| 1. |
- Bahnan, Wael, et al.
(författare)
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A human monoclonal antibody bivalently binding two different epitopes in streptococcal M protein mediates immune function
- 2023
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Ingår i: EMBO Molecular Medicine. - : EMBO. - 1757-4684 .- 1757-4676. ; 15:2, s. 1-21
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Tidskriftsartikel (refereegranskat)abstract
- Group A streptococci have evolved multiple strategies to evade human antibodies, making it challenging to create effective vaccines or antibody treatments. Here, we have generated antibodies derived from the memory B cells of an individual who had successfully cleared a group A streptococcal infection. The antibodies bind with high affinity in the central region of the surface-bound M protein. Such antibodies are typically non-opsonic. However, one antibody could effectively promote vital immune functions, including phagocytosis and in vivo protection. Remarkably, this antibody primarily interacts through a bivalent dual-Fab cis mode, where the Fabs bind to two distinct epitopes in the M protein. The dual-Fab cis-binding phenomenon is conserved across different groups of M types. In contrast, other antibodies binding with normal single-Fab mode to the same region cannot bypass the M protein's virulent effects. A broadly binding, protective monoclonal antibody could be a candidate for anti-streptococcal therapy. Our findings highlight the concept of dual-Fab cis binding as a means to access conserved, and normally non-opsonic regions, regions for protective antibody targeting.
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| 2. |
- Bahnan, Wael, et al.
(författare)
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Pathogenic Yersinia Promotes Its Survival by Creating an Acidic Fluid-Accessible Compartment on the Macrophage Surface
- 2015
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:8
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Tidskriftsartikel (refereegranskat)abstract
- Microbial pathogens and host immune cells each initiate events following their interaction in an attempt to drive the outcome to their respective advantage. Here we show that the bacterial pathogen Yersinia pseudotuberculosis sustains itself on the surface of a macrophage by forming acidic fluid-accessible compartments that are partially bounded by the host cell plasma membrane. These Yersinia-containing acidic compartments (YACs) are bereft of the early endosomal marker EEA1 and the lysosomal antigen LAMP1 and readily form on primary macrophages as well as macrophage-like cell lines. YAC formation requires the presence of the Yersinia virulence plasmid which encodes a type III secretion system. Unexpectedly, we found that the initial formation of YACs did not require translocation of the type III effectors into the host cell cytosol; however, the duration of YACs was markedly greater in infections using translocation-competent Y. pseudotuberculosis strains as well as strains expressing the effector YopJ. Furthermore, it was in this translocation- and YopJ-dependent phase of infection that the acidic environment was critical for Y. pseudotuberculosis survival during its interaction with macrophages. Our findings indicate that during its extracellular phase of infection Y. pseudotuberculosis initiates and then, by a separate mechanism, stabilizes the formation of a highly intricate structure on the surface of the macrophage that is disengaged from the endocytic pathway.
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| 3. |
- Bahnan, Wael, et al.
(författare)
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Spike-Dependent Opsonization Indicates Both Dose-Dependent Inhibition of Phagocytosis and That Non-Neutralizing Antibodies Can Confer Protection to SARS-CoV-2
- 2022
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Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Spike-specific antibodies are central to effective COVID19 immunity. Research efforts have focused on antibodies that neutralize the ACE2-Spike interaction but not on non-neutralizing antibodies. Antibody-dependent phagocytosis is an immune mechanism enhanced by opsonization, where typically, more bound antibodies trigger a stronger phagocyte response. Here, we show that Spike-specific antibodies, dependent on concentration, can either enhance or reduce Spike-bead phagocytosis by monocytes independently of the antibody neutralization potential. Surprisingly, we find that both convalescent patient plasma and patient-derived monoclonal antibodies lead to maximum opsonization already at low levels of bound antibodies and is reduced as antibody binding to Spike protein increases. Moreover, we show that this Spike-dependent modulation of opsonization correlate with the outcome in an experimental SARS-CoV-2 infection model. These results suggest that the levels of anti-Spike antibodies could influence monocyte-mediated immune functions and propose that non-neutralizing antibodies could confer protection to SARS-CoV-2 infection by mediating phagocytosis.
