Meeting report of the sixth annual tri-service microbiome consortium symposium.

The Tri-Service Microbiome Consortium (TSMC) was founded to enhance collaboration, coordination, and communication of microbiome research among DoD organizations and to facilitate resource, material and information sharing amongst consortium members, which includes collaborators in academia and industry. The 6th Annual TSMC Symposium was a hybrid meeting held in Fairlee, Vermont on 27-28 September 2022 with presentations and discussions centered on microbiome-related topics within seven broad thematic areas: (1) Human Microbiomes: Stress Response; (2) Microbiome Analysis & Surveillance; (3) Human Microbiomes Enablers & Engineering; (4) Human Microbiomes: Countermeasures; (5) Human Microbiomes Discovery - Earth & Space; (6) Environmental Micro & Myco-biome; and (7) Environmental Microbiome Analysis & Engineering. Collectively, the symposium provided an update on the scope of current DoD microbiome research efforts, highlighted innovative research being done in academia and industry that can be leveraged by the DoD, and fostered collaborative opportunities. This report summarizes the activities and outcomes from the 6th annual TSMC symposium.

Marine Biofilm Engineered to Produce Current in Response to Small Molecules.

Engineered electroactive bacteria have potential applications ranging from sensing to biosynthesis. In order to advance the use of engineered electroactive bacteria, it is important to demonstrate functional expression of electron transfer modules in chassis adapted to operationally relevant conditions, such as non-freshwater environments. Here, we use the Shewanella oneidensis electron transfer pathway to induce current production in a marine bacterium, Marinobacter atlanticus, during biofilm growth in artificial seawater. Genetically encoded sensors optimized for use in Escherichia coli were used to control protein expression in planktonic and biofilm attached cells. Significant current production required the addition of menaquinone, which M. atlanticus does not produce, for electron transfer from the inner membrane to the expressed electron transfer pathway. Current through the S. oneidensis pathway in M. atlanticus was observed when inducing molecules were present during biofilm formation. Electron transfer was also reversible, indicating that electron transfer into M. atlanticus could be controlled. These results show that an operationally relevant marine bacterium can be genetically engineered for environmental sensing and response using an electrical signal.

Automated SSHHPS Analysis Predicts a Potential Host Protein Target Common to Several Neuroinvasive (+)ssRNA Viruses.

Within the viral genome, short stretches of homologous host pathogen sequences (SSHHPS) span the protease cleavage sites. To identify host proteins that may be cleaved during infection, we searched the human proteome for viral protease cleavage sites (~20 amino acids). We developed a sequence-to-symptom tool, automating the search and pairing process. We used the viral protein sequence, PHI-BLAST, and UniProt database for gene ontologies and disease relationships. We applied the tool to nine neuroinvasive viruses: Venezuelan and Eastern Equine encephalitis virus (VEEV, EEEV); severe acute respiratory syndrome (SARS, SARS-CoV-2); Middle East respiratory syndrome (MERS); EV-71; Japanese encephalitis virus (JEV); West Nile (WNV); and Zika (ZIKV). A comparison of the hits identified a protein common to all nine viruses called ADGRA2 (GPR124). ADGRA2 was a predicted hit of the 3CL main protease and papain-like protease (PLpro) of SARS-CoV-2. ADGRA2 is an adhesion G protein-coupled receptor and a key endothelial regulator of brain-specific angiogenesis. It is a Wnt7A/Wnt7B specific coactivator of beta-catenin signaling and is essential for blood-brain barrier (BBB) integrity in central nervous system (CNS) diseases. We show the cleavage of the predicted sequences in MYOM1, VWF by the SARS-CoV-2 PLpro; DNAH8 (dynein) by the MERS PLpro; ADGRA2 by the alphaviral VEEV nsP2 protease; and POT1 by the SARS-CoV-2 and MERS PLpro.

Vibrio natriegens genome-scale modeling reveals insights into halophilic adaptations and resource allocation.

