Aleksandra Milewska

Inactivation of Epidermal Growth Factor by Porphyromonas gingivalis as a Potential Mechanism for Periodontal Tissue Damage

ABSTRACT Porphyromonas gingivalis is a Gram-negative bacterium associated with the development of periodontitis. The evolutionary success of this pathogen results directly from the presence of numerous virulence factors, including peptidylarginine deiminase (PPAD), an enzyme that converts arginine to citrulline in proteins and peptides. Such posttranslational modification is thought to affect the function of many different signaling molecules. Taking into account the importance of tissue remodeling and repair mechanisms for periodontal homeostasis, which are orchestrated by ligands of the epidermal growth factor receptor (EGFR), we investigated the ability of PPAD to distort cross talk between the epithelium and the epidermal growth factor (EGF) signaling pathway. We found that EGF preincubation with purified recombinant PPAD, or a wild-type strain of P. gingivalis, but not with a PPAD-deficient isogenic mutant, efficiently hindered the ability of the growth factor to stimulate epidermal cell proliferation and migration. In addition, PPAD abrogated EGFR-EGF interaction-dependent stimulation of expression of suppressor of cytokine signaling 3 and interferon regulatory factor 1. Biochemical analysis clearly showed that the PPAD-exerted effects on EGF activities were solely due to deimination of the C-terminal arginine. Interestingly, citrullination of two internal Arg residues with human endogenous peptidylarginine deiminases did not alter EFG function, arguing that the C-terminal arginine is essential for EGF biological activity. Cumulatively, these data suggest that the PPAD-activity-abrogating EGF function in gingival pockets may at least partially contribute to tissue damage and delayed healing within P. gingivalis-infected periodontia.

Human Coronavirus HKU1 Spike Protein Uses O-Acetylated Sialic Acid as an Attachment Receptor Determinant and Employs Hemagglutinin-Esterase Protein as a Receptor-Destroying Enzyme

ABSTRACT Human coronavirus (hCoV) HKU1 is one of six hCoVs identified to date and the only one with an unidentified cellular receptor. hCoV-HKU1 encodes a hemagglutinin-esterase (HE) protein that is unique to the group a betacoronaviruses (group 2a). The function of HKU1-HE remains largely undetermined. In this study, we examined binding of the S1 domain of hCoV-HKU1 spike to a panel of cells and found that the S1 could specifically bind on the cell surface of a human rhabdomyosarcoma cell line, RD. Pretreatment of RD cells with neuraminidase (NA) and trypsin greatly reduced the binding, suggesting that the binding was mediated by sialic acids on glycoproteins. However, unlike other group 2a CoVs, e.g., hCoV-OC43, for which 9-O-acetylated sialic acid (9-O-Ac-Sia) serves as a receptor determinant, HKU1-S1 bound with neither 9-O-Ac-Sia-containing glycoprotein(s) nor rat and mouse erythrocytes. Nonetheless, the HKU1-HE was similar to OC43-HE, also possessed sialate-O-acetylesterase activity, and acted as a receptor-destroying enzyme (RDE) capable of eliminating the binding of HKU1-S1 to RD cells, whereas the O-acetylesterase-inactive HKU1-HE mutant lost this capacity. Using primary human ciliated airway epithelial (HAE) cell cultures, the only in vitro replication model for hCoV-HKU1 infection, we confirmed that pretreatment of HAE cells with HE but not the enzymatically inactive mutant blocked hCoV-HKU1 infection. These results demonstrate that hCoV-HKU1 exploits O-Ac-Sia as a cellular attachment receptor determinant to initiate the infection of host cells and that its HE protein possesses the corresponding sialate-O-acetylesterase RDE activity. IMPORTANCE Human coronaviruses (hCoV) are important human respiratory pathogens. Among the six hCoVs identified to date, only hCoV-HKU1 has no defined cellular receptor. It is also unclear whether hemagglutinin-esterase (HE) protein plays a role in viral entry. In this study, we found that, similarly to other members of the group 2a CoVs, sialic acid moieties on glycoproteins are critical receptor determinants for the hCoV-HKU1 infection. Interestingly, the virus seems to employ a type of sialic acid different from those employed by other group 2a CoVs. In addition, we determined that the HKU1-HE protein is an O-acetylesterase and acts as a receptor-destroying enzyme (RDE) for hCoV-HKU1. This is the first study to demonstrate that hCoV-HKU1 uses certain types of O-acetylated sialic acid residues on glycoproteins to initiate the infection of host cells and that the HKU1-HE protein possesses sialate-O-acetylesterase RDE activity.

