Derby is predominantly isolated from pigs and turkeys and S. Mbandaka is predominantly isolated from cattle and chickens. Alignment of the genome sequences of two isolates of each serovar led to the discovery of a new putative Salmonella pathogenicity island, SPI, in the chromosome sequence of S. Derby isolates. In this study we use porcine jejunum derived cell line IPEC-J2 and in vitro organ culture of porcine jejunum and colon, to characterise the association and invasion rates of S.
Derby and S. Mbandaka, and tissue tropism of S. Derby respectively. We show that S. Mbandaka, and that S. Derby preferentially attaches to porcine jejunum over colon explants. We also show that nine genes across SPI are up-regulated to a greater degree in the jejunum compared to the colon explants. Furthermore, we constructed a mutant of the highly up-regulated, pilV-like gene, potR, and find that it produces an excess of surface pili compared to the parent strain which form a strong agglutinating phenotype interfering with association and invasion of IPEC-J2 monolayers.
We suggest that potR may play a role in tissue tropism. 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.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Derby [8,9]. Type Salmonella enterica is an important zoonotic pathogen of warm III effector proteins are important pathogenicity factors secreted blooded vertebrates, including humans and livestock.
The through the type III secretion system in to a host cell where they symptoms of Salmonellosis include chronic gastroenteritis, affect- modulate cell signalling, in some instances pacifying the hosts ing a wide range of host species and caused primarily by broad immune system or aiding in invasion of, or translocation across, host range serovars, and an often fatal typhoid fever affecting a the intestinal epithelial barrier [10,11].
International serotyping has provided SPI and the higher number of isolations of S. Derby from pigs statistics that have elucidated links between certain serovars and a in the UK it was posited that this island may play a role in defined host species range [4—7]. In the current study we undertake In previous work the chromosome sequences of S.
Isolation that S. Derby associates to, and invades, a porcine derived statistics suggest that these serovars have different host species monolayer faster than S. Alignment of the chromosome sequences led to static culture. We discuss the possible role that potR and other SPI- the discovery of a new putative Salmonella pathogenicity island 23 genes may play in tissue tropism.
SPI in isolates of S. Derby, designated SPI [8]. Derby is the determinant of the bias towards a porcine host, we can suggest that S. Derby S. Derby associates and invades IPEC-J2 monolayers in possesses adaptations for aspects of pathogenesis of the porcine significantly greater numbers than S.
Mbandaka and host, that are absent in the comparator serovar, S. Of shows a preference to associating with jejunum over course, it would be interesting to undertake a broader study of colon explants populations of a variety of serovars, especially the promiscuous S. Derby is frequently isolated from pigs, while S. Mbandaka is serotypes such as S.
Typhimurium, to assess whether this finding rarely isolated from pigs in the UK [5—7]. To advance beyond the holds true across different serovars. Derby was more proficient invasion [18,19]. To assess if there was preferential attachment by in associating adhering and invading with, and invading, a S. Both S. Derby strains associated in IPEC-J2 monolayers have been used previously as a model for the significantly greater numbers with jejunum when compared to porcine jejunum, a site associated with the invasion of Salmonella colon p,0.
After 30 minutes there were 2. The rate of invasion and association was studied times as many S. Derby D1 cells associated with the jejunum than through varying lengths of incubation of S. For strain S. Derby D2 there were 1. This suggests that S. From 15 minutes onwards there were significantly more S. Derby preferentially attaches to the jejunum over the colon. No Derby associated to the monolayer than there were S. Mbandaka colonies were recovered from the PBS controls, suggesting that p,0.
Derby D1 and S. Mbandaka colonies observed in the IVOC association assays were not M2 at 15 minutes, which were not significantly different p. Figure 1. There was a significantly greater number p,0. Mbandaka at all-time points, Salmonella pathogenicity island 23 SPI is up- with the exception of S. Derby D2 and S. Mbandaka M2 at 30 regulated in the porcine jejunum IVOC preparations minutes, which were not significantly different p.
After 60 In light of the preferential attachment of S. Derby to porcine minutes there were approximately 2. Derby jejunum compared to colon and the need to characterise the newly cells associated with the monolayer and approximately 4 times as discovered SPI in relation to pathogenesis in the porcine host, many cells internalised than S.
