The tetrapeptide sequence of IL-18 and IL-1ß regulates their recruitment and activation by inflammatory caspases.

Inflammasomes are multiprotein signaling complexes that activate the innate immune system. Canonical inflammasomes recruit and activate caspase-1, which then cleaves and activates IL-1ß and IL-18, as well as gasdermin D (GSDMD) to induce pyroptosis. In contrast, non-canonical inflammasomes, caspases-4/-5 (CASP4/5) in humans and caspase-11 (CASP11) in mice, are known to cleave GSDMD, but their role in direct processing of other substrates besides GSDMD has remained unknown. Here, we show that CASP4/5 but not CASP11 can directly cleave and activate IL-18. However, CASP4/5/11 can all cleave IL-1ß to generate a 27-kDa fragment that deactivates IL-1ß signaling. Mechanistically, we demonstrate that the sequence identity of the tetrapeptide sequence adjacent to the caspase cleavage site regulates IL-18 and IL-1ß recruitment and activation. Altogether, we have identified new substrates of the non-canonical inflammasomes and reveal key mechanistic details regulating inflammation that may aid in developing new therapeutics for immune-related disorders.

Human GBP1 facilitates the rupture of the Legionella-containing vacuole and inflammasome activation.

IMPORTANCE: Inflammasomes are essential for host defense against intracellular bacterial pathogens like Legionella, as they activate caspases, which promote cytokine release and cell death to control infection. In mice, interferon (IFN) signaling promotes inflammasome responses against bacteria by inducing a family of IFN-inducible GTPases known as guanylate-binding proteins (GBPs). Within murine macrophages, IFN promotes the rupture of the Legionella-containing vacuole (LCV), while GBPs are dispensable for this process. Instead, GBPs facilitate the lysis of cytosol-exposed Legionella. In contrast, the functions of IFN and GBPs in human inflammasome responses to Legionella are poorly understood. We show that IFN-γ enhances inflammasome responses to Legionella in human macrophages. Human GBP1 is required for these IFN-γ-driven inflammasome responses. Furthermore, GBP1 co-localizes with Legionella and/or LCVs in a type IV secretion system (T4SS)-dependent manner and promotes damage to the LCV, which leads to increased exposure of the bacteria to the host cell cytosol. Thus, our findings reveal species- and pathogen-specific differences in how GBPs function to promote inflammasome responses.

Inflammasomes primarily restrict cytosolic Salmonella replication within human macrophages.

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that utilizes its type III secretion systems (T3SSs) to inject virulence factors into the host cell and colonize the host. In turn, a subset of cytosolic immune receptors respond to T3SS ligands by forming multimeric signaling complexes called inflammasomes, which activate caspases that induce interleukin-1 (IL-1) family cytokine release and an inflammatory form of cell death called pyroptosis. Human macrophages mount a multifaceted inflammasome response to Salmonella infection that ultimately restricts intracellular bacterial replication. However, how inflammasomes restrict Salmonella replication remains unknown. We find that caspase-1 is essential for mediating inflammasome responses to Salmonella and subsequent restriction of bacterial replication within human macrophages, with caspase-4 contributing as well. We also demonstrate that the downstream pore-forming protein gasdermin D (GSDMD) and ninjurin-1 (NINJ1), a mediator of terminal cell lysis, play a role in controlling Salmonella replication in human macrophages. Notably, in the absence of inflammasome responses, we observed hyperreplication of Salmonella within the cytosol of infected cells, and we also observed increased bacterial replication within vacuoles, suggesting that inflammasomes control Salmonella replication primarily within the cytosol and also within vacuoles. These findings reveal that inflammatory caspases and pyroptotic factors mediate inflammasome responses that restrict the subcellular localization of intracellular Salmonella replication within human macrophages.

TLR priming licenses NAIP inflammasome activation by immunoevasive ligands.

