Yuen-Yan Chang

Macropinocytosis is a non-selective endocytic process by which cells take up large amounts of extracellular fluid into giant vesicles known as macropinosomes. This mechanism is used by immune cells to sample the surroundings for antigens and can be exploited by cancer cells for nutrient uptake. What determines macropinosome fate after internalization is largely unknown. Here, we investigate the role of the phosphatidylinositol 3-kinase VPS34 (also known as PIK3C3) and its product phosphatidylinositol 3-phosphate (PtdIns3P) in macropinosome fate determination. VPS34 inhibition reduced successful macropinosome maturation and fluid-phase uptake and prevented the recruitment of early endosomal factors, including the small GTPase RAB5A and its effectors, to forming macropinosomes. Instead, forming macropinosomes under VPS34 inhibition accumulated regulators of endocytic recycling, including RAB8A, RAB10, RAB11A and PtdIns4P, which led to fusion of macropinosomes with the plasma membrane. Whereas RAB5A was necessary for establishment of endosomal identity, macropinosome fusion with the plasma membrane depended on RAB8A. Thus, macropinosome maturation is regulated by a VPS34-dependent phosphoinositide and RAB transition that determines whether newly formed macropinosomes acquire endosomal identity or recycle to the plasma membrane.

Flagella propel pathogens through their environments, yet are expensive to synthesize and are immunogenic. Thus, complex hierarchical regulatory networks control flagellar gene expression. Spirochetes are highly motile bacteria, but peculiarly, the archetypal flagellar regulator σ²⁸ is absent in the Lyme spirochete Borrelia burgdorferi. Here, we show that gene bb0268 (flgV) in B. burgdorferi, previously and incorrectly annotated to encode the RNA-binding protein Hfq, is instead a structural flagellar component that modulates flagellar assembly. The flgV gene is broadly conserved in the flagellar superoperon alongside σ²⁸ in many Spirochaetae, Firmicutes and other phyla, with distant homologs in Epsilonproteobacteria. We find that B. burgdorferi FlgV is localized within flagellar basal bodies, and strains lacking flgV produce fewer and shorter flagellar filaments and are defective in cell division and motility. During the enzootic cycle, flgV-deficient B. burgdorferi survive and replicate in Ixodes ticks but are attenuated for infection and dissemination in mice. Our work defines infection timepoints when spirochete motility is most crucial and implicates FlgV as a broadly distributed structural flagellar component that modulates flagellar assembly.

Intravacuolar pathogens need to gradually expand their surrounding vacuole to accommodate the growing number of bacterial offspring during intracellular replication. Here we found that Legionella pneumophila controls vacuole expansion by fine-tuning the generation of lysophospholipids within the vacuolar membrane. Upon allosteric activation by binding to host ubiquitin, the type IVB (Dot/Icm) effector VpdC converts phospholipids into lysophospholipids which, at moderate concentrations, are known to promote membrane fusion but block it at elevated levels by generating excessive positive membrane curvature. Consequently, L. pneumophila overproducing VpdC were prevented from adequately expanding their surrounding membrane, trapping the replicating bacteria within spatially confined vacuoles and reducing their capability to proliferate intracellularly. Quantitative lipidomics confirmed a VpdC-dependent increase in several types of lysophospholipids during infection, and VpdC production in transiently transfected cells caused tubulation of organelle membranes as well as mitochondria fragmentation, processes that can be phenocopied by supplying cells with exogenous lysophospholipids. Together, these results demonstrate an important role for bacterial phospholipases in vacuolar expansion.

Macropinocytosis refers to the nonselective uptake of extracellular molecules into many different types of eukaryotic cells within large fluid-filled vesicles named macropinosomes. Macropinosomes are relevant for a wide variety of cellular processes, such as antigen sampling in immune cells, homeostasis in the kidney, cell migration or pathogen uptake. Understanding the molecular composition of the different macropinosomes formed during these processes has helped to differentiate their regulations from other endocytic events. Here, we present a magnetic purification protocol that segregates scarce macropinosomes from other endocytic vesicles at a high purity and in a low-cost and unbiased manner. Our protocol takes advantage of moderate-sized magnetic beads of 100 nm in diameter coupled to mass-spectrometry-based proteomic analysis. Passing the cell lysate through a table-top magnet allows the quick retention of the bead-containing macropinosomes. Unlike other cell-fractionation-based methodologies, our protocol minimizes sample loss and production cost without prerequisite knowledge of the macropinosomes and with minimal laboratory experience. We describe a detailed procedure for the isolation of infection-associated macropinosomes during bacterial invasion and the optimization steps to readily adapt it to various studies. The protocol can be performed in 3 d to provide highly purified and enriched macropinosomes for qualitative proteomic composition analysis.

