The facultative intracellular pathogen Salmonella enterica resides in a specific membrane-bound compartment termed the Salmonella-containing vacuole (SCV). Despite being segregated from access to metabolites in the host cell cytosol, Salmonella is able to efficiently proliferate within the SCV. We set out to unravel the nutritional supply of Salmonella in the SCV with focus on amino acids. We studied the availability of amino acids by the generation of auxotrophic strains for alanine, asparagine, aspartate, glutamine, and proline in a macrophage cell line (RAW264.7) and an epithelial cell line (HeLa) and examined access to extracellular nutrients for nutrition. Auxotrophies for alanine, asparagine, or proline attenuated intracellular replication in HeLa cells, while aspartate, asparagine, or proline auxotrophies attenuated intracellular replication in RAW264.7 macrophages. The different patterns of intracellular attenuation of alanine-or aspartate-auxotrophic strains support distinct nutritional conditions in HeLa cells and RAW264.7 macrophages. Supplementation of medium with individual amino acids restored the intracellular replication of mutant strains auxotrophic for asparagine, proline, or glutamine. Similarly, a mutant strain deficient in succinate dehydrogenase was complemented by the extracellular addition of succinate. Complementation of the intracellular replication of auxotrophic Salmonella by external amino acids was possible if bacteria were proficient in the induction of Salmonella-induced filaments (SIFs) but failed in a SIF-deficient background. We propose that the ability of intracellular Salmonella to redirect host cell vesicular transport provides access of amino acids to auxotrophic strains and, more generally, is essential to continuously supply bacteria within the SCV with nutrients.T he facultative intracellular life-style is a common virulence strategy among bacterial pathogens, and intracellular lifestyles are as diverse as the diseases caused by these pathogens. While some bacteria lyse the host cell membrane compartment and initiate replication in the cytosol, others remain in a host cell-derived membrane compartment that is modified to allow intravacuolar survival and replication. To understand the lifestyle of bacterial pathogens, it is of central importance to analyze which nutritional limitations are experienced by the pathogen in its intracellular habitat and how the pathogen adapts its metabolism in order to survive and proliferate despite these limitations.Salmonella enterica is an invasive, facultative, intracellular pathogen responsible for foodborne diseases ranging from localized gastroenteritis to systemic typhoid fever. Inside mammalian host cells, S. enterica resides in a specialized membrane-bound compartment, the Salmonella-containing vacuole (SCV). The SCV is modified by the action of virulence factors of S. enterica, and the function of the type III secretion system (T3SS) encoded by Salmonella pathogenicity island 2 (SPI2) and its effector proteins is of central impor...

The infectious intracellular lifestyle of Salmonella enterica relies on the adaptation to nutritional conditions within the Salmonella-containing vacuole (SCV) in host cells. We summarize latest results on metabolic requirements for Salmonella during infection. This includes intracellular phenotypes of mutant strains based on metabolic modeling and experimental tests, isotopolog profiling using 13C-compounds in intracellular Salmonella, and complementation of metabolic defects for attenuated mutant strains towards a comprehensive understanding of the metabolic requirements of the intracellular lifestyle of Salmonella. Helpful for this are also genomic comparisons. We outline further recent studies and which analyses of intracellular phenotypes and improved metabolic simulations were done and comment on technical required steps as well as progress involved in the iterative refinement of metabolic flux models, analyses of mutant phenotypes, and isotopolog analyses. Salmonella lifestyle is well-adapted to the SCV and its specific metabolic requirements. Salmonella metabolism adapts rapidly to SCV conditions, the metabolic generalist Salmonella is quite successful in host infection.

The human-pathogenic bacterium Salmonella enterica adjusts and adapts to different environments while attempting colonization. In the course of infection nutrient availabilities change drastically. New techniques, “-omics” data and subsequent integration by systems biology improve our understanding of these changes. We review changes in metabolism focusing on amino acid and carbohydrate metabolism. Furthermore, the adaptation process is associated with the activation of genes of the Salmonella pathogenicity islands (SPIs). Anti-infective strategies have to take these insights into account and include metabolic and other strategies. Salmonella infections will remain a challenge for infection biology.

We performed perturbation analyses of the tricarboxylic acid cycle of the gastrointestinal pathogen Salmonella enterica serovar Typhimurium. The defect of fumarase activity led to accumulation of fumarate but also resulted in a global alteration of carbon fluxes, leading to increased storage of glycogen. Gross alterations were observed in proteome and metabolome compositions of fumarase-deficient Salmonella. In turn, these changes were linked to aberrant motility patterns of the mutant strain and resulted in highly increased phagocytic uptake by macrophages. Our findings indicate that basic cellular functions and specific virulence functions in Salmonella critically depend on the proper function of the primary metabolism.

37The tricarboxylic acid cycle is a central metabolic hub in most cells. Virulence functions of 38 bacterial pathogens such as facultative intracellular Salmonella enterica serovar Typhimurium 39 (STM) are closely connected to cellular metabolism. During systematic analyses of mutant 40 strains with defects in TCA cycle, a strain deficient in all fumarase isoforms (ΔfumABC) elicited 41 a unique metabolic profile. Alongside fumarate STM ΔfumABC accumulates intermediates of 42 glycolysis and pentose phosphate pathway. Analyses by metabolomics and proteomics revealed 43 that fumarate accumulation redirects carbon fluxes towards glycogen synthesis due to high 44 (p)ppGpp levels. In addition, we observed reduced abundance of CheY, leading to altered 45 motility and increased phagocytosis of STM by macrophages. Deletion of glycogen synthase 46 restored normal carbon fluxes and phagocytosis, and partially levels of CheY. We propose that 47