Screening of Streptococcus pneumoniae ABC transporter mutants demonstrates that LivJHMGF, a branched-chain amino acid ABC transporter, is necessary for disease pathogenesis

Abstract

Bacterial ABC transporters are an important class of transmembrane transporters that have a wide variety of substrates and are important for the virulence of several bacterial pathogens, including Streptococcus pneumoniae. However, many S. pneumoniae ABC transporters have yet to be investigated for their role in virulence. Using insertional duplication mutagenesis mutants, we investigated the effects on virulence and in vitro growth of disruption of 9 S. pneumoniae ABC transporters. Several were partially attenuated in virulence compared to the wild-type parental strain in mouse models of infection. For one ABC transporter, required for full virulence and termed LivJHMGF due to its similarity to branched-chain amino acid (BCAA) transporters, a deletion mutant (DeltalivHMGF) was constructed to investigate its phenotype in more detail. When tested by competitive infection, the DeltalivHMGF strain had reduced virulence in models of both pneumonia and septicemia but was fully virulent when tested using noncompetitive experiments. The DeltalivHMGF strain had no detectable growth defect in defined or complete laboratory media. Recombinant LivJ, the substrate binding component of the LivJHMGF, was shown by both radioactive binding experiments and tryptophan fluorescence spectroscopy to specifically bind to leucine, isoleucine, and valine, confirming that the LivJHMGF substrates are BCAAs. These data demonstrate a previously unsuspected role for BCAA transport during infection for S. pneumoniae and provide more evidence that functioning ABC transporters are required for the full virulence of bacterial pathogens.

FIG. 1.

Schematic diagram showing the organization of loci encoding ABC transporter components investigated in the present study. Each arrow represents a single gene, which are not drawn to scale. Gaps between genes correspond to gaps of greater than 50 bp between the stop codon of the upstream gene and the ATG of the downstream gene. The TIGR4 gene number is given above each gene, and whether the gene encodes a putative SBP, ATPase or membrane protein (permease) is written beneath. The diagonally shaded gene in each locus (▒) was targeted by IDM using pID701 containing an internal portion of the target gene.

FIG. 2.

(A) Structure of the livJHMGF locus. Each arrows represents one gene and contains the Sp number within the arrow and the gene annotation above the arrow. The size in base pairs of each gene is given below the arrow, and the number of base pairs between the stop codon of the upstream gene and the ATG of the downstream gene is given above the arrows in the corresponding gap. (B) Results of RT-PCR assessment of the transcriptional linkage between the livJHMGF genes. The bar represents the target product relative to the genes shown in panel A for each primer pair (named beneath the bar). A picture of the ethidium bromide-stained agarose gels showing the PCR products obtained using these primers and either DNA or cDNA as the template is given with each primer pair. (C) Representation of the structure of the livJHMGF locus in the ΔlivHMGF strain. (D) Ethidium bromide-stained agarose gels showing the PCR products obtained from the ΔlivHMGF and wild-type strain, confirming replacement of the livHMGF with the erythromycin resistance cassette (ery^r in the figure) in the ΔlivHMGF strain. Bars represent expected products for the primer pairs given above the lanes for their corresponding PCR products.

FIG. 3.

Assessment of the virulence of the ΔlivHMGF strain in mouse models of infection. (A) Time course for the development of fatal infection for groups of 10 mice given 5 × 10⁶ CFU of either the wild-type or the ΔlivHMGF strain (P = 0.88, log-rank test). (B) Log₁₀ ml⁻¹ bacterial CFU recovered from the lungs of mice 24 h after inoculation with 5 × 10⁶ CFU of either the wild-type or ΔlivHMGF strain. The results are shown as box-and-whisker diagrams of the median log₁₀ ml⁻¹ bacterial CFU and the interquartile range for data obtained from two identical experiments, each with five mice per strain.

FIG. 4.

Tryptophan fluorescence spectroscopy of purified His₆-LivJ after addition of 0.32 μM isoleucine (A), 3.2 μM leucine (B), or 16 μM valine (C) (marked by the first arrow). The addition of excess ligand (marked by the second arrow) had no further effect on fluorescence, nor did the addition of 50 μM proline, glycine, or alanine (data not shown). Fluorescence was measured by using arbitary units and an excitation wavelength of 280 nm with an emission wavelength of 309 nm.

FIG. 5.

Radioactive binding assays to purified His₆-LivJ. (A) Degree of binding of ¹⁴C-AIB (negative control) and the BCAAs [¹⁴C]leucine and [¹⁴C]isoleucine to His₆-LivJ expressed as pmol of substrate per mg of His₆-LivJ. No significant binding to AIB was detected. For the differences between [¹⁴C]leucine and [¹⁴C]isoleucine, P = 0.023 (Student t test). (B) Competitive blocking of [¹⁴C]isoleucine binding to His₆-LivJ by the addition of excess (500 μM) AIB, leucine, valine, or threonine. Compared to AIB, P = 0.005 for leucine, 0.014 for valine, and <0.001 for threonine. Compared to leucine, P < 0.05 for both valine and threonine (one-way analysis of variance).