Christophe Bordi

Accurate bacterial quantification is critical in many biological fields, from clinical diagnostics to environmental microbiology. Here, we establish a robust workflow for absolute quantification of bacterial species within mixed communities using Crystal Digital PCR^TM. Using a synthetic consortium of Clostridium acetobutylicum and Nitratidesulfovibrio vulgaris, we optimized primer design for species-specific detection and demonstrated that Crystal Digital PCR^TM enables reliable quantification of low-abundance species, down to a 1:10,000 ratio. We further show that the presence of one species does not interfere with the quantification of another. Finally, we demonstrate that Crystal Digital PCR^TM can also be used to determine plasmid-to-chromosome copy number ratios in bacteria carrying megaplasmids.

Pseudomonas aeruginosa is a major cause of nosocomial infections, particularly in immunocompromised patients or in individuals with cystic fibrosis. Genome sequences reveal that most P. aeruginosa strains contain a significant number of accessory genes gathered in genomic islands. Those genes are essential for P. aeruginosa to invade new ecological niches with high levels of antibiotic usage, like hospitals, or to survive during host infection by providing pathogenicity determinants. P. aeruginosa pathogenicity island 1 (PAPI-1), one of the largest genomic islands, encodes several putative virulence factors, including toxins, biofilm genes and antibiotic-resistance traits. The integrative and conjugative element (ICE) PAPI-1 is horizontally transferable by conjugation via a specialized GI-T4SS, but the mechanism regulating this transfer is currently unknown. Here, we show that this GI-T4SS conjugative machinery is directly induced by TprA, a regulator encoded within PAPI-1. Our data indicate that the nucleotide associated protein NdpA2 acts in synergy with TprA, removing a repressive mechanism exerted by MvaT. In addition, using a transcriptomic approach, we unravelled the regulon controlled by Ndpa2/TprA and showed that they act as major regulators on the genes belonging to PAPI-1. Moreover, TprA and NdpA2 trigger an atypical biofilm structure and enhance ICE PAPI-1 transfer.

Biofilm formation is a complex process resulting from the action of imbricated pathways in response to environmental cues. In this study, we showed that biofilm biogenesis in the opportunistic pathogenPseudomonas aeruginosadepends on the availability of RpoS, the sigma factor regulating the general stress response in bacteria. Moreover, it was demonstrated that RpoS is post-translationally regulated by the HsbR-HsbA partner switching system as has been demonstrated for its CrsR-CrsA homolog inShewanella oneidensis. Finally, it was established that HsbA, the anti-sigma factor antagonist, has a pivotal role depending on its phosphorylation state since it binds HsbR, the response regulator, when phosphorylated and FlgM, the anti-sigma factor of FliA, when non-phosphorylated. The phosphorylation state of HsbA thus drives the switch between the sessile and planktonic way of life ofP.aeruginosaby driving the release or the sequestration of one or the other of these two sigma factors.

Pseudomonas aeruginosa is an opportunistic pathogenic bacterium responsible for both acute and chronic infections and has developed resistance mechanisms due to its ability to promote biofilm formation and evade host adaptive immune responses. Here, we investigate the functional role of the periplasmic detector domain (GacS_PD) from the membrane-bound GacS histidine kinase, which is one of the key players for biofilm formation and coordination of bacterial lifestyles. A gacS mutant devoid of the periplasmic detector domain is severely defective in biofilm formation. Functional assays indicate that this effect is accompanied by concomitant changes in the expression of the two RsmY/Z small RNAs that control activation of GacA-regulated genes. The solution NMR structure of GacS_PD reveals a distinct PDC/PAS α/β fold characterized by a three-stranded β-sheet flanked by α-helices and an atypical major loop. Point mutations in a putative ligand binding pocket lined by positively-charged residues originating primarily from the major loop impaired biofilm formation. These results demonstrate the functional role of GacS_PD, evidence critical residues involved in GacS/GacA signal transduction system that regulates biofilm formation, and document the evolutionary diversity of the PDC/PAS domain fold in bacteria.

Myeloablative treatment preceding hematopoietic stem cell (HSC) and progenitor cell (HS/PC) transplantation results in severe myeloid cytopenia and susceptibility to infections in the lag period before hematopoietic recovery. We have previously shown that macrophage colony-stimulating factor (CSF-1; M-CSF) directly instructed myeloid commitment in HSCs. In this study, we tested whether this effect had therapeutic benefit in improving protection against pathogens after HS/PC transplantation. M-CSF treatment resulted in an increased production of mature myeloid donor cells and an increased survival of recipient mice infected with lethal doses of clinically relevant opportunistic pathogens, namely the bacteria Pseudomonas aeruginosa and the fungus Aspergillus fumigatus. M-CSF treatment during engraftment or after infection efficiently protected from these pathogens as early as 3 days after transplantation and was effective as a single dose. It was more efficient than granulocyte CSF (G-CSF), a common treatment of severe neutropenia, which showed no protective effect under the tested conditions. M-CSF treatment showed no adverse effect on long-term lineage contribution or stem cell activity and, unlike G-CSF, did not impede recovery of HS/PCs, thrombocyte numbers, or glucose metabolism. These results encourage potential clinical applications of M-CSF to prevent severe infections after HS/PC transplantation.