Vibrio natriegens is a Gram-negative bacterium with an exceptional growth rate that has the potential to become a standard biotechnological host for laboratory and industrial bioproduction. Despite this burgeoning interest, the current lack of organism-specific qualitative and quantitative computational tools has hampered the community's ability to rationally engineer this bacterium. In this study, we present the first genome-scale metabolic model (GSMM) of V. natriegens. The GSMM (iLC858) was developed using an automated draft assembly and extensive manual curation and was validated by comparing predicted yields, central metabolic fluxes, viable carbon substrates, and essential genes with empirical data. Mass spectrometry-based proteomics data confirmed the translation of at least 76% of the enzyme-encoding genes predicted to be expressed by the model during aerobic growth in a minimal medium. iLC858 was subsequently used to carry out a metabolic comparison between the model organism Escherichia coli and V. natriegens, leading to an analysis of the model architecture of V. natriegens' respiratory and ATP-generating system and the discovery of a role for a sodium-dependent oxaloacetate decarboxylase pump. The proteomics data were further used to investigate additional halophilic adaptations of V. natriegens. Finally, iLC858 was utilized to create a Resource Balance Analysis model to study the allocation of carbon resources. Taken together, the models presented provide useful computational tools to guide metabolic engineering efforts in V. natriegens.

Comparative analysis of stalked and acorn barnacle adhesive proteomes.

Barnacles interest the scientific community for multiple reasons: their unique evolutionary trajectory, vast diversity and economic impact-as a harvested food source and also as one of the most prolific macroscopic hard biofouling organisms. A common, yet novel, trait among barnacles is adhesion, which has enabled a sessile adult existence and global colonization of the oceans. Barnacle adhesive is primarily composed of proteins, but knowledge of how the adhesive proteome varies across the tree of life is unknown due to a lack of genomic information. Here, we supplement previous mass spectrometry analyses of barnacle adhesive with recently sequenced genomes to compare the adhesive proteomes of Pollicipes pollicipes (Pedunculata) and Amphibalanus amphitrite (Sessilia). Although both species contain the same broad protein categories, we detail differences that exist between these species. The barnacle-unique cement proteins show the greatest difference between species, although these differences are diminished when amino acid composition and glycosylation potential are considered. By performing an in-depth comparison of the adhesive proteomes of these distantly related barnacle species, we show their similarities and provide a roadmap for future studies examining sequence-specific differences to identify the proteins responsible for functional differences across the barnacle tree of life.

The SARS-CoV-2 SSHHPS Recognized by the Papain-like Protease.

Viral proteases are highly specific and recognize conserved cleavage site sequences of ∼6-8 amino acids. Short stretches of homologous host-pathogen sequences (SSHHPS) can be found spanning the viral protease cleavage sites. We hypothesized that these sequences corresponded to specific host protein targets since >40 host proteins have been shown to be cleaved by Group IV viral proteases and one Group VI viral protease. Using PHI-BLAST and the viral protease cleavage site sequences, we searched the human proteome for host targets and analyzed the hit results. Although the polyprotein and host proteins related to the suppression of the innate immune responses may be the primary targets of these viral proteases, we identified other cleavable host proteins. These proteins appear to be related to the virus-induced phenotype associated with Group IV viruses, suggesting that information about viral pathogenesis may be extractable directly from the viral genome sequence. Here we identify sequences cleaved by the SARS-CoV-2 papain-like protease (PLpro) in vitro within human MYH7 and MYH6 (two cardiac myosins linked to several cardiomyopathies), FOXP3 (an X-linked Treg cell transcription factor), ErbB4 (HER4), and vitamin-K-dependent plasma protein S (PROS1), an anticoagulation protein that prevents blood clots. Zinc inhibited the cleavage of these host sequences in vitro. Other patterns emerged from multispecies sequence alignments of the cleavage sites, which may have implications for the selection of animal models and zoonosis. SSHHPS/nsP is an example of a sequence-specific post-translational silencing mechanism.

Engineered Escherichia coli Biofilms Produce Adhesive Nanomaterials Shaped by a Patterned 43 kDa Barnacle Cement Protein.

