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The RNA Complement of Outer Membrane Vesicles From Salmonella enterica Serovar Typhimurium Under Distinct Culture Conditions.

ABSTRACT: Bacterial outer membrane vesicles (OMVs), as well as OMV-associated small RNAs, have been demonstrated to play a role in host-pathogen interactions. The presence of larger RNA transcripts in OMVs has been less studied and their potential role in host-pathogen interactions remains largely unknown. Here we analyze RNA from OMVs secreted by Salmonella enterica serovar Typhimurium (S. Typhimurium) cultured under different conditions, which mimic host-pathogen interactions. S. Typhimurium was grown to exponential and stationary growth phases in minimal growth control medium (phosphate-carbon-nitrogen, PCN), as well as in acidic and phosphate-depleted PCN, comparable to the macrophage environment and inducing therefore the expression of Salmonella pathogenicity island 2 (SPI-2) genes. Moreover, Salmonella pathogenicity island 1 (SPI-1), which is required for virulence during the intestinal phase of infection, was induced by culturing S. Typhimurium to the stationary phase in Lysogeny Broth (LB). For each condition, we identified OMV-associated RNAs that are enriched in the extracellular environment relative to the intracellular space. All RNA classes could be observed, but a vast majority of rRNA was exported in all conditions in variable proportions with a notable decrease in LB SPI-1 inducing media. Several mRNAs and ncRNAs were specifically enriched in/on OMVs dependent on the growth conditions. Important to note is that some RNAs showed identical read coverage profiles intracellularly and extracellularly, whereas distinct coverage patterns were observed for other transcripts, suggesting a specific processing or degradation. Moreover, PCR experiments confirmed that distinct RNAs were present in or on OMVs as full-length transcripts (IsrB-1/2; IsrA; ffs; SsrS; CsrC; pSLT035; 10Sa; rnpB; STM0277; sseB; STM0972; STM2606), whereas others seemed to be rather present in a processed or degraded form. Finally, we show by a digestion protection assay that OMVs are able to prevent enzymatic degradation of given full-length transcripts (SsrS, CsrC, 10Sa, and rnpB). In summary, we show that OMV-associated RNA is clearly different in distinct culture conditions and that at least a fraction of the extracellular RNA is associated as a full-length transcripts with OMVs, indicating that some RNAs are protected by OMVs and thereby leaving open the possibility that those might be functionally active.

SUBMITTER: Malabirade A

PROVIDER: S-EPMC6125333 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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The RNA Complement of Outer Membrane Vesicles From Salmonella enterica Serovar Typhimurium Under Distinct Culture Conditions.

Malabirade Antoine A Habier Janine J Heintz-Buschart Anna A May Patrick P Godet Julien J Halder Rashi R Etheridge Alton A Galas David D Wilmes Paul P Fritz Joëlle V JV

Frontiers in microbiology 20180830

Bacterial outer membrane vesicles (OMVs), as well as OMV-associated small RNAs, have been demonstrated to play a role in host-pathogen interactions. The presence of larger RNA transcripts in OMVs has been less studied and their potential role in host-pathogen interactions remains largely unknown. Here we analyze RNA from OMVs secreted by Salmonella enterica serovar Typhimurium (S. Typhimurium) cultured under different conditions, which mimic host-pathogen interactions. S. Ty ...[more]

