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Plasmid curing

Suneeta Chimalapati, Marcela de Souza Santos, Kelly Servage, Nicole J De Nisco, Ankur B Dalia, Kim Orth
The Gram-negative bacterium Vibrio parahaemolyticus is an opportunistic human pathogen and the leading cause of seafood borne acute gastroenteritis worldwide. Recently, this bacterium was implicated as the etiologic agent of a severe shrimp disease with consequent devastating outcomes to shrimp farming. In both cases, acquisition of genetic material via horizontal transfer provided V. parahaemolyticus with new virulence tools to cause disease. Dissecting the molecular mechanisms of V. parahaemolyticus pathogenesis often requires manipulating its genome...
March 19, 2018: Journal of Bacteriology
M Giles, S A Cawthraw, M AbuOun, C M Thomas, D Munera, M K Waldor, R M La Ragione, J M Ritchie
Objectives: To assess stability and contribution of a large ESBL-encoding IncI1 plasmid to intestinal colonization by Escherichia coli O104:H4 in two different mammalian hosts. Methods: Specific-pathogen-free 3-4-day-old New Zealand White rabbits and conventionally reared 6-week-old weaned lambs were orally infected with WT E. coli O104:H4 or the ESBL-plasmid-cured derivative, and the recovery of bacteria in intestinal homogenates and faeces monitored over time...
February 28, 2018: Journal of Antimicrobial Chemotherapy
Yu Liu, Miaoxian Zhao, Mingxing Gong, Ying Xu, Cantao Xie, Haohui Deng, Xueying Li, Hongkai Wu, Zhanhui Wang
Chronic hepatitis B virus (HBV) infection is difficult to cure due to the presence of covalently closed circular DNA (cccDNA). Accumulating evidence indicates that the CRISPR/Cas9 system effectively disrupts HBV genome, including cccDNA, in vitro and in vivo. However, efficient delivery of CRISPR/Cas9 system to the liver or hepatocytes using an adeno-associated virus (AAV) vector remains challenging due to the large size of Cas9 from Streptococcus pyogenes (Sp). The recently identified Cas9 protein from Staphylococcus aureus (Sa) is smaller than SpCas9 and thus is able to be packaged into the AAV vector...
February 16, 2018: Antiviral Research
Simon van der Els, Jennelle K James, Michiel Kleerebezem, Peter A Bron
CRISPR-Cas9 technology has been exploited for the removal or replacement of genetic elements in a wide range of prokaryotes and eukaryotes. Here we describe the extension of the Cas9 application toolbox to the industrially important dairy species Lactococcus lactis The Cas9 expression vector pLABTarget was constructed, encoding the Streptocccus pyogenes Cas9 under control of a constitutive promoter, and allowing plug and play introduction of sgRNA sequences to target specific genetic loci. Introduction of a pepN -targeting derivative of pLABTarget into L...
February 16, 2018: Applied and Environmental Microbiology
Karolina Pierzynowska, Lidia Gaffke, Aleksandra Hać, Jagoda Mantej, Natalia Niedziałek, Joanna Brokowska, Grzegorz Węgrzyn
Huntington's disease (HD) is a monogenic disorder, caused by mutations in the HTT gene which result in expansion of CAG triplets. The product of the mutated gene is misfolded huntingtin protein that forms aggregates leading to impairment of neuronal function, neurodegeneration, motor abnormalities and cognitive deficits. No effective cure is currently available for HD. Here we studied effects of genistein (trihydroxyisoflavone) on a HD cellular model consisting of HEK-293 cells transfected with a plasmid bearing mutated HTT gene...
February 12, 2018: Neuromolecular Medicine
Xiaohong Liu, Chenglong Jiao, Yue Ma, Qiyao Wang, Yuanxing Zhang
Tiger puffer (Takifugu rubripes) is becoming an economic promising aquaculture species in China. However, the development of Tiger puffer breeding industry is restricted by some serious aquatic disease such as vibriosis. An effective live attenuated vaccine MVAV6203 was developed in our previous studies by curing the virulence plasmid pEIB1 and unmarked inframe-deletion of the aroC gene from the virulent V. anguillarum. Here, we evaluated whether this live vaccine was suitable for Tiger puffer against disease caused by Vibrio genus...
February 8, 2018: Vaccine
Xin Song, He Huang, Zhiqiang Xiong, Yongjun Xia, Guangqiang Wang, Boxing Yin, Lianzhong Ai
The cryptic plasmid pLC2W was isolated from Lactobacillus casei CP002616. Nucleotide sequence analysis revealed that 4 putative open reading frames (ORF) were responsible for DNA replication. Four Escherichia coli-Lactobacillus shuttle vectors were constructed using different lengths of the pLC2W replicon to identify the shortest functional replicon. The length of the pLC2W replicon did not affect the stability of the plasmids. Green fluorescent protein (GFP) as a reporter was expressed successfully in several lactobacilli using our constructed vectors...
