Cross-talk between Type Three Secretion System and Metabolism in Yersinia

Pathogenic yersiniae utilize a type three secretion system (T3SS) to inject Yop proteins into host cells in order to undermine their immune response. YscM1 and YscM2 proteins have been reported to be functionally equivalent regulators of the T3SS in Yersinia enterocolitica. Here, we show by affinity purification, native gel electrophoresis and small angle x-ray scattering that both YscM1 and YscM2 bind to phosphoenolpyruvate carboxylase (PEPC) of Y. enterocolitica. Under in vitro conditions, YscM1, but not YscM2, was found to inhibit PEPC with an apparent IC₅₀ of 4 μm (K_i = 1 μm). To analyze the functional roles of PEPC, YscM1, and YscM2 in Yop-producing bacteria, cultures of Y. enterocolitica wild type and mutants defective in the formation of PEPC, YscM1, or YscM2, respectively, were grown under low calcium conditions in the presence of [U-¹³C₆]glucose. The isotope compositions of secreted Yop proteins and nine amino acids from cellular proteins were analyzed by mass spectrometry. The data indicate that a considerable fraction of oxaloacetate used as precursor for amino acids was derived from [¹³C₃]phosphoenolpyruvate by the catalytic action of PEPC in the wild-type strain but not in the PEPC^- mutant. The data imply that PEPC is critically involved in replenishing the oxaloacetate pool in the citrate cycle under virulence conditions. In the YscM1^- and YscM2^- mutants, increased rates of pyruvate formation via glycolysis or the Entner-Doudoroff pathway, of oxaloacetate formation via the citrate cycle, and of amino acid biosynthesis suggest that both regulators trigger the central metabolism of Y. enterocolitica. We propose a “load-and-shoot cycle” model to account for the cross-talk between T3SS and metabolism in pathogenic yersiniae.Type three secretion systems (T3SSs)³ are used by several Gram-negative bacteria as microinjection devices to deliver effector proteins into host cells (1). The translocated effector proteins reprogram the host cell in favor of the microbial invader or symbiont. Pathogenic yersiniae (the enteropathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis and the plague bacillus Yersinia pestis) utilize a plasmid-encoded T3SS to undermine the host primary immune response (2). This is mediated by the injection of a set of effector proteins called Yops (Yersinia outer proteins) into host cells, in particular into cells with innate immune functions, such as macrophages, dendritic cells, and neutrophils (3). The concerted action of Yops, targeting multiple signaling pathways, results in actin cytoskeleton disruption, suppression of proinflammatory signaling, and induction of apoptosis. This strategy enables yersiniae to multiply extracellularly in host tissue.Expression of the Yersinia T3SS is up-regulated at 37 °C, and translocation of Yops across the host cell membrane is triggered by cell contact (4, 5). Pathogenic yersiniae cultivated under low calcium conditions at 37 °C express a phenotype referred to as “low calcium response” (LCR). The LCR is characterized by growth restriction as well as massive expression and secretion of Yops into the culture medium (6–9). The allocation of energy and metabolites for the massive synthesis and transport of Yops is demanding, and this burden is believed to be responsible for the observed growth inhibition (10). To give an idea of the metabolic requirements, Yops are secreted to the culture supernatant in 10-mg amounts per liter of culture within 2 h after calcium depletion of the medium. Furthermore, post-translationally secreted substrates need to be unfolded by a T3SS-specific ATPase prior to secretion (11–14). In addition, T3SS-dependent transport of Yops requires the proton motive force (15). However, there is evidence that growth cessation and Yop expression can be uncoupled (16, 17), suggesting a coordinated regulation of metabolism and protein transport rather than the LCR reflecting an inevitable physiological consequence.What are the candidate proteins that could be involved in such a coordination? YscM1 and YscM2 (57% identical to YscM1) are key candidates, since they act at a major nodal point of the T3SS regulatory network in Y. enterocolitica. In Y. pestis and Y. pseudotuberculosis, only the YscM1 homologue LcrQ exists (99% identical to YscM1). YscM1/LcrQ and YscM2 are secretion substrates of the T3SS that are involved in up-regulation of Yop expression after host cell contact. Upon cell contact, the decrease of intracellular levels of YscM1/LcrQ and YscM2 due to their translocation into host cells results in a derepression of Yop synthesis (18–21). The two yscM copies of Y. enterocolitica were presumed to be functionally equivalent, since deletion of either gene was found to be phenotypically silent (19, 22). Only deletion of both yscM genes could establish the lcrQ phenotype (19, 22), distinguished by temperature sensitivity for growth, derepressed Yop expression, and secretion of LcrV and YopD in the presence of calcium ions.YscM1/LcrQ as well as YscM2 exhibit homology to the N terminus of the effector YopH (19, 23, 24), a fact that may explain their shared assistance by SycH (specific Yop chaperone) (20, 25). It was shown that YscM1/LcrQ and YscM2 exert their influence on Yop expression in concert with the T3SS components SycH, SycD (LcrH in Y. pestis and Y. pseudotuberculosis), and YopD (21, 26–28). It is further described that YscM1 and/or YscM2 interact with several of the T3SS-specific chaperones, in particular with SycH, SycE, SycD, and SycO (20, 29–31). This has led to the model that YscM/LcrQ proteins might function as an interface that senses whether chaperones are loaded with Yops and transduces these signals into control of Yop expression (14).These features of YscM1/LcrQ and YscM2 prompted us to speculate about a key role of these proteins in coordination of metabolism and expression of T3SS components. Using recombinant GST-YscM1 and GST-YscM2 as bait for Y. enterocolitica cytosolic proteins, we identified phosphoenolpyruvate carboxylase (PEPC) as interaction partner of both YscM1 and YscM2. Under in vitro conditions, YscM1 down-regulated PEPC activity and bacterial growth/replication. Isotopologue profiling of Yop proteins and derived amino acids from Y. enterocolitica grown in the presence of [U-¹³C₆]glucose showed the functionality of the PEPC reaction under virulence conditions (isotopologues are molecular entities that differ only in isotopic composition (number of isotopic substitutions); e.g. CH₄, CH₃D, and CH₂D₂). Moreover, biosynthetic rates of amino acids were increased in mutants defective in YscM1 or YscM2, suggesting a general role of these regulators in the metabolism of Y. enterocolitica. Recently, evidence has been accumulating that the metabolic state contributes to the regulation of T3SSs of diverse pathogens, also including the flagellar T3SS in Pseudomonas and Salmonella (32–36).     

