RpoS subunit of RNA polymerase is a bacterial alternative sigma factor and major regulator important for response to a variety of stress conditions. However, RpoS-dependent genes in Burkholderia pseudomallei remained undefined. We identified the RpoS regulon of B. pseudomallei using a proteomics approach, which revealed 70 differentially expressed proteins between rpoS(+) and rpoS(-) strains. The RpoS-dependent genes were then classified into 14 functional categories, most of which were related to stress response. We then used Hidden Markov Model (HMM) for prediction of RpoS-dependent promoters in 51 genes encoding 63 down-regulated proteins in rpoS(-) strain and successfully defined such promoters, which were classified into three main groups based upon their consensus sequences. Groups 1 and 2, which had the highest potential, were mostly "stress response" genes, whereas Group 3, with the lowest potential, belonged to genes encoding "hypothetical proteins". The promoter prediction was confirmed by 5'-RACE-PCR sequencing. We also expanded and defined RpoS-controlled genes from RpoS regulon, based on proteomic data, RpoS-dependent promoter prediction, gene organization and operon prediction. We finally confirmed the co-expression of these RpoS-dependent genes in operons using RT-PCR. Our data lead to better understanding of RpoS regulation in B. pseudomallei, whose RpoS regulon differs from other Gram-negative bacteria.

No abstract

Burkholderia pseudomallei is an intracellular Gram-negative bacterial pathogen and the causative agent of melioidosis, a widespread disease in Southeast Asia. Reactive nitrogen, in an intermediate form of nitric oxide (NO), is one of the first lines of defense used by host cells to eliminate intracellular pathogens, through the stimulation of inducible nitric oxide synthase (iNOS). Studies in phagocytotic cells have shown that the iNOS response is muted in B. pseudomallei infection, and implicated the rpoS sigma factor as a key regulatory factor mediating suppression. The liver is a main visceral organ affected by B. pseudomallei, and there is little knowledge about the interaction of liver cells and B. pseudomallei. This study investigated the induction of iNOS, as well as autophagic flux and light-chain 3 (LC3) localization in human liver (HC04) cells in response to infection with B. pseudomallei and its rpoS deficient mutant. Results showed that the rpoS mutant was unable to suppress iNOS induction and that the mutant showed less induction of autophagy and lower co-localization with LC3, and this was coupled with a lower intracellular growth rate. Combining these results suggest that B. pseudomallei rpoS is an important factor in establishing infection in liver cells.

Surface TLRs have been well established to play an essential role in Burkholderia pseudomallei infection. However, the role of endosomal TLRs has not been elucidated.

The capability in holding the activities of α-glucosidase (1U/mL) of crude extract and mitragynine from Mitragyna speciosa Korth 10 mg/mL. Crude ethyl acetate extracts 61.52±0.47% (IC50 = 17.28 mg/mL) which closed to acarbose 78.94±2.97% (IC50  = 15.74 mg/mL). Minimum density acarbose 0.1562 mg/mL, 4.95±5.96%, crude hexane extracts 2.5 mg/mL, 0.78±11.52%, crude dichloromethane extracts 2.5 mg/mL, 4.40±7.73%, crude ethyl acetate extracts 0.3125 mg/mL, 0.51±5.20 %, crude ethanol extracts 1.25 mg/mL, 0.29±2.54%, crude methanol extracts 0.625 mg/mL, 0.71±0.49%, fractions c (Mitragynine) 5 mg/mL, 1.54±4.50%, crude water extracts, fractions a and b, it was found that there was no activity holding of glucosidase enzyme which meant that korth had the ability to hold the activity of α-glucosidase enzyme in extracting by ethyl acetate. It was also another treatment option and to reduce adverse effects from medication in patients with type 2 diabetes. According to research, the effects of consuming korth leaves should be cautious. Studies on the neuroprotective effects of mitragynine showed that mitragynine could stimulate the nervous system just like cocaine, thereby causing nausea and vomiting. Additionally, korth leaf extract had been reported to be toxic to the liver and kidneys.