Loop-Mediated Isothermal Amplification Detection of SARS-CoV-2 and Myriad Other Applications.

As the second year of the COVID-19 pandemic begins, it remains clear that a massive increase in the ability to test for SARS-CoV-2 infections in a myriad of settings is critical to controlling the pandemic and to preparing for future outbreaks. The current gold standard for molecular diagnostics is the polymerase chain reaction (PCR), but the extraordinary and unmet demand for testing in a variety of environments means that both complementary and supplementary testing solutions are still needed. This review highlights the role that loop-mediated isothermal amplification (LAMP) has had in filling this global testing need, providing a faster and easier means of testing, and what it can do for future applications, pathogens, and the preparation for future outbreaks. This review describes the current state of the art for research of LAMP-based SARS-CoV-2 testing, as well as its implications for other pathogens and testing. The authors represent the global LAMP (gLAMP) Consortium, an international research collective, which has regularly met to share their experiences on LAMP deployment and best practices; sections are devoted to all aspects of LAMP testing, including preanalytic sample processing, target amplification, and amplicon detection, then the hardware and software required for deployment are discussed, and finally, a summary of the current regulatory landscape is provided. Included as well are a series of first-person accounts of LAMP method development and deployment. The final discussion section provides the reader with a distillation of the most validated testing methods and their paths to implementation. This review also aims to provide practical information and insight for a range of audiences: for a research audience, to help accelerate research through sharing of best practices; for an implementation audience, to help get testing up and running quickly; and for a public health, clinical, and policy audience, to help convey the breadth of the effect that LAMP methods have to offer.

Saliva "Treat-and-Heat" Triplex Reverse Transcription Loop-Mediated Isothermal Amplification Assay for SARS-CoV-2.

The demand for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) molecular diagnostics that are faster, cheaper, and simpler to run than nasopharyngeal-based reverse transcription quantitative PCR (RT-qPCR) tests remains unmet in many parts of the world. In the Philippines, geographical and economic access to quality diagnostic testing remains out of reach for many communities. We describe the preclinical development of a fluorescence-based reverse transcription loop-mediated isothermal amplification test that uses drooled saliva as the biospecimen. Six treat-and-heat ("direct") procedures that inactivate the virus and release the target RNA were compared. Using duplexed As1e and E1 primers, protocols derived from Ben-Assa et al. (2020) using proteinase K or from Rabe and Cepko (2020) using TCEP (Tris(2-carboxyethyl)phosphine hydrochloride)/EDTA provided reliable RNA amplification. The TCEP/EDTA-based method in particular showed improvement in robustness in duplex vs. singleplex format. Inclusion of human ß-actin primers provided a triplex test with an internal amplification control that could be distinguished from SARS-CoV-2 amplicons based on melt curve analysis. After including the dUTP/uracil-DNA glycosylase system and implementing laboratory procedures to avoid cross-contamination, false positive amplification was acceptably rare. The duplex or triplex tests are predicted to reliably detect patient salivary viral loads >100 copies/µL and to yield equivocal results between 10 and 100 copies/µL. These viral loads, corresponding to RT-qPCR C t ∼29-32, are expected to identify the majority of infected and, particularly, of infectious patients. If clinically validated, the test would provide additional testing capacity requiring only a fraction of the time, cost, and infrastructure of the current nasopharyngeal swab-based RT-qPCR test, thereby improving access to testing for more Filipinos.

Disruption of fatty acid amide hydrolase activity prevents the effects of chronic stress on anxiety and amygdalar microstructure.

