Investigating the mechanisms of a lipoprotein chaperone essential to outer membrane biogenesis Restricted; Files Only
Smith, Hannah (Summer 2023)
Abstract
Antibiotic resistant infections are an emergent threat to global health, and the treatment of currently simple infections is becoming increasingly problematic. Gram-negative bacteria are particularly worrisome because they produce an essential, protective outer membrane (OM) that shields the cell from antibiotic treatment. Several conserved cellular machines are responsible for building the OM, and each of them require an essential lipoprotein component. Therefore, lipoprotein trafficking is essential to OM assembly, presenting a promising avenue for the development of novel antibiotics. In Escherichia coli, LolCDE is an inner membrane (IM) ATP-binding cassette transporter that extracts lipoproteins and delivers them to LolA, a periplasmic chaperone. LolA shuttles lipoproteins across the periplasm to LolB, which inserts lipoproteins into the OM. LolA is universally conserved across Gram-negative bacteria, but many Proteobacteria do not produce LolB. Thus, investigating the molecular mechanisms of LolA is paramount for understanding its essential cellular roles. This work assesses LolA function, ranging from molecular insights to evolutionary perspectives, highlighting its potential as a novel antibiotic target. First, the role of a hydrophobic patch on the surface of LolA is examined in vivo, clarifying the intermediate steps of lipoprotein release from the IM. Next, the activity of LolA in organisms lacking LolB is assessed, advancing our understanding of the lipoprotein trafficking pathway in organisms outside of E. coli. Finally, an allele-specific inhibition system of LolA is developed, enabling the validation of a biological signature for the inhibition of OM lipoprotein biogenesis. This establishes a pipeline for the design and confirmation of compounds that act as inhibitors of LolA and OM lipoprotein biogenesis more broadly. Altogether, this work rigorously investigates the mechanisms of LolA, furthering the field’s knowledge of an essential and conserved cellular process.
Table of Contents
Chapter I: Introduction…………………………………………………………………………1
Part I: Gram-negative bacteria are an imminent threat to global health…………………………………………………………………………1
Part II: OM Biogenesis…………………………………………………………………………2
Part III: Lipoprotein trafficking to the OM…………………………………………………………………………4
Part IV: Introduction to dissertation research…………………………………………………………………………8
Figure 1. Outer membrane biogenesis and a summary of this dissertation work…………………………………………………………………………10
References…………………………………………………………………………12
Chapter II: A hydrophobic patch on the surface of LolA is critical for interacting with LolC…………………………………………………………………………20
Preface…………………………………………………………………………20
Abstract…………………………………………………………………………23
Introduction…………………………………………………………………………24
Results…………………………………………………………………………27
Single mutants of the large hydrophobic surface patch (LP) of LolA have modest fitness defects…………………………………………………………………………27
Figure 1. The large hydrophobic surface patch (LP) of LolA is near C-terminal residues of LolA that interact with the pad of LolC…………………………………………………………………………29
Figure 2. Mutating the LP of LolA is well-tolerated…………………………………………………………………………32
A triple mutant of the hydrophobic surface patch (LolALP) is an inefficient lipoprotein chaperone…………………………………………………………………………33
Figure 3. A triple mutant of the LP of LolA (LolALP) causes OM permeability…………………………………………………………………………35
LolALP poorly releases Pal from the IM…………………………………………………………………………37
Figure 4. LolALP does not efficiently release Pal from the inner membrane in vivo…………………………………………………………………………38
Disrupting the LP of LolA likely impedes the interaction with the LolC pad…………………………………………………………………………39
Figure 5. The LP of LolA likely facilitates the interaction with the pad of LolC…………………………………………………………………………44
LolALP is unable to function in cells producing LolC(M175R)…………………………………………………………………………44
Discussion…………………………………………………………………………45
Materials & Methods…………………………………………………………………………47
Supplemental Materials…………………………………………………………………………51
Figure S1. Levels of LolA single LP mutants are comparable to wildtype LolA…………………………………………………………………………51
Figure S2. Levels of the LolA triple LP mutant are comparable to wildtype LolA and each of the single arginine mutants…………………………………………………………………………52
Figure S3. ∆lpp strains expressing LolALP grow similarly to strains expressing LolAWT…………………………………………………………………………53
Figure S4. lpp(∆K58) strains expressing LolALP grow slower than strains expressing LolAWT…………………………………………………………………………54
Figure S5. LolALP is not dominant…………………………………………………………………………55
Table S1. Strains used in this study…………………………………………………………………………56
Table S2. Plasmids used in this study…………………………………………………………………………62
Table S3. Oligonucleotides used in this study…………………………………………………………………………66
References…………………………………………………………………………69
Chapter III: Teasing apart the evolution of lipoprotein trafficking in gram-negative bacteria reveals a bifunctional LolA…………………………………………………………………………75
Preface…………………………………………………………………………75
Abstract…………………………………………………………………………78
Significance…………………………………………………………………………79
Introduction…………………………………………………………………………79
Figure 1. LolB is not widely conserved in Proteobacteria despite conservation of the Lol pathway and essential OM lipoproteins…………………………………………………………………………81
Results…………………………………………………………………………82
LolB is only present in ɣ- and β-Proteobacteria…………………………………………………………………………82
