Functional domains of SP110 that modulate its transcriptional regulatory function and cellular translocation.

BACKGROUND: SP110, an interferon-induced nuclear protein, belongs to the SP100/SP140 protein family. Very recently, we showed that SP110b, an SP110 isoform, controls host innate immunity to Mycobacterium tuberculosis infection by regulating nuclear factor-κB (NF-κB) activity. However, it remains unclear how the structure of SP110 relates to its cellular functions. In this study, we provide experimental data illustrating the protein domains that are responsible for its functions. METHODS: We examined the effects of SP110 isoforms and a series of deletion mutants of SP110 on transcriptional regulation by luciferase reporter assays. We also employed confocal microscopy to determine the cellular distributions of enhanced green fluorescent protein-tagged SP110 isoforms and SP110 mutants. In addition, we performed immunoprecipitation and Western blotting analyses to identify the regions of SP110 that are responsible for protein interactions. RESULTS: Using reporter assays, we first demonstrated that SP110 isoforms have different regulatory effects on NF-κB-mediated transcription, supporting the notion that SP110 isoforms may have distinct cellular functions. Analysis of deletion mutants of SP110 showed that the interaction of the N-terminal fragment (amino acids 1-276) of SP110 with p50, a subunit of NF-κB, in the cytoplasm plays a crucial role in the down-regulation of the p50-driven tumor necrosis factor-α (TNFα) promoter activity in the nucleus, while the middle and C-terminal regions of SP110 localize it to various cellular compartments. Surprisingly, a nucleolar localization signal (NoLS) that contains one monopartite nuclear localization signal (NLS) and one bipartite NLS was identified in the middle region of SP110. The identification of a cryptic NoLS in the SP110 suggests that although this protein forms nuclear speckles in the nucleoplasm, it may be directed into the nucleolus to carry out distinct functions under certain cellular conditions. CONCLUSIONS: The findings from this study elucidating the multidomain structure of the SP110 not only identify functional domains of SP110 that are required for transcriptional regulation, cellular translocation, and protein interactions but also implicate that SP110 has additional functions through its unexpected activity in the nucleolus.

SP110b Controls Host Immunity and Susceptibility to Tuberculosis.

RATIONALE: How host genetic factors affect Mycobacterium tuberculosis (Mtb) infection outcomes remains largely unknown. SP110b, an IFN-induced nuclear protein, is the nearest human homologue to the mouse Ipr1 protein that has been shown to control host innate immunity to Mtb infection. However, the function(s) of SP110b remains unclear. OBJECTIVES: To elucidate the role of SP110b in controlling host immunity and susceptibility to tuberculosis (TB), as well as to identify the fundamental immunological and molecular mechanisms affected by SP110b. METHODS: Using cell-based approaches and mouse models of Mtb infection, we characterized the function(s) of SP110b/Ipr1. We also performed genetic characterization of patients with TB to investigate the role of SP110 in controlling host susceptibility to TB. MEASUREMENTS AND MAIN RESULTS: SP110b modulates nuclear factor-κB (NF-κB) activity, resulting in downregulation of tumor necrosis factor-α (TNF-α) production and concomitant upregulation of NF-κB-induced antiapoptotic gene expression, thereby suppressing IFN-γ-mediated monocyte and/or macrophage cell death. After Mtb infection, TNF-α is also downregulated in Ipr1-expressing mice that have alleviated cell death, less severe necrotic lung lesions, more efficient Mtb growth control in the lungs, and longer survival. Moreover, genetic studies in patients suggest that SP110 plays a key role in modulating TB susceptibility in concert with NFκB1 and TNFα genes. CONCLUSIONS: These results indicate that SP110b plays a crucial role in shaping the inflammatory milieu that supports host protection during infection by fine-tuning NF-κB activity, suggesting that SP110b may serve as a potential target for host-directed therapy aimed at manipulating host immunity against TB.

Isoniazid Concentration and NAT2 Genotype Predict Risk of Systemic Drug Reactions during 3HP for LTBI.

Weekly rifapentine and isoniazid therapy (known as 3HP) for latent tuberculosis infection (LTBI) is increasingly used, but systemic drug reactions (SDR) remain a major concern. Methods: We prospectively recruited two LTBI cohorts who received the 3HP regimen. In the single-nucleotide polymorphism (SNP) cohort, we collected clinical information of SDRs and examined the NAT2, CYP2E1, and AADAC SNPs. In the pharmacokinetic (PK) cohort, we measured plasma drug and metabolite levels at 6 and 24 h after 3HP administration. The generalised estimating equation model was used to identify the factors associated with SDRs. Candidate SNPs predicting SDRs were validated in the PK cohort. A total of 177 participants were recruited into the SNP cohort and 129 into the PK cohort, with 14 (8%) and 13 (10%) in these two cohorts developing SDRs, respectively. In the SNP cohort, NAT2 rs1041983 (TT vs. CC+CT, odds ratio [OR] [95% CI]: 7.00 [2.03-24.1]) and CYP2E1 rs2070673 (AA vs. TT+TA, OR [95% CI]: 3.50 [1.02-12.0]) were associated with SDR development. In the PK cohort, isoniazid level 24 h after 3HP administration (OR [95% CI]: 1.61 [1.15-2.25]) was associated with SDRs. Additionally, the association between the NAT2 SNP and SDRs was validated in the PK cohort (rs1041983 TT vs. CC+CT, OR [95% CI]: 4.43 [1.30-15.1]). Conclusions: Isoniazid played a role in the development of 3HP-related SDRs. This could provide insight for further design of a more optimal regimen for latent TB infection.

