TY - JOUR

T1 - A random mutagenesis screen enriched for missense mutations in bacterial effector proteins

AU - Urbanus, Malene L

AU - Zheng, Thomas M

AU - Khusnutdinova, Anna N

AU - Banh, Doreen

AU - O'Connor Mount, Harley

AU - Gupta, Alind

AU - Stogios, Peter J

AU - Savchenko, Alexei

AU - Isberg, Ralph R

AU - Yakunin, Alexander F

AU - Ensminger, Alexander W

N1 - © The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.

PY - 2024/9/4

Y1 - 2024/9/4

N2 - To remodel their hosts and escape immune defenses, many pathogens rely on large arsenals of proteins (effectors) that are delivered to the host cell using dedicated translocation machinery. Effectors hold significant insight into the biology of both the pathogens that encode them and the host pathways that they manipulate. One of the most powerful systems biology tools for studying effectors is the model organism, Saccharomyces cerevisiae. For many pathogens, the heterologous expression of effectors in yeast is growth inhibitory at a frequency much higher than housekeeping genes, an observation ascribed to targeting conserved eukaryotic proteins. Abrogation of yeast growth inhibition has been used to identify bacterial suppressors of effector activity, host targets, and functional residues and domains within effector proteins. We present here a yeast-based method for enriching for informative, in-frame, missense mutations in a pool of random effector mutants. We benchmark this approach against three effectors from Legionella pneumophila, an intracellular bacterial pathogen that injects a staggering >330 effectors into the host cell. For each protein, we show how in silico protein modeling (AlphaFold2) and missense-directed mutagenesis can be combined to reveal important structural features within effectors. We identify known active site residues within the metalloprotease RavK, the putative active site in SdbB, and previously unidentified functional motifs within the C-terminal domain of SdbA. We show that this domain has structural similarity with glycosyltransferases and exhibits in vitro activity consistent with this predicted function.

AB - To remodel their hosts and escape immune defenses, many pathogens rely on large arsenals of proteins (effectors) that are delivered to the host cell using dedicated translocation machinery. Effectors hold significant insight into the biology of both the pathogens that encode them and the host pathways that they manipulate. One of the most powerful systems biology tools for studying effectors is the model organism, Saccharomyces cerevisiae. For many pathogens, the heterologous expression of effectors in yeast is growth inhibitory at a frequency much higher than housekeeping genes, an observation ascribed to targeting conserved eukaryotic proteins. Abrogation of yeast growth inhibition has been used to identify bacterial suppressors of effector activity, host targets, and functional residues and domains within effector proteins. We present here a yeast-based method for enriching for informative, in-frame, missense mutations in a pool of random effector mutants. We benchmark this approach against three effectors from Legionella pneumophila, an intracellular bacterial pathogen that injects a staggering >330 effectors into the host cell. For each protein, we show how in silico protein modeling (AlphaFold2) and missense-directed mutagenesis can be combined to reveal important structural features within effectors. We identify known active site residues within the metalloprotease RavK, the putative active site in SdbB, and previously unidentified functional motifs within the C-terminal domain of SdbA. We show that this domain has structural similarity with glycosyltransferases and exhibits in vitro activity consistent with this predicted function.

KW - Bacterial Proteins/genetics

KW - Mutagenesis

KW - Mutation, Missense

KW - Saccharomyces cerevisiae/genetics

KW - Legionella pneumophila/genetics

KW - Models, Molecular

U2 - 10.1093/g3journal/jkae158

DO - 10.1093/g3journal/jkae158

M3 - Article

C2 - 39028840

VL - 14

JO - G3: Genes, Genomes, Genetics

JF - G3: Genes, Genomes, Genetics

SN - 2160-1836

IS - 9

ER -