Table of Contents

Cover

Titles of the Series “Drug Discovery in Infectious Diseases”

Title Page

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
1. Introduction
2. Yersinia Life Cycles and Pathogenesis
3. Carbon Metabolism and Links to Yersinia Pathogenesis
4. Nutritional Virulence: Nutritional Adaptation Important for Pathogenesis
5. Coordinated Control of Carbon Metabolism and Virulence
6. Conclusions
7. Acknowledgments
8. References
Chapter 2: Crosstalk between Metabolism and Virulence of Legionella pneumophila
1. Introduction
2. Key Metabolic Features of L. pneumophila
3. Serine and Glucose Metabolism of L. pneumophila under In Vitro Conditions
4. Metabolism of L. pneumophila under Intracellular Conditions
5. Metabolic Adaptation during the Life Cycle of Intracellular L. pneumophila
6. Metabolic Host Cell Responses Triggered by Intracellular L. pneumophila
7. Conclusion
8. References
Chapter 3: Metabolism of Intracellular Salmonella enterica
1. Introduction
2. S. enterica – A Metabolic Generalist
3. Metabolism of Glucose by S. enterica
4. Connection to the TCA Cycle by Oxidative Decarboxylation of Pyruvate
5. The Important Role of the TCA Cycle and Anaplerotic Reactions
6. Metabolism of Intracellular S. enterica
7. The Metabolism of S. enterica Compared to Other Important Intracellular and Gastrointestinal Pathogens
8. Salmonella Induce Networks of Tubular Structures – Access to Host-Derived Nutrients?
9. S. enterica Has a Bimodal Lifestyle in Epithelial Cells
10. Salmonella Metabolism Limits Possibilities for New Antimicrobials
11. Conclusions
12. References
Chapter 4: The Human Microbiome in Health and Disease
1. Introduction
2. Methods for Characterizing the Microbiota
3. Diet and Geographical Factors
4. Functional Gastrointestinal Disorders
5. Manipulation of the Microbiota
6. Conclusions
7. Acknowledgments
8. References
Chapter 5: Mechanisms of Dysbiosis in the Inflamed Gut
1. Introduction
2. Dysbiosis during Inflammatory Diseases of the Gastrointestinal Tract
3. Nutrient Acquisition by Commensal Bacteria in the Normal Gut
4. Nutritional Mechanisms for Dysbiosis in the Inflamed Gut
5. Inflammation-Driven Bloom of Enteric Pathogens in the Gut Lumen
6. How Anaerobic Respiration Enhances Growth in the Inflamed Gut
7. Conclusions
8. References
Chapter 6: Strategies for Nutrient Acquisition by Magnaporthe oryzae during the Infection of Rice
1. Introduction
2. Adhesion and Germination
3. Appressorium Formation
4. Penetration
5. Growth in Planta: The Biotrophic Growth Phase
6. Growth in Planta: Necrotrophic Growth Phase
7. Growth in Planta: Sporulation
8. Life Outside of the Rice Plant
9. Conclusion
10. References

Part Two: New Inhibitors and Targets of Infectious Diseases

Chapter 7: Part B: New Inhibitors and Targets of Infectious Diseases
1. Introduction
2. Mannheimia haemolytica
3. Pathogenesis of Pneumonic Pasteurellosis
4. Virulence Factors of M. haemolytica
5. Antibiotic Use against M. haemolytica
6. Antibiotic Resistance
7. Resistance Mechanisms
8. Discovery of New Antibacterial Agents
9. The Importance of In Vivo Growth Conditions
10. Potential Outer Membrane Targets for Novel Antibiotics against M. haemolytica
11. Conclusion
12. References
Chapter 8: Identification of Anti-infective Compounds Using Amoebae
1. Introduction
2. Anti-virulence Compounds as an Alternative to Antibiotics
3. Amoebae as Model Host Systems in Compound Screening
4. The Amoeba-Resistant Pathogens L. pneumophila and M. marinum
5. Development of Novel Amoebae-Based Screening Systems
6. Palmostatin M – A Novel Antibacterial Compound Specific for Legionella and Mycobacterium Species
7. Conclusion
8. Acknowledgments
9. References
Chapter 9: Stress Biology in Fungi and “Omic” Approaches as Suitable Tools for Analyzing Plant–Microbe Interactions
1. Introduction
2. Stress Signaling Pathways Are Important in Plant–Microbe Interactions
3. High Osmolarity
4. Reactive Oxygen Species (ROS)
5. Plant Hormones (Phytoalexins)
6. Technologies for Studying Signaling Pathways
7. The Age of “Omics” – General Information
8. Transcriptomics
9. Transcriptomics in Use
10. Proteomics
11. Proteomics in Use
12. Metabolomics
13. Metabolomics in Use
14. Conclusion
15. References
Chapter 10: Targeting Plasmids: New Ways to Plasmid Curing
1. Introduction
2. Research Approaches for Plasmid Curing
3. Prerequisites for Clinical Use and Possible Application Methods
4. Common Methods and Compounds in Use
5. Approaches and Techniques to Identify Antiplasmid Effects/Compounds since 2000
6. Conclusion
7. References
Chapter 11: Regulation of Secondary Metabolism in the Gray Mold Fungus Botrytis cinerea
1. Botrytis cinerea, the Causal Agent of Gray Mold Disease
2. Repertoire of Secondary Metabolites Produced by B. cinerea
3. Expression of SM Genes during In Vitro and In Planta Conditions
4. Pathway-Specific Regulation by Transcription Factors
5. Concerted Regulation of Secondary Metabolism and Light-Dependent Development
6. Regulation of Secondary Metabolism by Conserved Signal Transduction Pathways
7. Role of the Chromatin Landscape in the Regulation of Secondary Metabolism?
8. Concluding Remarks
9. References
Index

