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An integrative structural biology approach in the study of bacterial persistence and virulence

Yurong Wen (UGent)
(2014)
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(UGent) and (UGent)
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Abstract
Antibiotics have provided an enormous contribution to humans’ fight against infectious microorganisms resulting in a dramatic decrease in mortality due to microbial infections around the globe. However, the widespread and uncontrolled use of antibiotics, particularly as growth promoters in feedstock caused an environmental stress for bacterial evolution and Multi Drug Resistance (MDR) and Multi Drug Tolerance (MDT) are two of the major challenges for further eradication of the bacterial infections. In recent years, this antibiotics crisis has become one of the most serious public health concerns and threats by renewed awareness. Therefore, how the bacteria could survive under the situation of antimicrobial treatment and other environmental stress, and how the bacteria infect the host organism are essential to develop novel antibacterial strategies. Finding the right strategies such as discovery of new antimicrobial drugs are urgent to fight with the current crisis of antibiotic therapy. In this PhD research, we picked up two major process concerning bacterial infections which are bacterial persistence and virulence respectively. Among others, we investigated Toxin-antitoxin systems of which the recent discovery of new families is one of the most exciting molecular microbial phenomena crucial to understand the bacterial behavior. Toxin-antitoxin systems are widespread two or three component systems encoded in the bacterial genome and highlighted to be involved in the persistence, biofilm formation, pathogenicity, programmed cell death and many other cellular processes in bacteria. Due to those characteristics, it also has potential as attractive targets in antimicrobial drug discovery. We applied multiple structural biology, biochemical and biophysical techniques to explore the molecular mechanism of the HipAB toxin-antitoxin system coded in S.oneidensis MR-1 genome, involved in biofilm and persistence. Multiple atomic structures including individual HipB as well as complexes of HipA with non-hydrolysable ATP analogues, Mg2+ and HipA-HipB with its operator DNA were solved. We showed that phosphorylated HipA can engage in an unexpected ternary complex with HipB and double stranded operator DNA that is distinct from the prototypical counterpart complex from E.coli. The structure of HipB in complex with its operator DNA reveals a flexible C-terminus that is sequestering HipA in the ternary complex, indicative of its role in binding HipA to abolish its function in persistence. The structure of a HipA mutant devoid of phosphorylation capacity in complex with a non-hydrolyzable ATP analogue uncovers that HipA autophosphorylation is coupled to an unusual conformational collapse of its pLoop. However, HipA is unable to phosphorylate the translation factor Elongation factor Tu (EF-Tu), contrary to previous reports but in agreement with more recent findings. Our studies suggest that the phosphorylation state of HipA is an important factor in persistence and that the structural and mechanistic diversity of HipAB modules as regulatory factors in bacterial persistence is broader than previously thought. Aside from the investigation of bacterial persistence, we also study the bacterial secretion systems which play a central role in modulating the interactions between the bacteria and its host organisms or extracellular environment by secreting diverse virulence factors. We focused on the opportunistic human pathogen Pseudomonas aeruginosa type 2 secretion system which is one of the most versatile systems used in gram negative bacteria. In P.aeruginosa, the T2SS machinery contains 12 protein subunits which can be distinguished into 5 subassemblies. Among those 12 protein subunits, XcpP is thought to be tightly involved in a procedure that bridges the inner membrane and the outer membrane secretin XcpQ; the assembly of XcpP and XcpQ is crucial to understand the transport machinery. We carried out multiple mass spectrometry experiments and tightly collaborated with a PhD student from Prof. Savvas Savvides’ group, Ruben Van der Meeren focusing on the biophysical, biochemical and functional analysis of the assembly between XcpP and XcpQ. We demonstrated that the P.aeruginosa T2SS secretin XcpQ periplasmic domain is a dimeric protein which oligomerize into a functional dodecameric assembly with C6 symmetry instead of the previous reported dodecameric assembly with C12 symmetry. We proposed the two transition states for XcpP and XcpQ periplasmic interaction which consist of the “open” and “closed” state of the Type 2 secretion system to transport the exoprotein from the periplasm to the extracellular milieu. Overall, we have successfully applied mass spectrometric based integrative structural biology to study the bacterial persistence and virulence. The approach combining the biochemical, structural biological, biophysical techniques and especially the mass spectrometric based techniques that could bridge those techniques are powerful for the structural and functional investigation of supermolecular assembly.
Keywords
Type 2 Secretion System, HipA-HipB, Toxin Antitoxin System, Virulence, Persistence, Integrative Structural Biology, Bacterial Infection

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MLA
Wen, Yurong. “An Integrative Structural Biology Approach in the Study of Bacterial Persistence and Virulence.” 2014 : n. pag. Print.
APA
Wen, Y. (2014). An integrative structural biology approach in the study of bacterial persistence and virulence. Ghent University. Faculty of Sciences, Ghent, Belgium.
Chicago author-date
Wen, Yurong. 2014. “An Integrative Structural Biology Approach in the Study of Bacterial Persistence and Virulence”. Ghent, Belgium: Ghent University. Faculty of Sciences.
Chicago author-date (all authors)
Wen, Yurong. 2014. “An Integrative Structural Biology Approach in the Study of Bacterial Persistence and Virulence”. Ghent, Belgium: Ghent University. Faculty of Sciences.
