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All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Media matters! Alterations in the loading and release of Histoplasma capsulatum extracellular vesicles in response to different nutritional milieus

Histoplasma capsulatum is a dimorphic fungus that most frequently causes pneumonia, but can also disseminate and proliferate in diverse tissues. H. capsulatum has a complex secretion system that mediates the release of macromolecule-degrading enzymes and virulence factors. The formation and release of extracellular vesicles (EVs) are an important mechanism for non-conventional secretion in both ascomycetes and basidiomycetes. H. capsulatum EVs contain diverse proteins associated with virulence and are immunologically active. Despite the growing knowledge of EVs from H. capsulatum and other pathogenic fungi, the extent that changes in the environment impact the sorting of organic molecules in EVs has not been investigated. In our research, we cultivated H. capsulatum with distinct culture media to investigate the potential plasticity in EV loading in response to differences in nutrition. Our findings reveal that nutrition plays an important role in EV loading and formation, which may translate into differences in biological activities of these fungi in various fluids and tissues.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Xylella fastidiosa modulates exopolysaccharide polymer length and the dynamics of biofilm development with a β-1,4-endoglucanase

Xylella fastidiosa is a Gram-negative bacterium that causes disease in many economically important crops. It colonizes the plant host xylem and the mouthparts of its insect vectors where it produces exopolysaccharide (EPS) and forms robust biofilms. Typically, the ability to form a biofilm enhances virulence, but X. fastidiosa does not fit neatly into that paradigm. Instead, X. fastidiosa enters into biofilms to attenuate its movement in the xylem, which, in turn, slows disease progression. In most of its over 600 known plant hosts, X. fastidiosa behaves as a benign commensal, but in some hosts like Vitis vinifera grapevines, it acts as a pathogen. Its ability to attenuate its own virulence in susceptible hosts may be a remnant of its commensal lifestyle in other hosts. Here, we demonstrate that X. fastidiosa utilizes a β-1,4 endoglucanase to cleave its self-produced β-1,4-glucan exopolysaccharide polymer to process it from a higher molecular weight to a lower molecular weight polymer. This processing mediates surface adherence of the cells and ultimately governs overall biofilm architecture, indicating enzymatic pruning of the EPS plays a key role in biofilm-mediated attenuation of X. fastidiosa in planta and, thus, is a key vestige that links its commensal behaviors to its parasitic behaviors in specific hosts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Predicting Antimicrobial Resistance Using Partial Genome Alignments

Antimicrobial resistance (AMR) is an important global health threat that impacts millions of people worldwide each year. Developing methods that can detect and predict AMR phenotypes can help to mitigate the spread of AMR by informing clinical decision making and appropriate mitigation strategies. Many bioinformatic methods have been developed for predicting AMR phenotypes from whole-genome sequences and AMR genes, but recent studies have indicated that predictions can be made from incomplete genome sequence data. In order to more systematically understand this, we built random forest-based machine learning classifiers for predicting susceptible and resistant phenotypes for Klebsiella pneumoniae (1,640 strains), Mycobacterium tuberculosis (2,497 strains), and Salmonella enterica (1,981 strains). We started by building models from alignments that were based on a reference chromosome for each species. We then subsampled each chromosomal alignment and built models for the resulting subalignments, finding that very small regions, representing approximately 0.1 to 0.2% of the chromosome, are predictive. In K. pneumoniae, M. tuberculosis, and S. enterica, the subalignments are able to predict multiple AMR phenotypes with at least 70% accuracy, even though most do not encode an AMR-related function. We used these models to identify regions of the chromosome with high and low predictive signals. Finally, subalignments that retain high accuracy across larger phylogenetic distances were examined in greater detail, revealing genes and intergenic regions with potential links to AMR, virulence, transport, and survival under stress conditions. IMPORTANCE Antimicrobial resistance causes thousands of deaths annually worldwide. Understanding the regions of the genome that are involved in antimicrobial resistance is important for developing mitigation strategies and preventing transmission. Machine learning models are capable of predicting antimicrobial resistance phenotypes from bacterial genome sequence data by identifying resistance genes, mutations, and other correlated features. They are also capable of implicating regions of the genome that have not been previously characterized as being involved in resistance. In this study, we generated global chromosomal alignments for Klebsiella pneumoniae, Mycobacterium tuberculosis, and Salmonella enterica and systematically searched them for small conserved regions of the genome that enable the prediction of antimicrobial resistance phenotypes. In addition to known antimicrobial resistance genes, this analysis identified genes involved in virulence and transport functions, as well as many genes with no previous implication in antimicrobial resistance.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Global Network Analysis of Neisseria gonorrhoeae Identifies Coordination between Pathways, Processes, and Regulators Expressed during Human Infection

