RFP-based method for real-time tracking of invasive bacteria in a heterogeneous population of cells.

Quantification of bacterial invasion into eukaryotic cells is a prerequisite to unfold the molecular mechanisms of this vector's function to obtain insights for improving its efficiency. Invasion is traditionally quantified by antibiotic protection assays that require dilution plating and counting of colony-forming units rescued from infected cells. However, to differentiate between attached and internalized bacteria vector, this assay requires supplementation by a time-consuming and tedious immunofluorescence staining, making it laborious and reduces its reliability and reproducibility. Here we describe a new red fluorescent protein (RFP)-based high-throughput and inexpensive method for tracking bacterial adherence and internalization through flow cytometry to provide a convenient and real-time quantification of bacterial invasiveness in a heterogeneous population of cells. We invaded MCF-7, A549, and HEK-293 cells with the E. coli vector and measured RFP using imaging flow cytometry. We found high cellular infection of up to 70.47% in MCF-7 compared to 27.4% and 26.2% in A549 and HEK-293 cells, respectively. The quantitative evaluation of internalized E. coli is rapid and cell-dependent, and it distinctively differentiates between attached and cytosolic bacteria while showing the degree of cellular invasiveness. This imaging flow cytometry approach can be applied broadly to study host-bacteria interaction.

Extracellular RNAs in Bacterial Infections: From Emerging Key Players on Host-Pathogen Interactions to Exploitable Biomarkers and Therapeutic Targets.

Non-coding RNAs (ncRNAs) are key regulators of post-transcriptional gene expression in prokaryotic and eukaryotic organisms. These molecules can interact with mRNAs or proteins, affecting a variety of cellular functions. Emerging evidence shows that intra/inter-species and trans-kingdom regulation can also be achieved with exogenous RNAs, which are exported to the extracellular medium, mainly through vesicles. In bacteria, membrane vesicles (MVs) seem to be the more common way of extracellular communication. In several bacterial pathogens, MVs have been described as a delivery system of ncRNAs that upon entry into the host cell, regulate their immune response. The aim of the present work is to review this recently described mode of host-pathogen communication and to foster further research on this topic envisaging their exploitation in the design of novel therapeutic and diagnostic strategies to fight bacterial infections.

Chemiosmosis, Evolutionary Conflict, and Eukaryotic Symbiosis.

Mutualistic symbiosis, in which individuals of different species cooperate and both benefit, has long been an evolutionary puzzle. Why should individuals of two different species cooperate? In this case, as in all others, cooperation is not automatic, but rather requires the mediation of evolutionary conflicts. In chemiosmosis, redox reactions produce a trans-membrane "proton-motive force" that powers energy-requiring reactions in most organisms. Chemiosmosis may also have a role in conflict mediation. Chemiosmosis rapidly produces considerable amounts of products, increasing the risk of end-product inhibition and the formation of dangerous by-products, such as reactive oxygen species. While several mechanisms can modulate chemiosmosis, potential negative effects can also be ameliorated by simply dispersing excess product into the environment. This "free lunch you are forced to make" can attract individuals of other species leading to groups, in which other organisms share the products that are released into the environment by the chemiosmotic cell or organism. Since the time of Darwin, evolutionary biology has recognized that groups are the key to the evolution of cooperation. With many small groups, chance associations of cooperators can arise, even if cooperation is selected against at the individual level. Groups of cooperators can then outcompete groups of defectors, which do not cooperate. Indeed, numerous symbioses may have arisen in this way, perhaps most notably the symbioses of host cells and chemiosmotic bacteria that gave rise to the eukaryotic cell. Other examples in which one partner relies on chemiosmotic products supplied by the other include lichens, corals or other metazoans and dinoflagellates, sap-feeding insects, and plant-rhizobia and plant-mycorrhiza interactions. More problematic are cases of gut microbiomes-for instance, those of termites, ruminants, and even human beings. Under some but not all circumstances, chemiosmosis can be co-opted into punishing defectors and enforcing cooperation, thus leading to mutualistic symbioses.

Chemosynthetic symbioses.

