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Zn2+ Uptake in Streptococcus pyogenes: Characterization of adcA and lmb Null Mutants.

Tedde V, Rosini R, Galeotti CL - PLoS ONE (2016)

Bottom Line: An effective regulation of metal ion homeostasis is essential for the growth of microorganisms in any environment and in pathogenic bacteria is strongly associated with their ability to invade and colonise their hosts.In contrast, Lmb and HtpA were expressed at detectable levels only during growth in the presence of low zinc concentrations or in the adcA mutant, when expression of lmb is required to compensate for the lack of adcA expression.In the latter case, Lmb and HtpA were overexpressed by several fold, thus indicating that also in GAS AdcA is a zinc-specific importer and, although it shares this function with Lmb, the two substrate-binding proteins do not show fully overlapping roles in zinc homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Research Centre, GlaxoSmithKline Vaccines S.r.l., Siena, Italy.

ABSTRACT
An effective regulation of metal ion homeostasis is essential for the growth of microorganisms in any environment and in pathogenic bacteria is strongly associated with their ability to invade and colonise their hosts. To gain a better insight into zinc acquisition in Group A Streptococcus (GAS) we characterized deletion mutants of the adcA and lmb genes of Streptococcus pyogenes strain MGAS5005 encoding the orthologues of AdcA and AdcAII, the two surface lipoproteins with partly redundant roles in zinc homeostasis in Streptococcus pneumoniae. Null adcA and lmb mutants were analysed for their capability to grow in zinc-depleted conditions and were found to be more susceptible to zinc starvation, a phenotype that could be rescued by the addition of Zn2+ ions to the growth medium. Expression of AdcA, Lmb and HtpA, the polyhistidine triad protein encoded by the gene adjacent to lmb, during growth under conditions of limited zinc availability was examined by Western blot analysis in wild type and mutant strains. In the wild type strain, AdcA was always present with little variation in expression levels between conditions of excess or limited zinc availability. In contrast, Lmb and HtpA were expressed at detectable levels only during growth in the presence of low zinc concentrations or in the adcA mutant, when expression of lmb is required to compensate for the lack of adcA expression. In the latter case, Lmb and HtpA were overexpressed by several fold, thus indicating that also in GAS AdcA is a zinc-specific importer and, although it shares this function with Lmb, the two substrate-binding proteins do not show fully overlapping roles in zinc homeostasis.

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Expression of Lmb and HtpA in ΔadcA mutants.Western blot analysis of total cell extracts from S. pyogenes MGAS5005 wild type and ΔadcA  mutant grown in complete medium (THY) or in zinc-depleted medium (THY + 35 μM TPEN) containing increasing amounts of ZnCl2. (A) Western blot using anti-AdcA specific antibodies. (B) Western blot using anti-Lmb specific antibodies. (C) Western blot using anti-HtpA specific antibodies.
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pone.0152835.g003: Expression of Lmb and HtpA in ΔadcA mutants.Western blot analysis of total cell extracts from S. pyogenes MGAS5005 wild type and ΔadcA mutant grown in complete medium (THY) or in zinc-depleted medium (THY + 35 μM TPEN) containing increasing amounts of ZnCl2. (A) Western blot using anti-AdcA specific antibodies. (B) Western blot using anti-Lmb specific antibodies. (C) Western blot using anti-HtpA specific antibodies.

Mentions: The increased susceptibility to zinc starvation of the adcA mutant strongly supports the notion that AdcA is indeed involved in zinc homeostasis in S. pyogenes, as already reported for other streptococci. In particular, in S. pneumoniae a recent characterization of AdcA has highlighted its complementarity in zinc homeostasis with AdcAII, the other Zn2+-binding SBP of streptococci, and demonstrated the cooperative functionality between the two importers [8]. Furthermore, immediately downstream of the gene coding for the AdcAII homologue Lmb in all the GAS strains characterized to date is located the gene coding for the histidine triad protein HtpA, a member of a family of proteins containing multiple copies of a histidine triad motif known to bind Zn2+ ions [33][34]. The gene coding for HtpA is highly conserved both in sequence and location also in GBS strains [33] and in S. pneumoniae, where adcAII is also located in the same operon as phtD, one of the four genes coding for a pneumococcal histidine triad protein [34][35]. For these reasons, we analysed the differences in expression levels of Lmb and HtpA in the ΔadcA mutant grown in zinc-depleted medium supplemented with increasing amounts of zinc ions (Fig 3). Firstly, it should be noted that in the wild type strain expression of AdcA was highest in medium supplemented with 10–15 μM of zinc ions, while at 20 μM ZnCl2 it was comparable with the level of expression observed in complete medium (Fig 3A). This suggests that limiting amounts of Zn2+ ions are able to induce only a small increase in expression of AdcA. On the contrary, Lmb and HtpA were expressed at significantly higher levels in both wild type and ΔadcA strains grown in the presence of 10–15 μM Zn2+ ions (Fig 3B and 3C). During growth in rich medium containing excess zinc ions (THB), expression of Lmb and HtpA was undetectable in wild type cells and low in the ΔadcA mutant. Moreover, Lmb and HtpA were expressed at higher levels in the ΔadcA mutant grown in the presence of 20 μM Zn2+ ions, a growth condition that presumably requires a functional AdcA for zinc homeostasis, which in a ΔadcA mutant could only be provided by an increased expression of Lmb and, consequently, of HtpA.


