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Antibiotic uptake in Gram-negative bacteria
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Daniel Daley)
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The increasing emergence and spread of antibiotic-resistant bacteria is a serious threat to public health. Of particular concern are Gram-negative bacteria such as Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae or Pseudomonas aeruginosa. Some of these strains are resistant to a large number of antibiotics and thus our treatment options are rapidly declining. In addition to the increasing number of antibiotic-resistant bacteria, a major problem is that many of the antibiotics at our disposal are ineffective against Gram-negative bacteria. This is partly due to the properties of the outer membrane (OM) which prevents efficient uptake. The overarching goal of this thesis was to investigate how the OM of the Gram-negative bacterium E. coli could be weakened to improve the activity of antibiotics.

In the first two papers of my thesis (paper I + II), I investigated the periplasmic chaperone network which consists of the two parallel pathways SurA and Skp/DegP. This network is essential for the integrity of the OM and strains lacking either SurA or Skp are defective in the assembly of the OM, which results in an increased sensitivity towards vancomycin and other antimicrobials. We identified a novel component of the periplasmic chaperone network, namely YfgM, and showed that it operates in the same network as Skp and SurA/DegP. In particular, we demonstrated that deletion of YfgM in strains with either a ΔsurA or Δskp background further compromised the integrity of the OM, as evidenced by an increased sensitivity towards vancomycin.

In the remaining two papers of my thesis (paper III + IV), the goal was to characterize small molecules that permeabilize the OM and thus could be used to improve the activity of antibiotics. Towards this goal, we performed a high-throughput screen and identified an inhibitor of the periplasmic chaperone LolA, namely MAC-13243, and showed that it can be used to permeabilize the OM of E. coli (paper III). We further demonstrated that MAC-13243 can be used to potentiate the activity of antibiotics which are normally ineffective against E. coli. In the last paper of my thesis (paper IV), we undertook a more specific approach and wanted to identify an inhibitor against the glycosyltransferase WaaG. This enzyme is involved in the synthesis of LPS and genetic inactivation of WaaG results in a defect in the OM, which leads to an increased sensitivity to various antibiotics. In this paper, we identified a small molecular fragment (compound L1) and showed that it can be used to inhibit the activity of WaaG in vitro.

To summarize, this thesis provides novel insights into how the OM of the Gram-negative bacterium E. coli can be weakened by using small molecules. We believe that the two identified small molecules represent important first steps towards the design of more potent inhibitors that could be used in clinics to enhance the activity of antibiotics.

Place, publisher, year, edition, pages
Department of Biochemistry and Biophysics, Stockholm University , 2017.
Keyword [en]
Gram-negative bacteria, Antibiotic uptake, Outer membrane, Lipopolysaccharide
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-148541ISBN: 978-91-7797-041-5 (print)ISBN: 978-91-7797-042-2 (electronic)OAI: oai:DiVA.org:su-148541DiVA, id: diva2:1153263
Public defence
2017-12-12, Magnélisalen Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.

Available from: 2017-11-17 Created: 2017-10-29 Last updated: 2017-11-17Bibliographically approved
List of papers
1. YfgM Is an Ancillary Subunit of the SecYEG Translocon in Escherichia coli
Open this publication in new window or tab >>YfgM Is an Ancillary Subunit of the SecYEG Translocon in Escherichia coli
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2014 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 289, no 27, p. 19089-19097Article in journal (Refereed) Published
Abstract [en]

Protein secretion in Gram-negative bacteria is essential for both cell viability and pathogenesis. The vast majority of secreted proteins exit the cytoplasm through a transmembrane conduit called the Sec translocon in a process that is facilitated by ancillary modules, such as SecA, SecDF-YajC, YidC, and PpiD. In this study we have characterized YfgM, a protein with no annotated function. We found it to be a novel ancillary subunit of the Sec translocon as it co-purifies with both PpiD and the SecYEG translocon after immunoprecipitation and blue native/SDS-PAGE. Phenotypic analyses of strains lacking yfgM suggest that its physiological role in the cell overlaps with the periplasmic chaperones SurA and Skp. We, therefore, propose a role for YfgM in mediating the trafficking of proteins from the Sec translocon to the periplasmic chaperone network that contains SurA, Skp, DegP, PpiD, and FkpA.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-106016 (URN)10.1074/jbc.M113.541672 (DOI)000339062900039 ()
Available from: 2014-07-18 Created: 2014-07-18 Last updated: 2017-12-05Bibliographically approved
2. Identification of Putative Substrates for the Periplasmic Chaperone YfgM in Escherichia coli Using Quantitative Proteomics
Open this publication in new window or tab >>Identification of Putative Substrates for the Periplasmic Chaperone YfgM in Escherichia coli Using Quantitative Proteomics
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2015 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 14, no 1, p. 216-226Article in journal (Refereed) Published
Abstract [en]

