Co-evolution and exploitation of host cell signaling pathways by bacterial pathogens

Bacterial pathogens have evolved by combinations of gene acquisition, deletion, and modification, which increases their fitness. Additionally, bacteria are able to evolve in "quantum leaps" via the ability to promiscuously acquire new genes. Many bacterial pathogens - especially Gram-negative enteric pathogens - have evolved mechanisms by which to subvert signal transduction pathways of eukaryotic cells by expressing genes that mimic or regulate host protein factors involved in a variety of signaling cascades. This results in the ability to cause diseases ranging from tumor formation in plants to gastroenteritis and bubonic plague. Here, we present recent advances on mechanisms of bacterial pathogen evolution, including specific signaling cascades targeted by their virulence genes with an emphasis on the ubiquitin modification system, Rho GTPase regulators, cytoskeletal modulators, and host innate immunity. We also comment briefly on evolution of host defense mechanisms in place that limit disease caused by bacterial pathogens.     

Leishmania donovani Infection Causes Distinct Epigenetic DNA Methylation Changes in Host Macrophages

Infection of macrophages by the intracellular protozoan Leishmania leads to down-regulation of a number of macrophage innate host defense mechanisms, thereby allowing parasite survival and replication. The underlying molecular mechanisms involved remain largely unknown. In this study, we assessed epigenetic changes in macrophage DNA methylation in response to infection with L. donovani as a possible mechanism for Leishmania driven deactivation of host defense. We quantified and detected genome-wide changes of cytosine methylation status in the macrophage genome resulting from L. donovani infection. A high confidence set of 443 CpG sites was identified with changes in methylation that correlated with live L. donovani infection. These epigenetic changes affected genes that play a critical role in host defense such as the JAK/STAT signaling pathway and the MAPK signaling pathway. These results provide strong support for a new paradigm in host-pathogen responses, where upon infection the pathogen induces epigenetic changes in the host cell genome resulting in downregulation of innate immunity thereby enabling pathogen survival and replication. We therefore propose a model whereby Leishmania induced epigenetic changes result in permanent down regulation of host defense mechanisms to protect intracellular replication and survival of parasitic cells.     

Genomic analysis of immunity in a Urochordate and the emergence of the vertebrate immune system: “waiting for Godot”

Genome-wide sequence analysis in the invertebrate chordate, Ciona intestinalis, has provided a comprehensive picture of immune-related genes in an organism that occupies a key phylogenetic position in vertebrate evolution. The pivotal genes for adaptive immunity, such as the major histocompatibility complex (MHC) class I and II genes, T-cell receptors, or dimeric immunoglobulin molecules, have not been identified in the Ciona genome. Many genes involved in innate immunity have been identified, including complement components, Toll-like receptors, and the genes involved in intracellular signal transduction of immune responses, and show both expansion and unexpected diversity in comparison with the vertebrates. In addition, a number of genes were identified which predicted integral membrane proteins with extracellular C-type lectin or immunoglobulin domains and intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and immunoreceptor tyrosine-based activation motifs (ITAMs) (plus their associated signal transduction molecules), suggesting that activating and inhibitory receptors have an MHC-independent function and an early evolutionary origin. A crucial component of vertebrate adaptive immunity is somatic diversification, and the recombination activating genes (RAG) and activation-induced cytidine deaminase (AID) genes responsible for the Generation of diversity are not present in Ciona. However, there are key V regions, the essential feature of an immunoglobulin superfamily VC1-like core, and possible proto-MHC regions scattered throughout the genome waiting for Godot.     

Identification of immunostimulatory DNA-induced genes by suppression subtractive hybridization

Bacterial DNA and related synthetic immunostimulatory oligodeoxyribo-nucleotides (ISS-ODN) have stimulatory effects on mammalian immune cells through a Toll-like receptor, TLR9. Genes upregulated in ISS-ODN-stimulated immune cells are obviously significant to delineate the mechanism of the induced innate immunity. Employing suppression subtractive hybridization (SSH), we have generated a profile of genes induced by ISS-ODN in spleen cells. Sequencing of 87 clones isolated by the SSH showed 39 clones corresponding to known mouse genes in the public database. Eleven clones appeared to possess 80-90% homology with known mouse genes and the remaining 37 clones showed no significant homology with any known mouse genes. A series of known genes which have not previously been reported to be induced with ISS-ODN were confirmed to be induced in ISS-ODN-stimulated bone marrow-derived macrophages: NF-kappaB p105, IRF-1, PA28beta, IRG2, and MyD88. These genes were suggested to be involved in the molecular process of innate host defense mechanisms.     

