Correspondence: Ivan Rychlik, Veterinary Research Institute, Hudcova 70, 621 00 Brno, Czech Republic. Tel.: +420 533 331 201; fax: +420 541 211 229; e-mail: firstname.lastname@example.org
In this study, we were interested in the association of attenuated mutants of Salmonella enterica serovar Enteritidis with subpopulations of porcine white blood cells (WBC). The mutants included those with inactivated aroA, phoP, rfaL, rfaG, rfaC and fliC genes and a mutant with five major pathogenicity islands removed (ΔSPI1-5 mutant). Using flow cytometry, we did not observe any difference in the interactions of the wild-type S. Enteritidis, aroA and phoP mutants with WBC. ΔSPI1-5 and fliC mutants had a minor defect in their association with granulocytes and monocytes, but not with T- or B-lymphocytes. All three rfa mutants associated with granulocytes, monocytes and B-lymphocytes more than the wild-type S. Enteritidis did. Electron microscopy confirmed that the association correlated with the intracellular presence of S. Enteritidis and that the Salmonella-containing vacuole in the WBC infected with the rfa mutants, unlike all other strains, did not develop into a spacious phagosome. Intact lipopolysaccharide, but not the type III secretion system encoded by SPI-1, SPI-2 or the flagellar operon, is important for the initial interaction of S. Enteritidis with porcine leukocytes. This information can be used for the design of live Salmonella vaccines preferentially targeting particular cell types including cancer or tumor cells.
Salmonella enterica is a facultative intracellular bacterial pathogen capable of infecting a wide range of mammals, birds and reptiles. Although there are quite remarkable differences in the course of infection depending on a combination of particular host and serovar of S. enterica, the infection always consists of oral ingestion, multiplication of S. enterica in the gut lumen, followed by the adhesion and invasion of nonprofessional phagocyte cells in the intestinal tract (M cells or gut epithelial cells). After translocation through the gut epithelium, S. enterica interacts with macrophages, which are believed to be responsible for S. enterica distribution across the host's body and into secondary sites of infection such as the liver or the spleen. However, there are many different cells present in the gut tissue, for example fibroblasts or neutrophils, which may also interact with S. enterica after its translocation across the gut epithelium. Moreover, S. enterica has been reported to temporarily exist extracellularly and, under such conditions, it can become exposed to additional cell types including the leukocytes infiltrating from the blood stream (Berndt et al., 2007; Pullinger et al., 2007). Despite this, the interaction of S. enterica with different cell types has been addressed only in a few studies. Geddes et al. (2007) showed that Salmonella enterica serovar Typhimurium preferentially interacted and associated with neutrophils and monocytes in Balb/C mice after intraperitoneal administration. Similarly, Cano et al. (2001) showed that S. Typhimurium may persist in fibroblasts and that the behavior of wild-type S. Typhimurium is quite different from the characteristics of the phoP mutant. Finally, we have recently shown that S. Enteritidis rfaL and rfaC mutants with modified lipopolysaccharide exhibit increased binding to porcine leukocytes in vitro (Matiasovic et al., 2011).
Animals and humans can be protected against infection with a particular serovar of S. enterica by vaccination and due to the course of the infection, live-attenuated vaccines are generally more effective than inactivated ones. There are several live-attenuated vaccines available for the protection of humans or farm animals against infection with particular S. enterica serovars. However, whether the vaccine strains have the same tropism for different cell types within a host's body as the appropriate wild-type strain is unknown, despite the fact that such characteristics may influence vaccine efficacy and so manipulation with a preference for a particular target cell population may be used for improvement of vaccine performance. Furthermore, in at least one vaccine, it is likely that the vaccine strain has an increased association with leukocytes – the protection of poultry against fowl typhoid is based on the rough strain of Salmonella enterica serovar Gallinarum, which may have a modified tropism similar to what we showed for the rfaL and rfaC mutants of S. Enteritidis (Matiasovic et al., 2011).