PMID: 30214435

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Project description:Enteric pathogens cycle between nutrient-rich host and nutrient-poor external environment. These pathogens compete for nutrients while cycling between host and external environment, and often experience starvation. In this context, we have studied the role of a global regulator (NtrC) of Salmonella Typhimurium. The ntrC knockout mutation caused extended lag phase (8 h) and slow growth in the minimal medium. In lag phase, the wild-type cells showed ~60-fold more expression of ntrC gene. Gene expression studies and biochemical assays showed that the extended lag phase and slow growth is due to slow metabolism, instead of nitrogen transport. Further, we observed that ntrC knockout mutation led extended lag phase and slow growth, made ΔntrC mutant unable to compete with wild-type S. Typhimurium in both static and fluctuating nutrient condition. In addition to this, ΔntrC knockout mutant was unable to survive long-term nitrogen starvation (150 days). The nutrient recycling assays and gene expression studies revealed that ntrC gene is essential for rapid recycling of nutrients from the dead cells. Moreover, in the absence of ntrC gene, magnesium limits the nutrient recycling efficiency of S. Typhimurium. Therefore, the ntrC gene, which is often studied with respect to nitrogen scavenging in a low nitrogen growing condition, is required even in the adequate supply of nitrogen to maintain optimal growth and fast exit from the lag phase. Hence, we conclude that, the ntrC expression is essential for competitive fitness of S. Typhimurium under the low and fluctuating nutrient condition. IMPORTANCE S. Typhimurium, both in host and external environment, faces enormous competition from other microorganisms. The competition may take place either in static or in fluctuating nutrient conditions. Thus, how S. Typhimurium survives under such overlapping stress conditions remained unclear. Therefore, using S. Typhimurium as model organism we report that a global regulator NtrC, found in enteric bacteria like Escherichia coli and Salmonella, activates the set of genes and operons involved in rapid adaptation and efficient nutrient recycling/scavenging. These properties enable cells to compete with other microbes under the characteristic feast-or-famine lifestyle of S. Typhimurium. Therefore, this work helps us to understand the starvation physiology of the enteric bacterial pathogen S. Typhimurium.

Project description:Typhoid caused by Salmonella enterica serovar Typhi remains a major health concern worldwide. The emergence of multidrug-resistant strains of Salmonella with increased virulence, communicability, and survivability leading to increased morbidity and mortality has further complicated its management. Currently available vaccines for typhoid have less-than-desired efficacy and certain unacceptable side effects, making it pertinent to search for new immunogens suitable for vaccine formulation. The outer membrane proteins (OMPs) of Salmonella have been considered possible candidates for conferring protection against typhoid. OMPs interface the cell with the environment, thus representing important virulence factors with a significant role in the pathobiology of gram-negative bacteria and bacterial adaptation. An OMP of Salmonella enterica serovar Typhimurium with an apparent molecular mass of 49 kDa that is highly immunogenic, evokes humoral and cell-mediated immune responses, and confers 100% protection to immunized rats against challenge with very high doses (up to 100 times the 50% lethal dose) of Salmonella enterica serovar Typhimurium has been identified. Further, very efficient clearance of bacteria from the reticuloendothelial systems of immunized animals was seen. This protein is recognized by the antibodies present in serum of typhoid patients. When sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel-eluted protein was further analyzed by high-performance liquid chromatography (HPLC) and two-dimensional electrophoresis, two polypeptides with the same molecular weight were resolved. These have different isoelectric points and gave two peaks with different retention times in reverse-phase HPLC. However, only one of the two bands interacted with patient serum. The immunogenicity studies (enzyme-linked immunosorbent assay and delayed-type hypersensitivity [DTH]) indicated that the immunoreactive protein evoked a strong immune response in rats. The N-terminal sequencing and analysis of the homology of this protein with sequences in the protein database of Salmonella resulted in a match with the N-terminal sequences of a protein in Salmonella enterica serovar Typhi (CT18 and Ty2 strains). The homology search further revealed it to be a hypothetical protein, whose gene had unidentified open reading frames in Salmonella serovar Typhi encoding 447 amino acid residues, corresponding to a molecular mass of 49 kDa. The nucleotide sequence of the encoding gene was deduced, and the gene was amplified by PCR using appropriate primers. An amplified 1.3-kb band was purified and sequenced to confirm its identity. These OMPs provide promising targets for the development of a candidate vaccine against typhoid.

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The RNA Complement of Outer Membrane Vesicles From Salmonella enterica Serovar Typhimurium Under Distinct Culture Conditions.

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The RNA Complement of Outer Membrane Vesicles From <i>Salmonella enterica</i> Serovar Typhimurium Under Distinct Culture Conditions.

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