The Adc/Lmb System Mediates Zinc Acquisition in Streptococcus agalactiae and Contributes to Bacterial Growth and Survival

Abstract

The Lmb protein of Streptococcus agalactiae is described as an adhesin that binds laminin, a component of the human extracellular matrix. In this study, we revealed a new role for this protein in zinc uptake. We also identified two Lmb homologs, AdcA and AdcAII, redundant binding proteins that combine with the AdcCB translocon to form a zinc-ABC transporter. Expression of this transporter is controlled by the zinc concentration in the medium through the zinc-dependent regulator AdcR. Triple deletion of lmb, adcA, and adcAII, or that of the adcCB genes, impaired growth and cell separation in a zinc-restricted environment. Moreover, we found that this Adc zinc-ABC transporter promotes S. agalactiae growth and survival in some human biological fluids, suggesting that it contributes to the infection process. These results indicated that zinc has biologically vital functions in S. agalactiae and that, under the conditions tested, the Adc/Lmb transporter constitutes the main zinc acquisition system of the bacterium.

Importance: A zinc transporter, composed of three redundant binding proteins (Lmb, AdcA, and AdcAII), was characterized in Streptococcus agalactiae This system was shown to be essential for bacterial growth and morphology in zinc-restricted environments, including human biological fluids.

FIG 1

Transcriptional organization of the lmb, adcA, adcAII, and adcRCB genes and a model of Zn-dependent regulation by AdcR in Streptococcus agalactiae. In the presence of Zn²⁺, AdcR can bind to the AdcR-binding motifs (TTAACNNGTTAA) located in the promoter regions of the lmb-sht, adcAII-shtII, and adcRCB operons and the adcA gene, causing repression of their expression. On the left of each sequence diagram is the promoter; the symbol on the right is the transcriptional terminator; black boxes are AdcR-binding boxes; the small circle with the dash inside it, prior to the sequence itself, indicates repression.

FIG 2

Amino acid sequence alignment of Lmb, AdcA, and AdcAII proteins of Streptococcus agalactiae. Identical residues are highlighted in gray. Potential zinc-binding residues are boxed in black. The His-rich loop of AdcA, which has been suggested to aid in recruiting Zn²⁺, is boxed by the black dotted lines. The Zin-T domain of AdcA, which may contain a supplementary zinc-binding site, is underlined. Sequence alignment was performed using the BioEdit program.

FIG 3

Regulation of the Streptococcus agalactiae lmb, adcA, and adcAII genes is Zn dependent. (A) lmb-sht operon promoter activity was measured in Zn-restricted CDM supplemented with various amounts of added metals (0 to 100 μM). A909 cells were grown until the mid-exponential phase of growth (OD₆₀₀, 0.5), and β-galactosidase assays were performed as described in Materials and Methods. The reference value (100%) is the lmb-sht promoter activity of cells grown in zinc-restricted CDM (0 μM Zn²⁺ added; white bar), and activities were calculated based on this reference. The values shown are mean results ± standard deviations. The asterisks indicate P values obtained using unpaired Student's t test, comparing promoter activity of willd-type (WT) cells grown in zinc-restricted CDM and cells grown in CDM with the various added Zn²⁺ concentrations. **, P < 0.01; ***, P < 0.001. (B) lmb, adcA, and adcAII expression levels were measured in zinc-restricted CDM containing 0 (white bars) or 10 μM added Zn²⁺ (black bars). qRT-PCR was performed on RNA extracts of S. agalactiae A909 grown until the mid-exponential phase. The amount of transcripts of each gene was normalized against recA transcript levels. The reference value (100%) is the level of lmb transcripts after growth in zinc-restricted CDM (0 μM added Zn²⁺). Gene expression is presented as the fold change. Results are presented as the means ± standard deviations of three independent experiments. The asterisks indicate P values obtained using an unpaired Student t test to compare gene expression of cells grown in zinc-restricted CDM versus cells grown with 10 μM added Zn²⁺. *, P < 0.05; **, P < 0.01.

FIG 4

AdcR is required for Zn-dependent repression of the lmb, adcA, and adcAII genes. (A) WT A909 (white bars) or its isogenic ΔadcR mutant (black bars) were grown either in zinc-restricted CDM or in medium supplemented with 10 μM Zn²⁺ until reaching the mid-exponential phase (OD₆₀₀, 0.5). β-Galactosidase assays were performed as described in Materials and Methods. The reference value (100) is the lmb-sht promoter activity of WT cells grown in zinc-restricted CDM, and the results are presented as the fold change compared to this reference. The values shown are the means ± standard deviations of three independent assays. (B) WT A909 (white bars) or its isogenic ΔadcR mutant (black bars) strains were grown in zinc-restricted CDM supplemented with 10 μM Zn²⁺ until reaching the mid-exponential phase (OD₆₀₀, 0.5). The amount of transcript of each gene was normalized against recA transcript levels. The reference value (1) is the level of lmb transcript of the wild-type strain. Gene expression is presented as the fold change. Results are presented as means ± standard deviations of three independent experiments. The asterisks indicate P values obtained using an unpaired Student t test to compare promoter activity (A) or gene expression (B) of the WT strain and that of its isogenic ΔadcR mutant. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 5

