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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2019

Critical Nodes of Virus-Host Interaction Revealed Through an Integrated Network Analysis

Viruses are one of the major causes of various acute and chronic infectious diseases and thus a major contributor to the global burden of disease. Several studies have shown how viruses have evolved to hijack basic cellular pathways and evade innate immune response by modulating key host factors and signalling pathways. A collective view of these multiple studies could advance our understanding of viral evasion mechanisms and provide new therapeutic perspectives for the treatment of viral diseases. Here, we performed an integrative meta-analysis to elucidate the 17 different host-virus interactomes. Network and bioinformatics analyses showed how viruses with small genomes efficiently achieve the maximal effect by targeting multifunctional and highly connected host proteins with a high occurrence of disordered regions. We also identified the core cellular process subnetworks that are targeted by all the viruses. Integration with functional RNA interference (RNAi) datasets showed that a large proportion of the targets are required for viral replication. Furthermore, we performed an interactome-informed drug re-purposing screen and identified novel activities for broad-spectrum antiviral agents against hepatitis C virus and human metapneumovirus. Altogether, these orthogonal datasets could serve as a platform for hypothesis generation and follow-up studies to broaden our understanding of the viral evasion landscape.

immunology↗

bioRxiv2019

Phospho-sRNA-seq reveals extracellular mRNA/lncRNA fragments as potential biomarkers in human plasma

Extracellular RNAs (exRNAs) in biofluids have attracted great interest as potential biomarkers. Whereas extracellular microRNAs (miRNAs) in blood plasma are extensively characterized, extracellular messenger RNAs (mRNA) and long noncoding RNAs (lncRNA) are less well-studied. We report that plasma contains fragmented mRNAs and lncRNAs that are largely missed by standard small RNA-seq protocols due to lack of 5 phosphate or presence of 3 phosphate. These fragments were revealed using a modified protocol ("phospho-sRNA-seq") incorporating RNA treatment with T4-polynucleotide kinase, which we compared with standard small RNA-seq for sequencing synthetic RNAs with varied 5 and 3 ends, as well as human plasma exRNA. Analyzing phospho-sRNA-seq data using a custom, high-stringency bioinformatic pipeline, we identified mRNA/lncRNA transcriptome fingerprints in plasma, including tissue-specific gene sets. In a longitudinal study of bone marrow transplant patients, bone marrow-and liver-enriched exRNA genes tracked with bone marrow recovery and liver injury, respectively, providing proof-of-concept validation as a biomarker approach. By enabling access to an unexplored realm of mRNA and lncRNA fragments, phospho-sRNA-seq opens up new possibilities for plasma transcriptomic biomarker development.

molecular biology↗

bioRxiv2019

Malignant liver cancers have distinct myeloid-derived suppressor cell signatures

Myeloid-derived suppressor cells (MDSCs) are immunosuppressive cells that are increased in patients with numerous malignancies including viral-derived hepatocellular carcinoma (HCC). Here, we report an elevation of MDSC in other hepatobiliary malignancies including non-viral HCC, neuroendocrine tumors (NET), colorectal carcinoma with liver metastases (CRLM), but not cholangiocarcinoma (CCA). Investigation of myeloid cell infiltration in HCC, NET and intrahepatic CCA tumors further established that the frequency of antigen-presenting cells was limited compared to benign lesions suggesting that primary and metastatic hepatobiliary cancers have distinct peripheral and tumoral myeloid signatures. Bioinformatics analysis of the Cancer Genome Atlas demonstrated that a high MDSC score in HCC patients predicted poor disease outcome. Mechanistic studies indicated that the oncometabolite D-2-hydroxyglutarate resulting from isocitrate dehydrogenase 1 mutation could be a limiting factor of MDSC accumulation in CCA patients. Given our observation that MDSCs are increased in non-CCA malignant liver cancers, they may comprise suitable targets for effective immunotherapy approaches.

