Quantification of the affinities of CRISPR-Cas9 nucleases for cognate protospacer adjacent motif (PAM) sequences.

The CRISPR/Cas9 nucleases have been widely applied for genome editing in various organisms. Cas9 nucleases complexed with a guide RNA (Cas9-gRNA) find their targets by scanning and interrogating the genomic DNA for sequences complementary to the gRNA. Recognition of the DNA target sequence requires a short protospacer adjacent motif (PAM) located outside this sequence. Given that the efficiency of target location may depend on the strength of interactions that promote target recognition, here we sought to compare affinities of different Cas9 nucleases for their cognate PAM sequences. To this end, we measured affinities of Cas9 nucleases from Streptococcus pyogenes, Staphylococcus aureus, and Francisella novicida complexed with guide RNAs (gRNAs) (SpCas9-gRNA, SaCas9-gRNA, and FnCas9-gRNA, respectively) and of three engineered SpCas9-gRNA variants with altered PAM specificities for short, PAM-containing DNA probes. We used a "beacon" assay that measures the relative affinities of DNA probes by determining their ability to competitively affect the rate of Cas9-gRNA binding to fluorescently labeled target DNA derivatives called "Cas9 beacons." We observed significant differences in the affinities for cognate PAM sequences among the studied Cas9 enzymes. The relative affinities of SpCas9-gRNA and its engineered variants for canonical and suboptimal PAMs correlated with previous findings on the efficiency of these PAM sequences in genome editing. These findings suggest that high affinity of a Cas9 nuclease for its cognate PAM promotes higher genome-editing efficiency.

Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity.

Cas12a, also known as Cpf1, is a type V-A CRISPR-Cas RNA-guided endonuclease that is used for genome editing based on its ability to generate specific dsDNA breaks. Here, we show cryo-EM structures of intermediates of the cleavage reaction, thus visualizing three protein regions that sense the crRNA-DNA hybrid assembly triggering the catalytic activation of Cas12a. Single-molecule FRET provides the thermodynamics and kinetics of the conformational activation leading to phosphodiester bond hydrolysis. These findings illustrate why Cas12a cuts its target DNA and unleashes unspecific cleavage activity, degrading ssDNA molecules after activation. In addition, we show that other crRNAs are able to displace the R-loop inside the protein after target DNA cleavage, terminating indiscriminate ssDNA degradation. We propose a model whereby the conformational activation of the enzyme results in indiscriminate ssDNA cleavage. The displacement of the R-loop by a new crRNA molecule will reset Cas12a specificity, targeting new DNAs.

Characterization of the outer membrane proteome of Francisella noatunensis subsp. orientalis.

AIMS: The aims of the current study were to characterize the outer membrane proteins (OMPs) of Francisella noatunensis subsp. orientalis (Fno) STIR-GUS-F2f7, and identify proteins recognized by sera from tilapia, Oreochromis niloticus, (L) that survived experimental challenge with Fno. METHODS AND RESULTS: The composition of the OMPs of a virulent strain of Fno (STIR-GUS-F2f7), isolated from diseased red Nile tilapia in the United Kingdom, was examined. The sarcosine-insoluble OMPs fraction was screened with tilapia hyperimmune sera by western blot analysis following separation of the proteins by 1D SDS-PAGE. Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) was used to identify the various proteins present in the OMP profile. Two hundred and thirty-nine proteins were identified, of which 44 were found in the immunogenic band recognized by the tilapia hyperimmune serum. In silico analysis was performed to predict the function and location of the OMPs identified by MS. CONCLUSIONS: Using a powerful proteomic-based approach in conjugation with western immunoblotting, proteins comprising the outer membrane fraction of Fno STIR-GUS-F2f7 were identified, catalogued and screened for immune recognition by tilapia sera. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study is the first report on the characterization of Fno-OMPs. The findings here provide preliminary data on bacterial surface proteins that exist in direct contact with the host's immune defences during infection and offer an insight into the pathogenesis of Fno.

Structural insights into the apo-structure of Cpf1 protein from Francisella novicida.

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 induced microparticulate caspase-1/gasdermin-D activation is regulated by NLRP3 independent of Pyrin.

