Niche-specific profiling reveals transcriptional adaptations required for the cytosolic lifestyle of <i>Salmonella enterica</i>

AbstractSalmonella enterica serovar Typhimurium (S. Typhimurium) is a zoonotic pathogen that causes diarrheal disease in humans and animals. During salmonellosis, S. Typhimurium colonizes epithelial cells lining the gastrointestinal tract. S. Typhimurium has an unusual lifestyle in epithelial cells that begins within an endocytic-derived Salmonella-containing vacuole (SCV), followed by escape into the cytosol, epithelial cell lysis and bacterial release. The cytosol is a more permissive environment than the SCV and supports rapid bacterial growth. The physicochemical conditions encountered by S. Typhimurium within the cytosol, and the bacterial genes required for cytosolic colonization, remain unknown. Here we have exploited the parallel colonization strategies of S. Typhimurium in epithelial cells to decipher the two niche-specific bacterial virulence programs. By combining a population-based RNA-seq approach with single-cell microscopic analysis, we identified bacterial genes/sRNAs with cytosol-specific or vacuole-specific expression signatures. Using these genes/sRNAs as environmental biosensors, we defined that Salmonella is exposed to iron and manganese deprivation and oxidative stress in the cytosol and zinc and magnesium deprivation in the SCV. Furthermore, iron availability was critical for optimal S. Typhimurium replication in the cytosol, as well as entC, fepB, soxS and sitA-mntH. Virulence genes that are typically associated with extracellular bacteria, namely Salmonella pathogenicity island 1 (SPI1) and SPI4, had a cytosolic-specific expression profile. Our study reveals that the cytosolic and vacuolar S. Typhimurium virulence gene programs are unique to, and tailored for, residence within distinct intracellular compartments. Therefore, this archetypical vacuole-adapted pathogen requires extensive transcriptional reprogramming to successfully colonize the mammalian cytosol.Author SummaryIntracellular pathogens reside either within a membrane-bound vacuole or are free-living in the cytosol and their virulence programs are tailored towards survival within a particular intracellular compartment. Some bacterial pathogens (such as Salmonella enterica) can successfully colonize both intracellular niches, but how they do so is unclear. Here we have exploited the parallel intracellular lifestyles of S. enterica in epithelial cells to identify the niche-specific bacterial expression profiles and environmental cues encountered by S. enterica. We have also discovered bacterial genes that are required for colonization of the cytosol, but not the vacuole. Our results advance our understanding of pathogen-adaptation to alternative replication niches and highlight an emerging concept in the field of bacteria-host cell interactions.</jats:sec

Role of a single noncoding nucleotide in the evolution of an epidemic African clade of Salmonella

Salmonella enterica serovar Typhimurium ST313 is a relatively newly emerged sequence type that is causing a devastating epidemic of bloodstream infections across sub-Saharan Africa. Analysis of hundreds ofSalmonellagenomes has revealed that ST313 is closely related to the ST19 group ofSTyphimurium that cause gastroenteritis across the world. The core genomes of ST313 and ST19 vary by only ∼1,000 SNPs. We hypothesized that the phenotypic differences that distinguish AfricanSalmonellafrom ST19 are caused by certain SNPs that directly modulate the transcription of virulence genes. Here we identified 3,597 transcriptional start sites of the ST313 strain D23580, and searched for a gene-expression signature linked to pathogenesis ofSalmonellaWe identified a SNP in the promoter of thepgtEgene that caused high expression of the PgtE virulence factor in AfricanS.Typhimurium, increased the degradation of the factor B component of human complement, contributed to serum resistance, and modulated virulence in the chicken infection model. We propose that high levels of PgtE expression by AfricanSTyphimurium ST313 promote bacterial survival and dissemination during human infection. Our finding of a functional role for an extragenic SNP shows that approaches used to deduce the evolution of virulence in bacterial pathogens should include a focus on noncoding regions of the genome

