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Response of methicillin-resistant Staphylococcus aureus to amicoumacin A.

Lama A, Pané-Farré J, Chon T, Wiersma AM, Sit CS, Vederas JC, Hecker M, Nakano MM - PLoS ONE (2012)

Bottom Line: Consistent with the notion that LrgA modulates murein hydrolase activity, COL grown in the presence of amicoumacin A showed reduced autolysis, which was primarily caused by lower hydrolase activity.The codon substitutions in EF-G that cause amicoumacin A resistance and fusidic acid resistance reside in separate domains and do not bring about cross resistance.Taken together, these results suggest that amicoumacin A might cause perturbation of the cell membrane and lead to energy dissipation.

View Article: PubMed Central - PubMed

Affiliation: Division of Environmental and Biomolecular Systems, Institute of Environmental Health, Oregon Health & Science University, Beaverton, Oregon, United States of America.

ABSTRACT
Amicoumacin A exhibits strong antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA), hence we sought to uncover its mechanism of action. Genome-wide transcriptome analysis of S. aureus COL in response to amicoumacin A showed alteration in transcription of genes specifying several cellular processes including cell envelope turnover, cross-membrane transport, virulence, metabolism, and general stress response. The most highly induced gene was lrgA, encoding an antiholin-like product, which is induced in cells undergoing a collapse of Δψ. Consistent with the notion that LrgA modulates murein hydrolase activity, COL grown in the presence of amicoumacin A showed reduced autolysis, which was primarily caused by lower hydrolase activity. To gain further insight into the mechanism of action of amicoumacin A, a whole genome comparison of wild-type COL and amicoumacin A-resistant mutants isolated by a serial passage method was carried out. Single point mutations generating codon substitutions were uncovered in ksgA (encoding RNA dimethyltransferase), fusA (elongation factor G), dnaG (primase), lacD (tagatose 1,6-bisphosphate aldolase), and SACOL0611 (a putative glycosyl transferase). The codon substitutions in EF-G that cause amicoumacin A resistance and fusidic acid resistance reside in separate domains and do not bring about cross resistance. Taken together, these results suggest that amicoumacin A might cause perturbation of the cell membrane and lead to energy dissipation. Decreased rates of cellular metabolism including protein synthesis and DNA replication in resistant strains might allow cells to compensate for membrane dysfunction and thus increase cell survivability.

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Northern blot analyses for SACOL0678 (A) and SACOL2176 (B) operon.Total RNA was isolated from S. aureus COL at 0 (t0), 10 (t10), and 40 (t40) min after the addition of amicoumacin A. 10 µg of total RNA isolated from each culture was separated in a formaldehyde-agarose gel and the RNA-blotted membrane was hybridized with SACOL0678- or SACOL2173(asp23)-specific digoxigenin-labeled probes. The sizes of the transcripts were determined by comparison to an RNA ladder on the same gel. The corresponding stained gels are shown underneath each blot. Schematic views of the gene loci based on NCBI COL genome site are shown with predicted transcripts. σB indicates locations of σB-controlled promoters. Microarray results of each operon's genes are summarized in the right panel of the corresponding Northern blot gels. Closed squares and open squares show samples taken at t10 and t40, respectively. The average of triplicates and standard deviations are indicated.
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pone-0034037-g003: Northern blot analyses for SACOL0678 (A) and SACOL2176 (B) operon.Total RNA was isolated from S. aureus COL at 0 (t0), 10 (t10), and 40 (t40) min after the addition of amicoumacin A. 10 µg of total RNA isolated from each culture was separated in a formaldehyde-agarose gel and the RNA-blotted membrane was hybridized with SACOL0678- or SACOL2173(asp23)-specific digoxigenin-labeled probes. The sizes of the transcripts were determined by comparison to an RNA ladder on the same gel. The corresponding stained gels are shown underneath each blot. Schematic views of the gene loci based on NCBI COL genome site are shown with predicted transcripts. σB indicates locations of σB-controlled promoters. Microarray results of each operon's genes are summarized in the right panel of the corresponding Northern blot gels. Closed squares and open squares show samples taken at t10 and t40, respectively. The average of triplicates and standard deviations are indicated.

