The Humanization and Maturation of an Anti-PrPc Antibody.

The cellular prion protein (PrPc) is a cell surface glycoprotein that is highly expressed in a variety of cancer tissues in addition to the nervous system, and its elevated expression is correlated to poor prognosis in many cancer patients. Our team previously found that patients with colorectal cancer (CRC) with high-level PrPc expression had significantly poorer survival than those with no or low-level PrPc expression. Mouse antibodies for PrPc inhibited tumor initiation and liver metastasis of PrPc-positive human CRC cells in mouse model experiments. PrPc is a candidate target for CRC therapy. In this study, we newly cloned a mouse anti-PrPc antibody (Clone 6) and humanized it, then affinity-matured this antibody using a CHO cell display with a peptide antigen and full-length PrPc, respectively. We obtained two humanized antibody clones with affinities toward a full-length PrPc of about 10- and 100-fold of that of the original antibody. The two humanized antibodies bound to the PrPc displayed significantly better on the cell surface than Clone 6. Used for Western blotting and immunohistochemistry, the humanized antibody with the highest affinity is superior to the two most frequently used commercial antibodies (8H4 and 3F4). The two new antibodies have the potential to be developed as useful reagents for PrPc detection and even therapeutic antibodies targeting PrPc-positive cancers.

Genetic factors associated with acquired phenotypic drug resistance and its compensatory evolution during tuberculosis treatment.

OBJECTIVES: We elucidated the factors, evolution, and compensation of antimicrobial resistance (AMR) in Mycobacterium tuberculosis (MTB) isolates under dual pressure from the intra-host environment and anti-tuberculosis (anti-TB) drugs. METHODS: This retrospective case-control study included 337 patients with pulmonary tuberculosis from 15 clinics in Tianjin, China, with phenotypic drug susceptibility testing results available for at least two time points between January 1, 2009 and December 31, 2016. Patients in the case group exhibited acquired AMR to isoniazid (INH) or rifampicin (RIF), while those in the control group lacked acquired AMR. The whole-genome sequencing (WGS) was conducted on 149 serial longitudinal MTB isolates from 46 patients who acquired or reversed phenotypic INH/RIF-resistance during treatment. The genetic basis, associated factors, and intra-host evolution of acquired phenotypic INH/RIF-resistance were elucidated using a combined analysis. RESULTS: Anti-TB interruption duration of ≥30 days showed association with acquired phenotypic INH/RIF resistance (aOR = 2·2, 95% CI, 1·0-5·1) and new rpoB mutations (p = 0·024). The MTB evolution was 1·2 (95% CI, 1·02-1·38) single nucleotide polymorphisms per genome per year under dual pressure from the intra-host environment and anti-TB drugs. AMR-associated mutations occurred before phenotypic AMR appearance in cases with acquired phenotypic INH (10 of 16) and RIF (9 of 22) resistances. DISCUSSION: Compensatory evolution may promote the fixation of INH/RIF-resistance mutations and affect phenotypic AMR. The TB treatment should be adjusted based on gene sequencing results, especially in persistent culture positivity during treatment, which highlights the clinical importance of WGS in identifying reinfection and AMR acquisition before phenotypic drug susceptibility testing.

Type-III-A structure of mycobacteria CRISPR-Csm complexes involving atypical crRNAs.

Tuberculosis (TB), a leading cause of mortality globally, is a chronic infectious disease caused by Mycobacterium tuberculosis that primarily infiltrates the lung. The mature crRNAs in M. tuberculosis transcribed from the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) locus exhibit an atypical structure featured with 5' and 3' repeat tags at both ends of the intact crRNA, in contrast to typical Type-III-A crRNAs that possess 5' repeat tags and partial crRNA sequences. However, this structural peculiarity particularly concerning the specific binding characteristics of the 3' repeat end within the mature crRNA within the Csm complex, has not been comprehensively elucidated. Here, our Mycobacteria CRISPR-Csm complexes structure represents the largest Csm complex reported to date. It incorporates an atypical Type-III-A CRISPR RNA (crRNA) (46 nt) with 5' 8-nt and 3' 4-nt repeat sequences in the stoichiometry of Mycobacteria Csm1125364151. The PAM-independent single-stranded RNAs (ssRNAs) are the most suitable substrate for the Csm complex. The 3'-repeat end trimming of mature crRNA was not necessary for its cleavage activity in Type-III-A Csm complex. Our work broadens our understanding of the Type-III-A Csm complex and identifies another mature crRNA processing mechanism in the Type-III-A CRISPR-Cas system based on structural biology.

