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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2018, Vol. 38 Issue (9): 1-11    DOI: 10.13523/j.cb.20180901
Orginal Article     
Effect of Glutamate Dehydrogenase Deletion on Biofilm Formation,Virulence and Extracellular Proteins Expression of Listeria monocytogenes
Wen-jing WANG,Li-yu YANG,Chan-juan LIU,Jin ZHAO,Qin LUO()
Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Sciences, Central China Normal University, Wuhan 430079, China
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Abstract  

Objective:To explore the effect of glutamate dehydrogenase deletion on biofilm formation, virulence and extracellular proteins expression of Listeria monocytogenes.Methods: The effect of glutamate dehydrogenase deletion on the formation of L.monocytogenes biofilms was examined using a microtiter plate method. The hemolytic activity, the semi-lethal dose of Helicoverpa armigera (Hübner)in wild-type, GDH-deficient and revertant strains were compared.The differentially expressed extracellular proteins between the wild strain and GDH-deficient strain were identified and analyzed by iTRAQ technology. Results: Compared with wild strains, the amount of biofilm formed by the GDH-deficient strains was significantly decreased (P≤0.01), as well as the hemolytic activity, but its semi-lethal dose on Helicoverpa armigera (Hübner) was increased 1.6 fold. The functions of all sixty-two differential expressed extracellular proteins identified by iTRAQ were cataloged into 16 functional groups, including carbohydrate transport and metabolism, energy synthesis and transport, transcription, cell membrane protein related to cell wall and so on. Among them, the most dominant groups including 24 proteins, accounting for 38.71% of total differential expressed proteins, are related to carbon and nitrogen metabolism as well as energy transport, suggesting that glutamate dehydrogenase is a key enzyme in carbon and nitrogen metabolism as well as energy synthesis and transport for L.monocytogenes. The slow growth and reduced biomass of EGDeΔgdhA compared to EGDe in minimal essential medium were in agreement well with these proteomic results. In addition, the expressions of three proteins from EGDeΔgdhA associated with bacterial adhesion and biofilm formation were also significantly decreased.Conclusion: Deletion of glutamate dehydrogenase reduces bacterial biofilm formation and virulence, affects significantly the extracellular protein expression in L.monocytogenes.

Key words： Listeria monocytogenes      Glutamate dehydrogenase      Biofilm      Virulence      iTRAQ      Extracellular protein     
Received: 10 April 2018      Published: 12 October 2018
Corresponding Authors: Qin LUO     E-mail: qinluo@mail.ccnu.edu.cn
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Wen-jing WANG
Li-yu YANG
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Jin ZHAO
Qin LUO
Cite this article:

Wen-jing WANG,Li-yu YANG,Chan-juan LIU,Jin ZHAO,Qin LUO. Effect of Glutamate Dehydrogenase Deletion on Biofilm Formation,Virulence and Extracellular Proteins Expression of Listeria monocytogenes. China Biotechnology, 2018, 38(9): 1-11.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180901     OR     https://manu60.magtech.com.cn/biotech/Y2018/V38/I9/1