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| 4. |
- de Neergaard, Therese, et al.
(författare)
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Invasive Streptococcal Infection Can Lead to the Generation of Cross-Strain Opsonic Antibodies
- 2022
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Ingår i: Microbiology spectrum. - : American Society for Microbiology. - 2165-0497. ; 10:6
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Tidskriftsartikel (refereegranskat)abstract
- The human pathogen Streptococcus pyogenes causes substantial morbidity and mortality. It is unclear if antibodies developed after infections with this pathogen are opsonic and if they are strain specific or more broadly protective. Here, we quantified the opsonic-antibody response following invasive S. pyogenes infection. Four patients with S. pyogenes bacteremia between 2018 and 2020 at Skåne University Hospital in Lund, Sweden, were prospectively enrolled. Acute- and convalescent-phase sera were obtained, and the S. pyogenes isolates were genome sequenced ( emm118, emm85, and two emm1 isolates). Quantitative antibody binding and phagocytosis assays were used to evaluate isolate-dependent opsonic antibody function in response to infection. Antibody binding increased modestly against the infecting isolate and across emm types in convalescent- compared to acute-phase sera for all patients. For two patients, phagocytosis increased in convalescent-phase serum both for the infecting isolate and across types. The increase was only across types for one patient, and one had no improvement. No correlation to the clinical outcomes was observed. Invasive S. pyogenes infections result in a modestly increased antibody binding with differential opsonic capacity, both nonfunctional binding and broadly opsonic binding across types. These findings question the dogma that an invasive infection should lead to a strong type-specific antibody increase rather than a more modest but broadly reactive response, as seen in these patients. Furthermore, our results indicate that an increase in antibody titers might not be indicative of an opsonic response and highlight the importance of evaluating antibody function in S. pyogenes infections. IMPORTANCE The bacterium Streptococcus pyogenes is a common cause of both mild and severe human diseases resulting in substantial morbidity and mortality each year. No vaccines are available, and our understanding of the antibody response to this human pathogen is still incomplete. Here, we carefully analyzed the opsonic antibody response following invasive infection in four patients. Unexpectedly, the patients did not always generate opsonic antibodies against the specific infecting strain. Instead, we found that some patients could generate cross-opsonic antibodies, leading to phagocytosis of bacteria across strains. The emergence of cross-opsonic antibodies is likely important for long-term immunity against S. pyogenes. Our findings question the dogma that mostly strain-specific immunity is developed after infection and add to our overall understanding of how immunity to S. pyogenes can evolve.
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| 5. |
- Engström, Patrik, et al.
(författare)
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Expansion of the Chlamydia trachomatis inclusion does not require bacterial replication
- 2015
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Ingår i: International Journal of Medical Microbiology. - : Elsevier BV. - 1438-4221 .- 1618-0607. ; 305:3, s. 378-382
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Tidskriftsartikel (refereegranskat)abstract
- Chlamydia trachomatis replication takes place inside of a host cell, exclusively within a vacuole known as the inclusion. During an infection, the inclusion expands to accommodate the increasing numbers of C. trachomatis. However, whether inclusion expansion requires bacterial replication and/or de novo protein synthesis has not been previously investigated in detail. Therefore, using a chemical biology approach, we herein investigated C. trachomatis inclusion expansion under varying conditions in vitro. Under normal cell culture conditions, inclusion expansion correlated with C trachomatis replication. When bacterial replication was inhibited using KSK120: an inhibitor that targets C. trachomatis glucose metabolism, inclusions expanded even in the absence of bacterial replication. In contrast, when bacterial protein synthesis was inhibited using chloramphenicol, expansion of inclusions was blocked. Together, these data suggest that de novo protein synthesis is necessary, whereas bacterial replication is dispensable for C trachomatis inclusion expansion. (C) 2015 The Authors. Published by Elsevier GmbH.
