Publications

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human caspase-4 detects tetra-acylated lps and cytosolic francisella and functions differently from murine caspase-11.caspase-4/5 in humans and caspase-11 in mice bind hexa-acylated lipid a, the lipid moeity of lipopolysaccharide (lps), to induce the activation of non-canonical inflammasome. pathogens such as francisella novicida express an under-acylated lipid a and escape caspase-11 recognition in mice. here, we show that caspase-4 drives inflammasome responses to f. novicida infection in human macrophages. caspase-4 triggers f. novicida-mediated, gasdermin d-dependent pyroptosis and activates the nlrp3 infla ...201829339744
establishing rna virus resistance in plants by harnessing crispr immune system.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 ...201829327438
detecting release of bacterial dsdna into the host cytosol using fluorescence microscopy.recognition of pathogens by the innate immune system relies on germline-encoded pattern recognition receptors (prrs) that recognize unique microbial molecules, so-called pathogen-associated molecular patterns (pamps). nucleic acids and their derivatives are one of the most important groups of pamps, and are recognized by a number of surface-associated as well as cytosolic prrs. cyclic gmp-amp synthase (cgas) recognizes the presence of pathogen- or host-derived dsdna in the cytosol and initiates ...201829177864
zinc acquisition mechanisms differ between environmental and virulent francisella species.zinc is an essential nutrient for bacterial growth. because host cells can restrict pathogen access to zinc as an antimicrobial defense mechanism, intracellular pathogens such as francisella must sense their environment and acquire zinc in response. in many bacteria, the conserved transcription factor zur is a key regulator of zinc acquisition. to identify mechanisms of zinc uptake in francisella novicida u112, transcriptome sequencing of wild-type and putative zur mutant bacteria was performed. ...201829109188
fncpf1: a novel and efficient genome editing tool for saccharomyces cerevisiae.cpf1 is a new class ii family of crispr-cas rna-programmable endonucleases with unique features that make it a very attractive alternative or complement to cas9 for genome engineering. using constitutively expressed cpf1 from francisella novicida, the present study demonstrates that fncpf1 can mediate rna-guided dna cleavage at targeted genomic loci in the popular model and industrial yeast saccharomyces cerevisiae. fncpf1 very efficiently and precisely promoted repair dna recombination with eff ...201729106617
silkworm model for francisella novicida infection.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, ...201729066381
the metabolic enzyme fructose-1,6-bisphosphate aldolase acts as a transcriptional regulator in pathogenic francisella.the enzyme fructose-bisphosphate aldolase occupies a central position in glycolysis and gluconeogenesis pathways. beyond its housekeeping role in metabolism, fructose-bisphosphate aldolase has been involved in additional functions and is considered as a potential target for drug development against pathogenic bacteria. here, we address the role of fructose-bisphosphate aldolase in the bacterial pathogen francisella novicida. we demonstrate that fructose-bisphosphate aldolase is important for bac ...201729021545
structure of the conserved francisella virulence protein fvfa.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 o ...201728994410
ifn-γ extends the immune functions of guanylate binding proteins to inflammasome-independent antibacterial activities during francisella novicida infection.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 ...201728968459
precise insertion and guided editing of higher plant genomes using cpf1 crispr nucleases.precise genome editing of plants has the potential to reshape global agriculture through the targeted engineering of endogenous pathways or the introduction of new traits. to develop a crispr nuclease-based platform that would enable higher efficiencies of precise gene insertion or replacement, we screened the cpf1 nucleases from francisella novicida and lachnospiraceae bacterium nd2006 for their capability to induce targeted gene insertion via homology directed repair. both nucleases, in the pr ...201728912524
crispr/cpf1 enables fast and simple genome editing of saccharomyces cerevisiae.cpf1 represents a novel single rna-guided crispr/cas endonuclease system suitable for genome editing with distinct features compared with cas9. we demonstrate the functionality of three cpf1 orthologues - acidaminococcus spp. bv3l6 (ascpf1), lachnospiraceae bacterium nd2006 (lbcpf1) and francisella novicida u112 (fncpf1) - for genome editing of saccharomyces cerevisiae. these cpf1-based systems enable fast and reliable introduction of donor dna on the genome using a two-plasmid-based editing app ...201728886218
shared features of cryptic plasmids from environmental and pathogenic francisella species.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 b ...201728837612
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 ...201728695850
structure of the cpf1 endonuclease r-loop complex after target dna cleavage.cpf1 is an rna-guided endonuclease that is emerging as a powerful genome-editing tool. here we provide insight into its dna-targeting mechanism by determining the structure of francisella novicida cpf1 with the triple-stranded r-loop generated after dna cleavage. the structure reveals the machinery involved in dna unwinding to form a crispr rna (crrna)-dna hybrid and a displaced dna strand. the protospacer adjacent motif (pam) is recognized by the pam-interacting domain. the loop-lysine helix-lo ...201728562584
structural basis for guide rna processing and seed-dependent dna targeting by crispr-cas12a.the crispr-associated protein cas12a (cpf1), which has been repurposed for genome editing, possesses two distinct nuclease activities: endoribonuclease activity for processing its own guide rnas and rna-guided dnase activity for target dna cleavage. to elucidate the molecular basis of both activities, we determined crystal structures of francisella novicida cas12a bound to guide rna and in complex with an r-loop formed by a non-cleavable guide rna precursor and a full-length target dna. corrobor ...201728431230
targeted activation of diverse crispr-cas systems for mammalian genome editing via proximal crispr targeting.bacterial crispr-cas systems comprise diverse effector endonucleases with different targeting ranges, specificities and enzymatic properties, but many of them are inactive in mammalian cells and are thus precluded from genome-editing applications. here we show that the type ii-b fncas9 from francisella novicida possesses novel properties, but its nuclease function is frequently inhibited at many genomic loci in living human cells. moreover, we develop a proximal crispr (termed proxy-crispr) targ ...201728387220
