56 results on '"David W. Lazinski"'
Search Results
2. Vibrio cholerae motility exerts drag force to impede attack by the bacterial predator Bdellovibrio bacteriovorus
- Author
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Miles C. Duncan, John C. Forbes, Y Nguyen, Lauren M. Shull, Rebecca K. Gillette, David W. Lazinski, Afsar Ali, Robert M. Q. Shanks, Daniel E. Kadouri, and Andrew Camilli
- Subjects
Science - Abstract
Prey bacteria have evolved different strategies to counteract predation but the genetic basis remains unclear. Here, Duncan et al. identify key genes involved in Vibrio cholerae sensitivity to Bdellovibrio bacteriovorus predation, providing new insights into prey resistance mechanisms.
- Published
- 2018
- Full Text
- View/download PDF
3. Identification of Spacer and Protospacer Sequence Requirements in the Vibrio cholerae Type I-E CRISPR/Cas System
- Author
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Jacob Bourgeois, David W. Lazinski, and Andrew Camilli
- Subjects
CRISPR/Cas ,Vibrio cholerae ,protospacer-adjacent motif ,Microbiology ,QR1-502 - Abstract
ABSTRACT The prokaryotic adaptive immune system CRISPR/Cas serves as a defense against bacteriophage and invasive nucleic acids. A type I-E CRISPR/Cas system has been detected in classical biotype isolates of Vibrio cholerae, the causative agent of the disease cholera. Experimental characterization of this system revealed a functional immune system that operates using a 5′-TT-3′ protospacer-adjacent motif (PAM) for interference. However, several designed spacers against the 5′-TT-3′ PAM do not interfere as expected, indicating that further investigation of this system is necessary. In this study, we identified additional conserved sequences, including a pyrimidine in the 5′ position of the spacer and a purine in the complementary position of the protospacer using 873 unique spacers and 2,267 protospacers mined from CRISPR arrays in deposited sequences of V. cholerae. We present bioinformatic evidence that during acquisition the protospacer purine is captured in the prespacer and that a 5′-RTT-3′ PAM is necessary for spacer acquisition. Finally, we demonstrate experimentally, by designing and manipulating spacer and cognate PAMs in a plasmid conjugation assay, that a 5′-RTT-3′ PAM is necessary for CRISPR interference, and we discover functional consequences for spacer efficacy related to the identity of the 5′ spacer pyrimidine. IMPORTANCE Bacterial CRISPR/Cas systems provide immunity by defending against phage and other invading elements. A thorough comprehension of the molecular mechanisms employed by these diverse systems will improve our understanding of bacteriophage-bacterium interactions and bacterial adaptation to foreign DNA. The Vibrio cholerae type I-E system was previously identified in an extinct classical biotype and was partially characterized for its function. Here, using both bioinformatic and functional assays, we extend that initial study. We have found that the type I-E system still exists in modern strains of V. cholerae. Furthermore, we defined additional sequence elements both in the CRISPR array and in target DNA that are required for immunity. CRISPR/Cas systems are now commonly used as precise and powerful genetic engineering tools. Knowledge of the sequences required for CRISPR/Cas immunity is a prerequisite for the effective design and experimental use of these systems. Our results greatly facilitate the effective use of one such system. Furthermore, we provide a publicly available software program that assists in the detection and validation of CRISPR/Cas immunity requirements when such a system exists in a bacterial species.
- Published
- 2020
- Full Text
- View/download PDF
4. High-Throughput Analysis of Gene Function in the Bacterial Predator Bdellovibrio bacteriovorus
- Author
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Miles C. Duncan, Rebecca K. Gillette, Micah A. Maglasang, Elizabeth A. Corn, Albert K. Tai, David W. Lazinski, Robert M. Q. Shanks, Daniel E. Kadouri, and Andrew Camilli
- Subjects
Bdellovibrio bacteriovorus ,Escherichia coli ,Tn-seq ,Vibrio cholerae ,predatory bacteria ,Microbiology ,QR1-502 - Abstract
ABSTRACT Bdellovibrio bacteriovorus is a bacterial predator capable of killing and replicating inside most Gram-negative bacteria, including antibiotic-resistant pathogens. Despite growing interest in this organism as a potential therapeutic, many of its genes remain uncharacterized. Here, we perform a high-throughput genetic screen with B. bacteriovorus using transposon sequencing (Tn-seq) to explore the genetic requirements of predation. Two hundred one genes were deemed essential for growth in the absence of prey, whereas over 100 genes were found to be specifically required for predative growth on the human pathogens Vibrio cholerae and Escherichia coli in both planktonic and biofilm states. To further this work, we created an ordered-knockout library in B. bacteriovorus and developed new high-throughput techniques to characterize the mutants by their stage of deficiency in the predator life cycle. Using microscopy and flow cytometry, we confirmed 10 mutants defective in prey attachment and eight mutants defective in prey rounding. The majority of these genes are hypothetical and previously uncharacterized. Finally, we propose new nomenclature to group B. bacteriovorus mutants into classes based on their stage of predation defect. These results contribute to our basic understanding of bacterial predation and may be useful for harnessing B. bacteriovorus to kill harmful pathogens in the clinical setting. IMPORTANCE Bdellovibrio bacteriovorus is a predatory bacterium that can kill a wide range of Gram-negative bacteria, including many human pathogens. Given the global rise of antibiotic resistance and dearth of new antibiotics discovered in the past 30 years, this predator has potential as an alternative to traditional antibiotics. For many years, B. bacteriovorus research was hampered by a lack of genetic tools, and the genetic mechanisms of predation have only recently begun to be established. Here, we comprehensively identify and characterize predator genes required for killing bacterial prey, as well as genes that interfere in this process, which may allow us to design better therapeutic predators. Based on our study, we and other researchers may ultimately be able to genetically engineer strains that have improved killing rates, target specific species of prey, or preferentially target prey in the planktonic or biofilm state.
- Published
- 2019
- Full Text
- View/download PDF
5. Author Correction: Antibiotic susceptibility signatures identify potential antimicrobial targets in the Acinetobacter baumannii cell envelope
- Author
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Edward Geisinger, Nadav J. Mortman, Yunfei Dai, Murat Cokol, Sapna Syal, Andrew Farinha, Delaney G. Fisher, Amy Y. Tang, David W. Lazinski, Stephen Wood, Jon Anthony, Tim van Opijnen, and Ralph R. Isberg
- Subjects
Science - Abstract
A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-20098-z
- Published
- 2020
- Full Text
- View/download PDF
6. Homopolymer tail-mediated ligation PCR: a streamlined and highly efficient method for DNA cloning and library construction
- Author
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David W. Lazinski and Andrew Camilli
- Subjects
molecular cloning ,annealing-assisted ligation ,DNA capture ,massively-parallel sequencing ,Biology (General) ,QH301-705.5 - Abstract
The amplification of DNA fragments, cloned between user-defined 5′ and 3′ end sequences, is a prerequisite step in the use of many current applications including massively parallel sequencing (MPS). Here we describe an improved method, called homopolymer tail-mediated ligation PCR (HTML-PCR), that requires very little starting template, minimal hands-on effort, is cost-effective, and is suited for use in high-throughput and robotic methodologies. HTML-PCR starts with the addition of homopolymer tails of controlled lengths to the 3′ termini of a double-stranded genomic template. The homopolymer tails enable the annealing-assisted ligation of a hybrid oligonucleotide to the template's recessed 5′ ends. The hybrid oligonucleotide has a user-defined sequence at its 5′ end. This primer, together with a second primer composed of a longer region complementary to the homopolymer tail and fused to a second 5′ user-defined sequence, are used in a PCR reaction to generate the final product. The user-defined sequences can be varied to enable compatibility with a wide variety of downstream applications. We demonstrate our new method by constructing MPS libraries starting from nanogram and sub-nanogram quantities of Vibrio cholerae and Streptococcus pneumoniae genomic DNA.
- Published
- 2013
- Full Text
- View/download PDF
7. Genes Contributing to Staphylococcus aureus Fitness in Abscess- and Infection-Related Ecologies
- Author
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Michael D. Valentino, Lucy Foulston, Ama Sadaka, Veronica N. Kos, Regis A. Villet, John Santa Maria, David W. Lazinski, Andrew Camilli, Suzanne Walker, David C. Hooper, and Michael S. Gilmore
- Subjects
Microbiology ,QR1-502 - Abstract
ABSTRACT Staphylococcus aureus is a leading cause of both community- and hospital-acquired infections that are increasingly antibiotic resistant. The emergence of S. aureus resistance to even last-line antibiotics heightens the need for the development of new drugs with novel targets. We generated a highly saturated transposon insertion mutant library in the genome of S. aureus and used Tn-seq analysis to probe the entire genome, with unprecedented resolution and sensitivity, for genes of importance in infection. We further identified genes contributing to fitness in various infected compartments (blood and ocular fluids) and compared them to genes required for growth in rich medium. This resulted in the identification of 426 genes that were important for S. aureus fitness during growth in infection models, including 71 genes that could be considered essential for survival specifically during infection. These findings highlight novel as well as previously known genes encoding virulence traits and metabolic pathways important for S. aureus proliferation at sites of infection, which may represent new therapeutic targets. IMPORTANCE Staphylococcus aureus continues to be a leading cause of antibiotic-resistant community and nosocomial infection. With the bacterium’s acquisition of resistance to methicillin and, more recently, vancomycin, the need for the development of new drugs with novel targets is urgent. Applying a highly saturated Tn-seq mutant library to analyze fitness and growth requirements in a murine abscess and in various infection-relevant fluids, we identified S. aureus traits that enable it to survive and proliferate during infection. This identifies potential new targeting opportunities for the development of novel therapeutics.
- Published
- 2014
- Full Text
- View/download PDF
8. Identification of a Membrane-Bound Transcriptional Regulator That Links Chitin and Natural Competence in Vibrio cholerae
- Author
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Ankur B. Dalia, David W. Lazinski, and Andrew Camilli
- Subjects
Microbiology ,QR1-502 - Abstract
ABSTRACT Vibrio cholerae is naturally competent when grown on chitin. It is known that expression of the major regulator of competence, TfoX, is controlled by chitin; however, the molecular mechanisms underlying this requirement for chitin have remained unclear. In the present study, we identify and characterize a membrane-bound transcriptional regulator that positively regulates the small RNA (sRNA) TfoR, which posttranscriptionally enhances tfoX translation. We show that this regulation of the tfoR promoter is direct by performing electrophoretic mobility shift assays and by heterologous expression of this system in Escherichia coli. This transcriptional regulator was recently identified independently and was named “TfoS” (S. Yamamoto et al., Mol. Microbiol., in press, doi:10.1111/mmi.12462). Using a constitutively active form of TfoS, we demonstrate that the activity of this regulator is sufficient to promote competence in V. cholerae in the absence of chitin. Also, TfoS contains a large periplasmic domain, which we hypothesized interacts with chitin to regulate TfoS activity. In the heterologous host E. coli, we demonstrate that chitin oligosaccharides are sufficient to activate TfoS activity at the tfoR promoter. Collectively, these data characterize TfoS as a novel chitin-sensing transcriptional regulator that represents the direct link between chitin and natural competence in V. cholerae. IMPORTANCE Naturally competent bacteria can take up exogenous DNA from the environment and integrate it into their genome by homologous recombination. This ability to take up exogenous DNA is shared by diverse bacterial species and serves as a mechanism to acquire new genes to enhance the fitness of the organism. Several members of the family Vibrionaceae become naturally competent when grown on chitin; however, a molecular understanding of how chitin activates competence is lacking. Here, we identify a novel membrane-bound transcriptional regulator that is required for natural transformation in the human pathogen Vibrio cholerae. We demonstrate that this regulator senses chitin oligosaccharides to activate the competence cascade, thus, uncovering the molecular link between chitin and natural competence in this Vibrio species.
- Published
- 2014
- Full Text
- View/download PDF
9. Vibrio cholerae motility exerts drag force to impede attack by the bacterial predator Bdellovibrio bacteriovorus
- Author
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Lauren M Shull, Daniel E. Kadouri, Andrew Camilli, Afsar Ali, Y Nguyen, Robert M. Q. Shanks, John C. Forbes, Rebecca K. Gillette, Miles C. Duncan, and David W. Lazinski
- Subjects
0301 basic medicine ,Movement ,Science ,General Physics and Astronomy ,Virulence ,Biology ,medicine.disease_cause ,Bacterial Adhesion ,Article ,General Biochemistry, Genetics and Molecular Biology ,Microbiology ,Predation ,03 medical and health sciences ,medicine ,lcsh:Science ,Vibrio cholerae ,Pathogen ,Predator ,Microbial Viability ,Multidisciplinary ,Viscosity ,O Antigens ,Reproducibility of Results ,General Chemistry ,Bdellovibrio bacteriovorus ,biology.organism_classification ,Biomechanical Phenomena ,030104 developmental biology ,Genes, Bacterial ,Mutation ,behavior and behavior mechanisms ,lcsh:Q ,Bacteria ,Genetic screen - Abstract
The bacterial predator Bdellovibrio bacteriovorus is evolved to attack and kill other bacteria, including the human intestinal pathogen Vibrio cholerae. Although B. bacteriovorus exhibit a broad prey range, little is known about the genetic determinants of prey resistance and sensitivity. Here we perform a genetic screen on V. cholerae and identify five pathways contributing to predation susceptibility. We find that the essential virulence regulators ToxR/S increase susceptibility to predation, as mutants of these genes are more resistant to predation. We observe by flow cytometry that lipopolysaccharide is a critical defense, as mutants lacking O-antigen are rapidly attacked by predatory B. bacteriovorus. Using polymer solutions to alter media viscosity, we find that when B. bacteriovorus attacks motile V. cholerae, increased drag forces slow its ability to prey. These results provide insights into key prey resistance mechanisms, and may be useful in the application of B. bacteriovorus in treating infections., Prey bacteria have evolved different strategies to counteract predation but the genetic basis remains unclear. Here, Duncan et al. identify key genes involved in Vibrio cholerae sensitivity to Bdellovibrio bacteriovorus predation, providing new insights into prey resistance mechanisms.
