Serena Ammendola

Salmonella enterica serovar Typhimurium can enter and colonize the apoplast of plants, including the edible ones, making them a potential reservoir for the pathogen and a route for human contamination. We previously showed that in Arabidopsis thaliana shoot colonization, S. Typhimurium takes advantage of its ability to export zinc primarily through the P_1B-type ATPase ZntA induced by zinc excess. Moreover, a plant line with reduced ability to translocate zinc from roots to shoots is more susceptible to S. Typhimurium shoot colonization. We, therefore, hypothesized that plants employ zinc intoxication as a defense strategy against invading bacteria. Here we show that, upon S. Typhimurium colonization, A. thaliana modulates the expression of zinc transporters, favoring the long-distance movement of the metal as well as lowering zinc storage into the plant vacuoles. Notably, we demonstrate that this strategy depends on the recognition of bacterial flagellin by the FLS2 receptor, a signal that triggers the PAMP-Triggered Immunity response in plants. Disrupting this interaction, either using an S. Typhimurium Δfla strain or an A. thaliana fls2 mutant line, reduces the zntA expression in shoot-colonizing bacteria. This observation is confirmed by a luminescent zinc biosensor assay, showing that the A. thaliana fls2 does not increase bioavailable zinc in the S. Typhimurium colonized shoots. Moreover, gene expression analyses in the colonized fls2 line revealed a downregulation of the root-to-shoot zinc translocation compared to Col-0. Overall, our results suggest that flagellin recognition by plants triggers zinc fluxes towards the invading bacteria as a facet of the PAMP-Triggered Immunity response, highlighting the importance of plant zinc homeostasis for the interaction with human pathogens.

Nutritional immunity, a key component of the vertebrate innate immune response, involves the modulation of zinc availability to limit the growth of pathogens. Pseudomonas aeruginosa counteracts host-imposed zinc starvation through metabolic adaptations, including reprogramming of gene expression and activating efficient metal uptake systems. To unravel how zinc shortage contributes to the complexity of bacterial adaptation to the host environment, it is critical to use model systems that mimic fundamental features of P. aeruginosa-related diseases in humans. Among available animal models, Galleria mellonella has recently emerged as a promising alternative to mammalian hosts. This study aims to evaluate whether G. mellonella can recapitulate the zinc-related nutritional immunity responses observed in mammalian infections. Our results show that, upon P. aeruginosa infection, the larvae upregulate several zinc transporters, suggesting an active redistribution of the metal in response to the pathogen. Additionally, P. aeruginosa colonizing the larvae induces Zn uptake regulator-controlled genes, consistent with bacterial adaptation to zinc starvation. Disruption of bacterial zinc uptake capability significantly reduces P. aeruginosa virulence, underscoring the importance of zinc acquisition in pathogenesis also within this model host. As a proof of concept, we also demonstrate that this in vivo model can serve as a viable preliminary screening tool to unveil novel players involved in P. aeruginosa response to zinc starvation, offering valuable insights into the host–pathogen battle for micronutrients.

Limiting the availability of transition metals at infection sites serves as a critical defense mechanism employed by the innate immune system to combat microbial infections. Pseudomonas aeruginosa exhibits a remarkable ability to thrive in zinc-deficient environments, facilitated by intricate cellular responses governed by numerous genes regulated by the zinc-responsive transcription factor Zur. Many of these genes have unknown functions, including those within the predicted PA2911-PA2914 and PA4063-PA4066 operons. A structural bioinformatics investigation revealed that PA2911-PA2914 comprises a TonB-dependent outer membrane receptor and inner membrane ABC-permeases responsible for importing metal-chelating molecules, whereas PA4063-PA4066 contains genes encoding a MacB transporter, likely involved in the export of large molecules. Molecular genetics and biochemical experiments, feeding assays, and intracellular metal content measurements support the hypothesis that PA2911-PA2914 and PA4063-PA4066 are engaged in the import and export of the pyochelin-cobalt complex, respectively. Notably, cobalt can reduce zinc demand and promote the growth of P. aeruginosa strains unable to import zinc, highlighting pyochelin-mediated cobalt import as a novel bacterial strategy to counteract zinc deficiency. These results unveil an unexpected role for pyochelin in zinc homeostasis and challenge the traditional view of this metallophore exclusively as an iron transporter. IMPORTANCE: The mechanisms underlying the remarkable ability of Pseudomonas aeruginosa to resist the zinc sequestration mechanisms implemented by the vertebrate innate immune system to control bacterial infections are still far from being fully understood. This study reveals that the Zur-regulated gene clusters PA2911-2914 and PA4063-PA4066 encode systems for the import and export of cobalt-bound pyochelin, respectively. This proves to be a useful strategy to counteract conditions of severe zinc deficiency since cobalt can replace zinc in many proteins. The discovery that pyochelin may contribute to cellular responses to zinc deficiency leads to a reevaluation of the paradigm that pyochelin is a siderophore involved exclusively in iron acquisition and suggests that this molecule has a broader role in modulating the homeostasis of multiple metals.