functional characterization of the dna gyrases in fluoroquinolone-resistant mutants of francisella novicida.fluoroquinolone (fq) resistance is a major health concern in the treatment of tularemia. because dna gyrase has been described as the main target of these compounds, our aim was to clarify the contributions of both gyra and gyrb mutations found in francisella novicida clones highly resistant to fqs. wild-type and mutated gyra and gyrb subunits were overexpressed so that the in vitro fq sensitivity of functional reconstituted complexes could be evaluated. the data obtained were compared to the mi ...201728167561
nkg2d promotes b1a cell development and protection against bacterial infection.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 ...201728087665
host-based lipid inflammation drives pathogenesis in francisella infection.mass spectrometry imaging (msi) was used to elucidate host lipids involved in the inflammatory signaling pathway generated at the host-pathogen interface during a septic bacterial infection. using francisella novicida as a model organism, a bacterial lipid virulence factor (endotoxin) was imaged and identified along with host phospholipids involved in the splenic response in murine tissues. here, we demonstrate detection and distribution of endotoxin in a lethal murine f. novicida infection mode ...201729109289
mass spectrometry analysis of intact francisella bacteria identifies lipid a structure remodeling in response to acidic ph stress.structural modification of lipid a, the lipid anchor of lps, is one of the strategies used by gram-negative bacteria to evade host innate immunity. francisella tularensis is a human pathogen that infects and replicates within phagocytic cells. it produces an atypical lipid a, whose structure precludes an efficient recognition by both innate immune players, tlr4 and cationic antimicrobial peptides. interestingly, a recent report indicates that the lipid a of francisella (lvs vaccinal strain) unde ...201728807561
differential substrate usage and metabolic fluxes in francisella tularensis subspecies holarctica and francisella novicida.francisella tularensis is an intracellular pathogen for many animals causing the infectious disease, tularemia. whereas f. tularensis subsp. holarctica is highly pathogenic for humans, f. novicida is almost avirulent for humans, but virulent for mice. in order to compare metabolic fluxes between these strains, we performed (13)c-labeling experiments with f. tularensis subsp. holarctica wild type (beaver isolate), f. tularensis subsp. holarctica strain lvs, or f. novicida strain u112 in complex m ...201728680859
glycosylation of a capsule-like complex (clc) by francisella novicida is required for virulence and partial protective immunity in mice.francisella tularensis is a gram-negative bacterium and the etiologic agent of tularemia. f. tularensis may appear encapsulated when examined by transmission electron microscopy (tem), which is due to production of an extracellular capsule-like complex (clc) when the bacterium is grown under specific environmental conditions. deletion of two glycosylation genes in the live vaccine strain (lvs) results in loss of apparent clc and attenuation of lvs in mice. in contrast, f. novicida, which is also ...201728611741
francisella novicida inhibits spontaneous apoptosis and extends human neutrophil lifespan.francisella novicida is a gram-negative bacterium that is closely related to the highly virulent facultative intracellular pathogen, francisella tularensis data published by us and others demonstrate that f. tularensis virulence correlates directly with its ability to impair constitutive apoptosis and extend human neutrophil lifespan. in contrast, f. novicida is attenuated in humans, and the mechanisms that account for this are incompletely defined. our published data demonstrate that f. novicid ...201728550119
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 ...201728478203
crispr-cpf1 assisted genome editing of corynebacterium glutamicum.corynebacterium glutamicum is an important industrial metabolite producer that is difficult to genetically engineer. although the streptococcus pyogenes (sp) crispr-cas9 system has been adapted for genome editing of multiple bacteria, it cannot be introduced into c. glutamicum. here we report a francisella novicida (fn) crispr-cpf1-based genome-editing method for c. glutamicum. crispr-cpf1, combined with single-stranded dna (ssdna) recombineering, precisely introduces small changes into the bact ...201728469274
identification of conserved abc importers necessary for intracellular survival of legionella pneumophila in multiple hosts.it is established that the human pathogen legionella pneumophila becomes significantly augmented for infection of macrophages after intracellular growth in amoebae when compared to like-strains cultivated in laboratory media. based on this observation, we reasoned that the most critical virulence determinants of l.p. are expressed by responding to stimuli generated by the protozoan host specifically; a process we term "protozoan-priming." we sought to identify l.p. virulence factors that were re ...201729250489
interferon-inducible guanylate-binding proteins at the interface of cell-autonomous immunity and inflammasome activation.guanylate-binding proteins (gbps) are essential components of cell-autonomous immunity. in response to ifn signaling, gbps are expressed in the cytoplasm of immune and nonimmune cells, where they unleash their antimicrobial activity toward intracellular bacteria, viruses, and parasites. recent studies have revealed that gbps are essential for mediating activation of the caspase-1 inflammasome in response to the gram-negative bacteria salmonella enterica serovar typhimurium, francisella novicida, ...201727418355
whole-genome relationships among francisella bacteria of diverse origins define new species and provide specific regions for detection.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 ...201727881415
advances with using crispr/cas-mediated gene editing to treat infections with hepatitis b virus and hepatitis c virus.chronic infections with hepatitis b and hepatitis c viruses (hbv and hcv) account for the majority of cases of cirrhosis and hepatocellular carcinoma. current therapies for the infections have limitations and improved efficacy is necessary to prevent complications in carriers of the viruses. in the case of hbv persistence, the replication intermediate comprising covalently closed circular dna (cccdna) is particularly problematic. licensed therapies have little effect on cccdna and hbv replicatio ...201728087399
