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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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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.

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., (© 2020 International Federation for Cell Biology.)

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 CO 2 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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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.

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.

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., (© 2020 John Wiley & Sons Ltd.)

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., (© 2020 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

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.

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.

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., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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/ .

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2019 American Society for Microbiology.)

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Recent studies evaluated the impact of dust exposure on pure and mixed cultures of Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, and Pseudomonas aeruginosa, revealing increased biofilm formation and altered sensitivities to H 2 O 2 . In this study, we examined the impact of lead (Pb), house, road, and combined dust on K. pneumoniae and P. aeruginosa in pure, mixed, or eukaryotic co-culture with human alveolar basal epithelial (A549) cells. Although no impact on pure or mixed culture growth was observed when bacteria were exposed to Pb, house, or road dust, increased biofilm was produced by P. aeruginosa in the presence of 0.8 μg/mL of Pb, while P. aeruginosa and K. pneumoniae both exhibited increased biofilm production in the presence of 100 μg/mL of house, road, and combined dust. When co-cultured with eukaryotic A549 cells, both bacteria demonstrated increased proliferation 6 h post-infection when challenged with house, road, or combined dust. However, when mixed bacteria were co-cultured with A549 cells, P. aeruginosa exhibited a significant ~ 1.5-fold increased proliferation in the presence of 100 μg/mL house, road, or combined dust. In sharp contrast, K. pneumoniae exhibited significantly reduced proliferation, when in mixed (with P. aeruginosa) A-549 co-culture, following exposure to 100 μg/mL house, road, or combined dust. To evaluate whether a host cell inflammatory response contributed to this disparity, NF-κB activation was evaluated in each co-culture infection. K. pneumoniae-A-549 co-culture, treated with 100 μg/mL of combined dust, exhibited no alterations in NF-κB translocation to the nucleus. Further, no differences in cytokine production were observed in the K. pneumoniae A-549 co-culture treated with 100 μg/mL of house dust. Taken together, these data suggest that within the lung environment, mixed infections exposed to dust or dust contaminants could benefit one organism at the expense of the other, independent of the activation of inflammatory pathways.

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.

ExoY is one of four well-characterized Pseudomonas aeruginosa type 3 secretion system (T3SS) effectors. It is a nucleotidyl cyclase toxin that is inactive inside the bacteria, but becomes potently activated once it is delivered into the eukaryotic target cells. Recently, filamentous actin was identified as the eukaryotic cofactor that stimulates specifically ExoY enzymatic activity by several orders of magnitude. In this review, we discuss recent advances in understanding the biochemistry of nucleotidyl cyclase activity of ExoY and its regulation by interaction with filamentous actin., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The adenylate cyclase toxin (CyaA) plays an essential role in the early stages of respiratory tract colonization by Bordetella pertussis, the causative agent of whooping cough. Once secreted, CyaA invades eukaryotic cells, leading to cell death. The cell intoxication process involves a unique mechanism of translocation of the CyaA catalytic domain directly across the plasma membrane of the target cell. Herein, we review our recent results describing how calcium is involved in several steps of this intoxication process. In conditions mimicking the low calcium environment of the crowded bacterial cytosol, we show that the C-terminal, calcium-binding Repeat-in-ToXin (RTX) domain of CyaA, RD, is an extended, intrinsically disordered polypeptide chain with a significant level of local, secondary structure elements, appropriately sized for transport through the narrow channel of the secretion system. Upon secretion, the high calcium concentration in the extracellular milieu induces the refolding of RD, which likely acts as a scaffold to favor the refolding of the upstream domains of the full-length protein. Due to the presence of hydrophobic regions, CyaA is prone to aggregate into multimeric forms in vitro, in the absence of a chaotropic agent. We have recently defined the experimental conditions required for CyaA folding, comprising both calcium binding and molecular confinement. These parameters are critical for CyaA folding into a stable, monomeric and functional form. The monomeric, calcium-loaded (holo) toxin exhibits efficient liposome permeabilization and hemolytic activities in vitro, even in a fully calcium-free environment. By contrast, the toxin requires sub-millimolar calcium concentrations in solution to translocate its catalytic domain across the plasma membrane, indicating that free calcium in solution is actively involved in the CyaA toxin translocation process. Overall, this data demonstrates the remarkable adaptation of bacterial RTX toxins to the diversity of calcium concentrations it is exposed to in the successive environments encountered in the course of the intoxication process., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Biological systems provide the richest spectrum of sophisticated design for materials engineering. We herein provide a paradigm of Au 22 (SG) 18 -engineered (SG, glutathione thiolate) and hydrogen bonds engaged assemblies for mimicking capsid protein self-assembly. The water-evaporation-induced self-assembly method allows discrete ultrasmall gold nanoclusters (GNCs) to be self-assembled into super-GNCs assemblies (SGNCs) ranging from nano-, meso- to microscale in water-dimethyl sulfoxide binary solvents in a template-free manner. After removing free and hydration layer water molecules, the formation of SGNCs is engaged by the collective cohesion of hydrogen bonds between glutathione ligands of gradually approaching GNCs. Then, a series of tightly orchestrated cellular events induced by the complexes of Au 22 (SG) 18 -engineered assemblies and folic acid are demonstrated to mimic the invasion of eukaryotic cells by pathogens. First, the activation of macropinocytosis mimics the macropinocytic entry used by the pathogens to invade host cells. Then the cytoplasmic vacuolization is a mimicry of vacuolating effects induced by the oligomeric vacuolating toxins secreted by some bacteria. Lastly, the escaping from macropinosomes into cytosol is in a vacuolating toxin's strategy. The findings demonstrate the capabilities of artificial pathogens to emulate the structures and functions of natural pathogens.

