Cloning and expression of human cyclophilin A and its interaction with human coronavirus NL63 nucleocapsid protein

Magister Scientiae (Medical Bioscience) - MSc(MBS)Coronaviridae family is composed of a number of ribonucleic acid (RNA)-containing viruses currently classified into two genera, the coronavirus and torovirus. The family is classified together with the Arteviridae in the order Nidovirales. Coronaviruses are enveloped single stranded positive sense RNA viruses about 80-160 nm in diameter. The coronavirus is, as in the case of all positive sense RNA virus, a messenger, and the naked RNA is infectious. The 5′-two thirds of the genome encodes for a polyprotein that contains all the enzymes necessary for replication, whereas the 3′-one third encodes for all the structural proteins that mediate viral entry into the host cell. The structural proteins include spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins.Nucleocapsid protein is one of the most crucial structural components of coronaviruses;hence major attention has been focused on characterization of this protein. Some laboratories have demonstrated that this protein interferes with different cellular pathways, thus implying it to be a key regulatory component of the virus (Zakhartchouk, Viswanathan et al. 2005). Furthermore, it has been shown that severe acute respiratory syndrome (SARS)-N protein interacts with cellular proteins, including cyclophilin A (CypA), heterogenous nuclear ribonucleoprotein (hnRNP) A1, human ubiquitin-conjugating enzyme, cyclin dependent kinase (CDK)-cyclin complex protein, Ikappaßalpha (IkBα), cytochrome (Cyt) P450 etc. For the purpose of this study, the focus is based on CypA interaction with human coronavirus (HCoV) NL63-N protein. These interactions might play a role in the pathology of HCoV-NL63. Using glutathione-S-transferase (GST), the interaction of CypA with the nucleocapsid protein can be clearly demonstrated to be direct and specific. Since the N protein is involved in viral RNA packaging to form a helical core, it is suffice to say that both NL63-N and CypA are possibly within the HCoV-NL63 replication/transcription complex and NL63-N/human CypA interaction might function in the regulation of HCoV-NL63 RNA synthesis. In addition, the results will demonstrate that HCoV-NL63-N has only a specific domain for interacting with CypA

On airway host defence during allergic inflammation

Asthma is a chronic inflammatory disease of the airways, affecting and disablingapproximately 300 million people worldwide. The inflammatory profile ischaracterized by infiltration of eosinophils, which are a rich source of factors thatare implicated in tissue remodeling. The chronic inflammatory response and theremodeled phenotype create a hospitable environment for secondary bacterialinfections. In recent years, systemic infections caused by Streptococcuspneumoniae in asthmatics have received global attention. The risk of acquiringpneumonia in patients suffering from asthma is 2-10 fold increased as compared tohealthy individuals. The cause is not known and in this thesis we hypothesized thatthe dysregulated allergic response may impair innate host defenses. Themechanisms being investigated may help to explain how the prolonged anddysregulated inflammatory response increases the vulnerability of asthmatics toinvasive pneumococcal disease. Initially, the regulation of chemokines, inparticular eotaxins, by mast cell proteases was investigated. From this study, wewere able to map the region of eotaxin-3/CCL26 that harbors antimicrobial(COOH-terminal) and anti-endotoxin (NH2-terminal) activity followingproteolytic cleavage with mast cell chymase and tryptase, respectively. However,the receptor activating properties (NH2-terminal) were lost. In a separate study, theanti-endotoxin fragment derived from CCL26 conferred therapeutic benefits in amouse model of LPS-induced inflammation. Furthermore, the interaction ofchemokines, particularly Th-2 chemokines, with osteopontin (OPN) wasinvestigated. OPN is an anionic glycoprotein that is upregulated in asthma and itsexpression increases with the severity of asthma. OPN bound to the COOHterminalof chemokines and completely abolished their antimicrobial activitywithout affecting their NH2-terminal localized functions, including LPSneutralizationand receptor activating properties. To ascertain if whether theeffects of OPN are generic or specific for Th-2 chemokines, we investigated itsinteraction with the classical antimicrobial peptides that are constitutivelyexpressed and upregulated during COPD. Interestingly, OPN bound andneutralized their antimicrobial activity but did not interfere with the muraminidaseactivity and protease inhibitory function of lysozyme and secretory leukocyteprotease inhibitor (SLPI), respectively. These studies suggest that chemokines andantimicrobial peptides can serve as host defense peptides but their actions aremodulated by mast cell proteases and OPN. Therefore, there is an urgent need forstudies focusing on modification of antimicrobial peptides to become resistant toproteolytic cleavage, altered pH and various salt conditions. Also, the elucidationof the novel roles of OPN during allergic inflammation could present potentialpharmaceutical targets. Taken together, this thesis explains several mechanismsthat impair innate host defenses during allergic inflammation

Midkine in Host Defense.

