Proteomics approaches for the analysis of enriched microbial subpopulations and visualization of complex functional information

Bernhardt, Jörg; Michalik, Stephan; Wollscheid, Bernd; Völker, Uwe; Schmidt, Frank

doi:10.1016/j.copbio.2012.10.009

Cited by 23 publications

(16 citation statements)

References 36 publications

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“…Finally, proteins which displayed an fold change >1.5 together with a P values <0.05 were regarded as significantly increased or decreased in level depending on the treatment compared to a control. Voronoi‐like treemaps were generated using the Paver software (DECODON GmbH, Greifswald, Germany) and the latest functional categorization of the SEED database of S. aureus NCTC8325 (SEED DB version 2.0).…”

Section: Methodsmentioning

confidence: 99%

Alternative fluorescent labeling strategies for characterizing gram‐positive pathogenic bacteria: Flow cytometry supported counting, sorting, and proteome analysis of Staphylococcus aureus retrieved from infected host cells

et al. 2016

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Staphylococcus aureus is a Gram-positive opportunistic pathogen that is able to cause a broad range of infectious diseases in humans. Furthermore, S. aureus is able to survive inside nonprofessional phagocytic host cell which serve as a niche for the pathogen to hide from the immune system and antibiotics therapies. Modern OMICs technologies provide valuable tools to investigate host-pathogen interactions upon internalization. However, these experiments are often hampered by limited capabilities to retrieve bacteria from such an experimental setting. Thus, the aim of this study was to develop a labeling strategy allowing fast detection and quantitation of S. aureus in cell lysates or infected cell lines by flow cytometry for subsequent proteome analyses. Therefore, S. aureus cells were labeled with the DNA stain SYTO V R 9, or Vancomycin BODIPY V R FL (VMB), a glycopeptide antibiotic binding to most Gram-positive bacteria which was conjugated to a fluorescent dye. Staining of S. aureus HG001 with SYTO 9 allowed counting of bacteria from pure cultures but not in cell lysates from infection experiments. In contrast, with VMB it was feasible to stain bacteria from pure cultures as well as from samples of infection experiments. VMB can also be applied for histocytochemistry analysis of formaldehyde fixed cell layers grown on coverslips. Proteome analyses of S. aureus labeled with VMB revealed that the labeling procedure provoked only minor changes on proteome level and allowed cell sorting and analysis of S. aureus from infection settings with sensitivity similar to continuous gfp expression. Furthermore, VMB labeling allowed precise counting of internalized bacteria and can be employed for downstream analyses, e.g., proteomics, of strains not easily amendable to genetic manipulation such as clinical isolates. V C 2016 International Society for Advancement of Cytometry

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Section: Methodsmentioning

confidence: 99%

Alternative fluorescent labeling strategies for characterizing gram‐positive pathogenic bacteria: Flow cytometry supported counting, sorting, and proteome analysis of Staphylococcus aureus retrieved from infected host cells

et al. 2016

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“…Box blots of functional groups were generated using the SEED database of S. aureus NCTC8325 [SEED DB version 2.0 (Overbeek et al, 2005)], and Voronoi treemaps were created using the Paver software (DECODON GmbH) (Bernhardt et al, 2013). …”

Section: Methodsmentioning

confidence: 99%

Comparative proteome analysis reveals conserved and specific adaptation patterns of Staphylococcus aureus after internalization by different types of human non-professional phagocytic host cells

et al. 2014

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Staphylococcus aureus is a human pathogen that can cause a wide range of diseases. Although formerly regarded as extracellular pathogen, it has been shown that S. aureus can also be internalized by host cells and persist within these cells. In the present study, we comparatively analyzed survival and physiological adaptation of S. aureus HG001 after internalization by two human lung epithelial cell lines (S9 and A549), and human embryonic kidney cells (HEK 293). Combining enrichment of bacteria from host-pathogen assays by cell sorting and quantitation of the pathogen's proteome by mass spectrometry we characterized S. aureus adaptation during the initial phase between 2.5 h and 6.5 h post-infection. Starting with about 2 × 106 bacteria, roughly 1450 S. aureus proteins, including virulence factors and metabolic enzymes were identified by spectral comparison and classical database searches. Most of the bacterial adaptation reactions, such as decreased levels of ribosomal proteins and metabolic enzymes or increased amounts of proteins involved in arginine and lysine biosynthesis, enzymes coding for terminal oxidases and stress responsive proteins or activation of the sigma factor SigB were observed after internalization into any of the three cell lines studied. However, differences were noted in central carbon metabolism including regulation of fermentation and threonine degradation. Since these differences coincided with different intracellular growth behavior, complementary profiling of the metabolome of the different non-infected host cell types was performed. This revealed similar levels of intracellular glucose but host cell specific differences in the amounts of amino acids such as glycine, threonine or glutamate. With this comparative study we provide an impression of the common and specific features of the adaptation of S. aureus HG001 to specific host cell environments as a starting point for follow-up studies with different strain isolates and regulatory mutants.

