Bacterial Outer Membrane Vesicles Induce a Transcriptional Shift in Arabidopsis Towards Immune System Activation Leading to Suppression of Pathogen Growth in Planta

Gram negative bacteria form spherical blebs on their cell periphery, which later dissociate and released into the surrounding environment. Previous studies have shown that these nano scale structures, derived primarily from the bacterial outer membrane and are termed outer membrane vesicles (OMVs), induce typical immune outputs in both mammals and plants. On the other hand, these same structures have been shown to promote infection and disease. To better understand the broad transcriptional change plants undergo following exposure to OMVs, we treated Arabidopsis thaliana (Arabidopsis) seedlings with OMVs purified from the Gram-negative plant pathogenic bacterium Xanthomonas campestris pv. campestris and performed RNA-seq analysis on OMV- and mock-treated samples at 2, 6 and 24 h post challenge. We found that the most pronounced transcriptional shift occurred in the first two time points, as was reflected by both the number of differentially expressed genes (DEGs) and the average fold change. Gene ontology enrichment analysis revealed that OMVs induce a major transcriptional shift in Arabidopsis towards immune system activation, upregulating a multitude of immune-related pathways including a variety of immune receptors and transcriptional factors. Comparing Arabidopsis response to OMVs and to single purified elicitors, revealed that while OMVs induce a similar suite of genes and pathways as single elicitors, some differential pathways activated by OMVs were detected including response to drug and apoptosis, which may indicate exposure to toxic compounds via OMV. To examine whether the observed transcriptional shift in Arabidopsis leads to an effective immune response, plants were pretreated with OMVs and then inoculated with a bacterial pathogen. OMV-mediated priming led to a significant reduction in bacterial titer in inoculated leaves two days following inoculation. Mutations in the elongation factor receptor (EFR), flagellin receptor (FLS2), or the brassinosteroid-insensitive 1-associated kinase (BAK1) receptor, did not significantly affect OMV-priming. All together these results show that OMV induce a broad transcriptional shift in Arabidopsis leading to upregulation of multiple immune pathways, and that this transcriptional change is reflected in the ability to better resist bacterial infection.

Soluble MAC is primarily released from MAC-resistant bacteria that potently convert complement component C5

The Membrane Attack Complex (MAC or C5b-9) is an important effector of the immune system to kill invading microbes. MAC is formed when complement enzymes on the bacterial surface convert complement component C5 into C5b. Although the MAC is a membrane-inserted complex, soluble forms of MAC (sMAC, or terminal complement complex (TCC)) are often detected in sera of patients suffering from infections. Consequently, sMAC has been proposed as a biomarker, but it remains unclear when and how it is formed during infections. Here, we studied mechanisms of MAC formation on bacteria and found that sMAC is primarily formed in human serum by bacteria resistant to MAC-dependent killing. Surprisingly, C5 was converted into C5b more potently by MAC-resistant compared to MAC-sensitive Escherichia coli strains. Both the increase in C5 conversion and sMAC generation were linked to the expression of lipopolysaccharide (LPS) O-Antigen in the bacterial outer membrane. In addition, we found that MAC precursors are released from the surface of MAC-resistant bacteria during MAC assembly. Release of MAC precursors from bacteria induced lysis of bystander human erythrocytes in the absence of other serum components. However, serum regulators vitronectin (Vn) and clusterin (Clu) can prevent this bystander lysis. Combining size exclusion chromatography with mass spectrometry profiling, we show that sMAC released from bacteria in serum is a heterogeneous mixture of complexes composed of C5b-8, up to 3 copies of C9 and multiple copies of Vn and Clu. Altogether, our data provide molecular insight into how sMAC is generated during bacterial infections. This fundamental knowledge could form the basis for exploring the use of sMAC as biomarker.

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 ‘Ca. 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 to CLas, suggesting a possible role on host-interaction. Therefore, in the current study, we have treated ACPs with different OmpA-derived peptides aiming to evaluate the 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, while 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 three months post the final application. Together, our results demonstrate the promising use of OmpA-derived peptides as a novel biotechnological tool to control CLas.

Acquisition of ionic copper by the bacterial outer membrane protein OprC through a novel binding site

Copper, while toxic in excess, is an essential micronutrient in all kingdoms of life due to its essential role in the structure and function of many proteins. Proteins mediating ionic copper import have been characterised in detail for eukaryotes, but much less so for prokaryotes. In particular, it is still unclear whether and how gram-negative bacteria acquire ionic copper. Here, we show that Pseudomonas aeruginosa OprC is an outer membrane, TonB-dependent transporter that is conserved in many Proteobacteria and which mediates acquisition of both reduced and oxidised ionic copper via an unprecedented CxxxM-HxM metal binding site. Crystal structures of wild-type and mutant OprC variants with silver and copper suggest that acquisition of Cu(I) occurs via a surface-exposed “methionine track” leading towards the principal metal binding site. Together with whole-cell copper quantitation and quantitative proteomics in a murine lung infection model, our data identify OprC as an abundant component of bacterial copper biology that may enable copper acquisition under a wide range of conditions.

Biomimetic Nanoparticles Coated with Bacterial Outer Membrane Vesicles as a New-Generation Platform for Biomedical Applications

The biomedical field is currently reaping the benefits of research on biomimetic nanoparticles (NPs), which are synthetic nanoparticles fabricated with natural cellular materials for nature-inspired biomedical applications. These camouflage NPs are capable of retaining not only the physiochemical properties of synthetic nanoparticles but also the original biological functions of the cellular materials. Accordingly, NPs coated with cell-derived membrane components have achieved remarkable growth as prospective biomedical materials. Particularly, bacterial outer membrane vesicle (OMV), which is a cell membrane coating material for NPs, is regarded as an important molecule that can be employed in several biomedical applications, including immune response activation, cancer therapeutics, and treatment for bacterial infections with photothermal activity. The currently available cell membrane-coated NPs are summarized in this review. Furthermore, the general features of bacterial OMVs and several multifunctional NPs that could serve as inner core materials in the coating strategy are presented, and several methods that can be used to prepare OMV-coated NPs (OMV-NPs) and their characterization are highlighted. Finally, some perspectives of OMV-NPs in various biomedical applications for future potential breakthrough are discussed. This in-depth review, which includes potential challenges, will encourage researchers to fabricate innovative and improvised, new-generation biomimetic materials through future biomedical applications.

An Intranasal Vaccine Based on Outer Membrane Vesicles Against SARS-CoV-2

The prevailing pandemic of SARS-CoV-2 highlights the desperate need of alternative vaccine-platforms, which are safe, effective, and can be modified to carry antigens of emerging pathogens. The current SARS-CoV-2 vaccines based on mRNA and adenoviral vector technology meet some of these criteria but still face limitations regarding administration route, mass production, stability, and storage. Herein, we introduce a novel SARS-CoV-2 vaccine candidate based on bacterial outer membrane vesicles (OMVs). Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) have been genetically modified to produce increased amounts of detoxified OMVs decorated with the receptor binding domain (RBD) of the SARS-CoV-2 Spike protein. Intranasal immunization with RBD-decorated OMVs induced not only a robust immune response against the bacterial outer membrane components but also detectable antibody titers against the Spike protein. Cell culture infection assays using a Spike-pseudotyped lentivirus confirmed the presence of SARS-CoV-2 neutralizing antibodies. Highest titers against the SARS-CoV-2 Spike protein and most potent neutralization activity were observed for an alternating immunization regimen using RBD-decorated OMVs from ETEC and V. cholerae in turn. These results highlight the versatile vaccine applications offered by OMVs via expression of heterologous antigens in the donor bacterium.