scholarly journals Transformation of Rickettsia prowazekiito Rifampin Resistance

1998 ◽  
Vol 180 (8) ◽  
pp. 2118-2124 ◽  
Author(s):  
Lyudmila I. Rachek ◽  
Aimee M. Tucker ◽  
Herbert H. Winkler ◽  
David O. Wood
Keyword(s):  
Genetic Manipulation ◽  
Single Point ◽  
Initial Step ◽  
Genetic System ◽  
Single Point Mutation ◽  
Rickettsia Prowazekii ◽  
Obligate Intracellular ◽  
Pcr Products ◽  
Epidemic Typhus ◽  
Obligate Intracellular Pathogen

ABSTRACT Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular parasitic bacterium that grows directly within the cytoplasm of the eucaryotic host cell. The absence of techniques for genetic manipulation hampers the study of this organism’s unique biology and pathogenic mechanisms. To establish the feasibility of genetic manipulation in this organism, we identified a specific mutation in the rickettsial rpoB gene that confers resistance to rifampin and used it to demonstrate allelic exchange in R. prowazekii. Comparison of the rpoB sequences from the rifampin-sensitive (Rifs) Madrid E strain and a rifampin-resistant (Rifr) mutant identified a single point mutation that results in an arginine-to-lysine change at position 546 of theR. prowazekii RNA polymerase β subunit. A plasmid containing this mutation and two additional silent mutations created in codons flanking the Lys-546 codon was introduced into the Rifs Madrid E strain of R. prowazekii by electroporation, and in the presence of rifampin, resistant rickettsiae were selected. Transformation, via homologous recombination, was demonstrated by DNA sequencing of PCR products containing the three mutations in the Rifrregion of rickettsial rpoB. This is the first successful demonstration of genetic transformation of Rickettsia prowazekii and represents the initial step in the establishment of a genetic system in this obligate intracellular pathogen.

scholarly journals S-Adenosylmethionine Transport in Rickettsia prowazekii

2003 ◽  
Vol 185 (10) ◽  
pp. 3031-3035 ◽  
Author(s):  
Aimee M. Tucker ◽  
Herbert H. Winkler ◽  
Lonnie O. Driskell ◽  
David O. Wood
Keyword(s):  
Evolutionary Relationship ◽  
Host Cells ◽  
Transport Systems ◽  
Rickettsia Prowazekii ◽  
Gene Encoding ◽  
E Coli ◽  
Obligate Intracellular ◽  
Gene Coding ◽  
Epidemic Typhus ◽  
Methionine Transport

ABSTRACT Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate, intracellular, parasitic bacterium that grows within the cytoplasm of eucaryotic host cells. Rickettsiae exploit this intracellular environment by using transport systems for the compounds available in the host cell's cytoplasm. Analysis of the R. prowazekii Madrid E genome sequence revealed the presence of a mutation in the rickettsial metK gene, the gene encoding the enzyme responsible for the synthesis of S-adenosylmethionine (AdoMet). Since AdoMet is required for rickettsial processes, the apparent inability of this strain to synthesize AdoMet suggested the presence of a rickettsial AdoMet transporter. We have confirmed the presence of an AdoMet transporter in the rickettsiae which, to our knowledge, is the first bacterial AdoMet transporter identified. The influx of AdoMet into rickettsiae was a saturable process with a KT of 2.3 μM. Transport was inhibited by S-adenosylethionine and S-adenosylhomocysteine but not by sinfungin or methionine. Transport was also inhibited by 2,4-dinitrophenol, suggesting an energy-linked transport mechanism, and by N-ethylmaleimide. AdoMet transporters with similar properties were also identified in the Breinl strain of R. prowazekii and in Rickettsia typhi. By screening Escherichia coli clone banks for AdoMet transport, the R. prowazekii gene coding for a transporter, RP076 (sam), was identified. AdoMet transport in E. coli containing the R. prowazekii sam gene exhibited kinetics similar to that seen in rickettsiae. The existence of a rickettsial transporter for AdoMet raises intriguing questions concerning the evolutionary relationship between the synthesis and transport of this essential metabolite.