Midkine shares several features in common with antibacterial proteins of the innate immune system. These include growth factor properties, heparin-binding regions, and effects on immune cells (i.e. recruitment and activation of neutrophils and macrophages). Indeed, recent research has demonstrated potent bactericidal and fungicidal activities of midkine. This protein is constitutively expressed at relevant concentrations at barriers of the body, such as in the skin and in the large airways, where the body first encounters potential pathogens. The antibacterial properties of midkine orthologues are preserved during evolution, as exemplified by miple2 of Drosophila. In addition to retinoic acid, gene expression can be promoted by additional factors present at sites of infection, reactive oxygen species, activation of the transcription factor NFκ-B, and hypoxia. In the light of the emerging resistance of pathogenic bacteria to conventional antibiotics, midkine is an interesting molecule that could serve as a template in developing novel pharmaceutical strategies against bacterial and fungal infections, either alone or in combination with conventional antibiotics

Osteopontin binds and modulate functions of eosinophil-recruiting chemokines.

Allergic asthma is characterized by eosinophilic inflammation and airway obstruction. There is also an increased risk of pulmonary infection caused by Streptococcus pneumoniae, in particular during severe asthma where high levels of the glycoprotein, osteopontin (OPN) are present in the airways. Eosinophils can be recruited by chemokines activating the receptor CCR3 including eotaxin-1/CCL11, eotaxin-2/CCL24, eotaxin-3/CCL26, RANTES/CCL5, and MEC/CCL28. In addition to inducing chemotaxis, several of these molecules have defensin-like antibacterial properties. This study set out to elucidate the functional consequences of OPN-binding to eosinophil-recruiting chemokines

Osteopontin That Is Elevated in the Airways during COPD Impairs the Antibacterial Activity of Common Innate Antibiotics.

Bacterial infections of the respiratory tract contribute to exacerbations and disease progression in chronic obstructive pulmonary disease (COPD). There is also an increased risk of invasive pneumococcal disease in COPD. The underlying mechanisms are not fully understood but include impaired mucociliary clearance and structural remodeling of the airways. In addition, antimicrobial proteins that are constitutively expressed or induced during inflammatory conditions are an important part of the airway innate host defense. In the present study, we show that osteopontin (OPN), a multifunctional glycoprotein that is highly upregulated in the airways of COPD patients co-localizes with several antimicrobial proteins expressed in the airways. In vitro, OPN bound lactoferrin, secretory leukocyte peptidase inhibitor (SLPI), midkine, human beta defensin-3 (hBD-3), and thymic stromal lymphopoietin (TSLP) but showed low or no affinity for lysozyme and LL-37. Binding of OPN impaired the antibacterial activity against the important bacterial pathogens Streptococcus pneumoniae and Pseudomonas aeruginosa. Interestingly, OPN reduced lysozyme-induced killing of S. pneumoniae, a finding that could be explained by binding of OPN to the bacterial surface, thereby shielding the bacteria. A fragment of OPN generated by elastase of P. aeruginosa retained some inhibitory effect. Some antimicrobial proteins have additional functions. However, the muramidase-activity of lysozyme and the protease inhibitory function of SLPI were not affected by OPN. Taken together, OPN can contribute to the impairment of innate host defense by interfering with the function of antimicrobial proteins, thus increasing the vulnerability to acquire infections during COPD

High Expression of Midkine in the Airways of Patients with Cystic Fibrosis.

Mutations in the CFTR gene result in impaired host defense during cystic fibrosis (CF), where Pseudomonas aeruginosa becomes a key pathogen. We investigated the expression pattern of the antibacterial growth factor midkine in CF and possible interference with its activity by the altered airway microenvironment. High midkine expression was found in CF lung tissue compared with controls, involving epithelium of the large and small airways, alveoli, and cells of the submucosa (i.e. neutrophils and mast cells). In CF sputum, midkine was present at 100-fold higher levels but was also subject to increased degradation, compared with midkine in sputum from healthy controls. Midkine had a bactericidal effect on P. aeruginosa but increasing salt concentrations and low pH impaired the activity. Molecular modeling suggested that the effects of salt and pH were due to electrostatic screening and a charge-neutralization of the membrane, respectively. Both neutrophil elastase and elastase of P. aeruginosa cleaved midkine to smaller fragments, resulting in impaired bactericidal activity. Thus, midkine is highly expressed in CF but its bactericidal properties may be impaired by the altered microenvironment as reflected by the in vitro conditions used in this study

OPN impairs the bactericidal activity of AMPs.

