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ObjectiveQuorum-sensing (QS) and small regulatory RNA (sRNA) play key regulatory roles in many signaling cascades of Pseudomonas aeruginosa. To investigate whether sRNA is involved in P. aeruginosa QS system, screening QS system-related sRNA, and to construct sRNA overexpression and deletion strains of Pseudomonas aeruginosa for further study of sRNA function.MethodsSRNA associated with the QS system was screened by qPCR and RNA-sequencing (RNA-seq). The target gene were amplified by PCR and inserted into the overexpression vector pROp200 or the homologous recombination vector pGSM-MR, respectively. The connection reaction solution of pROp200-sRNA and pGSM-ΔsRNA was transformed into Escherichia coli DH5a and SM10lp, respectively. The recombinant vectors were identified by PCR. The pROp200-sRNA was transformed into PAO1 by heat shock method, and the pGSM-ΔsRNA was transferred from SM10lp to PAO1 by conjugation. SRNA overexpression and deletion strains were identified by PCR, DNA sequencing and qPCR, the determination of the growth curves and the pyocyanin levels of strains.ResultsFive QS -associated sRNA P26, P5316.1, P30, P34 and AmiL were successfully screened by RNA-seq and qPCR. PCR, DNA sequencing and qPCR showed that sRNA of AmiL, P30 and P34 overexpression and knockout were successful. Compared with wild-type strain, sRNA overexpression and knockout had no significant effect on bacterial growth curve. It were notably that overexpression of AmiL and P30 inhibited and increase the production of pyocyanin, respectively (P0.05).ConclusionThe sRNA overexpression and deletion strains have been successfully constructed and can be used to study the regulatory relationship between sRNA and QS systems, and to further functional study.
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[Abstract] Objective To explore the roles of type VI secretion system (T6SS) and quorum sensing (QS) system in the biofilm formation in Pseudomonas aeruginosa. Methods The PAO1 biofilm bacteria and planktonic bacteria were established. The expression levels of T6SS associated genes hemolysin co-regulated protein (Hcp) gene (Hcp1, Hcp2, Hcp3), QS associated gene LasR, exopolysaccharide associated genes polysaccharide synthesis locus A (PslA) and pellicle A (PelA), type pili gene pilus A (PilA) and flagellin C (FliC) were detected by quantitative real-time PCR, and the expression levels of those genes were compared between PAO1 biofilm bacteria and planktonic bacteria using independent t-test. Results The expression levels of PslA, PelA, PilA and FliC genes in PAO1 biofilm bacteria were respectively 714, 274, 604 and 42 folds of those in the planktonic bacteria (all P0.05), suggesting that PAO1 bacteria formed mature biofilm with flagellum and pili. The expression levels of Hcp1, Hcp2, Hcp3, and LasR genes in PAO1 biofilm bacteria were respectively 1 045, 11 268, 6 654 and 1 226 folds of those in planktonic bacteria (all P0.05), suggesting that T6SS and QS systems were related to the biofilm formation of PAO1 bacteria. Conclusion T6SS and QS systems might be involved in biofilm formation in Pseudomonas aeruginosa, and its regulatory mechanism needs further study.
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Phenol-soluble modulins (PSMs) are a novel family of small amphipathic, alpha-heli-cal peptides with strong surfactant-like properties. PSMs have multiple roles in staphylococcal pathogenesis and are considered as important virulence-associated factors. They may cause lysis of many eukaryotic cells, such as human neutrophils,erythrocytes and dendritic cells;induce the expression of pro-inflammatory cyto-kines;facilitate the structuring and detachment of biofilms;influence the expression of other genes. This re-view summarizes the classification,structure,regulatory effect on gene expression and biological function of PSMs. The potential avenues to target PSMs for drug development against staphylococcal infections are also evaluated in this paper.
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Objective To investigate the effect of quorum sensing autoinducer 3OC12-HSL of Pseudomonas aeruginosa on the apoptosis of Caco-2 cells and its mechanism. Methods Caco-2 cells were incubated with 3OC12-HSL for 4 h and then examined by MTT method for cytoactivities. Flow cytometry was used to analyze apoptosis rate of Caco-2 cells. Expression of apoptosis associated proteins p-p38/MAPK and NF-κB were detected by Western blot. Results After exposure to 3OC12-HSL for 4 h, cytoactivities of Caco-2 cells was reduced(P<0.05) with dose-dependent pattern, and higher dose of 3OC12-HSL leaded to increasing apoptosis rate of Caco-2 cells(P<0.05). 3OC12-HSL raised expression of apoptosis associated proteins p-p38/MAPK and NF-κB detected by Western blot. Conclusion Quorum sensing autoinducer 3OC12-HSL can effect cytoactivities of Caco-2 cells and may induce its apoptosis by enhancing the expression of p-p38/MAPK and NF-κB protein.
