Cystic Fibrosis (CF) is caused by mutations in the CFTR gene, of which over 2000 have been reported to date. Mutations have yet to be analyzed in aggregate to assess their distribution across the tertiary structure of the CFTR protein, an approach that could provide valuable insights into the structure-function relationship of CFTR. In addition, the binding site of Class I correctors (VX-809, VX-661, and C18) is not well understood. In this study, exonic CFTR mutations and mutant allele frequencies described in 3 curated databases (ABCMdb, CFTR1, and CFTR2, comprising >130 000 data points) were mapped to 2 different structural models: a homology model of full-length CFTR protein in the open-channel state, and a cryo-electron microscopy core-structure of CFTR in the closed-channel state. Accordingly, residue positions of 6 high-frequency mutant CFTR alleles were found to spatially co-localize in CFTR protein, and a significant cluster was identified at the NBD1:ICL4 interdomain interface. In addition, immunoblotting confirmed the approximate binding site of Class I correctors, demonstrating that these small molecules act via a similar mechanism in vitro, and in silico molecular docking generated binding poses for their complex with the cryo-electron microscopy structure to suggest the putative corrector binding site is a multi-domain pocket near residues F374-L375. These results confirm the significance of interdomain interfaces as susceptible to disruptive mutation, and identify a putative corrector binding site. The structural pharmacogenomics approach of mapping mutation databases to protein models shows promise for facilitating drug discovery and personalized medicine for monogenetic diseases.

Edited by Gerald W. HartAspergillus fumigatus is an opportunistic fungal pathogen that causes both chronic and acute invasive infections. Galactosaminogalactan (GAG) is an integral component of the A. fumigatus biofilm matrix and a key virulence factor. GAG is a heterogeneous linear ␣-1,4 -linked exopolysaccharide of galactose and GalNAc that is partially deacetylated after secretion. A cluster of five co-expressed genes has been linked to GAG biosynthesis and modification. One gene in this cluster, ega3, is annotated as encoding a putative ␣-1,4-galactosaminidase belonging to glycoside hydrolase family 114 (GH114). Herein, we show that recombinant Ega3 is an active glycoside hydrolase that disrupts GAG-dependent A. fumigatus and Pel polysaccharide-dependent Pseudomonas aeruginosa biofilms at nanomolar concentrations. Using MS and functional assays, we demonstrate that Ega3 is an endo-acting ␣-1,4-galactosaminidase whose activity depends on the conserved acidic residues, Asp-189 and Glu-247. X-ray crystallographic structural analysis of the apo Ega3 and an Ega3-galactosamine complex, at 1.76 and 2.09 Å resolutions, revealed a modified (␤/␣) 8 -fold with a deep electronegative cleft, which upon ligand binding is capped to form a tunnel. Our structural analysis coupled with in silico docking studies also uncovered the molecular determinants for galactosamine specificity and substrate binding at the ؊2 to ؉1 binding subsites. The findings in this study increase the structural and mechanistic understanding of the GH114 family, which has >600 members encoded by plant and opportunistic human pathogens, as well as in industrially used bacteria and fungi.

Inhalation of conidia of the opportunistic mold Aspergillus fumigatus by immunocompromised hosts can lead to invasive pulmonary disease. Inhaled conidia that escape immune defenses germinate to form filamentous hyphae that invade lung tissues. Conidiation rarely occurs during invasive infection of the human host, allowing the bulk of fungal energy to be directed toward vegetative growth. We hypothesized that forced induction of conidiation during infection can suppress A. fumigatus vegetative growth, impairing the ability of this organism to cause disease. To study the effects of conidiation pathway dysregulation on A. fumigatus virulence, a key transcriptional regulator of conidiation (brlA) was expressed under the control of a doxycycline-inducible promoter. Time- and dose-dependent brlA overexpression was observed in response to doxycycline both in vitro and in vivo. Exposure of the inducible brlA overexpression strain to low doses of doxycycline under vegetative growth conditions in vitro induced conidiation, whereas high doses arrested growth. Overexpression of brlA attenuated A. fumigatus virulence in both an invertebrate and mouse model of invasive aspergillosis. RNA sequencing studies and phenotypic analysis revealed that brlA overexpression results in altered cell signaling, amino acid, and carbohydrate metabolism, including a marked upregulation of trehalose biosynthesis and a downregulation in the biosynthesis of the polysaccharide virulence factor galactosaminogalactan. This proof of concept study demonstrates that activation of the conidiation pathway in A. fumigatus can reduce virulence and suggests that brlA-inducing small molecules may hold promise as a new class of therapeutics for A. fumigatus infection. IMPORTANCE The mold Aspergillus fumigatus reproduces by the production of airborne spores (conidia), a process termed conidiation. In immunocompromised individuals, inhaled A. fumigatus conidia can germinate and form filaments that penetrate and damage lung tissues; however, conidiation does not occur during invasive infection. In this study, we demonstrate that forced activation of conidiation in filaments of A. fumigatus can arrest their growth and impair the ability of this fungus to cause disease in both an insect and a mouse model of invasive infection. Activation of conidiation was linked to profound changes in A. fumigatus metabolism, including a shift away from the synthesis of polysaccharides required for cell wall structure and virulence in favor of carbohydrates used for energy storage and stress resistance. Collectively, these findings suggest that activation of the conidiation pathway may be a promising approach for the development of new agents to prevent or treat A. fumigatus infection.

