EnP1, a Microsporidian Spore Wall Protein That Enables Spores To Adhere to and Infect Host Cells In Vitro

ABSTRACT Microsporidia are spore-forming fungal pathogens that require the intracellular environment of host cells for propagation. We have shown that spores of the genus Encephalitozoon adhere to host cell surface glycosaminoglycans (GAGs) in vitro and that this adherence serves to modulate the infection process. In this study, a spore wall protein (EnP1; Encephalitozoon cuniculi ECU01_0820) from E. cuniculi and Encephalitozoon intestinalis is found to interact with the host cell surface. Analysis of the amino acid sequence reveals multiple heparin-binding motifs, which are known to interact with extracellular matrices. Both recombinant EnP1 protein and purified EnP1 antibody inhibit spore adherence, resulting in decreased host cell infection. Furthermore, when the N-terminal heparin-binding motif is deleted by site-directed mutagenesis, inhibition of adherence is ablated. Our transmission immunoelectron microscopy reveals that EnP1 is embedded in the microsporidial endospore and exospore and is found in high abundance in the polar sac/anchoring disk region, an area from which the everting polar tube is released. Finally, by using a host cell binding assay, EnP1 is shown to bind host cell surfaces but not to those that lack surface GAGs. Collectively, these data show that given its expression in both the endospore and the exospore, EnP1 is a microsporidian cell wall protein that may function both in a structural capacity and in modulating in vitro host cell adherence and infection.

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Role of Sulfated Glycans in Adherence of the Microsporidian Encephalitozoon intestinalis to Host Cells In Vitro

Infection and Immunity ◽

10.1128/iai.73.2.841-848.2005 ◽

2005 ◽

Vol 73 (2) ◽

pp. 841-848 ◽

Cited By ~ 59

Author(s):

J. Russell Hayman ◽

Timothy R. Southern ◽

Theodore E. Nash

Keyword(s):

Cell Surface ◽

Host Cell ◽

Mutant Cell ◽

Host Cells ◽

Target Cells ◽

Cell Infection ◽

Encephalitozoon Intestinalis ◽

Successful Infection ◽

Host Cell Surface ◽

Spore Adherence

ABSTRACT Microsporidia are obligate intracellular opportunistic protists that infect a wide variety of animals, including humans, via environmentally resistant spores. Infection requires that spores be in close proximity to host cells so that the hollow polar tube can pierce the cell membrane and inject the spore contents into the cell cytoplasm. Like other eukaryotic microbes, microsporidia may use specific mechanisms for adherence in order to achieve target cell proximity and increase the likelihood of successful infection. Our data show that Encephalitozoon intestinalis exploits sulfated glycans such as the cell surface glycosaminoglycans (GAGs) in selection of and attachment to host cells. When exogenous sulfated glycans are used as inhibitors in spore adherence assays, E. intestinalis spore adherence is reduced by as much as 88%. However, there is no inhibition when nonsulfated glycans are used, suggesting that E. intestinalis spores utilize sulfated host cell glycans in adherence. These studies were confirmed by exposure of host cells to xylopyranoside, which limits host cell surface GAGs, and sodium chlorate, which decreases surface sulfation. Spore adherence studies with CHO mutant cell lines that are deficient in either surface GAGs or surface heparan sulfate also confirmed the necessity of sulfated glycans. Furthermore, when spore adherence is inhibited, host cell infection is reduced, indicating a direct association between spore adherence and infectivity. These data show that E. intestinalis specifically adheres to target cells by way of sulfated host cell surface GAGs and that this mechanism serves to enhance infectivity.

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Computational and in vitro experimental analyses of the anti-COVID-19 potential of Mortaparib and MortaparibPlus

Bioscience Reports ◽

10.1042/bsr20212156 ◽

2021 ◽

Vol 41 (10) ◽

Author(s):

Vipul Kumar ◽

Anissa Nofita Sari ◽

Hazna Noor Meidinna ◽

Jaspreet Kaur Dhanjal ◽

Chandru Subramani ◽

...

