PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 16256438

A new site-specific integration system for mycobacteria.

Jeffrey Murry·Christopher M Sassetti·Jonathan Moreira·James Lane·Eric J Rubin
View original source ↗Export metadata ↓

Abstract

Site-specific integration into the mycobacterial chromosome can produce stable transformants useful for understanding pathogenesis. However, gene expression can be problematic at certain sites of integration. We have used the Streptomyces phiC31 integration system to integrate vector DNA into Mycobacterium smegmatis, M. bovis BCG, and M. tuberculosis through site-specific recombination. A single dominant insertion site was found in M. smegmatis, as previously reported. Three different insertion sites were found in M. bovis BCG. In M. smegmatis, integrated vectors appear to be far more stable than episomal plasmids during unselected passage in vitro, although excision products are detectable. Plasmids based on the phiC31 integration system could make useful tools for the study of mycobacterial genetics.

Explore related subjects

BCG VaccineBase SequenceChromosomes, BacterialGenetic EngineeringGenetic VectorsMolecular Sequence DataMutagenesis, Site-DirectedMycobacteriumMycobacterium bovisMycobacterium smegmatisMycobacterium tuberculosisPlasmidsTransformation, BacterialJournal ArticleResearch Support, N.I.H., Extramural

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Jeffrey Murry, Christopher M Sassetti, Jonathan Moreira, James Lane, Eric J Rubin. 2005-10-26. A new site-specific integration system for mycobacteria.. https://doi.org/10.1016/j.tube.2005.08.016

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2007

Mycobacterium bovis bacillus Calmette-Guérin (BCG) induces human CC- and CXC-chemokines in vitro and in vivo.

Both CC- and CXC-chemokines are known to be potent leucocyte activators and chemoattractants and play important roles in inflammatory responses. However, chemokine response to bacillus Calmette-Guérin (BCG) infection remains incompletely defined. In this study, we investigated human CC- [macrophage-derived chemokine (MDC), monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1alpha and eosinophil chemoattractant activity (eotaxin)] and CXC-interferon-inducible protein (IP)-10 chemokine production in response to BCG stimulation. BCG efficiently induced all chemokines tested in the urine of four bladder cancer patients undergoing intravesical BCG immunotherapy. The peak urinary chemokine responses occurred generally between the fourth and sixth weekly treatment, except eotaxin, which was less predictable. To evaluate the effect of BCG on induction of chemokines in vitro, urothelial cell lines and peripheral blood mononuclear cells (PBMCs) were used. Although BCG induced no or marginal chemokines from urothelial SV-HUC-1, RT4 and T24 cells, BCG-derived cytokines [interleukin (IL)-1beta, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha] induced all chemokines tested except eotaxin from these cell lines. BCG also efficiently induced all chemokines tested except eotaxin from PBMCs of both BCG-naive and BCG-vaccinated subjects. MCP-1 and MIP-1alpha emerged at 4-5 h post-BCG exposure (early chemokines); IP-10 elevated at day 1 and peaked at day 2 (intermediate chemokine); and MDC elevated at day 1 and peaked at day 7 (late chemokine). This kinetic pattern was paralleled with that of BCG-induced cytokines [early: TNF-alpha; intermediate: IL-6 and IL-10; and late: IFN-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF)]. Taken together, these results indicate that BCG directly or indirectly induces human CC- and CXC-chemokine production, which may represent one of the mechanisms by which BCG exerts its anti-tumour activity.

BCG Vaccine↗
PubMed2007

Survival bias in observational studies of the impact of routine immunizations on childhood survival.

OBJECTIVES: In situations with vaccination coverage high enough to control pertussis, observational studies have reported divergent effects of diphtheria-tetanus-pertussis (DTP) vaccination on childhood survival. We examined whether this could be because of methodological differences. METHODS: Some studies of the impact of DTP updated information on vaccination retrospectively (retrospective updating approach) while others kept vaccination status fixed for the time between follow-up visits (landmark approach). First, we conducted simulations with these approaches to investigate the impact of different mortality levels, vaccination incidence rates, intervals between data collection visits, and the proportion of children whose vaccination card had not been seen after death. Second, we re-analysed data from Guinea-Bissau using the retrospective updating approach. RESULTS: In simulations, the retrospective updating approach produced rate ratio (vaccinated/unvaccinated) estimates biased towards zero because of a differential misclassification which created survival bias as risk-free observation time was allocated to the vaccinated group. The landmark approach produced rate ratio estimates biased towards one. Biases increased with interval between data collection visits and incidence of vaccination, but were unaffected by the underlying mortality level. Survival bias increased with the proportion of dead children whose vaccination status could not be updated. The re-analysis of data from Guinea-Bissau changed the estimated impact of DTP from an 84% higher mortality using the landmark approach to a 37% lower mortality using the retrospective updating approach. CONCLUSIONS: The apparent contradiction between DTP studies could be because of methodological differences. To assess child survival associated with routine vaccinations, studies minimizing the effect of biases are warranted.

BCG Vaccine↗