Primers Sequences
P1:gdhA A-F
P2:gdhA A-R
P3:gdhA B-F
P4:gdhA B-R
P5:gdhA-ck-F
P6:gdhA-ck-R
P7: gdhA-XbaI-F
P8: gdhA-BamHI-R
P9:pERL3-ck-F
P10:pERL3-ck-R		 ACAGGATCCGAACGCCTTAT
ATAAGTAGCCGATTTCACGAC
AAATCGGCTACTTATATAAAGTTCTATTCGG
GGTGAATTCTATTATTACGGGCTTC
AAGGTGGCTCTGACTTTGATC
ATAACAAGGTTACCAGGTGC
TAATCTAGATAAAACTCAACCAAGAGAAGG
ATGGGATCCCTGAGTTCTCAAATCTTACAG
GAAAACCGCTACGGATCACATC
CCAACCTGCCATCACGAGATTT
Table 1 The primers used in this study
Fig.1 Comparison of the growth of EGDe, EGDeΔgdhAand EGDeΔgdh A+pERL3-gdhA in minimal essential medium
Fig.2 Biofilm formation by EGDe, EGDeΔgdhA and EGDeΔgdhA+pERL3-gdhA (a) The biomass of the three strains after 24h and 48h of cultivation (b) Biofilm quantities for the three strains measured by microtiter plate assay after 24h and 48h of incubation (c) Biofilm formed in the 48h by the three strains observed under a 20× objective **:Significant differences of the biomass of biofilm betweent the mutant strain EGDeΔgdhA and the wild type strain EGDe during the same culture time (P≤0.01)
Fig.3 Hemolytic activity of EGDe, EGDeΔgdhA and EGDeΔgdhA+pERL3-gdhA
Strain LD50(cfu)
BHI (死亡条数为0)
EGDe 3.89×106
EGDeΔgdhA 6.34×106
EGDeΔgdhA+pERL3-gdhA 4.07×107
Table 2 The semi-lethal dose of EGDe, EGDeΔgdhA and EGDeΔgdhA+pERL3-gdhA on Helicoverpa armigera(Hübner)
Fig.4 Tissue section of the midgut of H. armigera after 72h injection of EGDe, EGDeΔgdhA and EGDeΔgdhA+pERL3-gdhABM:Basement membrane; EC:Epithelial cell; DEC:Dissolved epithelial cell
COG1) Locus2) Putative function3) MM/Pie4) Protein score Ratio5)
(G) Carbohydrate transport and metabolism
lmo0859 Uncharacterized protein 0.65/46.95 110.97 0.71
lmo2455 2-phosphoglycerate dehydratase 4.70/46.47 40.79 1.29
lmo2848 L-rhamnose isomerase 5.31/48.07 39.33 1.67
lmo2665 PTS galacticol transporter subunit IIC 8.74/45.57 36.51 1.48
lmo2734 Sugar hydrolase 5.14/100.90 67.64 0.19
lmo0401 Alpha-mannosidase 4.95/100.00 45.52 0.63
lmo0359 D-fructose-1,6-biphosphate aldolase 5.02 /31.60 41.25 0.51
lmo1217 Endo-1,4-beta-glucanase and to aminopeptidase 5.14/38.90 36.33 0.58
lmo2771 Beta-glucosidase 5.28/55.10 31.94 0.49
lmo2002 PTS sugar transporter 5.94/17.20 38.98 0.75
lmo2556 Fructose-1,6-bisphosphate aldolase 5.2/30.00 37.70 0.52
(C) Energy production and conversion
lmo0520 Transcriptional regulator 4.91/38.00 61.54 1.53
lmo1811 ATP-dependent DNA helicase RecG 5.69/78.00 98.2 0.52
lmo1183 Hypothetical protein 6.86/22.20 42.97 0.50
lmo0396 Pyrroline-5-carboxylate reductase 5.08/28.10 32.57 0.55
COG1) Locus2) Putative function3) MM/Pie4) Protein score Ratio5)
lmo0300 Phospho-beta-galactosidase 4.99/53.60 30.36 0.55
lmo0067 Dinitrogenase reductase ADP-ribosylation protein 4.89/37.70 328.92 0.48
(O) Posttranslational modification, protein turnover, chaperones
lmo2069 Class I heat-shock protein (chaperonin) GroES 4.60/100.60 241.72 2.26
lmo1059 Thioredoxin 4.83/199.20 41.1 3.58
lmo2206 Protease subunit B 64.97 /97.50 95.97 0.43
lmo0520 Transcriptional regulator 4.91/38.00 61.54 1.53
lmo1811 ATP-dependent DNA helicase RecG 5.69/78.00 98.2 0.52