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| 6. |
- Good, James A. D., 1985-, et al.
(författare)
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Thiazolino 2-Pyridone Amide Inhibitors of Chlamydia trachomatis Infectivity
- 2016
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 59:5, s. 2094-2108
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Tidskriftsartikel (refereegranskat)abstract
- The bacterial pathogen Chlamydia trachomatis is a global health burden currently treated with broad-spectrum antibiotics which disrupt commensal bacteria. We recently identified a compound through phenotypic screening that blocked infectivity of this intracellular pathogen without host cell toxicity (compound 1, KSK 120). Herein, we present the optimization of 1 to a class of thiazolino 2-pyridone amides that are highly efficacious (EC50 <= 100 nM) in attenuating infectivity across multiple serovars of C. trachomatis without host cell toxicity. The lead compound 21a exhibits reduced lipophilicity versus 1 and did not affect the growth or viability of representative commensal flora at 50 mu M. In microscopy studies, a highly active fluorescent analogue 37 localized inside the parasitiphorous inclusion, indicative of a specific targeting of bacterial components. In summary, we present a class of small molecules to enable the development of specific treatments for C. trachomatis.
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| 7. |
- Good, James A. D., et al.
(författare)
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Thiazolino 2-pyridone amide isosteres as inhibitors of Chlamydia trachomatis infectivity
- 2017
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 60:22, s. 9393-9399
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Tidskriftsartikel (refereegranskat)abstract
- Chlamydia trachomatis is a global health burden due to its prevalence as a sexually transmitted disease and as the causative agent of the eye infection trachoma. We recently discovered 3-amido thiazolino 2-pyridones which attenuated C. trachomatis infectivity without affecting host cell or commensal bacteria viability. We present here the synthesis and evaluation of nonhydrolyzable amide isosteres based on this class, leading to highly potent 1,2,3-triazole based infectivity inhibitors (EC50 ≤ 20 nM).
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| 8. |
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| 9. |
- Izadi, Arman, et al.
(författare)
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Subclass-switched anti-Spike IgG3 oligoclonal cocktails strongly enhance Fc-mediated opsonization
- 2023
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Ingår i: Proceedings of the National Academy of Sciences. - 1091-6490. ; 120:15
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies play a central role in the immune defense against SARS-CoV-2. Emerging evidence has shown that nonneutralizing antibodies are important for immune defense through Fc-mediated effector functions. Antibody subclass is known to affect downstream Fc function. However, whether the antibody subclass plays a role in anti-SARS-CoV-2 immunity remains unclear. Here, we subclass-switched eight human IgG1 anti-spike monoclonal antibodies (mAbs) to the IgG3 subclass by exchanging their constant domains. The IgG3 mAbs exhibited altered avidities to the spike protein and more potent Fc-mediated phagocytosis and complement activation than their IgG1 counterparts. Moreover, combining mAbs into oligoclonal cocktails led to enhanced Fc- and complement receptor-mediated phagocytosis, superior to even the most potent single IgG3 mAb when compared at equivalent concentrations. Finally, in an in vivo model, we show that opsonic mAbs of both subclasses can be protective against a SARS-CoV-2 infection, despite the antibodies being nonneutralizing. Our results suggest that opsonic IgG3 oligoclonal cocktails are a promising idea to explore for therapy against SARS-CoV-2, its emerging variants, and potentially other viruses.
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| 10. |
- Izadi, Arman, et al.
