Histoplasma capsulatum requires peroxisomes for multiple virulence functions including siderophore biosynthesis

ABSTRACT Peroxisomes are versatile eukaryotic organelles essential for many functions in fungi, including fatty acid metabolism, reactive oxygen species detoxification, and secondary metabolite biosynthesis. A suite of Pex proteins (peroxins) maintains peroxisomes, while peroxisomal matrix enzymes execute peroxisome functions. Insertional mutagenesis identified peroxin genes as essential components supporting the intraphagosomal growth of the fungal pathogen Histoplasma capsulatum. Disruption of the peroxins Pex5, Pex10, or Pex33 in H. capsulatum prevented peroxisome import of proteins targeted to the organelle via the PTS1 pathway. This loss of peroxisome protein import limited H. capsulatum intracellular growth in macrophages and attenuated virulence in an acute histoplasmosis infection model. Interruption of the alternate PTS2 import pathway also attenuated H. capsulatum virulence, although only at later time points of infection. The Sid1 and Sid3 siderophore biosynthesis proteins contain a PTS1 peroxisome import signal and localize to the H. capsulatum peroxisome. Loss of either the PTS1 or PTS2 peroxisome import pathway impaired siderophore production and iron acquisition in H. capsulatum, demonstrating compartmentalization of at least some biosynthetic steps for hydroxamate siderophore biosynthesis. However, the loss of PTS1-based peroxisome import caused earlier virulence attenuation than either the loss of PTS2-based protein import or the loss of siderophore biosynthesis, indicating additional PTS1-dependent peroxisomal functions are important for H. capsulatum virulence. Furthermore, disruption of the Pex11 peroxin also attenuated H. capsulatum virulence independently of peroxisomal protein import and siderophore biosynthesis. These findings demonstrate peroxisomes contribute to H. capsulatum pathogenesis by facilitating siderophore biosynthesis and another unidentified role(s) for the organelle during fungal virulence. IMPORTANCE The fungal pathogen Histoplasma capsulatum infects host phagocytes and establishes a replication-permissive niche within the cells. To do so, H. capsulatum overcomes and subverts antifungal defense mechanisms which include the limitation of essential micronutrients. H. capsulatum replication within host cells requires multiple distinct functions of the fungal peroxisome organelle. These peroxisomal functions contribute to H. capsulatum pathogenesis at different times during infection and include peroxisome-dependent biosynthesis of iron-scavenging siderophores to enable fungal proliferation, particularly after activation of cell-mediated immunity. The multiple essential roles of fungal peroxisomes reveal this organelle as a potential but untapped target for the development of therapeutics.

In this study, genes identified in a forward genetic screen for mutants of H. capsu latum that exhibited attenuated intracellular proliferation in phagocytes (4) highlight the essential role of peroxisomes for H. capsulatum intracellular growth. We discovered that disruption of both the PTS1 and PTS2 peroxisomal matrix protein import pathways impairs H. capsulatum virulence, although at different stages of infection, suggesting multiple peroxisome functions are required for H. capsulatum pathogenesis. Peroxin mutant characterizations demonstrate that peroxisomes contribute to H. capsulatum siderophore biosynthesis and reveal the existence of another peroxisome function controlled by the PTS1 peroxisome protein import pathway.

