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Characterization of Streptococcal Nucleases and Identification of their Host Interactomes

Kostaloupi, Styliani LU (2025) KEMR45 20251
Department of Chemistry
Abstract
Streptococcus pneumoniae and Streptococcus pyogenes are Gram-positive bacteria responsible for a wide spectrum of diseases, ranging from mild to even life-threatening. The annual global mortality rates of the bacteria amount to 1.68 million. While vaccines against S. pneumoniae are included in our medical arsenal, their efficacy is limited by poor immune responses in certain age groups or insufficient serotype coverage. Regarding S. pyogenes, no licensed vaccine exists as of today. These shortcomings, paired with the emergence of antibiotic resistance, significantly impair the effectiveness of current treatment strategies. Nucleases such as S. pyogenes Nuclease A (SpnA), extracellular streptodornase (Sda1) and S. pneumoniae DNA/RNA... (More)
Streptococcus pneumoniae and Streptococcus pyogenes are Gram-positive bacteria responsible for a wide spectrum of diseases, ranging from mild to even life-threatening. The annual global mortality rates of the bacteria amount to 1.68 million. While vaccines against S. pneumoniae are included in our medical arsenal, their efficacy is limited by poor immune responses in certain age groups or insufficient serotype coverage. Regarding S. pyogenes, no licensed vaccine exists as of today. These shortcomings, paired with the emergence of antibiotic resistance, significantly impair the effectiveness of current treatment strategies. Nucleases such as S. pyogenes Nuclease A (SpnA), extracellular streptodornase (Sda1) and S. pneumoniae DNA/RNA endonuclease (EndA), are virulence factors in streptococci that allow the bacteria to evade host immune response by degrading neutrophil extracellular traps (NETs).
This study investigates the nuclease activity of recombinantly expressed streptococcal nucleases SpnA, EndA and Sda1 and their mutants in vitro. We demonstrated that the EndA and Sda1 mutants show minimal to no enzymatic function, which can be restored through imidazole rescue. Contrary to current publications, our SpnA mutant exhibited enzymatic function. Furthermore, we utilized affinity purification - mass spectrometry to investigate the role of SpnA, EndA and Sda1 in host-pathogen interactions and how these differ between the nucleases, as well as how these differ in human and mouse plasma. Lastly, we determined that the nucleases retain their activity even when bound to human plasma proteins. The streptococcal nucleases demonstrated interactions with a wide range of human and mouse proteins. These findings indicate that SpnA, EndA and Sda1 interfere with a broad array of host responses, promoting bacterial survival and virulence.
This research project provides valuable insights into the function of streptococcal nucleases, highlighting their involvement in potential immune evasion mechanisms. Deciphering these mechanisms is key in understanding these enzymes and contributes to research towards antimicrobial drug development. (Less)
Popular Abstract
Meet the Pac-Men of Infection

Did you know that humans have more bacterial cells than mammalian cells? Those bacteria, called commensals, are beneficial to our health. However, not all bacteria are good. Some bacteria, like Streptococcus pneumoniae and Streptococcus pyogenes, are pathogenic and can cause a range of diseases from mild to even life threatening. Research shows that 924 million people will be infected annually by those bacteria. Our current treatment and prevention strategies face shortcomings. In fact, in 2024, the WHO listed the two bacteria among the 15 priority pathogens for which new antibiotics are urgently needed. To develop new vaccines and antibiotics, new attack points in the bacteria need to be explored. Some... (More)
Meet the Pac-Men of Infection

Did you know that humans have more bacterial cells than mammalian cells? Those bacteria, called commensals, are beneficial to our health. However, not all bacteria are good. Some bacteria, like Streptococcus pneumoniae and Streptococcus pyogenes, are pathogenic and can cause a range of diseases from mild to even life threatening. Research shows that 924 million people will be infected annually by those bacteria. Our current treatment and prevention strategies face shortcomings. In fact, in 2024, the WHO listed the two bacteria among the 15 priority pathogens for which new antibiotics are urgently needed. To develop new vaccines and antibiotics, new attack points in the bacteria need to be explored. Some major players in how bacteria make us sick are bacterial proteins called nucleases. Think of them as Pac-Men: they cut up human DNA into smaller pieces to avoid detection by our immune system. Blocking the function of these proteins with small molecules could stop the infection in its tracks.
In this study, we explored the activity of these nucleases and how this activity changes in the presence of molecules that either block or restore their function. Additionally, we analyzed how these nucleases interact with other proteins in the blood plasma of humans and mice. To do that, we employed a method called affinity-purification that can be likened to fishing: we used the nucleases as “bait” in a sea of plasma proteins. The proteins with a preference for this bait, like fish, specifically bind to it and are pulled out or enriched along with it. Then, we used advanced mass-spectrometry based tools to identify and quantify exactly which proteins are caught or enriched and mapped out the nuclease’s interacting host proteins.
Our findings shed light on how these bacteria fight back against our immune system and survive in our bodies. By uncovering these hidden defence strategies, we get a clearer picture of how bacteria outsmart our natural defences. Further understanding of these mechanisms is key to unlocking strategies to develop new antibacterial drugs that target these bacterial survival tactics directly. (Less)
Popular Abstract (Greek, Modern (1453-))
Γνωρίστε τους Πακ-Μεν των λοιμώξεων

