Dual proteomics of infected macrophages reveal bacterial and host players involved in the Francisella intracellular life cycle and cell to cell dissemination by merocytophagy.

Bacterial pathogens adapt and replicate within host cells, while host cells develop mechanisms to eliminate them. Using a dual proteomic approach, we characterized the intra-macrophage proteome of the facultative intracellular pathogen, Francisella novicida. More than 900 Francisella proteins were identified in infected macrophages after a 10-h infection. Biotin biosynthesis-related proteins were upregulated, emphasizing the role of biotin-associated genes in Francisella replication. Conversely, proteins encoded by the Francisella pathogenicity island (FPI) were downregulated, supporting the importance of the F. tularensis Type VI Secretion System for vacuole escape, not cytosolic replication. In the host cell, over 300 proteins showed differential expression among the 6200 identified during infection. The most upregulated host protein was cis-aconitate decarboxylase IRG1, known for itaconate production with antimicrobial properties in Francisella. Surprisingly, disrupting IRG1 expression did not impact Francisella's intracellular life cycle, suggesting redundancy with other immune proteins or inclusion in larger complexes. Over-representation analysis highlighted cell-cell contact and actin polymerization in macrophage deregulated proteins. Using flow cytometry and live cell imaging, we demonstrated that merocytophagy involves diverse cell-to-cell contacts and actin polymerization-dependent processes. These findings lay the groundwork for further exploration of merocytophagy and its molecular mechanisms in future research.Data are available via ProteomeXchange with identifier PXD035145.

Identification of an N-terminal tag (580N) that improves the biosynthesis of fluorescent proteins in Francisella tularensis and other Gram-negative bacteria.

Utilization of fluorescent proteins is widespread for the study of microbial pathogenesis and host-pathogen interactions. Here, we discovered that linkage of the 36 N-terminal amino acids of FTL_0580 (a hypothetical protein of Francisella tularensis) to fluorescent proteins increases the fluorescence emission of bacteria that express these recombinant fusions. This N-terminal peptide will be referred to as 580N. Western blotting revealed that the linkage of 580N to Emerald Green Fluorescent Protein (EmGFP) in F. tularensis markedly improved detection of this protein. We therefore hypothesized that transcripts containing 580N may be translated more efficiently than those lacking the coding sequence for this leader peptide. In support, expression of emGFPFt that had been codon-optimized for F. tularensis, yielded significantly enhanced fluorescence than its non-optimized counterpart. Furthermore, fusing emGFP with coding sequence for a small N-terminal peptide (Serine-Lysine-Isoleucine-Lysine), which had previously been shown to inhibit ribosomal stalling, produced robust fluorescence when expressed in F. tularensis. These findings support the interpretation that 580N enhances the translation efficiency of fluorescent proteins in F. tularensis. Interestingly, expression of non-optimized 580N-emGFP produced greater fluorescence intensity than any other construct. Structural predictions suggested that RNA secondary structure also may be influencing translation efficiency. When expressed in Escherichia coli and Klebsiella pneumoniae bacteria, 580N-emGFP produced increased green fluorescence compared to untagged emGFP (neither allele was codon optimized for these bacteria). In conclusion, fusing the coding sequence for the 580N leader peptide to recombinant genes might serve as an economical alternative to codon optimization for enhancing protein expression in bacteria.

A conserved but structurally divergent loop in acyl protein thioesterase 1 regulates its catalytic activity, ligand binding, and folded stability.

