Purification and proteomics of pathogen-modified vacuoles and membranes

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.

Patterns, Receptors, and Signals: Regulation of Phagosome Maturation

“…Considering the role of protein phosphorylation in regulating almost all cellular processes, it is likely that even ‘hard-wired’ processes, such as phagosome maturation, are influenced by intracellular signaling cascades. The molecular regulation of vesicle trafficking processes through post-translational modifications and their effect on innate immunity is understudied and, thus, more work is needed in this area.

Phagosome ProteomicsThrough optimization, latex bead phagosomes [99] or bacterial vacuoles [69] can be isolated at high purity using density gradient ultracentrifugation. In recent years, this high purity allowed the thorough characterization of the protein composition of latex bead phagosomes 1, 16, 39 and some bacterial phagosomes, such as Legionella [75] and Mycobacterium [117] vacuoles, by mass spectrometry-based proteomics.

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Space: A Final Frontier for Vacuolar Pathogens

“…In most cases, vacuolar pathogens can tailor host membrane to form hybrid organelles that are biochemically and morphologically distinct from compartments found in uninfected cells (3). They may also modify the subcellular compartments in which they reside to aid nutrient acquisition, to promote trafficking to a replicative niche or to limit exposure to the cytosolic immune surveillance pathways (4). Moreover, vacuolar pathogens can often be transported between subcellular compartments as hitchhikers in the host endomembrane system.…”

Elucidation of host–pathogen protein–protein interactions to uncover mechanisms of host cell rewiring

“…The first is to identify all secreted proteins upon infection, where in silico predictions and experimental findings don’t always corroborate [76]. To help the identification of virulence factors from membrane-contained intracellular bacterial pathogens, one could consider purifying intact pathogen-containing compartments or vacuoles, and characterizing their proteome by mass-spectrometry to find new virulence factors that associate with the host membranes [77]. Secondly, due to their increased genomic complexity compared to viruses, the generation of transgenic cell lines to ectopically express each putative secreted protein would be highly time-consuming.…”