H. capsulatum intramacrophage growth and virulence require peroxins
To identify H. capsulatum genes required for intracellular replication in macrophages, we screened insertion mutants of H. capsulatum for mutants with impaired intracellular growth (4). Insertional mutants were generated using Agrobacterium tumefaciens-medi ated transformation of a T-DNA element, and the intracellular growth of H. capsulatum was monitored directly by fluorescence using a strain of H. capsulatum expressing td-Tomato RFP or indirectly by measuring macrophage lysis that occurs following intracellular fungal replication. Mutants were selected that showed less than a 30% increase in RFP fluorescence over time and/or at least 30% reduction in macrophage lysis.
Three T-DNA insertions associated with attenuated H. capsulatum growth in macrophages mapped to genes encoding proteins homologous to the peroxins Pex5, Pex10, and Pex33. The disrupted H. capsulatum genes in mutants 79C10 and 07A9 were designated the orthologs of genes encoding Pex5 and Pex10, respectively, based on reciprocal top-hit BLAST of Pex5 and Pex10 from Saccharomyces cerevisiae. The T-DNA insertion in 79C10 was located within the third exon of the PEX5 gene (998 nucleotides downstream of the CDS start) and the insertion in 07A9 disrupted the first exon of PEX10 (82 nucleotides downstream of the CDS start). Although the H. capsulatum Pex33 protein shows homology to Pex14 of S. cerevisiae, the reciprocal top-hit BLAST search of N. crassa indicated that the gene mutated in mutant 502F2 encodes a protein orthologous to Pex33. The T-DNA insertion in mutant 502F2 mapped to the promoter region of PEX33 (168 bp upstream of the start codon). These mutations are predicted to cause loss of peroxin functions either through disruption of the coding sequence (79C10 and 07A9) or interference with peroxin-encoding gene transcription (502F2).
The independent identification of multiple peroxin-encoding genes strongly suggests that H. capsulatum requires peroxisome function(s) for the pathogenesis of macro phages. To directly determine the consequence of loss of peroxin functions on intra cellular proliferation, macrophages were infected with each mutant and the number of viable cells was determined. Early experiments failed to recover any viable CFUs from plating macrophage lysates on solid media, the reasons for which remain unclear. Consequently, to measure intracellular proliferation, macrophages were lysed and the number of yeasts was determined by counting yeasts directly by hemacytometer and scoring yeast viability using the membrane impermeant Hoechst 33342 dye. The vast majority of recovered yeasts, both wild type and mutant, were viable in macrophages, but the pex10 and pex33 mutants did not proliferate as well as wild-type H. capsulatum (Fig. 1A). Complementation of the pex10 and pex33 mutants restored their proliferation ability, confirming the decreased intracellular proliferation was linked to the loss of Pex10 and Pex33. Loss of Pex5 function similarly decreased intracellular proliferation (Fig. 1B). Independent depletion of Pex5 through RNAi of PEX5 phenocopied the intracellular proliferation defect of the pex5 mutant, demonstrating that the decreased growth was a consequence of the loss of Pex5. Impaired intracellular growth on the loss of Pex5, Pex10, and Pex33 protein functions strongly attenuated the ability of H. capsulatum to cause the death of host macrophages ( Fig. 1C and D), and this attenuation was restored by PEX10 and PEX33 expression in pex10 and pex33 mutants (Fig. 1C) or was similarly impaired by RNAi-based depletion of PEX5 expression (Fig. 1D).
To determine the necessity of H. capsulatum peroxisome function on pathogenesis in vivo, the peroxin mutants were tested in a murine respiratory model of infection. After 8 days of infection, the loss of pex5, pex10, or pex33 severely reduced the fungal burden in the lungs as determined by the recovery of CFUs ( Fig. 1E and F). Given the difficulty in recovering CFUs of pex5, pex10, and pex33 mutants from macrophage lysates, the decreased fungal burden in the lungs was independently confirmed by quantitative PCR (qPCR) quantification of fungal genomes in the lung homogenates and by histology of lung sections (GMS [Grocott's methenamine silver]-stained yeasts). Depletion of PEX5 (PEX5-RNAi) reduced H. capsulatum yeast proliferation in the lungs by 14-fold and 10-fold as measured by the number of fungal genomes within the lungs (Fig. S1A) or by the number of yeast cells in lung sections ( Fig. S1B and C), respectively. These data indicate the essentiality of Pex5, Pex10, and Pex33 for the virulence of H. capsulatum.

