Hierarchical Cell Death Program Disrupts the Intracellular Niche Required for Burkholderia thailandensis Pathogenesis

ABSTRACT Burkholderia infections can result in serious diseases with high mortality, such as melioidosis, and they are difficult to treat with antibiotics. Innate immunity is critical for cell-autonomous clearance of intracellular pathogens like Burkholderia by regulating programmed cell death. Inflammasome-dependent inflammatory cytokine release and cell death contribute to host protection against Burkholderia pseudomallei and Burkholderia thailandensis; however, the contribution of apoptosis and necroptosis to protection is not known. Here, we found that bone marrow-derived macrophages (BMDMs) lacking key components of pyroptosis died via apoptosis during infection. BMDMs lacking molecules required for pyroptosis, apoptosis, and necroptosis (PANoptosis), however, were significantly resistant to B. thailandensis-induced cell death until later stages of infection. Consequently, PANoptosis-deficient BMDMs failed to limit B. thailandensis-induced cell-cell fusion, which permits increased intercellular spread and replication compared to wild-type or pyroptosis-deficient BMDMs. Respiratory B. thailandensis infection resulted in higher mortality in PANoptosis-deficient mice than in pyroptosis-deficient mice, indicating that, in the absence of pyroptosis, apoptosis is essential for efficient control of infection in vivo. Together, these findings suggest both pyroptosis and apoptosis are necessary for host-mediated control of Burkholderia infection.

respiratory infection. Together, these findings suggest a key role for multiple programmed cell death pathways in restricting Burkholderia-induced cell-cell fusion, bacterial infection, and infection-induced mortality.
Casp8/Ripk3/Casp1/11 2/2 BMDMs fail to restrict B. thailandensis cell-cell fusion and replication. The significantly delayed cell death and extensive cell-cell fusion observed in B. thailandensis-infected Casp8/Ripk3/Casp1/11 2/2 BMDMs suggested that  Fig. 2 were also used to generate data presented in Fig. S1 in the supplemental material and Fig. 3B to D.
Programmed Cell Death Controls Burkholderia Infection ® bacterial infection is poorly controlled in these cells compared to WT. The physiological importance of cell-cell fusion in the infectious process of B. thailandensis, B. pseudomallei, B. mallei, and Burkholderia oklahomensis is still poorly understood, but is thought to contribute to intercellular spread and promote bacterial replication. To determine the impact of deleting key PANoptosis components, we first examined cell-cell fusion and the bacterial burden of BMDMs by confocal microscopy. As expected, cell-cell fusion and the intracellular bacterial burden were notably increased in Casp8/Ripk3/Casp1/ 11 2/2 BMDMs compared to WT (Fig. 3A), indicating that delayed cell death in Casp8/ Ripk3/Casp1/11 2/2 BMDMs allows for increased cell-cell fusion and B. thailandensis intercellular spread to neighboring cells.
