Host - Pathogen Interaction E-Book
129,99 €
Mehr erfahren.
- Herausgeber: Wiley-VCH
- Kategorie: Fachliteratur
- Serie: Drug Discovery in Infectious Diseases
- Sprache: Englisch
In light of the rapidity increasing incidence rate of bacterial and fungal infections with multi-resistant pathogens, the metabolic changes associated with host-pathogen interactions offer one of the most promising starting points for developing novel antibiotics. . Part one of this comprehensive guide describes the metabolic adaptation of pathogenic microbes in humans, while part two points to routes for the development of novel antibiotics. This is volume six of the book series on drug discovery in infectious diseases by Paul Selzer.
Sie lesen das E-Book in den Legimi-Apps auf:
Seitenzahl: 502
Veröffentlichungsjahr: 2016
Ähnliche
Table of Contents
Cover
Titles of the Series “Drug Discovery in Infectious Diseases”
Title Page
Copyright
Preface
Cover Legend
List of Contributors
Part One: Adaptation of Microbial Metabolism in Host/Pathogen Interaction
Chapter 1: Part A: Adaptation of Microbial Metabolism in Host/Pathogen Interaction
Introduction
Yersinia
Life Cycles and Pathogenesis
Carbon Metabolism and Links to
Yersinia
Pathogenesis
Nutritional Virulence: Nutritional Adaptation Important for Pathogenesis
Coordinated Control of Carbon Metabolism and Virulence
Conclusions
Acknowledgments
References
Chapter 2: Crosstalk between Metabolism and Virulence of Legionella pneumophila
Introduction
Key Metabolic Features of
L. pneumophila
Serine and Glucose Metabolism of
L. pneumophila
under
In Vitro
Conditions
Metabolism of
L. pneumophila
under Intracellular Conditions
Metabolic Adaptation during the Life Cycle of Intracellular
L. pneumophila
Metabolic Host Cell Responses Triggered by Intracellular
L. pneumophila
Conclusion
References
Chapter 3: Metabolism of Intracellular Salmonella enterica
Introduction
S
.
enterica
– A Metabolic Generalist
Metabolism of Glucose by
S
.
enterica
Connection to the TCA Cycle by Oxidative Decarboxylation of Pyruvate
The Important Role of the TCA Cycle and Anaplerotic Reactions
Metabolism of Intracellular
S
.
enterica
The Metabolism of
S
.
enterica
Compared to Other Important Intracellular and Gastrointestinal Pathogens
Salmonella
Induce Networks of Tubular Structures – Access to Host-Derived Nutrients?
S
.
enterica
Has a Bimodal Lifestyle in Epithelial Cells
Salmonella
Metabolism Limits Possibilities for New Antimicrobials
Conclusions
References
Chapter 4: The Human Microbiome in Health and Disease
Introduction
Methods for Characterizing the Microbiota
Diet and Geographical Factors
Functional Gastrointestinal Disorders
Manipulation of the Microbiota
Conclusions
Acknowledgments
References
Chapter 5: Mechanisms of Dysbiosis in the Inflamed Gut
Introduction
Dysbiosis during Inflammatory Diseases of the Gastrointestinal Tract
Nutrient Acquisition by Commensal Bacteria in the Normal Gut
Nutritional Mechanisms for Dysbiosis in the Inflamed Gut
Inflammation-Driven Bloom of Enteric Pathogens in the Gut Lumen
How Anaerobic Respiration Enhances Growth in the Inflamed Gut
Conclusions
References
Chapter 6: Strategies for Nutrient Acquisition by Magnaporthe oryzae during the Infection of Rice
Introduction
Adhesion and Germination
Appressorium Formation
Penetration
Growth in Planta: The Biotrophic Growth Phase
Growth in Planta: Necrotrophic Growth Phase
Growth in Planta: Sporulation
Life Outside of the Rice Plant
Conclusion
References
Part Two: New Inhibitors and Targets of Infectious Diseases
Chapter 7: Part B: New Inhibitors and Targets of Infectious Diseases
Introduction
Mannheimia haemolytica
Pathogenesis of Pneumonic Pasteurellosis
Virulence Factors of
M. haemolytica
Antibiotic Use against
M. haemolytica
Antibiotic Resistance
Resistance Mechanisms
Discovery of New Antibacterial Agents
The Importance of
In Vivo
Growth Conditions
Potential Outer Membrane Targets for Novel Antibiotics against
M. haemolytica
Conclusion
References
Chapter 8: Identification of Anti-infective Compounds Using Amoebae
Introduction
Anti-virulence Compounds as an Alternative to Antibiotics
Amoebae as Model Host Systems in Compound Screening
The Amoeba-Resistant Pathogens
L. pneumophila
and
M. marinum
Development of Novel Amoebae-Based Screening Systems
Palmostatin M – A Novel Antibacterial Compound Specific for
Legionella
and
Mycobacterium
Species
Conclusion
Acknowledgments
References
Chapter 9: Stress Biology in Fungi and “Omic” Approaches as Suitable Tools for Analyzing Plant–Microbe Interactions
Introduction
Stress Signaling Pathways Are Important in Plant–Microbe Interactions
High Osmolarity
Reactive Oxygen Species (ROS)
Plant Hormones (Phytoalexins)
Technologies for Studying Signaling Pathways
The Age of “Omics” – General Information
Transcriptomics
Transcriptomics in Use
Proteomics
Proteomics in Use
Metabolomics
Metabolomics in Use
Conclusion
References
Chapter 10: Targeting Plasmids: New Ways to Plasmid Curing
Introduction
Research Approaches for Plasmid Curing
Prerequisites for Clinical Use and Possible Application Methods
Common Methods and Compounds in Use
Approaches and Techniques to Identify Antiplasmid Effects/Compounds since 2000
Conclusion
References
Chapter 11: Regulation of Secondary Metabolism in the Gray Mold Fungus Botrytis cinerea
Botrytis cinerea
, the Causal Agent of Gray Mold Disease
Repertoire of Secondary Metabolites Produced by
B. cinerea
Expression of SM Genes during
In Vitro
and
In Planta
Conditions
Pathway-Specific Regulation by Transcription Factors
Concerted Regulation of Secondary Metabolism and Light-Dependent Development
Regulation of Secondary Metabolism by Conserved Signal Transduction Pathways
Role of the Chromatin Landscape in the Regulation of Secondary Metabolism?
Concluding Remarks
References
Index
End User License Agreement
Pages
vii
viii
ix
xi
xii
xiii
xiv
1
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
217
218
219
220
Guide
Cover
Table of Contents
Preface
Part One: Adaptation of Microbial Metabolism in Host/Pathogen Interaction
Begin Reading
List of Illustrations
Chapter 1: Part A: Adaptation of Microbial Metabolism in Host/Pathogen Interaction
Figure 1.1 Metabolic pathways and virulence factors of
Y. pestis,
which are significantly induced in the mammalian host and the flea gut. Specific metabolic pathways and pathogenicity traits upregulated
in vivo
are presented, which are considered to be crucial for the colonization of the lung or bubo of the mammalian host (a) and the flea gut (b). Abbreviations: BarA/UvrY (nutrient-responsive two-component system); Csr (carbon storage regulator); Crp (cAMP receptor protein); GADP (glyceraldehyde-3P); Hfq: RNA chaperone; KDGP (2-dehydro-3-deoxy-gluconate-6P); M-cell (microfold cell); 3-P-G (3-phosphoglycerate); 2PG (2-phosphoglycerate); PhoP/PhoQ (ion-responsive two-component system), PsaA (pH6 antigen); Yops (
Yersinia
outer proteins); T3SS (type III secretion system); and TCA (tricarboxylic acid cycle).
Figure 1.2 Schematic overview of the environmental sensing and signal transduction system and the regulatory cascade with implicated control factors that are known to coordinate expression of metabolic functions and virulence-associated traits of
Y. pseudotuberculosis
. All sensory and regulatory components are also encoded in the other human pathogenic
Yersinia
species, but the function of some of them has still not been experimentally verified.
