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Neuraminidase B controls neuraminidase A-dependent mucus production and evasion

Hammond, Alexandria J; Binsker, Ulrike; Aggarwal, Surya D; Ortigoza, Mila Brum; Loomis, Cynthia; Weiser, Jeffrey N
Binding of Streptococcus pneumoniae (Spn) to nasal mucus leads to entrapment and clearance via mucociliary activity during colonization. To identify Spn factors allowing for evasion of mucus binding, we used a solid-phase adherence assay with immobilized mucus of human and murine origin. Spn bound large mucus particles through interactions with carbohydrate moieties. Mutants lacking neuraminidase A (nanA) or neuraminidase B (nanB) showed increased mucus binding that correlated with diminished removal of terminal sialic acid residues on bound mucus. The non-additive activity of the two enzymes raised the question why Spn expresses two neuraminidases and suggested they function in the same pathway. Transcriptional analysis demonstrated expression of nanA depends on the enzymatic function of NanB. As transcription of nanA is increased in the presence of sialic acid, our findings suggest that sialic acid liberated from host glycoconjugates by the secreted enzyme NanB induces the expression of the cell-associated enzyme NanA. The absence of detectable mucus desialylation in the nanA mutant, in which NanB is still expressed, suggests that NanA is responsible for the bulk of the modification of host glycoconjugates. Thus, our studies describe a functional role for NanB in sialic acid sensing in the host. The contribution of the neuraminidases in vivo was then assessed in a murine model of colonization. Although mucus-binding mutants showed an early advantage, this was only observed in a competitive infection, suggesting a complex role of neuraminidases. Histologic examination of the upper respiratory tract demonstrated that Spn stimulates mucus production in a neuraminidase-dependent manner. Thus, an increase production of mucus containing secretions appears to be balanced, in vivo, by decreased mucus binding. We postulate that through the combined activity of its neuraminidases, Spn evades mucus binding and mucociliary clearance, which is needed to counter neuraminidase-mediated stimulation of mucus secretions.
PMID: 33819312
ISSN: 1553-7374
CID: 4838982

Type I Interferon Signaling Is a Common Factor Driving Streptococcus pneumoniae and Influenza A Virus Shedding and Transmission

Zangari, Tonia; Ortigoza, Mila B; Lokken-Toyli, Kristen L; Weiser, Jeffrey N
The dynamics underlying respiratory contagion (the transmission of infectious agents from the airways) are poorly understood. We investigated host factors involved in the transmission of the leading respiratory pathogen Streptococcus pneumoniae Using an infant mouse model, we examined whether S. pneumoniae triggers inflammatory pathways shared by influenza A virus (IAV) to promote nasal secretions and shedding from the upper respiratory tract to facilitate transit to new hosts. Here, we show that amplification of the type I interferon (IFN-I) response is a critical host factor in this process, as shedding and transmission by both IAV and S. pneumoniae were decreased in pups lacking the common IFN-I receptor (Ifnar1-/- mice). Additionally, providing exogenous recombinant IFN-I to S. pneumoniae-infected pups was sufficient to increase bacterial shedding. The expression of IFN-stimulated genes (ISGs) was upregulated in S. pneumoniae-infected wild-type (WT) but not Ifnar1-/- mice, including genes involved in mucin type O-glycan biosynthesis; this correlated with an increase in secretions in S. pneumoniae- and IAV-infected WT compared to Ifnar1-/- pups. S. pneumoniae stimulation of ISGs was largely dependent on its pore-forming toxin, pneumolysin, and coinfection with IAV and S. pneumoniae resulted in synergistic increases in ISG expression. We conclude that the induction of IFN-I signaling appears to be a common factor driving viral and bacterial respiratory contagion.IMPORTANCE Respiratory tract infections are a leading cause of childhood mortality and, globally, Streptococcus pneumoniae is the leading cause of mortality due to pneumonia. Transmission of S. pneumoniae primarily occurs through direct contact with respiratory secretions, although the host and bacterial factors underlying transmission are poorly understood. We examined transmission dynamics of S. pneumoniae in an infant mouse model and here show that S. pneumoniae colonization of the upper respiratory tract stimulates host inflammatory pathways commonly associated with viral infections. Amplification of this response was shown to be a critical host factor driving shedding and transmission of both S. pneumoniae and influenza A virus, with infection stimulating expression of a wide variety of genes, including those involved in the biosynthesis of mucin, a major component of respiratory secretions. Our findings suggest a mechanism facilitating S. pneumoniae contagion that is shared by viral infection.
PMID: 33593970
ISSN: 2150-7511
CID: 4799892

