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Loss of Septation Initiation Network (SIN) kinases blocks tissue invasion and unlocks echinocandin cidal activity against Aspergillus fumigatus

['Ana Camila Oliveira Souza', 'Department Of Clinical Pharmacy', 'Translational Science', 'College Of Pharmacy', 'University Of Tennessee Health Science Center', 'Memphis', 'Tennessee', 'United States Of America', 'Adela Martin-Vicente', 'Ashley V. Nywening']

Date: 2021-09

Although considered effective treatment for many yeast fungi, the therapeutic efficacy of the echinocandin class of antifungals for invasive aspergillosis (IA) is limited. Recent studies suggest intense kinase- and phosphatase-mediated echinocandin adaptation in A. fumigatus. To identify A. fumigatus protein kinases required for survival under echinocandin stress, we employed CRISPR/Cas9-mediated gene targeting to generate a protein kinase disruption mutant library in a wild type genetic background. Cell wall and echinocandin stress screening of the 118 disruption mutants comprising the library identified only five protein kinase disruption mutants displaying greater than 4-fold decreased echinocandin minimum effective concentrations (MEC) compared to the parental strain. Two of these mutated genes, the previously uncharacterized A. fumigatus sepL and sidB genes, were predicted to encode protein kinases functioning as core components of the Septation Initiation Network (SIN), a tripartite kinase cascade that is necessary for septation in fungi. As the A. fumigatus SIN is completely uncharacterized, we sought to explore these network components as effectors of echinocandin stress survival. Our data show that mutation of any single SIN kinase gene caused complete loss of hyphal septation and increased susceptibility to cell wall stress, as well as widespread hyphal damage and loss of viability in response to echinocandin stress. Strikingly, mutation of each SIN kinase gene also resulted in a profound loss of virulence characterized by lack of tissue invasive growth. Through the deletion of multiple novel regulators of hyphal septation, we show that the non-invasive growth phenotype is not SIN-kinase dependent, but likely due to hyphal septation deficiency. Finally, we also find that echinocandin therapy is highly effective at eliminating residual tissue burden in mice infected with an aseptate strain of A. fumigatus. Together, our findings suggest that inhibitors of septation could enhance echinocandin-mediated killing while simultaneously limiting the invasive potential of A. fumigatus hyphae.

Aspergillus fumigatus is a ubiquitous fungal pathogen and the major causative agent of a life-threatening infection known as invasive aspergillosis (IA). IA is a disease in which, typically immune compromised, patients experience unfettered growth of this pathogenic mold in the lungs with dissemination to other organs being common. Therapeutic options are limited by multiple factors and only three major antifungal drug classes are currently in use. Although effective as treatment for other fungal diseases, the echinocandin class of antifungals have limited usefulness against IA. Our overall goal is to identify novel fungal proteins that, if targeted for inhibition as part of a co-therapeutic approach, could improve the anti-Aspergillus activity of echinocandins and therefore lead to better patient outcomes. Here, we sought to identify A. fumigatus genes required for fungal survival under echinocandin stress by generating and screening an A. fumigatus mutant library composed of disruptions in genes encoding putative protein kinases. We found that protein kinases required for hyphal septation are essential for survival in the presence of echinocandins and, surprisingly, for the ability of A. fumigatus to invade lung tissue. Our results suggest that novel septation inhibitors could enhance echinocandin activity while simultaneously limiting A. fumigatus virulence.

As septa are considered essential for the limitation of cell wall damage to filamentous fungal hyphae and the putative SIN is unstudied in A. fumigatus, we sought to further characterize the importance of each A. fumigatus SIN kinase to survival under echinocandin-induced stress both in vitro and during invasive disease. Our data indicate that each SIN kinase is essential for septum formation and for survival under echinocandin-induced cell wall stress. Strikingly, each of the SIN kinase disruption mutants were avirulent in a corticosteroid model of invasive aspergillosis (IA) and two of the three mutants were also avirulent in a chemotherapeutic model of IA. This lack of virulence was characterized by loss of tissue invasion and inability to accumulate fungal burden. Nevertheless, using culture-based residual fungal tissue burden as a gold-standard determination of fungicidal activity, we show that echinocandin therapy was enhanced in mice infected with SIN kinase mutants. Further, we show that loss of additional regulators of septation also results in avirulence characterized by lack of tissue invasion and loss of viability under echinocandin stress, suggesting that our phenotypes are likely due to loss of septation and not septation-independent functions of the A. fumigatus SIN.

