Functional Analysis Of Superoxide Dismutase In Biofilm Formation In Listeria Monocytogenes

Listeria monocytogenes is a foodborne pathogen capable of causinglisteriosis in human. It is widely distributed in the environment and formsbiofilms on food processing equipments, which are communities ofmicroorganisms adhering to a surface. The resistance of L.monocytogenes in biofilm to adversity such as disinfectant is significantlyincreased, thus the pathogen can contaminate food repeatedly andthreaten public hygiene and human health. The mechanism of biofilmformation is not yet clear in L. monocytogenes, therefore, the mechanisticstudy has significant importance for food safety.In our previous work, a mutant library was constructed by insertingTn917into the genome of L. monocytogenes4b G. One mutant strain（LM-49） with increased biofilm formation capacity was obtained throughscreening in the library, and the insertion site of Tn917located inlm.G₁771gene encoding a putative ABC transporter permease wasidentified. Two-dimensional gel electrophoresis （2-DE） and microarrayanalyses revealed that superoxide dismutase （SOD） was also up-regulatedby about2-fold in the Δ1771mutant in comparison to the wild-type4b G. In this dissertation, we analyzed the function of the sod gene in biofilmformation and its relationship with the lm.G₁771gene, and explored therelationship between antioxidative genes and biofilm formation based onthe previous studies. The main contents and conclusions are shown as thefollowing:1. Vector construction for homologous recombination and screeningsod-deleted mutants.5’ and3’ flanking sequences of the sod gene werelinked into the temperature-sensitive shuttle vector pKSV7containing thechloromycetin resistant gene, and vector pKSV7::sod14of the sod genefor homologous recombination was constructed successfully. Therecombinant plasmid was transformed into the competent cells of thewild-type G and Δ1771respectively, positive transformants were obtained,and then the sod gene deletion mutant Δsod and double deletion mutantΔ1771Δsod were screened by double exchange and resistance selection.Recombination probability was0.46%（2/436） and0.38%（5/1300）,respectively. The successful mutant construction provided the necessaryexperimental materials for the function analysis of SOD anddetermination of its relationship with lm.G₁771.2. Determination of the relationship between the lm.G₁771geneand the sod gene. First, planktonic and biofilm cells of the wild-type Gand the mutant strain Δ1771were cultured respectively. Second,RT-qPCR and zymographic analysis were performed to determine the expression level of SOD in the wild type G and Δ1771. Research resultsshowed that there was no distinct change in the expression level of SODbetween the wild-type G and Δ1771in planktonic cells, but thetranscriptional level and activity of SOD were separately increased by2.2-fold and2.3-fold in Δ1771compared to the wild type G in biofilmcells. In addition, the expression level of lm.G₁771in Δsod mutant wasnearly consistent with in the wild type G. These results showed that theexpression level of the sod was increased due to the deletion of thelm.G₁771gene in biofilm cells but not obvious in planktonic cells, andthe sod gene had no distinct impact on the expression of lm.G₁771inbiofilm cells.3. Physiological function of SOD in biofilm formation and itsrelationship with lm.G₁771. First, the abilities of biofilm formation inthe wild type G and three mutants （Δ1771, Δsod and Δ1771Δsod） wereassessed using crystal violet staining with96-well plates and afluorescence microscope. Second, the growth ability, ROS output and theantioxidative capacity were determined and compared among these fourstrains. The main results are as follows:（1） In contrast to the increasedability of biofilm formation in Δ1771, the biofilm formation abilities werereduced in both sod and Δ1771, especially in Δ1771Δsod.（2） There wasno obvious difference in growth rate between Δ1771and the wild-type G,but the growth rates of Δsod and Δ1771Δsod were slower than the wild-type G and their colony sizes were also smaller than the wild type Gunder the same culture conditions.（3） There was no significant differencein ROS output between Δ1771and the wild type G, and the ROSproduction of Δsod and Δ1771Δsod was enhanced distinctly compared tothe wild type G, especially in Δ1771Δsod.（4） The sensitivity of Δ1771tomethyl viologen （MV） was similar to the wild type G. Both Δsod andΔ1771Δsod were more sensitive to MV than wild type G, and thesensitivity of Δ1771Δsod was higher than that of Δsod. These resultsindicated that SOD served as an important antioxidase playing animportant role in maintaining the normal cell growth of L. monocytogenesand restraining excess ROS output, and positively regulated biofilmformation together with the lm.G₁771gene. There was a synergisticeffect in oxidative stress resistance between SOD and lm.G₁771.4. Influence of methyl violet （MV） inducing ROS production onbiofilm formation. The biofilm formation capabilities of five serotypes often wild-type L. monocytogenes strains in TSB were compared with inTSB+MV. Results showed that1mM MV obviously inhibited biofilmformation and cell clustering on the sheet glasses, showing that ROS wentagainst biofilm formation, which provided an evidence for the hypothesisthat SOD regulated biofilm formation by suppressing ROS.5. Two-dimensional electrophoresis （2-DE） and mass spectrometrywere conducted to identify proteins regulated by SOD.2-DE protein gel images were analysed by PDQuest8.0software to identify differentialprotein spots. As a result,14differentially spots with at least2-foldchange in intensity were identified, of which10were down-regulated and4up-regulated in Δsod. The down-regulated proteins were some enzymesinvolved in cell regulator, metabolism, growth and stress response. Theup-regulated proteins were enzymes related to metabolism and cell wallsynthesis. Part of these differential proteins played important roles inbiofilm formation. These results indicate the action mechanism of sod ingrowth and resistance and the action pathway of SOD positivelyregulating biofilm formation.6. RT-qPCR was conducted to analyze the expression of theanti-oxidative related genes in the deletion mutants both in biofilm andplanktonic cells. The anti-oxidative related genes studied in this thesiswere divided into three classes: direct participants for antioxidation suchas kat and fri, regulators （perR and sigB） for stress response regulatingthe expression of anti-oxidative genes, and genes involved in repairingDNA damage caused by oxidization such as recA. The following resultswere obtained in this section.（1） In planktonic cells, the expression levelsof antioxidative genes and regulators were lower in Δ1771and Δsod thanin the wild-type G, showing that both lm.G₁771and sod genespositively regulated these antioxidative genes and did not influence theexpression of recA. The expression level of recA was obviously increased in Δ1771Δsod. Because recA could activate SOS, it was suggested thatsod and lm.G₁771played important roles together in maintaining cellnormal growth.（2） Biofilm formation caused the up-regulation of partialantioxidative genes, especially in Δsod, showing that biofilm formationcontributed to repairing antioxdative capacity in antioxidation deficientstrains.（3） MV could stimulate L. monocytogenes cells to generate excessendogenous ROS. Under MV treatment, the expressions of allabove-mentioned genes were up-regulated in the wild type G, indicatingthat the adaptive mechanism to oxidative stress generated in biofilmformation was more complicated than that caused by the stimulation ofonly oxidant.In conclusion, biofilm formation can be restrained by ROS, and someanti-oxidative stress related genes were involved in biofilm formation.SOD participates in cellular detoxification and protects microorganismsagainst ROS by organism metabolism. The expression of some enzymesrelated to resistance, metabolism and growth was influenced by SOD. Inaddition, the expression of SOD was influenced by lm.G₁771, and thetwo genes showed synergistic effects on oxidative stress resistance. Theseresults indicate that SOD together with an ABC transporter coded bylm.G₁771influences biofilm formation, and they play important roles inbiofilm formation.