Although the study of pathogen sensing by host defense systems continues to uncover a role for inflammasome components specific to particular pathogens, gaps remain in our knowledge. After internalization, Francisella escapes from the phagosome in mononuclear cells and is thought to be detected by intracellular pathogen-response-receptors pyrin and Aim2 in human and murine models, respectively. However, it remains controversial as to the role of pyrin in detecting Francisella. Our current work aims to study the contribution of inflammasome sensor, Pyrin in regulating microparticulate caspase-1/GSDM-D activation by Francisella. Our findings suggest that NLRP3 is central to the activation/release of active caspase-1/GSDM-D encapsulated in microparticles (MP) by Francisella. We also provide evidence that this regulation is independent of pyrin, implicated in sensing cytosolic Francisella in NLRP3-/- conditions where endogenous Pyrin is present. Absence of NLRP3 completely abrogated Francisella mediated MP caspase-1/GSDM-D activation and release both before and after internalization of the pathogen. However, deletion of pyrin not only enhanced both LPS and Francisella mediated MP active caspase-1/GSDM-D release, but pyrin overexpression resulted in a reduction of inflammasome activation and release; suggesting an inhibitory role of pyrin in LPS and Francisella mediated MP responses. This NLRP3 dependence and inhibitory effect of pyrin correlated with cytokine release as well. These observations also correlated with MPs ability to induce cell death; as LPS and Francisella-induced MPs from pyrin-deficient cells were more potent than wild-type monocytes whereas, NLRP3-/- MPs failed to induce cell death. Taken together, we report that NLPR3 not only mediates Francisella induced cytokine responses, but is also critical for cytokine-independent microparticle-induced inflammasome activation and endothelial cell injury independent of pyrin.

Assembly of Francisella novicida Cpf1 endonuclease in complex with guide RNA and target DNA.

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.

Effects of lipid A acyltransferases on the pathogenesis of F. novicida.

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 MSn 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.

Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems.

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.

Synthesis of zwitterionic 1,1'-glycosylphosphodiester: a partial structure of galactosamine-modified Francisella lipid A.

Synthesis of a "double glycosidic" phosphodiester comprising anomeric centers of two 2-amino-2-deoxy-sugars is reported. The carbohydrate epitope of Francisella lipid A modified with α-d-galactosamine at the anomerically linked phosphate has been stereoselectively prepared and coupled to maleimide-activated bovine serum albumin via an amide-linked thiol-terminated spacer group. H-Phosphonate and phosphoramidite approaches have been explored for the coupling of 4,6-DTBS-2-azido-protected GalN lactol and peracetylated spacer-equipped reducing ßGlcN(1→6)GlcN disaccharide via phosphodiester linkage. Deprotection conditions preserving the integrity of the labile glycosidic zwitterionic phosphodiester were elaborated.

A rapid one-step method for the characterization of membrane lipid remodeling in Francisella using matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry.

Lipids are essential components of all bacterial membranes. The most common membrane-associated lipids in Gram-negative bacteria are phospholipids and lipid A, the hydrophobic anchor of lipopolysaccharide. Diversity in these lipids arises through structural modifications that include changes in the length and location of fatty acids, and the addition of phosphate and carbohydrate moieties. Analysis of individual structural modifications normally requires large quantities of starting material and multiple methods for the isolation, hydrolysis, and analysis. In this study, we developed a novel one-step protocol for the combined isolation of phospholipids and lipid A from Francisella subspecies followed by analysis using matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry. The total time for lipid isolation and analysis was approximately 15 min and with a lower limit of detection of approximately 100 ng of purified lipid. This protocol identified the major lipid structures using both wild-type Ft subspecies strains and lipid A biosynthesis mutants. We also determined the relative levels of individual lipid A and phospholipids after growth under conditions that mimic the mammalian infection process. This analysis showed that the bacterial membranes remodeled rapidly to adapt to changes in environmental growth conditions and may be important for Francisella pathogenesis.

Characterization of the pathogenicity island protein PdpA and its role in the virulence of Francisella novicida.

