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Intracellular Salmonella Paratyphi A is motile and differs in the expression of flagella-chemotaxis, SPI-1 and carbon utilization pathways in comparison to intracellular S. Typhimurium

['Helit Cohen', 'The Infectious Diseases Research Laboratory', 'Sheba Medical Center', 'Tel-Hashomer', 'Claire Hoede', 'Université Fédérale De Toulouse', 'Inrae', 'Bioinfomics', 'Ur Miat', 'Genotoul Bioinformatics Facility']

Date: 2022-05

Although Salmonella Typhimurium (STM) and Salmonella Paratyphi A (SPA) belong to the same phylogenetic species, share large portions of their genome and express many common virulence factors, they differ vastly in their host specificity, the immune response they elicit, and the clinical manifestations they cause. In this work, we compared their intracellular transcriptomic architecture and cellular phenotypes during human epithelial cell infection. While transcription induction of many metal transport systems, purines, biotin, PhoPQ and SPI-2 regulons was similar in both intracellular SPA and STM, we identified 234 differentially expressed genes that showed distinct expression patterns in intracellular SPA vs. STM. Surprisingly, clear expression differences were found in SPI-1, motility and chemotaxis, and carbon (mainly citrate, galactonate and ethanolamine) utilization pathways, indicating that these pathways are regulated differently during their intracellular phase. Concurring, on the cellular level, we show that while the majority of STM are non-motile and reside within Salmonella-Containing Vacuoles (SCV), a significant proportion of intracellular SPA cells are motile and compartmentalized in the cytosol. Moreover, we found that the elevated expression of SPI-1 and motility genes by intracellular SPA results in increased invasiveness of SPA, following exit from host cells. These findings demonstrate unexpected flagellum-dependent intracellular motility of a typhoidal Salmonella serovar and intriguing differences in intracellular localization between typhoidal and non-typhoidal salmonellae. We propose that these differences facilitate new cycles of host cell infection by SPA and may contribute to the ability of SPA to disseminate beyond the intestinal lamina propria of the human host during enteric fever.

Salmonella enterica is a ubiquitous, facultative intracellular animal and human pathogen. Although non-typhoidal Salmonella (NTS) and typhoidal Salmonella serovars belong to the same phylogenetic species and share many virulence factors, the disease they cause in humans is very different. While the underlying mechanisms for these differences are not fully understood, one possible reason expected to contribute to their different pathogenicity is a distinct expression pattern of genes involved in host-pathogen interactions. Here, we compared the global gene expression and intracellular phenotypes, during human epithelial cell infection of S. Paratyphi A (SPA) and S. Typhimurium (STM), as prototypical serovars of typhoidal and NTS, respectively. Interestingly, we identified different expression patterns in key virulence and metabolic pathways, cytosolic motility and increased reinvasion of SPA, following exit from infected cells. We hypothesize that these differences contribute to the invasive and systemic disease developed following SPA infection in humans.

Funding: This work was supported by the Infect-Era program, SalHostTrop project (“Understanding the Human-Restricted Host Tropism of Typhoidal Salmonella”, 2016-2020) and by the France Génomique National infrastructure, funded as part of “Investissement d’avenir” program managed by Agence Nationale pour la Recherche (ANR-10-INBS-09 contract). The work at the Gal-Mor laboratory was supported by grant numbers: 2616/18 from the joint ISF-Broad Institute program; 3-12435 from Infect-Era /Chief Scientist Ministry of Health; I-41-416.6-2018 from the German-Israeli Foundation for Scientific Research and Development (GIF, awarded to OGM and MH); and A128055 from the Research Cooperation Lower Saxony – Israel (The Volkswagen Foundation, awarded to OGM, MH and GG). The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

Data Availability: The raw reads data for this study have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB46495 and as specified in S7 Table .

Copyright: © 2022 Cohen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Here, to further understand the different pathogenicity of SPA vs. STM as prototypic NTS, we set out to compare their transcriptional landscape during non-phagocytic host cell infection. Intriguingly, we show that in contrast to STM, intracellular SPA is motile, largely resides in the host cell cytosol and induces the expression of SPI-1 and flagellar genes. In addition, we demonstrate that SPA and STM diverge in the intracellular expression of genes for citrate and ethanolamine metabolism and in their ability to utilize these carbon sources in vitro. We hypothesize that these differences contribute to the distinct diseases resulting from SPA vs. STM infection, and their different interactions with the human host.