Circ_UBAP2 exacerbates proliferation and metastasis of OS via targeting miR-665/miR-370-3p/HMGA1 axis.

Circ_UBAP2 is extensively engaged in regulating the development of various malignancies, containing osteosarcoma (OS). However, its biological significance and function are not fully understood. In this study, we found that circ_UBAP2 and HMGA1 levels were up-regulated, and miR-370-3p and miR-665 expressions were decreased in osteosarcoma tissues. Inhibition of circ_UBAP2 or HMGA1 expression in OS cells, cell viability, invasion and migration abilitities were notably hindered, and cell apoptosis abilities were increased. Bioinformatics analysis predicted that miR-665 and miR-370-3p were the downstream targets of circ_UBAP2, and the dual luciferase experiment demonstrated the correlation between them. In addition, inhibition of miR-665 and miR-370-3p expression could significantly reverse the impact of knocking down circ_UBAP2 on OS cells. HMGA1 was discovered to become the downstream target of both miR-665 and miR-370-3p. It was shown that over-expression of miR-665 or miR-370-3p notably stimulated the cell growth, invasion, and migration of osteosarcoma cells, while hindered cell apoptosis. Nevertheless, this effect could be reversed by concurrent over-expression of HMGA1. Our data strongly prove that circ_UBAP2 makes a vital impact on promoting the proliferation, invasion as well as migration of osteosarcoma cells via down-regulating the level of miR-665 and miR-370-3p, and later up-regulating the level of HMGA1. In conclusion, circ_UBAP2 is upregulated in osteosarcoma, and it competitively adsorbs miR-370-3p and miR-665, resulting in up-regulation of HMGA1, thus promoting OS development.

EspB and HtpG interact with the type III-A CRISPR/Cas system of Mycobacterium tuberculosis.

Introduction: Mycobacterium tuberculosis (MTB) has a type III-A clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas) system consisting of a Csm1-5 and CRISPR RNA (crRNA) complex involved in the defense against invading nucleic acids. However, CRISPR/Cas system in the MTB still is clearly unknown and needs to be further explored. Methods: In our work, two non-Cas system proteins EspB and HtpG protein were found and identified by LC-MS/MS. The effect of EspB and HtpG on Type III-A CRISPR/Cas System of M. tuberculosis was examined by using Plasmid interference assay and Co-immunoprecipitation analyses. We explored that EspB could interact with the crRNA RNP complex, but HtpG could inhibit the accumulation of the MTB Csm proteins and defense the mechanism of CRISPR/Cas system. Results: The proteins ESAT-6 secretion system-1(Esx-1) secreted protein B (EspB) and high-temperature protein G (HtpG), which were not previously associated with CRISPR/Cas systems, are involved in mycobacterial CRISPR/Cas systems with distinct functions. Conclusion: EspB is a novel crRNA-binding protein that interacts directly with the MTB crRNP complex. Meanwhile, HtpG influences the accumulation of MTB Csm proteins and EspB and interferes with the defense mechanism of the crRNP complex against foreign DNA in vivo. Thereby, our study not only leads to developing more precise clinical diagnostic tool to quickly detect for MTB infection, but also knows these proteins merits for TB biomarkers/vaccine candidates.

ASPSCR-1 and Sirt-5 alleviate Clonorchis liver fluke rCsNOSIP-induced oxidative stress, proliferation, and migration in cholangiocarcinoma cells.