pathology↗

bioRxiv2019

BioCell2XML: A novel tool for converting cell lineage data from SIMI BioCell to MaMuT (Fiji)

Computer-assisted 4D manual cell tracking has been a valuable method for understanding spatial-temporal dynamics of embryogenesis (e.g., Stach & Anselmi, 2015; Vellutini et al., 2017; Wolff et al., 2018) since the method was introduced in the late 1990s. Since two decades SIMI(R) BioCell (Schnabel et al., 1997), a software which initially was developed for analyzing data coming from the, at that time new technique of 4D microscopy, is in use. Many laboratories around the world use SIMI BioCell for the manual tracing of cells in embryonic development of various species to reconstruct cell genealogies with high precision. However, the software has several disadvantages: Limits in handling very large data sets, the virtually no maintenance over the last ten years (bound to older Windows versions), the difficulty to access the created cell lineage data for analyses outside SIMI BioCell, and the high cost of the program. Recently, bioinformatics, in close collaboration with biologists, developed new lineaging tools that are freely available through the open source image processing platform Fiji. Here we introduce a software tool that allows conversion of SIMI BioCell lineage data to a format that is compatible with the Fiji plugin MaMuT (Wolff et al., 2018). Hereby we intend to maintain the usability of SIMI BioCell created cell lineage data for the future and, for investigators who wish to do so, facilitate the transition from this software to a more convenient program.

developmental biology↗

bioRxiv2019

Discovery of several thousand highly diverse circular DNA viruses

Although it is suspected that there are millions of distinct viral species, fewer than 9,000 are catalogued in GenBanks RefSeq database. We selectively enriched for and amplified the genomes of circular DNA viruses in over 70 animal samples, ranging from cultured soil nematodes to human tissue specimens. A bioinformatics pipeline, Cenote-Taker, was developed to automatically annotate over 2,500 circular genomes in a GenBank-compliant format. The new genomes belong to dozens of established and emerging viral families. Some appear to be the result of previously undescribed recombination events between ssDNA viruses and ssRNA viruses. In addition, hundreds of circular DNA elements that do not encode any discernable similarities to previously characterized sequences were identified. To characterize these "dark matter" sequences, we used an artificial neural network to identify candidate viral capsid proteins, several of which formed virus-like particles when expressed in culture. These data further the understanding of viral sequence diversity and allow for high throughput documentation of the virosphere.

microbiology↗

bioRxiv2019

Modulation of Insr and insulin receptor signaling by hyperinsulinemia in vitro and in vivo

Hyperinsulinemia is commonly viewed as a compensatory response to insulin resistance, yet studies have suggested that chronically elevated insulin may also drive insulin resistance. The molecular mechanisms underpinning this potentially cyclic process remain poorly defined, especially on a transcriptome-wide level. To study the direct effects of prolonged exposure to excess insulin in muscle cells, we incubated C2C12 myotubes with elevated insulin for 16 hours, followed by 6 hours of serum starvation, and established that acute AKT and ERK signaling were attenuated in this model of in vitro hyperinsulinemia. Global RNA-sequencing of cells both before and after nutrient withdrawal highlighted genes in the insulin signaling, FOXO signaling, and glucose metabolism pathways indicative of hyperinsulinemia and starvation programs. We observed that hyperinsulinemia led to a substantial reduction in insulin receptor (Insr) gene expression, and subsequently a reduced surface INSR and total INSR protein, both in vitro and in vivo. Transcriptomic meta-analysis in >450 human samples demonstrated that fasting insulin reliably and negatively correlated with insulin receptor (INSR) mRNA in skeletal muscle. Bioinformatic modeling combined with RNAi, identified SIN3A as a negative regulator of Insr mRNA (and JUND, MAX, and MXI as positive regulators of Irs2 mRNA). Together, our analysis identifies novel mechanisms which may explain the cyclic processes underlying hyperinsulinemia-induced insulin resistance in muscle, a process directly relevant to the etiology and disease progression of type 2 diabetes.