Typhoid fever disease and host-response to S. Typhi infection is largely attributed to the function of the Vi polysaccharide capsule and its associated regulator, TviA encoded on SPI-7 [ 17 – 20 ]. Nonetheless, since SPA does not harbor the SPI-7, nor expresses the Vi capsule, different mechanisms such as long O antigen chains in the LPS of SPA were suggested to play a role in evasion from the host-immune response and the clinically indistinguishable enteric fever disease caused by SPA and S. Typhi [ 21 , 22 ]. Previously, we showed that in Salmonella culture grown in LB to the late logarithmic phase, SPI-1 genes and T3SS-1 effectors are expressed and secreted at significantly lower levels by SPA compared to S. Typhimurium (STM) [ 23 ], and demonstrated differences in the regulatory setup of the flagella-chemotaxis pathway between these serovars [ 24 , 25 ].

Within host cells, Salmonella are first compartmentalized into a modified intracellular phagosome, known as the Salmonella containing vacuole (SCV). Inside the SCV Salmonella manipulates the phagosome-lysosome membrane fusion and other cellular pathways, and starts to replicate intracellularly [ 13 ]. These activities require a second T3SS encoded by genes on SPI-2 and the translocation of a distinct set of effectors proteins [ 15 ]. Later, a subpopulation of this pathogen can escape into the cytosol where environmental conditions are more prone to bacterial growth. This step may lead to hyper-replication, epithelial cell lysis and bacterial release [ 16 ].

Active invasion into eukaryotic non-phagocytic cells is one of the key virulence-associated phenotypes of all S. enterica serovars. This unique capability facilitates Salmonella intestinal epithelium crossing of the small intestine [ 10 ] and is mediated by a designated type three secretion system (T3SS) encoded within the Salmonella Pathogenicity Island (SPI)-1. This sophisticated syringe-like nanosystem is evolutionary related to the flagellar apparatus [ 11 ] and is used to translocate an array of effector proteins directly into the host cell cytoplasm. These effectors trigger cytoskeletal rearrangements and Salmonella penetration of intestinal barriers [ 12 ]. In addition, T3SS-1 and its associated effectors significantly contribute to intestinal inflammation [ 13 ] that helps Salmonella to compete with the gut microbiota [ 14 ].

S. enterica infections are still considered a significant cause of mortality and morbidity with an annual incidence of over 27 million cases of enteric fever [ 5 ], and 78.7 million cases of gastroenteritis [ 6 ] worldwide. In recent years, the global prevalence of S. Paratyphi A (SPA) is increasing and in some countries (especially in eastern and southern Asia), SPA infections are accountable for up to 50% of all enteric fever cases [ 7 , 8 ]. The lack of a commercial SPA vaccine and the increased occurrence of antibiotic resistant strains illuminate SPA as a significant public health concern that is still an understudied pathogen [ 9 ].

Salmonella enterica (S. enterica) is an abundant Gram-negative, facultative intracellular animal and human pathogen. This highly diverse bacterial species contains more than 2600 antigenically distinct serovars (biotypes) that are different in their host specificity and the disease they cause. The developed clinical manifestation is the result of multiple factors, but largely depends on the characteristics of the infecting serovar and the immunological status of the host [ 1 ]. Many of the S. enterica serovars, including the ubiquitous S. enterica serovar Typhimurium (S. Typhimurium) are generalist pathogens and are capable of infecting a broad range of host species. Infection of immunocompetent humans by non-typhoidal Salmonellae (NTS) typically leads to a self-limiting, acute inflammatory gastroenteritis, confined to the terminal ileum and colon. In contrast, a few serovars including Typhi, Paratyphi A and Sendai, collectively referred to as ‘typhoidal salmonellae’ are restricted to the human host and known as the causative agents of enteric (typhoid) fever. In most cases, enteric fever is a non-inflammatory, systemic life-threatening disease, presented as bacteremia and dissemination of the pathogen to systemic sites such as the spleen, liver and lymph nodes [ 2 – 4 ].

