Momchilo Vuyisich

A rapid multiplex assay for nucleic acid-based diagnostics

We have developed a rapid (under 4 hours), multiplex, nucleic acid assay, adapted to a microsphere array detection platform. We call this assay multiplex oligonucleotide ligation-PCR (MOL-PCR). Unlike other ligation-based assays that require multiple steps, our protocol consists of a single tube reaction, followed by hybridization to a Luminex microsphere array for detection. We demonstrate the ability of this assay to simultaneously detect diverse nucleic acid signatures (e.g., unique sequences, single nucleotide polymorphisms) in a single multiplex reaction. Detection probes consist of modular components that enable target detection, probe amplification, and subsequent capture onto microsphere arrays. To demonstrate the utility of our assay, we applied it to the detection of three biothreat agents, B. anthracis, Y. pestis, and F. tularensis. Combined with the ease and robustness of this assay, the results presented here show a strong potential of our assay for use in diagnostics and surveillance.

Forest floor community metatranscriptomes identify fungal and bacterial responses to N deposition in two maple forests

Anthropogenic N deposition alters patterns of C and N cycling in temperate forests, where forest floor litter decomposition is a key process mediated by a diverse community of bacteria and fungi. To track forest floor decomposer activity we generated metatranscriptomes that simultaneously surveyed the actively expressed bacterial and eukaryote genes in the forest floor, to compare the impact of N deposition on the decomposers in two natural maple forests in Michigan, USA, where replicate field plots had been amended with N for 16 years. Site and N amendment responses were compared using about 74,000 carbohydrate active enzyme transcript sequences (CAZymes) in each metatranscriptome. Parallel ribosomal RNA (rRNA) surveys of bacterial and fungal biomass and taxonomic composition showed no significant differences in either biomass or OTU richness between the two sites or in response to N. Site and N amendment were not significant variables defining bacterial taxonomic composition, but they were significant for fungal community composition, explaining 17 and 14% of the variability, respectively. The relative abundance of expressed bacterial and fungal CAZymes changed significantly with N amendment in one of the forests, and N-response trends were also identified in the second forest. Although the two ambient forests were similar in community biomass, taxonomic structure and active CAZyme profile, the shifts in active CAZyme profiles in response to N-amendment differed between the sites. One site responded with an over-expression of bacterial CAZymes, and the other site responded with an over-expression of both fungal and different bacterial CAZymes. Both sites showed reduced representation of fungal lignocellulose degrading enzymes in N-amendment plots. The metatranscriptome approach provided a holistic assessment of eukaryote and bacterial gene expression and is applicable to other systems where eukaryotes and bacteria interact.

Simultaneous pathogen detection and antibiotic resistance characterization using SNP-based multiplexed oligonucleotide ligation-PCR (MOL-PCR).

Extensive use of antibiotics in both public health and animal husbandry has resulted in rapid emergence of antibiotic resistance in almost all human pathogens, including biothreat pathogens. Antibiotic resistance has thus become a major concern for both public health and national security. We developed multiplexed assays for rapid, simultaneous pathogen detection and characterization of ciprofloxacin and doxycycline resistance in Bacillus anthracis, Yersinia pestis, and Francisella tularensis. These assays are SNP-based and use Multiplexed Oligonucleotide Ligation-PCR (MOL-PCR). The MOL-PCR assay chemistry and MOLigo probe design process are presented. A web-based tool - MOLigoDesigner (http://MOLigoDesigner.lanl.gov) was developed to facilitate the probe design. All probes were experimentally validated individually and in multiplexed assays, and minimal sets of multiplexed MOLigo probes were identified for simultaneous pathogen detection and antibiotic resistance characterization.

Binding and cell intoxication studies of anthrax lethal toxin.