Isotopologue Profiling of Legionella pneumophila: ROLE OF SERINE AND GLUCOSE AS CARBON SUBSTRATES*

Legionella pneumophila (Lp) is commonly found in freshwater habitats but is also the causative agent of Legionnaires'' disease when infecting humans. Although various virulence factors have been reported, little is known about the nutrition and the metabolism of the bacterium. Here, we report the application of isotopologue profiling for analyzing the metabolism of L. pneumophila. Cultures of Lp were supplied with [U-¹³C₃]serine, [U-¹³C₆]glucose, or [1,2-¹³C₂]glucose. After growth, ¹³C enrichments and isotopologue patterns of protein-derived amino acids and poly-3-hydroxybutyrate were determined by mass spectrometry and/or NMR spectroscopy. The labeling patterns detected in the experiment with [U-¹³C₃]serine showed major carbon flux from serine to pyruvate and from pyruvate to acetyl-CoA, which serves as a precursor of poly-3-hydroxybutyrate or as a substrate of a complete citrate cycle with Si specificity of the citrate synthase. Minor carbon flux was observed between pyruvate and oxaloacetate/malate by carboxylation and decarboxylation, respectively. The apparent lack of label in Val, Ile, Leu, Pro, Phe, Met, Arg, and Tyr confirmed that L. pneumophila is auxotrophic for these amino acids. Experiments with [¹³C]glucose showed that the carbohydrate is also used as a substrate to feed the central metabolism. The specific labeling patterns due to [1,2-¹³C₂]glucose identified the Entner-Doudoroff pathway as the predominant route for glucose utilization. In line with these observations, a mutant lacking glucose-6-phosphate dehydrogenase (Δzwf) did not incorporate label from glucose at significant levels and was slowly outcompeted by the wild type strain in successive rounds of infection in Acanthamoeba castellanii, indicating the importance of this enzyme and of carbohydrate usage in general for the life cycle of Lp.     

