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The first step of lipid A biosynthesis in Escherichia coli (E. coli) is catalyzed by LpxA (EcLpxA), an acyltransferase selective for UDP-N-acetylglucosamine (UDP-GlcNAc) and R-3-hydroxymyristoyl-acyl carrier protein (3-OH-C14-ACP), and is an essential step in majority of Gram-negative bacteria. Since the majority of lipid A species isolated from F. novicida contains 3-OH-C16 or 3-OH-C18 at its C3 and C3′ positions, FnLpxA was thought to be selective for longer acyl chain (3-OH-C16 and 3-OH-C18) over short acyl chain (3-OH-C14, 3-OH-C12, and 3-OH-C10). Here we demonstrate that Francisella novicida (F. novicida) lpxA functionally complements an E. coli lpxA knockout mutant and efficiently transfers 3-OH-C14 as well as 3-OH-C16 in E. coli. Our results implicate that the acyl chain length of lipid A is determined by several factors including acyl chain selectivity of LpxA and downstream enzymes, as well as the composition of the acyl-ACP pool in vivo. We also report the crystal structure of F. novicida LpxA (FnLpxA) at 2.06 A. The N-terminal parallel beta-helix (LβH) and C-terminal alpha-helical domain are similar to other reported structures of LpxAs. However, our structure indicates that the supposed ruler residues for hydrocarbon length, 171L in one monomer and 168H in the adjacent monomer in a functional trimer of FnLpxA, are located just 3.8 A apart that renders not enough space for binding of 3-OH-C12 or longer acyl chains. This implicates that FnLpxA may have an alternative hydrophobic pocket, or the acyl chain may bend while binding to FnLpxA. In addition, the FnLpxA structure suggests a potential inhibitor binding site for development of antibiotics.

UNLABELLED Previously, several essential genes from psychrophilic bacteria have been substituted for their homologues in mesophilic bacterial pathogens to make the latter temperature sensitive. It has been noted that an essential ligA gene from an extreme psychrophile, Colwellia sp. C1, yielded a gene product that is inactivated at 27°C, the lowest that has been observed for any psychrophilic enzyme, and hypothesized that other essential proteins of that strain would also have low inactivation temperatures. This work describes the partial sequencing of the genome of Colwellia sp. C1 strain and the identification of 24 open reading frames encoding homologues of highly conserved bacterial essential genes. The gene encoding porphobilinogen deaminase (hemC), which is involved in the pathway of haem synthesis, has been tested for its ability to convert Francisella novicida into a temperature-sensitive strain. The hybrid strain carrying the C1-derived hemC gene exhibited a temperature-sensitive phenotype with a restrictive temperature of 36°C. These results support the conclusion that Colwellia sp. C1 is a rich source of heat-labile enzymes. SIGNIFICANCE AND IMPACT OF THE STUDY The issue of biosafety is often raised when it comes to work with pathogenic organisms. The main concern is caused by the risk of researchers being exposed to infectious doses of dangerous microbes. This paper analyses essential genes identified in partial genomic sequence of the psychrophilic bacterium Collwelia sp. C1. These sequences can be used as a mean of generating temperature-sensitive strains of pathogenic bacteria. Such strains are incapable of surviving at the temperature of human body. This means they could be applied as vaccines or for safer work with dangerous organisms.

A library of 33 compounds was screened for potentiation of the antibiotic FR 900098 against the Francisella tularensis surrogate Francisella novicida. From the screen a highly potent 2-oxazoline adjuvant was discovered capable of potentiating FR 900098 with a 1000-fold reduction in MIC against the Francisella sub-species F. novicida and F. philomiragia.

Additional file 2. Review history.