Human Coronavirus NL63 Utilizes Heparan Sulfate Proteoglycans for Attachment to Target Cells

ABSTRACT Human coronavirus NL63 (HCoV-NL63) is an alphacoronavirus that was first identified in 2004 in the nasopharyngeal aspirate from a 7-month-old patient with a respiratory tract infection. Previous studies showed that HCoV-NL63 and the genetically distant severe acute respiratory syndrome (SARS)-CoV employ the same receptor for host cell entry, angiotensin-converting enzyme 2 (ACE2), but it is largely unclear whether ACE2 interactions are sufficient to allow HCoV-NL63 binding to cells. The present study showed that directed expression of angiotensin-converting enzyme 2 (ACE2) on cells previously resistant to HCoV-NL63 renders them susceptible, showing that ACE2 protein acts as a functional receptor and that its expression is required for infection. However, comparative analysis showed that directed expression or selective scission of the ACE2 protein had no measurable effect on virus adhesion. In contrast, binding of HCoV-NL63 to heparan sulfates was required for viral attachment and infection of target cells, showing that these molecules serve as attachment receptors for HCoV-NL63. IMPORTANCE ACE2 protein was proposed as a receptor for HCoV-NL63 already in 2005, but an in-depth analysis of early events during virus infection had not been performed thus far. Here, we show that the ACE2 protein is required for viral entry but that it is not the primary binding site on the cell surface. Conducted research showed that heparan sulfate proteoglycans function as adhesion molecules, increasing the virus density on cell surface and possibly facilitating the interaction between HCoV-NL63 and its receptor. Obtained results show that the initial events during HCoV-NL63 infection are more complex than anticipated and that a newly described interaction may be essential for understanding the infection process and, possibly, also assist in drug design.

Gingipains: Critical Factors in the Development of Aspiration Pneumonia Caused by Porphyromonas gingivalis

Aspiration pneumonia is a life-threatening infectious disease often caused by oral anaerobic and periodontal pathogens such as Porphyromonas gingivalis. This organism produces proteolytic enzymes, known as gingipains, which manipulate innate immune responses and promote chronic inflammation. Here, we challenged mice with P. gingivalis W83 and examined the role of gingipains in bronchopneumonia, lung abscess formation, and inflammatory responses. Although gingipains were not required for P. gingivalis colonization and survival in the lungs, they were essential for manifestation of clinical symptoms and infection-related mortality. Pathologies caused by wild-type (WT) P. gingivalis W83, including hemorrhage, necrosis, and neutrophil infiltration, were absent from lungs infected with gingipain-null isogenic strains or WT bacteria preincubated with gingipain-specific inhibitors. Damage to lung tissue correlated with systemic inflammatory responses, as manifested by elevated levels of TNF, IL-6, IL-17, and C-reactive protein. These effects were unequivocally dependent on gingipain activity. Gingipain activity was also implicated in the observed increase in IL-17 in lung tissues. Furthermore, gingipains increased platelet counts in the blood and activated platelets in the lungs. Arginine-specific gingipains made a greater contribution to P. gingivalis-related morbidity and mortality than lysine-specific gingipains. Thus, inhibition of gingipain may be a useful adjunct treatment for P. gingivalis-mediated aspiration pneumonia.

Novel polymeric inhibitors of HCoV-NL63

Abstract The human coronavirus NL63 is generally classified as a common cold pathogen, though the infection may also result in severe lower respiratory tract diseases, especially in children, patients with underlying disease, and elderly. It has been previously shown that HCoV-NL63 is also one of the most important causes of croup in children. In the current manuscript we developed a set of polymer-based compounds showing prominent anticoronaviral activity. Polymers have been recently considered as promising alternatives to small molecule inhibitors, due to their intrinsic antimicrobial properties and ability to serve as matrices for antimicrobial compounds. Most of the antimicrobial polymers show antibacterial properties, while those with antiviral activity are much less frequent. A cationically modified chitosan derivative, N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC), and hydrophobically-modified HTCC were shown to be potent inhibitors of HCoV-NL63 replication. Furthermore, both compounds showed prominent activity against murine hepatitis virus, suggesting broader anticoronaviral activity.

