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An Innovative Protocol for Metaproteomic Analyses of Microbial Pathogens in Cystic Fibrosis Sputum
(2021)
Hallmarks of cystic fibrosis (CF) are increased viscosity of mucus and impaired mucociliary clearance within the airways due to mutations of the cystic fibrosis conductance regulator gene. This facilitates the colonization of the lung by microbial pathogens and the concomitant establishment of chronic infections leading to tissue damage, reduced lung function, and decreased life expectancy. Although the interplay between key CF pathogens plays a major role during disease progression, the pathophysiology of the microbial community in CF lungs remains poorly understood. Particular challenges in the analysis of the microbial population present in CF sputum is (I) the inhomogeneous, viscous, and slimy consistence of CF sputum, and (II) the high number of human proteins masking comparably low abundant microbial proteins. To address these challenges, we used 21 CF sputum samples to develop a reliable, reproducible and widely applicable protocol for sputum processing, microbial enrichment, cell disruption, protein extraction and subsequent metaproteomic analyses. As a proof of concept, we selected three sputum samples for detailed metaproteome analyses and complemented and validated metaproteome data by 16S sequencing, metabolomic as well as microscopic analyses. Applying our protocol, the number of bacterial proteins/protein groups increased from 199-425 to 392-868 in enriched samples compared to nonenriched controls. These early microbial metaproteome data suggest that the arginine deiminase pathway and multiple proteases and peptidases identified from various bacterial genera could so far be underappreciated in their contribution to the CF pathophysiology. By providing a standardized and effective protocol for sputum processing and microbial enrichment, our study represents an important basis for future studies investigating the physiology of microbial pathogens in CF in vivo – an important prerequisite for the development of novel antimicrobial therapies to combat chronic recurrent airway infection in CF.
Summary
The susceptibility of Candida albicans biofilms to a non‐thermal plasma treatment has been investigated in terms of growth, survival and cell viability by a series of in vitro experiments. For different time periods, the C. albicans strain SC5314 was treated with a microwave‐induced plasma torch (MiniMIP). The MiniMIP treatment had a strong effect (reduction factor (RF) = 2.97 after 50 s treatment) at a distance of 3 cm between the nozzle and the superior regions of the biofilms. In addition, a viability reduction of 77% after a 20 s plasma treatment and a metabolism reduction of 90% after a 40 s plasma treatment time were observed for C. albicans. After such a treatment, the biofilms revealed an altered morphology of their cells by atomic force microscopy (AFM). Additionally, fluorescence microscopy and confocal laser scanning microscopy (CLSM) analyses of plasma‐treated biofilms showed that an inactivation of cells mainly appeared on the bottom side of the biofilms. Thus, the plasma inactivation of the overgrown surface reveals a new possibility to combat biofilms.
In food chain, Pseudomonas spp. cause spoilage by reducing shelf life of fresh products, especially during cold storage, with a high economic burden for industries. However, recent studies have shed new light on health risks occurring when they colonize immunocompromised patient tissues. Likewise to P. aeruginosa, they exhibit antibiotic resistance and biofilm formation, responsible for their spread and persistence in the environment. Biofilm formation might be induced by environmental stresses, such as temperature fluctuations causing physiological and metabolic changes exacerbating food spoilage (by protease and pigment synthesis), and the production of adhesion molecules, chemotactic or underestimated virulence factors. In order to provide a new insight into phenotypic biodiversity of Pseudomonas spoilers isolated from cold stored cheese, in this work 19 Pseudomonas spp. were investigated for biofilm, pigments, exopolysaccharide production and motility at low temperature. Only nine strains showed these phenotypic traits and the blue pigmenting cheese strain P. fluorescens ITEM 17298 was the most distinctive. In addition, this strain decreased the survival probability of infected Galleria mellonella larvae, showing, for the first time, a pathogenic potential. Genomic and proteomic analyses performed on the ITEM 17298 planktonic cells treated or not with lactoferrin derived antibiofilm peptides allowed to reveal specific biofilm related-pathways as well as proteins involved in pathogenesis. Indeed, several genes were found related to signaling system by cGMP-dependent protein kinases, cellulose, rhamnolipid and alginate synthesis, antibiotic resistance, adhesion and virulence factors. The proteome of the untreated ITEM 17298, growing at low temperature, showed that most of the proteins associated with biofilm regulation, pigmentation motility, antibiotic resistance and pathogenecity were repressed, or decreased their levels in comparison to that of the untreated cultures. Thus, the results of this work shed light on the complex pathways network allowing psychrotrophic pseudomonads to adapt themselves to food-refrigerated conditions and enhance their spoilage. In addition, the discovery of virulence factors and antibiotic resistance determinants raises some questions about the need to deeper investigate these underestimated bacteria in order to increase awareness and provide input to update legislation on their detection limits in foods.
