ABSTRACT
Fungal β-1,3-glucanosyltransferases are cell wall-remodeling enzymes implicated in stress response, cell wall integrity, and virulence, with most fungal genomes containing multiple members. The insect-pathogenic fungus Beauveria bassiana displays robust growth over a wide pH range (pH 4 to 10). A random insertion mutant library screening for increased sensitivity to alkaline (pH 10) growth conditions resulted in the identification and mapping of a mutant to a β-1,3-glucanosyltransferase gene (Bbgas3). Bbgas3 expression was pH dependent and regulated by the PacC transcription factor, which activates genes in response to neutral/alkaline growth conditions. Targeted gene knockout of Bbgas3 resulted in reduced growth under alkaline conditions, with only minor effects of increased sensitivity to cell wall stress (Congo red and calcofluor white) and no significant effects on fungal sensitivity to oxidative or osmotic stress. The cell walls of ΔBbgas3 aerial conidia were thinner than those of the wild-type and complemented strains in response to alkaline conditions, and β-1,3-glucan antibody and lectin staining revealed alterations in cell surface carbohydrate epitopes. The ΔBbgas3 mutant displayed alterations in cell wall chitin and carbohydrate content in response to alkaline pH. Insect bioassays revealed impaired virulence for the ΔBbgas3 mutant depending upon the pH of the media on which the conidia were grown and harvested. Unexpectedly, a decreased median lethal time to kill (LT50, i.e., increased virulence) was seen for the mutant using intrahemocoel injection assays using conidia grown at acidic pH (5.6). These data show that BbGas3 acts as a pH-responsive cell wall-remodeling enzyme involved in resistance to extreme pH (>9).
IMPORTANCE Little is known about adaptations required for growth at high (>9) pH. Here, we show that a specific fungal membrane-remodeling β-1,3-glucanosyltransferase gene (Bbgas3) regulated by the pH-responsive PacC transcription factor forms a critical aspect of the ability of the insect-pathogenic fungus Beauveria bassiana to grow at extreme pH. The loss of Bbgas3 resulted in a unique decreased ability to grow at high pH, with little to no effects seen with respect to other stress conditions, i.e., cell wall integrity and osmotic and oxidative stress. However, pH-dependent alternations in cell wall properties and virulence were noted for the ΔBbgas3 mutant. These data provide a mechanistic insight into the importance of the specific cell wall structure required to stabilize the cell at high pH and link it to the PacC/Pal/Rim pH-sensing and regulatory system.
INTRODUCTION
Known for their remarkable bioremediation, pathogenic, and/or secondary metabolite production capabilities, fungi have evolved the ability to thrive on a wide variety of substrates under diverse environmental conditions (1). Filamentous fungi (Ascomycota) typically prefer neutral-to-acidic growth conditions and, indeed, often acidify their surrounding media/environment during growth; however, many of these fungi are quite capable of robust growth under alkaline conditions. Environmental pH can affect nutrient and mineral availability, fungal enzyme activities, and fungal membrane integrity and function. The fungal cell wall has evolved specific structural features to protect the cell against adverse stresses resulting from fluctuating environmental conditions. Polymers of β-1,3-glucans form the major cell wall carbohydrate component of yeasts and filamentous fungi, constituting 40 to 70% of the cell wall mass, although cell walls (and particularly septae) contain more small amounts of chitin (long chains of β-1,4-linked N-acetylglucosamine) (2). In the process of cell wall synthesis, chitin and glucan synthase complexes on the plasma membrane assemble UDP-N-acetylglucosamine and UDP-glucose into linear chitin and β-1,3-glucan chains, respectively, that extrude into and out of the cell wall space (3). For the β-glucans, the linear chains are often branched, elongated, modified, and/or cross-linked to other cell wall components, forming an integrated and flexible steric structure. Moreover, the organized fungal cell wall is dynamic and constantly remodeled both as a function of growth and due to responses to fluctuating environmental conditions, including changes in ambient pH, temperature, and osmotic and oxidative stress (4–6). For fungal pathogens, cell wall epitopes can also change in response to host cues, in particular in order to evade host defense and immune responses (7, 8).
