Toward a Systemic Understanding of Listeria monocytogenes Metabolism during Infection

Listeria monocytogenes is a foodborne human pathogen that can cause invasive infection in susceptible animals and humans. For proliferation within hosts, this facultative intracellular pathogen uses a reservoir of specific metabolic pathways, transporter, and enzymatic functions whose expression requires the coordinated activity of a complex regulatory network. The highly adapted metabolism of L. monocytogenes strongly depends on the nutrient composition of various milieus encountered during infection. Transcriptomic and proteomic studies revealed the spatial–temporal dynamic of gene expression of this pathogen during replication within cultured cells or in vivo. Metabolic clues are the utilization of unusual C2- and C3-bodies, the metabolism of pyruvate, thiamine availability, the uptake of peptides, the acquisition or biosynthesis of certain amino acids, and the degradation of glucose-phosphate via the pentose phosphate pathway. These examples illustrate the interference of in vivo conditions with energy, carbon, and nitrogen metabolism, thus affecting listerial growth. The exploitation, analysis, and modeling of the available data sets served as a first attempt to a systemic understanding of listerial metabolism during infection. L. monocytogenes might serve as a model organism for systems biology of a Gram-positive, facultative intracellular bacterium

High binding affinity of repressor IoIR avoids costs of untimely induction of myo-inositol utilization by Salmonella Typhimurium

Growth of Salmonella enterica serovar Typhimurium strain 14028 with myo-inositol (MI) is characterized by a bistable phenotype that manifests with an extraordinarily long (34 h) and variable lag phase. When cells were pre-grown in minimal medium with MI, however, the lag phase shortened drastically to eight hours, and to six hours in the absence of the regulator IoIR. To unravel the molecular mechanism behind this phenomenon, we investigated this repressor in more detail. Flow cytometry analysis of the iolR promoter at a single cell level demonstrated bistability of its transcriptional activation. Electrophoretic mobility shift assays were used to narrow the potential binding region of IoIR and identified at least two binding sites in most iol gene promoters. Surface plasmon resonance spectroscopy quantified IolR binding and indicated its putative oligomerization and high binding affinity towards specific iol gene promoters. In competitive assays, the ioIR deletion mutant, in which iol gene repression is abolished, showed a severe growth disadvantage of -15% relative to the parental strain in rich medium. We hypothesize that the strong repression of iol gene transcription is required to maintain a balance between metabolic flexibility and fitness costs, which follow the inopportune induction of an unusual metabolic pathway

Analysis of carbon substrates used by Listeria monocytogenes during growth in J774A.1 macrophages suggests a bipartite intracellular metabolism

Intracellular bacterial pathogens (IBPs) are dependent on various nutrients provided by the host cells. Different strategies may therefore be necessary to adapt the intracellular metabolism of IBPs to the host cells. The specific carbon sources, the catabolic pathways participating in their degradation, and the biosynthetic performances of IBPs are still poorly understood. In this report, we have exploited the technique of C-13-isotopologue profiling to further study the carbon metabolism of Listeria monocytogenes by using the EGDe wild-type strain and mutants (defective in the uptake and/or catabolism of various carbon compounds) replicating in J774A.1 macrophages. For this goal, the infected macrophages were cultivated in the presence of [1, 2-C-13(2)]glucose, [U-C-13(3)]glycerol, [U-C-13(3)]pyruvate, [U-C-13(3)]lactate, or a mix of [U-C-13]amino acids. GC/MS-based isotopologue profiling showed efficient utilization of amino acids, glucose 6-phosphate, glycerol, and (at a low extent) also of lactate but not of pyruvate by the IBPs. Most amino acids imported from the host cells were directly used for bacterial protein biosynthesis and hardly catabolized. However, Asp was de novo synthesized by the IBPs and not imported from the host cell. As expected, glycerol was catabolized via the ATP-generating lower part of the glycolytic pathway, but apparently not used for gluconeogenesis. The intermediates generated from glucose 6-phosphate in the upper part of the glycolytic pathway and the pentose phosphate shunt likely serve primarily for anabolic purposes (probably for the biosynthesis of cell wall components and nucleotides). This bipartite bacterial metabolism which involves at least two major carbon substrates-glycerol mainly for energy supply and glucose 6-phosphate mainly for indispensible anabolic performances-may put less nutritional stress on the infected host cells, thereby extending the lifespan of the host cells to the benefit of the IBPs