EF-Tu and RNase E : Essential and Functionally Connected Proteins

The rate and accuracy of protein production is the main determinant of bacterial growth. Elongation Factor Tu (EF-Tu) provides the ribosome with aminoacylated tRNAs, and is central for its activity. In Salmonella enterica serovar Typhimurium, EF-Tu is encoded by the genes tufA and tufB. A bacterial cell depending on tufA499-encoded EF-Tu mutant Gln125Arg grows extremely slowly. We found evidence that this is caused by excessive degradation of mRNA, which is suggested to be the result of transcription-translation decoupling because the leading ribosome is ‘starved’ for amino acids and stalls on the nascent mRNA, which is thus exposed to Riboendonuclease RNase E. The slow-growth phenotype can be reversed by mutations in RNase E that reduce the activity of this enzyme. We found that the EF-Tu mutant has increased levels of ppGpp during exponential growth in rich medium. ppGpp is usually produced during starvation, and we propose that Salmonella, depending on mutant EF-Tu, incorrectly senses the resulting situation with ribosomes ‘starving’ for amino acids as a real starvation condition. Thus, RelA produces ppGpp which redirects gene expression from synthesis of ribosomes and favours synthesis of building blocks such as amino acids. When ppGpp levels are reduced, either by over-expression of SpoT or by inactivation of relA, growth of the mutant is improved. We suggest this is because the cell stays in a fast-growth mode. RNase E mutants with a conditionally lethal temperature-sensitive (ts) phenotype were used to address the long-debated question of the essential role of RNase E. Suppressor mutations of the ts phenotype were selected and identified, both in RNase E as well as in extragenic loci. The internal mutations restore the wild-type RNase E function to various degrees, but no single defect was identified that alone could account for the ts phenotype. In contrast, identifying three different classes of extragenic suppressors lead us to suggest that the essential role of RNaseIE is to degrade mRNA. One possibility to explain the importance of this function is that in the absence of mRNA degradation by RNase E, the ribosomes become trapped on defective mRNAs, with detrimental consequences for continued cell growth

Reducing ppGpp Level Rescues an Extreme Growth Defect Caused by Mutant EF-Tu

Salmonella enterica grows extremely slowly when it depends on tufA499 (encoding the Gln125Arg mutant form of EF-Tu) to drive protein synthesis. We screened a plasmid library for multi-copy suppressors of the slow growth phenotype and identified spoT as a candidate. The spoT gene encodes a dual function enzyme with both ppGpp synthetase and hydrolase activities. When spoT was cloned behind an arabinose-inducible promoter the growth rate of the mutant strain increased in response to arabinose addition. We found that the slow-growing mutant strain had a relatively high basal level of ppGpp during exponential growth in rich medium. Overexpression of spoT significantly reduced this level of ppGpp suggesting that inappropriately high ppGpp levels might cause the slow growth rate associated with tufA499. We tested this hypothesis by inactivating relA (codes for RelA, a ribosome-associated ppGpp synthetase) in the mutant strain. This inactivation decreased the level of ppGpp in the mutant strain and increased its growth rate. Based on these data we propose that ribosomes depending on tufA499 for their supply of ternary complex (EF-Tu•GTP•aa-tRNA) experience amino acid starvation and that RelA on these starving ribosomes produces an excess of the alarmone ppGpp. This results in a suboptimal partitioning of transcription activity between genes important for fast growth in rich medium and genes important for growth in a poor medium. Accordingly, mutant bacteria growing in a rich medium act physiologically as though they were growing in a nutrient-poor environment. We propose that this generates a vicious circle and contributes to the extreme slow-growth phenotype associated with mutant EF-Tu. Reducing the level of ppGpp increases the growth rate of the mutant because it breaks this circle and reduces the wasteful misdirection of resources in the cell.Jessica M. Bergman and Disa L. Hammarlöf contributed equally to this work.</p

Overexpression of <i>spoT</i> increases mutant growth rate and growth yield.

