Search

The Gram-positive, spore-forming, obligate anaerobic firmicute species that make up the Clostridium genus have broad feedstock consumption capabilities and produce value-added metabolic products, but genetic manipulation is difficult, limiting their broad appeal. CRISPR-Cas systems have recently been applied to Clostridium species, primarily using Cas9 as a counterselection marker in conjunction with plasmid-based homologous recombination. CRISPR interference is a method that reduces gene expression of specific genes via precision targeting of a nuclease deficient Cas effector protein. Here, we develop a dCas12a-based CRISPR interference system for transcriptional gene repression in multiple mesophilic Clostridium species. We show the Francisella novicida Cas12a-based system has a broader applicability due to the low GC content in Clostridium species compared to CRISPR Cas systems derived from other bacteria. We demonstrate >99% reduction in transcript levels of targeted genes in Clostridium acetobutylicum and >75% reduction in Clostridium pasteurianum. We also demonstrate multiplexed repression via use of a single synthetic CRISPR array, achieving 99% reduction in targeted gene expression and elucidating a unique metabolic profile for their reduced expression. Overall, this work builds a foundation for high throughput genetic screens without genetic editing, a key limitation in current screening methods used in the Clostridium community. [ABSTRACT FROM AUTHOR]

Abstract Tularemia is a zoonosis caused by CDC-declared Tier 1 threat agent Francisella tularensis. F. tularensis subsp. novicida (F. novicida) is virulent in mice but non-pathogenic in immunocompetent humans and serves as a potential surrogate organism. In a recent study, we established a silkworm (Bombyx mori) model of infection for F. novicida. Francisella secretes its virulence factors through various mechanisms that modify the intracellular environment to ensure its replication and survival. To identify new pathogenic factors, we focused on the type I secretory system (T1SS) of Francisella. In silico analysis revealed a RtxA (Repeats-in-toxin) like protein in the Francisella genome. The characteristics of RtxA like protein were investigated using mutant analysis. Firstly, the role of rtxA in silkworms was investigated by infecting them with F. novicida strains into the hemocoel. The rtxA mutant failed to kill the silkworms, whereas F. novicida wild-type (WT) strain killed silkworms within 3–7 days post infection. The arrested growth of the mutant strain in silkworms was observed using a whole-body CFU count assay. We also investigated the growth characteristics of the rtxA mutant in hemocytes, one of the primary multiplication sites of Francisella within silkworms. Interrupted growth of the rtxA mutant with significantly reduced cytotoxicity was observed in hemocytes via confocal microscopy. Next, we analyzed the effect of rtxA in human monocyte cell line THP-1. The mutant strain showed significantly decreased growth and reduced cytotoxicity compared with its parental strain in THP-1 cells. This study newly identified RtxA like protein of F. novicida as an important lethal pathogenic factor in silkworm and mammalian cells. Highlights • There is a RtxA like protein in Francisella novicida. • The deletion mutant of RtxA like protein failed to kill silkworm. • RtxA like protein contributes to intracellular growth in THP-1 cells. [ABSTRACT FROM AUTHOR]

Lysine-63-linked (K63-linked) polyubiquitination of TRAF3 coordinates the engagement of pattern-recognition receptors with recruited adaptor proteins and downstream activator TBK1 in pathways that induce type I IFN. Whether autoubiquitination or other E3 ligases mediate K63-linked TRAF3 polyubiquitination remains unclear. We demonstrated that mice deficient in the E3 ligase gene Hectd3 remarkably increased host defense against infection by intracellular bacteria Francisella novicida, Mycobacterium, and Listeria by limiting bacterial dissemination. In the absence of HECTD3, type I IFN response was impaired during bacterial infection both in vivo and in vitro. HECTD3 regulated type I IFN production by mediating K63-linked polyubiquitination of TRAF3 at residue K138. The catalytic domain of HECTD3 regulated TRAF3 K63 polyubiquitination, which enabled TRAF3-TBK1 complex formation. Our study offers insights into mechanisms of TRAF3 modulation and provides potential therapeutic targets against infections by intracellular bacteria and inflammatory diseases. [ABSTRACT FROM AUTHOR]

Summary: Recently, CRISPR‐Cas (clustered, regularly interspaced short palindromic repeats–CRISPR‐associated proteins) system has been used to produce plants resistant to DNA virus infections. However, there is no RNA virus control method in plants that uses CRISPR‐Cas system to target the viral genome directly. Here, we reprogrammed the CRISPR‐Cas9 system from Francisella novicida to confer molecular immunity against RNA viruses in Nicotiana benthamiana and Arabidopsis plants. Plants expressing FnCas9 and sgRNA specific for the cucumber mosaic virus (CMV) or tobacco mosaic virus (TMV) exhibited significantly attenuated virus infection symptoms and reduced viral RNA accumulation. Furthermore, in the transgenic virus‐targeting plants, the resistance was inheritable and the progenies showed significantly less virus accumulation. These data reveal that the CRISPR/Cas9 system can be used to produce plant that stable resistant to RNA viruses, thereby broadening the use of such technology for virus control in agricultural field. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Understanding the virulence and pathogenesis of human pathogens using insect models is an increasingly popular method. Francisella novicida , which is virulent in mice but non-pathogenic to immunocompetent humans, is widely used as an ideal candidate for Francisella research. In this study, we developed a silkworm ( Bombyx mori ) infection model for F. novicida by inoculating the hemocoels of silkworms with F. novicida . We found that silkworms died within 3–7 days of F. novicida infection. However, the deletion mutant of DotU, the core part of type VI secretion systems, failed to kill silkworm. In whole silkworm bodies, the bacterial load of the DotU deletion mutant was significantly less than that of the wild-type strain. Approximately 10-fold increase in bacterial load was recorded in hemolymph and subcutaneous tissues compared with that in the silk gland, Malpighian tubule, and reproductive organs. The CFU count of the DotU deletion mutant in all organs was similar results to the whole body CFU count. Confocal microscopy further confirmed the arrested growth of the mutant strain within hemocytes. The intracellular growth of F. novicida strains was also analyzed using the silkworm ovary-derived cell line BmN4. In BmN4, both CFU count assay and confocal microscopy revealed extensive growth of the wild-type strain compared with that of the mutant strain. Francisella DotU has already been proven as a virulence factor in mammals, and it was also found to be an essential virulence factor in our silkworm infection model. Therefore, this silkworm infection model is suitable for identifying new virulence factors of Francisella . [ABSTRACT FROM AUTHOR]