Keyword(s):

Cell Surface ◽

Host Cell ◽

Antiviral Drug ◽

Receptor Expression ◽

Control Measures ◽

Host Cells ◽

Cell Surface Receptor ◽

Viral Inhibition ◽

Host Cell Surface

Abstract Coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has become a global health emergency. Although new vaccines have been generated and being implicated, discovery and application of novel preventive and control measures are warranted. We aimed to identify compounds that may possess the potential to either block the entry of virus to host cells or attenuate its replication upon infection. Using host cell surface receptor expression (angiotensin-converting enzyme 2 (ACE2) and Transmembrane protease serine 2 (TMPRSS2)) analysis as an assay, we earlier screened several synthetic and natural compounds and identified candidates that showed ability to down-regulate their expression. Here, we report experimental and computational analyses of two small molecules, Mortaparib and MortaparibPlus that were initially identified as dual novel inhibitors of mortalin and PARP-1, for their activity against SARS-CoV-2. In silico analyses showed that MortaparibPlus, but not Mortaparib, stably binds into the catalytic pocket of TMPRSS2. In vitro analysis of control and treated cells revealed that MortaparibPlus caused down-regulation of ACE2 and TMPRSS2; Mortaparib did not show any effect. Furthermore, computational analysis on SARS-CoV-2 main protease (Mpro) that also predicted the inhibitory activity of MortaparibPlus. However, cell-based antiviral drug screening assay showed 30–60% viral inhibition in cells treated with non-toxic doses of either MortaparibPlus or Mortaparib. The data suggest that these two closely related compounds possess multimodal anti-COVID-19 activities. Whereas MortaparibPlus works through direct interactions/effects on the host cell surface receptors (ACE2 and TMPRSS2) and the virus protein (Mpro), Mortaparib involves independent mechanisms, elucidation of which warrants further studies.

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Computational and in vitro experimental analyses of the Anti-COVID-19 potential of Mortaparib and MortaparibPlus

Bioscience Reports ◽

10.1042/bcj20210626 ◽

2021 ◽

Author(s):

Vipul Kumar ◽

Anissa Nofita Sari ◽

Hazna Noor Meidinna ◽

Jaspreet Kaur Dhanjal ◽

Chandru Subramani ◽

...

Keyword(s):

Cell Surface ◽

Host Cell ◽

Receptor Expression ◽

Control Measures ◽

Host Cells ◽

Cell Surface Receptor ◽

Virus Protein ◽

Viral Inhibition ◽

Host Cell Surface

COVID-19 pandemic caused by SARS-CoV-2 virus has become a global health emergency. Although new vaccines have been generated and being implicated, discovery and application of novel preventive and control measures are warranted. We aimed to identify compound/s that may possess the potential to either block the entry of virus to host cells or attenuate its replication upon infection. Using host cell surface receptor expression (Angiotensin-converting enzyme 2 (ACE2) and Transmembrane protease serine 2 (TMPRSS2) analysis as an assay, we earlier screened several synthetic and natural compounds and identified candidates that showed ability to downregulate their expression. Here, we report experimental and computational analyses of two small molecules, Mortaparib and MortaparibPlus that were initially identified as dual novel inhibitors of mortalin and PARP-1, for their activity against SARS-CoV-2. In silico analyses showed that MortaparibPlus, but not Mortaparib, stably binds into the catalytic pocket of TMPRSS2. In vitro analysis of control and treated cells revealed that MortaparibPlus caused downregulation of ACE2 and TMPRSS2; Mortaparib did not show any effect. Furthermore, computational analysis on SARS-CoV-2 main protease (Mpro) that also predicted the inhibitory activity of MortaparibPlus. However, cell based anti-virus drug screening assay showed 30~60% viral inhibition in cells treated with non-toxic doses of either MortaparibPlus or Mortaparib. The data suggests that these two closely related compounds possess multimodal anti-COVID 19 activities. Whereas MortaparibPlus works through direct interactions/effects on the host cell surface receptors (ACE2 and TMPRSS2) and the virus protein (Mpro), Mortaparib involves independent mechanisms, elucidation of which warrants further studies.

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A Single-Pass Type I Membrane Protein from the Apicomplexan Parasite Cryptosporidium parvum with Nanomolar Binding Affinity to Host Cell Surface

Microorganisms ◽

10.3390/microorganisms9051015 ◽

2021 ◽

Vol 9 (5) ◽

pp. 1015

Author(s):

Tianyu Zhang ◽

Xin Gao ◽

Dongqiang Wang ◽

Jixue Zhao ◽

Nan Zhang ◽

...

