Purification and structural characterization of the tularemia membrane protein virulence factors CapB and CapC.

Diagnostic potential of recombinant SucB protein for tularemia: An ELISA approach.

Francisella tularensis Schu S4 is the most virulent strain responsible for tularemia, a highly infectious zoonotic disease. Despite its potential as a pathogen of concern, no effective vaccine has been developed for this pathogen. In this study, two multi-epitope subunit vaccine candidates were designed targeting the virulence factors DacD and DsbA to reduce disease severity caused by F. tularensis Schu S4. Using a comprehensive immunoinformatics approach, the most efficient B- and T-cell epitopes were identified from conserved sequences and linked with adjuvants and linkers to generate two vaccine constructs. These constructs were modeled, refined, and validated through molecular docking against Toll-Like Receptors (TLRs) and MHC alleles, followed by 200ns molecular dynamics simulations. Their immunogenic potential and expression efficiency were further evaluated through immune simulations and computational cloning into the pET30a( +) vector. Both the constructed vaccine candidates, TulaVac1 and TulaVac2, showed high antigenicity, immunogenicity, stability, and solubility. The epitope conservancy was nearly 100%, with global population coverage rates of 73.19% and 99.74% for MHC class I and II epitopes, respectively. Docking analyses revealed six CTL epitopes with binding energies of ≤ -9.0kcal/mol, while vaccine-TLR and MHC docking suggested superior performance of TulaVac1. MM/GBSA analysis yielded favorable binding energies ranging from - 87.22 to - 161.30kcal/mol, MD simulations confirmed the stable interaction for 200ns, and immune simulations showed robust immune responses with satisfactory codon adaptation. These findings position TulaVac1 and TulaVac2 as promising candidates for effective prophylaxis against tularemia.

A man in his early 70s presented with septic shock and severe ARDS, likely after aerosolising Francisella tularensis Initial empirical treatment for community-acquired pneumonia with ceftriaxone failed, resulting in clinical deterioration with stage 3 acute kidney injury requiring dialysis and necrotising pneumonia. After pneumonic tularaemia was identified, antimicrobial therapy was transitioned to renally dosed levofloxacin, along with aggressive ICU support including prone positioning and tube thoracostomy. The patient ultimately made a full recovery, returning to his functional baseline one month after discharge.This case underscores the importance of eliciting environmental exposures in patients with severe, culture-negative pneumonia unresponsive to beta-lactams, as this should prompt consideration of inhalational tularaemia. It also highlights how pathogen-induced cytokine storm can drive catastrophic multiorgan failure and supports fluoroquinolones as a safe and effective first-line alternative to aminoglycosides in the setting of renal failure.

Tularemia, caused by Francisella tularensis, is a zoonotic disease with various sources, transmission routes, and geographically different clinical signs. Diagnosis is challenging due to nonspecific symptoms, highlighting the importance of laboratory testing for accurate detection, effective outbreak management, and targeted treatment. Recent improvements have enhanced the accuracy of sensitive and specific immunological and molecular methods. Techniques such as enzyme-linked immunosorbent assay, immunochromatography, microagglutination tests, indirect immunofluorescence assays, and PCR continue to be key detection tools. Molecular typing methods such as whole-genome sequencing, single-nucleotide polymorphism analysis, multiple-locus variable-number tandem repeat analysis, pulsed-field gel electrophoresis, and matrix-assisted laser desorption ionization-time of flight enable precise genetic characterization of F. tularensis strains. These methods improve understanding of phylogeny, strain diversity, and transmission routes, while supporting outbreak investigations. This review aims to provide a comprehensive overview of current laboratory methods for diagnosing and typing tularemia as well as their application in clinical and research settings.

Francisella tularensis is a bacterial pathogen that can contaminate drinking water and food products, causing tularemia - a severe zoonotic disease that affects both humans and animals. This infection is dangerous to human health and may be fatal without diagnosis and treatment. Therefore, rapid, sensitive, and selective detection of this microorganism is in high demand for clinical diagnostics, environmental monitoring, and ensuring food safety. This study presents the development of immunochromatographic (lateral flow) tests for revealing F. tularensis cells in natural and drinking water samples. A special feature of the test system is catalytically active Au@Pt nanoparticles (a peroxidase-like nanozyme) used as a label for specific antibodies. Au@Pt nanozyme can catalyze the oxidation of the peroxidase substrate, followed by the formation of a colored product, which amplifies the colorimetric signal on the test strip and significantly improves detection sensitivity. The tests allow for the detection of F. tularensis cells in concentrations down to 102-103 cells per mL, depending on the strain, with visual result assessment. The application of Au@Pt nanozyme decreased the detection limits by 321-9600 times compared to gold nanoparticles commonly used in immunochromatography. The assay duration is 14 min, including the catalytic enhancement step. Monoclonal antibodies against bacterial lipopolysaccharide provide selective detection of virulent strains, excluding cross-reactions with non-pathogenic strains of F. tularensis and other microorganisms that may also contaminate water sources. The effective testing of natural and tap water samples conducted with no preliminary sample preparation has proven the relevance of the developed approach.

