Vibrio fluvialis, an emerging foodborne pathogen of increasing public health concern, contains two distinct gene clusters encoding type VI secretion system (T6SS), the most newly discovered secretion pathway in Gram-negative bacteria. Previously we have shown that one of the two T6SS clusters, namely VflT6SS2, is active and associates with anti-bacterial activity. However, how its activity is regulated is not completely understood. Here, we report that the global regulator integration host factor (IHF) positively modulates the expression and thus the function of VflT6SS2 through co-regulating its major cluster and tssD2-tssI2 (also known as hcp-vgrG) orphan clusters. Specifically, reporter gene activity assay showed that IHF transactivates the major and orphan clusters of VflT6SS2, while deletion of either ihfA or ihfB, the genes encoding the IHF subunits, decreased their promoter activities and mRNA levels of tssB2, vasH, and tssM2 for the selected major cluster genes and tssD2 and tssI2 for the selected orphan cluster genes. Subsequently, the direct bindings of IHF to the promoter regions of the major and orphan clusters were confirmed by electrophoretic mobility shift assay (EMSA). Site-directed mutagenesis combined with reporter gene activity assay or EMSA pinpointed the exact binding sites of IHF in the major and orphan cluster promoters, with two sites in the major cluster promoter, consisting with its two observed shifted bands in EMSA. Functional studies showed that the expression and secretion of hemolysin-coregulated protein (Hcp) and the VflT6SS2-mediated antibacterial virulence were severely abrogated in the deletion mutants of ΔihfA and ΔihfB, but restored when their trans-complemented plasmids were introduced, suggesting that IHF mostly contributes to environmental survival of V. fluvialis by directly binding and modulating the transactivity and function of VflT6SS2.

Vibrio fluvialis is an emerging enteric pathogen of increasing public health threat. Two quorum sensing (QS) systems, VfqI-VfqR and CqsA/LuxS-HapR, and two type VI secretion systems (T6SSs), VflT6SS1 and VflT6SS2, have been identified in V. fluvialis. VflT6SS2 is regulated by environmental signals and intracellular regulators. In Vibrio species, QS systems were frequently reported to regulate various physiological functions and systems. Therefore, we wonder if QS systems can function as functional regulators of VflT6SS2. Here, we investigated the effects of QS circuit on VflT6SS2. Our results showed that the QS response regulator LuxO represses while the major regulator HapR activates VflT6SS2. The effect of LuxO is more pronounced at low cell density and is HapR-dependent. Deletion of hapR abolished Hcp expression and alleviated antibacterial virulence. However, these effects were rescued by introducing HapR-expressing plasmid. Reporter fusion analyses showed that HapR is required for the promoter activities of VflT6SS2. Sequence inspection of the major cluster promoter revealed two potential Motif 1 HapR binding sites, and their direct bindings were confirmed by both electrophoretic mobility shift assay (EMSA) and DNase I footprinting assay. Meanwhile, two single Motif 2 HapR binding sites were identified in each of tssD2_a (hcpA) and tssD2_b (hcpB) promoter regions of the orphan cluster which are less conserved and displayed lower affinities to HapR. Together, our current study demonstrated that VflT6SS2 expression was under the control of QS circuit in V. fluvialis, and this finding will enhance our understanding of possible crosstalk between T6SS and QS in different microbes.

Background Lung resident mesenchymal stem cells (LR-MSCs) play an important role in idiopathic pulmonary fibrosis (IPF) by transforming into myofibroblasts, thereby losing their repair ability. Evidence suggests that key proteins of multiple signaling pathways are involved in myofibroblast differentiation of LR-MSCs, such as β-Catenin and GLI family zinc finger 1 (GLI1). These proteins are regulated by SUMO (small ubiquitin-like modifier) modification, which is a post-translational modification that promotes protein degradation, while Sumo specific protein 1 (SENP1)-mediated deSUMOylation produces the opposite biological effects. Therefore, we speculated that SENP1 might be a potential target for treating pulmonary fibrosis by preventing the myofibroblast differentiation of LR-MSCs. Methods LR-MSCs were isolated from mice by using immunomagnetic beads. The extracted LR-MSCs were identified by flow cytometric analysis and multilineage differentiation assays. Lentivirus packaged shRNA silenced the expression of SENP1 in vitro and vivo. The silencing efficacy of SENP1 was verified by real-time quantitative PCR. The effect of down-regulated SENP1 on the myofibroblast differentiation of LR-MSCs was assessed by Immunofluorescence and Western blot. Immunoprecipitation was used to clarify that SENP1 was a key target for regulating the activity of multiple signaling pathways in the direction of LR-MSCs differentiation. LR-MSCs resident in the lung was analyzed with in vivo imaging system. HE and Masson staining was used to evaluate the therapeutic effect of LR-MSCs with SENP1 down-regulation on the lung of BLM mice. Results In this study, we found that the myofibroblast differentiation of LR-MSCs in IPF lung tissue was accompanied by enhanced SENP1-mediated deSUMOylation. The expression of SENP1 increased in LR-MSCs transition of bleomycin (BLM)-induced lung fibrosis. Interfering with expression of SENP1 inhibited the transformation of LR-MSCs into myofibroblasts in vitro and in vivo and restored their therapeutic effect in BLM lung fibrosis. In addition, activation of the WNT/β-Catenin and Hedgehog/GLI signaling pathways depends on SENP1-mediated deSUMOylation. Conclusions SENP1 might be a potential target to restore the repair function of LR-MSCs and treat pulmonary fibrosis.

Background It has been demonstrated that aberrant expression of serum microRNAs is potential markers for the prognostic prediction of acute myeloid leukemia (AML). However, the clinical significance of serum miR‐22 remained uncovered. In this study, we aimed to explore the potential prognostic value of serum miR‐22 for AML. Methods Blood samples were collected from 124 patients with AML and 60 healthy individuals. Serum miR‐22 level was detected by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR), and its potential clinical value was investigated. Results Our results showed that serum miR‐22 expression was significantly downregulated in AML subjects compared to healthy controls. Serum miR‐22 levels were lowest in AML patients with M4/M5 subtypes, and low serum miR‐22 expression occurred more frequently in AML patients with higher white blood cell counts or poor cytogenetic risk. Receiver operating characteristic (ROC) analysis revealed that serum miR‐22 well differentiated AML cases from healthy controls. In addition, serum miR‐22 downregulation was closely associated with worse clinical features and shorter survival. Low serum miR‐22 expression was confirmed to be an independent predictor for overall survival and relapse‐free survival in AML patients. Moreover, the expression level of serum miR‐22 was dramatically increased following treatment. In addition, serum miR‐22 levels were significantly higher in AML patients achieving complete remission (CR) than those without CR. Conclusion Collectively, serum miR‐22 might serve as a novel and promising prognostic biomarker for AML.

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