Neisseria gonorrhoeae is a Gram-negative diplococcus that is responsible for the sexually transmitted infection gonorrhea, a high-morbidity disease in the United States and worldwide. Over the past several years, N. gonorrhoeae strains resistant to antibiotics used to treat this infection have begun to emerge across the globe. Thus, new treatment strategies are needed to combat this organism. Here, we utilized N. gonorrhoeae transcriptomic data sets, including those obtained from natural infection of the human genital tract, to infer the first global gene coexpression network of this pathogen. Interrogation of this network revealed genes central to the network that are likely critical for gonococcal growth, metabolism, and virulence, including genes encoding hypothetical proteins expressed during mucosal infection. In addition, network analysis revealed overlap in the response of N. gonorrhoeae to incubation with neutrophils and exposure to hydrogen peroxide stress in vitro. Network analysis also identified new targets of the gonococcal global regulatory protein Fur, while examination of the network neighborhood of genes allowed us to assign additional putative categories to several proteins. Collectively, the characterization of the first gene coexpression network for N. gonorrhoeae described here has revealed new regulatory pathways and new categories for proteins and has shown how processes important to gonococcal infection in both men and women are linked. This information fills a critical gap in our understanding of virulence strategies of this obligate human pathogen and will aid in the development of new treatment strategies for gonorrhea.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Cryptococcus neoformans Secretes Small Molecules That Inhibit IL-1 β Inflammasome-Dependent Secretion

Cryptococcus neoformans is an encapsulated yeast that causes disease mainly in immunosuppressed hosts. It is considered a facultative intracellular pathogen because of its capacity to survive and replicate inside phagocytes, especially macrophages. This ability is heavily dependent on various virulence factors, particularly the glucuronoxylomannan (GXM) component of the polysaccharide capsule. Inflammasome activation in phagocytes is usually protective against fungal infections, including cryptococcosis. Nevertheless, recognition of C. neoformans by inflammasome receptors requires specific changes in morphology or the opsonization of the yeast, impairing proper inflammasome function. In this context, we analyzed the impact of molecules secreted by C. neoformans B3501 strain and its acapsular mutant Δcap67 in inflammasome activation in an in vitro model. Our results showed that conditioned media derived from B3501 was capable of inhibiting inflammasome-dependent events (i.e., IL-1β secretion and LDH release via pyroptosis) more strongly than conditioned media from Δcap67, regardless of GXM presence. We also demonstrated that macrophages treated with conditioned media were less responsive against infection with the virulent strain H99, exhibiting lower rates of phagocytosis, increased fungal burdens, and enhanced vomocytosis. Moreover, we showed that the aromatic metabolite DL-Indole-3-lactic acid (ILA) and DL-p-Hydroxyphenyllactic acid (HPLA) were present in B3501’s conditioned media and that ILA alone or with HPLA is involved in the regulation of inflammasome activation by C. neoformans. These results were confirmed by in vivo experiments, where exposure to conditioned media led to higher fungal burdens in Acanthamoeba castellanii culture as well as in higher fungal loads in the lungs of infected mice. Overall, the results presented show that conditioned media from a wild-type strain can inhibit a vital recognition pathway and subsequent fungicidal functions of macrophages, contributing to fungal survival in vitro and in vivo and suggesting that secretion of aromatic metabolites, such as ILA, during cryptococcal infections fundamentally impacts pathogenesis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Strengthening of enterococcal biofilms by Esp