enhanced survival but not amplification of francisella spp. in the presence of free-living amoebae.transmission of francisella tularensis, the etiologic agent of tularemia, has been associated with various water sources. survival of many waterborne pathogens within free-living amoeba (fla) is well documented; however, the role of amoebae in the environmental persistence of f. tularensis is unclear. in this study, axenic fla cultures of acanthamoeba castellanii, acanthamoeba polyphaga, and vermamoeba vermiformis were each inoculated with virulent strains of f. tularensis (types a and b), the a ...201727929353
differential roles of caspase-1 and caspase-11 in infection and inflammation.caspase-1, also known as interleukin-1β (il-1β)-converting enzyme (ice), regulates antimicrobial host defense, tissue repair, tumorigenesis, metabolism and membrane biogenesis. on activation within an inflammasome complex, caspase-1 induces pyroptosis and converts pro-il-1β and pro-il-18 into their biologically active forms. "ice(-/-)" or "casp1(-/-)" mice generated using 129 embryonic stem cells carry a 129-associated inactivating passenger mutation on the caspase-11 locus, essentially making t ...201728345580
murine survival of infection with francisella novicida and protection against secondary challenge is critically dependent on b lymphocytes.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 us ...201727965147
francisella inflammasomes: integrated responses to a cytosolic stealth bacterium.francisella tularensis is a facultative intracellular bacterium causing tularemia, a zoonotic disease. francisella replicates in the macrophage cytosol and eventually triggers cytosolic immune responses. in murine macrophages, francisella novicida and francisella tularensis live vaccine strain lyse in the host cytosol and activate the cytosolic dna receptor aim2. here, we review the mechanisms leading or contributing to aim2 inflammasome activation, including the role of tlrs and of ifn signalin ...201627460813
reclassification of wolbachia persica as francisella persica comb. nov. and emended description of the family francisellaceae.the taxonomic status of the bacterium wolbachia persica is described, and based on the evidence presented, transfer of this species to the genus francisella as francisella persica comb. nov. is proposed. this reclassification is supported by data generated from genomic comparisons of w. persica atcc vr-331t ( = fsc845t = dsm 101678t) to other near neighbours, including francisella tularensis subsp. novicida. the full-length 16s rrna gene sequence of strain atcc vr-331t had 98.5 % nucleotide iden ...201626747442
morphological analysis of francisella novicida epithelial cell infections in the absence of functional fipa.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 formatio ...201626239909
the bacterial microbiome of dermacentor andersoni ticks influences pathogen susceptibility.ticks are of medical importance owing to their ability to transmit pathogens to humans and animals. the rocky mountain wood tick, dermacentor andersoni, is a vector of a number of pathogens, including anaplasma marginale, which is the most widespread tick-borne pathogen of livestock. although ticks host pathogenic bacteria, they also harbor bacterial endosymbionts that have a role in tick physiology, survival, as well as pathogen acquisition and transmission. the goal of this study was to charac ...201626882265
cpf1 is a versatile tool for crispr genome editing across diverse species of cyanobacteria.cyanobacteria are the ideal organisms for the production of a wide range of bioproducts as they can convert co2 directly into the desired end product using solar energy. unfortunately, the engineering of cyanobacteria to create efficient cell factories has been impaired by the cumbersome genetic tools that are currently available for these organisms; especially when trying to accumulate multiple modifications. we sought to construct an efficient and precise tool for generating numerous markerles ...201628000776
the power of crispr-cas9-induced genome editing to speed up plant breeding.genome editing with engineered nucleases enabling site-directed sequence modifications bears a great potential for advanced plant breeding and crop protection. remarkably, the rna-guided endonuclease technology (rgen) based on the clustered regularly interspaced short palindromic repeats (crispr) and crispr-associated protein 9 (cas9) is an extremely powerful and easy tool that revolutionizes both basic research and plant breeding. here, we review the major technical advances and recent applicat ...201628097123
gene editing approaches against viral infections and strategy to prevent occurrence of viral escape. 201627930735
efficient targeted mutagenesis of rice and tobacco genomes using cpf1 from francisella novicida.crispr/cas9 systems are nowadays applied extensively to effect genome editing in various organisms including plants. crispr from prevotella and francisella 1 (cpf1) is a newly characterized rna-guided endonuclease that has two distinct features as compared to cas9. first, cpf1 utilizes a thymidine-rich protospacer adjacent motif (pam) while cas9 prefers a guanidine-rich pam. cpf1 could be used as a sequence-specific nuclease to target at-rich regions of a genome that cas9 had difficulty accessin ...201627905529
patterns of crispr/cas9 activity in plants, animals and microbes.the crispr/cas9 system and related rna-guided endonucleases can introduce double-strand breaks (dsbs) at specific sites in the genome, allowing the generation of targeted mutations in one or more genes as well as more complex genomic rearrangements. modifications of the canonical crispr/cas9 system from streptococcus pyogenes and the introduction of related systems from other bacteria have increased the diversity of genomic sites that can be targeted, providing greater control over the resolutio ...201627614091
cpf1 nucleases demonstrate robust activity to induce dna modification by exploiting homology directed repair pathways in mammalian cells.cpf1 nucleases have recently been repurposed for site-specific genome modification. two members of the cpf1 family, the ascpf1 from acidaminococcus sp. and the lbcpf1 from lachnospiraceae bacterium were shown to induce higher indel frequencies than spcas9 when examining four randomly-selected target sequences for each type of nuclease. whether they are a real match for cas9 nucleases, however, remains to be verified.201627630115
