Salmonella enterica serovar Typhimurium STM1266 encodes a regulator of curli biofilm formation: the brfS gene.

The major biofilm pathway in Salmonella enterica serovar Typhimurium involves specific growth conditions that induce the csgA gene whose product forms surface curli fibers that mediate biofilm formation. We have found that the previously uncharacterized STM1266 gene in S. Typhimurium plays a role in regulating biofilm formation via the curli pathway. S. Typhimurium ΔSTM1266 strains display a biofilm defect, and overexpression of STM1266 results in enhanced biofilm formation. STM1266 deletion resulted in lowered csgA expression using promoter-reporter ß-galactosidase assays, and csgA and csgD deletions abrogate the effects of STM1266 overexpression on biofilm formation while deletion of bcsA (encoding an essential enzyme for cellulose formation) has no effect. In a mouse infection model, the ΔSTM1266 strain displayed results similar to those seen for previously reported ΔcsgA strains. The STM1266 gene is predicted to encode a DNA-binding transcriptional regulator of the MerR family and is homologous to the Escherichia coli BluR regulator protein. We respectfully propose to ascribe the name brfS (biofilm regulator for Salmonella Typhimurium) to the STM1266 gene.

DExD/H Box Helicases DDX24 and DDX49 Inhibit Reactivation of Kaposi's Sarcoma Associated Herpesvirus by Interacting with Viral mRNAs.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus that is the causative agent of primary effusion lymphoma and Kaposi's sarcoma. In healthy carriers, KSHV remains latent, but a compromised immune system can lead to lytic viral replication that increases the probability of tumorigenesis. RIG-I-like receptors (RLRs) are members of the DExD/H box helicase family of RNA binding proteins that recognize KSHV to stimulate the immune system and prevent reactivation from latency. To determine if other DExD/H box helicases can affect KSHV lytic reactivation, we performed a knock-down screen that revealed DHX29-dependent activities appear to support viral replication but, in contrast, DDX24 and DDX49 have antiviral activity. When DDX24 or DDX49 are overexpressed in BCBL-1 cells, transcription of all lytic viral genes and genome replication were significantly reduced. RNA immunoprecipitation of tagged DDX24 and DDX49 followed by next-generation sequencing revealed that the helicases bind to mostly immediate-early and early KSHV mRNAs. Transfection of expression plasmids of candidate KSHV transcripts, identified from RNA pull-down, demonstrated that KSHV mRNAs stimulate type I interferon (alpha/beta) production and affect the expression of multiple interferon-stimulated genes. Our findings reveal that host DExD/H box helicases DDX24 and DDX49 recognize gammaherpesvirus transcripts and convey an antiviral effect in the context of lytic reactivation.

C5a Activates a Pro-Inflammatory Gene Expression Profile in Human Gaucher iPSC-Derived Macrophages.

Gaucher disease (GD) is an autosomal recessive disorder caused by bi-allelic GBA1 mutations that reduce the activity of the lysosomal enzyme ß-glucocerebrosidase (GCase). GCase catalyzes the conversion of glucosylceramide (GluCer), a ubiquitous glycosphingolipid, to glucose and ceramide. GCase deficiency causes the accumulation of GluCer and its metabolite glucosylsphingosine (GluSph) in a number of tissues and organs. In the immune system, GCase deficiency deregulates signal transduction events, resulting in an inflammatory environment. It is known that the complement system promotes inflammation, and complement inhibitors are currently being considered as a novel therapy for GD; however, the mechanism by which complement drives systemic macrophage-mediated inflammation remains incompletely understood. To help understand the mechanisms involved, we used human GD-induced pluripotent stem cell (iPSC)-derived macrophages. We found that GD macrophages exhibit exacerbated production of inflammatory cytokines via an innate immune response mediated by receptor 1 for complement component C5a (C5aR1). Quantitative RT-PCR and ELISA assays showed that in the presence of recombinant C5a (rC5a), GD macrophages secreted 8-10-fold higher levels of TNF-α compared to rC5a-stimulated control macrophages. PMX53, a C5aR1 blocker, reversed the enhanced GD macrophage TNF-α production, indicating that the observed effect was predominantly C5aR1-mediated. To further analyze the extent of changes induced by rC5a stimulation, we performed gene array analysis of the rC5a-treated macrophage transcriptomes. We found that rC5a-stimulated GD macrophages exhibit increased expression of genes involved in TNF-α inflammatory responses compared to rC5a-stimulated controls. Our results suggest that rC5a-induced inflammation in GD macrophages activates a unique immune response, supporting the potential use of inhibitors of the C5a-C5aR1 receptor axis to mitigate the chronic inflammatory abnormalities associated with GD.

Downregulation of the human peripheral myelin protein 22 gene by miR-29a in cellular models of Charcot-Marie-Tooth disease.

