAT3 pathway reduces the development of gastric cancer [27]. In addition, the JAK2/STAT3 pathway is important for the development of pulmonary fibrosis in Mycobacterium tuberculosis infection [28]. Thus, the JAK2/STAT3 pathway is important for the infection and pathogenesis of various bacterial intracellular infections.

Francisella are ingested through the pseudopodia of macrophages and incorporated into spacious vacuoles with endosomal markers [7, 8]. The organism then escapes from the phagosomal membrane and replicates in the cytoplasm [9]. To examine which step of phagocytosis or escape from phagosome is the target of inhibitors, the ingestion of E. coli, a bacterium that cannot escape from the phagosome was evaluated. Subsequently, the number of ingested intracellular E. coli was also decreased by treatment with cucurbitacin I or Stattic. This result indicates that cucurbitacin I and Stattic inhibit the phagocytosis step of bacterial infection.

Phagocytosis results from polymerization, depolymerization, and rearrangement of actin [23], and we therefore evaluated the actin dynamics of F. novicida-infected cells and observed abnormal arrangements of actin in cucurbitacin I- or Stattic-treated cells. These results suggest that the JAK2/STAT3 pathway regulates actin dynamics followed by phagocytosis. This finding is consistent with a previous study where cucurbitacin I inhibited cell motility or proliferation of cancer cells by interfering with actin dynamics [29, 30].

Since cucurbitacin I exhibits an antitumor effect, cucurbitacin I and JAK2/STAT3 inhibitors have received increasing attention as potential cancer therapeutic agents [31, 32]. In this study, we identified cucurbitacin I as an inhibitor of F. novicida infection and demonstrated that the JAK2/STAT3 pathway is important for the actin dynamics that underlie phagocytosis. In infection by other intracellular bacteria such as Brucella and Mycobacterium, the JAK2/STAT3 pathway is important for the intracellular growth and pathogenesis [26, 28]. Moreover, cucurbitacin I exhibits an antimicrobial effect through induction of autophagy [33]. Therefore, inhibitors such as cucurbitacin I and Stattic can be utilized as antimicrobial agents, and the JAK2/STAT3 pathway can be a therapeutic target of infection with intracellular bacteria as well.

Supporting information

S1 Table List of inhibitors.

*Fluorescence intensity of GFP-expressing F. novicida >5000 higher than that of the DMSO control was determined as positively regulating inhibitors (+), and >4000 lower than that of the control was determined as negatively regulating inhibitors (−). **Same compounds but derived from different providers.

(PDF)

S1 Data (XLSX)

10.1371/journal.pone.0310120.r001
Decision Letter 0
Shokoohi Ebrahim Academic Editor
© 2024 Ebrahim Shokoohi
2024
Ebrahim Shokoohi
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version0
19 Aug 2024

PONE-D-24-27526Effect of the JAK2/STAT3 Pathway on Internalization of Francisella novicida.PLOS ONE

Dear Dr. Shimizu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Oct 03 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Ebrahim Shokoohi

Academic Editor

PLOS ONE

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and 

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2022.1027424/full

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. 

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

4. Thank you for stating the following financial disclosure: "SM: JST SPRING Grant Number JPMJSP2111

TS: JSPS KAKENHI Grant Number 22K07054, MW: JSPS KAKENHI Grant Number 21H02360".

Please state what role the funders took in the study.  If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

5. Thank you for stating the following in the Acknowledgments Section of your manuscript: "This work was supported by JST SPRING Grant Number JPMJSP2111, JSPS KAKENHI Grant Number 22K07054, JSPS KAKENHI Grant Number 21H02360, and Molecular Profiling Committee, Grant-in-Aid for Transformative Research Areas “Advanced Animal Model Support (AdAMS)” from JSPS KAKENHI Grant Number JP 22H04922."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. 

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: "SM: JST SPRING Grant Number JPMJSP2111

TS: JSPS KAKENHI Grant Number 22K07054, MW: JSPS KAKENHI Grant Number 21H02360".

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

6. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

Dear Authors

We have received the feedback for your work. You need to address the concerns of the Referees. The comments are given for you reference. Please note that I have acted as a reviewer for this manuscript, and you will find my comments below, under Reviewer 1.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The paper entitled Effect of the JAK2/STAT3 Pathway on Internalization of Francisella novicida; is very interning. It was written well and provided with sufficient data. However, some concern need to be addressed.

1-Why the authors choose Cucurbitacin I? What about the other inhibitors?

2-What is the source of Cucurbitacin I?

3-What is the purpose of providing merge, DIC ,and GFP?

