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

Abstract

Haemophilus influenzae is a Gram-negative human pathogen that resides in the upper respiratory tract. Encapsulated H. influenzae type b (Hib) and type f (Hif) are the most common serotypes associated with invasive disease. H. influenzae displays various strategies to circumvent the host innate immune response, including the bactericidal effect of the complement system. In this study, we identified an H. influenzae lipoprotein having the ability to bind factor H (FH), the major regulator of the alternative pathway of complement activation. This protein, named protein H (PH), was surface exposed and was found in all clinical Hib and Hif isolates tested. Deletion of the gene encoding for PH (lph) in Hib and Hif significantly reduced the interaction between bacteria and FH. When Hib and Hif PH variants were separately expressed in nontypeable (unencapsulated) H. influenzae, which did not bind FH, an increased FH affinity was observed. We recombinantly expressed the two PH variants in Escherichia coli, and despite sharing only 56% identical amino acids, both FH-binding Haemophilus proteins similarly interacted with the complement regulator FH short consensus repeats 7 and 18–20. Importantly, Hib and Hif resistance against the bactericidal effect of human serum was significantly reduced when bacterial mutants devoid of PH were tested. In conclusion, we have characterized a hitherto unknown bacterial protein that is crucial for mediating an interaction between the human pathogen H. influenzae and FH. This novel interaction is important for H. influenzae resistance against complement activation and will consequently promote bacterial pathogenesis.

Haemophilus influenzae Type f Hijacks Vitronectin Using Protein H To Resist Host Innate Immunity and Adhere to Pulmonary Epithelial Cells

“…The H. influenzae type f strain Hif M10 and its isogenic lph mutant Hif M10Dlph (21) were grown in liquid brain heart infusion medium containing 10 mg ml 21 NAD and hemin, or on chocolate agar plates, followed by incubation at 37˚C in a humid atmosphere with 5% CO 2 . The lph mutant was grown in the presence of 10 mg ml 21 kanamycin.…”

“…Cloning procedures were as described in detail previously (21). Briefly, the gene encoding the FH-binding protein (lph) in Hif was amplified by PCR excluding the 63 initial nucleotides encoding for the signal peptide.…”

“…In parallel, heat-inactivated serum was prepared by inactivation at 56˚C for 30 min. Susceptibility of Hif and E. coli BL21(DE3) to complement-mediated killing was tested, as described (21). The optimal concentrations of serum and time points used were as previously described (16,17,21), and were empirically determined for Hif (5% NHS and 15 min) and E. coli BL21 (DE3) (1% NHS and 10 min).…”

“…Susceptibility of Hif and E. coli BL21(DE3) to complement-mediated killing was tested, as described (21). The optimal concentrations of serum and time points used were as previously described (16,17,21), and were empirically determined for Hif (5% NHS and 15 min) and E. coli BL21 (DE3) (1% NHS and 10 min). We optimized our experiments and used 5% of NHS at 15 min, as this percentage of NHS caused killing of the PH mutant (Hif M10Dlph), and yet exerted the serum-resistant phenotype of the wild-type Hif M10.…”

“…We have recently defined a unique 35-kDa FH-binding protein designated protein H (PH), which is expressed mainly in Hib and Hif (21). The goal of this study was to further examine the role of Hif PH in interactions with the human host.…”