Type I interferonopathies cover a phenotypically heterogeneous group of rare genetic diseases including the recently described proteasome-associated autoinflammatory syndromes (PRAAS). By definition, PRAAS are caused by inherited and/or de novo loss-of-function mutations in genes encoding proteasome subunits such as PSMB8, PSMB9, PSMB7, PSMA3, or proteasome assembly factors including POMP and PSMG2, respectively. Disruption of any of these subunits results in perturbed intracellular protein homeostasis including accumulation of ubiquitinated proteins which is accompanied by a type I interferon (IFN) signature. The observation that, similarly to pathogens, proteasome dysfunctions are potent type I IFN inducers is quite unexpected and, up to now, the underlying molecular mechanisms of this process remain largely unknown. One promising candidate for triggering type I IFN under sterile conditions is the unfolded protein response (UPR) which is typically initiated in response to an accumulation of unfolded and/or misfolded proteins in the endoplasmic reticulum (ER) (also referred to as ER stress). The recent observation that the UPR is engaged in subjects carrying POMP mutations strongly suggests its possible implication in the cause-and-effect relationship between proteasome impairment and interferonopathy onset. The purpose of this present review is therefore to discuss the possible role of the UPR in the pathogenesis of PRAAS. We will particularly focus on pathways initiated by the four ER-membrane proteins ATF6, PERK, IRE1-α, and TCF11/Nrf1 which undergo activation under proteasome inhibition. An overview of the current understanding of the mechanisms and potential cross-talk between the UPR and inflammatory signaling casacades is provided to convey a more integrated picture of the pathophysiology of PRAAS and shed light on potential biomarkers and therapeutic targets.
Introduction: Inhibition of androgen synthesis by abiraterone acetate (AA) entails enhanced overall survival rates and clinical benefit for patients with locally advanced and metastasized prostate cancer (PC). The expression of heat shock protein 27 (HSP27) is generally associated with cytoprotection and was demonstrated to mediate chemoresistance under cytostatic therapy, for instance, docetaxel treatment. In this study, we investigated the impact of AA treatment on HSP27 expression and PC cell growth. Materials and Methods: HSP27 expression levels in docetaxel and AA-treated PC cell lines LNCaP and PC-3 were determined by SDS PAGE and Western blot analysis. Proliferation assays were performed using a CASY Cell Counter and Analyzer Model TT (Roche Applied Science). Results: Despite significantly increased HSP27 expression in PC cells incubated with docetaxel, Western blot analysis implicated a significant reduction of the cytoprotective HSP27 in AA-treated PC cells. Notably, HSP27 stably overexpressed in PC-3-HSP27 cells did not appear as an HSP27-mediated proliferation benefit in the presence of AA as shown in docetaxel incubation studies. Conclusion: In contrast to repeatedly demonstrated HSP27-driven chemoresistance related to chemotherapeutics, our results may constitute a broader molecular mode of action of AA chemotherapy. AA efficacy may exert an HSP27 suppressive role that goes beyond the primarily assumed inhibition of androgen biosynthesis.
