Sequentially activated death complexes regulate pyroptosis and IL-1ß release in response to Yersinia blockade of immune signaling.

The Yersinia virulence factor YopJ potently inhibits immune signaling in macrophages by blocking activation of the signaling kinases TAK1 and IKK. In response, macrophages trigger a backup pathway of host defense that mediates cell death via the apoptotic enzyme caspase-8 and pyroptotic enzyme caspase-1. While caspase-1 is normally activated within multiprotein inflammasome complexes that contain the adaptor ASC and NLRs, which act as sensors of pathogen virulence, caspase-1 activation following Yersinia blockade of TAK1/IKK surprisingly requires caspase-8 and is independent of all known inflammasome components. Here, we report that caspase-1 activation by caspase-8 requires both caspase-8 catalytic and auto-processing activity. Intriguingly, while caspase-8 serves as an essential initiator of caspase-1 activation, caspase-1 amplifies its own activation through a feed-forward loop involving auto-processing, caspase-1-dependent cleavage of the pore-forming protein GSDMD, and subsequent activation of the canonical NLRP3 inflammasome. Notably, while caspase-1 activation and cell death are independent of inflammasomes during Yersinia infection, IL-1ß release requires the canonical NLPR3 inflammasome. Critically, activation of caspase-8 and activation of the canonical inflammasome are kinetically and spatially separable events, as rapid capase-8 activation occurs within multiple foci throughout the cell, followed by delayed subsequent assembly of a single canonical inflammasome. Importantly, caspase-8 auto-processing normally serves to prevent RIPK3/MLKL-mediated necroptosis, and in caspase-8's absence, MLKL triggers NLPR3 inflammasome activation and IL-1ß release. Altogether, our findings reveal that functionally interconnected but temporally and spatially distinct death complexes differentially mediate pyroptosis and IL-1ß release to ensure robust host defense against pathogen blockade of TAK1 and IKK.

Selective whole-genome amplification reveals population genetics of Leishmania braziliensis directly from patient skin biopsies.

In Brazil, Leishmania braziliensis is the main causative agent of the neglected tropical disease, cutaneous leishmaniasis (CL). CL presents on a spectrum of disease severity with a high rate of treatment failure. Yet the parasite factors that contribute to disease presentation and treatment outcome are not well understood, in part because successfully isolating and culturing parasites from patient lesions remains a major technical challenge. Here we describe the development of selective whole genome amplification (SWGA) for Leishmania and show that this method enables culture-independent analysis of parasite genomes obtained directly from primary patient skin samples, allowing us to circumvent artifacts associated with adaptation to culture. We show that SWGA can be applied to multiple Leishmania species residing in different host species, suggesting that this method is broadly useful in both experimental infection models and clinical studies. SWGA carried out directly on skin biopsies collected from patients in Corte de Pedra, Bahia, Brazil, showed extensive genomic diversity. Finally, as a proof-of-concept, we demonstrated that SWGA data can be integrated with published whole genome data from cultured parasite isolates to identify variants unique to specific geographic regions in Brazil where treatment failure rates are known to be high. SWGA provides a relatively simple method to generate Leishmania genomes directly from patient samples, unlocking the potential to link parasite genetics with host clinical phenotypes.

Suppression of Ca2+ signals by EGR4 controls Th1 differentiation and anti-cancer immunity in vivo.

While the zinc finger transcription factors EGR1, EGR2, and EGR3 are recognized as critical for T-cell function, the role of EGR4 remains unstudied. Here, we show that EGR4 is rapidly upregulated upon TCR engagement, serving as a critical "brake" on T-cell activation. Hence, TCR engagement of EGR4-/- T cells leads to enhanced Ca2+ responses, driving sustained NFAT activation and hyperproliferation. This causes profound increases in IFNγ production under resting and diverse polarizing conditions that could be reversed by pharmacological attenuation of Ca2+ entry. Finally, an in vivo melanoma lung colonization assay reveals enhanced anti-tumor immunity in EGR4-/- mice, attributable to Th1 bias, Treg loss, and increased CTL generation in the tumor microenvironment. Overall, these observations reveal for the first time that EGR4 is a key regulator of T-cell differentiation and function.

The Ca2+ export pump PMCA clears near-membrane Ca2+ to facilitate store-operated Ca2+ entry and NFAT activation.

Ca2+ signals, which facilitate pluripotent changes in cell fate, reflect the balance between cation entry and export. We found that overexpression of either isoform of the Ca2+-extruding plasma membrane calcium ATPase 4 (PMCA4) pump in Jurkat T cells unexpectedly increased activation of the Ca2+-dependent transcription factor nuclear factor of activated T cells (NFAT). Coexpression of the endoplasmic reticulum-resident Ca2+ sensor stromal interaction molecule 1 (STIM1) with the PMCA4b splice variant further enhanced NFAT activity; however, coexpression with PMCA4a depressed NFAT. No PMCA4 splice variant dependence in STIM1 association was observed, whereas partner of STIM1 (POST) preferentially associated with PMCA4b over PMCA4a, which enhanced, rather than inhibited, PMCA4 function. A comparison of global and near-membrane cytosolic Ca2+ abundances during store-operated Ca2+ entry revealed that PMCA4 markedly depressed near-membrane Ca2+ concentrations, particularly when PMCA4b was coexpressed with STIM1. PMCA4b closely associated with both POST and the store-operated Ca2+ channel Orai1. Furthermore, POST knockdown increased the near-membrane Ca2+ concentration, inhibiting the global cytosolic Ca2+ increase. These observations reveal an unexpected role for POST in coupling PMCA4 to Orai1 to promote Ca2+ entry during T cell activation through Ca2+ disinhibition.

EGR-mediated control of STIM expression and function.

Ca2+ is a ubiquitous, dynamic and pluripotent second messenger with highly context-dependent roles in complex cellular processes such as differentiation, proliferation, and cell death. These Ca2+ signals are generated by Ca2+-permeable channels located on the plasma membrane (PM) and endoplasmic reticulum (ER) and shaped by PM- and ER-localized pumps and transporters. Differences in the expression of these Ca2+ homeostasis proteins contribute to cell and context-dependent differences in the spatiotemporal organization of Ca2+ signals and, ultimately, cell fate. This review focuses on the Early Growth Response (EGR) family of zinc finger transcription factors and their role in the transcriptional regulation of Stromal Interaction Molecule (STIM1), a critical regulator of Ca2+ entry in both excitable and non-excitable cells.

Hold the door: hPMCA1/neuroplastin interactions regulate Ca2+-binding site accessibility.

In a September 2018 paper published in Nature Communications, Gong et al. identified the domains through which human PMCA1 and neuroplastin (NPTN) interact. Upon binding, hPMCA1 TM domains separate T110 in TM1 and A370 in TM3 to reveal the Ca2+-binding site. Thus, NPTN is able to directly modulate the accessibility of cytosolic Ca2+ to PMCA.