The Leopoldina international symposium on parasitism,commensalism and symbiosis--common themes,different outcome

The development of new methods, including genomics, which can even be applied to unculturable microorganisms, has significantly increased our knowledge about bacterial pathogenesis and symbiosis and, in consequence, is profoundly modifying our views on the evolution and the genetic and physiological basis of bacteria-host interactions. The presentations at this symposium revealed conceptual links between bacterial pathogenesis and symbiosis. The close co-operation of experts in both fields will result in significant synergy and new insights into basic mechanisms of bacteria-host interactions and their evolution. The meeting provided fascinating news about the genetic and metabolic consequences that the change in their lifestyle had for bacteria that developed from free-living to permanent host-associated organisms exemplified by intracellular pathogens or symbionts. In addition, surprising similarities but also striking differences between the strategies involved in the establishment of a symbiotic versus a parasitic lifestyle can be noted. In the long run, the characterization of such differences might lead to lifestyle prediction or to an evaluation of the pathogenic potential of newly isolated bacteria via the definition of genetic and/or metabolic signatures characteristic for pathogenic or symbiotic organisms. Moreover, it is expected that these investigations will lead to new strategies for the treatment or prevention of bacterial infections, or the avoidance of pathogen transmission.     

Origin of the bacterial SET domain genes: vertical or horizontal?

The presence of Supressor of variegation-Enhanser of zeste-Trithorax (SET) domain genes in bacteria is a current paradigm for lateral genetic exchange between eukaryotes and prokaryotes. Because a major function of SET domain proteins is the chemical modification of chromatin and bacteria do not have chromatin, there is no apparent functional requirement for the existence of bacterial SET domain genes. Consequently, their finding in only a small fraction of pathogenic and symbiotic bacteria was taken as evidence that bacteria have obtained the SET domain genes from their hosts. Furthermore, it was proposed that the products of the genes would, most likely, be involved in bacteria-host interactions. The broadened scope of sequenced bacterial genomes to include also free-living and environmental species provided a larger sample to analyze the bacterial SET domain genes. By phylogenetic analysis, examination of individual chromosomal regions for signs of insertion, and evaluating the chromosomal versus SET domain genes' GC contents, we provide evidence that SET domain genes have existed in the bacterial domain of life independently of eukaryotes. The bacterial genes have undergone an evolution of their own unconnected to the evolution of the eukaryotic SET domain genes. Initial finding of SET domain genes in predominantly pathogenic and symbiotic bacteria resulted, most probably, from a biased sample. However, a lateral transfer of SET domain genes may have occurred between some bacteria and a family of Archaea. A model for the evolution and distribution of SET domain genes in bacteria is proposed.     

Large-scale identification of bacteria-host crosstalk by affinity chromatography: capturing the interactions of Streptococcus suis proteins with host cells

Protein-protein interactions between bacteria and their hosts are responsible for all types of infection processes. The investigation of the bacteria-host crosstalk can provide a comprehensive understanding of the pathogenesis of bacterial disease. Despite scattered efforts in this field, a systematic identification of interactions between host and bacterial proteins remains unavailable. Here, we develop ACSP (affinity chromatography-based surface proteomics), which combines affinity chromatography and shotgun proteomics (LC-MS/MS), to investigate the interactions on a large-scale. Using ACSP, the potential surface interacting proteins (SIPs) of Streptococcus suis serotype 2 (SS2) were captured by the chromatographic resin, which was immobilized with the native surface molecules of Hep-2 cells. And then 40 potential SIPs were identified from the preys by LC-MS/MS, including 3 SIPs that have been previously reported in the literature. We selected 8 important SIPs and confirmed their ability to adhere to Hep-2 cells. Additionally, 3 newly identified SIPs, or their polyclonal antibodies, were found to significantly inhibit the adherence of SS2 to Hep-2 cells, indicating their essential role in the interaction between SS2 and Hep-2 cells. Using this example, we show that ACSP represents a new valuable tool for investigating the bacteria-host interactions.     