Bacteria require polysaccharides for structure, survival, and virulence. Despite their central role in microbiology, few tools are available to manipulate their production. In E. coli, the glycosyltransferase complex PgaCD produces poly-N-acetylglucosamine (PNAG), an extracellular matrix polysaccharide required for biofilm formation. We report that C6-substituted (H, F, N 3 , SH, NH 2 ) UDP-GlcNAc substrate analogues are inhibitors of PgaCD. In vitro, the inhibitors cause PNAG chain termination, consistent with the mechanism of PNAG polymerization from the nonreducing terminus. In vivo, expression of the GlcNAc-1-kinase NahK in E. coli provided a non-native GlcNAc salvage pathway that produced the UDP-GlcNAc analogue inhibitors in situ. The 6-fluoro and 6-deoxy derivatives were potent inhibitors of biofilm formation in the transformed strain, providing a tool to manipulate this key exopolysaccharide. Characterization of the UDP-GlcNAc pool and quantification of PNAG generation support PNAG termination as the primary in vivo mechanism of biofilm inhibition by 6-fluoro UDP-GlcNAc.

The synthesis of exopolysaccharides as biofilm matrix components by pathogens is a crucial factor for chronic infections and antibiotic resistance. Many periplasmic proteins involved in polymer processing and secretion in Gram-negative synthase dependent exopolysaccharide biosynthetic systems have been individually characterized. The operons responsible for the production of PNAG, alginate, cellulose and the Pel polysaccharide each contain a gene that encodes an outer membrane associated tetratricopeptide repeat (TPR) domain containing protein. While the TPR domain has been shown to bind other periplasmic proteins, the functional consequences of these interactions for the polymer remain poorly understood. Herein, we show that the C-terminal TPR region of PgaA interacts with the de-N-acetylase domain of PgaB, and increases its deacetylase activity. Additionally, we found that when the two proteins form a complex, the glycoside hydrolase activity of PgaB is also increased. To better understand structure-function relationships we determined the crystal structure of a stable TPR module, which has a conserved groove formed by three repeat motifs. Tryptophan quenching, mass spectrometry analysis and molecular dynamics simulation studies suggest that the crystallized TPR module can bind PNAG/dPNAG via its electronegative groove on the concave surface, and potentially guide the polymer through the periplasm towards the porin for export. Our results suggest a scaffolding role for the TPR domain that combines PNAG/dPNAG translocation with the modulation of its chemical structure by PgaB.

Many bacterial species use the secondary messenger, c-di-GMP, to promote the production of biofilm matrix components. In Pseudomonas aeruginosa , c-di-GMP production is stimulated upon initial surface contact and generally remains high throughout biofilm growth. Transcription of several gene clusters, including the Sia signal transduction system, are induced in response to high cellular levels of c-di-GMP. The output of this system is SiaD, a diguanylate cyclase whose activity is induced in the presence of the detergent SDS. Previous studies demonstrated that Sia-mediated cellular aggregation is a key feature of P. aeruginosa growth in the presence of SDS. Here, we show that the Sia system is important for producing low levels of c-di-GMP when P. aeruginosa is growing planktonically. In addition, we show that Sia activity is important for maintaining cell-associated Psl in planktonic populations. We also demonstrate that Sia mutant strains have reduced cell-associated Psl and a surface attachment-deficient phenotype. The Sia system also appears to posttranslationally impact cell-associated Psl levels. Collectively, our findings suggest a novel role for the Sia system and c-di-GMP in planktonic populations by regulating levels of cell-associated Psl.

Abstract

Obverse Cover: The cover image, by Steven V. Molinski et al., is based on the Research Article Comprehensive mapping of cystic fibrosis mutations to CFTR protein identifies mutation clusters and molecular docking predicts corrector binding site, DOI: https://doi.org/10.1002/prot.25496.