Tracking the dynamic interplay between bacterial and host factors during pathogen-induced vacuole rupture in real time.
Cell Microbiol. 2010 Apr 1; 12(4):545-56
I found this article interesting because the authors developed a novel fluorescence microscopy methodology to detect rupture of the phagosome membrane by intracellular bacterial pathogens at the single cell level.
By combining the use of a chimeric protein AIDA-beta-lactamase, FRET, microscopy and an automated algorithm the authors were able to detect bacterial escape from phagosomes into the cytoplasm in real time. This novel procedure is temporally sensitive compared to other methods used to detect bacterial escape, like comet tail formation. Additionally, the use of fluorescent fusions to different host cell proteins helped them to identify multiple proteins recruited during the invasion and vacuole escape processes (i.e. in Shigella escape, RhoA, Rac1 and galectin-3 are recruited). Using this system, the authors were also able to show the role of Salmonella effectors PipB2 and SifA in vacuole stabilization. This method is limited so far to Gram-negative bacteria but could potentially be adapted for other pathogenic bacteria.
Steele-Mortimer O and Ibarra J: F1000Prime Recommendation of [Ray K et al., Cell Microbiol 2010, 12(4):545-56]. In F1000Prime, 13 Apr 2010; DOI: 10.3410/f.2886956.2555054. F1000Prime.com/2886956#eval2555054
F1000Prime Recommendations, Dissents and Comments for [Ray K et al., Cell Microbiol 2010, 12(4):545-56]. In F1000Prime, 08 Dec 2013; F1000Prime.com/2886956
J Exp Med. 2011 Dec 19; 208(13):2705-16
Cell Host Microbe. 2009 Aug 20; 6(2):137-49
Cell Microbiol. 2010 Apr 1; 12(4):530-44
Nature. 2013 Apr 4; 496(7443):106-9
Escape into the host cell cytosol following invasion of mammalian cells is a common strategy used by invasive pathogens. This requires membrane rupture of the vesicular or vacuolar compartment formed around the bacteria after uptake into the host cell. The mechanism of pathogen-induced disassembly of the vacuolar membrane is poorly understood. We established a novel, robust and sensitive fluorescence microscopy method that tracks the precise time point of vacuole rupture upon uptake of Gram-negative bacteria. This revealed that the enteroinvasive pathogen Shigella flexneri escapes rapidly, in less than 10 min, from the vacuole. Our method demonstrated the recruitment of host factors, such as RhoA, to the bacterial entry site and their continued presence at the point of vacuole rupture. We found a novel host marker for ruptured vacuoles, galectin-3, which appears instantly in the proximity of bacteria after escape into the cytosol. Furthermore, we show that the Salmonella effector proteins, SifA and PipB2, stabilize the vacuole membrane inhibiting bacterial escape from the vacuole. Our novel approach to track vacuole rupture is ideally suited for high-content and high-throughput approaches to identify the molecular and cellular mechanisms of membrane rupture during invasion by pathogens such as viruses, bacteria and parasites.
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