Multidrug-resistant (MDR) Enterococcus faecalis are major causes of hospital-acquired infections. Numerous clinical strains of E . faecalis harbor a large pathogenicity island that encodes enterococcal surface protein (Esp), which is suggested to promote biofilm production and virulence, but this remains controversial. To resolve this issue, we characterized the Esp N-terminal region, the portion implicated in biofilm production. Small angle X-ray scattering indicated that the N-terminal region had a globular head, which consisted of two DEv-Ig domains as visualized by X-ray crystallography, followed by an extended tail. The N-terminal region was not required for biofilm production but instead significantly strengthened biofilms against mechanical or degradative disruption, greatly increasing retention of Enterococcus within biofilms. Biofilm strengthening required low pH, which resulted in Esp unfolding, aggregating, and forming amyloid-like structures. The pH threshold for biofilm strengthening depended on protein stability. A truncated fragment of the first DEv-Ig domain, plausibly generated by a host protease, was the least stable and sufficient to strengthen biofilms at pH ≤ 5.0, while the entire N-terminal region and intact Esp on the enterococcal surface was more stable and required a pH ≤ 4.3. These results suggested a virulence role of Esp in strengthening enterococcal biofilms in acidic abiotic or host environments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Eating the brain - A multidisciplinary study provides new insights into the mechanisms underlying the cytopathogenicity of Naegleria fowleri

Naegleria fowleri , the causative agent of primary amoebic meningoencephalitis (PAM), requires increased research attention due to its high lethality and the potential for increased incidence as a result of global warming. The aim of this study was to investigate the interactions between N. fowleri and host cells in order to elucidate the mechanisms underlying the pathogenicity of this amoeba. A co-culture system comprising human fibrosarcoma cells was established to study both contact-dependent and contact-independent cytopathogenicity. Proteomic analyses of the amoebas exposed to human cell cultures or passaged through mouse brain were used to identify novel virulence factors. Our results indicate that actin dynamics, regulated by Arp2/3 and Src kinase, play a considerable role in ingestion of host cells by amoebae. We have identified three promising candidate virulence factors, namely lysozyme, cystatin and hemerythrin, which may be critical in facilitating N. fowleri evasion of host defenses, migration to the brain and induction of a lethal infection. Long-term co-culture secretome analysis revealed an increase in protease secretion, which enhances N. fowleri cytopathogenicity. Raman microspectroscopy revealed significant metabolic differences between axenic and brain-isolated amoebae, particularly in lipid storage and utilization. Taken together, our findings provide important new insights into the pathogenic mechanisms of N. fowleri and highlight potential targets for therapeutic intervention against PAM.

Malych, Ronald↗
DOE OSTI2020

Concurrent evaluation of autophagy induction and Burkholderia infection at the single cell level

Autophagy is a natural, regulated cellular process that "cleans up" cellular debris by degrading and recycling dysfunctional proteins. There is a high potential impact of exploiting the benefits of autophagy to complement existing treatments, but little has been done to date on bacterial pathogens of defense concern such as Burkholderia pseudomallei, a highly virulent Select Agent pathogen that is intrinsically resistant to most classes of antibiotics. Assessment of autophagy in the context of infection typically requires use of multiple technologies in combination (e.g., Western analysis paired with microscopy or flow cytometry) as applied to heterogeneous populations of cells. To address this, we have developed a dual target reporter cell line (RAW264.7 LC3-BFP:mPlum, GFP-RelA) that enables concurrent visualization of infection and autophagy induction. We assessed the effect of clinically approved small molecule inducers of autophagy on infection by Burkholderia thailendensis, a closely related but less virulent surrogate for B. pseudomallei. The reporter cells were first infected with a B. thailendensis strain that constitutively expresses GFP, then treated with one of four known autophagy inducers (rapamycin, niclosamide, bromhexine HC1, or flubendazole) for 4 hours. Confocal fluorescence imaging was used to quantify autophagy stimulation at the single cell level. Autophagy maturation was observed as a decrease in BFP LC3 puncta with a concurrent increase in mPlum LC3 puncta. B. thailendensis infection was assessed by monitoring translocation of GFP-RelA (an NFkB subunit) into the nucleus and through quantitating the intracellular bacterial presence in single cells. Preliminary results indicate that bromhexine HC1 and niclosamide may hinder B. thailendensis' ability to replicate intracellularly and reduce overall bacterial survival.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Deep Sequencing of MHC-Adapted Viral Lines Reveals Complex Recombinational Exchanges With Endogenous Retroviruses Leading to High-Frequency Variants

