Clinical and molecular epidemiology of enterovirus infections in cerebrospinal fluid samples, 2019-2023.

We performed a comparative, retrospective analysis (March 2019-April 2023) of children diagnosed with non-polio enterovirus (NPEV) central nervous system (CNS) infections (n = 47 vs. 129 contemporaneous controls without NPEV, all <18 years old), requiring cerebrospinal fluid (CSF) testing upon presentation to hospital. We found that showed that admissions decreased during pandemic restrictions (13% vs. controls 33%, p = 0.003). The median age of children with NPEV was 41 days (IQR: 18-72), most were male (n = 76, 59%) and were less likely to present with symptoms of irritability (11% vs. controls 26%, p = 0.04), but more likely to be febrile (93% vs. controls 73%, p = 0.007), have higher respiratory rates (mean 44 bpm, SD 11, vs. controls 36 bpm, SD 14, p = 0.001), higher heart rates (mean 171 bpm, SD 27 vs. controls 141 bpm, SD 36, p < 0.001), higher CSF protein (median 0.66 g/L, interquartile range [IQR] 0.46-1.01, vs. controls 0.53 mg/mL, IQR 0.28-0.89, p = 0.04), higher CSF white cell count (WCC) (median WCC 9.5×106/L, IQR 1-16 vs. controls 3.15×106/L, IQR 2.7-3.6, p < 0.001), but lower CSF glucose (median 2.8 mmol/L, IQR 2.4-3.1 vs. controls 3.1 mmol/L, IQR 2.7-3.6, p < 0.001). Phylogenetic analysis showed that these NPEVs originated from Europe (EV A71, CV B4, E21, E6, CV B3, CV B5, E7, E11, E18), North America (CV B4, E18), South America (E6), Middle East (CV B5), Africa (CV B5, E18), South Asia (E15), East/Southeast Asia (E25, CV A9, E7, E11, E18), and Australia (CV B5).

Profiling of the ß-glucosidases identified in the genome of Penicillium funiculosum: insights from genomics, transcriptomics, proteomics, and homology-modeling studies.

The enzymatic conversion of lignocellulosic biomass to bioethanol depends on efficient enzyme systems with ß-glucosidase as one of the key components. In this study, we performed in-depth profiling of the various ß-glucosidases present in the genome of the hypercellulolytic fungus Penicillium funiculosum using genomics, transcriptomics, proteomics, and molecular dynamics simulation approaches. Of the eight ß-glucosidase genes identified in the P. funiculosum genome, three were predicted to be extracellular based on signal peptide prediction and abundance in the secretome. Among the three secreted ß-glucosidases, two belonged to the GH3 family and one belonged to the GH1 family. Homology models of these proteins predicted a deep and narrow active site for the GH3 ß-glucosidases (PfBgl3A and PfBgl3B) and a shallow open active site for the GH1 ß-glucosidase (PfBgl1A). The enzymatic assays indicated that P. funiculosum-secreted proteins showed high ß-glucosidase activities with prominent bands on the 4-methylumbelliferyl ß-D-glucopyranoside zymogram. To understand the contributory effects of each of the three secreted ß-glucosidases (PfBgls), the corresponding gene was deleted separately, and the effect of the deletion on the ß-glucosidase activity of the secretome was examined. Although not the most abundant, PfBgl3A was found to be one of the most important ß-glucosidases, as evidenced by a 42% reduction in ß-glucosidase activity in the ΔPfBgl3A strain. Our results advance the understanding of the genetic and biochemical nature of all ß-glucosidases produced by P. funiculosum and pave the way to design a superior biocatalyst for the hydrolysis of lignocellulosic biomass. IMPORTANCE Commercially available cellulases are primarily produced from Trichoderma reesei. However, external supplementation of the cellulase cocktail from this host with exogenous ß-glucosidase is often required to achieve the desired optimal saccharification of cellulosic feedstocks. This challenge has led to the exploration of other cellulase-producing strains. The nonmodel hypercellulolytic fungus Penicillium funiculosum has been studied in recent times and identified as a promising source of industrial cellulases mainly due to its ability to produce a balanced concoction of cellulolytic enzymes, including ß-glucosidases. Various genetic interventions targeted at strain improvement for cellulase production have been performed; however, the ß-glucosidases of this strain have remained largely understudied. This study, therefore, reports profiling of all eight ß-glucosidases of P. funiculosum via molecular and computational approaches. The results of this study provide useful insights that will establish the background for future engineering strategies to transform this fungus into an industrial workhorse.