Intracellular Parasitism by Histoplasma capsulatum: Fungal Virulence and Calcium Dependence

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Science  17 Nov 2000:
Vol. 290, Issue 5495, pp. 1368-1372
DOI: 10.1126/science.290.5495.1368


Histoplasma capsulatum is an effective intracellular parasite of macrophages and causes the most prevalent fungal respiratory disease in the United States. A “dimorphic” fungus,H. capsulatum exists as a saprophytic mold in soil and converts to the parasitic yeast form after inhalation. Only the yeasts secrete a calcium-binding protein (CBP) and can grow in calcium-limiting conditions. To probe the relation between calcium limitation and intracellular parasitism, we designed a strategy to disrupt CBP1 in H. capsulatum using a telomeric linear plasmid and a two-step genetic selection. The resultingcbp1 yeasts no longer grew when deprived of calcium, and they were also unable to destroy macrophages in vitro or proliferate in a mouse model of pulmonary infection.

Histoplasma capsulatum is a pathogenic fungus that is a major cause of respiratory and systemic mycosis, especially in immunocompromised individuals (1). Histoplasmosis occurs worldwide but is endemic in the Mississippi and Ohio River valleys in the United States, where the organism thrives in soil in its mycelial (mold) form. As with most other dimorphic fungal pathogens, conversion to a unicellular haploid yeast form occurs after inhalation and exposure to the warmer temperature of the respiratory tract (2). There, H. capsulatum is readily engulfed by macrophages, in which the yeasts survive and proliferate within the normally hostile environment of phagolysosomes (3). The characteristics of this particular intracellular compartment are poorly understood, although we have previously demonstrated that Histoplasma-laden phagolysosomes fail to acidify (4). Studies with Salmonella typhimurium, which also survives within phagolysosomes of macrophages, have suggested that this compartment is low in Ca2+ concentration (5).

The latter observation may have particular relevance for H. capsulatum, as we have observed a major difference in calcium dependence between the saprophytic (mycelial) form and the parasitic (yeast) form. Histoplasma capsulatum yeasts are capable of growing in a calcium-deprived environment and secrete a 7.8-kD calcium-binding protein (CBP); in contrast, mycelial cultures do not secrete CBP and require calcium for growth (6). The CBP structural gene, CBP1, has been cloned and sequenced, and a potential calcium binding site is predicted from the secondary structure of CBP (7). Purified CBP has also been shown to increase the association of 45CaCl2with H. capsulatum yeasts after they have been transferred to low-calcium medium (7). To verify the functional role of CBP in calcium acquisition and/or virulence, we devised a generally applicable gene-disruption strategy forHistoplasma: Linear telomeric plasmids and a two-step genetic selection were used to inactivate the CBP1 gene by allelic replacement. This work showed that CBP is critical for virulence and for calcium uptake by H. capsulatum yeasts, suggesting that this intracellular parasite has evolved a means to cope effectively with a calcium-limiting environment in vivo.

For most pathogenic fungi, classical recombinational analysis is either impossible or extremely tedious (8). Transformation ofH. capsulatum with plasmid DNA usually results in random integration of the DNA into the genome, often accompanied by tandem amplification and rearrangement of the transforming DNA (9, 10). Illegitimate recombination events so greatly outnumber homologous recombination events that it is impractical to detect the desired gene disruption; to date, only the counterselectable URA5 gene has been successfully knocked out in H. capsulatum (11). To prevent the high frequency of nonhomologous recombination, we designed a two-step gene-disruption strategy that uses a linearized telomeric vector maintained extrachromosomally in high copy number (10,12, 13). This telomeric plasmid (pTS100) contains two selectable markers, a URA5 gene located on an arm of the vector and a hygromycin-resistance cassette (hph) located within the CBP1 gene (replacing a portion of the coding sequence) [Web fig. 1 (14)] (13, 15). In the first selection step, this construct was used to transform a uracil auxotroph (16, 17) of a virulent strain of H. capsulatum (18). G186ARura5 (pTS100) transformants were initially grown on HMM agar (19) lacking uracil to select for yeasts that were maintaining the transformed DNA as a freely replicating linear plasmid. Six colony-purified yeast isolates were then inoculated into HMM broth without uracil and maintained for 3 weeks with regular medium changes. This length of time allows the desired double-crossover event to occur; the use of a linear plasmid vector ensures that single crossovers into the genome would break the chromosome and presumably be lethal. In the second step of this strategy, a positive-negative selection was applied with hygromycin and 5-fluoro-orotic acid (5-FOA), which inhibits the growth of uracil prototrophs. This step enriches for recombinants at theCBP1 locus by simultaneously selecting for stable maintenance of the disrupted gene (containing the hygromycin-resistance cassette) and selecting against the URA5-containing plasmid vector. Each of the six broth cultures was plated on solid medium containing hygromycin and 5-FOA, and an isolated colony from each culture was grown in broth under the same selection conditions for three additional weeks. Total genomic DNA was prepared from six colony-purified putative mutants and used as a template for polymerase chain reaction (PCR) and Southern analysis (20,21). The results indicate that an allelic replacement ofCBP1 with cbp1::hph had occurred in two out of six of the putative mutants (Fig. 1). This knockout strategy was repeated in the same strain (G186ARura5) and in a genetically unrelated strain, G217Bura5, with the same plasmid construct. In both cases (and despite some sequence heterogeneity with CBP1 in strain G217Bura5), a similar frequency of allelic replacement withcbp1::hph was confirmed, indicating that this strategy is an efficient and reproducible method for gene disruption inH. capsulatum.