proteomic discovery of host kinase signaling in bacterial infections.protein phosphorylation catalyzed by protein kinases acts as a reversible molecular switch in signal transduction, providing a mechanism for the control of protein function in cellular processes. during microbial infection, cellular signaling essentially contributes to immune control to restrict the dissemination of invading pathogens within the host organism. however, pathogenic microbes compete for the control of host signaling to create a beneficial environment for successful invasion and inf ...201627440122
ge-crispr - an integrated pipeline for the prediction and analysis of sgrnas genome editing efficiency for crispr/cas system.genome editing by sgrna a component of crispr/cas system emerged as a preferred technology for genome editing in recent years. however, activity and stability of sgrna in genome targeting is greatly influenced by its sequence features. in this endeavor, a few prediction tools have been developed to design effective sgrnas but these methods have their own limitations. therefore, we have developed "ge-crispr" using high throughput data for the prediction and analysis of sgrnas genome editing effic ...201627581337
imipenem represses crispr-cas interference of dna acquisition through h-ns stimulation in klebsiella pneumoniae.analysis of the genome of klebsiella pneumoniae ntuh-k2044 strain revealed the presence of two clustered regularly interspaced short palindromic repeats (crispr) arrays separated with crispr-associated (cas) genes. carbapenem-resistant k. pneumoniae isolates were observed to be less likely to have crispr-cas than sensitive strains (5/85 vs. 22/132). removal of the transcriptional repressor, h-ns, was shown to prevent the transformation of plasmids carrying a spacer and putative proto-spacer adja ...201627531594
zbp1/dai is an innate sensor of influenza virus triggering the nlrp3 inflammasome and programmed cell death pathways.the interferon-inducible protein z-dna binding protein 1 (zbp1, also known as dna-dependent activator of ifn-regulatory factors (dai) and dlm-1) was identified as a dsdna sensor, which instigates innate immune responses. however, this classification has been disputed and whether zbp1 functions as a pathogen sensor during an infection has remained unknown. herein, we demonstrated zbp1-mediated sensing of the influenza a virus (iav) proteins np and pb1, triggering cell death and inflammatory respo ...201627917412
crispr system acquisition and evolution of an obligate intracellular chlamydia-related bacterium.recently, a new chlamydia-related organism, protochlamydia naegleriophila knic, was discovered within a naegleria amoeba. to decipher the mechanisms at play in the modeling of genomes from the protochlamydia genus, we sequenced the full genome of pr. naegleriophila, which includes a 2,885,090 bp chromosome and a 145,285 bp megaplasmid. for the first time within the chlamydiales order, we describe the presence of a clustered regularly interspaced short palindromic repeats (crispr) system, the imm ...201627516530
functional definition of bira suggests a biotin utilization pathway in the zoonotic pathogen streptococcus suis.biotin protein ligase is universal in three domains of life. the paradigm version of bpl is the escherichia coli bira that is also a repressor for the biotin biosynthesis pathway. streptococcus suis, a leading bacterial agent for swine diseases, seems to be an increasingly-important opportunistic human pathogen. unlike the scenario in e. coli, s. suis lacks the de novo biotin biosynthesis pathway. in contrast, it retains a bioy, a biotin transporter-encoding gene, indicating an alternative survi ...201627217336
recent advances in genome editing using crispr/cas9.the crispr (clustered regularly interspaced short palindromic repeat)-cas9 (crispr-associated nuclease 9) system is a versatile tool for genome engineering that uses a guide rna (grna) to target cas9 to a specific sequence. this simple rna-guided genome-editing technology has become a revolutionary tool in biology and has many innovative applications in different fields. in this review, we briefly introduce the cas9-mediated genome-editing method, summarize the recent advances in crispr/cas9 tec ...201627252719
superinfection exclusion of the ruminant pathogen anaplasma marginale in its tick vector is dependent on the time between exposures to the strains.the remarkable genetic diversity of vector-borne pathogens allows for the establishment of superinfection in the mammalian host. to have a long-term impact on population strain structure, the introduced strains must also be transmitted by a vector population that has been exposed to the existing primary strain. the sequential exposure of the vector to multiple strains frequently prevents establishment of the second strain, a phenomenon termed superinfection exclusion. as a consequence, superinfe ...201626994084
crystal structure of cpf1 in complex with guide rna and target dna.cpf1 is an rna-guided endonuclease of a type v crispr-cas system that has been recently harnessed for genome editing. here, we report the crystal structure of acidaminococcus sp. cpf1 (ascpf1) in complex with the guide rna and its target dna at 2.8 å resolution. ascpf1 adopts a bilobed architecture, with the rna-dna heteroduplex bound inside the central channel. the structural comparison of ascpf1 with cas9, a type ii crispr-cas nuclease, reveals both striking similarity and major differences, t ...201627114038
interferon-induced guanylate-binding proteins in inflammasome activation and host defense.traditional views of the inflammasome highlight the assembly of pre-existing core components shortly after infection or tissue damage. emerging work, however, suggests that the inflammasome machinery is also subject to 'tunable' or inducible signals that might accelerate its autocatalytic properties and dictate where inflammasome assembly takes place in the cell. many of these signals operate downstream of interferon receptors to elicit inflammasome regulators, including a new family of interfer ...201627092805
human gbp1 does not localize to pathogen vacuoles but restricts toxoplasma gondii.guanylate binding proteins (gbps) are a family of large interferon-inducible gtpases that are transcriptionally upregulated upon infection with intracellular pathogens. murine gbps (mgbps) including mgbp1 and 2 localize to and disrupt pathogen-containing vacuoles (pvs) resulting in the cell-autonomous clearing or innate immune detection of pv-resident pathogens. human gbps (hgbps) are known to exert antiviral host defense and activate the nlrp3 inflammasome, but it is unclear whether hgbps can d ...201626874079
