Context-dependent symbioses and their potential roles in wildlife diseases

It is well known in ecology, evolution and medicine that both the nature (commensal, parasitic and mutualistic) and outcome (symbiont fitness, survival) of symbiotic interactions are often context-dependent. Less is known about the importance of context-dependence in symbioses involved in wildlife disease. We review variable symbioses, and use the amphibian disease chytridiomycosis to demonstrate how understanding context-dependence can improve the understanding and management of wildlife diseases. In chytridiomycosis, the host-pathogen interaction is context-dependent; it is strongly affected by environmental temperature. Skin bacteria can also modify the interaction; some bacteria reduce amphibians' susceptibility to chytridiomycosis. Augmentation of protective microbes is being considered as a possible management tool, but informed application of bioaugmentation requires understanding of how the interactions between host, beneficial bacteria and pathogen depend upon environmental context. The community-level response of the amphibian skin microbiota to environmental conditions may explain the relatively narrow range of environmental conditions in which past declines have occurred. Environmental context affects virulence and the protection provided by mutualists in other host-pathogen systems, including threatened bats and corals. Increased focus on context-dependence in interactions between wildlife and their symbionts is likely to be crucial to the future investigation and management of emerging diseases of wildlife.     

Characterization of abalone Haliotis tuberculata–Vibrio harveyi interactions in gill primary cultures

The decline of European abalone Haliotis tuberculata populations has been associated with various pathogens including bacteria of the genus Vibrio. Following the summer mortality outbreaks reported in France between 1998 and 2000, Vibrio harveyi strains were isolated from moribund abalones, allowing in vivo and in vitro studies on the interactions between abalone H. tuberculata and V. harveyi. This work reports the development of primary cell cultures from abalone gill tissue, a target tissue for bacterial colonisation, and their use for in vitro study of host cell—V. harveyi interactions. Gill cells originated from four-day-old explant primary cultures were successfully sub-cultured in multi-well plates and maintained in vitro for up to 24 days. Cytological parameters, cell morphology and viability were monitored over time using flow cytometry analysis and semi-quantitative assay (XTT). Then, gill cell cultures were used to investigate in vitro the interactions with V. harveyi. The effects of two bacterial strains were evaluated on gill cells: a pathogenic bacterial strain ORM4 which is responsible for abalone mortalities and LMG7890 which is a non-pathogenic strain. Cellular responses of gill cells exposed to increasing concentrations of bacteria were evaluated by measuring mitochondrial activity (XTT assay) and phenoloxidase activity, an enzyme which is strongly involved in immune response. The ability of gill cells to phagocyte GFP-tagged V. harveyi was evaluated by flow cytometry and gill cells-V. harveyi interactions were characterized using fluorescence microscopy and transmission electron microscopy. During phagocytosis process we evidenced that V. harveyi bacteria induced significant changes in gill cells metabolism and immune response. Together, the results showed that primary cell cultures from abalone gills are suitable for in vitro study of host-pathogen interactions, providing complementary assays to in vivo experiments.     

Flow cytometric analysis of microorganisms

The application of flow cytometry to microorganisms is as old as the technique itself, but it has historically been underexploited for microbial applications. This is now being reversed and microbiologists are ideally placed to benefit from recent technological advances. While earlier papers demonstrated the use of flow cytometry for studies of viability and taxonomy, recent developments in bioinformatics and reporter gene technologies are leading to novel applications in microbiology. Variants of green fluorescent protein have been used for the study of conditional microbial gene regulation in medically important host-pathogen interactions and fluorescence-activated cell sorting is being applied to the isolation of novel mutants in directed evolution studies. This paper reviews the reasons for the delay in the application of flow cytometry to microbial problems, the range of applications, and their limitations and considers the progress made in developing new strategies for use in microbiological investigations.     

Lipidomics of host-pathogen interactions

The cell biology of intracellular pathogens (viruses, bacteria, eukaryotic parasites) has provided us with molecular information of host-pathogen interactions. As a result it is becoming increasingly evident that lipids play important roles at various stages of host-pathogen interactions. They act in first line recognition and host cell signaling during pathogen docking, invasion and intracellular trafficking. Lipid metabolism is a housekeeping function in energy homeostasis and biomembrane synthesis during pathogen replication and persistence. Lipids of enormous chemical diversity play roles as immunomodulatory factors. Thus, novel biochemical analytics in combination with cell and molecular biology are a promising recipe for dissecting the roles of lipids in host-pathogen interactions.     

