Abstract
A reporter plasmid pSRG has been developed which expresses red fluorescent protein (RFP) from a constitutive prokaryotic promoter within Pasteurella multocida B:2 and green fluorescent protein (GFP) from a constitutive eukaryotic promoter within mammalian cells. This construct has been used to determine the location and viability of the bacteria when moving from the extracellular environment into the intracellular compartment of mammalian cells. Invasion assays with embryonic bovine lung (EBL) cells and an attenuated AroA- derivative of Pasteurella multocida B:2 (strain JRMT12), harbouring the plasmid 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, indicating release of the plasmid into the intracellular environment. At 5 h post-invasion, more EBL cells were expressing GFP, while still harbouring RFP-expressing bacteria. Concurrently, some EBL cells were shown to express only GFP, indicating loss of viable bacteria within these cells. 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.
Highlights
The numerous serotypes of Pasteurella multocida are associated with a variety of disease syndromes in a wide range of agricultural, domestic and feral animal species [1]
P. multocida was grown on Brain Heart Infusion (BHI) agar or in BHI broth (Difco) media and E. coli was grown on Luria-Bertani (LB) agar or in LB broth (Difco) media at 37uC and shaken at 180 rpm
Plasmid pEGFP-N1TM (Clontech) encodes green fluorescent protein (GFP) from a constitutive eukaryotic promoter PCMVIE and is able to replicate in E. coli and eukaryotic cells
Summary
The numerous serotypes of Pasteurella multocida are associated with a variety of disease syndromes in a wide range of agricultural, domestic and feral animal species [1]. P. multocida serotypes B:2 and E:2 are associated with haemorrhagic septicaemia (HS) of cattle, water buffaloes and occasionally other species, resulting in major economic losses, mainly in South East Asia [2,3]. A live-attenuated vaccine for HS might be a better alternative vaccine as, by mimicking the ability of the pathogen to infect the host, but without causing disease, a more prolonged protective immune response may be induced. Othman et al [7] reported on the ability of the live vaccine strain to adhere to and to invade embryonic bovine lung (EBL) cells with an efficiency similar to the wild-type strain. The live vaccine strain was able to survive intracellularly in the EBL cells for at least 7 hours
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