Barnacles integrate multiple protein components into distinct amyloid-like nanofibers arranged as a bulk material network for their permanent underwater attachment. The design principle for how chemistry is displayed using adhesive nanomaterials, and fragments of proteins that are responsible for their formation, remains a challenge to assess and is yet to be established. Here, we use engineered bacterial biofilms to display a library of amyloid materials outside of the cell using full-length and subdomain sequences from a major component of the barnacle adhesive. A staggered charged pattern is found throughout the full-length sequence of a 43 kDa cement protein (AACP43), establishing a conserved sequence design evolved by barnacles to make adhesive nanomaterials. AACP43 domain deletions vary in their propensity to aggregate and form fibers, as exported extracellular materials are characterized through staining, immunoblotting, scanning electron microscopy, and atomic force microscopy. Full-length AACP43 and its domains have a propensity to aggregate into nanofibers independent of all other barnacle glue components, shedding light on its function in the barnacle adhesive. Curliated Escherichia coli biofilms are a compatible system for heterologous expression and the study of foreign functional amyloid adhesive materials, used here to identify the c-terminal portion of AACP43 as critical in material formation. This approach allows us to establish a common sequence pattern between two otherwise dissimilar families of cement proteins, laying the foundation to elucidate adhesive chemistries by one of the most tenacious marine fouling organisms in the ocean.

Proteomics Reveals Distinct Changes Associated with Increased Gamma Radiation Resistance in the Black Yeast Exophiala dermatitidis.

The yeast Exophiala dermatitidis exhibits high resistance to γ-radiation in comparison to many other fungi. Several aspects of this phenotype have been characterized, including its dependence on homologous recombination for the repair of radiation-induced DNA damage, and the transcriptomic response invoked by acute γ-radiation exposure in this organism. However, these findings have yet to identify unique γ-radiation exposure survival strategies-many genes that are induced by γ-radiation exposure do not appear to be important for recovery, and the homologous recombination machinery of this organism is not unique compared to more sensitive species. To identify features associated with γ-radiation resistance, here we characterized the proteomes of two E. dermatitidis strains-the wild type and a hyper-resistant strain developed through adaptive laboratory evolution-before and after γ-radiation exposure. The results demonstrate that protein intensities do not change substantially in response to this stress. Rather, the increased resistance exhibited by the evolved strain may be due in part to increased basal levels of single-stranded binding proteins and a large increase in ribosomal content, possibly allowing for a more robust, induced response during recovery. This experiment provides evidence enabling us to focus on DNA replication, protein production, and ribosome levels for further studies into the mechanism of γ-radiation resistance in E. dermatitidis and other fungi.

Meeting report of the third annual Tri-Service Microbiome Consortium symposium.

The Tri-Service Microbiome Consortium (TSMC) was founded to enhance collaboration, coordination, and communication of microbiome research among U.S. Department of Defense (DoD) organizations and to facilitate resource, material and information sharing among consortium members. The 2019 annual symposium was held 22-24 October 2019 at Wright-Patterson Air Force Base in Dayton, OH. Presentations and discussions centered on microbiome-related topics within five broad thematic areas: 1) human microbiomes; 2) transitioning products into Warfighter solutions; 3) environmental microbiomes; 4) engineering microbiomes; and 5) microbiome simulation and characterization. Collectively, the symposium provided an update on the scope of current DoD microbiome research efforts, highlighted innovative research being done in academia and industry that can be leveraged by the DoD, and fostered collaborative opportunities. This report summarizes the presentations and outcomes of the 3rd annual TSMC symposium.

Lactobacillus acidophilus Membrane Vesicles as a Vehicle of Bacteriocin Delivery.