Additional file 1: Figure S1. Analysis of the cleavage point of FnCas9 and SpCas9. Figure S2. Shared target sequence and editing method by using SpCas9(H840A)-RT and FnCas9(H969A)-RT. Figure S3. Various methods of prime editor delivery by using SpCas9(H840A)-RT or FnCas9(H969A)-RT expression plasmids. Figure S4. The target sequence and NGS analysis results commonly edited by SpCas9(H840A)-RT or FnCas9(H969A)-RT. Figure S5. Cell diversity experiment of prime editing using FnCas9(H969A)-RT. Figure S6. Comparative analysis of nickase activity of SpCas9(H840A)-RT and FnCas9(H969A)-RT. Figure S7. Targeting of multiple loci for direct comparison between SpCas9(H840A)-RT and FnCas9(H969A)-RT based prime editing. Figure S8. Comparative analysis of off-target editing of SpCas9(H840A)-RT and FnCas9(H969A)-RT based on in-silico predicted sites. Figure S9. Multiplexed prime editing with SpCas9(H840A)-RT and FnCas9(H969A)-RT system. Figure S10. Expansion of the range of prime editing in EMX1 locus by FnCas9(H969A)-RT. Figure S11. Result of the prime editing in target sequence induced by FnCas9(H969A)-RT. Figure S12. Expansion of the targetable range by prime editing with FnCas9(H969A)-RT or RHA-FnCas9(H969A)-RT.

The Class 2 Type V-A CRISPR effector protein Cas12a/Cpf1 has gained widespread attention in part because of the ease in achieving multiplexed genome editing, gene regulation, and DNA detection. Multiplexing derives from the ability of Cas12a alone to generate multiple guide RNAs from a transcribed CRISPR array encoding alternating conserved repeats and targeting spacers. While array design has focused on how to optimize guide-RNA sequences, little attention has been paid to sequences outside of the CRISPR array. Here, we show that a structured hairpin located immediately downstream of the 3ʹ repeat interferes with utilization of the adjacent encoded guide RNA by Francisella novicida (Fn)Cas12a. We first observed that a synthetic Rho-independent terminator immediately downstream of an array impaired DNA cleavage based on plasmid clearance in E. coli and DNA cleavage in a cell-free transcription-translation (TXTL) system. TXTL-based cleavage assays further revealed that inhibition was associated with incomplete processing of the transcribed CRISPR array and could be attributed to the stable hairpin formed by the terminator. We also found that the inhibitory effect partially extended to upstream spacers in a multi-spacer array. Finally, we found that removing the terminal repeat from the array increased the inhibitory effect, while replacing this repeat with an unprocessable terminal repeat from a native FnCas12a array restored cleavage activity directed by the adjacent encoded guide RNA. Our study thus revealed that sequences surrounding a CRISPR array can interfere with the function of a CRISPR nuclease, with implications for the design and evolution of CRISPR arrays.

Clustered regularly interspaced short palindromic repeats (CRISPRs) from Prevotella and Francisella 1 (Cpf1) are RNA-guided endonucleases that produce cohesive double-stranded breaks in DNA by specifically recognizing thymidine-rich protospacer-adjacent motif (PAM) sequences. Cpf1 is emerging as a powerful genome-editing tool. Despite previous structural studies on various Cpf1 proteins, the apo-structure of Cpf1 remains unknown. In the present study, we determined the solution structure of the Cpf1 protein from Francisella novicida (FnCpf1) with and without CRISPR RNA (crRNA) using small-angle X-ray scattering, providing the insights into the apo-structure of FnCpf1. The apo-structure of FnCpf1 was also visualized using negative staining electron microcopy. When we compared the apo-structure of FnCpf1 with crRNA-bound structure, their overall shapes (a closed form) were similar, suggesting that conformational change upon crRNA binding to FnCpf1 is not drastic, but a local induced fit might occur to recognize PAM sequences. In contrast, the apo Cpf1 from Moraxella bovoculi 237 (MbCpf1) was analyzed as an open form, implying that a large conformational change from an open to a closed form might be required for crRNA binding to MbCpf1. These results suggested that the crRNA-induced conformational changes in Cpf1 differ among species.