Figure 1

(A) PCR was performed with genomic DNA isolated from pTS100 transformants and from transformation recipient strain G186ARura5. Oligonucleotide primers were targeted to the 5′ and 3′ regions of CBP1: The forward primer spanned bases 100 to 117 of CBP1 and the reverse primer spanned bases 1727 to 1747 of the CBP1 gene. A transformant in which homologous recombination had not occurred [G186ARura5 (pTS100)] showed a PCR product that resulted from retention of the telomeric plasmid (2.9 kb) and its native chromosomal CBP1 locus (1.6 kb). Hygromycin- and 5-FOA–resistant transformants G186ARura5 cbp1::hph-6 and -7 have undergone an allelic replacement in CBP1 and therefore showed a single PCR product (2.9 kb). The recipient strain G186ARura5 showed only an intactCBP1 PCR product (1.6 kb). The predicted 1.6-kbCBP1 product was also amplified from wild-typeCBP1 on plasmid pJBP13, and a 2.9-kbcbp1::hph product was amplified from disruption plasmid pTS100. For Southern analysis, restriction digests of genomic DNAs from transformants, as well as the transformation recipient strain G186ARura5, were subjected to electrophoresis, blotted, and hybridized with three probes: a 167–base pair (bp) fragment spanning the Sma I–Msc I deletion that was replaced by the hph cassette (B), a 1.3-kb fragment spanning bases 1 to 1313 fromCBP1 (C), and a 1.0-kb fragment spanning bases 1 to 1023 of the hph cassette (D). Hybridization of the CBP1 probe to DNA from transformants G186ARura5 cbp1::hph-6 and -7 did not detect the native genomicCBP1 but instead detected an 11.5-kb Xho I band or a 7.7-kb Sal I band (C). This same probe detected bands corresponding to native CBP1(a 10.3-kb Xho I fragment or a 6.5-kb Sal I fragment) in recipient strain G186ARura5 DNA (C), which did not hybridize to anhph gene probe (D). DNA from transformants 6 and 7 also did not hybridize to a 167-bp probe spanning the Sma I/Msc ICBP1 deletion, whereas DNA from recipient strain G186ARura5 hybridized to this probe (B).

To demonstrate that CBP from H. capsulatum cbp1::hph isolates was no longer able to bind calcium, we prepared 45CaCl2 blots (22). Purified CBP and CBP in culture supernatants from parental strain G186ARura5 bound45CaCl2, but proteins in culture supernatants from H. capsulatum G186ARura5 cbp1::hph-6 and -7 did not bind (Fig. 2). These results were confirmed by Ruthenium red staining for calcium-binding proteins (22, 23): Although CBP could be detected in strain G186ARura5 culture supernatant, filtrates from cbp1::hph isolates had no detectable CBP (Fig. 2). Growth of cbp1-knockout yeasts in medium deprived of calcium was measured metabolically over time by monitoring reduction of 3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (24, 25). The parent strain G186ARura5grew well under all conditions tested; however, isolates disrupted inCBP1 were unable to grow in calcium-limited medium (Fig. 3).

Figure 2

(A) Ruthenium red staining of purified CBP or culture supernatant from H. capsulatum after SDS-PAGE and transfer to nitrocellulose. (B)45CaCl2 blot of purified CBP or culture supernatant after SDS-PAGE and transfer to nitrocellulose.

Figure 3

Histoplasma capsulatum cbp1::hph yeasts were grown in HMM medium in the presence or absence of EGTA. Metabolic activity as measured by MTT assays was used to monitor culture health and growth over time. (A) G186ARura5 cbp1::hph-6, -7, and G186ARura5 cbp1::hph-6 (pTS404) exhibited growth rates similar to that of parental strain G186ARura5in standard growth medium HMM (supplemented with uracil as needed). (B) In the presence of 150 μM EGTA, growth of strains G186ARura5 cbp1::hph-6 and -7 was inhibited. The CBP1-complemented strain, G186ARura5 cbp1::hph-6 (pTS404), grew as well as parental strain G186ARura5 in the presence of EGTA.

