Previous studies have demonstrated that the murine coronavirus mouse hepatitis virus (MHV) nonstructural protein 2 (ns2) is a 2=,5=-phosphodiesterase that inhibits activation of the interferon-induced oligoadenylate synthetase (OAS)-RNase L pathway. Enzymatically active ns2 is required for efficient MHV replication in macrophages, as well as for the induction of hepatitis in C57BL/6 mice. In contrast, following intranasal or intracranial inoculation, efficient replication of MHV in the brain is not dependent on an enzymatically active ns2. The replication of wild-type MHV strain A59 (A59) and a mutant with an inactive phosphodiesterase (ns2-H126R) was assessed in primary hepatocytes and primary central nervous system (CNS) cell types-neurons, astrocytes, and oligodendrocytes. A59 and ns2-H126R replicated with similar kinetics in all cell types tested, except macrophages and microglia. RNase L activity, as assessed by rRNA cleavage, was induced by ns2-H126R, but not by A59, and only in macrophages and microglia. Activation of RNase L correlated with the induction of type I interferon and the consequent high levels of OAS mRNA induced in these cell types. Pretreatment of nonmyeloid cells with interferon restricted A59 and ns2-H126R to the same extent and failed to activate RNase L following infection, despite induction of OAS expression. However, rRNA degradation was induced by treatment of astrocytes or oligodendrocytes with poly(I·C). Thus, RNase L activation during MHV infection is cell type specific and correlates with relatively high levels of expression of OAS genes, which are necessary but not sufficient for induction of an effective RNase L antiviral response.T he murine coronavirus mouse hepatitis virus (MHV) is an enveloped, positive-strand RNA virus of the coronavirus family within the nidovirus order. MHV is a collection of strains with tropisms for different organs, including the liver and central nervous system (CNS), and thus provides models for the study of acute encephalitis and hepatitis, as well as chronic demyelinating disease. The MHV-A59 strain (A59) used in this study induces mild encephalitis and moderate hepatitis. Studies of the pathogenesis of MHV strains and recombinant chimeric MHVs have shown that postentry virus-host interactions have significant impact on organ tropism and virulence in MHV-infected mice (1, 2).The type I interferon (IFN) response is an early innate response that is crucial to survival of mice following infection with many viruses, including MHV (3-5). During infection, viral doublestranded RNA (dsRNA) is recognized by pattern recognition receptors, such as MDA5 in the case of MHV in most cell types (3-5); this leads to the synthesis of type I IFN (Fig. 1). Alpha/beta IFN (IFN-␣/␤) induces expression of interferon-stimulated genes (ISGs) encoding pattern recognition receptors, transcription factors, and antiviral effectors, including multiple oligoadenylate synthetase (OAS) proteins. Viral dsRNA directly binds to and activates OAS to synthesize 2=,5=-linked oligoaden...

dCoxiella burnetii replicates within permissive host cells by employing a Dot/Icm type IV secretion system (T4SS) to translocate effector proteins that direct the formation of a parasitophorous vacuole. C57BL/6 mouse macrophages restrict the intracellular replication of the C. burnetii Nine Mile phase II (NMII) strain. However, eliminating Toll-like receptor 2 (TLR2) permits bacterial replication, indicating that the restriction of bacterial replication is immune mediated. Here, we examined whether additional innate immune pathways are employed by C57BL/6 macrophages to sense and restrict NMII replication. In addition to the known role of TLR2 in detecting and restricting NMII infection, we found that TLR4 also contributes to cytokine responses but is not required to restrict bacterial replication. Furthermore, the TLR signaling adaptors MyD88 and Trif are required for cytokine responses and restricting bacterial replication. The C. burnetii NMII T4SS translocates bacterial products into C57BL/6 macrophages. However, there was little evidence of cytosolic immune sensing of NMII, as there was a lack of inflammasome activation, T4SS-dependent cytokine responses, and robust type I interferon (IFN) production, and these pathways were not required to restrict bacterial replication. Instead, endogenous tumor necrosis factor (TNF) produced upon TLR sensing of C. burnetii NMII was required to control bacterial replication. Therefore, our findings indicate a primary role for TNF produced upon immune detection of C. burnetii NMII by TLRs, rather than cytosolic PRRs, in enabling C57BL/6 macrophages to restrict bacterial replication.T o initiate innate immune defense against bacterial pathogens, infected host cells utilize pattern recognition receptors (PRRs) to detect pathogen-associated molecular patterns (PAMPs) (1-3). Toll-like receptors (TLRs) located at the cell surface and within endosomes detect extracellular PAMPs such as bacterial lipoproteins and lipopolysaccharide (LPS) (4). Downstream of TLRs, the adaptor proteins MyD88 and Trif activate several signaling pathways, including NF-B, mitogen-activated protein kinases (MAPKs), and interferon (IFN) regulatory factor 3 (IRF3), which direct the expression of proinflammatory cytokines and other antimicrobial effectors (4). For intracellular bacterial pathogens, cytosolic PRRs, such as those of the nucleotide binding domain/ leucine-rich repeat (NLR) and RIG-I-like receptor (RLR) families, often are critical for host defense as they respond to PAMPs introduced into the host cell cytosol by bacterial pore-forming toxins or specialized secretion systems (5-8). In addition, cytosolic sensing can lead to the assembly of a multiprotein complex termed the inflammasome, which activates the host proteases caspase-1 and caspase-11, resulting in the release of IL-1 family cytokines and a form of cell death known as pyroptosis (9-16). These innate immune pathways collaborate to restrict intracellular bacterial infection through both cell-intrinsic and -extrinsic mechanisms (17)(18...

