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Nucleolar c-Myc recruitment by a Vibrio T3SS effector promotes host cell proliferation and bacterial virulence.

ABSTRACT: Pathogen type 3 secretion systems (T3SS) manipulate host cell pathways by directly delivering effector proteins into host cells. In Vibrio parahaemolyticus, the leading cause of bacterial seafood-borne diarrheal disease, we showed that a T3SS effector, VgpA, localizes to the host cell nucleolus where it binds Epstein-Barr virus nuclear antigen 1-binding protein 2 (EBP2). An amino acid substitution in VgpA (VgpA^L10A ) did not alter its translocation to the nucleus but abolished the effector's capacity to interact with EBP2. VgpA-EBP2 interaction led to the re-localization of c-Myc to the nucleolus and increased cellular rRNA expression and proliferation of cultured cells. The VgpA-EBP2 interaction elevated EBP2's affinity for c-Myc and prolonged the oncoprotein's half-life. Studies in infant rabbits demonstrated that VgpA is translocated into intestinal epithelial cells, where it interacts with EBP2 and leads to nucleolar re-localization of c-Myc. Moreover, the in vivo VgpA-EBP2 interaction during infection led to proliferation of intestinal cells and heightened V. parahaemolyticus' colonization and virulence. These observations suggest that direct effector stimulation of a c-Myc controlled host cell growth program can contribute to pathogenesis.

SUBMITTER: Hu M

PROVIDER: S-EPMC7809790 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Riemerella anatipestifer is a major pathogenic agent of duck septicemic and exudative diseases. Recent studies have shown that the R. anatipestifer type IX secretion system (T9SS) is a crucial factor in bacterial virulence. The AS87_RS04190 protein was obviously missing from the secreted proteins of the T9SS mutant strain Yb2ΔgldM. A bioinformatic analysis indicated that the AS87_RS04190 protein contains a T9SS C-terminal domain sequence and encodes a putative subtilisin-like serine protease (SspA). To determine the role of the putative SspA protein in R. anatipestifer pathogenesis and proteolysis, we constructed two strains with an sspA mutation and complementation, respectively, and determined their median lethal doses, their bacterial loads in infected duck blood, and their adherence to and invasion of cells. Our results demonstrate that the SspA protein functions in bacterial virulence. It is also associated with the bacterial protease activity and has a conserved catalytic triad structure (Asp126, His158, and Ser410), which is necessary for protein function. The optimal reactive pH and temperature were determined to be 7.0 and 50°C, respectively, and Km and Vmax were determined to be 10.15 mM and 246.96 U/mg, respectively. The enzymatic activity of SspA is activated by Ca2+, Mg2+, and Mn2+ and inhibited by Cu2+ and EDTA. SspA degrades gelatin, fibrinogen, and bacitracin LL-37. These results demonstrate that SspA is an effector protein of T9SS and functions in R. anatipestifer virulence and its proteolysis of gelatin, fibrinogen, and bacitracin LL-37. IMPORTANCE In recent years, Riemerella anatipestifer T9SS has been reported to act as a virulence factor. However, the functions of the proteins secreted by R. anatipestifer T9SS are not entirely clear. In this study, a secreted subtilisin-like serine protease SspA was shown to be associated with R. anatipestifer virulence, host complement evasion, and degradation of gelatin, fibrinogen, and LL-37. The enzymatic activity of recombinant SspA was determined, and its Km and Vmax were 10.15 mM and 246.96 U/mg, respectively. Three conserved sites (Asp126, His158, and Ser410) are necessary for the protein's function. The median lethal dose of the sspA-deleted mutant strain was reduced >10,000-fold, indicating that SspA is an important virulence factor. In summary, we demonstrate that the R. anatipestifer AS87_RS04190 gene encodes an important T9SS effector, SspA, which plays an important role in bacterial virulence.

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Nucleolar c-Myc recruitment by a Vibrio T3SS effector promotes host cell proliferation and bacterial virulence.

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