Sec22b regulates phagosomal maturation and antigen crosspresentation by dendritic cells.

Antigen (Ag) crosspresentation by dendritic cells (DCs) involves the presentation of internalized Ags on MHC class I molecules to initiate CD8+ T cell-mediated immunity in response to certain pathogens and tumor cells. Here, we identify the SNARE Sec22b as a specific regulator of Ag crosspresentation. Sec22b localizes to the ER-Golgi intermediate compartment (ERGIC) and pairs to the plasma membrane SNARE syntaxin 4, which is present in phagosomes (Phgs). Depletion of Sec22b inhibits the recruitment of ER-resident proteins to Phgs and to the vacuole containing the Toxoplasma gondii parasite. In Sec22b-deficient DCs, crosspresentation is compromised after Ag phagocytosis or endocytosis and after invasion by T. gondii. Sec22b silencing inhibited Ag export to the cytosol and increased phagosomal degradation by accelerating lysosomal recruitment. Our findings provide insight into an intracellular traffic pathway required for crosspresentation and show that Sec22b-dependent recruitment of ER proteins to Phgs critically influences phagosomal functions in DCs.

Caught in the act: In situ visualization of bacterial secretion systems by cryo-electron tomography.

Bacterial secretion systems, such as the type 3, 4, and 6 are multiprotein nanomachines expressed at the surface of pathogens with Gram-negative like envelopes. They are known to be crucial for virulence and to translocate bacteria-encoded effector proteins into host cells to manipulate cellular functions. This facilitates either pathogen attachment or invasion of the targeted cell. Effector proteins also promote evasion of host immune recognition. Imaging by cryo-electron microscopy in combination with structure determination has become a powerful approach to understand how these nanomachines work. Still, questions on their assembly, the precise secretion mechanisms, and their direct involvement in pathogenicity remain unsolved. Here, we present an overview of the recent developments in in situ cryo-electron microscopy. We discuss its potential for the investigation of the role of bacterial secretion systems during the host-bacterial crosstalk at the molecular level. These in situ studies open new perspectives for our understanding of secretion system structure and function.

Salmonella enters a dormant state within human epithelial cells for persistent infection.

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. 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 distinct S. Typhimurium lifestyles remains lacking. Here we developed a novel fluorescent reporter, Salmonella INtracellular Analyzer (SINA), compatible for fluorescence microscopy and flow cytometry in single-bacterium level quantification. This 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 subpopulation entered a dormant state in a vesicular compartment distinct from the conventional Salmonella-containing vacuoles (SCV) as well as the previously reported niche of dormant S. Typhimurium in macrophages. The dormant S. Typhimurium inside enterocytes were viable and expressed Salmonella Pathogenicity Island 2 (SPI-2) virulence factors at later time points. We found that the formation of these dormant S. 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 dormant S. Typhimurium represents an important pathogen niche and provides an alternative strategy for S. Typhimurium pathogenicity and its persistence.

A Role for Taok2 in Listeria monocytogenes Vacuolar Escape.

The bacterial pathogen Listeria monocytogenes invades host cells, ruptures the internalization vacuole, and reaches the cytosol for replication. A high-content small interfering RNA (siRNA) microscopy screen allowed us to identify epithelial cell factors involved in L. monocytogenes vacuolar rupture, including the serine/threonine kinase Taok2. Kinase activity inhibition using a specific drug validated a role for Taok2 in favoring L. monocytogenes cytoplasmic access. Furthermore, we showed that Taok2 recruitment to L. monocytogenes vacuoles requires the presence of pore-forming toxin listeriolysin O. Overall, our study identified the first set of host factors modulating L. monocytogenes vacuolar rupture and cytoplasmic access in epithelial cells.

Transcytosis subversion by M cell-to-enterocyte spread promotes Shigella flexneri and Listeria monocytogenes intracellular bacterial dissemination.

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.

Shigella hijacks the exocyst to cluster macropinosomes for efficient vacuolar escape.

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.

New methods to decrypt emerging macropinosome functions during the host-pathogen crosstalk.