We analyzed the proteomic response of the Gram-negative fish pathogen A. salmonicida to iron limitation, an elevated incubation temperature, and the antibiotic florfenicol. Proteins from different subcellular fractions (cytosol, inner membrane, outer membrane, extracellular and outer membrane vesicles) were enriched and analyzed. We identified several iron-regulated proteins that were not reported in the literature for A. salmonicida before. We could also show that hemolysin, an oxidative-stress-resistance chaperone, a putative hemin receptor, an M36 peptidase, and an uncharacterized protein were significantly higher in abundance not only under iron limitation but also with an elevated incubation temperature. This may indicate that these proteins involved in the infection process of A. salmonicida are induced by both factors. The analysis of the outer membrane vesicles (OMVs) with and without applied stresses revealed significant differences in the proteomes. OMVs were smaller and contained more cytoplasmic proteins after antibiotic treatment. After cultivation with low iron availability, several iron-regulated proteins were found in the OMVs, indicating that A. salmonicida OMVs potentially have a function in iron acquisition, as reported for other bacteria. The presence of iron-regulated transporters further indicates that OMVs obtained from ‘stressed’ bacteria might be suitable vaccine candidates that induce a protective anti-virulence immune response.
Permafrost-affected soil stores a significant amount of organic carbon. Identifying the biological constraints of soil organic matter transformation, e.g., the interaction of major soil microbial soil organic matter decomposers, is crucial for predicting carbon vulnerability in permafrost-affected soil. Fungi are important players in the decomposition of soil organic matter and often interact in various mutualistic relationships during this process. We investigated four different soil horizon types (including specific horizons of cryoturbated soil organic matter (cryoOM)) across different types of permafrost-affected soil in the Western Canadian Arctic, determined the composition of fungal communities by sequencing (Illumina MPS) the fungal internal transcribed spacer region, assigned fungal lifestyles, and by determining the co-occurrence of fungal network properties, identified the topological role of keystone fungal taxa. Compositional analysis revealed a significantly higher relative proportion of the litter saprotroph Lachnum and root-associated saprotroph Phialocephala in the topsoil and the ectomycorrhizal close-contact exploring Russula in cryoOM, whereas Sites 1 and 2 had a significantly higher mean proportion of plant pathogens and lichenized trophic modes. Co-occurrence network analysis revealed the lowest modularity and average path length, and highest clustering coefficient in cryoOM, which suggested a lower network resistance to environmental perturbation. Zi-Pi plot analysis suggested that some keystone taxa changed their role from generalist to specialist, depending on the specific horizon concerned, Cladophialophora in topsoil, saprotrophic Mortierella in cryoOM, and Penicillium in subsoil were classified as generalists for the respective horizons but specialists elsewhere. The litter saprotrophic taxon Cadophora finlandica played a role as a generalist in Site 1 and specialist in the rest of the sites. Overall, these results suggested that fungal communities within cryoOM were more susceptible to environmental change and some taxa may shift their role, which may lead to changes in carbon storage in permafrost-affected soil.
Microglia are the resident immune cells of the central nervous system (CNS) and play a major role in the regulation of brain homeostasis. To maintain their cellular protein homeostasis, microglia express standard proteasomes and immunoproteasomes (IP), a proteasome isoform that preserves protein homeostasis also in non-immune cells under challenging conditions. The impact of IP on microglia function in innate immunity of the CNS is however not well described. Here, we establish that IP impairment leads to proteotoxic stress and triggers the unfolded and integrated stress responses in mouse and human microglia models. Using proteomic analysis, we demonstrate that IP deficiency in microglia results in profound alterations of the ubiquitin-modified proteome among which proteins involved in the regulation of stress and immune responses. In line with this, molecular analysis revealed chronic activation of NF-κB signaling in IP-deficient microglia without further stimulus. In addition, we show that IP impairment alters microglial function based on markers for phagocytosis and motility. At the molecular level IP impairment activates interferon signaling promoted by the activation of the cytosolic stress response protein kinase R. The presented data highlight the importance of IP function for the proteostatic potential as well as for precision proteolysis to control stress and immune signaling in microglia function.