Interactions between bacteria and eukaryotes via small molecules

The interactions that occur between eukaryotes and bacteria have long been of interest, as knowledge of these processes could lead to the development of novel therapeutics and other potential applications in biotechnology. Many of these interactions are mediated by small molecules, which have subsequently formed the focus of numerous studies. An arsenal of small molecules exhibiting a wide range of activities has been isolated from various sources, including plants, animals and microorganisms. As a number of these compounds are pharmacologically active, there is a strong continued interest in natural product chemistry. Recent developments in this field have focused on two areas: evidence has been gathered to show that secondary metabolites are often produced by symbiotic bacteria, rather than by the eukaryotic host, and the importance of bacterial cell-to-cell signalling in bacteria-host interactions has been confirmed.     

Clathrin phosphorylation is required for actin recruitment at sites of bacterial adhesion and internalization

Bacterial pathogens recruit clathrin upon interaction with host surface receptors during infection. Here, using three different infection models, we observed that host-pathogen interactions induce tyrosine phosphorylation of clathrin heavy chain. This modification was critical for recruitment of actin at bacteria-host adhesion sites during bacterial internalization or pedestal formation. At the bacterial interface, clathrin assembled to form coated pits of conventional size. Because such structures cannot internalize large particles such as bacteria, we propose that during infection, clathrin-coated pits serve as platforms to initiate actin rearrangements at bacteria-host adhesion sites. We then showed that the clathrin-actin interdependency is initiated by Dab2 and depends on the presence of clathrin light chain and its actin-binding partner Hip1R, and that the fully assembled machinery can recruit Myosin VI. Together, our study highlights a physiological role for clathrin heavy chain phosphorylation and reinforces the increasingly recognized function of clathrin in actin cytoskeletal organization in mammalian cells.     

Bacterial virulence factors targeting Rho GTPases: parasitism or symbiosis?

In the past few years, an important question in microbiology has arisen from reports indicating that several pathogenic bacteria have evolved virulence factors directed towards a Ras subfamily of GTPases, namely the Rho GTPases. Progress made in studying both the virulence factors and the signaling pathways involving Rho GTPases has shed light on this crosstalk. One central question is raised by the findings that both activating and inactivating virulence factors that target Rho GTPases coexist in some pathogenic bacteria. Further studies on this peculiar aspect of the bacteria-host cell interactions, which leads to the outbreak of infectious diseases, might clarify whether this aspect of Rho GTPase activation or inactivation represents a finely adapted response of the pathogen for its own benefit or might lead to a reaction of the host against the bacteria.     

Insights into early mycobacterial pathogenesis from the zebrafish

Here we discuss the application of the zebrafish as a relatively new model host for the study of mycobacterial pathogenesis. Recent advances in our understanding of host-mycobacteria interactions from the zebrafish include insights into the role of the innate immune system in both controlling and facilitating infection. Analysis in the zebrafish has revealed that innate macrophages restrict initial bacterial growth, but also convey infecting bacteria into the granuloma, which serves as a place for bacterial growth and spread. Bacterial virulence determinants interact with these processes at different steps in pathogenesis, which can be dissected in these living see-through hosts. As these studies uncover new facets of the bacteria-host interactions in tuberculosis they raise even more questions for future investigation.     

Bacterial bug-out bags: outer membrane vesicles and their proteins and functions

Among the major bacterial secretions, outer membrane vesicles (OMVs) are significant and highly functional. The proteins and other biomolecules identified within OMVs provide new insights into the possible functions of OMVs in bacteria. OMVs are rich in proteins, nucleic acids, toxins and virulence factors that play a critical role in bacteria-host interactions. In this review, we discuss some proteins with multifunctional features from bacterial OMVs and their role involving the mechanisms of bacterial survival and defence. Proteins with moonlighting activities in OMVs are discussed based on their functions in bacteria. OMVs harbour many other proteins that are important, such as proteins involved in virulence, defence, and competition. Overall, OMVs are a power-packed aid for bacteria, harbouring many defensive and moonlighting proteins and acting as a survival kit in case of an emergency or as a defence weapon. In summary, OMVs can be defined as bug-out bags for bacterial defence and, therefore, survival.     