Mentions: Northern blot analysis was carried out with two independently isolated RNA samples to validate the microarray results. Five operons and one gene were randomly chosen and the results of two operons are shown in Figure 3. The operon of eight genes (SACOL0678 to SACOL0686) encodes a phage integrase family protein (SACOL0678) and monovalent cation/H+ antiporter subunits (SACOL0679 to 0686). A previous study showed that transcription of these genes is σB-dependent [27]. Based on the microarray result, all eight genes were induced at t10 and the increased level of transcription was sustained at similar levels at t40 (Table 1 and Table S1). In the Northern blot experiment, a low level of 6.8 kb transcript was detected in untreated cells and the transcript was highly elevated at t10 and t40 (Figure 3A). This result confirmed that the eight genes constitute an operon and that amicoumacin A upregulates the operon transcription as shown by the microarray hybridization result. Transcription of another operon starting from SACOL2176 was also upregulated at t10 in the microarray results but the transcription decreased to the untreated level at t40 (Table 1 and Table S1). SACOL2176 encodes an osmoprotectant transporter, SACOL2175 and SACOL2174 encode a protein of unknown function and a membrane protein, respectively, and SACOL2173 is asp23 that codes for alkaline shock protein 23 [35]. The Northern blot analysis detected three transcripts of 3.0 kb, 1.5 kb, and 0.6 kb, all of which increase at t10 but not at t40 (Figure 3B). The sizes of the transcripts correspond to the predicted transcripts initiated at the three σB-dependent promoters. In a similar way, we also validated the microarray result of SACOL0673-0672-0671, SACOL2596-2597, SACOL02554.1-2554-2553, and SACOL1062 (data not shown).


Response of methicillin-resistant Staphylococcus aureus to amicoumacin A.

Lama A, Pané-Farré J, Chon T, Wiersma AM, Sit CS, Vederas JC, Hecker M, Nakano MM - PLoS ONE (2012)

Northern blot analyses for SACOL0678 (A) and SACOL2176 (B) operon.Total RNA was isolated from S. aureus COL at 0 (t0), 10 (t10), and 40 (t40) min after the addition of amicoumacin A. 10 µg of total RNA isolated from each culture was separated in a formaldehyde-agarose gel and the RNA-blotted membrane was hybridized with SACOL0678- or SACOL2173(asp23)-specific digoxigenin-labeled probes. The sizes of the transcripts were determined by comparison to an RNA ladder on the same gel. The corresponding stained gels are shown underneath each blot. Schematic views of the gene loci based on NCBI COL genome site are shown with predicted transcripts. σB indicates locations of σB-controlled promoters. Microarray results of each operon's genes are summarized in the right panel of the corresponding Northern blot gels. Closed squares and open squares show samples taken at t10 and t40, respectively. The average of triplicates and standard deviations are indicated.
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Related In: Results  -  Collection