Vancouver
1.
Wen Y. An integrative structural biology approach in the study of bacterial persistence and virulence. [Ghent, Belgium]: Ghent University. Faculty of Sciences; 2014.
IEEE
[1]
Y. Wen, “An integrative structural biology approach in the study of bacterial persistence and virulence,” Ghent University. Faculty of Sciences, Ghent, Belgium, 2014.
@phdthesis{4299878,
  abstract     = {Antibiotics have provided an enormous contribution to humans’ fight against infectious microorganisms resulting in a dramatic decrease in mortality due to microbial infections around the globe. However, the widespread and uncontrolled use of antibiotics, particularly as growth promoters in feedstock caused an environmental stress for bacterial evolution and Multi Drug Resistance (MDR) and Multi Drug Tolerance (MDT) are two of the major challenges for further eradication of the bacterial infections. In recent years, this antibiotics crisis has become one of the most serious public health concerns and threats by renewed awareness. Therefore, how the bacteria could survive under the situation of antimicrobial treatment and other environmental stress, and how the bacteria infect the host organism are essential to develop novel antibacterial strategies. Finding the right strategies such as discovery of new antimicrobial drugs are urgent to fight with the current crisis of antibiotic therapy. 
In this PhD research, we picked up two major process concerning bacterial infections which are bacterial persistence and virulence respectively. Among others, we investigated Toxin-antitoxin systems of which the recent discovery of new families is one of the most exciting molecular microbial phenomena crucial to understand the bacterial behavior. Toxin-antitoxin systems are widespread two or three component systems encoded in the bacterial genome and highlighted to be involved in the persistence, biofilm formation, pathogenicity, programmed cell death and many other cellular processes in bacteria. Due to those characteristics, it also has potential as attractive targets in antimicrobial drug discovery. We applied multiple structural biology, biochemical and biophysical techniques to explore the molecular mechanism of the HipAB toxin-antitoxin system coded in S.oneidensis MR-1 genome, involved in biofilm and persistence. Multiple atomic structures including individual HipB as well as complexes of HipA with non-hydrolysable ATP analogues, Mg2+ and HipA-HipB with its operator DNA were solved. We showed that phosphorylated HipA can engage in an unexpected ternary complex with HipB and double stranded operator DNA that is distinct from the prototypical counterpart complex from E.coli. The structure of HipB in complex with its operator DNA reveals a flexible C-terminus that is sequestering HipA in the ternary complex, indicative of its role in binding HipA to abolish its function in persistence. The structure of a HipA mutant devoid of phosphorylation capacity in complex with a non-hydrolyzable ATP analogue uncovers that HipA autophosphorylation is coupled to an unusual conformational collapse of its pLoop. However, HipA is unable to phosphorylate the translation factor Elongation factor Tu (EF-Tu), contrary to previous reports but in agreement with more recent findings.  Our studies suggest that the phosphorylation state of HipA is an important factor in persistence and that the structural and mechanistic diversity of HipAB modules as regulatory factors in bacterial persistence is broader than previously thought. 
Aside from the investigation of bacterial persistence, we also study the bacterial secretion systems which play a central role in modulating the interactions between the bacteria and its host organisms or extracellular environment by secreting diverse virulence factors. We focused on the opportunistic human pathogen Pseudomonas aeruginosa type 2 secretion system which is one of the most versatile systems used in gram negative bacteria. In P.aeruginosa, the T2SS machinery contains 12 protein subunits which can be distinguished into 5 subassemblies. Among those 12 protein subunits, XcpP is thought to be tightly involved in a procedure that bridges the inner membrane and the outer membrane secretin XcpQ; the assembly of XcpP and XcpQ is crucial to understand the transport machinery. We carried out multiple mass spectrometry experiments and tightly collaborated with a PhD student from Prof. Savvas Savvides’ group, Ruben Van der Meeren focusing on the biophysical, biochemical and functional analysis of the assembly between XcpP and XcpQ. We demonstrated that the P.aeruginosa T2SS secretin XcpQ periplasmic domain is a dimeric protein which oligomerize into a functional dodecameric assembly with C6 symmetry instead of the previous reported dodecameric assembly with C12 symmetry. We proposed the two transition states for XcpP and XcpQ periplasmic interaction which consist of the “open” and “closed” state of the Type 2 secretion system to transport the exoprotein from the periplasm to the extracellular milieu.  Overall, we have successfully applied mass spectrometric based integrative structural biology to study the bacterial persistence and virulence. 
The approach combining the biochemical, structural biological, biophysical techniques and especially the mass spectrometric based techniques that could bridge those techniques are powerful for the structural and functional investigation of supermolecular assembly.},
  author       = {Wen, Yurong},
  isbn         = {9789059896857},
  keywords     = {Type 2 Secretion System,HipA-HipB,Toxin Antitoxin System,Virulence,Persistence,Integrative Structural Biology,Bacterial Infection},
  language     = {eng},
  pages        = {VI, 168},
  publisher    = {Ghent University. Faculty of Sciences},
  school       = {Ghent University},
  title        = {An integrative structural biology approach in the study of bacterial persistence and virulence},
  year         = {2014},
}