the role of airway macrophages in apoptotic cell clearance following acute and chronic lung inflammation.acute and chronic inflammatory responses in the lung are associated with the accumulation of large quantities of immune and structural cells undergoing apoptosis, which need to be engulfed by phagocytes in a process called 'efferocytosis'. apoptotic cell recognition and removal from the lung is mediated predominantly by airway macrophages, though immature dendritic cells and non-professional phagocytes, such as epithelial cells and mesenchymal cells, can also display this function. efficient cle ...201626957481
salmonella suppresses the trif-dependent type i interferon response in macrophages.salmonella enterica is an intracellular pathogen that causes diseases ranging from gastroenteritis to typhoid fever. salmonella bacteria trigger an autophagic response in host cells upon infection but have evolved mechanisms for suppressing this response, thereby enhancing intracellular survival. we recently reported that s. enterica serovar typhimurium actively recruits the host tyrosine kinase focal adhesion kinase (fak) to the surface of the salmonella-containing vacuole (scv) (k. a. owen et ...201626884434
structure and engineering of francisella novicida cas9.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 ...201626875867
quantitative proteomic analysis of shigella flexneri and shigella sonnei generalized modules for membrane antigens (gmma) reveals highly pure preparations.outer membrane blebs are naturally shed by gram-negative bacteria and are candidates of interest for vaccines development. genetic modification of bacteria to induce hyperblebbing greatly increases the yield of blebs, called generalized modules for membrane antigens (gmma). the composition of the gmma from hyperblebbing mutants of shigella flexneri 2a and shigella sonnei were quantitatively analyzed using high-sensitivity mass spectrometry with the label-free ibaq procedure and compared to the c ...201626746581
modification of the 1-phosphate group during biosynthesis of capnocytophaga canimorsus lipid a.capnocytophaga canimorsus, a commensal bacterium of dog's mouth flora causing severe infections in humans after dog bites or scratches, has a lipopolysaccharide (lps) (endotoxin) with low-inflammatory lipid a. in particular, it contains a phosphoethanolamine (p-etn) instead of a free phosphate group at the c-1 position of the lipid a backbone, usually present in highly toxic enterobacterial gram-negative lipid a. here we show that the c. canimorsus genome comprises a single operon encoding a lip ...201626644381
a crispr/cas9 and cre/lox system-based express vaccine development strategy against re-emerging pseudorabies virus.virus evolves rapidly to escape vaccine-induced immunity, posing a desperate demand for efficient vaccine development biotechnologies. here we present an express vaccine development strategy based on crispr/cas9 and cre/lox system against re-emerging pseudorabies virus, which caused the recent devastating swine pseudorabies outbreak in china. by crispr/cas9 system, the virulent genes of the newly isolated strain were simultaneously substituted by marker genes, which were subsequently excised usi ...201626777545
crystal structure and activity of francisella novicida udp-n-acetylglucosamine acyltransferase.the first step of lipid a biosynthesis in escherichia coli (e. coli) is catalyzed by lpxa (eclpxa), an acyltransferase selective for udp-n-acetylglucosamine (udp-glcnac) and r-3-hydroxymyristoyl-acyl carrier protein (3-oh-c14-acp), and is an essential step in majority of gram-negative bacteria. since the majority of lipid a species isolated from f. novicida contains 3-oh-c16 or 3-oh-c18 at its c3 and c3' positions, fnlpxa was thought to be selective for longer acyl chain (3-oh-c16 and 3-oh-c18) ...201627545601
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 pa ...201627041224
regulation of lysosomal dynamics and autophagy by ctsb/cathepsin b.cysteine cathepsins are responsible for driving proteolytic degradation within the lysosome and in the extralysosomal milieu. they also have an integral role in autophagy, antigen presentation, cellular stress signaling, metabolism and lysosome-dependent cell death. here, we discuss our findings on the role of ctsb (cathepsin b), a member of the cysteine cathepsin family, in regulating the bioavailability of lysosomes and autophagosomes and consider how this regulatory response influences host s ...201627786577
irgb10 liberates bacterial ligands for sensing by the aim2 and caspase-11-nlrp3 inflammasomes.the inflammasome is an intracellular signaling complex, which on recognition of pathogens and physiological aberration, drives activation of caspase-1, pyroptosis, and the release of the pro-inflammatory cytokines il-1β and il-18. bacterial ligands must secure entry into the cytoplasm to activate inflammasomes; however, the mechanisms by which concealed ligands are liberated in the cytoplasm have remained unclear. here, we showed that the interferon-inducible protein irgb10 is essential for acti ...201627693356
norharmane matrix enhances detection of endotoxin by maldi-ms for simultaneous profiling of pathogen, host, and vector systems.the discovery of novel pathogenic mechanisms engaged during bacterial infections requires the evolution of advanced techniques. here, we evaluate the dual polarity matrix norharmane (nrm) to improve detection of bacterial lipid a (endotoxin), from host and vector tissues infected with francisella novicida (fn). we evaluated nrm for improved detection and characterization of a wide range of lipids in both positive and negative polarities, including lipid a and phospholipids across a range of matr ...201627650574
rapid lipid a structure determination via surface acoustic wave nebulization and hierarchical tandem mass spectrometry algorithm.surface acoustic wave nebulization (sawn) is an easy to use sample transfer method for rapid mass spectrometric analysis. a new standing wave (sw) sawn chip, with higher ionization efficiency than our previously reported design, is used for rapid analysis of lipids.201627582344
cathepsin b modulates lysosomal biogenesis and host defense against francisella novicida infection.lysosomal cathepsins regulate an exquisite range of biological functions, and their deregulation is associated with inflammatory, metabolic, and degenerative diseases in humans. in this study, we identified a key cell-intrinsic role for cathepsin b as a negative feedback regulator of lysosomal biogenesis and autophagy. mice and macrophages lacking cathepsin b activity had increased resistance to the cytosolic bacterial pathogen francisella novicida genetic deletion or pharmacological inhibition ...201627551156
