Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (16): 3294-3304.doi: 10.3864/j.issn.0578-1752.2024.16.015

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles    

STM2503 Regulates Biofilm Formation and Stress Adaptability of S. Typhimurium

WANG NanWei(), LI LiLi, CHEN KaiFeng, ZHOU ZhouPing, PAN Peng, GUAN Jin, XU ChengGang, LIAO Ming, ZHANG JianMin()   

  1. School of Veterinary Medicine, South China Agricultural University/National and Regional Joint Engineering Laboratory for Medicament of Zoonosis Prevention and Control/Key Lab for Zoonosis of Ministry of Agriculture and Rural Affairs/Key Laboratory of Animal Vaccine Development of the Ministry of Agriculture/Guangdong Key Laboratory of Zoonotic Diseases of Animal Origin, Guangzhou 510642
  • Received:2024-03-05 Accepted:2024-06-03 Online:2024-08-16 Published:2024-08-27
  • Contact: ZHANG JianMin

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Abstract:

【Objective】S. Typhimurium is an important zoonotic pathogen, and its strong pressure adaptability brings great challenges to its prevention and control. In this study, the mechanism related to c-di-GMP pathway gene STM2503 regulating the biofilm formation, and thereby affects environmental stress capacity of S. Typhimurium, were investigated, so as to excavate the key factors regulating the stress adaptation of S. Typhimurium, and provide a theoretical basis for the formulation of new prevention and control strategies.【Method】The STM2503 gene deletion strain was constructed by λ-Red homologous recombination, and plasmid PBAD was used to express STM2503 to construct the gene complementation strain. Then, the effect of STM2503 on the intracellular c-di-GMP level of bacteria was revealed by detecting the signaling molecule c-di-GMP content in strains with different STM2503 expression levels. Next, the effect of STM2503 on bacterial motility and biofilm formation ability were detected by bacterial motility detection and crystal violet staining experiments, furthermore, the real-time fluorescence quantitative PCR was employed to elucidate its regulatory mechanism on bacterial motility and biofilm formation at the genetic level. Finally, the effect of STM2503 on the stress adaptability of S. Typhimurium was investigated through antibiotic treatment, oxidative stress, and disinfectant stress tests.【Result】Compared with the wild strain (WT269), the deletion of STM2503 increased the intracellular c-di-GMP content of the strain by 37.51%, and did not affect the normal growth of the strain. In addition, the deletion of STM2503 increased the expression level of extracellular matrix synthesis genes, and increased the content of extracellular polysaccharide, extracellular DNA and extracellular protein by 10.30% (P<0.01), 33.59% (P<0.001) and 27.60% (P<0.01), respectively, which ultimately led to a significant enhancement of biofilm formation ability by 1.63-fold (P<0.01). Moreover, 269ΔSTM2503 reduced the motile diameter by 17.22% (P<0.01) compared with the wild strain (WT269) at 6 h via decreasing the expression of flagella synthesis related genes fliA and flhC. These changes eventually led to a 1-3 fold reduction in the sensitivity of the 269ΔSTM2503 strain to β-lactam antibiotics, such as cefotaxime, cefepime, and amoxicillin, along with showed stronger adaptability under oxygen stress and SDS disinfectant stress. 【Conclusion】 In conclusion, STM2503 was involved in the degradation of the signaling molecule c-di-GMP and reduced the biofilm formation ability of S. Typhimurium by inhibiting the synthesis of extracellular substances and enhanced the motility of strains by upregulating the expression of flagellar synthesis genes, thereby reducing the drug resistance and environmental stress adaptability of S. Typhimurium. This study provided a theoretical basis for the excavation of the key targets for the prevention and control of S. Typhimurium.