Serratia marcescens Induces Apoptotic Cell Death in Host Immune Cells via a Lipopolysaccharide- and Flagella-dependent Mechanism

Injection of Serratia marcescens into the blood (hemolymph) of the silkworm, Bombyx mori, induced the activation of c-Jun NH₂-terminal kinase (JNK), followed by caspase activation and apoptosis of blood cells (hemocytes). This process impaired the innate immune response in which pathogen cell wall components, such as glucan, stimulate hemocytes, leading to the activation of insect cytokine paralytic peptide. S. marcescens induced apoptotic cell death of silkworm hemocytes and mouse peritoneal macrophages in vitro. We searched for S. marcescens transposon mutants with attenuated ability to induce apoptosis of silkworm hemocytes. Among the genes identified, disruption mutants of wecA (a gene involved in lipopolysaccharide O-antigen synthesis), and flhD and fliR (essential genes in flagella synthesis) showed reduced motility and impaired induction of mouse macrophage cell death. These findings suggest that S. marcescens induces apoptosis of host immune cells via lipopolysaccharide- and flagella-dependent motility, leading to the suppression of host innate immunity.     

Analysis of signal-dependent changes in the proteome of Drosophila blood cells during an immune response

Innate immunity is based on the recognition of cell-surface molecules of infecting agents. Microbial substances, such as peptidoglycan, lipopolysaccharide, and beta-1,3-glucans, produce functional responses in Drosophila hemocytes that contribute to innate immunity. We have used two-dimensional gel electrophoresis and MS to resolve lipopolysaccharide-induced changes in the protein profile of a Drosophila hemocytic cell line. We identified 24 intracellular proteins that were up- or down-regulated, or modified, in response to immune challenge. Several proteins with predicted immune functions, including lysosomal proteases, actin-binding/remodeling proteins, as well as proteins involved in cellular responses to oxidative stress, were affected by the immune assault. Intriguingly, a number of the proteins identified in this study have recently been implicated in phagocytosis in higher vertebrates. We suggest that phagocytosis is activated in Drosophila hemocytes by the presence of microbial substances, and that this activation constitutes an evolutionarily conserved arm of innate immunity. In addition, a number of proteins involved in calcium-regulated signaling, mRNA processing, and nuclear transport were affected, consistent with a possible role in reprogramming of gene expression. In conclusion, the present proteome analysis identified many proteins previously not linked to innate immunity, demonstrating that differential protein profiling of Drosophila hemocytes is a valuable tool for identification of new players in immune-related cellular processes.     

水稻免疫机制研究进展

植物先天免疫主要由两部分组成：一类是通过细胞膜上的病原菌分子模式识别受体识别病原微生物表面存在的分子特征激发的免疫反应（PTI）;另一类是专化性的抗病R蛋白识别病原微生物的效应蛋白,从而激发下游的病原菌小种特异性的防卫反应过程（ETI）．随着水稻抗病信号途径中越来越多的抗病基因以及关键的调控基因被克隆和功能鉴定,同时多种水稻病原菌效应蛋白的发现,水稻抗病机理的研究也越来越深入．本文阐述了水稻的PTI,ETI及其下游参与免疫信号转导的关键性组分,从而形成一个初步的水稻免疫调控网络．     

A galectin of unique domain organization from hemocytes of the Eastern oyster (Crassostrea virginica) is a receptor for the protistan parasite Perkinsus marinus

Invertebrates display effective innate immune responses for defense against microbial infection. However, the protozoan parasite Perkinsus marinus causes Dermo disease in the eastern oyster Crassostrea virginica and is responsible for catastrophic damage to shellfisheries and the estuarine environment in North America. The infection mechanisms remain unclear, but it is likely that, while filter feeding, the healthy oysters ingest P. marinus trophozoites released to the water column by the infected neighboring individuals. Inside oyster hemocytes, trophozoites resist oxidative killing, proliferate, and spread throughout the host. However, the mechanism(s) for parasite entry into the hemocyte are unknown. In this study, we show that oyster hemocytes recognize P. marinus via a novel galectin (C. virginica galectin (CvGal)) of unique structure. The biological roles of galectins have only been partly elucidated, mostly encompassing embryogenesis and indirect roles in innate and adaptive immunity mediated by the binding to endogenous ligands. CvGal recognized a variety of potential microbial pathogens and unicellular algae, and preferentially, Perkinsus spp. trophozoites. Attachment and spreading of hemocytes to foreign surfaces induced localization of CvGal to the cell periphery, its secretion and binding to the plasma membrane. Exposure of hemocytes to Perkinsus spp. trophozoites enhanced this process further, and their phagocytosis could be partially inhibited by pretreatment of the hemocytes with anti-CvGal Abs. The evidence presented indicates that CvGal facilitates recognition of selected microbes and algae, thereby promoting phagocytosis of both potential infectious challenges and phytoplankton components, and that P. marinus subverts the host's immune/feeding recognition mechanism to passively gain entry into the hemocytes.     

