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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 from heme-mediated lipid oxidation. Cultures of NZ131 (WT), ZE4951 (Mutant), ZE4951/pANKITA5b (Complement), and ZE4951/pKSM201 (Empty vector) strains were treated with 1 μM heme during the mid logarithmic phase of growth (60–70 Klett units). Culture samples were then collected at 30, 60, and 90 min post-heme exposure and allowed to react with TBA. The sample absorption at 532 nm was determined, and the formation of TBA-reactive-substances (TBARS) 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) calculated using student t-test (equal variance) at 0.05 levels of significance
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Fig4: The PefCD transporter protects GAS from heme-mediated lipid oxidation. Cultures of NZ131 (WT), ZE4951 (Mutant), ZE4951/pANKITA5b (Complement), and ZE4951/pKSM201 (Empty vector) strains were treated with 1 μM heme during the mid logarithmic phase of growth (60–70 Klett units). Culture samples were then collected at 30, 60, and 90 min post-heme exposure and allowed to react with TBA. The sample absorption at 532 nm was determined, and the formation of TBA-reactive-substances (TBARS) 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) calculated using student t-test (equal variance) at 0.05 levels of significance

Mentions: In order to uncover why the loss of the pefCD genes leads to reduced heme tolerance in GAS, we investigated the impact of heme on GAS cellular components in the wild type and mutant strains. In a recent study we demonstrated that exposure to low heme concentration was sufficient to damage the lipids in GAS envelope [15]. Oxidized lipids react with a thiobarbituric acid (TBA) reagent to form adducts (named TBARS) that can be monitored by spectroscopic methods and quantify with a standard curve [15, 47]. We compared the time course of TBARS formation between the wild type and mutant strain after the addition of 1 μM heme into the culture (Fig. 4). Analysis of samples collected 30 min post exposure revealed that the ZE4951 mutant exhibited higher level of lipid damage than the wild type strain (3 versus 1.5 nmol/ml TBRAS in the mutant and wild type strain respectively). Complementation of the pefC mutation with the pefRCD genes resulted in a significantly lower level of TBARS formation, in comparison to mutant cells harboring the negative control plasmid (0.97 and 2.49 nmol/ml respectively). Interestingly, the TBARS levels in the complemented mutant strain were reduced over time (from 0.98 to 0.38 nmol/ml in the 30 and 90 min samples respectively), while they remained approximately the same in the wild type, mutant, and the mutant cells harboring the control plasmid. Together these observations imply that the PefCD system defends GAS from heme-induced lipid damage and suggest that the expression of pefRCD genes under our experimental conditions may be limiting in the wild type strain.Fig. 4


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 from heme-mediated lipid oxidation. Cultures of NZ131 (WT), ZE4951 (Mutant), ZE4951/pANKITA5b (Complement), and ZE4951/pKSM201 (Empty vector) strains were treated with 1 μM heme during the mid logarithmic phase of growth (60–70 Klett units). Culture samples were then collected at 30, 60, and 90 min post-heme exposure and allowed to react with TBA. The sample absorption at 532 nm was determined, and the formation of TBA-reactive-substances (TBARS) 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) calculated using student t-test (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

Fig4: The PefCD transporter protects GAS from heme-mediated lipid oxidation. Cultures of NZ131 (WT), ZE4951 (Mutant), ZE4951/pANKITA5b (Complement), and ZE4951/pKSM201 (Empty vector) strains were treated with 1 μM heme during the mid logarithmic phase of growth (60–70 Klett units). Culture samples were then collected at 30, 60, and 90 min post-heme exposure and allowed to react with TBA. The sample absorption at 532 nm was determined, and the formation of TBA-reactive-substances (TBARS) 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) calculated using student t-test (equal variance) at 0.05 levels of significance
Mentions: In order to uncover why the loss of the pefCD genes leads to reduced heme tolerance in GAS, we investigated the impact of heme on GAS cellular components in the wild type and mutant strains. In a recent study we demonstrated that exposure to low heme concentration was sufficient to damage the lipids in GAS envelope [15]. Oxidized lipids react with a thiobarbituric acid (TBA) reagent to form adducts (named TBARS) that can be monitored by spectroscopic methods and quantify with a standard curve [15, 47]. We compared the time course of TBARS formation between the wild type and mutant strain after the addition of 1 μM heme into the culture (Fig. 4). Analysis of samples collected 30 min post exposure revealed that the ZE4951 mutant exhibited higher level of lipid damage than the wild type strain (3 versus 1.5 nmol/ml TBRAS in the mutant and wild type strain respectively). Complementation of the pefC mutation with the pefRCD genes resulted in a significantly lower level of TBARS formation, in comparison to mutant cells harboring the negative control plasmid (0.97 and 2.49 nmol/ml respectively). Interestingly, the TBARS levels in the complemented mutant strain were reduced over time (from 0.98 to 0.38 nmol/ml in the 30 and 90 min samples respectively), while they remained approximately the same in the wild type, mutant, and the mutant cells harboring the control plasmid. Together these observations imply that the PefCD system defends GAS from heme-induced lipid damage and suggest that the expression of pefRCD genes under our experimental conditions may be limiting in the wild type strain.Fig. 4

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