d-mannose treatment neither affects uropathogenic Escherichia coli properties nor induces stable FimH modifications

Abstract: Urinary tract infections (UTIs) are mainly caused by uropathogenic Escherichia coli (UPEC). Acute and recurrent UTIs are commonly treated with antibiotics, the efficacy of which is limited by the emergence of antibiotic resistant strains. The natural sugar d-mannose is considered as an alternative to antibiotics due to its ability to mask the bacterial adhesin FimH, thereby preventing its binding to urothelial cells. Despite its extensive use, the possibility that d-mannose exerts “antibiotic-like” activity by altering bacterial growth and metabolism or selecting FimH variants has not been investigated yet. To this aim, main bacterial features of the prototype UPEC strain CFT073 treated with d-mannose were analyzed by standard microbiological methods. FimH functionality was analyzed by yeast agglutination and human bladder cell adhesion assays. Our results indicate that high d-mannose concentrations have no effect on bacterial growth and do not interfere with the activity of different antibiotics. d-mannose ranked as the least preferred carbon source to support bacterial metabolism and growth, in comparison with d-glucose, d-fructose, and l-arabinose. Since small glucose amounts are physiologically detectable in urine, we can conclude that the presence of d-mannose is irrelevant for bacterial metabolism. Moreover, d-mannose removal after long-term exposure did not alter FimH’s capacity to bind to mannosylated proteins. Overall, our data indicate that d-mannose is a good alternative in the prevention and treatment of UPEC-related UTIs

Insights into the periplasmic proteins of Acinetobacter baumannii AB5075 and the impact of imipenem exposure. A proteomic approach

Carbapenem-resistant Acinetobacter baumannii strains cause life-threatening infections due to the lack of therapeutic options. Although the main mechanisms underlying antibiotic-resistance have been extensively studied, the general response to maintain bacterial viability under antibiotic exposure deserves to be fully investigated. Since the periplasmic space contains several proteins with crucial cellular functions, besides carbapenemases, we decided to study the periplasmic proteome of the multidrug-resistant (MDR) A. baumannii AB5075 strain, grown in the absence and presence of imipenem (IMP). Through the proteomic approach, 65 unique periplasmic proteins common in both growth conditions were identified: eight proteins involved in protein fate, response to oxidative stress, energy metabolism, antibiotic-resistance, were differentially expressed. Among them, ABUW_1746 and ABUW_2363 gene products presented the tetratricopeptide repeat motif, mediating protein-protein interactions. The expression switch of these proteins might determine specific protein interactions to better adapt to changing environmental conditions. ABUW_2868, encoding a heat shock protein likely involved in protection against oxidative stress, was upregulated in IMP-exposed bacteria. Accordingly, the addition of periplasmic proteins from A. baumannii cultured with IMP increased bacterial viability in an antioxidant activity assay. Overall, this study provides the first insights about the composition of the periplasmic proteins of a MDR A. baumannii strain, its biological response to IMP and suggests possible new targets to develop alternative antibiotic drugs

The Shigella flexneri OspB effector: an early immunomodulator

Through the action of the type three secretion system (T3SS) Shigella flexneri delivers several effectors into host cells to promote cellular invasion, multiplication and to exploit host-cell signaling pathways to modulate the host innate immune response. Although much progress has been made in the understanding of many type III effectors, the molecular and cellular mechanism of the OspB effector is still poorly characterized. In this study we present new evidence that better elucidates the role of OspB as pro-inflammatory factor at very early stages of infection. Indeed, we demonstrate that, during the first hour of infection, OspB is required for full activation of ERK1/2 and p38 MAPKs and the cytosolic phospholipase A2 (cPLA2). Activation of cPLA2 ultimately leads to the production and secretion of PMN chemoattractant metabolite(s) uncoupled with release of IL-8. Moreover, we also present evidence that OspB is required for the development of the full and promptly inflammatory reaction characteristic of S. flexneri wild-type infection in vivo. Based on OspB and OspF similarity (both effectors share similar transcription regulation, temporal secretion into host cells and nuclear localization) we hypothesized that OspB and OspF effectors may form a pair aimed at modulating the host cell response throughout the infection process, with opposite effects. A model is presented to illustrate how OspB activity would promote S. flexneri invasion and bacterial dissemination at early critical phases of infection

Polar localization of phon2, a periplasmic virulence-associated factor of Shigella flexneri, is required for proper IcsA exposition at the old bacterial pole