<p>(A) Interference of OPN with the bactericidal activity of AMPs was investigated using viable counts with <i>S</i>. <i>pneumoniae</i> and <i>P</i>. <i>aeruginosa</i>. Bacteria were grown to mid-logarithmic phase and incubated with AMPs alone (3 μM) or AMPs pre-incubated with OPN at ratio of 1:1 for one hour at 37°C. OPN caused inhibition of the bactericidal activity of all AMPs investigated except for LL-37. The histograms represent mean and standard deviation from three separate experiments. Two-way ANOVA with Sidak’s multiple comparisons test was used for statistical analysis. *<i>P</i>≤0.05, and ****<i>P</i> ≤ 0.0001. (B) Scanning electron microscopy images shows the morphology of bacteria after incubation with AMPs alone or AMPs pre-incubated with OPN (1:1). The AMPs alone permeabilized the membrane and caused leakage of intracellular contents indicating killing of the bacteria, which was confirmed in a parallel viable counts assay. Upon co-incubation of AMPs and OPN, the bacteria remained intact, except in the case of LL-37 and to some extent in the cases of <i>P</i>. <i>aeruginosa</i> (OPN with lactoferrin, lysozyme, and midkine). The scale bars in bottom figures in the right panels are 5 μm.</p

OPN does not influence the muramidase and protease inhibitory functions of lysozyme and SLPI respectively.

<p>(A) Effect of OPN on muramidase activity of lysozyme. Lysozyme was pre-incubated with or without OPN at equimolar concentrations and fluorogenic substrate was added to the mixture and incubated for 1 h at 37°C. The lysozyme activity was determined by the development of fluorescence, which is represented as relative fluorescence units (RFU). (B) Neutrophil elastase inhibitory property of SLPI was investigated in presence of OPN. Equimolar concentrations of OPN and SLPI were pre-incubated for 1 h at 37°C. This mixture was incubated with human neutrophil elastase (NE) (0.05 U/ml) for 20 min at RT. The NE activity was determined by a chromogenic substrate solution by recording the absorbance at 405 nm. The above experiments suggest that OPN cannot influence the enzymatic activities of lysozyme and SLPI.</p

Binding of OPN to antimicrobial proteins.

<p>(A) Surface plasmon resonance (SPR) sensorgrams illustrating interactions between different AMPs (analyte) and immobilized OPN (ligand). The curves were obtained after injection of different concentration of the AMPs (31.25, 62.5, 125, 250, and 500 μM respectively) and analysis shows binding incidence with association and dissociation curves between lactoferrin, SLPI, midkine, hBD-3, TSLP, and OPN. No significant binding to OPN was observed for lysozyme and LL-37. (B) The association rate constants (k_on), the dissociation rate constants (k_off), and the equilibrium binding constants (K_D) as calculated from the SPR. The association kinetics between OPN and neither lysozyme nor LL-37 could be determined (n. d.). (C) ELISA-based analysis of interaction between AMPs and OPN to confirm the binding pattern obtained using SPR. A 96 well plate was coated with 0.25, 1 and 2 μg of AMPs. Thereafter, the wells were incubated with 2 μg OPN, washed, followed by detection of bound OPN with antibodies. The histogram represents mean absorbance at 405 nm with standard deviation for each AMP. The incubations were performed in triplicates.</p

Impairment of the bactericidal activity of AMPs from the OPN-fragment VSS60 that is generated by elastase of <i>P</i>. <i>aeruginosa</i>.

<p>(A) Amino acid sequence of full length OPN and the VSS60-peptide (highlighted in green) which is generated by elastase of <i>P</i>. <i>aeruginosa</i>. The full-length OPN has an RGD-motif providing a binding site for integrins (highlighted in orange). (B) To investigate whether the VSS60-peptide retains the AMP-neutralizing properties of full-length OPN, <i>P</i>. <i>aeruginosa</i> were incubated in either buffer alone, with AMPs (3 μM), or AMPs pre-incubated with VSS60 peptide at a molecular ratio of 1:1 before addition to bacteria and incubated for one hour at 37°C. The antimicrobial activity was determined using viable counts. The VSS60-peptide reduced the bactericidal activity of SLPI, hBD-3, and TSLP. A similar but lower inhibitory activity compare with full length OPN. The histograms represent mean and standard deviation from three separate experiments. Two-way ANOVA with Sidak’s multiple comparisons test was used for statistical analysis. *<i>P</i>≤0.05, and ****<i>P</i> ≤ 0.0001. (C) Biophysical properties of OPN and peptide VSS60 derived from OPN showing number of amino acids, molecular weight, net charge and pI.</p