Francisella tularensis is a potent human pathogen that invades and survives in macrophage and epithelial cells. Two identical proteins, FTT_0924 from F. tularensis subsp. tularensis and FTL_1286 from F. tularensis subsp. holarctica LVS, have previously been identified as playing a role in protection of the bacteria from osmotic shock and its survival in macrophages. FTT_0924 has been shown to localize to the inner membrane, with its C-terminus exposed to the periplasm. Here, crystal structures of the F. novicida homologue FTN_0802, which we call FvfA, in two crystal forms are reported at 1.8 Å resolution. FvfA differs from FTT_0924 and FTL_1286 by a single amino acid. FvfA has a DUF1471 fold that closely resembles the Escherichia coli outer membrane lipoprotein RscF, a component of a phosphorelay pathway involved in protecting bacteria from outer membrane perturbation. The structural and functional similarities and differences between these proteins and their implications for F. tularensis pathogenesis are discussed. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The Francisella genus includes several recognized species, additional potential species, and other representatives that inhabit a range of incredibly diverse ecological niches, but are not closely related to the named species. Francisella species have been obtained from a wide variety of clinical and environmental sources; documented species include highly virulent human and animal pathogens, fish pathogens, opportunistic human pathogens, tick endosymbionts, and free-living isolates inhabiting brackish water. While more than 120 Francisella genomes have been sequenced to date, only a few contain plasmids, and most of these appear to be cryptic, with unknown benefit to the host cell. We have identified several putative cryptic plasmids in the sequenced genomes of three Francisella novicida and F. novicida-like strains (TX07-6608, AZ06-7470, DPG_3A-IS) and two new Francisella species (F. frigiditurris CA97-1460 and F. opportunistica MA06-7296). These plasmids were compared to each other and to previously identified plasmids from other Francisella species. Some of the plasmids encoded functions potentially involved in replication, conjugal transfer and partitioning, environmental survival (transcriptional regulation, signaling, metabolism), and hypothetical proteins with no assignable functions. Genomic and phylogenetic comparisons of these new plasmids to the other known Francisella plasmids revealed some similarities that add to our understanding of the evolutionary relationships among the diverse Francisella species. [ABSTRACT FROM AUTHOR]

Francisella novicida is a gram-negative pathogen commonly used to study infections by the potential bioterrorism agent, Francisella tularensis . The Francisella lipid A structure has been well characterized and showed to affect the pathogenesis of F. novicida . Previous work characterized two lipid A acyltransferases, LpxD1 and LpxD2, and constructed the lpxD1-null and lpxD2-null mutants. Mutational analysis showed the lpxD1-null mutant was attenuated in mice and subsequently exhibited protection against a lethal WT challenge. However, details as how the virulence has been changed have remained elusive. This study aims to analyze effects of lipid A acyltransferases on the pathogenesis of F. novicida . MS and MS n were conducted to confirm the lipid A structures of lpxD1-null and lpxD2-null mutants. The stress tolerance, Toll-like receptor 4 (TLR4) stimulation level, intracellular survival and replication ability and cytotoxicity of lpxD1-null and lpxD2-null mutants were analyzed. The results suggested the lpxD1-null mutant with shorter acyl chains in lipid A is more sensitive to various environmental stresses than F. novicida and lpxD2-null mutant. In addition, the lpxD1-null mutant fails to survive and replicate in cells and shows lower cytotoxicity to infected cells. This study provides insights into the pathogenesis of F. novicida . [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

NKG2D is a potent activating receptor that is expressed on cytotoxic immune cells such as CD8 T and NK cells, where it promotes cytotoxicity after binding stress ligands on infected or transformed cells. On NK cell precursors NKG2D modulates proliferation and maturation. Previously, we observed that NKG2D deficiency affects peripheral B cell numbers. In this study, we show that NKG2D regulates B1a cell development and function. We find that mice deficient for NKG2D have a strong reduction of B1a cell numbers. As a result, NKG2D-deficient mice produce significantly less Ag-specific IgM Abs upon immunization with T cell-independent Ags, and they are more susceptible to Gram-negative sepsis. Klrk1-/- B1a cells are also functionally impaired and they fail to provide protection against Francisella novicida upon adoptive transfer. Using mixed bone marrow chimeric mice, we show that the impact of NKG2D deficiency on B1a cell development is cell intrinsic. No changes in homeostatic turnover and homing of B cells were detectable, limiting the effects of NKG2D to modulation of the hematopoietic development of B1a cells. Using conditional ablation, we demonstrate that the effect of NKG2D on B1a cell development occurs at a developmental stage that precedes the common lymphoid progenitor. Our findings reveal an unexpected new role for NKG2D in the regulation of B1a cell development. The protective effects of this activating receptor therefore reach beyond that of cytotoxic cells, stimulating the immune system to fight bacterial infections by promoting development of innate-like B cells. [ABSTRACT FROM AUTHOR]

