IAI FigSearch
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Other Versions of this Article:
IAI.00304-07v1
75/6/2811    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bohse, M. L.
Right arrow Articles by Woods, J. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bohse, M. L.
Right arrow Articles by Woods, J. P.

 Previous Article  |  Next Article 

Infection and Immunity, June 2007, p. 2811-2817, Vol. 75, No. 6
0019-9567/07/$08.00+0     doi:10.1128/IAI.00304-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

RNA Interference-Mediated Silencing of the YPS3 Gene of Histoplasma capsulatum Reveals Virulence Defects{triangledown}

Megan L. Bohse and Jon P. Woods*

Department of Medical Microbiology and Immunology, University of Wisconsin Medical School, Madison, Wisconsin 53706

Received 23 February 2007/ Returned for modification 6 March 2007/ Accepted 28 March 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The YPS3 gene of Histoplasma capsulatum encodes a protein that is both surface localized in the cell wall of H. capsulatum and released into the culture medium. This protein is produced only during the pathogenic yeast phase of infection and is also expressed differentially in H. capsulatum strains of different virulence levels. In this study, we silenced the YPS3 transcript by using an interfering-RNA strategy and examined the silenced mutants for phenotypic differences in vitro and during infection. The mutants showed no growth defect during in vitro culture in a defined medium at 37°C and appeared to have normal virulence in a RAW 264.7 murine macrophage-like cell line. In a C57BL/6 mouse model of infection, however, the mutants caused significantly decreased fungal burdens, particularly in the peripheral phagocyte-rich tissues of livers and spleens. This defect in organ colonization was evident within 3 days of infection; however, it appeared to be exacerbated at later time points.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Histoplasma capsulatum is a pathogenic fungus with worldwide distribution. It is the causative agent of histoplasmosis, one of the most common fungal respiratory infections in the world, with an estimated 500,000 cases per year in the United States alone. The regions where histoplasmosis is endemic include the U.S. Midwest as well as areas of South America. In the United States, the prevalence of infection reaches the highest levels along the Ohio and Mississippi river valleys, where skin test reactivity to H. capsulatum antigens indicates that more than 90% of the population has been exposed to the organism.

Environmentally, H. capsulatum is a soil-dwelling organism, often associated with the nitrogen-rich environments of bird or bat guano. Despite having no known requirement for the infection of a mammalian host as part of its life cycle, H. capsulatum is well adapted to cause respiratory and systemic diseases in mammals. It is a thermally dimorphic fungus and exists in the soil (or in the laboratory at 25°C) as a mold. After the inhalational infection of mammalian tissues (or transfer to 37°C in the laboratory), H. capsulatum transforms into its pathogenic budding-yeast phase. The mold-to-yeast transition is essential for virulence and is controlled by DRK1, the dimorphism-regulating histidine kinase gene (14). In response to phase transition, H. capsulatum changes the mRNA levels corresponding to genes associated with nutrient acquisition, thermotolerance, cell structure, and stress response, as well as the aptly named yeast phase-specific genes, many of which have no known function (5, 8-10, 12, 13, 15, 19, 20). A shotgun microarray study which analyzed approximately one-third of the genes in the Histoplasma genome found nearly 500 genes that are differentially expressed in the mold and yeast phases (11).

YPS3 is a yeast phase-specific gene originally identified in a differential hybridization screen (13). The encoded protein is found both on the H. capsulatum cell wall and is also secreted from cells (21). On the cell wall, it is surface exposed, and it gets to the surface via secretion from the cell and binding to the polysaccharide chitin (3). Beyond its yeast-phase specificity, it is also expressed only in a subset of H. capsulatum strains, and these strains are typically North American isolates that have the highest virulence levels (13). The Yps3 protein of strain G217B is 137 amino acids in length and is characterized principally by an N-terminal secretion signal sequence and a C-terminal epidermal growth factor-like domain (3).