BACKGROUND: Clonorchiasis, caused by the infection of Clonorchis sinensis (C. sinensis), is a kind of neglected tropical disease, but it is highly related to cholangiocarcinoma. It has been well known that NO from chronic inflammation responses are thought to be a major component of the damage and ultimate carcinogenesis ESPs such as nitric oxide synthase interacting protein (NOSIP) are thought to enhance the damage. The objective of this study was to identify the protein candidates interact with recombinant CsNOSIP (rCsNOSIP) and explore their role involved in CCA development or progression. METHODS: We applied HuProt microarray containing 21,000 probe sets for a systematic identification of rCsNOSIP-binding proteins and grouped binding hits by gene function. Pull-down assays were used to confirm the interaction of rCsNOSIP with alveolar soft part sarcoma (ASPSCR-1) and sirtuins 5 (Sirt-5). ASPSCR-1/Sirt-5 over-expression and siRNA knockdown experiments were employed for obtain of ASPSCR-1/Sirt-5 high or low expression (ASP-oe/Sirt5-oe or ASP-si/Sirt5-si) cholangiocarcinoma cell line (CCLP-1) cells. Nitric oxide (NO) and reactive oxygen species assay (ROS) as well as cell proliferation and wound-healing assays were performed to observe the effect of rCsNOSIP on ASP-oe/Sirt5-oe or ASP-si/Sirt5-si CCLP-1 cells. RESULTS: Seventy candidate proteins protein "hits" were detected as rCsNOSIP-binding proteins by HuProt microarray and bioinformatics analysis. Pull down assay showed that ASPSCR-1 and Sirt-5 could interact with rCsNOSIP. In addition, endotoxin-free-rCsNOSIP could increase the production of NO and ROS and promote the migration of CCLP-1 cells, while its effect on enhancing cell proliferation was not significant. Furthermore, ROS/NO production, proliferation, or migration were increased in ASP-si or Sirt5-si CCLP-1 cells but decreased in Asp-oe or Sirt5-oe CCLP-1 cells when stimulated with rCsNOSIP. CONCLUSIONS: Our findings suggest that CsNOSIP as a component of CsESPs might promote the development and invasion of CCA and Sirt5/ ASPSCR1 as host molecules might play a novel protective role against adverse stimulus during C. sinensis infection. This work supports the idea that CsESPs induce the occurrence and progression of CCA through ROS/RNS-induced oxidative and nitrative DNA damage.

Altered hACE2 binding affinity and S1/S2 cleavage efficiency of SARS-CoV-2 spike protein mutants affect viral cell entry.

SARS-CoV-2 variants are constantly emerging, hampering public health measures in controlling the number of infections. While it is well established that mutations in spike proteins observed for the different variants directly affect virus entry into host cells, there remains a need for further expansion of systematic and multifaceted comparisons. Here, we comprehensively studied the effect of spike protein mutations on spike expression and proteolytic activation, binding affinity, viral entry efficiency and host cell tropism of eight variants of concern (VOC) and variants of interest (VOI). We found that both the full-length spike and its receptor-binding domain (RBD) of Omicron bind to hACE2 with an affinity similar to that of the wild-type. In addition, Alpha, Beta, Delta and Lambda pseudoviruses gained significantly enhanced cell entry ability compared to the wild-type, while the Omicron pseudoviruses showed a slightly increased cell entry, suggesting the vastly increased rate of transmission observed for Omicron variant is not associated with its affinity to hACE2. We also found that the spikes of Omicron and Mu showed lower S1/S2 cleavage efficiency and inefficiently utilized TMPRSS2 to enter host cells than others, suggesting that they prefer the endocytosis pathway to enter host cells. Furthermore, all variants' pseudoviruses we tested gained the ability to enter the animal ACE2-expressing cells. Especially the infection potential of rats and mice showed significantly increased, strongly suggesting that rodents possibly become a reservoir for viral evolution. The insights gained from this study provide valuable guidance for a targeted approach to epidemic control, and contribute to a better understanding of SARS-CoV-2 evolution.

