Influence of vegetation and substrate type on removal of emerging organic contaminants and microbial dynamics in horizontal subsurface constructed wetlands.

Constructed wetlands (CWs) offer an efficient alternative technology for removing emerging organic contaminants (EOCs) from wastewater. Optimizing CW performance requires understanding the impact of CW configuration on EOC removal and microbial community dynamics. This study investigated EOC removal and microbial communities in horizontal subsurface flow (HSSF) CWs over a 26-month operational period. Comparison between tuff-filled and gravel-filled CWs highlighted the superior EOC removal in tuff-filled CWs during extended operation, likely caused by the larger surface area of the tuff substrate fostering microbial growth, sorption, and biodegradation. Removal of partially positively charged EOCs, like atenolol (29-98 %) and fexofenadine (21-87 %), remained constant in the different CWs, and was mainly attributed to sorption. In contrast, removal rates for polar non-sorbing compounds, including diclofenac (3-64 %), acyclovir (9-85 %), and artificial sweeteners acesulfame (5-60 %) and saccharin (1-48 %), seemed to increase over time due to enhanced biodegradation. The presence of vegetation and different planting methods (single vs. mixed plantation) had a limited impact, underscoring the dominance of substrate type in the CW performance. Microbial community analysis identified two stages: a startup phase (1-7 months) and a maturation phase (19-26 months). During this transition, highly diverse communities dominated by specific species in the early stages gave way to more evenly distributed and relatively stable communities. Proteobacteria and Bacteroidetes remained dominant throughout. Alphaproteobacteria, Acidobacteria, Planctomycetes, Salinimicrobium, and Sphingomonas were enriched during the maturation phase, potentially serving as bioindicators for EOC removal. In conclusion, this study emphasizes the pivotal role of substrate type and maturation in the removal of EOCs in HSSF CW, considering the complex interplay with EOC physicochemical properties. Insights into microbial community dynamics underscore the importance of taxonomic and functional diversity in assessing CW effectiveness. This knowledge aids in optimizing HSSF CWs for sustainable wastewater treatment, EOC removal, and ecological risk assessment, ultimately contributing to environmental protection.

Identification and Analysis of Fungal-Specific Regions in the Aspergillus fumigatus Cu Exporter CrpA That Are Essential for Cu Resistance but Not for Virulence.

The opportunistic fungus Aspergillus fumigatus is the primary invasive mold pathogen in humans, and is responsible for an estimated 200,000 yearly deaths worldwide. Most fatalities occur in immunocompromised patients who lack the cellular and humoral defenses necessary to halt the pathogen's advance, primarily in the lungs. One of the cellular responses used by macrophages to counteract fungal infection is the accumulation of high phagolysosomal Cu levels to destroy ingested pathogens. A. fumigatus responds by activating high expression levels of crpA, which encodes a Cu+ P-type ATPase that actively transports excess Cu from the cytoplasm to the extracellular environment. In this study, we used a bioinformatics approach to identify two fungal-unique regions in CrpA that we studied by deletion/replacement, subcellular localization, Cu sensitivity in vitro, killing by mouse alveolar macrophages, and virulence in a mouse model of invasive pulmonary aspergillosis. Deletion of CrpA fungal-unique amino acids 1-211 containing two N-terminal Cu-binding sites, moderately increased Cu-sensitivity but did not affect expression or localization to the endoplasmic reticulum (ER) and cell surface. Replacement of CrpA fungal-unique amino acids 542-556 consisting of an intracellular loop between the second and third transmembrane helices resulted in ER retention of the protein and strongly increased Cu-sensitivity. Deleting CrpA N-terminal amino acids 1-211 or replacing amino acids 542-556 also increased sensitivity to killing by mouse alveolar macrophages. Surprisingly, the two mutations did not affect virulence in a mouse model of infection, suggesting that even weak Cu-efflux activity by mutated CrpA preserves fungal virulence.

Horizontal Gene Transfer of Triazole Resistance in Aspergillus fumigatus.

