Expression of a fungal-specific sub-telomeric gene, hrmA , in Aspergillus fumigatus is important for a colony biofilm morphology termed H-MORPH, increased hypoxic fitness, and virulence in a murine model of invasive pulmonary aspergillosis (IPA). How expression of hrmA contributes to virulence and worse disease progression is ill-defined. Increased hrmA expression results in reduced attachment of the extracellular matrix (ECM) to the fungal cell wall resulting in decreased strain adherence. Fungal strains that are less adherent in vitro are typically less virulent as the ECM heteropolysaccharide galactosaminogalactan (GAG) aids in adhesion to host cells and confers protection from host responses. Here we report that the UDP-glucose 4-epimerase encoding gene required for GAG biosynthesis, uge3 , is necessary for full virulence of the H-MORPH strain, hrmA REV (AF293:: hrmA D304G ). In contrast, loss of uge3 in the reference strain AF293 did not significantly impact virulence in the tested IPA murine model. Phenotypic, transcriptomic, and metabolic analyses of uge3 loss in the respective strain backgrounds revealed a key role for Uge3 in central carbon metabolism in a strain specific context that promotes disease progression. These results complement the known role of Uge3 in GAG biosynthesis and highlight strain specific metabolic differences in pathogenic A. fumigatus strains. IMPORTANCE:Aspergillus fumigatus forms adherent biofilms that contribute to its ability to persist and cause disease. However, significant strain diversity exists with regard to the morphology of A. fumigatus biofilms. A distinct colony morphotype associated with increased disease progression and low oxygen fitness, termed H-MORPH, was recently described. An additional defining feature of the H-MORPH biofilm morphotype is reduced in vitro adherence to surfaces. While reduced fungal strain adherence is most commonly associated with reductions in virulence, H-MORPH strains exhibit increased virulence relative to the well-studied N-MORPH reference strain AF293. Here we discover that the UDP-glucose 4-epimerase, Uge3, plays an important role in H-MORPH central carbon metabolism complementary to its role in production of the extracellular matrix polysaccharide galactosaminogalactan (GAG). In H-MORPH strains, this metabolic role for Uge3 becomes central to virulence. These data highlight A. fumigatus strain specific mechanisms of fungal carbon metabolism related to biofilm matrix production and fungal virulence.

Epizootic outbreaks of invasive pathogens, such as the fungal pathogen Batrachochytrium dendrobatidis (Bd), are increasingly recognized as a global threat to biodiversity. However, not all susceptible host populations are equally impacted by these disease outbreaks. While some populations persist despite intense infections and high mortality rates, others can transition to enzootic coexistence with the pathogen. Here, we use comparative peptidomics to identify differences in antimicrobial peptide (AMP) maturation in Bd-susceptible common midwife toads (Alytes obstetricans) through metamorphosis. Here, we show that animals that metamorphose before AMP immune maturation display a low-diversity AMP phenotype deficient in anti-Bd AMPs. Where a high incidence of postmetamorphic animals occur with immature AMPs, populations are associated with epizootic disease dynamics. Conversely, populations associated with mature AMPs are stable following invasion of the pathogen. Our results show that even intrinsically susceptible amphibian species can possess the tools that allow populations to recover following a severe Bd epizootic.

Exopolysaccharides are key matrix determinants that provide structural integrity and regulate biomechanical properties of microbial biofilms. Biofilm exopolysaccharides often undergo modifications that determine their functional properties and localization. In Bacillus cereus ATCC 10987, PelADA expressed from the pelDEADAFG operon is a putative deacetylase required for Pel-dependent biofilm formation. To understand the molecular basis of Pel deacetylation in B. cereus ATCC 10987, we determined the crystal structure of PelADA to 2.51 Å. PelADA adopts a distinct three-domain arrangement. We demonstrate in vitro that PelADA deacetylates α-1,4-linked GalNAc substrates in a length-dependent manner and that the N-terminal domain functions as a carbohydrate-binding module (CBM) capable of binding both GalNAc and partially deacetylated oligosaccharides. We found that the CBM domain together with the carbohydrate esterase (CE) domain forms an elongated carbohydrate binding cleft and that each domain is the founding member of two new CAZy families, CBM114 and CE26, respectively. Further, in vivo mutagenesis demonstrated that the catalytic activity of PelADA is required for Pel biosynthesis in B. cereus ATCC 10987. Employing AlphaFold, we propose a model wherein the N-terminal transmembrane helix of PelADA interacts with PelG. This interaction positions the protein to accept the polymer for deacetylation as it emerges from the cytoplasmic membrane. The work presented herein offers insight into the role of PelADA in Pel biosynthesis and modification in B. cereus ATCC 10987.

