Broad-spectrum inflammasome inhibition by thiomuscimol.

Inflammasome formation, arising from pathogen or internal activating signals, is a key step in canonical pyroptosis, a gasdermin-mediated inflammatory cell death. Inhibition of pyroptosis has great clinical relevance due to its involvement in many different disease states. Current inhibitors of pyroptosis either only inhibit the final lytic step, which still allows inflammatory signal release, or only inhibit a single inflammasome, which does not account for inherent redundancy in activation of other inflammatory pathways.

Muscimol inhibits plasma membrane rupture and ninjurin-1 oligomerization during pyroptosis.

Pyroptosis is a cell death process that causes inflammation and contributes to numerous diseases. Pyroptosis is mediated by caspase-1 family proteases that cleave the pore-forming protein gasdermin D, causing plasma membrane rupture and release of pathogenic cellular contents. We previously identified muscimol as a small molecule that prevents plasma membrane rupture during pyroptosis via an unidentified mechanism.

The unfolded protein response components IRE1α and XBP1 promote human coronavirus infection.

The cellular processes that support human coronavirus replication and contribute to the pathogenesis of severe disease remain incompletely understood. Many viruses, including coronaviruses, cause endoplasmic reticulum (ER) stress during infection. IRE1α is a component of the cellular response to ER stress that initiates non-conventional splicing of mRNA.

Molecular Mechanisms of Pyroptosis.

Pyroptosis is a regulated form of cell death that leads to inflammation and plays a role in many different diseases. Pyroptosis was initially defined by the dependence on caspase-1, a protease which is activated by innate immune signaling complexes called inflammasomes. Caspase-1 cleaves the protein gasdermin D, releasing the N-terminal pore-forming domain, which inserts into the plasma membrane.

Discovery of host-directed modulators of virus infection by probing the SARS-CoV-2-host protein-protein interaction network.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has highlighted the need to better understand virus-host interactions. We developed a network-based method that expands the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-host protein interaction network and identifies host targets that modulate viral infection. To disrupt the SARS-CoV-2 interactome, we systematically probed for potent compounds that selectively target the identified host proteins with high expression in cells relevant to COVID-19.

Simultaneous Detection of Inflammasome Activation and Membrane Damage During Pyroptosis.

Pyroptosis is a highly regulated inflammatory form of cell death that plays a role in many different diseases, including cancer. Pyroptosis was initially described to be mediated by caspase-1, which is activated by innate immune signaling complexes called inflammasomes. Inflammasomes trigger caspase-dependent activation of the pore-forming protein, gasdermin D, and plasma membrane disruption.

IRE1α Promotes Zika Virus Infection via XBP1.

Zika virus (ZIKV) is an emergent member of the family which causes severe congenital defects and other major sequelae, but the cellular processes that support ZIKV replication are incompletely understood. Related flaviviruses use the endoplasmic reticulum (ER) as a membranous platform for viral replication and induce ER stress during infection. Our data suggest that ZIKV activates IRE1α, a component of the cellular response to ER stress.

Diverse small molecules prevent macrophage lysis during pyroptosis.

Pyroptosis is a programmed process of proinflammatory cell death mediated by caspase-1-related proteases that cleave the pore-forming protein, gasdermin D, causing cell lysis and release of inflammatory intracellular contents. The amino acid glycine prevents pyroptotic lysis via unknown mechanisms, without affecting caspase-1 activation or pore formation. Pyroptosis plays a critical role in diverse inflammatory diseases, including sepsis.

Pyroptosis Induction and Detection.

Pyroptosis is a form of programmed pro-inflammatory cell death that plays a protective role in the host response to infection, but can also promote pathogenic inflammation. Pyroptosis is mediated by the cysteine protease, caspase-1. Caspase-1 cleaves gasdermin D, releasing the N-terminal pore-forming domain, which inserts into the plasma membrane and drives osmotic lysis.

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages.

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli.

PPARγ-mediated increase in glucose availability sustains chronic Brucella abortus infection in alternatively activated macrophages.

Eradication of persistent intracellular bacterial pathogens with antibiotic therapy is often slow or incomplete. However, strategies to augment antibiotics are hampered by our poor understanding of the nutritional environment that sustains chronic infection. Here we show that the intracellular pathogen Brucella abortus survives and replicates preferentially in alternatively activated macrophages (AAMs), which are more abundant during chronic infection.

Sensing of bacterial type IV secretion via the unfolded protein response.

Host cytokine responses to Brucella abortus infection are elicited predominantly by the deployment of a type IV secretion system (T4SS). However, the mechanism by which the T4SS elicits inflammation remains unknown. Here we show that translocation of the T4SS substrate VceC into host cells induces proinflammatory responses.

The small protein CydX is required for function of cytochrome bd oxidase in Brucella abortus.

