scholarly journals Chlamydia Genomic MinD Protein Has not the Function of Regulating Plasmid-dependent Genes as Pgp5

2018 ◽  
Vol 65 (3) ◽  
Author(s):  
Yina Sun ◽  
Jie Kong ◽  
Jingyue Ma ◽  
Manli Qi ◽  
Ying Zhang ◽  
...  
Keyword(s):  
Shuttle Vector ◽  
Immunofluorescence Assay ◽  
Developmental Cycle ◽  
Gene Products ◽  
Similar Function ◽  
Transformation System ◽  
Transcription Level ◽  
Chlamydia Muridarum ◽  
Plaque Purification ◽  
Chlamydial Genome

Chlamydia has a unique intracellular developmental cycle which has hindered the function study of Chlamydia. Transformation system of Chlamydia developed recently has greatly advanced the chlamydial function research and has been used to find chlamydial plasmid-encoded pgp5 can down-regulate plasmid-dependent genes. It is predicted that pgp5 has similar function with MinD protein encoded by chlamydial genome. However，it is unknown whether MinD has the function of regulating these plasmid-dependent genes. The pgp5 gene in the shuttle vector pGFP::CM was replaced with MinD gene of Chlamydia trachomatis (CT0582) or Chlamydia muridarum(TC0871). The recombinant plasmid was transformed into plasmid-free organisms-CMUT3.Real time PCR was used to detect the genes transcription level in these pgp5 replacement organisms. GlgA, one of the plasmid-regulated gene products was detected by the immunofluorescence assay. After recombination, transformation and plaque purification, the stable transformants CMUT3-pGFP::CM CT0582Rpgp5 and CMUT3-pGFP::CM TC0871Rpgp5 were generated. In these transformants, the plasmid-dependent genes were up-regulated, similar with those in the pgp5 premature stop mutant and pgp5 replacement with mCherry mutant. GlgA protein was also increased in all pgp5 mutants including CT0582Rpgp5 and TC0871Rpgp5. Thus, the transformation system has allowed us to identify the function of MinD that is useful for further understanding the chlamydiae. 

scholarly journals The Chlamydia muridarum plasmid revisited : new insights into growth kinetics

2018 ◽  
Vol 3 ◽  
pp. 25 ◽  
Author(s):  
Rachel J. Skilton ◽  
Yibing Wang ◽  
Colette O'Neill ◽  
Simone Filardo ◽  
Peter Marsh ◽  
...  
Keyword(s):  
Growth Kinetics ◽  
Shuttle Vector ◽  
Growth Curves ◽  
Developmental Cycle ◽  
Multiple Time ◽  
Chlamydia Muridarum ◽  
Growth Profile ◽  
Inclusion Phase ◽  
Multiple Time Points

Background:Research in chlamydial genetics is challenging because of its obligate intracellular developmental cycle.In vivosystems exist that allow studies of different aspects of basic biology of chlamydiae, the murineChlamydia muridarummodel is one of great importance and thus an essential research tool.C. muridarumcarries a plasmid that has a role in virulence.  Our aim was to compare and contrast theC. muridarumplasmid-free phenotype with that of a chromosomally isogenic plasmid-bearing strain, through the inclusion phase of the developmental cycle.Methods:We measured infectivity for plasmid bearing and plasmid-curedC. muridarumby inclusion forming assays in McCoy cells and in parallel bacterial chromosome replication by quantitative PCR, throughout the developmental cycle. In addition to these studies, we have carefully monitored chlamydial inclusion formation by confocal microscopy and transmission electron microscopy. A newE.coli/chlamydial shuttle vector (pNigg::GFP) was constructed using standard cloning technology and used to transformC. muridarumfor further phenotypic studies.Results:We have advanced the definition of the chlamydial phenotype away from the simple static observation of mature inclusions and redefined theC. muridarumplasmid-based phenotype on growth profile and inclusion morphology. Our observations on the growth properties of plasmid-curedC. muridarumchallenge the established interpretations, especially with regard to inclusion growth kinetics. Introduction of the shuttle plasmid pNigg::GFP into plasmid-curedC. muridarumrestored the wild-type plasmid-bearing phenotype and confirmed that loss of the plasmid was the sole cause for the changes in growth and chromosomal replication.Conclusions:Accurate growth curves and sampling at multiple time points throughout the developmental cycle is necessary to define plasmid phenotypes.  There are subtle but important (previously unnoticed) differences in the overall growth profile of plasmid-bearing and plasmid-freeC. muridarum.  We have proven that the differences described are solely due to the plasmid pNigg.

scholarly journals Establishment of a Tractable Genetic Transformation System in Veillonella spp.