The small RNA RssR regulates myo-inositol degradation by Salmonella enterica

Small noncoding RNAs (sRNAs) with putative regulatory functions in gene expression have been identified in the enteropathogen Salmonella enterica serovar Typhimurium (S. Typhimurium). Two sRNAs are encoded by the genomic island GEI4417/4436 responsible for myo-inositol (MI) degradation, suggesting a role in the regulation of this metabolic pathway. We show that a lack of the sRNA STnc2160, termed RssR, results in a severe growth defect in minimal medium (MM) with MI. In contrast, the second sRNA STnc1740 was induced in the presence of glucose, and its overexpression slightly attenuated growth in the presence of MI. Constitutive expression of RssR led to an increased stability of the reiD mRNA, which encodes an activator of iol genes involved in MI utilization, via interaction with its 5'-UTR. SsrB, a response regulator contributing to the virulence properties of salmonellae, activated rssR transcription by binding the sRNA promoter. In addition, the absence of the RNA chaperone Hfq resulted in strongly decreased levels of RssR, attenuated S. Typhimurium growth with MI, and reduced expression of several iol genes required for MI degradation. Considered together, the extrinsic RssR allows fine regulation of cellular ReiD levels and thus of MI degradation by acting on the reiD mRNA stability

Genome-wide association reveals host-specific genomic traits in Escherichia coli

Background Escherichia coli is an opportunistic pathogen which colonizes various host species. However, to what extent genetic lineages of E. coli are adapted or restricted to specific hosts and the genomic determinants of such adaptation or restriction is poorly understood. Results We randomly sampled E. coli isolates from four countries (Germany, UK, Spain, and Vietnam), obtained from five host species (human, pig, cattle, chicken, and wild boar) over 16 years, from both healthy and diseased hosts, to construct a collection of 1198 whole-genome sequenced E. coli isolates. We identified associations between specific E. coli lineages and the host from which they were isolated. A genome-wide association study (GWAS) identified several E. coli genes that were associated with human, cattle, or chicken hosts, whereas no genes associated with the pig host could be found. In silico characterization of nine contiguous genes (collectively designated as nan-9) associated with the human host indicated that these genes are involved in the metabolism of sialic acids (Sia). In contrast, the previously described sialic acid regulon known as sialoregulon (i.e. nanRATEK-yhcH, nanXY, and nanCMS) was not associated with any host species. In vitro growth experiments with a Δnan-9 E. coli mutant strain, using the sialic acids 5-N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) as sole carbon source, showed impaired growth behaviour compared to the wild-type. Conclusions This study provides an extensive analysis of genetic determinants which may contribute to host specificity in E. coli. Our findings should inform risk analysis and epidemiological monitoring of (antimicrobial resistant) E. coli

Shotgun sequencing of Yersinia enterocolitica strain W22703 (biotype 2, serotype O:9): genomic evidence for oscillation between invertebrates and mammals

<p>Abstract</p> <p>Background</p> <p><it>Yersinia enterocolitica </it>strains responsible for mild gastroenteritis in humans are very diverse with respect to their metabolic and virulence properties. Strain W22703 (biotype 2, serotype O:9) was recently identified to possess nematocidal and insecticidal activity. To better understand the relationship between pathogenicity towards insects and humans, we compared the W22703 genome with that of the highly pathogenic strain 8081 (biotype1B; serotype O:8), the only <it>Y. enterocolitica </it>strain sequenced so far.</p> <p>Results</p> <p>We used whole-genome shotgun data to assemble, annotate and analyse the sequence of strain W22703. Numerous factors assumed to contribute to enteric survival and pathogenesis, among them osmoregulated periplasmic glucan, hydrogenases, cobalamin-dependent pathways, iron uptake systems and the <it>Yersinia </it>genome island 1 (YGI-1) involved in tight adherence were identified to be common to the 8081 and W22703 genomes. However, sets of ~550 genes revealed to be specific for each of them in comparison to the other strain. The plasticity zone (PZ) of 142 kb in the W22703 genome carries an ancient flagellar cluster Flg-2 of ~40 kb, but it lacks the pathogenicity island YAPI_Ye, the secretion system <it>ysa </it>and <it>yts1</it>, and other virulence determinants of the 8081 PZ. Its composition underlines the prominent variability of this genome region and demonstrates its contribution to the higher pathogenicity of biotype 1B strains with respect to W22703. A novel type three secretion system of mosaic structure was found in the genome of W22703 that is absent in the sequenced strains of the human pathogenic <it>Yersinia </it>species, but conserved in the genomes of the apathogenic species. We identified several regions of differences in W22703 that mainly code for transporters, regulators, metabolic pathways, and defence factors.</p> <p>Conclusion</p> <p>The W22703 sequence analysis revealed a genome composition distinct from other pathogenic <it>Yersinia enterocolitica </it>strains, thus contributing novel data to the <it>Y. enterocolitica </it>pan-genome. This study also sheds further light on the strategies of this pathogen to cope with its environments.</p