physiology↗

bioRxiv2019

The opportunistic pathogen Stenotrophomonas maltophilia utilizes a type IV secretion system for interbacterial killing

Bacterial type IV secretion systems (T4SS) are a highly diversified but evolutionarily related family of macromolecule transporters that can secrete proteins and DNA into the extracellular medium or into target cells. They have been long known to play a fundamental role in bacterial conjugation and virulence of several species. It was recently shown that a subtype of T4SS harboured by the plant pathogenic bacterium Xanthomonas citri transfers toxins into other bacteria cells resulting in cell death. In this study, we show that a similar T4SS from the multi-drug-resistant global opportunistic pathogen Stenotrophomonas maltophilia is proficient in killing competitor bacterial species. T4SS-dependent duelling between S. maltophilia and X. citri was observed by time-lapse fluorescence microscopy. A bioinformatic search of the S. maltophilia K279a genome for proteins containing a C-terminal domain (XVIPCD) conserved in X. citri T4SS effectors identified eleven putative effectors secreted by the S. maltophilia T4SS. Six of these effectors have no recognizable domain except for the XVIPCD. We selected one of these new effectors (Smlt3024) and its cognate inhibitor (Smlt3025) for further characterization and confirmed that Smlt3024 is indeed secreted in a T4SS-dependent manner by S. maltophilia when in contact with a target bacterial species. Expression of Smlt3024 in the periplasm of E. coli resulted in greatly reduced growth rate and cell size, which could be counteracted by co-expression with its cognate periplasmic inhibitor, Smlt3025. This work expands our current knowledge about the diverse function of T4SSs subtypes and increases the panel of effectors known to be involved in T4SS-mediated interbacterial competition. Further elucidation of the mechanism of these antibacterial proteins could lead to the discovery of new antibacterial targets. The study also adds information about the molecular mechanisms possibly contributing to the establishment of S. maltophilia in different biotic and abiotic surfaces in both clinical and environmental settings. Author SummaryCompetition between microorganisms for nutrients and space determines which species will emerge and dominate or be eradicated from a specific habitat. Bacteria use a series of mechanisms to kill or prevent multiplication of competitor species. Recently, it was reported that a subtype of type IV secretion system (T4SS) works as a weapon to kill competitor bacterial species. In this study, we show that an important human opportunistic pathogen, Stenotrophomonas maltophilia, harbours a T4SS that promotes killing of competitor species. We also identified a series of new toxic proteins secreted by S. maltophilia via its T4SS to poison competitor species. We showed that two different bacterial species that harbour a bacteria-killing T4SS can kill each other; most likely due to differences in effector-immunity protein pairs. This work expands our current knowledge about the bacterial arsenal used in competitions with other species and expands the repertoire of antibacterial ammunition fired by T4SSs. In addition, the work contributes with knowledge on the possible mechanisms used by S. maltophilia to establish communities in different biotic and abiotic surfaces in both clinical and environmental settings.

microbiology↗

bioRxiv2019

Glutarate metabolism in Pseudomonas putida is regulated by two distinct glutarate sensing transcription factors

Transcription factor based biosensors can be leveraged to screen thousands of genetics designs for optimal production in engineered microbes. In this study we characterize two glutarate sensing transcription factors (CsiR and GcdR) from Pseudomonas putida. The genomic contexts of CsiR homologs were analyzed and DNA binding sites were bioinformatically predicted. Both CsiR and GcdR were purified and shown to bind upstream of their coding sequencing in vitro. CsiR was shown to dissociate from DNA in vitro when exogenous glutarate was added confirming it acts as a genetic repressor. Both transcription factors were then engineered into plasmid based biosensors and their respective sensing performance features calculated. Both sensor plasmids were then reintroduced into P. putida and were evaluated for their ability to sense flux through glutarate when grown on various lysine metabolites as sole carbon sources. These findings provide information describing glutarate flux in P. putida, and potentially useful tools for future metabolic engineering and synthetic biology efforts.

