| Listeria monocytogenes(LM), a facultative intracellular Gram-positive pathogenic bacterium, can infect both human and animals, causing abortion, meningoencephalitis, sepsis and other symptoms and leading to high mortality rate, which poses a serious threat to public health and safety of livestock production. Therefore, LM is classified as one of the four major foodborne pathogens by World Health Organization(WHO). LM possesses a strong environmental adaptability and can survive under the stress environments such as cold, hypertonic, acid, alkaline and fungicides. Previous studies have found that its virulence and environmental adaptability is closely related to its numerous virulence and environmental regulatory factors, including some protein regulatory factors and non-coding RNA(nc RNAs). As important regulatory factors, nc RNAs are involved in the regulation of virulence and environmental adaptability through regulating related genes expression. Currently, lots of nc RNAs have been found and identified in LM by bioinformatic and molecular biological techniques, which are involved in regulating LM virulence, environmental adaptability, metal ion transport, glucose metabolism, infection, biofilm formation as well as intracellular parasitic process. As a member of LM nc RNAs, however, the roles of Rli60 are unclear to date. The aim of this study is to explore the regulatory roles of nc RNA Rli60 in virulence and adaptability of LM to environmental stress by analyzing the differences of the phenotypes and regulated genes between LM-SB5 and LM-Drli60 deletion strains.1. Cloning, molecular characteristics of rli60 and hfq gene and construction of deletion strains of Listeria monocytogenesThe rli60 and hfq gene were amplified by PCR from LM-SB5 genome and then sequenced and analyzed, respectively. Secondary structure and putative targets of the Rli60 together with the secondary and tertiary structure of the Hfq protein were predicted and analyzed. The upstream and downstream homology arms were amplified and fragments of Δrli60 and Δhfq were generated by overlap extention PCR. Shuttle plasmid p KSV7-Δrli60 and p KSV7-Δhfq with chloramphenicol resistance were constructed. Then they were electroporated into the LM-SB5 competent cell for homologous recombinant under the pressure of 42 and chloramphenicol℃, respectively. The recombinant LM-Δrli60 and LM-Δhfq were screened and their genetic stability was examined by PCR. The results showed that the rli60 had a length of 246 bp and its putative targets includes hly, inl F, dlt A, rsb U, glt D and mpl genes which are related to the virulence and adaptability to environmental stresses. The Rli60 formed a “X” stem-loop structure which had 9-loop and 5 complementary double-stranded structures. The length of hfq gene is 234 bp, encoding 77 amino acids. The Hfq protein had an N-terminal α-helix followed five-stranded antiparallel β-barrel. It had RNA binding sites and hexamer interfaces which is related to the RNA binding and splicing process. The fragments of 1543 bp and 1421 bp were amplified from recombinant strains, which were matched with expected length, indicating that LM-Δrli60 and LM-Δhfq were successfully constructed and screened and had stable genetic characteristic. The results of q RT-PCR showed that the transcriptional level of rli60 gene was not significantly reduced for LM-Δhfq, indicating that the Rli60 plays roles in an Hfq-independent way.2. The regulatory roles of nc RNA Rli60 in adaptability of Listeria monocytogenes to environmental stress and biofilm generationTo understand the impacts of nc RNA Rli60 on the adaptability of LM to environmental stresses and biofilm generation, we compared LM-Δrli60 with LM-SB5 in the aspects of adaptability to environmental stresses by measuring their growth at different temperatures and p H and under hypertonic and alcohol conditions, and detected capability of biofilm generation by using crystal violet staining, as well as the transcriptional levels of genes(glt B and glt C) related to biofilm generation by real-time quantitative PCR(q RT-PCR). The results showed that 1) the growth of LM-Δrli60 was reduced under environmental stresses of low temperature(30°C), high temperature(42°C), as well as alkaline and alcoholic conditions; 2) the amount of biofilm produced by LM-Δrli60 was attenuated; and 3) the transcript levels of glt B and glt C genes at 24 h and 48 h were decreased. Overall, the results confirmed that nc RNA Rli60 plays important roles in regulating the adaptability of LM to environmental stresses, and regulates biofilm generation possibly through impacting the expression of glt B and glt C genes.3. The effects of nc RNA Rli60 deletion on virulence of Listeria monocytogenesTo understand the impacts of nc RNA Rli60 on LM’s virulence, we compared LM-Δrli60 with LM-SB5 in the following aspects: LD50 to Kunming mouse and amount in mouse liver and spleen as well as effects on pathology of mouse liver, spleen and kidney after inoculation. Hemolysis activity and phospholipase activity were determined by hemolysis activity assay experiment and phospholipase activity assay experiment. Adhesiveness, invasion ability, intracellular survival, proliferation and transcription of virulence genes were detected in mouse macrophage cell line RAW264.7. The results showed that 1) LD50 of LM-Drli60 to Kunming mouse was increased by 2.12 logarithmic magnitude, indicating strain LM-Drli60 has significantly decreased virulence; 2) LM-Drli60 amount in liver and spleen was significantly lower than that of LM-SB5; pathological damages of liver, spleen and kidney of mouse infected LM-Drli60 were declined compared to that of LM-SB5 infection. Hemolysis activity of LM-Drli60 decreases 4 times than that of LM-SB5. Both LM-SB5 and LM-Drli60 had phospholipase activity; 3) LM-Δrli60 had significantly higher adhesion rate and lower invasion rate in RAW264.7; and significantly lower intracellular survival and proliferation rates than that of LM-SB5. Inoculation of LM-Δrli60 significantly affected transcription of virulence genes, transcription level of prf A, inl A and inl B genes was significantly higher for LM-Δrli60, while transcription level of virulence genes plc A, hly and act A was significantly lower in LM-Δrli60 than in LM-SB5. Taking together, the results indicated that nc RNA Rli60 is crucial in regulating LM virulence.LM-Δrli60 was successfully constructed by homologous recombination in this study. LM-Δrli60 has weaker adaptability than LM-SB5 to low and high temperatures together with alkalineli and alcoholic conditions. The virulence, ability of invasion, intracellular survival and proliferation of the LM-Δrli60 were significantly decreased compared with the wildtype strain LM-SB5. This study confirmed that Rli60 could affect LM’s adaptability to environmental stresses and virulence. |
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