Results

Intracellular and extracellular STM and SPA exhibit distinct gene expression profiles Although STM and SPA belong to the same species, share about 89% of their genes and express many common virulence factors [26], the disease they cause in immunocompetent humans is very different. One possible reason, expected to contribute to their different pathogenicity is a distinct expression of genes involved in host-pathogen interactions. To test this hypothesis, we compared the transcriptomic architecture and gene activity of SPA or STM in a relevant context of epithelial cell infection. We chose to sample Salmonella infection at 8 h post infection (p.i.) when the pathogen is considered to be well engaged in replication and optimally adapted to the intracellular environment [27]. Thus, HeLa cells were infected with fluorescent STM and SPA for 8 h and sorted by flow cytometry to isolate Salmonella-infected cells. Overall, RNA was extracted from six independent HeLa cell cultures infected with SPA (N = 3) and STM (N = 3). In each experiment, 2–2.5×106 GFP-positive HeLa cells, (infected with either STM or SPA) were collected. In parallel, RNA was extracted from six independent extracellular STM (N = 3) and SPA (N = 3) cultures that were grown in LB to stationary phase microaerobically, prior to cells infection. Isolated RNA from these 12 samples was deep-sequenced (RNA-Seq), aligned to the corresponding Salmonella genome and compared between all conditions. Principal Component Analysis (PCA) of normalized gene read counts of these RNA-Seq data indicated a unique expression profile for each condition and good reproducibility between independent biological triplicates (Fig 1A). PPT PowerPoint slide

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TIFF original image Download: Fig 1. The number of STM and SPA genes expressed in LB medium and during intracellular growth. (A) Principal component analysis (PCA) of RNA-Seq data. The PCA was performed by using factomineR package on the normalized expression of 3851 shared genes of three independent SPA and STM cultures grown in LB medium (M1 to M3) and of three independent HeLa cell cultures (C1 to C3) infected with STM and SPA (as described in Materials and Methods). The percentages on each axis represent the percentages of variation explained by the principal components. Points that are closer together are more similar in gene expression patterns, and the barycenter of the triplicates used for each condition is shown by a larger symbol. (B and C) Salmonella RNA-Seq reads were aligned to S. Typhimurium 14028S and S. Paratyphi A 45157 genomes and normalized by transcripts per million (TPM) transformation. Genes with TPM ≥ 10 were considered as expressed. The number of expressed genes in STM (B) and SPA (C) growing in LB medium to stationary phase under microaerobic conditions and within HeLa cells at 8 h p.i. is shown. https://doi.org/10.1371/journal.ppat.1010425.g001

The intracellular transcriptomic landscape of S. Typhimurium RNA extracted from three independent Fluorescence-Activated Cell Sorting (FACS) HeLa cell cultures infected with STM have generated 496–575 million RNA-Seq reads (human and bacterial reads) per experiment. Of which, 4.9–6.6 million sequence reads, assigned to non-rRNA bacterial genes were used for intracellular bacterial transcriptome analysis. The three extracellular STM cultures grown in LB to stationary phase under microaerobic conditions, have generated 4.3–13.8 million RNA-Seq informative reads that were assigned to non-rRNA bacterial genes. To analyze the STM gene expression, we calculated for each gene the number of transcripts per kilobase million (TPM) from its feature read counts. The expression level threshold was set to a TPM value of 10 or above [28]. Altogether, we identified 1,018 distinct genes that were exclusively expressed by STM during intracellular infection of HeLa cells and 191 distinct genes that were expressed only when STM was grown in LB. 3,127 transcripts were commonly expressed at both conditions (Fig 1B and S1 Table). RNA-Seq successfully identified 365 upregulated and 465 downregulated genes that were changed by at least twofold inside epithelial cells at 8 h p.i. (adjusted p-value ≤ 0.05) by STM, relative to their expression in LB (Fig 2A and S2 Table). In agreement with previous studies [14,29,30], during STM infection, at least 27 genes belonging to the flagella-motility regulon and several genes from the type I fimbriae (fimAWYZ) were significantly repressed. Interestingly, the expression of genes encoding the Saf fimbriae (safABCD) was upregulated during HeLa cells infection by STM. PPT PowerPoint slide

