Role of the stress-responsive two-component system CpxAR in regulating fimbriae expression in Klebsiella pneumoniae CG43.

BACKGROUND: CpxAR is a two-component system that allows bacteria to reorganize envelope structures in response to extracellular stimuli. CpxAR negatively affects type 1 fimbriae expression in Klebsiella pneumoniae CG43, a hypervirulent strain. The involvement of CpxAR in the regulation of type 3 fimbriae expression was investigated. METHODS: cpxAR, cpxA, and cpxR gene-specific deletion mutants were generated. The deletion effects on the expression of type 1 and type 3 fimbriae were analyzed via measuring the promoter activity, mannose sensitive yeast agglutination activity, biofilm formation, and the production of the major pilins FimA and MrkA respectively. RNA sequencing analysis of CG43S3, ΔcpxAR, ΔcpxR and Δfur was employed to study the regulatory mechanism influencing the expression of type 3 fimbriae. RESULTS: Deletion of cpxAR increased type 1 and type 3 fimbrial expression. Comparative transcriptomic analysis showed that the expression of oxidative stress-responsive enzymes, type 1 and type 3 fimbriae, and iron acquisition and homeostasis control systems were differentially affected by cpxAR or cpxR deletion. Subsequent analysis revealed that the small RNA RyhB negatively affects the expression of type 3 fimbriae, while CpxAR positively controls ryhB expression. Finally, the site-directed mutation of the predicted interacting sequences of RyhB with the mRNA of MrkA attenuated the RyhB repression of type 3 fimbriae. CONCLUSION: CpxAR negatively regulates the expression of type 3 fimbriae by modulating cellular iron levels thereafter activating the expression of RyhB. The activated RyhB represses the expression of type 3 fimbriae by base-pairing binding to the 5'region of mrkA mRNA.

The role of urease in the acid stress response and fimbriae expression in Klebsiella pneumoniae CG43.

BACKGROUND/PURPOSE: Two urease operons were identified in Klebsiella pneumoniae CG43, ure-1 and ure-2. This study investigates whether a differential regulation of the expression of ure-1 and ure-2 exists and how urease activity influences the acid stress response and expression of type 1 and type 3 fimbriae. METHODS: The ureA1 and ureA2 gene specific deletion mutants were constructed. Promoter activity was assessed using a LacZ reporter system. The sensitivity to acid stress was determined by assessing the survival after pH 2.5 treatment. The influence on type 1 and type 3 fimbriae expression was assessed using western blotting and mannose-sensitive yeast agglutination and biofilm formation assay, respectively. RESULTS: Bacterial growth analysis in mM9-U or modified Stuart broth revealed that ure-1 was the principal urease system, and ure-2 had a negative effect on ure-1 activity. Deletion of the fur or nac gene had no apparent effect on the activity of Pure1, Pure2-1, and Pure2-2. The Pure2-2 activity was enhanced by deletion of the hns gene. ureA1 deletion increased acid stress sensitivity, whereas the deleting effect of ureA2 was notable without hns. Deletion of ureA1 or ureA2 significantly induced the expression of type 1 fimbriae but decreased MrkA production and biofilm formation. CONCLUSION: ure-1 is the primary expression system in K. pneumoniae CG43, while ure-2 is active in the absence of hns. Impairment of urease activity increases the sensitivity to acid stress, and the accumulation of urea induces the expression of type 1 fimbriae but represses type 3 fimbriae expression.

Enterovirus 71 induces mitochondrial reactive oxygen species generation that is required for efficient replication.

Redox homeostasis is an important host factor determining the outcome of infectious disease. Enterovirus 71 (EV71) infection has become an important endemic disease in Southeast Asia and China. We have previously shown that oxidative stress promotes viral replication, and progeny virus induces oxidative stress in host cells. The detailed mechanism for reactive oxygen species (ROS) generation in infected cells remains elusive. In the current study, we demonstrate that mitochondria were a major ROS source in EV71-infected cells. Mitochondria in productively infected cells underwent morphologic changes and exhibited functional anomalies, such as a decrease in mitochondrial electrochemical potential ΔΨ(m) and an increase in oligomycin-insensitive oxygen consumption. Respiratory control ratio of mitochondria from infected cells was significantly lower than that of normal cells. The total adenine nucleotide pool and ATP content of EV71-infected cells significantly diminished. However, there appeared to be a compensatory increase in mitochondrial mass. Treatment with mito-TEMPO reduced eIF2α phosphorylation and viral replication, suggesting that mitochondrial ROS act to promote viral replication. It is plausible that EV71 infection induces mitochondrial ROS generation, which is essential to viral replication, at the sacrifice of efficient energy production, and that infected cells up-regulate biogenesis of mitochondria to compensate for their functional defect.

MicroRNA-mediated somatic cell reprogramming.