Ήξερες ότι στο ανθρώπινο σώμα υπάρχουν περισσότερα βακτηριακά παρά ανθρώπινα κύτταρα; Αυτά τα βακτήρια ονομάζονται συμβιωτικά, και είναι ωφέλιμα για την υγεία μας. Όμως, δεν είναι όλα τα βακτήρια καλά. Μερικά βακτήρια, όπως ο πνευμονόκοκκος και ο στρεπτόκοκκος της ομάδας Α, είναι παθογόνα και προκαλούν ένα εύρος λοιμώξεων, από ήπιες έως και επικίνδυνες για τη ζωή. Σύμφωνα με έρευνες, 924 εκατομμύρια άνθρωποι θα μολυνθούν ετησίως από αυτά τα βακτήρια. Οι στρατηγικές που έχουμε σήμερα για θεραπεία και πρόληψη έχουν περιορισμούς. Μάλιστα, το 2024 ο ΠΟΥ τοποθέτησε αυτά τα δύο βακτήρια στην λίστα με τα 15 παθογόνα βακτήρια για τα οποία απαιτούνται επειγόντως νέα αντιβιοτικά. Για να αναπτυχθούν νέα εμβόλια... (More)
Γνωρίστε τους Πακ-Μεν των λοιμώξεων

Ήξερες ότι στο ανθρώπινο σώμα υπάρχουν περισσότερα βακτηριακά παρά ανθρώπινα κύτταρα; Αυτά τα βακτήρια ονομάζονται συμβιωτικά, και είναι ωφέλιμα για την υγεία μας. Όμως, δεν είναι όλα τα βακτήρια καλά. Μερικά βακτήρια, όπως ο πνευμονόκοκκος και ο στρεπτόκοκκος της ομάδας Α, είναι παθογόνα και προκαλούν ένα εύρος λοιμώξεων, από ήπιες έως και επικίνδυνες για τη ζωή. Σύμφωνα με έρευνες, 924 εκατομμύρια άνθρωποι θα μολυνθούν ετησίως από αυτά τα βακτήρια. Οι στρατηγικές που έχουμε σήμερα για θεραπεία και πρόληψη έχουν περιορισμούς. Μάλιστα, το 2024 ο ΠΟΥ τοποθέτησε αυτά τα δύο βακτήρια στην λίστα με τα 15 παθογόνα βακτήρια για τα οποία απαιτούνται επειγόντως νέα αντιβιοτικά. Για να αναπτυχθούν νέα εμβόλια και αντιβιοτικά, χρειάζεται να ανακαλύψουμε νέα “ευαίσθητα σημεία” των βακτηρίων. Μερικοί σημαντικοί «παίκτες» που προσβάλλουν τον οργανισμό και έχουν αρνητική επίδραση στην υγεία είναι κάποιες βακτηριακές πρωτεΐνες που ονομάζονται νουκλεάσες. Σκέψου τες σαν Πακ-Μεν: κόβουν το ανθρώπινο DNA σε μικρότερα κομμάτια για να κρυφτούν από το ανοσοποιητικό μας σύστημα. Αναστέλλοντας τη δράση αυτών των πρωτεϊνών με μικρά μόρια, μπορούμε να σταματήσουμε την λοίμωξη.
Σε αυτήν την μελέτη, εξερευνήσαμε την δραστικότητα αυτών των πρωτεϊνών, και πώς αυτή αλλάζει παρουσία μορίων που είτε σταματούν είτε επαναφέρουν την δράση τους. Επιπλέον, αναλύσαμε τις αλληλεπιδράσεις των νουκλεασών με άλλες πρωτεΐνες στο πλάσμα αίματος ανθρώπων και ποντικιών. Για να το πετύχουμε αυτό, εφαρμόσαμε μια μέθοδο, τον καθαρισμό συγγένειας, που μπορεί να παρομοιαστεί με το ψάρεμα: χρησιμοποιήσαμε τις νουκλεάσες ως το «δόλωμα» σε μία θάλασσα πρωτεϊνών του πλάσματος. Οι πρωτεΐνες με προτίμηση για αυτό το δόλωμα, σαν ψάρια, το αναγνωρίζουν και το «αρπάζουν», με αποτέλεσμα να παρασύρονται, ή να εμπλουτίζονται, μαζί του. Έπειτα, χρησιμοποιήσαμε μια προηγμένη τεχνική, την φασματομετρία μάζας, για να αναγνωρίσουμε ποιες ακριβώς πρωτεΐνες πιάστηκαν, ή εμπλουτίστηκαν, και χαρτογραφήσαμε τις πρωτεΐνες του ξενιστή που αλληλεπιδρούν με τη νουκλεάση.
Τα ευρήματά μας ρίχνουν φως στον τρόπο με τον οποίο αυτά τα βακτήρια αντιστέκονται στο ανοσοποιητικό μας σύστημα και επιβιώνουν στο σώμα μας. Αποκαλύπτοντας αυτές τις κρυμμένες αμυντικές στρατηγικές μπορούμε να έχουμε μια πιο καθαρή εικόνα για το πώς τα βακτήρια ξεγελούν τους φυσικούς μας μηχανισμούς άμυνας. Η περαιτέρω κατανόηση αυτών των μηχανισμών είναι το κλειδί για την ανάπτυξη νέων αντιβακτηριακών φαρμάκων που στοχεύουν άμεσα αυτές τις τακτικές επιβίωσης των βακτηρίων. (Less)