Francisella tularensis is a gram-negative facultative intracellular bacterial pathogen that in humans and animals causes the fulminating zoonotic disease tularemia. Free-living amoeba, such as A. castellanii and D. discoideum, serve as environmental hosts of various intracellular pathogens. Epidemiology of F. tularensis is commonly associated with water-borne transmission in some parts of the world, including mosquitoes and amoebae as the host reservoir of bacteria in aquatic environment resources. In vitro studies demonstrated intracellular replication of F. tularensis within D. discoideum and A. castellanii cells. It has been established that crucial step in Francisella infection is entering the target cells by receptor mediated phagocytosis. This process has been shown to be meaningful in macrophages, arthropod-derived cells and amoeba cells. We used a water-borne amoeba A. castellanii and D. discoideum as surrogate macrophages as amoeba model systems to study influence of metabolic inhibitor methylamine on results of intracellular and extracellular life of F. novicida. For the first time we showed that methylamine has inhibitory effect on F. novicida entry and replication in D. discoideum and A. castellanii. The influence of different levels of infection was also examined and showed a dose-dependent effect on intracellular and extracellular infection. Moreover, methylamine was analyzed to observe whether in concentration of 100 mM induce cytopathogenicity in amoebae mentioned before. We conclude that F. novicida cannot entry and replicates in D. discoideum and A. castellanii after treated with 100 mM methylamine and that this concentration of inhibitor is not toxic to amoeba itself and can be used to inhibit receptor-mediated phagocytosis during Francisella infection.

In many bacteria, the ability to modulate biofilm production relies on specific signaling molecules that are either self-produced or made by neighboring microbes within the ecological niche. We analyzed the potential interspecies signaling effect of the Burkholderia diffusible signal factor (BDSF) on Francisella novicida , a model organism for Francisella tularensis , and demonstrated that BDSF both inhibits the formation and causes the dispersion of Francisella biofilm. Specificity was demonstrated for the cis versus the trans form of BDSF. Using transcriptome sequencing, quantitative reverse transcription-PCR, and activity assays, we found that BDSF altered the expression of many F. novicida genes, including genes involved in biofilm formation, such as chitinases. Using a chitinase inhibitor, the antibiofilm activity of BDSF was also shown to be chitinase dependent. In addition, BDSF caused an increase in RelA expression and increased levels of (p)ppGpp, leading to decreased biofilm production. These results support our observation that exposure of F. novicida to BDSF causes biofilm dispersal. Furthermore, BDSF upregulated the genes involved in iron acquisition ( figABCD ), increasing siderophore production. Thus, this study provides evidence for a potential role and mechanism of diffusible signal factor (DSF) signaling in the genus Francisella and suggests the possibility of interspecies signaling between Francisella and other bacteria. Overall, this study suggests that in response to the interspecies DSF signal, F. novicida can alter its gene expression and regulate its biofilm formation.

Francisella novicida is a surrogate pathogen commonly used to study infections by the potential bioterrorism agent, Francisella tularensis. One of the primary sites of Francisella infections is the liver where >90 % of infected cells are hepatocytes. It is known that once Francisella enter cells it occupies a membrane-bound compartment, the Francisella-containing vacuole (FCV), from which it rapidly escapes to replicate in the cytosol. Recent work examining the Francisella disulfide bond formation (Dsb) proteins, FipA and FipB, have demonstrated that these proteins are important during the Francisella infection process; however, details as to how the infections are altered in epithelial cells have remained elusive. To identify the stage of the infections where these Dsbs might act during epithelial infections, we exploited a hepatocyte F. novicida infection model that we recently developed. We found that F. novicida ΔfipA-infected hepatocytes contained bacteria clustered within lysosome-associated membrane protein 1-positive FCVs, suggesting that FipA is involved in the escape of F. novicida from its vacuole. Our morphological evidence provides a tangible link as to how Dsb FipA can influence Francisella infections.

