Yu-Ching Su

Vitronectin in bacterial pathogenesis: a host protein used in complement escape and cellular invasion.

The multifunctional human glycoprotein vitronectin (Vn) plays a significant role in cell migration, tissue repair and regulation of membrane attack complex (MAC) formation. It also promotes neutrophil infiltration and, thus, enhances the inflammatory process during infection. In the host, a balanced homeostasis is maintained by Vn due to neutralization of the self‐reactivity of the MAC. On the other hand, Vn bound to the bacterial surface protects from MAC‐mediated lysis and enhances adhesion. Gram‐negative bacterial pathogens including Moraxella catarrhalis, Haemophilus influenzae and Neisseria gonorrhoeae use Vn recruitment to prevent MAC deposition at their surface. Moreover, Gram‐positive bacterial pathogens such as Streptococcus pneumoniae and S. pyogenes utilize Vn for effective adhesion to host cells and subsequent internalization. Vitronectin has an Arg–Gly–Asp (RGD) sequence for binding the host cell integrin receptors and a separate bacterial‐binding domain for pathogens, and thus more likely functions to cross‐link bacteria and epithelial cells. Once bacteria are attached to the vitronectin–integrin complex, various host cell‐signalling events are activated and promote internalization. In this review, we focus on the important roles of vitronectin in bacterial pathogenesis and describe different strategies used by pathogens to evade the host response by the help of this intriguing molecule.

Haemophilus influenzae acquires vitronectin via the ubiquitous Protein F to subvert host innate immunity

Acquisition of the complement inhibitor vitronectin (Vn) is important for the respiratory tract pathogen nontypeable Haemophilus influenzae (NTHi) to escape complement‐mediated killing. NTHi actively recruits Vn, and we previously showed that this interaction involves Protein E (PE). Here we describe a second Vn‐binding protein, a 30 kDa Yersinia YfeA homologue designated as Protein F (PF). An isogenic NTHi 3655Δhpf mutant devoid of PF displayed a reduced binding of Vn, and was consequently more sensitive to killing by human serum compared with the wild type. Surface expression of PF on Escherichia coli conferred binding of Vn that resulted in a serum resistant phenotype. Molecular analyses revealed that the N‐terminal of PF (Lys23‐Glu48) bound to the C‐terminal of Vn (Phe352‐Ser374) without disrupting the inhibitory role of Vn on the membrane attack complex. The PF–Vn complex actively delayed C9 deposition on PF‐expressing bacteria. Comparative studies of binding affinity and multiple mutants demonstrated that both PE and PF contribute individually to NTHi serum survival. PF was highly conserved and ubiquitously expressed in a series of randomly selected NTHi clinical isolates (n = 18). In conclusion, the multifaceted binding of Vn is beneficial for NTHi survival in serum and may contribute to successful colonization and consequently infection.

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

The mucosal pathogen nontypeable Haemophilus influenzae (NTHi) adheres to the respiratory epithelium or, in the case of epithelial damage, to the underlying basement membrane and extracellular matrix that, among other proteins, consists of laminin. We have recently identified protein F, an ABC transporter involved in NTHi immune evasion. Homology modeling of the protein F tertiary structure revealed a strong resemblance to the streptococcal laminin-binding proteins Lbp and Lmb. Here, we show that protein F promotes binding of NTHi to laminin and primary bronchial epithelial cells. Analyses with recombinant proteins and synthetic peptides revealed that the N-terminal part of protein F contains the host-interacting region. Moreover, protein F exists in all clinical isolates, and isogenic NTHi Δhpf mutants display significantly reduced binding to laminin and epithelial cells. We thus suggest protein F to be an important and ubiquitous NTHi adhesin.

Moraxella catarrhalis: from interactions with the host immune system to vaccine development.

Moraxella catarrhalis is a human-restricted commensal that over the last two decades has developed into an emerging respiratory tract pathogen. The bacterial species is equipped with various adhesins to facilitate its colonization. Successful evasion of the human immune system is a prerequisite for Moraxella infection. This strategy involves induction of an excessive proinflammatory response, intervention of granulocyte recruitment to the infection site, activation of selected pattern recognition receptors and cellular adhesion molecules to counteract the host bacteriolytic attack, as well as, finally, reprogramming of antigen presenting cells. Host immunomodulator molecules are also exploited by Moraxella to aid in resistance against complement killing and host bactericidal molecules. Thus, breaking the basis of Moraxella immune evasion mechanisms is fundamental for future invention of effective therapy in controlling Moraxella infection.