the crispr-associated dna-cleaving enzyme cpf1 also processes precursor crispr rna.crispr-cas systems that provide defence against mobile genetic elements in bacteria and archaea have evolved a variety of mechanisms to target and cleave rna or dna. the well-studied types i, ii and iii utilize a set of distinct crispr-associated (cas) proteins for production of mature crispr rnas (crrnas) and interference with invading nucleic acids. in types i and iii, cas6 or cas5d cleaves precursor crrna (pre-crrna) and the mature crrnas then guide a complex of cas proteins (cascade-cas3, ty ...201627096362
crispr-mediated epigenome editing.mounting evidence has called into question our understanding of the role that the central dogma of molecular biology plays in human pathology. the conventional view that elucidating the mechanisms for translating genes into proteins can account for a panoply of diseases has proven incomplete. landmark studies point to epigenetics as a missing piece of the puzzle. however, technological limitations have hindered the study of specific roles for histone post-translational modifications, dna modific ...201628018139
the bacteriophage carrier state of campylobacter jejuni features changes in host non-coding rnas and the acquisition of new host-derived crispr spacer sequences.incorporation of self-derived crispr dna protospacers in campylobacter jejuni pt14 occurs in the presence of bacteriophages encoding a crispr-like cas4 protein. this phenomenon was evident in carrier state infections where both bacteriophages and host are maintained for seemingly indefinite periods as stable populations following serial passage. carrier state cultures of c. jejuni pt14 have greater aerotolerance in nutrient limited conditions, and may have arisen as an evolutionary response to s ...201627047470
crispr-cas: biology, mechanisms and relevance.prokaryotes have evolved several defence mechanisms to protect themselves from viral predators. clustered regularly interspaced short palindromic repeats (crispr) and their associated proteins (cas) display a prokaryotic adaptive immune system that memorizes previous infections by integrating short sequences of invading genomes-termed spacers-into the crispr locus. the spacers interspaced with repeats are expressed as small guide crispr rnas (crrnas) that are employed by cas proteins to target i ...201627672148
survival and evolution of crispr-cas system in prokaryotes and its applications.prokaryotes have developed numerous innate immune mechanisms in order to fend off bacteriophage or plasmid attack. one of these immune systems is clustered regularly interspaced short palindromic repeats (crispr). crispr-associated proteins play a key role in survival of prokaryotes against invaders, as these systems cleave dna of foreign genetic elements. beyond providing immunity, these systems have significant impact in altering the bacterial physiology in term of its virulence and pathogenic ...201627725818
an approach for predicting essential genes using multiple homology mapping and machine learning algorithms.investigation of essential genes is significant to comprehend the minimal gene sets of cell and discover potential drug targets. in this study, a novel approach based on multiple homology mapping and machine learning method was introduced to predict essential genes. we focused on 25 bacteria which have characterized essential genes. the predictions yielded the highest area under receiver operating characteristic (roc) curve (auc) of 0.9716 through tenfold cross-validation test. proper features w ...201627660763
drug target identification and prioritization for treatment of ovine foot rot: an in silico approach.ovine foot rot is an infection of the feet of sheep, mainly caused by dichelobacter nodosus. in its virulent form, it is highly contagious and debilitating, causing significant losses in the form of decline in wool growth and quality and poor fertility. current methods of treatment are ineffective in complete eradication. effective antibiotic treatment of foot rot is hence necessary to ensure better outcomes during control phases by reduction in culling count and the possibility of carriers of t ...201627379247
comparative genomic analysis of mannheimia haemolytica from bovine sources.bovine respiratory disease is a common health problem in beef production. the primary bacterial agent involved, mannheimia haemolytica, is a target for antimicrobial therapy and at risk for associated antimicrobial resistance development. the role of m. haemolytica in pathogenesis is linked to serotype with serotypes 1 (s1) and 6 (s6) isolated from pneumonic lesions and serotype 2 (s2) found in the upper respiratory tract of healthy animals. here, we sequenced the genomes of 11 strains of m. hae ...201626926339
temperature sensitivity conferred by liga alleles from psychrophilic bacteria upon substitution in mesophilic bacteria and a yeast species.we have assembled a collection of 13 psychrophilic liga alleles that can serve as genetic elements for engineering mesophiles to a temperature-sensitive (ts) phenotype. when these liga alleles were substituted into francisella novicida, they conferred a ts phenotype with restrictive temperatures between 33 and 39°c. when the f. novicida liga hybrid strains were plated above their restrictive temperatures, eight of them generated temperature-resistant variants. for two alleles, the mutations that ...201626773080
type i interferons in bacterial infections: a balancing act.defense against bacterial infections requires activation of the immune response as well as timely reestablishment of tissue and immune homeostasis. instauration of homeostasis is critical for tissue regeneration, wound healing, and host recovery. recent studies revealed that severe infectious diseases frequently result from failures in homeostatic processes rather than from inefficient pathogen eradication. type i interferons (ifn) appear to play a key role in such processes. remarkably, the inv ...201628082986
inflammasome-independent nlrp3 restriction of a protective early neutrophil response to pulmonary tularemia.francisella tularensis (ft) causes a frequently fatal, acute necrotic pneumonia in humans and animals. following lethal ft infection in mice, infiltration of the lungs by predominantly immature myeloid cells and subsequent myeloid cell death drive pathogenesis and host mortality. however, following sub-lethal ft challenge, more mature myeloid cells are elicited and are protective. in addition, inflammasome-dependent il-1β and il-18 are important for protection. as nlrp3 appears dispensable for r ...201627926940