Mucosal-associated invariant T (MAIT) cells are among the most conserved and abundant innate-like T cells in humans that recognize microbial-derived riboflavin precursors and elicit potent antimicrobial responses. The foodborne pathogen Listeria monocytogenes is a broad host-range facultative intracellular pathogen that lacks the riboflavin biosynthetic pathway, leading us to hypothesize that this deficiency is pathoadaptive and allows the pathogen to evade MAIT cells. Here, we show that L. monocytogenes strains engineered to produce riboflavin (L. monocytogenes-ribDEAHT) are attenuated in wild-type mice but fully virulent in MAIT cell-deficient mice. Infection with L. monocytogenes-ribDEAHT prompted rapid and robust MAIT cell expansion in multiple tissues and required the cytolytic effector perforin to eliminate infected cells in vivo and in vitro. We also assessed the therapeutic potential of L. monocytogenes-ribDEAHT- stimulated MAIT cells in both infectious disease and cancer mouse models. Therapeutic administration of L. monocytogenes-ribDEAHT provided protection against Francisella tularensis in the lungs and inhibited tumor growth even in the absence of CD8+ T cells. These findings reveal the importance of MAIT cell evasion during L. monocytogenes infection and highlight the therapeutic potential of engineered L. monocytogenes to activate and harness MAIT cells for protection against infectious disease and cancer.

Tularemia is a rare zoonotic disease caused by Francisella tularensis, whose pulmonary manifestations occur only sporadically in Central Europe and can be particularly challenging to diagnose. We report on a 58-year-old patient presenting with B-symptoms in whom a thoracic mass was detected on computed tomography. Further imaging studies revealed a metabolically active mediastinal mass with lymphadenopathy, suggestive of bronchial carcinoma. Only surgical mediastinoscopy with evidence of granulomatous-histiocytic inflammation, as well as further infectious disease diagnostics, led to the diagnosis of pulmonary tularemia. Following a 20-day course of doxycycline, there was rapid clinical improvement and complete laboratory and radiological regression of the lymphadenopathy. This case underscores that pulmonary tularemia can resemble malignant processes morphologically and metabolically. Early consideration of infectious disease differential diagnoses, as well as the combination of histology, serology, and molecular biological methods, are crucial to avoid misdiagnoses and unnecessary invasive procedures.

Tularemia is a zoonotic infection caused by Francisella tularensis. Human cases in Germany are emerging and are usually associated with hare or tick exposure. Transmission via squirrel bites has only been reported once before in Germany. We describe a 23-year-old woman who developed tularemia 11 days after being bitten by an injured red squirrel (Sciurus vulgaris) in southern Germany. Initial symptoms included headache, chills and fatigue, followed by painful right axillary lymphadenopathy without fever. Empirical ciprofloxacin (500 mg twice daily) was initiated on day 10 post-bite. Francisellatularensis serology was negative at presentation but seroconversion was documented 19 days later, confirming the diagnosis. The patient recovered fully after a 10-day course of ciprofloxacin. This case underscores the need to consider tularemia after rodent bites in endemic areas, including squirrel bites, and highlights the favourable clinical course following early fluoroquinolone therapy.

Structural diversity of lipid A modulates neutrophil proteome and secretome responses.

