ABSTRACT
Using the Taguchi method to narrow experimental parameters, the antimicrobial efficiency of a cold atmospheric plasma jet (CAPJ) treatment was investigated. An L9 array with four parameters of CAPJ treatments, including the application voltage, CAPJ-sample distance, argon (Ar) gas flow rate, and CAPJ treatment time, were applied to examine the antimicrobial activity against Escherichia coli (E. coli). CAPJ treatment time was found to be the most influential parameter in its antimicrobial ability by evaluation of signal to noise ratios and analysis of variance. 100% bactericidal activity was achieved under the optimal bactericidal activity parameters including the application voltage of 8.5 kV, CAPJ-sample distance of 10 mm, Ar gas flow rate of 500 sccm, and CAPJ treatment time of 300 s, which confirms the efficacy of the Taguchi method in this design. In terms of the mechanism of CAPJ's antimicrobial ability, the intensity of hydroxyl radical produced by CAPJ positively correlated to its antimicrobial efficiency. The CAPJ antimicrobial efficiency was further evaluated by both DNA double-strand breaks analysis and scanning electron microscopy examination of CAPJ treated bacteria. CAPJ destroyed the cell wall of E. coli and further damaged its DNA structure, thus leading to successful killing of bacteria. This study suggests that optimal conditions of CPAJ can provide effective antimicrobial activity and may be grounds for a novel approach for eradicating bacterial infections.
ABSTRACT
CovR/CovS is an important two-component regulatory system in human pathogen group A Streptococcus (GAS). Epidemiological studies have shown that inactivation of the sensor kinase CovS is correlated with invasive clinical manifestations. The phosphorylation level of response regulator CovR decreases dramatically in the absence of CovS, resulting in the derepression of virulence factor expression and an increase in bacterial invasiveness. Streptococcal pyrogenic exotoxin B (SpeB) is a cysteine protease and is negatively regulated by CovR; however, the expression of SpeB is almost completely repressed in the covS mutant. The present study found that in the emm1-type A20 strain, non-phosphorylated CovR acts as a transcriptional repressor for SpeB-positive regulator Rgg. In addition, the expression of Rgg-negative regulator LacD.1 is upregulated in the covS mutant. These results suggest that inactivation of Rgg in the covS mutant would directly mediate speB repression. The current study showed that overexpression of rgg but not inactivation of lacD.1 in the covS mutant partially restores speB expression, indicating that only rgg repression, but not lacD.1 upregulation, contributes to the speB repression in the covS mutant.
ABSTRACT
MPT64, a secreted protein of Mycobacterium tuberculosis (MTB), stimulates the immune reactions within cells and is a protective antigen that is lost by the bacilli Calmette-Guérin (BCG) vaccine during propagation. To minimize the toxicity caused by MTB, we used the MPT64 gene encoded by nontoxic H37Ra MTB to carry out genetic expansion via polymerase chain reaction and gene clone MPT64. The plasmid DNA encoded MPT64 was expressed at 20°C for 22 H, and a large quantity of MPT64 was obtained. In the absence of urea, MPT64 multimers with subunits being covalently connected via disulfide bonds were detected by Western blot showing strong protein-protein interactions, as evidenced by the formation of MPT64 tetramers. Finally, with urea of decreasing concentrations, we refolded MPT64 purified in the presence of urea and determined its secondary structures using circular dichroism. MPT64 was found to contain 2.2% α-helix, 50.9% ß-sheet, 19.5% turn, and 27.4% random coil. The molecular weight of MPT64 was determined by a matrix-assisted laser desorption ionization-time of flight mass spectrometer and found to be 23,497 Da, very close to the theoretical molecular weight of MPT64. The results presented here provide a sound basis for future biochemical and biophysical studies of MPT64 or any other proteins encoded by nontoxic H37Ra MTB.
