Aspergillus fumigatus is a saprophytic mold that can cause infection in patients with impaired immunity or chronic lung diseases. The polysaccharide-rich cell wall of this fungus is a key point of ...contact with the host immune system. The availability of purified cell wall polysaccharides and mutant strains deficient in the production of these glycans has revealed that these glycans play an important role in the pathogenesis of A. fumigatus infections. Herein, we review our current understanding of the key polysaccharides present within the A. fumigatus cell wall, and their interactions with host cells and secreted factors during infection.
Aspergillus fumigatus is the most virulent species within the Aspergillus genus and causes invasive infections with high mortality rates. The exopolysaccharide galactosaminogalactan (GAG) contributes ...to the virulence of A. fumigatus. A co-regulated five-gene cluster has been identified and proposed to encode the proteins required for GAG biosynthesis. One of these genes, sph3, is predicted to encode a protein belonging to the spherulin 4 family, a protein family with no known function. Construction of an sph3-deficient mutant demonstrated that the gene is necessary for GAG production. To determine the role of Sph3 in GAG biosynthesis, we determined the structure of Aspergillus clavatus Sph3 to 1.25 Å. The structure revealed a (β/α)8 fold, with similarities to glycoside hydrolase families 18, 27, and 84. Recombinant Sph3 displayed hydrolytic activity against both purified and cell wall-associated GAG. Structural and sequence alignments identified three conserved acidic residues, Asp-166, Glu-167, and Glu-222, that are located within the putative active site groove. In vitro and in vivo mutagenesis analysis demonstrated that all three residues are important for activity. Variants of Asp-166 yielded the greatest decrease in activity suggesting a role in catalysis. This work shows that Sph3 is a glycoside hydrolase essential for GAG production and defines a new glycoside hydrolase family, GH135.
The pathways governing biosynthesis of the Aspergillus fumigatus exopolysaccharide galactosaminogalactan are poorly understood.
The structure of Sph3 revealed a (β/α)8 barrel fold. The enzyme hydrolyzes galactosaminogalactan and is required for the synthesis of this exopolysaccharide.
Sph3 is a glycoside hydrolase (GH) whose activity is essential for galactosaminogalactan biosynthesis.
Sph3 defines a new glycoside hydrolase superfamily, GH family 135.
Aspergillus fumigatus is a 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. Aspergillus biofilm formation requires the production of a cationic matrix exopolysaccharide, galactosaminogalactan (GAG). In this study, recombinant glycoside hydrolases (GH)s that degrade GAG were evaluated as antifungal agents in a mouse model of invasive aspergillosis. Intratracheal GH administration was well tolerated by mice. Pharmacokinetic analysis revealed that although GHs have short half-lives, GH prophylaxis resulted in reduced fungal burden in leukopenic mice and improved survival in neutropenic mice, possibly through augmenting pulmonary neutrophil recruitment. Combining GH prophylaxis with posaconazole treatment resulted in a greater reduction in fungal burden than either agent alone. This study lays the foundation for further exploration of GH therapy in invasive fungal infections.
The biofilm-forming mold Aspergillus fumigatus is a common causative agent of invasive fungal airway disease in patients with a compromised immune system or chronic airway disease. Treatment of A. fumigatus infection is limited by the few available antifungals to which fungal resistance is becoming increasingly common. The high mortality rate of A. fumigatus-related infection reflects a need for the development of novel therapeutic strategies. The fungal biofilm matrix is in part composed of the adhesive exopolysaccharide galactosaminogalactan, against which antifungals are less effective. Previously, we demonstrated antibiofilm activity with recombinant forms of the glycoside hydrolase enzymes that are involved in galactosaminogalactan biosynthesis. In this study, prophylaxis with glycoside hydrolases alone or in combination with the antifungal posaconazole in a mouse model of experimental aspergillosis improved outcomes. This study offers insight into the therapeutic potential of combining biofilm disruptive agents to leverage the activity of currently available antifungals.
Deacetylation of chitin by chitin deacetylases (Cda) results in the formation of chitosan. Chitosan, a polymer of β1,4 linked glucosamine, plays multiple roles in the function of the fungal cell ...wall, including virulence and evasion of host immune responses. In this study, the roles of chitosan and putative
s in cell wall structure and virulence of
were investigated. Low levels of chitosan were found in the conidial and cell wall of
. Seven putative
genes were identified, disrupted and the phenotype of the single mutants and the septuple mutants were investigated. No alterations in fungal cell wall chitosan levels, changes in fungal growth or alterations in virulence were detected in the single or septuple Δ
mutant strains. Collectively, these results suggest that chitosan is a minority component of the
cell wall, and that the seven candidate Cda proteins do not play major roles in fungal cell wall synthesis or virulence. However, Cda2 is involved in conidiation, suggesting that this enzyme may play a role in N-acetyl-glucosamine metabolism.