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TIFF original image Download: Fig 2. DEGs of intracellular S. Typhimurium compared to growth in LB medium. (A) a Volcano plot showing the fold change (log2 ratio) in the expression of STM genes grown intracellularly in HeLa cells (8 h p.i.) vs. the growth in LB medium (X-axis) plotted against the -log 10 adjusted p-value (Y-axis). Each dot on the plot represents the mean value (from three independent cultures) of one gene. Genes that were changed by more than twofold are colored in red. (B) Heatmap based on RNA-Seq results showing the relative transcription of STM genes of interest during intracellular infection (C1 to C3) vs. their extracellular expression in LB medium (M1 to M3). https://doi.org/10.1371/journal.ppat.1010425.g002 In contrast to the flagella and the fim genes, at least 31 genes from the PhoPQ regulon were highly induced intracellularly (Fig 2B). The two-component system PhoPQ, orchestrates Salmonella adaptation to the intracellular milieu and regulates a wide array of genes central for Salmonella virulence including those encoded on SPI-2 [31,32]. Correspondingly, we identified upregulation of the SPI-2 regulon including ssaBCDEGHIJKLMVNOPRSTU, sseABCDEFGIJL, sseK3, sspH2, pipB, pipB2, and steC genes (Fig 2B). These results are consistent with the known notion that SPI-2 expression is induced during intracellular replication of Salmonella [15,33] and with previous reports that studied STM expression during macrophages infection [34,35]. Other PhoP-regulated genes including pgtE (involved in bacterial resistance to antimicrobial peptides), pagN (encoding an adhesion/invasion protein), phoN (acid phosphatase) and pagCDJKO were also induced (Fig 2B). Iron is an essential micronutrient required by nearly all bacterial species, including Salmonella. Limiting the availability of the nutrient metals to intracellular pathogens is one of the main mechanisms of nutritional immunity. In return, Salmonella uses a variety of high-affinity iron uptake systems to compete with the host for essential transition metals required for its intracellular growth [36]. Congruently, we identified elevated expression of multiple iron (entABEFH and iroNBCDE), iron/ manganese (sitABCD), zinc (zntR, zitT and znuA), and magnesium (mgtAB) acquisition system genes, highlighting the essentiality of these metal ions for STM intracellular replication (Fig 2B). More than 100 suitable carbon substrates as well as various nitrogen, phosphorus and sulfur sources are available to invading pathogens, at different niches of the vertebrate hosts [37,38]. Interestingly, multiple genes belonging to the ethanolamine operon (eutQTMGAC) and genes involved in glycerol (glpABDK), and maltose (malEKM) metabolism were particularly induced during STM infection (Fig 2B). Nevertheless, the most highly induced metabolic gene was uhpT, encoding a hexose phosphate transporter that was upregulated by more than 40-fold at 8 h p.i. These results suggest that sugar phosphates are major substrates required for cytosolic intracellular proliferation of STM within HeLa cells, but also that ethanolamine, glycerol and maltose could be utilized as alternative carbon sources in epithelial cells. Similarly, biotin (bioABCDF), purine (nrdEFHI and purDEHKLT), and sulfate/thiosulfate (cysACHITW and sufABCDES) biosynthetic metabolic pathways were highly transcribed intracellularly by STM. Moreover, genes involved in biosynthesis of the amino acids histidine (hisACDFH and hutG), tryptophan (trpABDE), and glutamine (glnAGKL), and genes that catalyze the cleavage of N-acetylneuraminic acid (sialic acid; Neu5Ac) to form pyruvate and N-acetylmannosamine (ManNAc) (nanAEKT and nagB) were also upregulated by STM during HeLa cell infections (Fig 2B). A large number of genes involved in phosphate metabolism were identified to be induced within HeLa cells. For example, the transcriptional levels of pstABCS (encoding an ATP-dependent phosphate uptake system), which is responsible for inorganic phosphate uptake during phosphate starvation, ugpABCEQ, phoR and apeE were markedly increased. The ugp operon is involved in hydrolysis of diesters during their transport at the cytoplasmic side of the inner membrane, generating sn-glycerol-3-phosphate (G3P) which is used as a phosphate source [39]. The upg operon is regulated by a two-component system, PhoBR, which also regulates the outer membrane esterase, ApeE [40]. Additional genes belonging to the PhoB regulon, such as phnSVWU encoding the phosphonatase pathway, which is responsible for breaking down phosphonate and yielding cellular phosphate [41] were also significantly induced. Collectively, these results suggest that STM likely experiences iron, biotin, purine and phosphate deprivation in host epithelial cells and induces the expression of genes involved in their transport and utilization. In addition, these results imply that ethanolamine, glycerol and maltose are available sources of carbon for STM in epithelial cells.

