Biology subjects

Caspar, Y.

Publications and source records attributed to Caspar, Y..

7 recordsLinked to original sources

bioRxiv2026

Selective inhibition of anaerobic ubiquinone biosynthesis in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a metabolically versatile opportunistic pathogen capable of adapting to diverse and challenging environments, including the oxygen- and nutrient-limited conditions encountered during chronic infections. Its ability to utilize a broad range of carbon sources and to switch from aerobic to anaerobic respiration contributes to its persistence within the host and its resilience to therapeutic interventions. In particular, denitrification enables P. aeruginosa to grow under oxygen-limited conditions by using nitrate and nitrite as alternative electron acceptors, a process particularly relevant in biofilms and chronic infections such as cystic fibrosis. Ubiquinone (UQ) is essential for respiratory metabolism in P. aeruginosa, which possesses two distinct UQ biosynthetic pathways that ensure UQ production across a broad range of oxygen concentrations. Recent studies have identified the O2-independent UQ biosynthetic pathway as a key determinant of chronic infection. Because this pathway is restricted to a limited group of bacteria, we hypothesized that its selective inhibition could represent a strategy to specifically target P. aeruginosa under anaerobic conditions. To test this hypothesis, we screened analogues of 4-hydroxybenzoic acid (4-HB), the earliest precursor of the UQ biosynthetic pathway, and identified 4-aminobenzoic acid, also known as para-aminobenzoic acid (pABA), as a potential inhibitor. pABA primarily affects the anaerobic metabolism of P. aeruginosa by acting as a competing substrate for UQ biosynthetic enzymes, thereby interfering with UQ biosynthesis. This inhibitory effect positions pABA as a valuable tool for investigating, and potentially targeting, anaerobic metabolism in P. aeruginosa.

microbiology↗

bioRxiv2025

Non-destructive label-free automated identification of bacterial colonies at the species level directly on agar media using digital holography and convolutional neural network algorithms

Objectivesthis study aimed to develop a fully automated, non-destructive and label-free identification method of bacterial colonies, directly on agar plates, using a combination of digital holography and artificial intelligence and to evaluate its performances. Methodhigh-resolution holographic images of individual colonies on translucent brain-heart agar plates were taken every 30 minutes throughout an 18-hour incubation period (530 MPx for the full plate) using a large field 1x magnification system, a partially coherent LED light source and a high-resolution CMOS sensor. A database containing 49 490 digital holograms of individual colonies from 276 clinical strains belonging to ten of the most prevalent pathogenic bacterial species was used to train the convolutional neural network (CNN). Improvement in the accuracy of the prediction from the CNN algorithms was achieved using the information at different phylogenetic levels. Resultsthe performance of the BAIO-DX solution was assessed on 232 strains belonging to the 10 species used to train the algorithms but also on 64 strains from 8 species not included in the training database. For the species included in the training dataset, this new method identified 86.6% of the strains at the species level with a positive-percent agreement of 96.5%. An additional 48% of the strains not identified at the species level could be identified at the genus level thanks to the phylogenetic interpretation of the results. Conclusionsthese first results validate this approach as a candidate to obtain a fully automated non-destructive and label-free solution for bacterial identification in clinical microbiology laboratories. IMPORTANCE STATEMENTIdentification of pathogenic bacteria by culture-based methods are typically performed using MALDI-TOF mass spectrometry or biochemical systems. While automation and interpretive algorithms based on agar plate imaging and artificial intelligence (AI) has reduced manual steps, bacterial identification is still labor-intensive. Here we developed a fully automated, non-destructive and label-free identification method of bacterial colonies at the species level, directly on agar plates, using a combination of digital holography and convolutional neural network algorithms. After training the system with 276 strains belonging to ten of the most frequent pathogenic bacterial species, the BAIO-DX solution was able to identify 86.6% of new strains from these 10 species with a positive-percent agreement of 96.5%. These thorough proof of concept shows that imaging methods coupled to AI algorithms are promising to reach a fully automated identification of a significant proportion of pathogenic bacteria and has potential to enhance diagnostic workflows in clinical microbiology.

microbiology↗

bioRxiv2025

Avirulent Pseudomonas aeruginosa T3SS-negative strains belonging to Clade 5 produce variable quantities of secondary metabolites

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.

microbiology↗

bioRxiv2024

Protective human monoclonal antibodies that target the Type III Secretion System of Pseudomonas aeruginosa inhibit translocon function

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 PcrVs 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.

microbiology↗

bioRxiv2022

Aquatic long-term persistence of Francisella tularensis ssp. holarctica is driven by water temperature and transition to a viable but non-culturable state

