CoChat
Sign Up
Cited by 5Legionella and Acanthamoeba researchBacterial Genetics and BiotechnologyCRISPR and Genetic Engineering

The DOI matches its Crossref record; 1 of the 1 references it cites were found in a registry or an index.

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

Open access
Urbanus, Malene L, Zheng, Thomas M, Khusnutdinova, Anna N, Banh, Doreen, O’Connor Mount, Harley, Gupta, Alind, Stogios, Peter J, Savchenko, Alexei, Isberg, Ralph R, Yakunin, Alexander F, Ensminger, Alexander W·2024·G3: Genes, Genomes, Genetics
DOI 10.1093/g3journal/jkae158PMC PMC11373652

Published version of a preprint

Open this paper in CoChat — free account

Abstract

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.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1093/g3journal/jkae158

Cites

1 of 1 references verified0 citation errors · 0 not checkable

Statistics

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Flagged first, then by citations

Research works printed but not linked: 85

  1. Alberti, 2007, A suite of Gateway® cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae, Yeast, 24, 913, 10.1002/yea.1502 · DOI
  2. Bachran, 2013, Anthrax toxin-mediated delivery of the Pseudomonas exotoxin A enzymatic domain to the cytosol of tumor cells via cleavable ubiquitin fusions, Mbio, 4, e00201, 10.1128/mbio.00201-13 · DOI
  3. Berman, 2000, The protein data bank, Nucleic Acids Res, 28, 235, 10.1093/nar/28.1.235 · DOI · Paper page
  4. Black, 2019, Bacterial pseudokinase catalyzes protein polyglutamylation to inhibit the SidE-family ubiquitin ligases, Science, 364, 787, 10.1126/science.aaw7446 · DOI
  5. Bosis, 2011, A simple yeast-based strategy to identify host cellular processes targeted by bacterial effector proteins, PLoS One, 6, e27698, 10.1371/journal.pone.0027698 · DOI
  6. Brachmann, 1998, Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications, Yeast, 14, 115, 10.1002/(sici)1097-0061(19980130)14:2<115::aid-yea204>3.0.co;2-2 · DOI
  7. Brenner, 1988, The molecular evolution of genes and proteins: a tale of two serines, Nature, 334, 528, 10.1038/334528a0 · DOI
  8. Burstein, 2016, Genomic analysis of 38 Legionella species identifies large and diverse effector repertoires, Nat Genet, 48, 167, 10.1038/ng.3481 · DOI
  9. Burstein, 2009, Genome-scale identification of Legionella pneumophila effectors using a machine learning approach, Plos Pathog, 5, e1000508, 10.1371/journal.ppat.1000508 · DOI
  10. Campodonico, 2005, A yeast genetic system for the identification and characterization of substrate proteins transferred into host cells by the Legionella pneumophila Dot/Icm system, Mol Microbiol, 56, 918, 10.1111/j.1365-2958.2005.04595.x · DOI
  11. Chen, 2013, Fusion protein linkers: property, design and functionality, Adv Drug Deliver Rev, 65, 1357, 10.1016/j.addr.2012.09.039 · DOI
  12. Crouvoisier, 2007, Role of the amino acid invariants in the active site of MurG as evaluated by site-directed mutagenesis, Biochimie, 89, 1498, 10.1016/j.biochi.2007.06.011 · DOI
  13. Dalkas, 2010, Conformational dynamics of the anthrax lethal factor catalytic center, Biochemistry, 49, 10767, 10.1021/bi1017792 · DOI
  14. de Felipe, 2008, Legionella eukaryotic-like type IV substrates interfere with organelle trafficking, PLoS Pathog, 4, e1000117, 10.1371/journal.ppat.1000117 · DOI
  15. Di Tommaso, 2011, T-Coffee: a web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension, Nucleic Acids Res, 39, W13, 10.1093/nar/gkr245 · DOI
  16. Ensminger, 2012, Experimental evolution of Legionella pneumophila in mouse macrophages leads to strains with altered determinants of environmental survival, Plos Pathog, 8, e1002731, 10.1371/journal.ppat.1002731 · DOI
  17. Eschenfeldt, 2013, New LIC vectors for production of proteins from genes containing rare codons, J Struct Funct Genom, 14, 135, 10.1007/s10969-013-9163-9 · DOI