The intracellular transcriptomic landscape of S. Paratyphi A RNA-Seq analysis was similarly applied for intracellular SPA. 313.8 to 805.9 million RNA-Seq reads were obtained from three independent FACS-sorted HeLa cell cultures infected with SPA. Out of these, 14.6 to 36.6 million sequence reads were assigned to non-rRNA SPA genes and used to determine the intracellular SPA transcriptome (S3 Table). As a reference control, RNA extracted from extracellular SPA cultures grown in LB, generated 7.3 to 23 million informative reads (assigned to non-rRNA SPA genes). Overall, we identified 322 genes that were exclusively expressed (TPM ≥10) by SPA during infection of HeLa cells compared to 576 genes that were expressed only by extracellular SPA in LB culture. 2,995 SPA genes were expressed at both conditions (Fig 1C and S3 Table). At 8 h p.i. of HeLa cells, 322 SPA genes were upregulated and 579 genes were down-regulated (Fig 3A and S4 Table) by at least twofold (adjusted p-value ≤ 0.05). Like in intracellular STM, the genes for type I (Fim) fimbriae (fimCIWYZ) and additional fimbria clusters including Bcf (bcfDE), the Curli (csgBCDE), Stb (stbBE), Stc (stcABC), Stf (stfAD), Sth (sthABCD), and Stk (stkABC) were all strongly downregulated intracellularly (Fig 3B). Noteworthy, the downregulation of type I (fimACIZ), Curli (csgBDE) and Bcf (STM14_0037 and bcfB) fimbriae was more pronounced in SPA than in STM (S1 Fig), indicating that the SPA fimbriome is robustly repressed during intracellular replication, while some basal level of expression of these fimbrial genes is still maintained during STM infection. PPT PowerPoint slide

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TIFF original image Download: Fig 3. DEGs of intracellular S. Paratyphi A compared to growth in LB medium. (A) a volcano plot showing the fold change (log2 ratio) in the expression of SPA genes grown intracellularly in HeLa cells (8 h p.i.) vs. the growth in LB medium (X-axis) plotted against the -log 10 adjusted p-value (Y-axis). Each dot on the plot represents the mean value (from three independent cultures) of one gene. Genes that were changed by more than twofold are colored in red. (B) Heatmap based on RNA-Seq results showing the relative transcription of SPA genes of interest during intracellular infection (C1 to C3) vs. their extracellular expression in LB medium (M1 to M3). https://doi.org/10.1371/journal.ppat.1010425.g003 In contrast, but consistent with our findings for intracellular STM, genes belonging to the PhoPQ (including polymyxins resistance) and SPI-2 regulons were significantly induced by intracellular SPA. Moreover, various transition metals import systems including manganese and iron transporter (sitABCD), enterobactin biosynthesis system (entABCDEF and fepAG) and the iron ABC transporter IroC were considerably upregulated during HeLa cell infection by SPA (Fig 3B). In addition, the expression of several metabolic pathways that were significantly elevated during STM infection were induced by intracellular SPA as well, including biotin (bioABCDF) and purine (purCDEHKLT) biosynthesis genes. Further overlap in the intracellular transcriptome of these pathogens includes several amino acid biosynthesis pathways such as glutamine (glnAGKLPQ), tryptophan (trpABCDE) and histidine (hisAFGHI) that were upregulated in both intracellular STM and SPA. Nevertheless, comparison of the intracellular transcriptome between STM and SPA, identified 234 differentially expressed genes (DEGs; S5 Table), indicating that some key pathways are distinctly expressed during the intracellular phase of STM vs. SPA. Such differences included expression of several amino acids biosynthesis genes, which was more pronounced in intracellular SPA than in intracellular STM. These include biosynthesis genes of glycine (glyASU), isoleucine (ilvCDEG), leucine (leuABCD), lysine (lysAC and sucBD), methionine (metCEFHKLR), serine (serAC), and threonine (thrBC). In contrast, the threonine degradation pathway (tdcABCDEG) was found to be repressed in intracellular SPA. These results may suggest increased protein synthesis in SPA vs. STM during epithelial cell infection. Additional pathways that were upregulated in SPA, but not in STM are the oxidative phosphorylation (nuoEFGHIJKLMN and atpCD), and peptidoglycan cell wall formation (murACDEG) pathway genes (Fig 3B). Remarkably, further differences between the intracellular transcriptome of SPA vs. STM included different expression profiles of the SPI-1, motility and chemotaxis, and carbon utilization regulons as detailed in the following sections.