In yeasts and various fungi, several different nomenclature systems have been used to categorize the enzymes involved in β-1,3-glucan synthesis, i.e., β-1,3-glucanosyltransferases, including glycolipid anchored surface (Gas), glucan elongation (Gel) proteins, and pH response (PHR) proteins (6, 9, 10). Characterization of the five GAS proteins found in Saccharomyces cerevisiae has revealed differential, but sometimes overlapping, contributions of these proteins in cell morphogenesis, growth, cell wall integrity, and even transcriptional silencing (11–13). In the human yeast pathogen, Candida albicans, which also contains five β-1,3-glucanosyltransferases (PHR) genes, two, PHR1 and PHR2, are regulated in response to ambient pH (14). The expression of PHR1 is induced at neutral to alkaline pH (7 to 8) and repressed at pH <5.5, whereas PHR2 expression is induced under acidic conditions and repressed at neutral/alkaline pH (15). Both of these genes appear to be regulated by PacC/Rim101 that acts as an activator and repressor for PHR1 and PHR2 expression, respectively, by binding to the specific sites (GCCARG) in their promoter regions (16, 17). In the opportunistic fungal pathogen Pneumocystis jirovecii, PHR1 is also regulated in response to environmental pH and can rescue the S. cerevisiae Δgas1 mutant phenotype of lethality under conditions of cell wall stress (18). In the filamentous fungus Neurospora crassa, disruption of any of the five gel genes did not affect pH sensitivity (19). In terms of functional similarity, both gel1 and gel2 of the human pathogen Aspergillus fumigatus (containing seven gel genes) could rescue the growth and the morphological defects of the S. cerevisiae Δgas1 mutant (20). In A. fumigatus, Gel7 contributes to cell wall integrity under stress conditions, while Gel4 appears to be essential (21, 22). To the best of our knowledge, the contributions of β-1,3-glucanosyltransferases to alkaline adaptation have been examined exclusively in yeast and only under moderate alkaline conditions (up to pH 8), with no reports examining the functions of these enzymes in mediating growth under extreme alkaline conditions (pH 10).
The fungus-specific Pal/Rim signaling and response pathway is known to play an important role in the ability of fungi to cope with variation and changes in the ambient pH of the surrounding environment. Depending upon the fungus, the Pal/Rim pathway encodes sensor and transduction factors that are activated in response to specific pH signals, ultimately targeting the PacC/Rim101 transcription factor that regulates the expression of various genes, presumably involved in pH adaptation (23, 24). Under neutral or alkaline conditions, the Pal/Rim pathway stimulates the proteolytic cleavage of (inactive) cytoplasmic form of PacC, resulting in its activation and subsequent translocation into the nucleus, where it activates the transcription of “alkaline”-expressed genes, while repressing “acid”-expressed genes (25). In various fungi, PacC activity has also been shown to contribute to/regulate other processes, including secondary metabolite production, ion homeostasis, pathogenicity (in some, but not all cases), and cell wall synthesis and remodeling (26, 27). In the insect pathogen, Metarhizium robertsii, deletion of the pacC gene (MrpacC) resulted in reduced growth across a wide pH range (4 to 10), decreased sporulation, and decreased virulence (4). In contrast, in the insect pathogen Beauveria bassiana, the loss of pacC resulted in decreased growth under alkaline conditions, along with increased sensitivity to the cell wall stress, but it had little effect on virulence (28). However, few downstream targets of PacC that can account for the various phenotypes have been identified and characterized.
The insect-pathogenic fungus Beauveria bassiana represents a class of environmentally friendly fungal pathogens responsible for epizootic infections of insect populations in nature, and it is currently being used in various agricultural applications for the control of different insect pests (29). B. bassiana has also developed into a model system useful for exploring the genetics and evolution of fungal host-pathogen interactions, stress response, and development (30–32). Infection begins as spores attach, germinate, and penetrate the insect cuticle, with cuticle penetration involving the coordinated action of mechanical pressure, cuticle-degrading hydrolases, and other factors (33, 34). Changes in pH during the infection process are critical aspects manipulated by the fungus in order to successfully invade and infect the host. Initial acidification of the media, in part due to the production of acidic metabolites, including oxalate, gives way to alkalinization (via an as-yet-unknown mechanism) as the fungus penetrates the host integument and into the hemocoel, with specialized acid- and alkaline-active hydrolases having been characterized (35–38). B. bassiana is capable of robust growth over a wide pH range from 4 to 10.5 (39). The components of the Pal/Rim system, including PacC, have been characterized in B. bassiana, where although BbPacC was found to be required for acidification of media, e.g., oxalate production, and for growth at high pH (8 to 10.4), pacC mutants showed only minor impairment of virulence (28, 40).
Here, we report on a screen of a B. bassiana (random) transfer DNA (T-DNA) insertion library for mutants sensitive to extreme alkaline pH (10). One such mutant (T340) was mapped to a β-1,3-glucanosyltransferase annotated as gas3. Targeted gene knockout and complementation strains of Bbgas3 confirmed the initial phenotype, with the ΔBbgas3 mutant displaying poor growth at high pH (10). The ΔBbgas3 mutant showed little to no changes in resistance to most other stress conditions or in virulence, indicating a specific role for Bbgas3 in uniquely mediating alkaline resistance. Changes in cell wall epitopes were observed in the ΔBbgas3 mutant, and a regulatory pathway involving PacC was established.
