Priscilla Branchu

Les Escherichia coli entérohémorragiques (EHEC) sont responsables de toxi-infections alimentaires conduisant à des colites hémorragiques pouvant se compliquer d’un syndrome hémolytique et urémique. Une fois arrivés dans l’intestin, les EHEC adhèrent aux cellules épithéliales en causant des lésions d’attachement-effacement. Le système de sécrétion de type III et les protéines effectrices requis pour ce phénotype sont codés majoritairement par le locus d’effacement des entérocytes (LEE), constitué de plusieurs opérons (LEE1-5). Notre étude a permis de clarifier une des cascades de régulation contrôlant l’expression du LEE. Par des analyses en qRT-PCR et des immuno précipitations de la chromatine, nous avons déterminé que les régulateurs GadE et GadX sont des répresseurs indirects de l’expression du LEE. GadE active l’expression de gadX, et GadX réprime l’expression de ler, codant pour le principal activateur des opérons LEE2-5. De plus, GadE réprime aussi l’expression des opérons LEE4 et LEE5 indépendamment de Ler. En retour, Ler réprime l’expression de gadE et de gadX. Le monoxyde d’azote (NO) est un effecteur majeur de la réponse immune innée, produit en particulier par les cellules épithéliales intestinales. Il avait été montré que le NO réprime l’expression du LEE et active celle de gadE et de gadX. Notre étude a permis d’identifier le régulateur clé responsable de ces régulations, NsrR. NsrR réprime indirectement l’expression de gadE et gadX et active l’expression des opérons LEE1, LEE4 et LEE5 en se fixant sur leurs promoteurs respectifs. En présence de NO, NsrR devient inactif. Ainsi, le NO réprime directement l’expression du LEE en supprimant la fixation de NsrR aux promoteurs du LEE1, LEE4 et LEE5, et indirectement en activant l’expression de gadE et donc de gadX. Un modèle de régulation intégrant l’ensemble de ces résultats est proposé. D’autre part, nous avons identifié et caractérisé une nouvelle phosphodiestérase spécifique des EHEC les plus pathogènes, VmpA. Par son activité d’hydrolyse du di-GMPc, VmpA contrôle la mobilité bactérienne, la formation de biofilm, et probablement l’expression du LEE, mais aurait aussi un rôle plus général dans la physiologie des EHEC.

Pseudomonas aeruginosa , responsable d’infections sévères, souvent nosocomiales, est une bactérie dont la résistance aux antibiotiques constitue un enjeu majeur de santé publique. Le gène cprA , impliqué dans la résistance à la colistine, un antibiotique de dernier recours, joue également un rôle central dans la biogenèse de vésicules extracellulaires bactériennes spécifiques. Or, ces vésicules, produites en présence de colistine, possèdent un fort potentiel d’activation de l’inflammasome, en lien avec un blocage du flux autophagique dans les cellules hôtes. Ainsi, l’induction de l’expression de cprA par la colistine confère une résistance aux antibiotiques, mais augmente aussi la virulence bactérienne en exacerbant l’inflammation.

Outer membrane vesicles (OMVs) are nanosized vesicles naturally secreted by Gram‐negative bacteria and represent a promising platform for vaccine development. OMVs possess inherent immunostimulatory properties due to the presence of pathogen‐associated molecular patterns (PAMPs), providing self‐adjuvanting capabilities and the ability to elicit both innate and adaptive immune responses. This review outlines the advantages of OMVs over traditional vaccine strategies, including their safety, modularity, and the potential for genetic engineering to enable targeted antigen delivery. We describe approaches to enhance OMVs yield and immunogenicity, such as modifications to reduce lipopolysaccharide (LPS) toxicity and systems enabling antigen localization—either on the surface or within the lumen—using fusion constructs like ClyA, Lpp‐OmpA, AIDA‐I, Hbp, and Sec/Tat signal peptides. We further summarize preclinical applications of OMVs‐based vaccines targeting bacterial pathogens, viral infections, and cancer. In addition, we address key challenges in large‐scale production, purification, and long‐term stability, and explore strategies for conjugating or encapsulating heterologous antigens. Overall, OMVs offer a versatile and scalable extracellular vesicle‐based platform with strong potential for next‐generation vaccines targeting diverse infectious diseases and beyond.

