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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

Inactivation of the pefCD transporter leads to cellular accumulation of heme in cells grown in the presence of heme. a UV-visible spectra across wavelengths (250–700 nm) were recorded for organic fractions recovered after acidified chloroform extraction performed on a range of hemin chloride standards. b The observed absorbance at 388, 450, and 330 nms from UV scans (of organic fractions) were plugged into Ac = 2A388 − (A450 + A330) equation. The Ac values for standards extracted using chloroform for a range of hemin concentrations (0–4 μM, with 0.5 μM increments) were plotted against its hemin concentrations to generate a standard plot. The line equation of a standard plot was used to extrapolate hemin concentrations in the experimental samples. Cultures of NZ131 (WT), ZE4951 (Mutant), ZE4951/pANKITA5b (Complement), and ZE4951/pKSM201 (Empty vector) strains were treated with 3 μM heme during the mid logarithmic phase of growth (60–70 Klett units). Cells were harvested, washed, and were subjected to chloroform extraction. c UV-visible spectra across different wavelengths (250–700 nm) of the collected organic phases from tests samples were recorded. d Heme concentration in the test samples. The concentrations of heme in the test samples were calculated using the standard curve shown in B. 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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Fig6: Inactivation of the pefCD transporter leads to cellular accumulation of heme in cells grown in the presence of heme. a UV-visible spectra across wavelengths (250–700 nm) were recorded for organic fractions recovered after acidified chloroform extraction performed on a range of hemin chloride standards. b The observed absorbance at 388, 450, and 330 nms from UV scans (of organic fractions) were plugged into Ac = 2A388 − (A450 + A330) equation. The Ac values for standards extracted using chloroform for a range of hemin concentrations (0–4 μM, with 0.5 μM increments) were plotted against its hemin concentrations to generate a standard plot. The line equation of a standard plot was used to extrapolate hemin concentrations in the experimental samples. Cultures of NZ131 (WT), ZE4951 (Mutant), ZE4951/pANKITA5b (Complement), and ZE4951/pKSM201 (Empty vector) strains were treated with 3 μM heme during the mid logarithmic phase of growth (60–70 Klett units). Cells were harvested, washed, and were subjected to chloroform extraction. c UV-visible spectra across different wavelengths (250–700 nm) of the collected organic phases from tests samples were recorded. d Heme concentration in the test samples. The concentrations of heme in the test samples were calculated using the standard curve shown in B. 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: One interpretation of the experiments described above is that following the addition of heme to the culture medium GAS cells absorb it in surplus amounts; in the absence of a functional PefCD system, the bacterium accumulates heme or heme-related metabolites that damage the cell constituents. To directly test this hypothesis, we examined the cellular heme content by the acidified chloroform extraction method [50] described in the material and methods (Fig. 6). Culture samples of equal cell density were collected 90 min after the addition of 3 μM heme to the medium and the cells were washed extensively to remove surface bound heme prior to heme extraction. The presence of heme in the organic phase of the cell extracts was examined by spectroscopic analysis and the concentration of the extracted heme from each sample was extrapolated from a standard curve (Fig. 6b and d). We extracted in these experiments about 30 % more heme from the pefC mutant in comparison to the wild type strain. The heme concentration obtained from cells of the pefC mutant complemented with the pefRCD genes, was 7 % lower than that extracted from the wild type cells. On the other hand, the presence of the empty vector did not have a significant impact on the heme concentration in the extract from the pefC mutant. Therefore, the PefC accumulates more heme then the wild type strain. This observation suggests that the PefCD system expels surplus heme from GAS cells.Fig. 6


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

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

Inactivation of the pefCD transporter leads to cellular accumulation of heme in cells grown in the presence of heme. a UV-visible spectra across wavelengths (250–700 nm) were recorded for organic fractions recovered after acidified chloroform extraction performed on a range of hemin chloride standards. b The observed absorbance at 388, 450, and 330 nms from UV scans (of organic fractions) were plugged into Ac = 2A388 − (A450 + A330) equation. The Ac values for standards extracted using chloroform for a range of hemin concentrations (0–4 μM, with 0.5 μM increments) were plotted against its hemin concentrations to generate a standard plot. The line equation of a standard plot was used to extrapolate hemin concentrations in the experimental samples. Cultures of NZ131 (WT), ZE4951 (Mutant), ZE4951/pANKITA5b (Complement), and ZE4951/pKSM201 (Empty vector) strains were treated with 3 μM heme during the mid logarithmic phase of growth (60–70 Klett units). Cells were harvested, washed, and were subjected to chloroform extraction. c UV-visible spectra across different wavelengths (250–700 nm) of the collected organic phases from tests samples were recorded. d Heme concentration in the test samples. The concentrations of heme in the test samples were calculated using the standard curve shown in B. 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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Related In: Results  -  Collection

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Fig6: Inactivation of the pefCD transporter leads to cellular accumulation of heme in cells grown in the presence of heme. a UV-visible spectra across wavelengths (250–700 nm) were recorded for organic fractions recovered after acidified chloroform extraction performed on a range of hemin chloride standards. b The observed absorbance at 388, 450, and 330 nms from UV scans (of organic fractions) were plugged into Ac = 2A388 − (A450 + A330) equation. The Ac values for standards extracted using chloroform for a range of hemin concentrations (0–4 μM, with 0.5 μM increments) were plotted against its hemin concentrations to generate a standard plot. The line equation of a standard plot was used to extrapolate hemin concentrations in the experimental samples. Cultures of NZ131 (WT), ZE4951 (Mutant), ZE4951/pANKITA5b (Complement), and ZE4951/pKSM201 (Empty vector) strains were treated with 3 μM heme during the mid logarithmic phase of growth (60–70 Klett units). Cells were harvested, washed, and were subjected to chloroform extraction. c UV-visible spectra across different wavelengths (250–700 nm) of the collected organic phases from tests samples were recorded. d Heme concentration in the test samples. The concentrations of heme in the test samples were calculated using the standard curve shown in B. 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: One interpretation of the experiments described above is that following the addition of heme to the culture medium GAS cells absorb it in surplus amounts; in the absence of a functional PefCD system, the bacterium accumulates heme or heme-related metabolites that damage the cell constituents. To directly test this hypothesis, we examined the cellular heme content by the acidified chloroform extraction method [50] described in the material and methods (Fig. 6). Culture samples of equal cell density were collected 90 min after the addition of 3 μM heme to the medium and the cells were washed extensively to remove surface bound heme prior to heme extraction. The presence of heme in the organic phase of the cell extracts was examined by spectroscopic analysis and the concentration of the extracted heme from each sample was extrapolated from a standard curve (Fig. 6b and d). We extracted in these experiments about 30 % more heme from the pefC mutant in comparison to the wild type strain. The heme concentration obtained from cells of the pefC mutant complemented with the pefRCD genes, was 7 % lower than that extracted from the wild type cells. On the other hand, the presence of the empty vector did not have a significant impact on the heme concentration in the extract from the pefC mutant. Therefore, the PefC accumulates more heme then the wild type strain. This observation suggests that the PefCD system expels surplus heme from GAS cells.Fig. 6

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