RESULTS
Identification of gas3 and sequence analysis of Gas family in B. bassiana.A previously constructed B. bassiana T-DNA insertion library (41) was screened for mutants impaired for growth in potato-dextrose agar (PDA) buffered to pH 10. The insertion site of the mutant, designated T340, was mapped to a region containing two putative open reading frames, one encoding a function-unknown protein (accession no. KGQ07720) and a β-1,3-glucanosyltransferase (accession no. EJP67416) annotated as gas3. Complementation experiments indicated that that only the gas3 gene was able to rescue the T340 sensitivity to alkaline-growth-condition phenotype (see Fig. S1 and S2 in the supplemental material). Analyses of the available genomes for two B. bassiana strains (2860 and D1-5) indicated the presence of the following six putative β-1,3-glucanosyltransferase proteins: Gas1 (accession no. EJP66700), Gas2 (accession no. EJP62516), Gas3 (accession no. EJP67416), Gas4 (accession no. EJP61263), Gas5 (accession no. EJP68186), and Gas6 (accession no. EJP65749). As has been previously noted, phylogenetic analyses revealed the separation of the Gas proteins into two subfamilies (GH72− and GH72+) (Fig. S3). B. bassiana gas3, as well as gas4 and gas6 (GH72+), grouped with other filamentous fungal enzymes, including Magnaporthe oryzae gel4, A. fumigatus gel3, gel4, gel5, and gel7, C. albicans PHR1 and PHR2, and the yeast S. cerevisiae gas1 (Scgas1) and Scgas2. The GH72+ enzymes are characterized by containing an X8 (carbohydrate binding module containing six conserved cysteine residues [42]) domain absent in the GH72− proteins (Fig. S3). The B. bassiana Gas proteins contain conserved N-terminal signal peptides, a catalytic domain with two glutamic residue (E) catalytic residues, and C-terminal glycosylphosphatidylinositol pathogenicity island (GPI) anchor sites, as well as various Ser-rich and Gsn/Asp-rich regions. Variation in the number of gas genes identified in different strains was noted. As indicated above, our analysis of the two available B. bassiana genomes (2860 and D1-5) revealed six putative β-1,3-gluconosyltransfersae genes; however, the gas4 gene was not detectable in genomic DNA of Bb0062 (the strain used for construction and screening of the T-DNA mutant library) using PCR. Analyses of 10 different B. bassiana strains revealed the amplification of gas4 sequences in five of them. These included the B. bassiana GHA strain (registered product for insect control) and B. bassiana ATCC 90517, with ATCC 90517 being one of the most commonly used strains in the molecular characterization of this fungus and the strain used in this study (30–32) (Fig. S4).
B. bassiana gas3 is expressed in response to pH and is controlled by PacC.The expression pattern of the six B. bassiana gas genes was examined in response to environmental pH by quantitative real-time PCR (Fig. 1A). Bbgas1 expression was similar under acidic (pH 4) and neutral (pH 7) conditions, with a slight decrease in expression seen at alkaline pH (10). Bbgas2 expression was similar to that of Bbgas1, although its pattern gradually decreased from acidic to alkaline conditions. In contrast, Bbgas3 expression was low under acidic conditions but was induced 4-fold and 19-fold at pH 7 and 10, respectively. Bbgas5 and Bbgas6 expression levels were the reverse of Bbgas3, with high expression seen under acidic pH conditions, which was significantly decreased under neutral and alkaline pH conditions. BbpacC expression mirrored that of Bbgas3, with low expression at pH 4 and 7 and 6-fold induction at pH 10 (Fig. 1B). In order to examine whether Bbgas3 expression was dependent upon PacC, Bbgas3 and BbpacC expression was examined in response to pH (Fig. 1B), and Bbgas3 expression was examined in the wild type and a ΔBbpacC mutant background (Fig. 1C). These data showed coordinated expression of Bbgas3 and BbpacC and a failure of Bbgas3 induction in response to pH 7 and 10 in the ΔBbpacC mutant. These results are consistent with analyses of the Bbgas3 the promoter region that was found to contain six putative PacC binding sites (GCCARG), as well as putative self-regulation of PacC, as has been previously reported (28).
Transcriptional profiles of B. bassiana β-1,3-glucanosyltransferase genes. (A) Expression of Bbgas1 to Bbgas6 in wild-type B. bassiana as a function of external pH. (B) Expression of Bbgas3 and PacC in wild-type B. bassiana. (C) Expression of Bbgas3 in a B. bassiana ΔBbpacC background strain as a function of pH. Error bars are the standard deviation (SD) of the mean of the results from three replicate assays. Asterisks indicate significant differences.
Construction and analysis of Bbgas3 targeted gene deletion and complementation strains.In order to verify the initial T-DNA mutant phenotype and to further probe the functions of Bbgas3, targeted gene deletion (ΔBbgas3 mutant) and complementation (ΔBbgas3::Bbgas3 mutant) strains were constructed as detailed in Materials and Methods (Fig. S5). The phenotype of the ΔBbgas3 mutant strain was identical to the original T-DNA mutant in sensitivity to alkaline pH (Fig. 2A). Further phenotypic examination revealed small effects with regard to increased sensitivity to Congo red and calcofluor white (Fig. S6), but no apparent consequence of a loss of Bbgas3 on osmotic (salt and sorbitol), oxidative (H2O2 and menadione), or temperature (32°C) stress was noted (Fig. S7). As expected, the targeted gene deletion of Bbgas3 resulted in a loss of Bbgas3 transcript, with only minor effects on the expression of the remaining Bbgas genes (Fig. 2B). Expression of the Bbgas genes in the ΔBbgas3 mutant continued to be pH dependent (as seen in Fig. 1), with analyses of the ratio of ΔBbgas3 or wild-type Bbgas gene expression (which would be 1 if no change occurred) indicating a slight decrease in Bbgas1 expression at pH 10, which was found not to be statistically significant, a 1- to 2-fold decrease in Bbgas2 expression at pH 10, which was statistically significant (P < 0.05), as was Bbgas5 expression (decreased by 1- to 2-fold) at all pH values. In addition, Bbgas6 expression at pH 10 also appeared to be decreased (at pH 10), but the effect was not statistically significant.