Hyperactivation of the amygdala following chronic stress is believed to be one of the primary mechanisms underlying the increased propensity for anxiety-like behaviors and pathological states; however, the mechanisms by which chronic stress modulates amygdalar function are not well characterized. The aim of the current study was to determine the extent to which the endocannabinoid (eCB) system, which is known to regulate emotional behavior and neuroplasticity, contributes to changes in amygdalar structure and function following chronic stress. To examine the hypothesis, we have exposed C57/Bl6 mice to chronic restraint stress, which results in an increase in fatty acid amide hydrolase (FAAH) activity and a reduction in the concentration of the eCB N-arachidonylethanolamine (AEA) within the amygdala. Chronic restraint stress also increased dendritic arborization, complexity and spine density of pyramidal neurons in the basolateral nucleus of the amygdala (BLA) and increased anxiety-like behavior in wild-type mice. All of the stress-induced changes in amygdalar structure and function were absent in mice deficient in FAAH. Further, the anti-anxiety effect of FAAH deletion was recapitulated in rats treated orally with a novel pharmacological inhibitor of FAAH, JNJ5003 (50 mg per kg per day), during exposure to chronic stress. These studies suggest that FAAH is required for chronic stress to induce hyperactivity and structural remodeling of the amygdala. Collectively, these studies indicate that FAAH-mediated decreases in AEA occur following chronic stress and that this loss of AEA signaling is functionally relevant to the effects of chronic stress. These data support the hypothesis that inhibition of FAAH has therapeutic potential in the treatment of anxiety disorders, possibly by maintaining normal amygdalar function in the face of chronic stress.

A novel multiplex RT-PCR probe capture hybridization (RT-PCR-ELISA) for simultaneous detection of six viroids in four genera: Apscaviroid, Hostuviroid, Pelamoviroid, and Pospiviroid.

A rapid and sensitive assay was developed for the detection and identification of viroids by standard or multiplex reverse transcription-polymerase chain reaction (RT-PCR)-probe capture hybridization (RT-PCR-ELISA). The assay was applied successfully for the detection and identification of the following six viroid species from infected tissues: Potato spindle tuber viroid (Pospiviroid), Peach latent mosaic viroid (Pelamoviroid), Apple scar skin viroid (Apscaviroid), Apple dimple fruit viroid (Apscaviroid), Pear blister canker viroid (Apscaviroid), and Hop stunt viroid (Hostuviroid). Total RNA was obtained from infected tissue by the Qiagen RNeasy kit and, then viroid cDNA was synthesized using viroid specific complementary DNA primer. To identify and differentiate the amplicons of the six viroids, each amplicon was digoxigenin (DIG)-labelled during the amplification process, and then detected by a colorimetric system using a biotinylated cDNA capture probe specific for each viroid. The results revealed that each capture probe hybridized only to its complementary DIG-labelled amplicon. Thus the six viroids can be detected and differentiated in a multiplex RT-PCR-ELISA assay. In the multiplex assay, cDNAs of six viroids were synthesized simultaneously in one tube, DIG-labelled during amplification, then a portion of the DIG-labelled amplified products was hybridized with selected capture probe. All the six viroid capture probes hybridized to their respective complementary DIG-labelled RT-PCR-amplified product. These findings are important for viroid detection and identification for studying host-viroid interactions and for management and control viroid diseases.

Induction of hepatitis C virus-specific cytotoxic T lymphocytes in mice by immunization with dendritic cells treated with an anthrax toxin fusion protein.

As a novel and safe vaccine strategy, the anthrax toxin-mediated antigen delivery system composed of lethal factor (LF) fusion protein and protective antigen (PA) has been studied to prime hepatitis C virus (HCV) core-specific cytotoxic T lymphocytes (CTLs) in vivo. The core epitope fused to LF (LF-core) together with PA induces a negligible core-specific CTL response in mice, whereas core-specific CTL are effectively primed in mice by injecting dendritic cells (DCs) treated in vitro with LF-core and PA. These findings imply that LF fusion protein plus PA in combination with dendritic cells may be useful for a novel T cell vaccine against HCV infection.

Optochin resistance in Streptococcus pneumoniae: mechanism, significance, and clinical implications.