Symbioses between chemosynthetic bacteria and eukaryotic hosts can be found almost everywhere in the ocean, from shallow-water seagrass beds and coral reef sediments to the deep sea. Yet no one knew these existed until 45 years ago, when teeming communities of animals were found thriving at hydrothermal vents two and a half kilometers below the sea surface. The discovery of these lightless ecosystems revolutionized our understanding of the energy sources that fuel life on Earth. Animals thrive at vents because they live in a nutritional symbiosis with chemosynthetic bacteria that grow on chemical compounds gushing out of the vents, such as sulfide and methane, which animals cannot use on their own. The symbionts gain energy from the oxidation of these reduced substrates to fix CO2 and other simple carbon compounds into biomass, which is then transferred to the host. By associating with chemosynthetic bacteria, animals and protists can thrive in environments in which there is not enough organic carbon to support their nutrition, including oligotrophic habitats like coral reefs and seagrass meadows. Chemosymbioses have evolved repeatedly and independently in multiple lineages of marine invertebrates and bacteria, highlighting the strong selective advantage for both hosts and symbionts in forming these associations. Here, we provide a brief overview of chemosynthesis and how these symbioses function. We highlight some of the current research in this field and outline several promising avenues for future research.

Stoking the Fire: How Dying Cells Propagate Inflammatory Signalling through Extracellular Vesicle Trafficking.

Communication between dying cells and their environment is a critical process that promotes tissue homeostasis during normal cellular turnover, whilst during disease settings, it can contribute to inflammation through the release of intracellular factors. Extracellular vesicles (EVs) are a heterogeneous class of membrane-bound cell-derived structures that can engage in intercellular communication via the trafficking of bioactive molecules between cells and tissues. In addition to the well-described functions of EVs derived from living cells, the ability of dying cells to release EVs capable of mediating functions on target cells or tissues is also of significant interest. In particular, during inflammatory settings such as acute tissue injury, infection and autoimmunity, the EV-mediated transfer of proinflammatory cargo from dying cells is an important process that can elicit profound proinflammatory effects in recipient cells and tissues. Furthermore, the biogenesis of EVs via unique cell-death-associated pathways has also been recently described, highlighting an emerging niche in EV biology. This review outlines the mechanisms and functions of dying-cell-derived EVs and their ability to drive inflammation during various modes of cell death, whilst reflecting on the challenges and knowledge gaps in investigating this subgenre of extracellular vesicles research.

The Serratia grimesii outer membrane vesicles-associated grimelysin triggers bacterial invasion of eukaryotic cells.

Serratia grimesii are facultative pathogenic bacteria that can penetrate a wide range of host cells and cause infection, especially in immunocompromised patients. Previously, we have found that bacterial metalloprotease grimelysin is a potential virulence determinant of S. grimesii invasion (E. S. Bozhokina et al., (2011). Cell Biology International, 35(2), 111-118). Protease is characterized as an actin-hydrolyzing enzyme with a narrow specificity toward other cell proteins. It is not known, however, whether grimelysin is transported into eukaryotic cells. Here, we show, for the first time, that S. grimesii can generate outer membrane vesicles (OMVs) displayed specific proteolytic activity against actin, characteristic of grimelysin. The presence of grimelysin was also confirmed by the Western blot analysis of S. grimesii OMVs lysate. Furthermore, confocal microscopy analysis revealed that the S. grimesii grimelysin-containing OMVs attached to the host cell membrane. Finally, pretreatment of HeLa cells with S. grimesii OMVs before the cells were infected with bacteria increased the bacterial penetration several times. These data strongly suggest that protease grimelysin promotes S. grimesii internalization by modifying bacterial and/or host molecule(s) when it is delivered as a component of OMVs.

Cell Monolayer Translocation Assay.

An essential property associated with leptospiral virulence is the pathogen's ability to translocate across host cells, enabling Leptospira to evade the host immune response, disseminate, and establish infection. Cell monolayer translocation assay allows for the quantification of Leptospira strain's competence to cross cell barriers while measuring the integrity of the polarized eukaryotic cell monolayer during this process.

LITESEC-T3SS - Light-controlled protein delivery into eukaryotic cells with high spatial and temporal resolution.

Many bacteria employ a type III secretion system (T3SS) injectisome to translocate proteins into eukaryotic host cells. Although the T3SS can efficiently export heterologous cargo proteins, a lack of target cell specificity currently limits its application in biotechnology and healthcare. In this study, we exploit the dynamic nature of the T3SS to govern its activity. Using optogenetic interaction switches to control the availability of the dynamic cytosolic T3SS component SctQ, T3SS-dependent effector secretion can be regulated by light. The resulting system, LITESEC-T3SS (Light-induced translocation of effectors through sequestration of endogenous components of the T3SS), allows rapid, specific, and reversible activation or deactivation of the T3SS upon illumination. We demonstrate the light-regulated translocation of heterologous reporter proteins, and induction of apoptosis in cultured eukaryotic cells. LITESEC-T3SS constitutes a new method to control protein secretion and translocation into eukaryotic host cells with unparalleled spatial and temporal resolution.

