Jiazhen Chen

Mutations in panD encoding aspartate decarboxylase are associated with pyrazinamide resistance in Mycobacterium tuberculosis

Pyrazinamide (PZA) is a frontline anti-tuberculosis drug that plays a crucial role in the treatment of both drug susceptible and multidrug-resistant tuberculosis (MDR-TB). Resistance to PZA is most commonly associated with mutations in the pncA gene encoding nicotinamidase/pyrazinamidase which converts the prodrug PZA to the active form pyrazinoic acid (POA). RpsA (ribosomal protein S1) involved in trans-translation was recently shown to be a target of PZA and mutations in RpsA are found in some PZA-resistant TB strains. However, some other PZA-resistant strains lack mutations in either pncA or rpsA. To identify potential new mechanisms of PZA resistance, we isolated 174 in vitro mutants of M. tuberculosis H37Rv resistant to PZA to search for resistant isolates that do not have pncA or rpsA mutations. DNA sequencing revealed that 169 of the 174 (97.1%) PZA-resistant mutants had pncA mutations but 5 mutants lacked pncA or rpsA mutations. Whole genome sequencing analyses revealed that the 5 PZA-resistant mutants had different mutations all occurring in the same gene panD encoding aspartate decarboxylase, which is involved in synthesis of β-alanine that is a precursor for pantothenate and co-enzyme A biosynthesis. panD mutations were identified in naturally PZA-resistant Mycobacterium canetti strain and a PZA-resistant MDR-TB clinical isolate. Future studies are needed to address the role of panD mutations in PZA resistance and confirm PanD as a new target of PZA.

Analysis of microRNA expression profiling identifies miR-155 and miR-155* as potential diagnostic markers for active tuberculosis: a preliminary study

To explore biologic behaviors and disease relevance of microRNAs (miRNAs) in the development of active tuberculosis (ATB), we investigated the expression profile of Mycobacterium tuberculosis (MTB) purified protein derivative (PPD)-induced miRNAs to determine the specific miRNAs involved in the pathogenesis of ATB. The expression profile of miRNA under PPD challenge was first measured using microarray analysis in peripheral blood mononuclear cells isolated from ATB patients and healthy controls (HC). The remarkably reactive miRNAs were then validated in a larger cohort by quantitative real-time polymerase chain reaction (qRT-PCR). The receiver operating characteristic (ROC) curve was plotted to evaluate the diagnostic value of the determined PPD-responsive miRNAs. The potential targets for those miRNAs were also predicted by computational programs. Fourteen of 866 human miRNAs exhibited at least 1.8-fold difference in the ratio of expression level before and after stimulation with PPD between the ATB and HC groups. The qRT-PCR study validated the findings from microarray-based screening, in which miR-155 exhibited a fold change of 1.4 in the HC group and 3.7 in the ATB group upon PPD stimulation (p < 0.0001); miR-155* exhibited a fold change of 1.9 in the HC and 4.6 in the ATB group (p < 0.005). In ROC plots, the area under the curve was 0.8972 for miR-155 and 0.7945 for miR-155*. The background expression of these 2 microRNAs exhibited no differences between the ATB and HC groups. miR-155 and miR-155* exhibited characteristic expression by TB-specific antigen, suggesting that they can be potential diagnostic markers under the challenge of specific MTB antigens.

Novel Biomarkers Distinguishing Active Tuberculosis from Latent Infection Identified by Gene Expression Profile of Peripheral Blood Mononuclear Cells

Background Humans infected with Mycobacterium tuberculosis (MTB) can delete the pathogen or otherwise become latent infection or active disease. However, the factors influencing the pathogen clearance and disease progression from latent infection are poorly understood. This study attempted to use a genome-wide transcriptome approach to identify immune factors associated with MTB infection and novel biomarkers that can distinguish active disease from latent infection. Methodology/Principal Findings Using microarray analysis, we comprehensively determined the transcriptional difference in purified protein derivative (PPD) stimulated peripheral blood mononuclear cells (PBMCs) in 12 individuals divided into three groups: TB patients (TB), latent TB infection individuals (LTBI) and healthy controls (HC) (n = 4 per group). A transcriptional profiling of 506 differentially expressed genes could correctly group study individuals into three clusters. Moreover, 55- and 229-transcript signatures for tuberculosis infection (TB&LTBI) and active disease (TB) were identified, respectively. The validation study by quantitative real-time PCR (qPCR) performed in 83 individuals confirmed the expression patterns of 81% of the microarray identified genes. Decision tree analysis indicated that three genes of CXCL10, ATP10A and TLR6 could differentiate TB from LTBI subjects. Additional validation was performed to assess the diagnostic ability of the three biomarkers within 36 subjects, which yielded a sensitivity of 71% and specificity of 89%. Conclusions/Significance The transcription profiles of PBMCs induced by PPD identified distinctive gene expression patterns associated with different infectious status and provided new insights into human immune responses to MTB. Furthermore, this study indicated that a combination of CXCL10, ATP10A and TLR6 could be used as novel biomarkers for the discrimination of TB from LTBI.