Guanylate binding proteins (GBPs) are interferon-inducible proteins involved in the cell-intrinsic immunity against numerous intracellular pathogens. The molecular mechanisms underlying the potent antibacterial activity of GBPs are still unclear. GBPs have been functionally linked to the NLRP3, the AIM2 and the caspase-11 inflammasomes. Two opposing models are currently proposed to explain the GBPs-inflammasome link: i) GBPs would target intracellular bacteria or bacteria-containing vacuoles to increase cytosolic PAMPs release ii) GBPs would directly facilitate inflammasome complex assembly. Using Francisella novicida infection, we investigated the functional interactions between GBPs and the inflammasome. GBPs, induced in a type I IFN-dependent manner, are required for the F. novicida-mediated AIM2-inflammasome pathway. Here, we demonstrate that GBPs action is not restricted to the AIM2 inflammasome, but controls in a hierarchical manner the activation of different inflammasomes complexes and apoptotic caspases. IFN-γ induces a quantitative switch in GBPs levels and redirects pyroptotic and apoptotic pathways under the control of GBPs. Furthermore, upon IFN-γ priming, F. novicida-infected macrophages restrict cytosolic bacterial replication in a GBP-dependent and inflammasome-independent manner. Finally, in a mouse model of tularemia, we demonstrate that the inflammasome and the GBPs are two key immune pathways functioning largely independently to control F. novicida infection. Altogether, our results indicate that GBPs are the master effectors of IFN-γ-mediated responses against F. novicida to control antibacterial immune responses in inflammasome-dependent and independent manners., Author summary The cell-intrinsic immunity is defined as the mechanisms allowing a host cell infected by an intracellular pathogen to mount effective immune mechanisms to detect and eliminate pathogens without any help from other immune cells. In infected macrophages, the Guanylate Binding Proteins (GBPs) are immune proteins, induced at low levels in a cell autonomous manner by endogenous type I IFN or at high levels following IFN-γ production by innate and adaptive lymphocytes. The antibacterial activity of GBPs has been recently tightly linked to the inflammasomes. Inflammasomes are innate immune complexes leading to inflammatory caspases activation and death of the infected cell. Francisella novicida, a bacterium replicating in the macrophage cytosol, is closely related to F. tularensis, the agent of tularemia and is used as a model to study cytosolic immunity. GBPs contribute to F. novicida lysis within the host cytosol leading to DNA release and AIM2 inflammasome activation. In addition to their regulation of the AIM2 inflammasome, we identified that GBPs also control several other pyroptotic and apoptotic pathways activated in a hierarchical manner. Furthermore, we demonstrate that IFN-γ priming extends GBPs anti-microbial responses from the inflammasome-dependent control of cell death to an inflammasome-independent control of cytosolic bacterial replication. Our results, validated in a mouse model of tularemia, thus segregate the antimicrobial activities of inflammasomes and GBPs as well as highlight GBPs as the master effectors of IFN-γ-mediated cytosolic immunity.

The purification, reconstitution and crystallization of a complex of F. novicida Cpf1 with CRISPR RNA and target DNA is described., Bacteria and archaea use the CRISPR–Cas system as an adaptive response against infection by foreign nucleic acids. Owing to its remarkable flexibility, this mechanism has been harnessed and adopted as a powerful tool for genome editing. The CRISPR–Cas system includes two classes that are subdivided into six types and 19 subtypes according to conservation of the cas gene and loci organization. Recently, a new protein with endonuclease activity belonging to class 2 type V has been identified. This endonuclease, termed Cpf1, in complex with a single CRISPR RNA (crRNA) is able to recognize and cleave a target DNA preceded by a 5′-TTN-3′ protospacer-adjacent motif (PAM) complementary to the RNA guide. To obtain structural insight into the inner workings of Cpf1, the crystallization of an active complex containing the full extent of the crRNA and a 31-nucleotide dsDNA target was attempted. The gene encoding Cpf1 from Francisella novicida was cloned, overexpressed and purified. The crRNA was transcribed and purified in vitro. Finally, the ternary FnCpf1–crRNA–DNA complex was assembled and purified by preparative electrophoresis before crystallization. Crystals belonging to space group C2221, with unit-cell parameters a = 85.2, b = 137.6, c = 320.5 Å, were obtained and subjected to preliminary diffraction experiments.

Development of new therapeutics against Select Agents such as Francisella is critical preparation in the event of bioterrorism. Testing FDA-approved drugs for this purpose may yield new activities unrelated to their intended purpose and may hasten the discovery of new therapeutics. A library of 420 FDA-approved drugs was screened for antibiofilm activity against a model organism for human tularemia, Francisella (F.) novicida, excluding drugs that significantly inhibited growth. The initial screen was based on the 2-component system (TCS) dependent biofilm effect, thus, the QseC dependence of maprotiline anti-biofilm action was demonstrated. By comparing their FDA-approved uses, chemical structures, and other properties of active drugs, toremifene and polycyclic antidepressants maprotiline and chlorpromazine were identified as being highly active against F. novicida biofilm formation. Further down-selection excluded toremifene for its membrane active activity and chlorpromazine for its high antimicrobial activity. The mode of action of maprotiline against F. novicida was sought. It was demonstrated that maprotiline was able to significantly down-regulate the expression of the virulence factor IglC, encoded on the Francisella Pathogenicity Island (FPI), suggesting that maprotiline is exerting an effect on bacterial virulence. Further studies showed that maprotiline significantly rescued F. novicida infected wax worm larvae. In vivo studies demonstrated that maprotiline treatment could prolong time to disease onset and survival in F. novicida infected mice. These results suggest that an FDA-approved drug such as maprotiline has the potential to combat Francisella infection as an antivirulence agent, and may have utility in combination with antibiotics.