  18. Escoll, 2013, Molecular mechanisms in Legionella pathogenesis, Curr Top Microbiol, 376, 1, 10.1007/82_2013_351 · DOI
  19. Fields, 2002, Legionella and legionnaires’ disease: 25 years of investigation, Clin Microbiol Rev, 15, 506, 10.1128/cmr.15.3.506-526.2002 · DOI
  20. Finsel, 2015, Formation of a pathogen vacuole according to Legionella pneumophila: how to kill one bird with many stones, Cell Microbiol, 17, 935, 10.1111/cmi.12450 · DOI
  21. Gietz, 2007, High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method, Nat Protoc, 2, 31, 10.1038/nprot.2007.13 · DOI
  22. Gomez-Valero, 2019, More than 18,000 effectors in the Legionella genus genome provide multiple, independent combinations for replication in human cells, Proc Natl Acad Sci, 116, 2265, 10.1073/pnas.1808016116 · DOI
  23. Gomez-Valero, 2011, Comparative and functional genomics of Legionella identified eukaryotic like proteins as key players in host–pathogen interactions, Front Microbiol, 2, 208, 10.3389/fmicb.2011.00208 · DOI
  24. Gomez-Valero, 2014, Comparative analyses of Legionella species identifies genetic features of strains causing Legionnaires’ disease, Genome Biol, 15, 505, 10.1186/s13059-014-0505-0 · DOI
  25. Guo, 2014, A Legionella effector modulates host cytoskeletal structure by inhibiting actin polymerization, Microbes Infect, 16, 225, 10.1016/j.micinf.2013.11.007 · DOI
  26. Ha, 2000, The 1.9 Å crystal structure of Escherichia coli MurG, a membrane-associated glycosyltransferase involved in peptidoglycan biosynthesis, Protein Sci, 9, 1045, 10.1110/ps.9.6.1045 · DOI
  27. Heidtman, 2009, Large-scale identification of Legionella pneumophila Dot/Icm substrates that modulate host cell vesicle trafficking pathways, Cell Microbiol, 11, 230, 10.1111/j.1462-5822.2008.01249.x · DOI
  28. Holz, 2001, A human cDNA expression library in yeast enriched for open reading frames, Genome Res, 11, 1730, 10.1101/gr.181501 · DOI
  29. Hsieh, 2021, Dynamic remodeling of host membranes by self-organizing bacterial effectors, Science, 372, 935, 10.1126/science.aay8118 · DOI
  30. Hu, 2003, Crystal structure of the MurG:UDP-GlcNAc complex reveals common structural principles of a superfamily of glycosyltransferases, Proc National Acad Sci, 100, 845, 10.1073/pnas.0235749100 · DOI
  31. Huang, 2011, The E Block motif is associated with Legionella pneumophila translocated substrates, Cell Microbiol, 13, 227, 10.1111/j.1462-5822.2010.01531.x · DOI
  32. Huh, 2003, Global analysis of protein localization in budding yeast, Nature, 425, 686, 10.1038/nature02026 · DOI · Paper page
  33. Isberg, 2009, The Legionella pneumophila replication vacuole: making a cosy niche inside host cells, Nat Rev Microbiol, 7, 13, 10.1038/nrmicro1967 · DOI
  34. Kimura, 1994, Blasticidin S deaminase gene from Aspergillus terreus (BSD): a new drug resistance gene for transfection of mammalian cells, Biochim Biophys Acta (BBA)—Gene Struct Expr, 1219, 653, 10.1016/0167-4781(94)90224-0 · DOI
  35. Kozlov, 2018, Crystal structure of the Legionella effector Lem22, Proteins Struct Funct Bioinform, 86, 263, 10.1002/prot.25427 · DOI
  36. Kuzin, 2008, X-ray structure of the putative Zn-dependent peptidase Q74D82 at the resolution 1.7 A. Northeast Structural Genomics Consortium target GsR143A, RCSB Protein Data Bank, 10.2210/pdb3C37/pdb · DOI
  37. Lairson, 2008, Glycosyltransferases: structures, functions, and mechanisms, Annu Rev Biochem, 77, 521, 10.1146/annurev.biochem.76.061005.092322 · DOI
  38. Lesser, 2001, Expression of microbial virulence proteins in Saccharomyces cerevisiae models mammalian infection, EMBO J, 20, 1840, 10.1093/emboj/20.8.1840 · DOI
  39. Lin, 2018, Ravn is a member of a previously unrecognized group of Legionella pneumophila E3 ubiquitin ligases, Plos Pathog, 14, e1006897, 10.1371/journal.ppat.1006897 · DOI
  40. Liu, 2017, A Legionella effector disrupts host cytoskeletal structure by cleaving actin, PLoS Pathog, 13, e1006186, 10.1371/journal.ppat.1006186 · DOI
