The LspB Protein Is Involved in the Secretion of the LspA1 and LspA2 Proteins byHaemophilus ducreyi

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Abstract
The LspA1 and LspA2 proteins ofHaemophilus ducreyi35000 are two very large macromolecules that can be detected in concentrated culture supernatant fluid. Both of these proteins exhibit homology with the N-terminal region of theBordetella pertussisfilamentous hemagglutinin (FHA), which is involved in secretion of the latter macromolecule. ThelspA2open reading frame is flanked upstream by a gene,lspB, that encodes a predicted protein with homology to theB. pertussisFhaC outer membrane protein that is involved in secretion of FHA across the outer membrane. TheH. ducreyi lspBgene encodes a protein with a predicted molecular mass of 66,573 Da. Reverse transcription-PCR analysis suggested that thelspBgene was transcribed together with thelspA2gene on a single mRNA transcript. PolyclonalH. ducreyiLspB antiserum reacted with a 64-kDa antigen present in the Sarkosyl-insoluble cell envelope fraction ofH. ducreyi35000, which indicated that the LspB protein is likely an outer membrane protein. Concentrated culture supernatant fluids fromH. ducreyi lspBandlspA1 lspBmutants did not contain detectable LspA1 and detectable LspA2, respectively. However, complementation of thelspBmutant with the wild-typelspBgene on a plasmid restored LspB protein expression and resulted in release of detectable amounts of the LspA1 protein into culture supernatant fluid. When evaluated in the temperature-dependent rabbit model of infection, thelspBmutant was attenuated in the ability to cause lesions and was never recovered in a viable form from lesions. These results indicated that theH. ducreyiLspB protein is involved in secretion of the LspA1 and LspA2 proteins across the outer membrane.