Recent studies have shown that the phospho-proteome of Aspergilli is highly responsive to echinocandin-induced stress, implying extensive kinase- and phosphatase-mediated re-wiring of cellular physiology for survival during inhibition of β-1,3-glucan biosynthesis [ 28 – 30 ]. Further, multiple studies in A. fumigatus have implicated protein kinase and protein phosphatase activity as important to cell wall stress imposed by echinocandins [ 31 – 36 ]. Together, these reports suggest that the further study of phospho-regulatory events required for survival during echinocandin-induced stress could uncover novel avenues for combination therapies directed at enhancing echinocandin activity against Aspergilli and other human pathogenic fungi. Here, we utilized a CRISPR/Cas9-based rapid gene disruption technique to generate a protein kinase gene disruption library in a wild type genetic background of A. fumigatus. Screening of this library for cell wall stress and echinocandin sensitivity phenotypes uncovered multiple protein kinases contributing to growth under each condition. In addition to the previously characterized cell wall integrity pathway and cAMP-mediated signaling protein kinases, our screens identified orthologs of the Septation Initiation Network (SIN) kinases as essential for growth under echinocandin-induced stress [ 37 , 38 ].

The third major class of antifungals with anti-Aspergillus activity are the echinocandins, including caspofungin, micafungin and anidulafungin. These compounds are generally well-tolerated and are often used in salvage therapy for invasive infections [ 21 ]. Echinocandins are specific inhibitors of cell wall biosynthesis in fungi, as they inhibit the activity of the β-1,3-glucan synthase enzyme. This enzyme is encoded by the fksA gene in A. fumigatus and is the sole protein driving synthesis of the major cell wall component, β-1,3-glucan [ 22 ]. Whereas the activity of the echinocandins is fungicidal for the major yeast pathogens of the Candida genus, they are considered fungistatic against the Aspergilli [ 21 , 23 ]. Treatment of A. fumigatus with echinocandins causes lysis of hyphal tips and blunting of hyphal growth, but viability is maintained [ 24 ]. In addition, a caspofungin paradoxical effect (CPE) of growth inhibition has been described for caspofungin both in vitro and in vivo and is characterized by decreasing effectiveness of drug with increasing concentrations [ 21 , 25 ]. Current research suggests that the CPE is the result of the induction of tolerance mechanisms within hyphal compartments that survive caspofungin therapy. These mechanisms include remodeling of the cell wall, upregulation of cell wall integrity machinery and the induction of calcium-regulated stress pathways [ 21 , 25 ]. Although not conclusive, multiple studies using models of invasive aspergillosis have suggested that the CPE is not merely an in vitro phenomenon and may be an issue underlying treatment failure during caspofungin therapy of invasive aspergillosis in specific cases [ 25 ]. Likely underpinned by the fungistatic nature of the echinocandins against Aspergilli, breakthrough infections during echinocandin prophylaxis have been reported to be as high as 28% [ 26 ] and one study has identified echinocandin prophylaxis as an independent risk factor for breakthrough infections when compared with triazole prophylaxis [ 27 ]. Therefore, the echinocandins are mostly utilized where triazole therapy is contraindicated or has failed for invasive aspergillosis.

Therapy of invasive aspergillosis is limited to three currently available classes of antifungal compounds. The polyene class, of which Amphotericin B is the only member used for invasive disease, can be associated with acute infusion-related toxicities as well as nephrotoxicity with prolonged administration [ 7 ]. The triazole class are the frontline treatment for Aspergillus infections, with voriconazole considered the treatment of choice for this indication [ 8 ]. Treatment of aspergilloses is often prolonged, and mold-active antifungal prophylaxis employing triazole drugs is now common [ 9 – 13 ], both of which increase the potential for the development of drug-resistant organisms. Since the 1990s, triazole resistance in clinical isolates of this fungal pathogen has been increasing worldwide and is now the subject of significant research in the US and abroad [ 14 – 20 ]. Therefore, clinical use of the polyene and triazole classes is limited by patient toxicity and threatened by resistance, respectively.

