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BackgroundA nonsputum blood test capable of predicting progression of healthy individuals to active tuberculosis (TB) before clinical symptoms manifest would allow targeted treatment to curb transmission. We aimed to develop a proteomic biomarker of risk of TB progression for ultimate translation into a point-of-care diagnostic.Methods and findingsProteomic TB risk signatures were discovered in a longitudinal cohort of 6,363 Mycobacterium tuberculosis-infected, HIV-negative South African adolescents aged 12-18 years (68% female) who participated in the Adolescent Cohort Study (ACS) between July 6, 2005 and April 23, 2007, through either active (every 6 months) or passive follow-up over 2 years. Forty-six individuals developed microbiologically confirmed TB disease within 2 years of follow-up and were selected as progressors; 106 nonprogressors, who remained healthy, were matched to progressors. Over 3,000 human proteins were quantified in plasma with a highly multiplexed proteomic assay (SOMAscan). Three hundred sixty-one proteins of differential abundance between progressors and nonprogressors were identified. A 5-protein signature, TB Risk Model 5 (TRM5), was discovered in the ACS training set and verified by blind prediction in the ACS test set. Poor performance on samples 13-24 months before TB diagnosis motivated discovery of a second 3-protein signature, 3-protein pair-ratio (3PR) developed using an orthogonal strategy on the full ACS subcohort. Prognostic performance of both signatures was validated in an independent cohort of 1,948 HIV-negative household TB contacts from The Gambia (aged 15-60 years, 66% female), longitudinally followed up for 2 years between March 5, 2007 and October 21, 2010, sampled at baseline, month 6, and month 18. Amongst these contacts, 34 individuals progressed to microbiologically confirmed TB disease and were included as progressors, and 115 nonprogressors were included as controls. Prognostic performance of the TRM5 signature in the ACS training set was excellent within 6 months of TB diagnosis (area under the receiver operating characteristic curve [AUC] 0.96 [95% confidence interval, 0.93-0.99]) and 6-12 months (AUC 0.76 [0.65-0.87]) before TB diagnosis. TRM5 validated with an AUC of 0.66 (0.56-0.75) within 1 year of TB diagnosis in the Gambian validation cohort. The 3PR signature yielded an AUC of 0.89 (0.84-0.95) within 6 months of TB diagnosis and 0.72 (0.64-0.81) 7-12 months before TB diagnosis in the entire South African discovery cohort and validated with an AUC of 0.65 (0.55-0.75) within 1 year of TB diagnosis in the Gambian validation cohort. Signature validation may have been limited by a systematic shift in signal magnitudes generated by differences between the validation assay when compared to the discovery assay. Further validation, especially in cohorts from non-African countries, is necessary to determine how generalizable signature performance is.ConclusionsBoth proteomic TB risk signatures predicted progression to incident TB within a year of diagnosis. To our knowledge, these are the first validated prognostic proteomic signatures. Neither meet the minimum criteria as defined in the WHO Target Product Profile for a progression test. More work is required to develop such a test for practical identification of individuals for investigation of incipient, subclinical, or active TB disease for appropriate treatment and care.

Our understanding of mechanisms underlying progression from Mycobacterium tuberculosis infection to pulmonary tuberculosis disease in humans remains limited. To define such mechanisms, we followed M. tuberculosis-infected adolescents longitudinally. Blood samples from forty-four adolescents who ultimately developed tuberculosis disease (“progressors”) were compared with those from 106 matched controls, who remained healthy during two years of follow up. We performed longitudinal whole blood transcriptomic analyses by RNA sequencing and plasma proteome analyses using multiplexed slow off-rate modified DNA aptamers. Tuberculosis progression was associated with sequential modulation of immunological processes. Type I/II interferon signalling and complement cascade were elevated 18 months before tuberculosis disease diagnosis, while changes in myeloid inflammation, lymphoid, monocyte and neutrophil gene modules occurred more proximally to tuberculosis disease. Analysis of gene expression in purified T cells also revealed early suppression of Th17 responses in progressors, relative to M. tuberculosis-infected controls. This was confirmed in an independent adult cohort who received BCG re-vaccination; transcript expression of interferon response genes in blood prior to BCG administration was associated with suppression of IL-17 expression by BCG-specific CD4 T cells 3 weeks post-vaccination. Our findings provide a timeline to the different immunological stages of disease progression which comprise sequential inflammatory dynamics and immune alterations that precede disease manifestations and diagnosis of tuberculosis disease. These findings have important implications for developing diagnostics, vaccination and host-directed therapies for tuberculosis.Trial registrationClincialtrials.gov, NCT01119521.

