Effects of ClpP protease on biofilm formation of Enterococcus faecalis

Abstract Enterococcus faecalis (E. faecalis), one of the main pathogens responsible for refractory periapical periodontitis and nosocomial infections, exhibits markedly higher pathogenicity in biofilms. Objectives Studies have shown that caseinolytic protease P (ClpP) is involved in biofilm formation. However, to date, few studies have investigated the role of ClpP in the survival of E. faecalis, and in enhancing biofilm formation. Therefore, we investigated the role of ClpP in the formation of E. faecalis biofilms. Methodology In our study, we used homologous recombination to construct clpP deleted and clpP complement strains of E. faecalis ATCC 29212. A viable colony counting method was used to analyze the growth patterns of E. faecalis. Crystal violet staining (CV) and confocal scanning laser microscopy (CLSM) were used to characterize biofilm mass formation and scanning electron microscopy (SEM) was used to observe the biofilm microstructure. Data was statistically analyzed via Student's t-test or one-way analysis of variance (ANOVA). Results The results exhibited altered growth patterns for the clpP deletion strains and depleted polysaccharide matrix, resulting in reduced biofilm formation capacity compared to the standard strains. Moreover, ClpP was observed to increase biofilm formation in E. faecalis. Conclusion Our study shows that ClpP can increase biofilm formation in E. faecalis and emphasizes the importance of ClpP as a potential target against E. faecalis.

Clinical and genetic analysis of an infant with 3-methylglutaconic aciduria type VII / 中华医学遗传学杂志

OBJECTIVE@#To analyze the clinical and genetic characteristics of an infant girl featuring comprehensive developmental backwardness.@*METHODS@#The patient was subjected to clinical examination, gas chromatography mass spectrometry and next-generation sequencing (NGS).@*RESULTS@#The child was insensitive to sound, could not turn over, raise head, laugh or recognize his mother. Laboratory tests were all normal, but metabolic analysis suggested 3-methylglutaconic aciduria due to elevated 3-methylglutaconic acid and 3-methylglutaric acid. NGS has detected two compound heterozygous CLPB variants in the child, namely c.1085G>A and c.1700A>C, which were respectively inherited from her father and mother. Bioinformatic analysis predicted both variants to be pathogenic. The patient was diagnosed with 3-methylglutaconic aciduria type VII (MGCA7).@*CONCLUSION@#The MGCA7 in the child was probably caused by CLPB gene variants. NGS has provided a powerful diagnostic tool for this rare disorder.

The role of ClpX in erythropoietic protoporphyria

ABSTRACT Hemoglobin is an essential biological component of human physiology and its production in red blood cells relies upon proper biosynthesis of heme and globin protein. Disruption in the synthesis of these precursors accounts for a number of human blood disorders found in patients. Mutations in genes encoding heme biosynthesis enzymes are associated with a broad class of metabolic disorders called porphyrias. In particular, one subtype - erythropoietic protoporphyria - is caused by the accumulation of protoporphyrin IX. Erythropoietic protoporphyria patients suffer from photosensitivity and a higher risk of liver failure, which is the principle cause of morbidity and mortality. Approximately 90% of these patients carry loss-of-function mutations in the enzyme ferrochelatase (FECH), while 5% of cases are associated with activating mutations in the C-terminus of ALAS2. Recent work has begun to uncover novel mechanisms of heme regulation that may account for the remaining 5% of cases with previously unknown genetic basis. One erythropoietic protoporphyria family has been identified with inherited mutations in the AAA+ protease ClpXP that regulates ALAS activity. In this review article, recent findings on the role of ClpXP as both an activating unfoldase and degrading protease and its impact on heme synthesis will be discussed. This review will also highlight the role of ClpX dysfunction in erythropoietic protoporphyria.

Quantitative proteomics analysis of ClpS-mediated rifampicin resistance in Mycobacterium / 生物工程学报

Adaptor protein ClpS is an essential regulator of prokaryotic ATP-dependent protease ClpAP, which delivers certain protein substrates with specific amino acid sequences to ClpAP for degradation. However, ClpS also functions as the inhibitor of the ClpAP-mediated protein degradation for other proteins. Here, we constructed the clpS-overexpression Mycobacterium smegmatis strain, and showed for the first time that overexpression of ClpS increased the resistance of M. smegmatis to rifampicin that is one of most widely used antibiotic drugs in treatment of tuberculosis. Using quantitative proteomic technology, we systematically analyzed effects of ClpS overexpression on changes in M. smegmatis proteome, and proposed that the increased rifampicin resistance was caused by ClpS-regulated drug sedimentation and drug metabolism. Our results indicate that the changes in degradation related proteins enhanced drug resistance and quantitative proteomic analysis is an important tool for understanding molecular mechanisms responsible for bacteria drug resistance.