Proper protein localization is critical for bacterial virulence. PhoN2 is a virulence-associated ATP-diphosphohydrolase (apyrase) involved in IcsA-mediated actin-based motility of S. flexneri. Herein, by analyzing a ΔphoN2 mutant of the S. flexneri strain M90T and by generating phoN2::HA fusions, we show that PhoN2, is a periplasmic protein that strictly localizes at the bacterial poles, with a strong preference for the old pole, the pole where IcsA is exposed, and that it is required for proper IcsA exposition. PhoN2-HA was found to be polarly localized both when phoN2::HA was ectopically expressed in a Escherichia coli K-12 strain and in a S. flexneri virulence plasmid-cured mutant, indicating a conserved mechanism of PhoN2 polar delivery across species and that neither IcsA nor the expression of other virulence-plasmid encoded genes are involved in this process. To assess whether PhoN2 and IcsA may interact, two-hybrid and cross-linking experiments were performed. While no evidence was found of a PhoN2-IcsA interaction, unexpectedly the outer membrane protein A (OmpA) was shown to bind PhoN2-HA through its periplasmic-exposed C-terminal domain. Therefore, to identify PhoN2 domains involved in its periplasmic polar delivery as well as in the interaction with OmpA, a deletion and a set of specific amino acid substitutions were generated. Analysis of these mutants indicated that neither the (183)PAPAP(187) motif of OmpA, nor the N-terminal polyproline (43)PPPP(46) motif and the Y155 residue of PhoN2 are involved in this interaction while P45, P46 and Y155 residues were found to be critical for the correct folding and stability of the protein. The relative rapid degradation of these amino acid-substituted recombinant proteins was found to be due to unknown S. flexneri-specific protease(s). A model depicting how the PhoN2-OmpA interaction may contribute to proper polar IcsA exposition in S. flexneri is presented.Proper protein localization is critical for bacterial virulence. PhoN2 is a virulence-associated ATP-diphosphohydrolase (apyrase) involved in IcsA-mediated actin-based motility of S. flexneri. Herein, by analyzing a ΔphoN2 mutant of the S. flexneri strain M90T and by generating phoN2::HA fusions, we show that PhoN2, is a periplasmic protein that strictly localizes at the bacterial poles, with a strong preference for the old pole, the pole where IcsA is exposed, and that it is required for proper IcsA exposition. PhoN2-HA was found to be polarly localized both when phoN2::HA was ectopically expressed in a Escherichia coli K-12 strain and in a S. flexneri virulence plasmid-cured mutant, indicating a conserved mechanism of PhoN2 polar delivery across species and that neither IcsA nor the expression of other virulence-plasmid encoded genes are involved in this process. To assess whether PhoN2 and IcsA may interact, two-hybrid and cross-linking experiments were performed. While no evidence was found of a PhoN2-IcsA interaction, unexpectedly the outer membrane protein A (OmpA) was shown to bind PhoN2-HA through its periplasmic-exposed C-terminal domain. Therefore, to identify PhoN2 domains involved in its periplasmic polar delivery as well as in the interaction with OmpA, a deletion and a set of specific amino acid substitutions were generated. Analysis of these mutants indicated that neither the 183PAPAP187 motif of OmpA, nor the N-terminal polyproline 43PPPP46 motif and the Y155 residue of PhoN2 are involved in this interaction while P45, P46 and Y155 residues were found to be critical for the correct folding and stability of the protein. The relative rapid degradation of these amino acid-substituted recombinant proteins was found to be due to unknown S. flexneri-specific protease(s). A model depicting how the PhoN2-OmpA interaction may contribute to proper polar IcsA exposition in S. flexneri is presented. © 2014 Scribano et al

Strategie terapeutiche innovative basate sulla duplice attività, antibatterica e probiotica, del predatore batterico Bdellovibrio bacteriovorus