Large volumes of liquid and other materials from the extracellular environment are internalised by eukaryotic cells via an endocytic process called macropinocytosis. It is now recognised that this fundamental and evolutionarily conserved pathway is hijacked by numerous intracellular pathogens as an 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 internalisation. It emerges that the identity of macropinosomes can vary hugely and change rapidly during their lifetime. A deeper understanding of this important multi-faceted 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 characterisation 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.

SopB- and SifA-dependent shaping of the Salmonella-containing vacuole proteome in the social amoeba Dictyostelium discoideum.

The ability of Salmonella to survive and replicate within mammalian host cells involves the generation of a membranous compartment known as the Salmonella-containing vacuole (SCV). Salmonella employs a number of effector proteins that are injected into host cells for SCV formation using its type-3 secretion systems encoded in SPI-1 and SPI-2 (T3SS-1 and T3SS-2, respectively). Recently, we reported that S. Typhimurium requires T3SS-1 and T3SS-2 to survive in the model amoeba Dictyostelium discoideum. Despite these findings, the involved effector proteins have not been identified yet. Therefore, we evaluated the role of two major S. Typhimurium effectors SopB and SifA during D. discoideum intracellular niche formation. First, we established that S. Typhimurium resides in a vacuolar compartment within D. discoideum. Next, we isolated SCVs from amoebae infected with wild type or the ΔsopB and ΔsifA mutant strains of S. Typhimurium, and we characterised the composition of this compartment by quantitative proteomics. This comparative analysis suggests that S. Typhimurium requires SopB and SifA to modify the SCV proteome in order to generate a suitable intracellular niche in D. discoideum. Accordingly, we observed that SopB and SifA are needed for intracellular survival of S. Typhimurium in this organism. Thus, our results provide insight into the mechanisms employed by Salmonella to survive intracellularly in phagocytic amoebae.

The Shigella type III effector IpgD recodes Ca2+ signals during invasion of epithelial cells.

The role of second messengers in the diversion of cellular processes by pathogens remains poorly studied despite their importance. Among these, Ca2+ virtually regulates all known cell processes, including cytoskeletal reorganization, inflammation, or cell death pathways. Under physiological conditions, cytosolic Ca2+ increases are transient and oscillatory, defining the so-called Ca2+ code that links cell responses to specific Ca2+ oscillatory patterns. During cell invasion, Shigella induces atypical local and global Ca2+ signals. Here, we show that by hydrolyzing phosphatidylinositol-(4,5)bisphosphate, the Shigella type III effector IpgD dampens inositol-(1,4,5)trisphosphate (InsP3) levels. By modifying InsP3 dynamics and diffusion, IpgD favors the elicitation of long-lasting local Ca2+ signals at Shigella invasion sites and converts Shigella-induced global oscillatory responses into erratic responses with atypical dynamics and amplitude. Furthermore, IpgD eventually inhibits InsP3-dependent responses during prolonged infection kinetics. IpgD thus acts as a pathogen regulator of the Ca2+ code implicated in a versatility of cell functions. Consistent with this function, IpgD prevents the Ca2+-dependent activation of calpain, thereby preserving the integrity of cell adhesion structures during the early stages of infection.

The actin comet guides the way: How Listeria actin subversion has impacted cell biology, infection biology and structural biology.

The discovery of the role of ActA to polymerise actin at one pole of Listeria monocytogenes represents a key event in the field of cellular microbiology. It uncovered much more than the molecular principle behind actin-based motility of Listeria within the cytosol of infected cells, and it changed the way how actin dynamics could be studied and eventually understood. The ActA discovery took place at a time when cell biology, biochemistry and microbiology came together in a very fruitful fashion. Here, we provide an overview of the science that took place around this event. Then, we outline the wide array of research fields that have been impacted by this finding. This ranges from structural and biophysical investigations on actin and its dynamics, the role of actin polymerisation during infection with different pathogens, to actin-dynamics during various pathologies. Like a comet in the sky, Pascale Cossart's work on ActA has inspired and will inspire generations of (life) scientists.