Traditionally, Streptococcus pneumoniae is identified in the laboratory by demonstrating susceptibility to optochin. Between 1992 and 1998, 4 pneumococcal isolates exhibiting optochin resistance were recovered from patients at Children's National Medical Center. Three of the 4 isolates consisted of mixed populations of optochin-resistant and -susceptible organisms. Both subpopulations had identical antibiograms, serotypes, and restriction fragment profiles. The other isolate was uniformly resistant to optochin. Resistant strains had MICs of optochin 4-30-fold higher than susceptible strains, belonged to different serotypes, and had dissimilar restriction fragment profiles, indicating clonal unrelatedness. Resistance arose from single point mutations in either the a-subunit (W206S) or the c-subunit (G20S, M23I, and A49T) of H(+)-ATPase. There is speculation of a possible association between exposure to antimalarial drugs and evolution of optochin resistance. alpha-Hemolytic streptococci resistant to optochin, particularly invasive isolates, should be tested for bile solubility or with an S. pneumoniae DNA probe before identification as viridans streptococci.

Two regions of EpsL involved in species-specific protein-protein interactions with EpsE and EpsM of the general secretion pathway in Vibrio cholerae.

Extracellular secretion of proteins via the type II or general secretion pathway in gram-negative bacteria requires the assistance of at least 12 gene products that are thought to form a complex apparatus through which secreted proteins are translocated. Although this apparatus is specifically required only for the outer membrane translocation step during transport across the bacterial cell envelope, it is believed to span both membranes. The EpsE, EpsL, and EpsM proteins of the type II apparatus in Vibrio cholerae are thought to form a trimolecular complex that is required to either control the opening and closing of the secretion pore or to transduce energy to the site of outer membrane translocation. EpsL is likely to play an important role in this relay by interacting with both the cytoplasmic EpsE protein and the cytoplasmic membrane protein EpsM, which is predominantly exposed on the periplasmic side of the membrane. We have now extended this model and mapped the separate regions within EpsL that contain the EpsE and EpsM binding domains. By taking advantage of the species specificity of the type II pathway, we have used chimeric proteins composed of EpsL and its homologue, ExeL, from Aeromonas hydrophila together with either EpsE or its Aeromonas homologue, ExeE, to complement the secretion defect in both epsL and exeL mutant strains. These studies have mapped the species-specific EpsE binding site to the N-terminal cytoplasmic region between residues 57 and 216 of EpsL. In addition, the species-specific EpsM binding site was mapped to the C-terminal half of EpsL by coimmunoprecipitation of EpsM with different EpsL-ExeL chimeras. This site is present in the region between amino acids 216 and 296, which contains the predicted membrane-spanning segment of EpsL.

Development of a plant-derived subunit vaccine candidate against hepatitis C virus.

Hepatitis C virus (HCV) is a major cause of acute and chronic hepatitis with over 180 million cases worldwide. Vaccine development for HCV has been difficult. Presently, the virus cannot be grown in tissue culture and there is no vaccine or effective therapy against this virus. In this research, we describe the development of an experimental plant-derived subunit vaccine against HCV. A tobamoviral vector was engineered to encode a consensus sequence of hypervariable region 1 (HVR1), a potential neutralizing epitope of HCV, genetically fused to the C-terminal of the B subunit of cholera toxin (CTB). This epitope was selected from among the amino acid sequences of HVR1 "mimotopes" previously derived by phage display technology. The nucleotide sequence encoding this epitope was designed utilizing optimal plant codons. This mimotope is capable of inducing cross-neutralizing antibodies against different variants of the virus. Plants infected with recombinant tobacco mosaic virus (TMV) engineered to express the HVR1/CTB chimeric protein, contained intact TMV particles and produced the HVR1 consensus peptide fused to the functionally active, pentameric B subunit of cholera toxin. Plant-derived HVR1/CTB reacted with HVR1-specific monoclonal antibodies and immune sera from individuals infected with virus from four of the major genotypes of HCV. Intranasal immunization of mice with a crude plant extract containing the recombinant HVR1/CTB protein elicited both anti-CTB serum antibody and anti-HVR1 serum antibody which specifically bound to HCV virus-like particles. Using plant-virus transient expression to produce this unique chimeric antigen will facilitate the development and production of an experimental HCV vaccine. A plant-derived recombinant HCV vaccine can potentially reduce expenses normally associated with production and delivery of conventional vaccines.

