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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.
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.
Inflammatory bowel diseases (IBDs) have emerged as a public health problem worldwide with a limited number of efficient therapeutic options despite advances in medical therapy. Although changes in the gut microbiota composition are recognized as key drivers of dysregulated intestinal immunity, alterations in bile acids (BAs) have been shown to influence gut homeostasis and contribute to the pathogenesis of the disease. In this review, we explore the interactions involving BAs and gut microbiota in IBDs, and discuss how the gut microbiota–BA–host axis may influence digestive inflammation.
In the current era of precision oncology, it is widely acknowledged that CRC is a heterogeneous disease entity. Tumor location (right- or left-sided colon cancer or rectal cancer) is a crucial factor in determining disease progression as well as prognosis and influences disease management. In the last decade, numerous works have reported that the microbiome is an important element of CRC carcinogenesis, progression and therapy response. Owing to the heterogeneous nature of microbiomes, the findings of these studies were inconsistent. The majority of the studies combined colon cancer (CC) and rectal cancer (RC) samples as CRC for analysis. Furthermore, the small intestine, as the major site for immune surveillance in the gut, is understudied compared to the colon. Thus, the CRC heterogeneity puzzle is far from being solved, and more research is necessary for prospective trials that separately investigate CC and RC. Our prospective study aimed to map the colon cancer landscape using 16S rRNA amplicon sequencing in biopsy samples from the terminal ileum, healthy colon tissue, healthy rectal tissue and tumor tissue as well as in preoperative and postoperative stool samples of 41 patients. While fecal samples provide a good approximation of the average gut microbiome composition, mucosal biopsies allow for detecting subtle variations in local microbial communities. In particular, the small bowel microbiome has remained poorly characterized, mainly because of sampling difficulties. Our analysis revealed the following: (i) right- and left-sided colon cancers harbor distinct and diverse microbiomes, (ii) the tumor microbiome leads to a more consistent cancer-defined microbiome between locations and reveals a tumor microbiome–ileal microbiome association, (iii) the stool only partly reflects the microbiome landscape in patients with CC, and (iv) mechanical bowel preparation and perioperative antibiotics together with surgery result in major changes in the stool microbiome, characterized by a significant increase in the abundance of potentially pathogenic bacteria, such as Enterococcus. Collectively, our results provide new and valuable insights into the complex microbiome landscape in patients with colon cancer.
The early-life microbiome (ELM) interacts with the psychosocial environment, in particular during early-life adversity (ELA), defining life-long health trajectories. The ELM also plays a significant role in the maturation of the immune system. We hypothesised that, in this context, the resilience of the oral microbiomes, despite being composed of diverse and distinct communities, allows them to retain an imprint of the early environment. Using 16S amplicon sequencing on the EpiPath cohort, we demonstrate that ELA leaves an imprint on both the salivary and buccal oral microbiome 24 years after exposure to adversity. Furthermore, the changes in both communities were associated with increased activation, maturation, and senescence of both innate and adaptive immune cells, although the interaction was partly dependent on prior herpesviridae exposure and current smoking. Our data suggest the presence of multiple links between ELA, Immunosenescence, and cytotoxicity that occur through long-term changes in the microbiome.