BACKGROUND:Early diagnosis of invasive aspergillosis (IA) is critical for the initiation of effective antifungal therapy. Currently, detection of galactomannan (GM), a secreted fungal glycan, is the most used culture-independent diagnostic test for IA. However, limitations in the sensitivity and specificity of this test have led to interest in identifying other target molecules. Galactosaminogalactan (GAG), a polysaccharide cell wall component secreted by Aspergillus hyphae, is a potential diagnostic marker for IA. OBJECTIVES:To evaluate the utility of GAG as a diagnostic target, we generated a monoclonal antibody against GAG (mAb 1D1), established a GAG enzyme-linked immunosorbent assay (ELISA), evaluated its cross-reactivity with other respiratory pathogens, and compared the performance of the GAG detection ELISA with GM antigen detection in both an in vivo mouse model and human samples from patients with pulmonary aspergillosis. RESULTS:The GAG ELISA demonstrated strong reactivity with culture supernatants from Aspergillus fumigatus and Aspergillus flavus but limited reactivity with culture supernatants of other Aspergillus spp. and non-Aspergillus filamentous fungi. In a mouse model of IA, GAG was detected in lung tissue, serum, bronchoalveolar lavage fluid (BALF), and urine samples. Although GAG was detected by mAb 1D1 staining of Aspergillus hyphae in infected human lung tissue samples, it was not detectable in the serum, BALF, and urine of patients with pulmonary aspergillosis. CONCLUSIONS:Further studies are required to determine whether the failure to detect GAG in the serum, BALF, and urine of patients with pulmonary aspergillosis is due to absence or low GAG levels or other reasons.

A major biofilm matrix determinant of Pseudomonas aeruginosa is the partially deacetylated α-1,4 linked N-acetylgalactosamine polymer, Pel. After synthesis and transport of the GalNAc polysaccharide across the inner membrane, PelA partially deacetylates and hydrolyzes Pel before its export out of the cell via PelB. While the Pel modification and export proteins are known to interact in the periplasm, it is unclear how the interaction of PelA and PelB coordinates these processes. To determine how PelA modifies the polymer, we determined its structure to 2.1 Å and found a unique arrangement of four distinct domains. We have shown previously that the hydrolase domain exhibits endo-α-1,4-N-acetylgalactosaminidase activity. Characterization of the deacetylase domain revealed that PelA is the founding member of a new carbohydrate esterase family, CE21. Further, we found that the PelAB interaction enhances the deacetylation of N-acetylgalactosamine oligosaccharides. Using the PelA structure in conjunction with AlphaFold2 modeling of the PelAB complex, we propose a model wherein PelB guides Pel to the deacetylase domain of PelA and subsequently to the porin domain of PelB for export. Perturbation or loss of the PelAB interaction would result in less efficient deacetylation and potentially increased Pel hydrolysis. In PelA homologs across many phyla, the predicted structure and active sites are conserved, suggesting a common modification mechanism in Gram-negative bacterial species containing a functional pel operon.

