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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 pefRCD genes entail an operon prevalent in human and zoonotic streptococci. a The genomic arrangement and occurrence of the pefRCD genes in GAS (MGAS5005 strain) and GBS (NEM316 strain) is also conserved among members of the pyogenic cluster namely, S. equi (4047 strain), S. uberis (0140 J strain), and S. iniae (ISET0901). Additionally, this system is found in S. suis (JS14 strain). These PefCD homologs are referred to as SatAB in the literature [37]. The pef operon consists of a MarR-like transcriptional regulator (locus tag for strains tested: Spy_0195, gbs1402, SEQ_0290, SUB_1690, SSUJS14_1996, and DQ08_09375) controlling an adjacent ABC-type efflux system (Spy_0196-97, gbs1401-00, SEQ_0292-93, SUB_1689-88, SSUJS14_1995-94, and DQ08_09370-65). The neighboring genes upstream and downstream from the pef operon consist of lipid metabolism (gpsA) and unknown function, respectively. b The GAS pefRCD genes are co-transcribed. RT-PCR analysis was preformed with 0.8 μg RNA extracted from NZ131 strain. cDNA synthesized by use of the pefD antisense primer (ZE650), was amplified by PCR with gene specific primers and fractionated on 1.2 % agarose gel (lanes 2, 5, and 8). PCR was also conducted using genomic DNA (lanes 3, 6, and 9) or RNA (without RT reaction, lanes 4, 7, and 10) as templates. Fragments from the following genes were amplified: pefR, lanes 2, 3 and 4; pefC, lanes 5, 6 and 7; and pefD, lanes 8, 9, and 10. The lines above the gene diagram in Figure-1A depict the amplified regions and the primer sets used in the PCR are included on top. The sizes of molecular mass standards are indicated to the left of the gel
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Fig1: The pefRCD genes entail an operon prevalent in human and zoonotic streptococci. a The genomic arrangement and occurrence of the pefRCD genes in GAS (MGAS5005 strain) and GBS (NEM316 strain) is also conserved among members of the pyogenic cluster namely, S. equi (4047 strain), S. uberis (0140 J strain), and S. iniae (ISET0901). Additionally, this system is found in S. suis (JS14 strain). These PefCD homologs are referred to as SatAB in the literature [37]. The pef operon consists of a MarR-like transcriptional regulator (locus tag for strains tested: Spy_0195, gbs1402, SEQ_0290, SUB_1690, SSUJS14_1996, and DQ08_09375) controlling an adjacent ABC-type efflux system (Spy_0196-97, gbs1401-00, SEQ_0292-93, SUB_1689-88, SSUJS14_1995-94, and DQ08_09370-65). The neighboring genes upstream and downstream from the pef operon consist of lipid metabolism (gpsA) and unknown function, respectively. b The GAS pefRCD genes are co-transcribed. RT-PCR analysis was preformed with 0.8 μg RNA extracted from NZ131 strain. cDNA synthesized by use of the pefD antisense primer (ZE650), was amplified by PCR with gene specific primers and fractionated on 1.2 % agarose gel (lanes 2, 5, and 8). PCR was also conducted using genomic DNA (lanes 3, 6, and 9) or RNA (without RT reaction, lanes 4, 7, and 10) as templates. Fragments from the following genes were amplified: pefR, lanes 2, 3 and 4; pefC, lanes 5, 6 and 7; and pefD, lanes 8, 9, and 10. The lines above the gene diagram in Figure-1A depict the amplified regions and the primer sets used in the PCR are included on top. The sizes of molecular mass standards are indicated to the left of the gel

