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...
Intracellular Salmonella enterica serovar Typhimurium (STM) deploy the Salmonella Pathogenicity Island 2-encoded type III secretion system (SPI2-T3SS) for the massive remodeling of the endosomal system for host cells. This activity results in formation of an extensive interconnected tubular network of Salmonella -induced filaments (SIFs) connected to the Salmonella -containing vacuole (SCV). Such network is absent in cells infected with SPI2-T3SS-deficient mutant strains such as Δ ssaV . A tubular network with reduced dimensions is formed if SPI2-T3SS effector protein SseF is absent. Previous single cell live microscopy-based analyses revealed that intracellular proliferation of STM is directly correlated to the ability to transform the host cell endosomal system into a complex tubular network. This network may also abrogate host defense mechanisms such as delivery of antimicrobial effectors to the SCV. To test the role of SIFs in STM patho-metabolism, we performed quantitative comparative proteomics of STM recovered from infected murine macrophages. We infected RAW264.7 cells with STM wild type (WT), Δ sseF or Δ ssaV strains, recovered bacteria 12 h after infection and determined proteome compositions. Increased numbers of proteins characteristic for nutritional starvation were detected in STM Δ sseF and Δ ssaV compared to WT. In addition, STM Δ ssaV , but not Δ sseF showed signatures of increased exposure to stress by antimicrobial defenses, in particular reactive oxygen species, of the host cells. The proteomics analyses presented here support and extend the role of SIFs for the intracellular lifestyle of STM. We conclude that efficient manipulation of the host cell endosomal system by effector proteins of the SPI2-T3SS contributes to nutrition, as well as to resistance against antimicrobial host defense mechanisms.
Salmonella enterica serovar Typhimurium (STM) is exposed to reactive oxygen species (ROS) originating from aerobic respiration, antibiotic treatment, and the oxidative burst occurring inside the Salmonella -containing vacuole (SCV) within host cells. ROS damage cellular compounds, thereby impairing bacterial viability and inducing cell death. Proteins containing iron–sulfur (Fe–S) clusters are particularly sensitive and become non-functional upon oxidation. Comprising five enzymes with Fe–S clusters, the TCA cycle is a pathway most sensitive toward ROS. To test the impact of ROS-mediated metabolic perturbations on bacterial physiology, we analyzed the proteomic and metabolic profile of STM deficient in both cytosolic superoxide dismutases (Δ sodAB ). Incapable of detoxifying superoxide anions (SOA), endogenously generated SOA accumulate during growth. Δ sodAB showed reduced abundance of aconitases, leading to a metabolic profile similar to that of an aconitase-deficient strain (Δ acnAB ). Furthermore, we determined a decreased expression of acnA in STM Δ sodAB . While intracellular proliferation in RAW264.7 macrophages and survival of methyl viologen treatment were not reduced for STM Δ acnAB , proteomic profiling revealed enhanced stress response. We conclude that ROS-mediated reduced expression and damage of aconitase does not impair bacterial viability or virulence, but might increase ROS amounts in STM, which reinforces the bactericidal effects of antibiotic treatment and immune responses of the host.
Multidrug-resistant Enterobacterales (MDRE) are an emerging threat to global health, leading to rising health care costs, morbidity and mortality. Multidrug-resistance is commonly caused by different β-lactamases (e.g., ESBLs and carbapenemases), sometimes in combination with other resistance mechanisms (e.g., porin loss, efflux). The continuous spread of MDRE among patients in hospital settings and the healthy population require adjustments in healthcare management and routine diagnostics. Rapid and reliable detection of MDRE infections as well as gastrointestinal colonization is key to guide therapy and infection control measures. However, proper implementation of these strategies requires diagnostic methods with short time-to-result, high sensitivity and specificity. Therefore, research on new techniques and improvement of already established protocols is inevitable. In this review, current methods for detection of MDRE are summarized with focus on culture based and molecular techniques, which are useful for the clinical microbiology laboratory.
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.
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