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At least 109 records · Page 6
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes

Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a mu ltiplex, r andomized C RISPR i nterference s equencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 Legionella pneumophila virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with L. pneumophila bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. lpg2888 and lpg3000 were particularly fascinating for their apparent redundant functions during L. pneumophila human macrophage infection, while lpg3000 alone was essential for L. pneumophila virulence in the amoeban host Acanthamoeba castellanii . Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes

Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a multiplex, randomized CRISPR interference sequencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 Legionella pneumophila virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with L. pneumophila bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. lpg2888 and lpg3000 were particularly fascinating for their apparent redundant functions during L. pneumophila human macrophage infection, while lpg3000 alone was essential for L. pneumophila virulence in the amoeban host Acanthamoeba castellanii. Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.

79 ASTRONOMY AND ASTROPHYSICS↗
DOE OSTI2023

A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes

Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a mu ltiplex, r andomized C RISPR i nterference s equencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 Legionella pneumophila virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with L. pneumophila bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. lpg2888 and lpg3000 were particularly fascinating for their apparent redundant functions during L. pneumophila human macrophage infection, while lpg3000 alone was essential for L. pneumophila virulence in the amoeban host Acanthamoeba castellanii . Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.

Ellis, Nicole A. (ORCID:0000000195928415)↗
DOE OSTI2024

Characterization of Histophilus somni sialic acid uptake mutant (Δ nanP -Δ nanU ) using a mouse septicemia and mortality model

Histophilus somni is an important pathogen of the bovine respiratory disease complex, yet the mechanisms underlying its virulence remain poorly understood. It is known that H. somni can incorporate sialic acid into lipooligosaccharide (LOS), and sialylated H. somni is more resistant to phagocytosis and complement-mediated killing by serum compared to non-sialylated bacteria in vitro. However, the virulence of non-sialylated H. somni has not been evaluated in vivo using an animal model. In this study, we investigated the contribution of sialic acid to virulence by constructing an H. somni sialic acid uptake mutant (ΔnanP-ΔnanU) and comparing the parent and mutant strains in a mouse septicemia and mortality model. Intraperitoneal challenge of mice with wildtype H. somni (1 × 10 8 colony forming units/mouse, CFU) was lethal to all animals. Mice challenged with three different doses (1, 2, or 5 × 108 CFU/mouse) of an H. somni ΔnanP-ΔnanU sialic acid uptake mutant exhibited survival rates of 90 %, 60 %, and 0 % respectively. High-performance anion exchange chromatography analyses revealed that LOS prepared from both parent and the ΔnanP-ΔnanU mutant strains of H. somni were sialylated. These findings suggest the presence of de novo sialic acid synthesis pathway, although the genes associated with de novo sialic acid synthesis (neuB and neuC) were not identified by genomic analysis. The lower attenuation in mice is most likely attributed to the sialylated LOS of H. somni nanPU mutant.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A Single Amino Acid Substitution in the Matrix Protein (M51R) of Vesicular Stomatitis New Jersey Virus Impairs Replication in Cultured Porcine Macrophages and Results in Significant Attenuation in Pigs

In this study, we explore the virulence of vesicular stomatitis New Jersey virus (VSNJV) in pigs and its potential relationship with the virus’s ability to modulate innate responses. For this purpose, we developed a mutant of the highly virulent strain NJ0612NME6, containing a single amino acid substitution in the matrix protein (M51R). The M51R mutant of NJ0612NME6 was unable to suppress the transcription of genes associated with the innate immune response both in primary fetal porcine kidney cells and porcine primary macrophage cultures. Impaired viral growth was observed only in porcine macrophage cultures, indicating that the M51 residue is required for efficient replication of VSNJV in these cells. Furthermore, when inoculated in pigs by intradermal scarification of the snout, M51R infection was characterized by decreased clinical signs including reduced fever and development of less and smaller secondary vesicular lesions. Pigs infected with M51R had decreased levels of viral shedding and absence of RNAemia compared to the parental virus. The ability of the mutant virus to infect pigs by direct contact remained intact, indicating that the M51R mutation resulted in a partially attenuated phenotype capable of causing primary lesions and transmitting to sentinel pigs. Collectively, our results show a positive correlation between the ability of VSNJV to counteract the innate immune response in swine macrophage cultures and the level of virulence in pigs, a natural host of this virus. More studies are encouraged to evaluate the interaction of VSNJV with macrophages and other components of the immune response in pigs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Comparative Genomics Applied to Systematically Assess Pathogenicity Potential in Shiga Toxin-Producing Escherichia coli O145:H28

