Conjugation is a key mechanism for horizontal gene transfer and plays an important role in bacterial evolution, especially with respect to antibiotic resistance. However, little is known about the role of donor and recipient cells in regulation of conjugation. Here, using an Escherichia coli (SM10λπ)-Pseudomonas aeruginosa (PAO1) conjugation model, we demonstrated that deficiency of lasI/rhlI, genes associated with generation of the quorum sensing signals N-acyl homoserine lactones (AHLs) in PAO1, or deletion of the AHLs receptor SdiA in the donor SM10λπ both facilitated conjugation. When using another AHLs-non-producing E. coli strain EC600 as recipient cells, deficiency of sdiA in donor SM10λπ hardly affect the conjugation. More importantly, in the presence of exogenous AHLs, the conjugation efficiency between SMλπ and EC600 was dramatically decreased, while deficiency of sdiA in SMλπ attenuated AHLs-inhibited conjugation. These data suggest the conjugation suppression function of AHLs-SdiA chemical signaling. Further bioinformatics analysis, β-galactosidase reporter system and electrophoretic mobility shift assays characterized the binding site of SdiA on the promoter region of traI gene. Furthermore, deletion of lasI/rhlI or sdiA promoted traI mRNA expression in SM10λπ and PAO1 co-culture system, which was abrogated by AHLs. Collectively, our results provide new insight into an important contribution of quorum sensing system AHLs-SdiA to the networks that regulate conjugation.

Background/Aims: To investigate the role of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) in the clinical prognosis and cell biology of renal cell carcinoma (RCC). Methods: A total of 137 RCC tissues were evaluated by immunohistochemistry. The relationship between MTHFD2 overexpression and clinical parameters and vimentin expression was assessed. Kaplan-Meier curves and the log-rank test were applied for survival analysis according to MTHFD2 and vimentin expression in RCC tissues. The expression of MTHFD2 mRNA and protein was examined by quantitative reverse transcription PCR and western blotting, respectively. To determine further the biological activity of MTHFD2 in RCC, 786-O cells were transfected with short hairpin RNA specifically targeting MTHFD2 (shMTHFD2) with or without tumor necrosis factor (TNF)-α stimulation. Cell proliferation, cell migration and invasion and drug sensitivity were subsequently assessed using Cell Counting Kit-8, wound healing, and Transwell assays. Results: Immunohistochemical analysis demonstrated that both MTHFD2 and vimentin overexpression was positively associated with clinical staging, pathological grade, and poor overall survival (all P < 0.05). MTHFD2 expression was closely correlated with vimentin overexpression in RCC (r = 0.402, P < 0.001). After knocking down MTHFD2 expression in 786-O cells, decreased cell proliferation, migration, and invasion were observed and accompanied by the reduced expression of vimentin. The effects of MTHFD2 down-regulation could be partially restrained by TNF-α treatment. Vimentin expression and cell migration and invasion, but not cell proliferation, were reversed by TNF-α stimulation. Furthermore, treatment of 786-O cells with shMTHFD2 increased their sensitivity to chemotherapy drugs. Conclusion: The current results demonstrated that MTHFD2 was overexpressed in RCC and associated with poor clinical characteristics, vimentin expression, and cellular features connected to malignant disease, thus, implicating MTHFD2 as a potential target for RCC therapy.

Bladder cancer is the most common malignancy in urinary system and the ninth most common malignancy in the world. MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression by targeted repression of transcription and translation and play essential roles during cancer development. We investigated the expression of miR-135a in bladder cancer and explored its bio-function during bladder cancer progression. The expression of miR-135a in bladder cancer cells and tissues are performed by using Real-time PCR assay. Cell viability assay (MTT assay), colony formation assay, anchorage-independent growth ability assay and Bromodeoxyuridine labeling and immunofluorescence (BrdUrd) assay are used to examine cell proliferative capacity and tumorigenicity. Flow cytometry analysis is used to determine cell cycle progression. The expressions of p21, p27, CyclinD1, Ki67, PHLPP2 and FOXO1 are measured by Western blotting assay. Luciferase assay is used to confirm whether FOXO1 is the direct target of miR-135a. miR-135a is upregulated in bladder cancer cells and tissues. Enforced expression of miR-135a promotes bladder cancer cells proliferation, whereas inhibition of miR-135a reverses the function. Furthermore, for the first time we demonstrated PHLPP2 and FOXO1 are direct targets of miR-135a and transcriptionally down-regulated by miR-135a. Suppression of PHLPP2 or FOXO1 by miR-135a, consisted with dysregulation of p21, p27, Cyclin D1 and Ki67, play important roles in bladder cancer progression. Our study demonstrates that miR-135a promotes cell proliferation in bladder cancer by targeting PHLPP2 and FOXO1, and is performed as an onco-miR.

The effect of antibiotics on horizontal gene transfer (HGT) is controversial, and the underlying mechanism remains poorly understood. Here, using Escherichia coli SM10λπ as the donor strain, which carries a chromosomally integrated RP4 plasmid, we investigated the effect of antibiotics on conjugational transfer of a mobilizable gentamicin (Gm) resistance plasmid. The results showed that an exposure to gentamicin that restricted the survival of recipient cells significantly enhanced SM10λπ- Pseudomonas aeruginosa PAO1 conjugation, which was attenuated by a deficiency of lasI-rhlI , genes associated with the generation of the quorum sensing signals N -acyl homoserine lactones (AHLs) in PAO1, or the deletion of the AHL receptor SdiA in SM10λπ. Subsequent mechanistic investigations revealed that a treatment with Gm repressed the mRNA expression of lasI and rhlI in PAO1 and upregulated traI expression in SM10λπ. Moreover, PAO1 treated with other quorum sensing (QS)-inhibiting antibiotics such as azithromycin or chloramphenicol also showed a conjugation-promoting ability. On the other hand, when using non-AHL-producing E. coli strain EC600 as the recipient cells, the promoting effect of Gm on conjugation could not be observed. These data suggest that AHL-SdiA contributes to the effectiveness of antibiotics on plasmid conjugation. Collectively, our findings highlight the HGT-promoting effect of antibiotics and suggest quorum sensing as a promising target for controlling antibiotic resistance dissemination. These findings have implications for assessing the risks of antibiotic use and developing advisable antibiotic treatment protocols.

P. aeruginosa is a common nosocomial pathogen that causes diverse opportunistic infections in humans. The virulence crucial for infection is mainly regulated by QS.