Aspergillus fumigatus is among the most common causes of human invasive fungal infections in immunocompromised individuals, including solid organ transplant recipients, those undergoing hematopoietic stem cell transplant, and patients receiving highly immunosuppressive chemotherapies [ 1 – 3 ]. If untreated, these infections are almost always fatal and, even with proper diagnosis and treatment, are associated with an overall ~50% mortality rate [ 4 ]. Furthermore, the estimated annual cost of Aspergillus infections in the U.S. approaches $1 billion [ 5 ]. The most life-threatening Aspergillus infection occurs typically in the setting of profound and prolonged immune suppression and is known as invasive aspergillosis (IA). IA is initiated by the inhalation of A. fumigatus conidia from the environment [ 6 ]. In the immune compromised host, these conidia undergo a process of germination characterized by an initial phase of isotropic swelling followed by a switch to highly polarized growth leading to the formation of a germ tube. These germ tubes continue to extend through focused growth at the cell apex to generate the invasive hyphal forms that can invade surrounding tissue in search of nutrients, eventually reaching the pulmonary microvasculature system to disseminate [ 6 ]. Although decades of research have focused on A. fumigatus conidial adherence to and nutrient utilization in the host lung environment, as well as on the cellular and molecular processes essential for subsequent hyphal formation and invasion, our understanding of these processes remain incomplete.

Results

Generation of an A. fumigatus protein kinase disruption mutant library To identify protein kinase-driven pathways important for survival under echinocandin stress in A. fumigatus, we first generated a protein kinase disruption library in the A1163 (CEA10) wild type genetic background through coupling of CRISPR/Cas9-based gene targeting with a miniaturized protoplast transformation technique. Putative protein kinase genes were first identified through BLAST searches of the A. fumigatus A1163 (CEA10) genome database at FungiDB (fungidb.org) using the previously published known protein kinases of Aspergillus nidulans [39]. This search yielded 148 putative protein kinases representing 10 different protein kinase classes, as well as putative kinases falling into no known kinase class (S1 File). Of these 148 putative kinase genes, 142 were found to be encoded in the genomes of both sequenced laboratory strains, A1163 (CEA10) and Af293, and were therefore selected for disruption (S1 File). For library construction, we employed a miniaturized version of a CRISPR/Cas9-based gene editing technique, previously adapted in our lab, that provides up to 90% gene targeting efficiency in A. fumigatus [40,41]. Protospacer adjacent motif (PAM) sites for Cas9-induced double strand breaks and integration of hygromycin repair templates were selected using the Eukaryotic Pathogen CRISPR Guide RNA/DNA Design Tool (EuPaGDT, grna.ctegd.uga.edu) through batch upload analysis of all protein kinase coding sequences. Each PAM site was selected to direct double-strand DNA breaks near the putative transcriptional start site of each gene and integration of repair templates designed to disrupt readthrough of the first exon, when possible (S1 File). Cas9-ribonucleotides (RNPs) for gene targeting were assembled in vitro using custom designed guide RNAs (gRNAs) and commercially available Cas9 enzyme, as previously described [40]. Transformations were miniaturized into single wells of 96-well plates with a final well volume of 200 μl (Fig 1A). After transformation, total contents of individual wells were plated to osmotically stabilized agar and overlayed with hygromycin-containing top agar for selection (Fig 1B). Individual transformants were isolated to secondary selection plates and subsequently screened by multiple PCR reactions to confirm correct integration of the hygromycin repair template (Fig 1C). After three rounds of transformations, successful disruption of 118 protein kinase genes were confirmed (S1 File). The remaining 24 protein kinases for which disruptions were not achieved are largely orthologs of putatively essential genes in A. nidulans, suggesting conserved essentiality in A. fumigatus [39]. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Construction of a protein kinase disruption library in A. fumigatus by CRISPR/Cas9-mediated gene editing. A) Miniaturized protoplast transformations were carried out in 96-well plates, with a final total volume of 200 μl per well, and each well representing an attempted disruption of a single protein kinase gene. B) After the transformation process, the entire contents of each well were spread onto individual sorbitol minimal medium (SMM) agar plates and allowed to recover overnight at room temperature before overlaying with hygromycin-containing top agar for selection. Following these transformation procedures, typically 10 to 30 transformant colonies were evident on each selection plate after 3 to 4 days of incubation at 37°C. However, due to the high efficiency of gene targeting with the CRISPR/Cas9 system, only 3 to 4 colonies per transformation were required to be isolated for genotypic screening. C) Putative transformants were subjected to genotypic analyses by PCR to confirm proper integration of the repair template for gene disruption. These PCR analyses included screens with allele specific primer sets PF/PR and PF/IPR, pictured above. PF = Forward screening primer. PR = Reverse screening primer. IPR = Internal reverse screening primer complementary to HygR sequence. HygR = Hygromycin Resistance cassette, utilized as the repair template for gene disruption. All kinase genes were targeted for disruption at the 5’ end of the gene (within the first exon, where possible), as indicated by the placement of the protospacer and protospacer adjacent motif (PAM, red bold font) above. https://doi.org/10.1371/journal.ppat.1009806.g001