Human acyl protein thioesterases (APTs) catalyze the depalmitoylation of S-acylated proteins attached to the plasma membrane, facilitating reversible cycles of membrane anchoring and detachment. We previously showed that a bacterial APT homologue, FTT258 from the gram-negative pathogen Francisella tularensis, exists in equilibrium between a closed and open state based on the structural dynamics of a flexible loop overlapping its active site. Although the structural dynamics of this loop are not conserved in human APTs, the amino acid sequence of this loop is highly conserved, indicating essential but divergent functions for this loop in human APTs. Herein, we investigated the role of this loop in regulating the catalytic activity, ligand binding, and protein folding of human APT1, a depalmitoylase connected with cancer, immune, and neurological signaling. Using a combination of substitutional analysis with kinetic, structural, and biophysical characterization, we show that even in its divergent structural location in human APT1 that this loop still regulates the catalytic activity of APT1 through contributions to ligand binding and substrate positioning. We confirmed previously known roles for multiple residues (Phe72 and Ile74) in substrate binding and catalysis while adding new roles in substrate selectivity (Pro69), in catalytic stabilization (Asp73 and Ile75), and in transitioning between the membrane binding ß-tongue and substrate-binding loops (Trp71). Even conservative substitution of this tryptophan (Trp71) fulcrum led to complete loss of catalytic activity, a 13°C decrease in total protein stability, and drastic drops in ligand affinity, indicating that the combination of the size, shape, and aromaticity of Trp71 are essential to the proper structure of APT1. Mixing buried hydrophobic surface area with contributions to an exposed secondary surface pocket, Trp71 represents a previously unidentified class of essential tryptophans within α/ß hydrolase structure and a potential allosteric binding site within human APTs.

Systematic Review of Tularemia During Pregnancy.

BACKGROUND: Tularemia is caused by the gram-negative bacterium Francisella tularensis. Although rare, tularemia during pregnancy has been associated with pregnancy complications; data on efficacy of recommended antimicrobials for treatment are limited. We performed a systematic literature review to characterize clinical manifestations of tularemia during pregnancy and examine maternal, fetal, and neonatal outcomes with and without antimicrobial treatment. METHODS: We searched 9 databases, including Medline, Embase, Global Health, and PubMed Central, using terms related to tularemia and pregnancy. Articles reporting cases of tularemia with ≥1 maternal or fetal outcome were included. RESULTS: Of 5891 articles identified, 30 articles describing 52 cases of tularemia in pregnant patients met inclusion criteria. Cases were reported from 9 countries, and oropharyngeal and ulceroglandular tularemia were the most common presenting forms. A plurality (46%) of infections occurred in the second trimester. Six complications were observed: lymph node aspiration, lymph node excision, maternal bleeding, spontaneous abortion, intrauterine fetal demise, and preterm birth. No deaths among mothers were reported. Of 28 patients who received antimicrobial treatment, 1 pregnancy loss and 1 fetal death were reported. Among 24 untreated patients, 1 pregnancy loss and 3 fetal deaths were reported, including one where F. tularensis was detected in placental and fetal tissues. CONCLUSIONS: Pregnancy loss and other complications have been reported among cases of tularemia during pregnancy. However, risk of adverse outcomes may be lower when antimicrobials known to be effective are used. Without treatment, transplacental transmission appears possible. These data underscore the importance of prompt recognition and treatment of tularemia during pregnancy.

Confirmed Case of Longstanding Respiratory Francisella tularensis holarctica Infection: Nebraska, 2022.

A male patient with distant history of extensive rabbit contact and pulmonary nodules for 6 years developed empyema. Francisella tularensis holarctica was isolated from thoracentesis fluid. Retrospective immunohistochemical examination of a pulmonary nodule, biopsied 3 years prior, was immunoreactive for F. tularensis. These findings suggest the potential for chronic tularemia.

Tularemia From Veterinary Occupational Exposure.

Tularemia is a disease caused by Francisella tularensis, a highly infectious bacteria that can be transmitted to humans by direct contact with infected animals. Because of the potential for zoonotic transmission of F. tularensis, veterinary occupational risk is a concern. Here, we report on a human case of tularemia in a veterinarian after an accidental needlestick injury during abscess drainage in a sick dog. The veterinarian developed ulceroglandular tularemia requiring hospitalization but fully recovered after abscess drainage and a course of effective antibiotics. To systematically assess veterinary occupational transmission risk of F. tularensis, we conducted a survey of veterinary clinical staff after occupational exposure to animals with confirmed tularemia. We defined a high-risk exposure as direct contact to the infected animal's body fluids or potential aerosol inhalation without use of standard personal protective equipment (PPE). Survey data included information on 20 veterinary occupational exposures to animals with F. tularensis in 4 states. Veterinarians were the clinical staff most often exposed (40%), followed by veterinarian technicians and assistants (30% and 20%, respectively). Exposures to infected cats were most common (80%). Standard PPE was not used during 80% of exposures; a total of 7 exposures were categorized as high risk. Transmission of F. tularensis in the veterinary clinical setting is possible but overall risk is likely low. Veterinary clinical staff should use standard PPE and employ environmental precautions when handling sick animals to minimize risk of tularemia and other zoonotic infections; postexposure prophylaxis should be considered after high-risk exposures to animals with suspected or confirmed F. tularensis infection to prevent tularemia.