Metabolic adaptation of Chlamydia trachomatis to mammalian host cells

Metabolic adaptation is a key feature for the virulence of pathogenic intracellular bacteria. Nevertheless, little is known about the pathways in adapting the bacterial metabolism to multiple carbon sources available from the host cell. To analyze the metabolic adaptation of the obligate intracellular human pathogen Chlamydia trachomatis, we labeled infected HeLa or Caco‐2 cells with ¹³C‐marked glucose, glutamine, malate or a mix of amino acids as tracers. Comparative GC‐MS‐based isotopologue analysis of protein‐derived amino acids from the host cell and the bacterial fraction showed that C. trachomatis efficiently imported amino acids from the host cell for protein biosynthesis. FT‐ICR‐MS analyses also demonstrated that label from exogenous ¹³C‐glucose was efficiently shuffled into chlamydial lipopolysaccharide probably via glucose 6‐phosphate of the host cell. Minor fractions of bacterial Ala, Asp, and Glu were made de novo probably using dicarboxylates from the citrate cycle of the host cell. Indeed, exogenous ¹³C‐malate was efficiently taken up by C. trachomatis and metabolized into fumarate and succinate when the bacteria were kept in axenic medium containing the malate tracer. Together, the data indicate co‐substrate usage of intracellular C. trachomatis in a stream‐lined bipartite metabolism with host cell‐supplied amino acids for protein biosynthesis, host cell‐provided glucose 6‐phosphate for cell wall biosynthesis, and, to some extent, one or more host cell‐derived dicarboxylates, e.g. malate, feeding the partial TCA cycle of the bacterium. The latter flux could also support the biosynthesis of meso‐2,6‐diaminopimelate required for the formation of chlamydial peptidoglycan.     

Biosynthesis of the chromogen hermidin from Mercurialis annua L

Cut seedlings of Mercurialis annua L. were supplied with solutions containing 5.4mM [U-(13)C(6)]glucose and 50 mM unlabelled glucose. The pyridinone type chromogen, hermidin, was isolated and analyzed by NMR spectroscopy. (13)C NMR spectra revealed the presence of [4,5,6-(13)C(3)]hermidin in significant amount. NMR analysis of amino acids obtained by hydrolysis of labelled biomass showed the presence of [U-(13)C(3)]alanine, whereas aspartate was found to be virtually unlabelled. Photosynthetic pulse labelling of M. annua plants with (13)CO(2) followed by a chase period in normal air afforded [4,5,6-(13)C(3)]- and [2,3-(13)C(2)]hermidin with significant abundance. [U-(13)C(3)]Alanine and multiply (13)C-labelled aspartate isotopologues were also found in significant abundance. The labelling patterns of hermidin obtained in the present study closely resemble those observed for the pyridine ring of nicotine under similar experimental conditions. This suggests that hermidin, like nicotine, is biosynthesized via the nicotinic acid pathway from dihydroxyacetone phosphate and aspartate. The data show that pulse/chase labelling of plants with (13)CO(2) generates isotopologue patterns that are similar to those obtained with totally labelled carbohydrate as tracer, but with the added advantage that experiments can be conducted under strictly physiological conditions. This experimental concept appears ripe for application to a wide variety of problems in plant physiology.     

Pyruvate Carboxylase Plays a Crucial Role in Carbon Metabolism of Extra- and Intracellularly Replicating Listeria monocytogenes