Recent reports have shown that Gram-positive bacteria actively secrete spherical nanometer-sized proteoliposome membrane vesicles (MVs) into their surroundings. Though MVs are implicated in a broad range of biological functions, few studies have been conducted to examine their potential as delivery vehicles of antimicrobials. Here, we investigate the natural ability of Lactobacillus acidophilus MVs to carry and deliver bacteriocin peptides to the opportunistic pathogen, Lactobacillus delbrueckii. We demonstrate that upon treatment with lactacin B-inducing peptide, the proteome of the secreted MVs is enriched in putative bacteriocins encoded by the lab operon. Further, we show that purified MVs inhibit growth and compromise membrane integrity in L. delbrueckii, which is confirmed by confocal microscopy imaging and spectrophotometry. These results show that L. acidophilus MVs serve as conduits for antimicrobials to competing cells in the environment, suggesting a potential role for MVs in complex communities such as the gut microbiome. With the potential for controlling their payload through microbial engineering, MVs produced by L. acidophilus may be an interesting platform for effecting change in complex microbial communities or aiding in the development of new biomedical therapeutics.

Cassiosomes are stinging-cell structures in the mucus of the upside-down jellyfish Cassiopea xamachana.

Snorkelers in mangrove forest waters inhabited by the upside-down jellyfish Cassiopea xamachana report discomfort due to a sensation known as stinging water, the cause of which is unknown. Using a combination of histology, microscopy, microfluidics, videography, molecular biology, and mass spectrometry-based proteomics, we describe C. xamachana stinging-cell structures that we term cassiosomes. These structures are released within C. xamachana mucus and are capable of killing prey. Cassiosomes consist of an outer epithelial layer mainly composed of nematocytes surrounding a core filled by endosymbiotic dinoflagellates hosted within amoebocytes and presumptive mesoglea. Furthermore, we report cassiosome structures in four additional jellyfish species in the same taxonomic group as C. xamachana (Class Scyphozoa; Order Rhizostomeae), categorized as either motile (ciliated) or nonmotile types. This inaugural study provides a qualitative assessment of the stinging contents of C. xamachana mucus and implicates mucus containing cassiosomes and free intact nematocytes as the cause of stinging water.

The response of the melanized yeast Exophiala dermatitidis to gamma radiation exposure.

The melanized yeast Exophiala dermatitidis is resistant to many environmental stresses and is used as a model for understanding the diverse roles of melanin in fungi. Here, we describe the extent of resistance of E. dermatitidis to acute γ-radiation exposure and the major mechanisms it uses to recover from this stress. We find that melanin does not protect E. dermatitidis from γ-radiation. Instead, environmental factors such as nutrient availability, culture age and culture density are much greater determinants of cell survival after exposure. We also observe a dramatic transcriptomic response to γ-radiation that mobilizes pathways involved in morphological development, protein degradation and DNA repair, and is unaffected by the presence of melanin. Together, these results suggest that the ability of E. dermatitidis to survive γ-radiation exposure is determined by the prior and the current metabolic state of the cells as well as DNA repair mechanisms, and that small changes in these conditions can lead to large effects in radiation resistance, which should be taken into account when understanding how diverse fungi recover from this unique stress.

Melanin Produced by the Fast-Growing Marine Bacterium Vibrio natriegens through Heterologous Biosynthesis: Characterization and Application.

Melanin is a pigment produced by organisms throughout all domains of life. Due to its unique physicochemical properties, biocompatibility, and biostability, there has been an increasing interest in the use of melanin for broad applications. In the vast majority of studies, melanin has been either chemically synthesized or isolated from animals, which has restricted its use to small-scale applications. Using bacteria as biocatalysts is a promising and economical alternative for the large-scale production of biomaterials. In this study, we engineered the marine bacterium Vibrio natriegens, one of the fastest-growing organisms, to synthesize melanin by expressing a heterologous tyrosinase gene and demonstrated that melanin production was much faster than in previously reported heterologous systems. The melanin of V. natriegens was characterized as a polymer derived from dihydroxyindole-2-carboxylic acid (DHICA) and, similarly to synthetic melanin, exhibited several characteristic and useful features. Electron microscopy analysis demonstrated that melanin produced from V. natriegens formed nanoparticles that were assembled as "melanin ghost" structures, and the photoprotective properties of these particles were validated by their protection of cells from UV irradiation. Using a novel electrochemical reverse engineering method, we observed that melanization conferred redox activity to V. natriegens Moreover, melanized bacteria were able to quickly adsorb the organic compound trinitrotoluene (TNT). Overall, the genetic tractability, rapid division time, and ease of culture provide a set of attractive properties that compare favorably to current E. coli production strains and warrant the further development of this chassis as a microbial factory for natural product biosynthesis.IMPORTANCE Melanins are macromolecules that are ubiquitous in nature and impart a large variety of biological functions, including structure, coloration, radiation resistance, free radical scavenging, and thermoregulation. Currently, in the majority of investigations, melanins are either chemically synthesized or extracted from animals, which presents significant challenges for large-scale production. Bacteria have been used as biocatalysts to synthesize a variety of biomaterials due to their fast growth and amenability to genetic engineering using synthetic biology tools. In this study, we engineered the extremely fast-growing bacterium V. natriegens to synthesize melanin nanoparticles by expressing a heterologous tyrosinase gene with inducible promoters. Characterization of the melanin produced from V. natriegens-produced tyrosinase revealed that it exhibited physical and chemical properties similar to those of natural and chemically synthesized melanins, including nanoparticle structure, protection against UV damage, and adsorption of toxic compounds. We anticipate that producing and controlling melanin structures at the nanoscale in this bacterial system with synthetic biology tools will enable the design and rapid production of novel biomaterials for multiple applications.

