Abstract
Salmonella enterica are invasive intracellular pathogens that replicate within a membrane-bound compartment inside infected host cells known as the Salmonella-containing vacuole. How Salmonella obtains nutrients for growth within this intracellular niche despite the apparent isolation is currently not known. Recent studies have indicated the importance of glucose and related carbon sources for tissue colonization and intracellular proliferation within host cells during Salmonella infections, although none have been found to be essential. We found that wild-type Salmonella are capable of replicating within infected host cells in the absence of both exogenous sugars and/or amino acids. Furthermore, mutants defective in glucose uptake or dependent upon peptides for growth also showed no significant loss in intracellular replication, suggesting host-derived peptides can supply both carbon units and amino acids. Here, we show that intracellular Salmonella recruit the host proteins LAMP-2A and Hsc73, key components of the host protein turnover pathway known as chaperone-mediated autophagy involved in transport of cytosolic proteins to the lysosome for degradation. Host-derived peptides are shown to provide a significant contribution toward the intracellular growth of Salmonella The results reveal a means whereby intracellular Salmonella gain access to the host cell cytosol from within its membrane-bound compartment to acquire nutrients. Furthermore, this study provides an explanation as to how Salmonella evades activation of autophagy mechanisms as part of the innate immune response.
Highlights
Salmonella enterica are invasive intracellular pathogens that replicate within a membrane-bound compartment inside infected host cells known as the Salmonella-containing vacuole
To determine to what extent exogenous glucose and amino acid pools contribute to the intracellular growth of S. enterica serovar typhimurium (S. typhimurium), we infected host cells that had been deprived of either amino acids, glucose, or both for 24 h prior to and during infection
Statistically significant, these relatively minor effects and the absence of significant reductions in intracellular growth of Salmonella in intestinal epithelial cells indicated that intracellular growth of wild-type S. typhimurium can be independent of exogenous sources of both glucose and amino acids (Fig. 1, A and B)
Summary
To determine to what extent exogenous glucose and amino acid pools contribute to the intracellular growth of S. enterica serovar typhimurium (S. typhimurium), we infected host cells that had been deprived of either amino acids, glucose, or both for 24 h prior to and during infection. Continued interaction with early endocytic pathways [28, 39, 40] could maintain the supply of proteins to support intracellular growth To test this possibility, we pre-treated host cells that had been deprived of both glucose and free amino acids with broad specificity protease inhibitors prior to and during infection with either wild-type or peptide-dependent strains of Salmonella. The observations that chemical inhibition, neutralizing antibodies against Hsc, and knockdown expression of LAMP-2A and CMA activity were the only treatments found to affect the growth of the peptide-dependent Salmonella strain, as well as the observation that only the LAMP-2A isoform co-localized with the SCV, all supported the idea that CMA contributed to the intracellular growth of Salmonella. Consistent with the co-localization studies (Fig. 9), the known CMA substrate, GAPDH, was present in low but appreciable levels in the SCV preparations (Fig. 11, B and C)
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