Search

Direct pathogen detection in blood to diagnose active tuberculosis (TB) has been difficult due to low levels of circulating antigens or due to the lack of specific, high-affinity binding reagents and reliable assays with adequate sensitivity. We sought to determine whether slow off-rate modified aptamer (SOMAmer) reagents with subnanomolar affinity for Mycobacterium tuberculosis proteins (antigens 85A, 85B, 85C, GroES, GroEL2, DnaK, CFP10, KAD, CFP2, RplL, and Tpx) could be useful to diagnose tuberculosis. When incorporated into the multiplexed, array-based proteomic SOMAscan assay, limits of detection reached the subpicomolar range in 40% serum. Binding to native M. tuberculosis proteins was confirmed by using M. tuberculosis culture filtrate proteins and fractions from infected macrophages and via affinity capture assays and subsequent mass spectrometry. Comparison of serum from culture-positive pulmonary TB patients and TB suspects systematically ruled out for TB revealed small but statistically significant ( P < 0.0001) differences in the median M. tuberculosis signals and in specific pathogen markers, such as antigen 85B. Samples where many M. tuberculosis aptamers produced high signals were rare exceptions. In concentrated, protein-normalized urine from TB patients and non-TB controls, the CFP10 (EsxB) SOMAmer yielded the most significant differential signals ( P < 0.0276), particularly in TB patients with HIV coinfection. In conclusion, direct M. tuberculosis antigen detection proved difficult even with a sensitive method such as SOMAscan, likely due to their very low, subpicomolar abundance. The observed differences between cases and controls had limited diagnostic utility in serum and urine, but further evaluation of M. tuberculosis SOMAmers using other platforms and sample types is warranted.

Summary HtrA serine proteases are highly conserved and essential ATP-independent proteases with chaperone activity. Bacteria express a variable number of HtrA homologues that contribute to the virulence and pathogenicity of bacterial pathogens. Lyme disease spirochetes possess a single HtrA protease homologue, Borrelia burgdorferi HtrA (BbHtrA). Previous studies established that, like the human orthologue HtrA1, BbHtrA is proteolytically active against numerous extracellular proteins in vitro. In this study, we utilized size exclusion chromatography and blue native polyacrylamide gel electrophoresis (BN-PAGE) to demonstrate BbHtrA oligomeric structures that were substrate independent and salt sensitive. Examination of the influence of transition metals on the activity of BbHtrA revealed that this protease is inhibited by Zn2+ > Cu2+ > Mn2+. Extending this analysis to two other HtrA proteases, E. coli DegP and HtrA1, revealed that all three HtrA proteases were reversibly inhibited by ZnCl2 at all micro molar concentrations examined. Commercial inhibitors for HtrA proteases are not available and physiologic HtrA inhibitors are unknown. Our observation of conserved zinc inhibition of HtrA proteases will facilitate structural and functional studies of additional members of this important class of proteases.

New non-sputum biomarker tests for active tuberculosis (TB) diagnostics are of the highest priority for global TB control. We performed in-depth proteomic analysis using the 4,000-plex SOMAscan assay on 1,470 serum samples from seven countries where TB is endemic. All samples were from patients with symptoms and signs suggestive of active pulmonary TB that were systematically confirmed or ruled out for TB by culture and clinical follow-up. HIV coinfection was present in 34% of samples, and 25% were sputum smear negative. Serum protein biomarkers were identified by stability selection using L1-regularized logistic regression and by Kolmogorov-Smirnov (KS) statistics. A naive Bayes classifier using six host response markers (HR6 model), including SYWC, kallistatin, complement C9, gelsolin, testican-2, and aldolase C, performed well in a training set (area under the sensitivity-specificity curve [AUC] of 0.94) and in a blinded verification set (AUC of 0.92) to distinguish TB and non-TB samples. Differential expression was also highly significant ( P < 10 −20 ) for previously described TB markers, such as IP-10, LBP, FCG3B, and TSP4, and for many novel proteins not previously associated with TB. Proteins with the largest median fold changes were SAA (serum amyloid protein A), NPS-PLA2 (secreted phospholipase A2), and CA6 (carbonic anhydrase 6). Target product profiles (TPPs) for a non-sputum biomarker test to diagnose active TB for treatment initiation (TPP#1) and for a community-based triage or referral test (TPP#2) have been published by the WHO. With 90% sensitivity and 80% specificity, the HR6 model fell short of TPP#1 but reached TPP#2 performance criteria. In conclusion, we identified and validated a six-marker signature for active TB that warrants diagnostic development on a patient-near platform.