Francisella tularensis is a highly virulent, intracellular pathogen that causes the disease tularaemia. A research surrogate for F. tularensis is Francisella novicida, which causes a tularaemia-like disease in mice, grows similarly in macrophages, and yet is unable to cause disease in humans. Both Francisella species contain a cluster of genes referred to as the Francisella pathogenicity island (FPI). Pathogenicity determinant protein A (PdpA), encoded by the pdpA gene, is located within the FPI and has been associated with the virulence of Francisella species. In this work we examined the properties of PdpA protein expression and localization as well as the phenotype of a F. novicida pdpA deletion mutant. Monoclonal antibody detection of PdpA showed that it is a soluble protein that is upregulated in iron-limiting conditions and undetectable in an mglA or mglB mutant background. Deletion of pdpA resulted in a strain that was highly attenuated for virulence in chicken embryos and mice.

Discovery of new biosynthetic pathways: the lipid A story.

The outer monolayer of the outer membrane of Gram-negative bacteria consists of the lipid A component of lipopolysaccharide (LPS), a glucosamine-based saccharolipid that is assembled on the inner surface of the inner membrane. The first six enzymes of the lipid A pathway are required for bacterial growth and are excellent targets for the development of new antibiotics. Following assembly, the ABC transporter MsbA flips nascent LPS to the periplasmic side of the inner membrane, whereupon additional transport proteins direct it to the outer surface of the outer membrane. Depending on the bacterium, various covalent modifications of the lipid A moiety may occur during the transit of LPS to the outer membrane. These extra-cytoplasmic modification enzymes are therefore useful as reporters for monitoring LPS trafficking. Because of its conserved structure in diverse Gram-negative pathogens, lipid A is recognized as foreign by the TLR4/MD2 receptor of the mammalian innate immune system, resulting in rapid macrophage activation and robust cytokine production.

New species in the genus Francisella (Gammaproteobacteria; Francisellaceae); Francisella piscicida sp. nov. isolated from cod (Gadus morhua).

A Francisella strain, GM2212, previously isolated from moribund farmed Atlantic cod (Gadus morhua) in Norway, is closely related to Francisella philomiragia among Francisella spp. according to its complete 16S rDNA, 16S-23S intergenic spacer, 23S rDNA, 23S-5S intergenic spacer, 5S rDNA, FopA, lipoprotein TUL4 (LpnA), malate dehydrogenase and hypothetical lipoprotein (LpnB) sequences. A comparison between GM2212 and the type strain of Francisella philomiragia were performed by DNA-DNA hybridization and fatty acid analysis. The DNA-DNA hybridization showed a 70% similarity. The fatty acid analysis showed only minor differences between the Francisella isolates. Due to the inconclusive result from the DNA-DNA hybridisation, major emphasis concerning the status of this isolate is made on previously published molecular, phenotypic and biochemical characters. All characteristics taken together support the establishment of GM2212 as a novel species, for which the name Francisella piscicida sp. nov. is proposed (=CNCM I-3511(T) = DSM 18777(T) = LMG registration number not yet available).

The structure and function of Francisella lipopolysaccharide.

A key factor in the biology of Francisella spp. is lipopolysaccharide (LPS). Francisella LPS has many unique structural properties and poorly activates proinflammatory responses due to its lack of interaction with toll-like receptor 4 (TLR4). The LPS of this organism can be modified by various carbohydrates including glucose, mannose and galactosamine, which affect various aspects of virulence. Spontaneously occurring colony variants of F. tularensis have altered LPS. This altered LPS may account for the novel phenotypes of these variants that include effects on susceptibility to killing by normal human serum, intracellular survival and animal virulence. Mutants devoid of O-antigen (directed mutants in O-antigen biosynthetic genes) show reduced intracellular survival and mouse virulence. Thus, this surface component is important in F. tularensis pathogenesis, and the inability of the LPS to alarm the immune system coupled with its frequent modification/alteration likely aid the success of this pathogen during human infection.

Characterization of lipid A acylation patterns in Francisella tularensis, Francisella novicida, and Francisella philomiragia using multiple-stage mass spectrometry and matrix-assisted laser desorption/ionization on an intermediate vacuum source linear ion trap.