Experimental evolution (serial passage) of Friend virus complex (FVC) in mice demonstrates phenotypic adaptation to specific host major histocompatibility complex (MHC) genotypes. These evolved viral lines show increased fitness and virulence in their host-genotype-of-passage, but display fitness and virulence tradeoffs when infecting unfamiliar host MHC genotypes. Here, we deep sequence these viral lines in an attempt to discover the genetic basis of FVC adaptation. The principal prediction for genotype-specific adaptation is that unique mutations would rise to high frequency in viral lines adapted to each host MHC genotype. This prediction was not supported by our sequencing data as most observed high-frequency variants were present in each of our independently evolved viral lines. However, using a multi-variate approach to measure divergence between viral populations, we show that populations of replicate evolved viral lines from the same MHC congenic mouse strain were more similar to one another than to lines derived from different MHC congenic mouse strains, suggesting that MHC genotype does predictably act on viral evolution in our model. Sequence analysis also revealed rampant recombination with endogenous murine leukemia virus sequences (EnMuLVs) that are encoded within the BALB/c mouse genome. The highest frequency variants in all six lines contained a 12 bp insertion from a recombinant EnMuLV source, suggesting such recombinants were either being favored by selection or were contained in a recombinational hotspot. Interestingly, they did not reach fixation, as if they are low fitness. The amount of background mutations linked to FVC/EnMuLV variable sites indicated that FVC/EnMuLV recombinants had not reached mutation selection equilibrium and thus, that EnMuLV sequences are likely continuously introgressing into the replicating viral population. These discoveries raise the question: is the expression of EnMuLV sequences in mouse splenocytes that permit recombination with exogenous FVC a pathogen or host adaptation?

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Rapid Construction of an Infectious Clone of the Zika Virus, Strain ZKC2

Zika virus (ZIKV) has had detrimental effects on global public health in recent years. This is because the management of the disease has been limited, in part because its pathogenic mechanisms are not yet completely understood. Infectious clones are an important tool that utilize reverse genetics; these can be used to modify the ZIKV genomic RNA at the DNA level. A homologous recombination clone was used to construct pWSK29, a low copy plasmid that contained sequences for a T7 promoter, the whole genome of ZIKV ZKC2 strain, and a hepatitis delta virus ribozyme. High fidelity PCR was then used to amplify the T7 transcription template. The transcript was then transfected into susceptible cells via lipofection to recover the ZIKV ZKC2 strain. Finally, the virulence of rZKC2 was evaluated both in vitro and in vivo . The rZKC2 was successfully obtained and it showed the same virulence as its parent, the ZIKV ZKC2 strain (pZKC2), both in vitro and in vivo . The 3730 (NS2A-D62G) mutation site was identified as being important, since it had significant impacts on rZKC2 recovery. The 4015 (NS2A, A157V) mutation may reduce virus production by increasing the interferon type I response. In this study, one of the earliest strains of ZIKV that was imported into China was used for infectious clone construction and one possible site for antiviral medication development was discovered. The use of homologous recombination clones, of PCR products as templates for T7 transcription, and of lipofection for large RNA transfection could increase the efficiency of infectious clone construction. Our infectious clone provides an effective tool which can be used to explore the life cycle and medical treatment of ZIKV.