Codon harmonization reduces amino acid misincorporation in bacterially expressed P. falciparum proteins and improves their immunogenicity.

Codon usage frequency influences protein structure and function. The frequency with which codons are used potentially impacts primary, secondary and tertiary protein structure. Poor expression, loss of function, insolubility, or truncation can result from species-specific differences in codon usage. "Codon harmonization" more closely aligns native codon usage frequencies with those of the expression host particularly within putative inter-domain segments where slower rates of translation may play a role in protein folding. Heterologous expression of Plasmodium falciparum genes in Escherichia coli has been a challenge due to their AT-rich codon bias and the highly repetitive DNA sequences. Here, codon harmonization was applied to the malarial antigen, CelTOS (Cell-traversal protein for ookinetes and sporozoites). CelTOS is a highly conserved P. falciparum protein involved in cellular traversal through mosquito and vertebrate host cells. It reversibly refolds after thermal denaturation making it a desirable malarial vaccine candidate. Protein expressed in E. coli from a codon harmonized sequence of P. falciparum CelTOS (CH-PfCelTOS) was compared with protein expressed from the native codon sequence (N-PfCelTOS) to assess the impact of codon usage on protein expression levels, solubility, yield, stability, structural integrity, recognition with CelTOS-specific mAbs and immunogenicity in mice. While the translated proteins were expected to be identical, the translated products produced from the codon-harmonized sequence differed in helical content and showed a smaller distribution of polypeptides in mass spectra indicating lower heterogeneity of the codon harmonized version and fewer amino acid misincorporations. Substitutions of hydrophobic-to-hydrophobic amino acid were observed more commonly than any other. CH-PfCelTOS induced significantly higher antibody levels compared with N-PfCelTOS; however, no significant differences in either IFN-γ or IL-4 cellular responses were detected between the two antigens.

Adhesion of acorn barnacles on surface-active borate glasses.

Concerns about the bioaccumulation of toxic antifouling compounds have necessitated the search for alternative strategies to combat marine biofouling. Because many biologically essential minerals have deleterious effects on organisms at high concentration, one approach to preventing the settlement of marine foulers is increasing the local concentration of ions that are naturally present in seawater. Here, we used surface-active borate glasses as a platform to directly deliver ions (Na+, Mg2+ and BO43-) to the adhesive interface under acorn barnacles (Amphibalanus (=Balanus) amphitrite). Additionally, surface-active glasses formed reaction layers at the glass-water interface, presenting another challenge to fouling organisms. Proteomics analysis showed that cement deposited on the gelatinous reaction layers is more soluble than cement deposited on insoluble glasses, indicating the reaction layer and/or released ions disrupted adhesion processes. Laboratory experiments showed that the majority (greater than 79%) of adult barnacles re-attached to silica-free borate glasses for 14 days could be released and, more importantly, barnacle larvae did not settle on the glasses. The formation of microbial biofilms in field tests diminished the performance of the materials. While periodic water jetting (120 psi) did not prevent the formation of biofilms, weekly cleaning did dramatically reduce macrofouling on magnesium aluminoborate glass to levels below a commercial foul-release coating. This article is part of the theme issue 'Transdisciplinary approaches to the study of adhesion and adhesives in biological systems'.