Please use this url to cite or link to this publication:
author
Kostaloupi, Styliani LU
supervisor
organization
course
KEMR45 20251
year
type
H2 - Master's Degree (Two Years)
subject
keywords
Streptococcus pneumoniae, Streptococcus pyogenes, SpnA, EndA, Sda1, mass spectrometry, host-pathogen interactions, complement system, biochemistry
language
English
id
9198552
date added to LUP
2025-06-13 12:02:05
date last changed
2025-06-13 12:02:05
@misc{9198552,
  abstract     = {{Streptococcus pneumoniae and Streptococcus pyogenes are Gram-positive bacteria responsible for a wide spectrum of diseases, ranging from mild to even life-threatening. The annual global mortality rates of the bacteria amount to 1.68 million. While vaccines against S. pneumoniae are included in our medical arsenal, their efficacy is limited by poor immune responses in certain age groups or insufficient serotype coverage. Regarding S. pyogenes, no licensed vaccine exists as of today. These shortcomings, paired with the emergence of antibiotic resistance, significantly impair the effectiveness of current treatment strategies. Nucleases such as S. pyogenes Nuclease A (SpnA), extracellular streptodornase (Sda1) and S. pneumoniae DNA/RNA endonuclease (EndA), are virulence factors in streptococci that allow the bacteria to evade host immune response by degrading neutrophil extracellular traps (NETs).
This study investigates the nuclease activity of recombinantly expressed streptococcal nucleases SpnA, EndA and Sda1 and their mutants in vitro. We demonstrated that the EndA and Sda1 mutants show minimal to no enzymatic function, which can be restored through imidazole rescue. Contrary to current publications, our SpnA mutant exhibited enzymatic function. Furthermore, we utilized affinity purification - mass spectrometry to investigate the role of SpnA, EndA and Sda1 in host-pathogen interactions and how these differ between the nucleases, as well as how these differ in human and mouse plasma. Lastly, we determined that the nucleases retain their activity even when bound to human plasma proteins. The streptococcal nucleases demonstrated interactions with a wide range of human and mouse proteins. These findings indicate that SpnA, EndA and Sda1 interfere with a broad array of host responses, promoting bacterial survival and virulence. 
This research project provides valuable insights into the function of streptococcal nucleases, highlighting their involvement in potential immune evasion mechanisms. Deciphering these mechanisms is key in understanding these enzymes and contributes to research towards antimicrobial drug development.}},
  author       = {{Kostaloupi, Styliani}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Characterization of Streptococcal Nucleases and Identification of their Host Interactomes}},
  year         = {{2025}},
}