Editorial: Dealing with unusual hosts and unconventional habitats: versatile strategies of Salmonella enterica

Tea Tree Oil (TTO) is an essential oil obtained from the distillation of Melaleuca alternifolia leaves and branches. Due to its beneficial properties, TTO is widely used as an active ingredient in antimicrobial preparations for topical use or in cosmetic products and contains about 100 different compounds, with terpinen-4-ol, γ-terpinene and 1,8-cineole (or eucalyptol) being the molecules most responsible for its biological activities. In this work, the antimicrobial activity of whole TTO and these three major components was evaluated in vitro against fungi, bacteria and viruses. Molecular dynamics simulations were carried out on a bacterial membrane model and a Coxsackievirus B4 viral capsid, to propose an atomistic explanation of their mechanism of action. The obtained results indicate that the strong antimicrobial activity of TTO is attributable to the induction of an altered membrane functionality, mediated by the incorporation of its components within the lipid bilayer, and to a possible ability of the compounds to bind and alter the structural properties of the viral capsid.

Aims Death domain-associated protein (DAXX) and/or α-thalassemia/mental retardation X-linked (ATRX) genes mutations and alternative lengthening of telomeres (ALT) activation are associated with more aggressive behavior of non-functional pancreatic neuroendocrine tumors (NF-pNETs)[1] [2]. We aimed to evaluate their reliability on EUS-FNB specimens. Methods Patients who underwent EUS-FNB and subsequent surgical resection for PNETs between 2017 and 2019 were identified. Immunohistochemistry was performed to evaluate DAXX/ATRX expression and fluorescence in situ hybridization (FISH) for ALT status. The primary outcome was the concordance rate of markers expression between EUS-FNB and surgical specimens. Secondary aims were association between markers and aggressive lesions and their diagnostic performance in predicting aggressiveness. Results 41 NF-pNETs (mean diameter 36.1±26.5mm) were included. 24 showed features of lesion aggressiveness. Concordance of expressions of DAXX, ATRX, and ALT status between EUS-FNB and surgical specimens were 95.1% (κ=0.828; p<0.001), 92.7% (κ=0.626; p<0.001), and 100% (κ=1; p<0.001), respectively. DAXX/ATRX loss and ALT-positivity were significantly (p<0.05) associated with metastatic lymphnodes and lymphovascular invasion. The combination of all tumor markers (DAXX/ATRX loss + ALT-positivity + grade 2) reached an accuracy of 73.2% (95%CI 57.1–85.8) in identifying aggressive lesions (Table). Pre- and post-operative ki-67-based grading was concordant in 80.5% of cases (k=0.573; p<0.001) ([Table 1]). Conclusions DAXX/ATRX expression and ALT status can be accurately evaluated on EUS-FNB samples, potentially improving the identification of patients with increased risk and poorer prognosis. Article published online: 14 April 2023 © 2023. European Society of Gastrointestinal Endoscopy. All rights reserved. Georg Thieme Verlag KG Rüdigerstraße 14, 70469 Stuttgart, Germany