Aspergillus fumigatus is the primary mold pathogen in humans. It can cause a wide range of diseases in humans, with high mortality rates in immunocompromised patients. The first-line treatments for invasive A. fumigatus infections are the triazole antifungals that inhibit Cyp51 lanosterol demethylase activity, blocking ergosterol biosynthesis. However, triazole-resistant strains of A. fumigatus are increasingly encountered, leading to increased mortality. The most common triazole resistance mechanisms in A. fumigatus are alterations in the cyp51A gene or promoter. We tested the hypothesis that A. fumigatus can acquire triazole resistance by horizontal gene transfer (HGT) of resistance-conferring gene cyp51A. HGT has not been experimentally analyzed in filamentous fungi. Therefore, we developed an HGT assay containing donor A. fumigatus strains carrying resistance-conferring mutated cyp51A, either in its chromosomal locus or in a self-replicating plasmid, and recipient strains that were hygromycin resistant and triazole sensitive. Donor and recipient A. fumigatus strains were cocultured and transferred to selective conditions, and the recipient strain tested for transferred triazole resistance. We found that chromosomal transfer of triazole resistance required selection under both voriconazole and hygromycin, resulting in diploid formation. Notably, plasmid-mediated transfer was also activated by voriconazole or hypoxic stress alone, suggesting a possible route to HGT of antifungal resistance in A. fumigatus, both in the environment and during host infection. This study provides, for the first time, preliminary experimental evidence for HGT mediating antifungal resistance in a pathogenic fungus. IMPORTANCE It is well known that bacteria can transfer antibiotic resistance from one strain to another by horizontal gene transfer (HGT), leading to the current worldwide crisis of rapidly emerging antibiotic-resistant bacteria. However, in fungi, HGT events have only been indirectly documented by whole-genome sequencing. This study directly examined fungal HGT of antibiotic resistance in a laboratory setting. We show that HGT of antifungal triazole resistance occurs in the important human fungal pathogen Aspergillus fumigatus. Importantly, we show a plasmid-mediated transfer of triazole resistance occurs under conditions likely to prevail in the environment and in infected patients. This study provides an experimental foundation for future work identifying the drivers and mechanistic underpinnings of HGT in fungi.

Transcriptional response of Aspergillus fumigatus to copper and the role of the Cu chaperones.

Aspergillus fumigatus is the leading cause of life-threatening invasive mold infections in immunocompromised individuals. This ubiquitous saprophyte possesses several natural attributes allowing it to evade the immune system, including the ability to withstand high toxic Cu concentrations within the phagosomes of macrophages and neutrophils. We previously established that at high levels, Cu binds and activates the A. fumigatus transcription factor AceA, which upregulates the expression of the Cu exporter CrpA to expel excess Cu. Deletion of aceA or crpA result in extreme Cu sensitivity and attenuated virulence.To identify other elements participating in resistance to Cu, we performed a genome-wide analysis of the transcriptome by RNAseq to analyze the AceA-dependent response of A. fumigatus to excess Cu. We deleted key genes whose transcription was strongly upregulated by high Cu, including those encoding homologs of the three Cu chaperones cox17, atx1 and ccs1. Detailed analysis of these genes indicates that in A. fumigatus, cox17 is an essential gene with a possible role in respiration, the atxA gene product participates in reductive iron uptake and ccsA encodes the Cu chaperone activating A. fumigatus Sod1. Interestingly, although the ccsA-null strain was extremely sensitive to high Cu and oxidative stress, it was not attenuated in virulence in a mouse model of invasive pulmonary aspergillosis.Our work provides (i) a detailed view of the genome-wide transcriptional response of A. fumigatus to excess Cu, (ii) identification of the AceA-dependent transcriptome and (iii) analysis of the roles of the three Cu chaperones cox17, atxA and ccsA.

Fate and removal of bacteria and antibiotic resistance genes in horizontal subsurface constructed wetlands: Effect of mixed vegetation and substrate type.

This study aimed to investigate the influence of cropping method and substrate type on the fate and the removal of bacterial and antibiotic resistance genes (ARGs) indicators from primary wastewater by constructed wetlands (CWs) during startup and maturation stages. Four small-scale CWs differing in their plantation pattern (monoculture vs. polyculture) and substrate type were constructed and operated under field conditions. While for bacteria, the greatest impact of the cropping method and substrate type on removal was during the startup stage rather than the maturation stage, for ARGs, such impact was significant at both stages. During startup, the removal efficiencies of heterotrophic bacteria, fecal coliforms, E. coli, 16S rRNA genes and lacZ increased with the operation time. At maturation, the removal efficiencies were constant and were within the range of 89.2-99.4%, 93.7-98.9%, 89-98.8%, 94.1-99.6% and 92.9-98.7%, respectively. The removal efficiencies of intl1, tetM, intl1, sul1, ermB and total ARGs were also increased with the operation time. However, they were ARG type and configuration-dependent; at maturation they ranged between 50.7%-89.4%, 85.9%-97%, 49.6%-92.9%, 58.2%-96.7% and 79.9-94.3%, respectively. The tuff-filled serially planted CW was also the only one capable of removing these genes at similar high efficiency. Metagenomic analysis showed that none of the ARGs was among the most common ARGs in water and biofilm samples; rather most ARGs belonged to bacterial efflux transporter superfamilies. Although ARGs were removed, they were still detected in substrate biofilm and their relative concentrations were increased in the effluents. While the removal of both bacteria and ARGs was higher during summer compared to winter, the season had no effect on the removal pattern of ARGs. Hence, combination of the serial plantation with substrate having high surface area is a potential strategy that can be used to improve the performance of CWs.