- Published
- 2018
10. A Tail Fiber Protein and a Receptor-Binding Protein Mediate ICP2 Bacteriophage Interactions with Vibrio cholerae OmpU
- Author
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Andrew Camilli, Minmin Yen, David W. Lazinski, Kimberley D. Seed, Andrea Lim, and Comstock, Laurie E
- Subjects
medicine.medical_treatment ,Mutant ,medicine.disease_cause ,Medical and Health Sciences ,Bacteriophage ,tail fiber protein ,Cholera ,Models ,2.2 Factors relating to the physical environment ,2.1 Biological and endogenous factors ,Bacteriophages ,Aetiology ,Vibrio cholerae ,0303 health sciences ,Mutation ,biology ,Bacterial ,Viral Tail Proteins ,Biological Sciences ,Foodborne Illness ,Adhesins ,Infectious Diseases ,Phenotype ,arms race ,Models, Animal ,Rabbits ,Infection ,Research Article ,bacteriophages ,Phage therapy ,Inositol Phosphates ,Mutation, Missense ,Mutagenesis (molecular biology technique) ,Virulence ,Porins ,Microbiology ,Host Specificity ,Vaccine Related ,03 medical and health sciences ,Biodefense ,Genetics ,medicine ,Animals ,Humans ,Antigens ,OmpU ,Adhesins, Bacterial ,Molecular Biology ,Alleles ,030304 developmental biology ,Antigens, Bacterial ,Agricultural and Veterinary Sciences ,Host Microbial Interactions ,Animal ,030306 microbiology ,Prevention ,biology.organism_classification ,Bacterial adhesin ,Emerging Infectious Diseases ,Good Health and Well Being ,Capsid Proteins ,Missense ,Digestive Diseases - Abstract
ICP2 is a virulent bacteriophage (phage) that preys on Vibrio cholerae. ICP2 was first isolated from cholera patient stool samples. Some of these stools also contained ICP2-resistant isogenic V. cholerae strains harboring missense mutations in the trimeric outer membrane porin protein OmpU, identifying it as the ICP2 receptor. In this study, we identify the ICP2 proteins that mediate interactions with OmpU by selecting for ICP2 host range mutants within infant rabbits infected with a mixture of wild-type and OmpU mutant strains. ICP2 host range mutants that can now infect OmpU mutant strains have missense mutations in the putative tail fiber gene gp25 and the putative adhesin gene gp23. Using site-specific mutagenesis, we show that single or double mutations in gp25 are sufficient to generate the host range mutant phenotype. However, at least one additional mutation in gp23 is required for robust plaque formation on specific OmpU mutants. Mutations in gp23 alone were insufficient to produce a host range mutant phenotype. All ICP2 host range mutants retained the ability to form plaques on wild-type V. cholerae cells. The strength of binding of host range mutants to V. cholerae correlated with plaque morphology, indicating that the selected mutations in gp25 and gp23 restore molecular interactions with the receptor. We propose that ICP2 host range mutants evolve by a two-step process. First, gp25 mutations are selected for their broad host range, albeit accompanied by low-level phage adsorption. Subsequent selection occurs for gp23 mutations that further increase productive binding to specific OmpU alleles, allowing for near-wild-type efficiencies of adsorption and subsequent phage multiplication. IMPORTANCE Concern over multidrug-resistant bacterial pathogens, including Vibrio cholerae, has led to renewed interest in phage biology and the potential for phage therapy. ICP2 is a genetically unique virulent phage isolated from cholera patient stool samples. It is also one of three phages in a prophylactic cocktail that have been shown to be effective in animal models of infection and the only one of the three that requires a protein receptor (OmpU). This study identifies an ICP2 tail fiber and a receptor binding protein and examines how ICP2 responds to the selective pressures of phage-resistant OmpU mutants. We found that this particular coevolutionary arms race presents fitness costs to both ICP2 and V. cholerae.
- Published
- 2021
11. A Tail Fiber Protein and a Receptor-Binding Protein Mediate ICP2 Bacteriophage Interactions withVibrio choleraeOmpU
- Author
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Minmin Yen, Kimberley D. Seed, David W. Lazinski, Andrew Camilli, and Andrea Lim
- Subjects
Mutation ,biology ,Chemistry ,Mutant ,Wild type ,Mutagenesis (molecular biology technique) ,biology.organism_classification ,medicine.disease_cause ,Molecular biology ,Bacteriophage ,Vibrio cholerae ,Porin ,medicine ,Gene - Abstract
ICP2 is a virulent bacteriophage (phage) that preys onVibrio cholerae. ICP2 was first isolated from cholera patient stool samples. Some of these stools also contained ICP2-resistant isogenicV. choleraestrains harboring missense mutations in the trimeric outer membrane porin protein OmpU, identifying it as the ICP2 receptor. In this study, we identify the ICP2 proteins that mediate interactions with OmpU by selecting for ICP2 host-range mutants within infant rabbits infected with a mixture of wild type and OmpU mutant strains. ICP2 host-range mutants had missense mutations in putative tail fiber genegp25and putative adhesingp23. Using site-specific mutagenesis we show that single or double mutations ingp25are sufficient to generate the host-range mutant phenotype. However, at least one additional mutation ingp23is required for robust plaque formation on specific OmpU mutants. Mutations ingp23alone were insufficient to give a host-range mutant phenotype. All ICP2 host-range mutants retained the ability to plaque on wild typeV. choleraecells. The strength of binding of host-range mutants toV. choleraecorrelated with plaque morphology, indicating that the selected mutations ingp25andgp23restore molecular interactions with the receptor. We propose that ICP2 host-range mutants evolve by a two-step process where, first,gp25mutations are selected for their broad host-range, albeit accompanied by low level phage adsorption. Subsequent selection occurs forgp23mutations that further increase productive binding to specific OmpU alleles, allowing for near wild type efficiencies of adsorption and subsequent phage multiplication.ImportanceConcern over multidrug-resistant bacterial pathogens, includingVibrio cholerae, has led to a renewed interest in phage biology and their potential for phage therapy. ICP2 is a genetically unique virulent phage isolated from cholera patient stool samples. It is also one of three phages in a prophylactic cocktail shown to be effective in animal models of infection and the only one of the three that requires a protein receptor (OmpU). This study identifies a ICP2 tail fiber and a receptor binding protein and examines how ICP2 responds to the selective pressures of phage-resistant OmpU mutants. We found that this particular co-evolutionary arms race presents fitness costs to both ICP2 andV. cholerae.
- Published
- 2021
12. Antibiotic susceptibility signatures identify potential antimicrobial targets in the Acinetobacter baumannii cell envelope
- Author
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Nadav J. Mortman, Amy Y. Tang, Murat Cokol, Sapna Syal, Jon Anthony, Stephen Wood, Tim van Opijnen, Ralph R. Isberg, Delaney G. Fisher, Yunfei Dai, Andrew Farinha, Edward Geisinger, and David W. Lazinski
- Subjects
0301 basic medicine ,Science ,030106 microbiology ,Mutant ,General Physics and Astronomy ,Drug resistance ,DNA replication ,Biology ,medicine.disease_cause ,Article ,General Biochemistry, Genetics and Molecular Biology ,Bacterial genetics ,03 medical and health sciences ,polycyclic compounds ,medicine ,Cellular microbiology ,lcsh:Science ,Pathogen ,Gene ,Genetics ,Mutation ,Multidisciplinary ,Antimicrobials ,General Chemistry ,biochemical phenomena, metabolism, and nutrition ,bacterial infections and mycoses ,biology.organism_classification ,Acinetobacter baumannii ,030104 developmental biology ,bacteria ,lcsh:Q ,Cell envelope ,Microbial genetics - Abstract
A unique, protective cell envelope contributes to the broad drug resistance of the nosocomial pathogen Acinetobacter baumannii. Here we use transposon insertion sequencing to identify A. baumannii mutants displaying altered susceptibility to a panel of diverse antibiotics. By examining mutants with antibiotic susceptibility profiles that parallel mutations in characterized genes, we infer the function of multiple uncharacterized envelope proteins, some of which have roles in cell division or cell elongation. Remarkably, mutations affecting a predicted cell wall hydrolase lead to alterations in lipooligosaccharide synthesis. In addition, the analysis of altered susceptibility signatures and antibiotic-induced morphology patterns allows us to predict drug synergies; for example, certain beta-lactams appear to work cooperatively due to their preferential targeting of specific cell wall assembly machineries. Our results indicate that the pathogen may be effectively inhibited by the combined targeting of multiple pathways critical for envelope growth., A unique cell envelope contributes to the antibiotic resistance of the pathogen Acinetobacter baumannii. Here, Geisinger et al. identify A. baumannii mutants with altered antibiotic susceptibility, infer the function of uncharacterized proteins involved in envelope synthesis, and predict antibiotic synergies.
- Published
- 2020
13. Identification of spacer and protospacer sequence requirements in the Vibrio cholerae Type I-E CRISPR/Cas System
- Author
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David W. Lazinski, Jacob Bourgeois, and Andrew Camilli
- Subjects
0301 basic medicine ,DNA, Bacterial ,Molecular Biology and Physiology ,Base pair ,030106 microbiology ,Computational biology ,Biology ,medicine.disease_cause ,Microbiology ,Conserved sequence ,protospacer-adjacent motif ,Bacteriophage ,03 medical and health sciences ,chemistry.chemical_compound ,CRISPR/Cas ,Plasmid ,medicine ,CRISPR ,Clustered Regularly Interspaced Short Palindromic Repeats ,Molecular Biology ,Vibrio cholerae ,CRISPR interference ,biology.organism_classification ,Acquired immune system ,humanities ,QR1-502 ,Protospacer adjacent motif ,030104 developmental biology ,chemistry ,DNA, Intergenic ,CRISPR-Cas Systems ,human activities ,DNA ,Research Article - Abstract
Bacterial CRISPR/Cas systems provide immunity by defending against phage and other invading elements. A thorough comprehension of the molecular mechanisms employed by these diverse systems will improve our understanding of bacteriophage-bacterium interactions and bacterial adaptation to foreign DNA. The Vibrio cholerae type I-E system was previously identified in an extinct classical biotype and was partially characterized for its function. Here, using both bioinformatic and functional assays, we extend that initial study. We have found that the type I-E system still exists in modern strains of V. cholerae. Furthermore, we defined additional sequence elements both in the CRISPR array and in target DNA that are required for immunity. CRISPR/Cas systems are now commonly used as precise and powerful genetic engineering tools. Knowledge of the sequences required for CRISPR/Cas immunity is a prerequisite for the effective design and experimental use of these systems. Our results greatly facilitate the effective use of one such system. Furthermore, we provide a publicly available software program that assists in the detection and validation of CRISPR/Cas immunity requirements when such a system exists in a bacterial species., The prokaryotic adaptive immune system CRISPR/Cas serves as a defense against bacteriophage and invasive nucleic acids. A type I-E CRISPR/Cas system has been detected in classical biotype isolates of Vibrio cholerae, the causative agent of the disease cholera. Experimental characterization of this system revealed a functional immune system that operates using a 5′-TT-3′ protospacer-adjacent motif (PAM) for interference. However, several designed spacers against the 5′-TT-3′ PAM do not interfere as expected, indicating that further investigation of this system is necessary. In this study, we identified additional conserved sequences, including a pyrimidine in the 5′ position of the spacer and a purine in the complementary position of the protospacer using 873 unique spacers and 2,267 protospacers mined from CRISPR arrays in deposited sequences of V. cholerae. We present bioinformatic evidence that during acquisition the protospacer purine is captured in the prespacer and that a 5′-RTT-3′ PAM is necessary for spacer acquisition. Finally, we demonstrate experimentally, by designing and manipulating spacer and cognate PAMs in a plasmid conjugation assay, that a 5′-RTT-3′ PAM is necessary for CRISPR interference, and we discover functional consequences for spacer efficacy related to the identity of the 5′ spacer pyrimidine. IMPORTANCE Bacterial CRISPR/Cas systems provide immunity by defending against phage and other invading elements. A thorough comprehension of the molecular mechanisms employed by these diverse systems will improve our understanding of bacteriophage-bacterium interactions and bacterial adaptation to foreign DNA. The Vibrio cholerae type I-E system was previously identified in an extinct classical biotype and was partially characterized for its function. Here, using both bioinformatic and functional assays, we extend that initial study. We have found that the type I-E system still exists in modern strains of V. cholerae. Furthermore, we defined additional sequence elements both in the CRISPR array and in target DNA that are required for immunity. CRISPR/Cas systems are now commonly used as precise and powerful genetic engineering tools. Knowledge of the sequences required for CRISPR/Cas immunity is a prerequisite for the effective design and experimental use of these systems. Our results greatly facilitate the effective use of one such system. Furthermore, we provide a publicly available software program that assists in the detection and validation of CRISPR/Cas immunity requirements when such a system exists in a bacterial species.