HtrA serine proteases are highly conserved and essential ATP-independent proteases with chaperone activity. Bacteria express a variable number of HtrA homologues that contribute to the virulence and pathogenicity of bacterial pathogens. Lyme disease spirochetes possess a single HtrA protease homologue, Borrelia burgdorferi HtrA (BbHtrA). Previous studies established that, like the human orthologue HtrA1, BbHtrA is proteolytically active against numerous extracellular proteins in vitro. In this study, we utilized size exclusion chromatography and blue native polyacrylamide gel electrophoresis (BN-PAGE) to demonstrate BbHtrA oligomeric structures that were substrate independent and salt sensitive. Examination of the influence of transition metals on the activity of BbHtrA revealed that this protease is inhibited by Zn(2+) Cu(2+)Mn(2+). Extending this analysis to two other HtrA proteases, E. coli DegP and HtrA1, revealed that all three HtrA proteases were reversibly inhibited by ZnCl2 at all micro molar concentrations examined. Commercial inhibitors for HtrA proteases are not available and physiologic HtrA inhibitors are unknown. Our observation of conserved zinc inhibition of HtrA proteases will facilitate structural and functional studies of additional members of this important class of proteases.

Understanding the functions of multi-cellular organs in terms of the molecular networks within each cell is an important step in the quest to predict phenotype from genotype. B-lymphocyte population dynamics, which are predictive of immune response and vaccine effectiveness, are determined by individual cells undergoing division or death seemingly stochastically. Based on tracking single-cell time-lapse trajectories of hundreds of B cells, single-cell transcriptome, and immunofluorescence analyses, we constructed an agent-based multi-modular computational model to simulate lymphocyte population dynamics in terms of the molecular networks that control NF-κB signaling, the cell cycle, and apoptosis. Combining modeling and experimentation, we found that NF-κBcRel enforces the execution of a cellular decision between mutually exclusive fates by promoting survival in growing cells. But as cRel deficiency causes growing B cells to die at similar rates to non-growing cells, our analysis reveals that the phenomenological decision model of wild-type cells is rooted in a biased race of cell fates. We show that a multi-scale modeling approach allows for the prediction of dynamic organ-level physiology in terms of intra-cellular molecular networks.

Borrelia burgdorferi synthesizes an HtrA protease (BbHtrA) which is a surface-exposed, conserved protein within Lyme disease spirochetes with activity toward CheX and BmpD of Borrelia spp, as well as aggrecan, fibronectin and proteoglycans found in skin, joints and neural tissues of vertebrates. An antibody response against BbHtrA is observed in Lyme disease patients and in experimentally infected laboratory mice and rabbits. Given the surface location of BbHtrA on B. burgdorferi and its ability to elicit an antibody response in infected hosts, we explored recombinant BbHtrA as a potential vaccine candidate in a mouse model of tick-transmitted Lyme disease. We immunized mice with two forms of BbHtrA: the proteolytically active native form and BbHtrA ablated of activity by a serine to alanine mutation at amino acid 226 (BbHtrA(S226A)). Although inoculation with either BbHtrA or BbHtrA(S226A) produced high-titer antibody responses in C3H/HeJ mice, neither antigen was successful in protecting mice from B. burgdorferi challenge. These results indicate that the search for novel vaccine candidates against Lyme borreliosis remains a challenge.

African Americans with factor VII (FVII) deficiency, as defined by clinical laboratory values, are frequently asymptomatic. To date the genotypes underlying this FVII defect in asymptomatic African Americans have not been established. We show in 3 unrelated African American patients that the defect is due to a G to A nucleotide change resulting in an arginine to glutamine mutation in Factor VII amino acid 304. This defect results in low FVII coagulant activity levels using rabbit brain thromboplastin but not using human thromboplastin. This report may aid transfusion and hematology specialists to evaluate patient results and prevent unnecessary transfusions to treat patients with abnormal laboratory values.