Protein kinase-mediated regulation of A. fumigatus growth and asexual development Of the 118 disrupted protein kinase mutant strains generated in this study, twenty-six were unable to grow at the same rate as the parental strain when cultured on standard laboratory minimal media. Disruption of seven different protein kinases resulted in a reduction of growth of greater than 50% when compared to the parent strain, generating compact colonies that were unable to expand radially on minimal media (Figs 2A and S1A). Among these kinase disruptions were the cell wall integrity mitogen-activated protein kinase (MAPK), mpkA (AFUB_070630), and the upstream MAPK kinase (MAPKK), mkkA (AFUB_006190). Loss of either of these kinases has been previously shown to result in compact colony morphology [35]. Although deletion of the cell wall integrity MAPKK kinase (MAPKKK), bck1 (AFUB_038060), was previously shown to result in reduced growth as well, disruption of this kinase in our library was associated with only a mild reduction in colony growth (Figs 2B and S1A). In addition, significantly reduced growth was generated by disruption of the catalytic subunit of Protein Kinase A, pkaC1 (AFUB_027890), or the PAK-kinase, cla4 (AFUB_053440) (Figs 2A and S1A). Both kinases were also previously characterized as important for vegetative growth in A. fumigatus [42,43]. Previously uncharacterized A. fumigatus protein kinases causing > 50% growth reduction upon disruption included orthologs of the eukaryotic LAMMER kinase (lkh1; AFUB_016170), an S. cerevisiae kinase regulating the actin cytoskeleton (prk1; AFUB_006320), and a cyclin-dependent protein kinase (sgv1; AFUB_053070) (Figs 2A and S1A). Eighteen additional protein kinase disruptions resulted in mild-to-moderate growth reductions ranging between 10–50% of the parental strain (Figs 2B and S1A). Of the previously characterized kinase genes fitting into this category, we identified slow growth in disrupted orthologs of a phosphorelay sensor kinase (tscB; AFUB_017740) [44], the Cross-Pathway Control kinase (cpcC; AFUB_054310) [45], a p21-Activated Kinase (PAK) family protein (ste20/pakA; AFUB_021710) [32], a cyclin-dependent protein kinase (ssn3; AFUB_035220) [46], and the cell wall integrity MAP kinase kinase kinase (bck1; AFUB_038060) [35] (Figs 2B and S1A). Thirteen mutant strains also displayed a significant impairment in asexual differentiation, as evidenced by significantly reduced conidia production, with nine of these kinase disruptions resulting in severe loss of conidiation when compared to CEA10 (Figs 2C and S1B). Six of these kinases have been previously characterized as required for conidiation, including three that comprise an asexual developmental kinase cascade in A. fumigatus (steC: AFUB_053960), ste7/mkkB; AFUB_043130, and mpkB; AFUB_078810) [47]. The remaining previously characterized kinases were pkaC1, mpkA, and cla4, each of which negatively impact asexual development upon deletion [35,42,43]. The finding that disruption of previously characterized growth-mediating kinases resulted in growth retardation of strains in our library supported the validity of our gene disruption approach. Therefore, these initial studies identified multiple novel protein kinases regulating A. fumigatus growth and development. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Colony morphologies of selected A. fumigatus protein kinase disruption mutants. 96-hr colony morphologies of severely (A) and moderately (B) growth restricted protein kinase disruption mutants, as well as colony morphologies of mutants that are not growth restricted but display reduced conidiation (C). Ten thousand conidia from each strain were point inoculated onto the center of minimal media agar and cultured for 96 hrs at 37°C. https://doi.org/10.1371/journal.ppat.1009806.g002