Pex5, Pex10, and Pex33 facilitate peroxisomal matrix protein import in H. capsulatum
Previous studies in fungi have demonstrated roles for Pex5, Pex10, and Pex33 in the import of proteins which contain one of two peroxisomal targeting sequences, PTS1 or PTS2, into the peroxisomal matrix. Pex5 acts specifically as a cytosolic receptor for proteins in the cytosol that contain the C-terminal PTS1 (43,44), while Pex33 functions as a member of the protein translocon that imports PTS-containing proteins across the peroxisomal membrane (30). Pex10 functions in the ubiquitination of Pex5 to initiate its recycling back to the cytosol (34). To test if H. capsulatum Pex5, Pex33, and Pex10 contribute to the peroxisomal import of PTS1-containing proteins, the import of a PTS1tagged GFP (GFP:PTS1) in pex5, pex33, and pex10 mutants was assessed. For peroxisomal targeting of GFP, the C-terminal three amino acids from the peroxisomal catalase protein (CatP) that includes a recognizable PTS1 sequence (PRL) were fused to the C-terminus of GFP. In wild-type H. capsulatum, the GFP:PTS1 fusion protein localized to multiple puncta consistent with subcellular organelles (Fig. 2A). The fluorescent puncta do not co-localize with nuclei, endoplasmic reticulum, or mitochondria (Fig. S2). The localization of GFP depended on the C-terminal PTS1 sequence, as GFP lacking the PTS1 signal showed diffuse cytosolic fluorescence ( Fig. 2A). Loss of Pex5, Pex10, or Pex33 function prevented the localization of the GFP:PTS1 to puncta ( Fig. 2A and B), while complementation of Pex10 and Pex33 fully restored the punctate localization (Fig. S3). The dependence of the discrete subcellular compartmentalization of fluorescence on both the presence of a PTS1 sequence and peroxin functions indicates the puncta represent peroxisomes, and the import of the GFP:PTS1 into peroxisomes requires the PTS1-protein import pathway that includes Pex5, Pex33, and Pex10.

PTS2 protein import pathway is necessary for H. capsulatum virulence only during adaptive immunity
While the phenotypes of Pex5-deficient strains establish the role of PTS1-protein import in H. capsulatum virulence, they do not address the potential role of the PTS2-protein import pathway. To specifically examine the import of PTS2-containing proteins, we depleted the PTS2 cytosolic receptor, Pex7.  3A) and attenuated the ability of yeasts to cause macrophage death (Fig. 3B) similar to the loss of PTS1-protein import machinery (Fig. 1). Depletion of Pex7 function did not cause a reduction in the lung fungal burden compared to Pex7-producing H. capsulatum yeasts 8 days post-infection of mice (Fig. 3C) unlike the loss of PTS1-dependent protein import ( Fig. 1E and F). However, at 14 days post-infection, yeasts lacking Pex7 had lower Research Article mBio lung fungal burdens compared to Pex7-producing yeasts (Fig. 3D). Depletion of Pex7 did not result in a plating defect, further differentiating the PTS1 and PTS2 pathways in H. capsulatum. These data show that the PTS2-protein import pathway is also required for full H. capsulatum yeast virulence, but that the PTS2-protein import pathway plays a distinct role from the PTS1-protein import pathway, specifically during later stages of infection. Among the candidates in the PTS1-protein data set, two proteins (Sid1 and Sid3) from the H. capsulatum siderophore biosynthesis cluster (10) focused attention on the potential siderophore biosynthesis function of the H. capsulatum peroxisome. Studies in multiple fungi have established the role of siderophores in fungal acquisition of limited iron (23,24,(46)(47)(48)(49). To determine if peroxin mutants had decreased siderophore production, we tested the growth of peroxin-lacking H. capsulatum yeasts in limited iron. Wild-type H. capsulatum yeasts can grow in media containing the iron-chelator batho phenanthrolinedisulfonate (BPS) with an inhibitory concentration for 50% growth (IC 50 ) of 152 µM, whereas yeasts unable to synthesize siderophores due to the depletion of Sid1 (SID1-RNAi) show 20-fold greater sensitivity to BPS restriction of iron (IC 50 approxi mately 7 µM; Fig. 4A). Loss of PTS1-protein import without Pex5, Pex10, and Pex33 functions resulted in nearly identical inhibition of H. capsulatum growth by BPS as depletion of Sid1, indicating decreased ability to acquire limited iron (Fig. 4A). Depletion of the PTS2-protein import pathway (PEX7-RNAi) showed an intermediate sensitivity to BPS (IC 50 = 51 µM) between wild-type and Sid1-depleted yeasts (Fig. 4B), demonstrating differences in the roles of PTS1-and PTS2-protein import pathways in the acquisition of limited iron.

