Last data update: Dec 02, 2024. (Total: 48272 publications since 2009)
Records 1-14 (of 14 Records) |
Query Trace: Gallegos-Candela M[original query] |
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Comprehensive characterization of toxins during progression of inhalation anthrax in a non-human primate model
Boyer AE , Gallegos-Candela M , Lins RC , Solano MI , Woolfitt AR , Lee JS , Sanford DC , Knostman KAB , Quinn CP , Hoffmaster AR , Pirkle JL , Barr JR . PLoS Pathog 2022 18 (12) e1010735 Inhalation anthrax has three clinical stages: early-prodromal, intermediate-progressive, and late-fulminant. We report the comprehensive characterization of anthrax toxins, including total protective antigen (PA), total lethal factor (LF), total edema factor (EF), and their toxin complexes, lethal toxin and edema toxin in plasma, during the course of inhalation anthrax in 23 cynomolgus macaques. The toxin kinetics were predominantly triphasic with an early rise (phase-1), a plateau/decline (phase-2), and a final rapid rise (phase-3). Eleven animals had shorter survival times, meanstandard deviation of 58.77.6 hours (fast progression), 11 animals had longer survival times, 11334.4 hours (slow progression), and one animal survived. Median (lower-upper quartile) LF levels at the end-of-phase-1 were significantly higher in animals with fast progression [138 (54.9-326) ng/mL], than in those with slow progression [23.8 (15.6-26.3) ng/mL] (p = 0.0002), and the survivor (11.1 ng/mL). The differences were also observed for other toxins and bacteremia. Animals with slow progression had an extended phase-2 plateau, with low variability of LF levels across all time points and animals. Characterization of phase-2 toxin levels defined upper thresholds; critical levels for exiting phase-2 and entering the critical phase-3, 342 ng/mL (PA), 35.8 ng/mL (LF), and 1.10 ng/mL (EF). The thresholds were exceeded earlier in animals with fast progression (38.57.4 hours) and later in animals with slow progression (78.715.2 hours). Once the threshold was passed, toxin levels rose rapidly in both groups to the terminal stage. The time from threshold to terminal was rapid and similar; 20.87.4 hours for fast and 19.97.5 hours for slow progression. The three toxemic phases were aligned with the three clinical stages of anthrax for fast and slow progression which showed that anthrax progression is toxin- rather than time-dependent. This first comprehensive evaluation of anthrax toxins provides new insights into disease progression. |
Welder's Anthrax: A Tale of 2 Cases.
Hendricks K , Martines RB , Bielamowicz H , Boyer AE , Long S , Byers P , Stoddard RA , Taylor K , Kolton CB , Gallegos-Candela M , Roberts C , DeLeon-Carnes M , Salzer J , Dawson P , Brown D , Templeton-LeBouf L , Maves RC , Gulvik C , Lonsway D , Barr JR , Bower WA , Hoffmaster A . Clin Infect Dis 2022 75 S354-s363 Bacillus anthracis has traditionally been considered the etiologic agent of anthrax. However, anthrax-like illness has been documented in welders and other metal workers infected with Bacillus cereus group spp. harboring pXO1 virulence genes that produce anthrax toxins. We present 2 recent cases of severe pneumonia in welders with B. cereus group infections and discuss potential risk factors for infection and treatment options, including antitoxin. |
Zeptomole per milliliter detection and quantification of edema factor in plasma by LC-MS/MS yields insights into toxemia and the progression of inhalation anthrax
