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

Immunization with the recombinant Burkholderia pseudomallei outer membrane protein Omp85 induces protective immunity in mice

Burkholderia pseudomallei is resistant to a wide range of antibiotics, leading to relapse and recrudescence of melioidosis after cessation of antibiotic therapy. More effective immunotherapies are needed for better management of melioidosis. We evaluated the prophylactic potential of the immunogenic outer membrane protein Omp85 as a vaccine against murine melioidosis. Immunization of BALB/c mice with recombinant Omp85 (rOmp85) triggered a Th2-type immune response. Up to 70% of the immunized animals were protected against infectious challenge of B. pseudomallei with reduced bacterial load in extrapulmonary organs. Mouse anti-rOmp85 promoted complement-mediated killing and opsonophagocytosis of B. pseudomallei by human polymorphonuclear cells. In conclusion, we demonstrated that B. pseudomallei Omp85 is potentially able to induce protective immunity against melioidosis

Complete Genome Sequence of Encapsulated Haemophilus influenzae Type f KR494, an Invasive Isolate That Caused Necrotizing Myositis.

Haemophilus influenzae serotype f (Hif) is an etiologic agent of bacterial invasive disease. Here, we report the first annotated genome sequence of the Hif strain KR494, which was isolated from a patient suffering from sepsis and necrotizing myositis. The genome sequence will increase the understanding of Hif 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

A novel PBP3 substitution in Haemophilus influenzae confers reduced aminopenicillin susceptibility

BACKGROUND:Identification and characterization of non-typeable Haemophilus influenzae (NTHi) with reduced susceptibility to β-lactam antibiotics due to mutations in penicillin binding protein 3 (PBP3) is a clinical challenge. We analyzed a blood isolate, NTHi93-57485, that was categorized as aminopenicillin resistant but lacked key amino acid substitutions in PBP3 that have previously been associated with reduced aminopenicillin susceptibility. The significance of an alternative amino acid substitution (Y528H) in this isolate was examined.RESULTS:Site-directed mutagenesis of a β-lactam susceptible H. influenzae (NTHi3655) was performed to introduce the Y528H substitution into wild-type ftsI (encoding for PBP3). Disc diffusion screening and broth microdilution determination of MICs for β-lactam agents were done with the NTHi3655-PBP3Y528H mutant and were compared with the NTHi3655 wild-type as well as the original clinical isolate NTHi93-57485. Introduction of the Y528H substitution in NTHi3655 resulted in positive screening for β-lactam resistance. MICs for aminopenicillins were increased in the mutant compared to the wild-type. However, the mutant remained susceptible to aminopenicillins according to EUCAST clinical breakpoints (assuming intravenous treatment) and the introduction of Y528H alone did not increase the resistance to the same level as NTHi93-57485. None of the isolates had frame shift insertions in the acrR gene regulating the AcrAB efflux pump.CONCLUSIONS:In parallel to the previously well-described PBP3-substitutions R517H and N526K, we demonstrate that Y528H confers reduced aminopenicillin susceptibility

A novel vitronectin-binding protein of Pseudomonas aeruginosa for effective infection of the airways

Objectives Pseudomonas aeruginosa is a Gram-negative species that causes chronic and acute infections of the lung, skin, urinary tract and eyes. Most P. aeruginosa isolates are highly resistant to antibiotics and difficult to eradicate due to biofilm formation. The bacterium is known to utilize host proteins by diverse strategies in order to enhance its virulence. Vitronectin is a glycoprotein that is abundant in serum and the extracellular matrix, and is involved in cell adhesion, migration, tissue repair and regulation of the complement cascade. The concentration of vitronectin in the lung reflects the level of inflammation in patients with interstitial lung disease. Furthermore, the production is upregulated in patients with cystic fibrosis, which are often chronically colonised with P. aeruginosa. In this study, we analysed the vitronectin-binding capability of clinical strains and identified the P. aeruginosa surface proteins involved in vitronectin binding. Methods P. aeruginosa clinical isolates (n=64) from the airway (n=36), blood (n=15) and urine (n=13), in addition to the reference strain (PAO1) were analysed in a direct binding assay using [125I]-vitronectin. To identify the vitronectin-binding surface proteins of P. aeruginosa, the outer membrane proteins of PAO1 were separated by 2D-SDS-PAGE and western blotting. Vitronectin binding proteins of P. aeruginosa were recombinantly expressed in Escherichia coli and protein-protein interactions were evaluated by ELISA and flow cytometry. P. aeruginosa transposon mutants obtained from the “P. aeruginosa two-allele library” were analysed for vitronectin binding by [125I]-vitronectin or vitronectin coated to a glass surface. Results Our direct binding assay revealed that P. aeruginosa airway isolates bound significantly more vitronectin in comparison to blood (p=0.02) and urine isolates (p=0.04) (Fig. A). Using an approach consisting of 2D-SDS-PAGE and western blotting, we identified two outer membrane proteins that interacted with vitronectin (Fig. B). Expression of one of those (vitronectin binding protein 1; VnBp1) in an E. coli laboratory strain resulted in VnBp1 on the cell surface, and a vitronectin-binding phenotype. In addition, recombinantly expressed and purified VnBP1 showed a dose-dependent interaction with vitronectin in an ELISA (Fig. C). P. aeruginosa with a transposon insert in the vnBp1 gene bound significantly less vitronectin in comparison to the wild type (p=0.0009). Moreover, vnBp1 deficient mutants also showed significant reduced adherence to vitronectin coated glass slide (p≤0.001) in comparison to the wild type (Fig. D). Conclusions P. aeruginosa isolates cultured from the lung bind significantly more vitronectin in comparison to strains cultured from urine or blood. Vitronectin is recruited at the surface via VnBp1. This mechanism is likely to be of great importance for P. aeruginosa adhesion to the airway epithelial and basal lamina of disrupted airway epithelial cell layer and hence for the colonisation of the respiratory tract

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

The 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 for increased survival in the human host. The respiratory pathogen Moraxella catarrhalis that resides in the mucosa is highly serum resistant 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

The Laminin Interactome : A Multifactorial Laminin-Binding Strategy by Nontypeable Haemophilus influenzae for Effective Adherence and Colonization

Laminin is a well-defined component of the airway basement membrane (BM). Efficient binding of laminin via multiple interactions is important for nontypeable Haemophilusinfluenzae (NTHi) colonization in the airway mucosa. Here we identified elongation factor thermo-unstable (EF-Tu), L-lactate dehydrogenase (LDH), Protein D and peptidoglycan-associated lipoprotein P6 as novel laminin-binding proteins (Lbps) of NTHi. In parallel with other well-studied Lbps (P4, PE, PF and Hap), EF-Tu, LDH, PD and P6 exhibited interactions with laminin, and mediated NTHi laminin-dependent adherence to pulmonary epithelial cell lines. Importantly, the NTHi laminin interactome consisting of the well-studied and novel Lbps recognized laminin LG domains from the subunit α chains of laminin-111 and -332, of which the latter isoform is the main laminin in the airway BM. The NTHi interactome mainly targeted multiple heparin-binding domains of laminin. In conclusion, the NTHi interactome exhibited a high plasticity of interactions with different laminin isoforms via multiple heparin-binding sites