Large volumes of liquid and other material from the extracellular environment are internalized by eukaryotic cells via an endocytic process called macropinocytosis. It is now recognized that this fundamental and evolutionary conserved pathway is hijacked by numerous intracellular pathogens as entry portal to the host cell interior. Yet, an increasing number of additional cellular functions of macropinosomes in pathologic processes have been reported beyond this role for fluid internalization. It emerges that the identity of macropinosomes can vary hugely and change rapidly during their lifetime. A deeper understanding of this important multifaceted compartment is based on novel methods for their investigation. These methods are either imaging‐based for the tracking of macropinosome dynamics, or they provide the means to extract macropinosomes at high purity for comprehensive proteomic analyses. Here, we portray these new approaches for the investigation of macropinosomes. We document how these method developments have provided insights for a new understanding of the intracellular lifestyle of the bacterial pathogens Shigella and Salmonella. We suggest that a systematic complete characterization of macropinosome subversion with these approaches during other infection processes and pathologies will be highly beneficial for our understanding of the underlying cellular and molecular processes.

SalmonellaTyphimurium (S. Typhimurium) is an enteric bacterium capable of invading a wide range of hosts, including rodents and humans. It targets different host cell types showing different intracellular lifestyles.S. Typhimurium colonizes different intracellular niches and is able to either actively divide at various rates or remain dormant to persist. A comprehensive tool to determine these distinctS. Typhimurium lifestyles remains lacking. Here we developed a novel fluorescent reporter,SalmonellaINtracellular Analyzer (SINA), compatible for fluorescence microscopy and flow cytometry in single-bacterium level quantification. This identified aS. Typhimurium subpopulation in infected epithelial cells that exhibits a unique phenotype in comparison to the previously documented vacuolar or cytosolicS. Typhimurium. This subpopulation entered a dormant state in a vesicular compartment distinct from the conventionalSalmonella-containing vacuoles (SCV) as well as the previously reported niche of dormantS. Typhimurium in macrophages. The dormantS. Typhimurium inside enterocytes were viable and expressedSalmonellaPathogenicity Island 2 (SPI-2) virulence factors at later time points. We found that the formation of these dormantS. Typhimurium is not triggered by the loss of SPI-2 effector secretion but it is regulated by (p)ppGpp-mediated stringent response through RelA and SpoT. We predict that intraepithelial dormantS. Typhimurium represents an important pathogen niche and provides an alternative strategy forS. Typhimurium pathogenicity and its persistence.

Salmonella Typhimurium ( S. Typhimurium) is an enteric bacterium capable of invading a wide range of hosts, including rodents and humans. It targets different host cell types showing different intracellular lifestyles. Within the infected cells S. Typhimurium colonizes multiple intracellular niches, and it is able to either actively divide at various rates, or remain dormant to persist. A comprehensive tool to monitor these distinct S. Typhimurium lifestyles has not been available so far. Here we developed a novel fluorescent reporter, Salmonella Intracellular Analyzer (SINA), compatible for fluorescence microscopy and flow cytometry for quantification at the single-bacterium level. Using SINA, we identified a S. Typhimurium subpopulation in infected epithelial cells that exhibits a unique phenotype in comparison to the previously documented vacuolar or cytosolic S. Typhimurium. This newly identified subpopulation remained dormant within a vesicular compartment distinct from either conventional Salmonella -containing vacuoles (SCV) or the previously reported niche of dormant S. Typhimurium inside macrophages. The dormant S. Typhimurium inside enterocytes were viable and expressed Salmonella Pathogenicity Island 2 (SPI-2) virulence factors at later infection time points. We found that the formation of these dormant S. Typhimurium is not triggered by the loss of SPI-2 expression but it is regulated by (p)ppGpp-mediated stringent response through RelA and SpoT. We predict that intraepithelial dormant S. Typhimurium represents an important pathogen niche as it provides an alternative strategy for S. Typhimurium pathogenicity and persistence. Author Summary: Salmonella Typhimurium is a clinically relevant bacterial pathogen that causes Salmonellosis. It can actively or passively invade various host cell types and reside in a Salmonella -containing vacuole (SCV) within host cells. The SCV can be remodeled into a replicative niche with the aid of Salmonella Type III Secretion System 2 (T3SS2) effectors or else, the SCV is ruptured for the access of the nutrient-rich host cytosol. Depending on the infected host cell type, S. Typhimurium undertake different lifestyles that are distinct by their subcellular localization, replication rate and metabolic rate. We present here a novel fluorescent reporter system that rapidly detects S. Typhimurium lifestyles using fluorescence microscopy and flow cytometry. We identified a dormant S. Typhimurium population within enterocytes that displays capacities in host cell persistence, dormancy exit and antibiotic tolerance. We found that the molecular pathway suppressing S. Typhimurium dormancy in enterocytes is the one that has been shown to promote dormancy in macrophages. This suggests a divergent physiological consequence regulated by the same set of S. Typhimurium molecular mediators depending on the challenged host cell type. Altogether, our work demonstrates the potential of fluorescence reporters in facile bacterial characterization, and revealed a dormant S. Typhimurium population in human enterocytes that is distinct from those observed in macrophages and fibroblasts.