modulating endotoxin activity by combinatorial bioengineering of meningococcal lipopolysaccharide.neisseria meningitidis contains a very potent hexa-acylated lps that is too toxic for therapeutic applications. we used systematic molecular bioengineering of meningococcal lps through deletion of biosynthetic enzymes in combination with induction of lps modifying enzymes to yield a variety of novel lps mutants with changes in both lipid a acylation and phosphorylation. mass spectrometry was used for detailed compositional determination of the lps molecular species, and stimulation of immune cel ...201627841285
mitogen-activated protein kinases (mapks) are modulated during francisella tularensis infection, but inhibition of extracellular-signal-regulated kinases (erks) is of limited therapeutic benefit.francisella tularensis is a gram-negative intracellular bacterium that causes the disease tularemia. the disease can be fatal if left untreated and there is currently no licenced vaccine available; the identification of new therapeutic targets is therefore required. toll-like receptors represent an interesting target for therapeutic modulation due to their essential role in generating immune responses. in this study, we analysed the in vitro expression of the key mitogen-activated protein kinase ...201627714591
diverse functions of small rnas in different plant-pathogen communications.rna silencing is a conserved mechanism that utilizes small rnas (srnas) to direct the regulation of gene expression at the transcriptional or post-transcriptional level. plants utilizing rna silencing machinery to defend pathogen infection was first identified in plant-virus interaction and later was observed in distinct plant-pathogen interactions. rna silencing is not only responsible for suppressing rna accumulation and movement of virus and viroid, but also facilitates plant immune responses ...201627757103
francisella tularensis iglg belongs to a novel family of paar-like t6ss proteins and harbors a unique n-terminal extension required for virulence.the virulence of francisella tularensis, the etiological agent of tularemia, relies on an atypical type vi secretion system (t6ss) encoded by a genomic island termed the francisella pathogenicity island (fpi). while the importance of the fpi in f. tularensis virulence is clearly established, the precise role of most of the fpi-encoded proteins remains to be deciphered. in this study, using highly virulent f. tularensis strains and the closely related species f. novicida, iglg was characterized a ...201627602570
comparative transcriptional analyses of francisella tularensis and francisella novicida.francisella tularensis is composed of a number of subspecies with varied geographic distribution, host ranges, and virulence. in view of these marked differences, comparative functional genomics may elucidate some of the molecular mechanism(s) behind these differences. in this study a shared probe microarray was designed that could be used to compare the transcriptomes of francisella tularensis subsp. tularensis schu s4 (ftt), francisella tularensis subsp. holarctica or960246 (fth), francisella ...201627537327
tularemia vaccine development: paralysis or progress?francisella tularensis (ft) is a gram-negative intercellular pathogen and category a biothreat agent. however, despite 15 years of strong government investment and intense research focused on the development of a us food and drug administration-approved vaccine against ft, the primary goal remains elusive. this article reviews research efforts focused on developing an ft vaccine, as well as a number of important factors, some only recently recognized as such, which can significantly impact the d ...201627200274
m-cells contribute to the entry of an oral vaccine but are not essential for the subsequent induction of protective immunity against francisella tularensis.m-cells (microfold cells) are thought to be a primary conduit of intestinal antigen trafficking. using an established neutralizing anti-rankl (receptor activator of nf-κb ligand) antibody treatment to transiently deplete m-cells in vivo, we sought to determine whether intestinal m-cells were required for the effective induction of protective immunity following oral vaccination with δiglb (a defined live attenuated francisella novicida mutant). m-cell depleted, δiglb-vaccinated mice exhibited inc ...201627100824
nanoaerosols reduce required effective dose of liposomal levofloxacin against pulmonary murine francisella tularensis subsp. novicida infection.the institute of theoretical and experimental biophysics in moscow recently developed a new nanoaerosol generator. this study evaluated this novel technology, which has the potential to enhance therapeutic delivery, with the goal of using the generator to treat pulmonary francisella tularensis subsp. novicida (f. novicida) infections in balb/c mice.201627090889
francisella tularensis - immune cell activator, suppressor, or stealthy evader: the evolving view from the petri dish.one of the hallmarks of pulmonary tularemia, which results from inhalation of francisella tularensis - a significant bioterrorism concern, is the lack of an acute th1-biased inflammatory response in the early phase of disease (days 1-3) despite significant bacterial loads. in an effort to understand this apparent hypo-responsiveness, many laboratories have utilized in vitro cell-based models as tools to probe the nature and consequences of host cell interactions with f. tularensis. the first use ...201627695643
two parallel pathways for ferric and ferrous iron acquisition support growth and virulence of the intracellular pathogen francisella tularensis schu s4.iron acquisition mechanisms in francisella tularensis, the causative agent of tularemia, include the francisella siderophore locus (fsl) siderophore operon and a ferrous iron-transport system comprising outer-membrane protein fupa and inner-membrane transporter feob. to characterize these mechanisms and to identify any additional iron uptake systems in the virulent subspecies tularensis, single and double deletions were generated in the fsl and feo iron acquisition systems of the strain schu s4. ...201626918301
dissection of francisella-host cell interactions in dictyostelium discoideum.francisella bacteria cause severe disease in both vertebrates and invertebrates and include one of the most infectious human pathogens. mammalian cell lines have mainly been used to study the mechanisms by which francisella manipulates its host to replicate within a large variety of hosts and cell types, including macrophages. here, we describe the establishment of a genetically and biochemically tractable infection model: the amoeba dictyostelium discoideum combined with the fish pathogen franc ...201626712555
