Hot or Not? Novel Mechanisms of Innate Immune Discrimination Between Pathogens and Non-Pathogens
The innate immune system responds to infectious threats by detecting specific molecular structures conserved among microbes, such as bacterial lipopolysaccharide or flagellin. However, these conserved molecules are found on harmless and pathogenic microbes alike. In order to discriminate between harmful and harmless microbes, it has been proposed that the innate immune system may also sense `patterns of pathogenesis', the disruptions to host physiology orchestrated specifically by pathogens to infect, replicate within, and spread among their hosts. Immune recognition in plants is known to be based in part upon recognition of specific pathogen-associated activities, but few analogous examples have been described in mammals. The intracellular bacterial pathogen Legionella pneumophila can infect macrophages in the mammalian lung, causing a severe inflammatory pneumonia called Legionnaires' Disease. For virulence, L. pneumophila requires a Dot/Icm Type IV secretion system that translocates bacterial effectors to the host cytosol. In this dissertation, L. pneumophila was used as a tool to reveal two novel immunosurveillance mechanisms that can discriminate between virulent and avirulent bacteria. The two distinct pathways integrate detection of both microbial molecules and pathogen-associated activities to generate specific responses to Dot/Icm+ L. pneumophila. The first of these novel mechanisms leads to a potent transcriptional response, termed the `Effector-Triggered Response' (ETR), in macrophages infected with virulent L. pneumophila, but not an avirulent Dot/Icm- mutant. I demonstrate that this unique transcriptional response is due to secretion of five bacterial effector molecules that inhibit host protein synthesis. Upon infection of macrophages with Dot/Icm+ L. pneumophila, these five effectors caused a global decrease in host translation, thereby preventing synthesis of IkB, an inhibitor of the NF-kB transcription factor. Thus, macrophages infected with wildtype L. pneumophila exhibited
prolonged activation of NF-kB, which was associated with transcription of ETR target genes such as Il23a and Csf2. L. pneumophila mutants lacking the five effectors still activated TLRs and NF-kB, but because the mutants permitted normal IkB synthesis, NF-kB activation was more transient and was not sufficient to fully induce the ETR. Translation inhibition also activated other host pathways, including MAP kinase signaling. L. pneumophila mutants expressing enzymatically inactive effectors were also unable to fully induce the ETR, whereas multiple compounds or bacterial toxins that inhibit host protein synthesis via distinct mechanisms recapitulated the ETR when administered with TLR ligands. Thus, a a pathogen-encoded activity, namely translation inhibition, can elicit a specific immune response, both in cultured macrophages and in vivo.
The second novel mechanism consists of two different TNF-inducible inflammasomes that initiate an inflammatory host cell death, called pyroptosis, in macrophages infected with virulent L. pneumophila but not with an avirulent Dot/Icm- mutant. One of these inflammasomes begins with the previously reported detection of bacterial flagellin by the host proteins Naip5 and Nlrc4, but then leads to the activation of a novel downstream `death effector'. The other inflammasome involves activation of the protease Caspase-11 by cIAP1, a host protein that has not previously been implicated in inflammatory death. This latter form of cell death is antagonized by a bacterial effector, SdhA, which is required for growth of L. pneumophila within macrophages. The data presented here are consistent with a model in which increased host cell death upon infection with L. pneumophila aids in restriction of bacterial growth within these macrophages. The activity of these novel inflammasomes may explain the long-standing observation that TNF is crucial for complete restriction of L. pneumophila growth in macrophages.
Previous studies have demonstrated that the host response to bacterial infection is induced primarily by specific microbial molecules that activate TLRs or cytosolic pattern recognition receptors. Our results add to this model by providing several striking illustration of how the host immune response to a virulent pathogen can also be shaped by pathogen-encoded activities, such as (1) inhibition of host protein synthesis and (2) delivery of ligands to the
cytosol via specialized secretion systems. Elucidation of these immunosurveillance pathways increases our understanding of how the innate immune system may integrate multiple signals to sense a microbe, determine whether that microbe is a pathogen, and finally generate an appropriately scaled response.