Francisella tularensis is a highly virulent zoonotic pathogen that causes tularemia and, because of weaponization efforts in past world wars, is considered a tier 1 biothreat agent. Detection and surveillance of F. tularensis may be confounded by the presence of uncharacterized, closely related organisms. Through DNA-based diagnostics and environmental surveys, novel clinical and environmental Francisella isolates have been obtained in recent years. Here we present 7 new Francisella genomes and a comparison of their characteristics to each other and to 24 publicly available genomes as well as a comparative analysis of 16S rRNA and sdhA genes from over 90 Francisella strains. Delineation of new species in bacteria is challenging, especially when isolates having very close genomic characteristics exhibit different physiological features--for example, when some are virulent pathogens in humans and animals while others are nonpathogenic or are opportunistic pathogens. Species resolution within Francisella varies with analyses of single genes, multiple gene or protein sets, or whole-genome comparisons of nucleic acid and amino acid sequences. Analyses focusing on single genes (16S rRNA, sdhA), multiple gene sets (virulence genes, lipopolysaccharide [LPS] biosynthesis genes, pathogenicity island), and whole-genome comparisons (nucleotide and protein) gave congruent results, but with different levels of discrimination confidence. We designate four new species within the genus; Francisella opportunistica sp. nov. (MA06-7296), Francisella salina sp. nov. (TX07-7308), Francisella uliginis sp. nov. (TX07-7310), and Francisella frigiditurris sp. nov. (CA97-1460). This study provides a robust comparative framework to discern species and virulence features of newly detected Francisella bacteria. [ABSTRACT FROM AUTHOR]

Respiratory infection of mice with Francisella novicida has recently been used as a model for the highly virulent human pathogen Francisella tularensis . Similar to F. tularensis , even small doses of F. novicida administered by respiratory routes are lethal for inbred laboratory mice. This feature obviously limits study of infection-induced immunity. Parenteral sublethal infections of mice with F. novicida are feasible, but the resulting immune responses are incompletely characterized. Here we use parenteral intradermal (i.d.) and intraperitoneal (i.p.) F. novicida infections of C57BL/6J mice to determine the role of B cells in controlling primary and secondary F. novicida infections. Despite developing comparable levels of F. novicida -primed T cells, B cell knockout mice were much more susceptible to both primary i.d. infection and secondary i.p. challenge than wild type (normal) C57BL/6J mice. Transfer of F. novicida -immune sera to either wild type C57BL/6J mice or to B cell knockout mice did not appreciably impact survival of subsequent lethal F. novicida challenge. However, F. novicida -immune mice that were depleted of T cells after priming but just before challenge survived and cleared secondary i.p. F. novicida challenge. Collectively these results indicate that B cells, if not serum antibodies, play a major role in controlling F. novicida infections in mice. [ABSTRACT FROM AUTHOR]

The article reports on the occurence of sytemic visceral granuloma in cultured tilapia and marine fish including Piscirickettsiosis-like syndrome (PLOs). It also mentions that Francisella noatunensis subsp. noatunensis (Fnn) and Francisella noatunensis subsp. orientalis (Fno) have been identified as etiological agents of the francisellosis in the warm and cold water fishes respectively.

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-C 14 -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-C 16 or 3-OH-C 18 at its C3 and C3′ positions, FnLpxA was thought to be selective for longer acyl chain (3-OH-C 16 and 3-OH-C 18 ) over short acyl chain (3-OH-C 14 , 3-OH-C 12, and 3-OH-C 10 ). Here we demonstrate that Francisella novicida ( F. novicida ) lpxA functionally complements an E. coli lpxA knockout mutant and efficiently transfers 3-OH-C 14 as well as 3-OH-C 16 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 Å. 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 Å apart that renders not enough space for binding of 3-OH-C 12 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary CRISPR-Cas adaptive immune systems in prokaryotes boast a diversity of protein families and mechanisms of action, where most systems rely on protospacer-adjacent motifs (PAMs) for DNA target recognition. Here, we developed an in vivo, positive, and tunable screen termed PAM-SCANR (PAM screen achieved by NOT-gate repression) to elucidate functional PAMs as well as an interactive visualization scheme termed the PAM wheel to convey individual PAM sequences and their activities. PAM-SCANR and the PAM wheel identified known functional PAMs while revealing complex sequence-activity landscapes for the Bacillus halodurans I-C (Cascade), Escherichia coli I-E (Cascade), Streptococcus thermophilus II-A CRISPR1 (Cas9), and Francisella novicida V-A (Cpf1) systems. The PAM wheel was also readily applicable to existing high-throughput screens and garnered insights into SpyCas9 and SauCas9 PAM diversity. These tools offer powerful means of elucidating and visualizing functional PAMs toward accelerating our ability to understand and exploit the multitude of CRISPR-Cas systems in nature. [ABSTRACT FROM AUTHOR]

Summary The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets complementary to the guide RNA and has been applied to programmable genome editing. Cas9-mediated cleavage requires a protospacer adjacent motif (PAM) juxtaposed with the DNA target sequence, thus constricting the range of targetable sites. Here, we report the 1.7 Å resolution crystal structures of Cas9 from Francisella novicida (FnCas9), one of the largest Cas9 orthologs, in complex with a guide RNA and its PAM-containing DNA targets. A structural comparison of FnCas9 with other Cas9 orthologs revealed striking conserved and divergent features among distantly related CRISPR-Cas9 systems. We found that FnCas9 recognizes the 5′-NGG-3′ PAM, and used the structural information to create a variant that can recognize the more relaxed 5′-YG-3′ PAM. Furthermore, we demonstrated that the FnCas9-ribonucleoprotein complex can be microinjected into mouse zygotes to edit endogenous sites with the 5′-YG-3′ PAM, thus expanding the target space of the CRISPR-Cas9 toolbox. [ABSTRACT FROM AUTHOR]

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 Δ fipA-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. [ABSTRACT FROM AUTHOR]

Francisella noatunensis subsp. orientalis ( Fno ) is an emergent fish pathogen in both marine and fresh water environments. The bacterium is suspected to persist in the environment even without the presence of a suitable fish host. In the present study, the influence of different abiotic factors such as salinity and temperature were used to study the biofilm formation of different isolates of Fno including intracellular growth loci C (iglC) and pathogenicity determinant protein A (pdpA) knockout strains. Finally, we compared the susceptibility of planktonic and biofilm to three disinfectants used in the aquaculture and ornamental fish industry, namely Virkon ® , bleach and hydrogen peroxide. The data indicates that Fno is capable of producing biofilms within 24 h where both salinity as well as temperature plays a role in the growth and biofilm formation of Fno . Mutations in the iglC or pdpA , both known virulence factors, do not appear to affect the capacity of Fno to produce biofilms, and the minimum inhibitory concentration, and minimum biocidal concentration for the three disinfectants were lower than the minimum biofilm eradication concentration values. This information needs to be taken into account if trying to eradicate the pathogen from aquaculture facilities or aquariums. [ABSTRACT FROM AUTHOR]

