NontypeableHaemophilus influenzaeProtein E Binds Vitronectin and Is Important for Serum Resistance

Abstract

Nontypeable Haemophilus influenzae (NTHi) commonly causes local disease in the upper and lower respiratory tract and has recently been shown to interfere with both the classical and alternative pathways of complement activation. The terminal pathway of the complement system is regulated by vitronectin that is a component of both plasma and the extracellular matrix. In this study, we identify protein E (PE; 16 kDa), which is a recently characterized ubiquitous outer membrane protein, as a vitronectin-binding protein of NTHi. A PE-deficient NTHi mutant had a markedly reduced survival in serum compared with the PE-expressing isogenic NTHi wild type. Moreover, the PE-deficient mutant showed a significantly decreased binding to both soluble and immobilized vitronectin. In parallel, PE-expressing Escherichia coli bound soluble vitronectin and adhered to immobilized vitronectin compared with controls. Surface plasmon resonance technology revealed a KD of 0.4 μΜ for the interaction between recombinant PE and immobilized vitronectin. Moreover, the PE-dependent vitronectin-binding site was located at the heparin-binding domains of vitronectin and the major vitronectin-binding domain was found in the central core of PE (aa 84–108). Importantly, vitronectin bound to the surface of NTHi 3655 reduced membrane attack complex-induced hemolysis. In contrast to incubation with normal human serum, NTHi 3655 showed a reduced survival in vitronectin-depleted human serum, thus demonstrating that vitronectin mediates a protective role at the bacterial surface. Our findings show that PE, by binding vitronectin, may play an important role in NTHi pathogenesis.

Haemophilus influenzae stores and distributes hemin by using Protein E

“…E. coli harboring pET26bhpe or pET16bhpe (Hallstrom et al, 2009;Singh et al, 2013) were grown in LB broth in the presence of 50 µg/ml kanamycin or 100 µg/ml ampicillin, respectively.…”

“…PE was initially described as an adhesin that binds to epithelial cells of different origins including those from COPD patients (Ronander et al, 2009). In addition, PE simultaneously binds to extracellular matrix proteins and plasminogen, interactions that all contribute to bacterial virulence (Barthel et al, 2012;Hallstrom et al, 2009;Hallstrom et al, 2011). We recently crystallized PE and showed that it is a dimer with multiple binding sites for proteins derived from the host (Hallstrom et al, 2011;Singh et al, 2012;Singh et al, 2013).…”

Haemophilus influenzae Protein F Mediates Binding to Laminin and Human Pulmonary Epithelial Cells

“…NTHi has recently been reported to bind to ECM-proteins such as Ln and vitronectin [17,18,29]. Here, we report the NTHi ABC-transporter protein PF to be a novel Lnand cell-binding adhesin.…”

Identification of a Haemophilus influenzae Factor H–Binding Lipoprotein Involved in Serum Resistance

“…Bacteria loaded with FH are, however, still exposed to the other pathways of the complement system. Hence, to avoid serum killing from all pathways, H. influenzae also recruits the complement regulator C4b-binding protein, the major soluble regulator of the classical and the lectin pathway, in addition to vitronectin, which inhibits the formation of the lytic MAC (20,22,23,63). Another strategy to circumvent the deleterious effects of complement relies on masking the structures which interact with activators of complement.…”

“…H. influenzae has been shown to escape serum-mediated killing by binding FH and C4b-binding protein, the latter governs the classical pathway of complement activation, but also vitronectin, which results in inhibition of the terminal pathway and formation of the MAC (19)(20)(21)(22)(23).…”