Bacteria secrete RNAs, some of which have effects on other cells and on other species as signalling RNAs. Prokaryotic membrane vesicles (MVs) contain a range of RNA types. The MV structure offers protection from degradation as well as receptors to facilitate delivery to target cells. Microscopic imaging and molecular biology analyses have provided evidence to demonstrate that bacterial MVs deliver their RNA into eukaryotic cells. Moreover, in some cases the RNA cargo is demonstrably functional and phenotypic changes can be identified in MV-RNA treated target cells. The challenge now is to dissect the effect of MV-RNA on target cells away from the effects of non-RNA components of the MV such as lipopolysaccharide that can co-purify with RNA.

Background: Structural variation contributes substantially to polymorphism within species. Chromosomal rearrangements that impact genes can lead to functional variation among individuals and influence the expression of phenotypic traits. Genomes of fungal pathogens show substantial chromosomal polymorphism that can drive virulence evolution on host plants. Assessing the adaptive significance of structural variation is challenging, because most studies rely on inferences based on a single reference genome sequence., Results: We constructed and analyzed the pangenome of Zymoseptoria tritici, a major pathogen of wheat that evolved host specialization by chromosomal rearrangements and gene deletions. We used single-molecule real-time sequencing and high-density genetic maps to assemble multiple genomes. We annotated the gene space based on transcriptomics data that covered the infection life cycle of each strain. Based on a total of five telomere-to-telomere genomes, we constructed a pangenome for the species and identified a core set of 9149 genes. However, an additional 6600 genes were exclusive to a subset of the isolates. The substantial accessory genome encoded on average fewer expressed genes but a larger fraction of the candidate effector genes that may interact with the host during infection. We expanded our analyses of the pangenome to a worldwide collection of 123 isolates of the same species. We confirmed that accessory genes were indeed more likely to show deletion polymorphisms and loss-of-function mutations compared to core genes., Conclusions: The pangenome construction of a highly polymorphic eukaryotic pathogen showed that a single reference genome significantly underestimates the gene space of a species. The substantial accessory genome provides a cradle for adaptive evolution.