(författare)
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The hinge-engineered IgG1-IgG3 hybrid subclass IgGh47 potently enhances Fc-mediated function of anti-streptococcal and SARS-CoV-2 antibodies
- 2024
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Ingår i: Nature Communications. - 2041-1723. ; 15, s. 1-22
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Tidskriftsartikel (refereegranskat)abstract
- Streptococcus pyogenes can cause invasive disease with high mortality despite adequate antibiotic treatments. To address this unmet need, we have previously generated an opsonic IgG1 monoclonal antibody, Ab25, targeting the bacterial M protein. Here, we engineer the IgG2-4 subclasses of Ab25. Despite having reduced binding, the IgG3 version promotes stronger phagocytosis of bacteria. Using atomic simulations, we show that IgG3’s Fc tail has extensive movement in 3D space due to its extended hinge region, possibly facilitating interactions with immune cells. We replaced the hinge of IgG1 with four different IgG3-hinge segment subclasses, IgGhxx. Hinge-engineering does not diminish binding as with IgG3 but enhances opsonic function, where a 47 amino acid hinge is comparable to IgG3 in function. IgGh47 shows improved protection against S. pyogenes in a systemic infection mouse model, suggesting that IgGh47 has promise as a preclinical therapeutic candidate. Importantly, the enhanced opsonic function of IgGh47 is generalizable to diverse S. pyogenes strains from clinical isolates. We generated IgGh47 versions of anti-SARS-CoV-2 mAbs to broaden the biological applicability, and these also exhibit strongly enhanced opsonic function compared to the IgG1 subclass. The improved function of the IgGh47 subclass in two distant biological systems provides new insights into antibody function.
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| 11. |
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| 12. |
- Kulén, Martina, et al.
(författare)
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Methyl sulfonamide substituents improve the pharmacokinetic properties of bicyclic 2-pyridone based Chlamydia trachomatis inhibitors
- 2019
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Ingår i: MedChemComm. - : Royal Society of Chemistry. - 2040-2503 .- 2040-2511. ; 10:11, s. 1966-1987
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Tidskriftsartikel (refereegranskat)abstract
- Chlamydia trachomatis infections are a global health problem and new approaches to treat C. trachomatis with drugs of high specificity would be valuable. A library of substituted ring fused 2-pyridones has been synthesized and evaluated for their ability to attenuate C. trachomatis infectivity. In vivo pharmacokinetic studies were performed, with the best candidates demonstrating that a C8-methylsulfonamide substituent improved pharmacokinetic properties important for oral administration. C8-Methyl sulfonamide analogue 30 inhibited C. trachomatis infectivity in low micromolar concentrations. Further pharmacokinetic evaluation at an oral dose of 10 mg kg(-1) showed an apparent bioavailability of 41%, compared to C8-cyclopropyl and -methoxy analogues which had negligible oral uptake. In vitro ADME (absorption, distribution, metabolism and excretion) testing of solubility and Caco-2 cell permeability revealed that both solubility and permeability is greatly improved with the C8-methyl sulfonamide 30, effectively moving it from BCS (Biopharmaceutical Classification System) class IV to II.
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| 13. |
- McCormack, Ryan, et al.
(författare)
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Perforin-2 Protects Host Cells and Mice by Restricting the Vacuole to Cytosol Transitioning of a Bacterial Pathogen
- 2016
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Ingår i: Infection and Immunity. - 0019-9567 .- 1098-5522. ; 84:4, s. 1083-1091
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Tidskriftsartikel (refereegranskat)abstract
- The host-encoded Perforin-2 (encoded by the macrophage-expressed gene 1, Mpeg1), which possesses a pore-forming MACPF domain, reduces the viability of bacterial pathogens that reside within membrane-bound compartments. Here, it is shown that Perforin-2 also restricts the proliferation of the intracytosolic pathogen Listeria monocytogenes. Within a few hours of systemic infection, the massive proliferation of L. monocytogenes in Perforin-2(-/-) mice leads to a rapid appearance of acute disease symptoms. We go on to show in cultured Perforin-2(-/-) cells that the vacuole-to-cytosol transitioning of L. monocytogenes is greatly accelerated. Unexpectedly, we found that in Perforin-2(-/-) macrophages, Listeria-containing vacuoles quickly (<= 15 min) acidify, and that this was coincident with greater virulence gene expression, likely accounting for the more rapid translocation of L. monocytogenes to its replicative niche in the cytosol. This hypothesis was supported by our finding that a L. monocytogenes strain expressing virulence factors at a constitutively high level replicated equally well in Perforin-2(+/+) and Perforin-2(-/-) macrophages. Our findings suggest that the protective role of Perforin-2 against listeriosis is based on it limiting the intracellular replication of the pathogen. This cellular activity of Perforin-2 may derive from it regulating the acidification of Listeria-containing vacuoles, thereby depriving the pathogen of favorable intracellular conditions that promote its virulence gene activity.