The AIM2 inflammasome detects double-stranded DNA in the cytosol and induces caspase-1-dependent pyroptosis as well as release of the inflammatory cytokines interleukin 1β (IL-1β) and IL-18. AIM2 is critical for host defense against DNA viruses and bacteria that replicate in the cytosol, such as Francisella tularensis subspecies novicida (F. novicida). The activation of AIM2 by F. novicida requires bacteriolysis, yet whether this process is accidental or is a host-driven immunological mechanism has remained unclear. By screening nearly 500 interferon-stimulated genes (ISGs) through the use of small interfering RNA (siRNA), we identified guanylate-binding proteins GBP2 and GBP5 as key activators of AIM2 during infection with F. novicida. We confirmed their prominent role in vitro and in a mouse model of tularemia. Mechanistically, these two GBPs targeted cytosolic F. novicida and promoted bacteriolysis. Thus, in addition to their role in host defense against vacuolar pathogens, GBPs also facilitate the presentation of ligands by directly attacking cytosolic bacteria.

Francisella tularensis is considered a potential bioterrorism agent due to its low infectious dose, high mortality rate, and ability to be spread via the aerosol route. We characterized the F. tularensis subspecies novicida mutant strain FTN0109 as a potential vaccine candidate against tularemia. This strain, which lacks an outer membrane lipoprotein, is attenuated in vitro and in vivo, as it exhibits reduced replication within murine J774 macrophages and has a pulmonary LD50 in BALB/c and C57BL/6 mice of >105 CFU (compared to WT parental strain U112, LD50 FTN0109 also conferred complete protection in BALB/c mice against subsequent pulmonary challenge with 10 LD50 (60,000 CFU) of the murine virulent Francisella strain LVS. We also have demonstrated partial protection (50%) against the highly human virulent subspecies tularensis strain SCHU S4 (25 LD50, 12,500 CFU) following intratracheal vaccination in the Fischer 344 rat, a second rodent model for tularemia. Overall, our results suggest that FTN0109 serves as a potential putative vaccine candidate against pulmonary tularemia.

Fluoroquinolone (FQ) resistance is a major health concern in the treatment of tularemia. Because DNA gyrase has been described as the main target of these compounds, our aim was to clarify the contributions of both GyrA and GyrB mutations found in Francisella novicida clones highly resistant to FQs. Wild-type and mutated GyrA and GyrB subunits were overexpressed so that the in vitro FQ sensitivity of functional reconstituted complexes could be evaluated. The data obtained were compared to the MICs of FQs against bacterial clones harboring the same mutations and were further validated through complementation experiments and structural modeling. Whole-genome sequencing of highly FQ-resistant lineages was also done. Supercoiling and DNA cleavage assays demonstrated that GyrA D87 is a hot spot FQ resistance target in F. novicida and pointed out the role of the GyrA P43H substitution in resistance acquisition. An unusual feature of FQ resistance acquisition in F. novicida is that the first-step mutation occurs in GyrB, with direct or indirect consequences for FQ sensitivity. Insertion of P466 into GyrB leads to a 50% inhibitory concentration (IC 50 ) comparable to that observed for a mutant gyrase carrying the GyrA D87Y substitution, while the D487E-ΔK488 mutation, while not active on its own, contributes to the high level of resistance that occurs following acquisition of the GyrA D87G substitution in double GyrA/GyrB mutants. The involvement of other putative targets is discussed, including that of a ParE mutation that was found to arise in the very late stage of antibiotic exposure. This study provides the first characterization of the molecular mechanisms responsible for FQ resistance in Francisella .