Pathogenic bacteria induce eukaryotic cell damage which range from discrete modifications of signalling pathways, to morphological alterations and even to cell death. Accurate quantitative detection of these events is necessary for studying host-pathogen interactions and for developing strategies to protect host organisms from bacterial infections. Investigation of morphological changes is cumbersome and not adapted to high-throughput and kinetics measurements. Here, we describe a simple and cost-effective method based on automated analysis of live cells with stained nuclei, which allows real-time quantification of bacteria-induced eukaryotic cell damage at single-cell resolution. We demonstrate that this automated high-throughput microscopy approach permits screening of libraries composed of interference-RNA, bacterial strains, antibodies and chemical compounds in ex vivo infection settings. The use of fluorescently-labelled bacteria enables the concomitant detection of changes in bacterial growth. Using this method named CLIQ-BID (Cell Live Imaging Quantification of Bacteria Induced Damage), we were able to distinguish the virulence profiles of different pathogenic bacterial species and clinical strains.

Short linear motifs (SLiMs) are protein binding modules that play major roles in almost all cellular processes. SLiMs are short, often highly degenerate, difficult to characterize and hard to detect. The eukaryotic linear motif (ELM) resource (elm.eu.org) is dedicated to SLiMs, consisting of a manually curated database of over 275 motif classes and over 3000 motif instances, and a pipeline to discover candidate SLiMs in protein sequences. For 15 years, ELM has been one of the major resources for motif research. In this database update, we present the latest additions to the database including 32 new motif classes, and new features including Uniprot and Reactome integration. Finally, to help provide cellular context, we present some biological insights about SLiMs in the cell cycle, as targets for bacterial pathogenicity and their functionality in the human kinome., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Bacteria often coordinate virulence factors to fine-tune the host response during infection. These coordinated events can include toxins counteracting or amplifying effects of another toxin or though regulating the stability of virulence factors to remove their function once it is no longer needed. Multifunctional autoprocessing repeats-in toxin (MARTX) toxins are effector delivery toxins that form a pore into the plasma membrane of a eukaryotic cell to deliver multiple effector proteins into the cytosol of the target cell. The function of these proteins includes manipulating actin cytoskeletal dynamics, regulating signal transduction pathways and inhibiting host secretory pathways. Investigations into the molecular mechanisms of these effector domains are providing insight into how the function of some effectors overlap and regulate one another during infection. Coordinated crosstalk of effector function suggests that MARTX toxins are not simply a sum of all their parts. Instead, modulation of cell function by effector domains may depend on which other effector domain are co-delivered. Future studies will elucidate how these effectors interact with each other to modulate the bacterial host interaction., (© 2017 John Wiley & Sons Ltd.)

Following colonization of host tissues, bacterial pathogens encounter new niches in which they must gain access to nutrients and cope with stresses and defence signals generated by the host. For some pathogens, the adaptation to a new 'within-host' lifestyle involves modifications of envelope components that bear molecular patterns normally recognized by the host innate immune system. These new modified patterns limit host recognition, therefore promoting immune evasion and pathogenicity. In this review, we describe how envelope components like the peptidoglycan or lipopolysaccharide can be altered within the host to impair responses triggered by pattern recognition receptors (PRR). We also discuss the few cases reported to date of chemical modifications that occur in the envelope of some intracellular bacterial pathogens when they reside inside eukaryotic cells. These envelope alterations may have evolved due to the sentinel role performed by PRRs over pathogen-specific molecular patterns. The available data indicate that only selected pathogens seem to evade recognition due to 'within-host' envelope changes, with most of them displaying such patterns also in non host environments. Given the importance of these alterations, future studies should focus in the responsible pathogen regulators, most yet unknown, that could be targeted to prevent immune evasion.