Multiple protein kinases are required for growth under cell wall and echinocandin stress To identify protein kinases required for cell wall stress tolerance, we performed spot-dilution assays in the presence of the common cell wall stress agents calcofluor white (CFW) and congo red (CR) on minimal media (MM), as well as caspofungin minimum effective concentration (MEC) assays, for all 118 viable kinase disruption mutants. Whereas caspofungin is an echinocandin-class antifungal that directly inhibits the fungal β-glucan synthase, CFW and CR are known to interfere with cell wall assembly by interacting with nascent chitin chains to prevent crosslinking of chitin to glucan moieties [48]. Spot-dilution assays identified seven protein kinase disruptions that displayed increased susceptibility to both CFW and CR, and an additional seven mutants that were hypersusceptible to only CR (Fig 3A and 3B). Among those 14 mutants found to be hypersusceptible to either cell wall active compound were the disruptions of the cell wall integrity kinases mkkA and mpkA (Fig 2A) which have previously been shown to be required for survival under various forms of cell wall stress [35]. Interestingly, the MAPKKK at the head of the A. fumigatus cell wall integrity pathway, Bck1, was again not identified by our assays as producing a cell wall stress hypersensitivity phenotype upon disruption. This finding, coupled with the lack of a severe growth restriction phenotype in Fig 2 for the bck1 disruption mutant, indicated that some mutations in our library may be either non-disruptive or only partially disruptive to gene function. Our cell wall stress screens also uncovered protein kinases whose disruption generated resistance to either CFW (pkaC1) or to CR (kfsA, cmkA, and stk22), as evidenced by the increased ability to sustain colony formation under stress (Fig 2C). PPT PowerPoint slide