End User License Agreement

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-¹³C₃]serine into L. pneumophila under in vitro conditions [44]. The ¹³C-profiles are indicated by bold lines connecting ¹³C-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 ¹³C-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 ¹³C-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-¹³C₆]glucose into L. pneumophila under in vitro conditions [44]. The ¹³C-profiles are indicated by bold lines connecting ¹³C-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 ¹³C 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 ¹³C-isotopologue-profiling analysis (¹³C-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 ¹³C-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 ¹³C-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 ΔicmT 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. ^SIndicates 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)₂Cys₆ transcription factor (TF) is encoded by bcboa13 and a Nmr-A like regulator is putatively encoded by bcboa1. (b) While both single mutants Δbcboa6 and Δbcbot2, impaired in BOA and BOT production, respectively, are not altered in virulence compared to the wild type (WT) strain (not shown), the double ΔΔbcboa6-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 NaNO₃ as nitrogen source). BIK – accumulation of bikaverin in liquid cultures 7 dpi (minimal medium with NH₄NO₃ 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).

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

Wellcome Trust Centre for Molecular Parasitology, Institute of Infection Immunity and Inflammation

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.

The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at <http://dnb.d-nb.de>.

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.

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 Kaiserslautern
February 2016

Gottfried Unden
Eckhard Thines
Anja 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
robert.davies@glasgow.ac.uk

Petra Dersch^*
Helmholtz Centre for Infection Research
Department of Molecular Infection Biology
Inhoffenstr. 7
Braunschweig, 38124
Germany
petra.dersch@helmholtz-hzi.de

Wolfgang Eisenreich^*
Technische Universität München
Lehrstuhl für Biochemie
Lichtenbergstr. 4
85747 Garching
Germany
wolfgang.eisenreich@mytum.de

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
michael.hensel@biologie.uni-osnabrueck.de

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
hilbi@imm.uzh.ch

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
jacob@ibwf.de

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
pwotoole@ucc.ie

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 Schüffler^*}
Institut für Biotechnologie und Wirkstoff-Forschung gGmbH (IBWF)
Erwin-Schrödinger-Str. 56
67663, Kaiserslautern
Germany
schueffler@ibwf.de

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
n.j.talbot@exeter.ac.uk

Muriel Viaud^*
INRA, UMR 1290 BIOGER INRA-AgroParisTech
Avenue Lucien Brétignières
78850, Grignon
France
viaud@versailles.inra.fr

Sebastian E. Winter^*
University of Texas Southwestern Medical Center
Department of Microbiology
5323 Harry Hines Blvd
Dallas, TX 75390
USA
sebastian.winter@UTSouthwestern.edu

Alexander Yemelin
Institut für Biotechnologie und Wirkstoff-Forschung gGmbH (IBWF)
Erwin-Schrödinger-Str. 56
67663, Kaiserslautern
Germany

^*corresponding author

Host - Pathogen Interaction

Microbial Metabolism, Pathogenicity and Antiinfectives

Preface

Cover Legend

List of Contributors

Part One
Adaptation of Microbial Metabolism in Host/Pathogen Interaction