Growth of wild type (WT), ΔBbgas3 mutant, and complemented (ΔBbgas3::Bbgas3) strains on PDA buffered to indicated starting pH values, as described Materials and Methods. Plates were spot inoculated with a conidial suspension of each strain and incubated at 26°C for 7 days. (B) Quantitative real-time PCR (qRT-PCR) expression profile of Bbgas genes (1–6) in the B. bassiana ΔBbgas3 mutant as a function of the pH of the growth media. Fungal cultures were grown RNA extracted as detailed in Materials and Methods. Error bars are the SD of the mean of the results from three replicate assays; asterisks indicate significant differences (Tukey's HSD, P < 0.01).
In order to evaluate any effects of loss of Bbgas3 on cell wall composition, the concentrations of chitin, the alkali-soluble fraction (containing β-1,3/1,6-glucans), and the alkali-insoluble fraction (β-1,3/1,6-glucan covalently linked to chitin) were quantified in the various strains (Fig. 3A). Compared to the wild-type strain, the ΔBbgas3 mutant showed comparable levels of total sugar content as well as chitin and alkaline-soluble alkaline-insoluble at pH 7, but increases in the alkali-insoluble fraction and chitin content were observed when cells were responding to alkaline conditions (pH 10), with a decrease in the alkali-soluble component at pH 10. Calcofluor white staining of chitin revealed increased labeling of growing germling/hyphal tips in the ΔBbgas3 mutant strain under neutral and alkaline conditions compared to those of the wild-type control (Fig. 3B).
Effect of Bbgas3 deletion on B. bassiana cell wall components. (A) Total carbohydrate, alkaline-soluble and -insoluble, and chitin content of B. bassiana wild-type and ΔBbgas3 mutant cells grown in media buffered to pH 7 and pH 10, and analyzed as detailed in Materials and Methods. Error bars are the SD of the mean of the results from three replicate assays, and asterisks on column bars indicate significant difference (Tukey's HSD, P < 0.01). (B) Calcofluor white staining of wild-type and ΔBbgas3 mutant cells. Inset and arrow indicate enhanced chitin deposition in the hyphal tip of the mutant at pH. Scale bar = 500 μm.
Transmission electron microscopic visualization of the fungal cell wall revealed a significant (P < 0.05) reduction in the cell wall thickness of ΔBbgas3 aerial conidia grown at pH 10 but no effect on conidia harvested from plates buffered to pH 7 (Fig. 4A and B and S8). In addition, a significant difference (P < 0.05) in aberrant conidia, with apparent distorted and/or partially collapsed shapes and poor cytoplasmic staining, was seen for ΔBbgas3 mutant conidia at pH 7 (5% versus <0.1% for the wild type) that increased to ∼12% of the harvested conidia grown at pH 10 for the mutant, compared to ∼2% abnormal cells seen in the wild-type strain (Fig. 4C and D). In order to determine any effects of the loss of Bbgas3 on cell surface carbohydrate epitopes, lectin (wheat germ agglutinin [WGA] and concanavalin A [ConA]) and β-1,3-glucan antibody mapping of aerial conidia was performed using confocal microscopy and flow cytometry. Conidia derived from pH 7 growth conditions displayed a moderate (P < 0.05) decrease in cell surface β-1,3-glucan, as well as a large increase in β-N-acetylglucosamine (β-GlcNAc) and/or sialic acid epitopes (WGA reactive), with no change in α-glucose and/or α-N-GlcNAc epitopes (ConA reactive) (Fig. 5A). Similar to cells at pH 7, conidia derived from pH 10 growth conditions displayed a moderate (P < 0.05) decrease in cell surface β-1,3-glucan; however, decreases (P < 0.05) in both WGA-reactive and ConA-reactive cell surface epitopes were seen for these cells (Fig. 5B).
Analysis of conidial cell walls and morphology of the B. bassiana wild type (WT) and ΔBbgas3 mutant. (A and B) TEM images and quantification of conidial cell wall thickness. Scale bar = 0.2 μm. (C and D) Representative images and quantification of distorted conidial morphologies seen in the ΔBbgas3 mutant. Arrows indicate measurement of cell wall thickness. Scale bar = 0.5 μm. Asterisks on column bars indicate significant difference (Tukey's HSD, P < 0.01).
Flow cytometry distribution and quantification of lectin and β-glucan antibody staining in B. bassiana wild-type (black) and ΔBbgas3 mutant (green) aerial conidia. Error bars are the SD of the mean of the results from three replicate assays. ConA and WGA are the lectins of wheat germ agglutinin and concanavalin A, respectively. Stars on column bars indicate significant differences (Tukey's HSD, P < 0.01).