The oligoadenylate synthetase (OAS)-RNase L pathway is a potent interferon (IFN)-T he coronavirus mouse hepatitis virus (MHV) strain A59 (referred to here as A59) causes moderate hepatitis and mild encephalitis, followed by chronic demyelinating disease, in susceptible C57BL/6 (B6) mice (1-3). A59 is cleared from the liver and central nervous system (CNS) primarily by the T cell response 7 to 10 days postinfection (4, 5). However, type I interferon (IFN) production, an early innate immune response, is crucial for early control of MHV infection, as mice deficient in type I IFN receptor expression (Ifnar1 Ϫ/Ϫ ) uniformly die by 2 days after infection (6-8). Interestingly, A59 fails to induce IFN-␣/␤ in most cell types, with the notable exception of myeloid cells (7). Induction of IFN-␣/␤ in macrophages and brain-resident microglia during MHV infection is dependent on sensing of viral double-stranded RNA (dsRNA) by the cytosolic RNA helicase melanoma differentiation-associated gene 5 (MDA5), encoded by Ifih1 (7, 9, 10). IFN induces a large array of interferon-stimulated genes (ISGs), which include pattern recognition receptors (PRRs), signaling molecules, transcription factors, and antiviral effectors (11)(12)(13)(14)(15)(16) (Fig. 1, left, diagrams IFN synthesis and signaling in MHV-infected macrophages). The only other source of type I IFN during A59 infection, primarily IFN-␣, is induced through a TLR7-dependent pathway in plasmacytoid dendritic cells (pDCs) (17).Among the ISGs are several Oas genes encoding proteins that function as nucleic acid sensors to synthesize 2=,5=-oligoadenylates (2-5A) in response to viral dsRNA in the host cytosol (18). Mice express several oligoadenylate synthetase (OAS) proteins that produce 2-5A, including OAS1a/g, OAS2, and OAS3, as well

dVibrio cholerae is the causative agent of the diarrheal disease cholera. The ability of V. cholerae to colonize and cause disease requires the intricately regulated expression of a number of virulence factors during infection. One of the signals sensed by V. cholerae is the presence of oxygen-limiting conditions in the gut. It has been shown that the virulence activator AphB plays a key role in sensing low oxygen concentrations and inducing the transcription of another key virulence activator, TcpP. In this study, we used a bacterial two-hybrid system to further examine the effect of oxygen on different virulence regulators. We found that anoxic conditions enhanced the interaction between TcpP and ToxR, identified as the first positive regulator of V. cholerae virulence genes. We further demonstrated that the TcpP-ToxR interaction was dependent on the primary periplasmic protein disulfide formation enzyme DsbA and cysteine residues in the periplasmic domains of both ToxR and TcpP. Furthermore, we showed that in V. cholerae, an interaction between TcpP and ToxR is important for virulence gene induction. Under anaerobic growth conditions, we detected ToxR-TcpP heterodimers, which were abolished in the presence of the reducing agent dithiothreitol. Our results suggest that V. cholerae may sense intestinal anoxic signals by multiple components to activate virulence.

Bacteria utilize the tightly regulated stress response (SOS) pathway to respond to a variety of genotoxic agents, including antimicrobials. Activation of the SOS response is regulated by a key repressor-protease, LexA, which undergoes autoproteolysis in the setting of stress, resulting in derepression of SOS genes. Remarkably, genetic inactivation of LexA’s self-cleavage activity significantly decreases acquired antibiotic resistance in infection models and renders bacteria hypersensitive to traditional antibiotics, suggesting that a mechanistic study of LexA could help inform its viability as a novel target for combating acquired drug resistance. Despite structural insights into LexA, a detailed knowledge of the enzyme’s protease specificity is lacking. Here, we employ saturation and positional scanning mutagenesis on LexA’s internal cleavage region to analyze >140 mutants and generate a comprehensive specificity profile of LexA from the human pathogen Pseudomonas aeruginosa (LexAPa). We find that the LexAPa active site possesses a unique mode of substrate recognition. Positions P1–P3 prefer small hydrophobic residues that suggest specific contacts with the active site, while positions P5 and P1′ show a preference for flexible glycine residues that may facilitate the conformational change that permits autoproteolysis. We further show that stabilizing the β-turn within the cleavage region enhances LexA autoproteolytic activity. Finally, we identify permissive positions flanking the scissile bond (P4 and P2′) that are tolerant to extensive mutagenesis. Our studies shed light on the active site architecture of the LexA autoprotease and provide insights that may inform the design of probes of the SOS pathway.