Eric Faudry

Pseudomonas aeruginosa is a major human opportunistic pathogen associated with a high incidence of multi-drug resistance. The antibody-based blockade of P. aeruginosa virulence factors represents a promising alternative strategy to mitigate its infectivity. In this study, we employed single B cell sorting from cystic fibrosis patients to isolate human monoclonal antibodies (mAbs) targeting proteins from the P. aeruginosa Type 3 Secretion System (T3SS) and characterized a panel of mAbs directed at PscF and PcrV. Among those, two mAbs, P5B3 and P3D6, that bind to the injectisome tip protein PcrV, exhibited T3SS blocking activity. We solved the crystal structure of the P3D6 Fab-PcrV complex, which revealed that the Ab binds to the C-terminal region of PcrV. In addition, we compared the T3SS-blocking activity of three PcrV-targeting mAbs, including two from previous independent studies, using two distinct assays to evaluate pore formation and toxin injection. We conducted a mechanistic and structural analysis of their modes of action through modeling based on the known structure of a functional homolog, SipD from Salmonella typhimurium . The analysis suggests that anti-PcrV mAbs may act through different mechanisms, ranging from preventing PcrV oligomerization to disrupting PcrV’s scaffolding function, thereby inhibiting the assembly and function of the translocon pore. Our findings provide additional evidence that T3SS-targeting Abs, some capable of inhibiting virulence, are elicited in P. aeruginosa -infected patients. The results offer deeper insights into PcrV recognition by mAbs and their associated mechanisms of action, helping to identify which Abs are more likely to be therapeutically useful based on their mode of action and potency. This paves the way for the development of effective alternatives to traditional antibiotics in the fight against this resilient pathogen.

Pseudomonas aeruginosa is a major human opportunistic pathogen associated with a high incidence of multi-drug resistance. The antibody-based blockade of P. aeruginosa virulence factors represents a promising alternative strategy to mitigate its infectivity. In this study, we employed single B cell sorting from cystic fibrosis patients to isolate human monoclonal antibodies (mAbs) targeting proteins from the P. aeruginosa Type 3 Secretion System (T3SS) and characterized a panel of mAbs directed at PscF and PcrV. Among those, two mAbs, P5B3 and P3D6, that bind to the injectisome tip protein PcrV, exhibited T3SS blocking activity. We solved the crystal structure of the P3D6 Fab-PcrV complex, which revealed that the Ab binds to the C-terminal region of PcrV. In addition, we compared the T3SS-blocking activity of three PcrV-targeting mAbs, including two from previous independent studies, using two distinct assays to evaluate pore formation and toxin injection. We conducted a mechanistic and structural analysis of their modes of action through modeling based on the known structure of a functional homolog, SipD from Salmonella typhimurium. The analysis suggests that anti-PcrV mAbs may act through different mechanisms, ranging from preventing PcrV oligomerization to disrupting PcrV’s scaffolding function, thereby inhibiting the assembly and function of the translocon pore. Our findings provide additional evidence that T3SS-targeting Abs, some capable of inhibiting virulence, are elicited in P. aeruginosa-infected patients. The results offer deeper insights into PcrV recognition by mAbs and their associated mechanisms of action, helping to identify which Abs are more likely to be therapeutically useful based on their mode of action and potency. This paves the way for the development of effective alternatives to traditional antibiotics in the fight against this resilient pathogen.

The study of bacteria swimming behavior or their interaction with other bacteria or cells requires an efficient and flexible tool for bacteria manipulation. Optical tweezers have been shown to be perfectly adapted for this task. Here we report optical trapping of pathogen Pseudomonas aeruginosa bacteria using optical fiber tweezers with dedicated nanostructured optical fibers. Well-aligned straight chains of up to ten bacteria were observed with optical fiber tips, whereas contactless trapping was realized at distances of 100 and 45 µm for Fresnel lens fibers and TIROFs, respectively. Very efficient trapping at laser powers as low as 3.7 mW was achieved. The bacteria vitality is an important parameter in trapping experiments. Mean square displacement and speed autocorrelation methods were applied to obtain a vitality measure and to classify the free bacteria trajectories into free floating, running, and run-wrap-run categories. The high frame rates of our observation videos allow us to reveal a relation between bacteria speed and bacteria orientation oscillations.

Pseudomonas species are ubiquitous in the environment and serve as valuable source of enzymes and secondary metabolites for industrial applications. P. aeruginosa secretes metalloproteases such as elastase LasB and produces bioactive small molecules, including pyocyanin, rhamnolipids, and pyoverdine, with potential biotechnological applications. However, the interest in P. aeruginosa for industrial use has been limited due to the virulence-associated Type III Secretion System (T3SS), a key factor in host-pathogen interactions. In this study, we genotypically and phenotypically characterized a collection of P. aeruginosa strains naturally lacking T3SS-encoding genes. Phylogenetic analysis revealed that these strains belong to two distinct clades. Several strains exhibited low or no cytotoxicity on epithelial cell lines and were avirulent in the Galleria infection model. The level of LasB and the three metabolites — pyocyanin, rhamnolipids, and pyoverdine — varied independently of virulence profiles. Notably, we identified avirulent strains capable of producing at least two secondary metabolites, including monorhamnolipids, highlighting their potential for biotechnological applications.