CprA is a short‐chain dehydrogenase/reductase (SDR) that contributes to resistance against colistin and antimicrobial peptides. The cprA gene is conserved across Pseudomonas aeruginosa clades and its expression is directly regulated by the two‐component system PmrAB. We have shown that cprA expression leads to the production of outer membrane vesicles (OMVs) that block autophagic flux and have a greater capacity to activate the non‐canonical inflammasome pathway. In a murine model of sepsis, a P. aeruginosa strain deleted for cprA was less virulent than the wild‐type (WT) strain. These results demonstrate the important role of CprA in the pathogenicity of P. aeruginosa. It is worth noting that CprA is also a functional ortholog of hemolysin F (HlyF), which is encoded by virulence plasmids of Escherichia coli. We have shown that other cryptic SDRs encoded by mammalian and plant pathogens, such as Yersinia pestis and Ralstonia solanacearum are functional orthologs of CprA and HlyF. These SDRs also induce the production of OMVs which block autophagic flux. This study uncovers a new family of virulence determinants in Gram‐negative bacteria, offering potential for innovative therapeutic interventions and deeper insights into bacterial pathogenesis.

Enterohemorrhagic Escherichia coli (EHEC) are foodborne pathogens causing severe human infections including hemorrhagic colitis and hemolytic uremic syndrome (HUS), particularly in children. Ruminants are the main reservoir of EHEC which colonize their intestinal tract through a mechanism involving the bacterial outer membrane adhesin intimin. Vaccination of cattle has shown efficacy in reducing EHEC O157:H7 shedding in feces. However, most of these vaccines are based on purified proteins and/or require the addition of adjuvants, resulting in expensive vaccines that are not used by breeders. This study introduces the development of a new type of vaccine based on Outer Membrane Vesicles (OMVs) carrying the C-terminal domain of intimin (Int280). A vaccine which combines OMVs carrying luminal Int280 and OMVs displaying surface-exposed Int280 was produced using two addressing systems based on PelB peptide signal and Lpp-OmpA hybrid protein, respectively. This mixed vaccine was tested in a mouse model as a proof of concept using the murine host-specific intestinal pathogen Citrobacter rodentium which shares a similar intimin-based adhesion mechanism with EHEC. Vaccination of mice with OMV-Int280 elicited a strong anti-intimin IgG response. Interestingly, we observed a shortened C. rodentium fecal shedding duration in immunized mice compared to the control group. This OMVs-intimin vaccine therefore represents a promising candidate for the control of EHEC intestinal carriage and fecal shedding in ruminants. IMPORTANCE: Enterohemorrhagic Escherichia coli (EHEC) are foodborne pathogenic bacteria causing intestinal infection that may lead to hemorrhagic colitis and hemolytic uremic syndrome (HUS) particularly in young children. There is no effective treatment, and antibiotics are contraindicated because they promote the development of HUS. Vaccination of ruminants, the main reservoir of EHEC, has been proposed as an important strategy to reduce the fecal shedding of EHEC to reduce transmission to humans. Outer Membrane Vesicles (OMVs) derived from E. coli are a highly attractive vaccine platform. Here, we produced OMVs enriched with the C-terminal part of the intimin (Int280). As a proof of concept, we used a mice model of Citrobacter rodentium colonization as a surrogate for EHEC intestinal colonization. Vaccination elicited antibodies against intimin and decreased the duration of fecal shedding of C. rodentium . Therefore, this OMV-Int280 vaccine is a promising candidate to control EHEC intestinal carriage and fecal shedding in ruminants.

