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Guanylate-Binding Proteins are interferon-inducible GTPases that play a key role in cell autonomous responses against intracellular pathogens. Despite sharing high sequence similarity, subtle differences among GBPs translate into functional divergences that are still largely not understood. A key GBP feature is the formation of supramolecular GBP complexes on the bacterial surface. Such complexes are observed when GBP1 binds lipopolysaccharide (LPS) from Shigella and Salmonella and further recruits GBP2-4. Here, we compared GBP recruitment on two cytosol-dwelling pathogens, Francisella novicida and S. flexneri. Francisella novicida was coated by GBP1 and GBP2 and to a lower extent by GBP4 in human macrophages. Contrary to S. flexneri, F. novicida was not targeted by GBP3, a feature independent of T6SS effectors. Multiple GBP1 features were required to promote targeting to F. novicida while GBP1 targeting to S. flexneri was much more permissive to GBP1 mutagenesis suggesting that GBP1 has multiple domains that cooperate to recognize F. novicida atypical LPS. Altogether our results indicate that the repertoire of GBPs recruited onto specific bacteria is dictated by GBP-specific features and by specific bacterial factors that remain to be identified. Human Guanylate Binding Proteins target the cytosolic pathogen Francisella novicida in a different way as other cytosolic bacteria such as Shigella flexneri. [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]

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]

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 species secrete a polycarboxylate siderophore that resembles rhizoferrin to acquire ferric iron. Several of the Francisella siderophore synthesis genes are contained in a Fur-regulated operon (designated fig or fsl) comprised of at least seven ORFs including fur. Reverse transcriptase-PCR showed transcriptional linkage between figD and figE and between figE and figF. Mutations were constructed in four of these ORFs ( figB, figC, figD, and figE) in Francisella novicida U112. All four of these new mutants and a F. novicida figA mutant grew at rates comparable to that of wild type under iron-replete conditions but growth of all five mutants was stunted in iron-limiting media. When ferric rhizoferrin was added to the iron-limited media, growth of the figA, figB, figC, and figD mutants was restored to levels similar to those obtained in iron-replete media. However, this exogenously added siderophore could not rescue the figE mutant. When Chrome Azurol S assays were used to measure siderophore production, the figA, figB, and figC mutants were markedly deficient in their ability to synthesize siderophore whereas the figD and figE mutants produced siderophore at levels equivalent to the wild-type parent strain. [ABSTRACT FROM AUTHOR]

Five transposon mutants of Francisella novicida were isolated that are compromised in their ability to grow in mouse macrophages in vitro. Sequence analysis of the DNA flanking the transposon insertions identified the genes that were interrupted in these mutants. One of the inactivated loci corresponds to the Francisella tularensis gene that encodes a 23-kDa protein that is the most prominently induced protein following macrophage infection. Another insertion was localised to approximately 2 kb upstream of the gene encoding the 23-kDa protein. By analysis of the incomplete Francisella genome sequence it was surmised that these two insertions disrupt different portions of a putative operon that encodes four proteins, none of which have discernible functions. Three other interrupted loci associated with poor intramacrophage growth showed similarity at the deduced amino acid level to alanine racemase, the ClpB heat-shock protease, and the purine biosynthetic enzyme, glutamine phosphoribosylpyrophosphate amidotransferases. [Copyright &y& Elsevier]

F. novicida acts via the intrinsic and extrinsic pathways to prolong neutrophil viability by a mechanism independent of opsonins and phagocytosis. 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. novicida binds natural IgG that is present in normal human serum, which in turn, elicits NADPH oxidase activation that does not occur in response to F. tularensis. As it is established that phagocytosis and oxidant production markedly accelerate neutrophil death, we predicted that F. novicida may influence the neutrophil lifespan in an opsonin‐dependent manner. To test this hypothesis, we quantified bacterial uptake, phosphatidylserine (PS) externalization, and changes in nuclear morphology, as well as the kinetics of procaspase‐3, ‐8, and ‐9 processing and activation. To our surprise, we discovered that F. novicida not only failed to accelerate neutrophil death but also diminished and delayed apoptosis in a dose‐dependent, but opsonin‐independent, manner. In keeping with this, studies of conditioned media (CM) showed that neutrophil longevity could be uncoupled from phagocytosis and that F. novicida stimulated neutrophil secretion of CXCL8. Taken together, the results of this study reveal shared and unique aspects of the mechanisms used by Francisella species to manipulate neutrophil lifespan and as such, advance understanding of cell death regulation during infection. [ABSTRACT FROM AUTHOR]