  41. López-Pelegrín, 2014, Multiple stable conformations account for reversible concentration-dependent oligomerization and autoinhibition of a metamorphic metallopeptidase, Angewandte Chemie Int Ed, 53, 10624, 10.1002/anie.201405727 · DOI
  42. López-Pelegrín, 2013, A novel family of soluble minimal scaffolds provides structural insight into the catalytic domains of integral membrane metallopeptidases, J Biol Chem, 288, 21279, 10.1074/jbc.m113.476580 · DOI
  43. Luo, 2021, Exploitation of the host ubiquitin system: means by Legionella pneumophila, Front Microbiol, 12, 790442, 10.3389/fmicb.2021.790442 · DOI
  44. Luo, 2004, Multiple substrates of the Legionella pneumophila Dot/Icm system identified by interbacterial protein transfer, P Natl Acad Sci Usa, 101, 841, 10.1073/pnas.0304916101 · DOI
  45. Marchler-Bauer, 2017, CDD/SPARCLE: functional classification of proteins via subfamily domain architectures, Nucleic Acids Res, 45, D200, 10.1093/nar/gkw1129 · DOI
  46. Margolin, 2000, Green fluorescent protein as a reporter for macromolecular localization in bacterial cells, Methods, 20, 62, 10.1006/meth.1999.0906 · DOI
  47. Maxwell, 1999, A simple in vivo assay for increased protein solubility, Protein Sci, 8, 1908, 10.1110/ps.8.9.1908 · DOI
  48. Mondino, 2020, Legionnaires’ disease: state of the art knowledge of pathogenesis mechanisms of Legionella, Annu Rev Pathol: Mech Dis, 15, 1, 10.1146/annurev-pathmechdis-012419-032742 · DOI
  49. Morar, 2015, Crystal structure of the Legionella pneumophila lem10 effector reveals a new member of the HD protein superfamily, Proteins Struct Funct Bioinform, 83, 2319, 10.1002/prot.24933 · DOI
  50. Nevo, 2014, Identification of Legionella pneumophila effectors regulated by the LetAS-RsmYZ-CsrA regulatory cascade, many of which modulate vesicular trafficking, J Bacteriol, 196, 681, 10.1128/jb.01175-13 · DOI
  51. O’Connor, 2011, Minimization of the Legionella pneumophila genome reveals chromosomal regions involved in host range expansion, Proc National Acad Sci, 108, 14733, 10.1073/pnas.1111678108 · DOI
  52. Ollis, 1992, The α/β hydrolase fold, Protein Eng Des Sel, 5, 197, 10.1093/protein/5.3.197 · DOI
  53. Pinotsis, 2017, Structure of the WipA protein reveals a novel tyrosine protein phosphatase effector from Legionella pneumophila, J Biol Chem, 292, 9240, 10.1074/jbc.m117.781948 · DOI
  54. Qiu, 2017, Legionella and Coxiella effectors: strength in diversity and activity, Nat Rev Microbiol, 15, 591, 10.1038/nrmicro.2017.67 · DOI
  55. Rasila, 2009, Critical evaluation of random mutagenesis by error-prone polymerase chain reaction protocols, Escherichia coli mutator strain, and hydroxylamine treatment, Anal Biochem, 388, 71, 10.1016/j.ab.2009.02.008 · DOI
  56. Schrag, 1997, [4] Lipases and αβ hydrolase fold, Methods Enzym, 284, 85, 10.1016/s0076-6879(97)84006-2 · DOI
  57. Segal, 1998, Host cell killing and bacterial conjugation require overlapping sets of genes within a 22-kb region of the Legionella pneumophila genome, Proc National Acad Sci, 95, 1669, 10.1073/pnas.95.4.1669 · DOI
  58. Shames, 2023, Eat or be eaten: strategies used by Legionella to acquire host-derived nutrients and evade lysosomal degradation, Infect Immun, 91, e00441, 10.1128/iai.00441-22 · DOI
  59. Sheikh, 2017, Rapid screening of sugar-nucleotide donor specificities of putative glycosyltransferases, Glycobiology, 27, 206, 10.1093/glycob/cww114 · DOI
  60. Shen, 2009, Targeting eEF1A by a Legionella pneumophila effector leads to inhibition of protein synthesis and induction of host stress response, Cell Microbiol, 11, 911, 10.1111/j.1462-5822.2009.01301.x · DOI
  61. Sherwood, 2016, Autophagy evasion and endoplasmic Reticulum subversion: the Yin and Yang of Legionella intracellular infection, Annu Rev Microbiol, 70, 413, 10.1146/annurev-micro-102215-095557 · DOI
  62. Shohdy, 2005, Pathogen effector protein screening in yeast identifies Legionella factors that interfere with membrane trafficking, P Natl Acad Sci USA, 102, 4866, 10.1073/pnas.0501315102 · DOI