Background. Accurate outbreak analysis is essential for effective infectious disease control. While short-read Illumina sequencing is the current gold standard for genotyping pathogens, Oxford Nanopore Technologies (ONT) offers advantages such as portability and real-time sequencing. However, the accuracy of ONT for single nucleotide polymorphisms (SNP) detection and core genome multilocus sequence typing (cgMLST) remains poorly characterized, which is a critical issue when investigating outbreaks of highly dangerous biosafety-level-3 (BSL-3) agents.Aim. This study evaluates the potential of ONT sequencing for outbreak analysis of four BSL-3 bacterial species with low mutation rates: Bacillus (Ba.) anthracis, Brucella (Br.) melitensis, Brucella (Br.) suis and Francisella (F.) tularensis. Strains originating from epidemiologically defined outbreaks, with existing Illumina sequencing data, were selected for analysis. SNP calling was evaluated using three analytical strategies (PACU, clair3 and an assembly-based approach with snippy). The resulting genomic clusters were compared to epidemiologically defined outbreaks that had previously been reproduced using Illumina sequencing. Additionally, cgMLST was performed to evaluate genotyping resolution across the sequencing platforms.Results. Minor discrepancies in strain clustering were observed, particularly for F. tularensis, where homopolymeric regions contributed to false positives in read-based callers. The assembly-based snippy approach achieved the highest F1 score (0.96-0.99) across all species, followed closely by ab initio SNP callers PACU (F1 : 0.88-0.97) and clair3 (F1 : 0.83-0.98). Minor discrepancies in strain clustering were observed, particularly for F. tularensis, where homopolymeric regions contributed to false positives in read-based callers. CgMLST analysis showed high concordance between ONT and Illumina for Brucella spp., but greater variability for Ba. anthracis and F. tularensis.Conclusions. ONT sequencing, particularly when used with ONT assembly-based SNP calling, enables reliable outbreak analysis of highly pathogenic, low-diversity bacteria. While challenges remain for specific species and genomic features, ONT is a promising alternative for high-resolution bacterial genotyping in outbreak scenarios.

Salmonella infections, including typhoid and paratyphoid fevers, impose a substantial burden in low-income countries, contributing to significant morbidity and mortality associated with enteric and systemic infections. Developing effective treatments for Salmonella infections requires further consideration, especially due to widespread antibiotic resistance. Host-targeted therapies, such as AR-12, offer a promising solution to combat drug resistance. AR-12 has demonstrated broad-spectrum antimicrobial activity against various bacterial pathogens, including Salmonella enterica serovar Typhimurium, S. Typhi, Francisella tularensis, and F. novicida, as well as protozoan parasites and fungal pathogens. To expand its HDT potential against S. Typhimurium, we conducted a medicinal chemistry campaign using AR-12 as a scaffold with systematic optimization of various points of diversity and the pyrazole core to develop analogs informed by structure–activity relationship. This work led to the development of 81 AR-12 analogs. Primary screening identified 38 analogs that are both more potent and less cytotoxic than parent compound AR-12, while only three were less potent and more cytotoxic. Further, only seven compounds affected planktonic Salmonella growth below 20 μM, suggesting host-directed activity in most of the compounds. Twelve analogs were chosen for secondary screening in MDR S. Typhimurium. Compounds 372, 373, and 378 demonstrated remarkable selectivity, with values exceeding 1500 for both susceptible and MDR S. Typhimurium, compared to AR-12's selectivity of around 20. This approximately 100-fold improvement, coupled with improved potency against intracellular Salmonella, suggests these analogs have significantly greater host-directed activity than direct antibacterial effects. Proteomic analysis for the two most potent compounds, 341 and 370 revealed enrichment of vesicle-mediated transport proteins, specifically with respect to retrograde transport at the trans-Golgi-network and intra-Golgi traffic. These results suggest that the analogs reduce intracellular S. Typhimurium replication by disrupting its exploitation of the host cell’s vesicle-mediated transport system.

Type VI secretion systems (T6SSs) are widely distributed among Gram-negative bacteria, where they mostly act to promote bacterial warfare. Bacteria from the Francisella genus possess T6SSs that phylogenetically diverge from all other T6SSs and constitute the T6SSii subtype. Francisella tularensis, the agent of tularemia, relies on its T6SS to secrete effectors into host cells. Despite the key role of this nanomachine in Francisella virulence, the structure of T6SSii and the mechanism underlying its assembly are still poorly understood. Here, using Francisella novicida, we focused on understanding the structure and assembly of the spike, the most apical T6SS complex coupling effector delivery and membrane-puncturing activity. We solved the structure of the protein of unknown function, IglF, in complex with the N-terminal domain of IglG, the T6SSii PAAR protein. Interaction between IglF and IglG enabled the assembly of a mature T6SS spike complex both in Francisella and in a heterologous expression system. In contrast, disrupting IglF:IglG interactions prevented assembly of the PAAR protein with the central spike complex and invalidated T6SS assembly, as visualized by monitoring T6SS dynamics or secretion. Accordingly, IglF:IglG interactions were required for F. novicida virulence in vitro and in a mouse model of tularemia. Altogether, our findings shed light on the assembly mechanism of the Francisella T6SSii spike complex and its importance in virulence.