- Published
- 2020
14. Transposon Mutagenesis Screen of Klebsiella pneumoniae Identifies Multiple Genes Important for Resisting Antimicrobial Activities of Neutrophils in Mice
- Author
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David W. Lazinski, Anne L. McCabe, Joan Mecsas, Michelle K. Paczosa, Colin H. McLeish, Rebecca J. Silver, and Albert K. Tai
- Subjects
Transposable element ,Klebsiella ,Neutrophils ,Virulence Factors ,Klebsiella pneumoniae ,Immunology ,Mutant ,Virulence ,Microbiology ,Mice ,Pneumonia, Bacterial ,Animals ,Genetic Testing ,Gene ,Pathogen ,biology ,biology.organism_classification ,Molecular Pathogenesis ,Klebsiella Infections ,respiratory tract diseases ,Disease Models, Animal ,Mutagenesis, Insertional ,Infectious Diseases ,Host-Pathogen Interactions ,DNA Transposable Elements ,Parasitology ,Transposon mutagenesis - Abstract
Klebsiella pneumoniae is a Gram-negative bacterial pathogen that causes a range of infections, including pneumonias, urinary tract infections, and septicemia, in otherwise healthy and immunocompromised patients. K. pneumoniae has become an increasing concern due to the rise and spread of antibiotic-resistant and hypervirulent strains. However, its virulence determinants remain understudied. To identify novel K. pneumoniae virulence factors needed to cause pneumonia, a high-throughput screen was performed with an arrayed library of over 13,000 K. pneumoniae transposon insertion mutants in the lungs of wild-type (WT) and neutropenic mice using transposon sequencing (Tn-seq). Insertions in 166 genes resulted in K. pneumoniae mutants that were significantly less fit in the lungs of WT mice than in those of neutropenic mice. Of these, mutants with insertions in 51 genes still had significant defects in neutropenic mice, while mutants with insertions in 52 genes recovered significantly. In vitro screens using a minilibrary of K. pneumoniae transposon mutants identified putative functions for a subset of these genes, including in capsule content and resistance to reactive oxygen and nitrogen species. Lung infections in mice confirmed roles in K. pneumoniae virulence for the ΔdedA, ΔdsbC, ΔgntR, Δwzm-wzt, ΔyaaA, and ΔycgE mutants, all of which were defective in either capsule content or growth in reactive oxygen or nitrogen species. The fitness of the ΔdedA, ΔdsbC, ΔgntR, ΔyaaA, and ΔycgE mutants was higher in neutropenic mouse lungs, indicating that these genes encode proteins that protect K. pneumoniae against neutrophil-related effector functions.
- Published
- 2020
15. Antibiotic hypersensitivity signatures identify targets for attack in the Acinetobacter baumannii cell envelope
- Author
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Amy Y. Tang, Ralph R. Isberg, Nadav J. Mortman, David W. Lazinski, Sapna Syal, Murat Cokol, Tim van Opijnen, Stephen Wood, Edward Geisinger, Andrew Farinha, Delaney G. Fisher, Yunfei Dai, and Jon Anthony
- Subjects
0303 health sciences ,030306 microbiology ,medicine.drug_class ,Antibiotics ,Mutant ,Drug action ,Computational biology ,Biology ,biology.organism_classification ,Acinetobacter baumannii ,Cell wall ,03 medical and health sciences ,medicine ,Cell envelope ,Pathogen ,Function (biology) ,030304 developmental biology - Abstract
Acinetobacter baumannii is an opportunistic pathogen that is a critical, high-priority target for new antibiotic development. Clearing of A. baumannii requires relatively high doses of antibiotics across the spectrum, primarily due to its protective cell envelope. Many of the proteins that support envelope integrity and modulate drug action are uncharacterized, largely because there is an absence of orthologs for several proteins that perform essential envelope-associated processes, impeding progress on this front. To identify targets that can synergize with current antibiotics, we performed an exhaustive analysis of A. baumannii mutants causing hypersensitivity to a multitude of antibiotic treatments. By examining mutants with antibiotic hypersensitivity profiles that parallel mutations in proteins of known function, we show that the function of poorly annotated proteins can be predicted and used to identify candidate missing link proteins in essential A. baumannii processes. Using this strategy, we uncovered multiple uncharacterized proteins with critical roles in cell division or cell elongation, and revealed that a predicted cell wall D,D-endopeptidase has an unappreciated function in lipooligosaccharide synthesis. Moreover, we provide a genetic strategy that uses hypersensitivity signatures to predict drug synergies, allowing the identification of β-lactams that work cooperatively based on the cell wall assembly machineries that they preferentially target. These data reveal multiple pathways critical for envelope growth in A. baumannii that can be targeted in combination strategies for attacking the pathogen.
- Published
- 2020
16. Growth arrest and a persister state enable resistance to osmotic shock and facilitate dissemination of Vibrio cholerae
- Author
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Roberto C. Molina-Quiroz, Y Nguyen, Cecilia A. Silva-Valenzuela, Andrew Camilli, David W. Lazinski, and Shoshanna C. Kahne
- Subjects
0301 basic medicine ,Osmotic shock ,Multidrug tolerance ,Fresh Water ,Biology ,medicine.disease ,medicine.disease_cause ,Microbiology ,Cholera ,Virology ,03 medical and health sciences ,030104 developmental biology ,Osmotic Pressure ,Vibrio cholerae ,Aquatic environment ,Growth arrest ,medicine ,Humans ,Osmotic pressure ,Original Article ,Pathogen ,Ecology, Evolution, Behavior and Systematics - Abstract
Vibrio cholerae is a water-borne bacterial pathogen and causative agent of cholera. Although V. cholerae is a halophile, it can survive in fresh water, and this has a major role in cholera epidemics through consumption of contaminated water and subsequent fecal–oral spread. After dissemination from humans back into fresh water, V. cholerae encounters limited nutrient availability and an abrupt drop in conductivity but little is known about how V. cholerae adapts to, and survives in this environment. In this work, by abolishing or altering the expression of V. cholerae genes in a high-throughput manner, we observed that many osmotic shock tolerant mutants exhibited slowed or arrested growth, and/or generated a higher proportion of persister cells. In addition, we show that growth-arrested V. cholerae, including a persister subpopulation, are generated during infection of the intestinal tract and together allow for the successful dissemination to fresh water. Our results suggest that growth-arrested and persister subpopulations enable survival of V. cholerae upon shedding to the aquatic environment.
- Published
- 2017
17. High-Throughput Analysis of Gene Function in the Bacterial Predator Bdellovibrio bacteriovorus
- Author
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David W. Lazinski, Elizabeth A. Corn, Miles C. Duncan, Daniel E. Kadouri, Albert K. Tai, Rebecca K. Gillette, Robert M. Q. Shanks, Micah A. Maglasang, and Andrew Camilli
- Subjects
Genes, Viral ,Human pathogen ,Ecological and Evolutionary Science ,Microbiology ,predatory bacteria ,Predation ,03 medical and health sciences ,Gene Knockout Techniques ,Virology ,Escherichia coli ,Gene ,Vibrio cholerae ,Organism ,030304 developmental biology ,Genetics ,0303 health sciences ,biology ,030306 microbiology ,Biofilm ,Tn-seq ,High-Throughput Nucleotide Sequencing ,Bdellovibrio bacteriovorus ,biology.organism_classification ,QR1-502 ,Biofilms ,Mutation ,DNA Transposable Elements ,Bacteria ,Genetic screen ,Research Article - Abstract
Bdellovibrio bacteriovorus is a predatory bacterium that can kill a wide range of Gram-negative bacteria, including many human pathogens. Given the global rise of antibiotic resistance and dearth of new antibiotics discovered in the past 30 years, this predator has potential as an alternative to traditional antibiotics. For many years, B. bacteriovorus research was hampered by a lack of genetic tools, and the genetic mechanisms of predation have only recently begun to be established. Here, we comprehensively identify and characterize predator genes required for killing bacterial prey, as well as genes that interfere in this process, which may allow us to design better therapeutic predators. Based on our study, we and other researchers may ultimately be able to genetically engineer strains that have improved killing rates, target specific species of prey, or preferentially target prey in the planktonic or biofilm state., Bdellovibrio bacteriovorus is a bacterial predator capable of killing and replicating inside most Gram-negative bacteria, including antibiotic-resistant pathogens. Despite growing interest in this organism as a potential therapeutic, many of its genes remain uncharacterized. Here, we perform a high-throughput genetic screen with B. bacteriovorus using transposon sequencing (Tn-seq) to explore the genetic requirements of predation. Two hundred one genes were deemed essential for growth in the absence of prey, whereas over 100 genes were found to be specifically required for predative growth on the human pathogens Vibrio cholerae and Escherichia coli in both planktonic and biofilm states. To further this work, we created an ordered-knockout library in B. bacteriovorus and developed new high-throughput techniques to characterize the mutants by their stage of deficiency in the predator life cycle. Using microscopy and flow cytometry, we confirmed 10 mutants defective in prey attachment and eight mutants defective in prey rounding. The majority of these genes are hypothetical and previously uncharacterized. Finally, we propose new nomenclature to group B. bacteriovorus mutants into classes based on their stage of predation defect. These results contribute to our basic understanding of bacterial predation and may be useful for harnessing B. bacteriovorus to kill harmful pathogens in the clinical setting.
- Published
- 2019
18. The Landscape of Phenotypic and Transcriptional Responses to Ciprofloxacin in Acinetobacter baumannii: Acquired Resistance Alleles Modulate Drug-Induced SOS Response and Prophage Replication
- Author
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Zeyu Zhu, Elizabeth L. Wainwright, Tim van Opijnen, Ralph R. Isberg, Nadav J. Mortman, Germán Vargas-Cuebas, Stephen Wood, David W. Lazinski, Jon Anthony, Yunfei Dai, Sapna Syal, and Edward Geisinger
- Subjects
Acinetobacter baumannii ,DNA Topoisomerase IV ,antibiotic resistance ,prophage ,DNA damage ,Topoisomerase IV ,Prophages ,Microbial Sensitivity Tests ,Biology ,Virus Replication ,fluoroquinolone ,DNA gyrase ,Microbiology ,03 medical and health sciences ,Antibiotic resistance ,ciprofloxacin ,Virology ,Drug Resistance, Bacterial ,topoisomerase IV ,SOS response ,SOS Response, Genetics ,Prophage ,Alleles ,030304 developmental biology ,Genetics ,0303 health sciences ,Whole Genome Sequencing ,Acinetobacter ,030306 microbiology ,Topoisomerase ,Gene Expression Profiling ,Therapeutics and Prevention ,biology.organism_classification ,Editor's Pick ,QR1-502 ,3. Good health ,Anti-Bacterial Agents ,fitness ,Phenotype ,Mutation ,biology.protein ,DNA Damage ,Research Article - Abstract
Fluoroquinolones have been extremely successful antibiotics due to their ability to target multiple bacterial enzymes critical to DNA replication, the topoisomerases DNA gyrase and topo IV. Unfortunately, mutations lowering drug affinity for both enzymes are now widespread, rendering these drugs ineffective for many pathogens. To undermine this form of resistance, we examined how bacteria with target alterations differentially cope with fluoroquinolone exposures. We studied this problem in the nosocomial pathogen A. baumannii, which causes drug-resistant life-threatening infections. Employing genome-wide approaches, we uncovered numerous pathways that could be exploited to raise fluoroquinolone sensitivity independently of target alteration. Remarkably, fluoroquinolone targeting of topo IV in specific mutants caused dramatic hyperinduction of prophage replication and enhanced the mutagenic DNA damage response, but these responses were muted in strains with DNA gyrase as the primary target. This work demonstrates that resistance evolution via target modification can profoundly modulate the antibiotic stress response, revealing potential resistance-associated liabilities., The emergence of fluoroquinolone resistance in nosocomial pathogens has restricted the clinical efficacy of this antibiotic class. In Acinetobacter baumannii, the majority of clinical isolates now show high-level resistance due to mutations in gyrA (DNA gyrase) and parC (topoisomerase IV [topo IV]). To investigate the molecular basis for fluoroquinolone resistance, an exhaustive mutation analysis was performed in both drug-sensitive and -resistant strains to identify loci that alter ciprofloxacin sensitivity. To this end, parallel fitness tests of over 60,000 unique insertion mutations were performed in strains with various alleles in genes encoding the drug targets. The spectra of mutations that altered drug sensitivity were found to be similar in the drug-sensitive and gyrA parC double-mutant backgrounds, having resistance alleles in both genes. In contrast, the introduction of a single gyrA resistance allele, resulting in preferential poisoning of topo IV by ciprofloxacin, led to extreme alterations in the insertion mutation fitness landscape. The distinguishing feature of preferential topo IV poisoning was enhanced induction of DNA synthesis in the region of two endogenous prophages, with DNA synthesis associated with excision and circularization of the phages. Induction of the selective DNA synthesis in the gyrA background was also linked to heightened prophage gene transcription and enhanced activation of the mutagenic SOS response relative to that observed in either the wild-type (WT) or gyrA parC double mutant. Therefore, the accumulation of mutations that result in the stepwise evolution of high ciprofloxacin resistance is tightly connected to modulation of the SOS response and endogenous prophage DNA synthesis.