Treatment Outcome of Severe Respiratory Type B Tularemia Using Fluoroquinolones.

BACKGROUND: Fluoroquinolones lack approval for treatment of tularemia but have been used extensively for milder illness. Here, we evaluated fluoroquinolones for severe illness. METHODS: In an observational study, we identified case-patients with respiratory tularemia from July to November 2010 in Jämtland County, Sweden. We defined severe tularemia by hospitalization for >24 hours and severe bacteremic tularemia by Francisella tularensis subsp. holarctica growth in blood or pleural fluid. Clinical data and drug dosing were retrieved from electronic medical records. Chest images were reexamined. We used Kaplan-Meier curves to evaluate time to defervescence and hospital discharge. RESULTS: Among 67 case-patients (median age, 66 years; 81% males) 30-day mortality was 1.5% (1 of 67). Among 33 hospitalized persons (median age, 71 years; 82% males), 23 had nonbacteremic and 10 had bacteremic severe tularemia. Subpleural round consolidations, mediastinal lymphadenopathy, and unilateral pleural fluid were common on chest computed tomography. Among 29 hospitalized persons with complete outcome data, ciprofloxacin/levofloxacin (n = 12), ciprofloxacin/levofloxacin combinations with doxycycline and/or gentamicin (n = 11), or doxycycline as the single drug (n = 6) was used for treatment. One disease relapse occurred with doxycycline treatment. Treatment responses were rapid, with median fever duration 41.0 hours in nonbacteremic and 115.0 hours in bacteremic tularemia. Increased age-adjusted Charlson comorbidity index predicted severe bacteremic tularemia (odds ratio, 2.7 per score-point; 95% confidence interval, 1.35-5.41). A 78-year-old male with comorbidities and delayed ciprofloxacin/gentamicin treatment died. CONCLUSIONS: Fluoroquinolone treatment is effective for severe tularemia. Subpleural round consolidations and mediastinal lymphadenopathy were typical findings on computed tomography among case-patients in this study.

Modified activities of macrophages' deubiquitinating enzymes after Francisella infection.

Francisella tularensis influences several host molecular/signaling pathways during infection. Ubiquitination and deubiquitination are among the most important regulatory mechanisms and respectively occur through attachment or removal of the ubiquitin molecule. The process is necessary not only to mark molecules for degradation, but also, for example, to the activation of signaling pathways leading to pro-inflammatory host response. Many intracellular pathogens, including Francisella tularensis, have evolved mechanisms of modifying such host immune responses to escape degradation. Here, we describe that F. tularensis interferes with the host's ubiquitination system. We show increased total activity of deubiquitinating enzymes (DUBs) in human macrophages after infection, while confirm reduced enzymatic activities of two specific DUBs (USP10 and UCH-L5), and demonstrate increased activity of USP25. We further reveal the enrichment of these three enzymes in exosomes derived from F. tularensis-infected cells. The obtained results show the regulatory effect on ubiquitination mechanism in macrophages during F. tularensis infection.

Human Tularemia Epididymo-Orchitis Caused by Francisella tularensis Subspecies holartica, Austria.

A previously healthy man in Austria had tularemia epididymo-orchitis develop, leading to unilateral orchiectomy. Francisella tularensis subspecies holartica was detected by 16S rRNA gene sequencing analysis of inflamed granulomatous testicular tissue. Clinicians should suspect F. tularensis as a rare etiologic microorganism in epididymo-orchitis patients with relevant risk factors.

Francisella tularensis disrupts TLR2-MYD88-p38 signaling early during infection to delay apoptosis of macrophages and promote virulence in the host.

