A functional tonB gene is required for both utilization of heme and virulence expression by Haemophilus influenzae type b

Abstract

Haemophilus influenzae is nearly unique among facultatively anaerobic bacteria in its absolute requirement for exogenously supplied heme for aerobic growth. In this study, a mutant analysis strategy was used to facilitate identification of H. influenzae cell envelope components involved in the uptake of heme. Chemical mutagenesis was employed to produce a mutant of a nontypeable H. influenzae strain unable to utilize either protein-bound forms of heme or low levels of free heme. This mutant was transformed with a plasmid shuttle vector-based genomic library constructed from the same wild-type nontypeable H. influenzae strain, and a growth selection technique was used to obtain a recombinant clone that could utilize heme. Analysis of the DNA insert in the recombinant plasmid revealed the presence of several open reading frames, one of which encoded a 28-kDa protein with significant similarity to the TonB protein of Escherichia coli. This H. influenzae gene product was able to complement a tonB mutation in E. coli, allowing the E. coli tonB mutant to form single colonies on minimal medium containing vitamin B12. When this H. influenzae gene was inactivated by insertional mutagenesis techniques and introduced into the chromosome of wild-type strains of H. influenzae type b, the resultant transformants lost their abilities to utilize heme and produce invasive disease in an animal model. Genetic restoration of the ability to express this TonB homolog resulted in the simultaneous acquisition of both heme utilization ability and virulence. These results indicate that the H. influenzae TonB protein is required not only for heme utilization by this pathogen in vitro, but also for virulence of H. influenzae type b in an animal model.

Cloning and functional characterization of Neisseria gonorrhoeae tonB, exbB and exbD genes

“…Just 20 bp downstream of the stop codon of exbD was a predicted hairpin loop structure that may function as a transcriptional terminator for a putative tonB-exbB-exbD operon. The tonB and exbBD genes are also clustered in H. influenzae and P. putida, although the exbBD genes in these bacteria precede tonB (Bitter et al, 1993;Jarosik et al, 1994), as opposed to the situation in the gonococcus where the exbBD genes follow the tonB gene. An insertional mutation in the gonococcal tonB gene resulted in the failure of gonococci to utilize TF, LF, and HB.…”

“…Of the four amino acid residues in E. coli that are implicated in the TonB-ExbB interactions (Larsen et al, 1993), Ser-16 and Leu-27 were conserved in gonococcal TonB. The gonococcal TonB was only 26% identical to the E. coli TonB protein, but TonB proteins from a variety of bacteria including E. aerogenes , H. influenzae (Jarosik et al, 1994), K. pneumoniae P. aeruginosa (Poole et al, 1996), P. putida (Bitter et al, 1993), S. typhimurium (Hannavy et al, 1990), S. marcescens (Gaisser and Braun, 1991), and Y. pestis (Koebnik et al, 1993) are only 26-32% identical to E. coli and to each other, indicating limited conservation of this protein.…”

“…If this is the case, the gonococcal tonB mutants may be impaired in virulence. An H. influenzae tonB mutant proved to be avirulent in a rat model (Jarosik et al, 1994), while a V. cholerae tonB mutant failed to cause disease in infant mice (Henderson and Payne, 1994b). However, Tsolis et al (1996) examined the ability of a S. typhimurium tonB mutant to grow in different compartments of the host, and observed that the mutant was able to infect the liver and spleen of the mouse, indicating that additional Fe-acquisition systems are used during intracellular growth.…”

“…A hydropathy plot of the deduced ORF1 sequence revealed a hydrophobic N-terminal sequence similar to TonB of other systems (Gaisser and Braun, 1991). Pairwise comparisons between the DNA or amino acid sequences of ORF1 with the tonB genes or gene products from eight other bacteria, Enterobacter aerogenes, Haemophilus influenzae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas putida, Salmonella typhimurium, Serratia marcescens and Yersinia pestis, showed 39-44% identity at the DNA level and 26-32% identity at the protein level of TonB (pairwise values not shown) (Hannavy et al, 1990;Gaisser and Braun, 1991;Poole et al, 1996;Bitter et al, 1993;and Koebnik et al, 1993;Jarosik et al, 1994). An examination of the regions of amino acid sequence homology by multiple sequence alignment analysis, using PILEUP, showed extensive homology with TonB proteins, strongly suggesting that the deduced ORF1 protein was an E. coli TonB homologue (alignment not shown).…”

The hmu locus of Yersinia pestis is essential for utilization of free haemin and haem‐protein complexes as iron sources

“…Although the mechanisms for haemin uptake by most pathogens are still poorly understood, haem/haemoprotein utilization systems from V, cholerae, N. rneningitidis, H. influenzae, and Y. enterocolitica have been studied in greater detail. In most instances, binding and uptake of haemin from one or more host haemoproteins are mediated by iron-regulated outer membrane receptors in a Ton6 energy-dependent manner with the entire haem moiety being transported into the cell (Cope et a/., 1995;Henderson and Payne, 1993;Jarosik et a/., 1994;Stoljiljkovic and Hantke, 1992;Stoljiljkovic et a/., 1995).…”