Daqing Gao

Adherent-invasive E. coli (AIEC) is closely related to inflammatory bowel disease (IBD). However, its pathogenic mechanism has not yet been fully elucidated. Using a BLASTP search, we discovered that the amino acid sequence of a putative protein (UFP37798.1) in the AIEC LF82 strain is highly homologous to some regulators in the SlyA family. We named it EruA. We displayed the secondary structures of EruA using bioinformatics, overexpressed the His₆-tagged EruA protein using SDS-PAGE, and dissected the genetic organization of the eruA chromosomal region using 5′RACE. We constructed an eruA deletion mutant (ΔeruA) and a complementary strain (CΔeruA) of the LF82 strain. The transcriptomes of wild-type (WT) and ΔeruA bacteria were compared using RNA sequencing and qRT-PCR, thereby identifying 32 differentially expressed genes (DEGs). Based on YASARA software and EMSA analysis, EruA directly binds to the consensus sequences (P_fimA and P_tnaB) in the promoter region of the fimA and tnaB genes from these DEGs. By using a super-resolution confocal microscope (SCM), counting CFUs of colonies on plates, indole quantification, and crystal violet staining of biofilms adhered to tubes or 96-well plates, we found that EruA activates the fimA to promote bacterial adhesion to intestinal epithelial cells and activates the tnaB to enhance bacterial indole production and biofilm formation. Moreover, EruA helps AIEC resist environmental stress and enhances bacterial survival within macrophages as well as loading in mouse tissues. Notably, EruA promotes AIEC colonization in the colons of mice and exacerbates intestinal inflammation caused by bacterial infection in mice with DSS-induced inflammatory colitis, manifested by weight loss, colon length shortening, and pathological changes in colon tissues. Therefore, EruA plays a key role in the pathogenicity of AIEC.

Inflammatory bowel disease (IBD) involves the complex interplay among the mucosal barrier, microbiota, immunity and genetic factors. There are currently no satisfactory treatments for IBD. Administration of the probiotic Bacillus licheniformis (Bl) can improves colitis by regulating the gut microbiota. The phytohormone abscisic acid (ABA) treatment has favorable effects on immunity, as well as on inflammatory diseases like colitis. We hypothesized that the expression of an additional cyp gene by the Bl to increase its ABA production would enhance its effects. In this study, we found that a Bl-cyp strain overexpressing the cyp gene secreted more ABA into its supernatant than either the parental Bl stain or a Bl-pET82a strain expressing only a vector pET82a when these bacteria were grown in Nfb medium for 48 h. The prophylactic administration of the Bl-cyp strain culture more effectively attenuated dextran sodium sulfate (DSS)-induced colitis in mice compared to the Bl and Bl-pET28a strains. These findings were associated with significantly reduced epithelial barrier damage, as well as increased number of goblet cells and expression levels of occludin gene in the colonic epithelial layer, and decreased serum LPS levels in the Bl-cyp group. In addition, the administration of Bl-cyp strain effectively regulated the disordered gut microbiota by improving their diversity, richness and compositions more than the Bl or Bl-pET82a strain, including the ratio of Bacteroidota: Bacillota. It also inhibited the excessive growth of opportunistic pathogen Escherichia just like the Bl or Bl-pET82a strain. Moreover, the preventive administration of the Bl-cyp strain to mice following DSS-induced colitis enhanced the proportion of Treg cells and suppressed the proportion of Th17 cells in mesenteric lymph nodes (MLNs), decreased the levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-22, and increased the level of anti-inflammatory IL-10 in colon tissues, similar to treatment with a high concentration of the ABA standard (ABA-H). Notably, the treatment with the Bl-cyp strain more effectively regulated the disordered microbiota than the ABA-H. The administration of the Bl-cyp strain may provide a novel preventive approach for IBD, and may exert its effects by modulating the gut microbiota and host’s immune status.

Introduction: The targeted delivery of anti-cancer drugs to tumor tissue has been recognized as a promising strategy to increase their therapeutic efficacy and reduce side effects. Mesoporous silica-coated superparamagnetic Fe3O4 nanoparticles (NH2-MSNs), a kind of nanocarrier, can passively enter tumor tissues to enhance the permeability and retention of drugs. However, NH2-MSNs do not specifically bind to cancer cells. This drawback encouraged us to develop a more efficient nanocarrier for cancer therapy. Methods: Herein, we describe the development of an effective nanocarrier based on NH2-MSNs, which were modified with hyaluronic acid on their surface (HA-MSNs) and loaded with doxorubicin (DOX). We have successfully fabricated uniform spherical HA-MSNs nanocarriers. The targeting ability of this delivery system was evaluated through specific uptake by cells and IVIS imaging. Results: DOX-HA-MSNs nanocarriers displayed more dramatic cytotoxic activity against 4T1 breast cancer cells compared to GES-1 gastric mucosa cells. In vivo results revealed that once DOX-HA-MSNs nanocarriers are exposed to an external magnetic field, they could be rapidly attracted to the magnet and effectively cross the cytoplasmic membrane via CD44 receptor-mediated transcytosis. This allows them to access the cancer cell cytoplasm and release DOX based on changes in the physiological environment. Both in vitro and in vivo results demonstrated that the HA-MSNs nanocarriers provided better therapeutic efficacy. Conclusion: The HA-MSNs nanocarriers represent an effective new paradigm to treat cancers due to active targeting to the tumor cells. Moreover, the specific uptake by the tumor effectively protects normal tissues to reduce off-target side effects. The reported findings support further investigation of HA-MSNs for cancer therapy.

Edwardsiella tarda is distributed widely in a variety of hosts. Eha is its virulence regulator just found. In order to explore the mechanism of its regulation, we investigated the survival rates of wild type ET13, its eha mutant and complemented strains in RAW264.7 macrophages under light microscopic observation as well as by counting bacterial CFUs on the plates. All kinds of the strains could live within macrophage, however, the intracellular numbers of wild type were significantly higher than the mutant when the incubation time extended 4 h or 6h ( P < 0.05). Furthermore, more ROS was produced by the mutant-infected cells, indicating that Eha may enhance the ET13's capacity to detoxify ROS. Consistently, we found that the mutant exhibited more sensitivity and less survival ability to H2O2 by disk inhibitory assay and H2O2 treatment. We further demonstrated that the bacterial antioxidant enzymes SodC and KatG were regulated by Eha with qRT-PCR and β -Galactosidase assay. Collectively, our data show Eha is required for E. tarda toresist the oxidative stress from macrophages.