B. thailandensis, B. pseudomallei, B. mallei, and B. oklahomensis possess a unique type six secretion system (T6SS) effector protein, VgrG5, which is required for infectioninduced host cell-cell fusion, formation of MNGCs, and intercellular spread (63)(64)(65)(66). To distinguish between the role of bacterial-mediated cell-cell fusion and host-mediated cell death in regulating bacterial replication, we utilized WT B. thailandensis and a mutant strain of B. thailandensis vgrG5DCTD (indicated as BtDCTD) lacking the C-terminal domain of VgrG5 that is required for inducing eukaryotic cell-cell fusion (65). We first determined that cell-cell fusion in B. thailandensis-infected Casp8/Ripk3/Casp1/11 2/2 BMDMs required VgrG5 (Fig. 3B). To quantify intracellular bacterial replication, BMDMs lacking executioners of pyroptosis/necroptosis (Gsdmd/Mlkl 2/2 ) or PANoptosis (Casp8/ Ripk3/Casp1/11 2/2 ) were infected, and intracellular bacterial CFU counts were determined after 24 h. BMDMs deficient in the pyroptosis and necroptosis executioners GSDMD and MLKL were largely able to restrict intracellular bacterial replication, suggesting that apoptosis limited the intracellular replication of B. thailandensis ( Fig. 3B and C). In contrast, significantly increased bacterial replication was observed in Casp8/ Ripk3/Casp1/11 2/2 BMDMs compared to WT ( Fig. 3B and D). These findings indicate that the delayed cell death observed in Casp8/Ripk3/Casp1/11 2/2 BMDMs results in failure to restrict intracellular replication and cell-to-cell spread of B. thailandensis ( Fig. 3B and D). B. thailandensis lacking VgrG5 cell-cell fusion activity also replicated intracellularly to a higher degree in Casp8/Ripk3/Casp1/11 2/2 BMDMs than in WT (Fig. 3D), but loss of cell-cell fusion limited the overall replicative capacity, reinforcing the importance of cell-cell fusion and intercellular spread to the pathogenesis of the B. thailandensis. These data, together with the observation that pyroptosis is the primary cell death pathway activated in WT BMDMs (Fig. 2), suggest that intracellular replication and spread of B. thailandensis is restricted by pyroptosis but, in the absence of pyroptosis, caspase-8-dependent apoptosis compensates to mediate intracellular clearance.
To determine whether PANoptosis-deficient mice were more susceptible to lethal infection than pyroptosis-deficient mice, mice were challenged with a lower dose of B. thailandensis (500 CFU) and monitored daily. Mice lacking GSDMD survived infection similarly to WT mice (Fig. 4F). Mice lacking the pyroptotic caspases-1/11 were more susceptible to lethal B. thailandensis infection than both WT and Gsdmd 2/2 mice (Fig. 4F). However, mice deficient in PANoptosis were more susceptible to lethal infection than either Gsdmd 2/2 or Casp1/11 2/2 mice, suggesting that pyroptosis and apoptosis both mediate protection during B. thailandensis infection (Fig. 4F). These data also suggest that caspase-1/11 activity, independent from GSDMD cleavage, is important for limiting infection, likely due to cleavage of additional host proteins. PANoptosis-deficient mice also failed to control B. thailandensis bacterial replication in the lung and spleen during infection ( Fig. 4G and H). Consistent with mouse survival during infection, WT and Gsdmd 2/2 mice harbored less B. thailandensis, while Casp1/ 11 2/2 and Casp8/Ripk3/Casp1/11 2/2 mice harbored increased bacteria. Together, these data highlight the important role both pyroptosis and apoptosis play in restricting respiratory infection by B. thailandensis and further our understanding of the physiological role of Burkholderia-mediated cell-cell fusion in infection.

DISCUSSION
In this study, we found that pyroptosis and apoptosis both regulate cell death during intracellular infection by B. thailandensis (Fig. 5). These programmed cell death pathways are necessary for the host to restrict the intracellular replication of B. thailandensis. Pyroptosis is largely responsible for promoting intracellular clearance of B. thailandensis in WT BMDMs, but in BMDMs lacking critical pyroptotic machinery, cells undergo robust apoptotic cell death. Previous studies identified a critical role for pyroptotic cell death molecules in restricting B. thailandensis but the role of apoptotic caspases and necroptosis has not been examined (52)(53)(54)(55)(56)(57)(58). Pyroptosis alone carries out multiple distinct functions in mediating protection during Burkholderia infections. In lung epithelial cells, caspase-11 is required for host protection, while macrophages require caspase-1, suggesting important tissue-specific roles for cell death (60). In mice, caspase-1-dependent pyroptosis is required for the production of the cytokines IL-18 and IL-1b. IL-18-dependent production of gamma interferon (IFN-g) in mice is required for the control of B. thailandensis and B. pseudomallei, while the release of IL-1b is responsible for lethal inflammatory pathology (52,54), indicating that cell death must be carefully balanced by the host during Burkholderia infection.