Francisella novicida is a gram-negative pathogen that can induce disease in mice that mimics human tularemia, and is nearly identical to Francisella tularensis at the genomic level. In this work a number of antibiotic marker cassettes that incorporate a strong F. novicida promoter is constructed, which greatly enhances selection in F. novicida and F. tularensis. Two low-copy plasmid vectors based on a broad-host-range plasmid, and an integrating vector have also been made, and these can be used for genetic complementation. Two general approaches to deletion mutagenesis in F. novicida is also described. [ABSTRACT FROM AUTHOR]

Francisella tularensis is a highly pathogenic bacterium; however, little is known about its initial interactions with mucosal surfaces of the human respiratory tract. To investigate these interactions, we tested whether two Francisella strains could adhere to A549 human lung epithelial cells. We found that LVS adhered well to these cells while Francisella novicida adhered poorly. We used surface biotinylation to identify bacterial proteins that might mediate this adherence. We report the identification of the F. tularensis surface protein FsaP, which, when expressed in nonadherent Escherichia coli, confers recombinant bacteria with the ability to bind to A549 cells. [ABSTRACT FROM AUTHOR]

Objectives Francisella tularensis, a CDC class A potential bioterrorism agent, is a Gram-negative bacterium responsible for tularaemia. Understanding the mechanisms of resistance to antibiotics used as first-line treatment is of major security relevance. Methods We propagated the three parental reference strains Francisella tularensis subsp. holarctica live vaccine strain, Francisella novicida and Francisella philomiragia with increasing concentrations of ciprofloxacin, a fluoroquinolone used as curative and prophylactic treatment for tularaemia. This evolution procedure provided us with high-level ciprofloxacin-resistant mutants and all evolutionary intermediates towards high-level resistance. We determined the resistance levels to other fluoroquinolones (levofloxacin and moxifloxacin) and other antibiotic families (aminoglycosides, tetracyclines and macrolides) and characterized the genetic changes in the fluoroquinolone target genes encoding DNA gyrase and topoisomerase IV. Results All high-level resistant mutants shared cross-resistance to the tested fluoroquinolones, while some also revealed striking levels of cross-resistance to other clinically relevant antibiotic classes. High-level resistant mutants carried one to three mutations, including some not previously reported. We mapped all mutations onto known topoisomerase three-dimensional structures. Along the pathways towards high-level resistance, we identified complex evolutionary trajectories including polymorphic states and additional resistance mechanisms likely to be associated with efflux processes. Conclusions Our data demonstrated the efficiency and speed of in vitro production of mutants highly resistant to fluoroquinolones in Francisella species. They emphasize the urgent need to identify all antibiotic resistance mechanisms in these species, develop molecular tools for their detection and design new therapeutic alternatives for tularaemia. [ABSTRACT FROM PUBLISHER]

Cellular autofluorescence, though ubiquitous when imaging cells and tissues, is often assumed to be small in comparison to the signal of interest. Uniform estimates of autofluorescence intensity obtained from separate control specimens are commonly employed to correct for autofluorescence. While these may be sufficient for high signal-to-background applications, improvements in detector and probe technologies and introduction of spectral imaging microscopes have increased the sensitivity of fluorescence imaging methods, exposing the possibility of effectively probing the low signal-to-background regime. With spectral imaging, reliable monitoring of signals near or even below the noise levels of the microscope is possible if compensation for autofluorescence and background signals can be performed accurately. We demonstrate the importance of accurate autofluorescence modeling and the utility of spectral imaging and multivariate analysis methods using a case study focusing on fluorescence confocal spectral imaging of host-pathogen interactions. In this application fluorescent proteins are produced when Francisella novicida invade host macrophage cells. The resulting analyte signal is spectrally overlapped and typically weaker than the cellular autofluorescence. In addition to discussing the advantages of spectral imaging for following pathogen invasion, we present the spectral properties and cellular origin of macrophage autofluorescence. [ABSTRACT FROM AUTHOR]

