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Tularemia is a rare but potentially life threatening zoonotic disease caused by Francisella tularensis. F. novicida, previously considered a subspecies of F. tularensis, is currently considered a separate species. Human infections related to F. novicida are exceedingly rare but can cause morbidity and mortality in debilitated or immunocompromised individuals.A 42-year-old male presented at the hospital with vomiting, dehydration, constipation and pain in the right iliac fossa. He was first diagnosed with pancreatitis and admitted for further analysis. Chest computerized tomography scan showed the presence of parenchymal consolidation in the left upper and lower lobes of the lung with pleural effusion. Blood cultures isolated a Gram-negative coccobacillus, that was at first identified by MALDI-TOF as Francisella tularensis. Serological analysis for the detection of total antibodies against F. tularensis and Real-Time PCR targeting the gene coding for 23 kDa, resulting negative. Subsequently, PCR targeting helicases and tul4 genes, and the Regions of Difference RD1 and RD6 were performed allowing the identification of F. novicida. The isolate was further genetically characterized by whole genome sequencing (WGS).This is the first reported case of human infection caused by F. novicida in Italy.Given the rarity of human cases and the lack of specific symptoms, this pathogen is difficult to identify and the diagnosis can be extremely challenging. In this case report, despite the lack of amplification of the gene encoding for 23 kDa protein, the identification of Francisella species was achieved with the amplification of different genes and characterized by WGS.

Tularaemia is a zoonotic disease caused by Francisella tularensis (F. tularensis). Human infection is rare and can be life-threatening. F. tularensis subsp. novicida used to be a subspecies of F. tularensis, is now considered a different species, F. novicida. Though less virulent, F. novicida can cause morbidity and mortality among debilitated or immunocompromised patients. We reported that an adult with end-stage renal disease undergoing haemodialysis and a history of melioidotic aortic aneurysm developed F. novicida bacteraemic pneumonia, which was uneventfully treated by antimicrobial therapy. The microbiological confirmation of F. novicida infection relies on 16S rRNA sequencing. It is the first case of F. novicida infection in Taiwan.

Abstract Prime editing can induce a desired base substitution, insertion, or deletion in a target gene using reverse transcriptase after nick formation by CRISPR nickase. In this study, we develop a technology that can be used to insert or replace external bases in the target DNA sequence by linking reverse transcriptase to the Francisella novicida Cas9, which is a CRISPR-Cas9 ortholog. Using FnCas9(H969A) nickase, the targeting limitation of existing Streptococcus pyogenes Cas9 nickase [SpCas9(H840A)]-based prime editing is dramatically extended, and accurate prime editing is induced specifically for the target genes in human cell lines.

Bacterial infection tendentiously triggers inflammasome activation, whereas the roles of inflammasome activation in host defense against diverse infections remain unclear. Here, we identified that an ASC-dependent inflammasome activation played opposite roles in host defense against Francisella novicida wild-type (WT) U112 and mutant strain XWK4. Comparing with U112, XWK4 infection induced robust cytokine production, ASC-dependent inflammasome activation, and pyroptosis. Both AIM2 and NLRP3 were involved and played independent roles in XWK4-induced inflammasome activation. Type II interferon was partially required for XWK4-triggered inflammasome activation, which was different from type I interferon dependency in U112-induced inflammasome activation. Distinct from F. novicida U112 and Acinetobacter baumannii infection, Asc–/– mice were more resistant than WT mice response to XWK4 infection by limiting bacterial burden in vivo. The excessive inflammasome activation triggered by XWK4 infection caused dramatical cell death and pathological damage. Our study offers novel insights into mechanisms of inflammasome activation in host defense and provides potential therapeutic approach against bacterial infections and inflammatory diseases.

Francisella tularensis, the causative agent of tularemia, is capable of causing disease in a multitude of mammals and remains a formidable human pathogen due to a high morbidity, low infectious dose, lack of a FDA approved vaccine, and ease of aerosolization. For these reasons, there is concern over the use of F. tularensis as a biological weapon, and, therefore, it has been classified as a Tier 1 select agent. Fluoroquinolones and aminoglycosides often serve as the first line of defense for treatment of tularemia. However, high levels of resistance to these antibiotics has been observed in gram-negative bacteria in recent years, and naturally derived resistant Francisella strains have been described in the literature. The acquisition of antibiotic resistance, either natural or engineered, presents a challenge for the development of medical countermeasures. In this study, we generated a surrogate panel of antibiotic resistant F. novicida and Live Vaccine Strain (LVS) by selection in the presence of antibiotics and characterized their growth, biofilm capacity, and fitness. These experiments were carried out in an effort to (1) assess the fitness of resistant strains; and (2) identify new targets to investigate for the development of vaccines or therapeutics. All strains exhibited a high level of resistance to either ciprofloxacin or streptomycin, a fluoroquinolone and aminoglycoside, respectively. Whole genome sequencing of this panel revealed both on-pathway and off-pathway mutations, with more mutations arising in LVS. For F. novicida, we observed decreased biofilm formation for all ciprofloxacin resistant strains compared to wild-type, while streptomycin resistant isolates were unaffected in biofilm capacity. The fitness of representative antibiotic resistant strains was assessed in vitro in murine macrophage-like cell lines, and also in vivo in a murine model of pneumonic infection. These experiments revealed that mutations obtained by these methods led to nearly all ciprofloxacin resistant Francisella strains tested being completely attenuated while mild attenuation was observed in streptomycin resistant strains. This study is one of the few to examine the link between acquired antibiotic resistance and fitness in Francisella spp., as well as enable the discovery of new targets for medical countermeasure development.

