Eimeria acervulina Microneme Protein 3 Inhibits Apoptosis of the Chicken Duodenal Epithelial Cell by Targeting the Casitas B-Lineage Lymphoma Protein.

Eimeria acervulina (E. acervulina) causes coccidiosis in poultry which persists as economic pain worldwide. Most damage to the intestinal mucosa results from apoptosis of the infected intestinal epithelial cells. The Microneme protein 3 (MIC3) protein is a key virulence factor in some parasites involved in host cell apoptosis inhibition. Here, we studied whether and how MIC3 affects the apoptosis in E. acervulina infected chicken duodenal epithelial cells. Through flow cytometry (FCM), we found that the presence of merozoites and the overexpression of MIC3 significantly decreased apoptosis and the activity of caspase-3 in chicken duodenal epithelial cells at 4, 6, and 8 h post merozoite infection (P < 0.01). Silencing the Casitas B-lineage lymphoma (CBL) protein, a host receptor for MIC3 with shRNA was shown to promote apoptosis in the chicken duodenal epithelial cells. The early apoptotic rate of host cells in the lentiviral-MIC3 group was significantly lower than that in the lentiviral-MIC3 + shRNA CBL group at 4 h after MIC3 expression (P < 0.01), and it was moderately decreased in the lentiviral-MIC3 + shRNA CBL group compared with that in the shRNA CBL group. Our data indicated that MIC3 inhibited early apoptosis of E. acervulina infected chicken duodenal epithelial cells by targeting host receptor-CBL protein. These findings unveiled one of the mechanisms of how intracellular parasites affect the apoptosis of infected host cells, which provided a deeper understanding of their pathogenesis.

Regulated delayed attenuation improves vaccine efficacy in preventing infection from avian pathogenic Escherichia coli O78 and Salmonella typhimurium.

Avian pathogenic Escherichia coli (APEC) O78 and Salmonella typhimurium (S. Typhimurium) are two leading bacterial pathogens that cause significant economic loss in the poultry industry. O-antigen is an important immunogen of these two bacteria to induce host protective immune responses during infection. To develop a bivalent vaccine against APEC O78 and S. Typhimurium, the attenuated Salmonella ST01 (Δasd ΔrfbP Δcrp) was genetically constructed to deliver APEC O78 O-antigen polysaccharide (OPS), which stably expresses OPS with asd+ balanced-lethal system in vitro and in vivo. After oral immunization, the recombinant attenuated Salmonella vaccine (RASV) strain ST01 (pSS26-O78) provided insufficient protection against the APEC O78 challenge. Therefore, the regulated delayed attenuation strain ST02 (Δasd ΔrfbP ΔPcrp::TTaraC PBADcrp) was further constructed by regulating cyclic AMP receptor protein (crp) with araC PBAD cassette to better present the heterologous O-antigen to the host immune system. The innovative recombinant strain ST02 (pSS26-O78) stimulated robust antibody responses against APEC O78 and S. Typhimurium OPS, with serum titers over 1:800 for both IgG and IgA, thereby providing the complement-mediated bactericidal activity and stronger protection against APEC O78 and S. Typhimurium infection. Collectively, this study demonstrates a biologically-conjugated polysaccharide vaccine candidate that can enhance homologous protection against APEC O78 and S. Typhimurium.

Mycobacterial PPE13 activates inflammasome by interacting with the NATCH and LRR domains of NLRP3.

Pathogenic mycobacteria, such as Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium marinum, can trigger NLRP3 inflammasome activation leading to maturation and secretion of interleukin 1ß (IL-1ß). However, the mycobacterial factors involved in the activation of NLRP3 inflammasome are not fully understood. Here, we identified that the PPE family protein PPE13 was responsible for the induction of IL-1ß secretion in a NLRP3 inflammasome-dependent manner. We found that the recombinant Mycobacterium smegmatis expressing PPE13 activates NLRP3 inflammasome, thereby inducing caspase-1 cleavage and IL-1ß secretion in J774A.1, BMDMs, and THP-1 macrophages. To examine whether this inflammasome activation was triggered by PPE13 rather than components of M. smegmatis, PPE13 was introduced into the aforementioned macrophages by lentivirus as a delivery vector. Similarly, this led to the activation of NLRP3 inflammasome, indicating that PPE13 is a direct activator of NLRP3 cascade. We further demonstrated that the NLRP3 complex activated the inflammasome cascade, and the assembly of this complex was facilitated by PPE13 through interacting with the LRR and NATCH domains of NLRP3. Finally, we found that all PPE13 proteins isolated from M. tuberculosis, M. bovis, and M. marinum can activate NLRP3 inflammasome through binding to NLRP3, which requires C-terminal repetitive MPTR domain of PPE13. Thus, we, for the first time, revealed that PPE13 triggers the inflammasome-response by interacting with the MPTR domain of PPE13 and the LRR and NATCH domains of NLRP3. These findings provide a novel perspective on the function of PPE proteins in the immune system during mycobacteria invasion.