Francisella tularensis is a zoonotic pathogen and the causative agent of tularemia. F. tularensis replicates to high levels within the cytosol of macrophages and other host cells while subverting the host response to infection. Critical to the success of F. tularensis is its ability to delay macrophage apoptosis to maintain its intracellular replicative niche. However, the host-signaling pathway(s) modulated by F. tularensis to delay apoptosis are poorly characterized. The outer membrane channel protein TolC is required for F. tularensis virulence and its ability to suppress apoptosis and cytokine expression during infection of macrophages. We took advantage of the F. tularensis ∆tolC mutant phenotype to identify host pathways that are important for activating macrophage apoptosis and that are disrupted by the bacteria. Comparison of macrophages infected with wild-type or ∆tolC F. tularensis revealed that the bacteria interfere with TLR2-MYD88-p38 signaling at early times post infection to delay apoptosis, dampen innate host responses, and preserve the intracellular replicative niche. Experiments using the mouse pneumonic tularemia model confirmed the in vivo relevance of these findings, revealing contributions of TLR2 and MYD88 signaling to the protective host response to F. tularensis, which is modulated by the bacteria to promote virulence. IMPORTANCE Francisella tularensis is a Gram-negative intracellular bacterial pathogen and the causative agent of the zoonotic disease tularemia. F. tularensis, like other intracellular pathogens, modulates host-programmed cell death pathways to ensure its replication and survival. We previously identified the outer membrane channel protein TolC as required for the ability of F. tularensis to delay host cell death. However, the mechanism by which F. tularensis delays cell death pathways during intracellular replication is unclear despite being critical to pathogenesis. In the present study, we address this gap in knowledge by taking advantage of ∆tolC mutants of F. tularensis to uncover signaling pathways governing host apoptotic responses to F. tularensis and which are modulated by the bacteria during infection to promote virulence. These findings reveal mechanisms by which intracellular pathogens subvert host responses and enhance our understanding of the pathogenesis of tularemia.

Discovery of a glutathione utilization pathway in Francisella that shows functional divergence between environmental and pathogenic species.

Glutathione (GSH) is an abundant metabolite within eukaryotic cells that can act as a signal, a nutrient source, or serve in a redox capacity for intracellular bacterial pathogens. For Francisella, GSH is thought to be a critical in vivo source of cysteine; however, the cellular pathways permitting GSH utilization by Francisella differ between strains and have remained poorly understood. Using genetic screening, we discovered a unique pathway for GSH utilization in Francisella. Whereas prior work suggested GSH catabolism initiates in the periplasm, the pathway we define consists of a major facilitator superfamily (MFS) member that transports intact GSH and a previously unrecognized bacterial cytoplasmic enzyme that catalyzes the first step of GSH degradation. Interestingly, we find that the transporter gene for this pathway is pseudogenized in pathogenic Francisella, explaining phenotypic discrepancies in GSH utilization among Francisella spp. and revealing a critical role for GSH in the environmental niche of these bacteria.

Comparative evaluation of protective immunity against Francisella tularensis induced by subunit or adenovirus-vectored vaccines.

Tularemia is a highly contagious disease caused by infection with Francisella tularensis (Ft), a pathogenic intracellular gram-negative bacterium that infects a wide range of animals and causes severe disease and death in people, making it a public health concern. Vaccines are the most effective way to prevent tularemia. However, there are no Food and Drug Administration (FDA)-approved Ft vaccines thus far due to safety concerns. Herein, three membrane proteins of Ft, Tul4, OmpA, and FopA, and a molecular chaperone, DnaK, were identified as potential protective antigens using a multifactor protective antigen platform. Moreover, the recombinant DnaK, FopA, and Tul4 protein vaccines elicited a high level of IgG antibodies but did not protect against challenge. In contrast, protective immunity was elicited by a replication-defective human type 5 adenovirus (Ad5) encoding the Tul4, OmpA, FopA, and DnaK proteins (Ad5-Tul4, Ad5-OmpA, Ad5-FopA, and Ad5-DnaK) after a single immunization, and all Ad5-based vaccines stimulated a Th1-biased immune response. Moreover, intramuscular and intranasal vaccination with Ad5-Tul4 using the prime-boost strategy effectively eliminated Ft lung, spleen and liver colonization and provided nearly 80% protection against intranasal challenge with the Ft live vaccine strain (LVS). Only intramuscular, not intranasal vaccination, with Ad5-Tul4 protected mice from intraperitoneal challenge. This study provides a comprehensive comparison of protective immunity against Ft provided by subunit or adenovirus-vectored vaccines and suggests that mucosal vaccination with Ad5-Tul4 may yield desirable protective efficacy against mucosal infection, while intramuscular vaccination offers greater overall protection against intraperitoneal tularemia.