The Streptococcus milleri group (SMG), comprising Streptococcus intermedius, anginosus, and constellatus, can asymptomatically colonize various mucosal sites of healthy individuals. These bacteria are opportunistic pathogens that cause different types of infections across various anatomical sites. Although the pathogenic mechanisms leading to infections are not well defined in the SMG, auto-aggregation is a key driver of biofilm adhesion and cohesion in many Streptococci and Staphylococci. Here, we identify a S. intermedius gene cluster with significant homology to the Bacillus cereus and Pseudomonas aeruginosa pel operons, which are required for Pel exopolysaccharide production and biofilm formation in these species. This cluster contains five genes that are homologous to pelDEADAFG in other gram-positive species and four additional genes of unknown function. Characterization of a panel of clinical S. intermedius isolates identified a range of adherent biofilm and aggregation phenotypes , and aggregation in strain C1365 was dependent on each of the pelDEADAFG genes. Deletion of two of the additional genes, SIR_1592 and SIR_1594, reduced but did not abolish the aggregation phenotype. Furthermore, we demonstrate that SIR_1591 is a glycoside hydrolase and that C1365 produces a GalNAc-rich polymer as aggregates were disrupted by theα-1,4-N-acetylgalactosaminidases PelA and Sph3, but not the α-1,4-N-galactosaminidase Ega3. Using an abscess model of mouse infection, we show that loss of Pel production in a C1365 ΔpelF mutant allows for more effective bacterial clearance. The polymer also affects how S. intermedius interacts with the host immune system. Collectively, our data suggest that Pel biosynthesis contributes to S. intermedius pathogenicity.IMPORTANCESMG species are increasingly being recognized as pathogens. Despite their clinical relevance, little is known about how SMG members transition between asymptomatic colonization and infection. Herein, we show that clinical isolates of S. intermedius can be classified into four groups based on their aggregation and adherent biofilm phenotypes. We demonstrate that aggregation is dependent on the Pel polysaccharide and that Pel production allows bacteria not only to persist longer during infection but also modulates the immune responses of the host. Pel production requires the canonical pelDEADAFG genes. We also identified four additional genes in the S. intermedius pel cluster and found that under the conditions tested, two of these genes play a role in aggregation and Pel production. Functional homologs of the additional genes play major roles in host-pathogen interactions and stress responses in other bacteria, suggesting that these additional genes could play a role in Pel-related infections.

The Streptococcus Milleri Group (SMG) comprising of Streptococcus intermedius, anginosus and constellatus are commensal bacteria commonly found in healthy individuals. These bacteria are increasingly being recognized as opportunistic pathogens that can cause purulent infections at sterile body sites and have also been identified in the sputum of individuals with cystic fibrosis. Although the mechanisms of conversion to opportunistic pathogens are not well understood, auto-aggregation is a key driver of biofilm adhesion and cohesion in many Streptococci and Staphylococci. Here, we identify a gene cluster in the S. intermedius genome with significant homology to the pel operons in Bacillus cereus and Pseudomonas aeruginosa , which are required for Pel exopolysaccharide production and biofilm formation in these species. Characterization of a panel of clinical S. intermedius strains identified a range of aggregating phenotypes. Analysis of the pel operon in the hyper-aggregating C1365 strain revealed that each of the canonical pelDEADAFG genes, but not the four additional genes are required for aggregation. Further, we demonstrate that C1365 produces a GalNAc-rich exopolysaccharide and that aggregates can be disrupted by the α1,4 N- acetylgalactosaminidases, PelA and Sph3, but not other glycoside hydrolases, proteinase K or DNase I. Using an abscess model of mouse infection, we show that Pel driven aggregation leads to longer lasting infections, and that lack of Pel allows for the bacteria to be cleared more effectively. The polymer also affects how the bacteria interacts with the host immune system. Collectively, our data suggest that the pel operon has relevancy to S. intermedius pathogenicity. ### Competing Interest Statement The authors have declared no competing interest.

ABSTRACTA major biofilm matrix determinant ofPseudomonas aeruginosais the partially deacetylated α-1,4 linkedN-acetylgalactosamine polymer, Pel. After synthesis and transport of the GalNAc polysaccharide across the inner membrane, PelA partially deacetylates and hydrolyzes Pel before its export out of the cellviaPelB. While the Pel modification and export proteins are known to interact in the periplasm, it is unclear how the interaction of PelA and PelB coordinates these processes. To determine how PelA modifies the polymer, we determined its structure to 2.1 Å and found a unique arrangement of four distinct domains. We have shown previously that the hydrolase domain exhibits endo-α-1,4-N-acetylgalactosaminidase activity. Characterization of the deacetylase domain revealed that PelA is the founding member of a new carbohydrate esterase family, CE#. Further, we found that the PelAB interaction enhances the deacetylation ofN-acetylgalactosamine oligosaccharides. Using the PelA structure in conjunction with AlphaFold2 modelling of the PelAB complex, we propose a model wherein PelB guides Pel to the deacetylase domain of PelA and subsequently to the porin domain of PelB for export. Perturbation or loss of the PelAB interaction would result in less efficient deacetylation and potentially result in increased Pel hydrolysis. In PelA homologues across many phyla, the predicted structure and active sites are conserved, suggesting that there is a common modification mechanism in Gram-negative bacterial species that contain a functionalpeloperon.