A new twist on an old pathway--accessory Sec [corrected] systems

The export of proteins from their site of synthesis in the cytoplasm across the inner membrane is an important aspect of bacterial physiology. Because the location of extracytoplasmic proteins is ideal for host-pathogen interactions, protein export is also important to bacterial virulence. In bacteria, there are conserved protein export systems that are responsible for the majority of protein export: the general secretion (Sec) pathway and the twin-arginine translocation pathway. In some bacteria, there are also specialized export systems dedicated to exporting specific subsets of proteins. In this review, we discuss a specialized export system that exists in some Gram-positive bacteria and mycobacteria - the accessory Sec system. The common element to the accessory Sec system is an accessory SecA protein called SecA2. Here we present our current understanding of accessory Sec systems in Streptococcus gordonii, Streptococcus parasanguinis, Mycobacterium smegmatis, Mycobacterium tuberculosis and Listeria monocytogenes, making an effort to highlight apparent similarities and differences between the systems. We also review the data showing that accessory Sec systems can contribute to bacterial virulence.     

Host-pathogen interactions: a proteomic view

Host-pathogen interactions reflect the balance of host defenses and pathogen virulence mechanisms. Advances in proteomic technologies now afford opportunities to compare protein content between complex biologic systems ranging from cells to animals and clinical samples. Thus, it is now possible to characterize host-pathogen interactions from a global proteomic view. Most reports to date focus on cataloging protein content of pathogens and identifying virulence-associated proteins or proteomic alterations in host response. A more in-depth understanding of host-pathogen interactions has the potential to improve our mechanistic understanding of pathogenicity and virulence, thereby defining novel therapeutic and vaccine targets. In addition, proteomic characterization of the host response can provide pathogen-specific host biomarkers for rapid pathogen detection and characterization, as well as for early and specific detection of infectious diseases. A review of host-pathogen interactions focusing on proteomic analyses of both pathogen and host will be presented. Relevant genomic studies and host model systems will be also be discussed.     

Deconstructing host-pathogen interactions in Drosophila

Many of the cellular mechanisms underlying host responses to pathogens have been well conserved during evolution. As a result, Drosophila can be used to deconstruct many of the key events in host-pathogen interactions by using a wealth of well-developed molecular and genetic tools. In this review, we aim to emphasize the great leverage provided by the suite of genomic and classical genetic approaches available in flies for decoding details of host-pathogen interactions; these findings can then be applied to studies in higher organisms. We first briefly summarize the general strategies by which Drosophila resists and responds to pathogens. We then focus on how recently developed genome-wide RNA interference (RNAi) screens conducted in cells and flies, combined with classical genetic methods, have provided molecular insight into host-pathogen interactions, covering examples of bacteria, fungi and viruses. Finally, we discuss novel strategies for how flies can be used as a tool to examine how specific isolated virulence factors act on an intact host.     

Computational analysis of interactomes: Current and future perspectives for bioinformatics approaches to model the host-pathogen interaction space

Bacterial and viral pathogens affect their eukaryotic host partly by interacting with proteins of the host cell. Hence, to investigate infection from a systems' perspective we need to construct complete and accurate host-pathogen protein-protein interaction networks. Because of the paucity of available data and the cost associated with experimental approaches, any construction and analysis of such a network in the near future has to rely on computational predictions. Specifically, this challenge consists of a number of sub-problems: First, prediction of possible pathogen interactors (e.g. effector proteins) is necessary for bacteria and protozoa. Second, the prospective host binding partners have to be determined and finally, the impact on the host cell analyzed. This review gives an overview of current bioinformatics approaches to obtain and understand host-pathogen interactions. As an application example of the methods covered, we predict host-pathogen interactions of Salmonella and discuss the value of these predictions as a prospective for further research.     