H. capsulatum peroxisome function is required for siderophore production and growth in limited iron
To ensure that the inability of the peroxin mutants to grow in low-iron-containing medium resulted from deficient siderophore production, the ability of exogenous siderophores to restore growth in BPS-treated medium was tested. Culture filtrate extracts were collected from wild-type H. capsulatum yeasts grown in siderophoreinducing conditions (trace iron) and siderophore-repressing conditions (medium supplemented with 5 µM FeSO 4 ). Siderophores were extracted from culture filtrates via diaion resin extraction, and fractions containing siderophore activity (based on the Chrome Azurol S chromogenic assay) were collected. The siderophore activity-contain ing fraction (Fig. S4A) from the low-iron medium culture filtrate restored the growth of the siderophore-deficient yeasts (SID1-RNAi) in iron-limiting conditions (20 µM BPS) in a dose-dependent manner (Fig. S4B), confirming the fraction contained functional capsulatum (CFUs) in lungs 8 days following intranasal infection of mice with 3 × 10 4 yeasts. Individual data points are shown (n = 3-4 mice) with horizontal bars representing the mean fungal burden in the lungs. Asterisks indicate significant differences compared to the wild type by Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001).
Research Article mBio siderophore molecules. Addition of 0.25% (vol/vol) of this siderophore-containing fraction to media rescued the growth of the peroxin mutant strains in iron-limiting conditions, restoring the growth of each strain between 60% and 95% of wild-type levels ( Fig. 4C). A similar extract fraction from yeasts grown in siderophore-repressing condi tions did not rescue the growth of Sid1-deficient yeasts (Fig. S4B). The siderophorecontaining extract also restored growth of the PTS2-protein import-deficient strain (PEX7-RNAi) in an iron-limited medium (Fig. 4D). Complementation of Pex10 and Pex33 deficiencies also restored growth without siderophore extract supplementation, confirming the failure to grow was due to peroxisome deficiency (Fig. S4C). Together, these data show that H. capsulatum PTS1-and PTS2-protein import machinery are required for siderophore production and growth in iron-limiting conditions.

H. capsulatum Sid1 and Sid3 localize to peroxisomes
The presence of a PTS1 sequence at the C-terminus of Sid1 and Sid3, and the require ment for peroxisomes in siderophore production, suggests the Sid1 and Sid3 proteins should localize to the peroxisome matrix. Bioinformatic surveys of fungal genomes for homologs of siderophore-producing enzymes found PTS1 sequences on H. capsulatum Sid3 and an H. capsulatum homolog of the Aspergillus SidH protein (24). To confirm this, we constructed an N-terminal RFP fusion to Sid1 and Sid3 to preserve the C-terminal PTS1 and monitored their localization by fluorescence microscopy. To validate the RFP:Sid1 fusion protein retained function, we first generated a knockout of the SID1 gene using CRISPR-Cas9 (CRISPR-associated protein) methodology (50) and complemented the sid1 deletion mutation with the expression of the RFP:Sid1 fusion protein. Expression of the RFP:Sid1 fusion protein in the sid1 mutant restored the ability of yeasts to grow in limited iron media, confirming functional complementation (Fig. S5). The RFP:Sid1 protein localized to puncta similar to peroxisomes, and the peroxisomal localization was lost when the fusion protein was expressed in the Pex5-depleted strain that lacks PTS1protein import (Fig. 5A). The peroxisomal localization of RFP:Sid1 is also lost in pex10 and pex33 mutant strains and can be restored by complementation (Fig. S6). Furthermore, the RFP:Sid1 protein co-localized with the GFP:PTS1-marked peroxisomes (Fig. 5B), demon strating that Sid1 is transported to the peroxisome via the PTS1-import pathway. The H. capsulatum Sid3 fusion protein to RFP also localized in a punctate pattern and showed strong colocalization with GFP:PTS1-marked peroxisomes (Fig. 5B), linking two distinct proteins encoded in the siderophore biosynthetic gene cluster to peroxisomes.

Siderophore production is required for H. capsulatum virulence only during adaptive immunity
Although both PTS1-and PTS2-protein peroxisomal import pathways are important for H. capsulatum siderophore production, the requirement for each differs during mamma lian infection. To determine when siderophore production is necessary during host infection, we examined the lung fungal burden established by the Sid1-depleted strain, which is deficient for siderophore biosynthesis. Two previous studies of H. capsulatum Sid1 conflict as to when mammalian infection requires siderophore production (10,51).
In the current study, preventing siderophore biosynthesis (SID1-RNAi) attenuates H. capsulatum yeast growth in cultured macrophages (Fig. 5C) and decreased macrophage death (Fig. 5D). However, infection of mice with the Sid1-depleted strain produced an equivalent fungal burden in lungs as that of Sid1-expressing yeasts at 8 days postinfection ( Fig. 5E) but was strongly attenuated at 14 days ( Fig. 5F) similar to the results with the PTS2-protein import pathway-deficient strain (PEX7-RNAi; Fig. 3C and D).