Francisella tularensis is a highly virulent Gram-negative bacterial pathogen and the causative agent of tularemia. F. tularensis lacks secretion systems utilized by other intracellular bacterial pathogens but contains pil genes that encode for type IV pili (T4P) and may also function in protein secretion. T4P are observed on the surface of all Francisella spp. but pil -mediated protein secretion has only been reported for F. novicida , which is not normally pathogenic in humans. In this study, we used bio-orthogonal non-canonical amino acid tagging to identify proteins secreted by F. tularensis , for which there is limited information. We demonstrate that the F. tularensis pil system is capable of protein secretion and validate T4P-medeated secretion of the ChiA and ChiD chitinases and the KatG catalase. These results will facilitate investigation of Francisella virulence mechanisms and may provide targets for therapeutic intervention.

This work addresses critical gaps in our understanding of Francisella tularensis virulence by identifying lipid metabolism as a central determinant of intracellular survival and stress resistance. By integrating transposon mutagenesis, targeted gene deletion, and lipidomic profiling, this study provides mechanistic insight into how metabolic remodeling supports pathogenesis. Our identification and characterization of FTL_0690 as a long-chain acyl-CoA synthetase essential for lipid homeostasis, membrane integrity, and oxidative stress resistance reveals a previously unappreciated link between fatty acid metabolism and intramacrophage survival of F. tularensis .

Tick-borne diseases (TBDs) have become a great health concern worldwide. This study aimed to clarify the pathogenic and potentially pathogenic microorganisms carried by ticks that bite humans and to assess the risk of acquiring tick-borne infections in regions endemic for Japanese spotted fever. Tick specimens were prospectively collected from patients who presented with tick bites at 10 medical institutions in the Hiroshima, Okayama, and Kagawa prefectures, Japan between May 2023 and December 2024. The evaluated parameters included the estimated bite location, date of bite, patient age, tick species identification, and presence of TBD-associated pathogens in the collected ticks. Overall, 191 ticks were collected from 181 patients. Among patients with known sex, females were slightly more prevalent than males, and 45.9% of the patients were aged ≥ 70 years. Seasonal distribution demonstrated a peak incidence from April to July, with the highest number of cases observed in June (29.3%). Amblyomma testudinarium was the predominant species, accounting for 152 (79.6%) ticks, followed by Haemaphysalis hystricis (7.3%) and Haemaphysalis longicornis (6.8%). Nymphs represented the majority (83.8%), whereas adults and larvae accounted for 14.6% and 1.0%, respectively. A total of 27 ticks (14.1%) carried Rickettsia species, including two identified species (Rickettsia tamurae and Rickettsia monacensis) and one unclassified Rickettsia species. However, molecular analysis did not detect any known human pathogenic organisms, including Ehrlichia and Anaplasma species, Francisella tularensis, or Rickettsia japonica. Further epidemiological data regarding the abundance of tick-borne pathogens will provide valuable surveillance information with significant clinical utility for disease diagnosis and management.

This study aimed to serologically diagnose Brucella spp. and Francisella tularensis in patients presenting with overlapping clinical symptoms. A total of 467 serum samples were tested for antibodies against smooth Brucella spp., Brucella canis, and F. tularensis using Rose Bengal Plate Test (RBPT), Serum Agglutination Test (SAT), Microagglutination Test (MAT), and indirect ELISA (iELISA). Seropositivity for smooth Brucella spp. was 12.85% (60/467) by RBPT/SAT and 10.06% (47/467) by iELISA, with moderate agreement (κ = 0.505). The diagnostic titer-based (sole) seropositivity rate was 9.85% (46/467). For B. canis, seropositivity was 5.14% (24/467) by RBPT and 3.64% (17/467) by iELISA, showing substantial agreement (κ = 0.715). The sole seropositivity rate was 3.64% (17/467). For F. tularensis, both MAT and iELISA showed 3.64% (17/467) seropositivity, with moderate agreement (κ = 0.561), while the sole seropositivity rate was 3.43% (16/467). Co-seropositivity was observed in 1.07% (n= 5) of samples between B. canis RBPT and smooth Brucella iELISA, and in 4.07% (n= 19) between smooth Brucella spp. and F. tularensis. One sample (0.21%) was noteworthy, suggesting possible co-infection. These findings confirm the regional presence of brucellosis and tularemia and highlight the value of combining serological methods in patients with non-specific symptoms and low antibody titers.

