Reverse genetics through random mutagenesis in Histoplasma capsulatum
© Youseff et al; licensee BioMed Central Ltd. 2009
Received: 2 June 2009
Accepted: 17 November 2009
Published: 17 November 2009
The dimorphic fungal pathogen Histoplasma capsulatum causes respiratory and systemic disease in humans and other mammals. Progress in understanding the mechanisms underlying the biology and the pathogenesis of Histoplasma has been hindered by a shortage of methodologies for mutating a gene of interest.
We describe a reverse genetics process that combines the random mutagenesis of Agrobacterium-mediated transformation with screening techniques to identify targeted gene disruptions in a collection of insertion mutants. Isolation of the desired mutant is accomplished by arraying individual clones from a pool and employing a PCR-addressing method. Application of this procedure facilitated the isolation of a cbp1 mutant in a North American type 2 strain, a Histoplasma strain recalcitrant to gene knock-outs through homologous recombination. Optimization of cryopreservation conditions allows pools of mutants to be banked for later analysis and recovery of targeted mutants.
This methodology improves our ability to isolate mutants in targeted genes, thereby facilitating the molecular genetic analysis of Histoplasma biology. The procedures described are widely applicable to many fungal systems and will be of particular interest to those for which homologous recombination techniques are inefficient or do not currently exist.
The dimorphic fungal pathogen, Histoplasma capsulatum, parasitizes phagocytic cells of the mammalian immune system and causes one of the most common respiratory fungal infections world wide [1–3]. The mycelia-produced Histoplasma conidia are acquired by inhalation into the respiratory tract where exposure to mammalian body temperatures triggers their differentiation into pathogenic yeast cells [3, 4]. Histoplasma virulence requires this transition to the yeast phase and expression of the corresponding yeast-phase regulon [5–7]. This transcriptional profile includes genes encoding specific factors that promote Histoplasma virulence [7–9]. While mammalian alveolar macrophages efficiently phagocytose Histoplasma cells, they are unable to kill the yeast [10–12]. Within the macrophage, Histoplasma modifies the intracellular compartment to promote its survival and replication. The ability to subvert immune defenses and to survive within phagocytes enables Histoplasma to cause disease in both immunocompromised and immunocompetent individuals. This high potential for infection is reflected in the fact that histoplasmosis is one of the most common pulmonary fungal infections among healthy individuals .
The mechanistic details that underlie Histoplasma pathogenesis are still largely unknown owing to limited or inefficient genetic methodologies. The genome sequences of three phylogenetically distinct strains of Histoplasma have been completed (North American type 1, NAm 1; North American type 2, NAm 2, and a lineage from Panama, Pan)  which has accelerated the ability to identify, define, and analyze Histoplasma genes. However, demonstration that a gene product contributes to a particular facet of biology requires specific depletion of the candidate factor and comparison to a factor-replete strain in functional tests. Targeted deletion of candidate factors is most often accomplished through genetic means, employing homologous recombination to replace the wild-type gene with an engineered deletion or disruption allele. In Saccharomyces cerevisiae, homologous recombination is so efficient that gene deletion libraries have been compiled with mutants representing entire sets of genes or even the majority of the genes in the genome [15, 16]. In contrast, non-homologous or illegitimate recombination dominates in the dimorphic fungal pathogens , frustrating gene deletion attempts and impeding advancement of our molecular understanding of these fungi. Furthermore, Histoplasma can maintain introduced DNA (e.g. a deletion allele) as an extrachromosomal element which impedes efforts to incorporate alleles into the genome [18, 19].
Despite these obstacles, genes have been deleted in Histoplasma following development of a two-step procedure . Realization of the rare homologous recombination event necessitates a very large population as the frequency of allelic replacement is on the order of 1 in 1000 transformants . As typical transformation frequencies are insufficient, individual transformants harboring recombination substrates are instead cultured and repeatedly passaged to generate a large number of potential recombination events. In the second step, a dual positive and negative selection scheme enriches the population for the desired recombinant. In practice, only a portion of the isolated clones harbor the deletion requiring screening of many potential isolates. In Histoplasma, this process of reverse genetics (the generation of a mutant in a targeted gene) has been successfully accomplished for only six genes to date, the vast majority in the Panama phylogenetic group (URA5, CBP1, AGS1, AMY1, SID1) [20–24]. For reasons not well understood, this procedure has not been very successful in the Histoplasma NAm 2 lineage despite numerous attempts. Recently, a deletion of the gene encoding DPPIVA has been reported in the NAm 2 lineage .