Deletion of Bbgas3 does not affect acidification and has minor effect on virulence depending upon the pH of the growth media.Previous work in B. bassiana has shown a link between PacC and extracellular acidification, oxalate production, and increased sensitivity to alkaline pH (28). Acidification of the external media was assessed qualitatively via incorporation of the pH indicator dye bromocresol purple in agar plates, and quantitatively in the supernatant derived from liquid cultures (Fig. 6A and B). No differences in acidification were noted between any of the strains. In addition, the expression of Bbgas3 in the ΔBbpacC mutant background did not rescue the alkaline sensitivity phenotype of this mutant (Fig. 6C).
(A) Growth of B. bassiana wild-type, ΔBbgas3 mutant, and complemented (ΔBbgas3::Bbgas3) strains on CZA containing 0.05% bromocresol purple indicating ambient acidification. (B) Determination of the pH of growth media (CZB) inoculated with each respective fungal strain and measured at the indicated time course. (C) Growth of the wild-type, ΔBbpacC mutant, and ΔBbpacC::Bbgas3 complemented strains are shown on buffered PDA at indicated starting pH values, as described in Materials and Methods. Plates were inoculated with conidial suspensions and incubated at 26°C for 7 days.
The effect of Bbgas3 deletion on B. bassiana virulence was probed via insect bioassays using Galleria mellonella larvae. Suspensions of aerial conidia (1 × 107 conidia · ml−1) derived from the wild-type, ΔBbgas3 mutant, and ΔBbgas3::Bbgas3 complemented strains were isolated from three different growth conditions; namely, PDA buffer to pH values of 5.6 (acidic), 7.0 (neutral), and 10 (alkaline) were either (i) applied topically to the larvae, representing the natural route of infection, or (ii) injected into hemocoel cavity, thus bypassing penetration. Mortality was determined over a 12-day period, and the median lethal time (LT50) was estimated as detailed in Materials and Methods. Using conidia produced on acidic PDA media (pH 5.6), the deletion of Bbgas3 did not result in any significant changes of LT50 values using topical infections; however, a decrease in LT50 (i.e., increased mortality, P < 0.01) was seen in intrahemocoel injection assays (Fig. 7A and B and Table 1). Conidia produced on neutral PDA media showed no significant changes in mortality in either the topical or intrahemocoel injection bioassay (Fig. 7C and D and Table 1); however, the conidia derived from the ΔBbgas3 mutant harvested from alkaline PDA medium showed significantly increased (i.e., decreased virulence; P < 0.01) LT50 values, by ∼22% and 70%, in topical and intrahemocoel injection assays, respectively (Fig. 7C and D and Table 1). Insect cadavers appeared identical to the wild type, and no differences in sporulation on the cadavers were noted between the wild type and the ΔBbgas3 mutant (Fig. S9).
Insect bioassays. G. mellonella larvae were treated either topically (A, C, and E) or via intrahemocoel injection (B, D, and F) with conidia from wild-type B. bassiana (■), ΔBbgas3 mutant (●), ΔBbgas3::Bbgas3 complemented (◆) strains, or mock-treated controls (○), as described in Materials and Methods. (A to F) Conidia derived from growth on PDA at pH 5.6 (A and B). 7.0 (C and D), and pH 10 (E and F). The percent survival ± SD over the indicated time course is presented.
LT50 values for B. bassiana wild-type, ΔBbgas3 mutant, and complemented (ΔBbgas3::Bbgas3) strains in topical and intrahemocoel injection bioassays using G. mellonella larvae as the host
DISCUSSION
Remodeling of cell wall β-1,3-glucan via the activities of GPI-anchored β-1,3-glucanosyltransferases (Gas/Gel proteins) belonging to the glycoside hydrolase family 72 (GH72) is a critical process seen on all fungi (21). These proteins exist as gene families whose functions have diversified. For example, the previously characterized B. bassiana gas1 gene as well as the F. oxysporum gas1 gene (Fogas1) were unable to complement the yeast gas1 mutant, indicating divergence either in processing/targeting of the enzymes and/or in their enzymatic activities (5, 43). In the yeast C. albicans, two β-1,3-glucanosyltransferase genes, phr1 and phr2, have been shown to contribute to fungal adaptation to neutral/mildly alkaline (6 to 8) and acidic conditions, respectively (17, 44). Germ tubes produced by Prh1Δ mutants displayed abnormalities in elongation, accumulate chitin, and show constitutive activation of the Mkc1, Cek1, and Hog1 mitogen-activated protein (MAP) kinase signaling pathways. However, no functions for β-1,3-glycanosyltransferases in response to pH have yet been reported in filamentous fungi, and the effects at extreme alkaline pH values (10) have yet to be reported for any fungus. In M. oryzae, individual β-1,3-glycanosyltransferases (Gel protein) have been shown to be dispensable for pathogenicity, with none of the mutants affected in terms of fungal sensitivity to pH (2). However, M. oryzae Δgel1 Δgel3 Δgel4 triple mutants could not infect or sporulate on intact rice leaf. These and other studies indicate that some Gas/Gel proteins appear to show partially overlapping or redundant biological roles, with few reports of specific defined functions for these proteins (2, 19, 45). Previous characterization of β-1,3-glycanosyltransferases in B. bassiana (Bbgas1) did not show any effects with respect to pH, with Bbgas1 instead contributing to conidial thermotolerance and virulence (5).
