Biology subjects

Skurnik, D.

Publications and source records attributed to Skurnik, D..

6 recordsLinked to original sources

bioRxiv2022

Development of an antibody fused with an antimicrobial peptide targeting Pseudomonas aeruginosa: a new approach to prevent and treat bacterial infections

The increase of emerging drug resistant Gram-negative bacterial infections is of global concern. In addition, there is growing recognition that compromising the microbiota, through the use of broad spectrum antibiotics, may affect patient health in the long term. Therefore, there is the need to develop new -cidal strategies to combat Gram-negative infections that would consider these specific issues. In this study, we report and characterize one such approach, the antibody-drug conjugates (ADCs) that combine (i) targeting a specific pathogenic organism through a monoclonal antibody with (ii) the high killing activity of antimicrobial peptides. We focused on a major pathogenic Gram-negative bacterium associated with antibacterial resistance: Pseudomonas aeruginosa and designed an ADC by fusing an antimicrobial peptide at the C-terminal end of the VH and/or VL-chain of a monoclonal antibody, VSX, that targets the core of P. aeruginosa lipopolysaccharide (LPS). This ADC demonstrated appropriately minimal levels of toxicity to mammalian cells and rapidly kills P. aeruginosa strains through several mechanisms while protecting mice from P. aeruginosa lung infection when administered therapeutically. Furthermore, we found that the ADC was synergistic with several classes of antibiotics. This approach described in this study may result in a widely useful strategy to target specific pathogenic microorganisms without augmenting further antibiotic resistance. Author SummaryThe increasing of emerging drug resistant bacterial infections is a worldwide issue and infections caused by antibiotic resistant Gram-negative pathogens are particularly concerning. In addition, there is now growing recognition that disruption of the microbiota, through the use of broad spectrum antibiotics, may affect patient health in the long term. Therefore, there is the need to develop new -cidal strategies to combat Gram-negative infections while preserving the microbiota and also avoid enhancement of antibiotic resistance. We report and characterize here one such approach by using a specific monoclonal antibody associated with the potent killing activity of antimicrobial peptides in the form of an antibody-drug conjugate (ADC). The selected pathogenic bacterium was Pseudomonas aeruginosa, that presents numerous markers for both innate and acquired antibiotic resistance. The ADC lacked significant cytotoxicity against mammalian cells and was shown to be effective both in vitro and in vivo against P. aeruginosa.

microbiology↗

bioRxiv2022

Pre-clinical in vitro and in vivo characterization of a maternal vaccination before conception to protect against severe neonatal infections caused by Escherichia coli K1

Preterm birth remains the leading cause of neonatal morbidity and mortality today. Genetic, immunological, and infectious substrates are suspected. Preterm infants are at higher risk of severe neonatal infections and the main cause of bacterial infection in this population is Escherichia coli K1. Unfortunately, women with history of preterm birth have a high risk of recurrence. Therefore, these women constitute a target population for a vaccine, to date non-existent, against E. coli K1 to prevent these infections. In this study, we characterize the immunological and microbiological properties in adult female mice of a live attenuated vaccine candidate and the protection it conferred to newborn mice against severe infection caused by E. coli K1. We show that our E. coli K1 {Delta}aroA vaccine induces a strong immunity driven by polyclonal bactericidal antibodies. In our model of meningitis, pups born from mothers immunized before conception were strongly protected against different strains of E. coli K1 both in early-onset and late-onset diseases. Given the very high rate of mortality and neurological sequalae in neonatal meningitis caused by E. coli K1, this pre-clinical study provides a proof-of-concept for the development of a vaccine strategy against E. coli K1 severe infection in women at risk of preterm birth.

microbiology↗

bioRxiv2022

The HP1γ epigenetic silencer dampens IFN-γ response at the gut epithelial barrier

Interferon gamma (IFN-{gamma}) plays central roles in the pathophysiology of inflammatory bowel disease (IBD), both activating inflammatory responses and immunosuppressive functions. However, the epigenetic mechanisms controlling the expression of IFN-{gamma} responsive genes at the gut epithelial barrier are not well understood. In this study, we identified the epigenetic regulator HP1{gamma} as a transcriptional repressor of the IFN-{gamma}-responsive genes STAT1 (signal transducer and activator of transcription 1) and PD-L1 (Programmed Cell Death Ligand 1). Accordingly, HP1{gamma} gene inactivation in the mouse gut epithelium resulted in an immunopathology with a long-lasting up-regulation of STAT1 and PD-L1. Colon organoids models and in vitro cell lines showed that HP1{gamma} deficiency primed STAT1 and PD-L1 expressions, ultimately sensitizing epithelial cells to IFN-{gamma} stimulation. Chromatin immunoprecipitation experiments suggest that HP1 promoter tethering is involved in the silencing of gene expression. Overall, these results identify HP1{gamma} as an epigenetic silencing pathway controlling the IFN-{gamma} response at the epithelial barrier.