scholarly journals mariner-Based Transposon Mutagenesis of Rickettsia prowazekii

2007 ◽  
Vol 73 (20) ◽  
pp. 6644-6649 ◽  
Author(s):  
Zhi-Mei Liu ◽  
Aimee M. Tucker ◽  
Lonnie O. Driskell ◽  
David O. Wood
Keyword(s):  
Fluorescent Protein ◽  
Rickettsia Prowazekii ◽  
Obligate Intracellular ◽  
Transposon Insertion ◽  
Gene Coding ◽  
Epidemic Typhus ◽  
Intergenic Regions ◽  
Rifampin Resistance ◽  
Green Fluorescent ◽  
Random Transpositions

ABSTRACT Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular bacterium that grows directly within the cytoplasm of its host cell, unbounded by a vacuolar membrane. The obligate intracytoplasmic nature of rickettsial growth places severe restrictions on the genetic analysis of this distinctive human pathogen. In order to expand the repertoire of genetic tools available for the study of this pathogen, we have employed the versatile mariner-based, Himar1 transposon system to generate insertional mutants of R. prowazekii. A transposon containing the R. prowazekii arr-2 rifampin resistance gene and a gene coding for a green fluorescent protein (GFPUV) was constructed and placed on a plasmid expressing the Himar1 transposase. Electroporation of this plasmid into R. prowazekii resulted in numerous transpositions into the rickettsial genome. Transposon insertion sites were identified by rescue cloning, followed by DNA sequencing. Random transpositions integrating at TA sites in both gene coding and intergenic regions were identified. Individual rickettsial clones were isolated by the limiting-dilution technique. Using both fixed and live-cell techniques, R. prowazekii transformants expressing GFPUV were easily visible by fluorescence microscopy. Thus, a mariner-based system provides an additional mechanism for generating rickettsial mutants that can be screened using GFPUV fluorescence.

scholarly journals CRISPR-Based Genetic Manipulation of Candida Species: Historical Perspectives and Current Approaches

2021 ◽  
Vol 2 ◽  
Author(s):  
Deeva Uthayakumar ◽  
Jehoshua Sharma ◽  
Lauren Wensing ◽  
Rebecca S. Shapiro
Keyword(s):  
Large Scale ◽  
Gene Editing ◽  
Genetic Manipulation ◽  
Single Point ◽  
Antifungal Drug ◽  
Sexual Cycle ◽  
Single Point Mutation ◽  
Functional Genomic ◽  
Historical Perspectives ◽  
Genomic Studies

The Candida genus encompasses a diverse group of ascomycete fungi that have captured the attention of the scientific community, due to both their role in pathogenesis and emerging applications in biotechnology; the development of gene editing tools such as CRISPR, to analyze fungal genetics and perform functional genomic studies in these organisms, is essential to fully understand and exploit this genus, to further advance antifungal drug discovery and industrial value. However, genetic manipulation of Candida species has been met with several distinctive barriers to progress, such as unconventional codon usage in some species, as well as the absence of a complete sexual cycle in its diploid members. Despite these challenges, the last few decades have witnessed an expansion of the Candida genetic toolbox, allowing for diverse genome editing applications that range from introducing a single point mutation to generating large-scale mutant libraries for functional genomic studies. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology is among the most recent of these advancements, bringing unparalleled versatility and precision to genetic manipulation of Candida species. Since its initial applications in Candida albicans, CRISPR-Cas9 platforms are rapidly evolving to permit efficient gene editing in other members of the genus. The technology has proven useful in elucidating the pathogenesis and host-pathogen interactions of medically relevant Candida species, and has led to novel insights on antifungal drug susceptibility and resistance, as well as innovative treatment strategies. CRISPR-Cas9 tools have also been exploited to uncover potential applications of Candida species in industrial contexts. This review is intended to provide a historical overview of genetic approaches used to study the Candida genus and to discuss the state of the art of CRISPR-based genetic manipulation of Candida species, highlighting its contributions to deciphering the biology of this genus, as well as providing perspectives for the future of Candida genetics.