Shiga toxin-producing Escherichia coli (STEC) O145:H28 can cause severe disease in humans and is a predominant serotype in STEC O145 environmental isolates. Here, comparative genomics was applied to a set of clinical and environmental strains to systematically evaluate the pathogenicity potential in environmental strains. While the core genes-based tree separated all O145:H28 strains from the non O145:H28 reference strains, it failed to segregate environmental strains from the clinical. In contrast, the accessory genes-based tree placed all clinical strains in the same clade regardless of their genotypes or serotypes, apart from the environmental strains. Loss-of-function mutations were common in the virulence genes examined, with a high frequency in genes related to adherence, autotransporters, and the type three secretion system. Distinct differences in pathogenicity islands LEE, OI-122, and OI-57, the acid fitness island, and the tellurite resistance island were detected between the O145:H28 and reference strains. A great amount of genetic variation was detected in O145:H28, which was mainly attributed to deletions, insertions, and gene acquisition at several chromosomal “hot spots”. Finally, our study demonstrated a distinct virulence gene repertoire among the STEC O145:H28 strains originating from the same geographical region and revealed unforeseen contributions of loss-of-function mutations to virulence evolution and genetic diversification in STEC.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Evaluation in Swine of a Recombinant Georgia 2010 African Swine Fever Virus Lacking the I8L Gene

African swine fever virus (ASFV) is the causative agent of African swine fever, a disease currently causing significant economic losses in Europe and Asia. Specifically, the highly virulent ASFV strain Georgia 2010 (ASFV-G) is producing disease outbreaks in this large geographical region. The ASFV genome encodes for over 150 genes, most of which are still not experimentally characterized. I8L is a highly conserved gene that has not been studied beyond its initial description as a virus ORF. Transcriptional analysis of swine macrophages infected with ASFV-G demonstrated that the I8L gene is transcribed early during the virus replication cycle. To assess the importance of I8L during ASFV-G replication in vitro and in vivo, as well as its role in virus virulence in domestic swine, we developed a recombinant virus lacking the I8L gene (ASFV-G-ΔI8L). Replication of ASFV-G-ΔI8L was similar to parental ASFV-G replication in primary swine macrophage cultures, suggesting that the I8L gene is not essential for ASFV-G replication in vitro. Similarly, replication of ASFV-G-ΔI8L in swine intramuscularly inoculated with 102 HAD50 displayed replication kinetics similar to ASFV-G. In addition, animals inoculated with ASFV-G-ΔI8L presented with a clinical disease indistinguishable from that induced by the same dose of the virulent parental ASFV-G isolate. We conclude that deletion of the I8L gene from ASFV-G does not affect virus replication in vitro or in vivo, nor changes the disease outcome in swine.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Classical Swine Fever Virus Structural Glycoprotein E2 Interacts with Host Protein ACADM during the Virus Infectious Cycle

The E2 glycoprotein is one of the four structural proteins of the classical swine fever virus (CSFV) particle. E2 has been shown to be involved in many virus functions, including adsorption to host cells, virus virulence and interaction with several host proteins. Using a yeast two-hybrid screen, we have previously shown that the CSFV E2 specifically interacts with swine host protein medium-chain-specific acyl-Coenzyme A dehydrogenase (ACADM), an enzyme that catalyzes the initial step of the mitochondrial fatty acid beta-oxidation pathway. Here, we show that interaction between ACADM and E2 also happens in swine cells infected with CSFV using two different procedures: coimmunoprecipitation and a proximity ligation assay (PLA). In addition, the amino acid residues in E2 critically mediating the interaction with ACADM, M49 and P130 were identified via a reverse yeast two-hybrid screen using an expression library composed of randomly mutated versions of E2. A recombinant CSFV, E2ΔACADMv, harboring substitutions at residues M49I and P130Q in E2, was developed via reverse genomics from the highly virulent Brescia isolate. E2ΔACADMv was shown to have the same kinetics growth in swine primary macrophages and SK6 cell cultures as the parental Brescia strain. Similarly, E2ΔACADMv demonstrated a similar level of virulence when inoculated to domestic pigs as the parental Brescia. Animals intranasally inoculated with 10 5 TCID 50 developed a lethal form of clinical disease with virological and hematological kinetics changes undistinguishable from those produced by the parental strain. Therefore, interaction between CSFV E2 and host ACADM is not critically involved in the processes of virus replication and disease production.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