Development of loop-mediated isothermal amplification assay for detection of human coronavirus-NL63

Abstract Human coronavirus NL63 was identified in 2004 in the Netherlands. Due to the high prevalence and world-wide distribution of this pathogen, it is essential to develop a sensitive and specific detection assay suitable for use in a routine diagnostic laboratory. Techniques based on PCR or real-time PCR are laborious and expensive. Detailed analysis of the HCoV-NL63 genome permitted the identification of a conserved nucleic acid sequential motif, which was sufficient for the design of a loop-mediated isothermal amplification (LAMP) assay. Evaluation of the method showed that the test is specific to HCoV-NL63 and that it does not cross-react with other respiratory viruses. The detection limit was found to be 1 copy of RNA template per reaction in cell culture supernatants and clinical specimens.

HTCC: Broad Range Inhibitor of Coronavirus Entry

To date, six human coronaviruses have been known, all of which are associated with respiratory infections in humans. With the exception of the highly pathogenic SARS and MERS coronaviruses, human coronaviruses (HCoV-NL63, HCoV-OC43, HCoV-229E, and HCoV-HKU1) circulate worldwide and typically cause the common cold. In most cases, infection with these viruses does not lead to severe disease, although acute infections in infants, the elderly, and immunocompromised patients may progress to severe disease requiring hospitalization. Importantly, no drugs against human coronaviruses exist, and only supportive therapy is available. Previously, we proposed the cationically modified chitosan, N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC), and its hydrophobically-modified derivative (HM-HTCC) as potent inhibitors of the coronavirus HCoV-NL63. Here, we show that HTCC inhibits interaction of a virus with its receptor and thus blocks the entry. Further, we demonstrate that HTCC polymers with different degrees of substitution act as effective inhibitors of all low-pathogenic human coronaviruses.

Porphyromonas gingivalis enzymes enhance infection with human metapneumovirus in vitro

Relatively recently discovered, human metapneumovirus (HMPV) is a human pathogen with worldwide prevalence, accounting for a substantial percentage of respiratory tract diseases. Concurrent viral and bacterial infections enable intricate mechanisms of cooperation between pathogens, which complicate the symptoms and outcome of the disease. Such bilateral interactions are based on the modulation of bacterial growth on epithelium pathologically altered during viral illness and the modulation of immune responses, as well as the enhancement of virus replication by bacterial virulence factors. This study showed that proteases produced by Porphyromonas gingivalis, a Gram-negative bacterium implicated in the development of periodontitis, named gingipains, facilitated HMPV replication in LLC-MK2 cells and may contribute to HMPV pathogenicity in patients with periodontitis. Gingipains at low nanomolar concentrations enabled HMPV replication and allowed virus propagation in vitro. In contrast to previously published data for influenza virus, however, Staphylococcus aureus proteases and human neutrophil elastase did not affect virus replication.

Early events during human coronavirus OC43 entry to the cell

The Coronaviridae family clusters a number of large RNA viruses, which share several structural and functional features. However, members of this family recognize different cellular receptors and exploit different entry routes, what affects their species specificity and virulence. The aim of this study was to determine how human coronavirus OC43 enters the susceptible cell. Using confocal microscopy and molecular biology tools we visualized early events during infection. We found that the virus employs caveolin-1 dependent endocytosis for the entry and the scission of virus-containing vesicles from the cell surface is dynamin-dependent. Furthermore, the vesicle internalization process requires actin cytoskeleton rearrangements. With our research we strove to broaden the understanding of the infection process, which in future may be beneficial for the development of a potential therapeutics.

APOBEC3-mediated restriction of RNA virus replication

APOBEC3 family members are cytidine deaminases with roles in intrinsic responses to infection by retroviruses and retrotransposons, and in the control of other DNA viruses, such as herpesviruses, parvoviruses and hepatitis B virus. Although effects of APOBEC3 members on viral DNA have been demonstrated, it is not known whether they edit RNA genomes through cytidine deamination. Here, we investigated APOBEC3-mediated restriction of Coronaviridae. In experiments in vitro, three human APOBEC3 proteins (A3C, A3F and A3H) inhibited HCoV-NL63 infection and limited production of progeny virus, but did not cause hypermutation of the coronaviral genome. APOBEC3-mediated restriction was partially dependent on enzyme activity, and was reduced by the use of enzymatically inactive APOBEC3. Moreover, APOBEC3 proteins bound to the coronaviral nucleoprotein, and this interaction also affected viral replication. Although the precise molecular mechanism of deaminase-dependent inhibition of coronavirus replication remains elusive, our results further our understanding of APOBEC-mediated restriction of RNA virus infections.