An Infant Mouse Model of Influenza Virus Transmission Demonstrates the Role of Virus-Specific Shedding, Humoral Immunity, and Sialidase Expression by Colonizing Streptococcus pneumoniae

Ortigoza, Mila Brum; Blaser, Simone B; Zafar, M Ammar; Hammond, Alexandria J; Weiser, Jeffrey N
The pandemic potential of influenza A viruses (IAV) depends on the infectivity of the host, transmissibility of the virus, and susceptibility of the recipient. While virus traits supporting IAV transmission have been studied in detail using ferret and guinea pig models, there is limited understanding of host traits determining transmissibility and susceptibility because current animal models of transmission are not sufficiently tractable. Although mice remain the primary model to study IAV immunity and pathogenesis, the efficiency of IAV transmission in adult mice has been inconsistent. Here we describe an infant mouse model that supports efficient transmission of IAV. We demonstrate that transmission in this model requires young age, close contact, shedding of virus particles from the upper respiratory tract (URT) of infected pups, the use of a transmissible virus strain, and a susceptible recipient. We characterize shedding as a marker of infectiousness that predicts the efficiency of transmission among different influenza virus strains. We also demonstrate that transmissibility and susceptibility to IAV can be inhibited by humoral immunity via maternal-infant transfer of IAV-specific immunoglobulins and modifications to the URT milieu, via sialidase activity of colonizing Streptococcus pneumoniae Due to its simplicity and efficiency, this model can be used to dissect the host's contribution to IAV transmission and explore new methods to limit contagion.IMPORTANCE This study provides insight into the role of the virus strain, age, immunity, and URT flora on IAV shedding and transmission efficiency. Using the infant mouse model, we found that (i) differences in viral shedding of various IAV strains are dependent on specific hemagglutinin (HA) and/or neuraminidase (NA) proteins, (ii) host age plays a key role in the efficiency of IAV transmission, (iii) levels of IAV-specific immunoglobulins are necessary to limit infectiousness, transmission, and susceptibility to IAV, and (iv) expression of sialidases by colonizing S. pneumoniae antagonizes transmission by limiting the acquisition of IAV in recipient hosts. Our findings highlight the need for strategies that limit IAV shedding and the importance of understanding the function of the URT bacterial composition in IAV transmission. This work reinforces the significance of a tractable animal model to study both viral and host traits affecting IAV contagion and its potential for optimizing vaccines and therapeutics that target disease spread.
PMID: 30563897
ISSN: 2150-7511
CID: 3556562


Wu, Patrick B; Ortigoza, Mila; Eiras, Daniel
ISSN: 1525-1497
CID: 2481902

A novel small molecule inhibitor of influenza A viruses that targets polymerase function and indirectly induces interferon