Keyword(s):

Membrane Protein ◽

Cell Surface ◽

Host Cell ◽

Binding Affinity ◽

Cryptosporidium Parvum ◽

Host Cells ◽

Watery Diarrhea ◽

Type I ◽

Single Pass ◽

Host Cell Surface

Cryptosporidium parvum is a globally recognized zoonotic parasite of medical and veterinary importance. This parasite mainly infects intestinal epithelial cells and causes mild to severe watery diarrhea that could be deadly in patients with weakened or defect immunity. However, its molecular interactions with hosts and pathogenesis, an important part in adaptation of parasitic lifestyle, remain poorly understood. Here we report the identification and characterization of a C. parvum T-cell immunomodulatory protein homolog (CpTIPH). CpTIPH is a 901-aa single-pass type I membrane protein encoded by cgd5_830 gene that also contains a short Vibrio, Colwellia, Bradyrhizobium and Shewanella (VCBS) repeat and relatively long integrin alpha (ITGA) N-terminus domain. Immunofluorescence assay confirmed the location of CpTIPH on the cell surface of C. parvum sporozoites. In congruence with the presence of VCBS repeat and ITGA domain, CpTIPH displayed high, nanomolar binding affinity to host cell surface (i.e., Kd(App) at 16.2 to 44.7 nM on fixed HCT-8 and CHO-K1 cells, respectively). The involvement of CpTIPH in the parasite invasion is partly supported by experiments showing that an anti-CpTIPH antibody could partially block the invasion of C. parvum sporozoites into host cells. These observations provide a strong basis for further investigation of the roles of CpTIPH in parasite-host cell interactions.

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Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.649575 ◽

2021 ◽

Vol 8 ◽

Author(s):

Jingwen Yue ◽

Weihua Jin ◽

Hua Yang ◽

John Faulkner ◽

Xuehong Song ◽

...

Keyword(s):

Lung Cancer ◽

Cell Surface ◽

Heparan Sulfate ◽

Host Cell ◽

Host Cells ◽

Cell Surface Receptor ◽

Spike Protein ◽

Effective Prevention ◽

Heparin Derivatives ◽

Host Cell Surface

The severe acute respiratory syndrome (SARS)-like coronavirus disease (COVID-19) is caused by SARS-CoV-2 and has been a serious threat to global public health with limited treatment. Cellular heparan sulfate (HS) has been found to bind SARS-CoV-2 spike protein (SV2-S) and co-operate with cell surface receptor angiotensin-converting enzyme 2 (ACE2) to mediate SARS-CoV-2 infection of host cells. In this study, we determined that host cell surface SV2-S binding depends on and correlates with host cell surface HS expression. This binding is required for SARS-Cov-2 virus to infect host cells and can be blocked by heparin lyase, HS antagonist surfen, heparin, and heparin derivatives. The binding of heparin/HS to SV2-S is mainly determined by its overall sulfation with potential, minor contribution of specific SV2-S binding motifs. The higher binding affinity of SV2-S G614 mutant to heparin and upregulated HS expression may be one of the mechanisms underlying the higher infectivity of the SARS-CoV-2 G614 variant and the high vulnerability of lung cancer patients to SARS-CoV-2 infection, respectively. The higher host cell infection by SARS-CoV-2 G614 variant pseudovirus and the increased infection caused by upregulated HS expression both can be effectively blocked by heparin lyase and heparin, and possibly surfen and heparin derivatives too. Our findings support blocking HS-SV2-S interaction may provide one addition to achieve effective prevention and/treatment of COVID-19.

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Mechanism of CD150 (SLAM) Down Regulation from the Host Cell Surface by Measles Virus Hemagglutinin Protein

Journal of Virology ◽

10.1128/jvi.78.18.9666-9674.2004 ◽

2004 ◽

Vol 78 (18) ◽

pp. 9666-9674 ◽

Cited By ~ 23

Author(s):

G. Grant Welstead ◽

Eric C. Hsu ◽

Caterina Iorio ◽

Shelly Bolotin ◽

Christopher D. Richardson

Keyword(s):

Cell Surface ◽

Host Cell ◽

Measles Virus ◽

Host Cells ◽

Down Regulation ◽

Cellular Receptors ◽

Regulatory Molecule ◽

Hemagglutinin Protein ◽

Host Cell Surface ◽

H Protein

ABSTRACT Measles virus has been reported to enter host cells via either of two cellular receptors, CD46 and CD150 (SLAM). CD46 is found on most cells of higher primates, while SLAM is expressed on activated B, T, and dendritic cells and is an important regulatory molecule of the immune system. Previous reports have shown that measles virus can down regulate expression of its two cellular receptors on the host cell surface during infection. In this study, the process of down regulation of SLAM by measles virus was investigated. We demonstrated that expression of the hemagglutinin (H) protein of measles virus was sufficient for down regulation. Our studies provided evidence that interactions between H and SLAM in the endoplasmic reticulum (ER) can promote the down regulation of SLAM but not CD46. In addition, we demonstrated that interactions between H and SLAM at the host cell surface can also contribute to SLAM down regulation. These results indicate that two mechanisms involving either intracellular interactions between H and SLAM in the ER or receptor-mediated binding to H at the surfaces of host cells can lead to the down regulation of SLAM during measles virus infection.