immunology↗

bioRxiv2022

A high-throughput sequencing approach identifies immunotherapeutic targets for bacterial meningitis in neonates

BackgroundWorldwide, Escherichia coli is the leading cause of neonatal Gram-negative bacterial meningitis, but full understanding of the pathogenesis of this disease is not yet achieved. Moreover, to date, no vaccine is available against bacterial neonatal meningitis. MethodsHere, we used Transposon Sequencing of saturated banks of mutants (TnSeq) to evaluate E. coli K1 genetic fitness in murine neonatal meningitis. We identified E. coli K1 genes encoding for factors important for systemic dissemination and brain infection, and focused on products with a likely outer-membrane or extra-cellular localization, as these are potential vaccine candidates. We used in vitro and in vivo models to study the efficacy of active and passive immunization. ResultsWe selected for further study the conserved surface polysaccharide Poly-{beta}-(1-6)-N-Acetyl Glucosamine (PNAG), as a strong candidate for vaccine development. We found that PNAG was a virulence factor in our animal model. We showed that both passive and active immunization successfully prevented and/or treated meningitis caused by E. coli K1 in neonatal mice. We found an excellent opsonophagocytic killing activity of the antibodies to PNAG and in vitro these antibodies were also able to decrease binding, invasion and crossing of E. coli K1 through two blood brain barrier cell lines. Finally, to reinforce the potential of PNAG as a vaccine candidate in bacterial neonatal meningitis, we demonstrated that Group B Streptococcus, the main cause of neonatal meningitis in developed countries, also produced PNAG and that antibodies to PNAG could protect in vitro and in vivo against this major neonatal pathogen. InterpretationAltogether, these results indicate the utility of a high-throughput DNA sequencing method to identify potential immunotherapy targets for a pathogen, including in this study a potential broad-spectrum target for prevention of neonatal bacterial infections. FundingsANR Seq-N-Vaq, Charles Hood Foundation, Hearst Foundation. Groupe Pasteur Mutualite

microbiology↗

bioRxiv2022

The human-specific miR-6762-5p is an activator of RhoA GTPase enhancing Shigella flexneri intercellular spreading

MicroRNAs have recently emerged as major players in host-bacterial pathogens interaction, either as part of the host defense mechanism to neutralize infection or as a bacterial arsenal aimed at subverting host cell functions. Here we identify the newly evolutionary emerged human microRNA miR-6762-5p as a new player in the host-Shigella interplay. A microarray analysis in infected epithelial cells allowed the detection of this miRNA exclusively during the late phase of infection. Conditional expression of miR-6762-5p combined with a transcriptome analysis indicated a role in cytoskeleton remodeling. Likewise, miR-6762-5p enhanced stress fibers formation through RhoA activation and in silico analysis identified several regulators of RhoA activity as potential direct transcriptional targets. We further showed that miR-6762-5p expression induces an increase in Shigella intercellular spreading, while miR-6762-5p inhibition reduced bacterial dissemination. Overall, we have identified a human-specific miR-6762-5p acting specifically at the Shigella dissemination step. We propose a model in which the expression of miR-6762-5p induces cytoskeleton modifications through RhoA activation to achieve a successful dissemination of Shigella in the host.

microbiology↗

bioRxiv2021

A qnr-plasmid allows aminoglycosides to induce SOS in Escherichia coli

The plasmid-mediated quinolone resistance (PMQR) genes have been shown to promote high-level bacterial resistance to fluoroquinolone antibiotics, potentially leading to clinical treatment failures. In Escherichia coli, sub-inhibitory concentrations (sub-MIC) of the widely used fluoroquinolones are known to induce the SOS response. Interestingly, the expression of several PMQR qnr genes is controlled by the SOS master regulator. During the characterization of a small qnrD-plasmid carried in E. coli, we observed that the aminoglycosides become able to induce the SOS response in this species, thus leading to the transcription of qnrD. We found that induction of the SOS response is due to nitric oxide (NO) accumulation in presence of sub-MIC of aminoglycosides. We demonstrated that the NO accumulation is driven by two plasmid genes, ORF3 and ORF4, whose products act at two levels. ORF3 encode a FAD-binding oxidoreductase which helps NO synthesis, while ORF4 code for an FNR-type transcription factor, related to an O2-responsive regulator of hmp expression, able to repress the Hmp-mediated NO detoxification pathway of E. coli. Thus, this discovery, that other major classes of antibiotics may induce the SOS response could have worthwhile implications for antibiotic stewardship efforts in preventing the emergence of resistance.

microbiology↗