Peroxin Pex11 is required for H. capsulatum virulence in a siderophoreindependent manner
In addition to PEX5, PEX10, and PEX33, the genetic screen identified another peroxinencoding gene required for H. capsulatum intracellular growth. In this mutant (199G8), the T-DNA element was inserted into the third exon of the gene (879 nucleotides downstream of the CDS start) encoding the ortholog of the S. cerevisiae Pex11 peroxin. Unlike the pex5, pex10, and pex33 mutants, loss of Pex11 function did not impair the import of a PTS1-tagged GFP into peroxisomes (Fig. 6A), distinguishing the role of Pex11 from that of the other peroxin mutants isolated. Nonetheless, loss of Pex11 function impaired the intracellular proliferation of yeasts (Fig. 6B) and decreased macrophage killing by yeasts (Fig. 6C), which was restored by complementation of the pex11 mutation through the expression of the PEX11 gene. Importantly, Pex11-deficient yeasts had attenuated virulence in respiratory infections at 8 days compared to the PEX11-express ing strain (Fig. 6D). However, the pex11 mutant did not show decreased growth in ironlimited media or dependence on siderophore utilization ( Fig. 6E and F), confirming this peroxin contributes to H. capsulatum pathogenesis through a mechanism independent of the siderophore biosynthesis role of peroxisomes.  Research Article mBio capsulatum genome identified multiple components of siderophore biosynthesis. At least two siderophore biosynthetic enzymes, Sid1 and Sid3, localize to the peroxisome, and the peroxisomal localization of Sid1 is dependent on Pex5 (Fig. 5A and B). Depletion or disruption of multiple peroxisome import components, including the PTS1 and PTS2 pathways, impairs H. capsulatum acquisition of iron showing proper peroxisome function is necessary for H. capsulatum siderophore production (10, 53). Related H. capsulatum strains 505 and G184 produce fusarinine and coprogen-type hydroxamate siderophores in culture (54). The H. capsulatum genome encodes many proteins homologous to the Aspergillus fumigatus siderophore biosynthetic pathway (24,55) with most genes present in a single iron-regulated gene cluster (10), including the SID1, SID3, SID4, and NPS1 genes. SID1 encodes an N5-monooxygenase, which catalyzes the first committed step of siderophore biosynthesis by converting L-ornithine into N5hydroxy-L-ornithine. Interestingly, the H. capsulatum Sid1 protein also has a PTS1 signal (ARL) and shows peroxisomal localization ( Fig. 5A and B) unlike the orthologous A. fumigatus SidA protein that lacks a C-terminal PTS1 sequence (24,55). Sid1 orthologs from related dimorphic fungi, such as Blastomyces, Paracoccidioides, and Coccidioides contain putative PTS1 sequences as well. Close inspection of the Sid1:RFP localization shows that some Sid1 is also present in the cytosol in contrast to the Sid3:RFP (Fig. 5B). This might reflect different efficiency of PTS1 recognition and capture of the Sid3 and Sid1 PTS1 (SKL and ARL, respectively). Compartmentalization of the Sid1 N5-hydroxy-Lornithine monooxygenase within the peroxisome in Onygenales fungi with other siderophore biosynthetic enzymes (e.g., Sid3) may concentrate enzyme activity, sub strates, and products for more efficient siderophore synthesis. Although A. fumigatus encodes proteins for both reductive iron transport and production of hydroxamate siderophores, only the latter is required for virulence in vivo (48,56). The genome of the G217B strain of H. capsulatum used in this study does not encode a reductive iron acquisition system and thus relies on siderophores for the acquisition of limited iron (51). Together these findings indicate that fungal acquisition of iron from the mammalian host specifically requires the peroxisome-dependent biosynthesis of siderophores.