Chapter 2: Crosstalk between Metabolism and Virulence of Legionella pneumophila
Figure 2.1 Incorporation of [U-
13
C
3
]serine into
L. pneumophila
under
in vitro
conditions [44]. The
13
C-profiles are indicated by bold lines connecting
13
C-labeled atoms in a given molecule. The labeling patterns of the compounds in boxes were determined. The numbers indicate the molar abundances (mol%) of the respective isotopologues as determined by quantitative NMR or MS (M+1, M+2, M+3 indicates isotopomers with 1, 2, or 3
13
C-labels, respectively). The natural molar abundances of M+1, M+2, and M+3 isotopomers are approximately 1.1%, 0.01%, and 0.0001%, respectively. The detected enrichments for the multiply
13
C-labeled isotopologues are increased by several orders of magnitude in the labeling experiment and therefore underline the specificity and significance of these data. PEP, phosphoenolpyruvate; OAA, oxaloacetate; and α-KG, α-ketoglutarate.
Figure 2.2 Incorporation of [U-
13
C
6
]glucose into
L. pneumophila
under
in vitro
conditions [44]. The
13
C-profiles are indicated by bold lines connecting
13
C-labeled atoms in a given molecule. The labeling patterns of the compounds in boxes were determined. The numbers indicate the molar abundances (mol%) of the respective isotopologues as determined by quantitative NMR or MS. For more details, see legend to Figure 2.1.
Figure 2.3 Schematic drawing of the metabolism of
L. pneumophila
(
Lpp
) (Schunder et al. [50]; modified). A combination of data of
in silico
analysis (gray arrows),
in vitro
/AYE medium grown
Lpp
(blue arrows), and
in vivo
/
Acanthamoeba castellanii
grown
Lpp
(black arrows) using
13
C isotope (*)-labeled substrates, are shown.
In vitro,
glucose is mainly metabolized through the ED pathway and pyruvate enters the citric acid cycle or the PHB synthesis pathway (reaction 4) mainly through acetyl-CoA (Ac-CoA). The Embden–Meyerhof–Parnas (EMP) pathway is present but not used to metabolize glucose
in vitro
, but is thought to be used in gluconeogenesis (reaction 5). The glucoamylase (Lpp0489) is essential for
L. pneumophila
to generate glucose from glycogen [51]. The proteasome is involved in the generation of free amino acids in infected host cells [52]. The amino acids (red circles) reach the
A. castellanii Legionella
-containing vacuole (Ac-LCV) by a SLC1A5-like amino acid transport protein or by other yet not known amino acid transporters (black circles). Many amino acid transport proteins are known for
L. pneumophila
(green circles) and are expressed during intracellular growth. Amino acids of
in vitro
grown bacteria are given in blue and amino acids of
A. castellanii
and of
in vivo
grown
L. pneumophila
are shown in black. The amino acids Gln, Trp, Met, Cys, and Arg could not be analyzed by the isotopologue profiling method. Some amino acids (#) of LCV-grown bacteria showed a significant different isotopologue profile when compared with
L. pneumophila
infected
A. castellanii
-derived amino acids. This seems to be a result of the (co)-metabolism of these amino acids through the Glu-Asp (reaction 2) and the Glu-Pyr (reaction 3) transaminases or by
de novo
synthesis, for example, using Ser dehydratase (reaction 1) to metabolize serine. PHB of
L. pneumophila
(yellow circle) was also found to be labeled in
in vitro
and
in vivo
experiments. That glycerol metabolism is important for intracellular growth of
L. oakridgensis
[53] and a glycerol-3-P transporter complex is present within the genome sequence, as well as carbohydrates and fatty acid (FA) transport proteins [33]. The role of nutrients delivered to the LCV by the cargo of vesicles of the endoplasmic reticulum (ER) is not known yet. Genes (
lpp
gene numbers) are given within the green and blue circles. AAs, amino acids; Ac,
A. castellanii
; Hex-P, hexosephosphate transport protein; KG, ketoglutarate; Pyr, pyruvate; CIT, citrate; MAL, malate; OAA, oxaloacetate; Lpp,
L. pneumophila
strain Paris; reaction 1, serine dehydratase; reaction 2, Glu-Asp transaminase; reaction 3, Glu-Pyr transaminase; and reaction 4, PHB synthesis pathway (β-ketothiolase, acetoacetyl-CoA-reductase, PHB polymerase).
Chapter 3: Metabolism of Intracellular Salmonella enterica
Figure 3.1 Intracellular lifestyle of
Salmonella enterica
.
Salmonella
is taken up by host cells either by
Salmonella-
induced invasion (SPI1-T3SS-induced macropinocytosis) or by phagocytosis. By translocating effector proteins via the SPI2-T3SS into the host cell, the SCV undergoes an altered maturation process. The formation of
Salmonella
-induced filaments (SIFs) takes place with the start of
Salmonella
replication between 4 and 6 h after infection. SIF develop on a microtubule scaffold.
Figure 3.2 Model of intracellular metabolism of
Salmonella enterica
. Carbon substrates, catabolic reactions, and
de novo
synthesized amino acids deduced from differential genes expression profiling (DGEP) data and
13
C-isotopologue-profiling analysis (
13
C-IPA) data as well as
in vivo
studies are shown for intracellular growing
S. enterica
. Solid black arrows indicate pathways and reactions that may be required under all conditions, but the extent of their contribution to the metabolism of intracellular bacteria cannot be deduced from the data available. The blue boxes indicate carbon sources used for
S. enterica
and the red arrows show the active catabolic reactions for the major carbon source, which is glucose for
S. enterica
. Red boxes indicate amino acids that are synthesized
de novo
, according to the
13
C-IPA data. In
S. enterica
Ser, Gly, Ala, Val, Asp, and Glu are
de novo
synthesized more efficiently than the other amino acids. Dashed red arrows indicate metabolic reactions and pathways suggested from DGEP and
13
C-IPA data from mutants defective for the major carbon source and by mouse infection studies. Mutants defective for utilization of major carbon sources had to use nonglycogenic carbon substrates such as glycerol-3P or pyruvate for carbon metabolism. Besides the reversible reactions from glycolysis, this requires anaplerotic reactions and gluconeogenesis. Gluconeogenesis involves the reactions generating PEP from pyruvate (by PEP synthase, Pps) and fructose-6-phosphate from fructose-1,6-bisphosphate (by fructose-1,6-biphosphatase, Fbp). Green arrows show the anabolic step and the major catabolic intermediates from which they derive. Central metabolic pathways (blue boxes) comprise glycolysis and gluconeogenesis, the KDPGP, the pentose phosphate pathway (PPP), the tricarboxylic acid (TCA) cycle, and various anaplerotic reactions that replenish metabolic gaps, such as the generation of oxaloacetate by phosphoenolpyruvate carboxylase (Ppc), the glyoxylate shunt (which uses isocitrate lyase, AceA, and malate synthase, AceB), and the generation of pyruvate by decarboxylating malate dehydrogenase (SfcA). PTS, PEP-dependent phosphotransferase system; GlnT, gluconate transferase; Mdh, malate dehydrogenase; GltA, citrate synthase; AcnAB, aconitase AB; IcdA, isocitrate dehydrogenase A; SucAB, succinyl-CoA synthetase AB; IpdA, α-ketoglutarate dehydrogenase A; SdhCDAB, succinate dehydrogenase; FrdABCD, fumarate reductase; and FumA, B, C, fumarases A, B, C [4, 5].
Figure 3.3 Model of the dynamic extension and contraction of SIF and possible accession to membrane vesicles. (Reproduced from Rajashekar
et al.
