Identification of the Key Protein for Zinc Uptake in Hemophilus influenzae

Abstract

Very little is known about specific mechanisms for zinc accumulation and transport in bacteria. In this study a putative adhesin B in Hemophilus influenzae, the product of gene HI0119, has been identified as a periplasmic zinc-binding protein (PZP1). A pzp1-deficient mutant has been constructed which is defective for growth under aerobic conditions and grows poorly under anaerobic conditions. The growth defect is specifically rescued by supplementing the growth medium with high concentrations of zinc. Subcellular fractionation was used to localize PZP1 to the periplasmic region in a nontypeable H. influenzae strain and in a transfected recombinant Escherichia coli strain (TApzp1). Recombinant PZP1, purified from a periplasmic extract of E. coli strain TApzp1, contained ϳtwo zinc atoms/protein molecule as determined by neutron activation analysis and atomic absorption spectroscopy. The zinc atoms could be removed by incubation with EDTA, and, by further addition of zinc, a total of five zinc atoms/PZP1 could be bound. Direct binding of 65 Zn to the recombinant protein by Western blot was demonstrated. Taken together, these results provide direct evidence that PZP1 plays a key role in zinc uptake by H. influenzae.

A Novel Zinc Binding System, ZevAB, Is Critical for Survival of Nontypeable Haemophilus influenzae in a Murine Lung Infection Model

“…5A). Addition of 60 M zinc restored growth of the znuA mutant in rich medium to wild-type levels (data not shown), similar to results previously reported for znuA mutants of other H. influenzae strains (39). When grown in 0.1 mM EDTA, the znuA mutant exhibited both a 21-fold reduction in growth yield at 10 h and a 2.3-fold increase in generation time compared with growth of this strain in sBHI medium, whereas the parent strain, zevA mutant, and zevB mutant grew normally under this condition (Fig.…”

“…Aside from the ZevAB system presented here, only one other system has been implicated in zinc utilization of H. influenzae, the high-affinity zinc transporter ZnuABC. An H. influenzae znuA (originally called pzp1) mutant was shown to exhibit growth defects in normal medium that could be rescued by addition of zinc, and recombinant ZnuA bound up to 5 molecules of zinc per protein (39). The znuBC genes have also been demonstrated to participate in zinc utilization in other organisms (11,53), including Pasteurella multocida (21), a close relative of H. influenzae, and are thought to encode the ATPase (znuC) and membrane permease (znuB) components of the transporter (52,53).…”

Structure and metal binding properties of ZnuA, a periplasmic zinc transporter from Escherichia coli

“…Briefly, the imidazolate ring outer-shell scattering is approximated using the four most prominent scattering paths derived from the structure of Zn(imidazole) 4 , as calculated by FEFF [42]. The coordination number employed for a given path is adjusted to reflect the total number of scattering paths of similar length.…”

“…In addition, these cells are unable to take up 65 Zn 2+ [3]. Mutants deficient in the expression of ZnuABC in pathogens such as Hemophilus influenzae and Brucella abortus exhibit decreased virulence as well as retarded transformation and growth rates [4,5]. Thus, ZnuABC might play an important role in bacterial survival during host infection.…”

“…The stoichiometry of metal binding is also unresolved. For example, H. influenzae Pzp1 (Hin-Pzp1) can bind up to five Zn 2+ ions per molecule [4] and Syn-ZnuA up to eight, whereas the various crystal structures only show one metal binding site. To extend our understanding of metal binding and transport by and specificity of cluster 9 MBRs, we have determined the crystal structures of the apo, Zn 2+ -loaded, and Co 2+ -loaded forms of Eco-ZnuA and have characterized its metal binding properties by circular dichroism (CD), optical, electron paramagnetic resonance (EPR), and X-ray absorption spectroscopies.…”