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TIFF original image Download: Fig 3. Multiple protein kinases contribute to cell wall integrity in A. fumigatus. A) Protein kinase gene disruption mutants displaying increased susceptibility to both cell wall disrupting agents, calcofluor white (CFW) and congo red (CR), by spot-dilution assay when compared to the wild type parent (CEA10). B) Protein kinase gene disruption mutants displaying hyper-susceptibility to only CR when compared to the parent strain. C) Protein kinase gene disruptants displaying increased resistance to CFW (pkaC1-1) or to varying concentrations of CR (kfsA-1, cmkA-1, and stk22-1). For each target protein kinase gene, the systematic name is listed with the strain name given in parentheses. Strain names were designed using either the previously published or putative (based on homology to Aspergillus nidulans) gene names with the addition of “-1” to indicate a disruption mutation of that gene. GMM = glucose minimal media with no CFW or CR added. For all assays, conidial inocula were applied at 104, 103, 102, and 101 total conidia and plates were incubated at 37°C for 72 hrs. https://doi.org/10.1371/journal.ppat.1009806.g003 To see if the CFW and CR susceptibility phenotypes correlated with echinocandin susceptibility, modified caspofungin MEC analyses were performed by broth microdilution (BMD) [49]. Whereas only five protein kinase disruption mutants were identified to display ≥ 4-fold increased susceptibility to caspofungin (i.e., at least two dilution shift), an additional 44 mutants displayed a 2-fold (one dilution) increase in caspofungin susceptibility (S1 File). Of those mutants that we previously identified as hypersusceptible to CFW, CR, or both, only the AFUB_087120, AFUB_013090 (cak1), and AFUB_018600 (pom1) disruption mutants showed no shift in caspofungin MEC values. All other cell wall stress susceptible mutants displayed at least a 2-fold reduction in caspofungin MEC values (S1 File). Interestingly, the disruption mutants for AFUB_09320 (cmkA), AFUB_014350 (kfsA), and AFUB_027890 (pkaC1) which displayed increased resistance to either CFW or CR, also displayed increased susceptibility to caspofungin (2-fold reduced MEC). Importantly, among the caspofungin hypersusceptible kinase mutants that displayed ≥ 4-fold decreased MEC values were those known to be involved in cell wall integrity signaling (mpkA and mkk2) and the cAMP-activated protein kinase (pkaC1), of which mpkA and pkaC1 have been previously characterized as necessary for response to echinocandin stress [50,51]. Mutation of two additional kinases that are not members of the cell wall integrity pathway, AFUB_095460 (sidB) and AFUB_05070 (sepL), also displayed high levels of susceptibility in both of our cell wall stress and caspofungin MEC assays (Fig 3A and S1 File). These putative A. fumigatus kinases are orthologous to the A. nidulans SepL and SidB kinases that function as members of the Septation Initiation Network (SIN) kinase cascade. The core of the A. nidulans SIN pathway is composed of three kinases: the SteK-class proteins, SepH and SepL, and the AGC-class kinase, SidB (Fig 4A) [39]. Deletion of any single A. nidulans SIN kinase gene results in aseptate hyphae and reduction of conidiation whereas analysis of the Neurospora crassa SIN kinase orthologs has found only two of the three conserved kinases to be essential for the process of hyphal septation [39,52]. As the SIN pathway is completely uncharacterized in A. fumigatus, we sought to examine the importance of each SIN kinase to hyphal septation and to protection against echinocandin damage both in vitro and in vivo during invasive aspergillosis. PPT PowerPoint slide

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TIFF original image Download: Fig 4. The Septation Initiation Network (SIN) kinases are each required for hyphal septation and protection against cell wall damage in A. fumigatus. A) The putative core SIN pathway in A. fumigatus based on signal transduction models constructed for Schizosaccharomyces pombe and Aspergillus nidulans. A protein kinase cascade, initiated by activation of the SepH kinase through interaction with the GTP-bound GTPase, Spg1, leads to downstream activation of the SepL and SidB kinases to eventually promote initiation of septation. SepL and SidB are shown with their putative regulatory binding partners, SepM and Mob1, respectively. B) Deletion of sepH (ΔsepH) phenocopies sepL and sidB disruption (sepL-1 and sidB-1, respectively) as evidenced by restricted colony size and loss of conidiation (i.e., white colony formation). Complementation of SIN activity in the sepL-1 disruption mutant by gene replacement (sepL-1-C’) results in full growth recovery and conidiation. Ten thousand conidia from each strain were spot-inoculated onto the center of a GMM agar plate and cultured for 96 hrs at 37°C. C) Loss of any single SIN kinase results in absence of growth in the presence of the cell wall destabilizing compounds CFW or CR. Conidia from each strain were spot inoculated in descending concentrations onto GMM alone or GMM containing either 40 μg/ml CFW or CR. D) Loss of any single SIN kinase results in the absence of septa in mature hyphae. Conidia from each strain were cultured to mature hyphae (16 hrs at 37°C) and subsequently stained with calcofluor white (CFW) and propidium iodide (PI) to visualize septa and nuclei, respectively. White arrowheads indicate septa in the CEA10 (parent) and sepL-1 complemented (sepL-1-C’) strains. No septa were evident in the ΔsepH, sepL-1 or sidB-1 mutants. https://doi.org/10.1371/journal.ppat.1009806.g004

