Bio-imaging Studies on Host Pathogen Interactions

One of the primary aims of our research has been to elucidate interactions between pathogenic (zoonotic) bacteria and their hosts. In order to study these interactions during the infection process the development and use of appropriate in vivo and in vitro models and bio-imaging tools is essential. In our laboratory bio-imaging has been utilised to study enterohaemorrhagic Escherichia coli (EHEC) a sub set of pathogenic E.coli that can cause diarrhoea and haemorrhagic colitis in humans. In particular we have examined surface attachment of zoonotic verocytotoxigenic Escherichia coli (VTEC) O157:H7, O26 and O103. The localized adherence phenotype with actin rearrangements characteristic of attaching and effacing (AE) lesion formation in both cell culture and ruminants are demonstrated in Fig. 1 and Fig. 2. In a further study bio-imaging also revealed that these AE E. coli interact with non-bacterial zoonotic pathogens such as Cryptosporidium parvum. Our studies have also used bio-imaging to study the mechanisms of pathogenesis for Salmonella Enteritidis and Typhimurium. Using cultured HEp-2 cells and an aflagellate S. Enteritidis mutant EAV9 (fliC-) the role of flagella in the initiation of membrane ruffling and invasion in host cells was demonstrated.It is well established that there is a paucity of animal models available to investigate the pathogenesis of many zoonotic microorganisms and thus alternative in vitro models are urgently required. To facilitate and improve investigations three dimensional (3D) models of various cells lines (Int407, IPEC-J2 and HT29-16E) prepared on collagen coated microcarrier beads have been used for host-microbe interaction studies (Fig. 2). This has enabled continued investigation of host-pathogen interactions on cells which more realistically resemble in-vivo tissues/organs. In our laboratory this model has been used in combination with bio-imaging to study the intimate interactions between microorganisms and the host cell and for detailed studies on the efficacy of novel intervention agents. One such study aimed to determine potential mechanisms of Brachyspira pilosicoli pathogenesis and whether the probiotic lactobacilli could intervene in the Brachyspira pathogenesis. We found the pathology associated with the Brachyspira infection to the HT29-16E 3D cells included blebbing, loss of microvilli, disintegrated cytoplasm with vacuolation, chromatin condensation and fragmentation and cell sloughing. However when lactobacilli was coadministrated, fewer Brachyspira were observed by bio-imaging adhering to and invading the host cells and the array of pathology was greatly reduced. Furthermore, there was much better preservation of the brush border [1]. In a further study the prebiotic Bimuno®, which contains galactooligosaccharides, produced by the galactosyltransferase activity of Bifidobacterium bifidum NCIMB 41171, was shown to reduce the severity of Salmonella Typhimurium invasion and pathology in a gut loop model and significantly reduce membrane ruffling in 3D cultured cells. The reduced infection in mice is possibly due to stimulating members of the normal flora to proliferate which may subsequently reduce the invasion of the bacteria into host cells [2].These ongoing studies have demonstrated that bio-imaging combined with the appropriate in vivo and in vitro models are essential research tools for the study of interactions between pathogenic (zoonotic) bacteria and their hosts. It enables the mechanisms of adhesion/invasion and the efficacy of novel intervention agents to be further elucidated [3].