Analysis of carbon substrates used by Listeria monocytogenes during growth in J774A.1 macrophages suggests a bipartite intracellular metabolism (original) (raw)
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Life of Listeria monocytogenes in the host cells' cytosol
Microbes and Infection, 2007
In the past decades impressive knowledge has been accumulated concerning the basic mechanisms of interactions between intracellular bacteria and their host cells. Comparatively little is known on the metabolic requirements necessary for efficient replication of these bacteria within their specific host cell compartments. Recent developments in functional genomics have led to more extensive studies of the metabolic aspects that may be crucial for understanding the pathogenesis of intracellular bacteria. Here we summarize our present knowledge on the physiology of L. monocytogenes with emphasis on those parts that seem to be important for its ability to replicate in the cytosol of mammalian host cells.
Deciphering the intracellular metabolism of Listeria monocytogenes by mutant screening and modelling
BMC Genomics, 2010
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 genomewide 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.
Proceedings of the National Academy of Sciences, 2006
The carbon metabolism of Listeria monocytogenes (Lm) EGD and the two isogenic mutant strains Lm⌬prfA and Lm⌬prfApPRFA* (showing no or enhanced expression, respectively, of the virulence factor PrfA) was determined by 13 C isotopologue perturbation. After growth of the bacteria in a defined medium containing a mixture of [U-13 C6]glucose and glucose with natural 13 C abundance (1:25, wt͞wt), 14 amino acids were isolated and analyzed by NMR spectroscopy. Multiply 13 C-labeled isotopologues were determined quantitatively by signal deconvolution. The 13 C enrichments and isotopologue patterns allowed the reconstruction of most amino acid biosynthesis pathways and illustrated that overproduced PrfA may strongly influence the synthesis of some amino acids, notably that of the branched amino acids (Val, Ile, and Leu). Retrobiosynthetic analysis of the isotopologue compositions showed that degradation of glucose occurs to a large extent via the pentose phosphate pathway and that the citrate cycle is incomplete because of the absence of 2-oxoglutarate dehydrogenase activity. The reconstructed labeling pattern of oxaloacetate indicated its formation by carboxylation of pyruvate. This metabolic reaction seems to have a strong impact on the growth requirement in defined minimal medium. Bioinformatical steady-state network analyses and flux distribution predictions confirmed the experimental data and predicted metabolite fluxes through the enzymes of the pathways under study.
Applied and Environmental Microbiology, 2004
Although carbon dioxide (CO 2) is known to inhibit growth of most bacteria, very little is known about the cellular response. The food-borne pathogen Listeria monocytogenes is characterized by its ability to grow in high CO 2 concentrations at refrigeration temperatures. We examined the listerial responses of different strains to growth in air, 100% N 2 , and 100% CO 2. The CO 2-induced changes in membrane lipid fatty acid composition and expression of selected genes were strain dependent. The acid-tolerant L. monocytogenes LO28 responded in the same manner to CO 2 as to other anaerobic, slightly acidic environments (100% N 2 , pH 5.7). An increase in the expression of the genes encoding glutamate decarboxylase (essential for survival in strong acid) as well as an increased amount of branched-chain fatty acids in the membrane was observed in both atmospheres. In contrast, the acid-sensitive L. monocytogenes strain EGD responded differently to CO 2 and N 2 at the same pH. In a separate experiment with L. monocytogenes 412, an increased isocitrate dehydrogenase activity level was observed for cells grown in CO 2-containing atmospheres. Together, our findings demonstrate that the CO 2response is a partly strain-dependent complex mechanism. The possible links between the CO 2-dependent changes in isocitrate dehydrogenase activity, glutamate metabolism and branched fatty acid biosynthesis are discussed. MATERIALS AND METHODS Bacterial strains. L. monocytogenes 412 (obtained from the Danish Meat Research Institute, Roskilde, Denmark) was isolated from bacon. L. monocytogenes LO28, a clinical strain (isolated by P.
