Physiochemically Distinct Surface Properties of SU-8 Polymer Modulate Bacterial Cell-Surface Holdfast and Colonization.

SU-8 polymer is an excellent platform for diverse applications due to its high aspect ratio of micro/nanostructure fabrication and exceptional physicochemical and biocompatible properties. Although SU-8 polymer has often been investigated for various biological applications, how its surface properties influence the interaction of bacterial cells with the substrate and its colonization is poorly understood. In this work, we tailor SU-8 nanoscale surface properties to investigate single-cell motility, adhesion, and successive colonization of phytopathogenic bacteria, Xylella fastidiosa. Different surface properties of SU-8 thin films have been prepared using photolithography processing and oxygen plasma treatment. A more significant density of carboxyl groups in hydrophilic plasma-treated SU-8 surfaces promotes faster cell motility in the earlier growth stage. The hydrophobic nature of pristine SU-8 surfaces shows no trackable bacterial motility and 5-10 times more single cells adhered to the surface than its plasma-treated counterpart. In addition, plasma-treated SU-8 samples suppressed bacterial adhesion, with surfaces showing less than 5% coverage. These results not only showcase that SU-8 surface properties can impact the spatiotemporal bacterial behavior but also provide insights into pathogens' prominent ability to evolve and adapt to different surface properties.

Probing the Application of OmpA-Derived Peptides to Disrupt the Acquisition of 'Candidatus Liberibacter asiaticus' by Diaphorina citri.

Huanglongbing (HLB) is currently the most devastating disease of citrus worldwide. Both bacteria 'Candidatus Liberibacter asiaticus' (CLas) and 'Candidatus Liberibacter americanus' (CLam) are associated with HLB in Brazil but with a strong prevalence of CLas over CLam. Conventionally, HLB management focuses on controlling the insect vector population (Diaphorina citri; also known as Asian citrus psyllid [ACP]) by spraying insecticides, an approach demonstrated to be mostly ineffective. Thus, development of novel, more efficient HLB control strategies is required. The multifunctional bacterial outer membrane protein OmpA is involved in several molecular processes between bacteria and their hosts and has been suggested as a target for bacterial control. Curiously, OmpA is absent in CLam in comparison with CLas, suggesting a possible role in host interaction. Therefore, in the current study, we have treated ACPs with different OmpA-derived peptides, aiming to evaluate acquisition of CLas by the insect vector. Treatment of psyllids with 5 µM of Pep1, Pep3, Pep5, and Pep6 in artificial diet significantly reduced the acquisition of CLas, whereas increasing the concentration of Pep5 and Pep6 to 50 µM abolished this process. In addition, in planta treatment with 50 µM of Pep6 also significantly decreased the acquisition of CLas, and sweet orange plants stably absorbed and maintained this peptide for as long as 3 months post the final application. Together, our results demonstrate the promising use of OmpA-derived peptides as a novel biotechnological tool to control CLas.

Controlled spatial organization of bacterial growth reveals key role of cell filamentation preceding Xylella fastidiosa biofilm formation.

The morphological plasticity of bacteria to form filamentous cells commonly represents an adaptive strategy induced by stresses. In contrast, for diverse human and plant pathogens, filamentous cells have been recently observed during biofilm formation, but their functions and triggering mechanisms remain unclear. To experimentally identify the underlying function and hypothesized cell communication triggers of such cell morphogenesis, spatially controlled cell patterning is pivotal. Here, we demonstrate highly selective cell adhesion of the biofilm-forming phytopathogen Xylella fastidiosa to gold-patterned SiO2 substrates with well-defined geometries and dimensions. The consequent control of both cell density and distances between cell clusters demonstrated that filamentous cell formation depends on cell cluster density, and their ability to interconnect neighboring cell clusters is distance-dependent. This process allows the creation of large interconnected cell clusters that form the structural framework for macroscale biofilms. The addition of diffusible signaling molecules from supernatant extracts provides evidence that cell filamentation is induced by quorum sensing. These findings and our innovative platform could facilitate therapeutic developments targeting biofilm formation mechanisms of X. fastidiosa and other pathogens.

GC-TOF/MS-based metabolomics analysis to investigate the changes driven by N-Acetylcysteine in the plant-pathogen Xanthomonas citri subsp. citri.

N-Acetylcysteine (NAC) is an antioxidant, anti-adhesive, and antimicrobial compound. Even though there is much information regarding the role of NAC as an antioxidant and anti-adhesive agent, little is known about its antimicrobial activity. In order to assess its mode of action in bacterial cells, we investigated the metabolic responses triggered by NAC at neutral pH. As a model organism, we chose the Gram-negative plant pathogen Xanthomonas citri subsp. citri (X. citri), the causal agent of citrus canker disease, due to the potential use of NAC as a sustainable molecule against phytopathogens dissemination in citrus cultivated areas. In presence of NAC, cell proliferation was affected after 4 h, but damages to the cell membrane were observed only after 24 h. Targeted metabolite profiling analysis using GC-MS/TOF unravelled that NAC seems to be metabolized by the cells affecting cysteine metabolism. Intriguingly, glutamine, a marker for nitrogen status, was not detected among the cells treated with NAC. The absence of glutamine was followed by a decrease in the levels of the majority of the proteinogenic amino acids, suggesting that the reduced availability of amino acids affect protein synthesis and consequently cell proliferation.