The inefficient and laborious process of deleting genes in Histoplasma prompted development of RNA interference (RNAi) as an alternative method to determine the role of gene products in Histoplasma biology . To date, eight genes have been functionally defined by RNAi (AGS1, UGP1, DRK1, YPS3, RYP1, GGT1 DPPIVA, DPPIVB) [7, 8, 22, 23, 25–27]. However, RNAi can not generate a complete loss of function, and this potential for residual function imposes difficulties in interpreting negative results with RNAi (i.e. the absence of a phenotype). Unlike chromosomal mutations which are more permanent, plasmid-based RNAi effects must be constantly maintained with selection. For studies of Histoplasma virulence, this in vivo selection of plasmids requires use of the URA5 selection marker and ura5 auxotrophic Histoplasma strains.
To overcome these obstacles and limitations in our current techniques for genetic analysis of Histoplasma, we have developed a procedure for isolating chromosomally-located gene mutants without reliance on homologous recombination. We employed random mutagenesis to create collections of mutants. One approach to identify the desired gene disruption would be to characterize the mutation of each isolate in the collection of random mutants. However, this requires many resources, substantial time and effort, and thus is not well suited for studies targeting a particular gene. In forward genetics, random mutagenesis is successful because the desired mutant can be typically isolated or identified out of the much larger collection of mutants by growth phenotype or morphological changes. In reverse genetics, the mutant phenotype is the very aspect under investigation and thus mutants can not be identified by predicted changes. To enable reverse genetics following random mutagenesis in Histoplasma, we adapted PCR-based procedures employed for large scale screening in Arabidopsis and C. elegans [28–30]. We optimized a mutant pooling strategy and utilized PCR to efficiently identify mutant pools which contain the strain with the disrupted gene. To extract the strain with the targeted mutation, the pool is subsequently subdivided and individual clones addressed and screened by PCR. We demonstrate the effectiveness of this method by employing it to isolate a cbp1 mutant in the NAm 2 Histoplasma strain background.
Results and Discussion
Insertion mutant screening
Optimization of pool size for reliable detection of targeted mutations
As the successful isolation of a mutant in a targeted gene depends critically on the ability to identify a positive individual among a much larger population, we determined the PCR detection limit for different pool sizes. Histoplasma strain OSU4 harbors a T-DNA insertion in the AGS1 gene in which the T-DNA right border is oriented towards the 3' end of the AGS1 gene. Performance of PCR using a right border T-DNA primer and an AGS1 gene-specific primer produces a PCR amplicon of 1242 bp. To estimate the detection limit afforded by PCR in which a single strain could be found among a population of 200, 800, or 3200 mutants, 50 ng of nucleic acid purified from OSU 4 were diluted 1:200, 1:800, and 1:3200 with TE buffer and PCR performed on these templates with RB3 and AGS1-50 primers. With 30 cycles, PCR could consistently detect the OSU4 template when diluted as much as 1:800 (Figure 1B). To better approximate the condition where the desired mutant would be present among a much larger population of other T-DNA insertions, we mixed OSU4 with a pool of random T-DNA insertion mutants at a OSU4 yeast-to-mutant pool ratio of 1:200, 1:800, and 1:3200. Nucleic acids were purified from each pool and PCR was performed as before with 50 ng of total nucleic acid as templates. The positive 1242 bp amplicon was detected when OSU4 was present in as little as 1/800th of the total population of yeast (Figure 1B). A faint band representing the ags1::T-DNA PCR product was observed when OSU4 constituted 1/3200th of the template. No ags1::T-DNA PCR product was detected in templates prepared from the mutant pool alone or wild-type Histoplasma. A smaller PCR amplicon which is not specific to the ags1::T-DNA template was detected in all reactions derived from the random insertion mutant pool.