[58], with the permission of John Wiley and Sons.) (a)
Salmonella
within the SCV translocate effector proteins of the SPI2-T3SS (orange circles). (b) Membrane vesicles transported on microtubules (MTs) are recruited and fuse with the SCV by the activity of effector proteins. These events not only allow the enlargement of the SCV and delivery of luminal content to the SCV (indicating blue shading) but also lead to accumulation of motor proteins on the SCV. (c) Increased accumulation of motor proteins results in a pulling force on the SCV membrane and formation of tubular extension. (d) If pulling forces are too high, motor proteins could lose contact to SIF membranes or MT, resulting in contraction of SIF. Depending on the nature of the motor protein recruited, SIF extend toward the minus (−) or plus (+) end of MT. The extension toward the plus end appears to be dominant [58].
Chapter 4: The Human Microbiome in Health and Disease
Figure 4.1 The wide variety of different choices available to characterize the microbiota. Each method has advantages and disadvantages. Each step separating the sample from the endpoint is a potential source of bias.
Figure 4.2 The complex interactions between host, microbiota, and environment. Both the host and the microbiota have a range of characteristics unique to an individual. The health status of the host–microbiota superorganism can be altered by a number of external environmental factors, perhaps most significantly by diet. Host factors and the composition of the microbiota determine an individual's response to stressors. These may or may not alter the homeostasis of the superorganism. A number of microbial therapeutics are currently under investigation to restore health, including bacteriotherapy, altered diet, and fecal transplantation. Probiotics and prebiotics are also candidate interventions, but efficacy for microbiota modulation is still unclear due to lack of mechanisms of action and inconsistency between studies.
Chapter 5: Mechanisms of Dysbiosis in the Inflamed Gut
Figure 5.1 Generation of alternative electron acceptors as byproducts of the inflammatory response in the gut. Superoxide created by the host enzymes NADPH oxidase 1 (NOX1), dual oxidase 2 (DUOX2), and phagocyte oxidase (PHOX) is converted into peroxide by superoxide dismutase (SOD), which in return is used as a substrate for myeloperoxidase (MPO) to generate hypochlorite. Reactive oxygen species, in particular hypochlorite, oxidize endogenous thiosulfate to tetrathionate [46], a substrate for the
S
.
typhimurium
tetrathionate reductase. Nitric oxide produced by inducible nitric oxide synthase (iNOS) reacts with superoxide to form peroxynitrite. Peroxynitrite isomerizes to nitrate, which serves as the electron acceptor for bacterial nitrate respiration in the inflamed gut [47]. Breakdown of nitrate yields nitrite, which can be reduced by bacterial nitrite reductases. Organic sulfides and tertiary amines can be oxidized by reactive oxygen and nitrogen species to the respective sulfoxide or amine
N
-oxide. Structurally diverse sulfoxides or amine
N
-oxides can serve as terminal electron acceptors for Enterobacteriaceae [48].
Figure 5.2 Metabolic environment conducive to the outgrowth of commensal and pathogenic Enterobacteriaceae in the inflamed gut. Production of reactive oxygen (ROS) and nitrogen (RNS) species yields oxidation produces that can serve as electron acceptors for Enterobacteriaceae (for details see Figure 5.1). Oxygen levels increase as a result of ileostomy, a surgical opening of the otherwise anaerobic intestinal lumen [40]. Primary fermenters in the normal gut microbiota break down complex glycans to formate, hydrogen, lactate, succinate, and short-chain fatty acids, including propionate, acetate, and butyrate. Acetate is also produced by acetogenesis from carbon dioxide and exogenous electron donors (reviewed in [25]). In the presence of alternative electron acceptors, these fermentation end products could be used as carbon and energy sources for Enterobacteriaceae.
S. typhimurium
utilizes ethanolamine, possibly derived from the membrane lipids of extruded or exfoliated enterocytes, in the presence of tetrathionate [84].
Chapter 6: Strategies for Nutrient Acquisition by Magnaporthe oryzae during the Infection of Rice
Figure 6.1 Developmental changes in
Magnaporthe oryzae
during infection of rice. (a) Electron micrograph of
M. oryzae
Guy11 spores, which infect rice via an appressorium, shown in (b), laser scanning confocal microscopy, where rice cell walls are stained red with propidium iodide, and green fluorescence denotes the appressorium and invasive hypha. Panels (c) and (d) show cell-to-cell movement of aniline blue stained hyphae of
M. oryzae
during the colonization of tissue. After the biotrophic phase of infection has finished, colonization continues as hyphae grow extracellularly as shown in (e), where red signal is derived from propidium iodide fluorescence, staining plant cell walls, and green fluorescence is from WGA-Alexa 488 stained fungal cell walls.
M. oryzae
compatible infection on leaves is characterized by (f) lozenge-shaped lesions. Scale bars represent (a) 2.5 µm, (b, c, e) 10 µm, (d) 5 µm, and (f) 2.5 mm. White arrowheads show cell-to-cell crossing points.
Chapter 7: Part B: New Inhibitors and Targets of Infectious Diseases
Figure 7.1 Lung of calf after experimental challenge with
M. haemolytica
showing pneumonic lesions.
Figure 7.2 Generalized structure of Gram-negative cell envelope including the outer membrane, periplasm, and cytoplasmic membrane. The outer membrane comprises an outer leaflet of lipopolysaccharide (LPS), an inner leaflet of phospholipids, various transmembrane proteins which have characteristic β-barrel structures and include the porins, and lipoproteins.
Figure 7.3 Outer membrane protein profiles of bovine (Bov) and ovine (Ov)
M. haemolytica
isolates of serotypes A1, A2, A7, A11, and A13. The bacteria were grown under iron-limited growth conditions and show enhanced expression of iron-regulated proteins in the upper molecular mass region.
Figure 7.4 Schematic representation of the AcrAB–TolC efflux pump. AcrB represents the cytoplasmic membrane-spanning protein responsible for actively pumping antibiotics across the cell envelope; TolC represents the outer membrane channel through which the antibiotics are pumped; and AcrA represents the periplasmic adapter protein linking AcrB and TolC.
Figure 7.5 Schematic representation of generalized BAM complex, which is involved in folding and insertion of integral OMPs into the outer membrane. BamA is an integral membrane protein consisting of a transmembrane β-barrel domain and five polypeptide-transport-associated (POTRA) domains, which is required for insertion of proteins into the outer membrane; BamB to E are accessory lipoproteins localized in the inner leaflet of the outer membrane. BamA and BamD are essential proteins and inhibition of BamD may lead to interference with OMP assembly (128). Unfolded OMP precursors are transported across the cytoplasmic membrane by the Sec machinery and, in the periplasm, interact with the chaperone proteins SurA, Skp, and DegP (not shown).
Figure 7.6 Schematic representation of the iron-uptake transferrin receptor complex TbpAB and the TonB energy-transduction system comprising TonB, ExbB, and ExbD. Transport of essential iron across the outer membrane via the TonB-dependent TbpA protein, and other iron-transport proteins, could be blocked by inhibiting the TonB system.
Chapter 8: Identification of Anti-infective Compounds Using Amoebae
Figure 8.1 Potential targets of antivirulence compounds. Several antivirulence schemes can be exploited to restrict pathogens. Antibodies can bind to and neutralize secreted bacterial toxins. Small-molecule compounds can target bacterial secretion systems, preventing the injection of effector proteins, or modulate quorum sensing systems, thus blocking bacterial colonization and virulence. Lastly, compounds can be designed to bind to transcriptional regulators of virulence genes, thus inhibiting their expression.
Figure 8.2 Setup and results of amoebae screens. (a) Overview of the fluorescence-based
L. pneumophila
/
A. castellanii
growth assay. Ninety-six-well plates seeded with
A. castellanii
are infected with GFP-producing
L. pneumophila
. The increase in GFP fluorescence is monitored, indicating the progress of intracellular replication. (b) Fluorescence growth curve of wild-type
L. pneumophila
and the replication-defective Δ
icmT
mutant strain within
A. castellanii
indicating the typical phases (delay-growth-stationary) over 30 h. (c) Dose-response curves of
L. pneumophila
treated with the β-lactone palmostatin M both in broth (extracellular growth) and within
A. castellanii
or RAW 264.7 macrophages (intracellular growth). Graph indicates mean and standard deviation (SD) from more than 10 separate experiments. (d) Palmostatin M also demonstrated significant inhibition of
M. tuberculosis
extracellular growth over a long timescale (60 days). Graph indicates mean and SD of triplicate assays.