- Published
- 2019
19. Lon Protease Has Multifaceted Biological Functions in Acinetobacter baumannii
- Author
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Carly Ching, Veronica G. Godoy, David W. Lazinski, Chineme Onwubueke, Brendan Yang, and Andrew Camilli
- Subjects
0303 health sciences ,Proteases ,Protease ,030306 microbiology ,medicine.medical_treatment ,Biofilm ,Cellular homeostasis ,biochemical phenomena, metabolism, and nutrition ,Biology ,biology.organism_classification ,Microbiology ,Acinetobacter baumannii ,03 medical and health sciences ,Multicellular organism ,Antibiotic resistance ,medicine ,bacteria ,Molecular Biology ,Bacteria ,030304 developmental biology - Abstract
Acinetobacter baumannii is a Gram-negative opportunistic pathogen that is known to survive harsh environmental conditions and is a leading cause of hospital-acquired infections. Specifically, multicellular communities (known as biofilms) of A. baumannii can withstand desiccation and survive on hospital surfaces and equipment. Biofilms are bacteria embedded in a self-produced extracellular matrix composed of proteins, sugars, and/or DNA. Bacteria in a biofilm are protected from environmental stresses, including antibiotics, which provides the bacteria with selective advantage for survival. Although some gene products are known to play roles in this developmental process in A. baumannii, mechanisms and signaling remain mostly unknown. Here, we find that Lon protease in A. baumannii affects biofilm development and has other important physiological roles, including motility and the cell envelope. Lon proteases are found in all domains of life, participating in regulatory processes and maintaining cellular homeostasis. These data reveal the importance of Lon protease in influencing key A. baumannii processes to survive stress and to maintain viability. IMPORTANCEAcinetobacter baumannii is an opportunistic pathogen and is a leading cause of hospital-acquired infections. A. baumannii is difficult to eradicate and to manage, because this bacterium is known to robustly survive desiccation and to quickly gain antibiotic resistance. We sought to investigate biofilm formation in A. baumannii, since much remains unknown about biofilm formation in this bacterium. Biofilms, which are multicellular communities of bacteria, are surface attached and difficult to eliminate from hospital equipment and implanted devices. Our research identifies multifaceted physiological roles for the conserved bacterial protease Lon in A. baumannii. These roles include biofilm formation, motility, and viability. This work broadly affects and expands understanding of the biology of A. baumannii, which will permit us to find effective ways to eliminate the bacterium.
- Published
- 2019
20. Author Correction: Antibiotic susceptibility signatures identify potential antimicrobial targets in the Acinetobacter baumannii cell envelope
- Author
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Andrew Farinha, Nadav J. Mortman, Tim van Opijnen, Stephen Wood, David W. Lazinski, Amy Y. Tang, Murat Cokol, Edward Geisinger, Sapna Syal, Delaney G. Fisher, Yunfei Dai, Ralph R. Isberg, and Jon Anthony
- Subjects
Acinetobacter baumannii ,DNA, Bacterial ,medicine.drug_class ,Science ,DNA Mutational Analysis ,Antibiotics ,Cellular microbiology ,General Physics and Astronomy ,Microbial Sensitivity Tests ,DNA replication ,General Biochemistry, Genetics and Molecular Biology ,Microbiology ,Bacterial Proteins ,Cell Wall ,Drug Resistance, Multiple, Bacterial ,medicine ,Humans ,Author Correction ,Cross Infection ,Multidisciplinary ,biology ,Antimicrobials ,Drug Synergism ,General Chemistry ,Antimicrobial ,biology.organism_classification ,Anti-Bacterial Agents ,Mutation ,DNA Transposable Elements ,Microbial genetics ,Cell envelope ,Acinetobacter Infections - Abstract
A unique, protective cell envelope contributes to the broad drug resistance of the nosocomial pathogen Acinetobacter baumannii. Here we use transposon insertion sequencing to identify A. baumannii mutants displaying altered susceptibility to a panel of diverse antibiotics. By examining mutants with antibiotic susceptibility profiles that parallel mutations in characterized genes, we infer the function of multiple uncharacterized envelope proteins, some of which have roles in cell division or cell elongation. Remarkably, mutations affecting a predicted cell wall hydrolase lead to alterations in lipooligosaccharide synthesis. In addition, the analysis of altered susceptibility signatures and antibiotic-induced morphology patterns allows us to predict drug synergies; for example, certain beta-lactams appear to work cooperatively due to their preferential targeting of specific cell wall assembly machineries. Our results indicate that the pathogen may be effectively inhibited by the combined targeting of multiple pathways critical for envelope growth.
- Published
- 2020
21. Global Tn-seq analysis of carbohydrate utilization and vertebrate infectivity ofBorrelia burgdorferi
- Author
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David W. Lazinski, Andrew Camilli, Steven J. Norris, Linden T. Hu, Tao Lin, Lihui Gao, Erin B. Troy, and Maureen E. Lundt
- Subjects
0301 basic medicine ,Genetics ,Infectivity ,biology ,Mutagenesis (molecular biology technique) ,Mannose ,Carbohydrate metabolism ,biology.organism_classification ,Microbiology ,Phenotype ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,Biochemistry ,chemistry ,Transposon mutagenesis ,Borrelia burgdorferi ,Molecular Biology ,Gene - Abstract
Borrelia burgdorferi maintains a complex life cycle between tick and vertebrate hosts. Although some genes have been identified as contributing to bacterial adaptation in the different hosts, the list is incomplete. In this manuscript, we report the first use of transposon mutagenesis combined with high-throughput sequencing (Tn-seq) in B. burgdorferi. We utilize the technique to investigate mechanisms of carbohydrate utilization in B. burgdorferi and the role of carbohydrate metabolism during mouse infection. We performed genetic fitness analyses to identify genes encoding factors contributing to growth on glucose, maltose, mannose, trehalose and N-acetyl-glucosamine. We obtained insight into the potential functions of proteins predicted to be involved in carbohydrate utilization and identified additional factors previously unrecognized as contributing to the metabolism of the tested carbohydrates. Strong phenotypes were observed for the putative carbohydrate phosphotransferase transporters BB0408 and BBB29 as well as the response regulator Rrp1. We further validated Tn-seq for use in mouse studies and were able to correctly identify known infectivity factors as well as additional transporters and genes on lp54 that may contribute to optimal mouse infection. As such, this study establishes Tn-seq as a powerful method for both in vitro and in vivo studies of B. burgdorferi.
- Published
- 2016
22. The landscape of intrinsic and evolved fluoroquinolone resistance inAcinetobacter baumanniiincludes suppression of drug-induced prophage replication
- Author
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Ralph R. Isberg, Elizabeth L. Wainwright, Nadav J. Mortman, Zeyu Zhu, Stephen Wood, Sapna Syal, David W. Lazinski, Jon Anthony, Germán Vargas-Cuebas, Edward Geisinger, and Tim van Opijnen
- Subjects
Genetics ,0303 health sciences ,030306 microbiology ,Topoisomerase ,Mutant ,Biology ,biology.organism_classification ,DNA gyrase ,3. Good health ,Acinetobacter baumannii ,03 medical and health sciences ,biology.protein ,Insertion ,SOS response ,Gene ,Prophage ,030304 developmental biology - Abstract
The emergence of fluoroquinolone resistance in nosocomial pathogens has restricted the clinical efficacy of this antibiotic class. InAcinetobacter baumannii, the majority of clinical isolates now show high-level resistance due to mutations ingyrA(DNA gyrase) andparC(Topo IV). To investigate the molecular basis for fluoroquinolone resistance, an exhaustive mutation analysis was performed in both drug sensitive and resistant strains to identify loci that alter the sensitivity of the organism to ciprofloxacin. To this end, parallel fitness tests of over 60,000 unique insertion mutations were performed in strains with various alleles in genes encoding the drug targets. The spectrum of mutations that altered drug sensitivity was found to be similar in the drug sensitive and double mutantgyrAparCbackground having resistance alleles in both genes. In contrast, introduction of a singlegyrAresistance allele, resulting in preferential poisoning of Topo IV by ciprofloxacin, led to extreme alterations in the insertion mutation fitness landscape. The distinguishing feature of preferential Topo IV poisoning was induction of DNA synthesis in the region of two endogenous prophages, which appeared to occurin situ. Induction of the selective DNA synthesis in thegyrAbackground was also linked to enhanced activation of SOS response and heightened transcription of prophage genes relative to that observed in either the WT orgyrAparCdouble mutants. Therefore, the accumulation of mutations that result in the stepwise evolution of high ciprofloxacin resistance is tightly connected to suppression of hyperactivation of the SOS response and endogenous prophage DNA synthesis.ImportanceFluoroquinolones have been extremely successful antibiotics. Their clinical efficacy derives from the ability to target multiple bacterial enzymes critical to DNA replication, the topoisomerases DNA gyrase and Topo IV. Unfortunately, mutations lowering drug affinity for both enzymes are now widespread, rendering these drugs ineffective for many pathogens. To undermine this form of resistance, we sought to understand how bacteria with target alterations differentially cope with fluoroquinolone exposures. We studied this problem in the nosocomial pathogenA. baumannii, which causes resistant, life-threating infections. Employing genome-wide approaches, we uncovered numerous pathways that could be exploited to lower fluoroquinolone resistance independently of target alteration. Remarkably, fluoroquinolone targeting of Topo IV in specific mutants caused dramatic prophage hyperinduction, a response that was muted in strains with DNA gyrase as the primary target. This work demonstrates that resistance evolution via target modification can profoundly modulate the antibiotic stress response, revealing potential resistance-associated liabilities.
- Published
- 2018
23. A Mutation in the Bacillus subtilis rsbU Gene That Limits RNA Synthesis during Sporulation
- Author
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David M. Rothstein, Marcia S. Osburne, David W. Lazinski, and Abraham L. Sonenshein
- Subjects
0301 basic medicine ,Hot Temperature ,030106 microbiology ,Phosphatase ,Mutant ,Bacillus subtilis ,medicine.disease_cause ,Microbiology ,Phosphates ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,Sigma factor ,Stress, Physiological ,RNA polymerase ,medicine ,Molecular Biology ,Gene ,Genetics ,Spores, Bacterial ,Mutation ,biology ,Ethanol ,Point mutation ,Gene Expression Regulation, Bacterial ,biology.organism_classification ,Phosphoric Monoester Hydrolases ,RNA, Bacterial ,030104 developmental biology ,chemistry ,Phosphorus Radioisotopes ,Genome, Bacterial ,Research Article - Abstract
Mutants of Bacillis subtilis that are temperature sensitive for RNA synthesis during sporulation were isolated after selection with a 32 P suicide agent. Whole-genome sequencing revealed that two of the mutants carried an identical lesion in the rsbU gene, which encodes a phosphatase that indirectly activates SigB, the stress-responsive RNA polymerase sigma factor. The mutation appeared to cause RsbU to be hyperactive, because the mutants were more resistant than the parent strain to ethanol stress. In support of this hypothesis, pseudorevertants that regained wild-type levels of sporulation at high temperature had secondary mutations that prevented expression of the mutant rsbU gene. The properties of these RsbU mutants support the idea that activation of SigB diminishes the bacterium's ability to sporulate. IMPORTANCE Most bacterial species encode multiple RNA polymerase promoter recognition subunits (sigma factors). Each sigma factor directs RNA polymerase to different sets of genes; each gene set typically encodes proteins important for responses to specific environmental conditions, such as changes in temperature, salt concentration, and nutrient availability. A selection for mutants of Bacillus subtilis that are temperature sensitive for RNA synthesis during sporulation unexpectedly yielded strains with a point mutation in rsbU , a gene that encodes a protein that normally activates sigma factor B (SigB) under conditions of salt stress. The mutation appears to cause RsbU, and therefore SigB, to be active inappropriately, thereby inhibiting, directly or indirectly, the ability of the cells to transcribe sporulation genes.
- Published
- 2017
24. Immunity Provided by an Outer Membrane Vesicle Cholera Vaccine Is Due to O-Antigen-Specific Antibodies Inhibiting Bacterial Motility
- Author
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David W. Lazinski, Andrew Camilli, and Zhu Wang
- Subjects
0301 basic medicine ,Lipopolysaccharide ,030106 microbiology ,Immunology ,Motility ,medicine.disease_cause ,Microbiology ,Immunoglobulin G ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,Cholera ,Immunity ,Intestine, Small ,medicine ,Animals ,Vibrio cholerae ,Antigens, Bacterial ,Mice, Inbred BALB C ,biology ,O Antigens ,Cholera Vaccines ,Antibodies, Bacterial ,030104 developmental biology ,Infectious Diseases ,chemistry ,Microbial Immunity and Vaccines ,biology.protein ,Female ,Parasitology ,Antibody ,Cholera vaccine ,Bacterial outer membrane - Abstract
An outer membrane vesicle (OMV)-based cholera vaccine is highly efficacious in preventing intestinal colonization in the suckling mouse model. Immunity from OMVs comes from immunoglobulin (Ig), particularly IgG, in the milk of mucosally immunized dams. Anti-OMV IgG renders Vibrio cholerae organisms immotile, thus they pass through the small intestine without colonizing. However, the importance of motility inhibition for protection and the mechanism by which motility is inhibited remain unclear. By using both in vitro and in vivo experiments, we found that IgG inhibits motility by specifically binding to the O-antigen of V. cholerae . We demonstrate that the bivalent structure of IgG, although not required for binding to the O-antigen, is required for motility inhibition. Finally, we show using competition assays in suckling mice that inhibition of motility appears to be responsible for most, if not all, of the protection engendered by OMV vaccination, thus providing insight into the mechanism of immune protection.
