title: Activation of PrfA results in overexpression of virulence factors but does not rescue the pathogenicity of Listeria monocytogenes M7.
abstract: Listeria monocytogenes encodes a transcriptional activator, PrfA, to positively regulate the expression of virulence factors. Several mutations in PrfA (PrfA*) have been found to contribute to increased regulatory activity. Here, we describe a strain, M7, containing a PrfA*(G145S) that activates expression of virulence factors but with low pathogenicity. To study this contradictory relationship, we exchanged the prfA genes between strains EGDe and M7 (designated EGDe-prfA(M7) and M7-prfA(EGDe)). The phospholipase B (PlcB) and listeriolysin O (LLO) activities were significantly upregulated in the strain EGDe-prfA(M7) (PrfA*). Constitutive activation of PrfA potentiated virulence of the pathogenic strain EGDe, shown as increased adhesion and invasion as well as enhanced cell-to-cell spread in cultured cell lines. However, the strain M7, though PrfA-activated, had significant defects in these virulence-related phenotypes and low pathogenicity in the murine infection model, as compared with EGDe or EGDe-PrfA(M7). To further uncover the possible mechanisms, we analysed abundance and distributions of InlA, InlB, LLO and ActA proteins, all regulated by PrfA, in EGDe, M7 and their prfA mutants. Western blotting showed that the PrfA-regulated genes of constitutively activated PrfA strains were overexpressed in vitro, while different distributions were observed. In contrast to the virulent strain EGDe-prfA(M7), the majority of InlB in M7 was detected in the culture supernatant and not on the bacterial surface. We suppose that the low virulence of strain M7 is due to its defects in infecting host cells, possibly as a result of failed anchorage on the bacterial cells of surface proteins like InlB, a major protein involved in adhesion and invasion of pathogenic L. monocytogenes strains. Further research is warranted to address why InlB detaches from the bacterial cells of this particular strain.
authors.affiliation: Zhejiang University Institute of Preventive Veterinary Medicine, 388 Yuhangtang Road, Hangzhou, Zhejiang, 310058, PR China.|​ Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, 388 Yuhangtang Road, Hangzhou, Zhejiang, 310058, PR China.
authors.forename: Chun
authors.initials: C
authors.lastname: Fang
authors.affiliation: Zhejiang University Institute of Preventive Veterinary Medicine, 388 Yuhangtang Road, Hangzhou, Zhejiang, 310058, PR China.|​ Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, 388 Yuhangtang Road, Hangzhou, Zhejiang, 310058, PR China.
authors.forename: Tong
authors.initials: T
authors.lastname: Cao
authors.affiliation: ​ College of Animal Science and Technology, Zhejiang A&F University, 88 Huanbei Road, Lin'an, Zhejiang, 311300, PR China.
authors.forename: Changyong
authors.initials: C
authors.lastname: Cheng
authors.affiliation: Zhejiang University Institute of Preventive Veterinary Medicine, 388 Yuhangtang Road, Hangzhou, Zhejiang, 310058, PR China.|​ Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, 388 Yuhangtang Road, Hangzhou, Zhejiang, 310058, PR China.
authors.forename: Ye
authors.initials: Y
authors.lastname: Xia
authors.affiliation: Zhejiang University Institute of Preventive Veterinary Medicine, 388 Yuhangtang Road, Hangzhou, Zhejiang, 310058, PR China.|​ Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, 388 Yuhangtang Road, Hangzhou, Zhejiang, 310058, PR China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Shan
authors.affiliation: Zhejiang University Institute of Preventive Veterinary Medicine, 388 Yuhangtang Road, Hangzhou, Zhejiang, 310058, PR China.|​ Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, 388 Yuhangtang Road, Hangzhou, Zhejiang, 310058, PR China.
authors.forename: Yongping
authors.initials: Y
authors.lastname: Xin
authors.affiliation: Zhejiang University Institute of Preventive Veterinary Medicine, 388 Yuhangtang Road, Hangzhou, Zhejiang, 310058, PR China.|​ Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, 388 Yuhangtang Road, Hangzhou, Zhejiang, 310058, PR China.
authors.forename: Ningning
authors.initials: N
authors.lastname: Guo
authors.affiliation: Zhejiang University Institute of Preventive Veterinary Medicine, 388 Yuhangtang Road, Hangzhou, Zhejiang, 310058, PR China.|​ Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, 388 Yuhangtang Road, Hangzhou, Zhejiang, 310058, PR China.
authors.forename: Xiaoliang
authors.initials: X
authors.lastname: Li
authors.affiliation: ​ College of Animal Science and Technology, Zhejiang A&F University, 88 Huanbei Road, Lin'an, Zhejiang, 311300, PR China.
authors.forename: Houhui
authors.initials: H
authors.lastname: Song
authors.affiliation: Zhejiang University Institute of Preventive Veterinary Medicine, 388 Yuhangtang Road, Hangzhou, Zhejiang, 310058, PR China.|​ College of Animal Science and Technology, Zhejiang A&F University, 88 Huanbei Road, Lin'an, Zhejiang, 311300, PR China.|​ Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, 388 Yuhangtang Road, Hangzhou, Zhejiang, 310058, PR China.
authors.forename: Weihuan
authors.initials: W
authors.lastname: Fang
mesh_terms: D000818:Animals; D001422:Bacterial Adhesion; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004195:Disease Models, Animal; D004705:Endocytosis; D005260:Female; D006801:Humans; D008089:Listeria monocytogenes / Q000235:genetics / Q000472:pathogenicity*; D008088:Listeriosis / Q000382:microbiology* / Q000473:pathology*; D008813:Mice, Inbred ICR; D010454:Peptide Termination Factors / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation; D037521:Virulence Factors / Q000096:biosynthesis*
chemical_list: D001426:Bacterial Proteins; D010454:Peptide Termination Factors; C545356:PrfA protein, Listeria monocytogenes; D037521:Virulence Factors