The molecular and cellular basis of infection—Perspectives from the first advanced summer school in Africa (2008)

Bacteria, viruses and parasites are ubiquitous in our biosphere and as such we encounter numerous potential pathogens routinely in our everyday lives. For many of these organisms, the human being offers the ideal host environment with nearly limitless resources that can be used for energy production, proliferation and subsequent spread. Luckily for us, getting into the human body is not an easy task! Evolution, as it would be, has provided us with numerous mechanisms to resist pathogen invasion both through physical barriers and our multifactorial immune system. However, in spite of our fantastic defenses, hostile invaders still manage to establish infections. The molecular mechanisms that underpin these events have been the subject of intense study for many decades and also served as the central focus of a summer school in Africa. During the first week of March 2008, Hermanus, a small sleepy town in South Africa better known for whale watching, served as the venue for a summer school on the molecular and cellular basis of infection, where 50 students, PhDs and postdoctoral researchers, from around the world were assembled along with prominent researchers in field of viral, bacterial and parasite pathogenesis. This school, the first of its kind in Africa, was sponsored by the European Molecular Biology Organization (EMBO), the Federation of European Biochemical Societies (FEBS), the International Centre for Genetic Engineering and Biotechnology (ICGEB), the International Union of Biochemistry and Molecular Biology (IUBMB), the United Nations Conference on Trade and Development and United Nations Educational, Scientific and Cultural Organization (UNESCO) and the Pasteur Institute. Three main themes were extensively discussed and debated during this meeting: host mimicry and implications for pathogen entry and virulence; the molecular mechanisms of pathogen recognition by the innate immune system and subversion of these mechanisms by microorganisms; and finally the use of genomics to further understand pathogenesis. This report serves to summarize the scientific findings that were discussed and resulting implications for infection. For over a century, pathogens have been the subject of extensive studies and the close observation of microbes has revealed that viruses, bacteria and parasites possess incredibly diverse and resourceful mechanisms for invasion and survival within the host cell. Surprisingly, these organisms have evolved mechanisms of camouflage, hijacking and sabotage that can be modulated in response to the different environmental conditions they encounter. It has also become increasingly clear that many microorganisms have adapted for optimal pathogenesis in their respective hosts and consequently they can be used as “undercover agents” to understand mechanisms of host cell invasion and subsequent disease. These ideas and the resulting data were discussed at the summer school mainly by four researchers: Pascale Cossart and Hélène Bierne from the Pasteur Institute in Paris, France, Matthew Welch from Stanford University in California and Lucas Pelkmans from the Eidgenössische Technische Hochschule in Zurich, Switzerland. Pascale Cossart provided an exhaustive overview on the state of the art in Listeria-host interactions and the use of Listeria monocytogenes as a multifaceted model system. This bacterium is able to cross multiple host barriers during infection mainly due to the two major bacterial surface proteins, the internalins, InlA and InlB. These proteins mediate Listeria internalization in nonphagocytic cells through interaction with E-cadherin and the hepatocyte growth factor (HGF) receptor on the host cell surface. Dr. Cossart discussed studies on the mechanisms of InlA/E-cadherin-mediated bacterial internalization which showed that Listeria mimics the formation of adherent junctions for its internalization and more importantly, that E-cadherin is necessary to allow the bacterium to cross the intestinal barrier (1). The study of the InlB internalization pathway of Listeria revealed that the bacterium is internalized within host cells through a clathrin-dependent pathway involving the ubiquitination of the Met receptor, similarly to challenge with HGF, the natural ligand of Met (1). More recently, Cossart and co workers showed that clathrin-mediated endocytosis of Listeria is not an exception but is the preferred internalization pathway for a number of zippering bacteria (2). Dr. Cossart also discussed new findings regarding the Listeria pore-forming toxin, which is able to induce modifications in the levels of phosphorylation of host cell histones, resulting in transcriptional changes independently of bacterial internalization (3). Continuing with the same theme, Hélène Bierne discussed the recent identification of a listerial secreted protein likely involved in the modulation of host gene expression by hijacking gene silencing complexes. She extended the discussion to several other bacterial effectors shown to manipulate host cell nuclear processes, that is, gene expression and the cell cycle. For instance, the secreted OspF protein from Shigella flexneri translocates to the nucleus and induces histone dephosphorylation that eventually results in repression of NF-κB-regulated genes. Several Gram negative bacteria secrete cyclomodulins, a growing family of bacterial toxins and effectors that interfere with the eukaryotic cell cycle. Dr. Bierne also discussed the case of Agrobacterium, the only known bacterium capable of natural DNA transfer into a eukaryotic host, a process leading to uncontrolled tissue proliferation and expression by the host of molecules used by bacteria as a source of metabolites. Pathogens have also proven to be powerful tools to study actin-based motility processes