scholarly journals Harnessing machine learning to unravel protein degradation in Escherichia coli

2020 ◽  
Author(s):  
Natan Nagar ◽  
Noa Ecker ◽  
Gil Loewenthal ◽  
Oren Avram ◽  
Daniella Ben-Meir ◽  
...  
Keyword(s):  
Machine Learning ◽  
Escherichia Coli ◽  
Drug Targets ◽  
Isotope Labeling ◽  
Interaction Network ◽  
Culture Method ◽  
Mass Spectrometry Analysis ◽  
Antibacterial Drug ◽  
Spectrometry Analysis ◽  
Quality Control Mechanism

AbstractDegradation of intracellular proteins in Gram-negative bacteria regulates various cellular processes and serves as a quality control mechanism by eliminating damaged proteins. To understand what causes the proteolytic machinery of the cell to degrade some proteins while sparing others, we employed a quantitative pulsed-SILAC (Stable Isotope Labeling with Amino acids in Cell culture) method followed by mass spectrometry analysis to determine the half-lives for the proteome of exponentially growing Escherichia coli, under standard conditions. We developed a likelihood-based statistical test to find actively degraded proteins, and identified dozens of novel proteins that are fast-degrading. Finally, we used structural, physicochemical and protein-protein interaction network descriptors to train a machine-learning classifier to discriminate fast-degrading proteins from the rest of the proteome. Our combined computational-experimental approach provides means for proteomic-based discovery of fast degrading proteins in bacteria and the elucidation of the factors determining protein half-lives and have implications for protein engineering. Moreover, as rapidly degraded proteins may play an important role in pathogenesis, our findings could identify new potential antibacterial drug targets.

scholarly journals Harnessing Machine Learning To Unravel Protein Degradation in Escherichia coli

mSystems ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Natan Nagar ◽  
Noa Ecker ◽  
Gil Loewenthal ◽  
Oren Avram ◽  
Daniella Ben-Meir ◽  
...  
Keyword(s):  
Machine Learning ◽  
Escherichia Coli ◽  
Protein Degradation ◽  
Isotope Labeling ◽  
Characteristic Curve ◽  
Mass Spectrometry Analysis ◽  
Antibacterial Drug ◽  
Content Type ◽  
Spectrometry Analysis ◽  
E Coli

ABSTRACT Degradation of intracellular proteins in Gram-negative bacteria regulates various cellular processes and serves as a quality control mechanism by eliminating damaged proteins. To understand what causes the proteolytic machinery of the cell to degrade some proteins while sparing others, we employed a quantitative pulsed-SILAC (stable isotope labeling with amino acids in cell culture) method followed by mass spectrometry analysis to determine the half-lives for the proteome of exponentially growing Escherichia coli, under standard conditions. We developed a likelihood-based statistical test to find actively degraded proteins and identified dozens of fast-degrading novel proteins. Finally, we used structural, physicochemical, and protein-protein interaction network descriptors to train a machine learning classifier to discriminate fast-degrading proteins from the rest of the proteome, achieving an area under the receiver operating characteristic curve (AUC) of 0.72. IMPORTANCE Bacteria use protein degradation to control proliferation, dispose of misfolded proteins, and adapt to physiological and environmental shifts, but the factors that dictate which proteins are prone to degradation are mostly unknown. In this study, we have used a combined computational-experimental approach to explore protein degradation in E. coli. We discovered that the proteome of E. coli is composed of three protein populations that are distinct in terms of stability and functionality, and we show that fast-degrading proteins can be identified using a combination of various protein properties. Our findings expand the understanding of protein degradation in bacteria and have implications for protein engineering. Moreover, as rapidly degraded proteins may play an important role in pathogenesis, our findings may help to identify new potential antibacterial drug targets.

