Development of a novel bacterial surface display system using truncated OmpT as an anchoring motif.

OBJECTIVES: An efficient bacterial surface display system based on the anchoring motif derived from Escherichia coli (E. coli) outer membrane protease OmpT was developed in this study. RESULTS: Referring to the classical Lpp-OmpA (LOA) display system, the signal peptide and nine amino acids of mature Lpp were fused to the transmembrane domain comprising five ß-strands of truncated OmpT to generate a novel Lpp-OmpT (LOT) display system. The C-terminal fusion strategy was used to fuse a small peptide (His tag) and red fluorescent protein (mCherry) to the C-terminus of LOT. Cell surface exposure of His tag and mCherry were compared between the LOA and LOT display systems. E. coli expressing LOT-His tag adsorbed more Cu2+ than E. coli expressing LOA-His tag. E. coli expressing both LOT-mCherry-His tag and LOA-mCherry-His tag adhered to Cu2+ chelating sepharose beads, and adhered cells could be dissociated from the beads after excess Cu2+ treatment. More importantly, compared with the LOA system, a higher amount of LOT-mCherry-His tag hybrid protein was demonstrated to be localized at the outer membrane by both fluorescence spectrophotometric determination of cell fractions and cell-surface immunofluorescence assay. CONCLUSIONS: These results suggest that genetically modified OmpT can be used as a potential anchoring motif to efficiently and stably display polypeptides and proteins, and that the LOT system could be used in a variety of biotechnological and industrial processes.

Persistently high prevalence of primary resistance and multidrug resistance of tuberculosis in Heilongjiang Province, China.

BACKGROUND: The spread of multidrug-resistant tuberculosis (MDR-TB) Mycobacterium tuberculosis (M. tuberculosis) strains has been a big challenge to the TB control and prevention in China. Knowledge about patterns of drug resistance in TB high-burden areas of China is crucial to develop appropriate control strategies. We conducted a comprehensive investigation of the resistance pattern of M. tuberculosis in Heilongjiang Province. METHODS: 1427 M. tuberculosis clinical strains were isolated from pulmonary TB patients hospitalized between 2007 and 2012. The susceptibility of the isolates to the first-line anti-TB drugs and the resistance of MDR M. tuberculosis to fluoroquinolones were examined. We also performed a statistical analysis to identify the correlated risk factors for high burden of MDR-TB. RESULTS: The global resistance rates of 2007-2012 to the first-line drugs and MDR were 57.0 and 22.8 %, respectively. Notably, the primary MDR-TB and pan-resistance rates were as high as 13.6 and 5.0 %, respectively. Of MDR M. tuberculosis isolates (2009), approximately 13 % were not susceptible to any of the fluoroquinolones tested. Being age of 35 to 54, high re-treatment proportion, the presence of cavity lesion, and high proportion of shorter hospitalization are correlated with the development of MDR-TB. CONCLUSIONS: The high prevalence of drug resistant, MDR-TB, and fluoroquinolone-resistant MDR-TB is a big concern for TB control. More importantly, in order to control the development of MDR-TB effectively, we need to pay more attention to the primary resistance. Targeting reducing the prevalence of the risk factors may lead to better TB control in China.

Genotypes and characteristics of clustering and drug susceptibility of Mycobacterium tuberculosis isolates collected in Heilongjiang Province, China.

For the last decade China has occupied second place, after India, among the top five countries with high burdens of tuberculosis (TB). Heilongjiang Province is located in northeastern China. The prevalence of drug-resistant TB in Heilongjiang Province is higher than the average level in China. To determine the transmission characteristics of Mycobacterium tuberculosis strains isolated in this area and their genetic relationships, especially among the Beijing family strains, we investigated their genotypes. From May 2007 to October 2008, 200 M. tuberculosis isolates from patients presenting pulmonary TB were analyzed by molecular typing using PCR-based methods: spacer-oligonucleotide typing (spoligotyping), Beijing family-specific PCR (detection of the deletion of region of difference 105 [RD105]), and mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analysis. Different combinations of MIRU-VNTR loci were evaluated to define the genotypes and clustering characteristics of the local strains. We found that Beijing family strains represented 89.5% of the isolates studied. However, the rates of multidrug-resistant (MDR) M. tuberculosis among Beijing and non-Beijing family strains were not statistically different. The 15-locus set is considered the optimal MIRU-VNTR locus combination for analyzing the M. tuberculosis strains epidemic in this area, while the 10-locus set is an ideal set for first-line molecular typing. We found that the clustering rate of all the M. tuberculosis isolates analyzed was 10.0% using the 15-locus set typing. We conclude that the Beijing family genotype is predominant and that highly epidemic TB and MDR TB are less likely associated with the active transmission of M. tuberculosis in the study area.