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

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.

Keywords: DNA damage detection; Doxorubicin; Gram-positive; Heme content; Multi-drug exporter; Mutational analysis; PefRCD.

Fig. 1

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

Fig. 2

The construction of a polar mutation in pefC. a Schematic representation of the pefC::pMZ1 mutation in ZE4951 strain. The lines below the locus diagram denote the DNA fragments amplified by the analysis described in section B. The lines above the diagram denote the products the Q-PCR described in section C. Spec ^R signifies the spectinomycin resistance aad9 gene and ori represents pMZ1 origin of replication. The black regions in the pefC gene denote the internal fragment that was cloned into pMZ1 and is thus duplicated in the mutant chromosome. b PCR analysis of the pefRCD locus in the ZE4951 mutant. DNA was amplified by PCR and fractionated on 0.8 % agarose gel. The DNA ladder is shown in lane 1. For the analysis of the left chromosome/plasmid junction the reactions were done with the ZE553/SpecFw primer set and genomic DNA of NZ131 (lane 2), ZE4951 (lane 3), or no DNA (lane 4). For the right plasmid/chromosome junction, the reactions consist of the ZE554/SpecRev primer set and genomic DNA of NZ131 (lane 5), ZE4951 (lane 6), or no DNA (lane 7). For the spec ^R cassette, the reactions were preformed with the SpecFW/SpecRev primer set and genomic DNA of the NZ131 (lane 8), ZE4951 (lane 9), pMZ1 (lane 10, positive control), or no DNA (lane 11). c Relative expression of the pefC and pefD genes in ZE4951 and NZ131 strains. Total RNA was extracted and the relative expression of the pefC and pefD genes was evaluated by Q-PCR. The relative expression of the pefC and pefD genes was normalized to rpsL transcript levels. The asterisk (*) indicates P value of statistical significance (P <0.05) tested using student t-test (assuming equal variance) at 0.05 levels of significance

Fig. 3

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 (OD_{600 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

Fig. 4

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

Fig. 5

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

Fig. 6

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 A _c = 2A ₃₈₈ − (A ₄₅₀ + A ₃₃₀) equation. The A_c 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

Fig. 7

A schematic depiction of heme-tolerance transporters described in streptococci. Putative homologs of the HrtAB (Class-3 ABC-type transporter) were identified in GBS, and were shown to be induced by heme. The PefAB and PefCD are heme and PPIX efflux machineries used by GBS. PefAB is related to the drug/proton antiporter family, while PefCD consists of Class-1 ABC type transporter. Our data show that GAS employs PefCD to efflux heme and various antibacterial compounds