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Effects of antimicrobial peptides on methanogenic archaea - PubMed

Effects of antimicrobial peptides on methanogenic archaea

C Bang et al. Antimicrob Agents Chemother. 2012 Aug.

Abstract

As members of the indigenous human microbiota found on several mucosal tissues, Methanobrevibacter smithii and Methanosphaera stadtmanae are exposed to the effects of antimicrobial peptides (AMPs) secreted by these epithelia. Although antimicrobial and molecular effects of AMPs on bacteria are well described, data for archaea are not available yet. Besides, it is not clear whether AMPs affect them as the archaeal cell envelope differs profoundly in terms of chemical composition and structure from that of bacteria. The effects of different synthetic AMPs on growth of M. smithii, M. stadtmanae, and Methanosarcina mazei were tested using a microtiter plate assay adapted to their anaerobic growth requirements. All three tested methanoarchaea were highly sensitive against derivatives of human cathelicidin, of porcine lysin, and a synthetic antilipopolysaccharide peptide (Lpep); however, sensitivities differed markedly among the methanoarchaeal strains. The potent AMP concentrations affecting growth were below 10 μM, whereas growth of Escherichia coli WBB01 was not affected at peptide concentrations up to 10 μM under the same anaerobic growth conditions. Atomic force microscopy and transmission electron microscopy revealed that the structural integrity of the methanoarchaeal cells is destroyed within 4 h after incubation with AMPs. The disruption of the cell envelope of M. smithii, M. stadtmanae, and M. mazei within a few minutes of exposure was verified by using LIVE/DEAD staining. Our results strongly suggest that the release of AMPs by eukaryotic epithelial cells is a potent defense mechanism targeting not only bacteria, but also methanoarchaea.

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Figures

Fig 1
Fig 1

Growth inhibition of M. mazei, M. stadtmanae, and M. smithii by LL32. A total of 1 × 107 cells were incubated with the indicated concentrations of the human cathelicidin derivative LL32 at 37°C in 250 μl minimal medium under anaerobic conditions. Turbidity of cultures at 600 nm (OD600) was measured at different time points. Error bars represent standard deviations of three parallel cultures in one experimental setup. (A) M. mazei; (B) M. stadtmanae; (C) M. smithii.

Fig 2
Fig 2

Growth inhibition of M. mazei, M. stadtmanae, M. smithii, and E. coli WBB01 in cultures containing Lpep 19-2.5 and NK2. A tota1 of 1 × 107 cells were incubated with different concentrations of the peptide Lpep 19-2.5 (A) or NK2 (B) at 37°C in 250 μl minimal medium. The turbidity of control cultures at 600 nm (OD600) in the stationary phase (M. mazei, 50 h; M. stadtmanae, 72 h; M. smithii, 96 h; E. coli WBB01, 6 h) was set to 100%. Error bars represent standard abbreviations of three parallel cultures in one experimental setup.

Fig 3
Fig 3

Microscopic analysis of methanoarchaea after treatment with LL32 and Lpep 19-2.5. Cultures of M. mazei, M. stadtmanae, and M. smithii were grown to the mid-exponential growth phase, LIVE/DEAD staining kit reagents were added according to the manufacturer's protocol, and 1-ml aliquots of cultures were dispensed to anaerobic Hungate tubes. AMPs were added at the following concentrations: M. mazei, 1 μM LL32 or 1 μM Lpep 19-2.5; M. stadtmanae, 5 μM LL32 or 10 μM Lpep 19-2.5; M. smithii, 1 μM LL32 or 3 μM Lpep 19-2.5. (A) Fluorescent micrographs with filter sets for propidium iodide and SYTO 9 fluorescence taken after 10 min of incubation in the presence of AMPs (or in the absence for control cultures). (B) Phase-contrast micrographs taken after 60 min of incubation in the presence of AMPs (or in the absence for control cultures). Pictures are representative for the respective sample.

Fig 4
Fig 4

Effects of LL32 and Lpep 19.2-5 treatment on the morphology of M. stadtmanae and M. smithii examined by AFM. AFM images of untreated cultures (controls) and of cultures incubated with respective MICs in the presence of 5 μM LL32 or 10 μM Lpep 19-2.5 for M. stadtmanae and 1 μM LL32 or 3 μM Lpep 19-2.5 for M. smithii. Cultures were grown until the mid-exponential phase at 37°C, adjusted to 1 × 107 cells in fresh minimal medium, and incubated in the presence of AMPs for 4 h. Images were taken in air in alternating current (AC) (tapping) mode and are representative of the samples examined. The length (scale in μm) and height (color code) are indicated.

Fig 5
Fig 5

Effects of LL32 and Lpep 19.2-5 treatment on the morphology of M. stadtamanae and M. smithii examined by transmission electron microscopy (TEM). Shown are the TEM results from untreated cultures (controls) and cultures that were incubated with their respective MICs: 5 μM LL32 or 10 μM Lpep 19-2.5 for M. stadtmanae and 1 μM LL32 or 3 μM Lpep 19-2.5 for M. smithii. Cultures were grown until the mid-exponential phase at 37°C, adjusted to 1 × 107 cells in fresh minimal medium, and incubated with AMPs for 4 h. Images are representative of the respective samples.

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