Francisella tularensis is the causative agent of tularemia and has gained recent interest as it poses a significant biothreat risk. F. novicida is commonly used as a laboratory surrogate for tularemia research due to genetic similarity and susceptibility of mice to infection. Currently, there is no FDA-approved tularemia vaccine, and identifying therapeutic targets remains a critical gap in strategies for combating this pathogen. Here, we investigate the soluble lytic transglycosylase or Slt in F. novicida, which belongs to a class of peptidoglycan-modifying enzymes known to be involved in cell division. We assess the role of Slt in biology and virulence of the organism as well as the vaccine potential of the slt mutant. We show that the F. novicida slt mutant has a significant growth defect in acidic pH conditions. Further microscopic analysis revealed significantly altered cell morphology compared to wild-type, including larger cell size, extensive membrane protrusions, and cell clumping and fusion, which was partially restored by growth in neutral pH or genetic complementation. Viability of the mutant was also significantly decreased during growth in acidic medium, but not at neutral pH. Furthermore, the slt mutant exhibited significant attenuation in a murine model of intranasal infection and virulence could be restored by genetic complementation. Moreover, we could protect mice using the slt mutant as a live vaccine strain against challenge with the parent strain; however, we were not able to protect against challenge with the fully virulent F. tularensis Schu S4 strain. These studies demonstrate a critical role for the Slt enzyme in maintaining proper cell division and morphology in acidic conditions, as well as replication and virulence in vivo. Our results suggest that although the current vaccination strategy with F. novicida slt mutant would not protect against Schu S4 challenges, the Slt enzyme could be an ideal target for future therapeutic development.

Francisella tularensis is the causative agent of the life-threatening disease tularemia. However, the molecular tools to study Francisella are limited. Especially, expression plasmids are sparse and difficult to use, as they are unstable and prone to spontaneous loss. Most Francisella expression plasmids lack inducible promoters making it difficult to control gene expression levels. In addition, available expression plasmids are mainly designed for F. tularensis, however, genetic differences including restriction-modification systems impede the use of these plasmids in F. novicida, which is often used as a model organism to study Francisella pathogenesis. Here we report construction and characterization of two mobilizable plasmids (pFNMB1 and pFNMB2) designed for regulated gene expression in F. novicida. pFNMB plasmids contain a tetracycline inducible promoter to control gene expression levels and oriT for RP4 mediated mobilization. We show that both plasmids are stably maintained in bacteria for more than 40 generations over 4 days of culturing in the absence of selection against plasmid loss. Expression levels are dependent on anhydrotetracycline concentration and homogeneous in a bacterial population. pFNMB1 and pFNMB2 plasmids differ in the sequence between promoter and translation start site and thus allow to reach different maximum levels of protein expression. We used pFNMB1 and pFNMB2 for complementation of Francisella Pathogenicity Island mutants ΔiglF, ΔiglI, and ΔiglC in-vitro and pFNMB1 to complement ΔiglI mutant in bone marrow derived macrophages.

Francisella tularensis is a Gram-negative bacterium and the etiologic agent of tularemia. F. tularensis may appear encapsulated when examined by transmission electron microscopy (TEM), which is due to production of an extracellular capsule-like complex (CLC) when the bacterium is grown under specific environmental conditions. Deletion of two glycosylation genes in the live vaccine strain (LVS) results in loss of apparent CLC and attenuation of LVS in mice. In contrast, F. novicida, which is also highly virulent for mice, is reported to be non-encapsulated. However, the F. novicida genome contains a putative polysaccharide locus with homology to the CLC glycosylation locus in F. tularensis. Following daily subculture of F. novicida in Chamberlain's defined medium, an electron dense material surrounding F. novicida, similar to the F. tularensis CLC, was evident. Extraction with urea effectively removed the CLC, and compositional analysis indicated the extract contained galactose, glucose, mannose, and multiple proteins, similar to those found in the F. tularensis CLC. The same glycosylation genes deleted in LVS were targeted for deletion in F. novicida by allelic exchange using the same mutagenesis vector used for mutagenesis of LVS. In contrast, this mutation also resulted in the loss of five additional genes immediately upstream of the targeted mutation (all within the glycosylation locus), resulting in strain F. novicida Δ1212–1218. The subcultured mutant F. novicida Δ1212–1218 was CLC-deficient and the CLC contained significantly less carbohydrate than the subcultured parent strain. The mutant was severely attenuated in BALB/c mice inoculated intranasally, as determined by the lower number of F. novicida Δ1212–1218 recovered in tissues compared to the parent, and by clearance of the mutant by 10–14 days post-challenge. Mice immunized intranasally with F. novicida Δ1212–1218 were partially protected against challenge with the parent, produced significantly reduced levels of inflammatory cytokines, and their spleens contained only areas of lymphoid hyperplasia, whereas control mice challenged with the parent exhibited hypercytokinemia and splenic necrosis. Therefore, F. novicida is capable of producing a CLC similar to that of F. tularensis, and glycosylation of the CLC contributed to F. novicida virulence and immunoprotection.