and back in the late 90s the study of Listeria intracellular movement resulted in the identification of the bacterial protein ActA, responsible for the formation of the so called “actin comet tails” (4). The work of Matthew Welch has identified the downstream cellular effectors of ActA namely the actin related protein (Arp) 2/3 complex. Dr. Welch described how the listerial ActA protein mimics the Wiskott-Aldrich syndrome protein (WASP) in the activation of Arp2/3 for the formation of actin comet tails (4). The study of other bacteria that also move in the cytoplasm of infected cells by polymerizing host actin, has led to the conclusion that hijacking of the Arp2/3 complex is a common paradigm for several intracellular pathogens (4). It has also emerged that viruses can exploit actin for intracellular movement through a mechanism that is dependent on the Arp2/3 complex via WASP-like proteins. This notion is supported by the observation that despite the large divergence between bacteria and viruses, the baculovirus protein p78/83 (5) shares a high degree of similarity with the ActA protein from Listeria and the RickA protein from Rickettsia (4). In the case of viruses, actin based motility is not only required for cell-to-cell spread as in bacteria, but is also used as a mechanism of entry into the host nucleus for viral replication (6). Lucas Pelkmans discussed viral entry and detailed how viruses can be classified through characterization of the host signaling pathways which they hijack during internalization. This will allow for the reclassification of viruses according to their internalization pathways rather than on genome similarities which could be of great importance for the development of new drugs that can target the endocytic pathways hijacked in host cells rather than targeting the virus, which is subject to rapid adaptation through mutation. To achieve a complete characterization of the signaling cascades activated upon viral internalization, Pelkmans and colleagues have set up an innovative and high-throughput system to identify all the kinases implicated in the internalization of different viruses. Using a HeLa cell-based system they have performed a genome-wide analysis of human kinases involved in the internalization of two model viruses, namely the Vesicular Stomatitis Virus (VSV) and the Simian Virus 40 (SV40), which invade cells via clathrin- and caveolae-mediated endocytosis respectively. Remarkably they showed that, out of the nearly 600 kinases tested, about 90 were specifically regulating VSV infection and about 80 regulated SV40 infection. Moreover, the degree of overlap was limited to less than 40 kinases and even more surprisingly two-thirds of these kinases have opposing effects in viral internalization. The data collected allowed the researchers to perform a so-called “pheno-cluster” of all the kinases tested that revealed interesting links between different biosynthetic pathways (7). Dr. Pelkmans further discussed a computational system for the study of viral infectomes over a whole cell population which will allow for the generation of a predictive model for viral infections. The development of new vaccine strategies for malaria was discussed by Robert Menard from the Pasteur Institute in France and Syed Shams Yazdani from the ICGEB in India. The work of Robert Menard and co workers is mainly focused on the characterization of the pre-erythrocytic phases of malarial infection, when parasites have not yet invaded the liver and transformed into the cell-infecting form, since this stage of infection may be important for new vaccine strategies. Dr. Menard discussed a powerful technique for the in vivo imaging of live GFP-expressing sporozoites as they infect the dermis of a model host such as a rodent which has allowed the characterization and quantitative analysis of sporozoite gliding movement (8). He indicated that sporozoites that are injected in the host skin as the mosquito tries to find the nearest blood vessel, are not necessarily destined for death or clearance, but rather they move rapidly within the first 15 min upon injection trying to reach the blood or lymphatic system (9). Normal movement of sporozoites in the skin requires a process called “cell traversal” by which the parasite invades host cells by disrupting the plasma membrane. Indeed, when rodents are infected with mutant parasites impaired in movement, sporozoites remain in the skin where they are eventually cleared by phagocytic leukocytes (10). Dr. Menard and co workers found that sporozoites could also reside in the skin of the host where merozoites could be formed in keratinocytes and fibroblasts. Whether these skin-derived merozoites can lead to an efficient infection is still an open question. Dr. Yazdani provided a comprehensive overview on the current initiatives for the development of vaccines for malaria by discussing the different possible points of intervention during the parasite lifecycle. He also highlighted the important issues around the feasibility and other obstacles associated with the development of vaccines for malaria. The importance of the correct adjuvant was extensively discussed followed by a synopsis of the current vaccines that have undergone various human clinical trials along with their relative efficacies. The malaria vaccine development pathway with reference to a vaccine for Plasmodium vivax infections based on the receptor binding domain, PvRII, of the P. vivax Duffy binding protein was also discussed (11). It appears that essentially all eukaryotic organisms sense invading microorganisms through the recognition of similar microbial patterns, suggesting that these innate mechanisms for self versus non-self recognition are phylogenetically ancient. Jean-luc Imler, from the Institut de Biologie Moléculaire et Cellulaire-BMC, Strasbourg, France, uses antimicrobial mechanisms in the genus Drosophila