microbiology↗

bioRxiv2019

Systematic identification of circular RNAs and corresponding regulatory networks unveil their potential roles in the midgut of Apis cerana cerana workers

BackgroundCircular RNAs (circRNAs) are newly discovered noncoding RNAs (ncRNAs) that play key roles in various biological functions, such as the regulation of gene expression and alternative splicing. CircRNAs have been identified in some species, including western honeybees. However, the understanding of honeybee circRNA is still very limited, and to date, no study on eastern honeybee circRNA has been conducted. Here, the circRNAs in the midguts of Apis cerana cerana workers were identified and validated, and the regulatory networks were constructed. Differentially expressed circRNAs (DEcircRNAs) and the corresponding competitively endogenous RNA (ceRNA) networks in the development of the workers midgut were further investigated. ResultsHere, 7- and 10-day-old A. c. cerana workers midguts (Ac1 and Ac2) were sequenced using RNA-seq, and a total of 9589 circRNAs were predicted using bioinformatics. These circRNAs were approximately 201-800 nt in length and could be classified into six types; the annotated exonic circRNAs were the most abundant. Additionally, five novel A. c. cerana circRNAs were confirmed by PCR amplification and Sanger sequencing, indicating the authenticity of A. c. cerana circRNAs. Interestingly, novel_circ_003723, novel_circ_002714, novel_circ_002451 and novel_circ_001980 were the most highly expressed circRNAs in both Ac1 and Ac2, which is indicative of their key roles in the development of the midgut. Moreover, 55 DEcircRNAs were identified in the Ac1 vs Ac2 comparison group, including 34 upregulated and 21 downregulated circRNAs. Further investigation showed that the source genes of circRNAs were classified into 34 GO terms and were involved in 141 KEGG pathways. In addition, the source genes of DEcircRNAs were categorized into 10 GO terms and 15 KEGG pathways, which demonstrated that the corresponding DEcircRNAs may affect the growth, development, and material and energy metabolisms of the workers midgut by regulating the expression of the related source genes. Additionally, the circRNA-miRNA regulatory networks were constructed and analyzed, and the results demonstrated that 1060 circRNAs can bind to 74 miRNAs and that 71.51% of circRNAs can be linked to only one miRNA. Furthermore, the DEcircRNA-miRNA-mRNA networks were constructed and explored, and the results indicate that the 13 downregulated circRNAs can bind to eight miRNAs and to 29 target genes. In addition, the results indicate that the 16 upregulated circRNAs can bind to 9 miRNAs and to 29 target genes, demonstrating that DEcircRNAs are likely involved in the regulation of midgut development via ceRNA mechanisms. Moreover, the regulatory networks of miR-6001-y-targeted DEcircRNAs were analyzed, and the results showed that eight DEcircRNAs may affect the development of A. c. cerana workers midguts by targeting miR-6001-y. Finally, four randomly selected DEcircRNAs were verified via RT-qPCR, confirming the reliability of our sequencing data. ConclusionThis is the first systematic investigation of circRNAs and their corresponding regulatory networks in eastern honeybees. The identified circRNAs from the A. c. cerana workers midgut will enrich the known reservoir of honeybee ncRNAs. DEcircRNAs may play a comprehensive role during the development of the workers midgut via the regulation of source genes and the interaction with miRNAs by acting as ceRNAs. The eight DEcircRNAs that targeted miR-6001-y were likely to be vital for the development of the workers midgut. Our results provide a valuable resource for the future studies of A. c. cerana circRNA and lay a foundation to reveal the molecular mechanisms underlying the regulatory networks of circRNAs responsible for the workers midgut development; in addition, these findings facilitate a functional study on the key circRNAs involved in the developmental process. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

molecular biology↗

bioRxiv2019

Rapid, multiplexed, whole genome and plasmid sequencing of foodborne pathogens using long-read nanopore technology

