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Thursday, December 23, 2010
Imagine what would happen if we did not have antimicrobial defenses. Any minor injury to the physical integrity of our skin would cause life- threatening infection. We would be helpless against the myriad airborne microbes that we take in our lungs when we breathe. Any and all food that we ate would have to be completely sterile. Fortunately, we have evolved efficient defense mechanisms that hold microbes at bay. We often think of ourselves at constant war against this plethora of potentially harmful microorganisms that surround us, against the ‘non-self.’ In recent times, however, this view has evolved to incorporate what we now know about human biology. We increasingly appreciate that we are, in fact, ‘superorganisms,’ composed of our human cells and a diverse, and numerically astounding, stable population of microorganisms. These are essential for our development, they provide enhanced metabolic capabilities, and they keep inflammation at bay. Proper regulation of the microbiota, and of our responses to it, is crucial for our wellbeing. Imagine being able to tinker with this symbiosis in a highly specific manner, to reestablish harmony when it goes awry by infection or chronic inflammation, with minimal collateral damage.
Our work seeks to obtain fundamental insights on host-microbe interactions, focusing on intestinal epithelial cells, in an unbiased and comprehensive manner, and to use those insights to increase our understanding of human health and disease. Our overarching hypothesis is that intestinal epithelial cells, in addition to their physical barrier function, serve as sentinels and coordinators of the host response to luminal microbes. In support of this hypothesis, intestinal epithelial cells were recently shown to directly sense microbes, producing antimicrobial factors in response. Intestinal epithelial cells also produce signals that recruit and kickstart the innate and adaptive branches of the immune system (Fig. 1A). We use the nematode Caenorhabditis elegans as a simpler and defined tool to identify new and evolutionarily conserved epithelial host defense pathways, and to understand how they function during host- microbe interactions in vivo. Because innate immunity evolved before the split between invertebrates and vertebrates, many host signaling pathways are conserved between nematodes and humans (1). 80% of C. elegans genes have clear human homologs, and 90% of human disease genes evolved before the split with invertebrates. Therefore, it is reasonable to use C. elegans to identify defense pathways that are conserved in humans. In contrast to the complex human intestinal immune system, C. elegans relies entirely on innate epithelial cell responses to fight intestinal infection (Fig. 1A,B)(1). In the laboratory, C. elegans is propagated and maintained on agar plates with lawns of nonpathogenic bacteria as food source. Each animal produces ~300 genetically identical progeny within 3 days. Transgenic C. elegans can be easily generated by microinjection of DNA. C. elegans is transparent, facilitating direct real- time observation of infection and gene expression in vivo, in the whole live animal. Additionally, gene expression in C. elegans can be knocked down conveniently, by double-stranded RNA (dsRNA)-mediated RNA interference (RNAi), by feeding worms live non-pathogenic E. coli expressing dsRNA corresponding to each individual C. elegans gene (~90% of the genome is available as a dsRNA expression library). Besides fundamental studies of innate immunity and pathogen virulence, C. elegans has been used in translational research designed to identify novel targets for next generation antimicrobial compounds.
We have been focusing on human pathogenic bacteria such as Staphylococcus aureus, to identify host pathways that are important for defense against infection. Thus, as an added benefit, our studies also have the potential to illuminate virulence strategies that are used in vivo by S. aureus to manipulate epithelial cell biology and establish infection. Because both host and pathogen are genetically tractable, our system is the most powerful, highly defined, and simplified way to ask basic questions about fundamental mechanisms of host-microbe interaction in vivo, which determine health or disease of the human ‘superorganism.’ We take advantage of this to learn new lessons about host-pathogen interactions and then establishing how these primitive defense mechanisms function in the context of more complex human responses. We found that S. aureus causes extensive damage to the intestinal epithelial cells (Fig. 1C,D,E), indicative of active pathogenesis. We also determined the C. elegans host response to S. aureus infection, identifying ~200 S. aureus induced genes and ~200 repressed genes (2). So far, every induced gene we have studied is induced in the intestinal epithelium. Importantly, this host response is also induced by heat-killed S.aureus, which does not cause pathology; thus, we suspect that S. aureus sensing involves pathogen-associated molecules and not damage sensing. Our studies focus on three evolutionarily conserved pathways that we recently identified: hypoxia signaling, Wnt signal transduction, and a novel transcription factor.
As an example, we found a critical role for the protein ß-catenin in the regulation of the host response to infection (3). ß-catenin is a component of highly conserved signaling pathways downstream of Wnt-class glycoprotein ligands (Fig. 1I). We found that animals lacking bar-1 were hypersusceptible to S. aureus-mediated killing (Fig. 1F). Similarly, animals lacking bar-1 downstream targt gene egl-5 were also hypersusceptible (Fig. 1F). Consistent with the survival assays, bar-1(-) and egl-5(-) mutants were defective in the induction of the intestinal host response (e.g. Fig. 1G,H). To extrapolate these findings to human cells, we evaluated the effect of ß-catenin and HOX genes homologous to egl-5 on bacterially-triggered inflammation. We found that both ß-catenin and HOX modulated inflammatory signaling in human epithelial cells (3). Thus, these results established that ß-catenin signaling is essential for proper intestinal host defense. This is an example of using C. elegans to identify new components of human innate immunity signaling.
In collaboration with labs at MGH, HMS, and the Broad Institute, we are using C. elegans in high-throughput screens to identify novel host defense genes and bacterial virulence factors. Our ultimate goal is to understand the reciprocal regulation of human transcriptional networks and bacterial transcriptional networks, and relate the state of this meta-network to the physiological health state of the ‘superorganism.’ This understanding will enable us to design rational and specific therapies to correct deviations that cause infectious or inflammatory diseases in the intestine.
1. Irazoqui JE, Urbach JM, Ausubel FM. Evolution of host innate defence: insights from Caenorhabditis elegans and primitive invertebrates. Nat Rev Immunol. 2010;10(1):47-58.
2. Irazoqui JE, Troemel ER, Feinbaum RL, Luhachack LG, Cezairliyan BO, Ausubel FM. Distinct Pathogenesis and Host Responses during Infection of C. elegans by P. aeruginosa and S. aureus. PLoS Pathog. 2010;6(7):e1000982.
3. Irazoqui JE, Ng A, Xavier RJ, Ausubel FM. Role for beta-catenin and HOX transcription factors in Caenorhabditis elegans and mammalian host epithelial-pathogen interactions. Proc Natl Acad Sci USA. 2008;105(45):17469-74.
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