The Fungal Exopolysaccharide Galactosaminogalactan Mediates Virulence by Enhancing Resistance to Neutrophil Extracellular Traps

Of the over 250 Aspergillus species, Aspergillus fumigatus accounts for up to 80% of invasive human infections. A. fumigatus produces galactosaminogalactan (GAG), an exopolysaccharide composed of galactose and N-acetyl-galactosamine (GalNAc) that mediates adherence and is required for full virulence. Less pathogenic Aspergillus species were found to produce GAG with a lower GalNAc content than A. fumigatus and expressed minimal amounts of cell wall-bound GAG. Increasing the GalNAc content of GAG of the minimally pathogenic A. nidulans, either through overexpression of the A. nidulans epimerase UgeB or by heterologous expression of the A. fumigatus epimerase Uge3 increased the amount of cell wall bound GAG, augmented adherence in vitro and enhanced virulence in corticosteroid-treated mice to levels similar to A. fumigatus. The enhanced virulence of the overexpression strain of A. nidulans was associated with increased resistance to NADPH oxidase-dependent neutrophil extracellular traps (NETs) in vitro, and was not observed in neutropenic mice or mice deficient in NADPH-oxidase that are unable to form NETs. Collectively, these data suggest that cell wall-bound GAG enhances virulence through mediating resistance to NETs

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Background: Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. // Methods: We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung's disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. // Findings: We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung's disease) from 264 hospitals (89 in high-income countries, 166 in middle-income countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in low-income countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. // Interpretation: Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between low-income, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Inhibition or disruption of NETs attenuates the susceptibility of <i>A</i>. <i>nidulans</i> to killing by human neutrophil-mediated killing.

<p>(A) Neutrophil extracellular traps formation by primary human PMN as visualized by the DNA intercalating agent propidium iodide. Arrows indicate the increased binding of propidium iodide stained NETs on the surface of wild-type <i>A</i>. <i>nidulans</i> hyphae. Images were acquired using a 543 nm laser and detected by confocal microscopy at 600X magnification with 4X digital zoom. Scale bar represents 10 μm. (B) Susceptibility of fungal strains to injury by PMNs in the presence (gray bars) or absence (black bars) of micrococcal nuclease (MNase). (C) Susceptibility of fungal strains to injury by PMNs from healthy donor (black bars) or from a CGD patient (grey bars). (D) Susceptibility of fungal strains to injury by PMNs pre-treated with 10 μM dexamethasone (grey bars) or untreated (black bars). * indicates a significant difference between <i>A</i>. <i>nidulans</i> and <i>A</i>. <i>fumigatus</i> or An-Uge3 strains, p<0.05 by ANOVA with Tukey’s test for pairwise comparison. § indicates significant difference treatment groups of PMNs co-incubated with <i>A</i>. <i>nidulans</i>, p<0.05 by ANOVA with Tukey’s test for pairwise comparison. All data are represented as mean +/- SEM.</p

Overexpression of <i>uge3</i> or <i>ugeB</i> in <i>A</i>. <i>nidulans</i> increases the GalNAc content of GAG and enhances the formation of adherent biofilms.

<p>(A) Relative expression of <i>uge3</i> in the An-Uge3 strain and <i>ugeB</i> in the An-UgeB strain compared to the expression level of <i>ugeB</i> in wild-type <i>A</i>. <i>nidulans</i> grown in Brian medium and as measured by real-time RT-PCR. (B) Total amount of secreted GAG from the indicated strains. (C) GalNAc content of secreted GAG from the indicated strains as determined by gas chromatography and quantified by hexose or hexosamine assays. (D) Cell wall GalNAc staining with FITC-conjugated soybean agglutinin (SBA). SBA binding to mature hyphal mats of the indicated strains was quantified by fluorometry. (E) Scanning electron micrograph of hyphae of indicated species at 20,000X magnification. Arrows indicate surface decorations associated with cell wall-bound GAG. (F) Formation of adherent biofilms on tissue culture treated polystyrene plates by the indicated strains. After 24 hours growth, biofilms were washed and visualized by staining with 0.1% crystal violet. (G) Detection of β-1,3-glucan exposure on the surface of hyphae by immunostaining with Fc-dectin-1 antibody labeled with FITC secondary antibody and quantified by fluorometry at 495 nm. For all panels: An-Uge3 indicates the <i>A</i>. <i>nidulans</i> overexpressing <i>uge3</i> strain; An-UgeB indicates the <i>A</i>. <i>nidulans</i> overexpressing <i>ugeB</i> strain; and AnWT indicates wild type <i>A</i>. <i>nidulans</i>. Data are represented as mean +/- SEM and * indicates a significant difference between <i>A</i>. <i>nidulans</i>, and both overexpression strains, p<0.05 by ANOVA with Tukey’s test for pairwise comparison.</p

