Laura Nicolini

Role of ZnuABC and ZinT in Escherichia coli O157:H7 zinc acquisition and interaction with epithelial cells

BackgroundZinc is an essential element for all living cells. Recent studies have shown that the ZnuABC zinc uptake system significantly contributes to the ability of several pathogens to multiply in the infected host and cause disease, suggesting that zinc is scarcely available within different tissues of the host. To better understand the role of zinc in bacterial pathogenicity, we have undertaken a functional characterization of the role of the ZnuABC-mediated zinc uptake pathway in enterohemorrhagic Escherichia coli O157:H7.ResultsIn this work we have analyzed the expression and the role in metal uptake of ZnuA, the periplasmic component of the ZnuABC transporter, and of ZinT, another periplasmic protein which has been shown to contribute to zinc recruitment. We report that the expression of zinT and znuA, regulated by Zur, is induced in zinc-poor media, and that inactivation of either of the genes significantly decreases E. coli O157:H7 ability to grow in zinc depleted media. We also demonstrate that ZinT and ZnuA have not a redundant function in zinc homeostasis, as the role of ZinT is subordinated to the presence of ZnuA. Moreover, we have found that znuA and zinT are strongly induced in bacteria adhering to cultured epithelial cells and that lack of ZnuA affects the adhesion ability. In addition we have found that a fraction of apo-ZinT can be secreted outside the cell where the protein might sequester environmental zinc, inducing a condition of metal starvation in surrounding cells.ConclusionsThe here reported results demonstrate that ZnuABC plays a critical role in zinc uptake also in E. coli O157:H7 and that ZinT contributes to the ZnuA-mediated recruitment of zinc in the periplasmic space. Full functionality of the zinc import apparatus is required to facilitate bacterial adhesion to epithelial cells, indicating that the microbial ability to compete with the host cells for zinc binding is critical to establish successful infections. The observation that ZinT can be secreted when it is in the apo-form suggests that its presence in the extracellular environment may somehow contribute to metal uptake or facilitate bacterial colonization of the intestinal epithelia.

Solid state fermentation of orange peel and grape stalks by Pleurotus ostreatus, Agrocybe aegerita, and Armillariella mellea

SummarySolid state cultures of Pleurotus ostreatus, Agrocybe aegerita, and Armillariella mellea were carried out on orange peel (OP) and distillery grape stalks (GS), single or mixed, and compared with that on wheat straw (WS). Good levels of substrate colonization were achieved on OP and GS by P. ostreatus and A. aegerita, whereas A. mellea was grown on OP alone or mixed with GS. A. aegerita completed its life cycle producing basidiocarps on all substrates, while P. ostreatus fruited only on WS and OP+GS. A. mellea did not produce basidiocarps during the experiment. Indeed, P. ostreatus and A. aegerita depleted 50%–60% and 20%–30% of the lignin content, respectively, for OP and GS, while A. mellea degraded 22% of lignin only on GS. The latter fungus utilized only water soluble sugars on OP and OP+GS and so it would not be suitable for direct bioconversion of these raw materials. The results obtained, compared with those of the WS fermentation process, suggest the possibility of utilizing such lignocellulosic substrates as ruminant feed.

Distinctive functional features in prokaryotic and eukaryotic Cu, Zn superoxide dismutases

Abstract Bacterial and eukaryotic Cu,Zn superoxide dismutases show remarkable differences in the active site region and in their quaternary structure organization. We report here a functional comparison between four Cu,Zn superoxide dismutases from Gram-negative bacteria and the eukaryotic bovine enzyme. Our data indicate that bacterial dimeric variants are characterized by catalytic rates higher than that of the bovine enzyme, probably due to the solvent accessibility of their active site. Prokaryotic Cu,Zn superoxide dismutases also show higher resistance to hydrogen peroxide inactivation and lower HCO3 --dependent peroxidative activity. Moreover, unlike the eukaryotic enzyme, all bacterial variants are susceptible to inactivation by chelating agents and show variable sensitivity to proteolytic attack, with the E. coli monomeric enzyme showing higher rates of inactivation by EDTA and proteinase K. We suggest that differences between individual bacterial variants could be due to the influence of modifications at the dimer interface on the enzyme conformational flexibility.

Changes in in-vitro digestibility of orange peels and distillery grape stalks after solid-state fermentation by higher fungi

Abstract In-vitro digestibility of orange peels (OP) and distillery grape stalks (GS) was examined after solid-state fermentation by the basidiomycetes Pleurotus ostreatus, Agrocybe aegerita, and Armillariella mellea. The results obtained with these substrates, alone or mixed together, were compared with those from fermentation of wheat straw (WS). The digestibility of GS was enhanced by the solid-state growth of P. ostreatus (+ 16·22%) and A. aegerita (+ 4·24%); no improvement was obtained with A. mellea. The in-vitro-digestibility values of OP and OP + GS were similar to or lower than those of the unfermented substrates, and the only advantage observed was the loss of lignin content and a slight increase of crude protein in the end-products.