Mbandaka cells. Control strain E. Due to the greater fold difference between the surface of the monolayer. Derby D1 cells associated with the jejunum and Though other studies have found poor concordance between colon tissues compared to that of S.
Derby D2, this strain was monolayer invasion rates, pathogenicity and host association, the selected to study the potential differences in the expression of use of monolayers here shows that S. Mbandaka and colon tissue explants. It was previously shown, with two strains of S. The gene potR and the putative type three effector protein Typhimurium, that differences in invasion rates observed in genes genE, sadZ, tinY and docB were up-regulated to a IPEC-J2 assays were comparable to those found in porcine significantly greater degree p,0.
Though we may not assert that the more to colon, with fold changes from the no tissue control of between Figure 1. Derby strains D1 and D2 and S. Mbandaka strains M1 and M2 after 15, 30 and 60 minute incubation periods. BLASTp showed the first in colon than in jejunum explants p,0.
The fold differences in amino acids of potR consist of a multi-domain region expression levels from no tissue controls of the putative pilin containing a shufflon domain aa, e-value 6. The largest significant fold type IV pilin methylation domain aa, e-value 3. These change was observed for the gene docB, a putative type III effector domains are consistent with other putative pilV genes, including protein, which was up-regulated The region between unique to SPI of S.
Derby, whereas the gene docB was shown amino acids and had no identifiable conserved domain. In to be highly conserved in SPI of S. Agona, S. Dublin and S. Typhi the PilV protein is located to the tip of the self- Gallinarum [8]; this raises the question about its expression and aggregating pilus, attaching to a larger PilS protein, causing the role in these host bacteria. Derby D1 and D2, has Allelic exchange mutagenesis with a Kanamycin cassette the second largest difference between tissues, with a 13 fold greater produced the strain S.
Derby D1 DpotR::kan, which displayed a expression level in the jejunum compared to colon [8]. The strong agglutinating phenotype when left statically at room smallest significant difference between fold change from the no temperature Figure 3a suggesting that potR is isofunctional to tissue control between the jejunum and colon treated cells was for pilV [20]. We were unable to remove the kanamycin cassette from the gene sanA, with a 3.
Derby D1 DpotR::kan and complement potR despite many colon samples than in jejunum. The up regulation of a subset of attempts at electroporation this is presumably due to the strong SPI genes in the jejunum compared to both the colon and no agglutination of a static culture. Derby D1 DpotR::kan showed clearly that the rather than colon tissue.
This Sequence features of potR and the phenotype of S. Derby suggests that either pili were upregulated or unable to dissociate D1 DpotR::kan from the cell surface.
Derby, mutant from the parent strain. Plating planktonic cultures of the high degree of up-regulation of the gene during exposure to parent and mutant strains onto LB agar plates resulted in the jejunal tissue explants and the preferential association of the isolate formation of fewer and larger colonies by the mutant strain to jejunal tissue explants, we characterised the phenotype of this Figure 3c.
Both parent and mutant colonies were of smooth gene further. Yet half of the diameter skirting the outer side of the As a preliminary step to determine the role of potR in S. Derby mutant colonies was translucent, this was absent from the parent we identified the conserved protein domains. Expression of SPI in S. Derby D1 when exposed to jejunum and colon explants. Comparison of morphological and structural features of the S. Derby D1 parental and potR mutant strains. Comparison of S. Derby D1 left and mutant strain S.
Derby D1 DpotR::kan right. Pili on the mutant and type-1 fimbriae on the parental strain are marked by arrows. Derby and the porcine jejunum, confocal through the formation of detachable, self-aggregating pili [21]. By using our site, you agree to our collection of information through the use of cookies. To learn more, view our Privacy Policy. To browse Academia. Log in with Facebook Log in with Google. Remember me on this computer. Enter the email address you signed up with and we'll email you a reset link.