Clostridium difficile causes antibiotic-associated diarrhea and is a major public health concern. Current therapies disrupt the protective intestinal flora, do not reliably prevent recurrent infections, and will be decreasingly effective should less susceptible strains emerge. CRS3123 is an oral agent that inhibits bacterial methionyl-tRNA synthetase and has potent activity against C. difficile and aerobic Gram-positive bacteria but little activity against Gram-negative bacteria, including anaerobes. This first-in-human, double-blind, placebo-controlled, dose escalation study evaluated the safety and systemic exposure of CRS3123 after a single oral dose in healthy adults. Five cohorts of eight subjects each received CRS3123 or placebo in a 3:1 ratio. Doses for the respective active arms were 100 mg, 200 mg, 400 mg, 800 mg, and 1,200 mg. Blood and urine were collected for pharmacokinetic analysis. CRS3123 concentrations were measured with validated LC-MS/MS techniques. There were no serious adverse events or immediate allergic reactions during administration of CRS3123. In the CRS3123-treated groups, the most frequent adverse events were decreased hemoglobin, headache, and abnormal urine analysis; all adverse events in the active-treatment groups were mild to moderate, and their frequency did not increase with dose. Although CRS3123 systemic exposure increased at higher doses, the increase was less than dose proportional. The absorbed drug was glucuronidated at reactive amino groups on the molecule, which precluded accurate pharmacokinetic analysis of the parent drug. Overall, CRS3123 was well tolerated over this wide range of doses. This safety profile supports further investigation of CRS3123 as a treatment for C. difficile infections. (This study has been registered at ClinicalTrials.gov under identifier NCT01551004.).

BackgroundNew drug regimens of greater efficacy and shorter duration are needed for tuberculosis (TB) treatment. The identification of accurate, quantitative, non-culture based markers of treatment response would improve the efficiency of Phase 2 TB drug testing.MethodsIn an unbiased biomarker discovery approach, we applied a highly multiplexed, aptamer-based, proteomic technology to analyze serum samples collected at baseline and after 8 weeks of treatment from 39 patients with pulmonary TB from Kampala, Uganda enrolled in a Centers for Disease Control and Prevention (CDC) TB Trials Consortium Phase 2B treatment trial.ResultsWe identified protein expression differences associated with 8-week culture status, including Coagulation Factor V, SAA, XPNPEP1, PSME1, IL-11 Rα, HSP70, Galectin-8, α2-Antiplasmin, ECM1, YES, IGFBP-1, CATZ, BGN, LYNB, and IL-7. Markers noted to have differential changes between responders and slow-responders included nectin-like protein 2, EphA1 (Ephrin type-A receptor 1), gp130, CNDP1, TGF-b RIII, MRC2, ADAM9, and CDON. A logistic regression model combining markers associated with 8-week culture status revealed an ROC curve with AUC = 0.96, sensitivity = 0.95 and specificity = 0.90. Additional markers showed differential changes between responders and slow-responders (nectin-like protein), or correlated with time-to-culture-conversion (KLRK1).ConclusionsSerum proteins involved in the coagulation cascade, neutrophil activity, immunity, inflammation, and tissue remodeling were found to be associated with TB treatment response. A quantitative, non-culture based, five-marker signature predictive of 8-week culture status was identified in this pilot study.

In an unbiased approach to biomarker discovery, we applied a highly multiplexed proteomic technology (SOMAscan, SomaLogic, Inc, Boulder, CO) to understand changes in proteins from paired serum samples at enrollment and after 8 weeks of TB treatment from 39 patients with pulmonary TB from Kampala, Uganda enrolled in the Center for Disease Control and Preventions Tuberculosis Trials Consortium (TBTC) Study 29. This work represents the first large-scale proteomic analysis employing modified DNA aptamers in a study of active tuberculosis (TB). We identified multiple proteins that exhibit significant expression differences during the intensive phase of TB therapy. There was enrichment for proteins in conserved networks of biological processes and function including antimicrobial defense, tissue healing and remodeling, acute phase response, pattern recognition, protease/anti-proteases, complement and coagulation cascade, apoptosis, immunity and inflammation pathways. Members of cytokine pathways such as interferon-gamma, while present, were not as highly represented as might have been predicted. The top proteins that changed between baseline and 8 weeks of therapy were TSP4, TIMP-2, SEPR, MRC-2, Antithrombin III, SAA, CRP, NPS-PLA2, LEAP-1, and LBP. The novel proteins elucidated in this work may provide new insights for understanding TB disease, its treatment and subsequent healing processes that occur in response to effective therapy.