Introduzione: Bdellovibrio bacteriovorus preda batteri Gram-negativi ed è ubiquitario in vari ambienti, dove svolge un ruolo di equilibratore delle specie batteriche presenti. Molte sono le infezioni causate da biofilm microbici misti, di solito resistenti alle cure antibiotiche: ne è un esempio la Fibrosi Cistica (FC). Il primo obiettivo del presente studio è stato valutare l’attività antibatterica di B. bacteriovorus verso specie batteriche isolate da pazienti FC. Il secondo obiettivo è stato valutare l’utilizzo di B. bacteriovorus come probiotico, valutandone la presenza nell’ecosistema polimicrobico intestinale umano, in soggetti sani e affetti da Celiachia, Morbo di Crohn, Colite Ulcerosa, e FC. Metodi: L’attività contro biofilm preformati di Pseudomonas aeruginosa e Staphylococcus aureus è stata misurata sia in sistemi statici (piastre) che dinamici (bioflux). Con lo zymogramma è stata inoltre valutata l’attività litica contro tali ceppi. Al fine di valutare la presenza del B. bacteriovorus nell’ecosistema intestinale umano, il DNA totale di campioni fecali e bioptici collezionati da pazienti sani e malati è stato utilizzato per effettuare Real-time PCR con primers B. bacteriovorus-specifici. Risultati: I risultati ottenuti indicano una predazione di B. bacteriovorus non solo nei confronti del Gram-negativo P. aeruginosa, ma anche, e per la prima volta osservata, del Gram-positivo S. aureus. Le prove in statico hanno evidenziato una significativa diminuzione del 70% del biofilm dopo 24 ore di incubazione, mentre le prove in dinamico una diminuzione del 35% dopo 15 ore di incubazione. Inoltre, per la prima volta, abbiamo dimostrato la presenza di B. bacteriovorus a livello della mucosa intestinale e la sua significativa diminuzione nei soggetti malati, rispetto ai controlli. Conclusioni: I risultati ottenuti incoraggiano una più approfondita ricerca sul Bdellovibrio, per un suo eventuale utilizzo in strategie terapeutiche innovative volte sia a combattere infezioni sostenute da biofilm batterici (antibatterico), che al ripristino di ecosistemi microbici come quello intestinale (probiotico

Strategie terapeutiche innovative basate sulla duplice attivit\ue0, antibatterica e probiotica, del predatore batterico Bdellovibrio bacteriovorus

Introduzione: Bdellovibrio bacteriovorus preda batteri Gram-negativi ed \ue8 ubiquitario in vari ambienti, dove svolge un ruolo di equilibratore delle specie batteriche presenti. Molte sono le infezioni causate da biofilm microbici misti, di solito resistenti alle cure antibiotiche: ne \ue8 un esempio la Fibrosi Cistica (FC). Il primo obiettivo del presente studio \ue8 stato valutare l\u2019attivit\ue0 antibatterica di B. bacteriovorus verso specie batteriche isolate da pazienti FC. Il secondo obiettivo \ue8 stato valutare l\u2019utilizzo di B. bacteriovorus come probiotico, valutandone la presenza nell\u2019ecosistema polimicrobico intestinale umano, in soggetti sani e affetti da Celiachia, Morbo di Crohn, Colite Ulcerosa, e FC. Metodi: L\u2019attivit\ue0 contro biofilm preformati di Pseudomonas aeruginosa e Staphylococcus aureus \ue8 stata misurata sia in sistemi statici (piastre) che dinamici (bioflux). Con lo zymogramma \ue8 stata inoltre valutata l\u2019attivit\ue0 litica contro tali ceppi. Al fine di valutare la presenza del B. bacteriovorus nell\u2019ecosistema intestinale umano, il DNA totale di campioni fecali e bioptici collezionati da pazienti sani e malati \ue8 stato utilizzato per effettuare Real-time PCR con primers B. bacteriovorus-specifici. Risultati: I risultati ottenuti indicano una predazione di B. bacteriovorus non solo nei confronti del Gram-negativo P. aeruginosa, ma anche, e per la prima volta osservata, del Gram-positivo S. aureus. Le prove in statico hanno evidenziato una significativa diminuzione del 70% del biofilm dopo 24 ore di incubazione, mentre le prove in dinamico una diminuzione del 35% dopo 15 ore di incubazione. Inoltre, per la prima volta, abbiamo dimostrato la presenza di B. bacteriovorus a livello della mucosa intestinale e la sua significativa diminuzione nei soggetti malati, rispetto ai controlli. Conclusioni: I risultati ottenuti incoraggiano una pi\uf9 approfondita ricerca sul Bdellovibrio, per un suo eventuale utilizzo in strategie terapeutiche innovative volte sia a combattere infezioni sostenute da biofilm batterici (antibatterico), che al ripristino di ecosistemi microbici come quello intestinale (probiotico