An Homologue of the Human 100-kDa Protein (p100) Is Differentially Expressed by Histoplasma capsulatum during Infection of Murine Macrophages A. Porta,* ,1 S. Colonna-Romano,* I. Callebaut,† A. Franco,* L. Marzullo,* ,2 G. S. Kobayashi,‡ and B. Maresca* , ‡ ,3 *International Institute of Genetics and Biophysics, CNR, Via Marconi 12, 80125 Naples, Italy; †Laboratoire de Mineralogie–Cristallographie de Paris, Universite ´ P. & M. Curie, Paris, France; and ‡Division of Infectious Diseases, Washington University, School of Medicine, St. Louis, Missouri 63110 Received November 14, 1998 Using differential display reverse transcription- PCR (DDRT-PCR) we have identified several se- quences that are specifically expressed by His- toplasma capsulatum during infection of murine macrophages (M). Here, we report the characteriza- tion of a clone, pHc12, identified as a differentially expressed gene 1 hour after infection of M. Screening of a cDNA library of H. capsulatum allowed us to iso- late a clone, pHc12-E, that contains the complete cod- ing sequence. We show that after infection the level of transcription of this gene increases about 5 fold. Anal- ysis of its sequence revealed the presence of an open reading frame of 890 aa (ORF890) that shares respec- tively 30 and 33% identity with human and Caeno- rhabditis elegans p100 kD and rat p105 kD co-activator proteins. Using the two-dimensional Hydrophobic Cluster Analysis (HCA) method, we showed that H. capsulatum ORF890 and p100 kD co-activator proteins are clearly related. The H. capsulatum protein con- sists of a four-fold repeated module (domains I to IV) like the p100 kD co-activator proteins, whose three- dimensional (3D) structure is related to staphylococ- cal thermonuclease, followed by a modified fifth “hy- brid” domain which partially resembles the structure of the tudor domain found in multiple copies in the Drosophila melanogaster tudor protein. These data strongly suggest that ORF890 is homologous to human p100 kD and that this protein, named Hcp100, may play an essential role during infection by co-activating the expression of specific genes. © 1999 Academic Press Histoplasma capsulatum, a dimorphic fungal patho- gen, is the etiologic agent of histoplasmosis, a pulmo- nary disease that is usually asymptomatic or sub- clinical in healthy individuals but may be severe or fatal in patients who are immunosuppressed or other- wise debilitated (1). This fungal parasite is interesting in that it is able to survive and replicate within naive macrophages (M), most probably by activating spe- cific and as yet unknown mechanisms which effectively impair the natural killing activity of this cell popula- tion (2, 3, 4). To understand the genetic and regulatory processes that make H. capsulatum a successful para- site we hypothesized that, as in the case of Salmonella (5), H. capsulatum may activate specific genes whose products are effective in protecting the organisms from the natural killing response of the phagocyte. We recently applied the Differential Display Reverse Transcription-PCR (DDRT-PCR) technique to isolate several cDNAs specifically induced during attachment to—and internalization of—H. capsulatum into murine M (6, 7). One of these clones (pHc12), identified as a sequence differentially expressed as early as 15 min after infection when cells adhere to macrophage. How- ever, pHc12 showed maximal expression one hour after infection when 95% of cells where internalized. Thus a unidirectional cDNA library of H. capsulatum was used to identify the entire coding sequence. The se- quence was found to share a significant level of identity (30%) with the p100 kD co-activator proteins found in human (8), rat (9) and C. elegans (10). The nuclear p100 protein has been shown to bind to the nuclear antigen 2 (EBNA2) of the Epstein-Barr virus and co- activates gene expression mediated by the acidic do- main of the viral protein, probably by acting as a bridge between EBNA2 and the basal transcription machin- ery (8). To characterize further the H. capsulatum pro- tein, we applied Hydrophobic Cluster Analysis, HCA, 1 Present address: Georgetown University, School of Medicine, Dept. of Microbiology, Washington, DC. 2 Present address: National Institute for Basic Biology, Myodaiji- cho, Okazaki 444 Japan. 3 To whom correspondence should be addressed. Fax: (39-81) 593- 6123. E-mail: maresca@iigbna.iigb.na.cnr.it. Biochemical and Biophysical Research Communications 254, 605– 613 (1999) Article ID bbrc.1998.9894, available online at http://www.idealibrary.com on 605 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.