Omics analysis of Mycobacterium tuberculosis isolates uncovers Rv3094c, an ethionamide metabolism-associated gene.

Global control of the tuberculosis epidemic is threatened by increasing prevalence of drug resistant M. tuberculosis isolates. Many genome-wide studies focus on SNP-associated drug resistance mechanisms, but drug resistance in 5-30% of M. tuberculosis isolates (varying with antibiotic) appears unrelated to reported SNPs, and alternative drug resistance mechanisms involving variation in gene/protein expression are not well-studied. Here, using an omics approach, we identify 388 genes with lineage-related differential expression and 68 candidate drug resistance-associated gene pairs/clusters in 11 M. tuberculosis isolates (variable lineage/drug resistance profiles). Structural, mutagenesis, biochemical and bioinformatic studies on Rv3094c from the Rv3093c-Rv3095 gene cluster, a gene cluster selected for further investigation as it contains a putative monooxygenase/repressor pair and is associated with ethionamide resistance, provide insights on its involvement in ethionamide sulfoxidation, the initial step in its activation. Analysis of the structure of Rv3094c and its complex with ethionamide and flavin mononucleotide, to the best of our knowledge the first structures of an enzyme involved in ethionamide activation, identify key residues in the flavin mononucleotide and ethionamide binding pockets of Rv3094c, and F221, a gate between flavin mononucleotide and ethionamide allowing their interaction to complete the sulfoxidation reaction. Our work broadens understanding of both lineage- and drug resistance-associated gene/protein expression perturbations and identifies another player in mycobacterial ethionamide metabolism.

Influence of the COVID-19 pandemic on the vitamin D status of children: A cross-sectional study.

Coronavirus disease 2019 (COVID-19), as well as its prevention and control measures, seriously affected people's livehood, which may have affected the body's level of vitamin D (VD). This study aimed to investigate the effect of the COVID-19 pandemic on the VD status of children in Zhengzhou, China. In this study, we included 12 272 children in 2019 (before the COVID-19 pandemic) and 16 495 children in 2020 (during the COVID-19 pandemic) to examine the changes in VD levels and deficiency rates among children before and during the COVID-19 pandemic. Total VD levels in 2020 were significantly higher than those in 2019 (26.56 [18.15, 41.40] vs. 25.98 [17.92, 40.09] ng/ml, p < 0.001). Further analysis revealed that during the COVID-19 pandemic control period in 2020, the VD levels in February, March, and April were lower than those in the same months of 2019, while the VD deficiency rates were significantly higher. Additionally, our data revealed that VD levels decreased significantly with age. Among children older than 6 years, the VD deficiency rate exceeded 50%. These results indicate that we should pay close attention to VD supplementation during the COVID-19 pandemic control period and in children older than 6 years of age.

Development and Validation of the Random Forest Model via Combining CT-PET Image Features and Demographic Data for Distant Metastases among Lung Cancer Patients.

The work aimed at developing and validating a random forest model of CT-PET image features combined with demographic data to diagnose distant metastases among lung cancer patients. This study involved lung cancer patients from The Cancer Genome Atlas lung adenocarcinoma (TCGA-LUAD) dataset, the lung PET-CT dataset, the lung squamous cell carcinoma (LSCC) dataset, and the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium lung adenocarcinoma (CPTAC-LUAD) dataset and collected the information on 178 CT, 178 PET, and the patients' age, history of smoking, and gender. We conducted image processing and feature extraction. Finally, 4 computed tomography (CT) image features and 2 positron emission tomography (PET) image features were extracted. Four prediction models based on CT image features, PET image features, and demographic data were developed, and the area under the receiver operating characteristic (ROC) curve was used to evaluate the performance of prediction models. A total of 178 eligible samples were randomly divided into a training set (n = 134) and a testing set (n = 44) at a ratio of 3 : 1, with 2021 as a random number. ROC analyses illustrated that the predictive performance for distant metastases of combining CT-PET image features and demographic data for training and testing were 0.923 (95% confidence interval (CI): 0.873-0.973) and 0.873 (95% CI: 0.757-0.990). In addition, the predictive performance of the combined model in the testing set was significantly better than that of the CT-demographic data model (0.716, 95% CI: 0.531-0.902), PET-demographic data model (0.802, 95% CI: 0.633-0.970), and CT-PET model (0.797, 95% CI: 0.666-0.928). The random forest model via combining CT-PET image features and demographic data could have great performance in predicting distant metastases among lung cancer patients.