Regulatory and structural properties differentiating the chromosomal and the bacteriophage-associated Escherichia coli O157:H7 Cu, Zn Superoxide Dismutases

BackgroundHighly virulent enterohemorrhagic Escherichia coli O157:H7 strains possess three sodC genes encoding for periplasmic Cu, Zn superoxide dismutases: sodC, which is identical to the gene present in non-pathogenic E. coli strains, and sodC-F1 and sodC-F2, two nearly identical genes located within lambdoid prophage sequences. The significance of this apparent sodC redundancy in E. coli O157:H7 has not yet been investigated.ResultsWe report that strains deleted of one or more sodC genes are less resistant than the wild type strain to a challenge with hydrogen peroxide, thus confirming their involvement in the bacterial antioxidant apparatus. To understand if the different sodC genes have truly overlapping functions, we have carried out a comparison of the functional, structural and regulatory properties of the various E. coli O157:H7 SodC enzymes. We have found that the chromosomal and prophagic sodC genes are differentially regulated in vitro. sodC is exclusively expressed in aerobic cultures grown to the stationary phase. In contrast, sodC-F1 and sodC-F2 are expressed also in the logarithmic phase and in anaerobic cultures. Moreover, the abundance of SodC-F1/SodC-F2 increases with respect to that of SodC in bacteria recovered from infected Caco-2 cells, suggesting higher expression/stability of SodC-F1/SodC-F2 in intracellular environments. This observation correlates with the properties of the proteins. In fact, monomeric SodC and dimeric SodC-F1/SodC-F2 are characterized by sharp differences in catalytic activity, metal affinity, protease resistance and stability.ConclusionOur data show that the chromosomal and bacteriophage-associated E. coli O157:H7 sodC genes have different regulatory properties and encode for proteins with distinct structural/functional features, suggesting that they likely play distinctive roles in bacterial protection from reactive oxygen species. In particular, dimeric SodC-F1 and SodC-F2 possess physico-chemical properties which make these enzymes more suitable than SodC to resist the harsh environmental conditions which are encountered by bacteria within the infected host.

Virulence properties of type VII Streptococcus agalactiae (group B streptococci) and immunochemical analysis of capsular type polysaccharide.

Strains of a new polysaccharide type of group B streptococci (GBS), type VII, have been isolated from human carriers and invasive infections. Some of these strains bear the protein antigen c or R, as do other GBS serotypes. The capsular type polysaccharide is sialylated and this residue is involved in the immunodeterminant structure. All type VII strains examined were virulent in CD-1 mice; the LD50 after intraperitoneal (i.p.) challenge was 4.57 (SD 0.12) x10(7) cfu for the reference strain and 5.49 (SD 1.5) x10(7) cfu for clinical isolates. A particular feature of this serotype was the ability to induce septic arthritis not only when injected intravenously (i.v.), but also when injected i.p. Rabbit antiserum against the capsular type VII polysaccharide exhibited opsonic activity in a phagocytosis assay and protective activity against infection.

Sialic acid and biomass production by Streptococcus agalactiae under different growth conditions

A fermentation process to increase type capsular polysaccharide production by different serotypes of Streptococcus agalactiae (group B Streptococcus) was established. As sialic acid is an integral component of the polysaccharide, its synthesis was used to monitor polysaccharide, its synthesis was used to monitor polysaccharide production. Culture conditions, examined both on laboratory and pilot-plant scales, allowed optimal bacterial growth and high polysaccharide production in a medium composed of ultrafiltered Todd Hewitt broth supplemented with 2% (w/v) glucose and 1.5% (w/v) Na2HPO4, at a constant pH of 7.2. Studies using different gas atmospheres (air, CO2 or their mixture) showed that air greatly enhanced polysaccharide production.

Liver Enzyme Elevation During Darunavir-Based Antiretroviral Treatment in HIV-1–Infected Patients With or Without Hepatitis C Coinfection: Data from the ICONA Foundation Cohort

Abstract Objectives: To investigate differences in liver enzyme elevation (LEE) between HIV-infected patients with and without HCV coinfection who start a darunavir/ritonavir-containing regimen. Methods: HIV-infected patients enrolled in the Italian Cohort of Naïve to Antiretrovirals (ICONA) Foundation Study were included if they started darunavir/ritonavir for the first time. Patients were classified as not HCV coinfected, HCV active coinfected (HCV RNA positive), and HCV nonactive coinfected (HCV-Ab positive/HCV RNA negative). Time to LEE endpoint was defined using the ACTG toxicity scale, based on changes relative to baseline. Kaplan-Meier was used to estimate 1-year and 2-year probability of LEE. The incidence rate ratios (IRRs) of LEEs were estimated until the last follow-up (intention-to-treat analysis [ITT]) and up to darunavir/ritonavir discontinuation (on-treatment analysis [OT]). Results: Overall, 703 patients were included. Ninety-one were HCV-Ab positive; of those, 68 (9.7%) had active HCV coinfection. In 879 person-years of follow-up, 101 LEEs occurred (ITT). No severe hepatotoxicity event was registered in active HCV coinfected patients. HCV active coinfection was predictive of LEE in the overall population (OT: adjusted incidence rate ratio (IRR), 2.25; 95% CI, 0.70–7.24; P = .17; ITT: adjusted IRR, 3.62; 95% CI, 1.67–7.83; P .001) and in naïve patients (OT: adjusted IRR, 6.29; 95% CI, 2.54–15.55; P = .00; ITT: adjusted IRR, 3.87; 95% CI, 0.99–15.16; P = .05). Conclusions: No grade 3–4 LEEs occurred in HCV active coinfected patients. HCV active coinfected patients experienced low grade LEEs more frequently than HCV-Ab negative patients. Darunavir/ritonavir seems to be safe whatever the HCV status, when liver enzymes are carefully monitored.