NLR family, apoptosis inhibitory proteins (NAIPs) detect bacterial flagellin and structurally related components of bacterial type III secretion systems (T3SS), and recruit NLR family, CARD domain containing protein 4 (NLRC4) and caspase-1 into an inflammasome complex that induces pyroptosis. NAIP/NLRC4 inflammasome assembly is initiated by the binding of a single NAIP to its cognate ligand, but a subset of bacterial flagellins or T3SS structural proteins are thought to evade NAIP/NLRC4 inflammasome sensing by not binding to their cognate NAIPs. Unlike other inflammasome components such as NLRP3, AIM2, or some NAIPs, NLRC4 is constitutively present in resting macrophages, and not thought to be regulated by inflammatory signals. Here, we demonstrate that Toll-like receptor (TLR) stimulation upregulates NLRC4 transcription and protein expression in murine macrophages, which licenses NAIP detection of evasive ligands. TLR-induced NLRC4 upregulation and NAIP detection of evasive ligands required p38 MAPK signaling. In contrast, TLR priming in human macrophages did not upregulate NLRC4 expression, and human macrophages remained unable to detect NAIP-evasive ligands even following priming. Critically, ectopic expression of either murine or human NLRC4 was sufficient to induce pyroptosis in response to immunoevasive NAIP ligands, indicating that increased levels of NLRC4 enable the NAIP/NLRC4 inflammasome to detect these normally evasive ligands. Altogether, our data reveal that TLR priming tunes the threshold for NAIP/NLRC4 inflammasome activation and enables inflammasome responses against immunoevasive or suboptimal NAIP ligands.

Human and mouse NAIP/NLRC4 inflammasome responses to bacterial infection.

Intracellular immune complexes known as inflammasomes sense breaches of cytosolic sanctity. Inflammasomes promote downstream proinflammatory events, including interleukin-1 (IL-1) family cytokine release and pyroptotic cell death. The nucleotide-binding leucine-rich repeat family, apoptosis inhibitory protein/nucleotide-binding leucine-rich repeat family, caspase recruitment domain (CARD) domain-containing protein 4 (NAIP/NLRC4) inflammasome is involved in a range of pathogenic and protective inflammatory processes in mammalian hosts. In particular, the NAIP/NLRC4 inflammasome responds to flagellin and components of the virulence-associated type III secretion (T3SS) apparatus in the host cytosol, thereby allowing it to be a critical mediator of host defense during bacterial infection. Notable species- and cell type-specific differences exist in NAIP/NLRC4 inflammasome responses to bacterial pathogens. With a focus on Salmonella enterica serovar Typhimurium as a model pathogen, we review differences between murine and human NAIP/NLRC4 inflammasome responses. Differences in NAIP/NLRC4 inflammasome responses across species and cell types may have arisen in part due to evolutionary pressures.

The tetrapeptide sequence of IL-1ß regulates its recruitment and activation by inflammatory caspases.

The mammalian innate immune system uses germline-encoded cytosolic pattern-recognition receptors (PRRs) to detect intracellular danger signals. At least six of these PRRs are known to form multiprotein complexes called inflammasomes which activate cysteine proteases known as caspases. Canonical inflammasomes recruit and activate caspase-1 (CASP1), which in turn cleaves and activates inflammatory cytokines such as IL-1ß and IL-18, as well as the pore forming protein, gasdermin D (GSDMD), to induce pyroptotic cell death. In contrast, non-canonical inflammasomes, caspases-4/-5 (CASP4/5) in humans and caspase-11 (CASP11) in mice, are activated by intracellular LPS to cleave GSDMD, but their role in direct processing of inflammatory cytokines has not been established. Here we show that active CASP4/5 directly cleave IL-18 to generate the active species. Surprisingly, we also discovered that CASP4/5/11 cleave IL-1ß at D27 to generate a 27 kDa fragment that is predicted to be inactive and cannot signal to the IL-1 receptor. Mechanistically, we discovered that the sequence identity of the P4-P1 tetrapeptide sequence adjacent to the caspase cleavage site (D116) regulates the recruitment and processing of IL-1ß by inflammatory caspases to generate the bioactive species. Thus, we have identified new substrates of the non-canonical inflammasomes and reveal key mechanistic details regulating inflammation.

The Best of Both Worlds: Discovery-Driven Learning through a Lab-Seminar Approach.