  63. Siggers, 2008, The yeast Saccharomyces cerevisiae: a Versatile model system for the identification and characterization of bacterial virulence proteins, Cell Host Microbe, 4, 8, 10.1016/j.chom.2008.06.004 · DOI
  64. Sikorski, 1989, A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae, Genetics, 122, 19, 10.1093/genetics/122.1.19 · DOI
  65. Sisko, 2006, Multifunctional analysis of Chlamydia-specific genes in a yeast expression system, Mol Microbiol, 60, 51, 10.1111/j.1365-2958.2006.05074.x · DOI
  66. Slagowski, 2008, A functional genomic yeast screen to identify pathogenic bacterial proteins, Plos Pathog, 4, e9, 10.1371/journal.ppat.0040009 · DOI
  67. Sprouffske, 2016, Growthcurver: an R package for obtaining interpretable metrics from microbial growth curves, Bmc Bioinformatics, 17, 172, 10.1186/s12859-016-1016-7 · DOI
  68. Suarez, 2009, Stable expression of a GFP-BSD fusion protein in Babesia bovis merozoites, Int J Parasitol, 39, 289, 10.1016/j.ijpara.2008.08.006 · DOI
  69. Sulpizio, 2019, Protein polyglutamylation catalyzed by the bacterial calmodulin-dependent pseudokinase SidJ, Elife, 8, e51162, 10.7554/elife.51162 · DOI
  70. Toulabi, 2013, Identification and structural characterization of a Legionella phosphoinositide phosphatase, J Biol Chem, 288, 24518, 10.1074/jbc.m113.474239 · DOI
  71. Urbanus, 2016, Diverse mechanisms of metaeffector activity in an intracellular bacterial pathogen, Legionella pneumophila, Mol Syst Biol, 12, 893, 10.15252/msb.20167381 · DOI
  72. van Roessel, 2002, Imaging into the future: visualizing gene expression and protein interactions with fluorescent proteins, Nat Cell Biol, 4, E15, 10.1038/ncb0102-e15 · DOI
  73. Valdivia, 2004, Modeling the function of bacterial virulence factors in Saccharomyces cerevisiae, Eukaryot Cell, 3, 827, 10.1128/ec.3.4.827-834.2004 · DOI
  74. Valleau, 2018, Discovery of ubiquitin deamidases in the pathogenic arsenal of Legionella pneumophila, Cell Rep., 23, 568, 10.1016/j.celrep.2018.03.060 · DOI
  75. Varadi, 2021, AlphaFold protein structure database: massively expanding the structural coverage of protein-sequence space with high-accuracy models, Nucleic Acids Res, 50, D439, 10.1093/nar/gkab1061 · DOI · Paper page
  76. Vicente, 2023, Glycosyltransferase 8 domain-containing protein 1 (GLT8D1) is a UDP-dependent galactosyltransferase, Sci Rep, 13, 21684, 10.1038/s41598-023-48605-4 · DOI
  77. Vogel, 1998, Conjugative transfer by the virulence system of Legionella pneumophila, Science, 279, 873, 10.1126/science.279.5352.873 · DOI
  78. Voth, 2021, Structural and functional characterization of Legionella pneumophila effector MavL, Biomol, 11, 1802, 10.3390/biom11121802 · DOI
  79. Waterhouse, 2009, Jalview Version 2—a multiple sequence alignment editor and analysis workbench, Bioinformatics, 25, 1189, 10.1093/bioinformatics/btp033 · DOI
  80. Wong, 2015, Structure of the Legionella effector, lpg1496, suggests a role in nucleotide metabolism, J Biol Chem, 290, 24727, 10.1074/jbc.m115.671263 · DOI
  81. Xu, 2010, Inhibition of host vacuolar H+-ATPase activity by a Legionella pneumophila effector, PLoS Pathog, 6, e1000822, 10.1371/journal.ppat.1000822 · DOI
  82. Yang, 2023, Legionella pneumophila-mediated host posttranslational modifications, J Mol Cell Biol, 15, mjad032, 10.1093/jmcb/mjad032 · DOI
  83. Zhang, 2011, An improved method for whole protein extraction from yeast Saccharomyces cerevisiae, Yeast, 28, 795, 10.1002/yea.1905 · DOI
  84. Zhu, 2011, Comprehensive identification of protein substrates of the Dot/Icm type IV transporter of Legionella pneumophila, PLoS One, 6, e17638, 10.1371/journal.pone.0017638 · DOI
  85. Zimmermann, 2018, A completely reimplemented MPI bioinformatics toolkit with a new HHpred server at its core, J Mol Biol, 430, 2237, 10.1016/j.jmb.2017.12.007 · DOI · Paper page

