Xiaofang Zhu

The flagella of F18ab Escherichia coli is a virulence factor that contributes to infection in a IPEC-J2 cell model in vitro

Bacterial flagella contribute to pathogen virulence; however, the role of flagella in the pathogenesis of F18ab E. coli-mediated swine edema disease (ED) is not currently known. We therefore evaluated the role of flagella in F18ab E. coli adhesion, invasion, biofilm formation, and IL-8 production using an in vitro cell infection model approach with gene-deletion mutant and complemented bacterial strains. We demonstrated that the flagellin-deficient fliC mutant had a marked decrease in the ability to adhere to and invade porcine epithelial IPEC-J2 cells. Surprisingly, there was no difference in adhesion between the F18 fimbriae-deficient ΔfedA mutant and its parent strain. In addition, both the ΔfedA and double ΔfliCΔfedA mutants exhibited an increased ability to invade IPEC-J2 cells compared to the wild-type strain, although this may be due to increased expression of other adhesins following the loss of F18ab fimbriae and flagella. Compared to the wild-type strain, the ΔfliC mutant showed significantly reduced ability to form biofilm, whereas the ΔfedA mutant increased biofilm formation. Although ΔfliC, ΔfedA, and ΔfliCΔfedA mutants had a reduced ability to stimulate IL-8 production from infected Caco-2 cells, the ΔfliC mutant impaired this ability to a greater extent than the ΔfedA mutant. The results from this study clearly demonstrate that flagella are required for efficient F18ab E. coli adhesion, invasion, biofilm formation, and IL-8 production in vitro.

Flagella from F18+Escherichia coli play a role in adhesion to pig epithelial cell lines.

F18 fimbriae and toxins produced by F18 fimbriae-carrying Escherichia coli (E. coli) strains are known virulence factors responsible for post-weaning diarrhea (PWD) and edema disease (ED). In this study, we showed that fliC isogenic mutants constructed in two reference wild-type F18 fimbriae (F18+) E. coli were markedly impaired in adherence in vitro cell models (p < 0.05). Flagella purified from F18+E. coli could directly bind to cultured piglet epithelial cells and block adherence of F18+E. coli to cells when pre-incubated. In addition, the F18+E. coli fliC deletion mutants up-regulated the expression of type I fimbriae produced by F18+E. coli strains. These results demonstrated that expression of flagella is essential for the adherence of F18+E. coli in vitro.

Both flagella and F4 fimbriae from F4ac+ enterotoxigenic Escherichia coli contribute to attachment to IPEC-J2 cells in vitro.

The role of flagella in the pathogenesis of F4ac+ Enterotoxigenic Escherichia coli (ETEC) mediated neonatal and post-weaning diarrhea (PWD) is not currently understood. We targeted the reference C83902 ETEC strain (O8:H19:F4ac+ LT+ STa+ STb+), to construct isogenic mutants in the fliC (encoding the major flagellin protein), motA (encoding the flagella motor), and faeG (encoding the major subunit of F4 fimbriae) genes. Both the ΔfliC and ΔfaeG mutants had a reduced ability to adhere to porcine intestinal epithelial IPEC-J2 cells. F4 fimbriae expression was significantly down-regulated after deleting fliC, which revealed that co-regulation exists between flagella and F4 fimbriae. However, there was no difference in adhesion between the ΔmotA mutant and its parent strain. These data demonstrate that both flagella and F4 fimbriae are required for efficient F4ac+ ETEC adhesion in vitro.

Participation of Gab1 and Gab2 in IL-22-mediated keratinocyte proliferation, migration, and differentiation.

Interleukin-22 (IL-22) is one of the key mediators of keratinocyte alterations in psoriasis. IL-22 inhibits keratinocyte differentiation and induces the migration of human keratinocytes. Grb2-associated binder 1 (Gab1) has been shown to mediate epidermal growth factor-induced epidermal growth and differentiation via interaction with the Src homology-2-containing protein-tyrosine phosphatase (Shp2). In this investigation, we explore the role of Gab1 and Gab2 in IL-22-mediated keratinocyte activities. We show that both Gab1 and Gab2 were tyrosine phosphorylated in IL-22-stimulated HaCaT cells and human primary epidermal keratinocytes and contributed to the activation of Extracellular signal regulated kinase 1/2 (Erk1/2) through interaction with Shp2. We further demonstrate that HaCaT cells infected with adenoviruses expressing Shp2-binding-defective Gab1/2 mutants exhibited decreased cell proliferation and migration, as well as increased differentiation. Moreover, similar results were observed in HaCaT cells infected with adenovirus-based small interfering RNAs targeting Gab1 and/or Gab2. Altogether, these data underscore the critical roles of Gab1 and Gab2 in IL-22-mediated HaCaT cell proliferation, migration, and differentiation.