Lins RC , Boyer AE , Kuklenyik Z , Woolfitt AR , Goldstein J , Hoffmaster AR , Gallegos-Candela M , Leysath CE , Chen Z , Brumlow JO , Quinn CP , Bagarozzi DA Jr , Leppla SH , Barr JR . Anal Bioanal Chem 2019 411 (12) 2493-2509 Inhalation of Bacillus anthracis spores can cause a rapidly progressing fatal infection. B. anthracis secretes three protein toxins: lethal factor (LF), edema factor (EF), and protective antigen (PA). EF and LF may circulate as free or PA-bound forms. Both free EF (EF) and PA-bound-EF (ETx) have adenylyl cyclase activity converting ATP to cAMP. We developed an adenylyl cyclase activity-based method for detecting and quantifying total EF (EF+ETx) in plasma. The three-step method includes magnetic immunocapture with monoclonal antibodies, reaction with ATP generating cAMP, and quantification of cAMP by isotope-dilution HPLC-MS/MS. Total EF was quantified from 5PL regression of cAMP vs ETx concentration. The detection limit was 20 fg/mL (225 zeptomoles/mL for the 89 kDa protein). Relative standard deviations for controls with 0.3, 6.0, and 90 pg/mL were 11.7-16.6% with 91.2-99.5% accuracy. The method demonstrated 100% specificity in 238 human serum/plasma samples collected from unexposed healthy individuals, and 100% sensitivity in samples from 3 human and 5 rhesus macaques with inhalation anthrax. Analysis of EF in the rhesus macaques showed that it was detected earlier post-exposure than B. anthracis by culture and PCR. Similar to LF, the kinetics of EF over the course of infection were triphasic, with an initial rise (phase-1), decline (phase-2), and final rapid rise (phase-3). EF levels were ~ 2-4 orders of magnitude lower than LF during phase-1 and phase-2 and only ~ 6-fold lower at death/euthanasia. Analysis of EF improves early diagnosis and adds to our understanding of anthrax toxemia throughout infection. The LF/EF ratio may also indicate the stage of infection and need for advanced treatments. |
Accurate and selective quantification of anthrax protective antigen in plasma by immunocapture and isotope dilution mass spectrometry
Solano MI , Woolfitt AR , Boyer AE , Lins RC , Isbell K , Gallegos-Candela M , Moura H , Pierce CL , Barr JR . Analyst 2019 144 (7) 2264-2274 Anthrax protective antigen (83 kDa, PA83) is an essential component of two major binary toxins produced by Bacillus anthracis, lethal toxin (LTx) and edema toxin (ETx). During infection, LTx and ETx contribute to immune collapse, endothelial dysfunction, hemorrhage and high mortality. Following protease cleavage on cell receptors or in circulation, the 20 kDa (PA20) N-terminus is released, activating the 63 kDa (PA63) form which binds lethal factor (LF) and edema factor (EF), facilitating their entry into their cellular targets. Several ELISA-based PA methods previously developed are primarily qualitative or semi-quantitative. Here, we combined protein immunocapture, tryptic digestion and isotope dilution liquid chromatography-mass spectrometry (LC-MS/MS), to develop a highly selective and sensitive method for detection and accurate quantification of total-PA (PA83 + PA63) and PA83. Two tryptic peptides in the 63 kDa region measure total-PA and three in the 20 kDa region measure PA83 alone. Detection limits range from 1.3-2.9 ng mL-1 PA in 100 muL of plasma. Spiked recovery experiments with combinations of PA83, PA63, LF and EF in plasma showed that PA63 and PA83 were quantified accurately against the PA83 standard and that LF and EF did not interfere with accuracy. Applied to a study of inhalation anthrax in rhesus macaques, total-PA suggested triphasic kinetics, similar to that previously observed for LF and EF. This study is the first to report circulating PA83 in inhalation anthrax, typically at less than 4% of the levels of PA63, providing the first evidence that activated PA63 is the primary form of PA throughout infection. |
Validated MALDI-TOF-MS method for anthrax lethal factor provides early diagnosis and evaluation of therapeutics