Differences in carbon catabolism during intracellular infection of S. Paratyphi A and S. Typhimurium In order to survive, persist and replicate in host cells, intracellular pathogens must adapt their metabolic pathways to the specific physical conditions and available nutrients found in the intracellular environment. Carbon catabolism provides bacteria with energy by means of reducing equivalents, ATP and essential biosynthetic precursors. Different studies have suggested that hexose monosaccharides such as glucose and glucose-6P are the main source of carbon during Salmonella infection [42–46]. Comparison of the metabolic gene expression between intracellular STM and SPA revealed significant differences in the expression profile of several carbon catabolic pathways. For example, the gene uhpT encoding an antiporter for external hexose 6-phosphate and internal inorganic phosphate [47], presented about 90-fold higher expression by intracellular STM than SPA (Fig 4A). This different expression might be because the sensor-regulator system encoded by uhpABC is defective by pseudogene formation in SPA [48]. Similarly, the gene encoding for galactonate permeases (dgoT) was upregulated in intracellular STM, but not in SPA, suggesting that STM utilizes more hexose 6-phosphate and galactonate than SPA as a carbon source during intracellular growth. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Differences in carbon utilization gene expression between STM and SPA. (A) Heatmap of carbon catabolism gene expression by intracellular STM vs. intracellular SPA. Pseudogenes in the SPA genome are marked with an asterisk. (B) The fold change in the transcription of the glucose-6P (uhpT), citrate (citA and citX), and ethanolamine (eutP, eutQ and eutG) utilization pathways in intracellular STM relative to their expression in intracellular SPA was determined using qRT-PCR. RNA was extracted from FACS-sorted intracellular salmonellae 8 h p.i. The mean and the standard error of the mean (SEM) of 3–8 independent qRT-PCR reactions are shown. Student’s t-test was used to determine statistical significance. **, p < 0.01; ***, p < 0.001; ND, not detected. (C) Growth of STM and SPA in M9 in the presence (+) or absence (-) of casamino acids (AA) and ethanolamine (ETA), under anaerobic conditions. (D) Growth of STM and SPA in M9 supplemented with citrate as a sole carbon source. https://doi.org/10.1371/journal.ppat.1010425.g004 Similarly, RNA-Seq identified that at least seven genes involved in citrate metabolism including citCDEFTX and citC2 exhibited higher transcription levels in intracellular STM than SPA (Fig 4A). Independent qRT-PCR further confirmed that the intracellular expression of citA and citX encoding citrate-proton symporter and apo-citrate lyase phosphoribosyl-dephospho-CoA transferase, respectively is lower by about 3- and 10-fold in intracellular SPA vs. STM (Fig 4B). STM can utilize ethanolamine as a sole source of carbon, nitrogen, and energy in a cobalamin (vitamin B12)-dependent manner. Ethanolamine may be important to STM growth in the host, since it is derived from the membrane phospholipid phosphatidylethanolamine that is particularly prevalent in the gastrointestinal tract, in a process involving breakdown of the precursor molecule phosphatidylethanolamine by phosphodiesterases to glycerol and ethanolamine [49]. In STM, ethanolamine catabolism involves 17 genes organized in the eut operon, controlled by the transcriptional activator EutR [50]. At least six ethanolamine catabolism genes (eutSPQTGH) were expressed at higher levels by intracellular STM compared to SPA (Fig 4A). These differences were independently verified by qRT-PCR that showed significantly reduced expression of eutG, eutP and eutQ genes in intracellular SPA vs. STM (Fig 4B). Collectively, these observations suggest that STM is able to better utilize intracellular citrate, ethanolamine and galactonate as intracellular carbon sources than SPA, and that SPA may use an alternative carbon source such as amino acids during intracellular growth. To further test this hypothesis, we compared the growth of SPA and STM in a defined M9 medium supplemented with ethanolamine or citrate as a carbon source. Growth under anaerobic conditions in M9 medium supplemented with ethanolamine, without amino acids, did not allow bacterial growth of neither STM nor SPA. Adding casamino acids, without additional carbon source, resulted in a minimal and comparable growth of both STM and SPA, likely due to the utilization of amino acids as a restricted carbon source. Nevertheless, adding ethanolamine as a bona fide carbon source to the medium resulted in increased and rapid growth of STM, but only a minor improvement of SPA growth (relative to its growth on amino acids only; Fig 4C), indicating the ability of STM, but not SPA, to efficiently ferment ethanolamine. Replacing the carbon source with citrate under aerobic growth conditions allowed delayed, but substantial growth of STM, in comparison to restricted and slower growth of SPA (Fig 4D). These results are concurring with the transcriptomic data and indicate a better ability of STM than SPA to utilize both ethanolamine and citrate as a sole carbon source. These differences may also contribute to a higher intracellular replication pace of STM than SPA found in HeLa cells (S2 Fig).

