Role of host cell-derived amino acids in nutrition of intracellular Salmonella enterica - PubMed (original) (raw)
Role of host cell-derived amino acids in nutrition of intracellular Salmonella enterica
Jasmin Popp et al. Infect Immun. 2015 Dec.
Abstract
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.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Figures
FIG 1
Schematic overview of selected amino acid biosynthesis pathways in S. Typhimurium and their origin from central metabolic pathways. Genes that encode the relevant enzymes are indicated and are further referred to in the text. Intermediates in the proline biosynthesis pathway are indicated by numbers, as follows: ①, γ-glutamyl phosphate; ②, glutamate γ-semialdehyde; ③, Δ1-pyrroline-5-carboxylate. Solid arrows indicate single reaction steps, while dotted arrows represent several reactions, with the number of steps given in parentheses. The reaction from ② to ③ occurs spontaneously. αKG, α-ketoglutarate; αKV, α-keto-isovalerate; CIT, citrate; GLC, glucose; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PYR, pyruvate.
FIG 2
Role of amino acid biosynthesis pathways in intracellular proliferation of S. Typhimurium. Intracellular replication of S. Typhimurium WT, Δ_ssaV_, and various mutant strains impaired in amino acid biosynthesis in RAW264.7 (A) and HeLa (B) cells was determined by gentamicin protection assays. The Δ_ssaV_ strain defective in the SPI2-encoded T3SS served as a negative control. The Δ_avtA_ Δ_yfbQ_ Δ_yfdZ_ strain is abbreviated ayy. Infected cells were lysed at 2 h and 16 h p.i., and serial dilutions were plated onto agar plates for determination of CFU. _x_-fold intracellular proliferation is calculated as the ratio of CFU at 16 h p.i./CFU at 2 h p.i. The x_-fold proliferation of the WT strain was set as 100%, and intracellular proliferation of mutant strains is expressed as a percentage of the value for the WT. Experiments were performed in triplicates, and mean values and standard deviations are given for at least three biological replicates (two biological replicates for the Δ_asnAB strain in HeLa cells). Statistical significances were calculated by Student's t test and are indicated as follows: n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 3
Complementation of asparagine- and proline-auxotrophic strains by extracellular amino acids. Intracellular replication of the S. Typhimurium WT strain, the Δ_ssaV_ strain, and mutant strains auxotrophic for asparagine (two independently generated strains) (A) or proline (B) in RAW264.7 cells was determined as described in the legend of Fig. 2. Cell culture medium was not supplemented (−) or was supplemented (+) with 0.8 mM asparagine (A) or 0.8 mM proline (B). Amino acids were added at 1 h p.i., with a change to medium containing 10 μg/ml gentamicin. Experiments were performed in triplicates, and mean values and standard deviations are given for one biological experiment. Statistical significances were calculated by Student's t test and are indicated as follows: n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 4
Complementation of the glutamine-auxotrophic strain by extracellular amino acids. The effect of the addition of glutamine (Gln) or alanyl-glutamine (Glx) on the intracellular replication of S. Typhimurium in RAW264.7 cells (A) or HeLa cells (B) was determined as described in the legend of Fig. 2. S. Typhimurium WT, Δ_ssaV_, and glutamine-auxotrophic Δ_glnA_ strains were used for infection. Cell culture medium was supplemented with 4 mM glutamine (Gln) or alanyl-glutamine (Glx). Amino acids were added at 1 h p.i., i.e., with a change to medium containing 10 μg/ml gentamicin. Experiments were performed in triplicates, and mean values and standard deviations are given for two (WT strain with Glx, ssaV mutant with Gln, and ssaV mutant with Glx) or three (Δ_glnA_ strain with Gln and Δ_glnA_ strain with Glx) independent biological experiments. Statistical significances were calculated by Student's t test and are indicated as follows: n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 5
Effect of availability of extracellular succinate on intracellular replication of S. Typhimurium. RAW264.7 cells were infected with WT, Δ_ssaV_, and Δ_sucAB_ strains, and intracellular proliferation was determined as described in the legend of Fig. 2. Hybridoma express medium was used for the period of the infection experiment without (−) or with (+) the addition of 24.6 mM succinate (Suc), added at 1 h p.i. WT replication was set as 100%, and replication of the mutant strains is expressed as a percentage of WT replication. Experiments were performed in triplicates, and mean values and standard deviations are given for at least three biological experiments, with the exception of experiments with the WT strain with succinate and the Δ_ssaV_ strain with succinate (two replicates). Statistical significances were calculated by Student's t test and are indicated as follows: n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 6
Effect of Salmonella-induced filament formation on complementation by extracellular proline. The intracellular replication of S. Typhimurium WT, Δ_ssaV_, Δ_sifA_ Δ_sseJ_, and proline-auxotrophic Δ_proC_ or Δ_proC_ Δ_sifA_ Δ_sseJ_ strains in RAW264.7 cells was determined as described in the legend of Fig. 2. WT replication was set as 100%, and replication of the mutant strains was calculated as a percentage of WT replication. Cell culture media were used without (−) or with (+) 0.8 mM proline added to the medium at 1 h p.i. Experiments were performed in triplicates, and mean values and standard deviations are given for three biological replicates. Statistical significances were calculated by Student's t test and are indicated as follows: n.s., not significant; ***, P < 0.001.
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