Slaughterhouse Wastewater Treatment Plants (SWWTPs) are identified as hotspots for the dissemination of antimicrobial resistance, a global health concern. This study presents a novel investigation assessing the effects of advanced oxidation treatment on the removal of both antibiotic-resistant bacteria (ARBs) and antibiotic resistance genes (ARGs) from poultry slaughterhouse effluents, aiming to mitigate the environmental spread of antimicrobial resistance. Samples were collected from the influent and effluent of a poultry SWWTP located in Rio de Janeiro, Brazil.

Effluent samples underwent treatment with UV/H₂O₂ in a benchtop reactor at varied oxidizing agent concentrations and different pH levels. DNA and plasmid DNA (pDNA) were extracted from both influent and treated effluent samples and purified using the Wizard® DNA Clean-Up System (Promega) for qPCR analysis and shotgun sequencing on the MiSeq Illumina platform. Slaughterhouse wastewater harbors plasmid-linked ARGs associated with clinically relevant bacterial orders, such as Enterobacteriales. The SWWTP exhibited inefficient removal of these elements, emphasizing the role of slaughterhouse effluents in environmental AMR dissemination. UV/H₂O₂ treatment at pH 3 and 7 was notably effective in removing plasmids and reducing ARGs in the effluent. These results demonstrate that UV/H₂O₂ treatment is a potent strategy for reducing pathogen-associated ARGs.

Breast cancer is the most diagnosed and lethal cancer type among women worldwide, yet, despite recent advances in clinic research, therapies that present good efficacy and low toxicity and side effects are still needed to improve patient survival and overall life quality. The chaperone and proteasome networks, both part of the proteostasis system, are dysregulated in breast cancer and contribute to its malignancy and aggressiveness. In fact, proteasome inhibitors, such as oprozomib and bortezomib, have presented high potential as therapeutic options for breast cancer impairment. However, due recurrent drug-resistance, its efficacy is reduced. It this project, in which I participate as the main responsible for the experimental procedures, our objective is to explore the proteomic and transcriptomic landscape of the chaperone and proteasome networks in the disease and investigate the mechanisms responsible for the dysregulation of both systems in breast cancer, aiming to contribute to better understand its impact in breast cancer clinical and biological behavior, besides possible mechanism of proteasome-inhibitors resistance. Initially, we used bioinformatic approaches to predict transcriptional regulators of the proteostasis genes in breast cancer, and found NFY-A as a major regulator of chaperones, co-chaperones, and proteasome subunits.

We validated it impact in proteostasis gene expression by qPCR, and evaluated its impact in cell phenotypes by silencing NFY-A in vitro by siRNA and evaluating cell viability, proliferation, and colony formation, obtaining significant results (P-value < 0.05). After, we selected STIP1, a co-chaperone regulated by NFY-A, and performed a co-immunoprecipitation associated to mass-spectrometry to evaluate its interactome. We identified among its interaction partners the proteasome subunits PSMB2, PSME2, PSMB1, and PSMD2, and chaperones such as HSP90AB1, HSPA5, and HSP90B1. With our partial results, we could identify a common transcription regulator associated to the dysregulation of both chaperone and proteasome networks in breast cancer, shedding light on the origin of the super-activation of both systems in the disease. Also, we demonstrate that the proteostasis genes regulated by NFY-A compose an interactome, indicating a possible mechanism of interdependence of both systems. In the next steps, we intent to evaluate the proteasome activity in NFY-A silenced cells using the cell-based Proteasome-Glo™ assays (PROMEGA G8760), and determine the relationship between the aforementioned chaperones/co-chaperone with the activity and stability of the proteasome in vitro. In general, our project has potential to contribute to the understanding why breast tumors present dysregulated proteostasis and unravel the interplay of the chaperone and proteasome networks in the disease, providing insights of possible mechanisms of proteasome-inhibitors resistance.

Citrus canker disease is caused by Xanthomonas citri subsp. citri (Xcc), a bacterial pathogen that relies on the translocation of effector proteins called Transcription Activator-Like Effectors (TALEs) into host cells to successfully colonize citrus plants. The Xcc TALE PthA4, which is essential to cause canker, directly transactivates CsLOB1, a major citrus canker susceptibility gene. Recent studies have revealed that CsLOB1-INDUCED EXPANSIN 1 gene (CsLIEXP1) is highly and directly upregulated by CsLOB1 in Xcc-infected plants. Because expansins are associated with cell wall loosening, potentially facilitating bacterial colonization, the CsLOB1-dependent activation of CsLIEXP1 is thought to contribute to canker symptoms and disease progression. Thus, CsLIEXP1 likely represents a critical canker susceptibility gene. In this study, we employed CRISPR/Cas9 to disrupt the function of CsLIEXP1 by modifying its corresponding coding region in Citrus sinensis cv ‘Hamlin’ and evaluated the post-infection responses of edited plants.