To date, the function of Yps3 has not been determined, nor has its potential role in mammalian virulence. In this study, we silenced the YPS3 gene by using RNA interference (RNAi) and analyzed its effects on in vitro growth and mammalian infection. Our results indicate that YPS3-silenced mutants are defective in organ colonization in a mouse model of infection and that this deficiency is exacerbated in peripheral phagocyte-rich tissues.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and culture conditions. (i) Yeast strains. H. capsulatum strain G217B ura5-23 is a uracil auxotroph of the restriction fragment length polymorphism class 2 strain G217B (ATCC 26032) derived by UV radiation mutagenesis and has been described previously (18). H. capsulatum was grown in solid or broth Histoplasma-macrophage medium (HMM), a rich defined medium (23). Solid medium contained 0.5% agarose (SeaKem LE) and 10 µM supplemental FeSO4. In the case of uracil auxotrophs, the medium included 0.1 mg of uracil/ml. All cells were grown in a 5% CO2-95% air atmosphere. For growth curve analysis, yeast cells were taken from late-log-phase cultures and resuspended at a concentration of 3 x 105 cells/ml in 20 ml of HMM (A600 of 1 corresponds to 2.24 x 108 CFU/ml) (data not shown). Culture turbidity was monitored with a photoelectric colorimeter (Manostat Corporation, New York, NY).

(ii) Bacterial strain. Plasmids were cloned and propagated in the Escherichia coli strain JM109 [(F' traD36 proA+B+ lacIqZ{Delta}M15) {Delta}(lac-proAB) glnV44 e14– gyrA96 recA1 relA1 endA1 thi hsdR17].

(iii) Mammalian cells. The mammalian cell line used in this study was RAW 264.7 (ATCC TIB-71), a murine macrophage-like cell line acquired from the American Type Culture Collection. RAW 264.7 cells were grown in RPMI medium (Cellgro, Herndon, VA) supplemented with 10% heat-inactivated fetal calf serum (Invitrogen, Carlsbad, CA).

DNA preparation. Plasmids were prepared from E. coli by using an alkaline lysis QIAprep8 miniprep kit procedure according to the recommendations of the kit manufacturer (QIAGEN, Valencia, CA). DNA from agarose gels was purified by using the QIAquick silica gel extraction kit (QIAGEN). DNA was isolated from H. capsulatum by using a MasterPure yeast DNA purification kit according to the directions of the manufacturer (Epicenter, Madison, WI).

Plasmid construction. The RNAi plasmids pYPS610 and pYPS620 (Fig. 1) were based on the previously described telomeric shuttle plasmid pWU45 (18). This vector contains the Podospora anserina URA5 gene and a telomeric cassette for selection and maintenance in H. capsulatum and the ampicillin resistance gene for selection in E. coli. RNAi silencing vectors were created as follows. A fragment of 517 bp of the predicted YPS3 cDNA sequence was cloned in an inverted orientation into pBluescript SK(+) (Stratagene, La Jolla, CA) digested with KpnI-XhoI and SacII-NotI, leaving a 78-bp region of the pBluescript SK(+) plasmid as a spacer. The trpC terminator was then PCR amplified from the previously described vector pAn7-1 (16) and cloned into the construct after digestion with SacII and SacI. These inverted repeat constructs were then cloned behind the promoter region corresponding to either CBP1 (pYPS610) or H2B (pYPS620) and moved into plasmid pWU45 digested with NheI and SphI. Constructs were verified by sequencing. All PCR products were amplified using the high-fidelity polymerase Triplemaster (Eppendorf, Westbury, NY).


Figure 1
View larger version (20K):
[in this window]
[in a new window]

  		FIG. 1. YPS3-silencing plasmids. These plasmids contain 517 bp of the predicted YPS3 cDNA sequence cloned in an inverted orientation and driven by the CBP1 (pYPS610) or H2B (pYPS620) promoter. They include inverted telomeres and the Podospora anserina URA5 gene for selection in H. capsulatum.