Lipid A is the active center of lipopolysaccharide which also known as endotoxin. Monophosphoryl-lipid A (MPLA) has less toxicity but retains potent immunoadjuvant activity; therefore, it can be developed as adjuvant for improving the strength and duration of the immune response to antigens. However, MPLA cannot be chemically synthesized and can only be obtained by hydrolyzing lipopolysaccharide (LPS) purified from Gram-negative bacteria. Purifying LPS is difficult and time-consuming and can damage the structure of MPLA. In this study, Escherichia coli mutant strains HWB01 and HWB02 were constructed by deleting several genes and integrating Francisella novicida gene lpxE into the chromosome of E. coli wild type strain W3110. Compared with W3110, HWB01 and HWB02 synthesized very short LPS, Kdo2-monophosphoryl-lipid A (Kdo2-MPLA) and Kdo2-pentaacyl-monophosphoryl-lipid A (Kdo2-pentaacyl-MPLA), respectively. Structural changes of LPS in the outer membranes of HWB01 and HWB02 increased their membrane permeability, surface hydrophobicity, auto-aggregation ability and sensitivity to some antibiotics, but the abilities of these strains to activate the TLR4/MD-2 receptor of HKE-Blue hTLR4 cells were deceased. Importantly, purified Kdo2-MPLA and Kdo2-pentaacyl-MPLA differed from wild type LPS in their ability to stimulate the mammalian cell lines THP-1 and RAW264.7. The purification of Kdo2-MPLA and Kdo2-pentaacyl-MPLA from HWB01 and HWB02, respectively, is much easier than the purification of LPS from W3110, and these lipid A derivatives could be important tools for developing future vaccine adjuvants. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Inflammasomes are multi-protein signaling platforms that upon activation trigger the maturation of the pro-inflammatory cytokines, interleukin-1β ( IL-1β) and IL-18, and cell death. Inflammasome sensors detect microbial and host-derived molecules. Here, we review the mechanisms of inflammasome activation triggered by bacterial infection, primarily focusing on two model intracellular bacterial pathogens, Francisella novicida and Salmonella typhimurium. We discuss the complex relationship between bacterial recognition through direct and indirect detection by inflammasome sensors. We highlight regulation mechanisms that potentiate or limit inflammasome activation. We discuss the importance of caspase-1 and caspase-11 in host defense, and we examine the downstream consequences of inflammasome activation within the context of bacterial infections. [ABSTRACT FROM AUTHOR]

Sepsis is a complex immune disorder that is characterized by systemic hyperinflammation. Alarmins, which are multifunctional endogenous factors, have been implicated in exacerbation of inflammation in many immune disorders including sepsis. Here we show that Galectin-9, a host endogenous β-galactoside binding lectin, functions as an alarmin capable of mediating inflammatory response during sepsis resulting from pulmonary infection with Francisella novicida, a Gram negative bacterial pathogen. Our results show that this galectin is upregulated and is likely released during tissue damage in the lungs of F. novicida infected septic mice. In vitro, purified recombinant galectin-9 exacerbated F. novicida-induced production of the inflammatory mediators by macrophages and neutrophils. Concomitantly, Galectin-9 deficient (Gal-9-/-) mice exhibited improved lung pathology, reduced cell death and reduced leukocyte infiltration, particularly neutrophils, in their lungs. This positively correlated with overall improved survival of F. novicida infected Gal-9-/- mice as compared to their wild-type counterparts. Collectively, these findings suggest that galectin-9 functions as a novel alarmin by augmenting the inflammatory response in sepsis development during pulmonary F. novicida infection. [ABSTRACT FROM AUTHOR]

Type I IFN production is an important host immune response against viral and bacterial infections. However, little is known about the ligands and corresponding host receptors that trigger type I IFN production during bacterial infections. We used a model intracellular pathogen, Francisella novicida, to begin characterizing the type I IFN response to bacterial pathogens. F. novicida replicates in the cytosol of host cells and elicits a robust type I IFN response that is largely TLR independent, but is dependent on the adapter molecule STING, suggesting that the type I IFN stimulus during F. novicida infection is cytosolic. In this study, we report that the cytosolic DNA sensors, cyclic GMP-AMP synthase (cGAS) and Ifi204, are both required for the STING-dependent type I IFN response to F. novicida infection in both primary and immortalized murine macrophages. We created cGAS, Ifi204, and Sting functional knockouts in RAW264.7 macrophages and demonstrated that cGAS and Ifi204 cooperate to sense dsDNA and activate the STING-dependent type I IFN pathway. In addition, we show that dsDNA from F. novicida is an important type I IFN stimulating ligand. One outcome of cGAS-STING signaling is the activation of the absent in melanoma 2 inflammasome in response to F. novicida infection. Whereas the absent in melanoma 2 inflammasome is beneficial to the host during F. novicida infection, type I IFN signaling by STING and IFN regulatory factor 3 is detrimental to the host during F. novicida infection. Collectively, our studies indicate that cGAS and Ifi204 cooperate to sense cytosolic dsDNA and F. novicida infection to produce a strong type I IFN response. [ABSTRACT FROM AUTHOR]