Shigella flexneri invades host cells by entering within a bacteria-containing vacuole (BCV). In order to establish its niche in the host cytosol, the bacterium ruptures its BCV. Contacts between S. flexneri BCV and infection-associated macropinosomes (IAMs) formed in situ have been reported to enhance BCV disintegration. The mechanism underlying S. flexneri vacuolar escape remains however obscure. To decipher the molecular mechanism priming the communication between the IAMs and S. flexneri BCV, we performed mass spectrometry-based analysis of the magnetically purified IAMs from S. flexneri-infected cells. While proteins involved in host recycling and exocytic pathways were significantly enriched at the IAMs, we demonstrate more precisely that the S. flexneri type III effector protein IpgD mediates the recruitment of the exocyst to the IAMs through the Rab8/Rab11 pathway. This recruitment results in IAM clustering around S. flexneri BCV. More importantly, we reveal that IAM clustering subsequently facilitates an IAM-mediated unwrapping of the ruptured vacuole membranes from S. flexneri, enabling the naked bacterium to be ready for intercellular spread via actin-based motility. Taken together, our work untangles the molecular cascade of S. flexneri-driven host trafficking subversion at IAMs to develop its cytosolic lifestyle, a crucial step en route for infection progression at cellular and tissue level. Shigella flexneri is a clinically relevant bacterial pathogen that causes bacillary dysentery. It invades the host cell by injecting a repository of bacterial effectors through its type III secretion system. Upon its entry into host cell, S. flexneri resides shortly in a bacteria-containing vacuole (BCV), which is rapidly ruptured for the cytosolic propagation and infection progression of the pathogen. Infection-associated macropinosomes (IAMs) formed in situ during S. flexneri entry are found to promote the efficient vacuolar escape of S. flexneri with an unclear mechanism. We present here the molecular players involving in the BCV-IAM interactions obtained by proteomic analysis of magnetically-purified IAMs. We decipher the successive steps of S. flexneri BCV escape, pinpointing the bacterial effector-mediated hijacking of host trafficking pathways to promote the BCV disintegration and displacement of BCV membranes from the bacteria. This study sheds light on the mechanism by which bacterial pathogens modulate BCV vacuolar integrity through interaction with infection-induced vesicle compartments for the establishment of their intracellular replicative niches and pathogenicities.

Microfold (M) cell host-pathogen interaction studies would benefit from the visual analysis of dynamic cellular and microbial interplays. We adapted a human in vitro M cell model to physiological bacterial infections, expression of fluorescent localization reporters and long-term three-dimensional time-lapse microscopy. This approach allows following key steps of M cell infection dynamics at subcellular resolution, from the apical onset to basolateral epithelial dissemination. We focused on the intracellular pathogen Shigella flexneri, classically reported to transcytose through M cells to initiate bacillary dysentery in humans, while eliciting poorly protective immune responses. Our workflow was critical to reveal that S. flexneri develops a bimodal lifestyle within M cells leading to rapid transcytosis or delayed vacuolar rupture, followed by direct actin motility-based propagation to neighboring enterocytes. Moreover, we show that Listeria monocytogenes, another intracellular pathogen sharing a tropism for M cells, disseminates in a similar manner and evades M cell transcytosis completely. We established that actin-based M cell-to-enterocyte spread is the major dissemination pathway for both pathogens and avoids their exposure to basolateral compartments in our system. Our results challenge the notion that intracellular pathogens are readily transcytosed by M cells to inductive immune compartments in vivo, providing a potential mechanism for their ability to evade adaptive immunity. Microfold (M) epithelial cells are important for the onset of infections and induction of immune responses in many mucosal diseases. We extended a human in vitro M cell model to apical infections, expression of fluorescent host and microbial reporters and real-time fluorescence microscopy. Focusing on the human intracellular pathogen S. flexneri, responsible for bacillary dysentery, this workflow allowed us to uncover that the bacterium can subvert the immunological sampling function of M cells by promoting a cytosolic lifestyle and spreading directly to neighboring enterocytes. This mechanism was shared with the etiologic agent of listeriosis, the intracellular pathogen L. monocytogenes and allowed both pathogens to avoid exposure to underlying immune compartments. These results may provide a mechanism for the ability of intracellular pathogens to evade adaptive immunity in vivo, emphasizing the importance of advanced studies of M cell host-pathogen interactions to understand early steps of mucosal invasion and their consequences on immunity.