Microbiology courses are often designed as either a lecture class with a laboratory component or a seminar-style class. Each type of course provides students with unique learning opportunities. Lab courses allow students to perform simple experiments that relate to fundamental concepts taught in the corresponding lectures, while seminar courses challenge students to read and discuss primary literature. Microbiology courses offering a combination of seminar-style discussions and laboratory procedures are rare. Our goal in the "Microbial Diversity and Pathogenesis" undergraduate course is to integrate experiences of a seminar class with those of a discovery-driven lab course, thereby strengthening students' learning experiences through diversified didactic approaches. In the first half of the course, students read and discuss published peer-reviewed articles that cover major topics in both basic and applied microbiology, including antibiotic resistance, pathogenesis, and biotechnology applications. Complementing this primary literature, students perform microbiology experiments related to the topics covered in the readings. The assigned readings, discussions, and experiments provide a foundation in the second half of the course for inquiry-based exploratory research using student-designed transposon screens and selections. The course culminates in each student drafting a hypothesis-driven research proposal based on their literature review, their learned experimental techniques, and the preliminary data generated as a class. Through such first-hand experimental experience, students gain fundamental lab skills that are applicable beyond the realm of microbiology, such as sterile technique and learning how to support conclusions with scientific evidence. We observed a tremendous synergy between the seminar and lab aspects of our course. This unique didactic experience allows students to understand and connect primary literature to their experiments, while the discovery-driven aspect of this approach fosters active engagement of students with scientific research.

Human NAIP/NLRC4 and NLRP3 inflammasomes detect Salmonella type III secretion system activities to restrict intracellular bacterial replication.

Salmonella enterica serovar Typhimurium is a Gram-negative pathogen that uses two distinct type III secretion systems (T3SSs), termed Salmonella pathogenicity island (SPI)-1 and SPI-2, to deliver virulence factors into the host cell. The SPI-1 T3SS enables Salmonella to invade host cells, while the SPI-2 T3SS facilitates Salmonella's intracellular survival. In mice, a family of cytosolic immune sensors, including NAIP1, NAIP2, and NAIP5/6, recognizes the SPI-1 T3SS needle, inner rod, and flagellin proteins, respectively. Ligand recognition triggers assembly of the NAIP/NLRC4 inflammasome, which mediates caspase-1 activation, IL-1 family cytokine secretion, and pyroptosis of infected cells. In contrast to mice, humans encode a single NAIP that broadly recognizes all three ligands. The role of NAIP/NLRC4 or other inflammasomes during Salmonella infection of human macrophages is unclear. We find that although the NAIP/NLRC4 inflammasome is essential for detecting T3SS ligands in human macrophages, it is partially required for responses to infection, as Salmonella also activated the NLRP3 and CASP4/5 inflammasomes. Importantly, we demonstrate that combinatorial NAIP/NLRC4 and NLRP3 inflammasome activation restricts Salmonella replication in human macrophages. In contrast to SPI-1, the SPI-2 T3SS inner rod is not sensed by human or murine NAIPs, which is thought to allow Salmonella to evade host recognition and replicate intracellularly. Intriguingly, we find that human NAIP detects the SPI-2 T3SS needle protein. Critically, in the absence of both flagellin and the SPI-1 T3SS, the NAIP/NLRC4 inflammasome still controlled intracellular Salmonella burden. These findings reveal that recognition of Salmonella SPI-1 and SPI-2 T3SSs and engagement of both the NAIP/NLRC4 and NLRP3 inflammasomes control Salmonella infection in human macrophages.

Salmonella "RecAmends" self-healing.

Persistent bacteria contribute to antibiotic treatment failure, potentially fueling resistance. Persisters must survive host defenses while retaining infective capacity after antibiotic cessation. In this issue of Cell Host and Microbe, Hill et al. identify bacterial RecA as a DNA repair factor that allows intracellular bacteria survival, regrowth, and relapse.

Tales of diversity: Genomic and morphological characteristics of forty-six Arthrobacter phages.

The vast bacteriophage population harbors an immense reservoir of genetic information. Almost 2000 phage genomes have been sequenced from phages infecting hosts in the phylum Actinobacteria, and analysis of these genomes reveals substantial diversity, pervasive mosaicism, and novel mechanisms for phage replication and lysogeny. Here, we describe the isolation and genomic characterization of 46 phages from environmental samples at various geographic locations in the U.S. infecting a single Arthrobacter sp. strain. These phages include representatives of all three virion morphologies, and Jasmine is the first sequenced podovirus of an actinobacterial host. The phages also span considerable sequence diversity, and can be grouped into 10 clusters according to their nucleotide diversity, and two singletons each with no close relatives. However, the clusters/singletons appear to be genomically well separated from each other, and relatively few genes are shared between clusters. Genome size varies from among the smallest of siphoviral phages (15,319 bp) to over 70 kbp, and G+C contents range from 45-68%, compared to 63.4% for the host genome. Although temperate phages are common among other actinobacterial hosts, these Arthrobacter phages are primarily lytic, and only the singleton Galaxy is likely temperate.