In this study, we were therefore interested in determining whether attenuated mutants, which are frequently tested as live-attenuated Salmonella vaccines, may have an increased or a decreased tropism for a particular subpopulation of porcine peripheral white blood cells (WBC). The initial aim was to use this information for the future design of improved live Salmonella vaccines for the protection of animals against S. enterica infection. However, on analyzing the results, we realized that the same information might also be useful in two additional cases. Firstly, it can be used when selecting the most suitable S. enterica mutant as a vector for the targeted expression of heterologous antigen(s). Secondly, because S. enterica has also been used for cancer therapy (Zhao et al., 2005; Stritzker et al., 2010), modification of its preference for particular cells may influence either its delivery to the site of the tumor or its very interaction with tumor cells.
Materials and methods
Bacterial strains and growth conditions
Salmonella enterica serovar Enteritidis strain 147 spontaneously resistant to nalidixic acid was used in this study (Methner et al., 2004). The construction of isogenic aroA, phoP, rfaL, rfaG, rfaC and fliC mutants and the ΔSPI1-5 mutant has been described previously (Karasova et al., 2009; Rychlik et al., 2009), except for the fact that all the strains used in this study were transformed with the pFPV25.1 plasmid constitutively expressing green fluorescent protein (GFP) (Valdivia & Falkow, 1996). The strains were subcultured in Luria–Bertani (LB) broth or LB agar at 37 °C.
Isolation of porcine WBC and infection with S. Enteritidis
All these procedures have been described previously (Matiasovic et al., 2011). Briefly, peripheral blood was taken from the vena jugularis of four healthy pigs that were 3 months of age. After erythrocyte lysis and washing the leukocytes twice with Dulbecco's phosphate-buffered saline, WBC were resuspended in Hank's balanced salt solution at a concentration of 107 cells mL−1. If necessary, porcine heat-inactivated serum (Gibco) was added to the WBC preparation to reach a 10% concentration. WBC were infected with S. Enteritidis to reach a multiplicity of infection equal to 10. The viability of WBC was monitored based on the release of lactate dehydrogenase (LDH) into the culture medium using the CytoTox 96® Non-Radioactive Assay according to the instructions of the manufacturer (Promega, Madison) and propidium iodide staining, followed by flow cytometry.
Association of S. Enteritidis and WBC measured by flow cytometry
The association of GFP-labeled S. Enteritidis with WBC was determined by flow cytometry 60 min after infection. Four independent labelings were performed. In the first one, mouse monoclonal antibodies against CD172α [formerly SWC3, clone DH59B from Veterinary Medical Research and Development Inc., Pullman, WA, immunoglobulin G1 (IgG1)] and SWC8 (clone MIL-3, gift from Dr Joan Lunney, Animal Parasitology Institute, Beltsville, MD, IgM) were added to the infected WBC. Thereafter, bound monoclonal antibodies were detected by polyclonal goat anti-mouse antibodies against IgG1 and IgM conjugated with Alexa Fluor 647 (Molecular Probes) or phycoerythrin (Southern Biotechnology), respectively.
Together with flow cytometer light scattering, this analysis allowed the differentiation of granulocytes (CD172α+ and SWC8+), monocytes (CD172α+ and SWC8−) and lymphocytes (CD172α− and SWC8−). In an additional two analyses, WBC were labeled separately with mouse anti-IgM (clone K52 1C3 from Serotec, IgG1) and mouse anti-CD3 (clone 8E6 from VMRD, IgG1) monoclonal antibodies, followed by secondary antibodies as above. This allowed the determination of B- and T-lymphocytes, respectively. The analyses were performed using a FACSCalibur™ (Becton Dickinson) equipped with a 488 nm argon-ion laser and a 633 nm diode laser and cellquest™pro software (Becton Dickinson).