Invasive aspergillosis causes significant morbidity and mortality in immunocompromised patients. Natural killer (NK) cells are pivotal for antifungal defense. Thus far, CD56 is the only known pathogen recognition receptor on NK cells triggering potent antifungal activity against Aspergillus fumigatus. However, the underlying cellular mechanisms and the fungal ligand of CD56 have remained unknown. Using purified cell wall components, biochemical treatments, and ger mutants with altered cell wall composition, we herein found that CD56 interacts with the A. fumigatus cell wall carbohydrate galactosaminogalactan (GAG). This interaction induced NK-cell activation, degranulation, and secretion of immune-enhancing chemokines and cytotoxic effectors. Supernatants from GAG-stimulated NK cells elicited antifungal activity and enhanced antifungal effector responses of polymorphonuclear cells. In conclusion, we identified A. fumigatus GAG as a ligand of CD56 on human primary NK cells, stimulating potent antifungal effector responses and activating other immune cells.

Non-LEE-encoded Effector A (NleA) is a type III secreted effector protein of enterohaemorrhagic and enteropathogenic Escherichia coli as well as the related mouse pathogen Citrobacter rodentium. NleA translocation into host cells is essential for virulence. We previously published several lines of evidence indicating that NleA is modified by host-mediated mucin-type O-linked glycosylation, the first example of a bacterial effector protein modified in this way. In this study, we use lectins to provide direct evidence for the modification of NleA by O-linked glycosylation and determine that the interaction of NleA with the COPII complex is necessary for this modification to occur.

Pel exopolysaccharide biosynthetic loci are phylogenetically widespread biofilm matrix determinants in bacteria. In Pseudomonas aeruginosa, Pel is crucial for cell-to-cell interactions and reducing susceptibility to antibiotic and mucolytic treatments. While genes encoding glycoside hydrolases have long been linked to biofilm exopolysaccharide biosynthesis, their physiological role in biofilm development is unclear. Here we demonstrate that the glycoside hydrolase activity of P. aeruginosa PelA decreases adherent biofilm biomass and is responsible for generating the low molecular weight secreted form of the Pel exopolysaccharide. We show that the generation of secreted Pel contributes to the biomechanical properties of the biofilm and decreases the virulence of P. aeruginosa in Caenorhabditis elegans and Drosophila melanogaster. Our results reveal that glycoside hydrolases found in exopolysaccharide biosynthetic systems can help shape the soft matter attributes of a biofilm and propose that secreted matrix components be referred to as matrix associated to better reflect their influence.

AbstractInvasive aspergillosis causes significant morbidity and mortality in immunocompromised patients. Natural killer (NK) cells are pivotal for antifungal defense. Thus far, CD56 is the only knownpathogen recognition receptoron NK cells triggering potent antifungal activity againstAspergillus fumigatus. However, the underlying cellular mechanisms and the fungal ligand of CD56 have remained unknown. Using purified cell wall components, biochemical treatments, andA. fumigatusmutants with altered cell wall composition, we herein found that CD56 interacts with theA. fumigatuscell wall carbohydrate galactosaminogalactan (GAG). This interaction induced NK cell activation, degranulation, and secretion of immune-enhancing chemokines and cytotoxic effectors. Supernatants from GAG-stimulated NK cells elicited antifungal activity and enhanced antifungal effector responses of polymorphonuclear cells. In conclusion, we identifiedA. fumigatusGAG as a ligand of CD56 on human primary NK cells, stimulating potent antifungal effector responses and activating other immune cells.