biochemical and structural characterization of polyphosphate kinase 2 from the intracellular pathogen francisella tularensis.the metabolism of polyphosphate is important for the virulence of a wide range of pathogenic bacteria and the enzymes of polyphosphate metabolism have been proposed as an anti-bacterial target. in the intracellular pathogen francisella tularensis, the product of the gene ftt1564 has been identified as a polyphosphate kinase from the polyphosphate kinase 2 (ppk2) family. the isogenic deletion mutant was defective for intracellular growth in macrophages and was attenuated in mice, indicating an im ...201626582818
a structure-based classification of class a β-lactamases, a broadly diverse family of enzymes.for medical biologists, sequencing has become a commonplace technique to support diagnosis. rapid changes in this field have led to the generation of large amounts of data, which are not always correctly listed in databases. this is particularly true for data concerning class a β-lactamases, a group of key antibiotic resistance enzymes produced by bacteria. many genomes have been reported to contain putative β-lactamase genes, which can be compared with representative types. we analyzed several ...201626511485
a structure-based classification of class a β-lactamases, a broadly diverse family of enzymes.for medical biologists, sequencing has become a commonplace technique to support diagnosis. rapid changes in this field have led to the generation of large amounts of data, which are not always correctly listed in databases. this is particularly true for data concerning class a β-lactamases, a group of key antibiotic resistance enzymes produced by bacteria. many genomes have been reported to contain putative β-lactamase genes, which can be compared with representative types. we analyzed several ...201626511485
potentiation of the fosmidomycin analogue fr 900098 with substituted 2-oxazolines against francisella novicida.a library of 33 compounds was screened for potentiation of the antibiotic fr 900098 against the francisella tularensis surrogate francisella novicida. from the screen a highly potent 2-oxazoline adjuvant was discovered capable of potentiating fr 900098 with a 1000-fold reduction in mic against the francisella sub-species f. novicida and f. philomiragia.201628154750
use of essential gene, encoding prophobilinogen deaminase from extreme psychrophilic colwellia sp. c1, to generate temperature-sensitive strain of francisella novicida.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 ...201627248501
revisiting the gram-negative lipoprotein paradigm.the processing of lipoproteins (lpps) in gram-negative bacteria is generally considered an essential pathway. mature lipoproteins in these bacteria are triacylated, with the final fatty acid addition performed by lnt, an apolipoprotein n-acyltransferase. the mature lipoproteins are then sorted by the lol system, with most lpps inserted into the outer membrane (om). we demonstrate here that the lnt gene is not essential to the gram-negative pathogen francisella tularensis subsp. tularensis strain ...201525755189
outer-inner membrane vesicles naturally secreted by gram-negative pathogenic bacteria.outer-inner membrane vesicles (o-imvs) were recently described as a new type of membrane vesicle secreted by the antarctic bacterium shewanella vesiculosa m7t. their formation is characterized by the protrusion of both outer and plasma membranes, which pulls cytoplasmic components into the vesicles. to demonstrate that this is not a singular phenomenon in a bacterium occurring in an extreme environment, the identification of o-imvs in pathogenic bacteria was undertaken. with this aim, a structur ...201525581302
mouse genome engineering via crispr-cas9 for study of immune function.clustered regularly interspaced palindromic repeats (crispr)-associated (cas9) technology has proven a formidable addition to our armory of approaches for genomic editing. derived from pathways in archaea and bacteria that mediate the resistance to exogenous genomic material, the crispr-cas9 system utilizes a short single guide rna (sgrna) to direct the endonuclease cas9 to virtually anywhere in the genome. upon targeting, cas9 generates dna double-strand breaks (dsbs) and facilitates the repair ...201525607456
converging roles of caspases in inflammasome activation, cell death and innate immunity.inflammatory and apoptotic caspases are central players in inflammation and apoptosis, respectively. however, recent studies have revealed that these caspases have functions beyond their established roles. in addition to mediating cleavage of the inflammasome-associated cytokines interleukin-1β (il-1β) and il-18, inflammatory caspases modulate distinct forms of programmed cell death and coordinate cell-autonomous immunity and other fundamental cellular processes. certain apoptotic caspases assem ...201526655628
converging roles of caspases in inflammasome activation, cell death and innate immunity.inflammatory and apoptotic caspases are central players in inflammation and apoptosis, respectively. however, recent studies have revealed that these caspases have functions beyond their established roles. in addition to mediating cleavage of the inflammasome-associated cytokines interleukin-1β (il-1β) and il-18, inflammatory caspases modulate distinct forms of programmed cell death and coordinate cell-autonomous immunity and other fundamental cellular processes. certain apoptotic caspases assem ...201526655628
dangerous liaisons: caspase-11 and reactive oxygen species crosstalk in pathogen elimination.recently, the focus of murine caspase-11 and human orthologs caspase-4, -5 research has been on their novel function to induce noncanonical inflammasome activation in direct response to gram-negative bacterial infection. on the other hand, a new role in anti-bacterial autophagy has been attributed to caspase-11, -4 and -5, which currently stands largely unexplored. in this review, we connect lately emerged evidence that suggests these caspases have a key role in anti-bacterial autophagy and disc ...201526426007
roles of reactive oxygen species-degrading enzymes of francisella tularensis schu s4.francisella tularensis is a facultative intracellular bacterium utilizing macrophages as its primary intracellular habitat and is therefore highly capable of resisting the effects of reactive oxygen species (ros), potent mediators of the bactericidal activity of macrophages. we investigated the roles of enzymes presumed to be important for protection against ros. four mutants of the highly virulent schu s4 strain with deletions of the genes encoding catalase (katg), glutathione peroxidase (gpx), ...201525802058
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