Figure 8.3 Hypothetical mode of action of the β-lactone palmostatin M. (a) The (probable) interaction of the β-lactone palmostatin M with the active site of the Ras depalmitoylase APT1, leading to covalent modification and inhibition of the enzyme [[70, 71]]. (b) The hypothetical interaction of palmostatin M with a
Legionella
or
Mycobacterium
target, presumably being modified with a similar mechanism as APT1. The closely related β-lactone palmostatin B had no effect, and thus, likely does not modify a bacterial target.
Chapter 9: Stress Biology in Fungi and “Omic” Approaches as Suitable Tools for Analyzing Plant–Microbe Interactions
Figure 9.1 Flowchart of “omic” disciplines and their interplay in the postgenomic era.
Figure 9.2 Growth of annotated genomes in MycoCosm. The genomes sequenced by JGI are shown in blue and those sequenced by others are shown in red.
Chapter 10: Targeting Plasmids: New Ways to Plasmid Curing
Figure 10.1 Resistance to fluoroquinolones in
E. coli
, national data.
Figure 10.2 Resistance to third-generation cephalosporins in
K. pneumoniae
, national data.
Figure 10.3 Resistance to β-lactam antibacterial drugs in
S. aureus
(i.e., methicillin-resistant
S. aureus
, MRSA), national data.
Figure 10.4 Possible sites of action for plasmid curing agents. (A) Inhibition of replication. (B) Activation of plasmid coded antitoxin–toxin systems. (C) Inhibition of conjugation.
Figure 10.5 Structure of apramycin.
Figure 10.6 Structures of dehydrocrepenynic acid and linoleic acid.
Figure 10.7 Structures of ETIDRO and CLODRO.
Figure 10.8 Structure of 2-chloro-10-(2-dimethylaminoaethyl)-phenothiazine.
Figure 10.9 Structure of 8-epidiosbulbin E acetate (EEA).
Chapter 11: Regulation of Secondary Metabolism in the Gray Mold Fungus Botrytis cinerea
Figure 11.1 Repertoire of the predicted secondary metabolism key enzymes (KE) genes in
Botrytis cinerea
and their expression in different growth conditions. Conidial suspensions of the wild type strain B05.10 were incubated for 48 h on minimal medium (
M
), complete medium (
C
), grape Juice medium (
J
), grape berries (
G
;
Vitis vinifera
), or on bean leaves (
B
;
Phaseolus vulgaris
). Expressions (i.e., log2-normalized intensities) from NimbleGen array data were clustered and depicted by a color scale, where light gray represent weakly expressed genes and dark gray represent highly expressed genes.
S
Indicates that the KE is also present in the close species
Sclerotinia sclerotiorum
[5].
*
The KE responsible for ABA synthesis remains unknown, so the gene encoding the BcABA1 P450 monooxygenase was included in the analysis [12].
Figure 11.2 The two toxins botcinic acid (BOA) and botrydial (BOT) are produced from clusters of co-regulated genes and have a redundant role in virulence. (a) BOT and BOA clusters predicted in the wild strain B05.10 [13, 14] and surrounding AT-rich regions (J. van Kan
et al
., unpublished); chemical structures of BOT and BOA (PubChem). The KE for BOT synthesis is the Sesquiterpene cyclase (STC) encoded by
bcbot2/stc1.
Other co-regulated genes encode for P450 monooxygenases (
bcbot1, 3,
and
4
) also involved in BOT synthesis [12] (I. G. Collado
et al
., unpublished) and a putative acetyl transferase (
bcbot5
). Two KEs are required for BOA synthesis: the polyketide synthases (PKSs)
bcbot6/pks6
and
bcboa9/pks9
[14, 15]. Other co-regulated genes encode for putative monooxygenases (
bcboa2, 3, 4,
and
7
), dehydrogenases (
bcboa5
and
17
), a FAD-binding protein (
bcboa8
), a dehydratase (
bcboa16
), a thioesterase (
bcboa10
), a transferase (
bcboa11
), and unknown proteins (
bcboa12, 14,
and
16
). A pathway-specific Zn(II)
2
Cys
6
transcription factor (TF) is encoded by
bcboa13
and a Nmr-A like regulator is putatively encoded by
bcboa1
. (b) While both single mutants Δ
bcboa6
and Δ
bcbot2
, impaired in BOA and BOT production, respectively, are not altered in virulence compared to the wild type (WT) strain (not shown), the double ΔΔ
bcboa6-bcbot2
mutant, unable to produce any toxin, causes significant smaller necrotic lesions on several host plants. Here, conidial suspensions were inoculated on leaves of
Phaseolus vulgaris
(French bean) and
Vitis vinifera
(grape) berries.
Figure 11.3 Members of the VELVET complex (BcVEL1, BcLAE1) and the light-responsive transcription factor BcLTF1 control light-dependent differentiation, secondary metabolism, and virulence. (a) Phenotypes of the deletion mutants in wild strain 1750 (BIK producer) and the standard recipient strain B05.10. OA – detection of oxalic formation by acidification of the culture medium 6 dpi (bluish green: pH >7, yellow pH <6). DD – differentiation phenotype 12 dpi in constant darkness; the WT:B05.10 produces sclerotia while the other strains produce conidia. MEL – accumulation of DHN melanin in liquid cultures 7 dpi (minimal medium with NaNO
3
as nitrogen source). BIK – accumulation of bikaverin in liquid cultures 7 dpi (minimal medium with NH
4
NO
3
as nitrogen source). Virulence – lesion formation on primary leaves of
Phaseolus vulgaris
(French bean) 6 dpi. (b) Comparative gene expression studies of the mutants. Conidial suspensions of the wild type B05.10 and the three deletion mutants were incubated for 48 h on solid grape juice medium with cellophane overlays. Material from four biological replicates were used for hybridization of NimbleGen arrays. Statistical analyses revealed 18 KE-encoding genes that are differentially expressed in at least one mutant (
*
: fold change >2;
p
<0.05). Relative expression values of these genes (i.e., log2-normalized intensities scaled by gene) were clustered and depicted by color scale, in which shades of green and red represent under- and overexpressed genes, respectively. GEO accession GSE63021 and (A. Simon
et al.
, unpublished).
List of Tables
Chapter 3: Metabolism of Intracellular Salmonella enterica
Table 3.1 Nutrients that can be metabolized by
Salmonella
in infected tissues
Chapter 4: The Human Microbiome in Health and Disease
Table 4.1 Advantages and disadvantages of different characterization techniques
Titles of the Series “Drug Discovery in Infectious Diseases”
Selzer, P.M. (ed.)
Antiparasitic and Antibacterial
Drug Discovery
From Molecular Targets to Drug Candidates
2009
Print ISBN: 978-3-527-32327-2, also available in Adobe PDF format ISBN: 978-3-527-62682-3
Becker, K. (ed.)
Apicomplexan Parasites
Molecular Approaches toward Targeted Drug Development
2011
Print ISBN: 978-3-527-32731-7 also available in digital formats
Caffrey, C.R. (ed.)
Parasitic Helminths
Targets, Screens, Drugs and Vaccines
2012
Print ISBN: 978-3-527-33059-1 also available in digital formats
Jäger, T., Koch, O., Flohé, L. (eds.)
Trypanosomatid Diseases
Molecular Routes to Drug Discovery
2013
Print ISBN: 978-3-527-33255-7 also available in digital formats
Doerig, C., Späth, G., Wiese, M.
