Signalling pathway controlling cupD fimbrial genes expression and role in Pseudomonas aeruginosa pathogenesis

Most microbes in nature exist as surface associated communities called biofilms. Biofilms are described as a 'city of microbes' in which bacteria are hold together into a solid matrix. There is compelling evidence that the biofilm lifestyle is an efficient means for microorganisms to maintain a protected niche. Establishment of biofilms in humans leads to chronic infection that can persist for life and lead to host death. It is well described that stability and resilience of biofilms is a major medical and industrial problem. Biofilms have been shown to display resistance to antibiotic treatments and are recalcitrant to eradication via the immune system. The formation of the biofilm obeys to a series of events that start with initial attachment of the bacterium on the host tissue. The initial attachment requires bacterial surface adhesins that are frequently associated with long fibres called fimbriae. In the course of the infection process, the bacteria should adapt and either form a biofilm to persist or colonize the host more aggressively. For bacteria, these choices are based in large part on local environmental cues, and are effected through altered gene expression. For example whether the bacteria decide to stop motility and start attachment may result in the assembly of these long fibres-containing adhesins. In order to detect the environmental cues bacteria have evolved regulatory devices that are made of two-components, one for signal detection and one for affecting gene expression, both components, called sensor and response regulator being intimately linked. The opportunistic pathogen Pseudomonas aeruginosa is a prototype of free-living organism with the capacity to cause disease in a wide range of eukaryotic hosts. P. aeruginosa is responsible for some of the most serious infections in humans, including emerging nosocomial infections associated with impairment of host defences. It is responsible for severe lung and upper respiratory tract infections in patients with Cystic Fibrosis and acute pneumopathies. P. aeruginosa is equipped with numerous genes responsible for the assembly of the above-mentioned fimbriae (cup genes). Moreover, this bacterium has a wide repertoire of two-component systems that give this organism an ubiquitous and versatile character. Finally, P. aeruginosa displays a remarkable range of virulence, from weakly virulent isolates to highly virulent broad-spectrum strains. In the present study we will investigate the role of a two-component regulatory system (Rcs/Pvr) on expression of cupD fimbrial genes. We have chosen to study those genes, because they are located on the pathogenicity island of the highly virulent P. aeruginosa PA14 strain. These genes are otherwise not found in the genome of the laboratory strain P. aeruginosa PAO1. Pathogenicity islands are of mobile genetic elements, which play a pivotal role in the virulence of bacterial pathogens of humans, animals and plants. Our study will bring original, detailed and integrated molecular basis about a complex network of regulation, the Pvr/Rcs system, and the specific cupD gene target. The characterization of the role of these systems in biofilm and virulence will improve our basic knowledge of signalling and fimbriae assembly in P. aeruginosa and will provide molecular details for the search of lead molecules interfering with P. aeruginosa pathogenesis.

Technical Summary

The versatile and ubiquitous bacterium Pseudomonas aeruginosa is the quintessential opportunistic pathogen, because it can infect a broad range of hosts, from amoeba to humans, where it is found associated with burns, cystic fibrosis (CF), AIDS, and cancer. This pathogen produces an arsenal of virulence factors, among which a large number of secreted hydrolytic enzymes and toxins that contribute to survival within the host and cytotoxicity. In addition, cell surface adhesive structures, such as pili, contribute to attachment onto the host tissues. Such attachment frequently results in biofilm formation and may lead to recurrent and chronic infection. Chaperone-usher pathways have been identified in P. aeruginosa and named Cup. They are responsible for the assembly of cell surface fibres named fimbriae. The cupD gene cluster, not existing in the laboratory strain PAO1, is located on the PAPI-1 pathogenicity island of the highly virulent PA14 strain. The cupD cluster is adjacent to genes encoding two component system regulatory proteins (Rcs/Pvr). Two component systems are regulatory devices that detect environmental signal and switch gene expression to allow bacteria to adapt to new environments. The Rcs/Pvr system is similar to the previously described Bvg system in Bordetella pertussis, the master regulator for expression of virulence genes. Whereas RcsB is a transcriptional activator, PvrR is a putative phosphodiesterase capable of c-di-GMP degradation. Our study will bring original, detailed and integrated molecular basis about a complex network of regulation, the Pvr/Rcs system, and the specific cupD gene target. This system should be considered as an archetype to understand the combined role of direct gene expression and c-di-GMP signalling in the process of P. aeruginosa biofilm formation and virulence. Our study will yield basic knowledge and provide molecular details for the search of molecules interfering with P. aeruginosa pathogenesis.