Here, we report that B. bassiana gas3, encoding a putative β-1,3-glycanosyltransferase, is a key factor for fungal adaptation to extreme alkaline conditions by modulating cell wall integrity. The biological role of Bbgas3 was very specific, with no effects seen in the ΔBbgas3 mutant with respect to oxidative, osmotic, or high-temperature stress. Our prior work has documented that both the Msn2 and PacC transcription factors modulate pH effects, with PacC being the target of the Pal pH sensor/response pathway (28). Our data show that Bbgas3 shares a similar transcriptional pattern with and is positively regulated by PacC in response to ambient pH, with the Bbgas3 promoter sequence containing multiple putative PacC binding sites. These data are in contrast to observations in M. robertsii, where PacC has been shown to negatively regulate the transcription of several glycosyltransferase genes involved in cell wall synthesis/remodeling, although the regulation of β-glucanosyltransferases in M. robertsii has not yet been examined (4). In B. bassiana, PacC is also known to control the production or degradation of organic acids (particularly oxalate acid) and environmental acidification (28, 46), with oxalate implicated as an important virulence factor (47). However, the deletion of Bbgas3 did not affect environmental acidification, a not-too-surprising result, as the genes responsible for these phenotypes are other targets for PacC. In addition, overexpression of Bbgas3 in a ΔBbpacC mutant background failed to rescue the pH sensitivity of the parent mutant. This result implies that a loss of PacC results in pleiotropic effects on a loss of cell wall integrity, consistent with the broad phenotypic effects of the mutant (as opposed to the narrow phenotype seem in for the ΔBbgas3 mutant). The ΔBbgas3 mutant showed little to no defects in virulence via either topical infection, which represents the natural route of infection, or via injection of the fungal spores directly into the insect hemocoel cavity, thus bypassing the need for cuticle penetration when using conidia derived from PDA at neutral pH (7). A slight increase in virulence was noted for the ΔBbgas3 mutant in intrahemocoel injection assays when using conidia harvested from acidic (pH 5.6) PDA, whereas decreased virulence was seen for the ΔBbgas3 mutant harvested from alkaline (pH 10) PDA plates in both topical and intrahemocoel injection (particularly intrahemocoel injection) bioassays. Although at present there is little indication as to why we observed the increase in virulence in the mutant in injection assays using acid-condition derived conidia, the decreased virulence under alkaline conditions likely reflects the decreased cell wall robustness/integrity seen in the mutant. These results identify pH- and cell wall-dependent effects on virulence attributable to a cell wall-remodeling enzyme. When successful in infecting the host, sporulation on cadavers was normal, indicating that the mutant could successfully complete all stages of the infection cycle. Although tangential to the main results of this report, our data indicate some strain variation in the number of β-glucanosyltransferase genes identified, with approximately half (5/10) of the strains examined appearing to lack a gas4 gene homolog. As complete genomes for the strains lacking a gas4 homolog are not yet available, it cannot presently be ruled out that such a gene may still exist but has diverged enough from the test primers used to detect the gene that PCR amplification was not possible. Alternatively, redundancy or other factors may have made this gene expendable in some lineages.
The specific contribution of Bbgas3 to mediating resistance to extreme alkaline pH is, to our knowledge, the only example of such a strictly defined role for a fungal β-1,3-glycanosyltransferase. Further work determining the enzymatic mode of its action, i.e., what glucan cross-links are made by the enzyme that helps confer alkaline resistance to a membrane, is warranted. Our data show a decrease in cell wall thickness in the ΔBbgas3 mutant (at pH 10 but not pH 7), as well as global changes in cell wall carbohydrate content, including increased alkaline-insoluble glucan and chitin content and decreased alkaline-soluble content (again all only at pH 10), factors that affect cell wall integrity. In addition, increased deposition of chitin was seen at hyphal tips, and alterations in cell wall carbohydrate epitopes were seen that can help account for the alkaline sensitivity of the gas3 mutant. However, it should also be noted that aside from the role in glucan remodeling, at least one of Gas proteins, namely, S. cerevisiae GAS1, has been shown to affect transcriptional silencing via carbohydrate modification of chromatin and/or via maintenance of ribosomal DNA (rDNA) integrity (12, 48). Thus, it cannot be ruled out that the effects of Bbgas3 may also involve such mechanisms. In conclusion, we report the function of gas3 in the insect-pathogenic fungus B. bassiana as a specific modulator of fungal adaptation to extreme alkaline conditions. Bbgas3 is a downstream target of PacC and acts as a cell wall glucan-remodeling enzyme.
MATERIALS AND METHODS
Fungal strains and culture conditions.B. bassiana wild-type strains Bb0062 and ATCC 90517 and various mutants were routinely grown/maintained on potato dextrose broth/agar (PDB/PDA) or Czapek-dox broth/agar (CZB/CZA) (BD Difco, Sparks, MD, USA) at 26°C for 7 to 14 days. Conidia harvested from agar plates were suspended in sterile 0.05% Tween 80 solution and filtered through a single sheet of lens-wiping paper to remove any hyphae and/or mycelia. The final conidial concentration was determined using a hemocytometer. For pH adaptation determination, the PDA medium was buffered with NaHPO4·NaH2PO4 or citric acid-glycine, as described previously (39).