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pone-0034037-g003: Northern blot analyses for SACOL0678 (A) and SACOL2176 (B) operon.Total RNA was isolated from S. aureus COL at 0 (t0), 10 (t10), and 40 (t40) min after the addition of amicoumacin A. 10 µg of total RNA isolated from each culture was separated in a formaldehyde-agarose gel and the RNA-blotted membrane was hybridized with SACOL0678- or SACOL2173(asp23)-specific digoxigenin-labeled probes. The sizes of the transcripts were determined by comparison to an RNA ladder on the same gel. The corresponding stained gels are shown underneath each blot. Schematic views of the gene loci based on NCBI COL genome site are shown with predicted transcripts. σB indicates locations of σB-controlled promoters. Microarray results of each operon's genes are summarized in the right panel of the corresponding Northern blot gels. Closed squares and open squares show samples taken at t10 and t40, respectively. The average of triplicates and standard deviations are indicated.
Mentions: Northern blot analysis was carried out with two independently isolated RNA samples to validate the microarray results. Five operons and one gene were randomly chosen and the results of two operons are shown in Figure 3. The operon of eight genes (SACOL0678 to SACOL0686) encodes a phage integrase family protein (SACOL0678) and monovalent cation/H+ antiporter subunits (SACOL0679 to 0686). A previous study showed that transcription of these genes is σB-dependent [27]. Based on the microarray result, all eight genes were induced at t10 and the increased level of transcription was sustained at similar levels at t40 (Table 1 and Table S1). In the Northern blot experiment, a low level of 6.8 kb transcript was detected in untreated cells and the transcript was highly elevated at t10 and t40 (Figure 3A). This result confirmed that the eight genes constitute an operon and that amicoumacin A upregulates the operon transcription as shown by the microarray hybridization result. Transcription of another operon starting from SACOL2176 was also upregulated at t10 in the microarray results but the transcription decreased to the untreated level at t40 (Table 1 and Table S1). SACOL2176 encodes an osmoprotectant transporter, SACOL2175 and SACOL2174 encode a protein of unknown function and a membrane protein, respectively, and SACOL2173 is asp23 that codes for alkaline shock protein 23 [35]. The Northern blot analysis detected three transcripts of 3.0 kb, 1.5 kb, and 0.6 kb, all of which increase at t10 but not at t40 (Figure 3B). The sizes of the transcripts correspond to the predicted transcripts initiated at the three σB-dependent promoters. In a similar way, we also validated the microarray result of SACOL0673-0672-0671, SACOL2596-2597, SACOL02554.1-2554-2553, and SACOL1062 (data not shown).

Bottom Line: Consistent with the notion that LrgA modulates murein hydrolase activity, COL grown in the presence of amicoumacin A showed reduced autolysis, which was primarily caused by lower hydrolase activity.The codon substitutions in EF-G that cause amicoumacin A resistance and fusidic acid resistance reside in separate domains and do not bring about cross resistance.Taken together, these results suggest that amicoumacin A might cause perturbation of the cell membrane and lead to energy dissipation.

View Article: PubMed Central - PubMed

Affiliation: Division of Environmental and Biomolecular Systems, Institute of Environmental Health, Oregon Health & Science University, Beaverton, Oregon, United States of America.

ABSTRACT
Amicoumacin A exhibits strong antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA), hence we sought to uncover its mechanism of action. Genome-wide transcriptome analysis of S. aureus COL in response to amicoumacin A showed alteration in transcription of genes specifying several cellular processes including cell envelope turnover, cross-membrane transport, virulence, metabolism, and general stress response. The most highly induced gene was lrgA, encoding an antiholin-like product, which is induced in cells undergoing a collapse of Δψ. Consistent with the notion that LrgA modulates murein hydrolase activity, COL grown in the presence of amicoumacin A showed reduced autolysis, which was primarily caused by lower hydrolase activity. To gain further insight into the mechanism of action of amicoumacin A, a whole genome comparison of wild-type COL and amicoumacin A-resistant mutants isolated by a serial passage method was carried out. Single point mutations generating codon substitutions were uncovered in ksgA (encoding RNA dimethyltransferase), fusA (elongation factor G), dnaG (primase), lacD (tagatose 1,6-bisphosphate aldolase), and SACOL0611 (a putative glycosyl transferase). The codon substitutions in EF-G that cause amicoumacin A resistance and fusidic acid resistance reside in separate domains and do not bring about cross resistance. Taken together, these results suggest that amicoumacin A might cause perturbation of the cell membrane and lead to energy dissipation. Decreased rates of cellular metabolism including protein synthesis and DNA replication in resistant strains might allow cells to compensate for membrane dysfunction and thus increase cell survivability.

Show MeSH
Related in: MedlinePlus