A large number of hypothetical genes potentially encoding small proteins of unknown function are annotated in the Brucella abortus genome. Individual deletion of 30 of these genes identified four mutants, in BAB1_0355, BAB2_0726, BAB2_0470, and BAB2_0450 that were highly attenuated for infection. BAB2_0726, an YbgT-family protein located at the 3' end of the cydAB genes encoding cytochrome bd ubiquinal oxidase, was designated cydX.

Interactions of the human pathogenic Brucella species with their hosts.

Brucellosis is a zoonotic infection caused primarily by the bacterial pathogens Brucella melitensis and B. abortus. It is acquired by consumption of unpasteurized dairy products or by contact with infected animals.

Coordinated host responses during pyroptosis: caspase-1-dependent lysosome exocytosis and inflammatory cytokine maturation.

Activation of caspase-1 leads to pyroptosis, a program of cell death characterized by cell lysis and inflammatory cytokine release. Caspase-1 activation triggered by multiple nucleotide-binding oligomerization domain-like receptors (NLRs; NLRC4, NLRP1b, or NLRP3) leads to loss of lysosomes via their fusion with the cell surface, or lysosome exocytosis. Active caspase-1 increased cellular membrane permeability and intracellular calcium levels, which facilitated lysosome exocytosis and release of host antimicrobial factors and microbial products.

Putative ATP-binding cassette transporter is essential for Brucella ovis pathogenesis in mice.

Brucella ovis is a major cause of reproductive failure in sheep, which is associated with epididymitis and infertility in rams. Importantly, B. ovis is one of the few Brucella species that is not zoonotic.

Establishment of systemic Brucella melitensis infection through the digestive tract requires urease, the type IV secretion system, and lipopolysaccharide O antigen.

Human brucellosis is caused mainly by Brucella melitensis, which is often acquired by ingesting contaminated goat or sheep milk and cheese. Bacterial factors required for food-borne infection of humans by B. melitensis are poorly understood.

Identification of VceA and VceC, two members of the VjbR regulon that are translocated into macrophages by the Brucella type IV secretion system.

Survival and replication inside host cells by Brucella spp. requires a type IV secretion system (T4SS), encoded by the virB locus. However, the identity of the molecules secreted by the T4SS has remained elusive.

Laboratory maintenance of Brucella abortus.

This unit provides protocols for growth of Brucella abortus on solid or in liquid media and for long-term storage of laboratory stocks. Two issues affecting the culture and storage of isolates of this slow-growing bacterium are emphasized: contamination of cultures and outgrowth of attenuated variants lacking a complete lipopolysaccharide. Laboratories planning to work with B.

VirB3 to VirB6 and VirB8 to VirB11, but not VirB7, are essential for mediating persistence of Brucella in the reticuloendothelial system.

The Brucella abortus virB locus contains 12 open reading frames, termed virB1 through virB12, which encode a type IV secretion system. Polar mutations in the virB locus markedly reduce the ability of B. abortus to survive in cultured macrophages or to persist in organs of mice.

VirB12 is a serological marker of Brucella infection in experimental and natural hosts.

The Brucella species type IV secretion system, encoded by the virB1-12 locus, is required for intracellular replication and persistent infection in vivo. The requirement of VirB proteins for infection suggests that they are expressed in vivo and may therefore represent serological markers of infection. To test this idea, we purified recombinant VirB1, VirB5, VirB11, and VirB12 and tested for their recognition by antibodies in sera from experimentally infected mice and goats by using an indirect enzyme-linked immunosorbent assay.

Evaluation of novel Brucella melitensis unmarked deletion mutants for safety and efficacy in the goat model of brucellosis.

Pregnant goats were employed to assess unmarked deletion mutant vaccine candidates BMDeltaasp24, BMDeltacydBA, and BMDeltavirB2, as the target host species naturally infected with Brucella melitensis. Goats were assessed for the degree of pathology associated with the vaccine strains as well as the protective immunity afforded by each strain against abortion and infection after challenge with wild-type Brucella melitensis 16M. Both BMDeltaasp24 and BMDeltavirB2 were considered safe vaccine candidates in the pregnant goat model because they did not cause abortion or colonize fetal tissues.

Brucella abortus virB12 is expressed during infection but is not an essential component of the type IV secretion system.

The Brucella abortus virB operon, consisting of 11 genes, virB1 to virB11, and two putative genes, orf12 (virB12) and orf13, encodes a type IV secretion system (T4SS) that is required for intracellular replication and persistent infection in the mouse model. This study was undertaken to determine whether orf12 (virB12) encodes an essential part of the T4SS apparatus. The virB12 gene was found to encode a 17-kDa protein, which was detected in vitro in B.

Differential requirements for VirB1 and VirB2 during Brucella abortus infection.

The Brucella abortus virB operon, encoding a type IV secretion system (T4SS), is required for intracellular replication and persistent infection in the mouse model. The products of the first two genes of the virB operon, virB1 and virB2, are predicted to be localized at the bacterial surface, where they could potentially interact with host cells. Studies to date have focused on characterization of transposon mutations in these genes, which are expected to exert polar effects on downstream genes in the operon.