Biphasic activation of PI3K/Akt and MAPK/Erk1/2 signaling pathways in bovine herpesvirus type 1 infection of MDBK cells.

Many viruses have been known to control key cellular signaling pathways to facilitate the virus infection. The possible involvement of signaling pathways in bovine herpesvirus type 1 (BoHV-1) infection is unknown. This study indicated that infection of MDBK cells with BoHV-1 induced an early-stage transient and a late-stage sustained activation of both phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen activated protein kinases/extracellular signal-regulated kinase 1/2 (MAPK/Erk1/2) signaling pathways. Analysis with the stimulation of UV-irradiated virus indicated that the virus binding and/or entry process was enough to trigger the early phase activations, while the late phase activations were viral protein expression dependent. Biphasic activation of both pathways was suppressed by the selective inhibitor, Ly294002 for PI3K and U0126 for MAPK kinase (MEK1/2), respectively. Furthermore, treatment of MDBK cells with Ly294002 caused a 1.5-log reduction in virus titer, while U0126 had little effect on the virus production. In addition, the inhibition effect of Ly294002 mainly occurred at the post-entry stage of the virus replication cycle. This revealed for the first time that BoHV-1 actively induced both PI3K/Akt and MAPK/Erk1/2 signaling pathways, and the activation of PI3K was important for fully efficient replication, especially for the post-entry stage.

Quorum-sensing gene luxS regulates flagella expression and Shiga-like toxin production in F18ab Escherichia coli

To investigate the effect of the luxS gene on the expression of virulence factors in Shiga-like toxin producing and verotoxin-producing Escherichia coli, the luxS gene from E. coli 107/86 (wild type, O139:H1:F18ab, Stx2e) was deleted. The successful deletion of luxS was confirmed by bioluminescence assays. The luxS deletion mutant exhibited changed flagella-related phenotypes, like impaired expression of flagella, decreased flagella motility, reduced biofilm formation, and reduced ability to induce pro-immunity response in host cells, which were restored after complementation with the intact luxS gene. The mutant strain also displayed attenuated production of Stx2e. This study provides new information to the crucial function of luxS in regulating Shiga-like toxin producing E. coli virulence.

Contribution of flagellin subunit FliC to piglet epithelial cells invasion by F18ab E. coli.

Flagellar structures contribute to the virulence of multiple gastrointestinal pathogens either as the effectors of motility, as adhesins, or as a secretion apparatus for virulence factors. Escherichia coli F18ab variant strains are associated with edema disease (ED) in pig industries worldwide. These strains use flagella to increase the efficiency of epithelial cell invasion. In this study, we aimed to elucidate the mechanism by which flagella contribute to F18ab E. coli invasion. To explore the role of flagella in the invasion process, we performed invasion assays with either flagellated and motile, flagellated but non-motile, or non-flagellated non-motile bacteria. We observed that flagellated but non-motile bacteria invade piglet epithelial cells even more efficiently than the parent wild-type (WT) strain in vitro. By contrast, the non-flagellated bacteria have significantly reduced invasion as compared with the parent strain. These results demonstrate that flagella function mainly as adhesins to enhance the ability of F18ab E. coli to target piglet epithelial cells.

Identification of 987P Protein Receptors for Enterotoxigenic Escherichia coli

The 987P fimbriae of enterotoxigenic Escherichia coli (ETEC) mediates adhesive interactions with brush border vesicle (BBV) of the intestinal epithelial cells from the neonatal piglets. By adhering to intestinal epithelial cells, producing localized multiplication, the 987P ETEC can progress to mucosal surface colonization and concomitant effective enterotoxin delivery. To identify the receptors for the 987P, BBV proteins from piglet intestinal villous epithelial cells were separated by SDS-PAGE and analyzed by Ligand blot, protein bands with a set of 32-35 kD recognized by the 987P fimbriae were subjected to in gel proteolysis with trypsin. The tryptic fragments were separated by microbore reversed phase HPLC(RP-HPLC), samples shown to contain one major peak by MALDI-MS were submitted to Edman sequencing, three peptides were sequenced successfully and the all of three peptides matched the sequences of human or porcine histone H1 proteins. Porcine histone H1 proteins isolated from both piglet intestinal epithelial cells and BBV demonstrated the same SDS-PAGE migration pattern and 987P-binding properties as the 987P-specific protein receptors from piglet intestinal brush border did. The above results indicated that the 987P protein receptors are piglet BBV-derived Histone H1 proteins.