Qin, Zhiran↗
DOE OSTI2022

Antimicrobial resistance and genomic characterization of Escherichia coli from pigs and chickens in Zhejiang, China

Escherichia coli is considered an opportunistic pathogen and an indicator for antimicrobial resistance (AMR) monitoring. Despite many reports on its AMR monitoring, studies based on genome-based analysis of AMR genes are still insufficient. Here, 181 E. coli strains were isolated from anal swab samples collected from pigs and chickens of animal farms located in Eastern China and sequenced through the Illumina platform. The results showed that 87.85% (159/181) of the E. coli isolates were multidrug-resistant (MDR). Ampicillin (AMP)- spectinomycin (SPT)- tetracycline (TET)- florfenicol (FFC)- sulfisoxazole (SF)- trimethoprim/sulfamethoxazole (SXT) was the predominant AMR pattern. By whole-genome sequencing, we found that ST10 (10.49%, 19/181) and ST48 (7.18%, 13/181) were major sequence types. IncFIB and IncX1 were the most prevalent plasmid replicons. The AMR genes bla NDM-5 (1.10%, 2/181), mcr -1 (1.10%, 2/181), tet (X4) (1.10%, 2/181), and cfr (6.08%, 11/181) were also found in these isolates. In addition, among the 169 virulence genes detected, we identified ast A (37.02%, 67/181), hly A (1.66%, 3/181), hly B (1.66%, 3/181) and hly D (1.66%, 3/181), which were closely related to heat-stable enterotoxin 1 and α-hemolysin. In addition, there were 33 virulence genes associated with the iron uptake system, and 46 were adhesion-related genes. Our study highlighted the need for routine surveillance of AMR with advanced genomic approaches, providing up-to-date data on the prevalence of AMR for the development and execution of antimicrobial stewardship policy.

Zhou, Wei↗
DOE OSTI2022

The secreted FoAPY1 peptidase promotes Fusarium oxysporum invasion

The secretion of peptidases from several pathogens has been reported, but the biological function of these proteins in plant-pathogen interactions is poorly understood. Fusarium oxysporum , a soil-borne plant pathogenic fungus that causes Fusarium wilt in its host, can secrete proteins into host plant cells during the infection process to interfere with the host plant defense response and promote disease occurrence. In this study, we identified a peptidase, FoAPY1, that could be secreted from F. oxysporum depending on the N-terminal signal peptide of the protein. FoAPY1 belongs to the peptidase M28 family and exerts peptidase activity in vitro . Furthermore, the FoAYP1 gene knockout strain (∆FoAYP1) presented reduced virulence to tomato plants, but its mycelial growth and conidiation were unchanged. Moreover, FoAYP1 overexpression tomato seedlings exhibited enhanced susceptibility to F. oxysporum and Botrytis cinerea strains. These data demonstrated that FoAYP1 contributes to the virulence of F. oxysporum may through peptidase activity against host plant proteins.

Qian, Hengwei↗
DOE OSTI2024

The contribution of BvgR, RisA, and RisS to global gene regulation, intracellular cyclic-di-GMP levels, motility, and biofilm formation in Bordetella bronchiseptica

Bordetella bronchiseptica is a highly contagious respiratory bacterial veterinary pathogen. In this study the contribution of the transcriptional regulators BvgR, RisA, RisS, and the phosphorylation of RisA to global gene regulation, intracellular cyclic-di-GMP levels, motility, and biofilm formation were evaluated. Next Generation Sequencing (RNASeq) was used to differentiate the global gene regulation of both virulence-activated and virulence-repressed genes by each of these factors. The BvgAS system, along with BvgR, RisA, and the phosphorylation of RisA served in cyclic-di-GMP degradation. BvgR and unphosphorylated RisA were found to temporally regulate motility. Additionally, BvgR, RisA, and RisS were found to be required for biofilm formation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Peeling back the many layers of competitive exclusion

