3D correlative electron microscopy reveals continuity of Brucella-containing vacuoles with the endoplasmic reticulum

Entry of the facultative intracellular pathogen Brucella into host cells results in the formation of endosomal Brucella-containing vacuoles (eBCVs) that initially traffic along the endocytic pathway. eBCV acidification triggers the expression of a type IV secretion system that translocates bacterial effector proteins into host cells. This interferes with lysosomal fusion of eBCVs and supports their maturation to replicative Brucella-containing vacuoles (rBCVs). Bacteria replicate in rBCVs to large numbers, eventually occupying most of the cytoplasmic volume. As rBCV membranes tightly wrap each individual bacterium, they are constantly being expanded and remodeled during exponential bacterial growth. rBCVs are known to carry endoplasmic reticulum (ER) markers; however, the relationship of the vacuole to the genuine ER has remained elusive. Here, we have reconstructed the 3-dimensional ultrastructure of rBCVs and associated ER by correlative structured illumination microscopy (SIM) and focused ion beam/scanning electron microscopic tomography (FIB/SEM). Studying B. abortus-infected HeLa cells and trophoblasts derived from B. melitensis-infected mice, we demonstrate that rBCVs are complex and interconnected compartments that are continuous with neighboring ER cisternae, thus supporting a model that rBCVs are extensions of genuine ER.

Bacterial cell cycle dynamics: size regulation during exponential growth and role of polyphosphate during starvation response

Aster Louis K Vanhecke

Bacteria are the most diverse and abundant kingdom of life and have adapted to survive and thrive in habitats around the globe. When provided with ample nutrients they grow and divide at staggering rates, increasing their population exponentially. Upon nutrient depletion on the other hand, they quickly adapt by drastically altering their metabolism, halting growth to survive for a very long time. Since bacteria are tiny -about a few micrometers-, visualizing these processes requires microscopy. To measure the dynamics of their shape and inner structures precisely, one needs to choose a technique that balances spatial resolution, temporal resolution and photo-toxicity. In this thesis, I present two projects using advanced dynamic microscopy, first to study cell size regulation during exponential growth in the abundance of nutrients and then to elucidate the role and positioning of polyphosphate granules during cell cycle exit in response to nutrient starvation. During exponential growth, bacteria balance growth and division to regulate their size, resulting in a narrow size distribution, referred to as cell size homeostasis. Recent work tried to uncover what cells sense to decide to divide in order to achieve size homeostasis: time, size, growth or a combination of those. Control of cell division is often equated to control of constriction onset; however, the constriction period still accounts for up to 40% of cell growth and could thus contribute significantly to cell size regulation. We used SIM microscopy to measure constriction kinetics and their impact on cell size regulation in Caulobacter crescentus. We found that constriction rate regulation can determine cell size. Moreover, constriction rate modulation compensates for variability in elongation before constriction, allowing a higher fidelity cell size homeostasis. We suggest a parsimonious model where excess cell wall precursors accumulate proportionally to elongation before constriction and set the rate of constriction. This is the first direct demonstration that constriction rate can contribute to cell size control and homeostasis in bacteria. Upon nutrient starvation, bacteria exit their cell cycle to preserve energy and nutrients. In many bacteria, such as Pseudomonas aeruginosa, this is associated with the accumulation of polyphosphate (polyP) in intracellular granules. PolyP is created by polyphosphate kinases (ppkâs), which are required for successful cell cycle exit and survival of and recovery from long-term starvation. Interestingly, these polyP granules are regularly spaced within the nucleoid. To date, it is not known during which stage polyP is required for cell cycle exit, and what causes the spacing of the granules. Here, we use fluorescence microscopy to probe the cell cycle stage of Îppk cells arrested during nutrient starvation as well as the localization and dynamics of ppkâs. We show that a majority of Îppk cells are arrested with open replication forks. Furthermore, we find that ppkâs localize in distinct patterns, already prior to starvation and polyP granule production, which could be responsible for the positioning of polyP granules. To this end, we developed a background subtraction algorithm to remove cytoplasmic fluorescence, improving accuracy of spot detection and localization.

EPFL2019

Intestinal organoid cocultures with microbes

Devanjali Dutta

This protocol comprises various methods to coculture organoids (particularly human small intestinal and colon organoids) with microbes, including microinjection into the lumen and periphery of 3D organoids and exposure of organoids to microbes in a 2D layer. Adult-stem-cell-derived organoids model human epithelial tissues ex vivo, which enables the study of host-microbe interactions with great experimental control. This protocol comprises methods to coculture organoids with microbes, particularly focusing on human small intestinal and colon organoids exposed to individual bacterial species. Microinjection into the lumen and periphery of 3D organoids is discussed, as well as exposure of organoids to microbes in a 2D layer. We provide detailed protocols for characterizing the coculture with regard to bacterial and organoid cell viability and growth kinetics. Spatial relationships can be studied by fluorescence live microscopy, as well as scanning electron microscopy. Finally, we discuss considerations for assessing the impact of bacteria on gene expression and mutations through RNA and DNA sequencing. This protocol requires equipment for standard mammalian tissue culture, or bacterial or viral culture, as well as a microinjection device.

NATURE PORTFOLIO2021

Bacterial cell cycle dynamics: size regulation during exponential growth and role of polyphosphate during starvation response

Aster Louis K Vanhecke

EPFL2019