February 7, 2018: Journal of Dairy Science
Michael John Patton, Chih-Yu Chen, Chunfu Yang, Stuart McCorrister, Chris Grant, Garrett Westmacott, Xin-Yong Yuan, Estela Ochoa, Robert Fariss, William M Whitmire, John H Carlson, Harlan D Caldwell, Grant McClarty
Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes blinding trachoma and sexually transmitted disease. C. trachomatis isolates are classified into 2 biovars-lymphogranuloma venereum (LGV) and trachoma-which are distinguished biologically by their natural host cell infection tropism. LGV biovars infect macrophages and are invasive, whereas trachoma biovars infect oculo-urogenital epithelial cells and are noninvasive. The C. trachomatis plasmid is an important virulence factor in the pathogenesis of these infections...
January 30, 2018: MBio
Zhongliang Shen, Huijuan Yang, Sisi Yang, Wei Wang, Xiaoxian Cui, Xian Zhou, Wei Liu, Shaokun Pan, Yanfeng Liu, Junqi Zhang, Jiming Zhang, Youhua Xie, Jing Liu
Hepatitis B virus (HBV) generally causes self-limiting infection in immunocompetent adults, but establishes chronic infection in some adults and in most maternally infected infants. Factors determining clearance versus persistence are not fully understood. Hydrodynamic injection (HDI) of HBV replicon plasmid via tail vein generally results in quick clearance in immunocompetent adult mice. Here, we report the identification of strain-specific persistence of HBV in mice: one genotype B strain, designated BPS, persisted up to 33 weeks in ~50% of HDI mice...
December 14, 2017: Nature Communications
Moses Okee, Alice Bayiyana, Carol Musubika, Moses L Joloba, Fred Ashaba-Katabazi, Bernard Bagaya, Misaki Wayengera
Efficiency of artificial restriction enzymes toward curing HIV has only been separately examined, using differing delivery vehicles. We compared the in vitro transduction and target-mutagenesis efficiency of consortium plasmid and adenoviral vector delivered HIV-1 pol gene targeting zinc finger nuclease (ZFN) with CRISPR/Cas, Custom-ZFN, CRISPR-Cas-9, and plasmids and vectors (murCTSD_pZFN, pGS-U-gRNA, pCMV-Cas-D01A, Ad5-RGD); cell lines (TZM-bl and ACH-2/J-Lat cells); and the latency reversing agents prostratin, suberoylanilide hydroxamic acid, and phorbol myristate acetate...
January 2018: AIDS Research and Human Retroviruses
Koenraad Van Doorslaer, Dan Chen, Sandra Chapman, Jameela Khan, Alison A McBride
Human papillomavirus (HPV) genomes are replicated and maintained as extrachromosomal plasmids during persistent infection. The viral E2 proteins are thought to promote stable maintenance replication by tethering the viral DNA to host chromatin. However, this has been very difficult to prove genetically, as the E2 protein is involved in transcriptional regulation and initiation of replication, as well as its assumed role in genome maintenance. This makes mutational analysis of viral trans factors and cis elements in the background of the viral genome problematic and difficult to interpret...
November 21, 2017: MBio
K N Soujanya, R Siva, P Mohana Kumara, Amitava Srimany, G Ravikanth, F A Mulani, T Aarthy, H V Thulasiram, T R Santhoshkumar, Karaba N Nataraja, R Uma Shaanker
BACKGROUND: Camptothecin (CPT), a quinoline alkaloid, is a potent inhibitor of eukaryotic topoisomerase I. Because of this property, several derivatives of CPT are used as chemotherapeutic agents. CPT is produced by several plant species belonging to the Asterid clade as well as by a number of endophytic fungal associates of these plants. In this study, we report the production of CPT by four bacterial endophytes and show the possible role of a plasmid in the biosynthesis of CPT. METHODS: Endophytic bacteria were isolated from leaves, stems and fruits of Pyrenacantha volubilis Hook...
December 1, 2017: Phytomedicine: International Journal of Phytotherapy and Phytopharmacology
In Sun Hwang, Eom-Ji Oh, Donghyuk Kim, Chang-Sik Oh
Clavibacter michiganensis ssp. capsici is a Gram-positive plant-pathogenic bacterium causing bacterial canker disease in pepper. Virulence genes and mechanisms of C. michiganensis ssp. capsici in pepper have not yet been studied. To identify virulence genes of C. michiganensis ssp. capsici, comparative genome analyses with C. michiganensis ssp. capsici and its related C. michiganensis subspecies, and functional analysis of its putative virulence genes during infection were performed. The C. michiganensis ssp...