 
H. capsulatum transformation and screening. The G217B ura5-23 strain was electrotransformed with linearized plasmid as previously described (23). Briefly, cells were grown for 42 h with shaking at 37°C, washed once with 10% mannitol, and electroporated with HpaI-digested, ethanol-precipitated pYPS610, pYPS620, or pWU45 as an empty-vector control in a Gene Pulser electroporator (Bio-Rad, Hercules, CA). Following transformation, cells were spread onto HMM plates and grown for 2 to 3 weeks at 37°C. Following growth on plates, individual colonies were selected and grown in liquid medium. Three-day liquid cultures of 72 transformants were screened and assayed via Western blotting for secreted protein by using our previously described chitin binding assay (3). Briefly, a portion of the culture supernatant was mixed with the polysaccharide chitin (Sigma-Aldrich, St. Louis, MO), and the mixture was incubated for 1 h at room temperature. Following incubation, the adsorbed supernatant was separated by centrifugation and removed and the protein bound to the remaining chitin pellet was denatured, run on a sodium dodecyl sulfate (SDS)-polyacrylamide gel, and Western blotted as previously described (3). Briefly, samples were denatured by incubation in 50 mM Tris-Cl containing 2% SDS, 100 mM dithiothreitol, 10% glycerol, and 0.1% bromophenol blue for 5 min at 95°C, electrophoresed on an SDS-12% polyacrylamide gel, and electroblotted onto a nitrocellulose membrane. This membrane was blocked for 1 h with Tris-buffered saline (25 mM Tris, 123 mM NaCl, 2.7 mM KCl) containing 0.01% SDS, 0.05% Tween 20, and 5% dried milk. The blot was then incubated with a 1:10,000 dilution of Yps3-specific antiserum. Nonspecific antibody was removed by washing three times for 20 min each in 0.01% SDS and 0.05% Tween 20 in Tris-buffered saline. The washed nitrocellulose membrane was incubated in a 1:6,000 dilution of horseradish peroxidase-labeled goat anti-rabbit antibody (Bio-Rad). Strains that appeared to have low or undetectable levels of secreted Yps3 were rescreened, and three mutants with low to undetectable Yps3 levels were selected, analyzed by Southern blotting for confirmation of transformation, and colony purified.

Virulence assays. The virulence of H. capsulatum in RAW 264.7 cells was measured as the extent of host cell destruction as reflected by the percentage of residual viable host cells by monitoring the uptake of the thymidine analog bromodeoxyuridine (BrdU; Roche, Indianapolis, IN). RAW 264.7 cells were plated at a density of 5 x 104 cells per well in 96-well plates and allowed to adhere overnight. H. capsulatum yeast cultures at 37°C were grown for 48 h, diluted to a concentration of 5 x 106 cells/ml in serum-free RPMI medium, and added to the wells containing host cells at multiplicities of infection of 0.5, 5, and 10. The plates were placed at 37°C, and infection was allowed to progress for 4 h. After 4 h, uninternalized yeast cells were washed away with serum-free RPMI medium, and complete RPMI medium containing 10% fetal calf serum was added to each well. The plates were then incubated for 4 days at 37°C. On day 4, the proliferation assay was carried out per the manufacturer's protocol. Briefly, extracellular yeast cells were washed away with serum-free RPMI medium, and surviving RAW 264.7 cells were incubated with the BrdU reagent for 2 h. The cells were then fixed and incubated with anti-BrdU antibody conjugated to peroxidase and washed, substrate was added for 30 min, and an A370 reading was taken. H. capsulatum cells alone do not incorporate BrdU during this time frame (data not shown).

Infection of mice with H. capsulatum. Five- to 6-week-old female C57BL/6 mice were purchased from Harlan Laboratories (Madison, WI). For intranasal infection, mice were lightly anesthetized with isoflurane and infected with approximately 2 x 106 CFU of H. capsulatum in a 20-µl volume. For intraperitoneal infection, 6 x 107 CFU was administered in a 1-ml volume.

Organ culture for H. capsulatum. Groups of mice were euthanized by CO2 inhalation, followed by aseptic harvesting of their lungs, livers, and spleens after intranasal infection and their livers and spleens after intraperitoneal infection. Each organ was placed in sterile distilled water for a total volume of 10 ml and homogenized using a Stomacher 80 Biomaster (Seward, London, United Kingdom), followed by plating onto brain heart infusion agar for the enumeration of CFU. Colonies were counted after incubation at 22 to 24°C for 2 to 4 weeks. Plates with no colonies were assigned a value equal to the lower limit of detection (100 CFU/organ). Data are presented as numbers of CFU (expressed as log10 values) per organ.