The impact of arsenite [As(III)] on several levels of cellular metabolism and gene regulation was examined in Pseudomonas aeruginosa. P. aeruginosa isogenic mutants devoid of antioxidant enzymes or defective in various metabolic pathways, DNA repair systems, metal storage proteins, global regulators, or quorum sensing circuitry were examined for their sensitivity to As(III). Mutants lacking the As(III) translocator (ArsB), superoxide dismutase (SOD), catabolite repression control protein (Crc), or glutathione reductase (Gor) were more sensitive to As(III) than wild-type bacteria. The MICs of As(III) under aerobic conditions were 0.2, 0.3, 0.8, and 1.9 mM for arsB, sodA sodB, ctc, and gor mutants, respectively, and were 1.5- to 13-fold less than the MIC for the wild-type strain. A two-dimensional gel/matrix-assisted laser desorption ionization--time of flight analysis of As(III)-treated wild-type bacteria showed significantly (>40-fold) increased levels of a heat shock protein (IbpA) and a putative allo-threonine aldolase (GlyI). Smaller increases (up to 3.1-fold) in expression were observed for acetyl-coenzyme A acetyltransferase (AtoB), a probable aldehyde dehydrogenase (KauB), ribosomal protein L25 (RpIY), and the probable DNA-binding stress protein (PA0962). In contrast, decreased levels of a heme oxygenase (HemO/PigA) were found upon As(III) treatment. Isogenic mutants were successfully constructed for six of the eight genes encoding the aforementioned proteins. When treated with sublethal concentrations of As(III), each mutant revealed a marginal to significant lag period prior to resumption of apparent normal growth compared to that observed in the wild-type strain. Our results suggest that As(III) exposure results in an oxidative stress-like response in P. aeruginosa, although activities of classic oxidative stress enzymes are not increased. Instead, relief from As(III)-based oxidative stress is accomplished from the collective activities of ArsB, glutathione reductase, and the global regulator Crc. SOD appears to be involved, but its function may be in the protection of superoxide-sensitive sulfhydryl groups.

Under conditions of iron limitation, Pseudomonas aeruginosa secretes a high-affinity siderophore pyoverdine to scavenge Fe(III) in the extracellular environment and shuttle it into the cell. Uptake of the pyoverdine--Fe(III) complex is mediated by a specific outer-membrane receptor protein, FpvA (ferripyoverdine receptor). Three P. aeruginosa siderovars can be distinguished, each producing a different pyoverdine (type I-III) and a cognate FpvA receptor. Growth of an fpvA mutant of P. aeruginosa PAO1 (type I) under iron-limiting conditions can still be stimulated by its cognate pyoverdine, suggesting the presence of an alternative uptake route for type I ferripyoverdine. In silico analysis of the PAO1 genome revealed that the product of gene PA4168 has a high similarity with FpvA. Inactivation of PA4168 (termed fpvB) in an fpvA mutant totally abolished the capacity to utilize type I pyoverdine. The expression of fpvB is induced by iron limitation in Casamino acids (CAA) and in Mq-glucose medium, but, unlike fpvA, not in a complex deferrated medium containing glycerol as carbon source. The fpvB gene was also detected in other P. aeruginosa isolates, including strains producing type II and type III pyoverdines. Inactivation of the fpvB homologues in these strains impaired their capacity to utilize type I ferripyoverdine as a source of iron. Accordingly, introduction of fpvB in trans restored the capacity to utilize type I ferripyoverdine.

Results demonstrate that organic hydroperoxide resistance (ohr) gene encoded product is involved in the decomposition (resistance) of organic hydroperoxides. Data reveal that upon exposure to organic hydroperoxides, a 15-fold increase in the transcriptional activity of ohr is observed.

Results demonstrate that Pseudomonas aeruginosa mutants carrying xylE fusion plasmids loose greater than 95% catechol-2,3-dioxygenase on exposure to hydrogen peroxide. Data indicate that enzyme activity loss is due to the oxidation of the active-site ferrous-ion.

Results show that oxyR mutants of Pseudomonas aeruginosa are sensitive to hydrogen peroxide, can not survive aerobically, and tolerate aminoglycosides. However, the mutants survive in presence of either catalase KatA or under anaerobic conditions.

Results show the periplasmic ankyrin-like protein AnkB exhibit four 33-amino-acid ankyrin repeats in a 183 amino acids stretch of the protein. Data further indicate that AnkB together with a hydrogen peroxide-inducible KatB protein forms a protective lattice work for hydrogen peroxide detoxification.

