Understanding the role of hxc-type II secretion system of Pseudomonas aeruginosa in pathogenesis

Abstract

Cross-kingdom interactions are ubiquitous in nature and play a key role in the survival of species in polymicrobial environments. The communication between the organisms in mixed infections are always complex with the antagonistic or synergistic nature of this interaction difficult to determine. Pseudomonas aeruginosa and Candida albicans are two opportunistic human pathogens commonly found together in vivo. These two organisms have been associated with host colonisation in several diseases including cystic fibrosis (CF), severe burn wounds and ventilator-associated pneumonia; where the nature of their interactions potentially influences virulence properties of individual populations and in turn the eventual outcome of the polymicrobial disease. Notwithstanding the frequent co-isolation of P. aeruginosa and C. albicans from mixed infections, the nature of their interactions and the molecules involved are still unclear. Towards this aim we developed a fungal culture system in the presence of supernatants from different strains of P. aeruginosa to identify potential changes in fungal behaviour by measuring optical density (OD) at 600 nanometres (nm). Our preliminary data showed that wild type (WT) P. aeruginosa Lausanne strain (PAO1-L) induce filamentation of C. albicans, a key virulence attribute of this fungus. Using appropriate P. aeruginosa mutants, data showed that this observation was not due to the production of rhamnolipids, exotoxin A, or dependent on the virulence regulator ToxR. Interestingly, promotion of filamentous growth was attenuated when using supernatants from a PAO1 strain, PAAMP1. PAAMP1 has a 58kb chromosomal deletion including genes encoding the Hxc-Type II secretion system (Hxc-T2SS), low molecular weight alkaline phosphatase (LapA), cyclic-di-GMP phosphodiesterase (BifA), and ToxR; indicating that the gene(s) responsible for promoting hyphal growth in C. albicans was encoded within this region. Using biochemical approaches, the hyphal-inducing activity in WT PAO1-L supernatant was shown to be heat-labile (95°C, 10 min) and was retained in ultra-filtrates of relative molecular weights (rMW) above 10 kDa. Furthermore, protein gel analysis showed the presence of a band with rMW between 25-30 kDa specific for WT. Although the hyphal induction effect of PAO1-L secretions was lost in the course of the study and thus the observation inconclusive, the preliminary data put together suggest that WT POA1-L possibly secretes a protein capable of hyphal growth induction in C. albicans in vitro. Alongside employing biochemical techniques to identify the molecular nature of the hyphal-inducing agent(s) in P. aeruginosa secretion, genetic approach was used to ascertain gene(s) involved in the hyphal-inducing activity. Based on the preliminary data showing hyphal induction to be independent of ToxR gene regulation and the active agent having a rMW between 25–30kDa, it was hypothesised that the hyphal-inducing agent in the WT supernatant was Hxc-T2SS dependent LapA, which has a rMW between 38-to-40 kDa. Towards this aim, an in-frame deletion mutant lacking the hxc-operon (Δhxc) was constructed in a PAO1-L background. Phenotypic characterisation showed that bacterial growth and virulence traits like biofilm formation and pyocyanin production were not detectably affected by the hxc deletion. Addition of exogenous phosphate to culture medium significantly increased bacterial growth as well as biofilm formation, but pyocyanin secretion was markedly reduced in both the mutant and the wild type PAO1-L. Transcriptional analyses of the hxc-operon using a bioluminescent reporter mini-CTX-lux inserted into PAO1-L (this thesis) confirmed an earlier study by Ball et al (2002) that the operon is tightly regulated by two promoter regions, PhxccV and PhxcT. However, contrary to previous findings that PhxcV transcriptional activity is five times less than PhxcT under phosphate limiting conditions, this work demonstrated that PhxcV is highly expressed, though earlier, and peaks at 5 hours of incubation while PhxcT peaks at 10 hours. Further, the data indicated that expression of both promoters is sustained longer when bacteria is in contact with epithelial cells or in complete media such as X-vivo15 or DMEM-F12 compared to LB rich medium or phosphate-limiting proteose peptone medium. This supports earlier reports that the hxc operon is expressed during acute infection. Using an in vitro infection model, it was demonstrated that PAO1-LΔhxc detectably thrives better than the WT after contact with differentiated and undifferentiated human bronchial epithelial cells at a multiplicity of infection of 50. Assessment of bacterial-induced cytokine production showed that bronchial epithelial cells respond to P. aeruginosa infection through the production of cytokines and chemokines including TNF-α, GM-CSF, G-CSF, IL-6 IL-17C and IL-8. This pattern was the same for WT and Δhxc. Overall, the findings from this study show tight regulation and high dependency on culture conditions for the transcription of genes encoding the Hxc-T2SS, which implies that this system plays an important role on the adaptation of P. aeruginosa to its environment. Considering that the P. aeruginosa devotes Hxc-T2SS to a single substrate whose expression is not controlled by quorum sensing, this study provides a platform for further research into the hxc operon and how it may contribute to P. aeruginosa fitness under different conditions