Differences in salinity are boundaries that act as barriers for the dispersal of most aquatic organisms. This creates distinctive biota in freshwater and brackish water (mesohaline) environments. To test how saline boundaries influence the diversity and composition of host-associated microbiota, we analyzed the microbiome within the digestive tract of Theodoxus fluviatilis, an organism able to cross the freshwater and mesohaline boundary. Alpha-diversity measures of the microbiome in freshwater and brackish water were not significantly different. However, the composition of the bacterial community within freshwater T. fluviatilis differed significantly compared with mesohaline T. fluviatilis and typical bacteria could be determined for the freshwater and the mesohaline digestive tract microbiome. An artificial increase in salinity surrounding these freshwater snails resulted in a strong change in the bacterial community and typical marine bacteria became more pronounced in the digestive tract microbiome of freshwater T. fluviatilis. However, the composition of the digestive tract microbiome in freshwater snails did not converge to that found within mesohaline snails. Within mesohaline snails, no cardinal change was found after either an increase or decrease in salinity. In all samples, Pseudomonas, Pirellula, Flavobacterium, Limnohabitans, and Acinetobacter were among the most abundant bacteria. These bacterial genera were largely unaffected by changes in environmental conditions. As permanent residents in T. fluviatilis, they may support the digestion of the algal food in the digestive tract. Our results show that freshwater and mesohaline water host-associated microbiomes respond differently to changes in salinity. Therefore, the salinization of coastal freshwater environments due to a rise in sea level can influence the gut microbiome and its functions with currently unknown consequences for, e.g., nutritional physiology of the host.
Animals experience climatic variation in their natural habitats, which may lead to variation in phenotypic responses among populations through local adaptation or phenotypic plasticity. In ectotherm arthropods, the expression of thermoprotective metabolites such as free amino acids, sugars, and polyols, in response to temperature stress, may facilitate temperature tolerance by regulating cellular homeostasis. If populations experience differences in temperatures, individuals may exhibit population-specific metabolite profiles through differential accumulation of metabolites that facilitate thermal tolerance. Such thermoprotective metabolites may originate from the animals themselves or from their associated microbiome, and hence microbial symbionts may contribute to shape the thermal niche of their host. The social spider Stegodyphus dumicola has extremely low genetic diversity, yet it occupies a relatively broad temperature range occurring across multiple climate zones in Southern Africa. We investigated whether the metabolome, including thermoprotective metabolites, differs between populations, and whether population genetic structure or the spider microbiome may explain potential differences. To address these questions, we assessed metabolite profiles, phylogenetic relationships, and microbiomes in three natural populations along a temperature gradient. The spider microbiomes in three genetically distinct populations of S. dumicola showed no significant population-specific pattern, and none of its dominating genera (Borrelia, Diplorickettsia, and Mycoplasma) are known to facilitate thermal tolerance in hosts. These results do not support a role of the microbiome in shaping the thermal niche of S. dumicola. Metabolite profiles of the three spider populations were significantly different. The variation was driven by multiple metabolites that can be linked to temperature stress (e.g., lactate, succinate, or xanthine) and thermal tolerance (e.g., polyols, trehalose, or glycerol): these metabolites had higher relative abundance in spiders from the hottest geographic region. These distinct metabolite profiles are consistent with a potential role of the metabolome in temperature response.
Microbial metabolites measured using NMR may serve as markers for physiological or pathological host–microbe interactions and possibly mediate the beneficial effects of microbiome diversity. Yet, comprehensive analyses of gut microbiome data and the urine NMR metabolome from large general population cohorts are missing. Here, we report the associations between gut microbiota abundances or metrics of alpha diversity, quantified from stool samples using 16S rRNA gene sequencing, with targeted urine NMR metabolites measures from 951 participants of the Study of Health in Pomerania (SHIP). We detected significant genus–metabolite associations for hippurate, succinate, indoxyl sulfate, and formate. Moreover, while replicating the previously reported association between hippurate and measures of alpha diversity, we identified formate and 4-hydroxyphenylacetate as novel markers of gut microbiome alpha diversity. Next, we predicted the urinary concentrations of each metabolite using genus abundances via an elastic net regression methodology. We found profound associations of the microbiome-based hippurate prediction score with markers of liver injury, inflammation, and metabolic health. Moreover, the microbiome-based prediction score for hippurate completely mediated the clinical association pattern of microbial diversity, hinting at a role of benzoate metabolism underlying the positive associations between high alpha diversity and healthy states. In conclusion, large-scale NMR urine metabolomics delivered novel insights into metabolic host–microbiome interactions, identifying pathways of benzoate metabolism as relevant candidates mediating the beneficial health effects of high microbial alpha diversity.