Building peptidoglycan inside eukaryotic cells: A view from symbiotic and pathogenic bacteria.

The peptidoglycan (PG), as the exoskeleton of most prokaryotes, maintains a defined shape and ensures cell integrity against the high internal turgor pressure. These important roles have attracted researchers to target PG metabolism in order to control bacterial infections. Most studies, however, have been performed in bacteria grown under laboratory conditions, leading to only a partial view on how the PG is synthetized in natural environments. As a case in point, PG metabolism and its regulation remain poorly understood in symbiotic and pathogenic bacteria living inside eukaryotic cells. This review focuses on the PG metabolism of intracellular bacteria, emphasizing the necessity of more in vivo studies involving the analysis of enzymes produced in the intracellular niche and the isolation of PG from bacteria residing within eukaryotic cells. The review also points to persistent infections caused by some intracellular bacterial pathogens and the extent at which the PG could contribute to establish such physiological state. Based on recent evidences, I speculate on the idea that certain structural features of the PG may facilitate attenuation of intracellular growth. Lastly, I discuss recent findings in endosymbionts supporting a cooperation between host and bacterial enzymes to assemble a mature PG.

Cellular microbiology: Bacterial toxin interference drives understanding of eukaryotic cell function.

Intimate interactions between the armament of pathogens and their host dictate tissue and host susceptibility to infection also forging specific pathophysiological outcomes. Studying these interactions at the molecular level has provided an invaluable source of knowledge on cellular processes, as ambitioned by the Cellular Microbiology discipline when it emerged in early 90s. Bacterial toxins act on key cell regulators or membranes to produce major diseases and therefore constitute a remarkable toolbox for dissecting basic biological processes. Here, we review selected examples of recent studies on bacterial toxins illustrating how fruitful the discipline of cellular microbiology is in shaping our understanding of eukaryote processes. This ever-renewing discipline unveils new virulence factor biochemical activities shared by eukaryotic enzymes and hidden rules of cell proteome homeostasis, a particularly promising field to interrogate the impact of proteostasis breaching in late onset human diseases. It is integrating new concepts from the physics of soft matter to capture biomechanical determinants forging cells and tissues architecture. The success of this discipline is also grounded by the development of therapeutic tools and new strategies to treat both infectious and noncommunicable human diseases.

Prokaryotic and eukaryotic microbiomes associated with blooms of the ichthyotoxic dinoflagellate Cochlodinium (Margalefidinium) polykrikoides in New York, USA, estuaries.

While harmful algal blooms caused by the ichthyotoxic dinoflagellate, Cochlodinium (Margalefidinium) polykrikoides, are allelopathic and may have unique associations with bacteria, a comprehensive assessment of the planktonic communities associated with these blooms has been lacking. Here, we used high-throughput amplicon sequencing to assess size fractionated (0.2 and 5 µm) bacterial (16S) and phytoplankton assemblages (18S) associated with blooms of C. polykrikoides during recurrent blooms in NY, USA. Over a three-year period, samples were collected inside ('patch') and outside ('non-patch') dense accumulations of C. polykrikoides to assess the microbiome associated with these blooms. Eukaryotic plankton communities of blooms had significantly lower diversity than non-bloom samples, and non-bloom samples hosted 30 eukaryotic operational taxonomic units (OTUs) not found within blooms, suggesting they may have been allelopathically excluded from blooms. Differential abundance analyses revealed that C. polykrikoides blooms were significantly enriched in dinoflagellates (p<0.001) and the experimental enrichment of C. polykrikoides led to a significant increase in the relative abundance of eight genera of dinoflagellates but a significant decline in other eukaryotic plankton. Amoebophrya co-dominated both within- and near- C. polykrikoides blooms and was more abundant in bloom patches. The core bacterial microbiome of the >0.2µm fraction of blooms was dominated by an uncultured bacterium from the SAR11 clade, while the >5µm size fraction was co-dominated by an uncultured bacterium from Rhodobacteraceae and Coraliomargarita. Two bacterial lineages within the >0.2µm fraction, as well as the Gammaproteobacterium, Halioglobus, from the >5µm fraction were unique to the microbiome of blooms, while there were 154 bacterial OTUs only found in non-bloom waters. Collectively, these findings reveal the unique composition and potential function of eukaryotic and prokaryotic communities associated with C. polykrikoides blooms.