Pressure cycling technology for challenging proteomic sample processing: application to barnacle adhesive.

Successful proteomic characterization of biological material depends on the development of robust sample processing methods. The acorn barnacle Amphibalanus amphitrite is a biofouling model for adhesive processes, but the identification of causative proteins involved has been hindered by their insoluble nature. Although effective, existing sample processing methods are labor and time intensive, slowing progress in this field. Here, a more efficient sample processing method is described which exploits pressure cycling technology (PCT) in combination with protein solvents. PCT aids in protein extraction and digestion for proteomics analysis. Barnacle adhesive proteins can be extracted and digested in the same tube using PCT, minimizing sample loss, increasing throughput to 16 concurrently processed samples, and decreasing sample processing time to under 8 hours. PCT methods produced similar proteomes in comparison to previous methods. Two solvents which were ineffective at extracting proteins from the adhesive at ambient pressure (urea and methanol) produced more protein identifications under pressure than highly polar hexafluoroisopropanol, leading to the identification and description of >40 novel proteins at the interface. Some of these have homology to proteins with elastomeric properties or domains involved with protein-protein interactions, while many have no sequence similarity to proteins in publicly available databases, highlighting the unique adherent processes evolved by barnacles. The methods described here can not only be used to further characterize barnacle adhesive to combat fouling, but may also be applied to other recalcitrant biological samples, including aggregative or fibrillar protein matrices produced during disease, where a lack of efficient sample processing methods has impeded advancement. Data are available via ProteomeXchange with identifier PXD012730.

Response of Lactobacillus plantarum WCFS1 to the Gram-Negative Pathogen-Associated Quorum Sensing Molecule N-3-Oxododecanoyl Homoserine Lactone.

The bacterial quorum sensing phenomenon has been well studied since its discovery and has traditionally been considered to include signaling pathways recognized exclusively within either Gram-positive or Gram-negative bacteria. These groups of bacteria synthesize structurally distinct signaling molecules to mediate quorum sensing, where Gram-positive bacteria traditionally utilize small autoinducing peptides (AIPs) and Gram-negatives use small molecules such as acyl-homoserine lactones (AHLs). The structural differences between the types of signaling molecules have historically implied a lack of cross-talk among Gram-positive and Gram-negative quorum sensing systems. Recent investigations, however, have demonstrated the ability for AIPs and AHLs to be produced by non-canonical organisms, implying quorum sensing systems may be more universally recognized than previously hypothesized. With that in mind, our interests were piqued by the organisms Lactobacillus plantarum, a Gram-positive commensal probiotic known to participate in AIP-mediated quorum sensing, and Pseudomonas aeruginosa, a characterized Gram-negative pathogen whose virulence is in part controlled by AHL-mediated quorum sensing. Both health-related organisms are known to inhabit the human gut in various instances, both are characterized to elicit distinct effects on host immunity, and some studies hint at the putative ability of L. plantarum to degrade AHLs produced by P. aeruginosa. We therefore wanted to determine if L. plantarum cultures would respond to the addition of N-(3-oxododecanoyl)-L-homoserine lactone (3OC12) from P. aeruginosa by analyzing changes on both the transcriptome and proteome over time. Based on the observed upregulation of various two-component systems, response regulators, and native quorum sensing related genes, the resulting data provide evidence of an AHL recognition and response by L. plantarum.

Progress and Challenges in Ocean Metaproteomics and Proposed Best Practices for Data Sharing.