Effects of low molecular weight peptides from monkfish (Lophius litulon) roe on immune response in immunosuppressed mice.

This study aimed to investigate the immunomodulatory activation of low-molecular-weight peptides from monkfish (Lophius litulon) roe (named MRP) on cyclophosphamide (CTX)-induced immunosuppressed mice. Our results indicated that MRP (100 mg/kg/d BW) could significantly increase the body weight and immune organ index, and improve the morphological changes in the spleen and thymus of mice. These effects subsequently enhance the serum levels of interleukin (IL)-6, IL-1ß, tumor necrosis factor (TNF)-α, and immunoglobulin (Ig) A, IgM, and IgG. Furthermore, MRP could also improve CTX-induced oxidative stress, and activate the NF-κB and MAPK pathways in the spleen tissues. The findings reported herein indicate that MRP has a good immunomodulatory activation toward immunosuppressed mice, hence can potentially be developed as an immune adjuvant or functional food.

Practical application of European biological variation combined with Westgard Sigma Rules in internal quality control.

OBJECTIVES: Westgard Sigma Rules is a statistical tool available for quality control. Biological variation (BV) can be used to set analytical performance specifications (APS). The European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) regularly updates BV data. However, few studies have used robust BV data to determine quality goals and design a quality control strategy for tumor markers. The aim of this study was to derive APS for tumor markers from EFLM BV data and apply Westgard Sigma Rules to establish internal quality control (IQC) rules. METHODS: Precision was calculated from IQC data, and bias was obtained from the relative deviation of the External quality assurance scheme (EQAS) group mean values and laboratory-measured values. Total allowable error (TEa) was derived using EFLM BV data. After calculating sigma metrics, the IQC strategy for each tumor marker was determined according to Westgard Sigma Rules. RESULTS: Sigma metrics achieved for each analyte varied with the level of TEa. Most of these tumor markers except neuron-specific enolase reached 3σ or better based on TEamin. With TEades and TEaopt set as the quality goals, almost all analytes had sigma values below 3. Set TEamin as quality goal, each analyte matched IQC muti rules and numbers of control measurements according to sigma values. CONCLUSIONS: Quality goals from the EFLM BV database and Westgard Sigma Rules can be used to develop IQC strategy for tumor markers.

CD84 is a Suppressor of T and B Cell Activation during Mycobacterium tuberculosis Pathogenesis.