Both putative AdcR boxes within the lmb-sht operon promoter are required for full Zn-dependent promoter repression Transcriptional lacZ fusions with the lmb-sht promoter region carrying point mutations that destroyed an inverted repeat of the first putative AdcR-box (box₁*), or point mutations that destroyed an inverted repeat of the second putative AdcR-box (box₂*), or combined mutations (box_1-2*), were constructed. lacZ fusions containing the native lmb-sht promoter region and its derivatives were introduced into the WT strain. The native lmb-sht promoter region was also introduced in a ΔadcR mutant strain. β-Galactosidase assays were performed as described in Materials and Methods. The relative activity of the promoters was measured in zinc-restricted CDM containing 0 (white bars) or 10 μM added Zn²⁺ (black bars). The reference value (100) is the lmb-sht promoter activity of WT cells grown in zinc-restricted CDM, and the results are presented as the fold change against this reference. The values shown are the means ± standard deviations of three independent assays. The positions are numbered with respect to the start codon, and the transcription initiation site (with small right arrow) is labeled +1. Less relevant nucleotides are replaced with an N. Nucleotides in bold represent the two putative AdcR-binding sites, and underlined nucleotides indicate the introduced point mutations. The asterisks indicate P values obtained using an unpaired Student t test to compared promoter activity of the WT strain grown in zinc-restricted CDM and the other tested strains and conditions. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 6

The Lmb, AdcA, and AdcAII proteins are involved in zinc acquisition in association with the AdcCB translocon. A909 WT (gray) and Δlmb ΔadcA ΔadcAII mutant (black) strains (A) or WT (gray) and ΔadcCB (black) mutant strains (B) were grown in zinc-restricted CDM with various amounts of added Zn²⁺ (from 0 to 10 μM). The growth was monitored by assessing the OD₆₀₀ at 1-h intervals during 18 h. Data are representative mean OD₆₀₀ measurements from three independent experiments.

FIG 7

The Adc/Lmb zinc transporter affects cell morphology. Bacterial chain length of the A909 wild-type, Δlmb ΔadcA ΔadcAII, and ΔadcCB mutant strains was observed after growth in zinc-restricted CDM with 1 μM (top) or 10 μM (bottom) added zinc. Visualization of chain length was performed at a magnification of ×1,000. Cells were collected during the mid-exponential phase. Images were captured from three separate experiments, and at least 100 chains were counted from each set, for a total of 300 or more chains counted for each strain. Chain length values were distributed between arbitrarily set numerical categories and calculated as percentages of all counted chains. Results are presented as the means ± standard deviations for three independent counts. The asterisks indicate P values obtained using an unpaired Student t test to compare chain-length counts of the WT strain and the Δlmb ΔadcA ΔadcAII and ΔadcCB mutant strains. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 8

Intracellular survival of Streptococcus agalactiae in RAW 264.7 macrophages. RAW 264.7 cells were infected with A909 WT (black bars) or Δlmb ΔadcA ΔadcAII (white bars) strains at an MOI of 10, and phagocytosis was allowed to proceed for 1 h. Antibiotics were then added, and the cells were incubated for a period of 2 h to kill the extracellular bacteria. This initial antibiotic treatment, which represents time zero of the experiment, was extended for different times up to 24 h, and the cells were lysed to quantify the intracellular survival rates of the bacteria. Results are presented as the means ± standard deviations of three independent experiments performed in triplicate for each strain.

FIG 9

The Adc/Lmb zinc transporter is beneficial for Streptococcus agalactiae survival in two human biological fluids. A909 WT and Δlmb ΔadcA ΔadcAII mutant strains (A) or WT and ΔadcCB mutant strains (B) were inoculated in equivalent numbers into human cerebrospinal fluid (diamonds and black bars), amniotic fluid (squares and dark gray bars), or plasma (triangles and light gray bars). Cocultures were incubated at 37°C for 48 h without agitation, the CFU were counted, and the growth curves were traced. The proportion of each strain was monitored by plating diluted cultures on TH agar containing erythromycin (10 μg/ml) and X-Gal (60 μg/ml) (see Materials and Methods). Results are presented as the means ± standard deviations for three independent cocultures. The asterisks indicate P values obtained using an unpaired Student t test to compare the proportion of the strain at T0 and its proportion at the indicated times. *, P < 0.05; **, P < 0.01; ***, P < 0.001.