Here, we found that macrophages lacking key components of PANoptosis were resistant to infection-induced cell death until a later stage of B. thailandensis infection, resulting in extensive cell-cell fusion, MNGC formation, and increased intracellular bacterial replication. In the later stages of infection, macrophages underwent caspase-8-independent apoptosis characterized by activation of caspase-9 and caspase-3/7, suggesting intrinsic apoptosis can also be activated during B. thailandensis infection, consistent with previous reports (53). As a result of the defective activation of cell death in BMDMs lacking components of PANoptosis, intracellular B. thailandensis are able to persist for an extended period of time, allowing for increased infection-induced cell-cell fusion. The dramatic increase in cell-cell fusion in these cells is likely a result of the extended cell survival time, which increases the amount of time for intracellular bacteria to both replicate and form protrusions that increase contacts between cell membranes necessary for cell-cell fusion. These data also suggest that the eventual activation of caspase-9-dependent intrinsic apoptosis is insufficient to mediate protection from infection. These findings imply that infected macrophages are highly adaptable in activating programmed cell death pathways to clear intracellular pathogens, but also highlight the important role that pyroptosis and caspase-8-dependent apoptosis play in rapidly limiting B. thailandensis replication. In addition to the role cell death plays in controlling Burkholderia infection, questions remain as to why B. thailandensis, B. pseudomallei, B. mallei, and B. oklahomensis possess a unique VgrG5 C-terminal domain found only in Burkholderia spp., which is required for cell-cell fusion and virulence (64)(65)(66)(67). Our data suggest that this unique virulence strategy is required to counter host-mediated restriction of bacterial intracellular replication.
Previous work has identified a critical role for cross talk between cell death pathways in regulating cell death during bacterial and viral infections with Salmonella enterica serovar Typhimurium, Listeria monocytogenes, influenza A virus (IAV), and vesicular  (43). The involvement of pyroptosis, apoptosis, and necroptosis acting together established the concept of PANoptosis. Initial studies identified that activation of ZBP1-dependent PANoptosis is important for the control of IAV infection (23,68). Similarly, RIPK1-dependent PANoptosis was identified in macrophages lacking TAK1, which undergo spontaneous PANoptosis (45,48). Other studies found that Yersinia pestis, which secretes the T3SS effector protein YopJ, inhibits TAK1-dependent inflammatory signaling and consequently results in TAK1 inhibition-mediated inflammatory cell death (69,70). Additionally, Shigella flexneri utilizes T3SS effectors OspC1 and OspD3 to inhibit caspase-8 and RIPK1/RIPK3, respectively (71), further showing how bacterial pathogens can modulate cell death effectors. The evolutionary arms race between host cell death pathways and pathogen-mediated inhibition of various cell death components highlights the importance of studying the molecular host-pathogen interactions of multiple pathogens (40). While B. thailandensis predominantly activated pyroptosis and apoptosis during infection, it may possess a virulence factor that inhibits necroptosis, and other Burkholderia species may have unique abilities to modulate these programmed cell death pathways. Inflammatory cytokines or cell-mediated immunity may also change the cell death pathways engaged in B. thailandensisinfected cells. Furthermore, dysfunction of these cell death responses may be important to permit the chronic infections sometimes observed during B. pseudomallei infection in humans (72). Overall, these studies suggest a critical role for cell death and PANoptosis in limiting infection by many distinct microbial pathogens and highlight the role that pathogens themselves can play in dictating the cell death outcome (40,42,43).