4-Interfiering of the JAK2/STAT3 Pathway by Cucurbitacin, how it affect the phenotype of the bacteria?

5-The title of the paper is not in line with the context. It should be revised by the authors. Internalization, and withing the text is all about inhibiting the pathway by Cucurbitacin I.

Reviewer #2: The paper was checked as Effect of the JAK2/STAT3 Pathway on Internalization of Francisella novicida

It is an exciting paper on the plant extract main ingredient effect on the bacteria, which bring valuable insight into the bacterial physiology and biology within the cells. However, the authors must explain how they choose Cucurbitacin I, and what was the rate of inhibition? The title of the paper also confusing as only bacteria and the pathway included, but not the effect of plant extract on the pathway. Overall, the manuscript is well written, and I would recommend to be published in PLoS One after a minor revision.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

10.1371/journal.pone.0310120.r002
Author response to Decision Letter 0
Submission Version1
21 Aug 2024

Thank you for reviewing our paper entitled '' Effect of the JAK2/STAT3 Pathway on Internalization of Francisella novicida'' by Matsumoto et al. (PONE-D-24-27526), and giving us an opportunity to submit our manuscript again.

We corrected our manuscript according to comments of the editor and reviewers. We believe that the revised manuscript is improved significantly with the referees’ advice and now acceptable for publication in PLOS One.

1. When submitting your revision, we need you to address these additional requirements. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

--As you suggested, we checked the requirements and made small changes, e.g. to font sizes.

2. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2022.1027424/full

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

-- As you suggest, we checked our manuscript using iThenticate software and rephrase some sentences (Line 20-21, 91-92, 161-162).

3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

-- As you suggest, we arranged the ‘Funding Information’.

4. Thank you for stating the following financial disclosure: "SM: JST SPRING Grant Number JPMJSP2111 TS: JSPS KAKENHI Grant Number 22K07054, MW: JSPS KAKENHI Grant Number 21H02360".

Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

--As you suggested, we added the sentence in cover letter.

5. Thank you for stating the following in the Acknowledgments Section of your manuscript: "This work was supported by JST SPRING Grant Number JPMJSP2111, JSPS KAKENHI Grant Number 22K07054, JSPS KAKENHI Grant Number 21H02360, and Molecular Profiling Committee, Grant-in-Aid for Transformative Research Areas “Advanced Animal Model Support (AdAMS)” from JSPS KAKENHI Grant Number JP 22H04922."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: "SM: JST SPRING Grant Number JPMJSP2111

TS: JSPS KAKENHI Grant Number 22K07054, MW: JSPS KAKENHI Grant Number 21H02360".

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

--Thank you for checking our funding information. We removed Acknowledge section from the manuscript. The information listed above is correct. We added explanation about this information in cover letter.

6. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

--We checked references and found no problems.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: The paper entitled Effect of the JAK2/STAT3 Pathway on Internalization of Francisella novicida; is very interning. It was written well and provided with sufficient data. However, some concern need to be addressed.

--Thank you for your reviewing. As you suggested we corrected our manuscript.

1- Why the authors choose Cucurbitacin I? What about the other inhibitors?

--In this study, we identified 56 inhibitors that negatively regulate F. novicida infection. Among them 3 inhibitors were related to Jak-2/STAT3 pathway. Therefore we thought that Jak-2/STAT3 pathway might be involved in F. novicida infection. Cucurbitacin was used as a representative inhibitor of the Jak-2/Stat3 pathway. Additionally, other promising inhibitors have been identified, including those targeting mTOR and p53, which are currently undergoing analysis. To explain this we rewrote sentences (line 197-201).

2- What is the source of Cucurbitacin I?

--Cucurbitacin I is a natural cell-permeable triterpenoid isolated from Cucurbitaceae, and is a selective inhibitor of JAK2/STAT3. The Cucurbitacin I is explained in Discussion section (line334-335).

3- What is the purpose of providing merge, DIC ,and GFP?

--DIC was used to show the location of the cells; the intention was to show that the bacteria were intracellularly located by using merged images with the DIC.

4- Interfiering of the JAK2/STAT3 Pathway by Cucurbitacin, how it affect the phenotype of the bacteria?

--We thought cucurbitacin might have an antibiotic effect, so we added cucurbitacin to the medium and investigated growth, but found that cucurbitacin had no effect on bacterial growth (Fig 2). From these data we consider that cucurbitacin does not affect the phenotype of F. novicida.

5- The title of the paper is not in line with the context. It should be revised by the authors. Internalization, and withing the text is all about inhibiting the pathway by Cucurbitacin I.

--We changed the title as you suggested (line 1).

