Impact Of Rli87 Gene Deletion On Response Of Listeria Monocytogenes To Environmental Stress And It’s Virulence

Listeria monocytogenes(LM) is a kind of Gram-positive bacteria which also belongs tofacultative intracellular bacteria. This bacterum is widely found in soil, aquatic products and animals, which can survive and propagate under a variety of conditions such as low temperature, high salt, prolonged dry and pH5.6-9.8. LM mainly cause infection in animals and humans through the digestive tract,which is classified as an important zoonotic pathogen, The clinical symptoms is characterized by meningitis, septicemia and abortionin human and animal, which poses a huge threat for livestock and human health.Therefore, LM is identified as one of the four major food-borne pathogens by WHO.LM can express a large number of virulence factors which is involved inthe course of infection. The genes encoding virulence factors associated with infection and intracellular parasiticlife are mainly located in LM pathogenicity island 1(LIPI-1) and a pathogenicity island 2(LIPI-2). LIPI-1 contains six genes, followed prfA, plcA, hly, mpl, actA, plcB, while LIP-2, also known as internalization of island, mainly encodes small molecules internalization factors. Recent studies have found that noncoding RNAs(ncRNAs) in LMplay important regulatory roles in its growth, gene expression, glucose metabolism, metal ion transport, biofilm formation, intracellular parasitism and environmental stress.The regulation model of ncRNAs has become one of the most important ways in LM regulatory network. According to the regulatory mechanism of ncRNAs, LM ncRNAs can be divided into three kinds: cis-acting RNA(Cis-acting RNAs, such as riboswitches), antisense RNA(Anti-Sense RNAs, asRNAs) and trans-coding small RNA(Trans-encoded small RNAs, sRNAs). Cis-acting RNA is usually located in the mRNA 5 ’untranslated region(5’-UTR) or the 3’ untranslated region(3’-UTR), not only affecting the bacteria translation and transcripts, but also affecting the mRNA stability and folding structure. rli87 gene belongs to sRNAs, however, the biological function of the gene rli87 is unclear to date. In this study, rli87 gene deletion strains of LM-Δrli87was constructed by the homologous recombination technique, and the effects of ncRNA rli87 deficiency on growth characteristics,environmental stress and pathogenicity were explored. The main contents and results are as follows:1. Construction of LM Rli87 gene deletion strain and it’s growth characteristicsAccording to LM EGD-e genome sequence(accession number: AL591824) in GenBank, a conservative region was analyzed by DNAMAN software, and ownstream homology arms primers is designed with Primer 5.0 software. The Upstream and downstream homology arm containing rli87 gene were amplified by PCR technique respectively.Then rli87 gene deletion fragment was generated by the SOE-PCR techniquen and further cloned into pMD19-T. The plasmid pMD19-T-△ rli87 and pKSV7 were double digested, respectively, andrecovered rli87 gene deletion fragment was inserted into shuttle vector PKSV7 to generate recombinant shuttle plasmid pKSV7-△ rli87. pKSV7-△ rli87 was then transformed into LM EGD-e using electroporation method, and the strains after electroporation was screened by PCR and sequencing. Positive transformants under conditions of chloramphenicol and 42 ℃ was subcultured 10 generations to obtain a single exchanging of strains resistant to chloramphenicol.Dual exchanging strain ofLM-△ rli87 was then gained in the absence of chloramphenicol at 42 ℃ by subculture of 15 generations. LM-△ rli87 was cultured for 20 generations and identified by PCR, which was consistent with the expected results of a 584 bp fragment.It was confirmed that LM-△ rli87 deletion strains has good genetic stability. There was no significant difference in growth(p> 0.05) between LM EGD-e and LM-△ rli87 strains at 37 ℃, which confirmed that rli87 gene deletion had no effect on the growth characteristics at 37 ℃.2. Effects of rli87 deletion on environmental stress of Listeria monocytogenesThe wild strain LM EGD-e and deletion strain LM-△ rli87 were tested under different conditions of different temperatures, pH, osmotic pressure and high oxidative stress, and the expression of stress-related genes were detected.LM EGD-e and LM-△ rli87 LM were cultured under different conditions, andOD600 nm valueswere determinated.The growth curves of bacteria under different conditions at different time points were drawn.The results showed that growth rate of LM-△ rli87 was significantly higher than that of LM EGD-e(P<0.05); and the growth existed a significant difference(P<0.05) at different growth conditions of pH, pH = 9, The amount of LM-△ rli87 was decreased with the change of time when it was grown in 2% H2O2 oxidizing environment, while the amount of LM- △ rli87 was lower than LM EGD-e(P<0.05).Compared with LM EGD-e strain, LM-△ rli87 deletion strains rsbV, rsbW, hpt, clpP, ctsR 5 stress-related gene expression levels increased when it was grown in pH = 9, which showedncRNA rli87 played a role in regulation of these five stress-related genes in an alkaline environment.3. Roles of rli87 gene deletion on pathogenicity of Listeria monocytogenesRAW264.7 macrophages infected and BALB / C mice were infected by the strainsLM EGD-e and LM-△ rli87, respectively,the rate of cell adhesion and survival of bacteria in the test cells in micewere statistically counted to calculate. Expression levels of virulence genes were determinated by real-time RT-PCR,and pathogenic differences of deletion mutant and wild strains were detected by hemolysis test.Experimental results show that rli87 gene deletion strains adhesion rate and invasiveness decreased when compared with the LM EGD-e.Bacterial load capacity was reduced in liver, spleen. The median lethal dose of LM-△ rli87 is significantly increased by 103 fold than that of the wild strain.Transcription levels of virulence-related genes hly and PrfAwere significantly decreased. The ability of hemolysis of LM-△ rli87 was significantly reduced when compared with the LM EGD-e, which indicated that the virulence of LM-△ rli87 was decreased when ncRNA rli87 was deleted.In consideration of hemolytic capacity was determinated by hly gene, the results suggested that hly gene expression was regulatd byncRNA rli87.In all, rli87 gene deletion strain was successfully constructed by SOE-PCRand homologous recombinationin in this study, and effects of ncRNA rli87 deficiency on environmental stress and pathogenicity were studied.Compared with the LM EGD-e, environmental stress ability and pathogenityof LM-△ rli87 was decreased,which indicated that rli87 gene played regulatory rolesin LM virulence and environmental stress.