The human pathogen L. monocytogenes is a facultatively intracellular bacterium that survives and replicates in the cytosol of many mammalian cells. The listerial metabolism, especially under intracellular conditions, is still poorly understood. Recent studies analyzed the carbon metabolism of L. monocytogenes by the ¹³C isotopologue perturbation method in a defined minimal medium containing [U-¹³C₆]glucose. It was shown that these bacteria produce oxaloacetate mainly by carboxylation of pyruvate due to an incomplete tricarboxylic acid cycle. Here, we report that a pycA insertion mutant defective in pyruvate carboxylase (PYC) still grows, albeit at a reduced rate, in brain heart infusion (BHI) medium but is unable to multiply in a defined minimal medium with glucose or glycerol as a carbon source. Aspartate and glutamate of the pycA mutant, in contrast to the wild-type strain, remain unlabeled when [U-¹³C₆]glucose is added to BHI, indicating that the PYC-catalyzed carboxylation of pyruvate is the predominant reaction leading to oxaloacetate in L. monocytogenes. The pycA mutant is also unable to replicate in mammalian cells and exhibits high virulence attenuation in the mouse sepsis model.Listeria monocytogenes is a human pathogen that can cause systemic infections, especially in immunocompromised people, with symptoms such as septicemia, (encephalo)meningitis, placentitis, and stillbirth. These Gram-positive bacteria are able to enter the cytosol of many mammalian cells after being taken up via normal or induced phagocytosis by professional phagocytes, mainly macrophages and dendritic cells, and nonphagocytic cells, such as epithelial cells, fibroblasts, and endothelial cells (1, 8, 13). While the virulence genes and their regulation (4, 21), as well as the encoded virulence factors (20, 22), necessary for the various steps of the intracellular replication cycle of L. monocytogenes have been extensively studied in the past few decades, there is still little information concerning the metabolic capacities and the metabolic adaptation processes (10) that enable these bacteria to efficiently replicate in the cytosol of their host cells.The information on listerial metabolism obtained from the genome sequence (7) suggests that these heterotrophic bacteria are capable of utilizing a variety of carbohydrates as carbon sources, since a large number of genes encoding phosphoenolpyruvate (PEP)-phosphotransferase systems (PTS) were identified. Furthermore, all genes encoding the enzymes necessary for the catabolism of glycerol and dihydroxyacetone are present in the L. monocytogenes genome (7, 11). This genomic information is in accord with data from previous and more recent physiological studies (11, 17, 24).Most genes encoding the enzymes for the major catabolic pathways, namely, glycolysis, the citrate cycle, and the pentose phosphate cycle, are present in L. monocytogenes. The citrate cycle, however, seems to be interrupted, since the genes encoding 2-oxoglutarate dehydrogenase have not been identified in all L. monocytogenes strains sequenced so far, including EGD-e (7), or in Listeria innocua strain Clip 11262. This enzymatic gap in the citrate cycle was recently confirmed by ¹³C isotopologue perturbation studies using uniformly ¹³C-labeled glucose. The results showed that two C₄ amino acids, aspartate and threonine, are generated in L. monocytogenes, predominantly from building blocks comprising one or three ¹³C atoms, respectively (2). These data suggested that oxaloacetate, the direct or indirect precursor of both amino acids, is generated by an anaplerotic reaction assembling precursors composed of one and three carbon atoms, respectively. This can be afforded by the carboxylation of pyruvate catalyzed by the ATP-dependent pyruvate carboxylase (PYC) encoded by pycA.The genes encoding the enzymes for most anabolic pathways, but not those for the biosynthesis of thiamine (vitamin B₁), riboflavin (vitamin B₂), biotin, and thiotic acid (lipoate), were also identified in L. monocytogenes. However, these bacteria grow efficiently in a mineral salt medium containing a suitable carbon source (e.g., glucose) and these four cofactors only when the amino acids cysteine, methionine, glutamine, arginine, valine, isoleucine, and leucine are also added (17). According to Tsai and Hodgson, strain 10403S requires only methionine and cysteine (24). The missing sulfate reductase in L. monocytogenes readily explains the strict requirement for cysteine/methionine as a sulfur source, while the missing nitrate reductase may be the reason for the stimulatory growth effect of glutamine and arginine as reduced nitrogen sources. However, the need for the three branched-chain amino acids (BCAA) valine, isoleucine, and leucine for efficient growth of L. monocytogenes EGD-e (references 17 and 24 and our unpublished results) is less obvious, since L. monocytogenes has the complete genetic set for synthesis of the BCAA, indicating the role of metabolic intermediates in listerial growth.The central precursor for the biosynthesis of the BCAA is pyruvate, which is channeled into their biosynthetic pathways either directly, via oxidative decarboxylation of pyruvate to acetyl-coenzyme A (CoA), or more indirectly via oxaloacetate (generated by pyruvate carboxylation) to aspartate and further to threonine. Thus, biosynthesis of the BCAA may compete with the PYC-mediated generation of oxaloacetate for the common substrate pyruvate. These data suggest that PYC may play an important role in the carbon metabolism of L. monocytogenes.To more precisely determine the significance of this anaplerotic enzyme for listerial metabolism and pathogenesis, we generated a mutant of L. monocytogenes EGD-e defective in pycA, the gene encoding PYC, and studied the replication of this mutant under different extra- and intracellular growth conditions. The results show that PYC indeed plays a crucial role in the intracellular replication of L. monocytogenes and hence in the infection process.     

Carbon metabolism of Listeria monocytogenes growing inside macrophages

The intracellular metabolism of Listeria monocytogenes was studied by ¹³C-isotopologue profiling using murine J774A.1 macrophages as host cells. Six hours after infection, bacteria were separated from the macrophages and hydrolyzed. Amino acids were converted into tert-butyl-dimethylsilyl derivatives and subjected to gas chromatography/mass spectrometry. When the macrophages were supplied with [U-¹³C₆]glucose prior to infection, but not during infection, label was detected only in Ala, Asp and Glu of the macrophage and bacterial protein with equal isotope distribution. When [U-¹³C₆]glucose was provided during the infection period, ¹³C label was found again in Ala, Asp and Glu from host and bacterial protein, but also in Ser, Gly, Thr and Val from the bacterial fraction. Mutants of L. monocytogenes defective in the uptake and catabolism of the C₃-metabolites, glycerol and/or dihydroxyacetone, showed reduced incorporation of [U-¹³C₆]glucose into bacterial amino acids under the same experimental settings. The ¹³C pattern suggests that (i) significant fractions (50–100%) of bacterial amino acids were provided by the host cell, (ii) a C₃-metabolite can serve as carbon source for L. monocytogenes under intracellular conditions and (iii) bacterial biosynthesis of Asp, Thr and Glu proceeds via oxaloacetate by carboxylation of pyruvate.     

