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Innate immunity responds to pathogens by producing alarm signals and activating pathways that make host cells inhospitable for pathogen replication. The intracellular bacterium Burkholderia thailandensis invades the cytosol, hijacks host actin, and induces cell fusion to spread to adjacent cells, forming multinucleated giant cells (MNGCs) which promote bacterial replication. We show that type I interferon (IFN) restricts macrophage MNGC formation during B. thailandensis infection. Guanylate-binding proteins (GBPs) expressed downstream of type I IFN were required to restrict MNGC formation through inhibition of bacterial Arp2/3-dependent actin motility during infection. GTPase activity and the CAAX prenylation domain were required for GBP2 recruitment to B. thailandensis, which restricted bacterial actin polymerization required for MNGC formation. Consistent with the effects in in vitro macrophages, Gbp2−/−, Gbp5−/−, GbpChr3-KO mice were more susceptible to intranasal infection with B. thailandensis than wildtype mice. Our findings reveal that IFN and GBPs play a critical role in restricting cell-cell fusion and bacteria-induced pathology during infection., Author summary The intracellular bacterium Burkholderia thailandensis and its relatives B. pseudomallei and B. mallei each invade host cells and hijack the actin cytoskeleton polymerization machinery to transmit to neighboring cells by cell-cell fusion, a transmission strategy that is unique to this family. The high antibiotic resistance of the Burkholderia family underscores the need to understand how the immune system can control infections. Here, we show that the interferon immune response upregulates a family of immune proteins, the guanylate binding proteins (GBPs), to counter the bacterial intracellular motility and, as a consequence, cell-cell fusion. Infected macrophages extensively fuse when lacking key molecules in this immune pathway, and mice lacking the GBP2 or GBP5 proteins are 100-1000-fold more susceptible to infection than wildtype mice, highlighting the critical role this immune pathway plays in restricting bacterial infection and cell-cell fusion. We also found that mice lacking GBPs were protected if bacteria lacked a critical virulence factor, VgrG5, that is required for cell-cell fusion, highlighting that cell-cell fusion is dependent on both bacteria-mediated cytoskeleton remodeling as well as VgrG5. Together, this study provides insight into an intricate host-pathogen interaction key to the virulence of the Burkholderia family.

The cysteine protease caspase-7 has an established role in the execution of apoptotic cell death, but recent findings also suggest involvement of caspase-7 during the host response to microbial infection. Caspase-7 can be cleaved by the inflammatory caspase, caspase-1, and has been implicated in processing and activation of microbial virulence factors. Thus, caspase-7 function during microbial infection may be complex, and its role in infection and immunity has yet to be fully elucidated. Here we demonstrate that caspase-7 is cleaved during cytosolic infection with the intracellular bacterial pathogen, Listeria monocytogenes. Cleavage of caspase-7 during L. monocytogenes infection did not require caspase-1 or key adaptors of the primary pathways of innate immune signaling in this infection, ASC, RIP2 and MyD88. Caspase-7 protected infected macrophages against plasma membrane damage attributable to the bacterial pore-forming toxin Listeriolysin O (LLO). LLO-mediated membrane damage could itself trigger caspase-7 cleavage, independently of infection or overt cell death. We also detected caspase-7 cleavage upon treatment with other bacterial pore-forming toxins, but not in response to detergents. Taken together, our results support a model where cleavage of caspase-7 is a consequence of toxin-mediated membrane damage, a common occurrence during infection. We propose that host activation of caspase-7 in response to pore formation represents an adaptive mechanism by which host cells can protect membrane integrity during infection., Author Summary Macrophages are critical early responders recruited to sites of bacterial infection. Many intracellular bacterial pathogens subvert or bypass macrophage anti-microbial defenses by expression of virulence factors and toxins. The Gram-positive intracellular pathogen, Listeria monocytogenes, secretes a pore-forming toxin that can damage host membranes. We found that toxin-mediated damage during bacterial infection triggered activation of a cysteine protease, caspase-7. Activation of this protease was triggered by low levels of toxin alone, suggesting that sublethal toxin-mediated damage is sensed by the host cell. Infected viable macrophages lacking caspase-7 exhibited increased membrane permeability, suggesting that caspase-7 limits membrane damage in infected host cells. This study reveals an unanticipated cytoprotective role for caspase-7 during intracellular infection and provides evidence for a mechanism by which host cells can initiate an adaptive response to survive membrane-damaging toxins, which are commonly associated with pathogenic microbes.

