cretion of CBP is correlated not only with yeast/mycelial morphology but also with dependence on calcium for growth; H. capsulatum in the yeast phase is capable of growing in the presence of high levels of EGTA, whereas mycelial growth in limiting calcium is inhibited ( 11). This dependence may reflect a general problem faced by intracellular pathogens, because both Salmonella and Toxoplasma have specific responses to low calcium conditions that are duplicated during host cell parasitism ( 12, 13). Still, defining the role of CBP in Histoplasma virulence will depend on more than correlative and circumstantial evidence, and our efforts have focused on developing a formal molecular genetic proof.
In many fungi, the ability to evaluate protein function by gene disruption strategies is complicated by “illegitimate” recombination events. Simple allelic replacement is therefore difficult to detect among the high background of ectopic insertions, and marker-based selection strategies become complicated by duplications, rearrangements, and deletions that can accompany the desired recombination event. For molecular genetics in H. capsulatum, we have developed a range of strategies that are based on transformation with linear telomeric plasmids (14, 15). This genetic system has been adapted most recently as an allelic replacement tool, allowing us to disrupt the CBP1 locus in H. capsulatum and test its role in calcium acquisition and virulence.
To disrupt CBP1 in a virulent strain of H. capsulatum (G186AR), a linear telomeric plasmid was designed with several unique features to enrich for homologous recombination events. Most importantly, inverted telomeric repeats at each end of the linear plasmid help maintain the plasmid extrachromosomally and nearly eliminate ectopic integration events. For selection and disruption, the linear plasmid contains a URA5 gene, and the CBP1 gene has an internal fragment replaced by a hygromycin resistance marker (hph). In addition, over 5 kilobases of flanking DNA (upstream and downstream of the CBP1 coding sequence) was included to increase the frequency of the desired double crossover event. This construct was transformed into a uracil auxotroph of H. capsulatum G186AR, and transformants were selected initially as uracil prototrophs. Cultures were then grown in the presence of 5-fluoroorotic acid (selecting against URA5 on the plasmid vector) and hygromycin (selecting for retention of the disrupted CBP1 gene). After this two-step selection strategy, cbp1-null mutants were isolated at high frequency, and their genotypes were confirmed by PCR, Southern analysis, and protein gels (data not shown).
Two cbp1-null isolates were first tested for their ability to grow in the presence of EGTA. The growth of these knockout strains was inhibited in medium containing EGTA at a concentration as low as 150 µM (Fig. 2). (Normal growth medium used for Histoplasma contains 300 µM calcium.) In contrast, wild-type H. capsulatum strains were able to grow in medium containing greater than 1 mM EGTA. CBP1 expression was restored in one of the disrupted strains by transformation with another telomeric plasmid carrying wild-type CBP1. Complementing the knockout with CBP1 restores growth in calciumlimited medium (Fig. 2).
To determine the virulence of the CBP knockout strain, we evaluated its interaction with a macrophage-like cell line (P388D1 cells). The ability of yeast strains to kill P388D1 cells has been correlated previously with virulence as measured in a mouse model of histoplasmosis (7). When tested in this in vitro virulence model, the cbp1-null strain could be seen inside of the macrophages but was unable to destroy the macrophage monolayer. When the knockout strain was complemented with CBP1 in trans, the macrophages were killed as efficiently as they were by the wild-type virulent strain (Fig. 3).
These results describe the first successful disruption of a virulence determinant in H. capsulatum. The actual function of CBP in vivo still needs to be elucidated. CBP may act in a siderophore-like manner, binding calcium ions and providing