How proteins are trafficked, folded, and assembled into functional units in the cell envelope of Gram-negative bacteria is of significant interest. A number of chaperones have been identified, however, the molecular roles of these chaperones are often enigmatic because it has been challenging to assign substrates. Recently we discovered a novel periplasmic chaperone, called YfgM, which associates with PpiD and the SecYEG translocon and operates in a network that contains Skp and SurA. The aim of the study presented here was to identify putative substrates of YfgM. We reasoned that substrates would be incorrectly folded or trafficked when YfgM was absent from the cell, and thus more prone to proteolysis (the loss-of-function rationale). We therefore used a comparative proteomic approach to identify cell envelope proteins that were lower in abundance in a strain lacking yfgM, and strains lacking yfgM together with either skp or surA. Sixteen putative substrates were identified. The list contained nine inner membrane proteins (CusS, EvgS, MalF, OsmC, TdcB, TdcC, WrbA, YfhB, and YtfH) and seven periplasmic proteins (HdeA, HdeB, AnsB, Ggt, MalE, YcgK, and YnjE), but it did not include any lipoproteins or outer membrane proteins. Significantly, AnsB (an asparaginase) and HdeB (a protein involved in the acid stress response), were lower in abundance in all three strains lacking yfgM. For both genes, we ruled out the possibility that they were transcriptionally down-regulated, so it is highly likely that the corresponding proteins are misfolded/mistargeted and turned-over in the absence of YfgM. For HdeB we validated this conclusion in a pulse-chase experiment. The identification of HdeB and other cell envelope proteins as potential substrates will be a valuable resource for follow-up experiments that aim to delineate molecular the function of YfgM.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-113555 (URN)10.1074/mcp.M114.043216 (DOI)000347155500017 ()
Note

AuthorCount:6;

Available from: 2015-02-06 Created: 2015-02-04 Last updated: 2017-12-04Bibliographically approved
3. Increasing the permeability of Escherichia coli using MAC13243
Open this publication in new window or tab >>Increasing the permeability of Escherichia coli using MAC13243
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The outer membrane of gram-negative bacteria is a permeability barrier that prevents the efficient uptake of molecules with large scaffolds. As a consequence, a number of antibiotic classes are ineffective against gram-negative strains. Herein we carried out a high throughput screen for small molecules that make the outer membrane of Escherichia coli more permeable. We identified MAC13243, an inhibitor of the periplasmic chaperone LolA that traffics lipoproteins from the inner to the outer membrane. We observed that cells were (1) more permeable to the fluorescent probe 1-N-phenylnapthylamine, and (2) more susceptible to large-scaffold antibiotics when sub-inhibitory concentrations of MAC13243 were used. To exclude the possibility that the permeability was caused by an off-target effect, we genetically reconstructed the MAC13243-phenotype by depleting LolA levels using the CRISPRi system.

Keyword
Gram-negative bacteria, Antibiotic uptake, Lipopolysaccharide, MAC13243, Lipoprotein trafficking
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-148538 (URN)
Available from: 2017-10-29 Created: 2017-10-29 Last updated: 2017-10-30Bibliographically approved
4. Identification of a Fragment-Based Scaffold that Inhibits the Glycosyltransferase WaaG from Escherichia coli
Open this publication in new window or tab >>Identification of a Fragment-Based Scaffold that Inhibits the Glycosyltransferase WaaG from Escherichia coli
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2016 (English)In: Antibiotics, ISSN 0066-4774, E-ISSN 2079-6382, Vol. 5, no 1Article in journal (Refereed) Published
Abstract [en]

WaaG is a glycosyltransferase that is involved in the biosynthesis of lipopolysaccharide in Gram-negative bacteria. Inhibitors of WaaG are highly sought after as they could be used to inhibit the biosynthesis of the core region of lipopolysaccharide, which would improve the uptake of antibiotics. Herein, we establish an activity assay for WaaG using C-14-labeled UDP-glucose and LPS purified from a increment waaG strain of Escherichia coli. We noted that addition of the lipids phosphatidylglycerol (PG) and cardiolipin (CL), as well as the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) increased activity. We then use the assay to determine if three molecular scaffolds, which bind to WaaG, could inhibit its activity in vitro. We show that 4-(2-amino-1,3-thiazol-4-yl)phenol inhibits WaaG (IC50 1.0 mM), but that the other scaffolds do not. This study represents an important step towards an inhibitor of WaaG by fragment-based lead discovery.

Keyword
lipopolysaccharide, glucosyltransferase, Gram-negative bacteria, scaffold, fragment-based lead discovery
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-129989 (URN)10.3390/antibiotics5010010 (DOI)000373607800003 ()
Available from: 2016-05-13 Created: 2016-05-09 Last updated: 2017-10-30Bibliographically approved

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