Aspartate decarboxylase (PanD) as a new target of pyrazinamide in Mycobacterium tuberculosis.

Pyrazinamide (PZA) is a frontline anti-tuberculosis drug that plays a crucial role in the treatment of both drug-susceptible and multidrug-resistant tuberculosis (MDR-TB). PZA is a prodrug that is converted to its active form, pyrazinoic acid (POA), by a nicotinamidase/pyrazinamidase encoded by the pncA gene, the mutation of which is the major cause of PZA resistance. Although RpsA (ribosomal protein S1, involved in trans-translation) has recently been shown to be a target of POA/PZA, whole-genome sequencing has identified mutations in the panD gene encoding aspartate decarboxylase in PZA-resistant strains lacking pncA and rpsA mutations. To gain more insight into a possible new target of PZA, we isolated 30 POA-resistant mutants lacking mutations in pncA and rpsA from M. tuberculosis in vitro, and whole-genome sequencing of 3 mutants identified various mutations in the panD gene. Additionally, sequencing analysis revealed that the remaining 27 POA-resistant mutants all harbored panD mutations affecting the C-terminus of the PanD protein, with PanD M117I being the most frequent mutation (24/30, 80%). Conditional overexpression of panD from M. tuberculosis, M. smegmatis or E. coli, or of M. tuberculosis mutant PanD M117I, all conferred resistance to POA and PZA in M. tuberculosis. β-alanine and pantothenate, which are downstream products of PanD, were found to antagonize the antituberculosis activity of POA. In addition, the activity of the M. tuberculosis PanD enzyme was inhibited by POA at therapeutically relevant concentrations in a concentration-dependent manner but was not inhibited by the prodrug PZA or the control compound nicotinamide. These findings suggest that PanD represents a new target of PZA/POA. These results have implications for a better understanding of this peculiar persister drug and for the design of new drugs targeting M. tuberculosis persisters for improved treatment.

Evaluation of the Diagnostic Potential of IP-10 and IL-2 as Biomarkers for the Diagnosis of Active and Latent Tuberculosis in a BCG-Vaccinated Population

Background The Mycobacterium tuberculosis (Mtb)-specific T-cell interferon gamma release assays (IGRAs) are useful in detecting Mtb infection but perform poorly at distinguishing active tuberculosis disease (ATB) and latent tuberculosis infection (LTBI). This study is aimed at evaluating additional cytokines as biomarkers besides interferon-gamma (IFN-γ) to improve the identification of ATB and LTBI. Methodology/Principal Findings Sixty-six patients with ATB, 73 household contacts (HHC) of ATB patients and 76 healthy controls (HC) were recruited to undergo QuantiFERON TB GOLD in-tube assay (QFT) and the enzyme-linked immunosorbent assay (ELISA) where the release of IFN-γ, IFN-γ inducible protein 10 (IP-10), Interleukin 2 (IL-2) and Tumor Necrosis Factor-α (TNF-α) was determined in the whole blood with or without antigen-stimulation. The positive rates of the QFT, IP-10 and IL-2 tests were 86.4%, 89.4% and 86.4% for the ATB group with no difference between them (p>0.05). However, QFT in combination with IP-10 and IL-2 significantly increased the detection rate to 95.5% in the ATB group (p = 0.03) and the indeterminate rate of all samples decreased from 2.3% (5/215) to 0.4% (1/215). The un-stimulated level of IP-10 was significantly higher in the HHC than the ATB and HC groups. The IP-10 responses were strongly associated with extended Mtb exposure time and the degree of smear-positivity of the index cases. The IL-2/IFN-γ ratio in the antigen-stimulated plasma could discriminate LTBI from ATB with a sensitivity of 77.2% and a specificity of 87.2%. Conclusion The increased Mtb-specific antigen-stimulated expression of IP-10 and IL-2 may be useful for detecting both ATB and LTBI. Combining the QFT with IP-10 and IL-2 could increase the detection accuracy of active TB over the QFT alone.