Half a century after their discovery, polymers of N-acetylgalactosamine produced by the Aspergilli have garnered new interest as mediators of fungal virulence. Recent work has focused on the Aspergillus fumigatus secreted and cell wall-associated heteropolymer, galactosaminogalactan (GAG). This polymer, composed of galactose (Gal) and partially deacetylated N-acetylgalactosamine (GalNAc), plays a role in a variety of pathogenic processes including biofilm formation, immune modulation and evasion, and resistance to antifungals. Given its many potential contributions to fungal pathogenesis, GAG is a promising therapeutic target for novel antifungal strategies. As such, several studies have sought to elucidate the biosynthetic pathways required for GAG production and secretion. Herein we review the progress made in the understanding of the molecular mechanisms underlying GAG synthesis and identify several gaps in our understanding of this process.

The fungal cell wall and secreted exopolysaccharides play an important role in the interactions between fungi and their environment. Despite their central role in fungal biology, ecology, and host-pathogen interactions, the composition of these polymers and their synthetic pathways are not well understood. The protocols presented in this article describe an approach to isolate fungal cell wall polysaccharides and to identify and quantify the monosaccharide composition of these polymers by gas chromatography-mass spectrometry (GC-MS). © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: O-methyl trimethylsilyl monosaccharide derivatives composition analysis by GC-MS Support Protocol: Fungal cell wall extraction.

The filamentous fungus Aspergillus fumigatus is an ubiquitous mold that can cause invasive pulmonary infections in immunocompromised patients. Within the lung, A. fumigatus forms biofilms that can enhance resistance to antifungals and immune defenses, highlighting the importance of defining the mechanisms underlying biofilm development and associated emergent properties. A. fumigatus biofilms display a morphology and architecture that is distinct from bacterial and yeast biofilms. Moreover, A. fumigatus biofilms display unique characteristics in the composition of their extracellular matrix (ECM) and the regulatory networks governing biofilm formation. This review will discuss our current understanding of the form and function of A. fumigatus biofilms, including the unique components of ECM matrix, potential drug resistance mechanisms, the regulatory networks governing A. fumigatus biofilm formation, and potential therapeutics targeting these structures.

The mold Aspergillus fumigatus and bacterium Pseudomonas aeruginosa form biofilms in the airways of individuals with cystic fibrosis. Biofilm formation by A. fumigatus depends on the self-produced cationic exopolysaccharide galactosaminogalactan (GAG), while P. aeruginosa biofilms can contain the cationic exopolysaccharide Pel. GAG and Pel are rendered cationic by deacetylation mediated by either the secreted deacetylase Agd3 (A. fumigatus) or the periplasmic deacetylase PelA (P. aeruginosa). Given the similarities between these polymers, the potential for biofilm interactions between these organisms were investigated. P. aeruginosa were observed to adhere to A. fumigatus hyphae in a GAG-dependent manner and to GAG-coated coverslips of A. fumigatus biofilms. In biofilm adherence assays, incubation of P. aeruginosa with A. fumigatus culture supernatants containing de-N-acetylated GAG augmented the formation of adherent P. aeruginosa biofilms, increasing protection against killing by the antibiotic colistin. Fluorescence microscopy demonstrated incorporation of GAG within P. aeruginosa biofilms, suggesting that GAG can serve as an alternate biofilm exopolysaccharide for this bacterium. In contrast, Pel-containing bacterial culture supernatants only augmented the formation of adherent A. fumigatus biofilms when antifungal inhibitory molecules were removed. This study demonstrates biofilm interaction via exopolysaccharides as a potential mechanism of co-operation between these organisms in chronic lung disease.

Aspergillus fumigatus is a ubiquitous filamentous fungus that causes an array of diseases depending on the immune status of an individual, collectively termed aspergillosis. Antifungal therapy for invasive pulmonary aspergillosis (IPA) or chronic pulmonary aspergillosis (CPA) is limited and too often ineffective.

The genetic capacity to synthesize the biofilm matrix exopolysaccharide Pel is widespread among Gram-negative and Gram-positive bacteria. However, its exact chemical structure has been challenging to determine. Using a Pseudomonas aeruginosa strain engineered to overproduce Pel, improvements to the isolation procedure, and selective hydrolysis with the glycoside hydrolase PelA h , we demonstrate that Pel is a partially de- N- acetylated linear polymer of α-1,4- N- acetylgalactosamine comprised predominantly of dimeric repeats of galactosamine and N- acetylgalactosamine.