hide
Free keywords:
-
Abstract:
Pseudomonas aeruginosa is a ubiquitous, opportunistic pathogen causing life-threatening infections in immunosuppressed patients and patients with cystic fibrosis (CF). It has a remarkable ability to develop multi-resistance to a wide range of antibiotics, making it a significant concern for healthcare professionals. Also, P. aeruginosa is characterised by a high adaptability to various ecological niches. The latter is achieved due to the plasticity of the bacterial genome, which is rich in complex regulatory networks, and frequent mutations in regulatory genes. One of the known mutations described in clinical isolates of P. aeruginosa from patients with chronic infection is the mutation of the regulator gene retS. Inactivation of RetS leads to a decrease in the virulence of the bacterium due to the downregulation of the type III secretion system (T3SS); however, it upregulates the type VI secretion system (T6SS). The T6SS is a unique molecular machinery injecting a cocktail of protein toxins, also known as effectors, into neighbouring cells or the intercellular space and performing a variety of functions. Multiple T6SS effectors target prokaryotic and eukaryotic cells and also contribute
to the scavenging of scarce metal ions, thereby giving bacteria a competitive advantage and playing a role in virulence and adaptation to a nutrient-limited environment. The P. aeruginosa genome encodes three T6SS clusters, H1-, H2-, and H3-T6SS, with a specific set of effectors. In recent years, multiple regulatory pathways of these three T6SS clusters have been discovered. However, it is still unclear how highly complex regulatory networks fine tune the expression and activation of the T6SS clusters and which environmental cues play a role in the activation. Therefore, in this study, I tested which clusters are functionally active during
epithelial infection and which host environmental factors are responsible for activation. The analysis of the T6SS gene and protein expression using qPCR and western blot showed that H2-T6SS is not expressed on LB agar at the host temperature of 37°C. Also, a bacterial competition assay with fluorescently labelled strains confirmed the lack of competitive advantage mediated by the H2-T6SS effector TseT at 37°C. This is intriguing since H2-T6SS is also known to target eukaryotic cells, which often require a temperature of around 37°C to maintain their homeostasis. Therefore, the effect of physiologically-relevant conditions on the
activity of the H2-T6SS was tested here. First, the growth of the bacteria in less favoured
nutritional conditions of the cell medium DMEM compared to LB was sufficient toinduce the functional activity of the H2-T6SS. Second, analysis of protein expression in the bacteria collected from the infected differentiated pulmonary epithelium derived from murine lung
organoids demonstrated increasing Hcp2 production during the course of the infection. The H1- and H3-T6SS were functionally active both in laboratory conditions and under conditions mimicking the host environment and were not affected by temperature changes. Thus, the results of this study show the functional activity of all three clusters of T6SS during infection of the pulmonary epithelium. However, the expression of H2-T6SS can be inhibited by changes in environmental and nutritional conditions at 37°C. This opens up a new perspective on the role of H2-T6SS in adaptation to stress factors and available nutrients during colonisation of the host.