Gallegos-Candela M , Boyer AE , Woolfitt AR , Brumlow J , Lins RC , Quinn CP , Hoffmaster AR , Meister G , Barr JR . Anal Biochem 2017 543 97-107 Anthrax lethal factor (LF) is a zinc-dependent endoprotease and a critical virulence factor for Bacillus anthracis, the causative agent of anthrax. The mass spectrometry (MS) method for total-LF quantification includes three steps; 1) LF specific antibody capture/concentration, 2) LF-specific hydrolysis of a peptide substrate, and 3) detection and quantification of LF-cleaved peptides by isotope-dilution MALDI-TOF/MS. Recombinant LF spiked plasma was used for calibration and quality control (QC) materials. Specificity was 100% from analysis of serum and plasma from 383 non-infected humans, 31 rabbits, and 24 rhesus macaques. Sensitivity was 100% from 32 human clinical anthrax cases including infections by inhalation, ingestion, cutaneous and injection exposures and experimental infections for 29 rabbits and 24 rhesus macaques with inhalation anthrax. Robustness evaluation included sample storage, serum and plasma, antimicrobial and antitoxin effects and long-term performance. Data from 100 independent runs gave detection limits 0.01 ng/mL (111 amol/mL) for the 4-h method and 0.0027 ng/mL (30 amol/mL) for an alternate 20-h method. QC precision ranged from 7.7 to 14.8% coefficient of variation and accuracy from 0.2 to 9.8% error. The validated LF MS method provides sensitive quantification of anthrax total-LF using a robust high throughput platform for early diagnosis and evaluation of therapeutics during an anthrax emergency. |
Anthrax Toxin-Expressing Bacillus cereus Isolated from an Anthrax-Like Eschar.
Marston CK , Ibrahim H , Lee P , Churchwell G , Gumke M , Stanek D , Gee JE , Boyer AE , Gallegos-Candela M , Barr JR , Li H , Boulay D , Cronin L , Quinn CP , Hoffmaster AR . PLoS One 2016 11 (6) e0156987 Bacillus cereus isolates have been described harboring Bacillus anthracis toxin genes, most notably B. cereus G9241, and capable of causing severe and fatal pneumonias. This report describes the characterization of a B. cereus isolate, BcFL2013, associated with a naturally occurring cutaneous lesion resembling an anthrax eschar. Similar to G9241, BcFL2013 is positive for the B. anthracis pXO1 toxin genes, has a multi-locus sequence type of 78, and a pagA sequence type of 9. Whole genome sequencing confirms the similarity to G9241. In addition to the chromosome having an average nucleotide identity of 99.98% when compared to G9241, BcFL2013 harbors three plasmids with varying homology to the G9241 plasmids (pBCXO1, pBC210 and pBFH_1). This is also the first report to include serologic testing of patient specimens associated with this type of B. cereus infection which resulted in the detection of anthrax lethal factor toxemia, a quantifiable serum antibody response to protective antigen (PA), and lethal toxin neutralization activity. |
High-sensitivity MALDI-TOF MS quantification of anthrax lethal toxin for diagnostics and evaluation of medical countermeasures
Boyer AE , Gallegos-Candela M , Quinn CP , Woolfitt AR , Brumlow JO , Isbell K , Hoffmaster AR , Lins RC , Barr JR . Anal Bioanal Chem 2015 407 (10) 2847-58 Inhalation anthrax has a rapid progression and high fatality rate. Pathology and death from inhalation of Bacillus anthracis spores are attributed to the actions of secreted protein toxins. Protective antigen (PA) binds and imports the catalytic component lethal factor (LF), a zinc endoprotease, and edema factor (EF), an adenylyl cyclase, into susceptible cells. PA-LF is termed lethal toxin (LTx) and PA-EF, edema toxin. As the universal transporter for both toxins, PA is an important target for vaccination and immunotherapeutic intervention. However, its quantification has been limited to methods of relatively low analytic sensitivity. Quantification of LTx may be more clinically relevant than LF or PA alone because LTx is the toxic form that acts on cells. A method was developed for LTx-specific quantification in plasma using anti-PA IgG magnetic immunoprecipitation of PA and quantification of LF activity that co-purified with PA. The method was fast (<4 h total time to detection), sensitive at 0.033 ng/mL LTx in plasma for the fast analysis (0.0075 ng/mL LTx in plasma for an 18 h reaction), precise (6.3-9.9 % coefficient of variation), and accurate (0.1-12.7 %error; n ≥ 25). Diagnostic sensitivity was 100 % (n = 27 animal/clinical cases). Diagnostic specificity was 100 % (n = 141). LTx was detected post-antibiotic treatment in 6/6 treated rhesus macaques and 3/3 clinical cases of inhalation anthrax and as long as 8 days post-treatment. Over the course of infection in two rhesus macaques, LTx was first detected at 0.101 and 0.237 ng/mL at 36 h post-exposure and increased to 1147 and 12,107 ng/mL in late-stage anthrax. This demonstrated the importance of LTx as a diagnostic and therapeutic target. This method provides a sensitive, accurate tool for anthrax toxin detection and evaluation of PA-directed therapeutics. |