Mentions: We recently reported the transcriptional activation of a 3-gene cluster (MGAS5005 spy_0195, 0196, and 0197) ensuing GAS exposure to environmental heme. We named these GAS genes as pefRCD based on similarity in genetic organization, primary sequence, and regulation to the previously described pefRCD operon in GBS [15, 32] (Fig. 1a). Our analysis here showed that the pefRCD genes are highly conserved among GAS strains exhibiting 100 % identity for PefR, 98 % identity for PefC, and 99 % identity for PefD protein (for all 20 strains in the database). The pefCD genes are annotated in GAS genome as the subunits of a putative multi-drug resistance (MDR) system. Sequence examination indicated that the pefCD genes code for the two subunits of a heteroligomeric ATP-dependent exporter. Both ORFs contain, at the N terminus, an integral membrane (IM) permease domain from the superfamily of ATP binding cassettes fused to an ATP-hydrolyzing domain (ABC, also referred to as the nucleotide binding domain). This domain organization is of the Class-1 ABC-type transporter family [34]. This transporter class is comprised of systems with fused IM and ABC domains and contains the vast majority of ABC-type export systems [34]. Our recent BLAST analysis against the transporter classification (TC) database (http://www.tcdb.org) [35] revealed that the PefCD proteins consists of a transporter from the Drug Exporter-4 sub class (3.A.1.135). Importantly, this analysis revealed that PefCD exporter is identical to a previously described transporter from the GAS strain JRS4, named Rsc (regulated by stress and Cov) after its regulation by the response regulator CovR [36]. The substrate (s) of the Rsc system was not identified but the transporter was found to be required for bacterial growth at high temperature. The nearest relatives of the PefCD (RscAB) proteins are the components of the MDR transporters, SatAB of S. suis [37], PatAB from S. pneumonia [38], and LmrCD (aka YdaG and YdbA) from L. lactis [39]. For simplicity we continue in this manuscript to refer to the GAS exporter as the PefCD system. Further in-silico study performed on genomes of streptococci identified putative homologs of the pefRCD gene cluster in S. equi, S. uberis, S. suis and S. iniae [40–44]. A high degree of conservation in genetic organization and sequence exhibiting 54 % amino acid similarity for the PefR, 60 % for the PefC, and 64 % for the PefD protein among all the candidates tested by ClastalW pairwise alignment tool [45]. (Fig. 1a).Fig. 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 pefRCD genes entail an operon prevalent in human and zoonotic streptococci. a The genomic arrangement and occurrence of the pefRCD genes in GAS (MGAS5005 strain) and GBS (NEM316 strain) is also conserved among members of the pyogenic cluster namely, S. equi (4047 strain), S. uberis (0140 J strain), and S. iniae (ISET0901). Additionally, this system is found in S. suis (JS14 strain). These PefCD homologs are referred to as SatAB in the literature [37]. The pef operon consists of a MarR-like transcriptional regulator (locus tag for strains tested: Spy_0195, gbs1402, SEQ_0290, SUB_1690, SSUJS14_1996, and DQ08_09375) controlling an adjacent ABC-type efflux system (Spy_0196-97, gbs1401-00, SEQ_0292-93, SUB_1689-88, SSUJS14_1995-94, and DQ08_09370-65). The neighboring genes upstream and downstream from the pef operon consist of lipid metabolism (gpsA) and unknown function, respectively. b The GAS pefRCD genes are co-transcribed. RT-PCR analysis was preformed with 0.8 μg RNA extracted from NZ131 strain. cDNA synthesized by use of the pefD antisense primer (ZE650), was amplified by PCR with gene specific primers and fractionated on 1.2 % agarose gel (lanes 2, 5, and 8). PCR was also conducted using genomic DNA (lanes 3, 6, and 9) or RNA (without RT reaction, lanes 4, 7, and 10) as templates. Fragments from the following genes were amplified: pefR, lanes 2, 3 and 4; pefC, lanes 5, 6 and 7; and pefD, lanes 8, 9, and 10. The lines above the gene diagram in Figure-1A depict the amplified regions and the primer sets used in the PCR are included on top. The sizes of molecular mass standards are indicated to the left of the gel
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Show All Figures