The Interaction between the DOCK7 Protein and the E2 Protein of Classical Swine Fever Virus Is Not Involved with Viral Replication or Pathogenicity

The classical swine fever virus (CSFV) particle consists of three glycoproteins, all of which have been shown to be important proteins involved in many virus functions, including interaction with several host proteins. One of these proteins, E2, has been shown to be directly involved with adsorption to the host cell and important for virus virulence. Using the yeast two-hybrid system, we have previously shown that CSFV E2 specifically interacts with the (DOCK7) dedicator of cytokinesis, a scaffolding protein. In this report, the interaction between E2 and DOCK7 was evaluated. To confirm the yeast two-hybrid results and to determine that DOCK7 interacts in swine cells with E2, we performed co-immunoprecipitation and proximity ligation assay (PLA). After demonstrating the protein interaction in swine cells, E2 amino acid residues Y65, V283, and T149 were determined to be critical for interaction with Dock7 by using a random mutated library of E2 and a reverse yeast two-hybrid approach. That disruption of these three residues with mutations Y65F, V283D, and T149A abrogated the Dock7-E2 protein interaction. These mutations were then introduced into a recombinant CSFV, E2DOCK7v, by a reverse genomics approach using the highly virulent CSFV Brescia isolate as a backbone. E2DOCKv was shown to have similar growth kinetics in swine primary macrophages and SK6 cell cultures to the parental Brescia strain. Similarly, E2DOCK7v demonstrated a similar level of virulence to the parental Brescia when inoculated in domestic pigs. Animals intranasally inoculated with 10 5 TCID 50 developed a lethal form of clinical disease with virological and hematological kinetics changes indistinguishable from that produced by the parental strain. Therefore, interaction between CSFV E2 and host DOCK7 is not critically involved in the process of virus replication and disease production.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Mucus-derived glycans are inhibitory signals for Salmonella Typhimurium SPI-1-mediated invasion

Mucus forms a critical barrier against enteric pathogens like Salmonella enterica serovar Typhimurium. While in vivo studies indicate that secreted, gel-forming mucins and specifically core 3 glycosylation are protective against S. Typhimurium, the molecular mechanisms involved remain unclear. Here, we demonstrate that native intestinal mucins inhibit Salmonella invasion of colonic epithelial cells by downregulating the type 3 secretion system through suppression of the key virulence regulator, HilD. Our study identifies mucin glycans and specific mucin sugars, namely N-acetyl galactosamine and N-acetyl glucosamine, as the components responsible for mucin’s anti-virulence effect, likely via functional or direct interaction with HilD’s putative carbohydrate-binding domain. Notably, we find that the native presentation of these sugars is important for activity. These insights provide a mechanistic foundation for mucin-based strategies to combat enteric infections and, given the prevalence of homologous AraC-type regulators in other pathogens, suggest mucins’ potential as broad-spectrum anti-virulence agents.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Properties of protein unfolded states suggest broad selection for expanded conformational ensembles