Effect of TPA and HTLV-1 Tax on BRCA1 and ERE controlled genes expression.

Interference with the expression and/or functions of the multifunctional tumor suppressor BRCA1 leads to a high risk of breast and ovarian cancers. BRCA1 expression is usually activated by the estrogen (E2) liganded ERα receptor. Activated ERα is considered as a potent transcription factor which activates various genes expression by 2 pathways. A classical pathway, ERα binds directly to E2-responsive elements (EREs) in the promoters of the responsive genes and a non-classical pathway where ERα indirectly binds with the appropriate gene promoter. In our previous study, HTLV-1Tax was found to strongly inhibit ERα induced BRCA1 expression while stimulating ERα induced ERE dependent genes. TPA is a strong PKC activator which found to induce the expression of HTLV-1. Here we examined the effect of TPA on the expression of BRCA1 and genes controlled by ERE region in MCF-7 cells and on Tax activity on these genes. Our results showed strong stimulatory effect of TPA on both BRCA1 and ERE expression without treatment with E2. Tax did not show any significant effect on these TPA activities. It seems that TPA activation of BRCA1 and ERE expression is dependent on PKC activity but not through the NFκB pathway. However, 53BP1 may be involved in this TPA activity because its overexpression significantly reduced the TPA stimulatory effect on BRCA1 and ERE expression. Additionally, our Chip assay results probably exclude possible involvement of ERα pathway in this TPA activity because TPA did not interfere with the binding of ERα to both BRCA1 promoter and ERE region.

Differential effects of HTLV-1 Tax oncoprotein on the different estrogen-induced-ER α-mediated transcriptional activities.

The activated estrogen (E2) receptor α (ERα) is a potent transcription factor that is involved in the activation of various genes by 2 different pathways; a classical and non-classical. In classical pathway, ERα binds directly to E2-responsive elements (EREs) located in the appropriate genes promoters and stimulates their transcription. However, in non-classical pathway, the ERα can indirectly bind with promoters and enhance their activity. For instance, ERα activates BRCA1 expression by interacting with jun/fos complex bound to the AP-1 site in BRCA1 promoter. Interference with the expression and/or functions of BRCA1, leads to high risk of breast or/and ovarian cancer. HTLV-1Tax was found to strongly inhibit BRCA1 expression by preventing the binding of E2-ERα complex to BRCA1 promoter. Here we examined Tax effect on ERα induced activation of genes by the classical pathway by testing its influence on E2-induced expression of ERE promoter-driven luciferase reporter (ERE-Luc). Our findings showed that E2 profoundly stimulated this reporter expression and that HTLV-1Tax significantly induced this stimulation. This result is highly interesting because in our previous study Tax was found to strongly block the E2-ERα-mediated activation of BRCA1 expression. ERα was found to produce a big complex by recruiting various cofactors in the nucleus before binding to the ERE region. We also found that only part of the reqruited cofactors are required for the transcriptional activity of ERα complex. Chip assay revealed that the binding of Tax to the ERα complex, did not interfere with its link to ERE region.

HTLV-1 Tax oncoprotein inhibits the estrogen-induced-ER α-Mediated BRCA1 expression by interaction with CBP/p300 cofactors.