Since the first report of induced pluripotent stem cells (iPSCs) using somatic cell nuclear transfer (SCNT), much focus has been placed on iPSCs due to their great therapeutic potential for diseases such as abnormal development, degenerative disorders, and even cancers. Subsequently, Takahashi and Yamanaka took a novel approach by using four defined transcription factors to generate iPSCs in mice and human fibroblast cells. Scientists have since been trying to refine or develop better approaches to reprogramming, either by using different combinations of transcription factors or delivery methods. However, recent reports showed that the microRNA expression pattern plays a crucial role in somatic cell reprogramming and ectopic introduction of embryonic stem cell-specific microRNAs revert cells back to an ESC-like state, although, the exact mechanism underlying this effect remains unclear. This review describes recent work that has focused on microRNA-mediated approaches to somatic cell reprogramming as well as some of the pros and cons to these approaches and a possible mechanism of action. Based on the pivotal role of microRNAs in embryogenesis and somatic cell reprogramming, studies in this area must continue in order to gain a better understanding of the role of microRNAs in stem cells regulation and activity.

Identify intronic microRNA with bioinformatics.

MicroRNAs (miRNAs) are single-strand nonprotein coding RAN with 18 to 25-nucleotides long. With complementary sequence to target messenger RNA (mRNA), miRNA regulates mRNA degradation and protein translation. miRNAs have been identified in various organisms ranging from virus to human. Increasing evidence indicates that mammalian gene regulation has multiple layers and the availability of mRNA is not the sole regulation mechanism. The evolutionally conserved miRNA may be a primary regulation mechanism of gene expression. Its role in directing embryo development and stem cell differentiation should not be underestimated.Due to the small size of miRNA, identifying it with experimental approach (e.g., direct cloning) is difficult. The cell type and developmental-specific expression of miRNA make the experimental approach even more difficult. Consequently, bioinformatics approaches have been developed to identify novel miRNA. In human miRNA study, many studies search for the mostly complete human genomic sequence. Here, we report a rapid bioinformatics approach to mine miRNA from gene specific introns. Intronic miRNA may directly regulate the expression of its target genes during development. The reported bioinformatics approach not only identifies the potential miRNA, but also provides the intron location of these miRNA like sequence. This information is critical for studying the gene-gene interaction via miRNA, and facilitates the study of miRNA in gene expression regulation.

MiRNA targets of prostate cancer.

Prostate cancer (PC) is the most prevalent strain of cancer in men, but it is often slow-acting or undetected. Common diagnostic tools for PC include prostate biopsy and consequent analysis by the Gleason scoring of the tissue samples, as well as tests for the presence and levels of prostate-specific antigens. Common treatments for androgen-dependent PC include prostatectomy or irradiation, which can be invasive and significantly lower the patient's quality of life. Alternative treatments exist, such as androgen ablation therapy, which, though effective, causes relapse into androgen-independent PC, which is far more invasive and likely to metastasize to other parts of the body. MicroRNAs (miRNA) are short nucleotide sequences (between 19 and 25 nucleotides long) that bind to various targeted messenger RNA (mRNA) sequences post-transcriptionally through complementary binding and control gene expression, often through silencing or leading to the degradation of targeted mRNA. Studies have shown that miRNAs are expressed abnormally in various cancers, suggesting that they play a pivotal role in cancer development and progression. Some miRNAs are oncogenes that incite cancerous growth, while others are involved in tumor suppression and cell cycle controls. MiRNA expression also differs in various types of cancers. Studies of PC-specific miRNAs show potential for their utilization in the prevention, diagnosis, and treatment of PC to more effectively target tumor growth and provide patients with better therapeutic options.

Advances in microRNA-mediated reprogramming technology.

The use of somatic cells to generate induced-pluripotent stem cells (iPSCs), which have gene characteristic resembling those of human embryonic stem cells (hESCs), has opened up a new avenue to produce patient-specific stem cells for regenerative medicine. MicroRNAs (miRNAs) have gained much attention over the past few years due to their pivotal role in many biological activites, including metabolism, host immunity, and cancer. Soon after the discovery of embryonic-stem-cell- (ESC-) specific miRNAs, researchers began to investigate their functions in embryonic development and differentiation, as well as their potential roles in somatic cell reprogramming (SCR). Several approaches for ESC-specific miRNA-mediated reprogramming have been developed using cancer and somatic cells to generate ESC-like cells with similarity to iPSCs and/or hESCs. However, the use of virus-integration to introduce reprogramming factors limits future clinical applications. This paper discusses the possible underlying mechanism for miRNA-mediated somatic cell reprogramming and the approaches used by different groups to induce iPSCs with miRNAs.

A novel role of miR-302/367 in reprogramming.

Ever since the technique of coaxing ordinary skin cells into becoming pluripotent stem cells (iPSCs) has been developed, which have the potential to become any cell or tissue in the body, efforts were made to improve the approach because some major challenges. Increasing evidence suggests that several microRNAs (miRNAs) are involved in early embryonic development and embryonic stem cell formation, known as embryonic stem cell (ESC)-specific miRNAs, particularly the miR-302 family. We summarized here a novel approach to generate iPSCs by using miR-302 and its related miRNAs such as miR-367. The development of this miR-302/367-mediated iPSC (termed mirPSC) may provide tools to deal with the obstacles facing some current iPSC reprogramming methods. The mechanism by which miR-302/367 induce iPSC reprogramming is proposed.