Transcriptomic study of photobacterium damselae subsp. Damselaeroles of temperature and of the two-component system rstab as regulators of physiology and virulence

Resumo

Photobacterium damselae subsp. damselae is a heterogeneous and broad host-range pathogen that has been the causative agent of life-threatening infections in a great diversity of marine animals and in humans. Outbreaks caused by this bacterium occur predominantly in summer months or in unusually warm peaks throughout the year (with seawater temperatures around 25°C). It is acknowledged as one of the main zoonotic agents that can be transmitted from fish to humans. In humans, P. damselae subsp. damselae can cause fulminant septicaemias and necrotising fasciitis. The main virulence factors produced by this bacterium are cytotoxins Dly and PhlyP encoded by the pPHDD1 plasmid and PhlyC and PlpV by the chromosome. This thesis is focused on meaningful aspects that govern pathogenicity and fitness of P. damselae subsp. damselae strains of any type i.e., pPHDD1 harbouring and plasmidless strains. We aim to reveal weak points of P. damselae subsp. damselae pathobiology that can be targeted for mitigating the impact of infections. The first part of the thesis deals with the response of this bacterium to key temperatures in pathogenic processes and in the second part, we aimed to unravel the genetic network under the control of the Two Component System (TCS) RstAB, a recently-discovered major regulator of virulence. First, we focused on the comparison between P. damselae subsp. damselae cultured at 15°C, mimicking the free-swimming lifestyle of this bacterium at mid latitudes, vs at 25°C which has been proven to be a warm and risky temperature for the development of aquaculture outbreaks produced by this bacterium in summer months. We demonstrated that growth at 25°C is higher than at 15°C. To dive further into this comparison, we analysed by RNA sequencing (RNA-seq) the transcriptome produced by P. damselae subsp. damselae at both temperatures. Growth at 25°C upregulated mainly the expression of genes involved in nutrient acquisition such as porins, permeases and transporters. We found that genes encoding haemolysins Dly, PhlyP and PhlyC were among the most expressed of the whole transcriptome produced at both conditions. These data suggest that rather than an upregulation of cytotoxins, the increased likelihood of outbreaks in summer months lies in the upregulation of other virulence factors, motility, chemotaxis and an enhanced growth. We designed another comparison to address how P. damselae subsp. damselae responds to cultivation at human body temperature (37°C). As a control condition, we chose 25°C simulating warm waters from a global warming scenario. We found that growth at 37°C impairs cell viability and morphology. Strains isolated from humans are no more genetically similar to each other than they are to strains isolated from fish. These findings suggests that any genotype living in the marine environment can ultimately cause a human infection. Using an RNA-seq approach, we found that a temperature of 37°C upregulates the expression of genes related to heat-shock response, defence against reactive oxygen species and metabolic genes. One of the most striking findings of this work was to find that many functions related to virulence (e.g. damselysin) were downregulated at human body temperature. The findings presented in this section evidence that temperature is an environmental factor that govern physiological and virulence-related functions in P. damselae subsp. damselae, being 25°C around the optimum to trigger a virulent profile while 37°C constituting a stressful condition that makes human infections opportunistic rather than caused by well-adapted strains. Continuing with the study of processes implicated in fitness and virulence, the second part of this thesis deals with the study of the recently identified TCS RstAB, formed by the histidine kinase RstB and the response regulator RstA. Previous research demonstrated that this system positively regulates the expression of cytotoxins and virulence in P. damselae subsp. damselae. We used RNA-seq to compare the transcriptomic profile of P. damselae subsp. damselae strain RM-71 with that of deletion mutants ΔrstA and ΔrstB. We corroborate previous findings and found that the system also positively regulates genes involved in resistance to antimicrobial agents and survival within hosts, outer membrane proteins and synthesis of extracellular polysaccharides. The strong downregulation of 21 genes, distributed in two clusters, predictably involved in the synthesis of EPS and capsular polysaccharides (CPS) prompted us to conduct functional studies. Cluster I contains genes wza, wzb, wzc and sugar transferases among others; while cluster II contains genes yjbEFGH. The presence of a capsule around the wt strain RM-71 cells was demonstrated by transmission electron microscopy (TEM). In line with transcriptomic data, RstAB mutants showed acapsular phenotypes. In order to demonstrate that the synthesis and exportation of the capsular layer was carried out by the above-mentioned downregulated genes, we decided to construct deletion mutants by allelic exchange in genes belonging to both clusters: wza and wzc in cluster I, and yjbH in the divergently-transcribed cluster II. Mutation of wza and wzc abolished completely the synthesis of the capsular layer while mutation of yjbH resulted in the production of a significantly thinner capsule. Biofilm formation was higher in acapsular mutants Δwza and Δwzc, which suggests that other molecules underneath the capsular layer participate in cell adhesion and aggregation. Growth, motility and haemolytic activity were unaltered in mutants wza, wzc and yjbH. However, we show that acapsular mutants (Δwza and Δwzc) presented a significant reduction of fitness when growing in media supplemented with either turbot serum or mucus, and when they were injected in turbot and sea bream, most animals survived. The results presented here clearly indicate that the synthesis of a polysaccharidic capsular layer is essential for full virulence and immune evasion in P. damselae subsp. damselae.