BRCA1 is a multifunctional tumor suppressor, whose expression is activated by the estrogen (E2)-liganded ERα receptor and regulated by certain recruited transcriptional co-activators. Interference with BRCA1 expression and/or functions leads to high risk of breast or/and ovarian cancer. Another multifunctional protein, HTLV-1Tax oncoprotein, is widely regarded as crucial for developing adult T-cell leukemia and other clinical disorders. Tax profile reveals that it can antagonize BRCA1 expression and/or functionality. Therefore, we hypothesize that Tax expression in breast cells can sensitize them to malignant transformation by environmental carcinogens. Here we examined Tax effect on BRCA1 expression by testing its influence on E2-induced expression of BRCA1 promoter-driven luciferase reporter (BRCA1-Luc). We found that E2 strongly stimulated this reporter expression by liganding to ERα, which consequently associated with BRCA1 promoter, while ERα concomitantly recruited CBP/p300 to this complex for co-operative enhancement of BRCA1 expression. Introducing Tax into these cells strongly blocked this E2-ERα-mediated activation of BRCA1 expression. We noted, also, that Tax exerted this inhibition by binding to CBP/p300 without releasing them from their complex with ERα. Chip assay revealed that the binding of Tax to the CBP/p300-ERα complex, prevented its link to AP1 site. Interestingly, we noted that elevating the intracellular pool of CBP or p300 to excessive levels dramatically reduced the Tax-mediated inhibition of BRCA1 expression. Exploring the mechanism of this reduction revealed that the excessive co-factors were sufficient to bind separately the free Tax molecules, thus lowering their amount in the CBP/p300-ERα complex and relieving, thereby, the inhibition of BRCA1 expression.

Differential role of PKC-induced c-Jun in HTLV-1 LTR activation by 12-O-tetradecanoylphorbol-13-acetate in different human T-cell lines.

We have previously shown that TPA activates HTLV-1 LTR in Jurkat T-cells by inducing the binding of Sp1-p53 complex to the Sp1 site residing within the Ets responsive region 1 (ERR-1) of the LTR and that this activation is inhibited by PKCalpha and PKCepsilon. However, in H9 T-cells TPA has been noted to activate the LTR in two consecutive stages. The first stage is activation is mediated by PKCetta and requires the three 21 bp TRE repeats. The second activation mode resembles that of Jurkat cells, except that it is inhibited by PKCdelta. The present study revealed that the first LTR activation in H9 cells resulted from PKCetta-induced elevation of non-phosphorylated c-Jun which bound to the AP-1 site residing within each TRE. In contrast, this TRE-dependent activation did not occur in Jurkat cells, since there was no elevation of non-phosphorylated c-Jun in these cells. However, we found that PKCalpha and PKCepsilon, in Jurkat cells, and PKCetta and PKCdelta, in H9 cells, increased the level of phosphorylated c-Jun that interacted with the Sp1-p53 complex. This interaction prevented the Sp1-p53 binding to ERR-1 and blocked, thereby, the ERR-1-mediated LTR activation. Therefore, this PKC-inhibited LTR activation started in both cell types after depletion of the relevant PKCs by their downregulation. In view of these variable activating mechanisms we assume that there might be additional undiscovered yet modes of HTLV-1 LTR activation which vary in different cell types. Moreover, in line with this presumption we speculate that in HTLV-1 carriers the LTR of the latent provirus may also be reactivated by different mechanisms that vary between its different host T-lymphocyte subclones. Since this reactivation may initiate the ATL process, understanding of these mechanisms is essential for establishing strategies to block the possibility of reactivating the latent virus as preventive means for ATL development in carriers.

Role of caspase 9 in activation of HTLV-1 LTR expression by DNA damaging agents.

Adult T-cell leukemia (ATL) is caused by HTLV-I. The viral Tax oncoprotein plays a central role in initiating the process to ATL. However, after infection HTLV-1 enters into latency, during which virus gene expression is very low, so that the level of Tax is likely insufficient for exerting its oncogenic activities. Therefore only 5% of the infected individuals may develop ATL several decades after infection. It is assumed that the transition from latency to ATL development requires at least a temporary activation of the latent virus in order to elevate Tax to its oncogenic threshold. We have previously found that DNA damaging agents, which usually induce apoptosis, can also activate the viral LTR and that the anti apoptosis Bcl-2 protein not only avoid their apoptosis induction but concomitantly prevents their LTR activation effect. Therefore, the present study was designed to identify the factor that while participating in the apoptotic cascade acts also to activate the viral LTR. For this purpose we employed ectopic vectors expressing these apoptotic factors together with potent shRNAs against each of them and anti caspase peptide inhibitors. We have found that in addition to its function as initiator of the mitochondrial apoptotic cascade, caspase 9 can acts also as an executer which among other non-apoptotic functions it forms an Sp1-p53 complex that activates the LTR by binding to an Sp1 recognition site residing in the LTR. This finding can help in designing effective preventing strategies against ATL development in clinically latent HTLV-1 carriers.