Biology subjects

Zipfel, P. F.

Publications and source records attributed to Zipfel, P. F..

4 recordsLinked to original sources

bioRxiv2023

The role of pneumococcal extracellular vesicles on the pathophysiology of the kidney disease Hemolytic Uremic Syndrome

Streptococcus pneumoniae-induced hemolytic uremic syndrome (Sp-HUS) is a kidney disease characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury. This disease is frequently underdiagnosed and its pathophysiology is poorly understood. In this work, we compared clinical strains, isolated from infant Sp-HUS patients, to a reference pathogenic strain D39, for host cytotoxicity and further explored the role of Sp-derived extracellular vesicles (EVs) in the pathogenesis of a HUS infection. In comparison with the WT strain, pneumococcal HUS strains caused significant lysis of human erythrocytes and increased the release of hydrogen peroxide. Isolated Sp-HUS EVs were characterized by performing dynamic light-scattering microscopy and proteomic analysis. Sp-HUS strain released EVs at a constant concentration during growth, yet the size of the EVs varied and several subpopulations emerged at later time points. The cargo of the Sp-HUS EVs included several virulence factors at high abundance, i.e., the ribosomal subunit assembly factor BipA, the Pneumococcal Surface Protein A (PspA), the lytic enzyme LytC, several sugar utilization and fatty acid synthesis proteins. Sp-HUS EVs strongly downregulated the expression of the endothelial surface marker PECAM-1 and were internalized by human endothelial cells. Sp-HUS EVs elicited the release of pro-inflammatory cytokines (IL-1{beta}, IL-6) and chemokines (CCL2, CCL3, CXCL1) by human monocytes. These findings shed new light on the overall function of Sp-EVs, in the scope of infection-mediated HUS, and suggest new avenues of research for exploring the usefulness of Sp-EVs as therapeutic and diagnostic targets. ImportanceStreptococcus pneumoniae is a life-threatening human pathogen associated with severe illnesses in the upper respiratory tract. Disseminated infections also occur, as the kidney disease hemolytic uremic syndrome. Even though vaccination is available, this pathogen is responsible for a worldwide high mortality rate, especially among children from least developed countries, where vaccination strategies are poor or inexistent. It is estimated that 30% of invasive pneumococcal diseases are caused by antibiotic resistant bacteria, leading to the classification of "serious threat" by the World Health Organization. In order to prevent cases of severe illness, investigation in the direction of new vaccine candidates is of upmost importance. Pneumococcal extracellular vesicles pose as ideal candidates for a serotype-independent vaccine formulation. To this purpose, the aspects of vesicle formation, cargo allocation and function need to be understood in detail. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/526387v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@1f5faeborg.highwire.dtl.DTLVardef@2c6176org.highwire.dtl.DTLVardef@1b29245org.highwire.dtl.DTLVardef@a119ea_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2022

The choline-binding proteins PspA, PspC and LytA of Streptococcus pneumoniae and their role on host cellular adhesion and damage

Streptococcus pneumoniae is a Gram-positive opportunistic pathogen that can colonize the upper respiratory tract. It is a leading cause of a wide range of infectious diseases, including community-acquired pneumonia, meningitis, otitis media and bacteraemia. Pneumococcal infections cause 1-2 million deaths per year, most of which occur in developing countries, where this bacterial species is probably the most important pathogen during early infancy. Here, we focused on choline-binding proteins (CBPs), i.e., PspC, PspA and LytA, and their integration into and interaction with the cell wall of S. pneumoniae. The three pneumococcal proteins have different surface-exposed regions but share related choline-binding anchors. These surface-exposed pneumococcal proteins are in direct contact with host cells and have diverse functions. PspC and PspA bind several host plasma proteins, whereas LytA plays a role in cell division and the lytic phase. We explored the role of the three CBPs on adhesion and pathogenicity in a human host by performing relevant imaging and functional analyses, such as electron microscopy, confocal laser scanning microscopy and functional quantitative assays targeting biofilm formation and the haemolytic capacity of S. pneumoniae. In vitro biofilm formation assays and electron microscopy experiments were used to examine the ability of knockout mutant strains lacking the lytA, pspC or pspA genes to adhere to surfaces. The mutant strains were compared with the S. pneumoniae D39 reference strain. We found that LytA plays an important role in robust synthesis of the biofilm matrix. PspA and PspC appeared crucial for the haemolytic effects of S. pneumoniae on human red blood cells. Furthermore, all knockout mutants caused less damage to endothelial cells than wild-type bacteria, highlighting the significance of CPBs for the overall pathogenicity of S. pneumoniae. Hence, in addition to their structural function within the cell wall of S. pneumoniae, each of these three surface-exposed CBPs controls or mediates multiple steps during bacterial pathogenesis.