Ocean metaproteomics is an emerging field enabling discoveries about marine microbial communities and their impact on global biogeochemical processes. Recent ocean metaproteomic studies have provided insight into microbial nutrient transport, colimitation of carbon fixation, the metabolism of microbial biofilms, and dynamics of carbon flux in marine ecosystems. Future methodological developments could provide new capabilities such as characterizing long-term ecosystem changes, biogeochemical reaction rates, and in situ stoichiometries. Yet challenges remain for ocean metaproteomics due to the great biological diversity that produces highly complex mass spectra, as well as the difficulty in obtaining and working with environmental samples. This review summarizes the progress and challenges facing ocean metaproteomic scientists and proposes best practices for data sharing of ocean metaproteomic data sets, including the data types and metadata needed to enable intercomparisons of protein distributions and annotations that could foster global ocean metaproteomic capabilities.

Proteolytic cleavage of host proteins by the Group IV viral proteases of Venezuelan equine encephalitis virus and Zika virus.

The alphaviral nonstructural protein 2 (nsP2) cysteine proteases (EC 3.4.22.-) are essential for the proteolytic processing of the nonstructural (ns) polyprotein and are validated drug targets. A common secondary role of these proteases is to antagonize the effects of interferon (IFN). After delineating the cleavage site motif of the Venezuelan equine encephalitis virus (VEEV) nsP2 cysteine protease, we searched the human genome to identify host protein substrates. Here we identify a new host substrate of the VEEV nsP2 protease, human TRIM14, a component of the mitochondrial antiviral-signaling protein (MAVS) signalosome. Short stretches of homologous host-pathogen protein sequences (SSHHPS) are present in the nonstructural polyprotein and TRIM14. A 25-residue cyan-yellow fluorescent protein TRIM14 substrate was cleaved in vitro by the VEEV nsP2 protease and the cleavage site was confirmed by tandem mass spectrometry. A TRIM14 cleavage product also was found in VEEV-infected cell lysates. At least ten other Group IV (+)ssRNA viral proteases have been shown to cleave host proteins involved in generating the innate immune responses against viruses, suggesting that the integration of these short host protein sequences into the viral protease cleavage sites may represent an embedded mechanism of IFN antagonism. This interference mechanism shows several parallels with those of CRISPR/Cas9 and RNAi/RISC, but with a protease recognizing a protein sequence common to both the host and pathogen. The short host sequences embedded within the viral genome appear to be analogous to the short phage sequences found in a host's CRISPR spacer sequences. To test this algorithm, we applied it to another Group IV virus, Zika virus (ZIKV), and identified cleavage sites within human SFRP1 (secreted frizzled related protein 1), a retinal Gs alpha subunit, NT5M, and Forkhead box protein G1 (FOXG1) in vitro. Proteolytic cleavage of these proteins suggests a possible link between the protease and the virus-induced phenotype of ZIKV. The algorithm may have value for selecting cell lines and animal models that recapitulate virus-induced phenotypes, predicting host-range and susceptibility, selecting oncolytic viruses, identifying biomarkers, and de-risking live virus vaccines. Inhibitors of the proteases that utilize this mechanism may both inhibit viral replication and alleviate suppression of the innate immune responses.

Isolation and characterization of Lactobacillus-derived membrane vesicles.

Bacterial membrane vesicles have been implicated in a broad range of functions in microbial communities from pathogenesis to gene transfer. Though first thought to be a phenomenon associated with Gram-negative bacteria, vesicle production in Staphylococcus aureus, Lactobacillus plantarum, and other Gram-positives has recently been described. Given that many Lactobacillus species are Generally Regarded as Safe and often employed as probiotics, the engineering of Lactobacillus membrane vesicles presents a new avenue for the development of therapeutics and vaccines. Here we characterize and compare the membrane vesicles (MVs) from three different Lactobacillus species (L. acidophilus ATCC 53544, L. casei ATCC 393, and L. reuteri ATCC 23272), with the aim of developing future strategies for vesicle engineering. We characterize the vesicles from each Lactobacillus species comparing the physiochemical properties and protein composition of each. More than 80 protein components from Lactobacillus-derived MVs were identified, including some that were enriched in the vesicles themselves suggesting vesicles as a vehicle for antimicrobial delivery. Additionally, for each species vesicular proteins were categorized based on biological pathway and examined for subcellular localization signals in an effort to identify possible sorting mechanisms for MV proteins.