Photorhabdus: towards a functional genomic analysis of a symbiont and pathogen

Pathogenicity and symbiosis are central to bacteria-host interactions. Although several human pathogens have been subjected to functional genomic analysis, we still understand little about bacteria-invertebrate interactions despite their ecological prevalence. Advances in our knowledge of this area are often hindered by the difficulty of isolating and working with invertebrate pathogenic bacteria and their hosts. Here we review studies on pathogenicity and symbiosis in an insect pathogenic bacterium Photorhabdus and its entomopathogenic nematode vector and model insect hosts. Whilst switching between these hosts, Photorhabdus changes from a state of symbiosis with its nematode vector to one of pathogenicity towards its new insect host and both the bacteria and the nematode then cooperatively exploit the dying insect. We examine candidate genes involved in symbiosis and pathogenicity, their secretion and expression patterns in culture and in the host, and begin to dissect the extent of their genetic coregulation. We describe the presence of several large genomic islands, putatively involved in pathogenicity or symbiosis, within the otherwise Yersinia-like backbone of the Photorhabdus genome. Finally, we examine the emerging comparative genomics of the Photorhabdus group and begin to describe the interrelationship between anti-invertebrate virulence factors and those used against vertebrates.     

Insights into bacterial protein glycosylation in human microbiota

The study of human microbiota is an emerging research topic. The past efforts have mainly centered on studying the composition and genomic landscape of bacterial species within the targeted communities. The interaction between bacteria and hosts is the pivotal event in the initiation and progression of infectious diseases. There is a great need to identify and characterize the molecules that mediate the bacteria-host interaction. Bacterial surface exposed proteins play an important role in the bacteria- host interaction. Numerous surface proteins are glycosylated, and the glycosylation is crucial for their function in mediating the bacterial interaction with hosts. Here we present an overview of surface glycoproteins from bacteria that inhabit three major mucosal environments across human body: oral, gut and skin. We describe the important enzymes involved in the process of protein glycosylation, and discuss how the process impacts the bacteria-host interaction. Emerging molecular details underlying glycosylation of bacterial surface proteins may lead to new opportunities for designing anti-infective small molecules, and developing novel vaccines in order to treat or prevent bacterial infection.     

细菌外膜囊泡(OMV)研究进展

外膜囊泡(outer membrane vesicles,OMV)是在细菌生命活动中不断从细菌细胞表面脱离而形成的功能性囊泡,其内部含有蛋白质、脂质和核酸等成分,具有多种特殊的生物学功能,在细菌-细菌和细菌-宿主相互作用中起着关键作用.虽然大多数OMV的研究都是关于动物病原菌,但最近OMV在植物-细菌相作领域的作用已逐...     

Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection

Nod2 activates the NF-kappaB pathway following intracellular stimulation by bacterial products. Recently, mutations in Nod2 have been shown to be associated with Crohn's disease, suggesting a role for bacteria-host interactions in the etiology of this disorder. We show here that Nod2 is a general sensor of peptidoglycan through the recognition of muramyl dipeptide (MDP), the minimal bioactive peptidoglycan motif common to all bacteria. Moreover, the 3020insC frameshift mutation, the most frequent Nod2 variant associated with Crohn's disease patients, fully abrogates Nod2-dependent detection of peptidoglycan and MDP. Together, these results impact on the understanding of Crohn's disease development. Additionally, the characterization of Nod2 as the first pathogen-recognition molecule that detects MDP will help to unravel the well known biological activities of this immunomodulatory compound.     

The use of transepithelial models to examine host-pathogen interactions

Many pathogens must overcome an epithelial barrier in order to establish an infection. Unsurprisingly, such pathogens have evolved different mechanisms to overcome this obstacle, targeting specific epithelial structures or functions. These include disruption of epithelial barrier function, transcytosing from the apical to the basolateral membrane domain or inducing cell movement such as neutrophil recruitment. When studying these processes in vivo, animal models often fail to mimic the disease observed in humans and present a complex system in which many variables cannot be controlled. Therefore, in vitro transepithelial models that permit the study of a relevant biological surface have been developed, to integrate not only interactions between bacteria and epithelial cells but also, under certain conditions, to integrate a third cell type, such as neutrophils or dendritic cells. Such models are particularly useful for studying the bacteria-host relationship as it would occur in the microenvironment of the human epithelium and have enhanced our understanding of the unique strategies by which pathogenic bacteria exploit host cells to overcome the initial epithelial hurdle.     

