Mechanism of foreign DNA recognition by a CRISPR RNA-guided surveillance complex from Pseudomonas aeruginosa

The Type I-F CRISPR-mediated (clustered regularly interspaced short palindromic repeats) adaptive immune system in Pseudomonas aeruginosa consists of two CRISPR loci and six CRISPR-associated (cas) genes. Foreign DNA surveillance is performed by a complex of Cas proteins (Csy1-4) that assemble with a CRISPR RNA (crRNA) into a 350-kDa ribonucleoprotein called the Csy complex. Here, we show that foreign nucleic acid recognition by the Csy complex proceeds through sequential steps, initiated by detection of two consecutive guanine-cytosine base pairs (G-C/G-C) located adjacent to the complementary DNA target. We show that this motif, called the PAM (protospacer adjacent motif), must be double-stranded and that single-stranded PAMs do not provide significant discriminating power. Binding assays performed with G-C/G-C-rich competitor sequences indicate that the Csy complex interacts directly with this dinucleotide motif, and kinetic analyses reveal that recognition of a G-C/G-C motif is a prerequisite for crRNA-guided binding to a target sequence. Together, these data indicate that the Csy complex first interacts with G-C/G-C base pairs and then samples adjacent target sequences for complementarity to the crRNA guid

Murine and Bovine γδ T Cells Enhance Innate Immunity against Brucella abortus Infections

γδ T cells have been postulated to act as a first line of defense against infectious agents, particularly intracellular pathogens, representing an important link between the innate and adaptive immune responses. Human γδ T cells expand in the blood of brucellosis patients and are active against Brucella in vitro. However, the role of γδ T cells in vivo during experimental brucellosis has not been studied. Here we report TCRδ−/− mice are more susceptible to B. abortus infection than C57BL/6 mice at one week post-infection as measured by splenic colonization and splenomegaly. An increase in TCRγδ cells was observed in the spleens of B. abortus-infected C57BL/6 mice, which peaked at two weeks post-infection and occurred concomitantly with diminished brucellae. γδ T cells were the major source of IL-17 following infection and also produced IFN-γ. Depletion of γδ T cells from C57BL/6, IL-17Rα−/−, and GMCSF−/− mice enhanced susceptibility to B. abortus infection although this susceptibility was unaltered in the mutant mice; however, when γδ T cells were depleted from IFN-γ−/− mice, enhanced susceptibility was observed. Neutralization of γδ T cells in the absence of TNF-α did not further impair immunity. In the absence of TNF-α or γδ T cells, B. abortus-infected mice showed enhanced IFN-γ, suggesting that they augmented production to compensate for the loss of γδ T cells and/or TNF-α. While the protective role of γδ T cells was TNF-α-dependent, γδ T cells were not the major source of TNF-α and activation of γδ T cells following B. abortus infection was TNF-α-independent. Additionally, bovine TCRγδ cells were found to respond rapidly to B. abortus infection upon co-culture with autologous macrophages and could impair the intramacrophage replication of B. abortus via IFN-γ. Collectively, these results demonstrate γδ T cells are important for early protection to B. abortus infections

Nasal Acai Polysaccharides Potentiate Innate Immunity to Protect against Pulmonary Francisella tularensis and Burkholderia pseudomallei Infections

Pulmonary Francisella tularensis and Burkholderia pseudomallei infections are highly lethal in untreated patients, and current antibiotic regimens are not always effective. Activating the innate immune system provides an alternative means of treating infection and can also complement antibiotic therapies. Several natural agonists were screened for their ability to enhance host resistance to infection, and polysaccharides derived from the Acai berry (Acai PS) were found to have potent abilities as an immunotherapeutic to treat F. tularensis and B. pseudomallei infections. In vitro, Acai PS impaired replication of Francisella in primary human macrophages co-cultured with autologous NK cells via augmentation of NK cell IFN-γ. Furthermore, Acai PS administered nasally before or after infection protected mice against type A F. tularensis aerosol challenge with survival rates up to 80%, and protection was still observed, albeit reduced, when mice were treated two days post-infection. Nasal Acai PS administration augmented intracellular expression of IFN-γ by NK cells in the lungs of F. tularensis-infected mice, and neutralization of IFN-γ ablated the protective effect of Acai PS. Likewise, nasal Acai PS treatment conferred protection against pulmonary infection with B. pseudomallei strain 1026b. Acai PS dramatically reduced the replication of B. pseudomallei in the lung and blocked bacterial dissemination to the spleen and liver. Nasal administration of Acai PS enhanced IFN-γ responses by NK and γδ T cells in the lungs, while neutralization of IFN-γ totally abrogated the protective effect of Acai PS against pulmonary B. pseudomallei infection. Collectively, these results demonstrate Acai PS is a potent innate immune agonist that can resolve F. tularensis and B. pseudomallei infections, suggesting this innate immune agonist has broad-spectrum activity against virulent intracellular pathogens

