Abstract
Certain pathogenic bacteria adopt an intracellular lifestyle and proliferate in eukaryotic host cells. The intracellular niche protects the bacteria from cellular and humoral components of the mammalian immune system, and at the same time, allows the bacteria to gain access to otherwise restricted nutrient sources. Yet, intracellular protection and access to nutrients comes with a price, i.e., the bacteria need to overcome cell-autonomous defense mechanisms, such as the bactericidal endocytic pathway. While a few bacteria rupture the early phagosome and escape into the host cytoplasm, most intracellular pathogens form a distinct, degradation-resistant and replication-permissive membranous compartment. Intracellular bacteria that form unique pathogen vacuoles include Legionella, Mycobacterium, Chlamydia, Simkania, and Salmonella species. In order to understand the formation of these pathogen niches on a global scale and in a comprehensive and quantitative manner, an inventory of compartment-associated host factors is required. To this end, the intact pathogen compartments need to be isolated, purified and biochemically characterized. Here, we review recent progress on the isolation and purification of pathogen-modified vacuoles and membranes, as well as their proteomic characterization by mass spectrometry and different validation approaches. These studies provide the basis for further investigations on the specific mechanisms of pathogen-driven compartment formation.
Highlights
Proteomics of Pathogen-Host Interactions Mass spectrometry (MS)–based proteomics is a powerful technology, allowing the identification and quantification of hundreds of proteins from a single sample (Aebersold and Mann, 2003; Otto et al, 2014)
We focus on and compare membrane compartments modified by Legionella, Chlamydia, Simkania, or Salmonella spp., respectively, or phagosomes containing beads coated with distinct cell wall lipids purified from Mycobacterium tuberculosis
We identified a direct association of Simkania-containing vacuoles (SnCVs) with the endoplasmic reticulum (ER) of the host (Mehlitz et al, 2014)
Summary
Proteomics of Pathogen-Host Interactions Mass spectrometry (MS)–based proteomics is a powerful technology, allowing the identification and quantification of hundreds of proteins from a single sample (Aebersold and Mann, 2003; Otto et al, 2014). A striking feature of gel-free proteomics approaches is the superb sensitivity of high resolution and accurate mass MS Using this technological advances, it has become possible to investigate host compartments for the presence and composition of host and pathogen proteins, and to elucidate the dynamics of their interactions, as well as to explore post-translational modifications relating to the infection process (Bruckert and Abu Kwaik, 2014). Subcellular Pathogen Compartments As a pathogen is residing in a specific compartment in the host, the first step for most proteome studies in this field comprises selective enrichment of pathogen-containing vacuoles (PCVs) or internalized bacteria This may be achieved by subcellular/organellar fractionation based on physicochemical properties (Howe and Heinzen, 2008; He et al, 2012; Cheng et al, 2014), by immuno-affinity purification (Urwyler et al, 2010; Hoffmann et al, 2013; Vorwerk et al, 2015), or through single cell FACS enrichment by sorting internalized bacteria and lysed host cells/organelles (Becker et al, 2006; Pförtner et al, 2013; Surmann et al, 2014). The proteomic characterization of these unique pathogen niches by MS represents a comprehensive approach toward a detailed understanding of these compartments and provides the basis for further investigations on the specific mechanisms of pathogen-driven compartment formation and direct or indirect interactions of pathogen and host factors
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