The origin and evolution of cell-intrinsic antibacterial defenses in eukaryotes.

To survive in a world dominated by bacteria, eukaryotes have evolved numerous self-defense strategies. While some defenses are recent evolutionary innovations, others are ancient, with roots early in eukaryotic history. With a focus on antibacterial immunity, we highlight the evolution of pattern recognition receptors that detect bacteria, where diverse functional classes have been formed from the repeated use and reuse of a small set of protein domains. Next, we discuss core microbicidal strategies shared across eukaryotes, and how these systems may have been co-opted from ancient cellular mechanisms. We propose that studying antibacterial responses across diverse eukaryotes can reveal novel modes of defense, while highlighting the critical innovations that occurred early in the evolution of our own immune systems.

Cell-autonomous immunity by IFN-induced GBPs in animals and plants.

Inside host cells, guanylate binding proteins (GBPs) rapidly assemble into large antimicrobial defense complexes that combat a wide variety of bacterial pathogens. These massive nanomachines often completely coat targeted microbes where they act as recruitment platforms for downstream effectors capable of direct bactericidal activity. GBP-containing platforms also serve as sensory hubs to activate inflammasome-driven responses in the mammalian cytosol while in plants like Arabidopsis, GBP orthologues may facilitate intranuclear signaling for immunity against invasive phytopathogens. Together, this group of immune GTPases serve as a major defensive repertoire to protect the host cell interior from bacterial colonization across plant and animal kingdoms.

The Injectisome, a Complex Nanomachine for Protein Injection into Mammalian Cells.

Type III protein secretion systems (T3SSs), or injectisomes, are multiprotein nanomachines present in many Gram-negative bacteria that have a sustained long-standing close relationship with a eukaryotic host. These secretion systems have evolved to modulate host cellular functions through the activity of the effector proteins they deliver. To reach their destination, T3SS effectors must cross the multibarrier bacterial envelope and the eukaryotic cell membrane. Passage through the bacterial envelope is mediated by the needle complex, a central component of T3SSs that expands both the inner and outer membranes of Gram-negative bacteria. A set of T3SS secreted proteins, known as translocators, form a channel in the eukaryotic plasma membrane through which the effector proteins are delivered to reach the host cell cytosol. While the effector proteins are tailored to the specific lifestyle of the bacterium that encodes them, the injectisome is conserved among the different T3SSs. The central role of T3SSs in pathogenesis and their high degree of conservation make them a desirable target for the development of antimicrobial therapies against several important bacterial pathogens.

Machine-Learning-Based Predictor of Human-Bacteria Protein-Protein Interactions by Incorporating Comprehensive Host-Network Properties.

The large-scale identification of protein-protein interactions (PPIs) between humans and bacteria remains a crucial step in systematically understanding the underlying molecular mechanisms of bacterial infection. Computational prediction approaches are playing an increasingly important role in accelerating the identification of PPIs. Here, we developed a new machine-learning-based predictor of human- Yersinia pestis PPIs. First, three conventional sequence-based encoding schemes and two host network-property-related encoding schemes (i.e., NetTP and NetSS) were introduced. Motivated by previous human-pathogen PPI network analyses, we designed NetTP to systematically characterize the host proteins' network topology properties and designed NetSS to reflect the molecular mimicry strategy used by pathogen proteins. Subsequently, individual predictive models for each encoding scheme were inferred by Random Forest. Finally, through the noisy-OR algorithm, 5 individual models were integrated into a final powerful model with an AUC value of 0.922 in the 5-fold cross-validation. Stringent benchmark experiments further revealed that our model could achieve a better performance than two state-of-the-art human-bacteria PPI predictors. In addition to the selection of a suitable computational framework, the success of our proposed approach could be largely attributed to the introduction of two comprehensive host network-property-related feature sets. To facilitate the community, a web server implementing our proposed method has been made freely accessible at http://systbio.cau.edu.cn/intersppiv2/ or http://zzdlab.com/intersppiv2/ .

