In Crohn’s disease (CD) patients, the adherent-invasive Escherichia coli (AIEC) pathovar contributes to the chronic inflammation typical of the disease via its ability to invade gut epithelial cells and to survive in macrophages. We show that, in the AIEC strain LF82, inactivation of the pyrD gene, encoding dihydroorotate dehydrogenase (DHOD), an enzyme of the de novo pyrimidine biosynthetic pathway, completely abolished its ability of to grow in a macrophage environment-mimicking culture medium. In addition, pyrD inactivation reduced flagellar motility and strongly affected biofilm formation by downregulating transcription of both type 1 fimbriae and curli subunit genes. Thus, the pyrD gene appears to be essential for several cellular processes involved in AIEC virulence. Interestingly, vidofludimus (VF), a DHOD inhibitor, has been proposed as an effective drug in CD treatment. Despite displaying a potentially similar binding mode for both human and E. coli DHOD in computational molecular docking experiments, VF showed no activity on either growth or virulence-related processes in LF82. Altogether, our results suggest that the crucial role played by the pyrD gene in AIEC virulence, and the presence of structural differences between E. coli and human DHOD allowing for the design of specific inhibitors, make E. coli DHOD a promising target for therapeutical strategies aiming at counteracting chronic inflammation in CD by acting selectively on its bacterial triggers.

Antivirulence agents are considered a promising strategy to treat bacterial infections. Fluoropyrimidines possess antivirulence and antibiofilm activity against Gram-negative bacteria; however, their mechanism of action is yet unknown. Consistent with their known antibiofilm activity, fluoropyrimidines, particularly 5-fluorocytosine (5-FC), impair curli-dependent surface adhesion by Escherichia coli MG1655 via downregulation of curli fimbriae gene transcription. Curli inhibition requires fluoropyrimidine conversion into fluoronucleotides and is not mediated by c-di-GMP or the ymg-rcs envelope stress response axis, previously suggested as the target of fluorouracil antibiofilm activity in E. coli. In contrast, 5-FC hampered the transcription of curli activators RpoS and stimulated the expression of Fis, a curli repressor affected by nucleotide availability. This last observation suggested a possible perturbation of the de novo pyrimidine biosynthesis by 5-FC: indeed, exposure to 5-FC resulted in a ca. 2-fold reduction of UMP intracellular levels while not affecting ATP. Consistently, expression of the de novo pyrimidine biosynthesis genes carB and pyrB was upregulated in the presence of 5-FC. Our results suggest that the antibiofilm activity of fluoropyrimidines is mediated, at least in part, by perturbation of the pyrimidine nucleotide pool. We screened a genome library in search of additional determinants able to counteract the effects of 5-FC. We found that a DNA fragment encoding the unknown protein D8B36_18,480 and the N-terminal domain of the penicillin-binding protein 1b (PBP1b), involved in peptidoglycan synthesis, could restore curli production in the presence of 5-FC. Deletion of the PBP1b-encoding gene mrcB, induced csgBAC transcription, while overexpression of the gene encoding the D8B36_18,480 protein obliterated its expression, possibly as part of a coordinated response in curli regulation with PBP1b. While the two proteins do not appear to be direct targets of 5-FC, their involvement in curli regulation suggests a connection between peptidoglycan biosynthesis and curli production, which might become even more relevant upon pyrimidine starvation and reduced availability of UDP-sugars needed in cell wall biosynthesis. Overall, our findings link the antibiofilm activity of fluoropyrimidines to the redirection of at least two global regulators (RpoS, Fis) by induction of pyrimidine starvation. This highlights the importance of the de novo pyrimidines biosynthesis pathway in controlling virulence mechanisms in different bacteria and makes the pathway a potential target for antivirulence strategies.

Pseudomonas aeruginosa is the leading cause of death in cystic fibrosis (CF) patients, yet the genetic mechanisms driving its fitness in the host remain poorly defined. Previously collected transcriptomic data of clinical samples showed that expression of the gene PA14_RS04555 (sirB2) is stimulated in the CF lung environment. In this work, we show that sirB2 is regulated by the global transcriptional regulators Vfr and AmrZ. Loss of sirB2 markedly enhanced P. aeruginosa pathogenicity, increasing virulence in Galleria mellonella, and promoting bacterial translocation and biofilm formation in a differentiated airway epithelial infection model. Deletion of sirB2 triggered the emergence of biofilm-proficient rugose small colony variants (RSCVs), driven by elevated c-di-GMP and increased Pel polysaccharide production when cultures were grown in static conditions. The RSCV phenotype depends on suppressor mutations in the wsp operon, possibly as a response to redox imbalance caused by the lack of sirB2 under oxygen-limited conditions. Indeed, the sirB2 mutant exhibited impaired fitness during anaerobic respiration when nitrate was the sole electron acceptor, in a manner independent of the ubiquinone pool. Our findings show that sirB2 inactivation promotes RSCV emergence and identify sirB2 as a novel genetic determinant of metabolic fitness under host-relevant conditions, thereby underscoring the role of redox balance in chronic CF infections.