Francisella tularensis causes the disease tularemia. Human pulmonary exposure to the most virulent form, F. tularensis subsp. tularensis (Ftt), leads to high morbidity and mortality, resulting in this bacterium being classified as a potential biothreat agent. However, a closely-related species, F. novicida, is avirulent in healthy humans. No tularemia vaccine is currently approved for human use. We demonstrate that a single dose vaccine of a live attenuated F. novicida strain (Fn iglD) protects against subsequent pulmonary challenge with Ftt using two different animal models, Fischer 344 rats and cynomolgus macaques (NHP). The Fn iglD vaccine showed protective efficacy in rats, as did a Ftt iglD vaccine, suggesting no disadvantage to utilizing the low human virulent Francisella species to induce protective immunity. Comparison of specific antibody profiles in vaccinated rat and NHP sera by proteome array identified a core set of immunodominant antigens in vaccinated animals. This is the first report of a defined live attenuated vaccine that demonstrates efficacy against pulmonary tularemia in a NHP, and indicates that the low human virulence F. novicida functions as an effective tularemia vaccine platform. [ABSTRACT FROM AUTHOR]

The first common-source outbreak of Francisella novicida infections among humans is described, along with epidemiologic and laboratory evidence identifying contaminated ice as the likely source of infection.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. [ABSTRACT FROM AUTHOR]

Oral vaccination with the defined live attenuated Francisella novicida vaccine strain U112ΔiglB has been demonstrated to induce protective immunity against pulmonary challenge with the highly human virulent Francisella tularensis strain SCHU S4. However, this vaccination regimen requires a booster dose in mice and Exhibits 50% protective efficacy in the Fischer 344 rat model. To enhance the efficacy of this vaccine strain, we engineered U112ΔiglB to express the Salmonella typhimurium FljB flagellin D1 domain, a TLR5 agonist. The U112ΔiglB::fljB strain was highly attenuated for intracellular macrophage replication, and although the FljB protein was expressed within the cytosol, it exhibited TLR5 activation in a TLR5-expressing HEK cell line. Additionally, infection of splenocytes and lymphocytes with U112ΔiglB::fljB induced significantly greater TNF-α production than infection with U112ΔiglB. Oral vaccination with U112ΔiglB::fljB also induced significantly greater protection than U112ΔiglB against pulmonary SCHU S4 challenge in rats. The enhanced protection was accompanied by higher IgG2a production and serum-mediated reduction of Francisella infectivity. Thus, the U112ΔiglB::fljB strain may serve as a potential vaccine candidate against pneumonic tularemia. [ABSTRACT FROM AUTHOR]

Intracellular pathogens and other organisms have evolved mechanisms to exploit host cells for their life cycles. Virulence genes of some intracellular bacteria responsible for these mechanisms are located in pathogenicity islands, such as secretion systems that secrete effector proteins. The Francisella pathogenicity island is required for phagosomal escape, intracellular replication, evasion of host immune responses, virulence, and encodes a type 6 secretion system. We hypothesize that some Francisella novicida pathogenicity island proteins are secreted during infection of host cells. To test this hypothesis, expression plasmids for all Francisella novicida FPI-encoded proteins with C-terminal and N-terminal epitope FLAG tags were developed. These plasmids expressed their respective epitope FLAG-tagged proteins at their predicted molecular weights. J774 murine macrophage-like cells were infected with Francisella novicida containing these plasmids. The FPI proteins expressed from these plasmids successfully restored the intramacrophage growth phenotype in mutants of the respective genes that were deficient for intramacrophage growth. Using these expression plasmids, the localization of the Francisella pathogenicity island proteins were examined via immuno-fluorescence microscopy within infected macrophage-like cells. Several Francisella pathogenicity island encoded proteins (IglABCDEFGHIJ, PdpACE, DotU and VgrG) were detected extracellularly and they were co-localized with the bacteria, while PdpBD and Anmk were not detected and thus remained inside bacteria. Proteins that were co-localized with bacteria had different patterns of localization. The localization of IglC was dependent on the type 6 secretion system. This suggests that some Francisella pathogenicity island proteins were secreted while others remain within the bacterium during infection of host cells as structural components of the secretion system and were necessary for secretion. [ABSTRACT FROM AUTHOR]

Biofilms, multicellular communities of bacteria, may be an environmental survival and transmission mechanism of Francisella tularensis. Chitinases of F. tularensis ssp. novicida (Fn) have been suggested to regulate biofilm formation on chitin surfaces. However, the underlying mechanisms of how chitinases may regulate biofilm formation are not fully determined. We hypothesized that Fn chitinase modulates bacterial surface properties resulting in the alteration of biofilm formation. We analyzed biofilm formation under diverse conditions using chitinase mutants and their counterpart parental strain. Substratum surface charges affected biofilm formation and initial attachments. Biophysical analysis of bacterial surfaces confirmed that the chi mutants had a net negative-charge. Lectin binding assays suggest that chitinase cleavage of its substrates could have exposed the concanavalin A-binding epitope. Fn biofilm was sensitive to chitinase, proteinase and DNase, suggesting that Fn biofilm contains exopolysaccharides, proteins and extracellular DNA. Exogenous chitinase increased the drug susceptibility of Fn biofilms to gentamicin while decreasing the amount of biofilm. In addition, chitinase modulated bacterial adhesion and invasion of A549 and J774A.1 cells as well as intracellular bacterial replication. Our results support a key role of the chitinase(s) in biofilm formation through modulation of the bacterial surface properties. Our findings position chitinase as a potential anti-biofilm enzyme in Francisella species. [ABSTRACT FROM AUTHOR]

In this work, we describe the identification of synthetic, controllable promoters that function in the bacterial pathogen Francisella novicida, a model facultative intracellular pathogen. Synthetic DNA fragments consisting of the tetracycline operator (tetO) flanked by a random nucleotide sequence were inserted into a Francisella-Escherichia coli shuttle plasmid upstream of a promot-erless artificial operon containing the reporter genes cat and lacZ. Fragments able to promote transcription were selected for based on their ability to drive expression of the cat gene, conferring chloramphenicol resistance. Promoters of various strengths were found, many of which were repressed in the presence of the tetracycline repressor (TetR) and promoted transcription only in the presence of the TetR inducer anhydrotetracycline. A subset of both constitutive and inducible synthetic promoters were characterized to find their induction ratios and to identify their transcription start sites. In cases where tetO was located between or downstream of the -10 and -35 regions of the promoter, control by TetR was observed. If the tetO region was upstream of the -35 region by more than 9 bp, it did not confer TetR control. We found that three of three promoters isolated in F. novicida functioned at a comparable level in E. coli; however, none of the 10 promoters isolated in E. coli functioned at a significant level in F. novicida. Our results allowed us to isolate minimal F. novicida promoters of 47 and 48 bp in length. [ABSTRACT FROM AUTHOR]