Deciphering the intracellular metabolism of Listeria monocytogenes by mutant screening and modelling

Background: The human pathogen Listeria monocytogenes resides and proliferates within the cytoplasm of epithelial cells. While the virulence factors essentially contributing to this step of the infection cycle are well characterized, the set of listerial genes contributing to intracellular replication remains to be defined on a genome-wide level. Results: A comprehensive library of L. monocytogenes strain EGD knockout mutants was constructed upon insertion-duplication mutagenesis, and 1491 mutants were tested for their phenotypes in rich medium and in a Caco-2 cell culture assay. Following sequencing of the plasmid insertion site, 141 different genes required for invasion of and replication in Caco-2 cells were identified. Ten in-frame deletion mutants were constructed that confirmed the data. The genes with known functions are mainly involved in cellular processes including transport, in the intermediary metabolism of sugars, nucleotides and lipids, and in information pathways such as regulatory functions. No function could be ascribed to 18 genes, and a counterpart of eight genes is missing in the apathogenic species L. innocua. Mice infection studies revealed the in vivo requirement of IspE (Lmo0190) involved in mevalonate synthesis, and of the novel ABC transporter Lmo0135-0137 associated with cysteine transport. Based on the data of this genome-scale screening, an extreme pathway and elementary mode analysis was applied that demonstrates the critical role of glycerol and purine metabolism, of fucose utilization, and of the synthesis of glutathione, aspartate semialdehyde, serine and branched chain amino acids during intracellular replication of L. monocytogenes. Conclusion: The combination of a genetic screening and a modelling approach revealed that a series of transporters help L. monocytogenes to overcome a putative lack of nutrients within cells, and that a high metabolic flexibility contributes to the intracellular replication of this pathogen

Agnostic Pathway/Gene Set Analysis of Genome-Wide Association Data Identifies Associations for Pancreatic Cancer

Background Genome-wide association studies (GWAS) identify associations of individual single-nucleotide polymorphisms (SNPs) with cancer risk but usually only explain a fraction of the inherited variability. Pathway analysis of genetic variants is a powerful tool to identify networks of susceptibility genes. Methods We conducted a large agnostic pathway-based meta-analysis of GWAS data using the summary-based adaptive rank truncated product method to identify gene sets and pathways associated with pancreatic ductal adenocarcinoma (PDAC) in 9040 cases and 12 496 controls. We performed expression quantitative trait loci (eQTL) analysis and functional annotation of the top SNPs in genes contributing to the top associated pathways and gene sets. All statistical tests were two-sided. Results We identified 14 pathways and gene sets associated with PDAC at a false discovery rate of less than 0.05. After Bonferroni correction (P Conclusion Our agnostic pathway and gene set analysis integrated with functional annotation and eQTL analysis provides insight into genes and pathways that may be biologically relevant for risk of PDAC, including those not previously identified.Peer reviewe

Genome-wide meta-analysis identifies five new susceptibility loci for pancreatic cancer.

In 2020, 146,063 deaths due to pancreatic cancer are estimated to occur in Europe and the United States combined. To identify common susceptibility alleles, we performed the largest pancreatic cancer GWAS to date, including 9040 patients and 12,496 controls of European ancestry from the Pancreatic Cancer Cohort Consortium (PanScan) and the Pancreatic Cancer Case-Control Consortium (PanC4). Here, we find significant evidence of a novel association at rs78417682 (7p12/TNS3, P = 4.35 × 10-8). Replication of 10 promising signals in up to 2737 patients and 4752 controls from the PANcreatic Disease ReseArch (PANDoRA) consortium yields new genome-wide significant loci: rs13303010 at 1p36.33 (NOC2L, P = 8.36 × 10-14), rs2941471 at 8q21.11 (HNF4G, P = 6.60 × 10-10), rs4795218 at 17q12 (HNF1B, P = 1.32 × 10-8), and rs1517037 at 18q21.32 (GRP, P = 3.28 × 10-8). rs78417682 is not statistically significantly associated with pancreatic cancer in PANDoRA. Expression quantitative trait locus analysis in three independent pancreatic data sets provides molecular support of NOC2L as a pancreatic cancer susceptibility gene