The Agrobacterium-mediated transformation of epicotyls was performed using the EHA105 strain harboring the pDIRECT_22c vector carrying three sgRNAs targeting the first CsLIEXP1 exon. Sanger sequencing confirmed that CsLIEXP1-edited plants exhibited 26.4% of edited sequences. Three types of indels were obtained: a deletion of a thymine, a deletion of five nucleotides, and the insertion of a thymine CsLIEXP1-edited plants. For bacterial infection assays, leaves of edited and control plants were infiltrated with Xcc-GFP cell suspension at 104 UFC.mL-1 and analyzed by fluorescence and light microscopy 14 days after inoculation. Leaves of edited plants showed less canker symptoms with lesions limited to the site of bacterial inoculation and less pronounced cellular hypertrophy compared to control plants. Furthermore, CsLIEXP1 protein accumulation was reduced in CsLIEXP1-edited plants compared to wild-type plants when infected with Xcc.

These findings strengthen the notion that reduction of CsLIEXP1 protein levels confers tolerance to Xcc infection possibly by a mechanism involving reduced cell growth and expansion. Considering that CsLOB1 is both a susceptibility gene and a transcription factor associated with growth and development of plant organs, silencing of CsLOB1can be detrimental to plant growth, with potential impacts on development and productivity. Novel canker susceptibility genes downstream of CsLOB1, such as CsLIEXP1, can be better CRISPR targets for citrus canker control. Our results highlight the role of CsLIEXP1 as a canker susceptibility gene and provide new insights into plant-pathogen interactions. 

S. pneumoniae and the influenza virus are respiratory tract pathogens that cause high mortality worldwide. The influenza virus causes the flu, while the pneumococcus causes pneumonia and meningitis, as well as secondary infections in flu patients, worsening the clinical condition. Licensed pneumococcal vaccines are serotype-specific, which results in the replacement of circulating serotypes by non-vaccine serotypes. Therefore, aiming to develop a bivalent vaccine against these two pathogens, we constructed a recombinant influenza virus carrying the pneumococcal protein PspA (Flu-PspA), by reverse genetics. Thus, the objective of this work was to evaluate the effectiveness of a vaccine protocol using the Flu-PspA virus and recombinant PspA protein, in a murine model, via the intramuscular route. For this, C57BL/6 mice were immunized with Flu-PspA (1st dose) and with PspA protein adjuvanted with Alum (2nd dose), in the vaccine group; control virus Flu-CT (1st dose) and PspA protein adjuvanted with Alum or only Alum (2nd dose), or two doses of PBS, in the control groups. Fourteen days after the 2nd dose, anti-PspA and anti-influenza IgG antibodies were quantified by ELISA and the ability of anti-PspA antibodies to deposit complement (C3) on the surface of different pneumococcal strains was evaluated by flow cytometry.

Furthermore, 21 days after the last dose, the animals were lethally challenged with 7xLD50 of the invasive pneumococcal strain ATCC6303 or 100xLD50 of the influenza A/PR8/34 virus and survival monitored for ten days. Furthermore, three days after the lethal challenge, the bacterial load in bronchoalveolar lavage fluid (BALF) and blood were determined by titration and inflammatory cytokines in BALF were quantified by CBA. Thus, we observed that the animals in the vaccine group presented high levels of anti-PspA and anti-influenza IgG antibodies in the serum and the anti-PspA antibodies were effective in depositing complement in different pneumococcal strains, demonstrating a broad-spectrum vaccine response. Furthermore, we observed 100% protection after lethal challenges with pneumococcus and influenza, a reduction of two to three logs10 in pulmonary bacterial load and lower levels of inflammatory cytokines in BALF. Therefore, this vaccine protocol showed high protection and immunogenicity, being a promising bivalent vaccine strategy against influenza and pneumococcal pneumonia.

The present work deals with an innovative bivalent vaccine capable of protecting against the influenza virus and providing cross-protection against different pneumococcal serotypes. The technology is original and innovative and has a national application for invention to INPI with process number BR 1020220151636. Furthermore, it has a social and technological impact and can benefit the health of the Brazilian population, especially the portion of the population most susceptible and affected by the two diseases: children under 5 years of age, elderly , pregnant women, people with chronic diseases (e.g. asthmatics, people with obstructive lung diseases, heart disease).