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{degrees}C, the bacteria remained culturable for only one week, while culturability was extended to 6 weeks at 18{degrees}C and up to 11 weeks at 4{degrees}C. However, while the viability of the bacteria declined similarly to culturability at 37{degrees}C, the viability of the bacteria remained stable overtime at 18{degrees}C and 4{degrees}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{degrees}C) favors the persistence of the bacteria in a VBNC state, while a temperature above 30{degrees}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.

microbiology↗

bioRxiv2021

The biosynthetic pathway of ubiquinone contributes to pathogenicity of Francisella

Francisella tularensis is the causative agent of tularemia. Because of its extreme infectivity and high mortality rate, this pathogen was classified as a biothreat agent. Francisella spp are strict aerobe and ubiquinone (UQ) has been previously identified in these bacteria. While the UQ biosynthetic pathways were extensively studied in Escherichia coli allowing the identification of fifteen Ubi-proteins to date, little is known about Francisella spp. In this study, and using Francisella novicida as a surrogate organism, we first identified UQ8 as the major quinone found in the membranes of this bacterium. Then, we characterized the UQ biosynthetic pathway in F. novicida using a combination of bioinformatics, genetics and biochemical approaches. Our analysis disclosed the presence in Francisella of ten putative Ubi-proteins and we confirmed eight of them by heterologous complementation in E. coli. The UQ biosynthetic pathways from F. novicida and E. coli share a similar pattern. However, differences were highlighted: the decarboxylase remains unidentified in Francisella spp and homologs of the Ubi-proteins involved in the O2-independent UQ pathway are not present. This is in agreement with the strictly aerobic niche of this bacterium. Then, via two approaches, i.e. the use of an inhibitor (3-amino-4-hydroxybenzoic acid) and a transposon mutant, which both strongly impair the synthesis of UQ, we demonstrated that UQ is essential for the growth of F. novicida in a respiratory medium and contributes to its pathogenicity in Galleria mellonella used as an alternative animal model. ImportanceFrancisella tularensis is the causative bacterium of tularemia and is classified as a biothreat agent. Using multidisciplinary approaches, we investigated the ubiquinone (UQ) biosynthetic pathway that operates in F. novicida used as a surrogate. We showed that UQ8 is the major quinone identified in the membranes of Francisella novicida. We identified a new competitive inhibitor, which strongly decreased the biosynthesis of UQ. Our demonstration of the crucial role of UQ for the respiratory metabolism of F. novicida and for the involving in its pathogenicity in the Galleria mellonella model should stimulate the search for selective inhibitors of bacterial UQ biosynthesis.

microbiology↗

bioRxiv2020

Bacterial behavior in human blood reveals complement evaders with persister-like features

AbstractsBacterial bloodstream infections (BSI) are a major health concern and can cause up to 40% mortality. Pseudomonas aeruginosa BSI is often of nosocomial origin and is associated with a particularly poor prognosis. The mechanism of bacterial persistence in blood is still largely unknown. Here, we analyzed the behavior of a cohort of clinical and laboratory Pseudomonas aeruginosa strains in human blood. In this specific environment, complement was the main defensive mechanism, acting either by direct bacterial lysis or by opsonophagocytosis, which required recognition by immune cells. We found highly variable survival rates for different strains in blood, whatever their origin, serotype, or the nature of their secreted toxins and despite their detection by immune cells. We identified and characterized a complement-tolerant subpopulation of bacterial cells that we named "evaders". Evaders represented 0.1-0.001% of the initial bacterial load and displayed transient tolerance. Although evaders shared some features with bacterial persisters, which tolerate antibiotic treatment, they appear to have evolved distinct strategies to escape complement. We detected the evaders for five other major human pathogens: Acinetobacter baumannii, Burkholderia multivorans, enteroaggregative Escherichia coli, Klebsiella pneumoniae, and Yersinia enterocolitica. Thus, the evaders could allow the pathogen to persist within the bloodstream, and may be the cause of fatal bacteremia or dissemination, notably in the absence of effective antibiotic treatments. Author summary for "Complement evaders"Blood infections by antibiotic resistant bacteria, notably Pseudomonas aeruginosa, are major concerns in hospital settings. The complex interplay between P. aeruginosa and the innate immune system in the context of human blood is still poorly understood. By studying the behavior of various P. aeruginosa strains in human whole blood and plasma, we showed that bacterial strains display different rate of tolerance to the complement system. Despite the complement microbicide activity, most bacteria withstand elimination through phenotypic heterogeneity creating a tiny (<0.1%) subpopulation of transiently tolerant evaders. While genetically identical to the rest of the complement-sensitive population, evaders allow the bacteria to persist in plasma. This phenotypic heterogeneity thus prevents total elimination of the pathogen from the circulation, and represent a new strategy to disseminate within the organism.

microbiology↗