We report isolation of Francisella novicida–causing bacteremia in a woman from Thailand who was receiving chemotherapy for ovarian cancer. The organism was isolated from blood cultures and identified by 16S rDNA and PPIase gene analyses. Diagnosis and treatment were delayed due to unawareness of the disease in this region.

CRISPR-Cas systems are bacterial defenses against foreign nucleic acids derived from bacteriophages, plasmids or other sources. These systems are targeted in an RNA-dependent, sequence-specific manner, and are also adaptive, providing protection against previously encountered foreign elements. In addition to their canonical function in defense against foreign nucleic acid, their roles in various aspects of bacterial physiology are now being uncovered. We recently revealed a role for a Cas9-based Type II CRISPR-Cas system in the control of endogenous gene expression, a novel form of prokaryotic gene regulation. Cas9 functions in association with two small RNAs to target and alter the stability of an endogenous transcript encoding a bacterial lipoprotein (BLP). Since BLPs are recognized by the host innate immune protein Toll-like Receptor 2 (TLR2), CRISPR-Cas-mediated repression of BLP expression facilitates evasion of TLR2 by the intracellular bacterial pathogen Francisella novicida, and is essential for its virulence. Here we describe the Cas9 regulatory system in detail, as well as data on its role in controlling virulence traits of Neisseria meningitidis and Campylobacter jejuni. We also discuss potential roles of CRISPR-Cas systems in the response to envelope stress and other aspects of bacterial physiology. Since ~45% of bacteria and ~83% of Archaea encode these machineries, the newly appreciated regulatory functions of CRISPR-Cas systems are likely to play broad roles in controlling the pathogenesis and physiology of diverse prokaryotes.

Francisella tularensis is the causative agent of the acute disease tularemia. Due to its extreme infectivity and ability to cause disease upon inhalation, F. tularensis has been classified as a biothreat agent. Two subspecies of F. tularensis, tularensis and holarctica, are responsible for tularemia in humans. In comparison, the closely related species F. novicida very rarely causes human illness and cases that do occur are associated with patients who are immune compromised or have other underlying health problems. Virulence between F. tularensis and F. novicida also differs in laboratory animals. Despite this varying capacity to cause disease, the two species share ~97% nucleotide identity, with F. novicida commonly used as a laboratory surrogate for F. tularensis. As the F. novicida U112 strain is exempt from U.S. select agent regulations research studies can be carried out in non-registered laboratories lacking specialized containment facilities required for work with virulent F. tularensis strains. This review is designed to highlight phenotypic (clinical, ecological, virulence and pathogenic) and genomic differences between F. tularensis and F. novicida that warrant maintaining F. novicida and F. tularensis as separate species. Standardized nomenclature for F. novicida is critical for accurate interpretation of experimental results, limiting clinical confusion between F. novicida and F. tularensis and ensuring treatment efficacy studies utilize virulent F. tularensis strains.

Francisella tularensis is a gram negative facultative intracellular bacterium that causes the zoonotic disease tularemia. Free-living amoebae, such as Acanthamoeba and Hartmanella, are environmental hosts of several intracellular pathogens. Epidemiology of F. tularensis in various parts of the world is associated with water-borne transmission, which includes mosquitoes and amoebae as the potential host reservoirs of the bacteria in water resources. In vitro studies showed intracellular replication of F. tularensis within A. castellanii cells. Whether amoeba is a biological reservoir for Francisella in the environment is not known. We used Hartmanella vermiformis as an amoebal model system to study the intracellular life of F. novicida. For the first time we show that F. novicida survives and replicates within H. vermiformis. The iglC mutant strain of F. novicida is defective for survival and replication not only within A. castellanii but also in H. vermiformis cells. In contrast to mammalian cells, where bacteria replicate in the cytosol, F. novicida resides and replicates within membrane-bound vacuoles within the trophozoites of H. vermiformis. In contrast to the transient residence of F. novicida within acidic vacuoles prior to escaping to the cytosol of mammalian cells, F. novicida does not reside transiently or permanently in an acidic compartment within H. vermiformis when examined 30 min after initiation of the infection. We conclude that F. tularensis does not replicate within acidified vacuoles and does not escape into the cytosol of H. vermiformis. The Francisella pathogenicity island locus iglC is essential for intra-vacuolar proliferation of F. novicida within H. vermiformis. Our data show a distinct intracellular lifestyle for F. novicida within H. vermiformis compared to mammalian cells.