Circulating lethal toxin decreases the ability of neutrophils to respond to Bacillus anthracis
Weiner ZP , Ernst SM , Boyer AE , Gallegos-Candela M , Barr JR , Glomski IJ . Cell Microbiol 2014 16 (4) 504-18 Polymorphonuclear leucocytes (PMNs) play a protective role during Bacillus anthracis infection. However, B. anthracis is able to subvert the PMN response effectively as evidenced by the high mortality rates of anthrax. One major virulence factor produced by B. anthracis, lethal toxin (LT), is necessary for dissemination in the BSL2 model of mouse infection. While human and mouse PMNs kill vegetative B. anthracis, short in vitro half-lives of PMNs have made it difficult to determine how or if LT alters their bactericidal function. Additionally, the role of LT intoxication on PMN's ability to migrate to inflammatory signals remains controversial. LF concentrations in both serum and major organs were determined from mice infected with B. anthracis Sterne strain at defined stages of infection to guide subsequent administration of purified toxin. Bactericidal activity of PMNs assessed using ex vivo cell culture assays showed significant defects in killing B. anthracis. In vivo PMN recruitment to inflammatory stimuli was significantly impaired at 24 h as assessed by real-time analysis of light-producing PMNs within the mouse. The observations described above suggest that LT serves dual functions; it both attenuates accumulation of PMNs at sites of inflammation and impairs PMNs bactericidal activity against vegetative B. anthracis. |
Debridement increases survival in a mouse model of subcutaneous anthrax
Weiner ZP , Boyer AE , Gallegos-Candela M , Cardani AN , Barr JR , Glomski IJ . PLoS One 2012 7 (2) e30201 Anthrax is caused by infection with Bacillus anthracis, a spore-forming Gram-positive bacterium. A major virulence factor for B. anthracis is an immunomodulatory tripartite exotoxin that has been reported to alter immune cell chemotaxis and activation. It has been proposed that B. anthracis infections initiate through entry of spores into the regional draining lymph nodes where they germinate, grow, and disseminate systemically via the efferent lymphatics. If this model holds true, it would be predicted that surgical removal of infected tissues, debridement, would have little effect on the systemic dissemination of bacteria. This model was tested through the development of a mouse debridement model. It was found that removal of the site of subcutaneous infection in the ear increased the likelihood of survival and reduced the quantity of spores in the draining cervical lymph nodes (cLN). At the time of debridement 12 hours post-injection measurable levels of exotoxins were present in the ear, cLN, and serum, yet leukocytes within the cLN were activated; countering the concept that exotoxins inhibit the early inflammatory response to promote bacterial growth. We conclude that the initial entry of spores into the draining lymph node of cutaneous infections alone is not sufficient to cause systemic disease and that debridement should be considered as an adjunct to antibiotic therapy. |
Lethal factor toxemia and anti-protective antigen antibody activity in naturally acquired cutaneous anthrax