13CO2 as a universal metabolic tracer in isotopologue perturbation experiments

A tobacco plant was illuminated for 5h in an atmosphere containing (13)CO(2) and then maintained for 10 days under standard greenhouse conditions. Nicotine, glucose, and amino acids from proteins were isolated chromatographically. Isotopologue abundances of isolated metabolites were determined quantitatively by NMR spectroscopy and mass spectrometry. The observed non-stochastic isotopologue patterns indicate (i) formation of multiply labeled photosynthetic carbohydrates during the (13)CO(2) pulse phase followed by (ii) partial catabolism of the primary photosynthetic products, and (iii) recombination of the (13)C-labeled fragments with unlabeled intermediary metabolites during the chase period. The detected and simulated isotopologue profiles of glucose and amino acids reflect carbon partitioning that is dominated by the Calvin cycle and glycolysis/glucogenesis. Retrobiosynthetic analysis of the nicotine pattern is in line with its known formation from nicotinic acid and putrescine via aspartate, glyceraldehyde phosphate and alpha-ketoglutarate as basic building blocks. The study demonstrates that pulse/chase labeling with (13)CO(2) as precursor is a powerful tool for the analysis of quantitative aspects of plant metabolism in completely unperturbed whole plants.     

Growth Media Simulating Ileal and Colonic Environments Affect the Intracellular Proteome and Carbon Fluxes of Enterohemorrhagic Escherichia coli O157:H7 Strain EDL933

In this study, the intracellular proteome of Escherichia coli O157:H7 strain EDL933 was analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization–time-of-flight (MALDI-TOF) spectrometry after growth in simulated ileal environment media (SIEM) and simulated colonic environment media (SCEM) under aerobic and microaerobic conditions. Differentially expressed intracellular proteins were identified and allocated to functional protein groups. Moreover, metabolic fluxes were analyzed by isotopologue profiling with [U-¹³C₆]glucose as a tracer. The results of this study show that EDL933 responds with differential expression of a complex network of proteins and metabolic pathways, reflecting the high metabolic adaptability of the strain. Growth in SIEM and SCEM is obviously facilitated by the upregulation of nucleotide biosynthesis pathway proteins and could be impaired by exposition to 50 µM 6-mercaptopurine under aerobic conditions. Notably, various stress and virulence factors, including Shiga toxin, were expressed without having contact with a human host.     

Characterization of central carbon metabolism of Streptococcus pneumoniae by isotopologue profiling

The metabolism of Streptococcus pneumoniae was studied by isotopologue profiling after bacterial cultivation in chemically defined medium supplemented with [U-(13)C(6)]- or [1,2-(13)C(2)]glucose. GC/MS analysis of protein-derived amino acids showed lack of (13)C label in amino acids that were also essential for pneumococcal growth. Ala, Ser, Asp, and Thr displayed high (13)C enrichments, whereas Phe, Tyr, and Gly were only slightly labeled. The analysis of the labeling patterns showed formation of triose phosphate and pyruvate via the Embden-Meyerhof-Parnas pathway. The labeling patterns of Asp and Thr suggested formation of oxaloacetate exclusively via the phosphoenolpyruvate carboxylase reaction. Apparently, α-ketoglutarate was generated from unlabeled glutamate via the aspartate transaminase reaction. A fraction of Phe and Tyr obtained label via the chorismate route from erythrose 4-phosphate, generated via the pentose phosphate pathway, and phosphoenolpyruvate. Strikingly, the data revealed no significant flux from phosphoglycerate to Ser and Gly but showed formation of Ser via the reverse reaction, namely by hydroxymethylation of Gly. The essential Gly was acquired from the medium, and the biosynthesis pathway was confirmed in experiments using [U-(13)C(2)]glycine as a tracer. The hydroxymethyl group in Ser originated from formate, which was generated by the pyruvate formate-lyase. Highly similar isotopologue profiles were observed in corresponding experiments with pneumococcal mutants deficient in PavA, CodY, and glucose-6-phosphate dehydrogenase pointing to the robustness of the core metabolic network used by these facultative pathogenic bacteria. In conclusion, this study demonstrates the dual utilization of carbohydrates and amino acids under in vitro conditions and identifies the unconventional de novo biosynthesis of serine by pneumococci.