Nested PCR to reduce false-positives
To discriminate between true- and false-positive PCR products, we employed a secondary PCR reaction using a set of nested primers. Nested primers that do not overlap with the primary PCR primers were designed for both the T-DNA anchor and the AGS1 gene. Primary PCR reactions in which OSU4 represented 1/200th or 1/800th of the population were used as templates after 1:1000, 1:10,000, and 1:100,000 dilution in H2O. As shown in Figure 1C, this process eliminated the false-positive band observed in the primary PCR reactions. The ags1::T-DNA specific amplicon could be detected after either 1:1000 or 1:10,000 dilution of the primary PCR reaction. No ags1::T-DNA amplicon was produced when OSU4 was absent in the primary reaction template DNA. These data demonstrate that PCR can be an efficient screening technique to probe mutant pools for a clone in which a T-DNA element has inserted into a target gene. We selected a target pool size of approximately 200 insertion mutants as a balance between increased throughput afforded by larger pools but easier subdivision of smaller pools into individual clones to recover the detected mutant strain (see below).
Establishment of a bank of insertion mutants
Optimization of freezing conditions
Generation of mutant pools
Insertion mutants were generated in the NAm 2 Histoplasma strain WU15 by co-cultivation of Agrobacterium tumefaciens and Histoplasma yeast cells. Co-cultures were plated onto filters and Histoplasma transformants selected by transferring filters to medium containing hygromycin to which resistance is provided by sequences within the T-DNA element . Transformant yeast cells were collected and suspensions from individual plates combined to create pools derived from 100 to 200 independent mutant colonies. Yeast cell suspensions were diluted into fresh medium and allowed to grow for 24-48 hours. Twenty-four pools were prepared representing roughly 4000 insertion mutants. A portion of each culture was reserved for nucleic acid isolation and the remainder frozen in aliquots and stored at -80°C. Nucleic acids were purified from each pool, diluted to 50 ng/ul, and stored at -20°C until analysis by PCR.
With an estimated 9000-10,000 genes encoded by the Histoplasma genome, this collection does not represent the number of insertion mutants required for saturation of the genome. We used two probability functions to estimate the size of the library required for a 95% chance of isolating an insertion in a particular locus in the 40 megabase NAm 2 genome. Both calculations assume no bias in insertion sites. Based on the number of predicted genes, the Poisson approach estimates a library of approximately 30,000 insertions would be required. The single study in which multiple alleles of a single locus were isolated in Histoplasma (five AGS1 alleles isolated in a screen of 50,000 insertions; ) supports the Poisson calculation; five alleles would be the most probable number of alleles based on a 9000 or 10,000 target estimate. However, the AGS1 gene is an unusually large target and thus this number is probably an underestimate. An alternative calculation based solely on average gene size is provided by: P = 1-(1-x/G)n where P is the probability of the T-DNA inserting in a given target of size x in a genome of size G with n the total number of T-DNA insertion mutants . Assuming an average gene size of 2000 nucleotides, this calculation estimates a library of nearly 60,000 mutants would be required for a 95% probability of obtaining at least one insertion mutant in any given gene. Such a mutant bank would require 300 pools with an average pool size of 200 and PCR screening could be easily performed using three 96-well plates. Although our current collection of 4000 mutants is inadequate for complete genome coverage, it was sufficient to demonstrate proof-of-concept through identification and recovery of a mutant at the CBP1 locus.
Isolation of a cbp1 insertion mutant
Detection of a T-DNA insertion in CBP1
Recovery of the cbp1 insertion mutant
To further characterize the T-DNA insertion in OSU8, we amplified and sequenced the DNA flanking the T-DNA element. PCR amplicons were produced for both the left and right border flanking regions using T-DNA specific primers and CBP1 specific primers (data not shown). Alignment of the flanking regions with the Histoplasma G217B genome and T-DNA sequences showed truncation of the T-DNA imperfect direct repeats by 5 bp from the left border and 24 bp from the right border. Additionally, the T-DNA insertion event deleted 175 base pairs of the CBP1 promoter surrounding the site of insertion (Figure 3C). Due to T-DNA-induced genetic rearrangements that can occur, PCR-product sizes should be used only as an initial estimate of the location of T-DNA integration and the precise location of the insertion confirmed by sequencing the DNA flanking the T-DNA element. As our PCR screening method would not detect multiple T-DNA integrations, we performed a Southern blot using a T-DNA-specific probe to determine how many T-DNA elements were present in the OSU8 mutagenized genome. As shown in Figure 3D, only one band is detected indicating the OSU8 strain harbors a single T-DNA insertion. This 3.8 kb T-DNA probe-hybridizing fragment is the size predicted for the described insertion in the CBP1 promoter. No T-DNA sequences were detected in the parental WU15 strain.