Statistics. One-way analysis of variance was used for statistical analyses. In experiments in which two RNAi mutants were analyzed, one-way analysis of variance was followed by Dunnett's test. Both the statistical analysis data and graphs were compiled using Prism (GraphPad Software, San Diego, CA).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Suppression of Yps3 production by RNAi. We created vectors for RNAi silencing of YPS3 based on a hairpin design. This approach has previously been successful for the silencing of genes in Histoplasma (17). We utilized promoters from two different H. capsulatum genes, as heterologous expression in this organism is relatively uncharacterized and we wanted to increase the likelihood of effective silencing. pYPS610 uses the promoter region from CBP1, while pYPS620 utilizes the H2B promoter region. The plasmids are otherwise identical (Fig. 1). We screened 36 pYPS610 and 36 pYPS620 transformants for reductions in the levels of secreted Yps3. Three of the 36 pYPS610 transformants and 3 of the 36 pYPS620 transformants showed considerably reduced levels of secreted protein. As observed previously for H. capsulatum, as well as other fungal systems, we noted variability in the degree of silencing achieved (17). We identified 6 out of 72 transformants with grossly reduced levels of secreted Yps3 and did not further examine the other transformants that showed more modest or no reduction. We colony purified three of these transformants for more detailed analyses. The RNAi 4 transformant strain was of pYPS610 origin, while the RNAi 8 and RNAi 11 strains were transformed with pYPS620, and each had low to undetectable levels of secreted Yps3 (Fig. 2). In all three strains, transforming DNA was detectable by Southern blotting (data not shown); however, the plasmids appeared to have integrated, rather than being maintained as episomal. Empty-vector control strains also appeared to have undergone chromosomal integration of the transforming DNA. As described below, we examined multiple RNAi and empty-vector control transformants in phenotypic assays in order to address the potential effects of different sites of integration or other transformant-specific variations. Levels of secreted Yps3 remained low to undetectable in the RNAi transformants used for further study for at least 6 months of in vitro culture with continuous passage in HMM broth.


Figure 2
View larger version (95K):
[in this window]
[in a new window]

  		FIG. 2. RNAi-silenced YPS3 mutants show a reduction in the levels of secreted Yps3. A SYPRO ruby-stained polyacrylamide gel with filtered concentrated supernatants (top panel) reveals the disappearance of a band consistent with Yps3 protein (Yps3p) in three independent RNAi transformants compared to the empty-vector control transformant. A Western blot with anti-Yps3 antibody (bottom panel) shows the reduction in the levels of secreted Yps3. Molecular size standards (in kilodaltons) are indicated.

 
In vitro growth and infection of the murine macrophage-like cell line RAW 264.7. Two RNAi mutants, RNAi 4 and RNAi 11, were assayed for growth at 37°C in HMM, a rich defined medium. The RNAi mutants showed no growth defect versus an empty-vector control during in vitro growth (Fig. 3A). We tested the mutants for virulence in RAW 264.7 macrophages by using a previously described BrdU uptake assay (18). There was no significant difference between the mutants and the empty-vector control cells in the level of destruction of RAW 264.7 macrophages after 4 days of infection at any multiplicity of infection tested (Fig. 3B). Similar assays using crystal violet to stain for viable macrophages after 4 days of infection (18) likewise showed no difference in macrophage virulence, nor did assays which allowed the infection to progress for shorter (2 days) or longer (5 days) time frames (data not shown).


Figure 3
View larger version (21K):
[in this window]
[in a new window]

  		FIG. 3. (A) Yps3 RNAi mutants are not defective during in vitro yeast-phase growth in rich defined medium HMM. Growth was measured by using culture turbidity. (B) Yps3 RNAi mutants do not have reduced virulence in the murine macrophage-like cell line RAW 264.7. The rate of survival of the RAW 264.7 host cell monolayer after infection was measured by BrdU uptake and expressed as the percentage of the value for uninfected control cells. MOI, multiplicity of infection; EV, G217B ura5-23 strain transformed with pWU45, an empty vector. RNAi 4 and RNAi 11 are two independently generated G217B ura5-23 YPS3-silenced mutants. The assay was performed in triplicate, and results from a representative experiment are shown. Error bars indicate standard deviations.

