Two TonB systems in Actinobacillus pleuropneumoniae: their roles in iron acquisition and virulence

Abstract

Iron acquisition in vivo by Actinobacillus pleuropneumoniae depends upon a functional TonB system. Tonpitak et al. (W. Tonpitak, S. Thiede, W. Oswald, N. Baltes, and G.-F. Gerlach, Infect. Immun. 68:1164-1170, 2000) have described one such system, associated with tbpBA encoding the transferrin receptor, and here we report a second, termed tonB2. This gene cluster (exbB2-exbD2-tonB2) is highly homologous to those in other Pasteurellaceae, unlike the earlier system described (now termed tonB1), suggesting that it is the indigenous system for this organism. Both tonB2 and tonB1 are upregulated upon iron restriction. TonB2, but not TonB1, was found to be essential for growth in vitro when the sole source of iron was hemin, porcine hemoglobin, or ferrichrome. In the case of iron provided as iron-loaded porcine transferrin, neither tonB mutant was viable. The tonB1 phenotype could be explained by a polar effect of the mutation on transcription of downstream tbp genes. We propose that TonB2 is crucial for the acquisition of iron provided in this form, interacting with accessory proteins of the TonB1 system that have been demonstrated to be necessary by Tonpitak et al. TonB2 appears to play a much more important role in A. pleuropneumoniae virulence than TonB1. In an acute porcine infection model, the tonB2 mutant was found to be highly attenuated, while the tonB1 mutant was not. We hypothesize that acquisition of the tonB1-tbp gene cluster confers a biological advantage through its capacity to utilize transferrin-iron but that TonB1 itself plays little or no part in this process.

FIG. 1.

Genetic organization of two tonB systems in A. pleuropneumoniae, with tonB1 (as described by Tonpitak et al. [34]) shown at the top and tonB2 shown at the bottom. Hatched boxes represent genes in the same operon, while filled boxes represent other ORFs. Black triangles indicate the sites of insertional mutations in A. pleuropneumoniae strains 27A12 and 0F6. Arrows below the gene clusters represent the approximate binding positions of the indicated oligonucleotide primers.

FIG. 2.

Alignment of A. pleuropneumoniae TonB1 and TonB2 showing amino acid identities (lines) and biochemically conservative substitutions (dots). The identity for the proteins was 18%, and the similarity was 25%.

FIG. 3.

Schematic trees displaying the phylogenetic relatedness of protein sequences. (a) GroEL; (b) TonB (without A. pleuropneumoniae TonB1); (c) TonB (with A. pleuropneumoniae TonB1). For a full explanation, see the text. Sequences were obtained from GenBank or this work for the organisms. Abbreviations: A.a, A. actinomycetemcomitans; A.p, A. pleuropneumoniae; E.c, E. coli; H.i, H. influenzae; H.d, H. ducreyi; and N.m, N. meningitidis; A.p1, TonB1; A.p2, TonB2. Line lengths representing phylogenetic relatedness are to scale within but not between trees. The shaded areas enclose sequences from Pasteurellaceae.

FIG. 4.

RT-PCR analysis. (a) Products of RT-PCR of wild-type A. pleuropneumoniae grown under iron-restricted conditions, with primers tonB11 (exbB2) and tonB16 (tonB2) and templates as follows. Lane 1, total RNA treated with RNase A (negative control); lane 2, total RNA; lane 3, chromosomal DNA (positive control). M, size marker. (b) RT-PCR analysis of tonB expression in wild-type A. pleuropneumoniae, with primers tonBGER1 and tonBGER4 for tonB1 (lanes 1 to 4) and primers tonB10 and tonB18 for tonB2 (lanes 5 to 8), to generate bands between 200 and 400 bp as indicated. Templates were as follows. Lanes 1 and 5, RNA treated with RNase A, extracted from bacteria grown under iron-restricted conditions (negative control); lanes 2 and 6, RNA from bacteria grown under iron-replete conditions; lanes 3 and 7, RNA from bacteria grown under iron-restricted conditions; lanes 4 and 8, whole cellular DNA (positive control).

FIG. 5.

Zones of bacterial growth on iron-restricted solid medium around disks impregnated with different iron sources (clockwise from top left) as follows: hemin, Fe(NO₃)₃, Fe-pTf, porcine hemoglobin, and PBS (center). Panels show growth of wild-type A. pleuropneumoniae (a), the tonB1 mutant strain (b), the tonB1 mutant strain transformed with pLURO3 (c), and the tonB2 mutant strain (d).

FIG. 6.

RT-PCR analysis of tonB1 and tbpB expression. (a) RT-PCR analysis of tonB1 expression using primers tonBGER1 and tonBGER4. Lanes contain products from RNA templates under different growth conditions as follows: 1, wild type, iron replete; 2, wild type, iron restricted; 3, tonB1 mutant, iron restricted; 4, tonB1::pLURO3, iron restricted; 5, tonB2 mutant, iron restricted. M, size marker. (b) RT-PCR analysis of tbpB expression in A. pleuropneumoniae grown under conditions of iron restriction, using primers tbpB1 and tbpB2. Lanes contain products from RNA or DNA templates as follows: 1, wild-type RNA; 2, tonB1 mutant RNA; 3, wild-type RNA treated with RNase A (negative control); 4, tonB1 mutant RNA treated with RNase A (negative control); 5, wild-type DNA (positive control).

FIG. 7.

Immunoblotting analysis of wild-type and tonB1 mutants probed with MAb 1.48. Lanes contain separated whole-cell lysates of strains under different growth conditions as follows: 1, wild type, iron replete; 2, wild type, iron restricted; 3, tonB1 mutant, iron replete; 4, tonB1 mutant, iron restricted. The position of TbpB is indicated.

FIG. 8.

Bacterial growth visualized as colonies on the dark background of ferrichrome-stained agar, demonstrating ferrichrome utilization by A. pleuropneumoniae strains. (a) Wild type; (b) tonB1 mutant; (c) tonB2 mutant.