Listeriolysin O Pore-Forming Activity Is Required for ERK1/2 Phosphorylation During Listeria monocytogenes Infection.

Listeriolysin O (LLO) is a cholesterol-dependent cytolysin that mediates escape of L. monocytogenes from phagosomes and enables the bacteria to grow within the host. LLO is a versatile tool allowing Listeria to trigger several cellular responses. In this study, rapid phosphorylation of ERK1/2 on Caco-2 cells caused by Listeria infection was demonstrated to be highly dependent on LLO activity. The effect could be strongly induced by adding purified recombinant LLO alone and could be inhibited by exogenous cholesterol. Lack of the PEST sequence, known to tightly control cytotoxicity of LLO, did not affect ERK1/2 activation. However, the recombinant non-cytolytic LLOT515AL516A, with mutations in the cholesterol-binding motif, was unable to trigger this response. Recombinant LLON478AV479A, which lacks most of the cytolytic activity, also failed to activate ERK1/2 phosphorylation, and this effect could be rescued when the protein concentration reached a cytolytic level. Infection with an LLO-deficient mutant (Δhly) or the mutant complementing LLOT515AL516A abrogated the capacity of the bacteria to activate ERK1/2. However, infection with the Δhly mutant complementing LLON478AV479A, which retained partial pore-forming ability and could grow intracellularly, was capable of triggering ERK1/2 phosphorylation. Collectively, these data suggest that ERK1/2 activation by L. monocytogenes depends on the permeabilization activity of LLO and more importantly correlates with the cholesterol-binding motif of LLO.

An M29 Aminopeptidase from Listeria Monocytogenes Contributes to In Vitro Bacterial Growth but not to Intracellular Infection.

Aminopeptidases that catalyze the removal of N-terminal residues from polypeptides or proteins are crucial for physiological processes. Here, we explore the biological functions of an M29 family aminopeptidase II from Listeria monocytogenes (LmAmpII). We show that LmAmpII contains a conserved catalytic motif (EEHYHD) that is essential for its enzymatic activity and LmAmpII has a substrate preference for arginine and leucine. Studies on biological roles indicate that LmAmpII is required for in vitro growth in a chemically defined medium for optimal growth of L. monocytogenes but is not required for bacterial intracellular infection in epithelial cells and macrophages, as well as cell-to-cell spreading in fibroblasts. Moreover, LmAmpII is found as dispensable for bacterial pathogenicity in mice. Taken together, we conclude that LmAmpII, an M29 family aminopeptidase, can efficiently hydrolyze a wide range of substrates and is required for in vitro bacterial growth, which lays a foundation for in-depth investigations of aminopeptidases as potential targets to defend Listeria infection.

Fine mapping of a linear epitope on EDIII of Japanese encephalitis virus using a novel neutralizing monoclonal antibody.

The domain III (EDIII) of the envelope protein of Japanese encephalitis virus (JEV) is proposed to play an essential role in JEV replication and infection; it is involved in binding to host receptors and contains specific epitopes that elicit neutralizing antibodies. However, most previous studies have not provided detailed molecular information about the functional epitopes on JEV EDIII protein. In this study, we described a monoclonal antibody (mAb 2B4) we produced and characterized by IFA, PRNT, ELISA and Western blot analyses. The results showed that mAb 2B4 was specific to JEV EDIII protein and possessed high neutralization activity against JEV in vitro. Furthermore, we found that the motif, (394)HHWH(397), was the minimal unit of the linear epitope recognized by mAb 2B4 through screening a phage-displayed random 12-mer peptide library. Using sequence alignment analysis it was found that this motif was highly conserved among JEV strains and was present in West Nile Virus (WNV). Indeed, ELISA data showed that this epitope could be recognized by both JEV-positive swine serum and WNV-positive swine serum. Notably, this linear epitope was highly hydrophilic and was located within the terminal end of a ß-pleated sheet of EDIII. An analysis of the spatial conformation supported the possibility of inducing specific antibodies to this epitope. Taken together, we identified (394)HHWH(397) as an EDIII-specific linear epitope recognized by mAb 2B4, which would be beneficial for studying the pathogenic mechanism of JEV; and mAb 2B4 was also a potential diagnostic and therapeutic reagent.