Phosphorylation of BACH1 switches its function from transcription factor to mitotic chromosome regulator and promotes its interaction with HMMR

2018 ◽  
Vol 475 (5) ◽  
pp. 981-1002 ◽  
Author(s):  
Jie Li ◽  
Hiroki Shima ◽  
Hironari Nishizawa ◽  
Masatoshi Ikeda ◽  
Andrey Brydun ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Isotope Labeling ◽  
Binding Activity ◽  
Mass Spectrometry Analysis ◽  
Spindle Orientation ◽  
Mitotic Spindles ◽  
Spectrometry Analysis ◽  
Dna Binding Activity ◽  
Ejection Force ◽  
M Phase

The transcription repressor BACH1 performs mutually independent dual roles in transcription regulation and chromosome alignment during mitosis by supporting polar ejection force of mitotic spindle. We now found that the mitotic spindles became oblique relative to the adhesion surface following endogenous BACH1 depletion in HeLa cells. This spindle orientation rearrangement was rescued by re-expression of BACH1 depending on its interactions with HMMR and CRM1, both of which are required for the positioning of mitotic spindle, but independently of its DNA-binding activity. A mass spectrometry analysis of BACH1 complexes in interphase and M phase revealed that BACH1 lost during mitosis interactions with proteins involved in chromatin and gene expression but retained interactions with HMMR and its known partners including CHICA. By analyzing BACH1 modification using stable isotope labeling with amino acids in cell culture, mitosis-specific phosphorylations of BACH1 were observed, and mutations of these residues abolished the activity of BACH1 to restore mitotic spindle orientation in knockdown cells and to interact with HMMR. Detailed histological analysis of Bach1-deficient mice revealed lengthening of the epithelial fold structures of the intestine. These observations suggest that BACH1 performs stabilization of mitotic spindle orientation together with HMMR and CRM1 in mitosis, and that the cell cycle-specific phosphorylation switches the transcriptional and mitotic functions of BACH1.

Molecular Characterization and Genomic Analysis of Novel Phage vB_ ShiP-A7 Infecting Multidrug-Resistant Shiglla Fexneri and Escherichia Coli

2020 ◽  
Author(s):  
Jing Xu ◽  
Yu Gu ◽  
Xinyan Yu ◽  
Ruiyang Zhang ◽  
Xuesen Zhang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Case Reports ◽  
Burst Size ◽  
Shigella Flexneri ◽  
Genomic Analysis ◽  
Multidrug Resistant ◽  
Open Reading Frames ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Spherical Head

Abstract BackgroundPhage therapy has regained more attention due to the rise of multidrug-resistant (MDR) bacteria. Several case reports demonstrated clinical application of phage in resolving infections caused by MDR bacteria in recent years. ResultsWe isolated a new phage, vB_ShiP-A7, and then investigated its characteristics. Phage vB_ShiP-A7 is a member of Podoviridae that has an icosahedral spherical head and a short tail. vB_ShiP-A7 has large burst size and short replication time. vB_ShiP-A7’s genome is linear double stranded DNA composed of 40058 bp, encoding forty-three putative open reading frames. Comparative genome analysis demonstrated vB_ShiP-A7’s genome sequence is closely related to fifteen different phages (coverage 74-88%, identity 86-93%). Mass Spectrometry analysis revealed that twelve known proteins and six hypothetical proteins exist in particles of vB_ShiP-A7. Genome and proteome analyses confirmed the absence of lysogen-related proteins and toxic proteins in this phage. In addition, phage vB_ShiP-A7 can significantly reduce the growth of clinical MDR stains of Shigella flexneri and Escherichia coli in liquid culture. Furthermore, vB_ShiP-A7 can disrupt biofilms formed by Shigella flexneri or Escherichia coli in vitro. ConclusionPhage vB_ShiP-A7 is a stable novel phage, which has a strong application potential to inhibit MDR stains of Shigella flexneri and Escherichia coli. Comparing the genomes between vB_ShiP-A7 and other closely-related phages will help us better understand the evolutionary mechanism of phages.

scholarly journals Dissection of the Role of Pili and Type 2 and 3 Secretion Systems in Adherence and Biofilm Formation of an Atypical Enteropathogenic Escherichia coli Strain

2013 ◽  
Vol 81 (10) ◽  
pp. 3793-3802 ◽  
Author(s):  
Rodrigo T. Hernandes ◽  
Miguel A. De la Cruz ◽  
Denise Yamamoto ◽  
Jorge A. Girón ◽  
Tânia A. T. Gomes
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Coli Strain ◽  
Mass Spectrometry Analysis ◽  
Secretion Systems ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Rigid Rod