Using genetic mouse models deficient in key components of individual or multiple cell death pathways, we have now established a critical role for pyroptosis and apoptosis in controlling B. thailandensis infection (Fig. S4). Loss of these pathways rendered macrophages and mice highly susceptible to lethal infection by permitting unrestricted intracellular bacterial replication and intercellular spread by cell-cell fusion. Understanding these processes in the context of infection by fusogenic B. thailandensis, B. pseudomallei, B. mallei, and B. oklahomensis may have important implications for treating infections caused by these bacteria. The roles for programmed cell death in mediating protection against the more evolutionarily distant, nonfusogenic Burkholderia spp., including Burkholderia cepacia and Burkholderia cenocepacia, are likely distinct. Understanding the host immune response and virulence mechanisms of Burkholderia spp. is also necessary to improve treatment strategies, because Burkholderia spp. are inherently highly antibiotic resistant. Our data suggest more broadly that the cross talk between programmed cell death pathways may be important to restrict multiple other microbial pathogens by limiting their intracellular niche. Targeting programmed cell death pathways for therapeutic intervention needs to be carefully considered, as inhibitors may result in alternative and unfavorable forms of cell death during infection. This functional redundancy of cell death pathways also has important implications for treating other diseases ranging from cancer to autoimmunity. Despite the difficulty in targeting these highly interconnected cell death pathways, understanding the mechanisms by which these pathways are coordinated may identify new therapeutic targets which could benefit patients with acute or chronic infections that are currently difficult to treat.
Mouse infections. B. thailandensis was grown as described above. For mouse infection experiments, quantified frozen aliquots of B. thailandensis were diluted in phosphate-buffered saline (PBS) before infection to inoculate 5 Â 10 4 or 5 Â 10 2 bacteria per mouse. Mice were anesthetized with isoflurane and administered B. thailandensis in a 50 ml PBS suspension via the nares. After 2 days of infection, lungs were harvested for histology (tissues fixed in formalin and processed by the St. Jude Children's Research Hospital Veterinary Pathology Core) or CFU analysis. Histological scoring was performed by a board-certified veterinary pathologist (author P.V.) and assigned a semiquantitative score based on the severity grades 0 = within normal limits; 1 = minimal: rare or inconspicuous lesions; 2 = mild: multifocal or small, focal, or widely separated, but conspicuous lesions; 3 = moderate: multifocal, prominent lesions; 4 = marked: extensive to coalescing lesions or areas of inflammation with some loss of structure; 5 = severe: diffuse lesion with effacement of normal structure. Severity grades were converted to semiquantitative scores with the following criteria: 0 = 0; 1 = 1; 1.5 = 8; 2 = 15; 2.5 = 25; 3 = 40; 3.5 = 60; 4 = 80; 4.5 = 90; 5 = 100. CFU were determined by homogenizing tissue in PBS in Lysing Matrix C tubes on a FastPrep homogenizer (1169120050, MP Biomedicals) and plating on LB agar plates, then incubated overnight at 37°C.
Bone marrow-derived macrophage stimulations. BMDMs were differentiated as described above. Prior to stimulation, cells were washed with PBS, and PBS was replaced with fresh antibiotic-free DMEM containing 10% FBS. B. thailandensis (MOI 5) was pelleted onto cells at 300 Â g for 5 min; cells were washed after 1 h, incubated with DMEM containing 1,000 mg/ml kanamycin for 1 h to kill remaining extracellular B. thailandensis, washed, and finally incubated in DMEM containing 250 mg/ml kanamycin for the remainder of the experiment to restrict extracellular growth of B. thailandensis. After the final wash, SYTOX Green (25 nM, S7020, Thermo Fisher Scientific) or propidium iodide (PI) (250 ng/ml, P3566, Thermo Fisher Scientific) was added to media for IncuCyte experiments. To determine intracellular CFU counts, cells were washed with PBS, lysed with PBS containing 0.01% Triton X-100, serially diluted in PBS, and plated on LB agar plates, then incubated at 37°C.
Quantification and statistical analysis. GraphPad Prism 6.0 software was used for data analysis. Data are shown as mean 6 standard error of the mean (SEM). Statistical significance was determined by one-way or two-way ANOVA with the Dunn's, Tukey, or Holm-Sidak multiple-comparison test. The specific statistical testing for each experiment is indicated in the figure legends. Survival curves were compared using the log-rank test. P , 0.05 was considered statistically significant.
Data availability. Further information and requests for resources and reagents should be directed to and will be fulfilled by the corresponding author, Thirumala-Devi Kanneganti (thirumala-devi.kanneganti@stjude.org).

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. VIDEO S1, AVI file, 9.3 MB.