 
Evaluating the RNAi mutants in a C57BL/6 mouse model of infection. We tested the RNAi mutants in a C57BL/6 mouse model of either intranasal or intraperitoneal infection. For intranasal infection, mice were given 2 x 106 CFU of an empty-vector control strain or the mutant RNAi 4 or RNAi 11 strain. After 3, 7, 10, or 14 days, the mice were sacrificed and their lungs, livers, and spleens were examined for the presence of H. capsulatum via plate assays for the enumeration of CFU. For intraperitoneal infection, mice received 6 x 107 CFU of an empty-vector control strain or the mutant RNAi 4 or RNAi 11 strain. After 14 days of infection, the mice were sacrificed and their livers and spleens were analyzed for fungal burdens. During the 7-day intranasal infection (Fig. 4), eight mice were infected with an empty-vector control strain, eight mice were infected with the RNAi 4 strain, and seven mice were infected with the RNAi 11 strain. RNAi 4- and RNAi 11-infected lungs showed numbers of CFU lower by log10 of 1.28 and 1.66, respectively, than the numbers of CFU in lungs infected with the empty-vector control strain. The livers of RNAi 4- and RNAi 11-infected mice had numbers of CFU lower by log10 of 2.48 and 2.20 than the numbers in the livers of mice infected with the control strain. In the spleens of mutant-infected mice, the reductions in the fungal burdens were log10 of 2.06 CFU for RNAi 4 and log10 of 1.97 CFU for RNAi 11 compared to the fungal burdens in the spleens of control strain-infected mice (Fig. 4). Similar results were obtained in four independent experiments. Intranasal infections were also conducted with the mutant RNAi 8 strain and two other RNAi mutant strains derived from an additional independent transformation, all of which yielded results similar to those obtained with RNAi 4 and RNAi 11 strains, as well as an additional, independently derived, empty-vector control strain, which yielded results similar to those obtained with the original empty-vector control strain (data not shown). When the mice were infected intraperitoneally (n, four mice per strain tested), the RNAi 4 and RNAi 11 mutants yielded reductions in CFU of log10 of 1.83 and 1.73 in the livers and log10 of 1.28 and 1.5 in the spleens after 14 days of infection, respectively (Fig. 5). To assess colonization deficiency over time after intranasal infection with either an empty-vector control strain or the RNAi 11 strain, we allowed the infection to progress for 3, 10, and 14 days prior to sacrifice. The colonization deficiency of the RNAi mutant was detectable by 3 days but appeared to become amplified over time, particularly in the sites of dissemination. In the lungs, the reductions in CFU in the 3-, 10-, and 14-day samples were, respectively, log10 of 1.08, 0.73, and 1.68. In the livers, the reductions in CFU were log10 of 0.81, 1.03, and 2.35 as the infection progressed, while in the spleens, differences in CFU of log10 of 0.38, 2.05, and 2.89 were identified on days 3, 10, and 14 (Fig. 6). Several random isolates of empty-vector and RNAi strains from lungs, livers, and spleens of infected mice were transferred into HMM broth at 37°C and tested for Yps3 protein expression. All strains tested maintained a consistent control or silenced phenotype before and after mouse infection (data not shown).


Figure 4
View larger version (13K):
[in this window]
[in a new window]

  		FIG. 4. Silencing of YPS3 reduces fungal burdens in lungs, livers, and spleens in C57BL/6 mice following intranasal infection. Graphs represent numbers of CFU (expressed as log10 values) recovered from homogenates of organ tissues 7 days after intranasal infection with 2 x 106 CFU of H. capsulatum. Data are the pooled results of two separate experiments, each with three to four mice per fungal strain. Error bars indicate standard deviations. P values represent significant differences in comparison to the control empty-vector strain (EV).

 

Figure 5
View larger version (13K):
[in this window]
[in a new window]

  		FIG. 5. Silencing of YPS3 reduces the colonization of livers and spleens in C57BL/6 mice following intraperitoneal infection. Graphs represent numbers of CFU (expressed as log10 values) recovered from homogenates of organs 14 days after intraperitoneal infection with 6 x 107 CFU of Histoplasma capsulatum. Four mice per fungal strain were used, and error bars indicate standard deviations. P values represent significant differences in comparison to the control empty-vector strain (EV).