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems are known to mediate bacterial defence against foreign nucleic acids. We recently demonstrated a non-canonical role for a CRISPR-Cas system in controlling endogenous gene expression, which had not previously been appreciated. In the present article, we describe the studies that led to this discovery, beginning with an unbiased genome-wide screen to identify virulence genes in the intracellular pathogen Francisella novicida. A gene annotated as encoding a hypothetical protein, but which we now know encodes the Cas protein Cas9, was identified as one of the most critical to the ability of F. novicida to replicate and survive during murine infection. Subsequent studies revealed a role for this protein in evasion of the host innate immune response. Specifically, Cas9 represses the expression of a BLP (bacterial lipoprotein) that could otherwise be recognized by TLR2 (Toll-like receptor 2), a host protein involved in initiating an antibacterial pro-inflammatory response. By repressing BLP levels, Cas9 mediates evasion of TLR2, promoting bacterial virulence. Finally, we described the molecular mechanism by which Cas9 functions in complex with two small RNAs to target the mRNA encoding the BLP for degradation. This work greatly broadened the paradigm for CRISPR-Cas function, highlighting a role in gene regulation that could be conserved in numerous bacteria, and elucidating its integral contribution to bacterial pathogenesis. [ABSTRACT FROM AUTHOR]

Abstract: Previous results suggest that mutations in most genes in the Francisella pathogenicity island (FPI) attenuate the bacterium. Using a mouse model, here we determined the impact of mutations in pdpA, pdpC, and pdpD in Francisella novicida on in vitro replication in macrophages, and in vivo immunogenicity. In contrast to most FPI genes, deletion of pdpC (FnΔpdpC) and pdpD (FnΔpdpD) from F. novicida did not impact growth in mouse bone-marrow derived macrophages. Nonetheless, both FnΔpdpC and FnΔpdpD were highly attenuated when administered intradermally. Infected mice produced relatively normal anti-F. novicida serum antibodies. Further, splenocytes from infected mice controlled intramacrophage Francisella replication, indicating T cell priming, and mice immunized by infection with FnΔpdpC or FnΔpdpD survived secondary lethal parenteral challenge with either F. novicida or Francisella tularensis LVS. In contrast, deletion of pdpA (FnΔpdpA) ablated growth in macrophages in vitro. FnΔpdpA disseminated and replicated poorly in infected mice, accompanied by development of some anti-F. novicida serum antibodies. However, primed Th1 cells were not detected, and vaccinated mice did not survive even low dose challenge with either F. novicida or LVS. Taken together, these results suggest that successful priming of Th1 cells, and protection against lethal challenge, depends on expression of PdpA. [Copyright &y& Elsevier]

The inflammasome is a signaling platform that is central to the innate immune responses to bacterial infections. Francisella tularensis is a bacterium replicating within the host cytosol. During F. tularensis subspecies novicida infection, AIM2, an inflammasome receptor sensing cytosolic DNA, activates caspase-1 in an ASC-dependent manner, leading to both pyroptosis and release of the proinflammatory cytokines IL-1ß and IL-18. Activation of this canonical inflammasome pathway is key to limit F. novicida infection. In this study, by comparing the immune responses of AIM2 knockout (KO), ASCKO, and Casp1KO mice in response to F. novicida infection, we observed that IFN-γ levels in the serum of Casp1KO mice were much higher than the levels observed in AIM2KO and ASCKO mice. This difference in IFN-γ production was due to a large production of IFN-γ by NK cells in Casp1KO mice that was not observed in ASCKO mice. The deficit in IFN-γ production observed in ASCKO mice was not due to a reduced Dock2 expression or to an intrinsic defect of ASCKO NK cells. We demonstrate that in infected Casp1KO mice, IFN-γ production is due to an ASC-dependent caspase-1-independent pathway generating IL-18. Furthermore, we present in vitro data suggesting that the recently described AIM2/ASC/caspase-8 noncanonical pathway is responsible for the caspase-1-independent IL-18 releasing activity. To our knowledge, this study is the first in vivo evidence of an alternative pathway able to generate in a caspase-1-independent pathway bioactive IL-18 to boost the production of IFN-γ, a cytokine critical for the host antibacterial response. [ABSTRACT FROM AUTHOR]

Abstract: Francisella tularensis (F. tularensis), the causative agent of tularemia, has long been known to invade and occupy non-phagocytic epithelial cells. Many epithelial cell infection models have been developed to study this process; however, due to the lack of consensus on infection methods and precise experimental procedures to evaluate invasion and replication, selection of appropriate models to use based on the literature is challenging. To evaluate in vitro non-phagocytic cell infection models, we chose 8 epithelial cultured cell lines from published models to infect with F. tularensis subspecies novicida (F. novicida) and compared the results to a recently developed model that used the mouse hepatocyte BNL CL.2 cell line. We utilized classical gentamicin-based invasion assays to determine total intracellular bacterial loads and employed microscopic examination with staining techniques that distinguished between intracellular and extracellular bacteria to provide an accurate assessment of the proportion of invaded host cells and the degree of bacterial replication. We found that COS-7 cells exhibited the greatest invasion rates; CMT-93 cells contained the largest intracellular bacterial loads; ad HEK-293s were capable of invasion and replication rates at high levels, but required shorter infection incubation times. Although COS-7, CMT-93 and HEK-293 cell lines may be suited to study certain aspects of invasion or replication, we found that BNL CL.2 cells appeared the most appropriate to study the overall pathogenesis of F. novicida when examined in toto. [Copyright &y& Elsevier]