ABSTRACTAtypical enteropathogenicEscherichia coli(aEPEC) strains are diarrheal pathogens that lack bundle-forming pilus production but possess the virulence-associated locus of enterocyte effacement. aEPEC strain 1551-2 produces localized adherence (LA) on HeLa cells; however, its isogenic intimin (eae) mutant produces a diffuse-adherence (DA) pattern. In this study, we aimed to identify the DA-associated adhesin of the 1551-2eaemutant. Electron microscopy of 1551-2 identified rigid rod-like pili composed of an 18-kDa protein, which was identified as the major pilin subunit of type 1 pilus (T1P) by mass spectrometry analysis. Deletion offimAin 1551-2 affected biofilm formation but had no effect on adherence properties. Analysis of secreted proteins in supernatants of this strain identified a 150-kDa protein corresponding to SslE, a type 2 secreted protein that was recently reported to be involved in biofilm formation of rabbit and human EPEC strains. However, neither adherence nor biofilm formation was affected in a 1551-2sslEmutant. We then investigated the role of the EspA filament associated with the type 3 secretion system (T3SS) in DA by generating a doubleeae espAmutant. This strain was no longer adherent, strongly suggesting that the T3SS translocon is the DA adhesin. In agreement with these results, specific anti-EspA antibodies blocked adherence of the 1551-2eaemutant. Our data support a role for intimin in LA, for the T3SS translocon in DA, and for T1P in biofilm formation, all of which may act in concert to facilitate host intestinal colonization by aEPEC strains.

scholarly journals Specific Roles of the iroBCDEN Genes in Virulence of an Avian Pathogenic Escherichia coli O78 Strain and in Production of Salmochelins

2008 ◽  
Vol 76 (8) ◽  
pp. 3539-3549 ◽  
Author(s):  
Mélissa Caza ◽  
François Lépine ◽  
Sylvain Milot ◽  
Charles M. Dozois
Keyword(s):  
Escherichia Coli ◽  
Respiratory Infections ◽  
Mass Spectrometry Analysis ◽  
Liquid Chromatography Mass Spectrometry ◽  
Avian Pathogenic Escherichia Coli ◽  
Prevalence Studies ◽  
Spectrometry Analysis ◽  
E Coli ◽  
Pathogenic Escherichia Coli ◽  
Negative Mutant

ABSTRACT Avian pathogenic Escherichia coli (APEC) strains are a subset of extraintestinal pathogenic E. coli (ExPEC) strains associated with respiratory infections and septicemia in poultry. The iroBCDEN genes encode the salmochelin siderophore system present in Salmonella enterica and some ExPEC strains. Roles of the iro genes for virulence in chickens and production of salmochelins were assessed by introducing plasmids carrying different combinations of iro genes into an attenuated salmochelin- and aerobactin-negative mutant of O78 strain χ7122. Complementation with the iroBCDEN genes resulted in a regaining of virulence, whereas the absence of iroC, iroDE, or iroN abrogated restoration of virulence. The iroE gene was not required for virulence, since introduction of iroBCDN restored the capacity to cause lesions and colonize extraintestinal tissues. Prevalence studies indicated that iro sequences were associated with virulent APEC strains. Liquid chromatography-mass spectrometry analysis of supernatants of APEC χ7122 and the complemented mutants indicated that (i) for χ7122, salmochelins comprised 14 to 27% of the siderophores present in iron-limited medium or infected tissues; (ii) complementation of the mutant with the iro locus increased levels of glucosylated dimers (S1 and S5) and monomer (SX) compared to APEC strain χ7122; (iii) the iroDE genes were important for generation of S1, S5, and SX; (iv) iroC was required for export of salmochelin trimers and dimers; and (v) iroB was required for generation of salmochelins. Overall, efficient glucosylation (IroB), transport (IroC and IroN), and processing (IroD and IroE) of salmochelins are required for APEC virulence, although IroE appears to serve an ancillary role.

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