Staphylococcus aureus Virulence Factors Synthesis is Controlled by Central Metabolism

195 Pages·2017·3.13 MB·English

by  Yefei Zhu

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Dissertations & Theses in Veterinary and Veterinary and Biomedical Sciences,
Biomedical Science Department of
12-2010
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Yefei Zhu
School of Veterinary Medicine and Biomedical Sciences, [email protected]
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STAPHYLOCOCCUS AUREUS VIRULENCE FACTORS SYNTHESIS IS CONTROLLED BY
CENTRAL METABOLISM

by
Yefei Zhu

A DISSERTATION

Presented to the Faculty of
The Graduate College at the University of Nebraska
In Partial Fulfillment of Requirements
For the Degree of Doctor of Philosophy

Major: Integrative Biomedical Sciences
Under the Supervision of Professor Greg A. Somerville
Lincoln, Nebraska
December, 2010
STAPHYLOCOCCUS AUREUS VIRULENCE FACTORS SYNTHESIS IS CONTROLLED BY
CENTRAL METABOLISM
Yefei Zhu, Ph.D.
University of Nebraska, 2010
Advisor: Greg A. Somerville
Staphylococcus aureus is a versatile pathogen that can survive in diverse host
environments. This versatility depends on its ability to sense nutrients and respond by
modulating gene expression, including the synthesis of virulence determinants. In addition to its
ability to synthesize virulence factors, the capacity of S. aureus to form biofilms is an important
mediator of virulence in certain infections. Biofilms are a complex aggregation of bacteria
commonly encapsulated by an adhesive exopolysaccharide matrix (polysaccharide intercellular
adhesin; PIA). To study S. aureus biofilm formation, we assessed the metabolic requirements of
S. aureus growing in a biofilm and found the bacteria extracted glucose and accumulated lactate,
acetate, formate, and acetoin. Additionally, S. aureus selectively extracted six amino acids from
the culture medium (serine, proline, arginine, glutamine, glycine, and threonine). The major
staphylococcal exopolysaccharide, PIA, is synthesized when the tricarboxylic acid (TCA) cycle
is repressed. To better understand TCA cycle-dependent regulation of PIA and virulence factor
synthesis in S. aureus, we artificially induced the TCA cycle by limiting its ability to
exogenously acquire a TCA cycle-derived amino acid (i.e., glutamine) by inactivating the
glutamine permease gene (glnP) and assessed the effects on biofilm formation and virulence
factor synthesis. We found that inactivation of this major glutamine transporter increased TCA
cycle activity, transiently decreased PIA synthesis, and significantly reduced in vivo virulence in
a rabbit endocarditis model, establishing a causal relationship between TCA cycle activity and
virulence factor synthesis. This causal relationship between the TCA cycle and virulence factor
synthesis suggests there are regulatory proteins connecting metabolism and the regulation of
virulence factor synthesis. This regulation is likely to occur when a metabolite-responsive
regulator responds to changes in TCA cycle associated biosynthetic intermediates, the redox
status, and/or ATP. In related work, NMR metabolomic analysis of S. epidermidis indicated that
TCA cycle stress altered the intracellular concentration of ribose. Using this information, three
putative ribose-responsive RpiR-family regulators (orfs SAV0317, SAV0193 and SAV2315)
were identified in S. aureus strain UAMS-1. The proteins encoded by sav0317 and sav0193
regulate hexose monophosphate shunt transcription and alter virulence factor synthesis by
increasing the transcription or stability of RNAIII. These data confirm a close linkage of central
metabolism and virulence factor synthesis in S. aureus and establish that this metabolic linkage
can be manipulated to alter infectious outcomes.
iv

ACKNOWEDGMENTS

I owe my deepest gratitude to my advisor, Dr. Greg Somerville for his encouragement,
guidance and advice throughout my doctoral study and dissertation writing.

To the members of my supervisory committee: Dr. Raúl Barletta, Dr. Paul Fey, Dr.
Marjorie Lou and Dr. Scott McVey.

I am also grateful to the members of Dr. Somerville’s lab: Dr. Marat Sadykov, Erik
Jacobson, Melissa Lucas and Devon Kramer.

Lastly, I want to thank my wife, Yi Chen and my parents for giving me constant support.