Key words: S. Typhimurium, C-di-GMP, STM2503, biofilm, motility

Table 1

Primer sequences used for PCR"

引物名称 Primer 引物序列 Primer sequences (5′→3′)
STM2503-Red-F CCAGTGTGATTAAACGCCGTTTCCCCTCCCTTCGCCCATCTGCTAGTGTAGGCTGGAGCTGCTTC
STM2503-Red-R ACAGCCTGCCCACGACGAAGAGGAATAACGTCACACGCAGGAATCCATATGAATATCCTCCTTAG
STM2503-JD-F GTGCAGGTGCCGTGTATC
STM2503-JD-R CTATCAATCTGTTAGGCGTTTT
STM2503-HF AACTGCAGAACCAATGCATTGGGCTACGGATGGGCGAAAT
STM2503-HR GCCGGAATTCCGGCCACGCAGGAATCAAGTCT
pKD46-JD-F GCAACTTTATCCGCCTCC
pKD46-JD-R TCGCCCTTATTCCCTTTT
pCP20-JD-F CAGTGCTGCAATGATACCGC
pCP20-JD-R TCCTTGAGAGTTTTCGCCCC
PBAD-F GCGTCACACTTTGCTATGCC
PBAD-R ACGGCGTTTCACTTCTGAGT
BcsA-F TGCGGGCTGATTCTGCTGTTTGC
BcsA-R CGAAATGATTCACGCCCGCCGT
BcsB-F AAAAGGTATCGCACAAGGG
BcsB-R GCTACGCAGCAAATAGAGGT
CsgB-F TCGACTTTCGCCCGATTAT
CsgB-R AGGTCCAGGGTGACAGCAT
CsgA-F CCAGGGTGCGGATAACAGTA
CsgA-R CCAACCTGACGCACCATTAC
FliA-F GCAAGGAACGGCATTTAC
FliA-R AAAGTTGGCTGTTGTTGGTAT
FlhC-F GAAAGTGGGTTGCTTGAATTG
FlhC-R GCATCTCGGGAAAGTTTACG
FlhD-F TGATGATCGTCAAACCGGAAA
FlhD-R TGCCGCAGATGGTCAAACTG

Fig. 1

Identification of constructed STM2503 deletion and complementation strains A. Identification of STM2503 deletion, (M: 5000 bp DNA relative molecular weight; Lanes 1 and 2: deletion mutant strain 269 ΔSTM2503; Lanes 3 and 4: Wild strain WT269); B. Identification of complementation strain 269 ΔSTM2503R (M: 5000 bp DNA relative molecular weight; 1: Empty load of plasmid PBAD; 2: Complementation strain 269 ΔSTM2503R)"

Fig. 2

Effect of STM2503 deletion on bacterial growth"

Fig. 3

Effect of STM2503 on the intracellular c-di-GMP content of strains A: C-di-GMP standard curve; B: Identification of c-di-GMP content in strains with different STM2503 expression levels"

Fig. 4

Identification of multicellular behavioral phenotypes of strains with different expression levels of STM2503"

Fig. 5

Deletion of STM2503 enhanced the biofilm formation ability of S. Typhimurium"

Fig. 6

Quantification of extracellular matrix components in WT269, 269ΔSTM2503, and 269ΔSTM2503R strains A, B, C are quantitative analyses of extracellular proteins, extracellular DNA, and extracellular polysaccharides of strains with different STM2503 expression levels, respectively; D: Glucose standard curve"

Fig. 7

Regulation of S. Typhimurium motility by STM2503"

Fig. 8

QRT-PCR analysis of the effect of STM2503 on the expression levels of genes related to biofilm and motility"

Fig. 9

The role of STM2503 in stress adaptation of S. Typhimurium"

Table 2

Results of antibiotic susceptibility assay of strains with different expression levels of STM2503"

CTX FEP AMX CEF CAZ TET DOX KAN AMK GEN STR FFC CHL NAL CIP NOR OFX
269 1 0.25 1 4 4 1 2 2 2 2 16 4 2 4 0.5 1 1
269ΔSTM2503 2 1.00 2 8 8 1 2 2 2 2 16 4 2 4 0.5 1 1
269ΔSTM2503R 1 0.25 1 4 4 1 2 2 2 2 16 4 2 4 0.5 1 1
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