The availability of an ever-increasing diversity of prokaryotic genomes and metagenomes represents a major opportunity to understand and decipher the mechanisms behind the functional diversification of microbial biosynthetic pathways. However, it remains unclear to what extent a pathway producing a specific molecule from a specific precursor can diversify. In this study, we focus on the biosynthesis of ubiquinone (UQ), a crucial coenzyme that is central to the bioenergetics and to the functioning of a wide variety of enzymes in Eukarya and Pseudomonadota (a subgroup of the formerly named Proteobacteria). UQ biosynthesis involves three hydroxylation reactions on contiguous carbon atoms. We and others have previously shown that these reactions are catalyzed by different sets of UQ-hydroxylases that belong either to the iron-dependent Coq7 family or to the more widespread flavin monooxygenase (FMO) family. Here, we combine an experimental approach with comparative genomics and phylogenetics to reveal how UQ-hydroxylases evolved different selectivities within the constrained framework of the UQ pathway. It is shown that the UQ-FMOs diversified via at least three duplication events associated with two cases of neofunctionalization and one case of subfunctionalization, leading to six subfamilies with distinct hydroxylation selectivity. We also demonstrate multiple transfers of the UbiM enzyme and the convergent evolution of UQ-FMOs toward the same function, which resulted in two independent losses of the Coq7 ancestral enzyme. Diversification of this crucial biosynthetic pathway has therefore occurred via a combination of parallel evolution, gene duplications, transfers, and losses. [ABSTRACT FROM AUTHOR]

Gene targeting can be used to make modifications at a specific region in a plant's genome and create high-precision tools for plant biotechnology and breeding. However, its low efficiency is a major barrier to its use in plants. The discovery of CRISPR (clustered regularly interspaced short palindromic repeats)–Cas-based site-specific nucleases capable of inducing double-strand breaks in desired loci resulted in the development of novel approaches for plant gene targeting. Several studies have recently demonstrated improvements in gene targeting efficiency through cell-type-specific expression of Cas nucleases, the use of self-amplified gene-targeting-vector DNA, or manipulation of RNA silencing and DNA repair pathways. In this review, we summarize recent advances in CRISPR/Cas-mediated gene targeting in plants and discuss potential efficiency improvements. Increasing the efficiency of gene targeting technology will help pave the way for increased crop yields and food safety in environmentally friendly agriculture. [ABSTRACT FROM AUTHOR]

In only a few years, as a breakthrough technology, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) gene-editing systems have ushered in the era of genome engineering with a plethora of applications. One of the most promising CRISPR tools, so-called base editors, opened an exciting avenue for exploring new therapeutic approaches through controlled mutagenesis. However, the efficiency of a base editor guide varies depending on several biological determinants, such as chromatin accessibility, DNA repair proteins, transcriptional activity, factors related to local sequence context and so on. Thus, the success of genetic perturbation directed by CRISPR/Cas base-editing systems relies on an optimal single guide RNA (sgRNA) design, taking those determinants into account. Although there is 11 commonly used software to design guides specifically for base editors, only three of them investigated and implemented those biological determinants into their models. This review presents the key features, capabilities and limitations of all currently available software with a particular focus on predictive model-based algorithms. Here, we summarize existing software for sgRNA design and provide a base for improving the efficiency of existing available software suites for precise target base editing. [ABSTRACT FROM AUTHOR]

