Biology subjects

Vilhena, C.

Publications and source records attributed to Vilhena, C..

5 recordsLinked to original sources

bioRxiv2025

Environmental cues shape extracellular vesicles biogenesis and function in Streptococcus pneumoniae

The Gram-positive human pathogen Streptococcus pneumoniae adapts its metabolism to the environment during colonization and host invasion. Extracellular vesicles (EVs) are produced by S. pneumoniae in the process of infection but the exact interplay between metabolic adaptation and vesicle formation remains elusive. Here, we demonstrate that exposure to normal human serum induced rearrangement of the pneumococcal cell wall and significantly increased Sp-EVs production. Temperature and pH were critical factors for Sp-EVs formation: 37{degrees}C supported optimal EV production, while bacterial exposure to either basic or acidic environments slowed down pneumococcal EV biogenesis and led to a heterogeneous subpopulation profile. Proteomic analysis revealed that Sp-EVs are enriched in carbon metabolism-related proteins, specially those associated with glycolysis (e.g. eno, gapA, gapN, gpmA, pfkA, pykF, and tpi). Low glucose availability enhanced Sp-EVs production and intracellular ATP level, underlying a relation between metabolic status and EV biogenesis. Functionally, Sp-EVs promoted biofilm formation in both S. pneumoniae and Streptococcus pyogenes. Sp-EVs isolated under glucose-rich conditions enhanced S. pneumoniae biofilms, whereas Sp-EVs from glucose-poor conditions strongly stimulated S. pyogenes biofilm formation. These findings underscore the role of host and environmental cues in shaping pneumococcal EV production, composition, and function, highlighting their potential involvement in metabolic adaptation and interspecies interactions. ImportanceStreptococcus pneumoniae remains a leading cause of morbidity and mortality worldwide, responsible for diseases ranging from community-acquired pneumonia to meningitis and sepsis. It is of great concern in children under the age of five and in low-income countries, despite the existence of vaccination. The success of this pathogen relies on its capacity to adapt to diverse host niches, including the blood, nasopharynx and the lungs. Bacterial extracellular vesicles (EVs) have emerged as mediators of virulence and communication, yet their regulation by environmental and metabolic factors remains poorly understood. By exploring how environmental conditions and nutrient availability shape pneumococcal EV production and how these vesicles contribute to biofilm formation and interspecies interactions, our study contributes to the understanding of how S. pneumoniae applies complex strategies to persist and spread, with potential future applications in infection control and therapeutic strategies.

microbiology↗

bioRxiv2024

MftG is crucial for alcohol metabolism of mycobacteria by linking mycofactocin oxidation to respiration

Mycofactocin is a redox cofactor essential for the alcohol metabolism of mycobacteria.. While the biosynthesis of mycofactocin is well established, the gene mftG, which encodes an oxidoreductase of the glucose-methanol-choline superfamily, remained functionally uncharacterized. Here, we show that MftG enzymes are almost exclusively found in genomes containing mycofactocin biosynthetic genes and are present in 75% of organisms harboring these genes. Gene deletion experiments in Mycolicibacterium smegmatis demonstrated a growth defect of the {Delta}mftG mutant on ethanol as a carbon source, accompanied by an arrest of cell division reminiscent of mild starvation. Investigation of carbon and cofactor metabolism implied a defect in mycofactocin reoxidation. Cell-free enzyme assays and respirometry using isolated cell membranes indicated that MftG acts as a mycofactocin dehydrogenase shuttling electrons toward the respiratory chain. Transcriptomics studies also indicated remodeling of redox metabolism to compensate for a shortage of redox equivalents. In conclusion, this work closes an important knowledge gap concerning the mycofactocin system and adds a new pathway to the intricate web of redox reactions governing the metabolism of mycobacteria.

microbiology↗

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↗

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↗