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Identification of Haemophilus ducreyi genes expressed during human infection

Margaret E. Bauer^1,

Kate R. Fortney^2,

¹ Department of Microbiology and Immunology, Indiana University School of Medicine, 635 Barnhill Drive, Room MS420, Indianapolis, IN 46202, USA
² Department of Medicine, Indiana University School of Medicine, 545 Barnhill Drive, Room EH 435, Indianapolis, IN 46202, USA
³ The Center for Microbial Pathogenesis in The Research Institute at Nationwide Children's Hospital, 700 Children's Drive, Columbus, OH 43205-2696, USA
⁴ The Center for Microbial Interface Biology and Department of Pediatrics, The Ohio State University, 700 Children's Drive, Columbus, OH 43205-2696, USA
⁵ Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA
⁶ Center for Immunobiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA

Correspondence
Margaret E. Bauer
mebauer{at}iupui.edu

To identify Haemophilus ducreyi transcripts that are expressed during human infection, we used selective capture of transcribed sequences (SCOTS) with RNA isolated from pustules obtained from three volunteers infected with H. ducreyi, and with RNA isolated from broth-grown bacteria used to infect volunteers. With SCOTS, competitive hybridization of tissue-derived and broth-derived sequences identifies genes that may be preferentially expressed in vivo. Among the three tissue specimens, we identified 531 genes expressed in vivo. Southern blot analysis of 60 genes from each tissue showed that 87 % of the identified genes hybridized better with cDNA derived from tissue specimens than with cDNA derived from broth-grown bacteria. RT-PCR on nine additional pustules confirmed in vivo expression of 10 of 11 selected genes in other volunteers. Of the 531 genes, 139 were identified in at least two volunteers. These 139 genes fell into several functional categories, including biosynthesis and metabolism, regulation, and cellular processes, such as transcription, translation, cell division, DNA replication and repair, and transport. Detection of genes involved in anaerobic and aerobic respiration indicated that H. ducreyi likely encounters both microenvironments within the pustule. Other genes detected suggest an increase in DNA damage and stress in vivo. Genes involved in virulence in other bacterial pathogens and 32 genes encoding hypothetical proteins were identified, and may represent novel virulence factors. We identified three genes, lspA1, lspA2 and tadA, known to be required for virulence in humans. This is the first study to broadly define transcripts expressed by H. ducreyi in humans.

These authors contributed equally to this work.

A supplementary figure illustrating the SCOTS procedure, and three supplementary tables listing tissue samples used in this study, primers used in this study and genes expressed in samples from at least two volunteers, are available with the online version of this paper.