Bacteria, plants, and algae produce isoprenoids through the methylerythritol phosphate (MEP) pathway, an attractive pathway for antimicrobial drug development as it is present in prokaryotes and some lower eukaryotes but absent from human cells. The first committed step of the MEP pathway is catalyzed by 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR/MEP synthase). MEP pathway genes have been identified in many biothreat agents, including Francisella, Brucella, Bacillus, Burkholderia, and Yersinia. The importance of the MEP pathway to Francisella is demonstrated by the fact that MEP pathway mutations are lethal. We have previously established that fosmidomycin inhibits purified MEP synthase (DXR) from F. tularensis LVS. FR900098, the acetyl derivative of fosmidomycin, was found to inhibit the activity of purified DXR from F. tularensis LVS (IC50 = 230 nM). Fosmidomycin and FR900098 are effective against purified DXR from Mycobacterium tuberculosis as well, but have no effect on whole cells because the compounds are too polar to penetrate the thick cell wall. Fosmidomycin requires the GlpT transporter to enter cells, and this is absent in some pathogens, including M. tuberculosis. In this study, we have identified the GlpT homologs in F. novicida and tested transposon insertion mutants of glpT. We showed that FR900098 also requires GlpT for full activity against F. novicida. Thus, we synthesized several FR900098 prodrugs that have lipophilic groups to facilitate their passage through the bacterial cell wall and bypass the requirement for the GlpT transporter. One compound, that we termed "compound 1," was found to have GlpT-independent antimicrobial activity. We tested the ability of this best performing prodrug to inhibit F. novicida intracellular infection of eukaryotic cell lines and the caterpillar Galleria mellonella as an in vivo infection model. As a lipophilic GlpT-independent DXR inhibitor, compound 1 has the potential to be a broad-spectrum antibiotic, and should be effective against most MEP- dependent organisms. [ABSTRACT FROM AUTHOR]

Innate recognition systems, including the Toll-like receptors ( TLRs), play a critical role in activating host defences and proinflammatory pathways in response to infection. Pathogens have developed strategies to subvert TLRs in order to survive and replicate within the host. The model intracellular pathogen, Francisella novicida, modulates host defences to promote survival and replication in macrophages. TLR2, which recognizes bacterial lipoproteins ( BLPs), is critical for activating host defences and proinflammatory cytokine production in response to Francisella infection. Here we show that the F. novicida protein FTN_0757 acts to repress BLP production, dampening TLR2 activation. The Δ FTN_0757 mutant strain induced robust TLR2-dependent cytokine production in macrophages compared with wild-type bacteria, and produced increased amounts of BLPs. The deletion of one BLP ( FTN_1103) from Δ FTN_0757 decreased the total BLP concentration to near wild-type level sand correlated with a decrease in the inductionof TLR2 signalling. The overproduction of BLPs also contributed to the in vivo attenuation of the Δ FTN_0757 mutant, which was significantly rescued when FTN_1103 was deleted. Taken together, these data reveal a novel mechanism of immune evasion by the downregulation of BLP expression to subvert TLR2 activation, which is likely used by numerous other intracellular bacterial pathogens. [ABSTRACT FROM AUTHOR]

Type six secretion systems (T6SSs) are found in many Gram-negative bacteria and are important for their virulence or their ecological competitiveness. The multicomponent T6SSs are responsible for the translocation of effector molecules into target eukaryotic or prokaryotic cells. The Francisella pathogenicity island encodes a putative T6SS that Francisella novicida requires for intramacrophage growth and virulence during infection of rodents. Here, we present the X-ray crystal structure of the conserved type six secretion component TssL (DotU) from F. novicida. The structure of this protein, which is referred to as Ftn_TssL, revealed an all-α-helical fold that is a unique fusion of two 3-helix bundles. The sequence of Ftn_TssL shows low identity to presumed homologs that are found in most T6SSs. The structure of Ftn_TssL, however, has allowed us to provide bioinformatics evidence that the F. novicida TssL has a fold that is very likely representative for TssL forms from both T6SSs and from the distantly related B subclass of type four secretion systems. A map of sequence conservation on the TssL structure revealed a surface-exposed groove that may represent a functional site on the protein. [ABSTRACT FROM AUTHOR]

Background: Francisella tularensis is an intracellular pathogen that causes tularemia in humans and the public health importance of this bacterium has been well documented in recent history. Francisella philomiragia, a distant relative of F. tularensis, is thought to constitute an environmental lineage along with Francisella novicida. Nevertheless, both F. philomiragia and F. novicida have been associated with human disease, primarily in immune-compromised individuals. To understand the genetic relationships and evolutionary contexts among different lineages within the genus Francisella, the genome of Francisella spp. strain TX07-7308 was sequenced and compared to the genomes of F. philomiragia strains ATCC 25017 and 25015, F. novicida strain U112, and F. tularensis strain Schu S4. Results: The size of strain ATCC 25017 chromosome was 2,045,775 bp and contained 1,983 protein-coding genes. The size of strain TX07-7308 chromosome was 2,035,931 bp and contained 1,980 protein-coding genes. Pairwise BLAST comparisons indicated that strains TX07-7308 and ATCC 25017 contained 1,700 protein coding genes in common. NUCmer analyses revealed that the chromosomes of strains TX07-7308 and ATCC 25017 were mostly collinear except for a few gaps, translocations, and/or inversions. Using the genome sequence data and comparative analyses with other members of the genus Francisella (e.g., F. novicida strain U112 and F. tularensis strain Schu S4), several strain-specific genes were identified. Strains TX07-7308 and ATCC 25017 contained an operon with six open reading frames encoding proteins related to enzymes involved in thiamine biosynthesis that was absent in F. novicida strain U112 and F. tularensis strain Schu S4. Strain ATCC 25017 contained an operon putatively involved in lactose metabolism that was absent in strain TX07-7308, F. novicida strain U112, and F. tularensis strain Schu S4. In contrast, strain TX07-7308 contained an operon putatively involved in glucuronate metabolism that was absent in the genomes of strain ATCC 25017, F. novicida strain U112, and F. tularensis strain Schu S4. The polymorphic nature of polysaccharide biosynthesis/modification gene clusters among different Francisella strains was also evident from genome analyses. Conclusions: From genome comparisons, it appeared that genes encoding novel functions have contributed to the metabolic enrichment of the environmental lineages within the genus Francisella. The inability to acquire new genes coupled with the loss of ancestral traits and the consequent reductive evolution may be a cause for, as well as an effect of, niche selection of F. tularensis. Sequencing and comparison of the genomes of more isolates are required to obtain further insights into the ecology and evolution of different species within the genus Francisella. [ABSTRACT FROM AUTHOR]