Survival of Enterococcus faecalis in mouse peritoneal macrophages.

Enterococcus faecalis was tested for the ability to persist in mouse peritoneal macrophages in two separate studies. In the first study, the intracellular survival of serum-passaged E. faecalis 418 and two isogenic mutants [cytolytic strain FA2-2(pAM714) and non-cytolytic strain FA2-2(pAM771)] was compared with that of Escherichia coli DH5alpha by infecting BALB/c mice intraperitoneally and then monitoring the survival of the bacteria within lavaged peritoneal macrophages over a 72-h period. All E. faecalis isolates were serum passaged to enhance the production of cytolysin. E. faecalis 418, FA2-2(pAM714), and FA2-2(pAM771) survived at a significantly higher level (P = 0.0001) than did E. coli DH5alpha at 24, 48, and 72 h. Internalized E. faecalis 418, FA2-2(pAM714), and FA2-2(pAM771) decreased 10-, 55-, and 31-fold, respectively, over the 72-h infection period, while internalized E. coli DH5alpha decreased 20, 542-fold. The difference in the rate of survival of E. faecalis strains and E. coli DH5alpha was most prominent between 6 and 48 h postinfection (P = 0.0001); however, no significant difference in killing was observed between 48 and 72 h postinfection. In the second study, additional E. faecalis strains from clinical sources, including DS16C2, MGH-2, OG1X, and the cytolytic strain FA2-2(pAM714), were compared with the nonpathogenic gram-positive bacterium, Lactococcus lactis K1, for the ability to survive in mouse peritoneal macrophages. In these experiments, the E. faecalis strains and L. lactis K1 were grown in brain heart infusion (BHI) broth to ensure that there were equal quantities of injected bacteria. E. faecalis FA2-2(pAM714), DS16C2, MGH-2, and OG1X survived significantly better (P < 0.0001) than did L. lactis K1 at each time point. L. lactis K1 was rapidly destroyed by the macrophages, and by 24 h postinfection, viable L. lactis could not be recovered. E. faecalis FA2-2(pAM714), DS16C2, MGH-2, and OG1X declined at an equivalent rate over the 72-h infection period, and there was no significant difference in survival or rate of decline among the strains. E. faecalis FA2-2(pAM714), MGH-2, DS16C2, and OG1X exhibited an overall decrease of 25-, 55-, 186-, and 129-fold respectively, between 6 and 72 h postinfection. The overall reduction by 1.3 to 2.27 log units is slightly higher than that seen for serum-passaged E. faecalis strains and may be attributable to the higher level of uptake of serum-passaged E. faecalis than of E. faecalis grown in BHI broth. Electron microscopy of infected macrophages revealed that E. faecalis 418 was present within an intact phagocytic vacuole at 6 h postinfection but that by 24 h the infected macrophages were disorganized, the vacuolar membrane was degraded, and the bacterial cells had entered the cytoplasm. Macrophage destruction occurred by 48 h, and the bacteria were released. In conclusion, the results of these experiments indicate that E. faecalis can persist for an extended period in mouse peritoneal macrophages.

A conservative amino acid mutation in the chromosome-encoded dihydrofolate reductase confers trimethoprim resistance in Streptococcus pneumoniae.

