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. 2022 Feb 22;13(1):e0386921.
doi: 10.1128/mbio.03869-21. Epub 2022 Feb 22.

The Psychrotrophic Pseudomonas lundensis, a Non- aeruginosa Pseudomonad, Has a Type III Secretion System of the Ysc Family, Which Is Transcriptionally Active at 37°C

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The Psychrotrophic Pseudomonas lundensis, a Non- aeruginosa Pseudomonad, Has a Type III Secretion System of the Ysc Family, Which Is Transcriptionally Active at 37°C

Keerthikka Ravi et al. mBio. .

Abstract

The type III secretion system (T3SS) is a needle-like structure found in Gram-negative pathogens that directly delivers virulence factors like toxins and effector molecules into eukaryotic cells. The T3SS is classified into different families according to the type of effector and host. Of these, the Ysc family T3SS, found in Yersinia species and Pseudomonas aeruginosa, confers high virulence to bacteria against eukaryotic hosts. Here, we present the first identification and transcriptional analyses of a Ysc T3SS in a non-aeruginosa Pseudomonas species, Pseudomonas lundensis, an environmental psychrotrophic bacterium and important agent of frozen food spoilage. We have identified and sequenced isolates of P. lundensis from three very distinct ecological niches (Antarctic temporary meltwater pond, U.S. supermarket 1% pasteurized milk, and cystic fibrosis lungs) and compared these to previously reported food spoilage isolates in Europe. In this paper, we show that strains of P. lundensis isolated from these diverse environments with ambient temperatures ranging from below freezing to 37°C all possess a Ysc family T3SS secretion system and a T3S effector, ExoU. Using in vitro and in vivo transcriptomics, we show that the T3SS in P. lundensis is transcriptionally active, is expressed more highly at mammalian body temperature (37°C) than 4°C, and has even higher expression levels when colonizing a host environment (mouse intestine). Thus, this Ysc T3SS-expressing psychrotrophic Pseudomonad has an even greater range of growth niches than previously appreciated, including diseased human airways. IMPORTANCE P. lundensis strains have been isolated from environments that are distinct and diverse in both nutrient availability and environmental pressures (cold food spoilage, Antarctic melt ponds, cystic fibrosis lungs). As a species, this bacterium can grow in diverse niches that markedly vary in available nutrients and temperature, and in our study, we show that these various strains share greater than 99% sequence similarity. In addition, all isolates studied here encoded complete homologs of the Ysc family T3SS seen in P. aeruginosa. Until recently, P. aeruginosa has remained as the only Pseudomonas species to have a characterized functional Ysc (Psc) family T3SS. With the identification of a complete Ysc T3SS in P. lundensis that is expressed at 37°C in vivo, it is intriguing to wonder whether this bacterium may indeed have some level of symbiotic activity, of yet unknown type, when consumed by a mammalian host.

Keywords: 2T.2.5.2; AU1044; Antarctica; M101; M105; Pseudomonas fluorescens; milk; spoilage.

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Conflict of interest statement

The authors declare no conflict of interest.

We declare that we have no conflict of interest with the material presented in this literature review.

Figures

FIG 1
FIG 1
Ysc family T3SS in P. lundensis. (A) The T3SS of P. lundensis is most homologous to that of P. aeruginosa. Phylogenetic tree depicting the similarity between T3SS among Pseudomonas species discussed in this paper. The tree was built on the predicted amino acid sequences of the T3SS structural proteins PscS/Ysc, PscT/YscT, PscU/PscT, and their T3SS homologues in the other Pseudomonas species. Branch length is on the top side of branch and labeled in blue; confidence intervals based on 1,000 bootstraps of the tree are on the underside of the branch and are labeled in red. (B) pBLAST of T3SS protein from an environmental and clinical strain of P. lundensis, 2T.2.5.2 and AU1044, respectively. The BLAST analysis was done against the Psc family protein from P. aeruginosa strain PA14. (C) Gene maps of the type III secretion system from P. aeruginosa strain PA14 and P. lundensis strains DSM6252, AU104, AU11122, AU10414, M101, M105, MFPA15A1205, and 2T.2.5.2. DSM6252 is the type strain isolated from spoiled beef. AU1044, AU11122, and AU10414 are clinical isolates of P. lundensis from sputum samples of patients with cystic fibrosis. M101 and M105 are strains isolated from spoiled milk, MFPA15A1205 is a strain from a spoiled food source, and 2T.2.5.2 was isolated from an Antarctic temporary meltwater pond. In PA14, the exoU and spcU are not present immediately downstream to the core genes as seen in P. lundensis. The gene annotations seen for PA14 also apply to the P. lundensis strains.
FIG 2
FIG 2
Schematic representation of the structural components of the type III secretion system in P. lundensis. The component labels are color coded depending on the protein alignment with their homologs in P. aeruginosa PA14. Black indicates an alignment score >75%, blue indicates alignment score <75% but >50%, and red indicates <50% alignment score. The schematics are based on the current knowledge of the complex and also presumed localizations for components that have so far not been unambiguously localized. IM, inner membrane; OM, outer membrane. Created with BioRender.com.
FIG 3
FIG 3
Circular genome plot of AU1044 (A), 2T.2.5.2 (B), M101 (C), and M105 (D). Counting the tracks from outside to inside, tracks 1 and 2 show the coding genes in the positive and negative strands, respectively; track 3 shows the genomic islands (GI), predicted using Island4Viewer; track 4 shows predicted insertion sequence (IS) elements predicted made using ISEScan; and gene blocks in red in tracks 5 and 6 are the type III secretion system genes in the positive and negative strands, respectively. Track 7, generated using Alien_Hunter (https://www.sanger.ac.uk/tool/alien-hunter/), shows GI that are predicted to be acquired through horizontal gene transfer (HGT). Blue peaks correspond to the probability of an HGT phenomenon. Track 8 is the GC plot; purple peaks are a segment of genomes with GC% below average, and olive-green peaks are segments with GC% above average. The T3SS genes do not lie in any predicted region of HGT or genomic islands.
FIG 4
FIG 4
P. lundensis expresses its T3SS under both in vitro and in vivo conditions. (A) Expression profile of T3SS genes at 4°C and 37°C under in vitro growth conditions. The expression is shown in terms of FPKM reads that were normalized using DSEeq2. Here, we see an upregulation or increased FPKM levels of all T3SS genes at 37°C. Interestingly, some basal structure components, like PscU PscT, PscI, and PscL, have very small significant upregulation. Each line represents individual replicates/mice in either of the three conditions. (B) Scatterplot showing genes that have a significant change in expression at 37°C compared to 4°C. Open circles represent genes with a significant change in expression. Closed circles represent T3SS genes. (C) Levels of P. lundensis AU1044 in the intestinal tract of mice after oral gavage. Germfree BALB/c mice (n = 6) were given an oral gavage of 2.75 × 109 CFU of AU1044 at day 0 and analyzed at 2 weeks postgavage. (D) Scatterplot showing genes that have a significant change in expression in vivo in the cecum of germfree mice compared to aerobic in vitro growth at 37°C. Genes were identified as differentially expressed if their adjusted P value (Padj) was less than 0.05 at a log2 fold change of >|0| for the 37°C versus 4°C in vitro experiment and >|1| for the in vivo versus 37°C in vitro experiment.
FIG 5
FIG 5
Schematic representation showing the outline for the experiments set up for in vitro and in vivo RNA transcriptomics (A) and the bioinformatics pipeline used to analyze the data (B).

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