- Published
- 2017
25. Identification ofin vivoregulators of theVibrio cholerae xdsgene using a high-throughput genetic selection
- Author
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EmilyKate McDonough, David W. Lazinski, and Andrew Camilli
- Subjects
Transposable element ,Regulation of gene expression ,Genetics ,Mutant ,Artificial Gene Fusion ,Biology ,medicine.disease_cause ,Microbiology ,Vibrio cholerae ,Gene expression ,medicine ,Molecular Biology ,Gene ,Regulator gene - Abstract
Vibrio cholerae, the causative agent of cholera, remains a threat to public health in areas with inadequate sanitation. As a waterborne pathogen, V. cholerae moves between two dissimilar environments, aquatic reservoirs and the intestinal tract of humans. Accordingly, this pathogen undergoes adaptive shifts in gene expression throughout the different stages of its lifecycle. One particular gene, xds, encodes a secreted exonuclease that was previously identified as being induced during infection. Here we sought to identify regulators responsible for the in vivo-specific induction of xds. A transcriptional fusion of xds to two consecutive antibiotic resistance genes was used to select transposon mutants that had inserted within or adjacent to regulatory genes and thereby caused increased expression of the xds fusion under non-inducing conditions. Large pools of selected insertion sites were sequenced in a high throughput manner using Tn-seq to identify potential mechanisms of xds regulation. Our selection identified the two-component system PhoB/R as the dominant activator of xds expression. In vitro validation confirmed that PhoB, a protein which is only active during phosphate limitation, was responsible for xds activation. Using xds expression as a biosensor of the extracellular phosphate level, we observed that the mouse small intestine is a phosphate-limited environment.
- Published
- 2014
26. Understanding Barriers to Borrelia burgdorferi Dissemination during Infection Using Massively Parallel Sequencing
- Author
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Linden T. Hu, Erin B. Troy, Tao Lin, David W. Lazinski, Lihui Gao, Andrew Camilli, and Steven J. Norris
- Subjects
Lipoproteins ,Immunology ,Population ,Sensitivity and Specificity ,Microbiology ,Mice ,Lyme disease ,Immune system ,Bacterial Proteins ,Immunity ,medicine ,Animals ,Borrelia burgdorferi ,education ,Skin ,Lyme Disease ,education.field_of_study ,Microbial Viability ,Innate immune system ,biology ,High-Throughput Nucleotide Sequencing ,Reproducibility of Results ,Bacterial Infections ,medicine.disease ,biology.organism_classification ,Virology ,Bacterial Load ,Immunity, Innate ,Culture Media ,Mice, Inbred C57BL ,Mutagenesis, Insertional ,Infectious Diseases ,Population bottleneck ,Host-Pathogen Interactions ,Myeloid Differentiation Factor 88 ,Lyme disease microbiology ,Female ,Parasitology ,Carrier Proteins - Abstract
Borrelia burgdorferi is an invasive spirochete that can cause acute and chronic infections in the skin, heart, joints, and central nervous system of infected mammalian hosts. Little is understood about where the bacteria encounter the strongest barriers to infection and how different components of the host immune system influence the population as the infection progresses. To identify population bottlenecks in a murine host, we utilized Tn-seq to monitor the composition of mixed populations of B. burgdorferi during infection. Both wild-type mice and mice lacking the Toll-like receptor adapter molecule MyD88 were infected with a pool of infectious B. burgdorferi transposon mutants with insertions in the same gene. At multiple time points postinfection, bacteria were isolated from the mice and the compositions of the B. burgdorferi populations at the injection site and in distal tissues determined. We identified a population bottleneck at the site of infection that significantly altered the composition of the population. The magnitude of this bottleneck was reduced in MyD88 −/− mice, indicating a role for innate immunity in limiting early establishment of B. burgdorferi infection. There is not a significant bottleneck during the colonization of distal tissues, suggesting that founder effects are limited and there is not a strict limitation on the number of organisms able to initiate populations at distal sites. These findings further our understanding of the interactions between B. burgdorferi and its murine host in the establishment of infection and dissemination of the organism.
- Published
- 2013
27. Transposon-Sequencing Analysis Unveils Novel Genes Involved in the Generation of Persister Cells in Uropathogenic Escherichia coli
- Author
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Roberto C. Molina-Quiroz, Stuart B. Levy, David W. Lazinski, and Andrew Camilli
- Subjects
0301 basic medicine ,Transposable element ,Multidrug tolerance ,Mutant ,Microbial Sensitivity Tests ,Biology ,medicine.disease_cause ,Microbiology ,Cell wall ,03 medical and health sciences ,medicine ,Uropathogenic Escherichia coli ,Pharmacology (medical) ,Escherichia coli ,Mechanisms of Action: Physiological Effects ,Pharmacology ,Mutation ,Escherichia coli Proteins ,Gene Expression Regulation, Bacterial ,Anti-Bacterial Agents ,030104 developmental biology ,Infectious Diseases ,Membrane protein ,DNA Transposable Elements ,Ampicillin ,Cell envelope - Abstract
Persister cells are highly tolerant to different antibiotics and are associated with relapsing infections. In order to understand this phenomenon further, we exposed a transposon library to a lethal concentration of ampicillin, and mutants that survived were identified by transposon sequencing (Tn-Seq). We determined that mutations related to carbon metabolism, cell envelope (cell wall generation and membrane proteins), and stress response have a role in persister cell generation.
- Published
- 2016
28. Genome‐Wide Fitness and Genetic Interactions Determined by Tn‐seq, a High‐Throughput Massively Parallel Sequencing Method for Microorganisms
- Author
-
David W. Lazinski, Tim van Opijnen, and Andrew Camilli
- Subjects
Massive parallel sequencing ,Emerging technologies ,Virology ,Parasitology ,General Medicine ,Computational biology ,Biology ,Microbiology ,Throughput (business) ,Genome - Published
- 2015
29. A bacteriophage encodes its own CRISPR/Cas adaptive response to evade host innate immunity
- Author
-
David W. Lazinski, Stephen B. Calderwood, Kimberley D. Seed, and Andrew Camilli
- Subjects
Genes, Viral ,Genomic Islands ,viruses ,Molecular Sequence Data ,Genome, Viral ,Biology ,Article ,CRISPR Spacers ,Substrate Specificity ,Bacterial genetics ,Bacteriophage ,03 medical and health sciences ,Bacteriolysis ,Immune system ,CRISPR ,Bacteriophages ,Amino Acid Sequence ,Vibrio cholerae ,030304 developmental biology ,Genetics ,0303 health sciences ,Multidisciplinary ,Innate immune system ,Base Sequence ,030306 microbiology ,Inverted Repeat Sequences ,Chromosomes, Bacterial ,biology.organism_classification ,Acquired immune system ,Biological Evolution ,Virology ,Immunity, Innate ,Lytic cycle ,Gene Deletion - Abstract
Bacteriophages (or phages) are the most abundant biological entities on earth, and are estimated to outnumber their bacterial prey by tenfold. The constant threat of phage predation has led to the evolution of a broad range of bacterial immunity mechanisms that in turn result in the evolution of diverse phage immune evasion strategies, leading to a dynamic co-evolutionary arms race. Although bacterial innate immune mechanisms against phage abound, the only documented bacterial adaptive immune system is the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system, which provides sequence-specific protection from invading nucleic acids, including phage. Here we show a remarkable turn of events, in which a phage-encoded CRISPR/Cas system is used to counteract a phage inhibitory chromosomal island of the bacterial host. A successful lytic infection by the phage is dependent on sequence identity between CRISPR spacers and the target chromosomal island. In the absence of such targeting, the phage-encoded CRISPR/Cas system can acquire new spacers to evolve rapidly and ensure effective targeting of the chromosomal island to restore phage replication.
- Published
- 2013
30. Elevated activity of the large form of ADAR1 in vivo: Very efficient RNA editing occurs in the cytoplasm
- Author
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Swee Kee Wong, Shuji Sato, and David W. Lazinski
- Subjects
Cytoplasm ,Adenosine Deaminase ,Nuclear Localization Signals ,RNA-binding protein ,In Vitro Techniques ,Biology ,Article ,Cell Line ,Substrate Specificity ,Viral Proteins ,Genes, Reporter ,medicine ,Humans ,Molecular Biology ,Gene ,Sequence Deletion ,Cell Nucleus ,Base Sequence ,RNA-Binding Proteins ,Translation (biology) ,DNA-Directed RNA Polymerases ,Molecular biology ,Recombinant Proteins ,Protein Structure, Tertiary ,Cell biology ,Kinetics ,medicine.anatomical_structure ,Viral replication ,RNA editing ,Mutagenesis, Site-Directed ,RNA ,RNA Editing ,Nucleus ,Nuclear localization sequence - Abstract
Mammalian cells express small and large forms of the RNA editing enzyme ADAR1, referred to as ADAR1-S and ADAR1-L, respectively. Here we observed that ADAR1-L was >70-fold more active than was ADAR1-S when assayed with a substrate that could be edited in either the nucleus or cytoplasm, and was also much more active when assayed with a substrate that was generated in the cytoplasm during viral replication. In contrast, when a substrate that could only be edited within the nucleus was assayed, the activity of ADAR1-S was found to be somewhat higher than that of ADAR1-L. We show here not only that editing could occur in the cytoplasm but also that the process was extremely efficient, occurred rapidly, and could occur in the absence of translation. Consistent with the observation that editing in the cytoplasm can be very efficient, deletion of the nuclear localization signal from ADAR2 resulted in a protein with 15-fold higher activity when tested with a substrate that contained an editing site in the mature message. In addition to its potential role in an antiviral response, we propose that ADAR1-L is the form primarily responsible for editing mRNAs in which the editing site is retained after processing.
- Published
- 2003
31. Genes contributing to Staphylococcus aureus fitness in abscess- and infection-related ecologies
- Author
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Ama Sadaka, Veronica N. Kos, Régis Villet, Michael S. Gilmore, David W. Lazinski, Lucy Foulston, John P. Santa Maria, Suzanne Walker, Andrew Camilli, David C. Hooper, and Michael D. Valentino
- Subjects
Transposable element ,Male ,Staphylococcus aureus ,medicine.drug_class ,Virulence Factors ,Antibiotics ,Virulence ,Biology ,medicine.disease_cause ,Microbiology ,Genome ,Mice ,Antibiotic resistance ,Bacterial Proteins ,Virology ,Drug Resistance, Bacterial ,medicine ,Animals ,RNA, Antisense ,Gene ,Gene Library ,Computational Biology ,Sequence Analysis, DNA ,Staphylococcal Infections ,QR1-502 ,Abscess ,3. Good health ,Anti-Bacterial Agents ,DNA Transposable Elements ,Vancomycin ,Genome, Bacterial ,medicine.drug ,Research Article - Abstract
Staphylococcus aureus is a leading cause of both community- and hospital-acquired infections that are increasingly antibiotic resistant. The emergence of S. aureus resistance to even last-line antibiotics heightens the need for the development of new drugs with novel targets. We generated a highly saturated transposon insertion mutant library in the genome of S. aureus and used Tn-seq analysis to probe the entire genome, with unprecedented resolution and sensitivity, for genes of importance in infection. We further identified genes contributing to fitness in various infected compartments (blood and ocular fluids) and compared them to genes required for growth in rich medium. This resulted in the identification of 426 genes that were important for S. aureus fitness during growth in infection models, including 71 genes that could be considered essential for survival specifically during infection. These findings highlight novel as well as previously known genes encoding virulence traits and metabolic pathways important for S. aureus proliferation at sites of infection, which may represent new therapeutic targets., IMPORTANCE Staphylococcus aureus continues to be a leading cause of antibiotic-resistant community and nosocomial infection. With the bacterium’s acquisition of resistance to methicillin and, more recently, vancomycin, the need for the development of new drugs with novel targets is urgent. Applying a highly saturated Tn-seq mutant library to analyze fitness and growth requirements in a murine abscess and in various infection-relevant fluids, we identified S. aureus traits that enable it to survive and proliferate during infection. This identifies potential new targeting opportunities for the development of novel therapeutics.
- Published
- 2014
32. Genome-Wide Fitness and Genetic Interactions Determined by Tn-seq, a High-Throughput Massively Parallel Sequencing Method for Microorganisms
- Author
-
David W. Lazinski, Tim van Opijnen, and Andrew Camilli
- Subjects
Genetics ,Transposable element ,DNA, Bacterial ,Massive parallel sequencing ,Mutant ,High-Throughput Nucleotide Sequencing ,Bacterial genome size ,Sequence Analysis, DNA ,Biology ,Genome ,Article ,Streptococcus pneumoniae ,Transposon mutagenesis ,Molecular Biology ,Gene ,Selection (genetic algorithm) ,Genome, Bacterial - Abstract
The lagging annotation of bacterial genomes and the inherent genetic complexity of many phenotypes is hindering the discovery of new drug targets and the development of new antimicrobials and vaccines. Here we present the method Tn-seq, with which it has become possible to quantitatively determine fitness for most genes in a microorganism and to screen for quantitative genetic interactions on a genome-wide scale and in a high-throughput fashion. Tn-seq can thus direct studies in the annotation of genes and untangle complex phenotypes. The method is based on the construction of a saturated transposon insertion library. After library selection, changes in frequency of each insertion mutant are determined by sequencing of the flanking regions en masse. These changes are used to calculate each mutant's fitness. The method was originally developed for the Gram-positive bacterium Streptococcus pneumoniae, a causative agent of pneumonia and meningitis, but has now been applied to several different microbial species.