Two-step infection processes can lead to coevolution between functionally independent infection and resistance pathways

There is growing evidence that successful infection of hosts by pathogens requires a series of independent steps. However, how multistep infection processes affect host-pathogen coevolution is unclear. We present a coevolutionary model, inspired by empirical observations from a range of host-pathogen systems, where the infection process consists of the following two steps: the first is for the pathogen to recognize and locate a suitable host, and the second is to exploit the host while evading immunity. Importantly, these two steps conform to different models of infection genetics: inverse-gene-for-gene (IGFG) and gene-for-gene (GFG), respectively. We show that coevolution under this scenario can lead to coupled gene frequency changes across these two systems. In particular, selection often favors pathogens that are infective at the first, IGFG, step and hosts that are resistant at the second, GFG, step. Hence, there may be signals of positive selection between functionally independent systems whenever there are multistep processes determining resistance and infectivity. Such multistep infection processes are a fundamental, but overlooked feature of many host-pathogen interactions, and have important consequences for our understanding of host-pathogen coevolution.     

Flow cytometric evaluation of yeast–bacterial cell–cell interactions

Synthetic cell–cell interaction systems can be useful for understanding multicellular communities or for screening binding molecules. We adapt a previously characterized set of synthetic cognate nanobody–antigen pairs to a yeast–bacteria coincubation format and use flow cytometry to evaluate cell–cell interactions mediated by binding between surface-displayed molecules. We further use fluorescence-activated cell sorting to enrich a specific yeast-displayed nanobody within a mixed yeast-display population. Finally, we demonstrate that this system supports the characterization of a therapeutically relevant nanobody–antigen interaction: a previously discovered nanobody that binds to the intimin protein expressed on the surface of enterohemorrhagic Escherichia coli. Overall, our findings indicate that the yeast–bacteria format supports efficient evaluation of ligand–target interactions. With further development, this format may facilitate systematic characterization and high-throughput discovery of bacterial surface-binding molecules.     

Evaluation of invertebrate infection models for pathogenic corynebacteria

For several pathogenic bacteria, model systems for host-pathogen interactions were developed, which provide the possibility of quick and cost-effective high throughput screening of mutant bacteria for genes involved in pathogenesis. A number of different model systems, including amoeba, nematodes, insects, and fish, have been introduced, and it was observed that different bacteria respond in different ways to putative surrogate hosts, and distinct model systems might be more or less suitable for a certain pathogen. The aim of this study was to develop a suitable invertebrate model for the human and animal pathogens Corynebacterium diphtheriae, Corynebacterium pseudotuberculosis, and Corynebacterium ulcerans. The results obtained in this study indicate that Acanthamoeba polyphaga is not optimal as surrogate host, while both Caenorhabtitis elegans and Galleria larvae seem to offer tractable models for rapid assessment of virulence between strains. Caenorhabtitis elegans gives more differentiated results and might be the best model system for pathogenic corynebacteria, given the tractability of bacteria and the range of mutant nematodes available to investigate the host response in combination with bacterial virulence. Nevertheless, Galleria will also be useful in respect to innate immune responses to pathogens because insects offer a more complex cell-based innate immune system compared with the simple innate immune system of C.?elegans.     

Genome reduction in the alpha-Proteobacteria

More than 20 alpha-proteobacterial genomes are currently available. These range in size from 1-9 Mb and represent excellent model systems for evolutionary studies of the organizational features of bacterial genomes. Computational inferences have shown that genome reductions have occurred independently in lineages such as Rickettsia and Bartonella that are associated with intracellular lifestyles. Analyses of these reduced genomes have provided insights into the evolution of vector-borne transmission pathways. Further research into the population biology of bacteria, arthropods and vertebrate hosts will help to refine the biology of host-pathogen interactions and will facilitate the design of vaccines and vector-control programs.     

The type VI secretion system: a multipurpose delivery system with a phage-like machinery

Whether they live in the soil, drift in the ocean, survive in the lungs of human hosts or reside on the surfaces of leaves, all bacteria must cope with an array of environmental stressors. Bacteria have evolved an impressive suite of protein secretion systems that enable their survival in hostile environments and facilitate colonization of eukaryotic hosts. Collectively, gram-negative bacteria produce six distinct secretion systems that deliver proteins to the extracellular milieu or directly into the cytosol of host cells. The type VI secretion system (T6SS) was discovered recently and is encoded in at least one fourth of all sequenced gram-negative bacterial genomes. T6SS proteins are evolutionarily and structurally related to phage proteins, and it is likely that the T6SS apparatus is reminiscent of phage injection machinery. Most studies of T6SS function have been conducted in the context of host-pathogen interactions. However, the totality of data suggests that the T6SS is a versatile tool with roles in virulence, symbiosis, interbacterial interactions, and antipathogenesis. This review gives a brief history of T6SS discovery and an overview of the pathway's predicted structure and function. Special attention is paid to research addressing the T6SS of plant-associated bacteria, including pathogens, symbionts and plant growth-promoting rhizobacteria.     