Evaluation of Gamma Interferon Release Assays Using Mycobacterium tuberculosis Antigens for Diagnosis of Latent and Active Tuberculosis in Mycobacterium bovis BCG-Vaccinated Populations

ABSTRACT T-cell-based gamma interferon (IFN-γ) release assays (IGRAs) using Mycobacterium tuberculosis-specific antigens have shown higher sensitivity and specificity than the routine tuberculin skin test (TST). However, the effects of Mycobacterium bovis BCG vaccination and anti-tuberculosis (TB) treatment on dynamic T-cell responses to M. tuberculosis-specific antigens in active TB cases have rarely been investigated in regions where TB is endemic. Eighty-nine patients with active pulmonary TB (ATB) and 57 healthy controls (HC) from China were recruited and tested by sputum smear and culture, TSTs, and IGRAs with M. tuberculosis-specific antigens ESAT-6 and CFP-10 (T-SPOT.TB) as well as purified protein derivative (PPD) stimulation. All 146 participants were screened by the T-SPOT.TB assay at recruitment. T-SPOT.TB-positive rates in ATB and HC groups were 87.6% (78/89) and 21.1% (12/57), respectively. Of 38 ATB patients who were both TST and T-SPOT.TB tested, the positive rates were 73.7% (28/38) and 94.7% (36/38), respectively (P = 0.0215), and those in the HC group were 62.3% (33/53) and 18.9% (10/53), respectively (P < 0.0001). The T-SPOT.TB-positive rates declined during TB treatment and were 94.4% (51/54), 86.4% (19/22), and 61.5% (8/13) for ATB patients receiving 0- to 1-month, 1- to 3-month, and 3- to 6-month anti-TB treatment, respectively. The IGRA is a most promising test for both active TB and latent TB infection (LTBI) diagnosis due to the improvement of its specificity and convenience, especially in the Mycobacterium bovis BCG-vaccinated population. Furthermore, the T-SPOT.TB assay using ESAT-6 and CFP-10 in ATB patients during anti-TB treatment could serve as a potential predictor of therapeutic efficacy.

Identification of novel mutations associated with clofazimine resistance in Mycobacterium tuberculosis

OBJECTIVES Although clofazimine has been traditionally used to treat leprosy, there is recent interest in using clofazimine for the treatment of MDR-TB and drug-susceptible TB. However, the mechanisms of resistance to clofazimine are poorly understood. Here, we investigated the molecular basis of clofazimine resistance using resistant mutants isolated in vitro. METHODS We isolated 96 mutants of Mycobacterium tuberculosis resistant to clofazimine and performed WGS and Sanger sequencing to identify possible mutations associated with clofazimine resistance. RESULTS We found that 97% (93/96) of clofazimine-resistant mutants had a mutation in rv0678 encoding a transcription repressor for efflux pump MmpL5. Two mutational hot spots at nucleotide positions 193 and 466 in rv0678 accounted for 43.8% (42/96) and 11.5% (11/96) of the mutations, respectively. The previously reported A202G mutation (S68G) in rv0678 occurred less frequently, in 5 of 96 mutants. The remaining 34 mutations were scattered along the entire rv0678 gene. We discovered two new genes (rv1979c and rv2535c) associated with clofazimine resistance in mutants without rv0678 mutations. CONCLUSIONS Mutations in rv0678 are a major mechanism of clofazimine resistance. Our findings provide useful information for the design of new molecular tests for rapid detection of clofazimine resistance. Further studies are needed to address the role of rv1979c and rv2535c in clofazimine resistance and mechanisms of action.

Sputum microbiota associated with new, recurrent and treatment failure tuberculosis.