Caulobacter vibrioides LolA (cviLolA) can complement the deletion of E. coli LolA (ecoLolA) and LolB…………………………………………………………………………84
Figure 2. A modeled cviLolA structure suggests features of both LolA and LolB…………………………………………………………………………85
Figure 3. Caulobacter vibrioides LolA (cviLolAeco) can complement loss of E. coli LolA (ecoLolA) and LolB…………………………………………………………………………87
Introducing the LolB loop to ecoLolA results in a bifunctional chimera…………………………………………………………………………90
Figure 4. cviLolAeco requires a functional loop to complement E. coli LolB…………………………………………………………………………91
Loop mutants of ecoLolAloop can complement deletion of LolA but not LolB…………………………………………………………………………92
The bifunctional activity of ecoLolAloop in lpp+ cells is a liability…………………………………………………………………………93
Figure 5. ecoLolA which contains the loop of LolB can complement loss of both lolA and lolB…………………………………………………………………………94
Figure 6. Lipoprotein trafficking by ecoLolA and ecoLolAloop…………………………………………………………………………96
Discussion…………………………………………………………………………98
Materials and Methods…………………………………………………………………………101
Supplemental Materials…………………………………………………………………………105
Figure S1. Genetic organization of ispE, lolB, and TPR protein-encoding genes among select Proteobacteria…………………………………………………………………………106
Figure S2. ecoLolA interaction with LolC and the corresponding cviLolA region…………………………………………………………………………107
Figure S3. Levels of cviLolAeco are lower than wildtype ecoLolA…………………………………………………………………………108
Figure S4. cviLolA poorly complements deletion of lolA and introduction of ecoLolA C-terminal residues (cviLolAeco) enhances in vivo lipoprotein chaperone activity…………………………………………………………………………109
Figure S5. Levels of ecoLolAloop, ecoLolAloop(Asp), and ecoLolAloop(∆Leu) are comparable to wildtype ecoLolA…………………………………………………………………………110
Figure S6. Purification of Pal-LolA complexes…………………………………………………………………………111
Table S1. Strains used in this study…………………………………………………………………………112
Table S2. Plasmids used in this study…………………………………………………………………………119
Table S3. Oligonucleotides used in this study…………………………………………………………………………122
References…………………………………………………………………………126
Chapter IV: A biological signature for the inhibition of outer membrane lipoprotein biogenesis…………………………………………………………………………132
Preface…………………………………………………………………………132
Abstract…………………………………………………………………………135
Importance…………………………………………………………………………136
Introduction…………………………………………………………………………136
Figure 1. Lipoprotein maturation and trafficking and OM lipoprotein biogenesis inhibitors…………………………………………………………………………138
Results…………………………………………………………………………140
Depletion of OM lipoprotein biogenesis factors causes OM permeability…………………………………………………………………………140
Figure 2. Depletion of lipoprotein maturation or trafficking factors causes outer membrane permeability…………………………………………………………………………141
Loss of Lpp alleviates OM lipoprotein biogenesis defects…………………………………………………………………………142
Figure 3: Deletion of lpp protects against lipoprotein maturation and trafficking defects…………………………………………………………………………143
Depletion of OM lipoprotein biogenesis causes NlpE-dependent activation of Cpx…………………………………………………………………………144
Figure 4: Depletion of lipoprotein maturation or trafficking causes NlpE-dependent activation of the Cpx stress response…………………………………………………………………………145
Chemical inhibitors of OM lipoprotein biogenesis conform to the biological signature…………………………………………………………………………147
Figure 5: Chemical inhibitors of lipoprotein maturation or trafficking fit the expected profile of OM lipoprotein biogenesis inhibition…………………………………………………………………………148
Proposed LolA inhibitor MAC13243 does not fit the biological signature…………………………………………………………………………149
Table 1: Deletion of lpp increases resistance to OM lipoprotein biogenesis inhibitors…………………………………………………………………………151
Figure 6: An allele specific inhibitor of LolA causes OM permeability and activation of the Cpx stress response…………………………………………………………………………152
MAC13243 activity is LolA-independent…………………………………………………………………………153
Figure 7: MAC13243 activity is independent of LolA…………………………………………………………………………154
Discussion…………………………………………………………………………155
Materials and Methods…………………………………………………………………………159
Supplemental Materials…………………………………………………………………………162
Figure S1: Depletion of lipoprotein maturation or trafficking factors causes outer membrane permeability…………………………………………………………………………162
Figure S2: Deletion of lpp does not protect against general OM biogenesis defects…………………………………………………………………………163
Figure S3: Depletion of lipoprotein maturation or trafficking activates the Rcs stress response but does not activate general OM stress responses…………………………………………………………………………165
Figure S4: Chemical inhibitors of OM lipoprotein biogenesis cause Rcs activation but do not activate sigmaE or RpoD…………………………………………………………………………166
Figure S5: MTSES inhibits the activity of LolA(V24C) and resistance to MTSES increases in the absence of Lpp…………………………………………………………………………167
Figure S6: An allele specific inhibitor of LolA causes OM permeability to antibiotics…………………………………………………………………………168
Figure S7: MTSES treatment activates the Cpx stress response…………………………………………………………………………169
Figure S8: MTSES treatment causes activation of the Rcs stress response…………………………………………………………………………170
Table S1: Strains List…………………………………………………………………………171
Table S2: Plasmid and Oligonucleotide List…………………………………………………………………………178
References…………………………………………………………………………183
Chapter V: Conclusions and future directions…………………………………………………………………………195
References…………………………………………………………………………204
Glossary of Terms…………………………………………………………………………208
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