Reviewer #2: The paper was checked as Effect of the JAK2/STAT3 Pathway on Internalization of Francisella novicida. It is an exciting paper on the plant extract main ingredient effect on the bacteria, which bring valuable insight into the bacterial physiology and biology within the cells.

--Thank you for your review. We corrected our manuscript as you suggested.

However, the authors must explain how they choose Cucurbitacin I, and what was the rate of inhibition?

--In this study, we identified 56 inhibitors that negatively regulate F. novicida infection. Among them 3 inhibitors were related to Jak-2/STAT3 pathway. Therefore we thought that Jak-2/STAT3 pathway might be involved in F. novicida infection. Cucurbitacin was used as a representative inhibitor of the Jak-2/Stat3 pathway. Additionally, other promising inhibitors have been identified, including those targeting mTOR and p53, which are currently undergoing analysis. To explain this we rewrote sentences (line 197-201). In the first screening we used the intensity of GFP, so the rate of inhibition could only be expressed as a relative value compared to the control (table S1). Therefor, we measured the effect of Cucurbitacin I on F. novicida infection in Fig. 1.

The title of the paper also confusing as only bacteria and the pathway included, but not the effect of plant extract on the pathway.

--We changed the title as you suggest(line 1).

Overall, the manuscript is well written, and I would recommend to be published in PLoS One after a minor revision.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

--We checked figures with PACE and converted tiff files were uploaded to the journal online system.

Attachment Submitted filename: Response to Reviewers.docx

10.1371/journal.pone.0310120.r003
Decision Letter 1
Shokoohi Ebrahim Academic Editor
© 2024 Ebrahim Shokoohi
2024
Ebrahim Shokoohi
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version1
26 Aug 2024

Role of the JAK2/STAT3 Pathway on Infection of Francisella novicida.

PONE-D-24-27526R1

Dear Dr.Takashi Shimizu,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Ebrahim Shokoohi

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Authors improved the paper and answered all raised concerns by the Referees.

Reviewers' comments:

no comments

10.1371/journal.pone.0310120.r004
Acceptance letter
Shokoohi Ebrahim Academic Editor
© 2024 Ebrahim Shokoohi
2024
Ebrahim Shokoohi
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
30 Aug 2024

PONE-D-24-27526R1

PLOS ONE

Dear Dr. Shimizu,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

* All references, tables, and figures are properly cited

* All relevant supporting information is included in the manuscript submission,

* There are no issues that prevent the paper from being properly typeset

If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps.

Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

If we can help with anything else, please email us at customercare@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Ebrahim Shokoohi