Multidrug-resistant Streptococcus pneumoniae strains have emerged over the past decade at an alarming rate. The molecular mechanism of trimethoprim resistance was investigated in 5 pneumococcal strains isolated in the Washington, DC, area from patients with invasive infections. Cloning and sequencing of the trimethoprim resistance determinant from these pneumococci indicated that an altered chromosome-encoded dihydrofolate reductase (DHFR) was responsible for the observed resistance. Comparison of DHFR sequences from pneumococcal strains with various susceptibilities to trimethoprim, together with site-directed mutagenesis, revealed that substitution of isoleucine-100 with a leucine residue resulted in trimethoprim resistance. Hydrogen bonding between the carbonyl oxygen of isoleucine-100 and the 4-amino group of trimethoprim is proposed to play a critical role in the inhibition of DHFR by trimethoprim. This enzyme-substrate model should facilitate the design of new antibacterial agents with improved activity against S. pneumoniae.

Contribution of regulation by the bvg locus to respiratory infection of mice by Bordetella pertussis.

Whooping cough is an acute respiratory disease caused by the small, gram-negative bacterium Bordetella pertussis. B. pertussis expresses several factors that contribute to its ability to cause disease. These factors include surface-associated molecules, which are involved in the adherence of the organism to respiratory epithelial cells, as well as several extracellular toxins that inhibit host defenses and induce damage to host tissues. The expression of virulence factors in B. pertussis is dependent upon the bvg locus, which consists of three genes: bvgA, bvgS, and bvgR. The bvgAS genes encode a two-component regulatory system consisting of a sensor protein, BvgS, and a transcriptional activator, BvgA. Upon modification by BvgS, BvgA binds to the promoter regions of the bvg-activated genes and activates transcription. One of the bvg-activated genes, bvgR, is responsible for the regulation of the bvg-repressed genes, the functions of which are unknown. The fact that these genes are regulated by the bvg locus suggests that they play a role in the pathogenesis of the bacterium. In order to evaluate the contribution of bvg-mediated regulation to the virulence of B. pertussis and determine if expression of the bvg-repressed genes is required for the virulence of B. pertussis, we examined the ability of B. pertussis mutants, defective in their ability to regulate the expression of the bvg-activated and/or the bvg-repressed genes, to cause disease in the mouse aerosol challenge model. Our results indicate that the bvgR-mediated regulation of gene expression contributes to respiratory infection of mice.

Internalization of a Bacillus anthracis protective antigen-c-Myc fusion protein mediated by cell surface anti-c-Myc antibodies.

BACKGROUND: Anthrax toxin, secreted by Bacillus anthracis, consists of protective antigen (PA) and either lethal factor (LF) or edema factor (EF). PA, the receptor-binding component of the toxin, translocates LF or EF into the cytosol, where the latter proteins exert their toxic effects. We hypothesized that anthrax toxin fusion proteins could be used to kill virus-infected cells and tumor cells, if PA could be redirected to unique receptors found only on these cells. MATERIALS AND METHODS: To test this hypothesis in a model system, amino acids 410-419 of the human p62(c-myc) epitope were fused to the C-terminus of PA to redirect PA to the c-Myc-specific hybridoma cell line 9E10. RESULTS: The PA-c-Myc fusion protein killed both mouse macrophages and 9E10 hybridoma cells when administered with LF or an LF fusion protein (FP59), respectively. Similar results were obtained with PA, which suggests that PA-c-Myc used the endogenous PA receptor to enter the cells. By blocking the endogenous PA receptors on 9E10 cells with the competitive inhibitor PA SNKEDeltaFF, the PA-c-Myc was directed to an alternate receptor, i.e., the anti-c-Myc antibodies presented on the cell surface. The c-Myc IgG were proven to act as receptors because the addition of a synthetic peptide containing the c-Myc epitope along with PA SNKEDeltaFF further reduced the toxicity of PA-c-Myc + FP59. CONCLUSION: This study shows that PA can be redirected to alternate receptors by adding novel epitopes to the C-terminus of PA, enabling the creation of cell-directed toxins for therapeutic purposes.