Validation of the cbp1 mutant
We have developed a reverse genetics procedure employing random mutagenesis and PCR-based screening techniques to identify insertion mutants in a targeted gene in Histoplasma capsulatum without regard to a mutant phenotype. Since the mutagen creates a large insertion, the majority of mutations should reflect the knock-out mutant phenotype. However, insertions within the promoter of a gene may allow some residual transcription resulting in hypomorphic but not null phenotypes. In such cases, as demonstrated by our cbp1:T-DNA mutant, delineation of the minimal promoter of a targeted gene could resolve what type of phenotype the insertion mutation would likely produce. Thus, the regions most likely to produce mutant phenotypes are the proximal promoter and the coding region of the targeted gene. Consequently, we routinely design our PCR screening primers at the 3' end of the gene to amplify these regions in particular and maximize the targeted site for insertions. This process, as with any mutational approach, will not successfully isolate insertion mutations in genes essential for viability, and if the intended target is suspect to be such, RNAi may prove more beneficial. While integration of T-DNA into the Histoplasma genome appears relatively random, large scale studies in Magnaporthe, Leptosphaeria, and Arabidopsis indicate there is a bias for insertion of the T-DNA element into non-coding regions [37–40]. In addition, occurrence of large-scale deletions or rearrangement mutations will be missed by this approach. Thus, more insertion mutants may be required for saturation mutagenesis of the Histoplasma genome than calculated above.
The reverse genetics process detailed here increases the repertoire of methods available to disrupt gene functions in Histoplasma capsulatum. Since Agrobacterium-mediated transformation has been developed as an efficient mutagen for a variety of fungal species , this procedure should be readily applicable to those microorganisms as well. For intractable fungal systems where homologous recombination is very limited or allelic replacement unfeasible, this process provides the ability to disrupt gene functions necessary for functional genetic tests. The only requirement is an efficient insertional mutagen. The increased capability to disrupt gene functions in Histoplasma and in other fungi will greatly improve our mechanistic understanding of fungal biology.
Yeast strains and culture
(G217B) ura5-Δ42 ags1-5::T-DNA [hph]
(G217B) ura5-Δ42 cbp1-9::T-DNA [hph]
(G217B) ura5-Δ42/pCR473 [URA5, gfp-RNAi]
(G217B) ura5-Δ42/pCR475 [URA5, CBP1-RNAi]
Agrobacterium-mediated transformation of Histoplasma
Agrobacterium tumefaciens was used to transform Histoplasma capsulatum yeast using modifications to previously described protocols [23, 31]. A. tumefaciens strain LBA1100 was transformed with pCM41, an engineered plasmid containing a hygromycin resistance cassette flanked by the left and right border T-DNA sequences . A. tumefaciens harboring pCM41 was grown in LC media  containing 100 ug/ml kanamycin and 250 ug/ml spectinomycin to select for the T-DNA and Ti plasmids, respectively. Liquid LC media was inoculated with 10 colonies and grown overnight at 25°C with shaking (250 rpm). Bacteria were harvested by centrifugation (5000 × g) and resuspended in 5 volumes Agrobacterium induction medium [IM-- 44] and grown for approximately 16 hours at 25°C with shaking. Complete induction medium contained 0.2% glucose, antibiotics, and 200 uM acetosyringone and was buffered to pH 5.3 with MES. Bacteria were collected by centrifugation and resuspended in induction medium to an optical density at 600 nm of 1.5 (corresponding to roughly 1.5 × 109 bacteria/ml). Histoplasma WU15 yeast were harvested from solid HMM + uracil medium seeded 3 days earlier with 4 × 105 yeast/cm2. Yeast were collected by flooding plates with 5 mls HMM medium and scraping with a sterile spreader. Yeast were collected by centrifugation (1000 × g) and resuspended in induction medium at a density of 5 × 108 yeast/ml as determined by hemacytometer counts. For co-cultivation, 1.5 × 108 Agrobacterium cells were mixed with 5 × 107 Histoplasma yeast in a total volume of 400 ul and spread on Whatman #5 filter paper placed on top of solid induction medium supplemented with 0.7 mM cystine and 100 ug/ml uracil. Plates were incubated for 48 hrs at 25°C after which filters were transferred to selection medium (HMM + uracil + hygromycin + 200 uM cefotaxime) and incubated at 37°C with 5% CO2/95% air until Histoplasma transformants became visible (10-14 days). Six cm diameter plates were used so that roughly 50-100 transformants were obtained per plate.