Salmonella is probably the intracellular pathogen most extensively studied. Once inside the cell, this bacterium produces different proteins involved in the infection process known as effectors that translocate through its own secretion systems to the eukaryotic cytosol exerting diverse effects on the cell. Additionally, Salmonella can be engineered to include a protein expression system that, upon the addition of an inducer molecule, can produce heterologous proteins at a specific time during the course of the infection. The effect of such proteins on the eukaryotic (i.e., tumoral) cells can be detected following distinct approaches, which converts Salmonella in an effective tool to produce proteins inside eukaryotic cells with different purposes, such as killing tumoral cells. Here, we present diverse technics currently used to produce proteins by Salmonella inside tumoral cells and analyze its cytotoxic effect.

In this review we examine the use of secretion systems by bacteria to subvert host functions. Bacteria have evolved multiple systems to interact with and overcome their eukaryotic host and other prokaryotes. Secretion systems are required for the release of several effectors through the bacterial membrane(s) into the extracellular space or directly into the cytoplasm of the host. We review the secretion systems of Gram-positive and Gram-negative bacteria and describe briefly the structural composition of the seven secretion systems that have been associated with increased virulence through subversion of host functions. Some of the effects of such systems on eukaryotic host processes have been studied extensively. We also describe the best-characterized effectors of each secretion system to give an overview of the molecular mechanisms employed by bacteria to hide from the immune system and convert eukaryotic cells into optimal ecological niches for their replication.

Intracellular bacterial pathogens have developed versatile strategies to generate niches inside the eukaryotic cells that allow them to survive and proliferate. Making a home inside the host offers many advantages; however, intracellular bacteria must also overcome many challenges, such as disarming innate immune signaling and accessing host nutrient supplies. Gaining entry into the cell and avoiding degradation is only the beginning of a successful intracellular lifestyle. To establish these replicative niches, intracellular pathogens secrete various virulence proteins, called effectors, to manipulate host cell signaling pathways and subvert host defense mechanisms. Many effectors mimic host enzymes, whereas others perform entirely novel enzymatic functions. A large volume of work has been done to understand how intracellular bacteria manipulate membrane trafficking pathways. In this review, we focus on how intracellular bacterial pathogens target innate immune signaling, the unfolded protein response, autophagy, and cellular metabolism and exploit these pathways to their advantage. We also discuss how bacterial pathogens can alter host gene expression by directly modifying histones or hijacking the ubiquitination machinery to take control of several host signaling pathways., (© 2017 Cornejo et al.)

Ubiquitin E3 ligases control every aspect of eukaryotic biology by promoting protein ubiquitination and degradation. At the end of a three-enzyme cascade, ubiquitin ligases mediate the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to specific substrate proteins. Early investigations of E3s of the RING (really interesting new gene) and HECT (homologous to the E6AP carboxyl terminus) types shed light on their enzymatic activities, general architectures, and substrate degron-binding modes. Recent studies have provided deeper mechanistic insights into their catalysis, activation, and regulation. In this review, we summarize the current progress in structure-function studies of ubiquitin ligases as well as exciting new discoveries of novel classes of E3s and diverse substrate recognition mechanisms. Our increased understanding of ubiquitin ligase function and regulation has provided the rationale for developing E3-targeting therapeutics for the treatment of human diseases.

Some bacterial pathogens modulate signaling pathways of eukaryotic cells in order to subvert the host response for their own benefit, leading to successful colonization and invasion. Pathogenic bacteria produce multiple compounds that generate favorable conditions to their survival and growth during infection in eukaryotic hosts. Many bacterial toxins can alter the cell cycle progression of host cells, impairing essential cellular functions and impeding host cell division. This review summarizes current knowledge regarding cyclomodulins, a heterogeneous family of bacterial effectors that induce eukaryotic cell cycle alterations. We discuss the mechanisms of actions of cyclomodulins according to their biochemical properties, providing examples of various cyclomodulins such as cycle inhibiting factor, γ-glutamyltranspeptidase, cytolethal distending toxins, shiga toxin, subtilase toxin, anthrax toxin, cholera toxin, adenylate cyclase toxins, vacuolating cytotoxin, cytotoxic necrotizing factor, Panton-Valentine leukocidin, phenol soluble modulins, and mycolactone. Special attention is paid to the benefit provided by cyclomodulins to bacteria during colonization of the host.