Control of the HIV-1 DNA Reservoir Is Associated In Vivo and In Vitro with NKp46/NKp30 (CD335 CD337) Inducibility and Interferon Gamma Production by Transcriptionally Unique NK Cells

ABSTRACT The size of lentiviral DNA reservoirs reflects the effectiveness of immune responses against lentiviruses. So far, abundant information has been gathered on the control of HIV-1 replication. Understanding the innate mechanisms contributing to containment of the HIV DNA reservoir, however, are only partly clarified and are relevant to guiding interventions for reservoir containment or eradication. We studied the contribution of natural killer (NK) cell functional features in HIV patients controlling replication either spontaneously (HIV controllers [HIC]) or after progression and antiretroviral treatment (progressor patients [PP]). An inverse correlation between HIV DNA copy numbers (either total or integrated) in circulating CD4+ cells and NK cell function was observed. Induced interferon gamma (IFN-γ) production and NKp46/NKp30 activating receptor-induced expression correlated inversely with reservoir size. The correlation was present not only for a homogeneous cohort of HIC patients but also when PP were included in the analysis. Adaptive (NKG2C+ CD57+) NK cell features were not associated with reservoir size. However, a distinct set of 370 differentially expressed transcripts was found to underlie functional differences in NK cells controlling HIV DNA reservoir size. In proof-of-principle in vitro experiments of CD4+ cell infection with HIV-1, purified NK cells with the above-mentioned functional/transcriptional features displayed 10- and 30-fold higher abilities to control HIV replication and DNA burdens in vitro, respectively, than those of other NK cells. Thus, NK cells with a specific functional and transcriptional signature contribute to control of the HIV reservoir in CD4+ cells. Their selection, expansion, and/or adoptive transfer may support strategies to eradicate HIV-1 infection or to safely deescalate antiretroviral treatment. IMPORTANCE The most relevant feature of HIV-1 infection is represented by its DNA reservoir size in the body, which guarantees lifelong infection and resumption of virus replication after antiretroviral treatment interruption. So far, there has been little success in the identification of factors contributing to HIV-1 reservoir containment. In this study, by studying quantitative total and integrated HIV-1 DNA levels and NK cells in HIV-1 patients with either progressive or nonprogressive disease, we observed that inducible IFN-γ and natural cytotoxicity receptor (NCR) expression in a specific subset of NK cells with a characteristic transcriptional signature represents a correlate for HIV-1 reservoir control. This represents an advance in our understanding of the mechanism(s) that controls the lentivirus reservoir. Monitoring, selection, expansion, and adoptive transfer of these NK cells may allow monitoring of treatment efficacy and the likelihood of reservoir control and may support protocols for HIV-1 eradication.

Hepatitis C virus RNA levels at week-2 of telaprevir/boceprevir administration are predictive of virological outcome

BACKGROUND Triple therapy with telaprevir/boceprevir + pegylated-interferon+ribavirin can achieve excellent antiviral efficacy, but it can be burdened with resistance development at failure. AIMS To evaluate kinetics of hepatitis C virus (HCV) RNA decay and early resistance development, in order to promptly identify patients at highest risk of failure to first generation protease inhibitors. METHODS HCV-RNA was prospectively quantified in 158 patients receiving pegylated-interferon+ribavirin+telaprevir (N = 114) or+boceprevir (N = 44), at early time-points and during per protocol follow-up. Drug resistance was contextually evaluated by population sequencing. RESULTS HCV-RNA at week-2 was significantly higher in patients experiencing virological failure to triple-therapy than in patients with sustained viral response (2.3 [1.9-2.8] versus 1.2 [0.3-1.7]log IU/mL, p < 0.001). A 100 IU/mL cut-off value for week-2 HCV-RNA had the highest sensitivity (86%) in predicting virological success. Indeed, 23/23 (100%) patients with undetectable HCV-RNA reached success, versus 26/34 (76.5%) patients with HCV-RNA<100 IU/mL, and only 11/31 (35.5%) with HCV-RNA > 100 IU/mL (p < 0.001). Furthermore, differently from failing patients, none of the patient with undetectable HCV-RNA at week-2 had baseline/early resistance. CONCLUSIONS With triple therapy based on first generation protease inhibitors, suboptimal HCV-RNA decay at week-2 combined with early detection of resistance can help identifying patients with higher risk of virological failure, thus requiring a closer monitoring during therapy.