Baby chicks administered a fecal transplant from adult chickens are resistant to Salmonella colonization by competitive exclusion. A two-pronged approach was used to investigate the mechanism of this process. First, Salmonella response to an exclusive (Salmonella competitive exclusion product, Aviguard ® ) or permissive microbial community (chicken cecal contents from colonized birds containing 7.85 Log 10 Salmonella genomes/gram) was assessed ex vivo using a S. typhimurium reporter strain with fluorescent YFP and CFP gene fusions to rrn and hilA operon, respectively. Second, cecal transcriptome analysis was used to assess the cecal communities’ response to Salmonella in chickens with low (≤5.85 Log 10 genomes/g) or high (≥6.00 Log 10 genomes/g) Salmonella colonization. The ex vivo experiment revealed a reduction in Salmonella growth and hilA expression following co-culture with the exclusive community. The exclusive community also repressed Salmonella’s SPI-1 virulence genes and LPS modification, while the anti-virulence/inflammatory gene avrA was upregulated. Salmonella transcriptome analysis revealed significant metabolic disparities in Salmonella grown with the two different communities. Propanediol utilization and vitamin B12 synthesis were central to Salmonella metabolism co-cultured with either community, and mutations in propanediol and vitamin B12 metabolism altered Salmonella growth in the exclusive community. There were significant differences in the cecal community’s stress response to Salmonella colonization. Cecal community transcripts indicated that antimicrobials were central to the type of stress response detected in the low Salmonella abundance community, suggesting antagonism involved in Salmonella exclusion. This study indicates complex community interactions that modulate Salmonella metabolism and pathogenic behavior and reduce growth through antagonism may be key to exclusion.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

The Exometabolome of Xylella fastidiosa in Contact with Paraburkholderia phytofirmans Supernatant Reveals Changes in Nicotinamide, Amino Acids, Biotin, and Plant Hormones

Microbial competition within plant tissues affects invading pathogens’ fitness. Metabolomics is a great tool for studying their biochemical interactions by identifying accumulated metabolites. Xylella fastidiosa, a Gram-negative bacterium causing Pierce’s disease (PD) in grapevines, secretes various virulence factors including cell wall-degrading enzymes, adhesion proteins, and quorum-sensing molecules. These factors, along with outer membrane vesicles, contribute to its pathogenicity. Previous studies demonstrated that co-inoculating X. fastidiosa with the Paraburkholderia phytofirmans strain PsJN suppressed PD symptoms. Here, we further investigated the interaction between the phytopathogen and the endophyte by analyzing the exometabolome of wild-type X. fastidiosa and a diffusible signaling factor (DSF) mutant lacking quorum sensing, cultivated with 20% P. phytofirmans spent media. Liquid chromatography–mass spectrometry (LC-MS) and the Method for Metabolite Annotation and Gene Integration (MAGI) were used to detect and map metabolites to genomes, revealing a total of 121 metabolites, of which 25 were further investigated. These metabolites potentially relate to host adaptation, virulence, and pathogenicity. Notably, this study presents the first comprehensive profile of X. fastidiosa in the presence of a P. phytofirmans spent media. The results highlight that P. phytofirmans and the absence of functional quorum sensing affect the ratios of glutamine to glutamate (Gln:Glu) in X. fastidiosa. Additionally, two compounds with plant metabolism and growth properties, 2-aminoisobutyric acid and gibberellic acid, were downregulated when X. fastidiosa interacted with P. phytofirmans. These findings suggest that P. phytofirmans-mediated disease suppression involves modulation of the exometabolome of X. fastidiosa, impacting plant immunity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Genomic Characterization and Wetland Occurrence of a Novel Campylobacter Isolate from Canada Geese

