The response of type 2 quorum sensing in Klebsiella pneumoniae to a fluctuating culture environment.

Bacterial cells communicate with one another using chemical signaling molecules. This phenomenon is termed quorum sensing (QS). QS in Klebsiella pneumoniae is mediated by the synthesis of interspecies autoinducer 2 (AI-2), a furanosyl borate diester molecule. The response of Type 2 QS to environmental cues such as carbon sources, the initial pH of the medium, and boracic acid was investigated in the present study using a Vibrio harveyi BB170 reporter assay and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. The results show that glucose can affect AI-2 synthesis to the greatest extent, and 3.0% glucose can stimulate K. pneumoniae to produce more AI-2, with a four times increase in activity compared with that of the control culture. According to our previous research, Type 2 QS in K. pneumoniae is luxS dependent. Therefore, the close relationship between glucose concentration and luxS transcription level was confirmed with qRT-PCR technology. The results show that the response of QS to a fluctuating glucose concentration was observed as a change in the amount of luxS RNA transcripts. A maximum of luxS transcription appeared during the exponential growth phase when the glucose concentration was 30.0 g/L. At the same time, AI-2 production was also slightly impacted by the low initial pH. It is noteworthy that the addition of boracic acid at microdosage (0.1 g/L) can also induce AI-2 synthesis. Presumably, in K. pneumoniae, the 4,5-dihydroxy-2,3-pentanedione cyclizes by the addition of borate and loss of water, is hydrated, and is converted to the final AI-2 signaling molecule.

Inhibition of quorum sensing in the opportunistic pathogenic bacterium Chromobacterium violaceum by an extract from fruiting bodies of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (W.Curt.:Fr.) P. Karst. (higher Basidiomycetes).

Extracts of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum, inhibited quorum sensing in Chromobacterium violaceum CV026. G. lucidum fruiting bodies were milled and extracted with ethyl acetate. The crude extract was dissolved in an appropriate concentration of methanol, sterilized by filtration through a 0.22-µm membrane filter, and added to Ch. Violaceum CV026 cultures, which were used as an indicator to monitor quorum sensing inhibition. Inhibitory activity was measured by quantifying violacein production using a microplate reader. Methanol-soluble compounds extracted from G. lucidum significantly inhibited quorum sensing-controlled behavior in Ch. Violaceum in a concentration-dependent manner. The results suggest that compounds in G. lucidum might be useful to control and handle detrimental infections caused by human, animal, and plant pathogens. Further studies are in progress in our lab to isolate the specific compounds from G. lucidum extract, evaluate them as quorum sensing inhibitors, and analyze their mechanism of action.

A luxS-dependent transcript profile of cell-to-cell communication in Klebsiella pneumoniae.

Bacterial cells communicate with one another using chemical signaling molecules. The phenomenon is termed quorum sensing. The quorum sensing bacterium Klebsiella pneumoniae secretes a non-homoserine lactone autoinducer in the exponential phase of growth as detected by a Vibrio harveyi reporter assay for autoinducer 2 (AI-2). To further investigate regulation of AI-2 production in K. pneumoniae, the pfs and luxS promoter fusions to an operon luxCDABE reporter were constructed in a low copy number vector, which is derived from pBR322 and pET28a((+)) and allows an examination of transcription of the genes in the pathway for signal synthesis. In this study, comparisons were performed on the cell densities of wild-type and recombinant K. pneumoniae, on the transcription activity of pfs and luxS promoters, and on the synthesis of AI-2 as a function of culture time. The results show that luxS expression is constitutive and the transcription of luxS is tightly correlated to AI-2 production in K. pneumoniae because the peaks of AI-2 production and transcriptional level of luxS appear at the same time point. The close relation of the profiles of luxS transcription and AI-2 production was also confirmed with quantitative reverse transcription-PCR technology. These facts support the idea that the quorum sensing in K. pneumoniae is luxS dependent.