The critical first step in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)–associated (CRISPR–Cas) protein-mediated gene editing is recognizing a preferred protospacer adjacent motif (PAM) on target DNAs by the protein's PAM-interacting amino acids (PIAAs). Thus, accurate computational modeling of PAM recognition is useful in assisting CRISPR–Cas engineering to relax or tighten PAM requirements for subsequent applications. Here, we describe a universal computational protein design framework (UniDesign) for designing protein–nucleic acid interactions. As a proof of concept, we applied UniDesign to decode the PAM–PIAA interactions for eight Cas9 and two Cas12a proteins. We show that, given native PIAAs, the UniDesign-predicted PAMs are largely identical to the natural PAMs of all Cas proteins. In turn, given natural PAMs, the computationally redesigned PIAA residues largely recapitulated the native PIAAs (74% and 86% in terms of identity and similarity, respectively). These results demonstrate that UniDesign faithfully captures the mutual preference between natural PAMs and native PIAAs, suggesting it is a useful tool for engineering CRISPR–Cas and other nucleic acid-interacting proteins. UniDesign is open-sourced at https://github.com/tommyhuangthu/UniDesign. [ABSTRACT FROM AUTHOR]

Aims To establish a dual-function clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system combined genome editing and transcriptional repression for multiplex metabolic engineering of Pseudomonas mutabilis. Materials and results This CRISPR-Cas12a system consisted of two plasmids that enabled single gene deletion, replacement, and inactivation with efficiency >90% for most targets within 5 days. With the guidance of truncated crRNA containing 16 bp spacer sequences, a catalytically active Cas12a could be employed to repress the expression of the reporter gene eGFP up to 66.6%. When bdhA deletion and eGFP repression were tested simultaneously by transforming a single crRNA plasmid and Cas12a plasmid, the knockout efficiency reached 77.8% and the expression of eGFP was decreased by >50%. Finally, the dual-functional system was demonstrated to increase the production of biotin by 3.84-fold, with yigM deletion and birA repression achieved simultaneously. Conclusions This CRISPR-Cas12a system is an efficient genome editing and regulation tool to facilitate the construction of P. mutabilis cell factories. [ABSTRACT FROM AUTHOR]

Clustered regularly interspaced short palindromic repeats (CRISPRs) are known to provide adaptive immunity to bacteria against invading bacteriophages. In recent years, CRISPR-based technologies have been used for creating improved plant varieties; however, the indigenous CRISPR–Cas elements of plant growth-promoting bacteria are usually neglected. These indigenous genetic cassettes have evolved over millions of years and have shaped the bacterial genome. Therefore, these genetic loci can be used to study the adaptive capability of the bacteria in the environment. This study aims to bioinformatically analyze the genomes of a common free-living nitrogen-fixing Azotobacter spp. to assess their CRISPR–Cas diversity. Strains of Azotobacter vinelandii and Azotobacter chroococcum were found to harbor a large number of spacers. The phylogeny of different Cas and Cse1 proteins revealed a close evolutionary relationship among A. chroococcum B3, A. chroococcum NCIMB 8003 locus II, and A. vinelandii DJ locus I. The secondary structure of the hairpin loop of the repeat was also analyzed, and a correlation was derived between the structural stability of the hairpin loop and the number of spacers acquired by the CRISPR loci. These findings revealed the diversity and evolution of the CRISPR sequences and Cas proteins in Azotobacter species. Although the adaptive immune system of bacteria against bacteriophage, CRISPR–Cas, has been identified in many bacteria, studies of plant growth-promoting bacteria have been neglected. These indigenous CRISPRs have shaped the genome over millions of years and their study can lead to the understanding of the genome composition of these organisms. Our results strengthen the idea of using A. chroococcum and A. vinelandii as biofertilizer strains as they possess more spacers with highly stable repeat sequences, thereby imparting them higher chance of survival against mobile genetic elements like phages and plasmids. [ABSTRACT FROM AUTHOR]

Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) system has been developed into a precise and efficient genome editing tool. Since its discovery as an adaptive immune system in prokaryotes, it has been applied in many different research fields including biotechnology and medical sciences. The high demand for rapid, highly efficient and versatile genetic tools to thrive in bacteria‐based cell factories accelerates this process. This review mainly focuses on significant advancements of the CRISPR system in Bacillus subtilis, including the achievements in gene editing, and on problems still remaining. Next, we comprehensively summarize this genetic tool's up‐to‐date development and utilization in other Bacillus species, including B. licheniformis, B. methanolicus, B. anthracis, B. cereus, B. smithii and B. thuringiensis. Furthermore, we describe the current application of CRISPR tools in phages to increase Bacillus hosts' resistance to virulent phages and phage genetic modification. Finally, we suggest potential strategies to further improve this advanced technique and provide insights into future directions of CRISPR technologies for rendering Bacillus species cell factories more effective and more powerful. [ABSTRACT FROM AUTHOR]

Signal transducer and activator of transcription-1 (STAT1) signaling mediate most biological functions of IFNα, IFNβ and IFNγ although recent studies indicate that IFNγ can alter expression of several genes via a STAT1-independent pathway. STAT1 is critical for immunity against a variety of intracellular pathogens and some studies show that pathogens evade host immunity by interfering with STAT1 signaling. Here, we have investigated the role of STAT1 in host defense against pulmonary Francisella novicida infection using STAT1−/− mice. In addition, we examined the effect of F. novicida on STAT1 signaling in macrophages and on their ability to activate antigen-specific T cells. Both wild-type (WT) and STAT1−/− BALB/c mice were susceptible to aerosol challenge with 103 F. novicida and displayed 100% mortality. However, STAT1−/− mice developed more severe pneumonia, liver pathology and succumbed to infection faster than WT mice. The lungs, liver and hearts from F. novicida-infected STAT1−/− mice also contained more bacteria than WT mice at the time of death. In vitro studies showed that F. novicida suppressed IFNγRα (alpha subunit of IFNγ receptor) and MHC class II expression, down-regulated IFNγ-induced STAT1 activation and reduced nuclear binding of STAT1 in RAW264.7 macrophages. Furthermore, F. novicida-infected BMDM loaded with ovalbumin (OVA) were less efficient in activating OVA-specific CD4+ T cells in vitro. These findings demonstrate that STAT1-mediated signaling participates in the host defense against pulmonary F. novicida infection but it is not sufficient to prevent mortality associated with this infection. Moreover, our results show that F. novicida attenuates STAT1-mediated IFNγ signaling in macrophages and impairs their ability to activate antigen-specific CD4+ T cells. [ABSTRACT FROM AUTHOR]

Motivation Accumulated clinical studies show that microbes living in humans interact closely with human hosts, and get involved in modulating drug efficacy and drug toxicity. Microbes have become novel targets for the development of antibacterial agents. Therefore, screening of microbe–drug associations can benefit greatly drug research and development. With the increase of microbial genomic and pharmacological datasets, we are greatly motivated to develop an effective computational method to identify new microbe–drug associations. Results In this article, we proposed a novel method, Graph2MDA, to predict microbe–drug associations by using variational graph autoencoder (VGAE). We constructed multi-modal attributed graphs based on multiple features of microbes and drugs, such as molecular structures, microbe genetic sequences and function annotations. Taking as input the multi-modal attribute graphs, VGAE was trained to learn the informative and interpretable latent representations of each node and the whole graph, and then a deep neural network classifier was used to predict microbe–drug associations. The hyperparameter analysis and model ablation studies showed the sensitivity and robustness of our model. We evaluated our method on three independent datasets and the experimental results showed that our proposed method outperformed six existing state-of-the-art methods. We also explored the meaning of the learned latent representations of drugs and found that the drugs show obvious clustering patterns that are significantly consistent with drug ATC classification. Moreover, we conducted case studies on two microbes and two drugs and found 75–95% predicted associations have been reported in PubMed literature. Our extensive performance evaluations validated the effectiveness of our proposed method. Availability and implementation Source codes and preprocessed data are available at https://github.com/moen-hyb/Graph2MDA. Supplementary information Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide (LPS) is for most but not all Gram-negative bacteria an essential component of the outer leaflet of the outer membrane. LPS contributes to the integrity of the outer membrane, which acts as an effective permeability barrier to antimicrobial agents and protects against complement-mediated lysis. In commensal and pathogenic bacteria LPS interacts with pattern recognition receptors (e.g LBP, CD14, TLRs) of the innate immune system and thereby plays an important role in determining the immune response of the host. LPS molecules consist of a membrane-anchoring lipid A moiety and the surface-exposed core oligosaccharide and O-antigen polysaccharide. While the basic lipid A structure is conserved among different bacterial species, there is still a huge variation in its details, such as the number, position and chain length of the fatty acids and the decoration of the glucosamine disaccharide with phosphate, phosphoethanolamine or amino sugars. New evidence has emerged over the last few decades on how this lipid A heterogeneity confers distinct benefits to some bacteria because it allows them to modulate host responses in response to changing host environmental factors. Here we give an overview of what is known about the functional consequences of this lipid A structural heterogeneity. In addition, we also summarize new approaches for lipid A extraction, purification and analysis which have enabled analysis of its heterogeneity. [ABSTRACT FROM AUTHOR]

