A critical role of outer membrane vesicles in antibiotic resistance in carbapenem-resistant Klebsiella pneumoniae.

BACKGROUND: This study aimed to illustrate the status of carbapenem-resistant Enterobacterales (CRE) infections in a Chinese tertiary hospital and to investigate the role of outer membrane vesicles (OMVs) in antibiotic resistance in carbapenem-resistant Klebsiella pneumoniae (CRKP). METHODS: The data of CRE infections was collected from laboratory records, and the CRE isolates from two distinct periods (2015/07 to 2017/07 and 2020/04 to 2021/04) were enrolled to detect the carbapenemase genes by polymerase chain reaction (PCR). Multilocus sequence typing (MLST) was used to analyze the molecular characterization of CRKP. The conjugation assay was performed to verify the transmission of the antibiotic resistance plasmid. The OMVs of CRKP were isolated with a method combining an electrophoretic technique with a 300 kDa cut-off dialysis bag. The protein components in CRKP OMVs were analyzed by liquid chromatography tandem-mass spectrometry (LC-MS/MS), and the meropenem-hydrolyzing bioactivity of KPC in CRKP OMVs was determined with different treatments in vitro. RESULTS: A total of 178 CRE isolates, including 100 isolates from 2015/07 to 2017/07 and 78 isolates from 2020/04 to 2021/04, were collected for the detection of carbapenemase genes. We found that the carbapenemase gene blaKPC was the most prevalent, followed by blaNDM. By MLST, we found that sequence type (ST) 11 CRKP (96.1%) was the leading type during 2015/07 to 2017/07 and that the ST15 CRKP increased to 46.2% in the late period of 2020/04 to 2021/04. The diameters of Klebsiella pneumoniae OMVs ranged from 100 to 200 nm, and by proteomics analysis the most proteins from OMVs belonged to the "enzyme" group. The KPC enzyme was found in the OMVs from CRKP, and the OMVs could protect inside KPC from proteinase K digestion. Moreover, the KPC enzymes within OMVs, which could be released after Triton X-100 treatment, could hydrolyze meropenem. CONCLUSIONS: CRE has increasingly caused infections in hospitals, and blaKPC-positive CRKP infections have constituted a major proportion of infections in the past decade. The OMVs play a critical role in antibiotic resistance in CRKP.

LIM domain-containing protein Ajuba inhibits chemotherapy-induced apoptosis by negatively regulating p53 stability in colorectal cancer cells.

LIM protein-domain containing protein Ajuba (encoded by AJUBA) functions as a scaffold protein to regulate protein-protein interactions, signalling transduction and genes transcription. AJUBA expression is higher in colorectal cancer (CRC) tissues than normal tissues, but its specific molecular function in CRC progression is still not very clear. Here, we found that, in CRC cancer cell lines, overexpression of AJUBA decreased p53 levels, whereas knock-down of AJUBA significantly increased p53 levels. Although the presence of Ajuba did not influence p53 transcription, it formed a complex with p53 and MDM2 to promote the degradation of p53. AJUBA overexpression reduced the sensitivity of cancer cells to chemotherapeutic drugs and vice versa. In addition, chemotherapeutic drugs significantly induced AJUBA expression, which was largely dependent on the presence of p53. Therefore, Ajuba formed a negative feedback loop to regulate p53 expression and activity. In conclusion, as a novel p53-negative regulator, Ajuba inhibits the apoptosis of CRC cells induced by chemotherapeutic drugs and it may be a new therapeutic target for CRC treatment.

The LIM protein Ajuba recruits DBC1 and CBP/p300 to acetylate ERα and enhances ERα target gene expression in breast cancer cells.

Estrogen/ERα signaling is critical for breast cancer progression and therapeutic treatments. Thus, identifying new regulators of this pathway will help to develop new therapeutics to overcome chemotherapy resistance of the breast cancer cells. Here, we report Ajuba directly interacts with ERα to potentiate ERα target gene expression, and biologically Ajuba promotes breast cancer cell growth and contributes to tamoxifen resistance of these cells. Ajuba constitutively binds the DBD and AF2 regions of ERα, and these interactions can be markedly enhanced by estrogen treatment. Mechanistically, Ajuba recruits DBC1 and CBP/p300 and forms a ternary complex to co-activate ERα transcriptional activity and concomitantly enhances ERα acetylation. Moreover, components of this complex can be found at endogenous promoters containing functional ERα responsive elements. Taken together, these data demonstrate that Ajuba functions as a novel co-activator of ERα and that Ajuba/DBC1/CBP/p300 ternary complex may be a new target for developing therapeutics to treat breast cancer.