Biofilm can enhance antibiotic tolerance in bacteria, making treatment of biofilm-associated infections in clinical settings a significant challenge. 5-Fluorocytosine (5-FC), an FDA-approved drug mostly used as an antifungal, can hinder biofilm formation and production of virulence factors in Gram-negative bacteria. In this study, we tested 5-FC on nine uropathogenic Escherichia coli (UPEC) strains plus a fecal isolate. Our data indicated that 5-FC reduced curli fiber gene expression and inhibited virulence factors in UPEC strains. Unlike what was observed in other microorganisms, 5-FC antivirulence and antibiofilm properties were unaffected by either growth temperature or the medium pH, which might prove critical in urinary tract infection (UTI) treatment. Additionally, 5-FC impaired the expression of various UPEC virulence factors, including secreted toxins and type I and P fimbriae, thus leading to decreased UPEC adherence to bladder epithelial cells and improved survival of host cells. Finally, we found that a combination of 5-FC with β-lactams, but not other classes of antibiotics, significantly lowered the viability of bacteria in preformed biofilms. Despite a small set of pathogenic E. coli strains and an in vitro infection model, our findings strongly suggest that 5-FC might be a possible candidate as an antivirulence agent, particularly in a synergistic approach with β-lactam antibiotics.

Antimicrobial Blue Light (aBL) can be used to control the growth of pathogens in several applicative fields, from sanitization of inert surfaces to human skin treatment and from industry to food. Though the mechanism of action is still unknown, it has been hypothesized that specific wavelengths can activate potential endogenous photosensitizers in microbial cytoplasm and/or envelope. In turn, this photooxidative stress could induce inactivation of macromolecules resulting in bacterial killing. In this work, we investigated the effect of radiometric parameters of light at 410 nm on Escherichia coli K-12 MG1655, a strain rather tolerant to blue light irradiation. Interestingly, by changing the radiometric parameters of aBL protocol, different rates of killing were observed. Irradiation at 100 J/cm² caused a variable antimicrobial effect depending on the irradiance values. We observed an “irradiance effect”: namely, at higher irradiance values, the inhibitory effect is reduced. On the other hand, at increasing fluences the bactericidal rate increases. In addition, the shift from continuous to pulsed light could enhance the antimicrobial activity of protocols using higher irradiance values. Taken together, these results underline the importance of defining radiometric parameters to ensure the efficacy of aBL treatments and emphasize the importance of further research into the aBL mechanism.

Objectives: Biofilm increases bacterial antibiotic tolerance, posing a challenge for treating biofilm-associated infections in clinical settings. 5-fluorocytosine (5-FC), an FDA-approved antifungal and antitumor drug, has been shown to inhibit virulence factor production and biofilm formation in Gram-negative bacteria. This work aims to determine whether 5-FC antivirulence and antibiofilm activity are preserved in clinical E. coli isolates and to test possible synergies with antibiotics in treating preformed biofilm. Methods: 5-FC ability to inhibit biofilm formation, disrupt mature biofilm, and modulate virulence determinants was tested on uropathogenic E. coli (UPEC) clinical strains using the Crystal Violet-based biofilm adhesion and minimum biofilm eradication concentration assays. Gene expression was measured by RT-qPCR. The effect on bacterial viability within preformed biofilm was monitored using fluorescein diacetate and by determining colony-forming units in biofilm treated with or without 5-FC and specific antibiotics. Bladder epithelial cell cultures were used to assess the effect of 5-FC on UPEC adherence and cytotoxicity. Results: 5-FC inhibited biofilm formation in all tested UPEC strains. Gene expression analysis suggested that 5-FC concurrently inhibits the expression of genes encoding curli fibers, a major adhesion factor in E. coli , as well as other virulence determinants such as secreted toxins and type I and P fimbriae. Accordingly, 5-FC reduced UPEC adherence to epithelial cells and promoted host cell survival. Although 5-FC could not disrupt preformed biofilm, combining 5-FC with β-lactams drastically reduced the viability of biofilm-resident bacteria. Conclusions: 5-FC shows antibiofilm and antivirulence activity against uropathogenic E. coli strains by reducing the expression of several virulence factors and the overall UPEC pathogenic potential. In combination with β-lactams, 5-FC eradicates bacteria within mature biofilms, which are known to be highly refractory to antibiotic treatment. Our data suggest that 5-FC is an excellent candidate for preventing and treating bacterial infections associated with recalcitrant biofilms.