BACKGROUND Francisella novicida is a rare cause of human illness despite its close genetic relationship to Francisella tularensis, the agent of tularemia. During April-July 2011, 3 inmates at a Louisiana correctional facility developed F. novicida bacteremia; 1 inmate died acutely. METHODS We interviewed surviving inmates; reviewed laboratory, medical, and housing records; and conducted an environmental investigation. Clinical and environmental samples were tested by culture, real-time polymerase chain reaction (PCR), and multigene sequencing. Isolates were typed by pulsed-field gel electrophoresis (PFGE). RESULTS Clinical isolates were identified as F. novicida based on sequence analyses of the 16S ribosomal RNA, pgm, and pdpD genes. PmeI PFGE patterns for the clinical isolates were indistinguishable. Source patients were aged 40-56 years, male, and African American, and all were immunocompromised. Two patients presented with signs of bacterial peritonitis; the third had pyomyositis of the thigh. The 3 inmates had no contact with one another; their only shared exposures were consumption of municipal water and of ice that was mass-produced at the prison in an unenclosed building. Swabs from one set of ice machines and associated ice scoops yielded evidence of F. novicida by PCR and sequencing. All other environmental specimens tested negative. CONCLUSIONS To our knowledge, this is the first reported common-source outbreak of F. novicida infections in humans. Epidemiological and laboratory evidence implicate contaminated ice as the likely vehicle of transmission; liver disease may be a predisposing factor. Clinicians, laboratorians, and public health officials should be aware of the potential for misidentification of F. novicida as F. tularensis.

CRISPR/Cas9 systems are nowadays applied extensively to effect genome editing in various organisms including plants. CRISPR from Prevotella and Francisella 1 (Cpf1) is a newly characterized RNA-guided endonuclease that has two distinct features as compared to Cas9. First, Cpf1 utilizes a thymidine-rich protospacer adjacent motif (PAM) while Cas9 prefers a guanidine-rich PAM. Cpf1 could be used as a sequence-specific nuclease to target AT-rich regions of a genome that Cas9 had difficulty accessing. Second, Cpf1 generates DNA ends with a 5′ overhang, whereas Cas9 creates blunt DNA ends after cleavage. “Sticky” DNA ends should increase the efficiency of insertion of a desired DNA fragment into the Cpf1-cleaved site using complementary DNA ends. Therefore, Cpf1 could be a potent tool for precise genome engineering. To evaluate whether Cpf1 can be applied to plant genome editing, we selected Cpf1 from Francisella novicida (FnCpf1), which recognizes a shorter PAM (TTN) within known Cpf1 proteins, and applied it to targeted mutagenesis in tobacco and rice. Our results show that targeted mutagenesis had occurred in transgenic plants expressing FnCpf1 with crRNA. Deletions of the targeted region were the most frequently observed mutations. Our results demonstrate that FnCpf1 can be applied successfully to genome engineering in plants.

