A historical, economic, and technical-scientific approach to the current crisis in the development of antibacterial drugs: Promising role of antibacterial peptides in this scenario.

The emergence of antibiotic resistance (AMR) is a global public health problem. According to estimates, drug-resistant bacteria infect 2 million patients and perish 23,000 annually. To overcome this problem, antimicrobial peptides became a potential solution based on a new mechanism of action against bacteria. This article addresses the phenomenon of antibacterial resistance in most of its nuances, responding to historical, technical-scientific, and economic aspects. Likewise, it explores new therapeutic approaches to combat multi-resistant pathogens, specifically concerning antibacterial peptides, as a potential therapeutic tool to mitigate the current crisis of antibacterial drugs. It is expected that, with technological advances, especially with the advent and adoption of artificial intelligence, there will be an increase in diversified synthetic peptide production, which can face the challenges that we have in terms of antibacterial drugs.

Giving a Hand: Synthetic Peptides Boost the Antifungal Activity of Itraconazole against Cryptococcus neoformans.

Cryptococcus neoformans is a multidrug-resistant pathogen responsible for infections in immunocompromised patients. Here, itraconazole (ITR), a commercial antifungal drug with low effectiveness against C. neoformans, was combined with different synthetic antimicrobial peptides (SAMPs), Mo-CBP3-PepII, RcAlb-PepII, RcAlb-PepIII, PepGAT, and PepKAA. The Mo-CBP3-PepII was designed based on the sequence of MoCBP3, purified from Moringa oleifera seeds. RcAlb-PepII and RcAlb-PepIII were designed using Rc-2S-Alb, purified from Ricinus communis seed cakes. The putative sequence of a chitinase from Arabidopsis thaliana was used to design PepGAT and PepKAA. All SAMPs have a positive liquid charge and a hydrophobic potential ranging from 41-65%. The mechanisms of action responsible for the combined effect were evaluated for the best combinations using fluorescence microscopy (FM). The synthetic peptides enhanced the activity of ITR by 10-fold against C. neoformans. Our results demonstrated that the combinations could induce pore formation in the membrane and the overaccumulation of ROS on C. neoformans cells. Our findings indicate that our peptides successfully potentialize the activity of ITR against C. neoformans. Therefore, synthetic peptides are potential molecules to assist antifungal agents in treating Cryptococcal infections.

No Chance to Survive: Mo-CBP3-PepII Synthetic Peptide Acts on Cryptococcus neoformans by Multiple Mechanisms of Action.

Multidrug-resistant Cryptococcus neoformans is an encapsulated yeast causing a high mortality rate in immunocompromised patients. Recently, the synthetic peptide Mo-CBP3-PepII emerged as a potent anticryptococcal molecule with an MIC50 at low concentration. Here, the mechanisms of action of Mo-CBP3-PepII were deeply analyzed to provide new information about how it led C. neoformans cells to death. Light and fluorescence microscopies, analysis of enzymatic activities, and proteomic analysis were employed to understand the effect of Mo-CBP3-PepII on C. neoformans cells. Light and fluorescence microscopies revealed Mo-CBP3-PepII induced the accumulation of anion superoxide and hydrogen peroxide in C. neoformans cells, in addition to a reduction in the activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) in the cells treated with Mo-CBP3-PepII. In the presence of ascorbic acid (AsA), no reactive oxygen species (ROS) were detected, and Mo-CBP3-PepII lost the inhibitory activity against C. neoformans. However, Mo-CBP3-PepII inhibited the activity of lactate dehydrogenase (LDH) ergosterol biosynthesis and induced the decoupling of cytochrome c (Cyt c) from the mitochondrial membrane. Proteomic analysis revealed a reduction in the abundance of proteins related to energetic metabolism, DNA and RNA metabolism, pathogenicity, protein metabolism, cytoskeleton, and cell wall organization and division. Our findings indicated that Mo-CBP3-PepII might have multiple mechanisms of action against C. neoformans cells, mitigating the development of resistance and thus being a potent molecule to be employed in the production of new drugs against C. neoformans infections.

Antimicrobial susceptibility and production of virulence factors by bacteria recovered from bitches with pyometra.

Pyometra is one of the most common diseases in adult female dogs, characterized by a suppurative bacterial infection of the uterus with accumulation of inflammatory exudate and a variety of local and systemic clinical manifestations. This study aimed to identify the bacteria within the uterine content and vaginal canal of bitches with pyometra and evaluate their antimicrobial susceptibility and production of virulence factors. Uterine and vaginal content were collected with sterile swabs from 30 bitches diagnosed with pyometra. Bacteria were identified and assessed for their antimicrobial susceptibility and production of virulence factors, including biofilms, siderophores, proteases and hemolysins, both in planktonic and biofilm forms. A total of 82 bacterial isolates (35 uterus, 47 vagina), belonging to 21 species, were identified, with Escherichia coli as the most prevalent species (32/82, 39%). As for susceptibility, 39/79 (49.4%) isolates were resistant to one or more drugs, with resistance proportion among Gram-positive bacteria (87.5%) higher (p < .05) than that observed for Gram-negative bacteria (32.7%). Four coagulase-negative Staphylococcus species were resistant to methicillin. Regarding virulence, the isolates had low production of biofilms, siderophores, proteases and hemolysins, suggesting that the occurrence of pyometra might be more associated with host-related factors than bacterial virulence.

Skin-resident dendritic cells mediate postoperative pain via CCR4 on sensory neurons.