The innate immune responses of mammals to microbial infections include strategies based on manipulating the local concentration of metals such as iron (Fe) and zinc (Zn), commonly described as nutritional immunity. To evaluate whether these strategies are also present in zebrafish embryos, we have conducted a series of heart cavity-localized infection experiments with Pseudomonas aeruginosa strains characterized by a different ability to acquire Zn. We have found that, 48 h after infection, the bacterial strains lacking critical components of the Zn importers ZnuABC and ZrmABCD have a reduced colonization capacity compared to the wild-type strain. This observation, together with the finding of a high level of expression of Zur-regulated genes, suggests the existence of antimicrobial mechanisms based on Zn sequestration. However, we have observed that strains lacking such Zn importers have a selective advantage over the wild-type strain in the early stages of infection. Analysis of the expression of the gene that encodes for a Zn efflux pump has revealed that at short times after infection, P. aeruginosa is exposed to high concentrations of Zn. At the same time, zebrafish respond to the infection by activating the expression of the Zn transporters Slc30a1 and Slc30a4, whose mammalian homologs mediate a redistribution of Zn in phagocytes aimed at intoxicating bacteria with a metal excess. These observations indicate that teleosts share similar nutritional immunity mechanisms with higher vertebrates, and confirm the usefulness of the zebrafish model for studying host–pathogen interactions.

Salmonella capacity to colonize different environments depends on its ability to respond efficiently to fluctuations in micronutrient availability. Among micronutrients, Zn, besides playing an essential role in bacterial physiology, is a key element whose concentration can influence bacterial survival in a particular niche. Plant colonization by Salmonella enterica was described for several years, and some molecular determinants involved in this host-pathogen interaction have started to be characterized. However, it is still unclear if Zn plays a role in the outcome of this interaction, as well established for animal hosts that employ nutritional immunity strategies to counteract pathogens infections. In this study, we have investigated the involvement of Salmonella Typhimurium main effectors of zinc homeostasis in plant colonization, using Arabidopsis thaliana as a model host. The results show that to colonize plant tissues, Salmonella takes advantage of its ability to export excess metal through the efflux pumps ZntA and ZitB. In fact, the deletion of these Zn/Cd detoxification systems can affect bacterial persistence in the shoots, depending on metal availability in the plant tissues. The importance of Salmonella ability to export excess metal was enhanced in the colonization of plants grown in high Zn conditions. On the contrary, the bacterial disadvantage related to Zn detoxification impairment can be abrogated if the plant cannot efficiently translocate Zn to the shoots. Overall, our work highlights the role of Zn in Salmonella-plant interaction and suggests that modulation of plant metal content through biofortification may be an efficient strategy to control pathogen colonization.

The COVID-19 pandemic has highlighted the relevance of proper disinfection procedures and renewed interest in developing novel disinfectant materials as a preventive strategy to limit SARS-CoV-2 contamination. Given its widely known antibacterial, antifungal, and antiviral properties, Melaleuca alternifolia essential oil, also named Tea tree oil (TTO), is recognized as a potential effective and safe natural disinfectant agent. In particular, the proposed antiviral activity of TTO involves the inhibition of viral entry and fusion, interfering with the structural dynamics of the membrane and with the protein envelope components. In this study, for the first time, we demonstrated the virucidal effects of TTO against the feline coronavirus (FCoVII) and the human coronavirus OC43 (HCoV-OC43), both used as surrogate models for SARS-CoV-2. Then, to atomistically uncover the possible effects exerted by TTO compounds on the outer surface of the SARS-CoV-2 virion, we performed Gaussian accelerated Molecular Dynamics simulations of a SARS-CoV-2 envelope portion, including a complete model of the Spike glycoprotein in the absence or presence of the three main TTO compounds (terpinen-4-ol, γ-terpinene, and 1,8-cineole). The obtained results allowed us to hypothesize the mechanism of action of TTO and its possible use as an anti-coronavirus disinfectant agent.

Almost eighty years have passed since the publication of the studies by Arthur Schade and Leona Caroline, which we can consider as the first investigations that began to disclose the importance of metals in host–pathogen interactions