Loss of hyphal septation improves echinocandin-mediated fungal clearance during invasive disease Taken together, our in vitro and in vivo data suggest that A. fumigatus mutants lacking septa should be more susceptible to echinocandin therapy during infection. To examine the in vivo therapeutic relevance of our in vitro findings, we next employed the wild type CEA10 parent and the sepL-1 strain in a chemotherapeutic mouse model of invasive aspergillosis with and without echinocandin therapy. The sepL-1 mutant and chemotherapeutic model were chosen here as this combination was found to result in measurable mortality in our previous experiments. Ten mice per experimental arm were infected with 106 conidia of either the CEA10 or sepL-1 strain by intranasal inoculation on day 0. On Days +1, +2 and +3, mice were administered (or not) micafungin therapy (1 mg/kg/day or 2 mg/kg/day) by intraperitoneal injection (three total doses). Survival at the end of the 14-day infection was 0% for the CEA10 untreated arm and 75% for the sepL-1 untreated arm (p = 0.002) (Fig 9A). Strikingly, with micafungin therapy at 2 mg/kg/day, the sepL-1 mutant-infected mice exhibited 100% survival whereas the wild type strain-infected mice, treated in the same manner, produced 0% survival by day +9 (p < 0.0001) (Fig 9A). Although survival differences between treated and untreated CEA10 and sepL-1 strains were significant for each therapeutic regimen, we found no significant difference between sepL-1 treated and untreated experimental arms (Fig 10A). This was due to a combination of the low virulence of the sepL-1 mutant and the number of mice used per arm. However, in a separate experiment, the ability of micafungin therapy to enhance the reduction of residual tissue burden in sepL-1 infected mice was also examined. Mice (n = 8 / arm) were immune suppressed and inoculated identical to survival assays and were provided (or not) micafungin therapy at days +1, 2, and 3. Lungs were removed at day +0 or day +4, sectioned, and cultured on Inhibitory Mold Agar at 37°C for 48 hours. All non-micafungin treated mice infected with either the CEA10 or sepL-1 strains produced positive fungal cultures using tissue extracted at day +0 and day +4, indicating live fungus in the lung environment for both strains at this timepoint (Fig 10B). At day +4 with micafungin therapy (2 mg/kg/day), 75% of CEA10-infected mouse lung cultures (6 / 8) were still positive for fungal growth (Fig 10B). Compared to fungal colony morphologies arising from micafungin-free lungs (CEA10 day +0 or day +4, no micafungin), the micafungin-treated mouse lung cultures produced compact colony growth confirming presence of micafungin in tissues during therapy (Fig 10B). In contrast, lungs from sepL-1 infected, micafungin-treated mice extracted at day +4 contained no culturable fungal elements (Fig 10B). Together, these data suggest that loss of hyphal septation improves echinocandin therapy by enhancing the ability of this drug class to clear invading A. fumigatus hyphae from the lung. PPT PowerPoint slide

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TIFF original image Download: Fig 10. Loss of hyphal septation improves echinocandin therapy characterized by clearance of fungal burden from lung tissue. A) Survival analysis of mice infected with either the CEA10 or sepL-1 mutant strain with and without micafungin therapy. All mice were immune suppressed through intraperitoneal injection of cyclophosphamide on days -3, +1, +4, and +7 and a single subcutaneous injection of triamcinolone acetonide on day -1. Mice were inoculated with 1 X 106 conidia of the indicated strain suspended in 20 μl of sterile saline on day 0 and then received three separate intraperitoneal injections of micafungin at either 1 mg/kg (MFG1) or 2 mg/kg (MFG2) on days +1, +2 and +3. Statistical comparisons were made by Mantel-Cox log-rank test and represent each sepL-1 mutant experimental arm compared to its CEA10 control (i.e., CEA10 saline vs. sepL-1 saline, CEA10 MFG1 vs. sepL-1 MFG1, and CEA10 MFG2 vs. sepL-1 MFG2). B) CEA10 and sepL-1 residual lung tissue burden at day 0 and day 4 with and without micafungin 2 mg/kg therapy. Organ cultures are shown from two representative animals from each treatment group. Note that the sepL-1 infected mice treated with micafungin 2 mg/kg are culture negative at day 4 post-inoculation. MFG = micafungin. https://doi.org/10.1371/journal.ppat.1009806.g010

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