as a model to study innate immunity and its role in protection against infections using the interaction between microorganisms and the different Toll-like receptors (TLRs) (12). He highlighted the production of potent antimicrobial molecules by insects as a natural form of defense against invading pathogens and reviewed the structures of these antimicrobial peptides as well as the signaling cascades that lead to their induction upon detection of infectious non-self (13). A hallmark of the potent, multifaceted antimicrobial defense of Drosophila melanogaster is the challenge-induced synthesis of several families of antimicrobial peptides by cells in fat bodies (14). Biochemical analysis of the hemolymph of D. melanogaster and other diptera have led to the discovery of eight classes of antimicrobial peptides which were grouped into three families based on their main biological targets (13). In another lecture, Dr. Imler discussed the family of Dicer-like helicases/RIG-I-like helicases (DExD/H helicases) which sense viral nucleic acids in animals and plants and he indicated that higher eukaryotes have evolved broadly active mechanisms to sense viruses and suppress their proliferation (15). Finally, Dr. Imler reaffirmed that the parallels between the adult fly response and mammalian innate immune defenses, described above, point to a common ancestry and validate the relevance of the fly innate defense as a paradigm for innate immunity (12, 14). Since many internalization pathways terminate at the lysosomal compartment, pathogens have developed remarkable means to avoid interactions with this lytic organelle. Gisou Van der Goot, from Ecole Polytechnique de Lausanne in Switzerland discussed the activity of bacterial toxins, specifically the pore-forming toxins (PFTs), and their ability to promote colonization of the host and escape from the innate defense mechanisms (16). Dr. Van der Goot discussed one of the more fascinating aspects of PFTs which is their ability to adopt a water-soluble form at the beginning of the infectious cycle and subsequently transform into integral transmembrane proteins which insert in the membrane of target cells. There is a growing understanding of the sequence of events and the various conformational changes undergone by these toxins in order to bind to the host cell surface, to penetrate the cell membranes and to achieve pore formation (17). It appears that different classes of toxins trigger cellular changes that are sensed by the cells as danger signals which include, for example, mechanisms that sense variation in the cellular ion composition after membrane perforation by PFTs (16). The signaling pathways triggered through toxin-induced cell alterations likely play a role in modulating host responses to virulent bacteria. Dr. Van der Goot also discussed the different portals for toxin entry with particular emphasis on the mechanism of action of the Anthrax, Cholera, Shiga and Aerolysin toxins and further summarized the bacterial toxin “toolkit” required for host cell entry and manipulation (18). An important finding that was highlighted was the fact that many host cells sense bacterial toxins by their effects on cellular metabolism rather than molecular recognition of the toxin per se (17, 19). During his lectures, Jean-Pierre Gorvel, from Centre d'immunologie de Marseille Luminy, France, described his research, which is mainly focused on understanding the mechanisms that underpin Brucella infections. Dr. Gorvel detailed that contrary to other pathogens, Brucella infections take place without eliciting a significant immune response, which allows prolonged bacterial persistence resulting in chronic infection. Bacteria are able to penetrate dendritic cells facing the intestine lumen blocking both their maturation and their immune functions. These effects are mainly due to two Brucella proteins (Btp1 and 2) that target the TLR 2 activation pathway (20). Dr. Gorvel also explained that although the host receptor that mediates bacterial internalization has not yet been identified, it is clear that pathogenic Brucella invade phagocytic and nonphagocytic cells via lipid rafts by activating small GTPases of the Rho family, inducing the recruitment of actin filaments. Once inside cells, the bacteria reside within an early Brucella-containing vacuole (BCV) that matures and acquires typical endocytic markers via interactions with elements of the early endocytic pathway. BCVs escape fusion with lysosomes and fuse with the endoplasmic reticulum (ER) where Brucella finds a suitable environment for replication. During the early phases of infection, escape from lysosomes seems to be due to the high concentrations of cyclic glucans expressed at the bacterial surface that contribute to the maturation of BCVs. Later on, the Brucella type IV secretion system allows for the secretion of effectors that play a major role in escape from degradation, as mutants defective this system are avirulent in mouse models with bacterial intracellular replication being inhibited (21). Frank Brombacher, from ICGEB in South Africa discussed the role of cytokines, specifically interleukins (IL) 4 and 13 as promising candidates for therapy as they are involved in initiating, maintaining and regulating immunologic homeostasis and inflammatory processes. He also discussed new perspectives on the role of alternatively activated macrophages which are found in abundance during chronic T cell helper 2 (Th2) inflammatory responses to metazoan parasites. Dr. Brombacher presented evidence that alternative activation by type 2 cytokines (mainly IL-4 and IL-13) is an innate response to injury occurring even in the absence of an adaptive response. However, the class II-restricted CD4+ T helper pathway is essential to maintain alternatively activated macrophages in response to infection (22). 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