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Gasdermin B (GSDMB) belongs to a large family of pore forming cytolysins that execute inflammatory cell death programs. While genetic studies have linked GSDMB polymorphisms to human disease, its function in the immunological response to pathogens remains poorly understood. Here, we report a dynamic host-pathogen conflict between GSDMB and the IpaH7.8 effector protein secreted by enteroinvasive Shigella flexneri. We show that IpaH7.8 ubiquitinates and targets GSDMB for 26S proteasome destruction. This virulence strategy protects Shigella from the bacteriocidic activity of Natural Killer cells by suppressing Granzyme-A mediated activation of GSDMB. In contrast to the canonical function of most Gasdermin-family members, GSDMB does not inhibit Shigella by lysing host cells. Rather, it exhibits direct microbiocidal activity through recognition of phospholipids found on Gram-negative bacterial membranes. These findings place GSDMB as a central executioner of intracellular bacterial killing and reveals a mechanism employed by pathogens to counteract this host defense system.

Brucella abortus infects the placenta of pregnant cows, where it replicates to high levels and triggers abortion of the calf. The aborted material is highly infectious and transmits infection to both cows and humans, but very little is known about how B. abortus causes abortion. By studying this infection in pregnant mice, we discovered that B. abortus kills trophoblasts, which are important cells for maintaining pregnancy. This killing required an injected bacterial protein (VceC) that triggered an endoplasmic reticulum (ER) stress response in the trophoblast. By inhibiting ER stress or infecting mice that lack CHOP, a protein induced by ER stress, we could prevent death of trophoblasts, reduce inflammation, and increase the viability of the pups. Our results suggest that B. abortus injects VceC into placental trophoblasts to promote its transmission by abortion., Subversion of endoplasmic reticulum (ER) function is a feature shared by multiple intracellular bacteria and viruses, and in many cases this disruption of cellular function activates pathways of the unfolded protein response (UPR). In the case of infection with Brucella abortus, the etiologic agent of brucellosis, the unfolded protein response in the infected placenta contributes to placentitis and abortion, leading to pathogen transmission. Here we show that B. abortus infection of pregnant mice led to death of infected placental trophoblasts in a manner that depended on the VirB type IV secretion system (T4SS) and its effector VceC. The trophoblast death program required the ER stress-induced transcription factor CHOP. While NOD1/NOD2 expression in macrophages contributed to ER stress-induced inflammation, these receptors did not play a role in trophoblast death. Both placentitis and abortion were independent of apoptosis-associated Speck-like protein containing a caspase activation and recruitment domain (ASC). These studies show that B. abortus uses its T4SS to induce cell-type-specific responses to ER stress in trophoblasts that trigger placental inflammation and abortion. Our results suggest further that in B. abortus the T4SS and its effectors are under selection as bacterial transmission factors.

The enteric attaching and effacing (A/E) pathogens enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) and the invasive pathogens enteroinvasive E. coli (EIEC) and Shigella encode type III secretion systems (T3SS) used to inject effector proteins into human host cells during infection. Among these are a group of effectors required for NF-κB-mediated host immune evasion. Recent studies have identified several effector proteins from A/E pathogens and EIEC/ Shigella that are involved in suppression of NF-κB and have uncovered their cellular and molecular functions. A novel mechanism among these effectors from both groups of pathogens is to coordinate effector function during infection. This cooperativity among effector proteins explains how bacterial pathogens are able to effectively suppress innate immune defense mechanisms in response to diverse classes of immune receptor signaling complexes (RSCs) stimulated during infection.

The linear ubiquitin chain assembly complex (LUBAC) is a multimeric E3 ligase that catalyses M1 or linear ubiquitination of activated immune receptor signalling complexes (RSCs). Mutations that disrupt linear ubiquitin assembly lead to complex disease pathologies including immunodeficiency and autoinflammation in both humans and mice, but microbial toxins that target LUBAC function have not yet been discovered. Here, we report the identification of two homologous Shigella flexneri type III secretion system effector E3 ligases IpaH1.4 and IpaH2.5, which directly interact with LUBAC subunit Heme-oxidized IRP2 ubiquitin ligase-1 (HOIL-1L) and conjugate K48-linked ubiquitin chains to the catalytic RING-between-RING domain of HOIL-1-interacting protein (HOIP). Proteasomal degradation of HOIP leads to irreversible inactivation of linear ubiquitination and blunting of NF-κB nuclear translocation in response to tumour-necrosis factor (TNF), IL-1β and pathogen-associated molecular patterns. Loss of function studies in mammallian cells in combination with bacterial genetics explains how Shigella evades a broad spectrum of immune surveillance systems by cooperative inhibition of receptor ubiquitination and reveals the critical importance of LUBAC in host defence against pathogens.