The majority of hereditary neuropathies are caused by duplication of the peripheral myelin protein 22 (PMP22) gene. Therefore, mechanisms to suppress the expression of the PMP22 gene have high therapeutic significance. Here we asked whether the human PMP22 gene is a target for regulation by microRNA 29a (miR-29a). Using bioinformatics, we determined that the human PMP22 gene contains the conserved seed sequence of the miR-29a binding site and this regulatory motif is included in the duplicated region in neuropathic patients. Using luciferase reporter assays in HEK293 cells, we demonstrated that transient transfection of a miR-29a mimic is associated with reduction in PMP22 3'UTR reporter activity. Transfecting normal and humanized transgenic neuropathic mouse Schwann cells with a miR-29a expression plasmid effectively lowered both the endogenous mouse and the transgenic human PMP22 transcripts compared with control vector. In dermal fibroblasts derived from neuropathic patients, ectopic expression of miR-29a led to ~50% reduction in PMP22 mRNA, which corresponded to ~20% decrease in PMP22 protein levels. Significantly, miR-29a-mediated reduction in PMP22 mitigated the reduced mitotic capacity of the neuropathic cells. Together, these results support further testing of miR-29a and/or PMP22-targeting siRNAs as therapeutic agents for correcting the aberrant expression of PMP22 in neuropathic patients.

The Bacterial iprA Gene Is Conserved across Enterobacteriaceae, Is Involved in Oxidative Stress Resistance, and Influences Gene Expression in Salmonella enterica Serovar Typhimurium.

UNLABELLED: The iprA gene (formerly known as yaiV or STM0374) is located in a two-gene operon in the Salmonella enterica serovar Typhimurium genome and is associated with altered expression during spaceflight and rotating-wall-vessel culture conditions that increase virulence. However, iprA is uncharacterized in the literature. In this report, we present the first targeted characterization of this gene, which revealed that iprA is highly conserved across Enterobacteriaceae We found that S Typhimurium, Escherichia coli, and Enterobacter cloacae ΔiprA mutant strains display a multi-log-fold increase in oxidative stress resistance that is complemented using a plasmid-borne wild-type (WT) copy of the S Typhimurium iprA gene. This observation was also associated with increased catalase activity, increased S Typhimurium survival in macrophages, and partial dependence on the katE gene and full dependence on the rpoS gene. Our results indicate that IprA protein activity is sensitive to deletion of the N- and C-terminal 10 amino acids, while a region that includes amino acids 56 to 80 is dispensable for activity. RNA sequencing (RNA-Seq) analysis revealed several genes altered in expression in the S Typhimurium ΔiprA mutant strain compared to the WT, including those involved in fimbria formation, spvABCD-mediated virulence, ethanolamine utilization, the phosphotransferase system (PTS) transport, and flagellin phase switching from FlgB to FliC (likely a stochastic event) and several genes of hypothetical or putative function. IMPORTANCE: Overall, this work reveals that the conserved iprA gene measurably influences bacterial biology and highlights the pool of currently uncharacterized genes that are conserved across bacterial genomes. These genes represent potentially useful targets for bacterial engineering, vaccine design, and other possible applications.

Tissue-specific RNA-Seq in human evoked inflammation identifies blood and adipose LincRNA signatures of cardiometabolic diseases.

OBJECTIVE: Inappropriate transcriptional activation of innate immunity is a pathological feature of several cardiometabolic disorders, but little is known about inflammatory modulation of long intergenic noncoding RNAs (lincRNAs) in disease-relevant human tissues. APPROACH AND RESULTS: We applied deep RNA sequencing (>500 million filtered reads per sample) to blood and adipose during low-dose experimental endotoxemia (lipopolysaccharide) in a healthy human, with targeted replication in separate individuals undergoing endotoxemia (n=6), to identify inflammatory lincRNAs. A subset of these lincRNAs was examined for expression in adipocytes and monocytes, modulation in adipose of obese humans, and overlap with genome-wide association study signals for inflammatory and cardiometabolic traits. Of a stringent set of 4284 lincRNAs, ≈11% to 22% were expressed with 201 and 56 lincRNAs modulated by lipopolysaccharide in blood or adipose, respectively. Tissue-specific expression of a subset of 6 lipopolysaccharide-lincRNAs was replicated with lipopolysaccharide modulation confirmed for all 3 expressed in blood and 2 of 4 expressed in adipose. The broader generalizability of findings in blood of subject A was confirmed by RNA sequencing in 7 additional subjects. We confirmed adipocytes and monocytes as potential cell-sources of selective lipopolysaccharide-regulated lincRNAs, and 2 of these, linc-DMRT2 (P=0.002) and linc-TP53I13 (P=0.01), were suppressed in adipose of obese humans. Finally, we provide examples of lipopolysaccharide-modulated lincRNAs that overlap single nucleotide polymorphisms that are associated with cardiometabolic traits. CONCLUSIONS: Our findings provide novel insights into tissue-level, inflammatory transcriptome regulation in cardiometabolic diseases. These are complementary to more usual approaches limited to interrogation of DNA variations.