Electron microscopy of WBC infected with S. Enteritidis
One hour after the infection of WBC, the cells were pelleted by centrifugation at 2000 g for 10 min and resuspended in 30 μL of 4% gelatine warmed to 45 °C. After solidification, each sample was cut into 1–3 mm3 blocks, fixed with 3% glutaraldehyde and postfixed with 1% osmium tetroxide for 1 h. Samples were dehydrated with acetone and embedded in Epon 812 (Serva). Embedded samples were heat polymerized at 60 °C for 4 days and 100-nm ultrathin sections were prepared using an LKB ultramicrotome. Finally, the ultrathin sections were stained with uranyl acetate and lead citrate and observed using a Philips EM 208 transmission electron microscope under an acceleration of 90 kV. At least 300 different cells were viewed and the percentage of infected WBC was determined.
Data were evaluated using the nonparametric Mann–Whitney test comparing the WBC infected by different mutants with the WBC infected by the wild-type S. Enteritidis. All the statistical calculations were performed using prism statistical software (Graph Pad Software).
Salmonella association with porcine WBC
The purified porcine WBC consisted of T-lymphocytes (56% of all WBC, average from four animals), followed by granulocytes (33%), B-lymphocytes (8%) and monocytes (3%). The viability of the cells was over 90% and this did not change throughout the experiment, as determined by both propidium iodide staining and LDH release (not shown).
In the presence of serum, granulocytes exhibited the highest affinity for S. Enteritidis because between 40% and 50% of granulocytes were associated with the wild-type strain, aroA and phoP mutants. Granulocytes were associated significantly less with ΔSPI1-5 and fliC mutants and significantly more with all the rfa mutants when compared with the association with the wild-type S. Enteritidis (Fig. 1a).
When we gated for monocytes, in the case of infection with the wild-type S. Enteritidis, around 20% of all monocytes were positive for S. Enteritidis. Although S. Enteritidis association with monocytes was less frequent than with granulocytes, monocyte preferences for different S. Enteritidis mutants were very similar to those of granulocytes, i.e. there was a lower preference for ΔSPI1-5 and fliC mutants and a higher preference for all the rfa mutants (Fig. 1b).
Approximately 5% of all B-lymphocytes were associated with the wild-type S. Enteritidis in the presence of serum. Unlike granulocyte monocytes, B-lymphocytes did not exhibit a reduced preference for SPI1-5 and fliC mutants, but retained a significantly higher affinity for all three rfa mutants (Fig. 1c).
The T-lymphocytes bound to S. Enteritidis formed the least of all leukocyte subpopulations. Only 2.5% of all T-lymphocytes were positive for the wild-type S. Enteritidis and unlike all previous subpopulations, we did not observe any difference in preference for any of the mutants, i.e. all the mutants associated with a similar efficiency as the wild-type strain (Fig. 1d).
In the absence of serum, the number of WBC associated with S. Enteritidis decreased. Despite this, except for three cases, the associations of granulocytes, monocytes and B- and T-lymphocytes exhibited similar patterns as in the presence of serum. The first difference was the association of the ΔSPI1-5 mutant with granulocytes and monocytes, which, unlike the association in the presence of serum, did not reach any statistical significance when compared with the interaction of these cells with the wild-type strain. The second difference was that in the absence of serum, B-lymphocytes bound to rfaC and rfaG mutants significantly more than the wild-type S. Enteritidis or any other mutant including the rfaL mutant. The last difference from ‘serum included’ conditions was the association of T-lymphocytes with the rfaL mutant, which was significantly higher than that of the wild-type S. Enteritidis or any other mutant (Fig. 1).
