Abstract
Intracellular pathogens include all viruses, many bacteria and parasites capable of invading and surviving within host cells. Key to survival is the subversion of host cell pathways by the pathogen for the purpose of propagation and evading the immune system. The intracellular bacterium Shigella flexneri, the causative agent of bacillary dysentery, invades host cells in a vacuole that is subsequently ruptured to allow growth of the pathogen within the host cytoplasm. S. flexneri invasion has been classically described as a macropinocytosis-like process, however the underlying details and the role of macropinosomes in the intracellular bacterial lifestyle have remained elusive. We applied dynamic imaging and advanced large volume correlative light electron microscopy (CLEM) to study the highly transient events of S. flexneri’s early invasion into host epithelial cells and elucidate some of its fundamental features. First, we demonstrate a clear distinction between two compartments formed during the first step of invasion: the bacterial containing vacuole and surrounding macropinosomes, often considered identical. Next, we report a functional link between macropinosomes and the process of vacuolar rupture, demonstrating that rupture timing is dependent on the availability of macropinosomes as well as the activity of the small GTPase Rab11 recruited directly to macropinosomes. We go on to reveal that the bacterial containing vacuole and macropinosomes come into direct contact at the onset of vacuolar rupture. Finally, we demonstrate that S. flexneri does not subvert pre-existing host endocytic vesicles during the invasion steps leading to vacuolar rupture, and propose that macropinosomes are the major compartment involved in these events. These results provide the basis for a new model of the early steps of S. flexneri epithelial cell invasion, establishing a different view of the enigmatic process of cytoplasmic access by invasive bacterial pathogens.
Highlights
The lifestyle of intracellular bacterial pathogens is generally divided into the following steps: contact and entry into the host cell, residence within a vacuole, escape into the cytosol or establishment of a membrane encased niche, and cell-to-cell spreading [1]
Using large volume correlative light electron microscopy (CLEM) and dynamic microscopy we studied discrete and highly transient steps of S. flexneri early invasion in detail
We provide the first 3D high resolution view of the S. flexneri invasion site and of vacuolar rupture itself
Summary
The lifestyle of intracellular bacterial pathogens is generally divided into the following steps: contact and entry into the host cell, residence within a vacuole, escape into the cytosol or establishment of a membrane encased niche, and cell-to-cell spreading [1]. Effectors released upon cell contact induce major rearrangements of the host cell cytoskeleton, mainly polymerization of actin filaments to form bundles supporting membrane projections termed “ruffles” [8],[9],[10],[11] This leads to the formation of large membrane protrusions, which form a pocket enclosing the bacteria and facilitating entry [12]. In the case of Salmonella enterica, an invasive, T3SS-employing pathogen which shares many common aspects with S. flexneri entry into host cells, it was hypothesized that Salmonella containing vacuole and macropinosomes may be distinct, as they are sorted into different intracellular routes [21] It appears that the classic model for S. flexneri invasion may be too simplistic, and a revised model could include two parallel processes: (i) bacterial entry and (ii) membrane ruffling, whose precise biological role in invasion has not been studied in detail
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