Endoplasmic reticulum (ER) stress is a major contributor to inflammatory diseases, such as Crohn’s disease and type 2 diabetes1,2. ER stress induces the unfolded protein response (UPR), which involves activation of three transmembrane receptors, ATF6 (activating transcription factor 6), PERK (protein kinase RNA-like endoplasmic reticulum kinase) and IRE1α (inositol-requiring enzyme 1α)3 (Extended Data figure 1a). Once activated, IRE1α recruits TRAF2 (TNF receptor-associated factor 2) to the ER membrane to initiate inflammatory responses via the nuclear factor kappa B (NF-κB) pathway4. Inflammation is commonly triggered when pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) or nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), detect tissue damage or microbial infection. However, it is not clear which PRRs play a major role in inducing inflammation during ER stress. Here we show that NOD1 and NOD2, two members of the NLR family of PRRs, are important mediators of ER stress-induced inflammation. The ER stress inducers thapsigargin and dithiothreitol (DTT) triggered production of the pro-inflammatory cytokine interleukin (IL)-6 in a NOD1/2-dependent fashion. Inflammation and IL-6 production triggered by infection with Brucella abortus, which induces ER stress by injecting the type IV secretion system (T4SS) effector protein VceC into host cells5, was TRAF2, NOD1/2 and RIP2-dependent and could be blunted by treatment with the ER-stress inhibitor tauroursodeoxycholate (TUDCA) or an IRE1α kinase inhibitor. The association of NOD1 and NOD2 with pro-inflammatory responses induced by the IRE1α/TRAF2 signaling pathway provides a novel link between innate immunity and ER stress-induced inflammation.

Brucellosis is a global disease of domestic and wild mammals that is caused by intracellular bacteria of the genus Brucella. Although humans are not a natural reservoir for Brucella, infection in the human population is common in many countries, and brucellosis is one of the most common zoonotic infections. Brucella species have evolved to avoid the host’s immune system and infection is usually characterized by long-term persistence of the bacteria. One important Brucella virulence factor for intracellular survival and persistence in the host is the type IV secretion system. This review will discuss the Brucella type IV secretion system in detail, including current knowledge of architecture and regulation, as well as the newly identified effector substrates that this system transports into host cells.

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. We hypothesized that proteins translocated by the T4SS would be co-regulated with the virB operon. The LuxR family regulator VjbR, known to regulate virB, bound a fragment of the virB promoter containing an 18 bp palindromic motif (virB promoter box), showing that VjbR regulated the virB operon directly. To identify virB co-regulated genes, we searched the Brucella suis 1330 and B. abortus 2308 genomes for genes with an upstream virB promoter box. One hundred and forty-four promoters in the two genomes contained the virB promoter box, including those of fliC encoding flagellin and cgs encoding cyclic beta-glucan synthetase. Thirteen of these proteins were tested for VirB-dependent translocation into macrophages using a beta-lactamase reporter assay. This analysis resulted in the identification of the proteins encoded by BAB1_1652 (VceA) and BR1038/BAB1_1058 (VceC) as novel protein substrates of the Brucella T4SS. VceC could also be translocated by the Legionella pneumophila Dot/Icm T4SS into host cells. Our results suggest that VjbR co-ordinates expression of the T4SS and at least two of its secreted substrates.

Pathogenic bacteria introduce effector proteins directly into the cytosol of eukaryotic cells to promote invasion and colonization. OspG, a Shigella spp. effector kinase, plays a role in this process by helping to suppress the host inflammatory response. OspG has been reported to bind host E2 ubiquitin-conjugating enzymes activated with ubiquitin (E2∼Ub), a key enzyme complex in ubiquitin transfer pathways. A co-crystal structure of the OspG/UbcH5c∼Ub complex reveals that complex formation has important ramifications for the activity of both OspG and the UbcH5c∼Ub conjugate. OspG is a minimal kinase domain containing only essential elements required for catalysis. UbcH5c∼Ub binding stabilizes an active conformation of the kinase, greatly enhancing OspG kinase activity. In contrast, interaction with OspG stabilizes an extended, less reactive form of UbcH5c∼Ub. Recognizing conserved E2 features, OspG can interact with at least ten distinct human E2s∼Ub. Mouse oral infection studies indicate that E2∼Ub conjugates act as novel regulators of OspG effector kinase function in eukaryotic host cells.