Complete genome sequence of the emerging pathogen Cysteiniphilum spp. and comparative genomic analysis with genus Francisella: Insights into its genetic diversity and potential virulence traits.

Cysteiniphilum is a newly discovered genus in 2017 and is phylogenetically closely related to highly pathogenic Francisella tularensis. Recently, it has become an emerging pathogen in humans. However, the complete genome sequence of genus Cysteiniphilum is lacking, and the genomic characteristics of genetic diversity, evolutionary dynamics, and pathogenicity have not been characterized. In this study, the complete genome of the first reported clinical isolate QT6929 of genus Cysteiniphilum was sequenced, and comparative genomics analyses to Francisella genus were conducted to unveil the genomic landscape and diversity of the genus Cysteiniphilum. Our results showed that the complete genome of QT6929 consists of one 2.61 Mb chromosome and a 76,819 bp plasmid. The calculated average nucleotide identity and DNA-DNA hybridization values revealed that two clinical isolates QT6929 and JM-1 should be reclassified as two novel species in genus Cysteiniphilum. Pan-genome analysis revealed genomic diversity within the genus Cysteiniphilum and an open pan-genome state. Genomic plasticity analysis exhibited abundant mobile genetic elements including genome islands, insertion sequences, prophages, and plasmids on Cysteiniphilum genomes, which facilitated the broad exchange of genetic material between Cysteiniphilum and other genera like Francisella and Legionella. Several potential virulence genes associated with lipopolysaccharide/lipooligosaccharide, capsule, and haem biosynthesis specific to clinical isolates were predicted and might contribute to their pathogenicity in humans. Incomplete Francisella pathogenicity island was identified in most Cysteiniphilum genomes. Overall, our study provides an updated phylogenomic relationship of members of the genus Cysteiniphilum and comprehensive genomic insights into this rare emerging pathogen.

Tularemia in Pregnant Woman, Serbia, 2018.

Tularemia was diagnosed for a 33-year-old pregnant woman in Serbia after a swollen neck lymph node was detected at gestation week 18. Gentamicin was administered parenterally (120 mg/d for 7 d); the pregnancy continued with no complications and a healthy newborn was delivered. Treatment of tularemia optimizes maternal and infant outcomes.

[A rare and atypical form of tularemia in a context of immunodepression]. / Forme rare et atypique de tularémie dans un contexte d'immunodépression.

INTRODUCTION: We present an original severe case of tularemia with cutaneous damage, lymphadenopathy and pericarditis ; pathology of increasing incidence in Europe due to global warming. OBSERVATION: A 33-years-old women consulted emergency unit for altered general condition, anorexia, hyperthermia at 38,3°C, dyspnea and dry cough evolving for few days. Her only history was Crohn's disease with introduction of an anti-TNF alpha for 3 months. The interrogation found regular forest walks ¼. Treatment with Amoxicillin/clavulanic acid 1g 3 times daily and curative anticoagulation was started after the initial diagnosis of infectious pneumonia associated with pulmonary embolism. The patient reconsulted 2 weeks later for clinical deterioration associated with skin lesions. The chest CT scan showed increased mediastinal lymphadenopathy and a circumferential pericardial effusion ; quantified at 5mm on transthoracic ultrasound. Tularemia serology was positive in IgG at 400IU/mL. Despite an adapted antibiotic therapy with Ciprofloxacin, the patient presented a new brutal clinical deterioration. A pericardiocentesis was performed and the analysis revealed a predominantly neutrophilic exudate and a strongly positive PCR Francisella tularensis. Gentamicin 5mg/kg was associated allowing a resolution of the symptoms. CONCLUSION: Tularemia is one of the pathologies whose atypical presentation with pericarditis (favored by a certain immunodepression) worsens the prognosis. Global warming influences the epidemiology of inoculation diseases, including tularemia, making it more frequent.

[A Case of Oropharyngeal Tularemia Mimicking Lymphoma During Pregnancy]. / Gebelikte Lenfomayi Taklit Eden Bir Orofarengeal Tularemi Olgusu.