Background: Brucella species are Gram-negative intracellular bacteria that causes severe inflammatory diseases in animals and humans. Two major lipoproteins (L19 and L16) of Brucella outer membrane proteins were studied to explore the association with inflammatory response of human monocytes (THP-1).Methods: Activated THP-1 cells induced with recombinant L19 and L16 were analyzed in comparison with unlipidated forms (U19 and U16) and lipopolysaccharide (LPS) of Brucella melitensis, respectively.Results: Secretion of inflammatory factors tumor necrosis factor-α, interleukin (IL)-6, and IL-1β was significantly increased from L19, L16, or both stimulated THP-1 cells. High secretion of IL-18 was detected only from L19-induced cells. Signaling of those cytokine responses was identified mainly through the P38-mitogen-activated protein kinase pathway, and signaling of L19-induced IL-1β response partly occurred via necrosis factor-κB. While exploring different forms of IL-18, we found that L19-induced production of active IL-18 (18 kD) occurred through upregulating NLRP3 and activating caspase-1, whereas L16-induced production of inactive IL-18 fragments (15 kD and 16 kD) occurred through activating caspase-8/3. We also found that L19 upregulated phosphorylation of XIAP for inhibiting caspase-3 activity to cleave IL-18, whereas L16 activated caspase-3 for producing GSDME-N and leading to pyroptosis of THP-1 cells.Conclusions: Brucella L19 and L16 differentially induce IL-18 response or pyroptosis in THP-1 cells, respectively. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii is an insidious emerging nosocomial pathogen that has developed resistance to all available antimicrobials, including the last resort antibiotic, colistin. Colistin resistance often occurs due to mutations in the PmrAB two-component regulatory system. To better understand the regulatory mechanisms contributing to colistin resistance, we have biochemically characterized the A. baumannii PmrA response regulator. Initial DNA-binding analysis shows that A. baumannii PmrA bound to the Klebsiella pneumoniae PmrA box motif. This prompted analysis of the putative A. baumannii PmrAB regulon that indicated that the A. baumannii PmrA consensus box is 5′-HTTAAD N5 HTTAAD. Additionally, we provide the first structural information for the A. baumannii PmrA N-terminal domain through X-ray crystallography and we present a full-length model using molecular modelling. From these studies, we were able to infer the effects of two critical PmrA mutations, PmrA::I13M and PmrA::P102R, both of which confer increased colistin resistance. Based on these data, we suggest structural and dynamic reasons for how these mutations can affect PmrA function and hence encourage resistive traits. Understanding these mechanisms will aid in the development of new targeted antimicrobial therapies. Graphical Abstract [ABSTRACT FROM AUTHOR]

Historically, Francisella strains have been described as nonhemolytic. In this study, we show by use of solid and liquid hemolysis assays that some Francisella strains have hemolytic properties. The Francisella novicida type strain U112 is hemolytic to horse erythrocytes and Francisella philomiragia type strain FSC144 is hemolytic towards both human and horse erythrocytes. The F. novicida strain U112 released a protein (novilysin A) into the culture supernatant which cross-reacted with antiserum against Escherichia coli HlyA whereas there was no similar protein detectable with this cross-reactive property from the supernatant of the F. philomiragia strain. [Copyright &y& Elsevier]