Yefei Zhu
University of Nebraska-Lincoln
December 2010
v

Table of Contents

Abstract………………………………………………………………………………..…ii
Acknowledgements……………………………………………………….……………..iv
Table of Contents……………………………………………………………….………..v
Lists of Multimedia Objects…………………………………………………………...viii
Chapter I: Literature Review…………………………………………………………...1
Introduction……………………………………………………………………......2
Overview of Central Metabolism in Staphylococcus aureus……………………...3
Overview of Virulence Factors and their regulation in Staphylococcus aureus…..5
Metabolic Regulation of Virulence……………………………………………....13
Conclusions………………………………………………………………………28

Chapter II: Staphylococcus aureus Biofilm Metabolism and the Influence of
Arginine on Polysaccharide Intercellular Adhesin Synthesis, Biofilm Formation,
and Pathogenesis……………………………………………………………………….30
Abstract………………………………………………………………………….31
Introduction……………………………………………………………………...32
vi

Materials and Methods…………………………………………………………..35
Results……………………………………………………………………………41
Discussion……………………………………………..…………………………46
Tables and Figures……………………………………………………………….50

Chapter III: Tricarboxylic Acid Cycle-dependent Attenuation of Staphylococcus
aureus in vivo Virulence by Selective Inhibition of Amino Acid Transport………..57
Abstract……………………………………………………………………….….58
Introduction………………………………………………………………….…...59
Materials and Methods……………………………………………………….…..62
Results……………………………………………………………………………70
Discussion……………………………………………..…………………………75
Tables and Figures……………………………………………………………….80

Chapter IV: Rpir Homologues Link Staphylococcus aureus Virulence Factor
Synthesis to the Hexose Monophosphate Shunt………………………………………90
Abstract………………………………………………………………….……….91
Introduction………………………………………………………………….…...93
Materials and Methods……………………………………………………….…..96
vii

Results…………………………………………………………………………..104
Discussion……………………………………………..………………………..112
Tables and Figures……………………………………………………………...116

References……………………………………………………………………………...126

Appendices………………………………………………………………………………A
Appendix A: 2h post-inoculation proteomic analyses…………………………....A
Appendix B: 6h post-inoculation proteomic analyses……………………………K
viii

Lists of Multimedia Objects

Chapter II:
Table 1. Strains and plasmids used in this study……………………………...…………50
Table 2. Primers used in this study…………………………………..………………….51
Figure 1. Growth of S. aureus strains UAMS-1, UAMS-1182, and UAMS-1-arcD::ermB
in a 3-chamber flow cell……………………………………………………..…………..52
Figure 2. Metabolic extraction and accumulation by S. aureus strains UAMS-1 and
UAMS-1182 during biofilm growth in flow cells…………………………………….…53
Figure 3. Dissolved oxygen concentration in the culture medium effluent of strains
UAMS-1 and UAMS-1182 grown in flow cells…………………………………………55
Figure 4. Growth characteristics of strains UAMS-1 and UAMS-1-arcD::ermB grown
under biofilm and planktonic conditions………………………………………………...56

Chapter III:
Table 1. Strains and plasmids used in this study………………………………………..80
Table 2. Primers used in this study……………………………………………………...81
Table 3. S. aureus target tissue densities in rabbit endocarditis model at 48 h post-
infection………………………………………………………………………………….82
ix

Figure 1. Physiological and metabolic characteristics of S. aureus strains UAMS-1 and
UAMS-1-glnP::ermB……………………………………………………………………83
Figure 2. Inactivation of glnP transiently decreases icaADBC transcription and PIA
biosynthesis………………………………………………………………………………85
Figure 3. Growth of S. aureus strains UAMS-1 and UAMS-1-glnP::ermB in a 3-chamber
flow cell………………………………………………………………………………….87
Figure 4. Inactivation of glnP alters the temporal pattern of virulence factor synthesis..89

Chapter IV:
Figure 1. Southern blots demonstrating the inactivation of rpiRA, rpiRB and rpiRC in
strain UAMS-1 by the insertion of antibiotic cassettes ermB, cat, and tetM
respectively……………………………………………………………………..………116
Figure 2. Growth curves and culture medium pH profiles for strains UAMS-1, UAMS-1-
rpiRA, UAMS-1-rpiRB, UAMS-1-rpiRC mutants under aerobic growth conditions…..117
Figure 3. Inactivation of rpiR homologues alters hexose monophosphate shunt
activity………………………………………………………………………….….……118
Figure 4. Inactivation of rpiR homologues alters the expression of virulence factors and
delays biofilm formation…………………………………………………………….….119
Figure 5. RNAIII Northern blots……………………………………………………….121