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Infectivity and Drug Susceptibility Profiling of Different Leishmania-Host Cell Combinations

Pathogens ◽

10.3390/pathogens9050393 ◽

2020 ◽

Vol 9 (5) ◽

pp. 393

Author(s):

Kyung-Hwa Baek ◽

Laura Piel ◽

Thibault Rosazza ◽

Eric Prina ◽

Gerald F. Späth ◽

...

Keyword(s):

Host Cell ◽

Leishmania Donovani ◽

Early Stage ◽

Drug Susceptibility ◽

Host Cells ◽

Acute Monocytic Leukemia ◽

Cell Infection ◽

Monocytic Leukemia ◽

Vitro Assay

Protozoan parasites of the genus Leishmania are the causative agents of leishmaniasis, a spectrum of a disease that threatens public health worldwide. Although next-generation therapeutics are urgently needed, the early stage of the drug discovery process is hampered by very low hit rates from intracellular Leishmania phenotypic high-throughput screenings. Designing and applying a physiologically relevant in vitro assay is therefore in high demand. In this study, we characterized the infectivity, morphology, and drug susceptibility of different Leishmania and host cell infection combinations. Primary bone marrow-derived macrophage (BMDM) and differentiated human acute monocytic leukemia (THP-1) cells were infected with amastigote or promastigote forms of Leishmania amazonensis and Leishmania donovani. Regardless of host cell types, amastigotes were generally well phagocytosed and showed high infectivity, whereas promastigotes, especially those of L. donovani, had predominantly remained in the extracellular space. In the drug susceptibility test, miltefosine and sodium stibogluconate (SSG) showed varying ranges of activity with 14 and >10-fold differences in susceptibility, depending on the host-parasite pairs, indicating the importance of assay conditions for evaluating antileishmanial activity. Overall, our results suggest that combinations of Leishmania species, infection forms, and host cells must be carefully optimized to evaluate the activity of potential therapeutic compounds against Leishmania.

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Identification of a Neospora caninum Microneme Protein (NcMIC1) Which Interacts with Sulfated Host Cell Surface Glycosaminoglycans

Infection and Immunity ◽

10.1128/iai.70.6.3187-3198.2002 ◽

2002 ◽

Vol 70 (6) ◽

pp. 3187-3198 ◽

Cited By ~ 34

Author(s):

Nadine Keller ◽

Arunasalam Naguleswaran ◽

Angela Cannas ◽

Nathalie Vonlaufen ◽

Marianne Bienz ◽

...

Keyword(s):

Cell Surface ◽

Host Cell ◽

Solid Phase ◽

Neospora Caninum ◽

Host Cells ◽

Sequence Motif ◽

Host Cell Surface ◽

Adhesive Proteins ◽

Microneme Protein ◽

Microneme Proteins

ABSTRACT The invasive stages of apicomplexan parasites enter their host cells through mechanisms which are largely conserved throughout the phylum. Host cell invasion is divided into two distinct events, namely, adhesion onto the host cell surface and the actual host cell entry process. The former is mediated largely through microneme proteins which are secreted at the onset of establishing contact with the host cell surface. Many of the microneme proteins identified so far contain adhesive domains. We here present the genomic and corresponding cDNA sequences coding for a 460-amino-acid (aa) microneme protein in Neospora caninum tachyzoites which, due to its homology to MIC1 in Toxoplasma gondii (TgMIC1), was named NcMIC1. The deduced NcMIC1 polypeptide sequence contains an N-terminal signal peptide of 20 aa followed by two tandemly internal repeats of 48 and 44 aa, respectively. Integrated into each repeat is a CXXXCG sequence motif reminiscent of the thrombospondin-related family of adhesive proteins. The positioning of this motif is strictly conserved in TgMIC1 and NcMIC1. The C-terminal part, comprised of 278 aa, was expressed in Escherichia coli, and antibodies affinity purified on recombinant NcMIC1 were used to confirm the localization within the micronemes by immunofluorescence and immunogold transmission electron microscopy of tachyzoites. Immunohistochemistry of mouse brains infected with tissue cysts showed that expression of this protein is reduced in the bradyzoite stage. Upon initiation of secretion by elevating the temperature to 37°C, NcMIC1 is released into the medium supernatant. NcMIC1 binds to trypsinized, rounded Vero cells, as well as to Vero cell monolayers. Removal of glycosaminoglycans from the host cell surface and modulation of host cell surface glycosaminoglycan sulfation significantly reduces the binding of NcMIC1 to the host cell surface. Solid-phase binding assays employing defined glycosaminoglycans confirmed that NcMIC1 binds to sulfated glycosaminoglycans.

