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The GAS PefCD exporter is a MDR system that confers resistance to heme and structurally diverse compounds.

Sachla AJ, Eichenbaum Z - BMC Microbiol. (2016)

Bottom Line: This mutant was hypersensitive to heme, exhibiting significant growth inhibition already in the presence of 1 μM heme.Finally, the absence of the PefCD transporter potentiated the damaging effects of heme on GAS building blocks including lipids and DNA.This is the first heme resistance machinery described in GAS.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, College of Arts and Sciences, Georgia State University, P.O. Box 4010, Atlanta, GA, 30302-4010, USA.

ABSTRACT

Background: Group A streptococcus (GAS) is the etiological agent of a variety of local and invasive infections as well as post-infection complications in humans. This β-hemolytic bacterium encounters environmental heme in vivo in a concentration that depends on the infection type and stage. While heme is a noxious molecule, the regulation of cellular heme levels and toxicity is underappreciated in GAS. We previously reported that heme induces three GAS genes that are similar to the pefRCD (porphyrin regulated efflux) genes from group B streptococcus. Here, we investigate the contributions of the GAS pef genes to heme management and physiology.

Results: In silico analysis revealed that the PefCD proteins entail a Class-1 ABC-type transporter with homology to selected MDR systems from Gram-positive bacteria. RT-PCR experiments confirmed that the pefRCD genes are transcribed to polycistronic mRNA and that a pefC insertion inactivation mutant lost the expression of both pefC and pefD genes. This mutant was hypersensitive to heme, exhibiting significant growth inhibition already in the presence of 1 μM heme. In addition, the pefC mutant was more sensitive to several drugs and nucleic acid dyes and demonstrated higher cellular accumulation of heme in comparison with the wild type and the complemented strains. Finally, the absence of the PefCD transporter potentiated the damaging effects of heme on GAS building blocks including lipids and DNA.

Conclusion: We show here that in GAS, the pefCD genes encode a multi-drug efflux system that allows the bacterium to circumvent the challenges imposed by labile heme. This is the first heme resistance machinery described in GAS.

No MeSH data available.


Related in: MedlinePlus

The construction of a polar mutation in pefC. a Schematic representation of the pefC::pMZ1 mutation in ZE4951 strain. The lines below the locus diagram denote the DNA fragments amplified by the analysis described in section B. The lines above the diagram denote the products the Q-PCR described in section C. SpecR signifies the spectinomycin resistance aad9 gene and ori represents pMZ1 origin of replication. The black regions in the pefC gene denote the internal fragment that was cloned into pMZ1 and is thus duplicated in the mutant chromosome. b PCR analysis of the pefRCD locus in the ZE4951 mutant. DNA was amplified by PCR and fractionated on 0.8 % agarose gel. The DNA ladder is shown in lane 1. For the analysis of the left chromosome/plasmid junction the reactions were done with the ZE553/SpecFw primer set and genomic DNA of NZ131 (lane 2), ZE4951 (lane 3), or no DNA (lane 4). For the right plasmid/chromosome junction, the reactions consist of the ZE554/SpecRev primer set and genomic DNA of NZ131 (lane 5), ZE4951 (lane 6), or no DNA (lane 7). For the specR cassette, the reactions were preformed with the SpecFW/SpecRev primer set and genomic DNA of the NZ131 (lane 8), ZE4951 (lane 9), pMZ1 (lane 10, positive control), or no DNA (lane 11). c Relative expression of the pefC and pefD genes in ZE4951 and NZ131 strains. Total RNA was extracted and the relative expression of the pefC and pefD genes was evaluated by Q-PCR. The relative expression of the pefC and pefD genes was normalized to rpsL transcript levels. The asterisk (*) indicates P value of statistical significance (P <0.05) tested using student t-test (assuming equal variance) at 0.05 levels of significance
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Fig2: The construction of a polar mutation in pefC. a Schematic representation of the pefC::pMZ1 mutation in ZE4951 strain. The lines below the locus diagram denote the DNA fragments amplified by the analysis described in section B. The lines above the diagram denote the products the Q-PCR described in section C. SpecR signifies the spectinomycin resistance aad9 gene and ori represents pMZ1 origin of replication. The black regions in the pefC gene denote the internal fragment that was cloned into pMZ1 and is thus duplicated in the mutant chromosome. b PCR analysis of the pefRCD locus in the ZE4951 mutant. DNA was amplified by PCR and fractionated on 0.8 % agarose gel. The DNA ladder is shown in lane 1. For the analysis of the left chromosome/plasmid junction the reactions were done with the ZE553/SpecFw primer set and genomic DNA of NZ131 (lane 2), ZE4951 (lane 3), or no DNA (lane 4). For the right plasmid/chromosome junction, the reactions consist of the ZE554/SpecRev primer set and genomic DNA of NZ131 (lane 5), ZE4951 (lane 6), or no DNA (lane 7). For the specR cassette, the reactions were preformed with the SpecFW/SpecRev primer set and genomic DNA of the NZ131 (lane 8), ZE4951 (lane 9), pMZ1 (lane 10, positive control), or no DNA (lane 11). c Relative expression of the pefC and pefD genes in ZE4951 and NZ131 strains. Total RNA was extracted and the relative expression of the pefC and pefD genes was evaluated by Q-PCR. The relative expression of the pefC and pefD genes was normalized to rpsL transcript levels. The asterisk (*) indicates P value of statistical significance (P <0.05) tested using student t-test (assuming equal variance) at 0.05 levels of significance