Identification and functional analysis of antifungal immune response genes in Drosophila

Essential aspects of the innate immune response to microbial infection appear to be conserved between insects and mammals. Although signaling pathways that activate NF-kappaB during innate immune responses to various microorganisms have been studied in detail, regulatory mechanisms that control other immune responses to fungal infection require further investigation. To identify new Drosophila genes involved in antifungal immune responses, we selected genes known to be differentially regulated in SL2 cells by microbial cell wall components and tested their roles in antifungal defense using mutant flies. From 130 mutant lines, sixteen mutants exhibited increased sensitivity to fungal infection. Examination of their effects on defense against various types of bacteria and fungi revealed nine genes that are involved specifically in defense against fungal infection. All of these mutants displayed defects in phagocytosis or activation of antimicrobial peptide genes following infection. In some mutants, these immune deficiencies were attributed to defects in hemocyte development and differentiation, while other mutants showed specific defects in immune signaling required for humoral or cellular immune responses. Our results identify a new class of genes involved in antifungal immune responses in Drosophila.     

Host defense against oropharyngeal and vaginal candidiasis: Site-specific differences

Mucosal candidiasis is extremely common in immunocompromised patients. However, the prevalence of site-specific infection (i.e., oropharyngeal, vaginal, and esophageal candidiasis) can be quite variable depending on the immune status of the host. While vulvovaginal candidiasis is common in normal healthy women, oropharyngeal and esophageal candidiasis are more frequently encountered under immunocompromised states. Candida albicans, the causative agent in most cases of candidiasis, is a commensal organism of the gastrointestinal and lower female reproductive tracts. Thus, most healthy individuals have demonstrable Candida-specific immunity in the peripheral circulation. The pathogenic state is often precipitated by a deficiency or dysfunction in this immunity. Studies from animal models, women with recurrent vulvovaginal candidiasis, and HIV-infected individuals, however, suggest that distinct host defense mechanisms may function against oropharyngeal and vulvovaginal candidiasis. While cell-mediated immunity (CMI) appears important for protection against oropharyngeal candidiasis (OPC), there is little evidence to indicate that T cell-mediated immunity is protective against vulvovaginal candidiasis (VVC). Furthermore, whereas both local and systemically derived immune defenses appear important for protection against OPC, host defenses that protect against VVC appear limited to the local tissue and possibly restricted to innate mechanisms. Thus, current evidence suggests that VVC, unlike OPC, may not represent a strict opportunistic infection.     

Specific expression of inflammatory genes in macrophage subpopulations

Macrophages serve as an effective component of innate immunity in their ability to recognize, engulf and kill potential pathogens. They also coordinate additional host responses by synthesizing a range of inflammatory mediators that can activate the adaptive immune response and establish protective immunity. Although they are a key component of mammalian defense system, macrophage activity is not always beneficial to the host. The centrality of macrophages in disease processes makes macrophage regulation a major target in the prevention, control and cure of inflammatory processes. Consequently, macrophage-restricted genes may be crucial targets for therapeutic intervention. A review is presented of the use of large-scale cDNA microarrays to compare macrophage inflammatory genes differentially expressed in two distinct macrophages populations--bone marrow derived macrophages (bmm) and inflammatory thioglycolate-elicited peritoneal macrophages (tepm)--to non-macrophage cell populations consisting of primary embryonic fibroblast and spleen non-adherent cells. Expression profiles indicate that macrophage inflammatory genes are associated with expected functional categories, such as lysosomal degradation, phagocytosis, host defense and homeostasis.     

Physiological functions of hemocytes newly emerged from the cultured hematopoietic organs in the silkworm, Bombyx mori

Abstract  Cellular immunity is a very important part of insect innate immunity. It is not clear if hemocytes entering the hemolymph require a maturation process to become competent. The establishment of a tissue culture system for the insect hematopoietic organs would enable physiological function assays with hemocytes newly emerged from hematopoietic organs. To this end, we established a hematopoietic organ culture system for the purebred silkworm pnd pS and then studied the physiological functions of the newly emerged hemocytes. We found that Grace's medium supplemented with 10% heated silkworm larval plasma was better for culturing the hematopoietic organs of pnd pS . Newly emerged hemocytes phagocytosed propidium iodide-labeled bacteria and encapsulated the Iml-2 coated nickel beads as well as pupal tissue debris. This culture system is therefore capable of generating physiologically functional hemocytes. These hemocytes can be used to study the mechanisms of the hemocyte immune response among others.