Aims: We performed in silico analysis of CRISPRcas loci from Tenacibaculum maritimum, evaluated spoligotyping as a subtyping method and genotyped uncharacterized Turkish isolates from European sea bass by multilocus sequence typing (MLST). Methods and Results: Spoligotyping was performed with primers designed to allow amplification and sequencing of whole CRISPR‐arrays from 23 T. maritimum isolates. Twenty‐three completed/draft genomes were also downloaded from the NCBI database and analysed. MLST of Turkish isolates was achieved with a well‐established 7‐gene scheme. Tenacibaculum maritimum genomes carry a structurally complete but partially defective class II CRISPRcas locus due to known amino acid substitutions in encoded Cas9 proteins. Our spacer identification suggests that the host range of bacteriophage P2559Y and Vibrio phage nt‐1 include T. maritimum and that the most recurrent infection recorded by isolates has been with Tenacibaculum phage PTm5. Thirty‐eight isolates with this CRISPRcas locus belonged to 25 spoligotypes and to 24 sequence types by MLST, respectively. According to MLST, T. maritimum isolates from Turkey are most related to previously defined sequence types ST3, ST40 and ST41 isolates from Spain, Malta and France. Conclusions: The evaluated spoligotyping offers discriminatory power comparable to MLST. Significance and Impact of the Study: Spoligotyping has potential as a quick, easy and cheap tool for subtyping of T. maritimum isolates. [ABSTRACT FROM AUTHOR]

Humans are currently facing the problem of how to ensure that there is enough food to feed all of the world's population. Ensuring that the food supply is sufficient will likely require the modification of crop genomes to improve their agronomic traits. The development of engineered sequence-specific nucleases (SSNs) paved the way for targeted gene editing in organisms, including plants. SSNs generate a double-strand break (DSB) at the target DNA site in a sequence-specific manner. These DSBs are predominantly repaired via error-prone non-homologous end joining and are only rarely repaired via error-free homology-directed repair if an appropriate donor template is provided. Gene targeting (GT), i.e. the integration or replacement of a particular sequence, can be achieved with combinations of SSNs and repair donor templates. Although its efficiency is extremely low, GT has been achieved in some higher plants. Here, we provide an overview of SSN-facilitated GT in higher plants and discuss the potential of GT as a powerful tool for generating crop plants with desirable features. [ABSTRACT FROM AUTHOR]

Guanylate binding proteins (GBPs) are important effector molecules of autonomous response induced by proinflammatory stimuli, mainly IFNs. The murine GBPs clustered in chromosome 3 (GBPchr3) contains the majority of human homologous GBPs. Despite intense efforts, mycobacterial‐promoted diseases are still a major public health problem. However, the combined importance of GBPchr3 during mycobacterial infection has been overlooked. This study addresses the influence of the GBPchr3 in host immunity against mycobacterial infection to elucidate the relationship between cell‐intrinsic immunity and triggering of an efficient anti‐mycobacterial immune response. Here we show that all GBPchr3 are up‐regulated in lungs of mice during Mycobacterium bovis BCG infection, resembling tissue expression of IFN‐γ. Mice deficient in GBPchr3 (GBPchr3−/−) were more susceptible to infection, displaying diminished expression of autophagy‐related genes (LC3B, ULK1, and ATG5) in lungs. Additionally, there was reduced proinflammatory cytokine production complementary to diminished numbers of myeloid cells in spleens of GBPchr3−/−. Higher bacterial burden in GBPchr3−/− animals correlated with increased number of tissue granulomas. Furthermore, absence of GBPchr3 hampered activation and production of TNF‐α and IL‐12 by dendritic cells. Concerning macrophages, lack of GBPs impaired their antimicrobial function, diminishing autophagy induction and intracellular killing efficiency. In contrast, single GBP2 deficiency did not contribute to in vivo bacterial control. In conclusion, this study shows that GBPchr3 are important not only to stimulate cell‐intrinsic immunity but also for inducing an efficient immune response to control mycobacterial infection in vivo. [ABSTRACT FROM AUTHOR]