A Fine-Tuned Interaction between Trimeric Autotransporter Haemophilus Surface Fibrils and Vitronectin Leads to Serum Resistance and Adherence to Respiratory Epithelial Cells

ABSTRACT Haemophilus influenzae type b (Hib) escapes the host immune system by recruitment of the complement regulator vitronectin, which inhibits the formation of the membrane attack complex (MAC) by inhibiting C5b-C7 complex formation and C9 polymerization. We reported previously that Hib acquires vitronectin at the surface by using Haemophilus surface fibrils (Hsf). Here we studied in detail the interaction between Hsf and vitronectin and its role in the inhibition of MAC formation and the invasion of lung epithelial cells. The vitronectin-binding region of Hsf was defined at the N-terminal region comprising Hsf amino acids 429 to 652. Moreover, the Hsf recognition site on vitronectin consisted of the C-terminal amino acids 352 to 374. H. influenzae was killed more rapidly in vitronectin-depleted serum than in normal human serum (NHS), and increased MAC deposition was observed at the surface of an Hsf-deficient H. influenzae mutant. In parallel, Hsf-expressing Escherichia coli selectively acquired vitronectin from serum, resulting in significant inhibition of the MAC. Moreover, when vitronectin was bound to Hsf, increased bacterial adherence and internalization into epithelial cells were observed. Taking our findings together, we have defined a fine-tuned protein-protein interaction between Hsf and vitronectin that may contribute to increased Hib virulence.

Host–pathogen interactions of nontypeable Haemophilus influenzae: from commensal to pathogen

Nontypeable Haemophilus influenzae (NTHi) is a commensal microbe often isolated from the upper and lower respiratory tract. This bacterial species can cause sinusitis, acute otitis media in preschool children, exacerbations in patients suffering from chronic obstructive pulmonary disease, as well as conjunctivitis and bacteremia. Since the introduction of a vaccine against H. influenzae serotype b in the 1990s, the burden of H. influenzae‐related infections has been increasingly dominated by NTHi. Understanding the ability of NTHi to cause infection is currently an expanding area of study. NTHi is able to exert differential binding to the host tissue through the use of a broad range of adhesins. NTHi survival in the host is multifaceted, that is, using virulence factors involved in complement resistance, biofilm, modified immunoglobulin responses, and, finally, formation and utilization of host proteins as a secondary strategy of increasing the adhesive ability.

Haemophilus influenzae P4 Interacts With Extracellular Matrix Proteins Promoting Adhesion and Serum Resistance

Interaction with the extracellular matrix (ECM) is one of the successful colonization strategies employed by nontypeable Haemophilus influenzae (NTHi). Here we identified Haemophilus lipoprotein e (P4) as a receptor for ECM proteins. Purified recombinant P4 displayed a high binding affinity for laminin (Kd = 9.26 nM) and fibronectin (Kd = 10.19 nM), but slightly less to vitronectin (Kd = 16.51 nM). A P4-deficient NTHi mutant showed a significantly decreased binding to these ECM components. Vitronectin acquisition conferred serum resistance to both P4-expressing NTHi and Escherichia coli transformants. P4-mediated bacterial adherence to pharynx, type II alveolar, and bronchial epithelial cells was mainly attributed to fibronectin. Importantly, a significantly reduced bacterial infection was observed in the middle ear of the Junbo mouse model when NTHi was devoid of P4. In conclusion, our data provide new insight into the role of P4 as an important factor for Haemophilus colonization and subsequent respiratory tract infection.

Outer Membrane Protein OlpA Contributes to Moraxella catarrhalis Serum Resistance via Interaction With Factor H and the Alternative Pathway

Factor H is an important complement regulator of the alternative pathway commonly recruited by pathogens to achieve increased rates of survival in the human host. The respiratory pathogen Moraxella catarrhalis, which resides in the mucosa, is highly resistant to the bactericidal activity of serum and causes otitis media in children and respiratory tract infections in individuals with underlying diseases. In this study, we show that M. catarrhalis binds factor H via the outer membrane protein OlpA. M. catarrhalis serum resistance was dramatically decreased in the absence of either OlpA or factor H, demonstrating that this inhibition of the alternative pathway significantly contributes to the virulence of M. catarrhalis.

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

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.

Impact of immunization with Protein F on pulmonary clearance of nontypeable Haemophilus influenzae

Nontypeable Haemophilus influenzae (NTHi) is one of the main aetiologies of childhood bacterial infections as well as exacerbations in COPD patients. Currently, no licensed NTHi vaccine exists. In the present study, we evaluated the potential of the conserved and ubiquitous surface protein Haemophilus Protein F (PF) as a vaccine candidate. Our results show that incubation of NTHi with anti-PF antibodies significantly increased the opsonophygocytosis of human promyelocytic leukemia cell line-derived granulocytes, leading to efficient killing of the bacteria (P≤0.05). The presence of anti-PF IgG titers in healthy adults (n=60) was investigated, and we found that 26% of healthy blood donors carried antibodies with the main antigenic epitope being PF(23-48). Finally, mice immunized with PF(23-48) attained a significantly increased capacity to clear NTHi as compared to a control group immunized with a peptide derived from Moraxella catarrhalis β-lactamase (P≤0.05). Taken together, our results indicate that PF is a potential NTHi-vaccine candidate.