immunology↗

bioRxiv2021

The synthetic protein MFHR13 as efficient multitarget regulator against complement over-activation

The complement system constitutes the innate defense against pathogens. Its dysregulation leads to diseases and is a critical determinant in many viral infections, e.g.COVID-19. Factor H (FH) is the main regulator of the alternative pathway of complement activation and could be a therapy to restore homeostasis. However, recombinant FH is not available. Engineered FH versions may present alternative therapeutics. Here, we designed a synthetic protein, MFHR13, as a multitarget complement regulator. It combines the dimerization and C5-regulatory domains of human FH-related protein 1 (FHR1) with the C3-regulatory and cell surface recognition domains of human FH. MFHR13 includes the FH variant I62, which we characterized to induce improved C3b binding and cofactor activity compared to the variant V62. After comparative protein structure modelling, we introduced the SCR FH13, which includes an N-glycosylation site for higher protein stability. In summary, the fusion protein MFHR13 comprises SCRs FHR11-2:FH1-4:FH13:FH19-20. It shows an enhanced heparin binding and protects sheep erythrocytes from complement attack exhibiting 26 and 4-fold the regulatory activity of eculizumab and human FH, respectively. Furthermore, it also blocks the terminal pathway of complement activation and we demonstrate that MFHR13 and FHR1 bind to all proteins forming the membrane attack complex, which contributes to the mechanistic understanding of FHR1. We consider MFHR13 a promising candidate as a therapeutic for complement-associated diseases.

immunology↗

bioRxiv2020

Same, Same, but Different: Molecular Analyses of Streptococcus pneumoniae Immune Evasion Proteins Identifies new Domains and Reveals Structural Differences between PspC and Hic Variants

PspC and Hic proteins of Streptococcus pneumoniae are some of the most variable microbial immune evasion proteins identified to date. Due to structural similarities and conserved binding profiles it was assumed over a long time that these pneumococcal surface proteins represent a protein family, comprising eleven subgroups. Recently, however, by evaluating more proteins larger diversity of individual proteins became apparent. In contrast to previous assumptions a pattern evaluation of six PspC and five Hic variants, each representing one of the previously defined subgroups, revealed distinct structural and likely functionally regions of the proteins, and identified nine new domains and new domain alternates. Several domains are unique to PspC and Hic variants, while other domains are shared with other S. pneumoniae and bacterial virulent determinants. This understanding improved pattern evaluation on the level of full-length proteins, allowed a sequence comparison on the domain level and furthermore identified domains with a modular composition. This novel concept allows a better characterization of variability, and modular domain composition of individual proteins, enables a structural and functional characterization at the domain level and furthermore shows substantial structural differences between PspC and Hic proteins. Such knowledge will also be useful for molecular strain typing, characterizing PspC and Hic proteins from new clinical S. pneumoniae strains, including those derived from patients who present with pneumococcal hemolytic uremic syndrome. Furthermore this analysis explains the role of multifaceted intact PspC and Hic proteins in pathogen host interactions. and can provide a basis for rational vaccine design. Author SummaryThe human pathobiont Streptococcus pneumoniae expresses highly polymorphic PspC or Hic proteins, which bind a repertoire of host immune regulators and combine antigenic variation with conserved immune evasion features. Understanding domain composition of each protein encoded by more than 60 000 pspC or hic genes deposited in the data banks defines their diversity, a role in immune escape and can furthermore delineate structure function approach for single protein domains. PspC and Hic proteins show variable domain composition and sequence diversity, which explain differences in binding of human regulators and likely in immune escape. The results of our analyses provide insights in the domain composition of these diverse immune evasion proteins, identifies new domains, defines domains which are unique to PspC or Hic variants, and identifies domains which are shared with other bacterial immune evasion proteins. These data have implication on cell wall attachment, surface distribution and in immune escape.

microbiology↗