Production of GalNAc-rich GAG correlates with reported virulence of <i>Aspergillus spp</i>.

<p>(A) Scanning electron micrograph of hyphae of indicated species at 20,000X magnification. Arrows indicate surface decorations associated with cell wall-bound GAG. The GAG deficient <i>A</i>. <i>fumigatus</i> Δ<i>uge3</i> mutant [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005187#ppat.1005187.ref010" target="_blank">10</a>] and the <i>A</i>. <i>fumigatus</i> Δ<i>stuA</i> mutant [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005187#ppat.1005187.ref049" target="_blank">49</a>] which produces only minimal amounts of GAG are included for comparison purposes. (B) Cell wall GalNAc staining with FITC-conjugated soybean agglutinin (SBA). SBA binding to mature hyphal mats of the indicated species was quantified by fluorometry. Data are represented as mean +/- SEM. * indicates a significant difference between <i>A</i>. <i>fumigatus</i> and other species, p<0.05 by ANOVA and pairwise comparison.</p

GAG-mediated resistance to neutrophil killing is dependent on neutrophil lysate content.

<p>Susceptibility of fungal strains to injury by lysates derived from, (A) primary human neutrophils, (B) primary human neutrophils treated with DPI., (C) primary C57BL/6 mouse neutrophils or <i>gp91</i>^<i>phox</i> deficient (CGD) mouse neutrophils. * indicates a significant difference between <i>A</i>. <i>nidulans</i> and <i>A</i>. <i>fumigatus</i> or An-Uge3 strains, p<0.05 by ANOVA with Tukey’s test for pairwise comparison.</p

<i>nidulans</i> produces GalNAc-poor GAG which is associated with non-adherence.

<p><b><i>A</i>.</b> (A) Galactose and GalNAc content of secreted GAG from either <i>A</i>. <i>fumigatus</i> or <i>A</i>. <i>nidulans</i> as identified by gas chromatography and quantified by hexose or hexosamine assays. (B) Formation of adherent biofilms on tissue culture-treated polystyrene plates by <i>A</i>. <i>fumigatus</i> and <i>A</i>. <i>nidulans</i>. After 24 hours growth, biofilms were washed and visualized by staining with 0.1% crystal violet. (C) Detection of β-1,3-glucan exposure on the surface of hyphae by immunostaining with Fc-dectin-1 antibody by fluorometry. (D) Relative expression of <i>ugeB</i> in <i>A</i>. <i>nidulans</i> and <i>uge3</i> in <i>A</i>. <i>fumigatus</i>, during growth in Brian medium as measured by real-time RT-PCR. Expression of <i>tef1</i> from each respective species was used as an internal reference gene. Primer efficiency was verified, and was not different between species (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005187#ppat.1005187.s002" target="_blank">S2E Fig</a>). For all panels: data are represented as mean +/- SEM. AfWT indicates <i>A</i>. <i>fumigatus</i>, and AnWT indicates <i>A</i>. <i>nidulans</i>. * indicates a significant difference between <i>A</i>. <i>fumigatus</i> and <i>A</i>. <i>nidulans</i>, p<0.05 by Student <i>t</i> test or ANOVA with Tukey’s test for pairwise comparison, where applicable.</p