DISCUSSION
Depletion of the PTS1 and PTS2 pathways revealed distinct phenotypes for virulence in vivo, suggesting multiple roles for H. capsulatum peroxisomes in this context. Loss of the PTS1 pathway attenuates virulence early during infection, while loss of the PTS2 pathway only attenuates virulence at late stages of infection in a murine model. Sid4, encoded in the H. capsulatum siderophore gene cluster (10), contains a putative PTS2 localization sequence (RLQQTLNHI) as previously identified (24), linking the PTS2 import pathway and siderophore production in H. capsulatum (24). This, in combination with the late-stage attenuation defect seen in strains that lack Sid1 or Pex7, suggests that the virulence attenuation of H. capsulatum yeasts lacking the PTS2 protein import pathway can be explained primarily by the loss of siderophore production. The dynamic require ment for fungal siderophores during infection suggests that iron is available for H. capsulatum in the phagosome during the early stages of infection, but becomes limiting at later time points, necessitating the use of siderophores to acquire nutritional iron (10). This finding echoes a previous study that showed phagosomal copper availability to H. capsulatum becomes scarce only during adaptive immunity (4). This constitutes a trend of metal sequestration (both iron and copper) from intracellular H. capsulatum via adaptive immune responses as a form of nutritional immunity designed to limit the proliferation of intracellular pathogens.
While both PTS1-and PTS2-protein import into peroxisomes is required for sidero phore production and iron acquisition during infection, the early stage virulence attenuation resulting from the loss of PTS1-protein import indicates another role for peroxisomes in H. capsulatum pathogenesis distinct from siderophore biosynthesis. The difficulty in plating of PTS1-protein import-deficient strains but not PTS2-protein importdeficient strains further underscores the separable roles of peroxisomes in H. capsulatum biology. Synthesis of the vitamin biotin in A. fumigatus relies on the PTS1-protein import pathway in that organism, suggesting some essential vitamin synthesis requires peroxisomes (21). However, previous studies have determined that the loss of biotin biosynthesis does not significantly attenuate H. capsulatum growth in macrophages and virulence in vivo (57), which suggests biotin deficiency is not the cause of the peroxin mutants' attenuation. Peroxisomes are notable for sequestering peroxide-generating enzymatic reactions and consequently harbor a peroxisomal catalase. In H. capsulatum, the peroxisomal catalase, CatP, contains a PTS1 sequence which is sufficient to direct peroxisomal import ( Fig. 2A). However, depletion of the peroxisomal catalase CatP does not impair H. capsulatum virulence during infection due to a redundant cell-surfacelocalized catalase, CatB (42), indicating the loss of peroxisomal CatP localization does not underlie the strong virulence attenuation of the peroxisomal mutants. These data suggest a novel role for peroxisomes during mammalian fungal infection. The observation that the Pex11 protein is required for full virulence further indicates peroxisomal functions independent of siderophore biosynthesis are required for H. capsulatum pathogenesis and may give insight into the role of this organelle. Pex11 is central to the peroxisomal division process (58) and defects in Pex11 in both mam malian cells and yeasts generally result in a reduction of peroxisome abundance (59)(60)(61). However, the loss of Pex11 function in H. capsulatum did not eliminate peroxi somes (Fig. 6A) and did not prevent siderophore biosynthesis (Fig. 6E and F). Nonethe less, the absence of Pex11 function attenuated H. capsulatum virulence similar to the loss of PTS1-protein import machinery, indicating a distinct role from the function of peroxisomes in siderophore production. In addition, pex11 mutants do not have defects in plating efficiency, unlike the PTS1-protein import mutants. Thus, one or more specific PTS1-pathway imported matrix protein functions are necessary for H. capsulatum virulence with some linked to Pex11 function. This Pex11-dependent and PTS1-protein import-dependent function remains undetermined, but a report has suggested that Pex11 could be a non-selective pore-forming protein that transfers metabolites smaller than 400 Da across the peroxisomal membrane (62). In light of this, we hypothesize that Pex11 may provide substrates for a PTS1-containing matrix protein that enables H. capsulatum virulence during the early stages of infection.
This study demonstrates the multifaceted role of peroxisomes in the growth and replication of an intracellular fungal pathogen. The H. capsulatum peroxisome has at least three functions that are separable based on the distinct phenotypes of the isolated peroxin mutants: (i) siderophore production, which is essential for virulence during Asterisks indicate significant differences compared to the wild type (*P < 0.05, **P < 0.01, ***P < 0.001).
Research Article mBio the adaptive immune response, (ii) an undefined mechanism contributing to plating efficiency, and (iii) a novel virulence function essential for early stage infections that may involve transport of small molecules as a substrate for a peroxisomal enzyme. Future studies focused on PTS1-containing peroxisome matrix proteins should uncover the different functions of the peroxisome and how they contribute to intracellular prolif eration and virulence of H. capsulatum. Given the importance of fungal peroxisomes to multiple aspects of fungal virulence, including pathogenesis-enabling siderophore production, the peroxisome machinery could represent a potential target for antifun gal therapeutics, perhaps by targeting the Pex33 peroxin, which appears specific for ascomycetous fungi.