Metadata

Authors
Urbanus, M. L., Zheng, T. M., Khusnutdinova, A. N., Banh, D., O’Connor Mount, H., Gupta, A., Stogios, P. J., Savchenko, A., Isberg, R. R., Yakunin, A. F., & Ensminger, A. W.
Affiliations
  • University of Toronto (CA)
  • Bangor University (GB)
  • Department of Molecular Genetics, University of Toronto , Toronto, ON M5G 1M1, Canada
  • University of Calgary (CA)
  • Tufts University (US)
Corresponding author
Ensminger, Alexander W
ORCID
Venue
G3: Genes, Genomes, Genetics
Publisher
Oxford University Press (OUP)
Published
2024-07-19
Volume
14
Issue
9
ISSN
2160-1836 (ISSN-L)
DOI
10.1093/g3journal/jkae158
PMC
PMC11373652
OpenAlex
W4400809250
Cited by
5
References
1
Citations by year
2026: 2 · 2025: 1 · 2024: 2
FWCI
1.49
Type
journal article
Language
en
Open access
Licence
Locations
  • G3 Genes Genomes Genetics (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • Canadian Institutes of Health Research (PJT-162256)
  • NSERC Strategic Network
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Endocrinology › Legionella and Acanthamoeba research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
Keywords
bacterial effector proteins, missense mutations, Saccharomyces cerevisiae, Legionella pneumophila, effector translocation
MeSH terms
Bacterial Proteins, Models, Molecular, Saccharomyces cerevisiae, Mutagenesis, Legionella pneumophila, Mutation, Missense

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review