Much attention is being paid to conformational biases in the ensembles of intrinsically disordered proteins. However, it is currently unknown whether or how conformational biases within the disordered ensembles of foldable proteins affect function in vivo. Recently, we demonstrated that water can be a good solvent for unfolded polypeptide chains, even those with a hydrophobic and charged sequence composition typical of folded proteins. These results run counter to the generally accepted model that protein folding begins with hydrophobicity-driven chain collapse. Here we investigate what other features, beyond amino acid composition, govern chain collapse. We found that local clustering of hydrophobic and/or charged residues leads to significant collapse of the unfolded ensemble of pertactin, a secreted autotransporter virulence protein from Bordetella pertussis , as measured by small angle X-ray scattering (SAXS). Sequence patterns that lead to collapse also correlate with increased intermolecular polypeptide chain association and aggregation. Crucially, sequence patterns that support an expanded conformational ensemble enhance pertactin secretion to the bacterial cell surface. Similar sequence pattern features are enriched across the large and diverse family of autotransporter virulence proteins, suggesting sequence patterns that favor an expanded conformational ensemble are under selection for efficient autotransporter protein secretion, a necessary prerequisite for virulence. More broadly, we found that sequence patterns that lead to more expanded conformational ensembles are enriched across water-soluble proteins in general, suggesting protein sequences are under selection to regulate collapse and minimize protein aggregation, in addition to their roles in stabilizing folded protein structures.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Genomic Signatures of a Major Adaptive Event in the Pathogenic Fungus Melampsora larici-populina

The recent availability of genome-wide sequencing techniques has allowed systematic screening for molecular signatures of adaptation, including in nonmodel organisms. Host–pathogen interactions constitute good models due to the strong selective pressures that they entail. We focused on an adaptive event which affected the poplar rust fungus Melampsora larici-populina when it overcame a resistance gene borne by its host, cultivated poplar. Based on 76 virulent and avirulent isolates framing narrowly the estimated date of the adaptive event, we examined the molecular signatures of selection. Using an array of genome scan methods based on different features of nucleotide diversity, we detected a single locus exhibiting a consistent pattern suggestive of a selective sweep in virulent individuals (excess of differentiation between virulent and avirulent samples, linkage disequilibrium, genotype–phenotype statistical association, and long-range haplotypes). Our study pinpoints a single gene and further a single amino acid replacement which may have allowed the adaptive event. Although our samples are nearly contemporary to the selective sweep, it does not seem to have affected genome diversity further than the immediate vicinity of the causal locus, which can be explained by a soft selective sweep (where selection acts on standing variation) and by the impact of recombination in mitigating the impact of selection. Therefore, it seems that properties of the life cycle of M. larici-populina, which entails both high genetic diversity and outbreeding, has facilitated its adaptation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Hidden Allies: Decoding the Core Endohyphal Bacteriome of Aspergillus fumigatus

ABSTRACT Bacterial–fungal interactions that influence the behaviour of one or both organisms are common in nature. Well‐studied systems include endosymbiotic relationships that range from transient to long‐term associations. Diverse endohyphal bacteria associate with fungal hosts, emphasising the need to better comprehend the fungal bacteriome. We evaluated the hypothesis thatAspergillus fumigatusharbours an endohyphal community of bacteria that influence the host phenotype. We analysed whether 38A. fumigatusstrains show stable association with diverse endohyphal bacteria; all derived from single‐conidium cultures that were subjected to antibiotic and heat treatments. The fungal bacteriome, inferred through analysis of bacterial diversity within the fungal strains (short‐ and long‐ read sequencing methods), revealed the presence of core endohyphal bacterial genera. Microscopic analysis further confirmed the presence of endohyphal bacteria. The fungal strains exhibited high genetic diversity and phenotypic heterogeneity in drug susceptibility and in vivo virulence. No correlations were observed between genomic or functional traits and bacteriome diversity, but the abundance of some bacterial genera correlated with fungal virulence or posaconazole susceptibility. The observed endobacteriome may play functional roles, for example, nitrogen fixation. Our study emphasises the existence of complex interactions between fungi and endohyphal bacteria, possibly impacting the phenotype of the fungal host, including virulence.