H. capsulatum strains and growth
The H. capsulatum strains used in this study (

Isolation of H. capsulatum mutants with attenuated intramacrophage growth
H. capsulatum strain WU15 or strain OSU233 (63) was used as the genetic background for insertional mutagenesis using Agrobacterium tumefaciens strain LBA1100 harboring plasmid pBHt2 (9). Individual mutants were used to infect monolayers of P388D1 lacZ-expressing macrophage cells (64). Mutants with at least a 30% reduction in intramacrophage yeast growth (red fluorescence) or at least a 30% reduction in lysis of the macrophages were retained as intracellular-proliferation-deficient mutants.

Mapping and complementation of T-DNA insertional mutants
The location of the T-DNA insertion in individual mutants was determined by thermal asymmetric interlaced PCR (TAIL-PCR) as described previously (4). The sequences were aligned to the H. capsulatum genome sequence to determine the genomic context of the T-DNA insertions. The mutants characterized in this study included 79C10 (OSU197), 07A9 (OSU9), 502F2 (OSU131), and 199G8 (OSU377).
To complement the pex10 and pex33 mutants, a fragment consisting of the corre sponding gene sequence was amplified by PCR from H. capsulatum G217B genomic DNA using gene-specific primers and cloned into a URA5-based T-DNA plasmid (pCR628) for constitutive expression from the H. capsulatum H2B promoter. To complement the pex11 mutant, a 1,841-bp fragment consisting of the PEX11 gene sequence and 194 bp upstream and 562 bp downstream was amplified by PCR from H. capsulatum G217B genomic DNA using gene-specific primers. Complementation vectors or a control green fluorescent protein (GFP) expression vector (pCR628) were transformed by A. tumefa ciens-mediated transformation into the corresponding peroxin mutants.

Depletion of peroxin and siderophore biosynthesis gene functions by RNAi
Peroxin and siderophore biosynthesis gene functions were depleted from H. capsula tum yeasts by RNA interference (RNAi) (65). The RNAi vector was created by the insertion of two copies of a region of the targeted gene coding region [coding DNA sequence (CDS)] in an inverted orientation into the RNAi sentinel vector pED02 (66). RNAi vectors were transformed by Agrobacterium-mediated transformation into the GFP-expressing uracil auxotroph sentinel strain OSU194. Uracil prototroph (Ura+) transformants were recovered, and the sentinel GFP fluorescence was quantified using a modified gel documentation system and ImageJ software (v1.44p; http:// imagej.nih.gov.proxy.lib.ohio-state.edu/ij) to identify transformants with depletion of target gene expression.

Intramacrophage proliferation of H. capsulatum yeasts
Macrophage monolayers were established in 96-well plates and were infected with yeasts at an MOI of 1:1. After 2 hours, the medium was replaced to remove the remaining extracellular yeasts. Immediately or at 24 or 48 hours post-infection, intracellular yeasts were quantified by removal of macrophage culture supernatant followed by lysis of the macrophages with sterile H 2 O and plating of serial dilutions of the macrophage lysate on solid HMM to enumerate CFUs. For H. capsulatum strains unable to form CFUs, yeasts were instead enumerated directly by counting using a hemacytometer. Hoechst 33342 dye (1 µg/mL) was added to the macrophage lysate before counting, and unstained yeasts were deemed viable.