Electron microscopy of WBC infected with wild-type S. Enteritidis and rfaC mutant
Because the flow cytometry showed significant differences in the association of the rfa mutants and the rest of the strains, we verified this observation directly by electron microscopy. Using electron microscopy, only 2.63% of the WBC infected with wild-type S. Enteritidis in the absence of serum contained intracellular bacteria, while 8.3% of the WBC were positive when the rfaC mutant was used for the infection under the same conditions. The presence of serum increased the association (10.9% of WBC positive after infection with wild-type S. Enteritidis and 13.45% WBC positive after rfaC mutant infection), but otherwise did not influence the association profiles. Electron microscopy also showed that in the case of the wild-type S. Enteritidis uptake, the Salmonella-containing vacuoles (SCV) developed towards the spacious ones while in the case of all the rfa mutants, the vacuole closely fitted the S. Enteritidis cell inside and signs of bacterial cell disintegration could be observed inside the vacuole (Fig. 2).
In this study, we have characterized the interactions between attenuated S. Enteritidis mutants and porcine WBC in vitro. Such knowledge might be useful for the prediction of the properties of attenuated S. enterica mutants used as vaccines against salmonellosis itself or as vectors for targeting particular cell types including cancer cells. Three different types of association profiles have been found among the tested attenuated mutants. First, the phoP and aroA mutants did not differ from the wild-type S. Enteritidis in any of the assays. Second, the fliC and ΔSPI1-5 mutants, exhibited only minor differences when compared with the wild-type strain – likely due to the defect in chemotaxis in the fliC mutant (Khoramian-Falsafi et al., 1990; Jones et al., 1992) and the defect in cell invasion in the ΔSPI1-5 mutant (Kaniga et al., 1995). The last group comprising of rfaC, rfaG and rfaL mutants was characterized by a highly increased association with the host immune cells. The differences from the interaction with the wild-type strain could also be seen in the development of SCV, which, unlike the spacious one seen after the wild-type strain infection (Alpuche-Aranda et al., 1994; Boyen et al., 2006), fitted closely to the surface of the rfaC mutant (Fig. 2).
Our results show that type III secretion systems encoded by SPI-1, SPI-2 or flagellar operons have only a minor influence on the initial interactions of S. Enteritidis with porcine leukocytes. Instead, this interaction was dependent on the oligosaccharides exposed at the surface of S. Enteritidis. Interestingly, even within the rfa mutants, there were certain differences. In the absence of serum, the rfaL mutant expressing lipopolysaccharide without the O-antigen exhibited an increased affinity for T-lymphocytes while the rfaC and rfaG mutants expressing lipopolysaccharide without the outer and the inner oligosaccharide core, respectively, associated more than the rfaL mutant with B-lymphocytes.
All of these results might be used in rational vaccine design. However, if critically evaluated, live Salmonella vaccines for animal use are administered orally and therefore will be exposed to blood leukocytes only very rarely. On the other hand, attenuated S. enterica strains that were tested for tumor therapy in mice and humans were administered intravenously (Toso et al., 2002; Zhao et al., 2005; Leschner et al., 2009; Vendrell et al., 2011), i.e. they were immediately subjected to interactions with the blood leukocytes and via the circulation also to other cell types. In such a case, using the mutant's affinities towards particular cell types might be relevant. After association, the mutants with different affinities for neutrophils, B- or T-lymphocytes, for example the rfa mutants, might be differently transported throughout the host's body based on the B- or the T-lymphocyte tissue distribution. In addition, because the rfa mutants have different affinities to neutrophils, B- and T-lymphocytes, they may also have different affinities to other cell types including the tumor cells expressing aberrantly glycosylated antigens (Hakomori, 1996; Newsom-Davis et al., 2009). Finally, after differential binding to target cells, S. enterica with a different lipopolysaccharide structure may also induce a different response in the target cells (Fig. 2, see also Matiasovic et al., 2011), which further extends the potential for this system to be subjected to more detailed testing.
This work has been supported by the projects MZE0002716202 and QH81062 of the Czech Ministry of Agriculture, AdmireVet project CZ.1.05/2.1.00/01.0006 from the Czech Ministry of Education and 524/08/1606 project from the Czech Science Foundation.