Protein Phosphorylation in Parasites
Novel Targets for Antiparasitic Intervention
2013
Print-ISBN: 978-3-527-33235-9 also available in digital formats
Forthcoming Topics of the Series
Sylke Müller, Rachel Cerdan, Ovidiu Radulescu (eds.)
Comprehensive Analysis of Parasite Biology
Charles Q. Meng, Ann E. Sluder (eds.)
Ectoparasites: Drug Discovery AgainstMoving Targets
Edited by Gottfried Unden, Eckhard Thines, and Anja Schüffler
Host - Pathogen Interaction
Microbial Metabolism, Pathogenicity and Antiinfectives
The Editors
Volume Editors:
Prof. Dr. Gottfried Unden
University of Mainz
Institute for Microbiology and Wine Research, Johann-Joachim-Becherweg 15
55128 Mainz
Germany
Prof. Dr. Eckhard Thines
University of Mainz
Institute for Microbiology and Wine Research, Johann-Joachim-Becherweg 15
55128 Mainz
Germany
Dr. Anja Schüffler
Institute of Biotechnology and Drug Research, Erwin-Schrödinger-Straβe 2
67663 Kaiserslautern
Germany
Series Editor:
Prof. Dr. Paul M. Selzer
Boehringer Ingelheim Animal Health GmbH
Global Pharamceutical R&D AH
Binger Straβe 173
55216 Ingelheim am Rhein
Germany
and
University of Tübingen
Interfaculty Institute for Biochemstry
Tübingen
Germany
Wellcome Trust Centre for Molecular Parasitology, Institute of Infection Immunity and Inflammation
Faculty of Biomedical & Life Sciences
University of Glasgow
Glasgow
United Kingdom
Cover legend
See page IX
All books published by Wiley-VCH are carefully produced. Nevertheless, authors, editors, and publisher do not warrant the information contained in these books, including this book, to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.
Library of Congress Card No.: applied for
British Library Cataloguing-in-Publication Data
A catalogue record for this book is available from the British Library.
Bibliographic information published by the Deutsche Nationalbibliothek
The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at <http://dnb.d-nb.de>.
© 2016 Wiley-VCH Verlag GmbH & Co. KGaA,
Boschstr. 12, 69469 Weinheim, Germany
All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law.
Print ISBN: 978-3-527-33745-3
ePDF ISBN:} 978-3-527-68239-3
ePub ISBN: 978-3-527-68241-6
Mobi ISBN: 978-3-527-68240-9
oBook ISBN: 978-3-527-68238-6
Cover Design Adam-Design, Weinheim, Germany
Preface
Recent developments in microscopy, genomics, molecular biology, and metabolomic analysis allow a detailed analysis of the intracellular lifestyle of endosymbiotic bacteria. The studies showed changes in the cellular organization of the host cells and the bacteria, as well as new structures and cellular functions of the colonizing bacteria. Pathogenic bacteria not only require specific mechanisms for entering the host cell. Rather development of the intracellular and pathogenic lifestyle requires redirecting and adapting of central metabolic routes for successful survival under the changed metabolic conditions and for overcoming defense reactions of the host. Many central metabolic routes have to be redirected and adapted such as to allow their function under conditions of slow growth, limitation in the supply of oxygen, carbon sources, and metal ions, changes of pH and other adverse conditions. Interestingly, various metabolic traits that were known for a long time become obvious in their significance when considered in the context of bacteria/host metabolic interaction. Therefore, studies on the metabolism of bacteria growing in their host gained significant interest. Central metabolism and its adaptation mechanisms turned out to represent important virulence factors for the survival of the bacteria within their host. Understanding the specific metabolic pathways of the bacteria under conditions of host colonization opened new and unexpected views on bacterial physiology. Part A of the book presents some recent examples of this vast area of bacterial physiology. Part B shed lights on fungi–host interactions in human- and plant-pathogenic systems as well as on signaling processes of fungi involved in environmental changes.
The rapidly increasing number and severity of human and plant diseases caused by pathogenic fungi has recently led to many investigations concerning the pathogenic development and physiology of these organisms as well as interactions with their hosts. Most of our knowledge on pathogenic fungi originates from pathogens in terms of pathogenic development, infection, and spread within the host, the treatment of fungal infections, or the reduction of pathogenic effects. In recent years, the elucidation of host–fungus interaction was largely intensified. Fungi need to control their interaction with their hosts in various ways in penetration processes, survival inside hosts, and acquisition of nutrients. In addition, they have to cope with antifungal metabolites, the plant defense or the host immune system. The host may be confronted with toxic fungal metabolites demanding a response to the infection itself. In addition, this mutual interaction is affected by several parameters such as environmental changes or abiotic stress. In order to adapt to quickly changing environmental conditions, fungal pathogens have to respond to external signals. Understanding the signaling network and the chemical communication within this interaction could lead to new insights and define new targets to control pathogens. New methodologies contribute to understand essential processes during the life cycle of the pathogens and the initiation of host–pathogen interactions. The “omics” approach consisting of genome data, transcriptome analysis, proteomics, and metabolomics leads to many new possibilities to track pathological processes and elucidate their regulation and signaling.
The editors thank the contributing authors for their excellent work and the series editor Paul M. Selzer for his constructive advice and support.
Mainz and KaiserslauternFebruary 2016
Gottfried UndenEckhard ThinesAnja Schüffler
Cover Legend
GFP-picture in the background:
Fluorescent microscopic image of a GFP-expressing mutant of the grapevine trunk disease associated fungus Phaeomoniella chlamydospora growing in Vitis vinifera root tissue.
Picture: courtesy of the IBWF, Kaiserslautern, Germany.
Metabolic scheme part:
Host-adapted metabolism of Legionella pneumophila can be determined by 13C-labeling experiments. On the basis of the unique isotopologue patterns, pathways, and fluxes in the formation of metabolic products and their intermediates are reflected. Thereby, information on the core metabolism of the intracellular pathogen and its adaptation to host organisms is gleaned.
Picture: courtesy of Dr Eisenreich, see chapter 2 for details.