Academic Editor

PLOS ONE
==== Refs
References

1 Ellis J , Oyston PC , Green M , Titball RW . Tularemia. Clin Microbiol Rev. 2002;15 (4 ):631–46. Epub 2002/10/05. doi: 10.1128/CMR.15.4.631-646.2002 ; PubMed Central PMCID: PMC126859.12364373
2 McLendon MK , Apicella MA , Allen LA . Francisella tularensis: taxonomy, genetics, and Immunopathogenesis of a potential agent of biowarfare. Annu Rev Microbiol. 2006;60 :167–85. Epub 2006/05/18. doi: 10.1146/annurev.micro.60.080805.142126 ; PubMed Central PMCID: PMC1945232.16704343
3 Maurin M. Francisella tularensis as a potential agent of bioterrorism? Expert Rev Anti Infect Ther. 2015;13 (2 ):141–4. Epub 2014/11/22. doi: 10.1586/14787210.2015.986463 .25413334
4 Anthony LD , Burke RD , Nano FE . Growth of Francisella spp. in rodent macrophages. Infect Immun. 1991;59 (9 ):3291–6. Epub 1991/09/01. doi: 10.1128/iai.59.9.3291-3296.1991 ; PubMed Central PMCID: PMC258167.1879943
5 Kingry LC , Petersen JM . Comparative review of Francisella tularensis and Francisella novicida. Front Cell Infect Microbiol. 2014;4 :35. Epub 2014/03/25. doi: 10.3389/fcimb.2014.00035 ; PubMed Central PMCID: PMC3952080.24660164
6 Broms JE , Sjostedt A , Lavander M . The Role of the Francisella Tularensis Pathogenicity Island in Type VI Secretion, Intracellular Survival, and Modulation of Host Cell Signaling. Front Microbiol. 2010;1 :136. Epub 2010/01/01. doi: 10.3389/fmicb.2010.00136 ; PubMed Central PMCID: PMC3109350.21687753
7 Clemens DL , Lee BY , Horwitz MA . Virulent and avirulent strains of Francisella tularensis prevent acidification and maturation of their phagosomes and escape into the cytoplasm in human macrophages. Infect Immun. 2004;72 (6 ):3204–17. Epub 2004/05/25. doi: 10.1128/IAI.72.6.3204–3217.2004 ; PubMed Central PMCID: PMC415696.15155622
8 Clemens DL , Lee BY , Horwitz MA . Francisella tularensis enters macrophages via a novel process involving pseudopod loops. Infect Immun. 2005;73 (9 ):5892–902. Epub 2005/08/23. doi: 10.1128/IAI.73.9.5892–5902.2005 ; PubMed Central PMCID: PMC1231130.16113308
9 Golovliov I , Baranov V , Krocova Z , Kovarova H , Sjostedt A . An attenuated strain of the facultative intracellular bacterium Francisella tularensis can escape the phagosome of monocytic cells. Infect Immun. 2003;71 (10 ):5940–50. Epub 2003/09/23. doi: 10.1128/IAI.71.10.5940–5950.2003 ; PubMed Central PMCID: PMC201066.14500514
10 Checroun C , Wehrly TD , Fischer ER , Hayes SF , Celli J . Autophagy-mediated reentry of Francisella tularensis into the endocytic compartment after cytoplasmic replication. Proc Natl Acad Sci U S A. 2006;103 (39 ):14578–83. Epub 2006/09/20. doi: 10.1073/pnas.0601838103 ; PubMed Central PMCID: PMC1600002.16983090
11 Chong A , Celli J . The francisella intracellular life cycle: toward molecular mechanisms of intracellular survival and proliferation. Front Microbiol. 2010;1 :138. Epub 2010/01/01. doi: 10.3389/fmicb.2010.00138 ; PubMed Central PMCID: PMC3109316.21687806
12 Chong A , Wehrly TD , Child R , Hansen B , Hwang S , Virgin HW , et al . Cytosolic clearance of replication-deficient mutants reveals Francisella tularensis interactions with the autophagic pathway. Autophagy. 2012;8 (9 ):1342–56. Epub 2012/08/07. doi: 10.4161/auto.20808 ; PubMed Central PMCID: PMC3442881.22863802
13 Hofmann HD , Kirsch M . JAK2-STAT3 signaling: A novel function and a novel mechanism. JAKSTAT. 2012;1 (3 ):191–3. Epub 2012/07/01. doi: 10.4161/jkst.20446 ; PubMed Central PMCID: PMC3670243.24058769
14 Wu H , Huang M , Cao P , Wang T , Shu Y , Liu P . MiR-135a targets JAK2 and inhibits gastric cancer cell proliferation. Cancer Biol Ther. 2012;13 (5 ):281–8. Epub 2012/02/09. doi: 10.4161/cbt.18943 .22310976
15 Kiu H , Nicholson SE . Biology and significance of the JAK/STAT signalling pathways. Growth Factors. 2012;30 (2 ):88–106. Epub 2012/02/22. doi: 10.3109/08977194.2012.660936 ; PubMed Central PMCID: PMC3762697.22339650
16 Murray PJ . The JAK-STAT signaling pathway: input and output integration. J Immunol. 2007;178 (5 ):2623–9. Epub 2007/02/22. doi: 10.4049/jimmunol.178.5.2623 .17312100
17 Blaskovich MA , Sun J , Cantor A , Turkson J , Jove R , Sebti SM . Discovery of JSI-124 (cucurbitacin I), a selective Janus kinase/signal transducer and activator of transcription 3 signaling pathway inhibitor with potent antitumor activity against human and murine cancer cells in mice. Cancer Res. 2003;63 (6 ):1270–9. Epub 2003/03/22. .12649187
