Battista, M. | bioRxiv Science

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↗