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Fig1: The pefRCD genes entail an operon prevalent in human and zoonotic streptococci. a The genomic arrangement and occurrence of the pefRCD genes in GAS (MGAS5005 strain) and GBS (NEM316 strain) is also conserved among members of the pyogenic cluster namely, S. equi (4047 strain), S. uberis (0140 J strain), and S. iniae (ISET0901). Additionally, this system is found in S. suis (JS14 strain). These PefCD homologs are referred to as SatAB in the literature [37]. The pef operon consists of a MarR-like transcriptional regulator (locus tag for strains tested: Spy_0195, gbs1402, SEQ_0290, SUB_1690, SSUJS14_1996, and DQ08_09375) controlling an adjacent ABC-type efflux system (Spy_0196-97, gbs1401-00, SEQ_0292-93, SUB_1689-88, SSUJS14_1995-94, and DQ08_09370-65). The neighboring genes upstream and downstream from the pef operon consist of lipid metabolism (gpsA) and unknown function, respectively. b The GAS pefRCD genes are co-transcribed. RT-PCR analysis was preformed with 0.8 μg RNA extracted from NZ131 strain. cDNA synthesized by use of the pefD antisense primer (ZE650), was amplified by PCR with gene specific primers and fractionated on 1.2 % agarose gel (lanes 2, 5, and 8). PCR was also conducted using genomic DNA (lanes 3, 6, and 9) or RNA (without RT reaction, lanes 4, 7, and 10) as templates. Fragments from the following genes were amplified: pefR, lanes 2, 3 and 4; pefC, lanes 5, 6 and 7; and pefD, lanes 8, 9, and 10. The lines above the gene diagram in Figure-1A depict the amplified regions and the primer sets used in the PCR are included on top. The sizes of molecular mass standards are indicated to the left of the gel
Mentions: We recently reported the transcriptional activation of a 3-gene cluster (MGAS5005 spy_0195, 0196, and 0197) ensuing GAS exposure to environmental heme. We named these GAS genes as pefRCD based on similarity in genetic organization, primary sequence, and regulation to the previously described pefRCD operon in GBS [15, 32] (Fig. 1a). Our analysis here showed that the pefRCD genes are highly conserved among GAS strains exhibiting 100 % identity for PefR, 98 % identity for PefC, and 99 % identity for PefD protein (for all 20 strains in the database). The pefCD genes are annotated in GAS genome as the subunits of a putative multi-drug resistance (MDR) system. Sequence examination indicated that the pefCD genes code for the two subunits of a heteroligomeric ATP-dependent exporter. Both ORFs contain, at the N terminus, an integral membrane (IM) permease domain from the superfamily of ATP binding cassettes fused to an ATP-hydrolyzing domain (ABC, also referred to as the nucleotide binding domain). This domain organization is of the Class-1 ABC-type transporter family [34]. This transporter class is comprised of systems with fused IM and ABC domains and contains the vast majority of ABC-type export systems [34]. Our recent BLAST analysis against the transporter classification (TC) database (http://www.tcdb.org) [35] revealed that the PefCD proteins consists of a transporter from the Drug Exporter-4 sub class (3.A.1.135). Importantly, this analysis revealed that PefCD exporter is identical to a previously described transporter from the GAS strain JRS4, named Rsc (regulated by stress and Cov) after its regulation by the response regulator CovR [36]. The substrate (s) of the Rsc system was not identified but the transporter was found to be required for bacterial growth at high temperature. The nearest relatives of the PefCD (RscAB) proteins are the components of the MDR transporters, SatAB of S. suis [37], PatAB from S. pneumonia [38], and LmrCD (aka YdaG and YdbA) from L. lactis [39]. For simplicity we continue in this manuscript to refer to the GAS exporter as the PefCD system. Further in-silico study performed on genomes of streptococci identified putative homologs of the pefRCD gene cluster in S. equi, S. uberis, S. suis and S. iniae [40–44]. A high degree of conservation in genetic organization and sequence exhibiting 54 % amino acid similarity for the PefR, 60 % for the PefC, and 64 % for the PefD protein among all the candidates tested by ClastalW pairwise alignment tool [45]. (Fig. 1a).Fig. 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