2012 ◽  
Vol 78 (9) ◽  
pp. 3488-3491 ◽  
Author(s):  
Jinman Liu ◽  
Zhoujie Xie ◽  
Justin Merritt ◽  
Fengxia Qi
Keyword(s):  
Escherichia Coli ◽  
Genetic Transformation ◽  
Shuttle Vector ◽  
Transformation System ◽  
Genetic Studies ◽  
Content Type ◽  
Genetic Transformation System

ABSTRACTWe have constructed the firstEscherichia coli-Veillonellashuttle vector based on an endogenous plasmid (pVJL1) isolated from a clinicalVeillonellastrain. A highly transformableVeillonellastrain was also identified. Both the shuttle vector and the transformable strain should be valuable tools for futureVeillonellagenetic studies.

Identification of Plasmid-Free Chlamydia muridarum Organisms Using a Pgp3 Detection-Based Immunofluorescence Assay

2015 ◽  
Vol 25 (10) ◽  
pp. 1621-1628 ◽  
Author(s):  
Chaoqun Chen ◽  
Guangming Zhong ◽  
Lin Ren ◽  
Chunxue Lu ◽  
Zhongyu Li ◽  
...  
Keyword(s):  
Immunofluorescence Assay ◽  
Chlamydia Muridarum

scholarly journals Characterization of Murine Dendritic Cell Line JAWS II and Primary Bone Marrow-Derived Dendritic Cells in Chlamydia muridarum Antigen Presentation and Induction of Protective Immunity

2008 ◽  
Vol 76 (6) ◽  
pp. 2392-2401 ◽  
Author(s):  
Xiaozhou Jiang ◽  
Caixia Shen ◽  
Jose Rey-Ladino ◽  
Hong Yu ◽  
Robert C. Brunham
Keyword(s):  
Bone Marrow ◽  
Dendritic Cells ◽  
Antigen Presentation ◽  
Cell Line ◽  
Interleukin 12 ◽  
Developmental Cycle ◽  
Activation Markers ◽  
Chlamydia Muridarum ◽  
Primary Bone ◽  
Primary Bone Marrow

ABSTRACT Dendritic cells (DCs) appear to orchestrate much of the immunobiology of Chlamydia infection, but most studies of Chlamydia-DC interaction have been limited by the availability and heterogeneity of primary bone marrow-derived DCs (BMDCs). We therefore evaluated the immunobiology of Chlamydia muridarum infection in an immortal DC line termed JAWS II derived from BMDCs of a C57BL/6 p53-knockout mouse. JAWS II cells were permissive to the developmental cycle of Chlamydia. Infection-induced cell death was 50 to 80% less in JAWS II cells than in BMDCs. Chlamydia infected JAWS II cells and yielded infectious progeny 10-fold greater than that with primary BMDCs. JAWS II cells showed an expression pattern of cell activation markers and cytokine secretion following Chlamydia infection similar to that of primary BMDCs by up-regulating the expression of CD86, CD40, and major histocompatibility complex class II and secreting significant amounts of interleukin-12 (IL-12) but not IL-10. JAWS II cells pulsed with Chlamydia stimulated immune CD4+ T cells to secrete gamma interferon. Adoptive transfer of ex vivo Chlamydia-pulsed JAWS II cells conferred levels of immunity on C57BL/6 mice similar to those conferred by primary BMDCs. Taken together, the data show that JAWS II cells exhibit immunobiological characteristics and functions similar to those of primary BMDCs in terms of Chlamydia antigen presentation in vitro and antigen delivery in vivo. We conclude that the JAWS II cell line can substitute for primary BMDCs in Chlamydia immunobiological studies.

scholarly journals Autoregulation of the Kluyveromyces lactis pyruvate decarboxylase gene KlPDC1 involves the regulatory gene RAG3

Microbiology ◽  
2014 ◽  
Vol 160 (7) ◽  
pp. 1369-1378 ◽  
Author(s):  
Daniela Ottaviano ◽  
Chiara Micolonghi ◽  
Lorenza Tizzani ◽  
Marc Lemaire ◽  
Micheline Wésolowski-Louvel ◽  
...  
Keyword(s):  
Environmental Factors ◽  
Nuclear Localization ◽  
Transcriptional Activity ◽  
Kluyveromyces Lactis ◽  
Regulatory Gene ◽  
Pyruvate Decarboxylase ◽  
Genetic Screen ◽  
Gene Products ◽  
Transcription Level ◽  
Autoregulatory Mechanism

In the yeast Kluyveromyces lactis, the pyruvate decarboxylase gene KlPDC1 is strongly regulated at the transcription level by different environmental factors. Sugars and hypoxia act as inducers of transcription, while ethanol acts as a repressor. Their effects are mediated by gene products, some of which have been characterized. KlPDC1 transcription is also strongly repressed by its product – KlPdc1 – through a mechanism called autoregulation. We performed a genetic screen that allowed us to select and identify the regulatory gene RAG3 as a major factor in the transcriptional activity of the KlPDC1 promoter in the absence of the KlPdc1 protein, i.e. in the autoregulatory mechanism. We also showed that the two proteins Rag3 and KlPdc1 interact, co-localize in the cell and that KlPdc1 may control Rag3 nuclear localization.