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Interaction and Assembly of Two Novel Proteins in the Spore Wall of the Microsporidian Species Nosema bombycis and Their Roles in Adherence to and Infection of Host Cells

Infection and Immunity ◽

10.1128/iai.03155-14 ◽

2015 ◽

Vol 83 (4) ◽

pp. 1715-1731 ◽

Cited By ~ 18

Author(s):

Donglin Yang ◽

Guoqing Pan ◽

Xiaoqun Dang ◽

Yawei Shi ◽

Chunfeng Li ◽

...

Keyword(s):

Host Cell ◽

Spore Wall ◽

Host Cells ◽

Scaffolding Protein ◽

Environmental Pressures ◽

Content Type ◽

Nosema Bombycis ◽

Major Function ◽

Microsporidian Species

Microsporidia are obligate intracellular parasites with rigid spore walls that protect against various environmental pressures. Despite an extensive description of the spore wall, little is known regarding the mechanism by which it is deposited or the role it plays in cell adhesion and infection. In this study, we report the identification and characterization of two novel spore wall proteins, SWP7 and SWP9, in the microsporidian speciesNosema bombycis. SWP7 and SWP9 are mainly localized to the exospore and endospore of mature spores and the cytoplasm of sporonts, respectively. In addition, a portion of SWP9 is targeted to the spore wall of sporoblasts earlier than SWP7 is. Both SWP7 and SWP9 are specifically colocalized to the spore wall in mature spores. Furthermore, immunoprecipitation, far-Western blotting, unreduced SDS-PAGE, and yeast two-hybrid data demonstrated that SWP7 interacted with SWP9. The chitin binding assay showed that, within the total spore protein, SWP9 and SWP7 can bind to the deproteinated chitin spore coats (DCSCs) ofN. bombycis. However, binding of the recombinant protein rSWP7-His to the DCSCs is dependent on the combination of rSWP9–glutathioneS-transferase (GST) with the DCSCs. Finally, rSWP9-GST, anti-SWP9, and anti-SWP7 antibodies decreased spore adhesion and infection of the host cell. In conclusion, SWP7 and SWP9 may have important structural capacities and play significant roles in modulating host cell adherence and infectionin vitro. A possible major function of SWP9 is as a scaffolding protein that supports other proteins (such as SWP7) that form the integrated spore wall ofN. bombycis.

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Role of G2-S16 Polyanionic Carbosilane Dendrimer in the Prevention of Respiratory Syncytial Virus Infection In Vitro and In Vivo in Mice

Polymers ◽

10.3390/polym13132141 ◽

2021 ◽

Vol 13 (13) ◽

pp. 2141

Author(s):

Ignacio Rodriguez-Izquierdo ◽

Rafael Ceña-Diez ◽

Maria Jesús Serramia ◽

Rosa Rodriguez-Fernández ◽

Isidoro Martínez ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Cell Surface ◽

Host Cells ◽

Carbosilane Dendrimer ◽

Rsv Infection ◽

Host Cell Surface ◽

Syncytial Virus

The respiratory syncytial virus (RSV) causes respiratory infection and bronchiolitis, requiring hospitalization mainly in infants. The interaction between RSV, envelope glycoproteins G and F, and cell surface heparan sulfate proteoglycans (HSPG) is required for binding and entry into the host cells. A G2-S16 polyanionic carbosilane dendrimer was identified as a possible RSV inhibitor. We speculated that the G2-S16 dendrimer adheres to the host cell-surface HSPG, acts through binding to HS receptors, and prevents further RSV infection. The G2-S16 dendrimer was non-toxic when applied intranasally to Balb/c mice, and interestingly enough, this G2-S16 dendrimer inhibits 85% RSV. Therefore, our G2-S16 dendrimer could be a candidate for developing a new possible therapy against RSV infection.

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