List of Contributors
Robert L. Davies
*
University of Glasgow
Institute of Infection
Immunity, and Inflammation
College of Medical, Veterinary and Life Sciences
Sir Graeme Davies Building
120 University Place
Glasgow, G12 8TA
UK
Petra Dersch
*
Helmholtz Centre for Infection Research
Department of Molecular Infection Biology
Inhoffenstr. 7
Braunschweig, 38124
Germany
Wolfgang Eisenreich
*
Technische Universität München
Lehrstuhl für Biochemie
Lichtenbergstr. 4
85747 Garching
Germany
Andrew J. Foster
University of Exeter
School of Biosciences
College of Life and Environmental Sciences
Geoffrey Pope, Stocker Road
Exeter, EX4 4QD
UK
Christopher F. Harrison
Ludwig-Maximilians University
Department of Medicine
Max von Pettenkofer Institute
Pettenkoferstrasse 9a
80336, Munich
Germany
Michael Hensel
*
Universität Osnabrück
Abteilung Mikrobiologie
Fachbereich Biologie/Chemie
Barbarastr. 11
49076, Osnabrück
Germany
Ann Kathrin Heroven
Helmholtz Centre for Infection Research
Department of Molecular Infection Biology
Inhoffenstr. 7
Braunschweig, 38124
Germany
Klaus Heuner
Robert Koch Institute
Cellular Interactions of Bacterial Pathogens
ZBS 2, Seestraβe 10
13353 Berlin
Germany
Hubert Hilbi
*
University of Zürich
Department of Medicine
Institute of Medical Microbiology
Gloriastrasse 30/32
8006 Zürich
Switzerland
Cian Hill
University College Cork
National University of Ireland
School of Microbiology
Cork
Ireland
Peter Holtkötter
Universität Osnabrück
Abteilung Mikrobiologie
Fachbereich Biologie/Chemie
Barbarastr. 11
49076, Osnabrück
Germany
Stefan Jacob
*
Institut für Biotechnologie und Wirkstoff-Forschung gGmbH (IBWF)
Erwin-Schrödinger-Str. 56
67663, Kaiserslautern
Germany
Corinna Kübler
Institut für Biotechnologie und Wirkstoff-Forschung gGmbH (IBWF)
Erwin-Schrödinger-Str. 56
67663, Kaiserslautern
Germany
George R. Littlejohn
University of Exeter School of Biosciences
College of Life and Environmental Sciences
Geoffrey Pope, Stocker Road
Exeter, EX4 4QD
UK
Paul W. O'Toole
*
University College Cork
National University of Ireland
School of Microbiology, Cork
Ireland
and
University College Cork
National University of Ireland
Alimentary Pharmabiotic Centre
Cork
Ireland
Antoine Porquier
INRA, UMR 1290 BIOGER INRA-AgroParisTech
Avenue Lucien Brétignières
78850, Grignon
France
R. Paul Ross
Teagasc Food Research Centre
Food Biosciences Department
Moorepark
Fermoy
County Cork
Ireland
and
University College Cork
National University of Ireland
Alimentary Pharmabiotic Centre
Cork
Ireland
Anja Schü
ffler
*
}
Institut für Biotechnologie und Wirkstoff-Forschung gGmbH (IBWF)
Erwin-Schrödinger-Str. 56
67663, Kaiserslautern
Germany
Julia Schumacher
WWU Münster, IBBP
Schlossplatz 8
48143, Münster
Germany
Adeline Simon
INRA, UMR 1290 BIOGER INRA-AgroParisTech
Avenue Lucien Brétignières
78850, Grignon
France
Darren M. Soanes
University of Exeter, School of Biosciences
College of Life and Environmental Sciences
Geoffrey Pope, Stocker Road
Exeter, EX4 4QD
UK
Catherine Stanton
Teagasc Food Research Centre
Food Biosciences Department
Moorepark
Fermoy
County Cork
Ireland
and
University College Cork
National University of Ireland
Alimentary Pharmabiotic Centre
Cork
Ireland
Nicholas J. Talbot
*
University of Exeter
School of Biosciences
College of Life and Environmental Sciences
Geoffrey Pope, Stocker Road
Exeter, EX4 4QD
UK
Muriel Viaud
*
INRA, UMR 1290 BIOGER INRA-AgroParisTech
Avenue Lucien Brétignières
78850, Grignon
France
Sebastian E. Winter
*
University of Texas Southwestern Medical Center
Department of Microbiology
5323 Harry Hines Blvd
Dallas, TX 75390
USA
Alexander Yemelin
Institut für Biotechnologie und Wirkstoff-Forschung gGmbH (IBWF)
Erwin-Schrödinger-Str. 56
67663, Kaiserslautern
Germany
*corresponding author
Part OneAdaptation of Microbial Metabolism in Host/Pathogen Interaction
Chapter 1Metabolic Adaptation of Human Pathogenic Yersiniae
Ann Kathrin Heroven and Petra Dersch*
*Corresponding Author
Abstract
Colonization, subsequent penetration of epithelial layers as well as persistence and proliferation in subepithelial tissues of the host by bacterial pathogens demand the expression of special sets of virulence factors. In addition, the bacteria need to adapt their metabolism to survive and replicate within the specific host niches. Activated metabolic functions and physiological adaptation processes during their life cycle and the different stages of the infection reflect the complex and dynamic nutritional resources of their environments, interbacterial competition for energy sources and onslaught of bactericidal host responses. The enteric pathogenic Yersinia species Y. pseudotuberculosis and Y. enterocolitica and the causative agent of plague, Y. pestis, have adapted to grow in many different environmental reservoirs (e.g., soil, plants, insects) and in warm-blooded animals (e.g., rodents, pigs, humans) with a preference for lymphatic tissues. In the present book chapter, we discuss metabolic adaptations of human pathogenic yersiniae to successfully exploit available nutrients and metabolic functions during infection and illustrate the tight link between carbon metabolism and Yersinia virulence. Furthermore, current knowledge about the complex regulatory networks used to coordinate and fine-tune the control of metabolic and virulence functions are presented. Deciphering the mechanisms of the function and control of bacterial metabolism within host tissues will not only increase our understanding of host–pathogen interactions, it will also facilitate the identification of potential novel drug targets for future prevention and therapeutic strategies.
Introduction
Infections of human pathogenic yersiniae involves a large number of specific pathogenicity factors that mediate efficient resistance against the host defense systems and enable the bacteria to colonize, invade, and multiply successfully within host tissues. The structure, function, and expression of many of these classical virulence factors have been characterized, and their role in pathogenicity has been studied using different animal models. However, to become a successful pathogen, yersiniae must also adapt their metabolic functions to the nutrient/ion composition and the physical conditions (e.g., temperature, pH, oxygen tension) of their surrounding and coordinate their metabolism with their life cycle. These unspecific strategies were long neglected, but recent use of global omic-based profiling techniques, phenotypic microarrays, and the in vivo analysis of metabolic mutants allowed a deeper insight into nutrient sensing, sequestration, and utilization strategies that optimize the metabolism and biological fitness of Yersinia during infection.
Yersinia Life Cycles and Pathogenesis
Of the 17 species of the genus Yersinia only Y. pseudotuberculosis, Y. enterocolitica, and Y. pestis are known to cause diseases in mammals [1, 2]. The two enteric pathogens Y. pseudotuberculosis and Y. enterocolitica are the causative agents of yersiniosis, a gastrointestinal disease with a variety of symptoms such as enteritis, colitis, diarrhea, and mesenteric lymphadenitis, which becomes rarely systemic. Both enteropathogenic species are well adapted to survive long term in external habitats (e.g., ground water, soil, plants, and insects) and are able to persist and replicate in various wild and domestic animals [3, 4]. A recent study analyzing a large number of genomes revealed that they are heterotrophic pathogens that are able to utilize a large variety of C-/N-/energy sources [5]. In contrast, Y. pestis, the causal agent of plague, which has evolved as a separate clone from Y. pseudotuberculosis, shows a reduced metabolic flexibility based on functional gene loss. This may reflect its unique life cycle: (i) replication within the gastrointestinal tract (proventriculus) of infected fleas and (ii) proliferation in the lymphatic system, blood, or tissues of mammals, in particular rodents [6].
All yersiniae are zoonotic pathogens armored with diverse cell envelope–associated virulence structures that either promote host–pathogen interactions or contribute to Yersinia pathogenicity by suppression of the host immune response. In case of the enteric Yersinia species, initial attachment and invasion of the intestinal layer is mediated by the primary invasion factor invasin (InvA), but other adhesive surface-exposed proteins, for example, homologous Inv-type adhesins (InvB/Ifp, InvC), Ail, the autotransporter adhesin YadA and the PsaA (pH6 antigen)/Myf fimbriae appear to support the dissemination process at later stages of the infection [7, 8]. In Y. pestis mainly adhesins Ail and PsaA contribute to host–pathogen interactions, whereas other adhesin/invasin genes, for example, invA and yadA became unfunctional [9, 10]. Moreover, all pathogenic yersiniae evolved mechanisms that mediate resistance against the innate immune response. Several adhesins protect the bacteria against complement killing (e.g., Ail and YadA) or prevent phagocytosis (e.g., PsaA) [7]. Furthermore, they possess a 70-kDa virulence plasmid (pYV/pCD1) that encodes the Ysc (Yersinia secretion)-Yop type III secretion system (T3SS). This needle-like delivery machine (injectisome) enables the bacteria to inject different Yops (Yersinia outer proteins) effector toxins from the bacterial cytoplasm into the cytosol of host cells, in particular professional phagocytes [11]. Yersinia pathogenicity relies on the following crucial functions of translocated Yop effector proteins: (i) antiphagocytic activity by manipulation and destruction of the actin cytoskeleton; (ii) suppression of cytokine production by macrophages, dendritic cells, and neutrophils; and (iii) induction of host cell death [11].