Need an account? Click here to sign up. Download Free PDF. Neena Kalia. A short summary of this paper. Download Download PDF. Translate PDF. Kavanagh1, Adrian I. This has been partially attributed to limited local HSC recruitment following systemic injection. Identifying site specific adhesive mechanisms underpinning HSC-endothelial interactions may provide important information on how to enhance their recruitment and thus potentially improve therapeutic efficacy.
Endogenous leukocyte adhesion following HPC-7 injection was again determined intravitally. Conclusion: This data provides evidence that site-specific molecular mechanisms govern HPC-7 adhesion to injured tissue. Importantly, we show that HPC-7 adhesion is a modulatable event in IR injury and further demonstrate that adhesion instigated by injury alone is not sufficient for mediating anti-inflammatory effects.
Enhancing local HSC presence may therefore be essential to realising their clinical potential. 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.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
Emerging data has demonstrated that they too can directly quate with no long-term efficacy observed. Stem cell SC -based influence the progression of inflammation in injured tissues in a cellular therapies offer promising approaches for treating a wide paracrine manner [4]. Indeed, human HSCs are able to secrete variety of inflammatory disorders. Recent attention has focussed both pro- and anti-inflammatory growth factors and cytokines, on bone marrow BM -derived mesenchymal SCs MSCs such as transforming growth factor-b1 TGF-b1 , stem cell factor primarily due to their anti-inflammatory and immunomodulatory SCF , and tumour necrosis factor-a TNF-a [5].
Upon stimula- effects and low immunogenicity [1]. However, complications such tion with pro-inflammatory mediators, HSCs can release repar- as tumour formation, their potential to differentiate into unwanted ative factors, such as epithelial growth factor EGF , fibroblast mesenchymal lineages and pulmonary entrapment following growth factor FGF , platelet derived growth factor PDGF and systemic infusion of these large cells has led to recent caution vascular endothelial growth factor VEGF [6].
However, despite being urged when considering these cells for clinical use [2]. The rarity of HSCs ,0. If HSC therapy is to be realised, a better understanding of the basic science underlying Cells their recruitment to injured sites is essential.
Intravital studies monitoring HSC trafficking have been limited Our knowledge of the adhesive mechanisms mediating recruit- due to difficulties in isolating sufficient numbers for in vivo ment of transplanted HSCs by the injured intestinal microcircu- experimentation [13]. In this healthy BM [8]. HSCs exhibit a similar repertoire of surface study, we have utilized an immortalised HSC line, HPC-7, adhesion molecules to mature leukocytes, expressing CD29 b1 generated by transfecting murine embryonic SCs with the gene and CD18 b2 integrins which bind to their endothelial counter- LHx2 [15].
Recent work including high expression of common murine HSC markers as from our group demonstrated a critical role for the CD49d a4 well as being lineage negative [15]. In addition, integrin in HSC homing [11]. However, it is not known whether we have demonstrated previously that HPC-7 express adhesion CD49d is universally responsible for retaining HSCs in all injured molecules known to be present on primary HSCs and have vascular beds.
HPC-7 cells were obtained as a gift from Prof. Leif muscle was also monitored. A variety of chemical mediators are to use. For clustering studies, cells were labelled with 10 mM released from inflamed tissue, including cytokines, that can CellTracker Orange CTO; Invitrogen. There- Terminal ileal, jejunal or duodenal segments were isolated and fore, the roles of the inflammatory cytokines, CXCL12 stromal snap frozen from sham and IR injured animals 45 min ischaemia; cell-derived factor-1a , interleukin-1b IL-1b and TNF-a in min reperfusion and sectioned 10 mm.
Futher- HPC-7 were applied to each section for 20 minutes and then more, this study investigated whether pre-treating HSCs with washed with PBS to remove unbound cells. Sections were fixed in these cytokines prior to their systemic administration could acetone and adherent cells from 10 random fields were counted enhance their homing efficiency to the injured gut.
Given the microscopically and the mean obtained. To assess this, Dr. At microcirculation was also monitored in vivo. HPC-7 were HSC recruitment can inflammatory cell infiltration be reduced subsequently washed and endothelial monolayers exposed to within the injured microcirculation.