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Objective To investigate the biological effects of Pseudomonas aeruginosa quorum sensing molecule OdDHL on murine mast cells. Methods The molecule structure and purity of synthesized OdDHL were confirmed by mass spectrum or proton nuclear magnetic resonance (NMR) and high-performance liquid chromatography, respectively. Its biological activity was checked using a quorum sensing sensor bacterial strain. The viability, apoptosis and intracellular calcium changes of P815 cell line in response to different concentration of OdDHL were determined. Results The biological active OdDHL was synthesized successfully. OdDHL inhibited proliferation of P815 cells in a dose, and time dependent manner. It also induced apoptasis and intracellular calcium release in P815 cells. Conclusion Psendomonas aeruginosa quorum sensing molecule OdDHL induces apoptosis and intracellular calcium release in murine mast cell line P815.
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Objective To investigate the immunity effects of quorum sensing in the pulmonary infection in rats due to Pseudomonas aeruginosa.Method(1)300 healthy,clean Sprague-Dawley rats were divided into 3 groups randomly:two different Pseudomonas aeruginosa(the wide-type Pseudomonas aeruginosa PAO1 and its double mutant PAO-JP2,in which the quorum sensing systems were defective,embedded in minute seaweed algiante beads which was made by an ejection set with an acuminate hole were inoculated to Sprague-Dawley rats to establish animal model of chronic pulmonary infection.The control group were dealed with the same methods using the sterile brine instead.(2)The levels of antibody IgG,IgM,IgG1,IgG2a to Pseudomonas aeruginosa in sera and cytokines including interferon-?(IFN-?),Interleukin-4(IL-4)in lung homogenate was measured at 3,7,14,28 days after infection.Results(1)The mortality in the PAO1-JP2-infected group(11.0%)was significantly lower than that of the PAO1-infected group(29.7%)and the control group(4.21%).(2) Compared with the PAO-JP2 group,during the early stages of infection(3 days after infection),the IFN-?level in lung homogenates was significantly higher;during the middle stages of infection(7 days after infection),the levels of IFN-?and IL-4 of PAO1-infected rats were significantly higher;In the later stages of infection(14 to 28 days after infection),levles of IFN-?,IgG2a were lower,while levels of IL-4,IgG,IgG1 were higher persistantly in PAO1-infected rats.Conclusions Quorum sensing system play an important role in pathogenesis and immunity effects of pseudomonas aeruginosa infection via modulating the expression of virulence factors and interfering with the immune system of hosts.
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The intraspecies quorum sensing system of Streptococcus mutans is involved with com genes family.ComE is a kind of response regulator and act as a promoter to the quorum sensing genes.S.mutans comE mutant strain IFD140?comE was constructed using the inframe-deletion system via twice homologous recombination.In current genetic studies of S.mutans,insertion duplication and allelic exchange mutagenesis techniques routinely create polar effects to downstream genes.In-frame deletions are essentially free of these polar effects because the mutation does not introduce any genetic markers.By PCR,sequencing and RT-PCR,it was confirmed that IFD140?comE has only 717bp deleted within the comE gene and the transcription of the downstream gene comD was not interfered.The research of the morphological characteristics indicated that IFD140?comE formed clumps and cells accumulated at the bottom of the glass tubes and light-microscopic observations displayed that the mutant strain formed significantly longer chains compared to those formed by the wild strain.The successfully construction of IFD140?comE lay a foundation for further quorum sensing research.
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New strategies are needed for prevention of Pseudomonas aeruginosa (P. aeruginosa) infections, a widespread disease caused by P. aeruginosa with strong drug resistance. The immunoprotective capacity of the receptor of autoinducers protein LasR/RhlR was examined in the BALB/c mice. At first, specialized expression plasmids were developed to facilitate expression of LasR/RhlR proteins in Escherichia coli (E. coli). Then, biofilms were grown from clinical isolated P. aeruginosa PA0305 to investigate the relative contributions of cell signaling for biofilm formation. Morphological characters of biofilm were quantified using Image-Pro Plus software. Fluorescence analysis demonstrated that cell signal molecule LasR/RhlR significantly (P ＜ 0.05) influenced development of P. aeruginosa biofilm. Active immunization of mice with LasR/RhlR was found to provide significant protection against homologous challenge with P. aeruginosa in mice lungs. In 10 days after lungs inoculation, the bacterial clearance rate of the immunized mice was clearly higher than that of non-immunized groups on the basis of microbiological and histological assays. The protective effects of immunization with LasR and Rh1R together were the same as the result of LasR or Rh1R immunized mice alone. These data indicate that the manner ofLasR, Rh1R or both is an important determinant of immunoprotection in mice lungs infection.