Development of in vitro infection model methods for Gram-negative bacteria to assess potential broad-spectrum anti-virulence inhibitors

Bacteria exhibit common virulence targets which can be inhibited for potential broad-spectrum activity across a range of bacterial infections. The macrophage infectivity potentiator (Mip) is a bacterial protein that has peptidylprolyl cis/trans isomerase activity and is part of the FK506-binding protein subgroup, in the superfamily of immunophilins. They have previously been identified to be involved in intracellular virulence of Legionella pneumophila and Burkholderia pseudomallei and inhibition of the protein with pipecolic acid derived small-molecule inhibitors resulted in a decrease in virulence. Due to the highly conserved nature of Mip proteins it is believed that these proteins exist in Klebsiella pneumoniae and Burkholderia cenocepacia; two pathogens contributing to a rise in multi-drug resistant infections. Therefore, this study aims to examine the potential broad-spectrum activity of the Mip inhibitors in K. pneumoniae and B. cenocepacia by developing in vitro model methods for inhibitor evaluation. The presence of putative Mip proteins in K. pneumoniae and B. cenocepacia were confirmed with the use of online bioinformatics tools. The Mip inhibitors were then examined by first developing in vitro cell based methods to identify the magnitude at which K. pneumoniae and B. cenocepacia adhered to macrophages, and internalised, survived and replicated within the macrophages. An optimised adherence assay, and internalisation, survival and replication assay were then used to test the efficacy of the Mip inhibitors in K. pneumoniae and B. cenocepacia. The results obtained demonstrated that K. pneumoniae and B. cenocepacia were able to adhere to the macrophages, however, the concentration at which they adhered varied between strains. Only two strains of K. pneumoniae were internalised into the macrophages and only one strain was able to replicate at low concentrations within the macrophages during the 24 hours post-infection. All strains of B. cenocepacia were internalised into the macrophages, however, only one strain suggested replication within the macrophages during the 24 hours post-infection. The Mip inhibitors had little effect on adherence of K. pneumoniae to macrophages, however, trends in the data suggested that the Mip inhibitors had an effect on the intracellular virulence of K. pneumoniae and B. cenocepacia. Therefore from this preliminary study, it seems that Mip inhibitors have potential broad-spectrum activity in intracellular virulence of these pathogens. These findings provide insight into Mip proteins in K. pneumoniae and B. cenocepacia and the potential they have as common bacterial drug targets