November 14, 2017: New Phytologist
Anna Pöntinen, Mariella Aalto-Araneda, Miia Lindström, Hannu Korkeala
Listeria monocytogenes is one of the most heat-resistant non-spore-forming food-borne pathogens and poses a notable risk to food safety, particularly when mild heat treatments are used in food processing and preparation. While general heat stress properties and response mechanisms of L. monocytogenes have been described, accessory mechanisms providing particular L. monocytogenes strains with the advantage of enhanced heat resistance are unknown. Here, we report plasmid-mediated heat resistance of L. monocytogenes for the first time...
November 2017: MSphere
Teng Li, Yun Liu, Yanming Sun
Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide and the second most common cause of cancer-related mortalities. With a high potential for metastasis and recurrence, HCC is refractory to cure. The present study aimed to explore the role of a recent-discovered LncRNA, AB209630, in human HCC, in order to provide new insights useful for clinical HCC diagnosis and treatment. Reverse transcription-quantitative polymerase chain reaction was performed to examine the expression of AB209630 in clinical HCC samples and the adjacent non-cancerous tissues...
October 2017: Experimental and Therapeutic Medicine
Diana C Garcia-Ramon, Colin Berry, Carmen Tse, Alberto Fernández-Fernández, Antonio Osuna, Susana Vílchez
Bacillus pumilus strain 15.1 was previously found to cause larval mortality in the Med-fly Ceratitis capitata and was shown to produce crystals in association with the spore. As parasporal crystals are well-known as invertebrate-active toxins in entomopathogenic bacteria such as Bacillus thuringiensis (Cry and Cyt toxins) and Lysinibacillus sphaericus (Bin and Cry toxins), the B. pumilus crystals were characterized. The crystals were composed of a 45 kDa protein that was identified as an oxalate decarboxylase by peptide mass fingerprinting, N-terminal sequencing and by comparison with the genome sequence of strain 15...
October 12, 2017: Microbial Biotechnology
Vicente Soriano
The huge success of current antiretroviral therapy is mediated by a triple effect: (i) Halting progression to AIDS in infected persons; (ii) reducing the risk of transmission to contacts (treatment as prevention); and (iii) minimizing the risk of HIV acquisition treating uninfected persons at risk (pre-exposure prophylaxis). However, UNAIDS has estimated that only 70% of infected people globally are diagnosed, only 53% are treated, and overall 44% have undetectable viral load, which is the necessary request for ensuring any antiretroviral benefit...
October 2017: AIDS Reviews
Robert L Kruse, Thomas Shum, Xavier Legras, Mercedes Barzi, Frank P Pankowicz, Stephen Gottschalk, Karl-Dimiter Bissig
Current therapies against hepatitis B virus (HBV) do not reliably cure chronic infection, necessitating new therapeutic approaches. The T cell response can clear HBV during acute infection, and the adoptive transfer of antiviral T cells during bone marrow transplantation can cure patients of chronic HBV infection. To redirect T cells to HBV-infected hepatocytes, we delivered plasmids encoding bispecific antibodies directed against the viral surface antigen (HBsAg) and CD3, expressed on almost all T cells, directly into the liver using hydrodynamic tail vein injection...
December 15, 2017: Molecular Therapy. Methods & Clinical Development
Alejandra Gutierrez-Guerrero, Sabina Sanchez-Hernandez, Giuseppe Galvani, Javier Pinedo-Gomez, Rocio Martin-Guerra, Almudena Sanchez-Gilabert, Araceli Aguilar-González, Marién Cobo, Philip Gregory, Michael Holmes, Karim Benabdellah, Francisco Martin
Primary immunodeficiencies, including Wiskott-Aldrich syndrome (WAS), are a main target for genome-editing strategies using specific nucleases (SNs) because a small number of corrected hematopoietic stem cells could cure patients. In this work, we have designed various WAS gene-specific CRISPR/Cas9 systems and compared their efficiency and specificity with homodimeric and heterodimeric WAS-specific zinc finger nucleases (ZFNs), using K-562 cells as a cellular model and plasmid nucleofection or integration-deficient lentiviral vectors (IDLVs) for delivery...
October 25, 2017: Human Gene Therapy
Jacob D Palmer, Emma Piattelli, Beth A McCormick, Mark W Silby, Christopher J Brigham, Vanni Bucci
Complications arising from antibiotic-resistant bacteria are becoming one of the key issues in modern medicine. Members of drug-resistant Enterobacteriaceae spp. include opportunistic pathogens (e.g., Salmonella spp.) that are among the leading causes of morbidity and mortality worldwide. Overgrowth of these bacteria is considered a hallmark of intestinal dysbiosis. Microcins (small antimicrobial peptides) produced by some gut commensals can potentially cure these conditions by inhibiting these pathogens and have been proposed as a viable alternative to antibiotic treatment...
September 20, 2017: ACS Infectious Diseases
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