Pseudomonas species are ubiquitous in the environment and serve as valuable source of enzymes and secondary metabolites for industrial applications. Pseudomonas aeruginosa secretes metalloproteases, such as elastase LasB and produces bioactive small molecules, including pyocyanin, rhamnolipids, and pyoverdine, with potential biotechnological applications. However, the interest in P. aeruginosa for industrial use has been limited due to the virulence-associated Type III Secretion System (T3SS), a key factor in host–pathogen interactions. In this study, we genotypically and phenotypically characterized a collection of P. aeruginosa strains naturally lacking T3SS-encoding genes. Phylogenetic analysis revealed that these strains belong to two distinct clades. Several strains exhibited low or no cytotoxicity on epithelial cell lines and were avirulent in the Galleria infection model. The level of LasB and the three metabolites—pyocyanin, rhamnolipids, and pyoverdine—varied independently of virulence profiles. Notably, we identified avirulent strains capable of producing at least two secondary metabolites, including mono-rhamnolipids, highlighting their potential for biotechnological applications.

Hydrogels with antibacterial activities have the potential for many biomedical applications, such as wound healing, because of their capacity to maintain a moist environment and prevent infections. In this work, an ultrasound-induced supramolecular hydrogel consisting of easily accessible reducing-end-free glucosaminylbarbiturate-based hydrogelators that serve the in situ fabrication of silver nanoparticles (AgNPs), excluding the addition of any external reducing or stabilizing agents, has been developed. The innovative synthetic approach relied on the use of N,N'-disubstituted barbituric acid derivatives as a versatile chemical platform that site-selectively reacted with the amino function of glucosamine. A series of glucosaminylbarbiturate were synthesized, and we identified one carbohydrate-based hydrogelator that produced a thixotropic supramolecular hydrogel after ultrasound-mediated breaking of an intralocked hydrogen bond. AgNPs@hydrogels were prepared through in situ reduction of silver ions mediated by the reducing properties of carbohydrates. The AgNPs@hydrogel composite revealed good antimicrobial properties toward both Gram-positive and Gram-negative bacteria. These findings contribute to the development of carbohydrate-based supramolecular hydrogels and make them promising efficient and safe soft materials for antimicrobial therapy.

Pseudomonas aeruginosa is a major human opportunistic pathogen associated with a high incidence of multi-drug resistance. The antibody-based blockade of P. aeruginosa virulence factors represents a promising alternative strategy to mitigate its infectivity. In this study, we employed single B cell sorting from cystic fibrosis patients to isolate human monoclonal antibodies (mAbs) targeting proteins from the P. aeruginosa Type 3 Secretion System (T3SS) and characterized a panel of mAbs directed at PscF and PcrV. Among those, two mAbs, P5B3 and P3D6, that bind to the injectisome tip protein PcrV, exhibited T3SS blocking activity. We solved the crystal structure of the P3D6 Fab-PcrV complex, which revealed that the Ab binds to the C-terminal region of PcrV. In addition, we compared the T3SS-blocking activity of three PcrV-targeting mAbs, including two from previous independent studies, using two distinct assays to evaluate pore formation and toxin injection. We conducted a mechanistic and structural analysis of their modes of action through modeling based on the known structure of a functional homolog, SipD from Salmonella typhimurium . The analysis suggests that anti-PcrV mAbs may act through different mechanisms, ranging from preventing PcrV oligomerization to disrupting PcrV’s scaffolding function, thereby inhibiting the assembly and function of the translocon pore. Our findings provide additional evidence that T3SS-targeting Abs, some capable of inhibiting virulence, are elicited in P. aeruginosa -infected patients. The results offer deeper insights into PcrV recognition by mAbs and their associated mechanisms of action, helping to identify which Abs are more likely to be therapeutically useful based on their mode of action and potency. This paves the way for the development of effective alternatives to traditional antibiotics in the fight against this resilient pathogen.

Francisella tularensis is a highly virulent bacterium causing tularemia zoonosis. An increasing proportion of infections occur through contaminated hydro-telluric sources, especially for the subspecies holarctica ( Fth ). Although this bacterium has been detected in several aquatic environments, the mechanisms of its long-term persistence in water are not yet elucidated. We evaluated the culturability and the viability of a virulent Fth strain in independent microcosms filled with nutrient-poor water. At 37°C, the bacteria remained culturable for only one week, while culturability was extended to 6 weeks at 18°C and up to 11 weeks at 4°C. However, while the viability of the bacteria declined similarly to culturability at 37°C, the viability of the bacteria remained stable overtime at 18°C and 4°C for more than 24 months, long after loss of culturability. We identified water temperature as one of the major factors driving the aquatic survival of Fth through a transition of the whole Fth population in a viable but non-culturable (VBNC) state. Low temperature of water (≤18°C) favors the persistence of the bacteria in a VBNC state, while a temperature above 30°C kills culturable and VBNC Fth bacteria. These findings provide new insights into the environmental cycle of Francisella tularensis that suggest that the yet unidentified primary reservoir of the subspecies holarctica may be the aquatic environment itself in which the bacteria could persist for months or years without the need for a host.

Pseudomonas aeruginosa (P.a) is one of the most critical antibiotic resistant bacteria in the world and is the most prevalent pathogen in cystic fibrosis (CF), causing chronic lung infections that are considered one of the major causes of mortality in CF patients. Although several studies have contributed to understanding P.a within-host adaptive evolution at a genomic level, it is still difficult to establish direct relationships between the observed mutations, expression of clinically relevant phenotypes, and clinical outcomes. Here, we performed a comparative untargeted LC/HRMS-based metabolomics analysis of sequential isolates from chronically infected CF patients to obtain a functional view of P.a adaptation. Metabolic profiles were integrated with expression of bacterial phenotypes and clinical measurements following multiscale analysis methods. Our results highlighted significant associations between P.a “metabotypes”, expression of antibiotic resistance and virulence phenotypes, and frequency of clinical exacerbations, thus identifying promising biomarkers and therapeutic targets for difficult-to-treat P.a infections