Ortigoza, Mila Brum; Dibben, Oliver; Maamary, Jad; Martinez-Gil, Luis; Leyva-Grado, Victor H; Abreu, Pablo Jr; Ayllon, Juan; Palese, Peter; Shaw, Megan L
Influenza viruses continue to pose a major public health threat worldwide and options for antiviral therapy are limited by the emergence of drug-resistant virus strains. The antiviral cytokine, interferon (IFN) is an essential mediator of the innate immune response and influenza viruses, like many viruses, have evolved strategies to evade this response, resulting in increased replication and enhanced pathogenicity. A cell-based assay that monitors IFN production was developed and applied in a high-throughput compound screen to identify molecules that restore the IFN response to influenza virus infected cells. We report the identification of compound ASN2, which induces IFN only in the presence of influenza virus infection. ASN2 preferentially inhibits the growth of influenza A viruses, including the 1918 H1N1, 1968 H3N2 and 2009 H1N1 pandemic strains and avian H5N1 virus. In vivo, ASN2 partially protects mice challenged with a lethal dose of influenza A virus. Surprisingly, we found that the antiviral activity of ASN2 is not dependent on IFN production and signaling. Rather, its IFN-inducing property appears to be an indirect effect resulting from ASN2-mediated inhibition of viral polymerase function, and subsequent loss of the expression of the viral IFN antagonist, NS1. Moreover, we identified a single amino acid mutation at position 499 of the influenza virus PB1 protein that confers resistance to ASN2, suggesting that PB1 is the direct target. This two-pronged antiviral mechanism, consisting of direct inhibition of virus replication and simultaneous activation of the host innate immune response, is a unique property not previously described for any single antiviral molecule.
PMID: 22577360
ISSN: 1553-7374
CID: 2707492

Identification of small molecules with type I interferon inducing properties by high-throughput screening

Martinez-Gil, Luis; Ayllon, Juan; Ortigoza, Mila Brum; Garcia-Sastre, Adolfo; Shaw, Megan L; Palese, Peter
The continuous emergence of virus that are resistant to current anti-viral drugs, combined with the introduction of new viral pathogens for which no therapeutics are available, creates an urgent need for the development of novel broad spectrum antivirals. Type I interferon (IFN) can, by modulating the cellular expression profile, stimulate a non-specific antiviral state. The antiviral and adjuvant properties of IFN have been extensively demonstrated; however, its clinical application has been so far limited. We have developed a human cell-based assay that monitors IFN-beta production for use in a high throughput screen. Using this assay we screened 94,398 small molecules and identified 18 compounds with IFN-inducing properties. Among these, 3 small molecules (C3, E51 and L56) showed activity not only in human but also in murine and canine derived cells. We further characterized C3 and showed that this molecule is capable of stimulating an anti-viral state in human-derived lung epithelial cells. Furthermore, the IFN-induction by C3 is not diminished by the presence of influenza A virus NS1 protein or hepatitis C virus NS3/4A protease, which make this molecule an interesting candidate for the development of a new type of broad-spectrum antiviral. In addition, the IFN-inducing properties of C3 also suggest its potential use as vaccine adjuvant.
PMID: 23145065
ISSN: 1932-6203
CID: 2707482

Human host factors required for influenza virus replication

Konig, Renate; Stertz, Silke; Zhou, Yingyao; Inoue, Atsushi; Hoffmann, H-Heinrich; Bhattacharyya, Suchita; Alamares, Judith G; Tscherne, Donna M; Ortigoza, Mila B; Liang, Yuhong; Gao, Qinshan; Andrews, Shane E; Bandyopadhyay, Sourav; De Jesus, Paul; Tu, Buu P; Pache, Lars; Shih, Crystal; Orth, Anthony; Bonamy, Ghislain; Miraglia, Loren; Ideker, Trey; Garcia-Sastre, Adolfo; Young, John A T; Palese, Peter; Shaw, Megan L; Chanda, Sumit K
Influenza A virus is an RNA virus that encodes up to 11 proteins and this small coding capacity demands that the virus use the host cellular machinery for many aspects of its life cycle. Knowledge of these host cell requirements not only informs us of the molecular pathways exploited by the virus but also provides further targets that could be pursued for antiviral drug development. Here we use an integrative systems approach, based on genome-wide RNA interference screening, to identify 295 cellular cofactors required for early-stage influenza virus replication. Within this group, those involved in kinase-regulated signalling, ubiquitination and phosphatase activity are the most highly enriched, and 181 factors assemble into a highly significant host-pathogen interaction network. Moreover, 219 of the 295 factors were confirmed to be required for efficient wild-type influenza virus growth, and further analysis of a subset of genes showed 23 factors necessary for viral entry, including members of the vacuolar ATPase (vATPase) and COPI-protein families, fibroblast growth factor receptor (FGFR) proteins, and glycogen synthase kinase 3 (GSK3)-beta. Furthermore, 10 proteins were confirmed to be involved in post-entry steps of influenza virus replication. These include nuclear import components, proteases, and the calcium/calmodulin-dependent protein kinase (CaM kinase) IIbeta (CAMK2B). Notably, growth of swine-origin H1N1 influenza virus is also dependent on the identified host factors, and we show that small molecule inhibitors of several factors, including vATPase and CAMK2B, antagonize influenza virus replication.
PMID: 20027183
ISSN: 1476-4687
CID: 2707502