Intracellular bacterial pathogens replicate within eukaryotic cells and display unique adaptations that support key infection events including invasion, replication, immune evasion, and dissemination. From invasion to dissemination, all stages of the intracellular bacterial life cycle share the same three-dimensional cytosolic space containing the host cytoskeleton. For successful infection and replication, many pathogens hijack the cytoskeleton using effector proteins introduced into the host cytosol by specialized secretion systems. A subset of effectors contains eukaryotic-like motifs that mimic host proteins to exploit signaling and modify specific cytoskeletal components such as actin and microtubules. Cytoskeletal rearrangement promotes numerous events that are beneficial to the pathogen, including internalization of bacteria, structural support for bacteria-containing vacuoles, altered vesicular trafficking, actin-dependent bacterial movement, and pathogen dissemination. This review highlights a diverse group of obligate intracellular bacterial pathogens that manipulate the host cytoskeleton to thrive within eukaryotic cells and discusses underlying molecular mechanisms that promote these dynamic host-pathogen interactions.

Mitochondria and chloroplasts are now called organelles, but they used to be bacteria. As they transitioned from endosymbionts to organelles, they became more and more integrated into the biochemistry and cell biology of their hosts. Work over the last 15 years has shown that other symbioses show striking similarities to mitochondria and chloroplasts. In particular, many sap-feeding insects house intracellular bacteria that have genomes that overlap mitochondria and chloroplasts in terms of size and coding capacity. The massive levels of gene loss in some of these bacteria suggest that they, too, are becoming highly integrated with their host cells. Understanding these bacteria will require inspiration from eukaryotic cell biology, because a traditional microbiological framework is insufficient for understanding how they work., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Bacterial pathogens employ type IV secretion systems (T4SSs) for various purposes to aid in survival and proliferation in eukaryotic hosts. One large T4SS subfamily, the conjugation systems, confers a selective advantage to the invading pathogen in clinical settings through dissemination of antibiotic resistance genes and virulence traits. Besides their intrinsic importance as principle contributors to the emergence of multiply drug-resistant "superbugs," detailed studies of these highly tractable systems have generated important new insights into the mode of action and architectures of paradigmatic T4SSs as a foundation for future efforts aimed at suppressing T4SS machine function. Over the past decade, extensive work on the second large T4SS subfamily, the effector translocators, has identified a myriad of mechanisms employed by pathogens to subvert, subdue, or bypass cellular processes and signaling pathways of the host cell. An overarching theme in the evolution of many effectors is that of molecular mimicry. These effectors carry domains similar to those of eukaryotic proteins and exert their effects through stealthy interdigitation of cellular pathways, often with the outcome not of inducing irreversible cell damage but rather of reversibly modulating cellular functions. This article summarizes the major developments for the actively studied pathogens with an emphasis on the structural and functional diversity of the T4SSs and the emerging common themes surrounding effector function in the human host.

Intracellular bacterial pathogens have evolved to exploit the protected niche provided within the boundaries of a eukaryotic host cell. Upon entering a host cell, some bacteria can evade the adaptive immune response of its host and replicate in a relatively nutrient-rich environment devoid of competition from other host flora. Growth within a host cell is not without their hazards, however. Many pathogens enter their hosts through receptor-mediated endocytosis or phagocytosis, two intracellular trafficking pathways that terminate in a highly degradative organelle, the phagolysosome. This usually deadly compartment is maintained at a low pH and contains degradative enzymes and reactive oxygen species, resulting in an environment to which few bacterial species are adapted. Some intracellular pathogens, such as Shigella, Listeria, Francisella, and Rickettsia, escape the phagosome to replicate within the cytosol of the host cell. Bacteria that remain within a vacuole either alter the trafficking of their initial phagosomal compartment or adapt to survive within the harsh environment it will soon become. In this chapter, we focus on the mechanisms by which different vacuolar pathogens either evade lysosomal fusion, as in the case of Mycobacterium and Chlamydia, or allow interaction with lysosomes to varying degrees, such as Brucella and Coxiella, and their specific adaptations to inhabit a replicative niche.