Microbiota have recently been shown to be associated with many disease conditions. However, the microbiota associated with tuberculosis (TB) infection, recurrence and treatment outcome have not been systematically characterized. Here, we used high throughput 16S RNA sequencing to analyze the sputum microbiota associated with Mycobacterium tuberculosis infection and also to identify the microorganisms associated with different outcomes of TB treatment. We recruited 25 new TB patients, 30 recurrent TB patients and 20 TB patients with treatment failure, as well as 20 healthy controls. Streptococcus, Gramulicatella and Pseudomonas were more abundant in TB patients while Prevotella, Leptotrichia, Treponema, Catonella and Coprococcus were less abundant in TB patients than in the healthy controls. We found reduced frequency and abundance of some genera such as Bulleidia and Atopobium in recurrent TB patients compared with those in new TB patients. In addition, the ratio of Pseudomonas / Mycobacterium in recurrent TB was higher than that in new TB while the ratio of Treponema / Mycobacterium in recurrent TB was lower than that in new TB, indicating that disruption of these bacteria may be a risk factor of TB recurrence. Furthermore, Pseudomonas was more abundant and more frequently present in treatment failure patients than in cured new patients, and the ratio of Pseudomonas / Mycobacterium in treatment failure was higher than that in new TB. Our data suggest that the presence of certain bacteria and the disorder of lung microbiota may be associated with not only onset of TB but also its recurrence and treatment failure. These findings indicate that lung microbiota may play a role in pathogenesis and treatment outcome of TB and may need to be taken into consideration for improved treatment and control of TB in the future.

Novel recombinant RD2- and RD11-encoded Mycobacterium tuberculosis antigens are potential candidates for diagnosis of tuberculosis infections in BCG-vaccinated individuals.

Proteins encoded by region of deletions (RD) of Mycobacterium tuberculosis are useful in development of vaccines and diagnostic reagents. In the present study, six M. tuberculosis genes from RD2 and RD11, rv1978, nrdf1, mpt64, cfp-21, ppe57 and ppe59, were cloned and overexpressed in Escherichia coli. All six purified recombinant proteins could distinguish tuberculosis (TB) patients and latent TB infected subjects (LTBI), or called subclinical TB infection, from BCG-vaccinated healthy controls by T-cell IFN-gamma releasing ELISPOT. ELISPOT of Rv1978, NrdF1, Mpt64, CFP-21, Ppe57 and Ppe59 achieved sensitivities of 59%, 60%, 82%, 48%, 59% and 47% respectively in the detection of active TB and specificities of 94%, 90%, 76%, 93%, 100% and 93% respectively in BCG-vaccinated healthy controls. Combination of Ppe57 or NrdF1 with early secreted antigen target 6 (ESAT-6) or 10-kDa culture filtrate protein (CFP-10) in the IFN-gamma releasing ESLIPOT assay could increase the sensitivities in detecting active TB, for ESAT-6 from 82.1% to 85.7% or 92.9% (P=0.5 or 0.03, respectively) and for CFP-10 from 67.9% to 78.6% or 83.9%, respectively (both P<0.05). The high sensitivities, specificities and promising antigenic combination of NrdF1 and Ppe57 in detection of TB in BCG-vaccinated controls suggest their potential application in TB diagnosis.

PPE protein (Rv3425) from DNA segment RD11 of Mycobacterium tuberculosis : a novel immunodominant antigen of Mycobacterium tuberculosis induces humoral and cellular immune responses in mice

Subtractive DNA hybridization of pathogenic M. bovis and BCG, and comparative genome‐wide DNA microarray analysis of M. tuberculosis H37Rv and BCG identified several RD, designated as RD1 to RD16, between M. tuberculosis and M. bovis on the one hand and BCG on the other. These regions cover 108 ORF of M. tuberculosis H37Rv, and are deleted from all 13 BCG sub‐strains currently used as anti‐tuberculosis vaccines in different parts of the world. In this study, we evaluated cellular and humoral immune response in C57BL/6 mice immunized with the PPE protein Rv3425, encoded by an ORF found in RD11 of M. tuberculosis. Rv3425 protein induced an increased Th1/Th2 type immune response in mice, characterized by an elevated concentration of IFN‐γ in antigen stimulated splenocyte culture and a strong IgG1 antibody response. These results provide evidence on the immunogenicity of the PPE protein Rv3425 which, together with its reported immunodominant characteristics, imply that it may be a candidate for development of a vaccine for the control of TB.