scholarly journals Directed Mutagenesis of the Rickettsia prowazekii pld Gene Encoding Phospholipase D

2009 ◽  
Vol 77 (8) ◽  
pp. 3244-3248 ◽  
Author(s):  
Lonnie O. Driskell ◽  
Xue-jie Yu ◽  
Lihong Zhang ◽  
Yan Liu ◽  
Vsevolod L. Popov ◽  
...  
Keyword(s):  
Vaccine Strain ◽  
Phospholipase D ◽  
Genetic Manipulation ◽  
Wild Type ◽  
Rickettsia Prowazekii ◽  
Gene Encoding ◽  
Linear Dna ◽  
Epidemic Typhus ◽  
Type Gene ◽  
Guinea Pig Model

ABSTRACT Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligately intracytoplasmic bacterium, a lifestyle that imposes significant barriers to genetic manipulation. The key to understanding how this unique bacterium evades host immunity is the mutagenesis of selected genes hypothesized to be involved in virulence. The R. prowazekii pld gene, encoding a protein with phospholipase D activity, has been associated with phagosomal escape. To demonstrate the feasibility of site-directed knockout mutagenesis of rickettsial genes and to generate a nonrevertible vaccine strain, we utilized homologous recombination to generate a pld mutant of the virulent R. prowazekii strain Madrid Evir. Using linear DNA for transformation, a double-crossover event resulted in the replacement of the rickettsial wild-type gene with a partially deleted pld gene. Linear DNA was used to prevent potentially revertible single-crossover events resulting in plasmid insertion. Southern blot and PCR analyses were used to confirm the presence of the desired mutation and to demonstrate clonality. While no phenotypic differences were observed between the mutant and wild-type strains when grown in tissue culture, the pld mutant exhibited attenuated virulence in the guinea pig model. In addition, animals immunized with the mutant strain were protected against subsequent challenge with the virulent Breinl strain, suggesting that this transformant could serve as a nonrevertible, attenuated vaccine strain. This study demonstrates the feasibility of generating site-directed rickettsial gene mutants, providing a new tool for understanding rickettsial biology and furthering advances in the prevention of epidemic typhus.

scholarly journals Rickettsia prowazekii Transports UMP and GMP, but Not CMP, as Building Blocks for RNA Synthesis

1999 ◽  
Vol 181 (10) ◽  
pp. 3238-3241 ◽  
Author(s):  
Herbert H. Winkler ◽  
Robin Daugherty ◽  
Fuquan Hu
Keyword(s):  
Rna Synthesis ◽  
De Novo ◽  
Building Blocks ◽  
Nucleotide Metabolism ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Rickettsia Prowazekii ◽  
Free Living ◽  
Obligate Intracellular ◽  
Epidemic Typhus

ABSTRACT Rickettsia prowazekii, the etiological agent of epidemic typhus, is an obligate intracellular bacterium and is apparently unable to synthesize ribonucleotides de novo. Here, we show that as an alternative, isolated, purified R. prowazekiiorganisms transported exogenous uridyl- and guanylribonucleotides and incorporated these labeled precursors into their RNA in a rifampin-sensitive manner. Transport systems for nucleotides, which we have shown previously and show here are present in rickettsiae, have never been reported in free-living bacteria, and the usual nucleobase and nucleoside transport systems are absent in rickettsiae. There was a clear preference for the monophosphate form of ribonucleotides as the transported substrate. In contrast, rickettsiae did not transport cytidylribonucleotides. The source of rickettsial CTP appears to be the transport of UMP followed by its phosphorylation and the amination of intrarickettsial UTP to CTP by CTP synthetase. A complete schema of nucleotide metabolism in rickettsiae is presented that is based on a combination of biochemical, physiological, and genetic information.

