Flagellar and needle complex (injectosome) loop as anti bacterial drug target
The present invention relates to a method for screening a compound that inhibits secretion of toxins into host-cell cytoplasm by virulent bacteria using a needle type III secretion system. The compound of the invention is selected by screening for a compound which interacts with a loop region of the cytoplasmic domain of the membrane protein FlhB from Salmonella typhimurium or a paralog thereof. Compositions including the compound of the invention, use of the compound, and methods of treating disorders caused by virulent bacteria are also provided.
1. A method for screening a compound that inhibits secretion of toxins into the host-cell cytoplasm by virulent bacteria using a needle type III secretion system, wherein the toxins are secreted by the needle type III secretion system and wherein the virulent bacteria is Salmonella typhimurium , the method comprising the steps of:
contacting a candidate compound with a C-terminal cytoplasmic domain of the membrane protein FlhB (SEQ ID NO: 1) from Salmonella typhimurium or an amino acid sequence variant that is at least 90% identical to SEQ ID NO: 1,
analyzing the interaction of the candidate compound with a loop region consisting of amino acids Glu, Asn, Lys, Met and Ser at positions 281 to 285 of FlhB (SEQ ID NO:1) or with the loop region of said variant that is at least 90% identical to SEQ ID NO: 1, wherein the loop region of said variant consists of amino acids Glu, Asn, Lys, Met and Ser, which correspond to positions 281 to 285 of FlhB (SEQ ID NO: 1), and
selecting a compound that reduces the flexibility of the loop region of FlhB or the amino acid sequence variant.
2. The method of claim 1 , wherein the interaction of the compound with the loop region of the cytoplasmic domain of FlhB or the amino acid sequence variant is determined by whether or not the compound differentially binds to the membrane protein FlhB from Salmonella typhimurium and its Δ(281-285) mutant protein.
3. The method of claim 1 , wherein the compound that inhibits the secretion of toxins by virulent bacteria is an antibody or a fragment thereof, an aptamer or a small molecular compound.
4. A method for screening a compound that inhibits secretion of toxins into the host-cell cytoplasm by virulent bacteria using a needle type III secretion system, wherein the toxins are secreted by the needle type III secretion system and wherein the virulent bacteria is Salmonella typhimurium , the method comprising the steps of:
i) selecting a set of candidate compounds capable of interacting with a membrane protein FlhB (SEQ ID NO: 1) or an amino acid sequence variant that is at least 90% identical to SEQ ID NO: 1 from Salmonella typhimurium , by a hydrogen bond between at least one side chain of a loop region consisting of Glu, Asn, Lys, Met and Ser at positions 281 to 285 of FlhB (SEQ ID NO:1) or with the loop region of said variant that is at least 90% identical to SEQ ID NO: 1, wherein the loop region of said variant consists of amino acids Glu, Asn, Lys, Met and Ser, which correspond to positions 281 to 285 of FlhB (SEQ ID NO: 1), and the candidate compound,
ii) contacting the candidate compounds with bacteria having a flagellar and needle type III secretion system, and
iii) selecting a compound from the set of candidate compounds that reduces secretion of proteins secreted by the type III secretion system from the bacteria and/or motility of the bacteria using a flagellum.