Boyer AE , Quinn CP , Beesley CA , Gallegos-Candela M , Marston CK , Cronin LX , Lins RC , Stoddard RA , Li H , Schiffer J , Hossain MJ , Chakraborty A , Rahman M , Luby SP , Shieh WJ , Zaki S , Barr JR , Hoffmaster AR . J Infect Dis 2011 204 (9) 1321-7 Cutaneous anthrax outbreaks occurred in Bangladesh from August to October 2009. As part of the epidemiological response and to confirm anthrax diagnoses, serum samples were collected from suspected case patients with observed cutaneous lesions. Anthrax lethal factor (LF), anti-protective antigen (anti-PA) immunoglobulin G (IgG), and anthrax lethal toxin neutralization activity (TNA) levels were determined in acute and convalescent serum of 26 case patients with suspected cutaneous anthrax from the first and largest of these outbreaks. LF (0.005-1.264 ng/mL) was detected in acute serum from 18 of 26 individuals. Anti-PA IgG and TNA were detected in sera from the same 18 individuals and ranged from 10.0 to 679.5 mcg/mL and 27 to 593 units, respectively. Seroconversion to serum anti-PA and TNA was found only in case patients with measurable toxemia. This is the first report of quantitative analysis of serum LF in cutaneous anthrax and the first to associate acute stage toxemia with subsequent antitoxin antibody responses. |
Quantitative mass spectrometry for bacterial protein toxins - a sensitive, specific, high-throughput tool for detection and diagnosis
Boyer AE , Gallegos-Candela M , Lins RC , Kuklenyik Z , Woolfitt A , Moura H , Kalb S , Quinn CP , Barr JR . Molecules 2011 16 (3) 2391-413 Matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometry (MS) is a valuable high-throughput tool for peptide analysis. Liquid chromatography electrospray ionization (LC-ESI) tandem-MS provides sensitive and specific quantification of small molecules and peptides. The high analytic power of MS coupled with high-specificity substrates is ideally suited for detection and quantification of bacterial enzymatic activities. As specific examples of the MS applications in disease diagnosis and select agent detection, we describe recent advances in the analyses of two high profile protein toxin groups, the Bacillus anthracis toxins and the Clostridium botulinum neurotoxins. The two binary toxins produced by B. anthracis consist of protective antigen (PA) which combines with lethal factor (LF) and edema factor (EF), forming lethal toxin and edema toxin respectively. LF is a zinc-dependent endoprotease which hydrolyzes specific proteins involved in inflammation and immunity. EF is an adenylyl cyclase which converts ATP to cyclic-AMP. Toxin-specific enzyme activity for a strategically designed substrate, amplifies reaction products which are detected by MALDI-TOF-MS and LC-ESI-MS/MS. Pre-concentration/purification with toxin specific monoclonal antibodies provides additional specificity. These combined technologies have achieved high specificity, ultrasensitive detection and quantification of the anthrax toxins. We also describe potential applications to diseases of high public health impact, including Clostridium difficile glucosylating toxins and the Bordetella pertussis adenylyl cyclase. |
Antibody responses to a spore carbohydrate antigen as a marker of non-fatal inhalation anthrax in Rhesus macaques
Saile E , Boons GJ , Buskas T , Carlson RW , Kannenberg EL , Barr JR , Boyer AE , Gallegos-Candela M , Quinn CP . Clin Vaccine Immunol 2011 18 (5) 743-8 The Bacillus anthracis exosporium protein BclA contains an O-linked antigenic tetrasaccharide whose terminal sugar is known as anthrose (3). We hypothesized that serologic responses to anthrose may have diagnostic value in confirming exposure to aerosolized B. anthracis. We evaluated the serologic responses to a synthetic anthrose-containing trisaccharide (ATS) in a group of five Rhesus macaques (RM) that survived inhalation anthrax following exposure to B. anthracis Ames spores. Two of five animals were treated with ciprofloxacin starting at 48 (RM2, RM3) hours and two at 72 hours (RM4, RM5) post-exposure; one animal was untreated (RM1). Infection was confirmed by blood culture and detection of anthrax toxin lethal factor (LF) in plasma. Anti-ATS IgG responses were determined at 14, 21, 28, and 35 days post-exposure