Intracellular bacterial pathogens have evolved many ways to manipulate host cells for successful infection. Many of these pathogens use specialized secretion systems to inject bacterial proteins into the host cytosol that manipulate cellular processes to favor infection. Autophagy is a eukaryotic cellular remodeling process with a critical role in many diseases, including bacterial clearance. A growing field of research highlights mechanisms used by intracellular bacteria to manipulate autophagy as a pro-survival strategy. This review focuses on a select group of bacterial pathogens with diverse intracellular lifestyles that exploit autophagy-derived nutrients and membrane for survival. This group of pathogens uses secretion systems and specific effectors to subvert distinct components of autophagy. By understanding how intracellular pathogens manipulate autophagy, we gain insight not only into bacterial pathogenesis but also host cell signaling and autophagolysosome maturation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Many bacterial pathogens have evolved the ability to subvert and exploit host functions in order to enter and replicate in eukaryotic cells. For example, bacteria have developed specific mechanisms to target eukaryotic organelles such as the nucleus, the mitochondria, the endoplasmic reticulum and the Golgi apparatus. In this Review, we highlight the most recent advances in our understanding of the mechanisms that bacterial pathogens use to target these organelles. We also discuss how these strategies allow bacteria to manipulate host functions and to ultimately enable bacterial infection.

The Type VI secretion system is a widespread bacterial nanomachine, used to deliver toxins directly into eukaryotic or prokaryotic target cells. These secreted toxins, or effectors, act on diverse cellular targets, and their action provides the attacking bacterial cell with a significant fitness advantage, either against rival bacteria or eukaryotic host organisms. In this review, we discuss the delivery of diverse effectors by the Type VI secretion system, the modes of action of the so-called 'anti-bacterial' and 'anti-eukaryotic' effectors, the mechanism of self-resistance against anti-bacterial effectors and the evolutionary implications of horizontal transfer of Type VI secretion system-associated toxins. Whilst it is likely that many more effectors remain to be identified, it is already clear that toxins delivered by this secretion system represent efficient weapons against both bacteria and eukaryotes., (© 2015 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.)

The actin cytoskeleton is a key target of numerous microbial pathogens, including protozoa, fungi, bacteria and viruses. In particular, bacterial pathogens produce and deliver virulence effector proteins that hijack actin dynamics to enable bacterial invasion of host cells, allow movement within the host cytosol, facilitate intercellular spread or block phagocytosis. Many of these effector proteins directly or indirectly target the major eukaryotic actin nucleator, the Arp2/3 complex, by either mimicking nucleation promoting factors or activating upstream small GTPases. In contrast, this review is focused on a recently identified class of effector proteins from Gram-negative bacteria that function as direct actin nucleators. These effector proteins mimic functional activities of formins, WH2-nucleators and Ena/VASP assembly promoting factors demonstrating that bacteria have coopted the complete set of eukaryotic actin assembly pathways. Structural and functional analyses of these nucleators have revealed several motifs and/or mechanistic activities that are shared with eukaryotic actin nucleators. However, functional effects of these proteins during infection extend beyond plain actin polymerization leading to interference with other host cell functions such as vesicle trafficking, cell cycle progression and cell death. Therefore, their use as model systems could not only help in the understanding of the mechanistic details of actin polymerization but also provide novel insights into the connection between actin dynamics and other cellular pathways., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Mitochondria originated endosymbiotically from an Alphaproteobacteria-like ancestor. However, it is still uncertain which extant group of Alphaproteobacteria is phylogenetically closer to the mitochondrial ancestor. The proposed groups comprise the order Rickettsiales, the family Rhodospirillaceae, and the genus Rickettsia. In this study, we apply a new complex network approach to investigate the evolutionary origins of mitochondria, analyzing protein sequences modules in a critical network obtained through a critical similarity threshold between the studied sequences. The dataset included three ATP synthase subunits (4, 6, and 9) and its alphaproteobacterial homologs (b, a, and c). In all the subunits, the results gave no support to the hypothesis that Rickettsiales are closely related to the mitochondrial ancestor. Our findings support the hypothesis that mitochondria share a common ancestor with a clade containing all Alphaproteobacteria orders, except Rickettsiales.