Anthrax lethal toxin (LT) is a major virulence factor of Bacillus anthracis. The vast majority of the anthrax toxin-related literature describes the assembly of LT as a cell-dependent process. However, some reports have provided evidence for the existence of a fully assembled LT, either in vitro or in the bloodstream of anthrax-infected animals. To follow up on this work, we present studies on fully-assembled LT. We first demonstrate facile and cell-free assembly and purification of LT. We then show that fully assembled LT binds an anthrax toxin receptor with almost 100-fold higher affinity than the protective antigen (PA) alone. Quantitative cell intoxication assays were used to determine the LD50 (lethal dose 50) for LT. The cell-binding studies revealed that LT binds mammalian cells using a different mode from PA. Even when PA-specific receptors were blocked, fully assembled LT was able to bind the cell surface. Our studies support the existing evidence that LT fully assembles in the blood stream and can bind and intoxicate mammalian cells with very high affinity and efficacy. More importantly, the data presented here invoke the possibility that LT may bind cells in a receptor-independent fashion, or recognize receptors that do not interact with PA. Hence, blood borne LT may emerge as a novel therapeutic target for combating anthrax.

Molecular Dissection of Induced Platinum Resistance through Functional and Gene Expression Analysis in a Cell Culture Model of Bladder Cancer

We report herein the development, functional and molecular characterization of an isogenic, paired bladder cancer cell culture model system for studying platinum drug resistance. The 5637 human bladder cancer cell line was cultured over ten months with stepwise increases in oxaliplatin concentration to generate a drug resistant 5637R sub cell line. The MTT assay was used to measure the cytotoxicity of several bladder cancer drugs. Liquid scintillation counting allowed quantification of cellular drug uptake and efflux of radiolabeled oxaliplatin and carboplatin. The impact of intracellular drug inactivation was assessed by chemical modulation of glutathione levels. Oxaliplatin- and carboplatin-DNA adduct formation and repair was measured using accelerator mass spectrometry. Resistance factors including apoptosis, growth factor signaling and others were assessed with RNAseq of both cell lines and included confirmation of selected transcripts by RT-PCR. Oxaliplatin, carboplatin, cisplatin and gemcitabine were significantly less cytotoxic to 5637R cells compared to the 5637 cells. In contrast, doxorubicin, methotrexate and vinblastine had no cell line dependent difference in cytotoxicity. Upon exposure to therapeutically relevant doses of oxaliplatin, 5637R cells had lower drug-DNA adduct levels than 5637 cells. This difference was partially accounted for by pre-DNA damage mechanisms such as drug uptake and intracellular inactivation by glutathione, as well as faster oxaliplatin-DNA adduct repair. In contrast, both cell lines had no significant differences in carboplatin cell uptake, efflux and drug-DNA adduct formation and repair, suggesting distinct resistance mechanisms for these two closely related drugs. The functional studies were augmented by RNAseq analysis, which demonstrated a significant change in expression of 83 transcripts, including 50 known genes and 22 novel transcripts. Most of the transcripts were not previously associated with bladder cancer chemoresistance. This model system and the associated phenotypic and genotypic data has the potential to identify some novel details of resistance mechanisms of clinical importance to bladder cancer.

Biostimulation and microbial community profiling reveal insights on RDX transformation in groundwater

Hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine (RDX) is a high explosive released to the environment as a result of weapons manufacturing and testing worldwide. At Los Alamos National Laboratory, the Technical Area (TA) 16 260 Outfall discharged high‐explosives‐bearing water from a high‐explosives‐machining facility to Cañon de Valle during 1951 through 1996. These discharges served as a primary source of high‐explosives and inorganic‐element contamination in the area. Data indicate that springs, surface water, alluvial groundwater, and perched‐intermediate groundwater contain explosive compounds, including RDX (hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine); HMX (octahydro‐1,3,5,7‐tetranitro‐1,3,5,7‐tetrazocine); and TNT (2,4,6‐trinitrotoluene). RDX has been detected in the regional aquifer in several wells, and a corrective measures evaluation is planned to identify remedial alternatives to protect the regional aquifer. Perched‐intermediate groundwater at Technical Area 16 is present at depths from 650 ft to 1200 ft bgs. In this study, we examined the microbial diversity in a monitoring well completed in perched‐intermediate groundwater contaminated by RDX, and examined the response of the microbial population to biostimulation under varying geochemical conditions. Results show that the groundwater microbiome was dominated by Actinobacteria and Proteobacteria. A total of 1,605 operational taxonomic units (OTUs) in 96 bacterial genera were identified. Rhodococcus was the most abundant genus (30.6%) and a total of 46 OTUs were annotated as Rhodococcus. One OTU comprising 25.2% of total sequences was closely related to a RDX ‐degrading strain R. erythropolis HS4. A less abundant OTU from the Pseudomonas family closely related to RDX‐degrading strain P. putida II‐B was also present. Biostimulation significantly enriched Proteobacteria but decreased/eliminated the population of Actinobacteria. Consistent with RDX degradation, the OTU closely related to the RDX‐degrading P. putida strain II‐B was specifically enriched in the RDX‐degrading samples. Analysis of the accumulation of RDX‐degradation products reveals that during active RDX degradation, there is a transient increase in the concentration of the degradation products MNX, DNX, TNX, and NDAB. The accumulation of these degradation products suggests that RDX is degraded via sequential reduction of the nitro functional groups followed by abiotic ring‐cleavage. The results suggest that strict anaerobic conditions are needed to stimulate RDX degradation under the TA‐16 site‐specific conditions.

Genome Sequence of Aeribacillus pallidus Strain GS3372, an Endospore-Forming Bacterium Isolated in a Deep Geothermal Reservoir

ABSTRACT The genome of strain GS3372 is the first publicly available strain of Aeribacillus pallidus. This endospore-forming thermophilic strain was isolated from a deep geothermal reservoir. The availability of this genome can contribute to the clarification of the taxonomy of the closely related Anoxybacillus, Geobacillus, and Aeribacillus genera.

Functional and Structural Analysis of a Highly-Expressed Yersinia pestis Small RNA following Infection of Cultured Macrophages

Non-coding small RNAs (sRNAs) are found in practically all bacterial genomes and play important roles in regulating gene expression to impact bacterial metabolism, growth, and virulence. We performed transcriptomics analysis to identify sRNAs that are differentially expressed in Yersinia pestis that invaded the human macrophage cell line THP-1, compared to pathogens that remained extracellular in the presence of host. Using ultra high-throughput sequencing, we identified 37 novel and 143 previously known sRNAs in Y. pestis. In particular, the sRNA Ysr170 was highly expressed in intracellular Yersinia and exhibited a log2 fold change ~3.6 higher levels compared to extracellular bacteria. We found that knock-down of Ysr170 expression attenuated infection efficiency in cell culture and growth rate in response to different stressors. In addition, we applied selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE) analysis to determine the secondary structure of Ysr170 and observed structural changes resulting from interactions with the aminoglycoside antibiotic gentamycin and the RNA chaperone Hfq. Interestingly, gentamicin stabilized helix 4 of Ysr170, which structurally resembles the native gentamicin 16S ribosomal binding site. Finally, we modeled the tertiary structure of Ysr170 binding to gentamycin using RNA motif modeling. Integration of these experimental and structural methods can provide further insight into the design of small molecules that can inhibit function of sRNAs required for pathogen virulence.

First Complete Genome Sequence of Felis catus Gammaherpesvirus 1.

ABSTRACT We sequenced the complete genome of Felis catus gammaherpesvirus 1 (FcaGHV1) from lymph node DNA of an infected cat. The genome includes a 121,556-nucleotide unique region with 87 predicted open reading frames (61 gammaherpesvirus conserved and 26 unique) flanked by multiple copies of a 966-nucleotide terminal repeat.

Genome Sequence of Anoxybacillus geothermalis Strain GSsed3, a Novel Thermophilic Endospore-Forming Species

ABSTRACT Anoxybacillus geothermalis strain GSsed3 is an endospore-forming thermophilic bacterium isolated from filter deposits in a geothermal site. This novel species has a larger genome size (7.2 Mb) than that of any other Anoxybacillus species, and it possesses genes that support its phenotypic metabolic characterization and suggest an intriguing link to metals.