 

Figure 6
View larger version (18K):
[in this window]
[in a new window]

  		FIG. 6. The YPS3-silenced mutants show an initial colonization defect in lungs, livers, and spleens. This defect is amplified at the later time points in the peripheral tissues of livers and spleens. Graphs represent numbers of CFU (expressed as log10 values) recovered from homogenates of organ tissues 3, 10, or 14 days after intranasal infection with 2 x 106 CFU of H. capsulatum. Each time point is represented by the mean of results for four to five mice, and error bars indicate standard deviations. EV, empty-vector control strain.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The role of Yps3 in virulence has remained speculative since the identification of this protein in 1989. There is a correlation of YPS3 expression with pathogenicity, since only the most virulent strains of Histoplasma express YPS3 and only in the host-adapted yeast morphotype (2, 13). These observations were made with nonisogenic H. capsulatum strains, and the levels of Yps3 production were only one of many differences among the strains. In this study, we provide the first genetic evidence that the YPS3 gene of the restriction fragment length polymorphism class 2 strains of H. capsulatum influences virulence. We demonstrate that RNAi-silenced YPS3 mutants have a virulence defect in a mouse model of infection, although they grow normally as yeasts in vitro, can transition to the mycelial morphotype when the growth temperature is shifted to 25°C (data not shown), and appear normally virulent during the in vitro infection of RAW 264.7 macrophages.

In mice, Yps3 RNAi mutants were able to infect the lungs and disseminate to livers and spleens and showed initial proliferation, followed by clearance. But the RNAi downregulation of Yps3 resulted in significant quantitative defects relative to the wild type early in infection that expanded over time and defects at mononuclear phagocytic system sites of dissemination that were greater than the defect at the primary site of infection in the lungs. There are many possible mechanisms for this attenuation of virulence, which are not mutually exclusive. For instance, intranasally administered Yps3 mutants may not efficiently get to the lungs or establish or maintain infection once there. Alternately, Yps3 RNAi mutants may infect the lungs normally after intranasal infection but be defective for dissemination to the liver or spleen or for proliferation or the avoidance of clearance at these sites once dissemination is achieved. The mutants’ more rapid clearance from organs correlates with the onset of acquired immunity and may suggest that Yps3 interacts with the host immune system to perpetuate infection.

The few H. capsulatum genes so far demonstrated to influence infection have generally shown concordant results in mouse and in vitro macrophage infection models. For example, ura5 (18) and ags1 (17) mutants are relatively defective both for mouse infection and for in vitro infection of RAW 264.7 and U937 cells or P388D1 cells, respectively. In contrast, Yps3 RNAi mutants displayed normal virulence in RAW 264.7 macrophages in vitro, although their virulence was compromised during mouse infection. This finding is consistent with a role for Yps3 specifically in the in vivo animal infection environment or one involving host mechanisms or responses that are present or fully manifested only in the animal. Of course there are many differences between these models, and the elucidation of the mechanistic basis awaits further work.

The Yps3 mutants displayed lower fungal burdens in the lungs after intranasal infection, and this defect became amplified as the infection progressed. A defect in initial lung colonization or growth in the lungs may exist but is probably not adequate to explain all our findings. While the levels of lung colonization were lower for the RNAi mutants, these differences were not as great as those in livers and spleens, and the mutants maintained higher fungal burdens in the lungs than in the livers or spleens throughout the course of infection.

The empty-vector control strains of H. capsulatum were detectable in the livers and spleens of mice by 3 days, the earliest time point sampled, after intranasal infection, and increasing levels of fungi were present through 14 days postinfection. The Yps3 RNAi mutants disseminated to these peripherial tissues, but there were consistently at least 10-fold fewer cells per organ in tissues from mutant-infected mice than in those from control strain-infected mice. The mechanism for the impaired dissemination or reduced growth or faster clearance after dissemination remains to be determined. The mutant yeast cells may be defective at leaving the lungs or entering the bloodstream, extracellularly or within migratory host cells, perhaps due to an altered cellular or subcellular localization pattern, or perhaps they are cleared from the bloodstream more easily. In the related fungus Blastomyces dermatitidis, surface-localized Bad1, which is a Yps3 homolog, prevents complement molecule C3 deposition onto yeast cells (24). We have noticed a similar defect in complement deposition after coating strains of H. capsulatum that do not normally produce Yps3 with exogenous protein (our unpublished results). Increased opsonization due to reduced Yps3 production may be a potential mechanism of clearance.

Intraperitoneal infection bypasses the issues of lung infection and exit and directly addresses the question of whether the RNAi mutants can infect peripheral tissues as well as control transformants. When H. capsulatum cells were injected intraperitoneally, the RNAi mutants did not achieve wild-type levels of infection in the livers or spleens. This result implies that in addition to showing reduced levels in the lungs and a potential defect in dissemination from the lungs after intranasal infection, the RNAi mutant does not survive, proliferate, or resist clearance as well as the control strain in the peripheral tissues.