Inflammatory pain, such as hypersensitivity resulting from surgical tissue injury, occurs as a result of interactions between the immune and nervous systems with the orchestrated recruitment and activation of tissue-resident and circulating immune cells to the site of injury. Our previous studies identified a central role for Ly6Clow myeloid cells in the pathogenesis of postoperative pain. We now show that the chemokines CCL17 and CCL22, with their cognate receptor CCR4, are key mediators of this response. Both chemokines are up-regulated early after tissue injury by skin-resident dendritic and Langerhans cells to act on peripheral sensory neurons that express CCR4. CCL22, and to a lesser extent CCL17, elicit acute mechanical and thermal hypersensitivity when administered subcutaneously; this response abrogated by pharmacological blockade or genetic silencing of CCR4. Electrophysiological assessment of dissociated sensory neurons from naïve and postoperative mice showed that CCL22 was able to directly activate neurons and enhance their excitability after injury. These responses were blocked using C 021 and small interfering RNA (siRNA)-targeting CCR4. Finally, our data show that acute postoperative pain is significantly reduced in mice lacking CCR4, wild-type animals treated with CCR4 antagonist/siRNA, as well as transgenic mice depleted of dendritic cells. Together, these results suggest an essential role for the peripheral CCL17/22:CCR4 axis in the genesis of inflammatory pain via direct communication between skin-resident dendritic cells and sensory neurons, opening therapeutic avenues for its control.

Toxicity of spike fragments SARS-CoV-2 S protein for zebrafish: A tool to study its hazardous for human health?

Despite the significant increase in the generation of SARS-CoV-2 contaminated domestic and hospital wastewater, little is known about the ecotoxicological effects of the virus or its structural components in freshwater vertebrates. In this context, this study evaluated the deleterious effects caused by SARS-CoV-2 Spike protein on the health of Danio rerio, zebrafish. We demonstrated, for the first time, that zebrafish injected with fragment 16 to 165 (rSpike), which corresponds to the N-terminal portion of the protein, presented mortalities and adverse effects on liver, kidney, ovary and brain tissues. The conserved genetic homology between zebrafish and humans might be one of the reasons for the intense toxic effects followed inflammatory reaction from the immune system of zebrafish to rSpike which provoked damage to organs in a similar pattern as happen in severe cases of COVID-19 in humans, and, resulted in 78,6% of survival rate in female adults during the first seven days. The application of spike protein in zebrafish was highly toxic that is suitable for future studies to gather valuable information about ecotoxicological impacts, as well as vaccine responses and therapeutic approaches in human medicine. Therefore, besides representing an important tool to assess the harmful effects of SARS-CoV-2 in the aquatic environment, we present the zebrafish as an animal model for translational COVID-19 research.

Molecular basis of carrageenan-induced cytokines production in macrophages.

BACKGROUND: Low molecular weight carrageenan (Cg) is a seaweed-derived sulfated polysaccharide widely used as inflammatory stimulus in preclinical studies. However, the molecular mechanisms of Cg-induced inflammation are not fully elucidated. The present study aimed to investigate the molecular basis involved in Cg-induced macrophages activation and cytokines production. METHODS: Primary culture of mouse peritoneal macrophages were stimulated with Kappa Cg. The supernatant and cell lysate were used for ELISA, western blotting, immunofluorescence. Cg-induced mouse colitis was also developed. RESULTS: Here we show that Cg activates peritoneal macrophages to produce pro-inflammatory cytokines such as TNF and IL-1ß. While Cg-induced TNF production/secretion depends on TLR4/MyD88 signaling, the production of pro-IL-1ß relies on TLR4/TRIF/SYK/reactive oxygen species (ROS) signaling pathway. The maturation of pro-IL1ß into IL-1ß is dependent on canonical NLRP3 inflammasome activation via Pannexin-1/P2X7/K+ efflux signaling. In vivo, Cg-induced colitis was reduced in mice in the absence of NLRP3 inflammasome components. CONCLUSIONS: In conclusion, we unravel a critical role of the NLRP3 inflammasome in Cg-induced pro-inflammatory cytokines production and colitis, which is an important discovery on the pro-inflammatory properties of this sulfated polysaccharide for pre-clinical studies. Video abstract Carrageenan (Cg) is one the most used flogistic stimulus in preclinical studies. Nevertheless, the molecular basis of Cg-induced inflammation is not totally elucidated. Herein, Lopes et al. unraveled the molecular basis for Cg-induced macrophages production of biological active IL-1ß. The Cg-stimulated macrophages produces pro-IL-1ß depends on TLR4/TRIF/Syk/ROS, whereas its processing into mature IL-1ß is dependent on the canonical NLRP3 inflammasome.

Regulatory T cells counteract neuropathic pain through inhibition of the Th1 response at the site of peripheral nerve injury.

The inflammatory/immune response at the site of peripheral nerve injury participates in the pathophysiology of neuropathic pain. Nevertheless, little is known about the local regulatory mechanisms underlying peripheral nerve injury that counteracts the development of pain. Here, we investigated the contribution of regulatory T (Treg) cells to the development of neuropathic pain by using a partial sciatic nerve ligation model in mice. We showed that Treg cells infiltrate and proliferate in the site of peripheral nerve injury. Local Treg cells suppressed the development of neuropathic pain mainly through the inhibition of the CD4 Th1 response. Treg cells also indirectly reduced neuronal damage and neuroinflammation at the level of the sensory ganglia. Finally, we identified IL-10 signaling as an intrinsic mechanism by which Treg cells counteract neuropathic pain development. These results revealed Treg cells as important inhibitory modulators of the immune response at the site of peripheral nerve injury that restrains the development of neuropathic pain. In conclusion, the boosting of Treg cell function/activity might be explored as a possible interventional approach to reduce neuropathic pain development after peripheral nerve damage.