Caspase-Mediated Regulation and Cellular Heterogeneity of the cGAS/STING Pathway in Kaposi's Sarcoma-Associated Herpesvirus Infection.

As a result of the ongoing virus-host arms race, viruses have evolved numerous immune subversion strategies, many of which are aimed at suppressing the production of type I interferons (IFNs). Apoptotic caspases have recently emerged as important regulators of type I IFN signaling both in noninfectious contexts and during viral infection. Despite being widely considered antiviral factors since they can trigger cell death, several apoptotic caspases promote viral replication by suppressing innate immune response. Indeed, we previously discovered that the AIDS-associated oncogenic gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) exploits caspase activity to suppress the antiviral type I IFN response and promote viral replication. However, the mechanism of this novel viral immune evasion strategy is poorly understood, particularly with regard to how caspases antagonize IFN signaling during KSHV infection. Here, we show that caspase activity inhibits the DNA sensor cGAS during KSHV lytic replication to block type I IFN induction. Furthermore, we used single-cell RNA sequencing to reveal that the potent antiviral state conferred by caspase inhibition is mediated by an exceptionally small percentage of IFN-ß-producing cells, thus uncovering further complexity of IFN regulation during viral infection. Collectively, these results provide insight into multiple levels of cellular type I IFN regulation that viruses co-opt for immune evasion. Unraveling these mechanisms can inform targeted therapeutic strategies for viral infections and reveal cellular mechanisms of regulating interferon signaling in the context of cancer and chronic inflammatory diseases. IMPORTANCE Type I interferons are key factors that dictate the outcome of infectious and inflammatory diseases. Thus, intricate cellular regulatory mechanisms are in place to control IFN responses. While viruses encode their own immune-regulatory proteins, they can also usurp existing cellular interferon regulatory functions. We found that caspase activity during lytic infection with the AIDS-associated oncogenic gammaherpesvirus Kaposi's sarcoma-associated herpesvirus inhibits the DNA sensor cGAS to block the antiviral type I IFN response. Moreover, single-cell RNA sequencing analyses unexpectedly revealed that an exceptionally small subset of infected cells (<5%) produce IFN, yet this is sufficient to confer a potent antiviral state. These findings reveal new aspects of type I IFN regulation and highlight caspases as a druggable target to modulate cGAS activity.

PepN is a non-essential, cell wall-localized protein that contributes to neutrophil elastase-mediated killing of Streptococcus pneumoniae.

Streptococcus pneumoniae (Spn) is an asymptomatic colonizer of the human nasopharynx but can also cause disease in the inner ear, meninges, lung and blood. Although various mechanisms contribute to the effective clearance of Spn, opsonophagocytosis by neutrophils is perhaps most critical. Upon phagocytosis, Spn is exposed to various degradative molecules, including a family of neutrophil serine proteases (NSPs) that are stored within intracellular granules. Despite the critical importance of NSPs in killing Spn, the bacterial proteins that are degraded by NSPs leading to Spn death are still unknown. In this report, we identify a 90kDa protein in a purified cell wall (CW) preparation, aminopeptidase N (PepN) that is degraded by the NSP neutrophil elastase (NE). Since PepN lacked a canonical signal sequence or LPxTG motif, we created a mutant expressing a FLAG tagged version of the protein and confirmed its localization to the CW compartment. We determined that not only is PepN a CW-localized protein, but also is a substrate of NE in the context of intact Spn cells. Furthermore, in comparison to wild-type TIGR4 Spn, a mutant strain lacking PepN demonstrated a significant hyper-resistance phenotype in vitro in the presence of purified NE as well as in opsonophagocytic assays with purified human neutrophils ex vivo. Taken together, this is the first study to demonstrate that PepN is a CW-localized protein and a substrate of NE that contributes to the effective killing of Spn by NSPs and human neutrophils.