The emergence of multidrug-resistant bacteria and the limited availability of new antibiotics are of increasing clinical concern. A compounding factor is the ability of microorganisms to form biofilms (communities of cells encased in a protective extracellular matrix) that are intrinsically resistant to antibiotics. Enterococcus faecalis is an opportunistic pathogen that readily forms biofilms and also has the propensity to acquire resistance determinants via horizontal gene transfer. There is intense interest in the genetic basis for intrinsic and acquired antibiotic resistance in E. faecalis, since clinical isolates exhibiting resistance to multiple antibiotics are not uncommon. We performed a genetic screen using a library of transposon (Tn) mutants to identify E. faecalis biofilm-associated antibiotic resistance determinants. Five Tn mutants formed wild-type biofilms in the absence of antibiotics but produced decreased biofilm biomass in the presence of antibiotic concentrations that were subinhibitory to the parent strain. Genetic determinants responsible for biofilm-associated antibiotic resistance include components of the quorum-sensing system (fsrA, fsrC, and gelE) and two glycosyltransferase (GTF) genes (epaI and epaOX). We also found that the GTFs play additional roles in E. faecalis resistance to detergent and bile salts, maintenance of cell envelope integrity, determination of cell shape, polysaccharide composition, and conjugative transfer of the pheromone-inducible plasmid pCF10. The epaOX gene is located in a variable extended region of the enterococcal polysaccharide antigen (epa) locus. These data illustrate the importance of GTFs in E. faecalis adaptation to diverse growth conditions and suggest new targets for antimicrobial design.
Chronic airway infections by the opportunistic pathogen Pseudomonas aeruginosa are a major cause of mortality in cystic fibrosis (CF) patients. Although this bacterium has been extensively studied for its virulence determinants, biofilm growth, and immune evasion mechanisms, comparatively little is known about the nutrient sources that sustain its growth in vivo. Respiratory mucins represent a potentially abundant bioavailable nutrient source, although we have recently shown that canonical pathogens inefficiently use these host glycoproteins as a growth substrate. However, given that P. aeruginosa, particularly in its biofilm mode of growth, is thought to grow slowly in vivo, the inefficient use of mucin glycoproteins may be relevant to its persistence within the CF airways. To this end, we used whole-genome fitness analysis, combining transposon mutagenesis with high-throughput sequencing, to identify genetic determinants required for P. aeruginosa growth using intact purified mucins as a sole carbon source. Our analysis reveals a biphasic growth phenotype, during which the glyoxylate pathway and amino acid biosynthetic machinery are required for mucin utilization. Secondary analyses confirmed the simultaneous liberation and consumption of acetate during mucin degradation and revealed a central role for the extracellular proteases LasB and AprA. Together, these studies describe a molecular basis for mucin-based nutrient acquisition by P. aeruginosa and reveal a host-pathogen dynamic that may contribute to its persistence within the CF airways.KEYWORDS cystic fibrosis, glyoxylate pathway, mucin, Pseudomonas aeruginosa, TnSeq C ystic fibrosis (CF), a common and lethal autosomal recessive disease, results from mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein (1). Impaired CFTR function leads to abnormal transepithelial ion transport and a thickened, dehydrated mucus layer that overlays the epithelium of several organs, including the lungs (2, 3). Within the airways, defective mucociliary clearance facilitates chronic colonization by a complex bacterial community, and the ensuing inflammatory response leads to bronchiectasis, progressive lung damage, and eventual respiratory failure in a majority of CF patients (2). Despite the recent surge in studies describing a polymicrobial etiology of CF, Pseudomonas aeruginosa continues to be widely recognized as the primary driver of disease progression (4). This opportunistic pathogen can reach densities of 10 7 to 10 9 CFU/g of sputum, particularly in late stages of disease, suggesting that the mucus environment of the lower airways provides the bacterium an ideal growth environment (5, 6). A deeper understanding of this milieu, and its ability to sustain P. aeruginosa growth in vivo is critically important for improved disease management.The mucosal layer covering the respiratory epithelium is a complex mixture of water,
Achromobacter xylosoxidans has attracted increasing attention as an emerging pathogen in patients with cystic fibrosis. Intrinsic resistance to several classes of antimicrobials and the ability to form robust biofilms in vivo contribute to the clinical manifestations of persistent A. xylosoxidans infection. Still, much of A. xylosoxidans biofilm formation remains uncharacterized due to the scarcity of existing genetic tools. Here we demonstrate a promising genetic system for use in A. xylosoxidans ; generating a transposon mutant library which was then used to identify genes involved in biofilm development in vitro. We further described the effects of one of the genes found in the mutagenesis screen, encoding a putative enoyl-CoA hydratase, on biofilm structure and tolerance to antimicrobials. Through additional analysis, we find that a fatty acid signaling compound is essential to A. xylosoxidans biofilm ultrastructure and maintenance. This work describes methods for the genetic manipulation of A. xylosoxidans and demonstrated their use to improve our understanding of A. xylosoxidans pathophysiology.