Francisella tularensis is a highly infectious human pathogen that must replicate inside macrophage to cause disease. The ribosomes of F. tularensis can incorporate one of three different paralogs for the small ribosomal subunit protein bS21. One of these paralogs positively impacts translation of key virulence genes and promotes intramacrophage replication. Although ribosomal bS21 content influences F. tularensis virulence, the factors that control bS21 paralog production are not well understood. Here, we reveal that all three bS21 proteins influence the transcript abundance of the paralog important for virulence, bS21-2. In contrast, the other bS21 paralogs (bS21-1 and bS21-3) do not affect their own production. We further determined that the leader sequence of the bS21-2 mRNA is sufficient for bS21-mediated repression of mRNA abundance, suggesting that bS21-2 is autogenously regulated. Counterintuitively, we found that in cells lacking bS21-2, the increase in bS21-2-encoding mRNA does not lead to significant increases in protein production. This reduction in translation efficiency suggests that translation of the bS21-2 mRNA is controlled by other factors. Finally, we determined that bS21-2 exerts at least some of its effects on the bS21-2 transcript by decreasing its stability. Together, our findings suggest that F. tularensis may integrate multiple signals into a regulatory network to control the appropriate production of each bS21 paralog, and particularly the paralog important for virulence, bS21-2. This raises the possibility of a regulatory network that can both control ribosome composition in F. tularensis as well as virulence.

Francisella tularensis is the causative agent of tularemia, a zoonotic disease named after the Tulare County, California. Symptoms include sudden fever, chills, fatigue, and swollen lymph nodes, among others, and without treatment it is very serious or even fatal. In addition, F. tularensis is considered a potential bioterrorism threat due to its high infectivity and lethality. Ribosomes are key targets for many classes of antibiotics. In this study, we examined the F. tularensis ribosome and determined its structure at 2.5Å resolution using cryo-electron microscopy. Notably, we observed the stress-induced ribosome-associated inhibitor A (RaiA) protein bound to the ribosome. RaiA functions as a molecular hibernation factor, inhibiting bacterial translation in response to stress or nutrient deprivation. This mechanism parallels that described in the model organism Escherichia coli and in several pathogenic bacteria, such as Staphylococcus aureus. Furthermore, we solved structures of the antibiotics chloramphenicol and gentamicin bound to the F. tularensis ribosome. Collectively, these results provide structural insights that highlight previously unexplored opportunities for therapeutic intervention.

Francisella tularensis is a highly infectious Tier 1 select agent that causes tularemia, a potentially fatal disease. F. tularensis subspecies tularensis (type A), including subtypes A.I and A.II, and subspecies holarctica (type B) differ considerably in pathogenicity, with A.I strains being recognized as some of the most virulent bacterial pathogens known. We previously revealed that only the A.I clade can synthesize spermidine de novo. In this study, we show that deletion of spermidine synthase speE (ΔspeE) in prototype A.I strain SCHU S4 reduced spermidine levels relative to wild-type SCHU S4, type B live vaccine strain (LVS), and LVS ΔspeE. In conventional spermidine-containing chemically defined medium (cCDM), SCHU S4 and SCHU S4 ΔspeE grew substantially faster than LVS and LVS ΔspeE; however, SCHU S4 ΔspeE grew slower than SCHU S4. In modified cCDM without spermidine, SCHU S4 ΔspeE grew in a biphasic manner, which was alleviated by methylthioadenosine, adenine, or adenosine supplementation. Conversely, spermidine addition caused diauxic growth by LVS and LVS ΔspeE and was inhibitory above 0.1 mM, while SCHU S4 replication was substantially and similarly promoted by spermidine or spermine supplementation, with 0.02 mM spermidine being sufficient for maximum growth enhancement. These previously unknown traits demonstrate that de novo spermidine synthesis by A.I strains salvages adenine and promotes fitness, while exogenous spermidine and spermine further increase the rapid replication rate of hypervirulent A.I SCHU S4. These F. tularensis A.I attributes are likely an adaptation to an intracellular environment that contributes to persistence, while outcompeting an effective immune response during an infection.IMPORTANCEFrancisella tularensis is one of the most pathogenic bacteria known due to a low infectious dose, expansive host range, and high rate of mortality. This zoonotic pathogen poses a serious risk to public health and, therefore, is considered a potential bioterrorism agent. Our study revealed that hypervirulent strains of F. tularensis endogenously produce spermidine, which promotes fitness and salvages adenine, while exogenous spermidine or spermine further enhances the rapid replication rate of this intracellular pathogen. A better understanding of the inherent traits that allow F. tularensis to persist and outcompete an effective immune response is needed and may provide insight into preventing lethal infections by other intracellular pathogens.