Time course experiments revealed that the control strain increased or sustained fungal burdens in lungs, livers, and spleens during the progression of infection over 2 weeks. With the Yps3 RNAi mutants, infection levels in all organs dropped between the 10- and 14-day time points, which correlates with the onset of acquired immunity and may be consistent with Yps3 involvement in some aspect of this process. Acquired immunity, particularly a potent T-cell response and the production of the cytokines gamma interferon and tumor necrosis factor alpha, is critical for reducing fungal burdens and promoting organ clearance (1, 4, 22). In Blastomyces dermatitidis, the Bad1 protein imparts virulence via the modulation of host tumor necrosis factor alpha (6, 7). This immunomodulation has been ascribed to an intracellular domain not encoded by the expressed YPS3 genes (2, 3), however, suggesting that if Yps3 is modulating virulence or host cytokine responses, it is doing so via a different mechanism.


   ACKNOWLEDGMENTS
 
We thank Bruce Klein for helpful discussions and Robert Zarnowski for critical reading of the manuscript.

This work was supported by National Institutes of Health grants R01s HL55949 and AI52303 (to J.P.W.) and a traineeship on National Institutes of Health T32 AI055397 (M.L.B.).


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Medical Microbiology and Immunology, 420 SMI, University of Wisconsin Medical School, 1300 University Ave., Madison, WI 53706. Phone: (608) 265-6292. Fax: (608) 265-6132. E-mail: jpwoods{at}wisc.edu Back