United States public health agencies are focusing on next-generation sequencing (NGS) to quickly identify and characterize foodborne pathogens. Here, the MinION nanopore, long-read sequencer was used to simultaneously sequence the entire chromosome and plasmids of Salmonella enterica subsp. enterica serovar Bareilly and Escherichia coli O157:H7. A rapid, random sequencing approach, coupled with de novo genome assembly within a customized data analysis workflow, that can resolve highly-repetitive genomic regions, was developed. In sequencing runs, as short as four hours, using nanopore data alone, full-length genomes were obtained with an average identity of 99.87% for Salmonella Bareilly and 99.89% for E. coli in comparison to the respective MiSeq references. These long-read assemblies provided information on serotype, virulence factors, and antimicrobial resistance genes. Using a custom-developed, SNP-selection workflow, the potential of the nanopore-only assemblies (after only 30 minutes of sequencing) for rapid phylogenetic inference, with identical topology compared to the published dataset, was demonstrated. To achieve maximum quality assemblies, the developed bioinformatics workflow employed additional polishing steps to correct the systematic errors produced by the nanopore-only assemblies. Nanopore sequencing provided a shorter (10 hours library preparation and sequencing) turnaround time compared to other NGS technologies.

microbiology↗

bioRxiv2019

Structure-guided function discovery of an NRPS-like glycine betaine reductase for choline biosynthesis in fungi

Nonribosomal peptide synthetases (NRPS) and NRPS-like enzymes have diverse functions in primary and secondary metabolism. By using a structure-guided approach, we uncovered the function of an NRPS-like enzyme with unusual domain architecture, catalyzing two sequential two-electron reductions of glycine betaine to choline. Structural analysis based on homology model suggests cation-{pi} interactions as the major substrate specificity determinant, which was verified using substrate analogs and inhibitors. Bioinformatic analysis indicates this NRPS-like glycine betaine reductase is highly conserved and widespread in fungi kingdom. Genetic knockout experiments confirmed its role in choline biosynthesis and maintaining glycine betaine homeostasis in fungi. Our findings demonstrate that the oxidative choline-glycine betaine degradation pathway can operate in a fully reversible fashion and provide new insights in understanding fungal choline metabolism. The use of an NRPS-like enzyme for reductive choline formation is energetically efficient compared to known pathways. Our discovery also underscores the capabilities of structure-guided approach in assigning function of uncharacterized multidomain proteins, which can potentially aid functional discovery of new enzymes by genome mining.

biochemistry↗

bioRxiv2019

OxyR senses reactive sulfane sulfur and activates genes for its removal in Escherichia coli

Reactive sulfane sulfur species such as hydrogen polysulfide and organic persulfide are newly recognized as normal cellular components, involved in signaling and protecting cells from oxidative stress. Their production is extensively studied, but their removal is less characterized. Herein, we showed that reactive sulfane sulfur is toxic at high levels, and it is mainly removed via reduction by thioredoxin and glutaredoxin with the release of H2S in Escherichia coli. OxyR is best known to respond to H2O2, and it also played an important role in responding to reactive sulfane sulfur under both aerobic and anaerobic conditions. It was modified by hydrogen polysulfide to OxyR C199-SSH, which activated the expression of thioredoxin 2 and glutaredoxin 1. This is a new type of OxyR modification. Bioinformatics analysis showed that OxyRs are widely present in bacteria, including strict anaerobic bacteria. Thus, the OxyR sensing of reactive sulfane sulfur may represent a conserved mechanism for bacteria to deal with sulfane sulfur stress.

microbiology↗

bioRxiv2019

Quantitative proteomic profiling of tumor-associated vascular endothelial cells in colorectal cancer