Murine model of pulmonary histoplasmosis
Wild-type C57BL/6 mice were infected with wild-type, mutant, or complemented H. capsulatum strains by intranasal delivery of approximately 3 × 10 4 yeast cells. At 8 or 14 days post-infection, mice were euthanized and their lungs were collected for analysis. For CFU enumeration, serial dilutions of lung homogenates were plated on solid HMM to determine the fungal burden. For qPCR enumeration of fungal genomes, lungs were homogenized in water and passaged 20 times through a 25-gauge needle to lyse remaining mammalian cells. Yeasts were collected by centrifugation (16,000× g for 2 minutes), and the yeast-containing pellet was resuspended in 200 µL water. DNA was extracted by mechanical disruption of yeasts with 0.5-mm-diameter glass beads followed by isopropanol precipitation of DNA. H. capsulatum DNA was quantified by probe-based qPCR using a custom TaqMan probe (TTCTAGACGCTCTCAAGGGCGTTCTCAAG with 5′ 6-FAM (6-carboxyfluorescein) label and ZEN/Iowa Black dual quencher; Integrated DNA Technologies) targeting the H. capsulatum RPS12 gene. Forward and reverse RPS12 primers with sequences TCGGACGGAGAGACCGCT and TCACGGAGGACAACGCAAGAGC, respectively, were used for qPCR with the RPS12 TaqMan probe, with Entrans 2X qPCR Master Mix (Abclonal). For histology analysis, lungs were fixed in 10% formalin, paraf fin-embedded, and sectioned. Grocott's methenamine silver stain was performed on three non-consecutive lung sections from each mouse to allow the identification of H. capsulatum in lung tissue. A semi-quantitative measure of H. capsulatum fungal burden was obtained by counting yeast from 20 different non-overlapping 40× image fields from each lung section (three sections per lung). For CFU enumeration, serial dilutions of the homogenates were plated on solid HMM to determine the fungal burden (CFU). Animal experiments were performed in compliance with the National Research Council's Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) at Ohio State University (protocol 2007A0241).

Localization of Sid1 and Sid3 prot9eins
The wild-type H. capsulatum SID1 gene and SID3 genes were amplified by PCR and cloned into a T-DNA plasmid (pAG22) fusing the coding sequences to sequence to an N-terminal td-Tomato red-fluorescence protein (RFP) gene. The resultant plasmids (pCS22 and pCS12) were transformed into H. capsulatum yeasts by A. tumefaciens-medi ated transformation. The RFP:Sid1 expression construct was used to complement a sid1 deletion mutation.
Directed mutation of the SID1 locus was done by CRISPR/Cas9-mediated gene editing using a Cas9 and CRISPR guide RNA (gRNA) expression plasmid [derived from pPTS608 (67)] and optimized methodology (50). The gRNA protospacer sequences GAACGCGAC GCTGAATCCCG and CGAAGAGTTGACATACACCA were used to target the H. capsulatum SID1 locus.

Extraction of siderophores from conditioned H. capsulatum media
H. capsulatum strain WU15 was grown in 250 mL of 3M media (63) with 2% glucose, 30 mM glutamate, 100 µg/mL uracil, and with either 5 µM FeSO 4 (siderophore-repressing condition) or no supplemented FeSO 4 (siderophore-inducing condition). After 10 days of growth, culture filtrate was recovered by removal of yeasts by centrifugation (16,000× g for 5 minutes) and filtration of the supernatant (0.2-µm-diameter pore membrane). Diaion HP20 synthetic adsorbent resin (Alfa Aesar) was added to each sample (1 g/50 mL) and mixed at 30°C for 1 hour. The resin was collected, transferred to a gravity column, and washed with 10 mL of ultrapure H 2 O. Elution fractions were then obtained by flowing 2 mL of increasing aqueous methanol (10% to 100%) through the column. Fractions were evaporated at 60°C overnight, and the material was resuspended in 1 mL of H 2 O. Siderophore-containing fractions were identified via a modified Chrome Azurol S assay (68). Briefly, 0.1 mL of each fraction was mixed with 0.3 mL of water and 0.4 mL of CAS assay solution (600 µM hexadecyltrimethyl ammonium, 15 µM FeCl 3 , 150 µM Chrome Azurol S, 200 mM MES [2-(N-morpholino) ethanesulfonic acid], 0.5 mM HCl, and 5 mM 5-sulfosalicylic acid) and incubated for 1 hour. Siderophore scavenging of iron from the iron-containing CAS complex was determined as the absorbance ratio (420 nm:630 nm) measured with a spectrophotometer. For siderophore rescue assays, the extract originating from the 50% methanol fraction was added to HMM media with 20-µM BPS to a final concentration of 0.25% (vol/vol). Corresponding fractions from cultures grown in siderophore synthesis-repressing conditions (i.e., fractions from cultures supplemented with 5 µM FeSO 4 ) were used as the control. The growth of H. capsulatum yeasts in siderophore-lacking and siderophore-supplemented media was measured by optical density (595 nm) after 72 hours and normalized to the growth of each strain in HMM with no BPS and no siderophores were added.