with pre-exposure serum as a control. All animals, irrespective of ciprofloxacin treatment, mounted a specific, measurable anti-ATS IgG response. The earliest detectable responses were on day 14 (RM1, RM2, RM5) and at days 21 (RM4) and 28 (RM3). Specificity of the anti-ATS responses was demonstrated by competitive inhibition enzyme immunoassay (CIEIA) in which a two-fold excess of carbohydrate (wt/wt) in a bovine serum albumin (BSA) conjugate of the oligosaccharide (ATS-BSA) effected >94% inhibition, whereas a structural analog lacking the 3-hydroxy-3-methyl-butyryl moiety at the C-4" of the anthrosyl residue had no inhibition activity. These data suggest that anti-ATS antibody responses may be used to identify aerosol exposure to B. anthracis spores. The anti-ATS antibody responses were detectable during administration of cipropfloxacin. |
Comparison of MALDI-TOF-MS and HPLC-ESI-MS/MS for endopeptidase activity-based quantification of anthrax lethal factor in serum
Kuklenyik Z , Boyer AE , Lins R , Quinn CP , Gallegos-Candela M , Woolfitt A , Pirkle JL , Barr JR . Anal Chem 2011 83 (5) 1760-5 Diagnosing and treating anthrax at the earliest stage of disease is critical. We developed a method to diagnose anthrax at early stages of infection by detecting anthrax lethal factor (LF) at the attomol/mL level in plasma or serum. This method uses antibody capture and quantification of LF endoproteinase activity by isotope dilution matrix-assisted laser-desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). Many public health laboratories do not use MALDI-TOF-MS; thus, we have adapted the LF method for detection by electrospray ionization (ESI) tandem MS (MS/MS), which allowed comparison of both MS platforms for LF quantification. Calibration curves were linear from 0.05-2.5 ng/mL when measured after 2 h and from 0.005-1.0 ng/mL after 18 h incubation time. The limit of detection was 0.005 ng/mL using a 200 muL sample. The coefficient of variation for quality control samples was 6-12% for both MS platforms. Samples used to perform cross-validation included 158 serum samples from a study in rabbits exposed to anthrax spores by inhalation. Some were treated with anthrax immune globulin before exposure. Concentrations measured by ESI-MS/MS matched those by MALDI-TOF-MS with p = 0.99 (r(2) = 0.997) and -0.25% mean relative difference (+/-9% standard deviation). This study shows that isotope dilution MALDI-TOF-MS is a robust and precise quantitative MS platform. |
Studies on botulinum neurotoxins type/C1 and mosaic/DC using endopep-MS and proteomics
Moura H , Terilli RR , Woolfitt AR , Gallegos-Candela M , McWilliams LG , Solano MI , Pirkle JL , Barr JR . FEMS Immunol Med Microbiol 2010 61 (3) 288-300 Botulinum neurotoxins (BoNTs) are very potent toxins and category A biological threat agents. BoNT serotypes/C1 and/D affect birds and mammals and can be potentially lethal to humans. We have previously described the usefulness of the Endopep-MS method to detect the activity of BoNT A through G. This report was followed by the application of the method to clinical samples. The activity of the BoNT serotypes associated with human disease (/A,/B,/E, and/F) was successfully detected. However, BoNT/C and/D require different conditions for fast substrate cleavage and a comprehensive description of a method to study BoNT/C and/D has not yet been reported. This work describes a new, optimized version of the Endopep-MS method to detect BoNTs/C1 and/DC either spiked directly in 20 muL of reaction buffer or spiked in a larger volume of buffer and further extracted using antibody-coated magnetic beads. It was found that the incubation temperature at 42 degrees C was more effective for both toxin serotypes, but each toxin serotype has an optimum cleavage pH. Additionally, we describe for the first time a proteomics study using a fast trypsin digestion method and label-free quantification of these toxin serotypes. |
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