A Legionella pneumophila Kinase Phosphorylates the Hsp70 Chaperone Family to Inhibit Eukaryotic Protein Synthesis.

Legionella pneumophila (L.p.), the microbe responsible for Legionnaires' disease, secretes ∼300 bacterial proteins into the host cell cytosol. A subset of these proteins affects a wide range of post-translational modifications (PTMs) to disrupt host cellular pathways. L.p. has 5 conserved eukaryotic-like Ser/Thr effector kinases, LegK1-4 and LegK7, which are translocated during infection. Using a chemical genetic screen, we identified the Hsp70 chaperone family as a direct host target of LegK4. Phosphorylation of Hsp70s at T495 in the substrate-binding domain disrupted Hsp70's ATPase activity and greatly inhibited its protein folding capacity. Phosphorylation of cytosolic Hsp70 by LegK4 resulted in global translation inhibition and an increase in the amount of Hsp70 on highly translating polysomes. LegK4's ability to inhibit host translation via a single PTM uncovers a role for Hsp70 in protein synthesis and directly links it to the cellular translational machinery.

Taming the Triskelion: Bacterial Manipulation of Clathrin.

The entry of pathogens into nonphagocytic host cells has received much attention in the past three decades, revealing a vast array of strategies employed by bacteria and viruses. A method of internalization that has been extensively studied in the context of viral infections is the use of the clathrin-mediated pathway. More recently, a role for clathrin in the entry of some intracellular bacterial pathogens was discovered. Classically, clathrin-mediated endocytosis was thought to accommodate internalization only of particles smaller than 150 nm; however, this was challenged upon the discovery that Listeria monocytogenes requires clathrin to enter eukaryotic cells. Now, with discoveries that clathrin is required during other stages of some bacterial infections, another paradigm shift is occurring. There is a more diverse impact of clathrin during infection than previously thought. Much of the recent data describing clathrin utilization in processes such as bacterial attachment, cell-to-cell spread and intracellular growth may be due to newly discovered divergent roles of clathrin in the cell. Not only does clathrin act to facilitate endocytosis from the plasma membrane, but it also participates in budding from endosomes and the Golgi apparatus and in mitosis. Here, the manipulation of clathrin processes by bacterial pathogens, including its traditional role during invasion and alternative ways in which clathrin supports bacterial infection, is discussed. Researching clathrin in the context of bacterial infections will reveal new insights that inform our understanding of host-pathogen interactions and allow researchers to fully appreciate the diverse roles of clathrin in the eukaryotic cell.

Emerging insights into bacterial deubiquitinases.

Bacterial pathogens utilize eukaryotic cellular systems in various ways for their own benefits. To counteract host immune responses and survive in cells, bacteria modify host signaling pathways. For this aim, they have evolved virulence secretion systems. Bacteria-encoded effector proteins delivered via these secretion systems are the key players in bacterial pathogenesis. Ubiquitination is a post-translational modification that governs eukaryotic cellular systems. Recent studies have revealed that many bacterial effector proteins target the host ubiquitin system, often acting as ubiquitin-modulating enzymes such as ubiquitin ligases and deubiquitinases. Emerging lines of evidence have unveiled the diversity of bacterial deubiquitinases and have provided insights into the bacterial strategy to exploit the host ubiquitin system.

Bacterial type III secretion systems: a complex device for the delivery of bacterial effector proteins into eukaryotic host cells.

Virulence-associated type III secretion systems (T3SS) serve the injection of bacterial effector proteins into eukaryotic host cells. They are able to secrete a great diversity of substrate proteins in order to modulate host cell function, and have evolved to sense host cell contact and to inject their substrates through a translocon pore in the host cell membrane. T3SS substrates contain an N-terminal signal sequence and often a chaperone-binding domain for cognate T3SS chaperones. These signals guide the substrates to the machine where substrates are unfolded and handed over to the secretion channel formed by the transmembrane domains of the export apparatus components and by the needle filament. Secretion itself is driven by the proton motive force across the bacterial inner membrane. The needle filament measures 20-150 nm in length and is crowned by a needle tip that mediates host-cell sensing. Secretion through T3SS is a highly regulated process with early, intermediate and late substrates. A strict secretion hierarchy is required to build an injectisome capable of reaching, sensing and penetrating the host cell membrane, before host cell-acting effector proteins are deployed. Here, we review the recent progress on elucidating the assembly, structure and function of T3SS injectisomes.