Zn2+ Uptake in Streptococcus pyogenes: Characterization of adcA and lmb Null Mutants.

Tedde V, Rosini R, Galeotti CL - PLoS ONE (2016)

Expression of Lmb and HtpA in ΔadcA mutants.Western blot analysis of total cell extracts from S. pyogenes MGAS5005 wild type and ΔadcA  mutant grown in complete medium (THY) or in zinc-depleted medium (THY + 35 μM TPEN) containing increasing amounts of ZnCl2. (A) Western blot using anti-AdcA specific antibodies. (B) Western blot using anti-Lmb specific antibodies. (C) Western blot using anti-HtpA specific antibodies.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4816340&req=5

pone.0152835.g003: Expression of Lmb and HtpA in ΔadcA mutants.Western blot analysis of total cell extracts from S. pyogenes MGAS5005 wild type and ΔadcA mutant grown in complete medium (THY) or in zinc-depleted medium (THY + 35 μM TPEN) containing increasing amounts of ZnCl2. (A) Western blot using anti-AdcA specific antibodies. (B) Western blot using anti-Lmb specific antibodies. (C) Western blot using anti-HtpA specific antibodies.
Mentions: The increased susceptibility to zinc starvation of the adcA mutant strongly supports the notion that AdcA is indeed involved in zinc homeostasis in S. pyogenes, as already reported for other streptococci. In particular, in S. pneumoniae a recent characterization of AdcA has highlighted its complementarity in zinc homeostasis with AdcAII, the other Zn2+-binding SBP of streptococci, and demonstrated the cooperative functionality between the two importers [8]. Furthermore, immediately downstream of the gene coding for the AdcAII homologue Lmb in all the GAS strains characterized to date is located the gene coding for the histidine triad protein HtpA, a member of a family of proteins containing multiple copies of a histidine triad motif known to bind Zn2+ ions [33][34]. The gene coding for HtpA is highly conserved both in sequence and location also in GBS strains [33] and in S. pneumoniae, where adcAII is also located in the same operon as phtD, one of the four genes coding for a pneumococcal histidine triad protein [34][35]. For these reasons, we analysed the differences in expression levels of Lmb and HtpA in the ΔadcA mutant grown in zinc-depleted medium supplemented with increasing amounts of zinc ions (Fig 3). Firstly, it should be noted that in the wild type strain expression of AdcA was highest in medium supplemented with 10–15 μM of zinc ions, while at 20 μM ZnCl2 it was comparable with the level of expression observed in complete medium (Fig 3A). This suggests that limiting amounts of Zn2+ ions are able to induce only a small increase in expression of AdcA. On the contrary, Lmb and HtpA were expressed at significantly higher levels in both wild type and ΔadcA strains grown in the presence of 10–15 μM Zn2+ ions (Fig 3B and 3C). During growth in rich medium containing excess zinc ions (THB), expression of Lmb and HtpA was undetectable in wild type cells and low in the ΔadcA mutant. Moreover, Lmb and HtpA were expressed at higher levels in the ΔadcA mutant grown in the presence of 20 μM Zn2+ ions, a growth condition that presumably requires a functional AdcA for zinc homeostasis, which in a ΔadcA mutant could only be provided by an increased expression of Lmb and, consequently, of HtpA.

Bottom Line: An effective regulation of metal ion homeostasis is essential for the growth of microorganisms in any environment and in pathogenic bacteria is strongly associated with their ability to invade and colonise their hosts.In contrast, Lmb and HtpA were expressed at detectable levels only during growth in the presence of low zinc concentrations or in the adcA mutant, when expression of lmb is required to compensate for the lack of adcA expression.In the latter case, Lmb and HtpA were overexpressed by several fold, thus indicating that also in GAS AdcA is a zinc-specific importer and, although it shares this function with Lmb, the two substrate-binding proteins do not show fully overlapping roles in zinc homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Research Centre, GlaxoSmithKline Vaccines S.r.l., Siena, Italy.

ABSTRACT
An effective regulation of metal ion homeostasis is essential for the growth of microorganisms in any environment and in pathogenic bacteria is strongly associated with their ability to invade and colonise their hosts. To gain a better insight into zinc acquisition in Group A Streptococcus (GAS) we characterized deletion mutants of the adcA and lmb genes of Streptococcus pyogenes strain MGAS5005 encoding the orthologues of AdcA and AdcAII, the two surface lipoproteins with partly redundant roles in zinc homeostasis in Streptococcus pneumoniae. Null adcA and lmb mutants were analysed for their capability to grow in zinc-depleted conditions and were found to be more susceptible to zinc starvation, a phenotype that could be rescued by the addition of Zn2+ ions to the growth medium. Expression of AdcA, Lmb and HtpA, the polyhistidine triad protein encoded by the gene adjacent to lmb, during growth under conditions of limited zinc availability was examined by Western blot analysis in wild type and mutant strains. In the wild type strain, AdcA was always present with little variation in expression levels between conditions of excess or limited zinc availability. In contrast, Lmb and HtpA were expressed at detectable levels only during growth in the presence of low zinc concentrations or in the adcA mutant, when expression of lmb is required to compensate for the lack of adcA expression. In the latter case, Lmb and HtpA were overexpressed by several fold, thus indicating that also in GAS AdcA is a zinc-specific importer and, although it shares this function with Lmb, the two substrate-binding proteins do not show fully overlapping roles in zinc homeostasis.

Show MeSH
Related in: MedlinePlus