Interactions of an attenuated AroA- derivative of Pasteurella multocida B:2 with mammalian cells and its potential for DNA vaccine delivery

The primary aim of this study was to investigate the potential of an aroA mutant of Pasteurella multocida B:2 (vaccine strain JRMT12) as a candidate for DNA vaccine delivery (bactofection). First, the invasive property of the vaccine strain was assessed for its interaction with different mammalian cell lines. Next, a eukaryotic expression plasmid that could be maintained in Pasteurella was modified to contain a prokaryotic reporter gene to help in determining the location and viability of the bacteria when moving from the extracellular environment into the intracellular compartment of the mammalian cells. This plasmid was further developed to function with a dual prokaryotic and eukaryotic reporter system in order to demonstrate expression of the plasmid DNA in the mammalian cells. During interaction of strain JRMT12 with mammalian cell lines, the ability of the bacterium to adhere, invade and survive intracellularly was monitored and assessed. Three mammalian cell lines were used: a mouse macrophage-like cell line, J774.2; a bovine-lymphoma cell line, BL-3; and an embryonic bovine lung cell line, EBL. The JRMT12 strain was compared with strains of the wild-type P. multocida B:2 (85020), bovine P. multocida A:3, Mannheimia haemolytica A:1 and Escherichia coli XL-1 BLUE. Both P. multocida B:2 strains were capable of adhering to and invading J774.2, BL-3 and EBL cells. All of the Pasteurella and Mannheimia strains tested were able to adhere to EBL cells but only B:2 strains were taken up intracellularly in significant numbers. The vaccine strain, JRMT12 was found to survive intracellularly in EBL cells for at least 7 h although a steady decline in the number of viable intracellular bacteria was noted with time. In an invasion inhibition assay, the use of the microfilament formation inhibitor cytochalasin D suggested that the entry into mammalian cells was by an actin-dependent process. Cell viability assessment by trypan blue staining indicated that none of the bacterial strains was toxic for the mammalian cells. Upon entry into the mammalian cells the JRMT12 strain resided in a vacuolar compartment, as demonstrated by transmission electron microscopy. However, P. multocida A:3 and M. haemolytica A:1 were only found loosely adhering to the cell surface of EBL cells and were not detected intracellularly. Further morphological assessment by TEM showed that only a low percentage of mammalian cells appeared to contain one or more JRMT12, suggesting that only certain cells in the population were capable of being invaded by, or taking up, the bacteria. Attempts were made to construct a Pasteurella eukaryotic expression plasmid using a gene sequence from the Pasteurella shuttle plasmid pAKA16, developed previously in this laboratory, and the commercial eukaryotic expression plasmid pCMV-sCRIPT, but these were only partially successful. The origin of replication gene (oriP) in the Pasteurella shuttle plasmid was isolated and sequenced. Analysis of oriP showed sequence similarity with the known origins of replication in other Pasteurella plasmids. The E. coli plasmid origin of replication (oriE) was removed from pCMV-sCRIPT and the oriP gene was ligated into the oriE-free pCMV-sCRIPT but attempts to transform the resulting plasmid into P. multocida B:2 were not successful. An alternative approach to plasmid development was made using another commercial eukaryotic expression vector, pEGFP-N1. This plasmid has the same properties as pCMV-sCRIPT but has an additional, fluorescent reporter gene under the control of a eukaryotic promoter. It was found to be able to replicate in P. multocida B:2 but positive transformants were only recovered after prolonged incubation after electroporation. The plasmid was stably maintained in strain JRMT12 for at least 14 days with or without antibiotic selection. It was also successfully transfected into EBL cells, as shown by expression of green fluorescent protein (GFP) in individual cells. The P. multocida vaccine strain JRMT12 was also able to deliver the plasmid into EBL cells, although the number of EBL cells expressing GFP after bacterial delivery was lower than by direct transfection of the plasmid. Next, plasmid pMK-Express, a Pasteurellaceae prokaryotic expression vector with a gfp reporter gene, was used. When this was electroporated into the vaccine strain, the strain was shown to express GFP maximally as measured by fluorimetry, during the early exponential phase of bacterial growth. The DsRed.M1 gene coding for red fluorescent protein (RFP) from plasmid pDsRed-Monomer was then used to replace the gfp gene in pMK-Express to make the construct pMK-RED. After electroporation of pMK-RED into the JRMT12, RFP expression was detected maximally during the early exponential phase of bacterial growth. The same strain expressing RFP could also be detected in the intracellular compartment of the EBL cells by fluorescence microscopy at 3 h post-invasion. Finally, plasmid pSRG, our so-called “traffic light” plasmid with a dual reporter system was constructed. This was made from plasmid pEGFP-N1 (with its existing eukaryotic expression system for GFP expression) and the sodRED fragment (with a Pasteurella promoter controlling the DsRED.M1 gene for RFP expression) isolated from plasmid pMK-RED. This plasmid was stable in strain JRMT12 with or without antibiotic selection for 14 passages. RFP expression from JRMT12 was detected maximally during the early exponential phase of bacterial growth. Transfection of pSRG into EBL cells gave individual cells expressing GFP. Invasion assays with EBL cells and P. multocida B:2 JRMT12 pSRG+ showed that RFP-expressing bacteria could be detected intracellularly at 3 h post-invasion. At this stage, some EBL cells harbouring RFP-expressing bacteria were observed to express GFP simultaneously. At 5 h post-invasion, some of the EBL cells were still harbouring RFP-expressing bacteria and at the same time expressing GFP themselves. Concurrently, some Pasteurella free-EBL cells were shown to express GFP. These experiments proved the functionality of the pSRG dual reporter system and the potential of P. multocida B:2 JRMT12 for bactofection and delivery of a DNA vaccine. An apparent immunosuppressive effect of P. multocida B:2 on the proliferative response to concanavalin A (ConA) of peripheral blood mononuclear cells (PBMC) had been reported by Ataei (2007). The PBMC had been taken from calves infected with P. multocida B:2 or from normal calves and treated in vitro with extracts of P. multocida B:2. In the present study, in vitro assays with PBMC from normal calves were undertaken in an attempt to confirm these findings. A cell-free extract (CFE) of the vaccine strain JRMT12 was found to suppress the subsequent proliferation of PBMC in response to ConA in a dose-dependent manner. However, the results were not consistently reproducible and the same effect could not be demonstrated with CFE from the wild-type strain 85020.