PCR-based screening of T-DNA insertion mutants
Hygromycin-resistant transformants of Histoplasma were collected by flooding plates with HMM and suspending cells with a sterile spreader. Suspensions from individual plates were combined to obtain pools representing 100-200 independent transformant colonies. Yeast suspensions were diluted 1:10 into 10 mls HMM + uracil and grown for 24-48 hours. Two milliliters of culture were collected for nucleic acid isolation and the remaining culture frozen in 1 ml aliquots for later recovery of yeast.
To purify Histoplasma nucleic acid for PCR, cells were collected by centrifugation (2000 × g) and nucleic acids released by mechanical disruption of yeast in the presence of detergents and organic solvent . 250 ul of lysis buffer (20 mM Tris pH 8.0, 200 mM NaCl, 2 mM EDTA, 2% SDS, 4% Triton X-100) and 250 ul of phenol:chloroform:isoamyl alcohol (25:24:1) were added to cells and nucleic acids released by bead beating cells with 0.5 mm-diameter acid-washed glass beads. Phases were separated by centrifugation (5 minutes at 14,000 × g) and the aqueous phase transferred to new tubes. Nucleic acids were recovered by precipitation of the aqueous phase with 2.5 volumes of ethanol. As no efforts were taken to remove RNA co-purifying with the DNA, total nucleic acids were quantified by spectrophotometric readings at 260 nm
Oligonucleotides used for screening T-DNA insertion pools
Addressing and recovery of T-DNA insertion mutants
Yeast from pools showing a positive PCR were thawed from frozen stocks and dilutions were plated on solid HMM + uracil medium to obtain individual clones. One millimeter-diameter colonies were individually picked into 150 ul of HMM + uracil medium in 96-well plates and 25 ul from the wells of each row and column pooled using a multi-channel pipettor. The remaining yeast suspension in the 96-well plate was grown at 37°C with 5% CO2/95% air while addressing PCR was performed. Nucleic acids were prepared from the row and column pools and used as template for PCR. Yeast were recovered from positive wells and plated on solid medium. Single clones were isolated and template nucleic acids prepared for use in PCR. PCR amplicons were purified and sequenced to confirm and localize the insertion in the gene of interest.
Southern blot analysis of T-DNA insertion mutants
T-DNA mutant and WU15 genomic DNAs were prepared and digested overnight with Hind III. Nucleic acid fragments were separated by agarose gel electrophoresis and transferred to a Nytran membrane using a vacuum blot apparatus. Fragments were fixed to the membrane by ultraviolet irradiation (254 nm wavelength, 120,000 uJ/cm2; Stratalinker UV Crosslinker, Stratagene). A nucleic acid probe from the right side of the T-DNA element was prepared by PCR and labeled using the AlkPhos Direct Labeling System (Amersham). The T-DNA probe was hybridized to the membrane and was detected by chemiluminescence using the CDP-Star reagent (Amersham).
Cryopreservation of Histoplasma yeast
Histoplasma yeast were collected from exponential or early stationary phase cultures and added to vials containing either glycerol or dimethylsulfoxide (DMSO). For rapid freezing, vials were mixed and then placed immediately in a rack at -80°C. For gradual freezing, vials were placed within a styrofoam container which was then placed at -80°C. After 24 hours, vials were transferred to racks and stored at -80°C. For recovery, vials were thawed by incubation in a 37°C water bath followed by addition of 2 volumes 37°C HMM. Serial 5-fold dilutions were plated on solid HMM + uracil medium to enumerate viable colony forming units (cfu) for each freezing condition and results were compared to cfu counts before freezing.
Cbp1 production assay
Histoplasma yeast were grown in liquid HMM media to an optical density at 595 nm of 3.2 - 3.8. Histoplasma yeast were removed by centrifugation for 5 minutes at 2000 × g. The supernatant was further clarified by centrifugation for 5 minutes at 15,000 × g. SDS- and DTT-containing protein sample buffer was added to culture supernatants and the proteins separated by 12% poly-acrylamide gel electrophoresis using a Tris-tricine buffer system. The major culture filtrate proteins were visualized by silver staining of gels.
We thank Bill Goldman and members of the Goldman laboratory for providing the WU15 uracil auxotroph and the Agrobacterium strain and vector. This work was supported by an American Heart Association research grant (0865450D) for the analysis of Histoplasma pathogenesis.
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