SP110 Polymorphisms Are Genetic Markers for Vulnerability to Latent and Active Tuberculosis Infection in Taiwan.

One-fourth of the human population is estimated to have been exposed to Mycobacterium tuberculosis (Mtb) and carries the infection in its latent form. This latent infection presents a lifelong risk of developing active tuberculosis (TB) disease, and persons with latent TB infection (LTBI) are significant contributors to the pool of active TB cases. Genetic polymorphisms among hosts have been shown to contribute to the outcome of Mtb infection. The SP110 gene, which encodes an interferon-induced nuclear protein, has been shown to control host innate immunity to Mtb infection. In this study, we provide experimental data demonstrating the ability of the gene to control genetic susceptibility to latent and active TB infection. Genetic variants of the SP110 gene were investigated in the Taiwanese population (including 301 pulmonary TB patients, 68 LTBI individuals, and 278 healthy household contacts of the TB patients), and their association with susceptibility to latent and active TB infection was examined by performing an association analysis in a case-control study. We identified several SNPs (rs7580900, rs7580912, rs9061, rs11556887, and rs2241525) in the SP110 gene that are associated with susceptibility to LTBI and/or TB disease. Our studies further showed that the same SNPs may have opposite effects on the control of susceptibility to LTBI versus TB. In addition, our analyses demonstrated that the SP110 rs9061 SNP was associated with tumor necrosis factor-α (TNFα) levels in plasma in LTBI subjects. The results suggest that the polymorphisms within SP110 have a role in controlling genetic susceptibility to latent and active TB infection in humans. To the best of our knowledge, this is the first report showing that the SP110 variants are associated with susceptibility to LTBI. Our study also demonstrated that the identified SP110 SNPs displayed the potential to predict the risk of LTBI and subsequent TB progression in Taiwan.

Rapid Sputum Multiplex Detection of the M. tuberculosis Complex (MTBC) and Resistance Mutations for Eight Antibiotics by Nucleotide MALDI-TOF MS.

The increasing incidence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis (MTB) adds further urgency for rapid and multiplex molecular testing to identify the MTB complex and drug susceptibility directly from sputum for disease control. A nucleotide matrix-assisted-laser-desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based assay was developed to identify MTB (MTBID panel) and 45 chromosomal mutations for resistance to eight antibiotics (MTBDR panel). We conducted a 300 case trial from outpatients to evaluate this platform. An MTBID panel specifically identified MTB with as few as 10 chromosome DNA copies. The panel was 100% consistent with an acid-fast stain and culture for MTB, nontuberculous mycobacteria, and non-mycobacteria bacteria. The MTBDR panel was validated using 20 known MDR-MTB isolates. In a 64-case double-blind clinical isolates test, the sensitivity and specificity were 83% and 100%, respectively. In a 300-case raw sputum trial, the MTB identification sensitivity in smear-negative cases using MALDI-TOF MS was better than the COBAS assay (61.9% vs. 46.6%). Importantly, the failure rate of MALDI-TOF MS was better than COBAS (11.3% vs. 26.3%). To the best of our knowledge, the test described herein is the only multiplex test that predicts resistance for up to eight antibiotics with both sensitivity and flexibility.

HOXA5 inhibits metastasis via regulating cytoskeletal remodelling and associates with prolonged survival in non-small-cell lung carcinoma.

Homeobox genes comprise a family of regulatory genes that contain a common homeobox domain and act as transcription factors. Recent studies indicate that homeobox A5 (HOXA5) may serve as a tumour suppressor gene in breast cancers. However, the precise role and the underlying mechanism of HOXA5 in lung cancer remain unclear. Oligonucleotide microarrays and an invasion/metastasis lung adenocarcinoma cell line model were used to determine the correlation between HOXA5 expression and cancer cell invasion ability. We found that ectopic expression of HOXA5 in highly invasive cancer cells suppressed cell migration, invasion, and filopodia formation in vitro and inhibited metastatic potential in vivo. Knockdown of HOXA5 promoted the invasiveness of lung cancer cells. In addition, HOXA5 expression was associated with better clinical outcome in non-small cell lung cancer patients with wild-type EGFR. Furthermore, genome-wide transcriptomic and pathway analyses were performed to identify the potential molecular mechanisms. Our data showed that HOXA5 may bind to the promoters of the cytoskeleton-related genes and downregulate their mRNA and protein expression levels. Our studies provide new insights into how HOXA5 may contribute to the suppression of metastasis in lung cancer via cytoskeleton remodelling regulation. Therefore, targeted induction of HOXA5 may represent a promising approach for non-small-cell lung cancer therapy.