Populations of resident, non-migratory Canada geese are rapidly increasing. Canada geese are known to transmit viral and bacterial diseases, posing a possible threat to human health. The most prevalent pathogens vectored by geese are Campylobacter species, yet the current understanding of the identity and virulence of these pathogens is limited. In our previous study, we observed a high prevalence of Campylobacter spp. in the Banklick Creek wetland—a constructed treatment wetland (CTW) located in northern KY (USA) used to understand sources of fecal contamination originating from humans and waterfowl frequenting the area. To identify the types of Campylobacter spp. found contaminating the CTW, we performed genetic analyses of Campylobacter 16s ribosomal RNA amplified from CTW water samples and collected fecal material from birds frequenting those areas. Our results showed a high occurrence of a Campylobacter canadensis-like clade from the sampling sites. Whole-genome sequence analyses of an isolate from Canada goose fecal material, called MG1, were used to confirm the identity of the CTW isolates. Further, we examined the phylogenomic position, virulence gene content, and antimicrobial resistance gene profile of MG1. Lastly, we developed an MG1-specific real-time PCR assay and confirmed the presence of MG1 in Canada goose fecal samples surrounding the CTW. Our findings reveal that the Canada goose-vectored Campylobacter sp. MG1 is a novel isolate compared to C. canadensis that possesses possible zoonotic potential, which may be of human health concern.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Identification and Characterization of ten Escherichia coli Strains Encoding Novel Shiga Toxin 2 Subtypes, Stx2n as Well as Stx2j, Stx2m, and Stx2o, in the United States

The sharing of genome sequences in online data repositories allows for large scale analyses of specific genes or gene families. This can result in the detection of novel gene subtypes as well as the development of improved detection methods. Here, we used publicly available WGS data to detect a novel Stx subtype, Stx2n in two clinical E. coli strains isolated in the USA. During this process, additional Stx2 subtypes were detected; six Stx2j, one Stx2m strain, and one Stx2o, were all analyzed for variability from the originally described subtypes. Complete genome sequences were assembled from short- or long-read sequencing and analyzed for serotype, and ST types. The WGS data from Stx2n- and Stx2o-producing STEC strains were further analyzed for virulence genes pro-phage analysis and phage insertion sites. Nucleotide and amino acid maximum parsimony trees showed expected clustering of the previously described subtypes and a clear separation of the novel Stx2n subtype. WGS data were used to design OMNI PCR primers for the detection of all known stx1 (283 bp amplicon), stx2 (400 bp amplicon), intimin encoded by eae (221 bp amplicon), and stx2f (438 bp amplicon) subtypes. These primers were tested in three different laboratories, using standard reference strains. An analysis of the complete genome sequence showed variability in serogroup, virulence genes, and ST type, and Stx2 pro-phages showed variability in size, gene composition, and phage insertion sites. The strains with Stx2j, Stx2m, Stx2n, and Stx2o showed toxicity to Vero cells. Stx2j carrying strain, 2012C-4221, was induced when grown with sub-inhibitory concentrations of ciprofloxacin, and toxicity was detected. Taken together, these data highlight the need to reinforce genomic surveillance to identify the emergence of potential new Stx2 or Stx1 variants. The importance of this surveillance has a paramount impact on public health. Per our description in this study, we suggest that 2017C-4317 be designated as the Stx2n type-strain.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Isolation, Characterization, and Pathogenicity of Two Pseudomonas syringae Pathovars from Populus trichocarpa Seeds

Pseudomonas syringae is a ubiquitous plant pathogen, infecting both woody and herbaceous plants and resulting in devastating agricultural crop losses. Characterized by a remarkable specificity for plant hosts, P. syringae pathovars utilize a number of virulence factors including the type III secretion system and effector proteins to elicit disease in a particular host species. Here, two Pseudomonas syringae strains were isolated from diseased Populustrichocarpa seeds. The pathovars were capable of inhibiting poplar seed germination and were selective for the Populus genus. Sequencing of the newly described organisms revealed similarity to phylogroup II pathogens and genomic regions associated with woody host-associated plant pathogens, as well as genes for specific virulence factors. The host response to infection, as revealed through metabolomics, is the induction of the stress response through the accumulation of higher-order salicylates. Combined with necrosis on leaf surfaces, the plant appears to quickly respond by isolating infected tissues and mounting an anti-inflammatory defense. This study improves our understanding of the initial host response to epiphytic pathogens in Populus and provides a new model system for studying the effects of a bacterial pathogen on a woody host plant in which both organisms are fully genetically sequenced.

59 BASIC BIOLOGICAL SCIENCES↗