Interest in host-directed therapies as alternatives/adjuncts to antibiotic treatment has resurged with the increasing prevalence of antibiotic-resistant tuberculosis (TB). Immunotherapies that reinvigorate immune responses by targeting immune checkpoints like PD-1/PD-L1 have proved successful in cancer therapy. Immune cell inhibitory receptors that trigger Mycobacterium tuberculosis-specific immunosuppression, however, are unknown. Here, we show that the levels of CD84, a SLAM family receptor, increase in T and B cells in lung tissues from M. tuberculosis-infected C57BL/6 mice and in peripheral blood mononuclear cells (PBMCs) from pulmonary TB patients. M. tuberculosis challenge experiments using CD84-deficient C57BL/6 mice suggest that CD84 expression likely leads to T and B cell immunosuppression during M. tuberculosis pathogenesis and also plays an inhibitory role in B cell activation. Importantly, CD84-deficient mice showed improved M. tuberculosis clearance and longer survival than M. tuberculosis-infected wild-type (WT) mice. That CD84 is a putative M. tuberculosis infection-specific inhibitory receptor suggests it may be a suitable target for the development of TB-specific checkpoint immunotherapies. IMPORTANCE Immune checkpoint therapies, such as targeting checkpoints like PD-1/PD-L1, have proved successful in cancer therapy and can reinvigorate immune responses. The potential of this approach for treating chronic infectious diseases like TB has been recognized, but a lack of suitable immunotherapeutic targets, i.e., immune cell inhibitory receptors that trigger immunosuppression specifically during Mycobacterium tuberculosis pathogenesis, has limited the application of this strategy in the development of new TB therapies. Our focus in this study was to address this gap and search for an M. tuberculosis-specific checkpoint target. Our results suggest that CD84 is a putative inhibitory receptor that may be a suitable target for the development of TB-specific checkpoint immunotherapies.

M. tuberculosis CRISPR/Cas proteins are secreted virulence factors that trigger cellular immune responses.

The role of prokaryotic CRISPR/Cas system proteins as a defensive shield against invasive nucleic acids has been studied extensively. Non-canonical roles in pathogenesis involving intracellular targeting of certain virulence-associated endogenous mRNA have also been reported for some Type I and Type II CRISPR/Cas proteins, but no such roles have yet been established for Type III system proteins. Here, we demonstrate that M. tuberculosis (Type III-A system) CRISPR/Cas proteins Csm1, Csm3, Csm5, Csm6, and Cas6 are secreted and induce host immune responses. Using cell and animal experiments, we show that Cas6, in particular, provokes IFN-γ release from PBMCs from active tuberculosis (TB) patients, and its deletion markedly attenuates virulence in a murine M. tuberculosis challenge model. Recombinant MTBCas6 induces apoptosis of macrophages and lung fibroblasts, and interacts with the surface of cells in a caspase and TLR-2 independent manner. Transcriptomic and signal pathway studies using THP-1 macrophages stimulated with MTBCas6 indicated that MTBCas6 upregulates expression of genes associated with the NF-κB pathway leading to higher levels of IL-6, IL-1ß, and TNF-α release, cytokines known to activate immune system cells in response to M. tuberculosis infection. Our findings suggest that, in addition to their intracellular shielding role, M. tuberculosis CRISPR/Cas proteins have non-canonical extracellular roles, functioning like a virulent sword, and activating host immune responses.

relA Inactivation Converts Sulfonamides Into Bactericidal Compounds.

Folates are required for the de novo biosynthesis of purines, thymine, methionine, glycine, and pantothenic acid, key metabolites that bacterial cells cannot survive without. Sulfonamides, which inhibit bacterial folate biosynthesis and are generally considered as bacteriostats, have been extensively used as broad-spectrum antimicrobials for decades. Here we show that, deleting relA in Escherichia coli and other bacterial species converted sulfamethoxazole from a bacteriostat into a bactericide. Not as previously assumed, the bactericidal effect of SMX was not caused by thymine deficiency. When E. coli ∆relA was treated with SMX, reactive oxygen species and ferrous ion accumulated inside the bacterial cells, which caused extensive DNA double-strand breaks without the involvement of incomplete base excision repair. In addition, sulfamethoxazole showed bactericidal effect against E. coli O157 ∆relA in mice, suggesting the possibility of designing new potentiators for sulfonamides targeting RelA. Thus, our study uncovered the previously unknown bactericidal effects of sulfonamides, which advances our understanding of their mechanisms of action, and will facilitate the designing of new potentiators for them.

Efficacy of Dual-Targeting Combined Anti-Tuberculosis Drug Delivery System in the Treatment of Tuberculous Meningitis.