Cilj istraživanja: Francisella novicida je unutarstanična bakterija koja može inficirati brojne stanice uključujući i stanice ameba. Pretpostavlja se da amebe imaju važnu ulogu za održavanje francizela u vodenom okolišu. Za razliku od stanica sisavaca gdje citosolnom razmnožavanju ove bakterije prethodi kratki ulazak u fagosom, u stanicama ameba bakterija se razmnožava u vakuoli. Postavljena je hipoteza da boravak bakterije u fagosomu/vakuoli i u stanicama makrofaga i u stanicama ameba, ima bitnu ulogu u unutarstaničnom životu bakterije, njenoj virulenciji i patogenezi tularemije. Zbog toga su ciljevi rada bili: uspostaviti okolišnu amebu Dictyostelium discoideum kao modelni organizam za proučavanje unutarstaničnog života F. novicida, razviti metodu za izolaciju fagosoma/vakuole u kojima se nalazi F. novicida iz stanica D. discoideum i primarnih stanica humanih makrofaga, kao i identificirati proteine uključene u formiranje vakuole u kojoj se nalazi F. novicida nakon infekcije D. discoideum. Materijal i metode: U ovom radu istražen je endosomalno – lizosomalni put F. novicida u D. discoideum metodama konfokalne i elektronske mikroskopije te Western blot analizom. U svrhu analize fagosoma/vakuole u kojima se nalazi F. novicida nakon infekcije stanica ameba i humanih makrofaga, uspostavljena je metoda za njihovu izolaciju. Identifikacija proteina koji su uključeni u sastav vakuole provedena je masenom spektrometrijom. Rezultati: Praćenjem kinetike rasta pokazano je da se F. novicida razmnožava u D. discoideum. Bakterija se nakon ulaska u amebu nalazi u ranom vakuolarnom odjeljku, gdje se i razmnožava. Uspostavljena je metoda za izolaciju fagosoma/vakuole u kojoj se nalazi F. novicida i njihove membrane su visoko očuvane. Kvantitativnom proteomskom analizom uzoraka frakcije vakuola u kojima se nalazi F. novicida izoliranih iz D. discoideum 30 minuta nakon infekcije identificirano je ukupno 974 proteina s visokom grupnom specifičnošću. Zaključak: Rezultati ovog istraživanja pokazali su da F. novicida raste i razmnožava se u D. discoideum te ameba može služiti kao modelni organizam za proučavanje virulencije vrsta Francisella i patogeneze tularemije. Bakterija se nakon infekcije razmnožava u vakuoli. Uspješno je uspostavljena metoda za izolaciju fagosoma/vakuole u kojoj se nalazi F. novicida nakon infekcije D. discoideum i humanih makrofaga. Masenom spektrometrijom identificirani su proteini rane vakuole endosomalno – lizosomalnog puta što dokazuje lokalizaciju bakterije u vakuoli., Objectives: Francisella novicida is an intracellular bacterium that can infect multiple range of cells, including amoeba cells. It has been suggested that amoeba have important role for survival of Francisella in the water environment. After infection of mammalian cells, bacterium enters the phagosome and escapes in the cytosol where it replicates. In contrast, in amoeba cells bacterium replicates in vacuoles. We hypothesised that localization of Francisella in the phagosome/vacuole in mammalian and amoeba cells has an important role in the intracellular life cycle of Francisella, bacterial virulence as well as the pathogenesis of tularemia. The aim of this study was to establish Dictyostelium discoideum as a model organism for studying intracellular life cycle of Francisella. Specific aims were to establish a method for isolation of phagosome/vacuole containing Francisella from D. discoideum and human macrophages, and identification of proteins important in formation of the vacuole containing Francisella after infection of D. discoideum. Material and Methods: In this thesis, the endocytic pathway of F. novicida in D. discoideum was investigated using confocal microscopy, electron microscopy and Western blot analysis. The method for isolation of phagosome/vacuole containing Francisella was established. Proteins included in formation of vacuole containing Francisella were identified using mass spectrometry. Results: Growth kinetics study showed that F. novicida replicates in D. discoideum. After entry, bacterium is localized in early vacuolar compartment where it replicates. The method for isolation of phagosome/vacuole containing Francisella enabled the isolation of highly intact vacuoles. Proteomic analysis of fraction with vacuoles containing Francisella isolated from D. discoideum, 30 minutes after infection, identified 974 proteins with high group confidence. Conclusion: Results of this thesis showed that F. novicida replicates in D. discoideum, and amoeba can be used as a model organism for studying virulence of Francisella and pathogenesis of tularemia. After infection, bacterium replicates in the vacuoles. The method for isolation of phagosome/vacuole containing Francisella was successfully established. Mass spectrometry analysis identified proteins included in the formation of early vacuolar compartment in endocytic pathway, which confirmed localization of Francisella in vacuole.

[This corrects the article DOI: 10.3389/fmicb.2017.00935.].