In response to environmental changes, Pseudomonas aeruginosa is able to switch from a planktonic (free swimming) to a sessile (biofilm) lifestyle. The two-component system (TCS) GacS/GacA activates the production of two small non-coding RNAs, RsmY and RsmZ, but four histidine kinases (HKs), RetS, GacS, LadS and PA1611, are instrumental in this process. RetS hybrid HK blocks GacS unorthodox HK autophosphorylation through the formation of a heterodimer. PA1611 hybrid HK, which is structurally related to GacS, interacts with RetS in P. aeruginosa in a very similar manner to GacS. LadS hybrid HK phenotypically antagonizes the function of RetS by a mechanism that has never been investigated. The four sensors are found in most Pseudomonas species but their characteristics and mode of signaling may differ from one species to another. Here, we demonstrated in P. aeruginosa that LadS controls both rsmY and rsmZ gene expression and that this regulation occurs through the GacS/GacA TCS. We additionally evidenced that in contrast to RetS, LadS signals through GacS/GacA without forming heterodimers, either with GacS or with RetS. Instead, we demonstrated that LadS is involved in a genuine phosphorelay, which requires both transmitter and receiver LadS domains. LadS signaling ultimately requires the alternative histidine-phosphotransfer domain of GacS, which is here used as an Hpt relay by the hybrid kinase. LadS HK thus forms, with the GacS/GacA TCS, a multicomponent signal transduction system with an original phosphorelay cascade, i.e. H1_LadS→D1_LadS→H2_GacS→D2_GacA. This highlights an original strategy in which a unique output, i.e. the modulation of sRNA levels, is controlled by a complex multi-sensing network to fine-tune an adapted biofilm and virulence response. P. aeruginosa is able to switch from a planktonic to a sessile lifestyle by regulating the two small RNAs RsmY and RsmZ. The GacS/GacA TCS is the main system involved in their regulation; however, three HKs, RetS, LadS and PA1611, modulate this TCS. Here we elucidate the relationship between the LadS signaling pathway and the GacS/GacA TCS. We indeed show that LadS modulates GacS/GacA through an original phosphorelay mechanism where LadS utilizes its H1 and D1 domains and the H2 domain of the unorthodox GacS HK, thereby forming with GacS HK, a multicomponent signal transduction system. This multicomponent signal transduction system, first evidenced in this work, probably functions in a similar manner in some other pseudomonas species like P. fluorescens but not in all pseumonad genera as illustrated by P. syringae. Multicomponent signaling is an original and pertinent strategy for bacteria, in which a complex multi-sensing network controls a similar output (i.e. the modulation of levels of central regulatory RsmY and RsmZ sRNAs). This mechanism provides P. aeruginosa with the possibility of integrating at least two different signals, one from GacS and the other from LadS, to engage P. aeruginosa in a sessile lifestyle.

Pseudomonas aeruginosa is a Gram-negative environmental species and an opportunistic microorganism, establishing itself in vulnerable patients, such as those with cystic fibrosis (CF) or those hospitalized in intensive care units (ICU). It has become a major cause of nosocomial infections worldwide and a serious threat to Public Health because of overuse and misuse of antibiotics that have selected highly resistant strains against which very few therapeutic options exist. Herein is illustrated the intraclonal evolution of the genome of sequential isolates collected in a single CF patient from the early phase of pulmonary colonization to the fatal outcome. We also examined at the whole genome scale a pair of genotypically-related strains made of a drug susceptible, environmental isolate recovered from an ICU sink and of its multidrug resistant counterpart found to infect an ICU patient. Multiple genetic changes accumulated in the CF isolates over the disease time course including SNPs, deletion events and reduction of whole genome size. The strain isolated from the ICU patient displayed an increase in the genome size of 4.8% with major genetic rearrangements as compared to the initial environmental strain. The annotated genomes are given in free access in an interactive web application WallGene designed to facilitate large-scale comparative analysis and thus allowing investigators to explore homologies and syntenies between P. aeruginosa strains, here PAO1 and the five clinical strains described.

Although the completion and annotation of the entire genomic DNA sequence of Pseudomonas aeruginosa PAO1 strain has been carried out, an important number of genes are still of unknown function and many genetic elements involved in various regulatory pathways like small RNA are still unrevealed. One of the strategies to identify gene function or genetic elements is the construction and utilization of DNA genomic library. Here, we describe the construction a P. aeruginosa DNA genomic library.

In Pseudomonas aeruginosa, identification of new partners of a protein of interest could give precious clues to decipher a biological process in which this protein is involved. However, genes encoding for partners of a protein of interest are unknown and frequently scattered throughout the genome. We describe herein the construction and the use of pan-genomic bacterial two-hybrid libraries to identify new partners of a protein of interest encoded by P. aeruginosa.

Bacterial gene regulation is controlled by complex regulatory cascades which integrate input environmental signals and adapt specific and adequate output cellular responses. These complex networks are far from being elucidated, in particular in Pseudomonas aeruginosa. In the present study, we developed bacterial two-hybrid genome fragment libraries of the P. aeruginosa PAO1 strain to identify potential partners involved in the HptB/HsbR/HsbA pathway. This powerful tool, validated by the interaction previously described between HsbR and HsbA proteins, allowed us to demonstrate that the HsbR response regulator dimerizes through its PP2C/ATPase C-terminal effector domain, an observation further confirmed by pull-down experiments. This will also allow us to identify further new partners in this cascade.