- Published
- 2014
33. Identification of a membrane-bound transcriptional regulator that links chitin and natural competence in Vibrio cholerae
- Author
-
David W. Lazinski, Andrew Camilli, and Ankur B. Dalia
- Subjects
DNA, Bacterial ,Regulator ,Chitin ,Electrophoretic Mobility Shift Assay ,macromolecular substances ,Biology ,medicine.disease_cause ,Microbiology ,03 medical and health sciences ,chemistry.chemical_compound ,Virology ,medicine ,Transcriptional regulation ,Escherichia coli ,Vibrio cholerae ,030304 developmental biology ,Regulation of gene expression ,0303 health sciences ,030306 microbiology ,DNA Transformation Competence ,fungi ,Natural competence ,Gene Expression Regulation, Bacterial ,QR1-502 ,Recombinant Proteins ,Cell biology ,carbohydrates (lipids) ,chemistry ,Heterologous expression ,Protein Binding ,Transcription Factors ,Research Article - Abstract
Vibrio cholerae is naturally competent when grown on chitin. It is known that expression of the major regulator of competence, TfoX, is controlled by chitin; however, the molecular mechanisms underlying this requirement for chitin have remained unclear. In the present study, we identify and characterize a membrane-bound transcriptional regulator that positively regulates the small RNA (sRNA) TfoR, which posttranscriptionally enhances tfoX translation. We show that this regulation of the tfoR promoter is direct by performing electrophoretic mobility shift assays and by heterologous expression of this system in Escherichia coli. This transcriptional regulator was recently identified independently and was named “TfoS” (S. Yamamoto et al., Mol. Microbiol., in press, doi:10.1111/mmi.12462). Using a constitutively active form of TfoS, we demonstrate that the activity of this regulator is sufficient to promote competence in V. cholerae in the absence of chitin. Also, TfoS contains a large periplasmic domain, which we hypothesized interacts with chitin to regulate TfoS activity. In the heterologous host E. coli, we demonstrate that chitin oligosaccharides are sufficient to activate TfoS activity at the tfoR promoter. Collectively, these data characterize TfoS as a novel chitin-sensing transcriptional regulator that represents the direct link between chitin and natural competence in V. cholerae., IMPORTANCE Naturally competent bacteria can take up exogenous DNA from the environment and integrate it into their genome by homologous recombination. This ability to take up exogenous DNA is shared by diverse bacterial species and serves as a mechanism to acquire new genes to enhance the fitness of the organism. Several members of the family Vibrionaceae become naturally competent when grown on chitin; however, a molecular understanding of how chitin activates competence is lacking. Here, we identify a novel membrane-bound transcriptional regulator that is required for natural transformation in the human pathogen Vibrio cholerae. We demonstrate that this regulator senses chitin oligosaccharides to activate the competence cascade, thus, uncovering the molecular link between chitin and natural competence in this Vibrio species.
- Published
- 2014
34. A host-specific function is required for ligation of a wide variety of ribozyme-processed RNAs
- Author
-
Carl E. Reid and David W. Lazinski
- Subjects
viruses ,Molecular Sequence Data ,Virus ,Cell Line ,Viral Proteins ,Circular RNA ,RNA Precursors ,Humans ,RNA, Catalytic ,Ligase ribozyme ,Multidisciplinary ,Base Sequence ,biology ,Ribozyme ,RNA ,DNA-Directed RNA Polymerases ,Biological Sciences ,Blotting, Northern ,Molecular biology ,Cell biology ,Polynucleotide Ligases ,Rolling circle replication ,Mutation ,biology.protein ,Nucleic Acid Conformation ,RNA, Viral ,Hepatitis Delta Virus ,Ligation ,Hairpin ribozyme - Abstract
Hepatitis δ virus (HDV) replicates its circular RNA genome via a rolling circle mechanism. During this process, cis-acting ribozymes cleave adjacent upstream sequences and thereby resolve replication intermediates to unit-length RNA. The subsequent ligation of these 5′OH and 2′,3′-cyclic phosphate termini to form circular RNA is an essential step in the life cycle of the virus. Here we present evidence for the involvement of a host activity in the ligation of HDV RNA. We used both HDV and hammerhead ribozymes to generate a panel of HDV and non-HDV RNA substrates that bear 5′ hydroxyl and 2′,3′- cyclic phosphate termini. We found that ligation of these substrates occurred in host cells, but not in vitro or in Escherichia coli . The host-specific ligation activity was capable of joining RNA in both bimolecular and intramolecular reactions and functioned in a sequence-independent manner. We conclude that mammalian cells contain a default pathway that efficiently circularizes ribozyme processed RNAs. This pathway could be exploited in the delivery of stable antisense and decoy RNA to the nucleus.
- Published
- 2000
35. Effects of nucleotide changes on the ability of hepatitis delta virus to transcribe, process, and accumulate unit-length, circular RNA
- Author
-
H J Netter, David W. Lazinski, John M. Taylor, and Ting-Ting Wu
- Subjects
Time Factors ,Transcription, Genetic ,Molecular Sequence Data ,Immunology ,Mutant ,Microbiology ,Cell Line ,Terminal loop ,Open Reading Frames ,Transcription (biology) ,Circular RNA ,Virology ,Animals ,Humans ,Hepatitis delta Antigens ,Messenger RNA ,Base Sequence ,biology ,Hepatitis Antigens ,Ribozyme ,Wild type ,RNA ,Molecular biology ,Mutagenesis ,Insect Science ,COS Cells ,biology.protein ,Nucleic Acid Conformation ,RNA, Viral ,Hepatitis Delta Virus ,Research Article - Abstract
The circular RNA genome of hepatitis delta virus (HDV) can fold into an unbranched rodlike structure. We mutagenized the two ends of this structure and assayed the effects on the ability of the genomes to replicate and accumulate processed RNA transcripts in transfected cells. The top end, defined as that nearest to the 5' end of the putative mRNA for delta antigen, was much more sensitive than the other end, defined as the bottom. Most of the 22 mutants made at the bottom were able to accumulate RNA as well as the wild type. For deletions extending as close as 2 nucleotides (nt) from the predicted domains needed for the two ribozymes, the accumulation levels dropped to
- Published
- 1997
36. A core microbiome associated with the peritoneal tumors of pseudomyxoma peritonei
- Author
-
Traci L. Testerman, David W. Lazinski, Andrew Camilli, Kip L Bodi, Andre Dubois, D. Scott Merrell, Christopher J. Friedline, Thomas J. McAvoy, Carol Nieroda, Jeremy J. Gilbreath, Jennifer Francis, Cristina Semino-Mora, Armando Sardi, and Hui Liu
- Subjects
medicine.drug_class ,Firmicutes ,Antibiotics ,Microbiology ,RNA, Ribosomal, 16S ,medicine ,Pseudomyxoma peritonei ,Humans ,Genetics(clinical) ,Pharmacology (medical) ,Microbiome ,Genetics (clinical) ,In Situ Hybridization ,Peritoneal Neoplasms ,Medicine(all) ,Mucin-2 ,biology ,Bacteria ,Microbiota ,Research ,Mucins ,General Medicine ,Bacteria Present ,Bacterial Infections ,Sequence Analysis, DNA ,Amplicon ,medicine.disease ,biology.organism_classification ,Prognosis ,Pseudomyxoma Peritonei ,PMP ,Anti-Bacterial Agents ,Culture Media ,Survival Rate ,Treatment Outcome ,Proteobacteria ,Peritoneum ,Peritoneal cancer - Abstract
Background Pseudomyxoma peritonei (PMP) is a malignancy characterized by dissemination of mucus-secreting cells throughout the peritoneum. This disease is associated with significant morbidity and mortality and despite effective treatment options for early-stage disease, patients with PMP often relapse. Thus, there is a need for additional treatment options to reduce relapse rate and increase long-term survival. A previous study identified the presence of both typed and non-culturable bacteria associated with PMP tissue and determined that increased bacterial density was associated with more severe disease. These findings highlighted the possible role for bacteria in PMP disease. Methods To more clearly define the bacterial communities associated with PMP disease, we employed a sequenced-based analysis to profile the bacterial populations found in PMP tumor and mucin tissue in 11 patients. Sequencing data were confirmed by in situ hybridization at multiple taxonomic depths and by culturing. A pilot clinical study was initiated to determine whether the addition of antibiotic therapy affected PMP patient outcome. Main results We determined that the types of bacteria present are highly conserved in all PMP patients; the dominant phyla are the Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. A core set of taxon-specific sequences were found in all 11 patients; many of these sequences were classified into taxonomic groups that also contain known human pathogens. In situ hybridization directly confirmed the presence of bacteria in PMP at multiple taxonomic depths and supported our sequence-based analysis. Furthermore, culturing of PMP tissue samples allowed us to isolate 11 different bacterial strains from eight independent patients, and in vitro analysis of subset of these isolates suggests that at least some of these strains may interact with the PMP-associated mucin MUC2. Finally, we provide evidence suggesting that targeting these bacteria with antibiotic treatment may increase the survival of PMP patients. Conclusions Using 16S amplicon-based sequencing, direct in situ hybridization analysis and culturing methods, we have identified numerous bacterial taxa that are consistently present in all PMP patients tested. Combined with data from a pilot clinical study, these data support the hypothesis that adding antimicrobials to the standard PMP treatment could improve PMP patient survival.
- Published
- 2013
37. Genotyping 1000 yeast strains by next-generation sequencing
- Author
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Wu Wei, Stefan Wilkening, David W. Lazinski, Manu M. Tekkedil, Andrew Camilli, Gen Lin, Emilie S. Fritsch, Julien Gagneur, and Lars M. Steinmetz
- Subjects
Genotyping Techniques ,lcsh:QH426-470 ,lcsh:Biotechnology ,DNA fragmentation ,Biology ,Genome ,DNA sequencing ,Meiosis ,Yeasts ,lcsh:TP248.13-248.65 ,Genetics ,High throughput ,DNA Barcoding, Taxonomic ,Genomic library ,DNA, Fungal ,Illumina dye sequencing ,Gene Library ,Recombination, Genetic ,Massive parallel sequencing ,Methodology Article ,High-Throughput Nucleotide Sequencing ,Aneuploidy ,DNA isolation ,Yeast ,Recombination ,lcsh:Genetics ,Heat inactivation ,Next-generation sequencing ,Homologous recombination ,Biotechnology - Abstract
Background The throughput of next-generation sequencing machines has increased dramatically over the last few years; yet the cost and time for library preparation have not changed proportionally, thus representing the main bottleneck for sequencing large numbers of samples. Here we present an economical, high-throughput library preparation method for the Illumina platform, comprising a 96-well based method for DNA isolation for yeast cells, a low-cost DNA shearing alternative, and adapter ligation using heat inactivation of enzymes instead of bead cleanups. Results Up to 384 whole-genome libraries can be prepared from yeast cells in one week using this method, for less than 15 euros per sample. We demonstrate the robustness of this protocol by sequencing over 1000 yeast genomes at ~30x coverage. The sequence information from 768 yeast segregants derived from two divergent S. cerevisiae strains was used to generate a meiotic recombination map at unprecedented resolution. Comparisons to other datasets indicate a high conservation of recombination at a chromosome-wide scale, but differences at the local scale. Additionally, we detected a high degree of aneuploidy (3.6%) by examining the sequencing coverage in these segregants. Differences in allele frequency allowed us to attribute instances of aneuploidy to gains of chromosomes during meiosis or mitosis, both of which showed a strong tendency to missegregate specific chromosomes. Conclusions Here we present a high throughput workflow to sequence genomes of large number of yeast strains at a low price. We have used this workflow to obtain recombination and aneuploidy data from hundreds of segregants, which can serve as a foundation for future studies of linkage, recombination, and chromosomal aberrations in yeast and higher eukaryotes.
