SUMMARY Endoplasmic reticulum (ER) stress is observed in many human diseases, often associated with inflammation. ER stress can trigger inflammation through nucleotide-binding domain and leucine-rich repeat containing (NLRP3) inflammsome, which may stimulate inflammasome formation by association with damaged mitochondria. How ER stress triggers mitochondrial dysfunction and inflammasome activation is ill defined. Here we have used an infection model to show that the IRE1α ER stress sensor regulates regulated mitochondrial dysfunction through an NLRP3-mediated feed-forward loop, independently of ASC. IRE1α activation increased mitochondrial reactive oxygen species, promoting NLRP3 association with mitochondria. NLRP3 was required for ER stress-induced cleavage of caspase-2 and the pro-apoptotic factor, Bid, leading to subsequent release of mitochondrial contents. Caspase-2 and Bid were necessary for activation of the canonical inflammasome by infection-associated or general ER stress. These data identify an NLRP3-caspase-2 dependent mechanism that relays ER stress to the mitochondria to promote inflammation, integrating cellular stress and innate immunity.

No abstract

Vibrio cholerae is a gram-negative bacterium that is the causative agent of cholera, a severe diarrheal illness. The two biotypes of V. cholerae O1 capable of causing cholera, classical and El Tor, require different in vitro growth conditions for induction of virulence gene expression. Growth under the inducing conditions or infection of a host initiates a complex regulatory cascade that results in production of ToxT, a regulatory protein that directly activates transcription of the genes encoding cholera toxin (CT), toxin-coregulated pilus (TCP), and other virulence genes. Previous studies have shown that sodium bicarbonate induces CT expression in the V. cholerae El Tor biotype. However, the mechanism for bicarbonate-mediated CT induction has not been defined. In this study, we demonstrate that bicarbonate stimulates virulence gene expression by enhancing ToxT activity. Both the classical and El Tor biotypes produce inactive ToxT protein when they are cultured statically in the absence of bicarbonate. Addition of bicarbonate to the culture medium does not affect ToxT production but causes a significant increase in CT and TCP expression in both biotypes. Ethoxyzolamide, a potent carbonic anhydrase inhibitor, inhibits bicarbonate-mediated virulence induction, suggesting that conversion of CO 2 into bicarbonate by carbonic anhydrase plays a role in virulence induction. Thus, bicarbonate is the first positive effector for ToxT activity to be identified. Given that bicarbonate is present at high concentration in the upper small intestine where V. cholerae colonizes, bicarbonate is likely an important chemical stimulus that V. cholerae senses and that induces virulence during the natural course of infection.Cholera is a human disease that is characterized by massive loss of water and electrolytes, which leads to severe dehydration and hypovolemic shock if the condition is not treated. The causative agent of cholera is Vibrio cholerae, a highly motile, gram-negative, curved rod having a single polar flagellum. V. cholerae strains are classified into serogroups based on the lipopolysaccharide O antigen, and more than 200 serogroups have been identified to date. Only serogroups O1 and O139 are responsible for epidemic and pandemic cholera (46, 47). Serogroup O1 can be further divided into two biotypes, classical and El Tor, based on biochemical properties and susceptibility to bacteriophages (11,47). Classical biotype V. cholerae strains are thought to have caused the first six cholera pandemics, beginning in 1817, whereas the El Tor biotype has been responsible for the seventh pandemic, which has been ongoing since 1961 (11, 47).A major difference between the classical and El Tor biotypes is that they require different in vitro growth conditions for virulence gene induction. The classical biotype is cultured in LB medium at 30°C and pH 6.5 for maximal virulence gene expression and is cultured in LB medium at 37°C and pH 8.5 for minimal virulence gene expression (41). The El Tor biotype is cultured under biphasic condi...

The human diarrheal disease cholera is caused by the aquatic bacterium Vibrio cholerae. V. cholerae in the environment is associated with several varieties of aquatic life, including insect egg masses, shellfish, and vertebrate fish. Here we describe a novel animal model for V. cholerae, the zebrafish. Pandemic V. cholerae strains specifically colonize the zebrafish intestinal tract after exposure in water with no manipulation of the animal required. Colonization occurs in close contact with the intestinal epithelium and mimics colonization observed in mammals. Zebrafish that are colonized by V. cholerae transmit the bacteria to naive fish, which then become colonized. Striking differences in colonization between V. cholerae classical and El Tor biotypes were apparent. The zebrafish natural habitat in Asia heavily overlaps areas where cholera is endemic, suggesting that zebrafish and V. cholerae evolved in close contact with each other. Thus, the zebrafish provides a natural host model for the study of V. cholerae colonization, transmission, and environmental survival. Vibrio cholerae, the cause of the severe human diarrheal disease cholera, is also a ubiquitous inhabitant of coastal regions around the globe. As is the case for all species within the Vibrio genus, V. cholerae is an aquatic bacterium that may be found both freely swimming and in association with various forms of aquatic flora and fauna (1-5). The environmental lifestyle and reservoirs of V. cholerae have only in recent years become the subject of vigorous research and remain poorly understood.Over 200 V. cholerae serogroups have been identified from environmental sampling. However, only the O1 and O139 serogroups are capable of causing cholera. The O1 serogroup is further subdivided into two biotypes, classical and El Tor (6). Classical biotype V. cholerae is thought to have caused the first six of the seven known cholera pandemics beginning in 1817 and produces a more severe form of cholera. El Tor V. cholerae is responsible for the seventh pandemic, which began in 1961 and continues to the present day. El Tor strains are thought to be better suited for environmental survival, although the reasons for this are not clear. However, classical biotype strains are currently very difficult, if not impossible, to isolate from the environment, suggesting that El Tor strains have fully occupied the V. cholerae environmental niche. O139 serogroup strains, which caused large cholera outbreaks in the 1990s, have been shown to be derived from El Tor strains (7). In recent years some hybrid strains that closely resemble El Tor strains but also contain genetic material from classical strains have been isolated from cholera patients (8-10).To become a human pathogen, V. cholerae must be ingested in contaminated water or seafood. After ingestion, V. cholerae senses numerous signals resulting in production of virulence factors that permit colonization of the human intestine and ultimately cause the diarrhea that will transmit V. cholerae back into the environment...

Bacterial infection can trigger cellular stress programs, such as the unfolded protein response (UPR), which occurs when misfolded proteins accumulate within the endoplasmic reticulum (ER). Here, we used the human pathogen methicillin-resistant Staphylococcus aureus (MRSA) as an infection model to probe how ER stress promotes antimicrobial function. MRSA infection activated the most highly conserved unfolded protein response sensor, inositol-requiring enzyme 1α (IRE1α), which was necessary for robust bacterial killing in vitro and in vivo. The macrophage IRE1-dependent bactericidal activity required reactive oxygen species (ROS). Viable MRSA cells excluded ROS from the nascent phagosome and strongly triggered IRE1 activation, leading to sustained generation of ROS that were largely Nox2 independent. In contrast, dead MRSA showed early colocalization with ROS but was a poor activator of IRE1 and did not trigger sustained ROS generation. The global ROS stimulated by IRE1 signaling was necessary, but not sufficient, for MRSA killing, which also required the ER resident SNARE Sec22B for accumulation of ROS in the phagosomal compartment. Taken together, these results suggest that IRE1-mediated persistent ROS generation might act as a fail-safe mechanism to kill bacterial pathogens that evade the initial macrophage oxidative burst.