Tularemia is a zoonotic bacterial infectious disease caused by a gram-negative coccobacillus namely Francisella tularensis. In humans, disease leads to several different clinical forms (ulceroglandular, glandular, oculoglandular, respiratory, typhoidal and oropharyngeal). Since the main mode of transmission of the disease to humans in Türkiye is by drinking water contaminated with F.tularensis, the oropharyngeal form is the most common clinical manifestation. Since tularemia cases with pregnancy are rare, the literatüre about maternal and fetal complications of tularemia is sparse. In this report, a case of oropharyngeal tularemia mimicking lymphoma during pregnancy was presented. A 33-year-old 11-week pregnant patient living in a village in Sivas province admitted to the infectious diseases and clinical microbiology outpatient clinic with the complaint of swelling in the neck region that continued for six days. The patient, who was engaged in animal husbandry stated that she consumed raw milk and admitted to the otorhinolaryngology outpatient clinic of a hospital 10 days ago with the complaints of fever, chills, and sore throat. She stated that her complaints did not regress with the amoxicillin-clavulanate treatment recommended by her doctor and she noticed the swelling in her neck on the 4th day of the treatment. Upon further questioning, it was understood that the patient had a history of consumption of unchlorinated spring water. Her vital signs were normal and physical examination revealed non-fluctuant lymph nodes with the largest of 5 x 2 cm in the right posterior cervical region, and 3 x 2 cm in the left. Laboratory tests revealed a blood leukocyte count of 13.32 x 103/mm3 (75% granulocytes), a blood hemoglobin of 11.4 g/dL, an erythrocyte sedimentation rate of 45 mm/hour, and C-reactive protein of 90 mg/L. A non-contrast MRI examination revealed wall thickening of the nasopharynx and enlarged lymph nodes which were suspicious for lymphoma with significant diffusion restriction on diffusionweighted images. As the past medical history and clinical findings were suggestive for tularemia, the microagglutination test (MAT) was studied, but it was reported as negative with a titer at 1/80. Since the patient's complaints continued and tularemia cases were encountered in our region in the past years, the repeated MAT after two weeks was reported as positive with a titer at 1/320. An oropharyngeal form of tularemia was diagnosed and oral ciprofloxacin (2 x 750 mg) was given for three weeks by starting at the 14th gestational week. Lymphoma was excluded by histopathological examination of the fine needle aspiration biopsy performed on the patient's cervical lymph nodes, but the biopsy sample was compatible with granulomatous diseases. Histopathological findings of diagnostic biopsies of the larynx and nasopharynx were reactive. A healthy male baby, 2425 grams, 47 cm, was delivered by cesarean section from the patient who presented with labor contractions at the 37th week of pregnancy. There was no sign of congenital infection in the newborn. The patient and the baby were followed up to the end of one year and no abnormality was found. The evaluation of 17 cases reported in the literatüre including this case, suggest that tularemia may progress to involve serious obstetric complications during pregnancy, such as abortion, premature birth and intrauterine fetal death when appropriate and effective antibiotic treatment is not given.

Identification of pyrC gene as an immunosuppressive factor in Francisella novicida infection.

Francisella tularensis, a bacterial causative agent of the zoonosis tularemia, is highly pathogenic to humans. The pathogenicity of this bacterium is characterized by intracellular growth in immune cells, like macrophages, and host immune suppression. However, the detailed mechanism of immune suppression by F. tularensis is still unclear. To identify the key factors causing Francisella-mediated immunosuppression, large-scale screening using a transposon random mutant library containing 3552 mutant strains of F. tularensis subsp. novicida (F. novicida) was performed. Thirteen mutants that caused stronger tumor necrosis factor (TNF)-α production in infected U937 human macrophage cells than the wild-type F. novicida strain were isolated. Sequencing analysis of transposon insertion sites revealed 10 genes, including six novel genes, as immunosuppressive factors of Francisella. Among these, the relationship of the pyrC gene, which encodes dihydroorotase in the pyrimidine biosynthesis pathway, with Francisella-mediated immunosuppression was investigated. The pyrC deletion mutant strain (ΔpyrC) induced higher TNF-α production in U937 host cells than the wild-type F. novicida strain. The ΔpyrC mutant strain was also found to enhance host interleukin-1ß and interferon (IFN)-ß production. The heat-inactivated ΔpyrC mutant strain could not induce host TNF-α production. Moreover, the production of IFN-ß resulting from ΔpyrC infection in U937 cells was repressed upon treatment with the stimulator of interferon genes (STING)-specific inhibitor, H-151. These results suggest that pyrC is related to the immunosuppressive activity and pathogenicity of Francisella via the STING pathway.