Francisella tularensis je gram-negativna asporogena bakterija iz porodice Franciselaceae. Bakterije roda Francisella fakultativni su unutarstanični patogeni izolirani iz velikog broja različitih domaćina, uključujući i sisavce. F.tularensis subsp. tularensis uzročnik je akutne bolesti tularemije, a zbog ekstremne infektivnosti centar za kontrolu i prevenciju bolesti klasificirao ju je kao sredstvo bioterorizma kategorije A. Bolest kod ljudi s relativno niskom infektivnom dozom mogu izazvati i vrste F. philomiragia i F. holarctica. Podvrste F. noatunensis prilagođene su različitim tjelesnim temperaturama domaćina stoga F. noatunensis subsp. orientalis izaziva granulome u ribama toplih voda, a F. noatunensis subsp. noatunensis u ribama koje žive u hladnim vodama F. novicida visoko je virulentna za miševe i zamorce pa je vrlo dobar model istraživanja humane tularemije, ali može izazvati bolest kod imunokompromitiranih osoba. Ne postoje dokazi koji bi sugerirali prijenos F. novicida artropodnim vektorima ili životinjama te F. novicida nikada nije identificirana u divljim životinjama. Pretpostavlja se da je njen životni ciklus vezan za vodeni mikrookoliš jer je i sama bakterija izolirana iz slane vode. Apatogeni soj F. novicida U112, koji je korišten i u našem istraživanju, sve se češće koristi u svrhu laboratorijskih istraživanja zbog svoje niske virulencije i male mogućnosti razvoja bolesti kod ljudi. Cilj ovog istraživanja bio je pratiti utjecaj različitih temperatura na preživljavanje F. novicida in vitro. Također, utvrditi jesu li uzgojni uvjeti bolji u inkubator-tresilici ili u samom inkubatoru bez tresilice. Bakterijski soj U112 podvrgnut je različitim temperaturnim uzgojnim uvjetima u razdoblju od 5 dana. Od ukupno pet, tri su uzorka inkubirana pri temperaturama od 25 ℃, 37 ℃ i 42 ℃, a dva uzorka pri temperaturama od 25 ℃ i 37 ℃ inkubirana su u inkubator-tresilici. Svakih 24 sata praćena je kinetika rasta te bakterijsko razmnožavanje. Naši rezultati pokazuju da bakterije F. Novicida nisu termofilne te pri temperaturi od 42 ℃ ugibaju već nakon dva dana inkubacije. Pri temperaturama od 37 ℃ i 25 ℃ bez trešnja bakterije svakim danom rastu znatnom brzinom te im odgovaraju takvi uvjeti uzgoja. Uvjeti, koje pruža tresilica, bolji su za rast i razvoj bakterije F. novicida od onih koje pruža sam inkubator. Bakterije se bez tresilice bolje razmnožavaju pri temperaturi od 25 ℃ nego na 37 ℃ jer su im životni ciklus i mehanizmi preživljavanja više vezani za okoliš s relativno nižom temperaturom Francisella tularensis is a gram-negative asporogenous bacterium from the Franciselaceae family. The bacteria from the Francisella genus are facultative intracellular bacteria isolated from a large number of different hosts, including mammals. F. tularensis subsp. tularensis is the cause of the acute disease tularemia and is classified as a Category A biological agent by the CDC due to its extreme infectivity. The disease can also be caused by F. philomiragia and F. holarctica in humans with a relatively low infectious dose. Subspecies F. noatunensis are adapted to the different body temperatures of the host which is why F. noatunensis subsp. orientalis causes granuloma in warm water fish and F. noatunensis subsp. noatunensis does the same in cold water fish. F. novicida is highly virulent to mice and guinea pigs so it is a very good research model for human tularemia, but it can cause the disease in immunocompromised people. There is no evidence to suggest that the transmission of F. novicida by arthropod vectors or animals nor has it ever been identified in wild animals. It is presumed that its life cycle is tied to a water microenvironment because the very bacteria were isolated from salt water. The apathogenic strain F. novicida U112, which was also used in our research, is used more and more often in laboratory research due to its low virulence and small possibility of causing a disease in humans. The aim of this work was to study the effect of different temperatures on the survival of F. novicida in vitro, and to establish whether the growing conditions are better in an incubator with and without a shaker. The bacterial strain, U112, was subject to different temperature growing conditions through a period of 5 days. From a total of five, three samples were incubated at temperatures of 25 °C, 37 °C and 42 °C, and two samples at temperatures of 25 °C and 37 °C with shaking. The growth kinetics and bacterial reproduction were measured every 24 hours. Our results show that the F. novicida are not able to survive at 42 °C after two days of incubation. The bacteria grow at a considerable speed at 37 °C and 25 °C in an incubator without a shaker, these growing conditions benefit the bacteria. The conditions in shaker are more favorable to the F. novicida . The bacteria replicate better at 25 °C than at 37 °C because their lifecycles and survival mechanisms are more connected to an environment niche with a relatively lower temperature.

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.