Legionella pneumophila (L. pneumophila), the causative agent of a severe form of pneumonia called Legionnaires' disease, replicates in human monocytes and macrophages. Most inbred mouse strains are restrictive to L. pneumophila infection except for the A/J, Nlrc4−/− (Ipaf−/−), and caspase-1−/− derived macrophages. Particularly, caspase-1 activation is detected during L. pneumophila infection of murine macrophages while absent in human cells. Recent in vitro experiments demonstrate that caspase-7 is cleaved by caspase-1. However, the biological role for caspase-7 activation downstream of caspase-1 is not known. Furthermore, whether this reaction is pertinent to the apoptosis or to the inflammation pathway or whether it mediates a yet unidentified effect is unclear. Using the intracellular pathogen L. pneumophila, we show that, upon infection of murine macrophages, caspase-7 was activated downstream of the Nlrc4 inflammasome and required caspase-1 activation. Such activation of caspase-7 was mediated by flagellin and required a functional Naip5. Remarkably, mice lacking caspase-7 and its macrophages allowed substantial L. pneumophila replication. Permissiveness of caspase-7−/− macrophages to the intracellular pathogen was due to defective delivery of the organism to the lysosome and to delayed cell death during early stages of infection. These results reveal a new mechanism for caspase-7 activation downstream of the Nlrc4 inflammasome and present a novel biological role for caspase-7 in host defense against an intracellular bacterium., Author Summary Legionella pneumophila causes a severe form of pneumonia called Legionnaires' disease. In human macrophages, L. pneumophila establishes special vacuoles that do not fuse with the lysosome and grows intracellularly. However, in mouse macrophages, the bacteria are efficiently delivered to the lysosome for degradation. Importantly, caspase-1 is activated when L. pneumophila infects mouse macrophages, but not when it infects human cells. Caspase-1 activation promotes the fusion of the L. pneumophila vacuole with the lysosome and macrophage death. However, the caspase-1 substrate mediating such effects is unknown. Experiments performed in vitro demonstrate that caspase-7 is a substrate of caspase-1. Yet, it is not known if the reaction takes place within the macrophage, and it is unclear if it has any biological effect. In this study we show that, in mouse macrophages, caspase-7 is activated by L. pneumophila downstream of caspase-1 and requires the host receptors Nlrc4 and Naip5. Remarkably, caspase-7 activation during L. pneumophila infection restricts growth by promoting early macrophage death and efficient delivery of the organism to the lysosome. Consequently, L. pneumophila grows in the macrophages and the lungs of caspase-7−/− mice. Therefore, we demonstrate a novel caspase-7 activation pathway that contributes to the restriction of L. pneumophila infection.

Animal and plant eukaryotic pathogens, such as the human malaria parasite Plasmodium falciparum and the potato late blight agent Phytophthora infestans, are widely divergent eukaryotic microbes. Yet they both produce secretory virulence and pathogenic proteins that alter host cell functions. In P. falciparum, export of parasite proteins to the host erythrocyte is mediated by leader sequences shown to contain a host-targeting (HT) motif centered on an RxLx (E, D, or Q) core: this motif appears to signify a major pathogenic export pathway with hundreds of putative effectors. Here we show that a secretory protein of P. infestans, which is perceived by plant disease resistance proteins and induces hypersensitive plant cell death, contains a leader sequence that is equivalent to the Plasmodium HT-leader in its ability to export fusion of green fluorescent protein (GFP) from the P. falciparum parasite to the host erythrocyte. This export is dependent on an RxLR sequence conserved in P. infestans leaders, as well as in leaders of all ten secretory oomycete proteins shown to function inside plant cells. The RxLR motif is also detected in hundreds of secretory proteins of P. infestans, Phytophthora sojae, and Phytophthora ramorum and has high value in predicting host-targeted leaders. A consensus motif further reveals E/D residues enriched within ~25 amino acids downstream of the RxLR, which are also needed for export. Together the data suggest that in these plant pathogenic oomycetes, a consensus HT motif may reside in an extended sequence of ~25–30 amino acids, rather than in a short linear sequence. Evidence is presented that although the consensus is much shorter in P. falciparum, information sufficient for vacuolar export is contained in a region of ~30 amino acids, which includes sequences flanking the HT core. Finally, positional conservation between Phytophthora RxLR and P. falciparum RxLx (E, D, Q) is consistent with the idea that the context of their presentation is constrained. These studies provide the first evidence to our knowledge that eukaryotic microbes share equivalent pathogenic HT signals and thus conserved mechanisms to access host cells across plant and animal kingdoms that may present unique targets for prophylaxis across divergent pathogens., Synopsis Microbial interactions with host cells frequently involve utilization of pathogenic effectors that cause virulent infection and disease in the host. How these eukaryotic pathogenic effectors appear in the host cell is largely unknown. Recent studies have identified the first host-targeting (HT) signal for a eukaryotic pathogen in the human malaria parasite Plasmodium falciparum. Bhattacharjee, Haldar, and colleagues show that the Plasmodium HT-signal is conserved in the biotrophic oomycete Phytophthora infestans that caused the Irish potato famine. Like its malarial counterpart, the Phytophthora HT signal is present in major, known, virulence proteins, and predicts a pathogenic “host-targeted-secretome” of hundreds of putative effectors to colonize the host cell. Since Plasmodium and Phytophthora belong to distinct evolutionary groups, the study establishes for the first time that different eukaryotic microbes can share similar strategies in delivering toxic proteins to their hosts. This may present shared targets for controlling vastly different infections of both animals and plants. The present work has implications for agriculture and human health, since Phytophthora species devastate a wide range of food and commercial crops and Plasmodium species cause malaria, which kills more than one million children each year.