Methods and products for treating staphylococcal infections
AssigneeThe Brigham and Women's Hospital, Inc.
InventorsGerald B. Pier, Kimberly Jefferson
Life of this patent
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Abstract
The invention relates to nucleic acid sequences and related compositions for producing over-expression of the polysaccharide PNAG of Staphylococci. PNAG may be isolated and formulated into vaccines or used to generate antibodies. Binding agents of the nucleic acids are also described. The invention also relates to diagnostic and therapeutic methods using the compositions.
Claims133
A method of making a polysaccharide over-producing bacterium comprising
- introducing into a bacterium an ica nucleic acid operably linked to an ica regulatory nucleic acid,
- wherein the ica regulatory nucleic acid comprises
- (a) nucleic acid molecules which hybridize under stringent conditions to a nucleic acid molecule having a sequence of SEQ ID NO:2, have an addition, deletion or substitution in a region between and including nucleotides 9 and 43 of SEQ ID NO:2, and that enhance production of a polysaccharide from an ica locus, and
- (b) complements thereof.
A method of making a polysaccharide over-producing bacterium comprising
- introducing into a bacterium an ica nucleic acid operably linked to an ica regulatory nucleic acid, wherein the ica regulatory nucleic acid comprises a mutant icaR nucleic acid, and
- measuring polysaccharide production from the bacterium, wherein a high level of polysaccharide production is indicative of a polysaccharide over-producing bacterium.
A method of making a polysaccharide over-producing bacterium comprising
- recombinantly down-regulating wildtype IcaR protein production, and selecting a polysaccharide over-producing bacterium.
A method of making a polysaccharide over-producing bacterium comprising
- recombinantly altering the TATTT nucleotide sequence in the ica promoter region.
A recombinant polysaccharide over-producing bacterium comprising an ica nucleic acid operably linked to an ica regulatory nucleic acid,
- wherein the ica regulatory nucleic acid comprises
- (a) nucleic acid molecules which hybridize under stringent conditions to a nucleic acid molecule having a sequence of SEQ ID NO:2, have an addition, deletion or substitution in a region between and including nucleotides 9 and 43 of SEQ ID NO:2, and that enhance production of a polysaccharide from an ica locus, and
- (b) complements thereof.
- wherein the bacterium is not MN8m.
A recombinant polysaccharide over-producing bacterium comprising a mutant icaR nucleic acid.
An isolated nucleic acid molecule, comprising
- (a) nucleic acid molecules which hybridize under stringent conditions to a nucleic acid molecule having a sequence of SEQ ID NO:2, have an addition, deletion or substitution in a region between and including nucleotides 9 and 43 of SEQ ID NO:2, and that enhance production of a polysaccharide from an ica locus, and
- (b) complements thereof.
An isolated nucleic acid molecule selected from the group consisting of
- (a) a fragment of a nucleic acid molecule having a sequence of SEQ ID NO:1, and
- (b) complements of (a),
- wherein the fragment spans a MN8m mutation and enhances production of a polysaccharide from an ica locus when operably linked to an ica nucleic acid.
A method for identifying an isolated binding agent, comprising
- contacting a first nucleic acid molecule having the sequence of SEQ ID NO:2 or a functionally equivalent fragment thereof with a candidate molecule and determining whether the candidate molecule binds to the first nucleic acid molecule, and
- contacting a second nucleic acid molecule having the sequence of SEQ ID NO:1 or a functionally equivalent fragment thereof with the candidate molecule and determining whether the candidate molecule binds to the second nucleic acid molecule,
- wherein a candidate molecule that binds to either the first or the second nucleic acid molecule but not both is indicative of an isolated binding agent.
A method of identifying an ica promoter sequence associated with polysaccharide overproduction comprising
- detecting a nucleic acid molecule having a sequence alteration from wildtype in a region between and including nucleotides 9 and 43 of SEQ ID NO:2.
A method for identifying an ica regulatory nucleic acid molecule that enhances polysaccharide production comprising
- altering a nucleic acid molecule having a sequence of SEQ ID NO:2, and
- determining a level of reporter production by a bacterium that comprises the altered nucleic acid molecule operably linked to reporter nucleic acid,
- wherein a higher than wildtype level of reporter protein production is indicative of an ica regulatory nucleic acid molecule that enhances polysaccharide production.
A composition comprising
- an isolated binding agent that binds to a nucleic acid having a sequence of SEQ ID NO:1 with greater affinity than to SEQ ID NO:2.
A composition comprising
- an isolated binding agent that binds to a nucleic acid having a sequence of SEQ ID NO:2 with greater affinity than to SEQ ID NO:1.
A method of over-producing a protein in a bacterium comprising
- introducing into a bacterium a nucleic acid operably linked to an ica regulatory nucleic acid,
- wherein the ica regulatory nucleic acid comprises
- (a) nucleic acid molecules which hybridize under stringent conditions to a nucleic acid molecule having a sequence of SEQ ID NO:2, have an addition, deletion or substitution in a region between and including nucleotides 9 and 43 of SEQ ID NO:2, and that enhance production of a polysaccharide from an ica locus, and
- (b) complements thereof, and
- wherein the nucleic acid encodes a protein to be over-produced.
A method of over-producing a protein in a bacterium comprising
- introducing into a bacterium a nucleic acid operably linked to an ica regulatory nucleic acid, wherein the ica regulatory nucleic acid comprises a mutant icaR nucleic acid,
- wherein the nucleic acid encodes a protein to be over-produced.
Dependencies are inferred from the claim text.