To confirm that the phenotypes associated with thecbp1-knockouts are the result of the targeted mutation, we constructed an isogenic strain that contains CBP1 in trans. The plasmid, pTS404, was designed in the same manner as pTS100 [Web fig. 1 (14)], including 5′- and 3′-untranslated regions flanking the intact CBP1 gene. The complementation plasmid also included the Podospora URA5 gene and contained telomeric repeats for stable maintenance in Histoplasma. This plasmid was introduced by electrotransformation, and its extrachromosomal replication was subsequently confirmed by Southern blot analysis (26). The complemented strain G186ARura5 cbp1::hph-6 (pTS404) regained the ability to secrete CBP in quantities comparable to that secreted by wild-type yeasts (Fig. 2). Introduction of CBP1 in trans also restored this knockout strain's ability to grow in media deprived of calcium by EGTA (Fig. 3).

Because H. capsulatum yeasts are normally capable of proliferating in and destroying macrophages, we have developed a quantitative in vitro macrophage model for virulence that uses P388D1.D2 cells (a macrophage-like cell line) (3,27). This assay is based on the relative ability of a defined number of yeasts to kill macrophages in a given period of time (27). Strain-specific differences in the ability to kill P388D1.D2 cells correlate with virulence as measured in standard animal models of histoplasmosis. In its current version, this in vitro assay measures the amount of macrophage DNA remaining in a monolayer after infection by H. capsulatum yeasts (28), using an ultrasensitive fluorescent stain specific for double-stranded DNA. The cbp1-knockout strain was unable to destroy P388D1.D2 cells after 7 to 10 days of infection, whereas the parental strain G186ARura5 did. Complementation of thiscbp1-knockout strain with CBP1 in trans restored virulence to a level similar to that of the parent strain (Fig. 4A). Phenotypic complementation was not achieved with pTS105 (26), which is an identical plasmid contruct except for a short deletion within the CBP1coding sequence [Web fig. 1 (14)].

Figure 4

(A) Eight days after inoculation of macrophages with H. capsulatum, the host cells were lysed and then fluorescently quantitated for dsDNA. Macrophages infected with the avirulent G186ASura5 strain or thecbp1-knockout strains (G186ARura5 cbp1::hph-6 and -7) were not killed and therefore showed similar levels of relative fluorescent units (rfu). Acbp1-knockout strain complemented with wild-typeCBP1 [G186ARura5 cbp1::hph-6 (pTS404)] was virulent for macrophages, yielding results comparable to those for infection with the G186ARura5parental strain. Data shown are representative of three experiments and are expressed as the mean ± SE. (B) In this mouse model of pulmonary colonization, relative virulence of each strain can be evaluated by comparing the mean colony-forming units (CFU) inoculated intranasally and later recovered from mice at 8 days after infection. Data are representative of two experiments and are expressed as the mean ± SE per lung. The telomeric plasmid pWU55, which containsURA5, was transformed into strains G186ARura5 and G186ARura5 cbp1::hph-6 to restore uracil prototrophy, which is required for virulence in vivo (32).

Because CBP proved to be vital for Histoplasmapathogenesis in macrophages, we evaluated the virulence of acbp1-null strain of H. capsulatum in a murine model of respiratory infection (29). Mice, like many other mammals, are natural hosts for H. capsulatum, and the progression of pathology, dissemination, and immunity closely parallels human histoplasmosis (30). For animal infections, mice were inoculated intranasally with the H. capsulatum strains indicated in Fig. 4B. After 8 days, the lungs were removed to count the number of viable H. capsulatum yeasts. The number of G186ARura5 (pWU55) yeasts recovered from the lungs was more than 10-fold higher than the number administered intranasally 8 days earlier. Disrupting CBP1 in G186ARura5rendered these yeasts unrecoverable from lung tissue, except when the infecting dose was increased more than 1000-fold; even then, the number of yeasts recovered from the lung was greatly reduced from the original intranasal inoculum. These results do not reflect a simple growth defect in the cbp1-null strain, because all strains tested in mice have a similar generation time when grown in broth culture (26). When the cbp1-knockout strain was complemented by addition of CBP1 in trans, pulmonary colonization by the yeasts was restored to a level comparable to that of the virulent G186ARura5 (pWU55) strain.

In summary, CBP was indispensible for the virulence of H. capsulatum yeasts in vitro and in vivo, as well as for the growth of H. capsulatum in limiting calcium conditions. How CBP links both of these phenotypes remains unknown, but the simplest hypothesis is that calcium acquisition is an important strategy for microbial survival in this intracellular compartment. Alternatively, CBP may bind calcium in order to modulate phagolysosomal conditions that might otherwise inhibit yeast survival. For example, chelation of calcium could restrict the destructive power of some lysosomal enzymes, and a recent report shows that limiting calcium during formation of endosomes inhibits their normal acidification (31). This correlates with the failure of phagosomes containingHistoplasma to acidify (4), potentially pointing to a mechanism of avoiding intracellular destruction by lysosomal enzymes that typically have a low pK.

This study also presents a formal genetic proof of a virulence determinant in H. capsulatum. This gene-targeting strategy should be generally applicable to probing gene function in H. capsulatum and other closely related dimorphic fungal pathogens, which have similar problems in genetic manipulation that pose formidable barriers in testing the roles of putative virulence factors.

  • * To whom correspondence should be addressed. E-mail: goldman{at}


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