The RNA-binding protein Hfq is recognized as an important regulatory factor in a variety of cellular processes, including stress resistance and pathogenesis. Hfq has been shown in several bacteria to interact with small regulatory RNAs and act as a post-transcriptional regulator of mRNA stability and translation. Here we examined the impact of Hfq on growth, stress tolerance, and gene expression in the intracellular pathogen Francisella novicida. We present evidence of Hfq involvement in the ability of F. novicida to tolerate several cellular stresses, including heat-shock and oxidative stresses, and alterations in hfq gene expression under these conditions. Furthermore, expression of numerous genes, including several associated with virulence, is altered in a hfq mutant strain suggesting they are regulated directly or indirectly by Hfq. Strikingly, we observed a delayed entry into stationary phase and increased biofilm formation in the hfq mutant. Together, these data demonstrate a critical role for Hfq in F. novicida growth and survival. [ABSTRACT FROM AUTHOR]

[ABSTRACT FROM PUBLISHER]

Abstract: The ability of human beta-defensins hBD-1, hBD-2, and hBD-3 to exert direct in vitro antimicrobial effects was evaluated using Francisella tularensis Live Vaccine Strain (LVS) and Francisella novicida. While hBD-2 showed some antimicrobial activity in these assays, only hBD-3 demonstrated significant potency against Francisella. Francisella tularensis LVS infection induced elevated levels of hBD-2 mRNA in human airway epithelial (A549) cells, while having no significant impact on the levels of hBD-3 and only a moderate effect on the level of hBD-1 mRNA. Francisella infection avoided stimulating the production of the most potent anti-Francisella host peptide, hBD-3, in A549 cells, although hBD-3 is stimulated by other treatments. The differential induction of beta-defensins in Francisella infected lung epithelial cells suggests a complex dynamic in the expression of antimicrobial peptides and the innate immune response. [Copyright &y& Elsevier]

Francisella tularensis causes pulmonary tularemia and death in humans when left untreated. Here, using a novel aerosol infection model, we show that acute pulmonary Francisella novicida infection not only causes pneumonia and liver damage, but also induces dystrophic cardiac calcinosis (DCC) in BALB/c mice. C57BL/6 mice also develop pneumonia and hepatic damage, but fail to develop DCC. Development of DCC in BALB/c mice is associated with significant induction of RANKL but not osteopontin in their organs. Depletion of lung macrophages prior to infection markedly reduces pericarditis and calcification in BALB/c mice but does not increase their susceptibility to infection. [ABSTRACT FROM AUTHOR]

Francisella novicida (U112), a close relative of the highly virulent bacterium F. tularensis, is known to produce a lipopolysaccharide that is significantly different in biological properties from the LPS of F. tularensis. Here we present the results of the structural analysis of the F. novicida LPS core part, which is found to be similar to that of F. tularensis, differing only by one additional α-Glc residue:where R is an O-chain, linked via a β-bacillosamine (2,4-diamino-2,4,6-trideoxyglucose) residue. The lipid part of F. novicida LPS contains no phosphate substituent and apparently has a free reducing end, a feature also noted in F. tularensis LPS. [Copyright &y& Elsevier]

The article discusses the role of thermal changes in outer membrane remodelling of the intracellular pathogens, Francisella novicida, in response to a comment by Y. Li et al. It is shown that lipid fatty acid composition in Francisella changes at both low and high temperature within the genetic and enzymatic levels. The N-linked acyltransferases, lpxD1 and lpxD2, are important in this changes because of their ability to catalyze acylation and preserve Francisella.

In addition to defense against foreign DNA, the CRISPR-Cas9 system of Francisella novicida represses expression of an endogenous immunostimulatory lipoprotein. We investigated the specificity and molecular mechanism of this regulation, demonstrating that Cas9 controls a highly specific regulon of four genes that must be repressed for bacterial virulence. Regulation occurs through a protospacer adjacent motif (PAM)-dependent interaction of Cas9 with its endogenous DNA targets, dependent on a non-canonical small RNA (scaRNA) and tracrRNA. The limited complementarity between scaRNA and the endogenous DNA targets precludes cleavage, highlighting the evolution of scaRNA to repress transcription without lethally targeting the chromosome. We show that scaRNA can be reprogrammed to repress other genes, and with engineered, extended complementarity to an exogenous target, the repurposed scaRNA:tracrRNA-FnoCas9 machinery can also direct DNA cleavage. Natural Cas9 transcriptional interference likely represents a broad paradigm of regulatory functionality, which is potentially critical to the physiology of numerous Cas9-encoding pathogenic and commensal organisms. • FnoCas9 (Francisella novicida Cas9) uses scaRNA to bind endogenous DNA and repress transcription • The limited length of scaRNA:target complementarity prevents DNA cleavage • Cleavage-competent FnoCas9 uses distinct RNAs for repression versus cleavage • scaRNA can be reprogrammed to guide FnoCas9 to repress a new target Ratner et al. elucidate the mechanism of natural gene repression by Cas9, which is required for the virulence of Francisella novicida. Cas9 interferes with transcription by RNA-directed binding to DNA targets. Transcriptional repression may be a common feature of CRISPR-Cas9 systems that leads to diverse functions in bacterial physiology. [ABSTRACT FROM AUTHOR]