{triangledown} Published ahead of print on 2 April 2007. Back

Editor: A. Casadevall


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Allendoerfer, R., and G. S. Deepe, Jr. 1998. Blockade of endogenous TNF-alpha exacerbates primary and secondary pulmonary histoplasmosis by differential mechanisms. J. Immunol. 160:6072-6082.[Abstract/Free Full Text]
2. Bohse, M. L., and J. P. Woods. 2007. Expression and interstrain variability of the YPS3 gene of Histoplasma capsulatum. Eukaryot. Cell 6:609-615.[Abstract/Free Full Text]
3. Bohse, M. L., and J. P. Woods. 2005. Surface localization of the Yps3p protein of Histoplasma capsulatum. Eukaryot. Cell 4:685-693.[Abstract/Free Full Text]
4. Clemons, K. V., W. C. Darbonne, J. T. Curnutte, R. A. Sobel, and D. A. Stevens. 2000. Experimental histoplasmosis in mice treated with anti-murine interferon-gamma antibody and in interferon-gamma gene knockout mice. Microbes Infect. 2:997-1001.[CrossRef][Medline]
5. Di Lallo, G., S. Gargano, and B. Maresca. 1994. The Histoplasma capsulatum cdc2 gene is transcriptionally regulated during the morphologic transition. Gene 140:51-57.[CrossRef][Medline]
6. Finkel-Jimenez, B., M. Wuthrich, T. Brandhorst, and B. S. Klein. 2001. The WI-1 adhesin blocks phagocyte TNF-alpha production, imparting pathogenicity on Blastomyces dermatitidis. J. Immunol. 166:2665-2673.[Abstract/Free Full Text]
7. Finkel-Jimenez, B., M. Wuthrich, and B. S. Klein. 2002. BAD1, an essential virulence factor of Blastomyces dermatitidis, suppresses host TNF-alpha production through TGF-beta-dependent and -independent mechanisms. J. Immunol. 168:5746-5755.[Abstract/Free Full Text]
8. Gargano, S., G. Di Lallo, G. S. Kobayashi, and B. Maresca. 1995. A temperature-sensitive strain of Histoplasma capsulatum has an altered delta 9-fatty acid desaturase gene. Lipids 30:899-906.[Medline]
9. Harris, G. S., E. J. Keath, and J. Medoff. 1989. Characterization of alpha and beta tubulin genes in the dimorphic fungus Histoplasma capsulatum. J. Gen. Microbiol. 135:1817-1832.[Medline]
10. Harris, G. S., E. J. Keath, and J. Medoff. 1989. Expression of alpha- and beta-tubulin genes during dimorphic-phase transitions of Histoplasma capsulatum. Mol. Cell. Biol. 9:2042-2049.[Abstract/Free Full Text]
11. Hwang, L., D. Hocking-Murray, A. K. Bahrami, M. Andersson, J. Rine, and A. Sil. 2003. Identifying phase-specific genes in the fungal pathogen Histoplasma capsulatum using a genomic shotgun microarray. Mol. Biol. Cell 14:2314-2326.[Abstract/Free Full Text]
12. Johnson, C. H., M. G. Klotz, J. L. York, V. Kruft, and J. E. McEwen. 2002. Redundancy, phylogeny and differential expression of Histoplasma capsulatum catalases. Microbiology 148:1129-1142.[Abstract/Free Full Text]
13. Keath, E. J., A. A. Painter, G. S. Kobayashi, and G. Medoff. 1989. Variable expression of a yeast-phase-specific gene in Histoplasma capsulatum strains differing in thermotolerance and virulence. Infect. Immun. 57:1384-1390.[Abstract/Free Full Text]
14. Nemecek, J. C., M. Wuthrich, and B. S. Klein. 2006. Global control of dimorphism and virulence in fungi. Science 312:583-588.[Abstract/Free Full Text]
15. Patel, J. B., J. W. Batanghari, and W. E. Goldman. 1998. Probing the yeast phase-specific expression of the CBP1 gene in Histoplasma capsulatum. J. Bacteriol. 180:1786-1792.[Abstract/Free Full Text]
16. Punt, P. J., R. P. Oliver, M. A. Dingemanse, P. H. Pouwels, and C. A. van den Hondel. 1987. Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli. Gene 56:117-124.[CrossRef][Medline]
17. Rappleye, C. A., J. T. Engle, and W. E. Goldman. 2004. RNA interference in Histoplasma capsulatum demonstrates a role for alpha-(1,3)-glucan in virulence. Mol. Microbiol. 53:153-165.[CrossRef][Medline]
18. Retallack, D. M., E. L. Heinecke, R. Gibbons, G. S. Deepe, Jr., and J. P. Woods. 1999. The URA5 gene is necessary for Histoplasma capsulatum growth during infection of mouse and human cells. Infect. Immun. 67:624-629.[Abstract/Free Full Text]
19. Tian, X., and G. Shearer, Jr. 2001. Cloning and analysis of mold-specific genes in the dimorphic fungus Histoplasma capsulatum. Gene 275:107-114.[CrossRef][Medline]
20. Tian, X., and G. Shearer, Jr. 2002. The mold-specific MS8 gene is required for normal hypha formation in the dimorphic pathogenic fungus Histoplasma capsulatum. Eukaryot. Cell 1:249-256.[Abstract/Free Full Text]
21. Weaver, C. H., K. C. Sheehan, and E. J. Keath. 1996. Localization of a yeast-phase-specific gene product to the cell wall in Histoplasma capsulatum. Infect. Immun. 64:3048-3054.[Abstract]
22. Williams, D. M., J. R. Graybill, and D. J. Drutz. 1979. Experimental chemotherapy of histoplasmosis in nude mice. Am. Rev. Respir. Dis. 120:837-842.[Medline]
23. Woods, J. P., E. L. Heinecke, and W. E. Goldman. 1998. Electrotransformation and expression of bacterial genes encoding hygromycin phosphotransferase and beta-galactosidase in the pathogenic fungus Histoplasma capsulatum. Infect. Immun. 66:1697-1707.[Abstract/Free Full Text]
24. Zhang, M. X., T. T. Brandhorst, T. R. Kozel, and B. S. Klein. 2001. Role of glucan and surface protein BAD1 in complement activation by Blastomyces dermatitidis yeast. Infect. Immun. 69:7559-7564.[Abstract/Free Full Text]

Infection and Immunity, June 2007, p. 2811-2817, Vol. 75, No. 6
0019-9567/07/$08.00+0     doi:10.1128/IAI.00304-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Other Versions of this Article:
IAI.00304-07v1
75/6/2811    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bohse, M. L.
Right arrow Articles by Woods, J. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bohse, M. L.
Right arrow Articles by Woods, J. P.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
J. Bacteriol. J. Virol. Eukaryot. Cell
Microbiol. Mol. Biol. Rev. Clin. Vaccine Immunol. All ASM Journals