Targeting HIV proteins to the major histocompatibility complex class I processing pathway with a novel gp120-anthrax toxin fusion protein.

A challenge for subunit vaccines whose goal is to elicit CD8(+) cytotoxic T lymphocytes (CTLs) is to deliver the antigen to the cytosol of the living cell, where it can be processed for presentation by major histocompatibility complex (MHC) class I molecules. Several bacterial toxins have evolved to efficiently deliver catalytic protein moieties to the cytosol of eukaryotic cells. Anthrax lethal toxin consists of two distinct proteins that combine to form the active toxin. Protective antigen (PA) binds to cells and is instrumental in delivering lethal factor (LF) to the cell cytosol. To test whether the lethal factor protein could be exploited for delivery of exogenous proteins to the MHC class I processing pathway, we constructed a genetic fusion between the amino-terminal 254 aa of LF and the gp120 portion of the HIV-1 envelope protein. Cells treated with this fusion protein (LF254-gp120) in the presence of PA effectively processed gp120 and presented an epitope recognized by HIV-1 gp120 V3-specific CTL. In contrast, when cells were treated with the LF254-gp120 fusion protein and a mutant PA protein defective for translocation, the cells were not able to present the epitope and were not lysed by the specific CTL. The entry into the cytosol and dependence on the classical cytosolic MHC class I pathway were confirmed by showing that antigen presentation by PA + LF254-gp120 was blocked by the proteasome inhibitor lactacystin. These data demonstrate the ability of the LF amino-terminal fragment to deliver antigens to the MHC class I pathway and provide the basis for the development of novel T cell vaccines.

Decreased susceptibility to imipenem among penicillin-resistant Streptococcus pneumoniae.

We assessed the antimicrobial susceptibilities of 59 penicillin-intermediate or penicillin-resistant pneumococci. All strains were susceptible to vancomycin and rifampicin. The frequency of strains with decreased susceptibility to cefotaxime, chloramphenicol, imipenem and meropenem was 15, 31, 47 and 49% respectively. The high percentage of penicillin-intermediate or penicillin-resistant Streptococcus pneumoniae with decreased susceptibility to third-generation cephalosporins, chloramphenicol and carbapenems limits the therapeutic options for the treatment of invasive pneumococcal infections and particularly of meningitis.

Delivery of antigens to the MHC class I pathway using bacterial toxins.

Cytotoxic T lymphocytes (CTL) recognize antigens derived from endogenously expressed proteins presented on the cell surface in the context of major histocompatibility complex (MHC) class I molecules. Because CTL are effective in antiviral and antitumor responses, the delivery of antigens to the class I pathway has been the focus of numerous efforts. Generating CTL by immunization with exogenous proteins is often ineffective because these antigens typically enter the MHC class II pathway. This review focuses on the usefulness of bacterial toxins for delivering antigens to the MHC class I pathway. Several toxins naturally translocate into the cytosol, where they mediate their cytopathic effects, and the mechanisms by which this occurs has been elucidated. Molecular characterization of these toxins identified the functional domains and enabled the generation of modified proteins that were no longer toxic but retained the ability to translocate into the cytosol. Thus, these modified toxins could be examined for their ability to carry peptides or whole proteins into the cytosolic processing pathway. Of the toxins studied-diphtheria, pertussis, Pseudomonas, and anthrax-the anthrax toxin appears the most promising in its ability to deliver large protein antigens and its efficiency of translocation.

Flattening the hierarchy. A hospital streamlines managerial layers to meet market demands.