Mentions: In order to gain insights into the role of the pefCD genes in GAS physiology, we created a polar mutation in pefC by Campbell insertion (ZE4951 strain) in the background of the wild type strain, NZ131 (Table 1 and Fig. 2a). The formation of the pefC::pMZ1 mutation was confirmed by PCR analysis. This analysis established the presence of the specR cassette in ZE4951 chromosome (the 0.7 kb band in lane 9, Fig. 2b). In addition, these experiments verified the formation of the two expected plasmid/chromosome junctions; specifically, the region spanning specR and downstream up to the pefR gene (the 2.5 kb band in lane 3, Fig. 2b) and the region covering specR and upstream up to the pefC portion that is outside of the fragment that was cloned into pMZ1 (the 3.5 kb band in lane 6, Fig. 2b). The same PCR reactions did not produce any product when performed with the chromosomal DNA of the parental strain, NZ131 (lanes 4 and 7 in Fig. 2b). Together, this analysis confirmed that pMZ1 integrated into the correct site in the chromosome forming ZE4951 strain.Table 1


The GAS PefCD exporter is a MDR system that confers resistance to heme and structurally diverse compounds.

Sachla AJ, Eichenbaum Z - BMC Microbiol. (2016)

The construction of a polar mutation in pefC. a Schematic representation of the pefC::pMZ1 mutation in ZE4951 strain. The lines below the locus diagram denote the DNA fragments amplified by the analysis described in section B. The lines above the diagram denote the products the Q-PCR described in section C. SpecR signifies the spectinomycin resistance aad9 gene and ori represents pMZ1 origin of replication. The black regions in the pefC gene denote the internal fragment that was cloned into pMZ1 and is thus duplicated in the mutant chromosome. b PCR analysis of the pefRCD locus in the ZE4951 mutant. DNA was amplified by PCR and fractionated on 0.8 % agarose gel. The DNA ladder is shown in lane 1. For the analysis of the left chromosome/plasmid junction the reactions were done with the ZE553/SpecFw primer set and genomic DNA of NZ131 (lane 2), ZE4951 (lane 3), or no DNA (lane 4). For the right plasmid/chromosome junction, the reactions consist of the ZE554/SpecRev primer set and genomic DNA of NZ131 (lane 5), ZE4951 (lane 6), or no DNA (lane 7). For the specR cassette, the reactions were preformed with the SpecFW/SpecRev primer set and genomic DNA of the NZ131 (lane 8), ZE4951 (lane 9), pMZ1 (lane 10, positive control), or no DNA (lane 11). c Relative expression of the pefC and pefD genes in ZE4951 and NZ131 strains. Total RNA was extracted and the relative expression of the pefC and pefD genes was evaluated by Q-PCR. The relative expression of the pefC and pefD genes was normalized to rpsL transcript levels. The asterisk (*) indicates P value of statistical significance (P <0.05) tested using student t-test (assuming equal variance) at 0.05 levels of significance
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4837585&req=5