18 Mc Gann P , Rozak DA , Nikolich MP , Bowden RA , Lindler LE , Wolcott MJ , et al . A novel brain heart infusion broth supports the study of common Francisella tularensis serotypes. J Microbiol Methods. 2010;80 (2 ):164–71. Epub 2009/12/17. doi: 10.1016/j.mimet.2009.12.005 .20005265
19 Nagle SC Jr. , Anderson RE , Gary ND . Chemically defined medium for the growth of Pasteurella tularensis. J Bacteriol. 1960;79 (4 ):566–71. Epub 1960/04/01. doi: 10.1128/jb.79.4.566–571.1960 ; PubMed Central PMCID: PMC278733.14425793
20 Nakamura T , Shimizu T , Inagaki F , Okazaki S , Saha SS , Uda A , et al . Identification of Membrane-Bound Lytic Murein Transglycosylase A (MltA) as a Growth Factor for Francisella novicida in a Silkworm Infection Model. Front Cell Infect Microbiol. 2020;10 :581864. Epub 2021/02/09. doi: 10.3389/fcimb.2020.581864 ; PubMed Central PMCID: PMC7862118.33553001
21 Chen K , Yoshimura T , Gong W , Tian C , Huang J , Trinchieri G , et al . Requirement of CRAMP for mouse macrophages to eliminate phagocytosed E. coli through an autophagy pathway. J Cell Sci. 2021;134 (5 ). Epub 2021/01/21. doi: 10.1242/jcs.252148 ; PubMed Central PMCID: PMC7970306.33468624
22 Drevets DA , Canono BP , Campbell PA . Measurement of bacterial ingestion and killing by macrophages. Curr Protoc Immunol. 2015;109 :14 6 1–6 7. Epub 2015/04/08. doi: 10.1002/0471142735.im1406s109 .25845563
23 May RC , Machesky LM . Phagocytosis and the actin cytoskeleton. J Cell Sci. 2001;114 (Pt 6 ):1061–77. Epub 2001/03/03. doi: 10.1242/jcs.114.6.1061 .11228151
24 Cai Y , Fang X , He C , Li P , Xiao F , Wang Y , et al . Cucurbitacins: A Systematic Review of the Phytochemistry and Anticancer Activity. Am J Chin Med. 2015;43 (7 ):1331–50. Epub 2015/10/28. doi: 10.1142/S0192415X15500755 .26503558
25 Schust J , Sperl B , Hollis A , Mayer TU , Berg T . Stattic: a small-molecule inhibitor of STAT3 activation and dimerization. Chem Biol. 2006;13 (11 ):1235–42. Epub 2006/11/23. doi: 10.1016/j.chembiol.2006.09.018 .17114005
26 Yi J , Wang Y , Zhang J , Xu J , Li T , Chen C . Effects of JAK2 / STAT3 Signaling Pathway Activation on Intracellular Survival of Brucella. Pak Vet J. 2018;38 (2 ):153–8. doi: 10.29261/pakvetj/2018.048
27 Judd LM , Menheniott TR , Ling H , Jackson CB , Howlett M , Kalantzis A , et al . Inhibition of the JAK2/STAT3 pathway reduces gastric cancer growth in vitro and in vivo. PLoS One. 2014;9 (5 ):e95993. Epub 2014/05/09. doi: 10.1371/journal.pone.0095993 ; PubMed Central PMCID: PMC4013079.24804649
28 Izuhara K , Conway SJ , Moore BB , Matsumoto H , Holweg CT , Matthews JG , et al . Roles of Periostin in Respiratory Disorders. Am J Respir Crit Care Med. 2016;193 (9 ):949–56. Epub 2016/01/13. doi: 10.1164/rccm.201510-2032PP ; PubMed Central PMCID: PMC4872656.26756066
29 Guo H , Kuang S , Song QL , Liu M , Sun XX , Yu Q . Cucurbitacin I inhibits STAT3, but enhances STAT1 signaling in human cancer cells in vitro through disrupting actin filaments. Acta Pharmacol Sin. 2018;39 (3 ):425–37. Epub 2017/11/10. doi: 10.1038/aps.2017.99 ; PubMed Central PMCID: PMC5843842.29119966
30 Knecht DA , LaFleur RA , Kahsai AW , Argueta CE , Beshir AB , Fenteany G . Cucurbitacin I inhibits cell motility by indirectly interfering with actin dynamics. PLoS One. 2010;5 (11 ):e14039. Epub 2010/12/03. doi: 10.1371/journal.pone.0014039 ; PubMed Central PMCID: PMC2991314.21124831
31 Lee DH , Iwanski GB , Thoennissen NH . Cucurbitacin: ancient compound shedding new light on cancer treatment. ScientificWorldJournal. 2010;10 :413–8. Epub 2010/03/09. doi: 10.1100/tsw.2010.44 ; PubMed Central PMCID: PMC5763727.20209387
32 Alghasham AA . Cucurbitacins—a promising target for cancer therapy. Int J Health Sci (Qassim). 2013;7 (1 ):77–89. Epub 2013/04/06. doi: 10.12816/0006025 ; PubMed Central PMCID: PMC3612419.23559908
33 Wu Y , Chen H , Li R , Wang X , Li H , Xin J , et al . Cucurbitacin-I induces hypertrophy in H9c2 cardiomyoblasts through activation of autophagy via MEK/ERK1/2 signaling pathway. Toxicol Lett. 2016;264 :87–98. Epub 2016/11/12. doi: 10.1016/j.toxlet.2016.11.003 .27836799
