Biology subjects

Wagener, J.

Publications and source records attributed to Wagener, J..

5 recordsLinked to original sources

bioRxiv2025

Rho2-dependent cell wall remodeling boosts the fungistatic activity of manogepix

Fosmanogepix is a new antifungal agent currently undergoing phase 3 clinical trials. Its active moiety, manogepix, inhibits Gwt1 which is an enzyme essential for the assembly and attachment of glycosylphosphatidylinositol anchors to cell wall proteins. Manogepix has a strong fungistatic activity against most human pathogenic fungal species. Here we characterized the activity of manogepix against the major human pathogenic mold Aspergillus fumigatus. We show that manogepix susceptibility is linked to the expression of gwt1, the gene encoding Gwt1, thereby demonstrating that overexpression of gwt1 can confer resistance. In agreement with a previous study conducted with a different fungal pathogen, we observed an increase of cell well chitin after manogepix treatment. Using a luciferase-based reporter assay, we show that manogepix activates the cell wall integrity stress response pathway. However, manogepix only occasionally causes cell lysis. Mutants that lack the cell wall stress sensor Wsc1 or the Rho GTPase Rho4, which is important for septum formation, are slightly more susceptible to manogepix. In contrast, mutants that lack the Rho GTPase Rho2 or the cell wall stress sensor MidA, both of which are key for A. fumigatus to survive cell wall stress caused by granulocytes, heat and other cell wall perturbing chemical compounds, grow better than wild-type when exposed to manogepix. We show that both mutants, especially {Delta}rho2 but also {Delta}midA, fail to upregulate cell wall chitin to wild-type-like levels in response to manogepix. These results implicate a role for the Rho2-dependent stress response in the fungistatic activity of manogepix against A. fumigatus.

microbiology↗

bioRxiv2025

Rho2 regulates granulocyte-triggered stress adaptation and cell wall remodeling in Aspergillus fumigatus

The airborne opportunistic fungal pathogen Aspergillus fumigatus poses a deadly threat to immunocompromised patients. Neutrophil granulocytes play a key role in the defense against invasive infections caused by this pathogen. The mechanisms by which Aspergillus defends itself against attacks by the immune system are only partially understood. Here we show that human granulocytes activate the cell wall integrity (CWI) pathway of A. fumigatus and that key components of the CWI such as the cell wall stress sensor MidA and the Rho GTPases Rho2 and Rho4 are important for the survival of Aspergillus hyphae under granulocyte attacks. A more detailed investigation of the role of Rho2 revealed that a mutant lacking rho2 is less virulent in a Galleria mellonella infection model. Overexpression of Rho2 increases the resistance of A. fumigatus hyphae to killing by granulocytes. While a mutant lacking Rho2 has a normal cell wall composition, overexpression or constitutive activation of Rho2 leads to an altered cell wall composition and impairs growths of the pathogen. The fungicidal effect of constitutive activation of Rho2 signaling, which correlates with the formation of cell wall chitin bulges, depends on the CWI MAP kinase MpkA. However, Rho2 itself does not appear to be a direct activator of the CWI MAP kinase module. Our results support a model where Rho2 in A. fumigatus actively counteracts granulocyte attacks by upregulating cell wall biosynthesis, thereby strengthening the cell wall and aiding the fungus in surviving the stress condition.

microbiology↗

bioRxiv2024

Toxic eburicol accumulation drives the antifungal activity of azoles against Aspergillus fumigatus

Azole antifungals inhibit the sterol C14-demethylase (CYP51/Erg11), a key enzyme in the ergosterol biosynthesis pathway. They have fungistatic effects against yeasts but fungicidal effects against molds. The molecular basis for this difference remained unknown. The sequence of enzymatic steps required for ergosterol biosynthesis is different in yeasts and molds. Here we show that the azole-induced synthesis of fungicidal cell wall carbohydrate patches in the pathogenic mold Aspergillus fumigatus strictly correlates with the accumulation of the CYP51 substrate eburicol. A lack of other essential ergosterol biosynthesis enzymes, such as sterol C24-methyltransferase (Erg6A), squalene synthase (Erg9) or squalene epoxidase (Erg1) does not result in comparable cell wall alterations. Partial repression of Erg6A, which converts lanosterol into eburicol, increases azole resistance. The sterol C5-desaturase (ERG3)-dependent conversion of eburicol into 14-methylergosta-8,24(28)-dien-3{beta},6-diol, the "toxic diol" responsible for the antifungal effects of azoles in yeasts, is not required for the fungicidal effects in A. fumigatus. In contrast to yeast, where a lack of ERG3 functionality causes azole resistance, A. fumigatus lacking ERG3 becomes more azole susceptible. Mutants lacking mitochondrial complex III functionality, which are less susceptible to the fungicidal effects of azoles, but get strongly inhibited in growth, convert eburicol much more efficiently into the supposedly "toxic diol". Our results support a mechanistic model where the mode of action of azoles against the pathogenic mold A. fumigatus, other than in yeast, relies on the accumulation of eburicol which exerts fungicidal effects by triggering the formation of cell wall carbohydrate patches.

microbiology↗

bioRxiv2022

Toxoplasma gondii ROP1 subverts murine and human innate immune restriction

Toxoplasma gondii is an intracellular parasite that can infect many different host species and is a cause of significant human morbidity worldwide. T. gondii secretes a diverse array of effector proteins into the host cell which are critical for infection; however, the vast majority of these secreted proteins are uncharacterised. Here, we carried out a pooled CRISPR knockout screen in the T. gondii Prugniaud strain in vivo to identify secreted proteins that contribute to parasite immune evasion in the host. We identify 22 putative virulence factors and demonstrate that ROP1, the first-identified rhoptry protein of T. gondii, has a previously unrecognised role in parasite resistance to interferon gamma-mediated innate immune restriction. This function is conserved in the highly virulent RH strain of T. gondii and contributes to parasite growth in both murine and human macrophages. While ROP1 affects the morphology of rhoptries, from where the protein is secreted, it does not affect rhoptry secretion. ROP1 interacts with the host cell protein C1QBP, which appears to facilitate parasite immune evasion. In summary, we identify 22 secreted proteins which contribute to parasite growth in vivo and show that ROP1 is an important and previously overlooked effector in counteracting both murine and human innate immunity.

microbiology↗

bioRxiv2019

Differential requirements of cyclase associated protein (CAP) for actin turnover during the lytic cycle of Toxoplasma gondii

Toxoplasma gondii contains a limited subset of actin binding proteins. Here we show that ablation of the putative actin regulator cyclase-associated protein (TgCAP) leads to significant defects in some but not all actin dependent processes, including a defect in cell-cell communication, but surprisingly not synchronicity of division. Two CAP isoforms originate from alternative translational start sites and are beneficial for parasite fitness while a single isoform is sufficient for virulence in mice. Examination of the mutant parasites by 3D electron microscopy reveals that loss of CAP results in a defect to form a normal residual body, but all parasites remain connected within the vacuole. This dissociates synchronicity of division and parasite rosetting and reveals that establishment and maintenance of the residual body may be more complex than previously thought. These results highlight the different spatial requirements for actin turnover in Toxoplasma, controlled by a reduced subset of actin binding proteins.

microbiology↗