In the early 1990s it became clear to the leaders of St. Edward Mercy Medical Center, Fort Smith, AR, that the traditional ¿functional¿ model of organization, on which their hospital was based, did not allow it to meet new market demands. A core group of managers was formed to design a new organizational model and engineer the move toward it. Analyzing the hospital's structure, the core group found that it had too many administrative layers above too many specialized departments. In 1994 the group decided to adopt a span-of-control model of organization, which would give St. Edward a higher ratio of workers per manager. In 1995 the core group streamlined the hospital's managerial layers, deciding there would be no more than five. It reduced the number of supervisory positions by 36, including one vice president's slot. No manager was fired, though some were reassigned. St. Edward's reorganization continues at present. The new structure, which has cut personnel costs, fosters more open communication and empowers its workers, leading them to think in terms of ¿us and our hospital¿ rather than ¿me and my department.¿

Expression and mutagenesis of recombinant cholera toxin A subunit.

ADP-ribosylating protein exotoxins from Vibrio cholerae (CT) and Escherichia coli (LT-I) share two short regions of sequence similarity with Bordetella pertussis toxin (PT). Previous studies have indicated that substitution of arginine for lysine 7 within the first region of CT drastically decreases ADP ribosyltransferase activity. We have more closely defined the role of other amino acids in this region by generating modified proteins in which arginine 7 was replaced with lysine (R7K), aspartate 9 was replaced with arginine (D9R), glycine was substituted for proline 12 (P12G), amino acids 6 to 13 were deleted (delta 613) or the C-terminal KDEL sequence was changed to NEDL. The modified proteins R7K, D9R and delta 613 exhibited undetectable ADP ribosyltransferase activity. Comparison of the tryptic digest of R7K with native CT suggested that changes in protein conformation may be responsible for the loss of ADP-ribosylation activity.

Toxicity of Bordetella avium beta-cystathionase toward MC3T3-E1 osteogenic cells.

Bordetella avium is the etiological agent of an upper respiratory disease in birds which, symptomatically and pathologically, resembles bordetellosis in humans. Studies of the virulence of this organism revealed a novel cytotoxic protein, designated osteotoxin, that was lethal for MC3T3-E1 osteogenic cells, fetal bovine trabecular cells, UMR106-01(BSP) rat osteosarcoma cells, and embryonic bovine tracheal cells. The osteotoxin lacked dermonecrotic toxin activity, exhibited no cross-reactivity with antibody against B. avium dermonecrotic toxin, and was non-proteolytic. Osteotoxin (M(r) approximately 80,000 by gel filtration, pI 5.4) was purified to electrophoretic homogeneity from B. avium 197. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and spectrophotometric analyses showed that the native protein was a homodimer and that each of the non-covalently linked subunits (M(r) approximately 41,000) contained one molecule of pyridoxal 5'-phosphate. Microsequencing of the first 32 amino acids from the NH2 terminus allowed the synthesis of two oligonucleotide probes, which, together with polyclonal antibody to the purified protein, facilitated cloning, sequencing, and expression of the osteotoxin gene product in Escherichia coli. The open reading frame encodes a polypeptide of 396 amino acid residues (M(r) = 42,606, calculated pI 5.9), whose sequence exhibits approximately 38% identity (approximately 60% similarity) to pyridoxal 5'-phosphate-dependent beta-cystathionase(s) from E. coli, Salmonella typhimurium, and rat liver. The characteristic motif, TKYXXGHSD, associated with binding the cofactor in these enzymes is also present in osteotoxin. Physicochemical and enzymatic analyses established the coidentity of osteotoxin with beta-cystathionase. The region upstream of the beta-cystathionase (metC) gene in B. avium 197 lacked regulatory sequences ("Met boxes") described for metC in enteric species, and enzyme production was not repressed by methionine. Incubation of MC3T3-E1 osteogenic cells in medium containing L-[35S]cystine and purified beta-cystathionase resulted in 35S-labeling of the enzyme and at least one major MC3T3-E1 cell protein (M(r) approximately 50,000). cytotoxicity can be attributed to: 1) beta-cystathionase-catalyzed cleavage of L-cystine in the medium and formation of reactive sulfane-containing derivative(s), and 2) transfer of sulfane sulfur to metabolically sensitive or structurally important proteins in the osteogenic cells.