lmo1183 Hypothetical protein 6.86/22.20 42.97 0.50
lmo0396 Pyrroline-5-carboxylate reductase 5.08/28.10 32.57 0.55
lmo0300 Phospho-beta-galactosidase 4.99/53.60 30.36 0.55
lmo0067 Dinitrogenase reductase ADP-ribosylation protein 4.89/37.70 328.92 0.48
(M) Cell wall/membrane/envelope biogenesis
lmo0582 Peptidase P60 8.56/42.70 515.01 2.13
lmo2555 Glycosyl transferase family 1 6.91/47.89 55.32 0.377
lmo2754 D-alanyl-D-alanine carboxypeptidase 6.93/48.07 100.2 0.77
lmo2185 Sortase B 5.033/21.05 59.56 0.78
(I) Lipid transport and metabolis
lmo0354 Fatty-acid--CoA ligase 6.268/58.10 30.86 1.36
lmo1753 Diacylglycerol kinase 5.64/34.02 37.46 0.47
(K)Transcription
lmo0630 PTS fructose transporter subunit IIA 5.57/77.72 52.06 3.05
lmo2606 DNA-directed RNA polymerase subunit alpha 4.797/34.91 42.97 1.37
lmo1408 PadR family transcriptional regulator 8.82/20.72 97.05 0.36
lmo1878 Transcriptional regulator MntR 5.46/16.39 45.26 0.68
lmo0258 DNA polymerase III subunit beta 4.70/42.43 30.84 0.58
lmo1899 ATP-dependent helicase DinG 5.78/106.06 45.71 0.30
lmo2107 DeoR family transcriptional regulator 5.28/28.40 38.58 0.30
(D) Cell cycle control, cell division, chromosome partitioning
lmo1606 DNA translocase FtsK 5.317/87.79 50.78 1.46
(E) Amino acid transport and metabolism
lmo1663 Asparagine synthetase 5.02/48.99 38.39 1.20
lmo1628 Tryptophan synthase beta chain 5.50/43.70 62.16 0.51
lmo2539 Serine hydroxymethyltransferase 5.53/45.12 41.06 0.76
(J)Translation, ribosomal structure and biogenesis
lmo1530 Queuine tRNA-ribosyltransferase 7.21/42.92 38.02 1.31
lmo1459 Glycyl-tRNA synthetase subunit alpha 4.85/34.30 83.53 0.65
lmo2656 30S ribosomal protein S12 11.24/15.10 54.09 0.66
(F) Nucleotide transport and metabolism
lmo2155 Ribonucleotide-diphosphatereductase alpha 5.59/87.39 115.43 0.36
COG1) Locus2) Putative function3) MM/Pie4) Protein score Ratio5)
(L) Replication, recombination and repair
lmo1320 DNA polymerase III PolC 5.26/162.70 80.30 0.76
(V) Defense mechanisms
lmo0607 ABC transporter ATP-binding 5.56/63.3 0 52.10 0.35
lmo1964 lABC-type multidrug transport system 6.35/33.29 34.51 0.44
lmo0028 Similar to E.coli microcin C7 self-immunity protein 4.48/34.38 61.81 0.67
(S)Function unknown
lmo2181 Sortase 5.35/28.62 47.73 0.79
(H) Coenzyme transport and metabolism
lmo1042 Molybdopterin biosynthesis protein 5.167/45.00 38.26 0.44
lmo1093 NH(3)-dependent NAD(+) synthetase 5.07/30.59 32.26 0.68
lmo2770 Glutathione synthetase 4.92/88.56 36.36 0.47
(T) Signal transduction mechanisms
lmo1935 Protein-tyrosine/serine phosphatase 4.92/36.63 36.18 0.63
(R) General function prediction only
lmo0919 Antibiotic ABC transporter ATP-binding protein 8.929/59.10 74.01 1.44
lmo1760 Geranylgeranylglyceryl phosphate synthase-like protein 5.109/25.36 37.53 0.42
lmo1226 LacI family transcription regulator 5.79/38.81 43.98 0.72
lmo1537 Predicted GTPase 5.12/47.1 0 37.98 0.63
lmo2232 Hypothetical protein 4.70/49.10 38.21 0.77
lmo2359 Hypothetical protein 4.89/31.07 31.27 1.58
Table 3 Differentially expressed proteins identified by iTRAQ
Fig.5 COG clustering analysis of differentially expressed proteins
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