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. Ectopically expressed VceC interacted with the endoplasmic reticulum (ER) chaperone BiP/Grp78 and localized to the ER of HeLa cells. ER localization of VceC required a transmembrane domain in its N terminus. Notably, the expression of VceC resulted in reorganization of ER structures. In macrophages, VceC was required for B. abortus-induced inflammation by induction of the unfolded protein response by a process requiring inositol-requiring transmembrane kinase/endonuclease 1. Altogether, these findings suggest that translocation of the T4SS effector VceC induces ER stress, which results in the induction of proinflammatory host cell responses during B. abortus infection., IMPORTANCE Brucella species are pathogens that require a type IV secretion system (T4SS) to survive in host cells and to maintain chronic infection. By as-yet-unknown pathways, the T4SS also elicits inflammatory responses in infected cells. Here we show that inflammation caused by the T4SS results in part from the sensing of a T4SS substrate, VceC, that localizes to the endoplasmic reticulum (ER), an intracellular site of Brucella replication. Possibly via binding of the ER chaperone BiP, VceC causes ER stress with concomitant expression of proinflammatory cytokines. Thus, induction of the unfolded protein response may represent a novel pathway by which host cells can detect pathogens deploying a T4SS.

Netting the Bad Guys Antimicrobial peptides are an evolutionarily conserved component of innate immunity in the intestine. One family, α-defensins, typically exert their antimicrobial effects through microbicidal activity against bacteria. Humans express only two α-defensins, human defensin 5 (HD5) and HD6. HD5 exhibits bactericidal activity and plays a role in shaping the bacterial composition of the gut. HD6, on the other hand, does not show bactericidal activity and its function in the gut is unclear. Now, Chu et al. (p. 477 , published online 21 June; see the Perspective by Ouellette and Selsted ) show that HD6 protects against bacterial pathogens. Rather than killing them directly, HD6 binds to bacteria surface proteins and, through a process of self-assembly, forms fibrils and nanonets that ensnare invading bacterial pathogens.

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. A B. abortus cydX mutant lacked cytochrome bd oxidase activity, as shown by increased sensitivity to H(2)O(2), decreased acid tolerance and increased resistance to killing by respiratory inhibitors. The C terminus, but not the N terminus, of CydX was located in the periplasm, suggesting that CydX is an integral cytoplasmic membrane protein. Phenotypic analysis of the cydX mutant, therefore, suggested that CydX is required for full function of cytochrome bd oxidase, possibly via regulation of its assembly or activity.

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. Globally, it is one of the most widespread zoonoses, with 500,000 new cases reported each year. In endemic areas, Brucella infections represent a serious public health problem that results in significant morbidity and economic losses. An important feature of the disease is persistent bacterial colonization of the reticuloendothelial system. In this review we discuss recent insights into mechanisms of intracellular survival and immune evasion that contribute to systemic persistence by the pathogenic Brucella species.

In humans, pathogenic Brucella species cause a febrile illness known as brucellosis. A key pathogenic trait of this group of organisms is their ability to survive in immune cells and persist in tissues of the reticuloendothelial system, a process that requires the function of a Type IV secretion system. In contrast to other well-studied Gram-negative bacteria, Brucella spp. do not cause inflammation at the site of invasion, but have a latency period of 2-4 weeks before the onset of symptoms. This review discusses several mechanisms that allow Brucella spp. both to evade detection by pattern recognition receptors of the innate immune system and suppress their signalling. In contrast to these stealth features, the VirB Type IV secretion system, which mediates survival within phagocytic cells, stimulates innate immune responses in vivo. The responses stimulated by this virulence factor are sufficient to check bacterial growth, but not to elicit sterilizing immunity. The result is a stand-off between host and pathogen that results in persistent infection.

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. While a nonpolar deletion of the virB2 gene reduces survival in cultured macrophages and in organs of mice, a nonpolar deletion of virB1 only reduces survival in macrophages, whereas virB12 is dispensable for either virulence trait. Here we investigated the role of the remaining genes in the virB locus during survival in macrophages and virulence in mice. Mutants carrying nonpolar deletions of the virB3 , virB4 , virB5 , virB6 , virB7 , virB8 , virB9 , virB10 , or virB11 gene were constructed and characterized. All mutations reduced the ability of B. abortus to survive in J774A.1 mouse macrophage-like cells to a degree similar to that caused by a deletion of the entire virB locus. Deletion of virB3 , virB4 , virB5 , virB6 , virB8 , virB9 , virB10 , or virB11 markedly reduced the ability of B. abortus to persist in the spleens of mice at 8 weeks after infection. Interestingly, deletion of virB7 did not reduce the ability of B. abortus to persist in spleens of mice. We conclude that virB2 , virB3 , virB4 , virB5 , virB6 , virB8 , virB9 , virB10 , and virB11 are essential for virulence of B. abortus in mice, while functions encoded by the virB1 , virB7 , and virB12 genes are not required for persistence in organs with this animal model.