Gene deletion, complementation, and expression.Targeted gene deletion of B. bassiana was performed by homologous recombination via Agrobacterium-mediated fungal transformation, as described previously (39, 49). In brief, the coding region of Bbgas3 was replaced by the bar cassette conferring resistance to phosphinothricin to generate the ΔBbgas3 knockout mutant. The 3′-flanking (1,030 bp) and 5′-flanking (601 bp) sequences of Bbgas3 were amplified from B. bassiana genomic DNA via PCR using primer pairs gas3-LB-F/gas3-LB-R and gas3-RB-F/gas3-RB-R, respectively. The PCR products were inserted into the EcoRI and XbaI sites of pK2bar to form pK2bar-gas3-ko for fungal transformation. For ΔBbgas3 complementation, a fragment containing the complete open reading frame (ORF) (1,694 bp), plus promoter (2.1 kb upstream) and terminator (0.6 kb downstream) sequences, was amplified using the gas3-C-F/gas3-C-R primer pair. The product was inserted into pK2sur (39) at the XbaI restriction site, forming pK2sur-gas3 (sur marker conferring resistance to chlorimuron-ethyl). A constitutive Bbgas3 expression vector, pK2sur-pb3::gas3, was constructed as follows: a fragment containing the transcriptional terminator trpC from Aspergillus nidulans and the multiple-cloning site (MCS) from the vector pBARGPE1 was amplified using the MCS-F/TtrpC-R primer pair. The constitutive promoter derived from the glyceraldehyde phosphate dehydrogenase gene (Bbgpdp) was amplified from B. bassiana genomic DNA using the Pb-F/Pb-R primer pair. The above-mentioned two PCR fragments were digested with BglII/BamHI and XbaI/BamHI, respectively, and subsequently ligated into the vector pK2sur at the BamHI/XbaI site to produce pK2sur-pb3. Subsequently, the Bbgas3 coding region was amplified from B. bassiana cDNA using the OE-gas3-F/OE-gas3-R primer pair and inserted into the BamHI/EcoRV site of pK2sur-pb3 to form pK2sur-pb3::gas3.
RNA isolation and real-time PCR analysis.B. bassiana conidia were inoculated in PDB for 3 days, and mycelia were harvested by filtration, transferred to PDB buffered to pH values of 4, 7, and 10, and incubated with aeration for 2 h. Cells were harvested by centrifugation, and samples were ground in liquid nitrogen. Total RNA was isolated using the TRIzol reagent (Invitrogen) and treated with DNase I (TaKaRa, Dalian, China). The cDNA of each sample was synthesized using the RevertAid first-strand cDNA synthesis kit (MBI Fermentas). Quantitative real-time PCR analysis was performed using the CFX Connect real-time PCR detection system (Bio-Rad, USA) using SYBR Premix (Bio-Rad). PCRs were performed as follows: a 10-min denaturation at 95°C was followed by 40 cycles of 30 s at 95°C, 30 s at 56°C, and 30 s at 72°C. The relative expression of each gene was normalized against the expression of the β-tubulin gene (GenBank accession no. AJ312228) using the TubF/TubR primer pair.
Phenotypic assays.Fungal growth was assessed by spotting conidial suspensions (3 μl of 1 ×107 conidia/ml in 0.05% Tween 80) on buffered PDA at indicated pH values with or without Congo red (100 μg/ml) or calcofluor white (100 μg/ml) and incubating at 26°C for 7 days. To determine the effects of oxidative, osmotic, and temperature stress on fungal growth, conidial suspensions (3 μl of 1 ×107 conidia/ml in 0.05% Tween 80) of each strain were spotted on CZA plates or CZA containing menadione (60 μM), H2O2 (20 μM), 0.8 M NaCl, 1.0 M KCl, or 1.2 M sorbitol and incubated at 26°C or 32°C over a 10-day time course. The pH indicator dye bromocresol purple (0.01%) was added to the CZA to monitor acidification of the media during fungal growth. Colony morphologies and diameters were determined at the end of each time period.
Chitin and glucan determination.Fungal cells were grown in PDB for 72 h, after which the cells were transferred to fresh PDB buffered to pH 7 or pH 10. The total fungal mass of the cultures was harvested by filtration. Chitin content determination was performed as described previously (50, 51), with a slight modification. Briefly, samples were washed three times using 1 M NaCl and then ground in liquid nitrogen before SDS extraction (0.3 g) via boiling in 5 ml isolation solution (1.35 mM SDS, 2 mM EDTA, and 3% [vol/vol] β-mercaptoethanol) for 5 min. Samples were centrifuged at 12,000 rpm for 3 min and the resultant pellet suspended in 6 N HCl (6 ml), followed by hydrolysis at 100°C for 6 h and evaporation at 65°C. The samples were then dissolved in 1 ml water, and aliquots of equal volume (0.1 ml) of sample and solution A (1.5 N Na2CO3 in 4% acetylacetone) were mixed and incubated at 100°C for 20 min, followed by the addition of 0.7 ml of 96% ethanol and then 0.1 ml solution B (1.6 g of p-dimethylaminobenzaldehyde in 30 ml of concentrated HCl and 30 ml of ethanol). After 1 h of incubation at room temperature in the dark, the absorbance of the mixture at 520 nm was measured. Chitin content was quantified using a standard curve of 0 to 200 ng glucosamine.