Carbon Metabolism and Links to Yersinia Pathogenesis
External reservoirs, vector and animal environments colonized by Yersinia have likely driven the evolution of metabolic pathways to maximize present nutritional opportunities. Variations in certain metabolic functions might thus be a consequence of the adaptation to a specific host or host niche. A selective advantage can be gained either by acquisition of new metabolic functions, for example, by horizontal gene transfer, or by loss of function mutations that change the metabolic abilities of the pathogen. Furthermore, changes in the control mechanisms implicated in metabolic adaptation and regulatory strategies linking metabolic and virulence traits could manipulate the pathogen's response to varying nutrient availabilities in the environment.
Food Sources, Nutrient Sequestration, and Utilization
Animal tissues contain a large variety of different energy sources (e.g., sugars, amino acids, lipids, proteins) and can be regarded as a rich source of food for bacteria. In particular the digestive tract of mammals is nutrient rich and contains a large diversity of different nutritional substrates, which can be metabolized by enteric yersiniae. However, the pathogens have to compete successfully with the perfectly adapted resident microbiota. About 1014 bacteria form a complex microbial ecosystem of more than 400 species, in which strictly anaerobic bacteria degrade complex polysaccharides into simple carbohydrates, which are readily absorbed by the mammalian small intestine or used by other (facultative anaerobic) commensals such as Escherichia coli [12]. Furthermore, the host can rapidly change the availability of nutrients in host tissues based on the induction of inflammation and hypoxic conditions triggered by the immune response [13], and it can restrict access to essential ions such as magnesium, manganese, zinc, and iron [14, 15]. As a consequence, Yersinia needs to sense, retrieve, and metabolize nutrients more efficiently, or alternatively it must grow on available substrates, which are not used by other members of the competing microbiota. An important characteristic of many bacterial pathogens, including Yersinia, is their ability to sense and initiate use of readily digestible carbon sources by sophisticated global regulatory systems: (i) carbon catabolite repression (CCR) triggered in response of the availability of simple sugars, for example, glucose [16, 17] and (ii) the carbon storage regulator/regulator of secondary metabolites system (Csr/Rsm) [18, 19] (see also below: Coordinated control of carbon metabolism and virulence).
Metabolic Pathways of Yersinia Crucial for Virulence
All pathogenicYersinia species possess a highly flexible and robust metabolic system with many redundant or alternative catabolic and biosynthetic pathways, which allow them to respond very rapidly and efficiently to changing nutrient concentrations. Simple sugars can be utilized via glycolysis (Embden–Meyerhof pathway), the pentose phosphate pathway and the Entner–Doudoroff pathway. They can further be catabolized by aerobic or anaerobic respiration via a complete tricarboxylic acid (TCA) cycle and a functional glyoxylate bypass, or via fermentation [20–22]. Many enzymes and metabolic pathways are conserved among the different Yersinia species, but several characteristic differences were also observed. Due to the loss of multiple metabolic genes, for example, the glucose 6-phosphate dehydrogenase gene zwf Y. pestis is unable to use glucose via the pentose phosphate pathway [20]. It further lacks the methionine salvage and the urease pathway, aspartase to mediate catabolism of glutamate to aspartate and is unable to synthesize several amino acids, including glycine, threonine, L-valine and L-isoleucine, L-phenylalanine, and L-methionine [23, 24], which makes the pathogen more dependent on mechanisms accessing host nutrients. An important specific feature of Y. enterocolitica is its ability to metabolize 1,2-propanediol and ethanolamine by cobalamin-dependent enzymes under anaerobiosis using tetrathionate as terminal electron acceptor [5]. Tetrathionate production is strongly induced upon inflammation [25], indicating that these metabolic properties are advantageous for Y. enterocolitica to outcompete the microbiota of the intestine. In contrast, Y. pseudotuberculosis and Y. pestis are able to metabolize itaconate by converting it into pyruvate and acetyl-CoA. Itaconate contributes to the antimicrobial activity of macrophages as it inhibits isocitrate lyase, a key enzyme of the glyoxylate cycle. Thus, itaconate degradation could allow Yersinia to persist in macrophages [26].
Nutritional Virulence: Nutritional Adaptation Important for Pathogenesis
Various “omic” approaches and transcriptional profiling studies with pathogenic yersiniae grown in vitro under different virulence-relevant conditions revealed numerous metabolic pathways and adaptive metabolic responses, which could contribute to pathogenesis. Important initial studies addressed temporal changes during a temperature shift from 26 to 37 °C, mimicking transmission of Y. pestis from the flea to mammals. They revealed that not only virulence genes but also numerous metabolic functions are under thermal control [27, 28]. Genes encoding for enzymes involved in nitrogen assimilation were strongly downregulated, whereas those required for efficient catabolism of amino acids were induced in Y. pestis grown in vitro at 37 °C. Some of these enzymes are responsible for the majority of released metabolic ammonia via reactions that directly or indirectly promote deamination during formation of α-keto acids entering the TCA cycle. A thermal upshift caused a downregulation of glycolysis, whereby terminal oxidation of the available energy sources (carbohydrates, amino acids, and lipids) in the nutrient-rich medium was favored. This first in vitro study indicated that, in nature, Y. pestis prefers fermentative pathways in the flea vector, while oxidative catabolism is favored during rapid proliferation in the lymphatic systems of the mammalian host [27]. Moreover, differential expression of catabolic enzymes suggests that different sugars (e.g., maltose, gluconate, ribose) are utilized after temperature transition, and this metabolic switch appears to be crucial to trigger virulence. Two equivalent transcriptomic studies were directed to identify metabolic functions of Y. pestis required during septicaemic plague in humans and of Y. pseudotuberculosis during systemic infections. In vitro growth in media containing human plasma showed that in particular genes related to purine/pyrimidine metabolism were upregulated in plague bacilli and supported a previous report demonstrating that purine metabolism is crucial for Y. pestis pathogenicity [29, 30]. In Y. pseudotuberculosis, genes supporting the consumption of the plasma glucose (e.g., the glucose-specific phosphotransferase system (PTS)) were strongly upregulated [31]. This indicated that high growth rate aerobic cultivations on glucose induce an “overflow metabolism” channeling the carbon flow toward byproduct formation and secretion to balance accumulation of reducing equivalents (NADH) through the TCA cycle. In fact, our recent fluxome approach revealed that Y. pseudotuberculosis does not accumulate and excrete acetate like E. coli when grown on glucose; it spills large amount of pyruvate (46% of the glucose uptake). Preliminary results indicate that excretion of pyruvate by Y. pseudotuberculosis is achieved by a sustained glycolytic flux that is accompanied by a bottleneck in the TCA and a downregulation of acetate formation (Bücker et al., [32]).
Over the past years also in vivo gene expression profiling was performed to gain a better insight into host–pathogen interactions and the metabolic activities that support persistence and replication of Y. pestis in the flea [33] and the mammalian host [34–36]. Numerous metabolic genes involved in the catabolism of amino acids, in particular the L-glutamate group (e.g., glutamine, histidine, arginine, proline) were found to be upregulated in Y. pestis located in the proventriculus of infected fleas [33] (Figure 1.1). This was interpreted as a special adaptation to the flea gut, which contains protein and lipid rich meals with relatively low amount of carbohydrates. Utilization of the L-glutamate group amino acids involves enzymes of the TCA cycle, which are upregulated in the flea vector [33]. In contrast, catabolism of carbohydrates seems less important as most sugar uptake systems are repressed or only slightly expressed. Only chitobiose, a PTS sugar present in the flea's proventriculus spines, is efficiently imported and metabolized (Figure 1.1).
