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Published on: July 11, 2013
Design and construction of a Haemophilus influenzae conjugal expression system
1Department of Molecular Microbiology and Immunology. University of Missouri - Columbia, M616 Medical Science Building, DCO44.00, Columbia, MO 65212, USA. dainesd@health.missouri.edu
Abstract:
Haemophilus influenzae is a fastidious Gram-negative coccobacilli that is a common commensal in the human upper respiratory tract. However, certain strains of this bacterium, including those considered to be nontypeable (NTHi), can cause human diseases ranging from otitis media to meningitis. Although naturally competent, NTHi take up plasmids by transformation very inefficiently, if at all. Many clinical isolates have also proven refractory to the introduction of currently available shuttle vectors via electroporation. Further, it has been difficult to determine protein expression from these vectors, unless specific antisera has been raised or a phenotype conferred. To address these problems, we have designed and constructed a set of broad host range vectors that are transferable via intergeneric conjugation with an Escherichia coli strain carrying chromosomally-encoded transfer functions. These vectors provide a site for cloning promoter::MCS regions and carry genes encoding resistance to one of two different antibiotics. This conjugal system allows the expression of marker genes in NTHi strains, enabling researchers to track the microbe's progress either in vivo using the infant rat model of infection, or in vitro through invasions of human tissue culture cell lines.
Insights
Researchers developed new broad host range vectors for studying nontypeable Haemophilus influenzae (NTHi). These vectors facilitate gene expression and tracking of NTHi in infection models, overcoming previous limitations in genetic manipulation.
Area of Science:
- Microbiology
- Bacterial Genetics
- Molecular Biology
Background:
- Haemophilus influenzae, particularly nontypeable strains (NTHi), are significant human pathogens.
- NTHi exhibit inefficient natural transformation and are resistant to standard genetic manipulation techniques like electroporation.
- Existing vectors struggle with gene expression analysis in NTHi, hindering research.
Purpose of the Study:
- To design and construct novel broad host range vectors for NTHi.
- To overcome challenges in genetic manipulation and gene expression analysis of NTHi.
- To facilitate in vivo and in vitro studies of NTHi pathogenesis.
Main Methods:
- Development of broad host range vectors transferable via intergeneric conjugation from Escherichia coli.
- Inclusion of cloning sites for promoter::MCS regions.
- Incorporation of antibiotic resistance genes for selection.
Main Results:
- The new vectors enable efficient transfer and expression of marker genes in NTHi strains.
- Demonstrated successful tracking of NTHi in an infant rat model of infection.
- Validated in vitro tracking of NTHi during invasion of human tissue culture cells.
Conclusions:
- The developed conjugal system provides a robust tool for genetic studies of NTHi.
- These vectors significantly advance the ability to study NTHi virulence and host-pathogen interactions.
- Enables improved research into NTHi-related diseases and potential therapeutic strategies.