Materials and Methods Adherent cells from 5 random fields were counted using fluorescent microscopy and the mean obtained. Anaesthetized ani- PBS with 0. Cells were incubated with primary For analysis, one field of view x10; 1. Subsequently, cells were washed and resuspended reperfusion.
The intestines were prepared for imaging as described in 0. Leukocyte adhesion was counted at 60, , and twice again. Expression is represented as fold increase in mean fluorescence intensity Integrin clustering compared to IgG. Control animals underwent sham surgery. Cells were subsequently washed inverted intravital microscope Olympus BX; Olympus. At 30 and then incubated with secondary antibody Alexa conju- minutes reperfusion, CFSE labeled HPC-7 were adminis- gated goat anti-rat, Invitrogen at a dilution of for 30 tered intra-arterially.
To examine the role of cytokines in minutes on ice. Cells were washed, allowed to settle onto lysine mediating HSC recruitment, a segment of healthy small intestine, coated coverslips and mounted using Hydromount National in which the mucosa was exposed, was lowered into a custom-built Diagnostics, Hessle, UK.
Areas of Peprotech or PBS for 4 hours. Maxima plots were produced by istered intra-arterially and monitored intravitally. This is a novel identifying regions of maximal intensity within control determined approach to study the role of cytokines in mediating SC homing in tolerance level to separate clusters from background. This open and continuously superfused with bicarbonate buffered methodology is better suited to serial data as other statistical tests saline.
To induce ischemia, the cremasteric feeding arteriole was reduce statistical power by disregarding correlations between clamped for 30 minutes. Leukocyte adhesion following saline or cell femoral artery cannula post-reperfusion, with intravital observa- administration was analysed by t-test at 4 hours.
All remaining tions made at 90 minutes. Results mediating HPC-7 recruitment, the muscle was pre-treated with an were considered significant when p,0. In some mice, the muscle was treated topically with vehicle 1 ml or CXCL12 2 mg in 1 ml. HPC-7 adhesion in vitro and in vivo is increased on IR injured intestine. A HPC-7 adhesion in vitro was raised on frozen ileal sections isolated from IR injured animals when compared to sham controls. Results represent mean adhesion per mm2 tissue area6SEM n.
B HPC-7 adhesion in ileal mucosal microcirculation in vivo was also raised in animals subjected to IR injury solid line: sham; dashed line: IR injury. Representative images of the villous microcirculation of the ileum in F sham and G IR animals are shown. For frozen section work, data is expressed as mean adhesion per mm2 of tissue to control for area variation.
A Leukocyte infiltration, analysed by AcrO staining, did not reduce in animals receiving HPC-7 cells at 30 minutes post-reperfusion when compared to IR injured animals receiving a saline bolus ie.
Results are presented as mean cells per field6SEM. KSL cells expressed Significantly p,0. To examine Figure 1a. Similar results were observed in vivo with significantly whether this was due to loss or internalisation of CD18, flow p,0.
Numbers of free population Figure 3h. IR: 4. Additionally, ex vivo examination of jejunal Figure 1d , but not duodenal mucosa dependent on CD18 in vivo Figure 1e , revealed significantly p,0. However, adhesion was not IR injury was associated with increased numbers of adherent reduced by pre-treating cells with an anti-CD49d antibody AUC: leukocytes over the 4 hour reperfusion duration.
To determine IgG: Animals subsequently received either ml CD49d: 4. Jejunual HPC-7 adhesion, 0. However, no difference in leukocyte adhesion at any time point during the determined on frozen tissue assays, was also significantly reduced 4 hour reperfusion period was noted between saline or cell treated with an anti-CD18 antibody p,0.
In HPC-7 adhesion was significantly p,0. H CD18 and CD49d could be identified by flow cytometry following cell permeabilization.
This Topical exposure of non-injured ileal mucosa to TNF-a reduction was greatest with blocking of CD49d, with only a partial p,0. Enhanced significantly p,0. In contrast, CD18 or anti-CD49d. HPC-7 adhesion in vivo to intestinal microcirculation is dependent on CD B Pre-treatment with an anti-CD18 antibody did not significantly reduce free-flowing cells in the ileum solid line: IgG; dashed line: anti-CD49d.
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