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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 PefCD transporter protects GAS chromosome from heme-mediated damage. Cultures of NZ131 (WT), ZE4951 (Mutant), ZE4951/pANKITA5b (Complement), and ZE45/pKSM201 (Empty vector) strains were treated with 5 μM heme during the mid logarithmic phase of growth (60–70 Klett units). Genomic DNA was extracted from samples collected at 0, 30 and 90 min post exposure was allowed to react with ARP-biotin and analyzed. The sample absorption at 650 nm was determined and AP site formation was calculated using the standard curve shown in A. All samples were standardized with respect to cell number. The data are derived from two independent experiments each done in triplicates. The asterisk (*) denotes that the observed P value is statistically significant (P < 0.05) and is calculated using student t-test (equal variance) at 0.05 levels of significance
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Fig5: The PefCD transporter protects GAS chromosome from heme-mediated damage. Cultures of NZ131 (WT), ZE4951 (Mutant), ZE4951/pANKITA5b (Complement), and ZE45/pKSM201 (Empty vector) strains were treated with 5 μM heme during the mid logarithmic phase of growth (60–70 Klett units). Genomic DNA was extracted from samples collected at 0, 30 and 90 min post exposure was allowed to react with ARP-biotin and analyzed. The sample absorption at 650 nm was determined and AP site formation was calculated using the standard curve shown in A. All samples were standardized with respect to cell number. The data are derived from two independent experiments each done in triplicates. The asterisk (*) denotes that the observed P value is statistically significant (P < 0.05) and is calculated using student t-test (equal variance) at 0.05 levels of significance

Mentions: Studies performed with eukaryotic systems showed that heme could harm nucleic acids [48]. We asked if environmental heme can damage GAS genome and if the pef system offers protection from this harm. The repair of chemically altered bases (due to oxidation, deamination or alkylation) in the DNA is often mediated by repair mechanisms that involve the formation apurinic/apyrimidinic (AP) sites. Therefore, the amount of AP sites serves as a good indicator for DNA damage and repair. To quantitate the formation of AP sites in GAS chromosome, we used a reagent (ARP) that reacts specifically with the aldehyde group in the open ring form of the AP sites [49]. Chromosomal DNA was extracted from culture samples that were harvested at different time points and was allowed to react with ARP reagent. The formation of AP sites was detected by ELISA and quantified using a standard curve (Fig. 5). Analysis of chromosomal DNA from culture samples of the wild type strain collected 30 min after the addition of heme revealed about 90 % increase in the number of AP sites compared to the background level (11 and 5.8 AP sites per 105 bp respectively). The AP sites level was reduced over time, but remained 40 % above the background (8 AP sites per 105 bp) in the samples collected 90 min after treatment. Interestingly, in pefC mutant the background level of AP sites was about twice as high compared with the wild type strain (11.5 AP sites per 105 bp). Heme exposures resulted in a transient 117 % increase in the number of AP sites, which was reduced to background level within 60 min (25 and 11.5 AP sites per 105 bp in the 30 and 90 min respectively). The high AP levels observed in the mutant strain were complemented to those observed in the wild type strain by the pefRCD genes expressed from a plasmid in all samples. The levels of AP sites in the mutant strain carrying the empty vector were comparable to that of the mutant strain alone. These experiments indicate that environmental heme led to DNA damage in GAS and that the pefCD genes offered protection from DNA damage during growth in laboratory medium as well as in medium containing externally added heme. In addition, our data highlights the presence of DNA repair mechanisms in GAS.Fig. 5


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 PefCD transporter protects GAS chromosome from heme-mediated damage. Cultures of NZ131 (WT), ZE4951 (Mutant), ZE4951/pANKITA5b (Complement), and ZE45/pKSM201 (Empty vector) strains were treated with 5 μM heme during the mid logarithmic phase of growth (60–70 Klett units). Genomic DNA was extracted from samples collected at 0, 30 and 90 min post exposure was allowed to react with ARP-biotin and analyzed. The sample absorption at 650 nm was determined and AP site formation was calculated using the standard curve shown in A. All samples were standardized with respect to cell number. The data are derived from two independent experiments each done in triplicates. The asterisk (*) denotes that the observed P value is statistically significant (P < 0.05) and is calculated using student t-test (equal variance) at 0.05 levels of significance
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Related In: Results  -  Collection

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Fig5: The PefCD transporter protects GAS chromosome from heme-mediated damage. Cultures of NZ131 (WT), ZE4951 (Mutant), ZE4951/pANKITA5b (Complement), and ZE45/pKSM201 (Empty vector) strains were treated with 5 μM heme during the mid logarithmic phase of growth (60–70 Klett units). Genomic DNA was extracted from samples collected at 0, 30 and 90 min post exposure was allowed to react with ARP-biotin and analyzed. The sample absorption at 650 nm was determined and AP site formation was calculated using the standard curve shown in A. All samples were standardized with respect to cell number. The data are derived from two independent experiments each done in triplicates. The asterisk (*) denotes that the observed P value is statistically significant (P < 0.05) and is calculated using student t-test (equal variance) at 0.05 levels of significance
Mentions: Studies performed with eukaryotic systems showed that heme could harm nucleic acids [48]. We asked if environmental heme can damage GAS genome and if the pef system offers protection from this harm. The repair of chemically altered bases (due to oxidation, deamination or alkylation) in the DNA is often mediated by repair mechanisms that involve the formation apurinic/apyrimidinic (AP) sites. Therefore, the amount of AP sites serves as a good indicator for DNA damage and repair. To quantitate the formation of AP sites in GAS chromosome, we used a reagent (ARP) that reacts specifically with the aldehyde group in the open ring form of the AP sites [49]. Chromosomal DNA was extracted from culture samples that were harvested at different time points and was allowed to react with ARP reagent. The formation of AP sites was detected by ELISA and quantified using a standard curve (Fig. 5). Analysis of chromosomal DNA from culture samples of the wild type strain collected 30 min after the addition of heme revealed about 90 % increase in the number of AP sites compared to the background level (11 and 5.8 AP sites per 105 bp respectively). The AP sites level was reduced over time, but remained 40 % above the background (8 AP sites per 105 bp) in the samples collected 90 min after treatment. Interestingly, in pefC mutant the background level of AP sites was about twice as high compared with the wild type strain (11.5 AP sites per 105 bp). Heme exposures resulted in a transient 117 % increase in the number of AP sites, which was reduced to background level within 60 min (25 and 11.5 AP sites per 105 bp in the 30 and 90 min respectively). The high AP levels observed in the mutant strain were complemented to those observed in the wild type strain by the pefRCD genes expressed from a plasmid in all samples. The levels of AP sites in the mutant strain carrying the empty vector were comparable to that of the mutant strain alone. These experiments indicate that environmental heme led to DNA damage in GAS and that the pefCD genes offered protection from DNA damage during growth in laboratory medium as well as in medium containing externally added heme. In addition, our data highlights the presence of DNA repair mechanisms in GAS.Fig. 5

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