Sensitivity of eukaryotic cells to facultative pathogens can depend on physiological state of host cells. Previously we have shown that pretreatment of HeLa cells with N-acetylcysteine (NAC) makes the cells 2-3-fold more sensitive to invasion by the wild-type Serratia grimesii and recombinant Escherichia coli expressing gene of actin-specific metalloprotease grimelysin [1]. To evaluate the impact of chemically different antioxidants, in the present work we studied the effects of α-Lipoic acid (LA) and dihydrolipoic acid (DHLA) on efficiency of S. grimesii and recombinant E. coli expressing grimelysin gene to penetrate into HeLa and CaCo cells. Similarly to the effect of NAC, pretreatment of HeLa and CaCo cells with 0.6 or 1.25 mM DHLA increased the entry of grimelysin producing bacteria by a factor of 2.5 and 3 for the wild-type S. grimesii and recombinant E. coli, respectively. In contrast, pretreatment of the cells with 0.6 or 1.25 mM LA did not affect the bacteria uptake. The increased invasion of HeLa and CaCo cells correlated with the enhanced expression of E-cadherin and β-catenin genes, whereas expression of these genes in the LA-treated cells was not changed. Comparison of these results suggests that it is sulfhydryl group of DHLA that promotes efficient modification of cell properties assisting bacterial uptake. We assume that the NAC- and DHLA-induced stimulation of the E-cadherin-catenin pathway contributes to the increased internalization of the grimelysin producing bacteria within transformed cells., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Toxin-antitoxin (TA) modules contribute to the generation of non-growing cells in response to stress. These modules abound in bacterial pathogens although the bases for this profusion remain largely unknown. Using the intracellular bacterial pathogen Salmonella enterica serovar Typhimurium as a model, here we show that a selected group of TA modules impact bacterial fitness inside eukaryotic cells. We characterized in this pathogen twenty-seven TA modules, including type I and type II TA modules encoding antisense RNA and proteinaceous antitoxins, respectively. Proteomic and gene expression analyses revealed that the pathogen produces numerous toxins of TA modules inside eukaryotic cells. Among these, the toxins HokST, LdrAST, and TisBST, encoded by type I TA modules and T4ST and VapC2ST, encoded by type II TA modules, promote bacterial survival inside fibroblasts. In contrast, only VapC2ST shows that positive effect in bacterial fitness when the pathogen infects epithelial cells. These results illustrate how S. Typhimurium uses distinct type I and type II TA modules to regulate its intracellular lifestyle in varied host cell types. This function specialization might explain why the number of TA modules increased in intracellular bacterial pathogens.

Campylobacter (C.) jejuni is the most important reported cause for bacterial diarrhoea. The infection can be accompanied by fever and abdominal cramps and in rare cases the Guillain-Barré syndrome or reactive arthritis can develop as a post-infection complication. Several biological properties of Cjejuni, e. g. motility and chemotaxis, contribute to the biological fitness of the pathogen. For this, deficiencies in the function of these features clearly reduce the pathogenicity of C. jejuni without being a virulence factor per se. Opposing to this, there are two essential requirements to determine the virulence of C. jejuni which represent the adherence to, and the invasion of host cells. Thereby, adherence, as a virulence factor, is mediated by many different bacterial-derived components like proteins but also by several oligo- and polysaccharide structures that are linked to surface proteins but also to the flagella. In addition, several invasion-relevant features of C. jejuni have been detected so far. Whereas some of them are described functionally to modulate cytoskeleton arrangement of the host cell, others are only described as invasion relevant. Indeed, investigations with respect to the pathogenic potential of some adherence- and invasion-relevant components did not give identical results indicating that their relevance might depend on the interplay of the respective C. jejuni strains used in these studies with the corresponding host cells. This review summarizes the C. jejuni components for adherence to and invasion of host cells together with their particular mode of action if known.

