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Influenza A Virus (IAV) infection followed by bacterial pneumonia often leads to hospitalization and death in individuals from high risk groups. Following infection, IAV triggers the process of viral RNA replication which in turn disrupts healthy gut microbial community, while the gut microbiota plays an instrumental role in protecting the host by evolving colonization resistance. Although the underlying mechanisms of IAV infection have been unraveled, the underlying complex mechanisms evolved by gut microbiota in order to induce host immune response following IAV infection remain evasive. In this work, we developed a novel Maximal-Clique based Community Detection algorithm for Weighted undirected Networks (MCCD-WN) and compared its performance with other existing algorithms using three sets of benchmark networks. Moreover, we applied our algorithm to gut microbiome data derived from fecal samples of both healthy and IAV-infected pigs over a sequence of time-points. The results we obtained from the real-life IAV dataset unveil the role of the microbial families Ruminococcaceae, Lachnospiraceae, Spirochaetaceae and Prevotellaceae in the gut microbiome of the IAV-infected cohort. Furthermore, the additional integration of metaproteomic data enabled not only the identification of microbial biomarkers, but also the elucidation of their functional roles in protecting the host following IAV infection. Our network analysis reveals a fast recovery of the infected cohort after the second IAV infection and provides insights into crucial roles of Desulfovibrionaceae and Lactobacillaceae families in combating Influenza A Virus infection. Source code of the community detection algorithm can be downloaded from https://github.com/AniBhar84/MCCD-WN.
The anaerobic, gastrointestinal pathogen Clostridioides difficile can cause severe forms of enterocolitis which is mainly mediated by the toxins it produces. The RNA polymerase inhibitor Fidaxomicin is the current gold standard for the therapy of C. difficile infections due to several beneficial features including its ability to suppress toxin synthesis in C. difficile. In contrast to the Rifamycins, Fidaxomicin binds to the RNA polymerase switch region, which is also the binding site for Myxopyronin B. Here, serial broth dilution assays were performed to test the susceptibility of C. difficile and other anaerobes to Myxopyronin B, proving that the natural product is considerably active against C. difficile and that there is no cross-resistance between Fidaxomicin and Myxopyronin B in a Fidaxomicin-resistant C. difficile strain. Moreover, mass spectrometry analysis indicated that Myxopyronin B is able to suppress early phase toxin synthesis in C. difficile to the same degree as Fidaxomicin. Conclusively, Myxopyronin B is proposed as a new lead structure for the design of novel antibiotics for the therapy of C. difficile infections.
The anaerobic pathogen Clostridioides difficile is perfectly equipped to survive and persist inside the mammalian intestine. When facing unfavorable conditions C. difficile is able to form highly resistant endospores. Likewise, biofilms are currently discussed as form of persistence. Here a comprehensive proteomics approach was applied to investigate the molecular processes of C. difficile strain 630Δerm underlying biofilm formation. The comparison of the proteome from two different forms of biofilm-like growth, namely aggregate biofilms and colonies on agar plates, revealed major differences in the formation of cell surface proteins, as well as enzymes of its energy and stress metabolism. For instance, while the obtained data suggest that aggregate biofilm cells express both flagella, type IV pili and enzymes required for biosynthesis of cell-surface polysaccharides, the S-layer protein SlpA and most cell wall proteins (CWPs) encoded adjacent to SlpA were detected in significantly lower amounts in aggregate biofilm cells than in colony biofilms. Moreover, the obtained data suggested that aggregate biofilm cells are rather actively growing cells while colony biofilm cells most likely severely suffer from a lack of reductive equivalents what requires induction of the Wood-Ljungdahl pathway and C. difficile’s V-type ATPase to maintain cell homeostasis. In agreement with this, aggregate biofilm cells, in contrast to colony biofilm cells, neither induced toxin nor spore production. Finally, the data revealed that the sigma factor SigL/RpoN and its dependent regulators are noticeably induced in aggregate biofilms suggesting an important role of SigL/RpoN in aggregate biofilm formation.