Intracellular S. Paratyphi A expresses SPI-1 genes at higher levels than intracellular S. Typhimurium Previously we showed that in LB medium, under aerobic growth conditions to the late logarithmic phase, SPI-1 gene expression, as well as secretion of SPI-1-T3SS effector proteins, occur at significantly lower levels in SPA compared to STM [23]. Here, in sharp contrast, we found that intracellular SPA transcribed significantly higher levels of SPI-1 and T3SS-1-effector genes than intracellular STM (Fig 5A). This was clearly evident (log 2 change ≥ 2 and p-value ≤ 0.05) for at least 14 SPI-1 genes including the SPI-1 regulators hilA and hilD, the effector genes sopBDF, the T3SS-1 structural genes invCHIJ and spaOPQ, sigE encoding a chaperon (a.k.a. pipC) and iagB (S5 Table). PPT PowerPoint slide

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TIFF original image Download: Fig 5. Intracellular SPA expresses higher levels of SPI-1 genes than intracellular STM. (A) Heat map of RNA-Seq results showing the relative transcription of SPI-1 genes in three independent HeLa cell infections with STM and SPA. (B) The fold change in the transcription of six SPI-1 genes (hilA, hilD, invH, sopB, sopD and spaO) in intracellular SPA relative to their expression in intracellular STM was determined by qRT-PCR. RNA was extracted from FACS-sorted intracellular salmonellae 8 h p.i. The results show the mean of 3–6 qRT-PCR reactions and the error bars indicate the SEM. (C) SPA and STM strains expressing GFP and a 2HA-tagged version of the SPI-1 effectors SopE2 and SopB were used to infect HeLa cells. At 8 h p.i., cells were fixed and FACS-sorted for infected (GFP-positive cells). Equal number of infected HeLa cells (1×106 for each sample) were collected and separated by 12% SDS-PAGE. Western blotting using anti-hemagglutinin (HA) tag antibodies was used to detect the intracellular expression of SopE2 and SopB. Anti-DnaK antibodies were used as a loading control. https://doi.org/10.1371/journal.ppat.1010425.g005 Independent qRT-PCR was used to verify these observations and demonstrated that intracellular SPA transcribed hilA, hilD, invH, sopB, sopD and spaO at 4- to 27-fold higher levels than STM (Fig 5B). Moreover, Western blotting against a 2HA-tagged SopB and SopE2 further indicated elevated translation of these two SPI-1 effectors in intracellular SPA vs. STM (Fig 5C). Collectively, these results indicate that SPA expresses SPI-1 genes at higher levels during infection of non-phagocytic cells, in comparison to intracellular STM.

[END]

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