Up to 25% of the E. coli strains isolated from the feces of healthy humans harbor the pks genomic island encoding the synthesis of colibactin, a genotoxic metabolite. Evidence is accumulating for an etiologic role of colibactin in colorectal cancer. Little is known about the conditions of expression of colibactin in the gut. The intestine is characterized by a unique oxygenation profile, with a steep gradient between the physiological hypoxic epithelial surface and the anaerobic lumen, which favors the dominance of obligate anaerobes. Here, we report that colibactin production is maximal under anoxic conditions and decreases with increased oxygen concentration. We show that the aerobic respiration control (ArcA) positively regulates colibactin production and genotoxicity of pks+ E. coli in response to oxygen availability. Thus, colibactin synthesis is inhibited by oxygen, indicating that the pks biosynthetic pathway is adapted to the anoxic intestinal lumen and to the hypoxic infected or tumor tissue.

Colibactin is a genotoxin produced primarily by Escherichia coli harboring the genomic pks island (pks⁺ E. coli). Pks⁺ E. coli cause host cell DNA damage, leading to chromosomal instability and gene mutations. The signature of colibactin-induced mutations has been described and found in human colorectal cancer (CRC) genomes. An inflamed intestinal environment drives the expansion of pks⁺ E. coli and promotes tumorigenesis. Mesalamine (i.e., 5-aminosalycilic acid), an effective anti-inflammatory drug, is an inhibitor of the bacterial polyphosphate kinase (PPK). This drug not only inhibits the production of intestinal inflammatory mediators and the proliferation of CRC cells, but also limits the abundance of E. coli in the gut microbiota and diminishes the production of colibactin. Here, we describe the link between intestinal inflammation and colorectal cancer induced by pks⁺ E. coli. We discuss the potential mechanisms of the pleiotropic role of mesalamine in treating both inflammatory bowel diseases and reducing the risk of CRC due to pks⁺ E. coli.

The pks island codes for the enzymes necessary for synthesis of the genotoxin colibactin, which contributes to the virulence of Escherichia coli strains and is suspected of promoting colorectal cancer. From a collection of 785 human and bovine E. coli isolates, we identified 109 strains carrying a highly conserved pks island, mostly from phylogroup B2, but also from phylogroups A, B1 and D. Different scenarios of pks acquisition were deduced from whole genome sequence and phylogenetic analysis. In the main scenario, pks was introduced and stabilized into certain sequence types (STs) of the B2 phylogroup, such as ST73 and ST95, at the asnW tRNA locus located in the vicinity of the yersiniabactin-encoding High Pathogenicity Island (HPI). In a few B2 strains, pks inserted at the asnU or asnV tRNA loci close to the HPI and occasionally was located next to the remnant of an integrative and conjugative element. In a last scenario specific to B1/A strains, pks was acquired, independently of the HPI, at a non-tRNA locus. All the pks-positive strains except 18 produced colibactin. Sixteen strains contained mutations in clbB or clbD, or a fusion of clbJ and clbK and were no longer genotoxic but most of them still produced low amounts of potentially active metabolites associated with the pks island. One strain was fully metabolically inactive without pks alteration, but colibactin production was restored by overexpressing the ClbR regulator. In conclusion, the pks island is not restricted to human pathogenic B2 strains and is more widely distributed in the E. coli population, while preserving its functionality.

The emergence of new bacterial pathogens is a continuing challenge for agriculture and food safety. Salmonella Typhimurium is a major cause of foodborne illness worldwide, with pigs a major zoonotic reservoir. Two phylogenetically distinct variants, U288 and ST34, emerged in UK pigs around the same time but present different risk to food safety. Here we show using genomic epidemiology that ST34 accounts for over half of all S. Typhimurium infections in people while U288 less than 2%. That the U288 clade evolved in the recent past by acquiring AMR genes, indels in the virulence plasmid pU288-1, and accumulation of loss-of-function polymorphisms in coding sequences. U288 replicates more slowly and is more sensitive to desiccation than ST34 isolates and exhibited distinct pathogenicity in the murine model of colitis and in pigs. U288 infection was more disseminated in the lymph nodes while ST34 were recovered in greater numbers in the intestinal contents. These data are consistent with the evolution of S. Typhimurium U288 adaptation to pigs that may determine their reduced zoonotic potential.