Environmental Sciences & Ecology↗
DOE OSTI2023

Immune activation during Pseudomonas infection causes local cell wall remodeling and alters AGP accumulation

The plant cell boundary generally comprises constituents of the primary and secondary cell wall (CW) that are deposited sequentially during development. Although it is known that the CW acts as a barrier against phytopathogens and undergoes modifications to limit their invasion, the extent, sequence, and requirements of the pathogen-induced modifications of the CW components are still largely unknown, especially at the level of the polysaccharide fraction. To address this significant knowledge gap, we adopted the compatible Pseudomonas syringae–Arabidopsis thaliana system. We found that, despite systemic signaling actuation, Pseudomonas infection leads only to local CW modifications. Furthermore, by utilizing a combination of CW and immune signaling-deficient mutants infected with virulent or non-virulent bacteria, we demonstrated that the pathogen-induced changes in CW polysaccharides depend on the combination of pathogen virulence and the host's ability to mount an immune response. This results in a pathogen-driven accumulation of CW hexoses, such as galactose, and an immune signaling-dependent increase in CW pentoses, mainly arabinose, and xylose. Our analyses of CW changes during disease progression also revealed a distinct spatiotemporal pattern of arabinogalactan protein (AGP) deposition and significant modifications of rhamnogalacturonan sidechains. Furthermore, genetic analyses demonstrated a critical role of AGPs, specifically of the Arabinoxylan Pectin Arabinogalactan Protein1, in limiting pathogen growth. Collectively, our results provide evidence for the actuation of significant remodeling of CW polysaccharides in a compatible host-pathogen interaction, and, by identifying AGPs as critical elements of the CW in plant defense, they pinpoint opportunities to improve plants against diverse pathogens.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Sensor Domain of Histidine Kinase VxrA of Vibrio cholerae : Hairpin-Swapped Dimer and Its Conformational Change

VxrA and VxrB are cognate histidine kinase (HK)-response regulator (RR) pairs of a two-component signaling system (TCS) found in Vibrio cholerae, a bacterial pathogen that causes cholera. The VxrAB TCS positively regulates virulence, the type VI secretion system, biofilm formation, and cell wall homeostasis in V. cholerae, providing protection from environmental stresses and contributing to the transmission and virulence of the pathogen. The VxrA HK has a unique periplasmic sensor domain (SD) and, remarkably, lacks a cytoplasmic linker domain between the second transmembrane helix and the dimerization and histidine phosphotransfer (DHp) domain, indicating that this system may utilize a potentially unique signal sensing and transmission TCS mechanism. Here, we have determined several crystal structures of VxrA-SD and its mutants. These structures reveal a novel structural fold forming an unusual β hairpin-swapped dimer. A conformational change caused by relative rotation of the two monomers in a VxrA-SD dimer could potentially change the association of transmembrane helices and, subsequently, the pairing of cytoplasmic DHp domains. Based on the structural observation, we propose a putative scissor-like closing regulation mechanism for the VxrA HK. IMPORTANCE V. cholerae has a dynamic life cycle, which requires rapid adaptation to changing external conditions. Two-component signal transduction (TCS) systems allow V. cholerae to sense and respond to these environmental changes. The VxrAB TCS positively regulates a number of important V. cholerae phenotypes, including virulence, the type six secretion system, biofilm formation, and cell wall homeostasis. Here, we provide the crystal structures of the VxrA sensor histidine kinase sensing domain and propose a mechanism for signal transduction. The cognate signal for VxrAB remains unknown; however, in this work we couple our structural analysis with functional assessments of key residues to further our understanding of this important TCS.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

CRISPR-prime editing, a versatile genetic tool to create specific mutations with a single nucleotide resolution in Leptospira