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| 14. |
- Mojica, Sergio, 1987-, et al.
(författare)
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Red Fluorescent Chlamydia trachomatis Applied to Live Cell Imaging and Screening for Antibacterial Agents
- 2018
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Ingår i: Frontiers in Microbiology. - : Frontiers Media S.A.. - 1664-302X. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- In this study, we describe the application of a transformed Chlamydia trachomatis strain constitutively expressing the red fluorescent protein mCherry, to allow real-time monitoring of the infection cycle and screening for agents that block replication of C. trachomatis. The red fluorescent C. trachomatis strain was detected autonomously without antibody staining and was equally susceptible to doxycycline as the wild type strain. A high-throughput screening assay was developed using the transformed strain and automated fluorescence microscopy. The assay was used in a pilot screen of a 349 compound library containing natural products from Australian flora and fauna. Compounds with anti-chlamydial activity were tested for dose response and toxicity to host cells and two non-toxic compounds had 50% effective concentration (EC50) values in the low micromolar range. Natural products are valuable sources for drug discovery and the identified Chlamydia growth inhibition may be starting points for future drug development. Live cell imaging was used to visualize growth of the red fluorescent C. trachomatis strain over time. The screening assay reduced workload and reagents compared to an assay requiring immunostaining and could further be used to monitor the development of Chlamydia inclusions and anti-chlamydial effect in real time.
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| 15. |
- Neumann, Ariane, et al.
(författare)
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Streptococcal protein SIC activates monocytes and induces inflammation
- 2021
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Ingår i: iScience. - : Elsevier BV. - 2589-0042. ; 24:4
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Tidskriftsartikel (refereegranskat)abstract
- Streptococcus pyogenes is a major bacterial pathogen in the human population and isolates of the clinically important M1 serotype secrete protein Streptococcal inhibitor of complement (SIC) known to interfere with human innate immunity. Here we find that SIC from M1 bacteria interacts with TLR2 and CD14 on monocytes leading to the activation of the NF-κB and p38 MAPK pathways and the release of several pro-inflammatory cytokines (e.g. TNFα and INFγ). In human plasma, SIC binds clusterin and histidine-rich glycoprotein, and whole plasma, and these two purified plasma proteins enhanced the activation of monocytes by SIC. Isolates of the M55 serotype secrete an SIC homolog, but this protein did not activate monocytes. M1 isolates are common in cases of invasive S. pyogenes infections characterized by massive inflammation, and the results of this study indicate that the pro-inflammatory property of SIC contributes to the pathology of these severe clinical conditions.
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| 16. |
- Núñez-Otero, Carlos, et al.
(författare)
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A 2-pyridone amide inhibitor of transcriptional activity in Chlamydia trachomatis
- 2021
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Ingår i: Antimicrobial Agents and Chemotherapy. - : American Society for Microbiology. - 0066-4804 .- 1098-6596. ; 65:5
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Tidskriftsartikel (refereegranskat)abstract
- Chlamydia trachomatis is a strict intracellular bacterium that causes sexually transmitted infections and eye infections that can lead to lifelong sequelae. Treatment options are limited to broad-spectrum antibiotics that disturb the commensal flora and contribute to selection of antibiotic-resistant bacteria. Hence, development of novel drugs that specifically target C. trachomatis would be beneficial. 2-Pyridone amides are potent and specific inhibitors of Chlamydia infectivity. The first-generation compound KSK120 inhibits the developmental cycle of Chlamydia, resulting in reduced infectivity of progeny bacteria. Here, we show that the improved, highly potent second-generation 2-pyridone amide KSK213 allowed normal growth and development of C. trachomatis, and the effect was only observable upon reinfection of new cells. Progeny elementary bodies (EBs) produced in the presence of KSK213 were unable to activate transcription of essential genes in early development and did not differentiate into the replicative form, the reticulate body (RB). The effect was specific to C. trachomatis since KSK213 was inactive in the closely related animal pathogen Chlamydia muridarum and in Chlamydia caviae. The molecular target of KSK213 may thus be different in C. trachomatis or nonessential in C. muridarum and C. caviae. Resistance to KSK213 was mediated by a combination of amino acid substitutions in both DEAD/DEAH RNA helicase and RNase III, which may indicate inhibition of the transcriptional machinery as the mode of action. 2-Pyridone amides provide a novel antibacterial strategy and starting points for development of highly specific drugs for C. trachomatis infections.