Bacteriophage Cooperation Suppresses CRISPR-Cas3 and Cas9 Immunity

Bacteria utilize CRISPR-Cas adaptive immune systems for protection from bacteriophages (phages), and some phages produce anti-CRISPR (Acr) proteins that inhibit immune function. Despite thorough mechanistic and structural information for some Acr proteins, how they are deployed and utilized by a phage during infection is unknown. Here, we show that Acr production does not guarantee phage replication when faced with CRISPR-Cas immunity, but instead, infections fail when phage population numbers fall below a critical threshold. Infections succeed only if a sufficient Acr dose is contributed to a single cell by multiple phage genomes. The production of Acr proteins by phage genomes that fail to replicate leave the cell immunosuppressed, which predisposes the cell for successful infection by other phages in the population. This altruistic mechanism for CRISPR-Cas inhibition demonstrates inter-virus cooperation that may also manifest in other host-parasite interactions

Cytoprotective Nrf2 pathway is induced in chronically txnrd 1-deficient hepatocytes

Metabolically active cells require robust mechanisms to combat oxidative stress. The cytoplasmic thioredoxin reductase/thioredoxin (Txnrd1/Txn1) system maintains reduced protein dithiols and provides electrons to some cellular reductases, including peroxiredoxins. Here we generated mice in which the txnrd1 gene, encoding Txnrd1, was specifically disrupted in all parenchymal hepatocytes. Txnrd1-deficient livers exhibited a transcriptome response in which 56 mRNAs were induced and 12 were repressed. Based on the global hybridization profile, this represented only 0.3% of the liver transcriptome. Since most liver mRNAs were unaffected, compensatory responses were evidently effective. Nuclear pre-mRNA levels indicated the response was transcriptional. Twenty-one of the induced genes contained known antioxidant response elements (AREs), which are binding sites for the oxidative and chemical stress-induced transcription factor Nrf2. Txnrd1-deficient livers showed increased accumulation of nuclear Nrf2 protein and chromatin immunoprecipitation on the endogenous nqo1 and aox1 promoters in fibroblasts indicated that Txnrd1 ablation triggered in vivo assembly of Nrf2 on each. Chronic deletion of Txnrd1 results in induction of the Nrf2 pathway, which contributes to an effective compensatory response

Cas1 and the Csy complex are opposing regulators of Cas2/3 nuclease activity

The type I-F CRISPR adaptive immune system in Pseudomonas aeruginosa (PA14) consists of two CRISPR loci and six CRISPR-associated (cas) genes. Type I-F systems rely on a CRISPR RNA (crRNA)-guided surveillance complex (Csy complex) to bind foreign DNA and recruit a trans-acting nuclease (i.e., Cas2/3) for target degradation. In most type I systems, Cas2 and Cas3 are separate proteins involved in adaptation and interference, respectively. However, in I-F systems, these proteins are fused into a single polypeptide. Here we use biochemical and structural methods to show that two molecules of Cas2/3 assemble with four molecules of Cas1 (Cas2/32:Cas14) into a four-lobed propeller-shaped structure, where the two Cas2 domains form a central hub (twofold axis of symmetry) flanked by two Cas1 lobes and two Cas3 lobes. We show that the Cas1 subunits repress Cas2/3 nuclease activity and that foreign DNA recognition by the Csy complex activates Cas2/3, resulting in bidirectional degradation of DNA targets. Collectively, this work provides a structure of the Cas1-2/3 complex and explains how Cas1 and the target-bound Csy complex play opposing roles in the regulation of Cas2/3 nuclease activity