ABSTRACT Leptospirosis, caused by pathogenic bacteria from the genusLeptospira, is a global zoonosis responsible for more than one million human cases and 60,000 deaths annually. The disease also affects many domestic animal species. Historically, genetic manipulation ofLeptospirahas been difficult to perform, resulting in limited knowledge on pathogenic mechanisms of disease and the identification of virulence factors. The application of CRISPR/Cas9 and its variations have helped fill these gaps but the generation of knockout mutants remains challenging because double-strand breaks (DSBs) inflicted by Cas9 nuclease are lethal toLeptospiracells. The novel CRISPR prime editing (PE) strategy is the first precise genome-editing technology that allows deletions, insertions, and base substitutions without introducing DSBs. This revolutionary technique utilizes a nickase Cas9 that cleaves a single strand of DNA, coupled with an engineered reverse transcriptase and a modified single-guide RNA (termed prime editing guide RNA) containing an extended 3′ end with the desired edits. We demonstrate the application of CRISPR-PE in both saprophytic and pathogenicLeptospirafrom multiple species and serovars by introducing deletions or insertions into target DNA with a remarkable precision of just one nucleotide. Additionally, we demonstrate the ability to genetically manipulateLeptospira borgpetersenii, a prevalent pathogenic species of humans, domestic cattle, and wildlife animals. Rapid plasmid loss by mutated strains in liquid culture allows for the generation of knockout strains without selective markers, which can be readily used to elucidate virulence factors and develop optimized bacterin and/or live vaccines against leptospirosis. IMPORTANCE Leptospirosis is a geographically widespread bacterial zoonosis. Genetic manipulation of pathogenicLeptospiraspp. has been laborious and difficult to perform, limiting our ability to understand how leptospires cause disease. The application of the CRISPR/Cas9 system toLeptospiraenhanced our ability to generate knockdown and knockout mutants; however, the latter remains challenging. Here, we demonstrate the application of the CRISPR prime editing technique inLeptospira, allowing the generation of knockout mutants in several pathogenic species, with mutations comprising just a single nucleotide resolution. Notably, we generated a mutant in theLeptospira borgpeterseniibackground, a prevalent pathogenic species of humans and cattle. Our application of this method opens new avenues for studying pathogenic mechanisms ofLeptospiraand the identification of virulence factors across multiple species. These methods can also be used to facilitate the generation of marker-less knockout strains for updated and improved bacterin and/or live vaccines.

Microbiology↗
DOE OSTI2020

Social Evolution and Cheating in Plant Pathogens

Plant pathogens are a critical component of the microbiome that exist as populations undergoing ecological and evolutionary processes within their host. Many aspects of virulence rely on social interactions mediated through multiple forms of public goods, including quorum-sensing signals, exoenzymes, and effectors. Virulence and disease progression involve life-history decisions that have social implications with large effects on both host and microbe fitness, such as the timing of key transitions. Considering the molecular basis of sequential stages of plant–pathogen interactions highlights many opportunities for pathogens to cheat, and there is evidence for ample variation in virulence. Case studies reveal systems where cheating has been demonstrated and others where it is likely occurring. Harnessing the social interactions of pathogens, along with leveraging novel sensing and -omics technologies to understand microbial fitness in the field, will enable us to better manage plant microbiomes in the interest of plant health.

Plant Sciences↗
DOE OSTI2021

A Targeted Sequencing Assay for Serotyping Escherichia coli Using AgriSeq Technology

The gold standard method for serotyping Escherichia coli has relied on antisera-based typing of the O- and H-antigens, which is labor intensive and often unreliable. In the post-genomic era, sequence-based assays are potentially faster to provide results, could combine O-serogrouping and H-typing in a single test, and could simultaneously screen for the presence of other genetic markers of interest such as virulence factors. Whole genome sequencing is one approach; however, this method has limited multiplexing capabilities, and only a small fraction of the sequence is informative for subtyping or identifying virulence potential. A targeted, sequence-based assay and accompanying software for data analysis would be a great improvement over the currently available methods for serotyping. The purpose of this study was to develop a high-throughput, molecular method for serotyping E. coli by sequencing the genes that are required for production of O- and H-antigens, as well as to develop software for data analysis and serotype identification. To expand the utility of the assay, targets for the virulence factors, Shiga toxins ( stx 1 , and stx 2 ) and intimin ( eae ) were included. To validate the assay, genomic DNA was extracted from O-serogroup and H-type standard strains and from Shiga toxin-producing E. coli , the targeted regions were amplified, and then sequencing libraries were prepared from the amplified products followed by sequencing of the libraries on the Ion S5™ sequencer. The resulting sequence files were analyzed via the SeroType Caller™ software for identification of O-serogroup, H-type, and presence of stx 1 , stx 2 , and eae . We successfully identified 169 O-serogroups and 41 H-types. The assay also routinely detected the presence of stx 1a,c,d (3 of 3 strains), stx 2c−e,g (8 of 8 strains), stx 2f (1 strain), and eae (6 of 6 strains). Taken together, the high-throughput, sequence-based method presented here is a reliable alternative to antisera-based serotyping methods for E. coli .

Elder, Jacob R.↗