Interactions between Mytilus haemocytes and different strains of Escherichia coli and Vibrio cholerae O1 El Tor: role of kinase-mediated signalling

Marine bivalves accumulate large amounts of bacteria from the environment (mainly Vibrionaceae and coliforms). Although persistence of different bacteria in bivalve tissues largely depends on their sensitivity to the bactericidal activity of circulating haemocytes and haemolymph soluble factors, the mechanisms involved in bacteria-host cell interactions in these invertebrates are largely unknown. In the mussel Mytilus, differences in interactions between haemocytes and different Escherichia coli and Vibrio cholerae strains [E. coli MG155, a wild-type strain carrying type 1 fimbriae, and its unfimbriated derivative, AAEC072 Deltafim; V. cholerae O1 El Tor biotype strain N16961, carrying the mannose-sensitive haemagglutinin (MSHA), and its MSHA mutant] lead to differences in bactericidal activity in the presence of serum. Here we show that different bacteria induced distinct patterns of phosphorylation of mitogen-activated protein kinases (MAPKs), in particular of the stress-activated MAPKs involved in the immune response. Differences in phosphorylation of PKC-like proteins were also observed. The results support the hypothesis that, like in mammalian host cells, different bacteria can modulate the signalling pathways of mussel haemocytes. The lower anti-bacterial activity towards the mutant E. coli strain and wild-type V. cholerae compared with wild E. coli may result from a reduced capacity of activating MAPKs. Moreover, the mutant V. cholerae strain that was the most resistant to the haemocyte bactericidal activity induced downregulation of cell signalling and showed the strongest effect on lysosomal membrane stability, evaluated as a marker of bivalve cell stress. These data suggest that certain bacteria could evade the bactericidal activity of mussel haemocytes through disruption of the host signalling pathways.     

Common molecular mechanisms of symbiosis and pathogenesis

Traditionally, symbiotic and pathogenic interactions were considered different manifestations of the bacteria-host interaction. However, the molecular mechanisms that mediate communication between and cellular modulation of the involved partners are quite similar. With this review we aim to contribute to a reduction of the traditional gap between symbiosis and pathogenesis research.     

Endocytosymbiosis of microorganisms in mammal eukaryotic cells and factors of its regulation

Morphofunctional equivalents of the process of long-term intracellular prokaryotes--eukaryotes interaction were studied by light and electron microscopy. The mechanisms for adaptation, elaborated in the course of evolution of bacteria-host interaction, were analysed on the ultrastructural level. A concept on the role of hypothalamic nonapeptides, as factors of regulation of intracellular persistence and symbiosis of prokaryotes, is discussed.     

ClpV, a unique Hsp100/Clp member of pathogenic proteobacteria

Hsp100/Clp proteins are key players in the protein quality control network of prokaryotic cells and function in the degradation and refolding of misfolded or aggregated proteins. Here we report the identification of a new class of Hsp100/Clp proteins, termed ClpV (virulent strain), that are present in bacteria interacting with eukaryotic cells, including human pathogens. The ClpV proteins are most similar to ClpB proteins within the Hsp100/Clp family, but cluster in a separate phylogenetic tree with a remarkable distance to ClpB. ClpV representatives from Salmonella typhimurium and enteropathogenic Escherichia coli form oligomeric assemblies and display ATP hydrolysis rates comparable to ClpB. However, unlike ClpB, both ClpV proteins failed to solubilize aggregated proteins. This lack of disaggregation activity correlated with the inability of ClpB model substrates to stimulate the ATPase activity of ClpV proteins, indicating differences in substrate selection. Furthermore, we show that clpV genes are generally organized in a conserved gene cluster, encoding a potential secretion system, and we demonstrate that increased levels of a dominant negative variant of either S. typhimurium or Yersinia pseudotuberculosis ClpV strongly reduce the ability of these pathogenic bacteria to invade epithelial cells. We propose a role of this novel and unique class of AAA+ proteins in bacteria-host cell interactions.     