The genus Rickettsia (Alphaproteobacteria, Rickettsiales, Rickettsiaceae) is comprised of obligate intracellular parasites, with virulent species of interest both as causes of emerging infectious diseases and for their potential deployment as bioterrorism agents. Currently, there are no effective commercially available vaccines, with treatment limited primarily to tetracycline antibiotics, although others (e.g. josamycin, ciprofloxacin, chloramphenicol, and azithromycin) are also effective. Much of the recent research geared toward understanding mechanisms underlying rickettsial pathogenicity has centered on characterization of secreted proteins that directly engage eukaryotic cells. Herein, we review all aspects of the Rickettsia secretome, including six secretion systems, 19 characterized secretory proteins, and potential moonlighting proteins identified on surfaces of multiple Rickettsia species. Employing bioinformatics and phylogenomics, we present novel structural and functional insight on each secretion system. Unexpectedly, our investigation revealed that the majority of characterized secretory proteins have not been assigned to their cognate secretion pathways. Furthermore, for most secretion pathways, the requisite signal sequences mediating translocation are poorly understood. As a blueprint for all known routes of protein translocation into host cells, this resource will assist research aimed at uniting characterized secreted proteins with their apposite secretion pathways. Furthermore, our work will help in the identification of novel secreted proteins involved in rickettsial 'life on the inside'., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Salmonella is a facultative intracellular bacterium found within a variety of phagocytic and nonphagocytic cells in vitro and in vivo For decades, it has been accepted that Salmonella can enter cells only through a Trigger mechanism mediated by a type three secretion system, called T3SS-1. However, recent researches have shown that this bacterium can use other invasion pathways mediating either Trigger or Zipper entry processes. Following eukaryotic cell invasion, Salmonella has to ensure its survival and proliferation within host cells. To do so, this bacterium resides either within a membrane-bound vacuole or freely within host cell cytosol. It is not clear why Salmonella has developed these alternate mechanisms for cell invasion and proliferation, but this provides a new insight into the mechanisms leading to Salmonella-induced diseases. Thus, the aim of this review is to show the evolution of Salmonella-host cell interaction paradigms by summarizing the different strategies used by Salmonella serotypes to invade and proliferate into eukaryotic cells., (© 2014 Federation of European Microbiological Societies.)

Intracellular bacterial pathogens have evolved distinct lifestyles inside eukaryotic cells. Some pathogens coexist with the infected cell in an obligate intracellular state, whereas others transit between the extracellular and intracellular environment. Adaptation to these intracellular lifestyles is regulated in both space and time. Non-coding small RNAs (sRNAs) are post-transcriptional regulatory molecules that fine-tune important processes in bacterial physiology including cell envelope architecture, intermediate metabolism, bacterial communication, biofilm formation, and virulence. Recent studies have shown production of defined sRNA species by intracellular bacteria located inside eukaryotic cells. The molecules targeted by these sRNAs and their expression dynamics along the intracellular infection cycle remain, however, poorly characterized. Technical difficulties linked to the isolation of "intact" intracellular bacteria from infected host cells might explain why sRNA regulation in these specialized pathogens is still a largely unexplored field. Transition from the extracellular to the intracellular lifestyle provides an ideal scenario in which regulatory sRNAs are intended to participate; so much work must be done in this direction. This review focuses on sRNAs expressed by intracellular bacterial pathogens during the infection of eukaryotic cells, strategies used with these pathogens to identify sRNAs required for virulence, and the experimental technical challenges associated to this type of studies. We also discuss varied techniques for their potential application to study RNA regulation in intracellular bacterial infections.