The Expression of the fim Operon Is Crucial for the Survival of Streptococcus parasanguinis FW213 within Macrophages but Not Acid Tolerance

Abstract

The acquisition of transition metal ions is essential for the viability and in some cases the expression of virulence genes in bacteria. The fimCBA operon of Streptococcus parasanguinis FW213 encodes a Mn(2+)/Fe(2+)-specific ATP-binding cassette transporter. FimA, a lipoprotein in the system, is essential for the development of endocarditis, presumably by binding to fibrin monolayers on the damaged heart tissue. Recent sequence analysis revealed that Spaf_0344 was homologous to Streptococcus gordonii scaR, encoding a metalloregulatory protein for the Sca Mn(2+)-specific transporter. Based on the homology, Spaf_0344 was designated fimR. By using various fim promoter (p fim ) derivatives fused with a promoterless chloramphenicol acetyltransferase gene, the functions of the cis-elements of p fim were analyzed in the wild-type and fimR-deficient hosts. The result indicated that FimR represses the expression of p fim and the palindromic sequences 5' to fimC are involved in repression of p fim . A direct interaction between FimR and the palindromic sequences was further confirmed by in vitro electrophoresis gel mobility shift assay and in vivo chromatin immunoprecipitation assay (ChIP)-quantitative real-time PCR (qPCR). The result of the ChIP-qPCR analysis also indicated that FimR is activated by Mn(2+) and, to a lesser degree, Fe(2+). Functional analysis indicated that the expression of FimA in S. parasanguinis was critical for wild-type levels of survival against oxidative stress and within phagocytes, but not for acid tolerance. Taken together, in addition to acting as an adhesin (FimA), the expression of the fim operon is critical for the pathogenic capacity of S. parasanguinis.

Figure 1. Schematic diagram of the fim operon and its flanking regions of S. parasanguinis FW213.

The relative location and transcription direction of each ORF are shown. Spaf_0345 and Spaf_0344 are indicated as 0345 and fimR, respectively. The limits of the sequence present in Figure 2A are indicated by two vertical arrows. The position of the erm in strain ΔfimR is indicated by an inverted triangle above the gene. The putative terminators for Spaf_0345 and fimR are indicated. The sizes of Spaf_0345 and fimR in nt, predicted molecular weight in kDa and pI of the gene products are shown.

Figure 2. The regulation of FimR on p_fim expression.

(A) The nt sequence of the 5′ flanking region of fimC. The pepO and fimC are transcribed from the opposite DNA strands, thus the sequence of pepO presented here is the noncoding strand, and the sequence of fimC is the coding strand. The transcription initiation sites (+1) of fimC and pepO are shown by a solid triangle above the sequence, and two open triangles below the sequence, respectively. The putative −10 and −35 sequences of p_fim are shaded. The potential Per box is underlined. The inverted repeat sequences are shown by horizontal arrows above the sequence. The sequence of the probe used in EMSA is boxed. The limits of the deletion derivatives are indicated by the numbers. (B) The CAT activities in wild-type FW213 and ΔfimR harboring various p_fim-cat fusions. All strains were grown in TH. Values shown are means and standard deviations of three independent experiments. All experiments were done in triplicate reactions and negative controls were reactions carried out in the absence of Cm.

Figure 3. Effect of Mn²⁺ and Fe²⁺ on p_fim expression.

Wild-type FW213 and ΔfimR harboring p_fim(445 b)-cat were grown in FMC containing 0.01 µM MnCl₂ and 0.1 µM FeSO₄ (I), 0.01 µM MnCl₂ and 50 µM FeSO₄ (II), 50 µM MnCl₂ and 0.1 µM FeSO₄ (III), 50 µM MnCl₂ and 50 µM FeSO₄ (IV). All cultures were supplemented with 1 mM MgSO₄ and 1 mM CaCl₂. Values are means and standard deviations of three independent experiments. Significant differences between samples were determined by two-way ANOVA using SPSS Statistic 17.0. The P values between the wild-type strain and ΔfimR under all four conditions are less than 0.01. P values between condition I and II are indicated in the figure. *, P<0.05; **, P<0.01.

Figure 4. EMSA demonstrating the interaction between FimR and p_fim .

Lanes 1 to 4 are reactions containing 0, 20, 40, and 80 µM His-FimR, respectively; lane 5 is reaction containing 80 µM His-FimR and unlabeled tcrB. The positions of the FimR-probe complexes are indicated by triangles.

Figure 5. ChIP-qPCR demonstrating the relative quantity of p_fim bound by FimR.

Cells were grown under 0.01 µM MnCl₂ and 0.1 µM FeSO₄ (I), 0.01 µM MnCl₂ and 50 µM FeSO₄ (II), 50 µM MnCl₂ and 0.1 µM FeSO₄ (III), and 50 µM MnCl₂ and 50 µM FeSO₄ (IV). The ΔCq of the sample from III was used as the reference. Significant differences between samples were determined using one-way ANOVA. A significant difference (P<0.05) was detected between all pairs of comparison.

Figure 6. Growth kinetics of the wild-type S. parasanguinis, VT930, ΔfimR, and VT930_ΔfimR grown in TH (A), TH containing 2 mM (B) and 4 mM (C) paraquat.

A representative figure of at least three experiments under each condition is shown.

Figure 7. The survival rate of the wild-type S. parasanguinis (I), VT930 (II), ΔfimR (III), and VT930_ΔfimR (IV) in THP1 (A) and RAW264.7 (B).

The numbers are means and standard deviations of three independent experiments. All experiments were done with triplicate samples. Significant differences between wild-type and recombinant strains were analyzed by one-way ANOVA. ***, P<0.01; **, P<0.05; *, P<0. 1.

Figure 8. Acid killing assay.

The means and standard deviations for three independent samples are shown. Significant differences between the wild-type and recombinant strains at 45 min were analyzed by one-way ANOVA. **, P<0.05; *, P<0. 1.