Characterization of a sialic acid- and P-selectin glycoprotein ligand-1-independent adhesin activity in the granulocytotropic bacterium Anaplasma phagocytophilum

Anaplasma phagocytophilum, the aetiologic agent of human granulocytic anaplasmosis, is an obligate intracellular bacterium that colonizes neutrophils and neutrophil precursors. The granulocytotropic bacterium uses multiple adhesins that cooperatively bind to the N-terminal region of P-selectin glycoprotein ligand-1 (PSGL-1) and to sialyl Lewis x (sLe(x)) expressed on myeloid cell surfaces. Recognition of sLe(x) occurs through interactions with alpha2,3-sialic acid and alpha1,3-fucose. It is unknown whether other bacteria-host cell interactions are involved. In this study, we have enriched for A. phagocytophilum organisms that do not rely on sialic acid for cellular adhesion and entry by maintaining strain NCH-1 in HL-60 cells that are severely undersialylated. The selected bacteria, termed NCH-1A, also exhibit lessened dependencies on PSGL-1 and alpha1,3-fucose. Optimal adhesion and invasion by NCH-1A require interactions with the known determinants (sialic acid, PSGL-1 and alpha1,3-fucose), but none of them is absolutely necessary. NCH-1A binding to sLe(x)-modified PSGL-1 requires recognition of the known determinants in the same manners as other A. phagocytophilum strains. These data suggest that A. phagocytophilum expresses a separate adhesin from those targeting sialic acid, alpha1,3-fucose and the N-terminal region of PSGL-1. We propose that NCH-1A upregulates expression of this adhesin.     

Properties of probiotic bacteria explored by proteomic approaches

The study of health-beneficial effects that probiotic bacteria can exert on humans and animals is at its beginning. Pending scientific questions include the identification of molecular markers of the health-promoting activity of specific strains, which may be used to select novel probiotic strains and to gain understanding of the mechanisms underlying their effects. In that perspective, the role of bacterial proteins must be evaluated, placing proteomics-based approaches at the core of the field. Until now, most proteomic analyses focused on the dynamics of abundant cytoplasmic proteins during adaptation of bacteria to conditions mimicking the gastro-intestinal tract environment. The development of in silico and experimental procedures allowing identification and quantification of surface-exposed and secreted proteins should boost our understanding of bacteria-host crosstalk.     

Gram-negative bacteria aggravate murine small intestinal Th1-type immunopathology following oral infection with Toxoplasma gondii

Oral infection of susceptible mice with Toxoplasma gondii results in Th1-type immunopathology in the ileum. We investigated gut flora changes during ileitis and determined contributions of gut bacteria to intestinal inflammation. Analysis of the intestinal microflora revealed that ileitis was accompanied by increasing bacterial load, decreasing species diversity, and bacterial translocation. Gram-negative bacteria identified as Escherichia coli and Bacteroides/Prevotella spp. accumulated in inflamed ileum at high concentrations. Prophylactic or therapeutic administration of ciprofloxacin and/or metronidazole ameliorated ileal immunopathology and reduced intestinal NO and IFN-gamma levels. Most strikingly, gnotobiotic mice in which cultivable gut bacteria were removed by quintuple antibiotic treatment did not develop ileitis after Toxoplasma gondii infection. A reduction in total numbers of lymphocytes was observed in the lamina propria of specific pathogen-free (SPF), but not gnotobiotic, mice upon development of ileitis. Relative numbers of CD4(+) T cells did not differ in naive vs infected gnotobiotic or SPF mice, but infected SPF mice showed a significant increase in the frequencies of activated CD4(+) T cells compared with gnotobiotic mice. Furthermore, recolonization with total gut flora, E. coli, or Bacteroides/Prevotella spp., but not Lactobacillus johnsonii, induced immunopathology in gnotobiotic mice. Animals recolonized with E. coli and/or total gut flora, but not L. johnsonii, showed elevated ileal NO and/or IFN-gamma levels. In conclusion, Gram-negative bacteria, i.e., E. coli, aggravate pathogen-induced intestinal Th1-type immunopathology. Thus, pathogen-induced acute ileitis may prove useful to study bacteria-host interactions in small intestinal inflammation and to test novel therapies based on modulation of gut flora.