Abnormal protein aggregation within neurons is a key pathologic feature of Parkinson’s disease (PD). The spread of brain protein aggregates is associated with clinical disease progression, but how this occurs remains unclear. Mutations in glucosidase, beta acid 1 (GBA), which encodes glucocerebrosidase (GCase), are the most penetrant common genetic risk factor for PD and dementia with Lewy bodies and associate with faster disease progression. To explore how GBA mutations influence pathogenesis, we previously created a Drosophila model of GBA deficiency (Gba1b) that manifests neurodegeneration and accelerated protein aggregation. Proteomic analysis of Gba1b mutants revealed dysregulation of proteins involved in extracellular vesicle (EV) biology, and we found altered protein composition of EVs from Gba1b mutants. Accordingly, we hypothesized that GBA may influence pathogenic protein aggregate spread via EVs. We found that accumulation of ubiquitinated proteins and Ref(2)P, Drosophila homologue of mammalian p62, were reduced in muscle and brain tissue of Gba1b flies by ectopic expression of wildtype GCase in muscle. Neuronal GCase expression also rescued protein aggregation both cell-autonomously in brain and non-cell-autonomously in muscle. Muscle-specific GBA expression reduced the elevated levels of EV-intrinsic proteins and Ref(2)P found in EVs from Gba1b flies. Perturbing EV biogenesis through neutral sphingomyelinase (nSMase), an enzyme important for EV release and ceramide metabolism, enhanced protein aggregation when knocked down in muscle, but did not modify Gba1b mutant protein aggregation when knocked down in neurons. Lipidomic analysis of nSMase knockdown on ceramide and glucosylceramide levels suggested that Gba1b mutant protein aggregation may depend on relative depletion of specific ceramide species often enriched in EVs. Finally, we identified ectopically expressed GCase within isolated EVs. Together, our findings suggest that GCase deficiency promotes accelerated protein aggregate spread between cells and tissues via dysregulated EVs, and EV-mediated trafficking of GCase may partially account for the reduction in aggregate spread.
Abnormal protein aggregation within neurons is a key pathologic feature of Parkinson's disease (PD). The spread of protein aggregates in the brain is associated with clinical disease progression, but how this occurs remains unclear. Mutations in the gene glucosidase, beta acid 1 (GBA), which encodes the lysosomal enzyme glucocerebrosidase (GCase), are the most penetrant common genetic risk factor for PD and dementia with Lewy bodies, and also associate with faster disease progression. To explore the mechanism by which mutations in GBA influence pathogenesis of these diseases, we previously created a Drosophila model of GBA deficiency (Gba1b) that manifests neurodegeneration, motor and cognitive deficits, and accelerated protein aggregation. Proteomic analysis of Gba1b mutants revealed dysregulation of proteins involved in extracellular vesicle (EV) biology, and we found altered protein composition of EVs from Gba1b mutants. To further investigate this novel mechanism, we hypothesized that GBA may influence the spread of pathogenic protein aggregates throughout the brain via EVs. We found that protein aggregation is reduced cellautonomously and non-cell-autonomously by expressing wildtype GCase in specific tissues. In particular, accumulation of insoluble ubiquitinated proteins and Ref (2)P in the brains of Gba1b flies are reduced by ectopic expression of GCase in muscle tissue.Neuronal expression of GCase also cell-autonomously rescued protein aggregation in brain as well as non-cell-autonomously rescued protein aggregation in muscle. Musclespecific GBA expression rescued the elevated levels of EV-intrinsic proteins and Ref(2)P found in EVs from Gba1b flies. Genetically perturbing EV biogenesis in specific tissues in the absence of GCase revealed differential cell-autonomous effects on protein aggregation but could not replicate the non-cell-autonomous rescue observed with tissuespecific GBA expression. Additionally, we identified ectopically expressed GCase within isolated EVs. Together, our findings suggest that GCase deficiency mediates accelerated spread of protein aggregates between cells and tissues via dysregulated EVs, and EVmediated trafficking of GCase may partially account for the reduction in aggregate spread.
A deeper understanding of complex biological processes, including tumor development and immune response, requires ultra high-plex, spatial interrogation of multiple 'omes'. Here we present the development and implementation of a novel spatial proteogenomic (SPG) assay on the GeoMx® Digital Spatial Profiler platform with NGS readout that enables ultra high-plex digital quantitation of proteins (> 100-plex) and RNA (whole transcriptome, > 18,000-plex) from a single FFPE sample. This study highlighted the high concordance, R > 0.85, and <11% change in sensitivity between SPG assay and the single analyte assays on various cell lines and tissues from human and mouse. Furthermore, we demonstrate that the SPG assay was reproducible across multiple users. When used in conjunction with advanced cellular neighborhood segmentation, distinct immune or tumor RNA and protein targets were spatially resolved within individual cell subpopulations in human colorectal cancer and non-small cell lung cancer. We used the SPG assay to interrogate 23 different glioblastoma multiforme samples across 4 pathologies. The study revealed distinct clustering of both RNA and protein based on pathology and anatomic location. The in-depth investigation of giant cell glioblastoma multiforme revealed distinct protein and RNA expression profiles compared to that of the more common glioblastoma multiforme. More importantly, the use of spatial proteogenomics allowed simultaneous interrogation of critical protein post-translational modifications alongside whole transcriptomic profiles within the same distinct cellular neighborhoods.
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