Figure 1.1 Metabolic pathways and virulence factors of Y. pestis, which are significantly induced in the mammalian host and the flea gut. Specific metabolic pathways and pathogenicity traits upregulated in vivo are presented, which are considered to be crucial for the colonization of the lung or bubo of the mammalian host (a) and the flea gut (b). Abbreviations: BarA/UvrY (nutrient-responsive two-component system); Csr (carbon storage regulator); Crp (cAMP receptor protein); GADP (glyceraldehyde-3P); Hfq: RNA chaperone; KDGP (2-dehydro-3-deoxy-gluconate-6P); M-cell (microfold cell); 3-P-G (3-phosphoglycerate); 2PG (2-phosphoglycerate); PhoP/PhoQ (ion-responsive two-component system), PsaA (pH6 antigen); Yops (Yersinia outer proteins); T3SS (type III secretion system); and TCA (tricarboxylic acid cycle).
Transcriptional profiling of Y. pestis located in the bubo in a rat model as well as in the lung of a murine pneumonic infection model was used to characterize the metabolic adaptation of Y. pestis to its mammalian host [34–36]. Notable is the strong induction of genes involved in iron acquisition (e.g., hemin uptake operon) and amino acid biosynthesis (e.g., histidine, glutamate, and aspartate), and downregulation of the TCA cycle and the ATP-proton motive force during pneumonic plague development [35, 36] (Figure 1.1). In parallel, genes encoding the Y. pestis specific antiphagocytic F1 protein capsule (Caf1), as well as the T3SS/Yop apparatus important for resistance against the innate immune response are highly expressed. A similar strong induction of the Caf1 capsule and the T3SS/Yop machinery was also observed in the rat bubo [34]. Furthermore, Y. pestis induces a protective response to reactive nitrogen species (RNS), which are released by polymorphonuclear neutrophils (PMNs) in the buboes [34]. This is reflected by an upregulation of the ribonucleotide reductase genes (nrdHIEF operon) and hmp, which encodes a flavohemoglobin that detoxifies RNS. To further investigate the importance of genes upregulated during bubonic plague, a mutant library was constructed and tested in a rodent model of bubonic plague [34, 37]. Virulence testing revealed that Y. pestis depends mainly on the catabolism of carbohydrates (i.e., glucose, galactans, and gluconate) [37] (Figure 1.1). Since the terminal part (gpmA, aceEF), but not the upper part (pgi, pfkA) of the glycolysis pathway was essential for competition with the wildtype in vivo, it was suspected that gluconate is metabolized to glyceraldehyde-3-phosphate, pyruvate, acetyl-CoA, and acetate, whereby the galactans and glucose are most likely channeled toward UDP-glucose synthesis [37] (Figure 1.1). Additional results, demonstrating unimportance of certain TCA cycle genes (e.g., gltA, acnA, and fumC) and constitutive expression of the glyoxylate shunt suggest that Y. pestis shifts to anaerobic respiration or fermentation during colonization of rodents [34].
Coordinated Control of Carbon Metabolism and Virulence
Rapid changes in environments encountered by yersiniae in their external habitats, during the vector-associated lifestyle and within the intestine/lymphatic tissues in mammals request a fast bacterial response to adjust metabolic and virulence traits. To overcome this challenge, it is no wonder that Yersinia and other bacteria use the availability of ions and nutrients as well as certain metabolic cues to coordinately control their metabolism and virulence function. For example, virulence factors can be activated via the stringent response through (p)ppGpp under nutrient-limiting conditions, such as amino acid and fatty acid starvation [38]. Furthermore, the synthesis and activity of certain transcriptional regulators and RNA elements (e.g., Fur, Zur, riboswitches) can be controlled by metal ions or small metabolites to modulate expression of metabolic or virulence functions. Many virulence genes are also under CCR control and are regulated by the global transcription factors cyclic adenosine monophophate (cAMP) receptor protein (Crp) and CsrA. They coordinate the uptake and utilization of alternative carbon sources and enable the bacteria to adjust their pathogenic properties in accordance to the availability of readily utilizable sugars [16, 18].
Importance of Ions
All pathogenic Yersinia species are characterized by a strong induction of numerous iron uptake and sequestration systems during the infection of mammals, indicating the importance for Yersinia to acquire iron [31, 34–36, 39]. The ferric uptake regulator Fur represses most of the iron uptake systems in the presence of iron and controls genes of various noniron metabolic and physiological functions including biofilm formation in Y. pestis [40–42]. Although Fur was also shown to control expression of the T3SS in related pathogens [43, 44], Fur-mediated regulation of T3S in Yersinia has not been described. However, most recently, a new regulator, IscR, was found to control expression of LcrF, the major regulator of the T3SS-associated genes in Y. pseudotuberculosis. It has been suggested that IscR senses iron, O2, and/or reactive oxygen species concentrations in order to optimize T3S synthesis [45].
Sensing of magnesium ions is another important feature of Yersinia to adapt virulence and metabolic gene expression. The pleiotropic two-component system (TCS) PhoP/PhoQ is composed of the membrane-bound sensor kinase PhoQ that responds to low magnesium and phosphorylates the cytoplasmic response regulator PhoP. It further recognizes low pH environments and host-secreted cationic antimicrobial peptides (CAMPs) [46]. Transcription of the Y. pestis phoP gene is significantly upregulated in the lung in an intranasally challenged plague model in mice [36] and in infected fleas [33, 47] and is essential for the formation of a normal foregut-blocking flea infection [33, 47]. Although the PhoP/PhoQ system was shown to be essential for the survival and proliferation of all pathogenic Yersinia species in macrophages and neutrophils in vitro [48–50], the role of the PhoP/PhoQ system for Yersinia pathogenesis is less clear. phoP mutants of Y. pestis GB and the Y. pseudotuberculosis derivative 32 777 were strongly attenuated in virulence, whereas loss of a functional phoP gene did not affect the pathogenicity of Y. pestis CO92 and the Y. pseudotuberculosis strain YPIII [48, 50–52]. This strongly suggests that the different outcomes are the result of strain-specific differences that remodel regulation and/or composition of the PhoP/PhoQ regulon. This is supported by recent findings from our laboratory, demonstrating the presence of strain-specific variations in the PhoP-mediated control of the Csr system affecting expression of numerous metabolic, stress adaptation, and virulence functions in Y. pseudotuberculosis [53].
Importance of the Csr System
The important global posttranscriptional Csr system is composed of the RNA-binding protein CsrA and Csr-type sRNAs (CsrB and CsrC in Y. pseudotuberculosis). CsrA recognizes conserved (N)GGA motifs in the loop portions of RNA hairpin structures that are mostly found in close vicinity to the ribosomal binding site in the target mRNA. Binding of CsrA affects translation and/or stability of the mRNA. The Csr-RNAs contain several CsrA-binding sites and can eliminate CsrA function by sequestration of CsrA from its target mRNAs [18, 19]. The Csr system controls many genes involved in metabolism and virulence in Yersinia similar to many other pathogens [18, 19]. A recent transcriptomic approach revealed that about 20% of the CsrA-dependent genes of Y. pseudotuberculosis are involved in metabolic processes [18] (Figure 1.2). The Y. pseudotuberculosis Csr system is further implicated in the first steps of the infection process through regulation of the global virulence gene regulator RovA, which activates the synthesis of the primary entry factor invasin and the PsaA fimbriae (Figure 1.2) [54, 55]. Preliminary data further indicate that the Csr system is also crucial for the expression of the Yersinia Ysc-Yop/T3SS machinery (R. Steinmann, unpublished results).
Figure 1.2 Schematic overview of the environmental sensing and signal transduction system and the regulatory cascade with implicated control factors that are known to coordinate expression of metabolic functions and virulence-associated traits of Y. pseudotuberculosis. All sensory and regulatory components are also encoded in the other human pathogenic Yersinia species, but the function of some of them has still not been experimentally verified.
Based on the crucial role of the Csr system, it is not surprising that the expression of the Csr components is tightly regulated in response to environmental parameters. Both Csr-RNAs are controlled by different regulatory mechanisms in response to ions and availability of C-sources. The TCS PhoP/PhoQ activates csrC transcription in a Mg2+
