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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

Insertion inactivation of pefC in GAS leads to impaired growth and heme hypersensitivity. a Growth of the NZ131 (WT), ZE4951 (Mutant), ZE4951/pANKITA5b (Complement), and ZE4951/pKSM201 (Empty vector) strains in THYB. Fresh media were inoculated with GAS cells (OD600 nm = 0.05) and the cultures were grown statically at 37 °C. Cell growth was monitored colorimetrically and expressed in Klett units. b-c: Growth of ZE4951/pANKITA5b (Complement) and ZE4951/pKSM201 (Empty vector) strains in THYB containing varying heme concentration. The same as with A, only that heme was added to the culture at the early logarithmic phase in final concentration of: b 1 μM; c 5 μM; and d 10 μM. The data are representative of at least two independent experiments
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Fig3: Insertion inactivation of pefC in GAS leads to impaired growth and heme hypersensitivity. a Growth of the NZ131 (WT), ZE4951 (Mutant), ZE4951/pANKITA5b (Complement), and ZE4951/pKSM201 (Empty vector) strains in THYB. Fresh media were inoculated with GAS cells (OD600 nm = 0.05) and the cultures were grown statically at 37 °C. Cell growth was monitored colorimetrically and expressed in Klett units. b-c: Growth of ZE4951/pANKITA5b (Complement) and ZE4951/pKSM201 (Empty vector) strains in THYB containing varying heme concentration. The same as with A, only that heme was added to the culture at the early logarithmic phase in final concentration of: b 1 μM; c 5 μM; and d 10 μM. The data are representative of at least two independent experiments

Mentions: For complementation analysis, we cloned the pefRCD operon with its native promoter into a shuttle vector (pKSM201). E. coli cells harboring the plasmid carrying the GAS pefRCD operon (pANKITA5b) did not exhibit any growth phenotype. However, despite our multiple attempts, we failed to create a sub clone that expresses only the pefCD genes (under the pef promoter). We therefore used the entire pefRCD operon (carried by pANKITA5b) to complement the ZE4951 mutant (Table 1). Analysis of ZE4951 growth in THYB media demonstrated that inactivation of the pef system led to a pronounced phenotype; the mutant cells grew slower and reached the stationary phase of growth at a lower cell density in comparison to the isogenic NZ131 wild type strain (Fig. 3a). In addition, cell viability of the mutant strain was significantly reduced during overnight incubation in stationary phase (data not shown). The growth defect of the ZE4951 mutant was reversed in the presence the pefRCD genes expressed in trans (pANKITA5b) but not in the presence of an empty shuttle vector (pKSM201, Fig. 3a). This analysis suggests that the pefCD genes are required for optimal GAS growth in standard laboratory medium.Fig. 3


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

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

Insertion inactivation of pefC in GAS leads to impaired growth and heme hypersensitivity. a Growth of the NZ131 (WT), ZE4951 (Mutant), ZE4951/pANKITA5b (Complement), and ZE4951/pKSM201 (Empty vector) strains in THYB. Fresh media were inoculated with GAS cells (OD600 nm = 0.05) and the cultures were grown statically at 37 °C. Cell growth was monitored colorimetrically and expressed in Klett units. b-c: Growth of ZE4951/pANKITA5b (Complement) and ZE4951/pKSM201 (Empty vector) strains in THYB containing varying heme concentration. The same as with A, only that heme was added to the culture at the early logarithmic phase in final concentration of: b 1 μM; c 5 μM; and d 10 μM. The data are representative of at least two independent experiments
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Insertion inactivation of pefC in GAS leads to impaired growth and heme hypersensitivity. a Growth of the NZ131 (WT), ZE4951 (Mutant), ZE4951/pANKITA5b (Complement), and ZE4951/pKSM201 (Empty vector) strains in THYB. Fresh media were inoculated with GAS cells (OD600 nm = 0.05) and the cultures were grown statically at 37 °C. Cell growth was monitored colorimetrically and expressed in Klett units. b-c: Growth of ZE4951/pANKITA5b (Complement) and ZE4951/pKSM201 (Empty vector) strains in THYB containing varying heme concentration. The same as with A, only that heme was added to the culture at the early logarithmic phase in final concentration of: b 1 μM; c 5 μM; and d 10 μM. The data are representative of at least two independent experiments
Mentions: For complementation analysis, we cloned the pefRCD operon with its native promoter into a shuttle vector (pKSM201). E. coli cells harboring the plasmid carrying the GAS pefRCD operon (pANKITA5b) did not exhibit any growth phenotype. However, despite our multiple attempts, we failed to create a sub clone that expresses only the pefCD genes (under the pef promoter). We therefore used the entire pefRCD operon (carried by pANKITA5b) to complement the ZE4951 mutant (Table 1). Analysis of ZE4951 growth in THYB media demonstrated that inactivation of the pef system led to a pronounced phenotype; the mutant cells grew slower and reached the stationary phase of growth at a lower cell density in comparison to the isogenic NZ131 wild type strain (Fig. 3a). In addition, cell viability of the mutant strain was significantly reduced during overnight incubation in stationary phase (data not shown). The growth defect of the ZE4951 mutant was reversed in the presence the pefRCD genes expressed in trans (pANKITA5b) but not in the presence of an empty shuttle vector (pKSM201, Fig. 3a). This analysis suggests that the pefCD genes are required for optimal GAS growth in standard laboratory medium.Fig. 3

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