The success of pregnancy depends on precisely adjusted, local immune mechanisms. In early pregnancy, fetal trophoblast cells implant into the endometrium to build and anchor the placenta. Simultaneously, they mediate fetal tolerance and defense against infections. To cover these versatile requirements, local immune factors must be in balance. A too tolerogenic milieu can lead to an inadequate placentation; while a too inflammatory milieu can cause rejection of the semi-allogenic fetus. Bacterial infections can provoke these inflammatory pregnancy complications as well. Therefore, the pregnant uterus was long thought to be sterile. Descriptions of a placental microbiome opened a scientific discourse, which is unsolved due to contrary studies. The colonization of the non-pregnant endometrium is, however, confirmed. It is supposed to affect both, uterine pathologies and fertility. Precise data are lacking. Aim of this work was to assess if and under which circumstances a bacterial colonization would be tolerable.
One of the described species in placental and endometrial samples is Fusobacterium nucleatum. It is an opportunistic bacterium, which is known from the human oral cavity and associated with the development of colon carcinomas. F. nucleatum supports tumorigenesis by the induction of epithelial proliferation, survival, migration and invasion as well as angiogenesis and tumor tolerance. Since similar processes are required for implantation and placentation, F. nucleatum might support these as well. In this work, the effects of F. nucleatum on leukocyte-trophoblast-interactions, especially of macrophages and innate lymphoid cells type 3 (ILC3), were assessed.
The monocytic cells (THP-1) were differentiated into inflammatory M1 (IFN-γ) or tissue-repairing and tolerogenic M2a (IL-4) and M2c (TGF-β) macrophages. Inactivated F. nucleatum, LPS or E. coli was added. Only small concentrations of inactivated bacteria were used (bacteria:leukocyte ratio of 0.1 or 1), since it was not the aim to analyze infections. Conditioned medium of treated leukocytes was added to trophoblastic cells (HTR-8/SVneo). Migratory, invasive and tube formation behavior of trophoblastic cells was quantified.
Treated M1 macrophages impaired trophoblast function, whereas M2a macrophages induced trophoblast invasion. M2c macrophages supported trophoblast migration and tube formation if treated with the smaller, but not with the higher concentration of F. nucleatum. This treatment induced the accumulation of HIF-1α and the secretion of VEGF-A in M2c macrophages as well. Moreover, the higher concentration of F. nucleatum caused rather inflammatory responses (NF-κB activation and cytokine expression). The activation of the HIF-1α-VEGF-A axis under the influence of TGF-β might serve as a mild immune stimulation by low abundant commensal bacteria supporting placentation.
In contrast to macrophages, the function of ILC3s during pregnancy is still unknown. In general, ILC3s are located in mucosal tissue, such as the gut. They participate in tolerance mechanisms and form the local micromilieu by the secretion of cytokines and the presentation of antigens. In order to characterize local, uterine ILC3s, murine ILC3s were compared to peripheral, splenic ILC3s. Uterine ILC3s were more activated and produced higher levels of IL-17 compared to splenic ILC3s. However, uterine ILC3s barely expressed MHCII on their surface. A reduced antigen presentation potential was confirmed in human ILC3s differentiated from cord blood stem cells by the addition of TGF-β or hCG. The treatment with bacteria increased MHCII expression, but not to the initial level. The higher bacterial concentration induced IL-8 secretion and led to an increased trophoblast invasion. ILC3s were less sensitive to bacterial stimulation than macrophages.
Recent studies on the uterine or placental presence of bacteria during pregnancy are discrepant. The results of this project indicate that bacteria or bacterial residues might serve as a mild stimulus under certain circumstances to support implantation without negative effects. The current discussion must therefore not only be expanded by additional studies, but especially include differentiated local conditions. In this context, the sheer presence of bacteria or bacterial components must not be equated with an infection representing a known hazard.