Acidobacteria represents one of the most dominant bacterial groups across diverse ecosystems. However, insight into their ecology and physiology has been hampered by difficulties in cultivating members of this phylum. Previous cultivation efforts have suggested an important role of trace elements for the proliferation of Acidobacteria, however, the impact of these metals on their growth and metabolism is not known. In order to gain insight into this relationship, we evaluated the effect of trace element solution SL10 on the growth of two strains (5B5 and WH15) of Acidobacteria belonging to the genus Granulicella and studied the proteomic responses to manganese (Mn). Granulicella species had highest growth with the addition of Mn, as well as higher tolerance to this metal compared to seven other metal salts. Variations in tolerance to metal salt concentrations suggests that Granulicella sp. strains possess different mechanisms to deal with metal ion homeostasis and stress. Furthermore, Granulicella sp. 5B5 might be more adapted to survive in an environment with higher concentration of several metal ions when compared to Granulicella sp. WH15. The proteomic profiles of both strains indicated that Mn was more important in enhancing enzymatic activity than to protein expression regulation. In the genomic analyses, we did not find the most common transcriptional regulation of Mn homeostasis, but we found candidate transporters that could be potentially involved in Mn homeostasis for Granulicella species. The presence of such transporters might be involved in tolerance to higher Mn concentrations, improving the adaptability of bacteria to metal enriched environments, such as the decaying wood-rich Mn environment from which these two Granulicella strains were isolated.
Swine are regarded as promising biomedical models, but the dynamics of theirgastrointestinal microbiome have been much less investigated than that of humans or mice. The aimof this study was to establish an integrated multi-omics protocol to investigate the fecal microbiomeof healthy swine. To this end, a preparation and analysis protocol including integrated samplepreparation for meta-omics analyses of deep-frozen feces was developed. Subsequent data integrationlinked microbiome composition with function, and metabolic activity with protein inventories, i.e.,16S rRNA data and expressed proteins, and identified proteins with corresponding metabolites.16S rRNA gene amplicon and metaproteomics analyses revealed a fecal microbiome dominated byPrevotellaceae,Lactobacillaceae,Lachnospiraceae,RuminococcaceaeandClostridiaceae.Similar microbiomecompositions in feces and colon, but not ileum samples, were observed, showing that feces can serveas minimal-invasive proxy for porcine colon microbiomes. Longitudinal dynamics in composition,e.g., temporal decreased abundance ofLactobacillaceaeandStreptococcaceaeduring the experiment,were not reflected in microbiome function. Instead, metaproteomics and metabolomics showed arather stable functional state, as evident from short-chain fatty acids (SCFA) profiles and associatedmetaproteome functions, pointing towards functional redundancy among microbiome constituents.In conclusion, our pipeline generates congruent data from different omics approaches on the taxonomyand functionality of the intestinal microbiome of swine.
For many years now, Bacillus megaterium serves as a microbial workhorse for the high-level production of recombinant proteins in the g/L-scale. However, efficient and stable production processes require the knowledge of the molecular adaptation strategies of the host organism to establish optimal environmental conditions. Here, we interrogated the osmotic stress response of B. megaterium using transcriptome, proteome, metabolome, and fluxome analyses. An initial transient adaptation consisted of potassium import and glutamate counterion synthesis. The massive synthesis of the compatible solute proline constituted the second longterm adaptation process. Several stress response enzymes involved in iron scavenging and reactive oxygen species (ROS) fighting proteins showed higher levels under prolonged osmotic stress induced by 1.8 M NaCl. At the same time, the downregulation of the expression of genes of the upper part of glycolysis resulted in the activation of the pentose phosphate pathway (PPP), generating an oversupply of NADPH. The increased production of lactate accompanied by the reduction of acetate secretion partially compensate for the unbalanced (NADH/NAD+) ratio. Besides, the tricarboxylic acid cycle (TCA) mainly supplies the produced NADH, as indicated by the higher mRNA and protein levels of involved enzymes, and further confirmed by 13C flux analyses. As a consequence of the metabolic flux toward acetyl-CoA and the generation of an excess of NADPH, B. megaterium redirected the produced acetyl-CoA toward the polyhydroxybutyrate (PHB) biosynthetic pathway accumulating around 30% of the cell dry weight (CDW) as PHB. This direct relation between osmotic stress and intracellular PHB content has been evidenced for the first time, thus opening new avenues for synthesizing this valuable biopolymer using varying salt concentrations under non-limiting nutrient conditions.