Journal of Biological Chemistry, 2018
Outer membrane vesicles produced by Gram-negative bacteria have been studied for half a century but the possibility that Gram-positive bacteria secrete extracellular vesicles (EVs) was not pursued until recently due to the assumption that the thick peptidoglycan cell wall would prevent their release to the environment. However, following their discovery in fungi, which also have cell walls, EVs have now been described for a variety of Gram-positive bacteria. EVs purified from Gram-positive bacteria are implicated in virulence, toxin release, and transference to host cells, eliciting immune responses, and spread of antibiotic resistance. Listeria monocytogenes is a Gram-positive bacterium that causes listeriosis. Here we report that L. monocytogenes produces EVs with diameters ranging from 20 to 200 nm, containing the pore-forming toxin listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC). Cell-free EV preparations were toxic to mammalian cells, the murine macrophage cell line J774.16, in a LLO-dependent manner, evidencing EV biological activity. The deletion of plcA increased EV toxicity, suggesting PI-PLC reduced LLO activity. Using simultaneous metabolite, protein, and lipid extraction (MPLEx) multiomics we characterized protein, lipid, and metabolite composition of bacterial cells and secreted EVs and found that EVs carry the majority of listerial virulence proteins. Using immunogold EM we detected LLO at several organelles within infected human epithelial cells and with high-resolution fluorescence imaging we show that dynamic lipid structures are released from L. monocytogenes during infection. Our findings demonstrate that L. monocytogenes uses EVs for toxin release and implicate these structures in mammalian cytotoxicity. The pathogenic Gram-positive bacterium Listeria monocytogenes is the etiological agent of listeriosis, a disease with serious consequences for pregnant women, newborns, and immunocompromised persons. Healthy individuals who have ingested large amounts of L. monocytogenes can suffer from gastroenteritis when the bacterium passes through the gastrointestinal barrier (1-4). L. monocytogenes can cause spontaneous abortions in pregnant women and meningoencephalitis by crossing the placental and blood-brain barriers, respectively (5). To invade cells, cross these barriers, and evade the immune system, L. monocytogenes has a sophisticated intracellular life cycle and pathogenic strategy (6, 7). Initially, L. monocytogenes invades various cell types, including nonphagocytic cells, by utilizing two internalins, internalin A (InlA) and internalin B (InlB), with a minor contribution by the pore-forming toxin listeriolysin O (LLO), 7 to induce uptake of the bacterium (1, 5, 8-11). Once internalized in the host vacuole, L. monocytogenes employs LLO, phosphatidylcholinespecific phospholipase (PC-PLC), and phosphatidylinositolspecific phospholipase C (PI-PLC) to disrupt the single vacuolar membrane, releasing the bacterium into the cytoplasm The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This article contains Movie S1, Tables S1-S4, and Figs. S1-S4. Multiomics data were deposited in the MassIVE repository under accession numbers MSV000081402, MSV000081403, and MSV000081404.
MCB 303) MICROBIAL PHYSIOLOGY AND METABOLISM {Dr. Chy. Ezemba
Since we are familiar with the structure of prokaryotic cells, we can discuss the activities that enable these microbes to thrive. The life-support processes of even the most structurally simple organism involve a large number of complex biochemical reactions. Most, although not all, of the biochemical processes of bacteria also occur in eukaryotic microbes and in the cells of multicellular organisms, including humans. However, the reactions that are unique to bacteria are fascinating because they allow microorganisms to do things we cannot do. For example, some bacteria can live on cellulose, whereas others can live on petroleum. Through their metabolism, bacteria recycle elements after other organisms have used them. Still other bacteria can live on diets of inorganic substances such as carbon dioxide, iron, sulfur, hydrogen gas, and ammonia. Microbial metabolism allows some microorganisms to grow in or on the human body as shown in dental plaque. 1.0 Concept: Physiology is the study of the processes which go no in living organisms. While on the other hand the term Metabolism to refer to the sum of all chemical reactions within the cells of a living organism. Because chemical reactions and the processes involve building up (Anabolism) while breaking down of nutrients within a cell is termed (Catabolism). Metabolism can be viewed as an energy-balancing act and this balanced flow of chemicals and energy maintains the life of a cell. These chemical reactions provide energy and create substances that sustain life. So Pathways can be categorized into two general types— catabolic and anabolic.
PLoS ONE, 2010
Analysis of the genome sequences of the major human bacterial pathogens has provided a large amount of information concerning their metabolic potential. However, our knowledge of the actual metabolic pathways and metabolite fluxes occurring in these pathogens under infection conditions is still limited. In this study, we analysed the intracellular carbon metabolism of enteroinvasive Escherichia coli (EIEC HN280 and EIEC 4608-58) and Salmonella enterica Serovar Typhimurium (Stm 14028) replicating in epithelial colorectal adenocarcinoma cells (Caco-2). To this aim, we supplied [U-13 C 6 ]glucose to Caco-2 cells infected with the bacterial strains or mutants thereof impaired in the uptake of glucose, mannose and/or glucose 6-phosphate. The 13 C-isotopologue patterns of protein-derived amino acids from the bacteria and the host cells were then determined by mass spectrometry. The data showed that EIEC HN280 growing in the cytosol of the host cells, as well as Stm 14028 replicating in the Salmonella-containing vacuole (SCV) utilised glucose, but not glucose 6-phosphate, other phosphorylated carbohydrates, gluconate or fatty acids as major carbon substrates. EIEC 4608-58 used C 3compound(s) in addition to glucose as carbon source. The labelling patterns reflected strain-dependent carbon flux via glycolysis and/or the Entner-Doudoroff pathway, the pentose phosphate pathway, the TCA cycle and anapleurotic reactions between PEP and oxaloacetate. Mutants of all three strains impaired in the uptake of glucose switched to C 3 -substrate(s) accompanied by an increased uptake of amino acids (and possibly also other anabolic monomers) from the host cell. Surprisingly, the metabolism of the host cells, as judged by the efficiency of 13 C-incorporation into host cell amino acids, was not significantly affected by the infection with either of these intracellular pathogens.