To preliminarily understand the pathogenic mechanism of Mycoplasma genitalium (Mg) GroEL protein, we used bioinformatics tools to predict the structure and function of Mg GroEL protein and then constructed the recombinant plasmid pET-28a-GroEL. The protein expression was induced by 0.2 mmol/L IPTG, and the expressed protein was purified by Ni-iminodicitic acid (IDA) column affinity. Tohoku Hospital Pediatrics-1 (THP-1) cells were exposed to 2 μg/mL Mg rGroEL. The levels of interleukin (IL)-1β and tumor necrosis factor (TNF)-α in the cell supernatant were measured by ELISA, and that of IL-6 was measured by an automatic chemiluminescence instrument. The activation of the nuclear factor-kappa B (NF-κB) signaling pathway was visualized by immunofluorescence and Western blotting. The results showed that Mg GroEL was a stable hydrophilic protein composed of 543 amino acid residues, with the relative molecular mass of 58.44 kDa, an isoelectric point of 5.68, and a molecular formula of C₂₅₆₈H₄₃₀₀N₇₀₀O₈₂₅S₈. The secondary structure was mainly composed of α-helices and random coils. Mg GroEL contained 12 B-cell dominant epitopes and 10 T-cell dominant epitopes. It exhibited high homology with the GroEL proteins from Mycoplasma pneumoniae, M. agalactiae, M. arthritidis, M. hyopneumoniae, and M. bovis. Mg rGroEL activated the NF-κB signaling pathway and promoted the secretion of IL-1β, IL-6, and TNF-α in THP-1 cells. These results suggest that Mg GroEL exhibits substantial antigenicity and possesses the capability of triggering inflammation in host cells. This study establishes a theoretical basis for future investigations pertaining to the role and pathogenic mechanisms of Mg GroEL.
Mycoplasma genitalium/metabolism* ; Chaperonin 60/metabolism* ; Computational Biology ; Bacterial Proteins/genetics* ; Humans ; NF-kappa B/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Interleukin-1beta/genetics* ; Inflammation ; Interleukin-6/genetics* ; Recombinant Proteins/genetics* ; THP-1 Cells ; Signal Transduction ; Escherichia coli/metabolism*

Amino Acid Sequence ; Animals ; Bacterial Proteins ; genetics ; metabolism ; Bacterial Toxins ; genetics ; metabolism ; Base Sequence ; Community-Acquired Infections ; microbiology ; Gene Expression Regulation, Bacterial ; Humans ; Lung ; microbiology ; pathology ; Mycoplasma pneumoniae ; Pneumonia, Mycoplasma ; microbiology ; Respiratory Distress Syndrome, Adult ; microbiology

BACKGROUNDMycoplasma pneumoniae is a common pathogen that caused community-acquired pneumonia (CAP). P1 protein served as major adhesion and immunodominant protein in Mycoplasma pneumoniae, but little about P1 gene was learned and the relationship between P1 genotype and macrolide resistance has yet to be explored.

METHODSThe DNA sequence of the entire P1 gene from 35 strains isolated from clinical specimens collected in Beijing, China, in 2010 was determined. The resulting sequences were checked for known macrolide resistance mutations, such as A2063G, A2064G, C2617G in domain V of 23S rRNA. Antibiotic susceptibility test was done to further identify macrolide resistant strains.

RESULTSThirty-four clinical strains were type 1, and were identical to type 1 reference strain MP129. Only one clinical strain, MpYYM22, was type 2, and proved to be variant 2c. One synonymous point mutation in the P1 type 1 gene from two isolates was identified relative to the MP129 P1 sequence at nucleotide position (nt) 552 (C>A), while another two isolates had missense mutations at nt 2504 (G>A). This point mutation caused an amino acid change from glycine to glutamic acid. An AGT tri-nucleotide variable-number tandem repeat (VNTR), coding for serine and repeating 6-11 times, up to 15-16 times, was found in the region between the RepMP4 and RepMP2/3 elements in the 35 isolates examined. All 35 clinical strains, including MpYYM22, demonstrated macrolide resistance with the range of minimum inhibitory concentration (MIC) of erythromycin from 64 to 256 µg/ml, having an A2063G transition in domain V of the 23S rRNA gene.

CONCLUSIONSP1 type 1 was the dominant type of Mycoplasma pneumoniae in Beijing in 2010, although variant 2c strains were present. More samples are needed to determine whether there is a relationship between the P1 genotype and macrolide resistance, as the 35 strains examined did not allow a conclusive result. However, the AGT tri-nucleotide VNTR may be a more informative locus for multi-locus VNTR analysis.

Anti-Bacterial Agents ; pharmacology ; DNA, Bacterial ; Drug Resistance, Bacterial ; Genotype ; Humans ; Macrolides ; pharmacology ; Microbial Sensitivity Tests ; Mycoplasma pneumoniae ; drug effects ; genetics ; metabolism

Infection by microorganisms may cause fatally erroneous interpretations in the biologic researches based on cell culture. The contamination by microorganism in the cell culture is quite frequent (5% to 35%). However, current approaches to identify the presence of contamination have many limitations such as high cost of time and labor, and difficulty in interpreting the result. In this paper, we propose a model to predict cell infection, using a microarray technique which gives an overview of the whole genome profile. By analysis of 62 microarray expression profiles under various experimental conditions altering cell type, source of infection and collection time, we discovered 5 marker genes, NM_005298, NM_016408, NM_014588, S76389, and NM_001853. In addition, we discovered two of these genes, S76389, and NM_001853, are involved in a Mycolplasma-specific infection process. We also suggest models to predict the source of infection, cell type or time after infection. We implemented a web based prediction tool in microarray data, named Prediction of Microbial Infection (http://www.snubi.org/software/PMI).
Algorithms ; Cell Line ; Chondrocytes/cytology/metabolism/microbiology ; Databases, Genetic ; Gene Expression Profiling ; Host-Pathogen Interactions ; Humans ; Keratinocytes/cytology/metabolism/microbiology ; *Models, Genetic ; Mycoplasma/genetics/metabolism ; Oligonucleotide Array Sequence Analysis