Glucan content was determined as described previously (52), with minor modifications. Briefly, harvested cells were ground in liquid nitrogen, and samples (10 mg) were suspended in 1 ml 98% formic acid and incubated at 100°C for 20 min. After centrifugation at 12,000 rpm for 3 min, the supernatant was collected and dried in a vacuum desiccator. The dried residue was then dissolved in 2 ml water. Neutral carbohydrate content was quantified using the phenol-sulfuric acid method of Dubois et al. (53). For the detection of alkali-soluble and -insoluble fractions, ground samples (10 mg) were suspended in 1 ml water followed by incubation at 100°C for 20 min. The suspension was centrifuged at 12,000 rpm for 2 min and the supernatant removed from the pellet. The pellet was resuspended either in (i) 1 ml KOH (for determination of the alkali-soluble fraction), followed by incubation at 72°C for 30 min, or (ii) 1 ml of 98% formic acid (for determination of the alkali-insoluble fraction), followed by incubation at 100°C for 20 min. Samples were centrifuged at 12,000 rpm for 2 min, and the supernatants of each treatment were used to determine carbohydrate (glucan) content as described above.
Microscopy and β-1,3-antibody/lectin binding assay.Samples for transmission electron microscopy (TEM) were prepared as described previously (5). Sections were examined using a Hitachi H-7000 TEM and digital images acquired using a Veleta camera and the iTEM software. Lectin binding assays were performed as described previously (54). Briefly, conidia of each strain were harvested by centrifugation and suspended in the appropriate lectin binding buffer, as recommended by the manufacturer's protocols. Alexa Fluor 488-labeled concanavalin A (ConA) and wheat germ agglutinin (WGA) lectins were obtained from Molecular Probes (Invitrogen). Cell wall β-1,3-glucan was labeled using a mouse monoclonal antibody (Biosupplies, Parkville, Australia), as described previously (33). Briefly, conidia were fixed in 3% (vol/vol) formaldehyde for 30 min and then washed three times using phosphate-buffered saline (PBS) buffer before being suspended in PBS buffer containing 1% (vol/vol) Tween 20 (PBS-T). Subsequently, the conidial suspensions were incubated with the anti-β-1,3-glucan antibody (0.1 mg · ml−1 in PBS buffer) overnight at 4°C. Samples were then washed three times using chilled (4°C) PBS before incubation with a fluorescein isothiocyanate (FITC) goat anti-mouse IgG (H+L) antibody (Proteintech) (0.1 mg · ml−1 in PBS-T buffer) as the secondary antibody for another 2 h in the dark. Samples were then rinsed 3 times (PBS) and resuspended in PBS. Fluorescence was observed using a confocal laser scanning microscope (FV1000; Olympus). Data acquisition and manipulation were performed with FACSCalibur (BD) and FACSDiva (BD), and the fluorescence was measured for ∼106 conidia.
Insect bioassays.Fungal virulence bioassays were performed using Galleria mellonella larvae as the host. Conidia harvested from PDA (pH 5.6) or PDA buffered to pH values of 7 and 10 after 10 to 14 days of growth were used for topical infection and intrahemocoel injection assays. Briefly, (i) larvae were dipped into conidial suspensions (107 conidia · ml−1) for about 5 s and placed on dry paper towels to remove the excess liquid on the insect surface. Larvae of the control were treated with 0.05% Tween 80. (ii) Larvae were injected with 5 μl of 105 conidia · ml−1 directly into the hemocoel cavity. Control larvae were treated with 0.05% Tween 80. All treated larvae were placed in 150-mm petri dishes and incubated in a manual climatic box at 26°C. Each treatment including the control contained 90 to 100 larvae, and all experiments were repeated three times with independent batches of conidia. The number of dead larvae was recorded daily, and the median lethal time (LT50) was calculated using Probit analysis of the SPSS 11 software. Statistical analyses for mortality experiments were performed using one-way analysis of variance (ANOVA) (SPSS).
ACKNOWLEDGMENTS
This work was jointly supported by the National Key R&D Program of China (grant 2017YFD0201202), the Natural Science Foundation of Chongqing (grant CSTC2017jcyjAX0342), and the Fundamental Research Funds for the Central Universities (grants XDJK2015B032 and XDJK2017D196) to Z.L. and T.Z., and a U.S. National Science Foundation grant (IOS-1557704) to N.O.K.
We declare no conflicts of interest.
FOOTNOTES
- Received 7 May 2018.
- Accepted 17 May 2018.
- Accepted manuscript posted online 25 May 2018.
Supplemental material for this article may be found at https://doi.org/10.1128/AEM.01086-18.
- Copyright © 2018 American Society for Microbiology.