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| 17. |
- Shrestha, Niraj, et al.
(författare)
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The Host-Encoded Heme Regulated Inhibitor (HRI) Facilitates Virulence-Associated Activities of Bacterial Pathogens
- 2013
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:7, s. e68754-
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Tidskriftsartikel (refereegranskat)abstract
- Here we show that cells lacking the heme-regulated inhibitor (HRI) are highly resistant to infection by bacterial pathogens. By examining the infection process in wild-type and HRI null cells, we found that HRI is required for pathogens to execute their virulence-associated cellular activities. Specifically, unlike wild-type cells, HRI null cells infected with the gram-negative bacterial pathogen Yersinia are essentially impervious to the cytoskeleton-damaging effects of the Yop virulence factors. This effect is due to reduced functioning of the Yersinia type 3 secretion (T3S) system which injects virulence factors directly into the host cell cytosol. Reduced T3S activity is also observed in HRI null cells infected with the bacterial pathogen Chlamydia which results in a dramatic reduction in its intracellular proliferation. We go on to show that a HRI-mediated process plays a central role in the cellular infection cycle of the Gram-positive pathogen Listeria. For this pathogen, HRI is required for the post-invasion trafficking of the bacterium to the infected host cytosol. Thus by depriving Listeria of its intracellular niche, there is a highly reduced proliferation of Listeria in HRI null cells. We provide evidence that these infection-associated functions of HRI (an eIF2 alpha kinase) are independent of its activity as a regulator of protein synthesis. This is the first report of a host factor whose absence interferes with the function of T3S secretion and cytosolic access by pathogens and makes HRI an excellent target for inhibitors due to its broad virulence-associated activities.
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| 18. |
- Wrighton, Sebastian, et al.
(författare)
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Group A streptococci induce stronger M protein-fibronectin interaction when specific human antibodies are bound
- 2023
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Ingår i: Frontiers in Microbiology. - : Frontiers Media SA. - 1664-302X. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- Group A streptococcus (GAS) is a highly adapted, human-specific pathogen that is known to manipulate the immune system through various mechanisms. GAS’ M protein constitutes a primary target of the immune system due to its spatial configuration and dominance on the bacterial surface. Antibody responses targeting the M protein have been shown to favor the conserved C region. Such antibodies (Abs) circumvent antigenic escape and efficiently bind to various M types. The ability of GAS to bind to fibronectin (Fn), a high molecular weight glycoprotein of the extracellular matrix, has long been known to be essential for the pathogen’s evolutionary success and fitness. However, some strains lack the ability to efficiently bind Fn. Instead, they have been found to additionally bind Fn via the A-B domains of their M proteins. Here, we show that human Abs can induce increased Fn-binding affinity in M proteins, likely by enhancing the weak A-B domain binding. We found that this enhanced Fn binding leads to a reduction in Ab-mediated phagocytosis, indicating that this constitutes a GAS immune escape mechanism. We could show that the Fc domain of Abs is necessary to trigger this phenomenon and that Ab flexibility may also play a key role. We, moreover, saw that our Abs could enhance Fn binding in 3 out of 5 emm type strains tested, belonging to different clades, making it likely that this is a more generalizable phenomenon. Together our results suggest a novel synergistic interplay of GAS and host proteins which ultimately benefits the bacterium.
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