scholarly journals Widespread Mutations in Voltage-Gated Sodium Channel Gene of Cimex lectularius (Hemiptera: Cimicidae) Populations in Paris

2021 ◽  
Vol 18 (2) ◽  
pp. 407
Author(s):  
Mohammad Akhoundi ◽  
Dahlia Chebbah ◽  
Denis Sereno ◽  
Anthony Marteau ◽  
Julie Jan ◽  
...  
Keyword(s):  
Single Point ◽  
Cimex Lectularius ◽  
Single Point Mutation ◽  
Homozygous Mutation ◽  
Knockdown Resistance ◽  
Bed Bugs ◽  
Bed Bug ◽  
Channel Gene ◽  
Blood Sucking ◽  
The Status

Bed bugs, Cimex lectularius and C. hemipterus, are common blood-sucking ectoparasites of humans with a large geographical distribution, worldwide. In France, little is known about the status of bed bugs’ infestation and their resistance to insecticides, particularly, pyrethroids. Here, we aimed to find mutations in the kdr gene, known to be involved in resistance to insecticides. We gathered bed bugs from various infested locations, including 17 private houses, 12 HLM building complex, 29 apartments, 2 EHPAD, and 2 immigrants’ residences. A total of 1211 bed bugs were collected and morphologically identified as C. lectularius. Two fragments of the kdr gene, encompassing codons V419L and L925I, were successfully amplified for 156 specimens. We recorded sense mutation in the first amplified fragment (kdr1) in 89 out of 156 (57%) samples, in which in 61 out of 89 (68.5%) sequences, a change of valine (V) into leucine (L) V419L was observed. Within the second fragment (kdr2), a homozygous mutation was recorded in 73 out of 156 (46.7%) specimens at the codon 925. At this position, 43 out of 73 (58.9%) specimens had a sense mutation leading to the replacement of leucine (L) by isoleucine (I). Among 162 mutant sequences analyzed (89 for the kdr1 fragment and 73 for the kdr2 one), we detected single point mutation in 26.6%, while 73.4% presented the mutation in both kdr1 and kdr2 fragments. All modifications recorded in bed bug populations of Paris are described to be involved in the knockdown resistance (kdr) against pyrethroids.

scholarly journals A Fungal Defensin Targets the SARS−CoV−2 Spike Receptor−Binding Domain

2021 ◽  
Vol 7 (7) ◽  
pp. 553
Author(s):  
Bin Gao ◽  
Shunyi Zhu
Keyword(s):  
Receptor Binding ◽  
Viral Entry ◽  
Single Point ◽  
Binding Domain ◽  
Host Cells ◽  
Single Point Mutation ◽  
Binding Motif ◽  
Microsporum Canis ◽  
Receptor Binding Domain ◽  
Fungal Defensin

Coronavirus Disease 2019 (COVID−19) elicited by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS−CoV−2) is calling for novel targeted drugs. Since the viral entry into host cells depends on specific interactions between the receptor−binding domain (RBD) of the viral Spike protein and the membrane−bound monocarboxypeptidase angiotensin converting enzyme 2 (ACE2), the development of high affinity RBD binders to compete with human ACE2 represents a promising strategy for the design of therapeutics to prevent viral entry. Here, we report the discovery of such a binder and its improvement via a combination of computational and experimental approaches. The binder micasin, a known fungal defensin from the dermatophytic fungus Microsporum canis with antibacterial activity, can dock to the crevice formed by the receptor−binding motif (RBM) of RBD via an extensive shape complementarity interface (855.9 Å2 in area) with numerous hydrophobic and hydrogen−bonding interactions. Using microscale thermophoresis (MST) technique, we confirmed that micasin and its C−terminal γ−core derivative with multiple predicted interacting residues exhibited a low micromolar affinity to RBD. Expanding the interface area of micasin through a single point mutation to 970.5 Å2 accompanying an enhanced hydrogen bond network significantly improved its binding affinity by six−fold. Our work highlights the naturally occurring fungal defensins as an emerging resource that may be suitable for the development into antiviral agents for COVID−19.

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