SummeryTo investigate the global proteomic profiles of vascular endothelial cells (VECs) in the tumor microenvironment and antiangiogenic therapy for colorectal cancer (CRC), matched pairs of normal (NVECs) and tumor-associated VECs (TVECs) were purified from CRC tissues by laser capture microdissection and subjected to iTRAQ based quantitative proteomics analysis. Here, 216 differentially expressed proteins (DEPs) were identified and performed bioinformatics analysis. Interestingly, these proteins were implicated in epithelial mesenchymal transition (EMT), ECM-receptor interaction, focal adhesion, PI3K-Akt signaling pathway, angiogenesis and HIF-1 signaling pathway, which may play important roles in CRC angiogenesis. Among these DEPs, Tenascin-C (TNC) was found to upregulated in the TVECs of CRC and be correlate with CRC multistage carcinogenesis and metastasis. Furthermore, the reduction of tumor-derived TNC could attenuate human umbilical vein endothelial cell (HUVEC) proliferation, migration and tube formation through ITGB3/FAK/Akt signaling pathway. Based on the present work, we provided a large-scale proteomic profiling of VECs in CRC with quantitative information, a certain number of potential antiangiogenic targets and a novel vision in the angiogenesis bio-mechanism of CRC. Summery statementWe provided large-scale proteomic profiling of vascular endothelial cells in colorectal cancer with quantitative information, a number of potential antiangiogenic targets and a novel vision in the angiogenesis bio-mechanism of CRC.

cancer biology↗

bioRxiv2019

High-coverage, long-read sequencing of Han Chinese trio reference samples.

Single-molecule long-read sequencing datasets were generated for a son-father-mother trio of Han Chinese descent that is part of the Genome In a Bottle (GIAB) consortium portfolio. The dataset was generated using the Pacific Biosciences Sequel System. The son and each parent were sequenced to an average coverage of 60 and 30, respectively, with N50 subread lengths between 16 and 18 kb. Raw reads and reads aligned to both the GRCh37 and GRCh38 are available at the NCBI GIAB ftp site (ftp://ftp-trace.ncbi.nlm.nih.gov/giab/ftp/data/ChineseTrio/) and the raw read data is archived in NCBI SRA (SRX4739017, SRX4739121, and SRX4739122). This dataset is available for anyone to develop and evaluate long-read bioinformatics methods.

genomics↗

bioRxiv2019

MicroRNA-138 negatively regulates the hypoxia-inducible factor 1α to suppress melanoma growth and metastasis

Melanoma with rapid progression towards metastasis becomes the deadliest form of skin cancer. However, the mechanism of melanoma growth and metastasis is still unclear. Here, we found that miRNA-138 was low expression and hypoxia-inducible factor 1 (HIF1) was high expression in the patients melanoma tissue, and they had a significant negative correlation (r=-0.937, P < 0.001). Patients with miRNA-138low/HIF1high signature were predominant in late stage. Further, bioinformatic analysis demonstrated that miRNA-138 directly targeted HIF1. We found that the introduction of miRNA-138 mimics to A375 cells could reduced HIF1 mRNA expression, and suppressed the cell proliferation, migration and invasion. Overexpression of miRNA-138 or inhibition of HIF1 significantly suppressed the growth and metastasis of melanoma in vivo. Our study demonstrates the role and clinical relevance of miRNA-138 and HIF1 in melanoma cell growth and metastasis, providing a novel therapeutic target for suppression of melanoma growth and metastasis.

cancer biology↗

bioRxiv2019

Single-Cell RNA Sequencing Reveals Regulatory Mechanism for Trophoblast Cell-Fate Divergence in Human Peri-Implantation Embryo