For many years now, Bacillus megaterium serves as a microbial workhorse for the high-level production of recombinant proteins in the g/L-scale. However, efficient and stable production processes require the knowledge of the molecular adaptation strategies of the host organism to establish optimal environmental conditions. Here, we interrogated the osmotic stress response of B. megaterium using transcriptome, proteome, metabolome, and fluxome analyses. An initial transient adaptation consisted of potassium import and glutamate counterion synthesis. The massive synthesis of the compatible solute proline constituted the second longterm adaptation process. Several stress response enzymes involved in iron scavenging and reactive oxygen species (ROS) fighting proteins showed higher levels under prolonged osmotic stress induced by 1.8 M NaCl. At the same time, the downregulation of the expression of genes of the upper part of glycolysis resulted in the activation of the pentose phosphate pathway (PPP), generating an oversupply of NADPH. The increased production of lactate accompanied by the reduction of acetate secretion partially compensate for the unbalanced (NADH/NAD+) ratio. Besides, the tricarboxylic acid cycle (TCA) mainly supplies the produced NADH, as indicated by the higher mRNA and protein levels of involved enzymes, and further confirmed by 13C flux analyses. As a consequence of the metabolic flux toward acetyl-CoA and the generation of an excess of NADPH, B. megaterium redirected the produced acetyl-CoA toward the polyhydroxybutyrate (PHB) biosynthetic pathway accumulating around 30% of the cell dry weight (CDW) as PHB. This direct relation between osmotic stress and intracellular PHB content has been evidenced for the first time, thus opening new avenues for synthesizing this valuable biopolymer using varying salt concentrations under non-limiting nutrient conditions.
For many years now, Bacillus megaterium serves as a microbial workhorse for the high-level production of recombinant proteins in the g/L-scale. However, efficient and stable production processes require the knowledge of the molecular adaptation strategies of the host organism to establish optimal environmental conditions. Here, we interrogated the osmotic stress response of B. megaterium using transcriptome, proteome, metabolome, and fluxome analyses. An initial transient adaptation consisted of potassium import and glutamate counterion synthesis. The massive synthesis of the compatible solute proline constituted the second longterm adaptation process. Several stress response enzymes involved in iron scavenging and reactive oxygen species (ROS) fighting proteins showed higher levels under prolonged osmotic stress induced by 1.8 M NaCl. At the same time, the downregulation of the expression of genes of the upper part of glycolysis resulted in the activation of the pentose phosphate pathway (PPP), generating an oversupply of NADPH. The increased production of lactate accompanied by the reduction of acetate secretion partially compensate for the unbalanced (NADH/NAD+) ratio. Besides, the tricarboxylic acid cycle (TCA) mainly supplies the produced NADH, as indicated by the higher mRNA and protein levels of involved enzymes, and further confirmed by 13C flux analyses. As a consequence of the metabolic flux toward acetyl-CoA and the generation of an excess of NADPH, B. megaterium redirected the produced acetyl-CoA toward the polyhydroxybutyrate (PHB) biosynthetic pathway accumulating around 30% of the cell dry weight (CDW) as PHB. This direct relation between osmotic stress and intracellular PHB content has been evidenced for the first time, thus opening new avenues for synthesizing this valuable biopolymer using varying salt concentrations under non-limiting nutrient conditions.
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.