A basic obligation of a commercial clinical laboratory is to validate a test before offering it for sale to diagnose patients. Advanced Laboratory Services offers a culture method to diagnose patients with a Borrelia species infection ([1][1]), but we do not find the evidence provided in support of

A novel method of culturing spirochetes from the serum of U.S. Lyme disease patients was recently reported by Sapi and colleagues to have 94% sensitivity and 100% specificity for Borrelia species as assessed by microscopy and DNA sequence analysis of the pyrG gene (E. Sapi, N. Pabbati, A. Datar, E. M. Davies, A. Rattelle, and B. A. Kuo, Int. J. Med. Sci. 10:362–376, 2013). The majority of the spirochetes described were related by pyrG sequences to species of Borrelia previously undetected in North American patients without a reported history of travel to Europe or Asia. To better understand these unexpected findings, we determined pyrG sequences of the laboratory reference strains used by the investigators for method development and testing of culture medium. Eighty percent (41/51) of the reported patient-derived pyrG sequences were identical to one of the laboratory strains, and an additional 12% (6/51) differed by only a single nucleotide across a 603-bp region of the pyrG gene. Thus, false positivity due to laboratory contamination of patient samples cannot be ruled out, and further validation of the proposed novel culture method is required.

Summary The Lyme disease spirochaete, Borrelia burgdorferi, causes damage to diverse host tissues and induces inflammation but the mechanisms of injury are poorly understood. We recently reported that a surface-exposed B. burgdorferi protease, which is expressed during human disease and is conserved within the major Lyme disease spirochaete species, degrades the extracellular matrix proteoglycan, aggrecan. Here we demonstrate that BbHtrA also degrades fibronectin and numerous proteoglycans found in skin, joints and neural tissues. BbHtrA degradation of fibronectin released known pro-inflammatory fibronectin fragments FnIII13–14 and Fnf-29, which may amplify the inflammatory processes triggered by the presence of the bacteria. When this hypothesis was tested directly by exposing chondrocytes to BbHtrA in vitro, inflammatory cytokines (sICAM-1 and IL-6) and chemokines (CXCL1, CCL1, CCL2 and CCL5) that are hallmarks of Lyme disease were induced. These results provide the first evidence that, by utilizing BbHtrA, B. burgdorferi may actively participate in its dissemination and in the tissue damage and inflammation observed in Lyme disease.

Connective tissues are the most common area of colonization for the Lyme disease spirochaete Borrelia burgdorferi. Colonization is aided by the interaction between numerous bacterial adhesins with components of the extracellular matrix (ECM). Here we describe a novel interaction between B. burgdorferi and the major ECM proteoglycan found in joints, aggrecan. Using affinity chromatography and mass spectrometry we identify two borrelial aggrecan-binding proteins: the known ECM ligand Bgp (BB0588) and an uncharacterized protease BbHtrA (BB0104). Proteinase K studies demonstrate that BbHtrA is surface exposed. Immunoblots using sera from patients with both early and late Lyme disease establish that BbHtrA is expressed during human disease, immunogenic, and conserved in the three major Lyme disease spirochaete species. Consequences of the interaction between aggrecan and BbHtrA were examined by proteolysis assays. BbHtrA cleaves aggrecan at a site known to destroy aggrecan function and which has been previously observed in the synovial fluid of patients with Lyme arthritis. These data demonstrate that B. burgdorferi possess aggrecan-binding proteins which may provide the organism with additional capability to colonize connective tissues. Moreover, our studies provide the first evidence that B. burgdorferi possess proteolytic activity which may contribute to the pathogenesis of Lyme arthritis.

Severe coagulant factor VII (FVII) deficiency in postpubertal dizygotic twin males results from two point mutations in the FVII gene, a promoter region T→C transition at -60 and a His-to-Arg substitution at amino acid 348; both mutations prevent persistence of plasma functional FVII. This report documents longitudinal laboratory measurements from infancy to adulthood of FVII coagulant activity (FVII:C) in the twin FVII-deficient patients; it also details specific biochemical analyses of the -60 T→C mutation. The results revealed FVII:C levels of less than 1% in infancy that remain severely decreased through puberty and into adulthood. In-vitro analyses utilizing hepatocyte nuclear factor 4α (HNF4α) co-transfection and a chromatin immunoprecipitation assay indicate that the -60 T→C mutation severely diminishes functional interaction between the FVII promoter and transcription factor HNF4α. The importance of interaction between the FVII gene and HNF4α in normal FVII expression provides an in-vivo illustration of the regulated expression of an autosomal gene encoding a coagulation protein. The constancy of FVII:C and peripubertal patient symptomatology reported here illustrates androgen-independent expression in contrast to expression with an analogous mutation in the promoter region of the gene encoding coagulation FIX.