Metallobiology of host-pathogen interactions: an intoxicating new insight

Iron, zinc and copper, among others, are transition metals with multiple biological roles that make them essential elements for life. Beyond the strict requirement of transition metals by the vertebrate immune system for its proper functioning, novel mechanisms involving direct metal intoxication of microorganisms are starting to be unveiled as important components of the immune system, in particular against Mycobacterium tuberculosis. In parallel, metal detoxification systems in bacteria have been recently characterized as crucial microbial virulence determinants. Here, we will focus on these exciting advancements implicating copper- and zinc-mediated microbial poisoning as a novel innate immune mechanism against microbial pathogens, shedding light on an emerging field in the metallobiology of host-pathogen interactions.     

Covalent attachment of proteins to peptidoglycan

Bacterial surface proteins are key players in host-symbiont or host-pathogen interactions. How these proteins are targeted and displayed at the cell surface are challenging issues of both fundamental and clinical relevance. While surface proteins of Gram-negative bacteria are assembled in the outer membrane, Gram-positive bacteria predominantly utilize their thick cell wall as a platform to anchor their surface proteins. This surface display involves both covalent and noncovalent interactions with either the peptidoglycan or secondary wall polymers such as teichoic acid or lipoteichoic acid. This review focuses on the role of enzymes that covalently link surface proteins to the peptidoglycan, the well-known sortases in Gram-positive bacteria, and the recently characterized l,d-transpeptidases in Gram-negative bacteria.     

Quantification of bacterial invasion into adherent cells by flow cytometry

Quantification of invasive, intracellular bacteria is critical in many areas of cellular microbiology and immunology. We describe a novel and fast approach to determine invasion of bacterial pathogens in adherent cell types such as epithelial cells or fibroblasts based on flow cytometry. Using the CEACAM-mediated uptake of Opa-expressing Neisseria gonorrhoeae as a well-characterized model of bacterial invasion, we demonstrate that the flow cytometry-based method yields results comparable to a standard antibiotic protection assay. Furthermore, the quantification of intracellular bacteria by the novel approach is not biased by intracellular killing of the microbes and correctly discriminates between cell-associated extracellular and bona fide intracellular bacteria. As flow cytometry-based quantification is also applicable to other pathogen-host interactions such as the integrin-mediated internalization of Staphylococcus aureus, this approach provides a fast and convenient alternative for the quantification of bacterial uptake and should be particularly useful in elucidating the molecular mechanisms of pathogen-triggered host cell invasion.     

Bacterial pili: molecular mechanisms of pathogenesis

Gram-negative bacteria produce a diverse array of pili that mediate microbe-microbe and host-pathogen interactions important in the development of disease. The structural and functional characterization of these organelles, particularly their role in triggering signals in both the bacterium and the host upon attachment, has begun to reveal the molecular mechanisms of bacterial diseases.     

Evaluation of interactions between strains of S. aureus isolated from different clinical specimens with peripheral blood phagocytic cells

The aim of this study was to investigate the interactions occurring between peripheral blood phagocytes and strains of S. aureus isolated from different clinical specimens (blood, respiratory tract, pus). To evaluate the sensitivity of microorganisms to bactericidal activity of phagocytes, monocytes and granulocytes separated from peripheral blood by standard density gradient and by counter-current centrifugal elutriation were incubated with suspensions of opsonized bacteria. In parallel, the viability of phagocytes was examined by flow cytometry, and the ability of bacteria to trigger reactive oxygen intermediates (ROI) production was evaluated by chemiluminescence measurement. To investigate efficiency of phagocytosis, bacteria were labelled with fluorescein isothiocyanate (FITC) and the percentage of cells containing FITC-labelled bacteria was analysed by flow cytometry. The data obtained show that strains of S. aureus originated from different clinical specimens, differ in their sensitivity to bactericidal activity of phagocytes--strains isolated from the blood show the highest, but strains isolated from respiratory tract show the lowest sensitivity for killing. These strains differ too in their ability to trigger monocyte CL response. Contrary, there was no difference in toxicity of bacteria against phagocytes. Strains isolated from peripheral blood showed significant negative correlation between the ability to trigger CL response and toxicity against phagocytes.