Tuberculous meningitis (TBM) is an incurable disease with high mortality. It is an extrapulmonary tuberculosis caused by mycobacterium tuberculosis which penetrated the blood-brain barrier and infected the meninges. Mycobacterium tuberculosis lurking in the body mainly reside in macrophages. Anti-tuberculous drugs usually can not target the blood-brain barrier and macrophages, the drug concentration in the lesion is low, which cannot effectively kill mycobacterium tuberculosis, making TBM difficult to treat. Targeted drug delivery systems can target drugs to specific nidus. In the study, we constructed a drug delivery system, which was a cell penetrate peptide B6 and phosphatidylserine (PS) modified polyethylene glycol (PEG) nanomaterial to target the blood-brain barrier and to target macrophages. This nanomaterial was a combined anti-tuberculosis drug delivery system encapsulating antituberculosis drugs rifampicin and pyrazinamide, designed to target macrophages in the brain and kill mycobacterium tuberculosis lurking in the macrophages. We have physically characterized the drug delivery system, and verified the bactericidal ability at cellular and animal level. Results have shown that the targeted drug delivery system had a remarkable efficacy to treat TBM in mice.

Genetic composition and evolution of the prevalent Mycobacterium tuberculosis lineages 2 and 4 in the Chinese and Zhejiang Province populations.

BACKGROUND: There are seven human-adaptation lineages of Mycobacterium tuberculosis (Mtb). Tuberculosis (TB) dissemination is strongly influenced by human movements and host genetics. The detailed lineage distribution evolution of Mtb in Zhejiang Province is unknown. We aim to determine how different sub-lineages are transmitted and distributed within China and Zhejiang Province. METHODS: We analysed whole-genome sequencing data for a worldwide collection of 1154 isolates and a provincial collection of 1296 isolates, constructed the best-scoring maximum likelihood phylogenetic tree. Bayesian evolutionary analysis was used to calculate the latest common ancestor of lineages 2 and 4. The antigenic diversity of human T cell epitopes was evaluated by calculating the pairwise dN/dS ratios. RESULTS: Of the Zhejiang isolates, 964 (74.38%) belonged to lineage 2 and 332 (25.62%) belonged to lineage 4. The distributions of the sub-lineages varied across the geographic regions of Zhejiang Province. L2.2 is the most ancient sub-lineage in Zhejiang, first appearing approximately 6897 years ago (95% highest posterior density interval (HDI): 6513-7298). L4.4 is the most modern sub-lineage, first appearing approximately 2217 years ago (95% HDI: 1864-2581). The dN/dS ratios showed that the epitope and non-epitope regions of lineage 2 strains were significantly (P < 0.001) more conserved than those of lineage 4. CONCLUSIONS: An increase in the frequency of lineage 4 may reflect its successful transmission over the last 20 years. The recent common ancestors of the sub-lineages and their transmission routes are relevant to the entry of humans into China and Zhejiang Province. Diversity in T cell epitopes may prevent Mycobacterium tuberculosis from being recognized by the immune system.

Identification and architecture of a putative secretion tube across mycobacterial outer envelope.

Tuberculosis-causing mycobacteria have thick cell-wall and capsule layers that are formed from complex structures. Protein secretion across these barriers depends on a specialized protein secretion system, but none has been reported. We show that Mycobacterium tuberculosis Rv3705c and its homologous MSMEG_6251 in Mycobacterium smegmatis are tube-forming proteins in the mycobacterial envelope (TiME). Crystallographic and cryo-EM structures of these two proteins show that both proteins form rotationally symmetric rings. Two layers of TiME rings pack together in a tail-to-tail manner into a ring-shaped complex, which, in turn, stacks together to form tubes. M. smegmatis TiME was detected mainly in the cell wall and capsule. Knocking out the TiME gene markedly decreased the amount of secreted protein in the M. smegmatis culture medium, and expression of this gene in knocked-out strain partially restored the level of secreted protein. Our structure and functional data thus suggest that TiME forms a protein transport tube across the mycobacterial outer envelope.