Biology subjects

Bugert, J. J.

Publications and source records attributed to Bugert, J. J..

3 recordsLinked to original sources

bioRxiv2025

DepoCatalog: Mapping the Diversity of 105 Recombinant Klebsiella Phage Depolymerases Across Sequence, Structure, and Substrate Specificity

A validated catalog of 105 recombinant depolymerases from Klebsiella phages covers 58 KL-types. 46 novel enzymes from prophages, jumbo phages, and common phages are linked to any known enzymatic activity against 14 classical serotypes and 12 genome-defined KL-types. Using activity-based profiling, structure prediction, and domain dissection, we developed a function-guided classification and a five-class structural catalog. This framework reveals highly specific enzymes active against up to three capsule types. K47 CPS was degraded by three diverse protein groups. Structurally similar depolymerases degrading particular CPS were found in distinct phage taxa, with highly conserved enzymes in Drulisvirus specific to K1-, K2-types. The exclusive depolymerases were found in siphoviruses targeting K2 and K62 serotypes. A case study of five structurally similar enzymes degrading KL22/KL37/KL111 and KL25/KL119 capsules suggested specificity switching via amino acid changes or C-domain modification. Klebsiella phage depolymerases catalog sheds light on their diversity, evolution, and potential application.

microbiology↗

bioRxiv2023

Appelmans Protocol for in vitro Klebsiella pneumoniae phage host range expansion leads to induction of a novel temperate linear plasmid prophage vB_KpnS-KpLi5

Adjuvant therapy with bacteriophage (phage) cocktails in combination with antibiotics is a therapeutic approach currently considered for treatment of infections with encapsulated, biofilm forming, and multidrug-resistant Klebsiella pneumoniae (Kp). Klebsiella phage are highly selective in targeting a bacterial capsule type. Considering the numerous Kp capsule types and other Kp host restriction factors, phage treatment could be facilitated when generating phages with a broad host range A modified Appelmans protocol was used to create phages with an extended host range via in vitro forced DNA recombination. Three T7-like Kp phages with highly colinear genomes were subjected to successive propagation on their susceptible host strains representing the capsule types K64, K27, and K23, and five Kp isolates of the same capsule types initially unsusceptible for phage lysis. After 30 propagation cycles, five phages were isolated via plaque assay. Four output phages represented the original input phages, while the fifth lysed a previously non-permissible Kp isolate, which was not lysed by any of the input phages. Surprisingly, sequence analysis revealed a novel N15/phiKO2-like phage genome (vB_KpnS_KpLi5) lacking substantial homologies to any of the used T7-like phages. This temperate phage was only induced in the presence of all input phages (cocktail), but not by any of them individually. Induction of temperate phages may be a stress response caused by using multiple phages simultaneously. Successive use of different phages for therapeutic purposes may be preferable over simultaneous application in cocktail formulations to avoid undesired induction of temperate phages. (243)

microbiology↗

bioRxiv2022

Recombinant reporter phage rTUN1::nLuc enables rapid detection and real-time antibiotic susceptibility testing of Klebsiella pneumoniae K64 strains

The emergence of multi drug resistant (MDR) Klebsiella pneumoniae (Kp) strains constitutes an enormous threat to global health as MDR associated treatment failure causes high mortality rates in nosocomial infections. Rapid pathogen detection and antibiotic resistance screening is therefore crucial for successful therapy and thus, patient survival. Reporter phage-based diagnostics offer a way to speed up pathogen identification and resistance testing, as integration of reporter genes into highly specific phages allow real-time detection of phage replication and thus, living host cells. Kp specific phages use the hosts capsule, a major virulence factor of Kp, as receptor for adsorption. To date, 80 different Kp capsule types (K-serotypes) have been described with predominant capsule types varying between different countries and continents. Therefore, reporter phages need to be customized according to the locally prevailing variants. Recently, we described the autographivirus vB_KpP_TUN1 (TUN1), which specifically infects Kp K64 strains, the most predominant capsule type at the military hospital in Tunis (MHT) that is also associated with high mortality rates. In this work, we developed the highly specific recombinant reporter phage rTUN1::nLuc, which produces Nanoluciferase (nLuc) upon host infection and thus, enables rapid detection of Kp K64 cells in clinical matrices such as blood and urine. At the same time, rTUN1::nLuc allows for rapid antibiotic susceptibility testing and therefore identification of suitable antibiotic treatment in less than 3 hours.

synthetic biology↗