The differential binding and activity of PRO 2000 against diverse HIV-1 envelopes

Sachdev, Darpun D; Zerhouni-Layachi, Bouchra; Ortigoza, Mila; Profy, Albert T; Tuen, Michael; Hioe, Catarina E; Klotman, Mary E
OBJECTIVE: PRO 2000 is a polyanionic microbicide that binds directly to the glycoprotein 120 (gp120) envelope protein to inhibit HIV-1 entry. We studied the breadth of PRO 2000 activity against HIV-1 derived from recently transmitted R5 viruses. We also investigated the interaction of this compound with X4 and R5 HIV-1 envelope glycoproteins using an epitope-mapping strategy. METHODS: The anti-HIV activity of PRO 2000 against subtype B and C Env-pseudotyped viruses was assessed in saline and cervicovaginal lavage fluid. Competitive binding assays were performed with X4 and R5 monomeric and virus-associated gp120. RESULTS: PRO 2000 was found to be active against recently transmitted subtype B and C viruses tested in vitro, however, at 1 microg/mL in saline, activity against subtype C was decreased compared with subtype B. Epitope mapping using anti-V3 region antibodies showed that PRO 2000 binds to the V3 region of monomeric and virus-associated X4 gp120 with a higher affinity than to V3 of R5 gp120. In contrast, the interaction of PRO 2000 with the CD4-binding site was similar for both X4 and R5 monomeric and virus-associated gp120. CONCLUSIONS: PRO 2000 has significant activity against recently transmitted viruses, although some activity is lost at low concentrations. Epitope binding studies suggest that this broad activity is due to direct and indirect interactions with multiple gp120 sites rather than V3 binding alone
PMID: 19349871
ISSN: 1525-4135
CID: 107749

Influenza A virus PB1-F2 protein contributes to viral pathogenesis in mice

Zamarin, Dmitriy; Ortigoza, Mila B; Palese, Peter
The influenza virus PB1-F2 protein is a novel protein previously shown to be involved in induction of cell death. Here we characterize the expression and the function of the protein within the context of influenza viral infection in tissue culture and a mouse model. We show that the C-terminal region of the protein can be expressed from a downstream initiation codon and is capable of interaction with the full-length protein. Using this knowledge, we generated influenza viruses knocked out for the expression of PB1-F2 protein and its downstream truncation products. Knocking out the PB1-F2 protein had no effect on viral replication in tissue culture but diminished virus pathogenicity and mortality in mice. The viruses replicated to similar levels in mouse lungs by day 3 postinfection, suggesting that the knockout did not impair viral replication. However, while the PB1-F2 knockout viruses were cleared after day 5, the wild-type viruses were detectable in mouse lungs until day 7, implying that expression of PB1-F2 resulted in delayed clearance of the viruses by the host immune system. Based on our findings and on the fact that the PB1 genomic segment was always newly introduced into some pandemic influenza viruses of the last century, we speculate that the PB1-F2 protein plays an important role in pathogenesis of influenza virus infection and may be an important contributor to pathogenicity of pandemic influenza viruses.
PMID: 16873254
ISSN: 0022-538x
CID: 2707512