Heme transport contributes to in vivo fitness of Bordetella pertussis during primary infection in mice.

Bordetella pertussis is a versatile pathogen with diverse high-affinity transport systems for the assimilation of nutritional iron required for multiplication (4). It is hypothesized that specific iron transport systems serve distinct roles in responding to changes in iron source availability in the host environment during the course of infection. The heme utilization systems encoded by the hurIR bhuRSTUV genes of B. pertussis and Bordetella bronchiseptica (11, 12, 13) are novel hybrid systems combining the heme transport machinery of gram-negative pathogens with extracytoplasmic function (ECF) regulators of the iron starvation subfamily (12). Transcriptional regulation of Bordetella heme utilization genes is complex. BhuR, the TonB-dependent outer membrane heme receptor that is required for heme uptake and utilization, participates directly in regulation of heme system gene expression. BhuR has a predicted molecular mass of 90 kDa and has conserved characteristics of microbial heme receptors, including a TonB box C motif and the FRAP/NPNL amino acid sequence motif common to many hemin/hemoglobin receptors (11). Unique among heme receptor proteins, however, BhuR also has the N-terminal extension domain characteristic of TonB-dependent receptors that communicate with ECF regulators (11). Expression of the regulatory hurIR genes is Fur and iron repressible, and expression of the transport and utilization genes bhuRSTUV is heme inducible by a mechanism involving transmembrane transduction of the BhuR heme receptor occupancy signal via the anti-sigma factor membrane regulator HurR to the ECF sigma factor HurI for transcriptional activation of the bhuRSTUV promoter (12, 13). The global response of a microbial pathogen to iron starvation in the host contributes fundamentally to in vivo bacterial fitness (6). Mixed infection competition experiments (1, 5, 10) use the mouse or other appropriate host organism as a selective environment to assess the relative fitness of coinfecting strains for survival and multiplication in the host environment. The goal of this study was to determine the importance of B. pertussis heme utilization for multiplication and survival in mice using mixed infection competition experiments with strain UT25Sm1 (BhuR+ Strr) (3) and the isogenic ΔbhuR::kan mutant strain PM5 (BhuR− Strr Kanr) (11). In previous studies, PM5 was found to have no measurable in vitro growth defect, except in heme utilization during iron starvation (11). The ΔbhuR::kan mutation of PM5 exerts polar effects on the downstream, cotranscribed heme utilization genes bhuSTUV (11) (Fig. ​(Fig.1);1); thus, PM5 is defective in expression of all functions required for utilization of heme as an iron source. Receptor mutants are well suited to analysis by mixed infection competition since they cannot be trans-complemented by a receptor-proficient coinfecting strain; the receptor does not function as a diffusible factor and can only transport substrates into the receptor-producing cell. In this study, the ability of a bhuR heme utilization mutant to colonize and persist was determined relative to that of an isogenic coinfecting wild-type strain. The results demonstrate that heme utilization functions contribute fundamentally to the fitness of B. pertussis in the experimental murine host system. FIG. 1. Genetic organization of the 8-kb heme utilization gene cluster of B. pertussis. Arrows indicate the transcriptional orientations and spatial limits of the ECF regulator pair genes hurIR and the bhuRSTUV heme utilization operon. Circles represent the known ...