- Published
- 2013
38. Gene fitness landscapes of Vibrio cholerae at important stages of its life cycle
- Author
-
David W. Lazinski, Faith Wallace-Gadsden, Andrew Camilli, Bharathi Patimalla-Dipali, and Heather D. Kamp
- Subjects
lcsh:Immunologic diseases. Allergy ,Immunology ,Genetic Fitness ,Mutagenesis (molecular biology technique) ,Biology ,medicine.disease_cause ,Microbiology ,Genome ,03 medical and health sciences ,Virology ,Genetics ,medicine ,Animals ,Vibrio cholerae ,Molecular Biology ,Gene ,lcsh:QH301-705.5 ,030304 developmental biology ,Life Cycle Stages ,0303 health sciences ,Microbial Viability ,Massive parallel sequencing ,Organisms, Genetically Modified ,030306 microbiology ,High-Throughput Nucleotide Sequencing ,Water ,medicine.disease ,Adaptation, Physiological ,Cholera ,3. Good health ,Animals, Newborn ,lcsh:Biology (General) ,Host-Pathogen Interactions ,Parasitology ,Transposon mutagenesis ,Rabbits ,lcsh:RC581-607 ,Genome, Bacterial ,Research Article - Abstract
Vibrio cholerae has evolved to adeptly transition between the human small intestine and aquatic environments, leading to water-borne spread and transmission of the lethal diarrheal disease cholera. Using a host model that mimics the pathology of human cholera, we applied high density transposon mutagenesis combined with massively parallel sequencing (Tn-seq) to determine the fitness contribution of >90% of all non-essential genes of V. cholerae both during host infection and dissemination. Targeted mutagenesis and validation of 35 genes confirmed our results for the selective conditions with a total false positive rate of 4%. We identified 165 genes never before implicated for roles in dissemination that reside within pathways controlling many metabolic, catabolic and protective processes, from which a central role for glycogen metabolism was revealed. We additionally identified 76 new pathogenicity factors and 414 putatively essential genes for V. cholerae growth. Our results provide a comprehensive framework for understanding the biology of V. cholerae as it colonizes the small intestine, elicits profuse secretory diarrhea, and disseminates into the aquatic environment., Author Summary Cholera is a deadly diarrheal disease that spreads in explosive epidemics and is caused by the water-borne bacterium Vibrio cholerae. Pathogenic strains of V. cholerae can be found in both fresh and salt water estuaries in-between cholera outbreaks. Cholera infections are frequently derived from contaminated fresh water sources. In this study, we sought to determine on a genome-wide scale how V. cholerae is able to colonize and proliferate in the nutrient-rich environment of the small intestine, but then also survive dissemination and persist in the nutrient-limited aquatic environment. Using a host model that mimics the pathology of human cholera, we utilized genome-wide transposon mutagenesis and massively parallel sequencing of the insertion junctions to obtain the relative fitness of V. cholerae mutants during infection and dissemination. This extensive data set represents the first genetic screen of any kind to identify genes important for dissemination into the environment and has broad significance for understanding and controlling the spread and persistence of Vibrio cholerae and potentially other water-borne pathogens in the environment.
- Published
- 2013
39. Intracellular cleavage and ligation of hepatitis delta virus genomic RNA: regulation of ribozyme activity by cis-acting sequences and host factors
- Author
-
David W. Lazinski and John M. Taylor
- Subjects
Molecular Sequence Data ,Immunology ,RNA-dependent RNA polymerase ,Genome, Viral ,Nucleic Acid Denaturation ,Microbiology ,Virology ,Genes, Regulator ,Escherichia coli ,RNA Precursors ,Humans ,RNA, Catalytic ,Ligase ribozyme ,Base Sequence ,biology ,Ribozyme ,RNA ,RNA, Circular ,Molecular biology ,GlmS glucosamine-6-phosphate activated ribozyme ,Insect Science ,biology.protein ,RNA, Viral ,Mammalian CPEB3 ribozyme ,Hepatitis Delta Virus ,Hairpin ribozyme ,VS ribozyme ,Research Article - Abstract
During replication, a ribozyme within the genomic RNA of hepatitis delta virus cleaves multimeric precursors to release a unit-length linear intermediate. Intramolecular ligation of this intermediate produces the circular genomic RNA. Although one copy of the ribozyme is reconstituted by such ligation, it does not subsequently cleave and destroy the circular conformation. We have identified cis-acting attenuator sequences that prevent self-cleavage of the circular product by base pairing with and inactivating the ribozyme. Furthermore, we have shown that during the initial processing of the multimeric precursor RNA, host-specific factors activate the ribozyme by preventing its association with the attenuator sequences. Thus, we demonstrate a novel switching mechanism that regulates ribozyme activity inside the cell.
- Published
- 1995
40. Identification of essential genes of the periodontal pathogen Porphyromonas gingivalis
- Author
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Elizabeth L Tenorio, David W. Lazinski, Margaret J. Duncan, Linden T. Hu, Andrew Camilli, and Brian A. Klein
- Subjects
Transposable element ,lcsh:QH426-470 ,lcsh:Biotechnology ,Molecular Sequence Data ,Mutagenesis (molecular biology technique) ,Bacterial genome size ,Polymerase Chain Reaction ,Genome ,Essential genes ,03 medical and health sciences ,lcsh:TP248.13-248.65 ,Genetics ,Gene ,Porphyromonas gingivalis ,DNA Primers ,Gene Library ,030304 developmental biology ,0303 health sciences ,Genes, Essential ,Base Sequence ,biology ,030306 microbiology ,Tn-seq ,Chromosome Mapping ,Computational Biology ,High-Throughput Nucleotide Sequencing ,biology.organism_classification ,lcsh:Genetics ,Mutagenesis ,Essential gene ,DNA Transposable Elements ,Transposon mutagenesis ,Periodontal disease ,Research Article ,Biotechnology - Abstract
Background Porphyromonas gingivalis is a Gram-negative anaerobic bacterium associated with periodontal disease onset and progression. Genetic tools for the manipulation of bacterial genomes allow for in-depth mechanistic studies of metabolism, physiology, interspecies and host-pathogen interactions. Analysis of the essential genes, protein-coding sequences necessary for survival of P. gingivalis by transposon mutagenesis has not previously been attempted due to the limitations of available transposon systems for the organism. We adapted a Mariner transposon system for mutagenesis of P. gingivalis and created an insertion mutant library. By analyzing the location of insertions using massively-parallel sequencing technology we used this mutant library to define genes essential for P. gingivalis survival under in vitro conditions. Results In mutagenesis experiments we identified 463 genes in P. gingivalis strain ATCC 33277 that are putatively essential for viability in vitro. Comparing the 463 P. gingivalis essential genes with previous essential gene studies, 364 of the 463 are homologues to essential genes in other species; 339 are shared with more than one other species. Twenty-five genes are known to be essential in P. gingivalis and B. thetaiotaomicron only. Significant enrichment of essential genes within Cluster of Orthologous Groups ‘D’ (cell division), ‘I’ (lipid transport and metabolism) and ‘J’ (translation/ribosome) were identified. Previously, the P. gingivalis core genome was shown to encode 1,476 proteins out of a possible 1,909; 434 of 463 essential genes are contained within the core genome. Thus, for the species P. gingivalis twenty-two, seventy-seven and twenty-three percent of the genome respectively are devoted to essential, core and accessory functions. Conclusions A Mariner transposon system can be adapted to create mutant libraries in P. gingivalis amenable to analysis by next-generation sequencing technologies. In silico analysis of genes essential for in vitro growth demonstrates that although the majority are homologous across bacterial species as a whole, species and strain-specific subsets are apparent. Understanding the putative essential genes of P. gingivalis will provide insights into metabolic pathways and niche adaptations as well as clinical therapeutic strategies.
- Published
- 2012
41. Homopolymer tail-mediated ligation PCR: a streamlined and highly efficient method for DNA cloning and library construction
- Author
-
David W. Lazinski and Andrew Camilli
- Subjects
Genetics ,DNA, Bacterial ,Massive parallel sequencing ,Oligonucleotide ,Polynucleotides ,Computational biology ,Molecular cloning ,Biology ,Polymerase Chain Reaction ,General Biochemistry, Genetics and Molecular Biology ,Article ,Sequencing by ligation ,genomic DNA ,chemistry.chemical_compound ,Streptococcus pneumoniae ,chemistry ,Genomic library ,Primer (molecular biology) ,Cloning, Molecular ,Vibrio cholerae ,DNA ,Biotechnology ,DNA Primers ,Gene Library - Abstract
The amplification of DNA fragments, cloned between user-defined 5′ and 3′ end sequences, is a prerequisite step in the use of many current applications including massively parallel sequencing (MPS). Here we describe an improved method, called homopolymer tail-mediated ligation PCR (HTML-PCR), that requires very little starting template, minimal hands-on effort, is cost-effective, and is suited for use in high-throughput and robotic methodologies. HTML-PCR starts with the addition of homopolymer tails of controlled lengths to the 3′ termini of a double-stranded genomic template. The homopolymer tails enable the annealing-assisted ligation of a hybrid oligonucleotide to the template's recessed 5′ ends. The hybrid oligonucleotide has a user-defined sequence at its 5′ end. This primer, together with a second primer composed of a longer region complementary to the homopolymer tail and fused to a second 5′ user-defined sequence, are used in a PCR reaction to generate the final product. The user-defined sequences can be varied to enable compatibility with a wide variety of downstream applications. We demonstrate our new method by constructing MPS libraries starting from nanogram and sub-nanogram quantities of Vibrio cholerae and Streptococcus pneumoniae genomic DNA.
- Published
- 2012
42. Expression of hepatitis delta virus RNA deletions: cis and trans requirements for self-cleavage, ligation, and RNA packaging
- Author
-
John M. Taylor and David W. Lazinski
- Subjects
viruses ,Immunology ,Genome, Viral ,Virus Replication ,Cleavage (embryo) ,Microbiology ,Virus ,Structure-Activity Relationship ,Virology ,RNA, Antisense ,Antigens, Viral ,Sequence Deletion ,Ribonucleoprotein ,Hepatitis delta Antigens ,Hepatitis B Surface Antigens ,biology ,Virion ,Ribozyme ,RNA ,biochemical phenomena, metabolism, and nutrition ,Molecular biology ,Viral replication ,Virion assembly ,Insect Science ,biology.protein ,Nucleic Acid Conformation ,RNA, Viral ,Hepatitis Delta Virus ,Research Article - Abstract
The hepatitis delta virus (HDV) genome is a circular, single-stranded, rod-shaped, 1.7-kb RNA that replicates via a rolling-circle mechanism. Viral ribozymes function to cleave replication intermediates which are then ligated to generate the circular product. HDV expresses two forms of a single protein, the small and large delta antigens (delta Ag-S and delta Ag-L), which associate with viral RNA in a ribonucleoprotein (RNP) structure. While delta Ag-S is required for RNA replication, delta Ag-L inhibits this process but promotes the assembly of the RNP into mature virions. In this study, we have expressed full-length and deleted HDV RNA inside cells to determine the minimal RNA sequences required for self-cleavage, ligation, RNP packaging, and virion assembly and to assess the role of either delta antigen in each of these processes. We report the following findings. (i) The cleavage and ligation reactions did not require either delta antigen and were not inhibited in their presence. (ii) delta Ag-L, in the absence of delta Ag-S, formed an RNP with HDV RNA which could be assembled into secreted virus-like particles. (iii) Full-length HDV RNAs were stabilized in the presence of either delta antigen and accumulated to much higher levels than in their absence. (iv) As few as 348 nucleotides of HDV RNA were competent for circle formation, RNP assembly, and incorporation into virus-like particles. (v) An HDV RNA incapable of folding into the rod-like structure was not packaged by delta Ag-L.
- Published
- 1994
43. Relating structure to function in the hepatitis delta virus antigen
- Author
-
David W. Lazinski and John M. Taylor
- Subjects
DNA Mutational Analysis ,Molecular Sequence Data ,Immunology ,Fluorescent Antibody Technique ,RNA-binding protein ,Protein Sorting Signals ,Biology ,Virus Replication ,Microbiology ,DNA polymerase delta ,Virus ,Structure-Activity Relationship ,Virus antigen ,Virology ,Antigens, Viral ,Cell Nucleus ,Hepatitis delta Antigens ,Base Sequence ,RNA-Binding Proteins ,Molecular biology ,Cell Compartmentation ,Viral replication ,Insect Science ,Helper virus ,Nucleic Acid Conformation ,Hepatitis Delta Virus ,Nuclear localization sequence ,Research Article - Abstract
Hepatitis delta virus expresses two forms of a single protein, the small (delta Ag-S) and large (delta Ag-L) antigens, which are identical except for an additional 19 residues present at the C terminus of delta Ag-L. While delta Ag-S is required to promote genome replication, delta Ag-L potently inhibits this process and also facilitates packaging of the viral genome by envelope proteins of the helper virus (hepatitis B virus). Regions within the antigens responsible for nuclear localization, RNA binding, and dimerization have been identified, yet it is not clear how these particular activities contribute to the ultimate replication and packaging phenotypes. Here we report the following findings. (i) Although the removal of the nuclear localization signal from either antigen resulted in significant cytoplasmic accumulation, both proteins still had access to the nucleus. As a consequence, no functional defect was observed with either mutant. (ii) The RNA-binding domain, although necessary for delta Ag-S function, could be deleted from delta Ag-L without compromising its ability to either inhibit replication or promote packaging. (iii) In contrast, the coiled-coil dimerization domain was required for both the activation of replication by delta Ag-S and the inhibition of replication by delta Ag-L. This region, with an additional 20 amino acids C-terminal to it, was necessary and sufficient to potently inhibit replication by interacting with the small antigen. (iv) The packaging property of delta Ag-L required a C-terminal Pro/Gly-rich region which is hypothesized to interact with the hepatitis B virus envelope proteins during the assembly process.