Fig2: The construction of a polar mutation in pefC. a Schematic representation of the pefC::pMZ1 mutation in ZE4951 strain. The lines below the locus diagram denote the DNA fragments amplified by the analysis described in section B. The lines above the diagram denote the products the Q-PCR described in section C. SpecR signifies the spectinomycin resistance aad9 gene and ori represents pMZ1 origin of replication. The black regions in the pefC gene denote the internal fragment that was cloned into pMZ1 and is thus duplicated in the mutant chromosome. b PCR analysis of the pefRCD locus in the ZE4951 mutant. DNA was amplified by PCR and fractionated on 0.8 % agarose gel. The DNA ladder is shown in lane 1. For the analysis of the left chromosome/plasmid junction the reactions were done with the ZE553/SpecFw primer set and genomic DNA of NZ131 (lane 2), ZE4951 (lane 3), or no DNA (lane 4). For the right plasmid/chromosome junction, the reactions consist of the ZE554/SpecRev primer set and genomic DNA of NZ131 (lane 5), ZE4951 (lane 6), or no DNA (lane 7). For the specR cassette, the reactions were preformed with the SpecFW/SpecRev primer set and genomic DNA of the NZ131 (lane 8), ZE4951 (lane 9), pMZ1 (lane 10, positive control), or no DNA (lane 11). c Relative expression of the pefC and pefD genes in ZE4951 and NZ131 strains. Total RNA was extracted and the relative expression of the pefC and pefD genes was evaluated by Q-PCR. The relative expression of the pefC and pefD genes was normalized to rpsL transcript levels. The asterisk (*) indicates P value of statistical significance (P <0.05) tested using student t-test (assuming equal variance) at 0.05 levels of significance
Mentions: In order to gain insights into the role of the pefCD genes in GAS physiology, we created a polar mutation in pefC by Campbell insertion (ZE4951 strain) in the background of the wild type strain, NZ131 (Table 1 and Fig. 2a). The formation of the pefC::pMZ1 mutation was confirmed by PCR analysis. This analysis established the presence of the specR cassette in ZE4951 chromosome (the 0.7 kb band in lane 9, Fig. 2b). In addition, these experiments verified the formation of the two expected plasmid/chromosome junctions; specifically, the region spanning specR and downstream up to the pefR gene (the 2.5 kb band in lane 3, Fig. 2b) and the region covering specR and upstream up to the pefC portion that is outside of the fragment that was cloned into pMZ1 (the 3.5 kb band in lane 6, Fig. 2b). The same PCR reactions did not produce any product when performed with the chromosomal DNA of the parental strain, NZ131 (lanes 4 and 7 in Fig. 2b). Together, this analysis confirmed that pMZ1 integrated into the correct site in the chromosome forming ZE4951 strain.Table 1

Bottom Line: This mutant was hypersensitive to heme, exhibiting significant growth inhibition already in the presence of 1 μM heme.Finally, the absence of the PefCD transporter potentiated the damaging effects of heme on GAS building blocks including lipids and DNA.This is the first heme resistance machinery described in GAS.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, College of Arts and Sciences, Georgia State University, P.O. Box 4010, Atlanta, GA, 30302-4010, USA.

ABSTRACT

Background: Group A streptococcus (GAS) is the etiological agent of a variety of local and invasive infections as well as post-infection complications in humans. This β-hemolytic bacterium encounters environmental heme in vivo in a concentration that depends on the infection type and stage. While heme is a noxious molecule, the regulation of cellular heme levels and toxicity is underappreciated in GAS. We previously reported that heme induces three GAS genes that are similar to the pefRCD (porphyrin regulated efflux) genes from group B streptococcus. Here, we investigate the contributions of the GAS pef genes to heme management and physiology.

Results: In silico analysis revealed that the PefCD proteins entail a Class-1 ABC-type transporter with homology to selected MDR systems from Gram-positive bacteria. RT-PCR experiments confirmed that the pefRCD genes are transcribed to polycistronic mRNA and that a pefC insertion inactivation mutant lost the expression of both pefC and pefD genes. This mutant was hypersensitive to heme, exhibiting significant growth inhibition already in the presence of 1 μM heme. In addition, the pefC mutant was more sensitive to several drugs and nucleic acid dyes and demonstrated higher cellular accumulation of heme in comparison with the wild type and the complemented strains. Finally, the absence of the PefCD transporter potentiated the damaging effects of heme on GAS building blocks including lipids and DNA.

Conclusion: We show here that in GAS, the pefCD genes encode a multi-drug efflux system that allows the bacterium to circumvent the challenges imposed by labile heme. This is the first heme resistance machinery described in GAS.

No MeSH data available.


Related in: MedlinePlus