Results demonstrate that expression of oxidative stress defence genes katB-ankB, ahpB, and ahpC-ahpF is regulated by oxyR gene product and is located upstream of DNA repair enzyme coding gene recG. Data indicate that the oxyR-recG locus governs oxidative stress and DNA repair function in Pseudomonas aeruginosa.

Results demonstrate identification of haem iron uptake gene cluster consisting of Pseudomonas haem uptake (phu) locus and haem-acquisition system (has) locus in Pseudomonas aeruginosa. Data indicate that phu and has are controlled by Fur protein and individually encode two distinct systems for the acquisition of haem and haemoglobin.

(B-F) Frequencies at 21 days after PBMC engraftment in spleens of human huCD45+ (B), CD3+ T cells (C), CD19+ B lineage cells (D), CD19+CD138- B cells (E), and CD19+CD138+ plasma cells (F). IL-10 induces IgG4 production in NOD-scid Il2r null mice humanized by engraftment of peripheral blood mononuclear cells To differentiate between B cells and plasma cells, we gated B cells as CD19+CD138- (Figure 1E,G) and plasma cells as CD138+ (Figure 1E,H). [Extracted from the article]

A unique regulator of the exotoxin A gene (toxA) in Pseudomonas aeruginosa, PtxR, has been previously identified. Because toxA expression and siderophore production in this bacterium are coordinately regulated by the ferric uptake regulator (Fur) and the Fur-regulated alternative sigma factor PvdS, regulation of ptxR itself in the context of these regulators was studied. Findings showed that the iron-regulated expression of ptxR under microaerobic conditions from the P2 promoter of ptxR is controlled directly by Fur through the iron-regulated expression of pvdS.

The ferric uptake regulatory protein (Fur)-associated gene (fagA) of Pseudomonas aeruginosa was cloned and analyzed to determine the role of Fur in the regulation of alginate biosynthesis. Sequence analysis of the cloned Pseudomonas aeruginosa fagA gene indicated the presence of two iron boxes or Fur binding sites in the upstream region of the gene. Furthermore, the gene was directly regulated and indirectly regulated by the alternative sigma factor AlgT(U) which was mediated by iron homeostasis.

The A4 and C6 ferric uptake regulator (Fur) mutants of Pseudomonas aeruginosa are incapable of proper siderophore-mediated iron uptake and aerobic growth. This is due to a decrease in total superoxide dismutase (SOD) and catalase activity. The mutants have a higher manganese-SOD (Mn-SOD) activity, contain altered Fur proteins, and constitutively produce the siderophores pyochelin and pyoverdin. Mutants of strain PAO1, with normal pyochelin and defective pyoverdin biosynthesis, have increased Mn-SOD activity. Sensitivity to redox cycling agent paraquat and hydrogen peroxide is greater in the mutants.

A T7 expression system is used to overexpress the clone Pseudomonas aeruginosa fur gene on P. aeruginosa while ion-exchange chromatography is employed in conjunction with metal affinity chromatography to purify the gene's fur protein. Results suggest that the iron regulation of exotoxin production in P. aeruginosa involves additional factors that may be under the control of PA-fur.

The formation of the extracellular virulence factor rhamnolipid by the human pathogen, Pseudomonas aeruginosa, is controlled by the regulatory protein RhlR, rhamnosyltransferase RhlAB and the gene rhlI. The structure of the protein RhlI is similar to that of the autoinducer synthetase LuxI. Mutants whose rhlI has been disrupted are unable to produce rhamnolipids and do not show RhlAB activity. Multiple copies of the lasR gene in these mutants causes the production of rhamnolipids showing that the LasR-LasI elastase regulatory system can compensate for the RhlR-RhlI rhamnolipid regulatory system.

Sequential analysis of a 16-kDa extracellular protein, produced by Pseudomonas aeruginosa PG201 in n-hexadecane medium, reveals the similarity of this protein with a protein-like activator for n-alkane oxidation (PA). Cloning and sequential analysis of the PA-encoding (pra) gene reveals the presence of a protein-coding region of 162 amino acids. Insertional mutagenesis leads to inactivation of pra gene and mutant strains lack the extracellular PA protein. Escherichia coli expresses the pra gene, and metal chelate affinity chromatography helps purity the recombinant protein present in the extracellular growth medium.

Rhamnolipid biosynthesis in Pseudomonas aeruginosa is activated by rhlR regulatory gene. The 2-kb long gene encodes a 28-kDa protein (RhlR) that functions as a transcriptional activator. The gene has a putative target promoter in the upstream. Rhamnolipid-deficient mutations are produced when wild-type rhlR genes are disrupted indicating the pleiotropic regulatory action of the RhlR protein.