<p>(A) Growth as a function of overexpression of <i>spoT</i>. TH7507 (<i>tufA+</i>), TH7509 (<i>tufA499</i>), and TH7964 (<i>tufA499</i>/pBAD-<i>spoT</i>) grown with or without added arabinose (0.2%) in LB. Growth curves are from a single, representative experiment. (B) TH7964 (<i>tufA499</i>/pBAD-<i>spoT</i>) grown on LA plates for 16 h at 37°C. Left panel: no arabinose added; Right panel: 0.2% arabinose added to induce expression.</p

Inactivation of <i>relA</i> reduces ppGpp level and increases mutant growth rate.

<p>(A) Reduction in ppGpp levels associated with inactivation of <i>relA</i>. Thin layer chromatography of guanine nucleotides isolated from strains with the <i>tufA499</i> allele. Lane 1: TH7509 (<i>tufA499</i>). Lane 2: TH7975 (<i>tufA499</i>, <i>relA21</i>::Tn<i>10</i>). The positions of ppGpp, pppGpp and GTP are indicated. (B) Growth curves of TH7507 (<i>tufA+</i>), TH7975 (<i>tufA499 relA</i>::Tn<i>10</i>), TH7964 (<i>tufA499</i>/pBAD-<i>spoT</i>) grown with 0.2% arabinose to cause overexpression of <i>spoT</i> or 0% arabinose as a control, and TH7509 (<i>tufA499</i>), all grown in LB (Bioscreen). Growth curves are from a single, representative, experiment. (C) Strains grown on an LA plate for 18 h at 37°C. Left panel: TH7509 (<i>tufA499</i>); Right panel: TH7975 (<i>tufA499 relA21</i>::Tn<i>10</i>).</p

Inactivating <i>relA</i> does not alter step-time of the <i>tufA499</i> mutant.

a<p>All strains carried the F-factor <i>F′128 pro</i>⁺<i>lac</i>⁺<i>zzf-1831</i>::Tn<i>10d-spc</i>.</p>b<p><b>Step time ± standard deviation (sec).</b></p>c<p>p-values calculated by unpaired t-tests, comparing the step-time of the mutant strains to the TH7480 <i>tufA</i> wild-type.</p>d<p>p-values calculated by unpaired t-tests, comparing <i>relA21</i>::Tn<i>10</i> strains to the corresponding <i>relA</i>+ strain.</p

The <i>tufA499</i> mutation is associated with a reduced expression of four tRNA aminoacyl synthetases.

<p>Expression levels of the synthetase genes <i>thrS</i>, <i>cysS</i>, <i>valS</i> and <i>proS</i> were measured by quantitative real-time PCR in wild-type TH7507 (<i>tufA</i>+) and TH7509 (<i>tufA499</i>). Values are averages of six independent replicates and normalized to the wild-type levels. Standard deviations represented as error bars. The differences between the mRNA levels in the wild-type and the <i>tufA499</i> strains are statistically significant according to an unpaired t-test, thrS: p = 0.0005, cysS: p = 0.0105, valS: p = 0.0225, proS: p = 0.0169.</p

The <i>tufA499</i> mutation is associated with a low expression of 16S rRNA, which can be compensated for by inactivation of <i>relA</i>.

a<p>Relative quantity of 16S rRNA expression compared to tmRNA expression.</p>b<p>Number of independent RNA preparations.</p>c<p>p-values calculated by unpaired t-tests, comparing strains to TH7507.</p

Reduction in ppGpp levels associated with induction of <i>spoT</i> overexpression.

<p>Thin layer chromatography of guanine nucleotides isolated from TH7964 (<i>tufA499</i>/pBAD-<i>spoT</i>) grown with different concentrations of arabinose as indicated. The positions of ppGpp, pppGpp and GTP are indicated.</p