Multipotent trophoblasts undergo dynamic morphological movement and cellular differentiation after embryonic implantation to generate placenta. However, the mechanism controlling trophoblast development and differentiation during peri-implantation development remains elusive. In this study, we modeled human embryo peri-implantation development from blastocyst to early post-implantation stages by using an in vitro coculture system, and profiled the transcriptome of individual trophoblast cells from these embryos. We revealed the genetic networks regulating peri-implantation trophoblast development. While determining when trophoblast differentiation happens, our bioinformatic analysis identified T-box transcription factor 3 (TBX3) as a key regulator for the differentiation of cytotrophoblast into syncytiotrophoblast. The function of TBX3 in trophoblast differentiation is then validated by a loss-of-function experiment. In conclusion, our results provided a valuable resource to study the regulation of trophoblasts development and differentiation during human peri-implantation development.

cell biology↗

bioRxiv2019

Pulcherrimin formation controls growth arrest of the Bacillus subtilis biofilm

Biofilm formation by Bacillus subtilis is a communal process that culminates in the formation of architecturally complex multicellular communities. Here we reveal that the transition of the biofilm into a non-expanding phase constitutes a distinct step in the process of biofilm development. Using genetic analysis we show that B. subtilis strains lacking the ability to synthesize pulcherriminic acid form biofilms that sustain the expansion phase, thereby linking pulcherriminic acid to growth arrest. However, production of pulcherriminic acid is not sufficient to block expansion of the biofilm. It needs to be secreted into the extracellular environment where it chelates Fe3+ from the growth medium in a non-enzymatic reaction. Utilizing mathematical modelling and a series of experimental methodologies we show that when the level of freely available iron in the environment drops below a critical threshold, expansion of the biofilm stops. Bioinformatics analysis allows us to identify the genes required for pulcherriminic acid synthesis in other Firmicutes but the patchwork presence both within and across closely related species suggests loss of these genes through multiple independent recombination events. The seemingly counterintuitive self-restriction of growth led us to explore if there were any benefits associated pulcherriminic acid production. We identified that pulcherriminic acid producers can prevent invasion from neighbouring communities through the generation of an \"iron free\" zone thereby addressing the paradox of pulcherriminic acid production by B. subtilis.\n\nSignificanceUnderstanding the processes that underpin the mechanism of biofilm formation, dispersal, and inhibition are critical to allow exploitation and to understand how microbes thrive in the environment. Here, we reveal that the formation of an extracellular iron chelate restricts the expansion of a biofilm. The countering benefit to self-restriction of growth is protection of an environmental niche. These findings highlight the complex options and outcomes that bacteria need to balance in order to modulate their local environment to maximise colonisation, and therefore survival.

microbiology↗

bioRxiv2019

Identification and characterization of putative Aeromonas spp. T3SS effectors

The genetic determinants of bacterial pathogenicity are highly variable between species and strains. However, a factor that is commonly associated with virulent Gram-negative bacteria, including many Aeromonas spp., is the type 3 secretion system (T3SS), which is used to inject effector proteins into target eukaryotic cells. In this study, we developed a bioinformatics pipeline to identify T3SS effector proteins, applied this approach to the genomes of 105 Aeromonas strains isolated from environmental, mutualistic, or pathogenic contexts and evaluated the cytotoxicity of the identified effectors through their heterologous expression in yeast. The developed pipeline uses a two-step approach, where candidate families are initially selected using HMM profiles with minimal similarity scores against the Virulence Factors DataBase (VFDB), followed by strict comparisons against positive and negative control datasets, greatly reducing the number of false positives. Using our approach, we identified 21 Aeromonas T3SS likely effector groups, of which 8 represented known or characterized effectors, while the remaining 13 had not previously been described in Aeromonas. We experimentally validated our in silico findings by assessing the cytotoxicity of representative effectors in Saccharomyces cerevisiae BY4741, with 15 out of 21 assayed proteins eliciting a cytotoxic effect in yeast. The results of this study demonstrate the utility of our approach, combining a novel in silico search method with in vivo experimental validation, and will be useful in future research aimed at identifying and authenticating bacterial effector proteins from other genera.

microbiology↗