Aspergillus fumigatus is a ubiquitous environmental mold. When conidia of A. fumigatus are inhaled by immunosuppressed individuals they can germinate to form filamentous hyphae that invade lung ...tissue to cause a necrotizing pneumonia which is associated with a high mortality rate. One of the major virulence factors of A. fumigatus is the production of the secreted cationic exopolysaccharide galactosaminogalactan (GAG), which is essential for biofilm formation. GAG is predicted to be synthesized by the products of a five gene cluster, one of which, ega3, is predicted to encode a glycoside hydrolase anchored to the cell membrane of A. fumigatus.Through mass spectrometry studies, we established that Ega3 cleaves mature deacetylated GAG. We hypothesized that like the other proteins in the cluster, Ega3 is necessary for GAG synthesis. To test this hypothesis, we sought to disrupt the ega3 by allele replacement with a drug resistance marker and characterize the phenotype of the resulting ega3 null mutant. Initial attempts to disrupt ega3 by our usual approach were unsuccessful but two ega3 null mutants were finally recovered, one using CRISPR/Cas9 and another using the conventional split marker protocol. Like other mutants in the GAG cluster, these mutants were deficient in biofilm formation and did not produce deacetylated GAG. Surprisingly, complementation with an ega3 allele failed to restore biofilm formation in both strains, despite restoration of Ega3 protein production as demonstrated by Western blot. These findings were consistent with the presence of a secondary mutation impairing GAG production. Analysis of the expression of the GAG cluster genes revealed that agd3, encoding the GAG deacetylase required for the production of mature, cationic GAG, was not expressed in the ∆ega3CRISPR mutant. Genome sequencing revealed that uge3, required for GAG production, was mutated in the other ega3 null mutant. Since we were only able to disrupt ega3 in the absence of the production of cationic GAG, we hypothesized that ega3 is conditionally essential in the presence of cationic GAG. To test this hypothesis, agd3 was expressed in the ∆ega3CRISPR mutant under the control of a tetracycline-inducible promoter (∆ega3CRISPR::agd3Tet on). Under agd3- expressing conditions, GAG production was restored but fungal growth was inhibited.Since GAG is a large cationic polymer and Ega3 cleaves cationic GAG, we hypothesized that cationic GAG may be toxic to the cell membrane of A. fumigatus and that membrane-bound Ega3 degrades GAG near the membrane. To test whether cationic GAG induces damage to the cell membrane in the absence of Ega3, ATP was measured in fungal culture supernatants as a proxy for cell leakage. Induction of agd3 expression in the (∆ega3CRISPR::agd3Teton) mutant resulted in the release of high levels of ATP suggesting that cationic GAG disrupts the cell membrane of A. fumigatus. We therefore hypothesized that secreted GAG may also mediate host cell injury during infection. To test this hypothesis, A549 pulmonary epithelial cells were loaded with radioactive chromium and exposed to culture supernatants from wild- type A. fumigatus+/- Ega3. Exposure to GAG-containing culture supernatants (CS) induced epithelial cell injury, but cells were almost completely protected from damage in the presence of Ega3. Propidium iodide (PI) staining of BMDMs exposed to CS mirrored these results, as did PI/annexin V staining of human NK cells.
Methionine dependence, the inability to grow in culture when methionine in the medium is replaced by its metabolic precursor homocysteine, occurs in many tumor cell lines. In most affected lines, the ...cause of methionine dependence is not known. An exception is the melanoma-derived cell line MeWo-LC1, in which hypermethylation of the MMACHC gene is associated with decreased MMACHC expression. Decreased expression results in decreased provision of the methylcobalamin cofactor required for activity of methionine synthase and thus decreased conversion of homocysteine to methionine. Analysis of data in the Cancer Cell Line Encyclopedia Archive demonstrated that MMACHC hypermethylation and decreased MMACHC expression occurred more frequently in melanoma cell lines when compared to other tumor cell lines. We further investigated methionine dependence and aspects of MMACHC function in a panel of six melanoma lines, including both melanoma lines with known methionine dependence status (MeWo, which is methionine independent, and A375, which is methionine dependent). We found that the previously unclassified melanoma lines HMCB, Colo829 and SH-4 were methionine dependent, while SK-Mel-28 was methionine independent. However, despite varying levels of MMACHC methylation and expression, none of the tested lines had decreased methylcobalamin and adenosylcobalamin synthesis as seen in MeWo-LC1, and the functions of both cobalamin-dependent enzymes methionine synthase and methylmalonyl-CoA mutase were intact. Thus, while melanoma lines were characterized by relatively high levels of MMACHC methylation and low expression, the defect in metabolism observed in MeWo-LC1 was unique, and decreased MMACHC expression was not a cause of methionine dependence in the other melanoma lines.
Aspergillus 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 α-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 α-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 α-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 (β/α)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.
The ulnar collateral ligament is commonly injured in overhead-throwing athletes, particularly baseball pitchers. Pitch movement (break) is a critical aspect to pitching performance. The primary ...purpose of this study was to determine the changes in pitch velocity, pitch break, angle of break, and pitch performance metrics before and after ulnar collateral ligament reconstruction (UCLR) in Major League Baseball (MLB) pitchers. The secondary purpose was to determine changes in pitch performance metrics before and after UCLR. We hypothesized that pitch break and pitch performance metrics would be unchanged following UCLR.
This was a retrospective case-series study of pitchers who had undergone primary UCLR between 2008 and 2014. Velocity, horizontal movement (Hmov), and vertical movement (Vmov) of each pitch were collected from the PITCHf/x system for each pitcher 12-24 months before surgery, 12-24 months after surgery, and 24-36 months after surgery. Overall break was calculated by taking the Pythagorean sum of Hmov and Vmov. Angle of break was determined by taking the inverse tangent of Vmov divided by Hmov. Repeated-measures analysis of covariance was performed to determine differences in pitch velocity, movement, angle of movement, and performance metrics between preoperative and postoperative time frames. Performance metrics included balls, strikes, swings, fouls, swings and misses, ground balls, line drives, pop-ups, fly balls, and home runs. Covariates included age at surgery, time from MLB debut to surgery, innings pitched as a starter, innings pitched as a reliever, and total pitches thrown.
In a cohort of 46 pitchers who underwent UCLR between 2008 and 2014, pitch velocity, movement, and angle were not significantly changed with respect to preoperative or postoperative time frames. In addition, postoperative time frames had clinically insignificant differences in pitch performance metrics.
Pitch break and performance metrics are not significantly affected in pitchers who return after UCLR.
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