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria. The lipid A region of LPS stimulates the immune system in a structure-dependent manner. We have previously identified the two major lipid A species from Francisella tularensis as asymmetric tetraacylated structures containing four long acyl chains (16 and 18 carbons) and a single phosphate group that is partially modified by galactosamine (Phillips, N. J.; Schilling B.; McLendon, M. K.; Apicella, M. A.; Gibson, B. W. Infect. Immun. 2004, 72, 5340-5348). In the current study, we used matrix-assisted laser desorption/ionization on an intermediate vacuum source (vMALDI) coupled to a linear ion trap (LIT) mass spectrometer in multiple-stage mass fragmentation mode (MSn) to determine the structures of several minor and low abundant lipid A species present in F. tularensis, Francisella novicida, and Francisella philomiragia LPS that have not been previously characterized. Comprehensive vMALDI-MSn fragmentation studies allowed us to deduce the composition and the position of the fatty acid substituents within the lipid A moieties. Unexpectedly, most of these minor lipid A species consisted of multiple isobaric species with acyl chains of various lengths. Moreover, we found that a small portion of these lipid A species may be modified by the addition of a hexose or hexosamine sugar, in addition to the galactosamine that was previously identified. Overall, we found that MSn analysis on the vMALDI-LIT-MS platform was highly efficient and sensitive, allowing for thorough analysis of very minor lipid A species.

Comparative proteome analysis of fractions enriched for membrane-associated proteins from Francisella tularensis subsp. tularensis and F. tularensis subsp. holarctica strains.

The facultative intracellular pathogen Francisella tularensis is the causative agent of the serious infectious disease tularemia. Despite intensive research, the virulence factors and pathogenetic mechanisms remain largely unknown. To identify novel putative virulence factors, we carried out a comparative proteome analysis of fractions enriched for membrane-associated proteins isolated from the highly virulent subspecies tularensis strain SCHU S4 and three representatives of subspecies holarctica of different virulence including the live vaccine strain. We identified six proteins uniquely expressed and four proteins expressed at significantly higher levels by SCHU S4 compared to the ssp. holarctica strains. Four other protein spots represented mass and charge variants and seven spots were charge variants of proteins occurring in the ssp. holarctica strains. The genes encoding proteins of particular interest were examined by sequencing in order to confirm and explain the findings of the proteome analysis. Our studies suggest that the subspecies tularensis-specific proteins represent novel potential virulence factors.

H2BC: a new technique for NMR analysis of complex carbohydrates.

It is demonstrated that the H2BC NMR pulse sequence (J. Am. Chem. Soc.2005, 127, 6154, Magn. Reson. Chem.2005, 43, 971-974) offers unambiguous assignments and significant simplification of NMR spectra of large and complex carbohydrates compared to other techniques for the establishment of correlations over more than one bond. H2BC almost exclusively correlates protons and proton-bearing carbon spins separated by two covalent bonds and is independent of occasionally vanishing (2)J(CH) coupling constants, which alleviates the problem of missing two-bond correlations in HMBC spectra. H2BC also solves the problem of distinguishing two- and three-bond correlations in HSQC-TOCSY or HMBC. It is a further asset of H2BC that the experiment is significantly shorter than HMBC and HSQC-TOCSY, and hence less sensitive to transverse relaxation. The H2BC experiment is demonstrated on an approximately 30-residue oligosaccharide from Francisella victoria.

Characterization of the lipopolysaccharide O-antigen of Francisella novicida (U112).

Francisella novicida (U112), a close relative of the highly virulent bacterium F. tularensis, was shown to produce a lipopolysaccharide in which the antigenic O-polysaccharide component was found by chemical, 1H and 13C NMR and MS analyses to be an unbranched neutral linear polymer of a repeating tetrasaccharide unit composed of 2-acetamido-2-deoxy-D-galacturonamide (D-GalNAcAN) and 2,4-diacetamido-2,4,6-trideoxy-D-glucose (D-Qui2NAc4NAc, di-N-acetylbacillosamine) residues (3:1) and had the structure: -->4)-alpha-D-GalNAcAN-(1-->4)-alpha-D-GalNAcAN-(1-->4)-alpha-D-GalNAcAN-(1-->3)-alpha-D-QuiNAc4NAc-(1-->. With polyclonal murine antibody, the F. novicida O-antigen did not show serological cross-reactivity with the O-antigen of F. tularensis despite the occurrence of a common -->4)-D-GalpNAcAN-(1-->4)-alpha-D-GalpNAcAN-(1--> disaccharide unit in their respective O-antigens. Thus, O-PS serology offers a practical way to distinguish between the two Francisella species.