scholarly journals Host Chemokine and Cytokine Response in the Endocervix within the First Developmental Cycle of Chlamydia muridarum

2009 ◽  
Vol 78 (1) ◽  
pp. 536-544 ◽  
Author(s):  
Roger G. Rank ◽  
H. Marie Lacy ◽  
Anna Goodwin ◽  
James Sikes ◽  
Judy Whittimore ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Host Response ◽  
Microscopic Analysis ◽  
Developmental Cycle ◽  
Time Interval ◽  
Expression Array ◽  
Electron Microscopic ◽  
Chlamydia Muridarum ◽  
Stage Of Development

ABSTRACT The initial host response in a primary chlamydial infection is the onset of acute inflammation. However, we still know very little about the early temporal events in the induction of the acute inflammatory response and how these events relate to the initial chlamydial developmental cycle in an actual genital infection. Because it was critical to initiate a synchronous infection in the endocervix in the first 24 h to evaluate the sequential expression of the host response, we developed the surgical methodology of depositing Chlamydia muridarum directly on the endocervix. Cervical tissue was collected at 3, 12, and 24 h after inoculation and the expression array of chemokines, cytokines, and receptors was assessed to characterize the response during the initial developmental cycle. Polymorphonuclear leukocyte (PMN) infiltration was first observed at 12 h after inoculation, and a few PMNs could be seen in the epithelium at 24 h. Electron microscopic analysis at 24 h showed that virtually all inclusions were at the same stage of development, indicating a synchronous infection. Several chemokine and cytokine genes were expressed as early as 3 h after infection, but by 12 h, 41 genes were expressed. Thus, activation of the host response occurs both with the introduction of elementary bodies into the host and early replication of reticulate bodies. No significant response was observed when UV-inactivated organisms were inoculated into the cervix at any time interval. This model provides an ideal opportunity to investigate the mechanisms by which the early inflammatory response is induced in vivo.

scholarly journals Shuttle Vector-Based Transformation System for Pyrococcus furiosus

2010 ◽  
Vol 76 (10) ◽  
pp. 3308-3313 ◽  
Author(s):  
Ingrid Waege ◽  
Georg Schmid ◽  
Sybille Thumann ◽  
Michael Thomm ◽  
Winfried Hausner
Keyword(s):  
Pyrococcus Furiosus ◽  
Shuttle Vector ◽  
Gel Filtration ◽  
Model Organism ◽  
Vector System ◽  
Transformation System ◽  
Reductase Gene ◽  
Genetic Transformation System ◽  
Coa Reductase ◽  
Pyrococcus Abyssi

ABSTRACT Pyrococcus furiosus is a model organism for analyses of molecular biology and biochemistry of archaea, but so far no useful genetic tools for this species have been described. We report here a genetic transformation system for P. furiosus based on the shuttle vector system pYS2 from Pyrococcus abyssi. In the redesigned vector, the pyrE gene from Sulfolobus was replaced as a selectable marker by the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene (HMG-CoA) conferring resistance of transformants to the antibiotic simvastatin. Use of this modified plasmid resulted in the overexpression of the HMG-CoA reductase in P. furiosus, allowing the selection of strains by growth in the presence of simvastatin. The modified shuttle vector replicated in P. furio s us, but the copy number was only one to two per chromosome. This system was used for overexpression of His6-tagged subunit D of the RNA polymerase (RNAP) in Pyrococcus cells. Functional RNAP was purified from transformed cells in two steps by Ni-NTA and gel filtration chromatography. Our data provide evidence that expression of transformed genes can be controlled from a regulated gluconeogenetic promoter.

scholarly journals Induction of the Chlamydia muridarum Stress/Persistence Response Increases Azithromycin Treatment Failure in a Murine Model of Infection

2013 ◽  
Vol 58 (3) ◽  
pp. 1782-1784 ◽  
Author(s):  
R. Phillips-Campbell ◽  
J. Kintner ◽  
R. V. Schoborg
Keyword(s):  
Stressed State ◽  
Treatment Failure ◽  
Murine Model ◽  
Developmental Cycle ◽  
Content Type ◽  
Persistently Infected ◽  
Chlamydia Muridarum

ABSTRACTViable but noninfectious (stressed/persistent) chlamydiae are more resistant to azithromycin (AZM) in culture than are organisms in the normal developmental cycle.Chlamydia muridarum-infected mice were exposed to amoxicillin to induce the organisms to enter the persistent/stressed state and subsequently treated with AZM. AZM treatment failure was observed in 22% of persistently infected mice, with an average of 321,667 inclusion-forming units (IFU) shed after AZM treatment. Productively infected mice had a 9% rate of AZM treatment failure and shed an average of 12,083 IFU. These data suggest that stressed chlamydiae are more resistant to frontline antichlamydial drugsin vivo.

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