- Published
- 1993
44. Editing on the genomic RNA of human hepatitis delta virus
- Author
-
Haoqiang Zheng, John M. Taylor, Tie-Bo Fu, and David W. Lazinski
- Subjects
Transcription, Genetic ,viruses ,Genetic Vectors ,Molecular Sequence Data ,Immunology ,RNA-dependent RNA polymerase ,Genome, Viral ,Biology ,Transfection ,Virus Replication ,Polymerase Chain Reaction ,Microbiology ,Virus ,Cell Line ,Transcription (biology) ,Virology ,Animals ,Humans ,Base Composition ,Base Sequence ,RNA ,Molecular biology ,Stop codon ,Open reading frame ,Oligodeoxyribonucleotides ,Viral replication ,RNA editing ,Insect Science ,RNA, Viral ,sense organs ,Hepatitis Delta Virus ,Research Article ,Plasmids - Abstract
It has been shown previously that during replication of the genome of human hepatitis delta virus (HDV), a specific nucleotide change occurs to eliminate the termination codon for the small delta antigen (G. Luo, M. Chao, S.-Y. Hsieh, C. Sureau, K. Nishikura, and J. Taylor, J. Virol. 64:1021-1027, 1990). This change creates an extension in the length of the open reading frame for the delta antigen from 195 to 214 amino acids. These two proteins, the small and large delta antigens, have important and distinct roles in the life cycle of HDV. To further investigate the mechanism of this specific nucleotide alteration, we developed a sensitive assay involving the polymerase chain reaction to monitor changes on HDV RNA sequences as they occurred in transfected cells. We found that the substrate for the sequence change was the viral genomic RNA rather than the antigenomic RNA. This sequence change occurred independently of genome replication or the presence of the delta antigen. Less than full-length genomic RNA could act as a substrate, but only if it also contained a corresponding RNA sequences from the other side of the rodlike structure, which is characteristic of HDV. We were also able to reproduce the HDV base change in vitro, by addition of purified viral RNA to nuclear extracts of cells from a variety of species.
- Published
- 1992
45. Roles of Carboxyl-Terminal and Farnesylated Residues in the Functions of the Large Hepatitis Delta Antigen
- Author
-
Brendan O'Malley and David W. Lazinski
- Subjects
Cytoplasm ,viruses ,Immunology ,Mutant ,Molecular Sequence Data ,Protein Prenylation ,Biology ,Microbiology ,Structure-Activity Relationship ,Viral envelope ,Virology ,Humans ,Amino Acid Sequence ,Peptide sequence ,Cells, Cultured ,Myristoylation ,Hepatitis delta Antigens ,Structure and Assembly ,Virus Assembly ,Virion ,RNA ,Fusion protein ,Biochemistry ,Insect Science ,Protein prenylation ,RNA, Viral ,lipids (amino acids, peptides, and proteins) - Abstract
The large hepatitis delta antigen (HDAg-L) mediates hepatitis delta virus (HDV) assembly and inhibits HDV RNA replication. Farnesylation of the cysteine residue within the HDAg-L carboxyl terminus is required for both functions. Here, HDAg-L proteins from different HDV genotypes and genotype chimeric proteins were analyzed for their ability to incorporate into virus-like particles (VLPs). Observed differences in efficiency of VLP incorporation could be attributed to genotype-specific differences within the HDAg-L carboxyl terminus. Using a novel assay to quantify the extent of HDAg-L farnesylation, we found that genotype 3 HDAg-L was inefficiently farnesylated when expressed in the absence of the small hepatitis delta antigen (HDAg-S). However, as the intracellular ratio of HDAg-S to HDAg-L was increased, so too was the extent of HDAg-L farnesylation for all three genotypes. Single point mutations within the carboxyl terminus of HDAg-L were screened, and three mutants that severely inhibited assembly without affecting farnesylation were identified. The observed assembly defects persisted under conditions where the mutants were known to have access to the site of VLP assembly. Therefore, the corresponding residues within the wild-type protein are likely required for direct interaction with viral envelope proteins. Finally, it was observed that when HDAg-S was artificially myristoylated, it could efficiently inhibit HDV RNA replication. Hence, a general association with membranes enables HDAg to inhibit replication. In contrast, although myristoylated HDAg-S was incorporated into VLPs far more efficiently than HDAg-S or nonfarnesylated HDAg-L, it was incorporated far less efficiently than wild-type HDAg-L; thus, farnesylation was required for efficient assembly.
- Published
- 2005
46. By Inhibiting Replication, the Large Hepatitis Delta Antigen Can Indirectly Regulate Amber/W Editing and Its Own Expression
- Author
-
Shuji Sato, David W. Lazinski, and Cromwell Cornillez-Ty
- Subjects
Gene Expression Regulation, Viral ,viruses ,Immunology ,Mutant ,Replication ,Biology ,medicine.disease_cause ,Virus Replication ,Microbiology ,Open Reading Frames ,Replication factor C ,Virology ,medicine ,Humans ,Codon ,Cells, Cultured ,Hepatitis delta Antigens ,Mutation ,Ter protein ,Wild type ,RNA ,Cell biology ,RNA editing ,Virion assembly ,Insect Science ,RNA, Viral ,RNA Editing ,Hepatitis Delta Virus - Abstract
Hepatitis delta virus (HDV) expresses two essential proteins with distinct functions. The small hepatitis delta antigen (HDAg-S) is expressed throughout replication and is needed to promote that process. The large form (HDAg-L) is farnesylated, is expressed only at later times via RNA editing of the amber/W site, and is required for virion assembly. When HDAg-L is artificially expressed at the onset of replication, it strongly inhibits replication. However, there is controversy concerning whether HDAg-L expressed naturally at later times as a consequence of editing and replication can similarly inhibit replication. Here, by stabilizing the predicted secondary structure downstream from the amber/W site, a replication-competent HDV mutant that exhibited levels of editing higher than those of the wild type was created. This mutant expressed elevated levels of HDAg-L early during replication, and at later times, its replication aborted prematurely. No further increase in amber/W editing was observed following the cessation of replication, indicating that editing was coupled to replication. A mutation in HDAg-L and a farnesyl transferase inhibitor were both used to abolish the ability of HDAg-L to inhibit replication. Such treatments rescued the replication defect of the overediting mutant, and even higher levels of amber/W editing resulted. It was concluded that when expressed naturally during replication, HDAg-L is able to inhibit replication and thereby inhibit amber/W editing and its own synthesis. In addition, the structure adjacent to the amber/W site is suboptimal for editing, and this creates a window of time in which replication can occur in the absence of HDAg-L.
- Published
- 2004
47. Determination of the multimerization state of the hepatitis delta virus antigens in vivo
- Author
-
David W. Lazinski and Cromwell Cornillez-Ty
- Subjects
Hepatitis delta Antigens ,Stereochemistry ,C-terminus ,Immunology ,Molecular Sequence Data ,Virion ,Biology ,Antiparallel (biochemistry) ,Virus Replication ,Microbiology ,Virus-Cell Interactions ,Biochemistry ,Antigen ,In vivo ,Virology ,Insect Science ,Humans ,Protein quaternary structure ,Amino Acid Sequence ,Hepatitis Delta Virus ,Peptide sequence ,Dimerization ,Cells, Cultured ,Cysteine - Abstract
Hepatitis delta virus expresses two essential proteins, the small and large delta antigens, and both are required for viral propagation. Proper function of each protein depends on the presence of a common amino-terminal multimerization domain. A crystal structure, solved using a peptide fragment that contained residues 12 to 60, depicts the formation of an octameric ring composed of antiparallel coiled-coil dimers. Because this crystal structure was solved for only a fragment of the delta antigens, it is unknown whether octamers actually form in vivo at physiological protein concentrations and in the context of either intact delta antigen. To test the relevance of the octameric structure, we developed a new method to probe coiled-coil structures in vivo. We generated a panel of mutants containing cysteine substitutions at strategic locations within the predicted monomer-monomer interface and the dimer-dimer interface. Since the small delta antigen contains no cysteine residues, treatment of cell extracts with a mild oxidizing reagent was expected to induce disulfide bond formation only when the appropriate pairs of cysteine substitution mutants were coexpressed. We indeed found that, in vivo, both the small and large delta antigens assembled as antiparallel coiled-coil dimers. Likewise, we found that both proteins could assume an octameric quaternary structure in vivo. Finally, during the course of these experiments, we found that unprenylated large delta antigen molecules could be disulfide cross-linked via the sole cysteine residue located within the carboxy terminus. Therefore, in vivo, the C terminus likely provides an additional site of protein-protein interaction for the large delta antigen.
- Published
- 2003
48. Replicating hepatitis delta virus RNA is edited in the nucleus by the small form of ADAR1
- Author
-
David W. Lazinski and Swee Kee Wong
- Subjects
DNA Replication ,Small interfering RNA ,Transcription, Genetic ,Adenosine Deaminase ,viruses ,RNA-binding protein ,Genome, Viral ,Biology ,Transfection ,Virus Replication ,Cell Line ,Humans ,RNA, Small Interfering ,DNA Primers ,Multidisciplinary ,Base Sequence ,DNA replication ,RNA ,RNA-Binding Proteins ,Biological Sciences ,Molecular biology ,Isoenzymes ,Viral replication ,RNA editing ,ADAR ,RNA, Viral ,RNA Editing ,Hepatitis Delta Virus - Abstract
Hepatitis delta virus (HDV) uses a host-encoded RNA-editing activity to express its two essential proteins from the same coding sequence. Adenosine deaminase that acts on RNA (ADAR)1 and ADAR2 are enzymes that catalyze such reactions, and each, when overexpressed, are capable of editing HDV RNA in vivo . However, the enzyme responsible for editing HDV RNA during replication has not been determined. Mammalian cells express two forms of ADAR1, a large form (ADAR1-L) that mainly localizes to the cytoplasm and a small form (ADAR1-S) that resides in the nucleus. Recently, we found that the specific activity of ADAR1-L within cells is much higher than that of ADAR1-S but only when the substrate can be edited in the cytoplasm. Here we observed that although both ADAR1-S and ADAR1-L were expressed throughout HDV replication, no ADAR2 could be observed at any time. Using expression vectors that individually overexpress either form of ADAR1, we found that ADAR1-S could stimulate editing during replication more efficiently. We next reduced ADAR1 levels during HDV replication. After transfection of an ADAR1-L-specific small interfering RNA (siRNA), we observed a significant loss of that protein and its associated cytoplasmic editing activity while the level of ADAR1-S remained unchanged. Transfection of this siRNA, however, did not reduce editing during HDV replication. In contrast, transfection of an siRNA that targets both forms of ADAR1 greatly reduced the expression of both proteins and potently inhibited editing during replication. We conclude that ADAR1-S edits HDV RNA during replication and that editing occurs in the nucleus.
- Published
- 2002
49. A Hepatitis B Surface Antigen Mutant That Lacks the Antigenic Loop Region Can Self-Assemble and Interact with the Large Hepatitis Delta Antigen
- Author
-
Brendan O'Malley and David W. Lazinski
- Subjects
Hepatitis B virus DNA polymerase ,Immunology ,Mutant ,Molecular Sequence Data ,Biology ,Microbiology ,Virus ,Hepatitis B virus PRE beta ,Antigen ,Mutant protein ,Virology ,Amino Acid Sequence ,Hepatitis delta Antigens ,Hepatitis B Surface Antigens ,Structure and Assembly ,Virus Assembly ,Hepatitis Antigens ,Virion ,Molecular biology ,Ribonucleoproteins ,Insect Science ,Mutation ,Hepatitis Delta Virus - Abstract
A novel hepatitis B virus surface antigen mutant harboring a deletion of most of the major antigenic loop region was competent for self-assembly and secretion. Although the mutant protein was competent for interaction with and incorporation of free large hepatitis delta antigen, it was partially defective in hepatitis delta virus RNP incorporation.
- Published
- 2002
50. Hepatitis delta virus minimal substrates competent for editing by ADAR1 and ADAR2
- Author
-
David W. Lazinski, Shuji Sato, and Swee Kee Wong
- Subjects
Adenosine Deaminase ,viruses ,Immunology ,Molecular Sequence Data ,Replication ,RNA-binding protein ,Biology ,Microbiology ,Cell Line ,Genes, Reporter ,Virology ,Humans ,Gene ,Hepatitis delta Antigens ,Base Sequence ,Hepatitis Antigens ,RNA ,RNA-Binding Proteins ,Molecular biology ,Stop codon ,Open reading frame ,RNA editing ,Research Design ,Insect Science ,ADAR ,Mutagenesis, Site-Directed ,RNA, Viral ,RNA Editing ,Hepatitis Delta Virus - Abstract
A host-mediated RNA-editing event allows hepatitis delta virus (HDV) to express two essential proteins, the small delta antigen (HDAg-S) and the large delta antigen (HDAg-L), from a single open reading frame. One or several members of the ADAR (adenosine deaminases that act on RNA) family are thought to convert the adenosine to an inosine (I) within the HDAg-S amber codon in antigenomic RNA. As a consequence of replication, the UIG codon is converted to a UGG (tryptophan [W]) codon in the resulting HDAg-L message. Here, we used a novel reporter system to monitor the editing of the HDV amber/W site in the absence of replication. In cultured cells, we observed that both human ADAR1 (hADAR1) and hADAR2 were capable of editing the amber/W site with comparable efficiencies. We also defined the minimal HDV substrate required for hADAR1- and hADAR2-mediated editing. Only 24 nucleotides from the amber/W site were sufficient to enable efficient editing by hADAR1. Hence, the HDV amber/W site represents the smallest ADAR substrate yet identified. In contrast, the minimal substrate competent for hADAR2-mediated editing contained 66 nucleotides.
- Published
- 2001
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