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“…Protein abundances in in vivo versus in vitro biofilms were illustrated using treemaps (56). Genes/proteins were functionally classified according TIGRFAMs and visualized in multiple level hierarchically organized regions (e.g.…”

Section: Methodsmentioning

confidence: 99%

A Metaproteomics Approach to Elucidate Host and Pathogen Protein Expression during Catheter-Associated Urinary Tract Infections (CAUTIs)

Lassek

Burghartz

Chaves‐Moreno

et al. 2015

Molecular & Cellular Proteomics

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Long-term catheterization inevitably leads to a catheterassociated bacteriuria caused by multispecies bacterial biofilms growing on and in the catheters. The overall goal of the presented study was (1) to unravel bacterial community structure and function of such a uropathogenic biofilm and (2) to elucidate the interplay between bacterial virulence and the human immune system within the urine. To this end, a metaproteomics approach combined with in vitro proteomics analyses was employed to investigate both, the pro-and eukaryotic protein inventory. Our proteome analyses demonstrated that the biofilm of the investigated catheter is dominated by three bacterial species, that is, Pseudomonas aeruginosa, Morganella morganii, and Bacteroides sp., and identified iron limitation as one of the major challenges in the bladder environment. In vitro proteome analysis of P. aeruginosa and M. morganii isolated from the biofilm revealed that these opportunistic pathogens are able to overcome iron restriction via the production of siderophores and high expression of corresponding receptors. Notably, a comparison of in vivo and in vitro protein profiles of P. aeruginosa and M. morganii also indicated that the bacteria employ different strategies to adapt to the urinary tract. Although P. aeruginosa seems to express secreted and surfaceexposed proteases to escape the human innate immune system and metabolizes amino acids, M. morganii is able to take up sugars and to degrade urea. Most interestingly, a comparison of urine protein profiles of three long-term catheterized patients and three healthy control persons demonstrated the elevated level of proteins associated with neutrophils, macrophages, and the complement system in the patient's urine, which might point to a specific activation of the innate immune system in response to biofilm-associated urinary tract infections. We thus hypothesize that the often asymptomatic nature of catheter-associated urinary tract infections might be based on a fine-tuned balance between the expression of bacterial virulence factors and the human immune system. Molecular & Cellular Proteomics 14: 10.1074/mcp.M114.043463, 989-1008, 2015.Catheter-associated urinary tract infections (CAUTIs) 1 account for up to 40% of all nosocomial infections and are thus the most prevalent source of hospital-acquired infectious diseases (1, 2). CAUTIs are mostly "asymptomatic" and characterized by less than 10 5 colony-forming units per milliliter urine, which do not cause any signs of infection or symptoms. A symptomatic CAUTI, usually correlated to a number of colony-forming units (CFUs) exceeding the above mentioned threshold, is diagnosed when symptoms commonly associated with urinary tract infections (e.g. fever, dysuria, urgency, flank pain, or leukocytosis) occur (3). The risk that CAUTIs become symptomatic increases dramatically during catheterFrom the ‡Institute

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Proteomics approaches for the analysis of enriched microbial subpopulations and visualization of complex functional information

Cited by 23 publications

References 36 publications

Alternative fluorescent labeling strategies for characterizing gram‐positive pathogenic bacteria: Flow cytometry supported counting, sorting, and proteome analysis of Staphylococcus aureus retrieved from infected host cells

Alternative fluorescent labeling strategies for characterizing gram‐positive pathogenic bacteria: Flow cytometry supported counting, sorting, and proteome analysis of Staphylococcus aureus retrieved from infected host cells

Comparative proteome analysis reveals conserved and specific adaptation patterns of Staphylococcus aureus after internalization by different types of human non-professional phagocytic host cells

A Metaproteomics Approach to Elucidate Host and Pathogen Protein Expression during Catheter-Associated Urinary Tract Infections (CAUTIs)

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