Iron-Modified Blood Culture Media Allow for the Rapid Diagnosis and Isolation of the Slow-Growing Pathogen Francisella tularensis.

The life-threatening disease tularemia is caused by Francisella tularensis, an intracellular Gram-negative bacterial pathogen. Due to the high mortality rates of the disease, as well as the low respiratory infectious dose, F. tularensis is categorized as a Tier 1 bioterror agent. The identification and isolation from clinical blood cultures of F. tularensis are complicated by its slow growth. Iron was shown to be one of the limiting nutrients required for F. tularensis metabolism and growth. Bacterial growth was shown to be restricted or enhanced in the absence or addition of iron. In this study, we tested the beneficial effect of enhanced iron concentrations on expediting F. tularensis blood culture diagnostics. Accordingly, bacterial growth rates in blood cultures with or without Fe2+ supplementation were evaluated. Growth quantification by direct CFU counts demonstrated significant improvement of growth rates of up to 6 orders of magnitude in Fe2+-supplemented media compared to the corresponding nonmodified cultures. Fe2+ supplementation significantly shortened incubation periods for successful diagnosis and isolation of F. tularensis by up to 92 h. This was achieved in a variety of blood culture types in spite of a low initial bacterial inoculum representative of low levels of bacteremia. These improvements were demonstrated with culture of either Francisella tularensis subsp. tularensis or subsp. holarctica in all examined commercial blood culture types routinely used in a clinical setup. Finally, essential downstream identification assays, such as matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS), immunofluorescence, or antibiotic susceptibility tests, were not affected in the presence of Fe2+. To conclude, supplementing blood cultures with Fe2+ enables a significant shortening of incubation times for F. tularensis diagnosis, without affecting subsequent identification or isolation assays. IMPORTANCE In this study, we evaluated bacterial growth rates of Francisella tularensis strains in iron (Fe)-enriched blood cultures as a means of improving and accelerating bacterial growth. The shortening of the culturing time should facilitate rapid pathogen detection and isolation, positively impacting clinical diagnosis and enabling prompt onset of efficient therapy.

Neutrophil Survival Signaling During Francisella tularensis Infection.

Neutrophils are the most abundant and shortest-lived leukocytes in humans and tight regulation of neutrophil turnover via constitutive apoptosis is essential for control of infection and resolution of inflammation. Accordingly, aberrant neutrophil turnover is hallmark of many disease states. We have shown in previous work that the intracellular bacterial pathogen Francisella tularensis markedly prolongs human neutrophil lifespan. This is achieved, in part, by changes in neutrophil gene expression. Still unknown is the contribution of major neutrophil pro-survival signaling cascades to this process. The objective of this study was to interrogate the contributions of ERK and p38 MAP kinase, Class I phosphoinositide 3-kinases (PI3K), AKT, and NF-κB to neutrophil survival in our system. We demonstrate that both ERK2 and p38α were activated in F. tularensis-infected neutrophils, but only p38α MAPK was required for delayed apoptosis and the rate of cell death in the absence of infection was unchanged. Apoptosis of both infected and uninfected neutrophils was markedly accelerated by the pan-PI3K inhibitor LY2094002, but AKT phosphorylation was not induced, and neutrophil death was not enhanced by AKT inhibitors. In addition, isoform specific and selective inhibitors revealed a unique role for PI3Kα in neutrophil survival after infection, whereas only simultaneous inhibition of PI3Kα and PI3kδ accelerated death of the uninfected controls. Finally, we show that inhibition of NF-κB triggered rapid death of neutrophil after infection. Thus, we defined roles for p38α, PI3Kα and NF-κB delayed apoptosis of F. tularensis-infected cells and advanced understanding of Class IA PI3K isoform activity in human neutrophil survival.