An Orthogonal T7 Replisome for Continuous Hypermutation and Accelerated Evolution in E. coli.

Systems that perform continuous hypermutation of designated genes without compromising the integrity of the host genome can dramatically accelerate the evolution of new or enhanced protein functions. We describe an orthogonal DNA replication system in E. coli based on the controlled expression of the replisome of bacteriophage T7. The system replicates circular plasmids that enable high transformation efficiencies and seamless integration into standard molecular biology workflows. Engineering of T7 DNA polymerase yielded variant proteins with mutation rates of 1.7 × 10 -5 substitutions per base in vivo - 100,000-fold above the genomic mutation rate. Continuous evolution using the mutagenic T7 replisome was demonstrated by expanding the substrate scope of TEM-1 ß-lactamase and increase activity 1,000-fold against clinically relevant monobactam and cephalosporin antibiotics in less than one week.

A parameterized two-domain thermodynamic model explains diverse mutational effects on protein allostery.

New experimental findings continue to challenge our understanding of protein allostery. Recent deep mutational scanning study showed that allosteric hotspots in the tetracycline repressor (TetR) and its homologous transcriptional factors are broadly distributed rather than spanning well-defined structural pathways as often assumed. Moreover, hotspot mutation-induced allostery loss was rescued by distributed additional mutations in a degenerate fashion. Here, we develop a two-domain thermodynamic model for TetR, which readily rationalizes these intriguing observations. The model accurately captures the in vivo activities of various mutants with changes in physically transparent parameters, allowing the data-based quantification of mutational effects using statistical inference. Our analysis reveals the intrinsic connection of intra- and inter-domain properties for allosteric regulation and illustrate epistatic interactions that are consistent with structural features of the protein. The insights gained from this study into the nature of two-domain allostery are expected to have broader implications for other multi-domain allosteric proteins.

A parametrized two-domain thermodynamic model explains diverse mutational effects on protein allostery.

New experimental findings continue to challenge our understanding of protein allostery. Recent deep mutational scanning study showed that allosteric hotspots in the tetracycline repressor (TetR) and its homologous transcriptional factors are broadly distributed rather than spanning well-defined structural pathways as often assumed. Moreover, hotspot mutation-induced allostery loss was rescued by distributed additional mutations in a degenerate fashion. Here, we develop a two-domain thermodynamic model for TetR, which readily rationalizes these intriguing observations. The model accurately captures the in vivo activities of various mutants with changes in physically transparent parameters, allowing the data-based quantification of mutational effects using statistical inference. Our analysis reveals the intrinsic connection of intra- and inter-domain properties for allosteric regulation and illustrate epistatic interactions that are consistent with structural features of the protein. The insights gained from this study into the nature of two-domain allostery are expected to have broader implications for other multidomain allosteric proteins.

Effects of the 5'-Triphosphate Metabolites of Ribavirin, Sofosbuvir, Vidarabine, and Molnupiravir on CTP Synthase Catalysis and Filament Formation: Implications for Repurposing Antiviral Agents against SARS-CoV-2.

Repurposing of antiviral drugs affords a rapid and effective strategy to develop therapies to counter pandemics such as COVID-19. SARS-CoV-2 replication is closely linked to the metabolism of cytosine-containing nucleotides, especially cytidine-5'-triphosphate (CTP), such that the integrity of the viral genome is highly sensitive to intracellular CTP levels. CTP synthase (CTPS) catalyzes the rate-limiting step for the de novo biosynthesis of CTP. Hence, it is of interest to know the effects of the 5'-triphosphate (TP) metabolites of repurposed antiviral agents on CTPS activity. Using E. coli CTPS as a model enzyme, we show that ribavirin-5'-TP is a weak allosteric activator of CTPS, while sofosbuvir-5'-TP and adenine-arabinofuranoside-5'-TP are both substrates. ß-d-N4 -Hydroxycytidine-5'-TP is a weak competitive inhibitor relative to CTP, but induces filament formation by CTPS. Alternatively, sofosbuvir-5'-TP prevented CTP-induced filament formation. These results reveal the underlying potential for repurposed antivirals to affect the activity of a critical pyrimidine nucleotide biosynthetic enzyme.

Molecular investigation of the tandem Tudor domain and plant homeodomain histone binding domains of the epigenetic regulator UHRF2.

Ubiquitin-like containing PHD and ring finger (UHRF)1 and UHRF2 are multidomain epigenetic proteins that play a critical role in bridging crosstalk between histone modifications and DNA methylation. Both proteins contain two histone reader domains, called tandem Tudor domain (TTD) and plant homeodomain (PHD), which read the modification status on histone H3 to regulate DNA methylation and gene expression. To shed light on the mechanism of histone binding by UHRF2, we have undergone a detailed molecular investigation with the TTD, PHD and TTD-PHD domains and compared the binding activity to its UHRF1 counterpart. We found that unlike UHRF1 where the PHD is the primary binding contributor, the TTD of UHRF2 has modestly higher affinity toward the H3 tail, while the PHD has a weaker binding interaction. We also demonstrated that like UHRF1, the aromatic amino acids within the TTD are important for binding to H3K9me3 and a conserved aspartic acid within the PHD forms an ionic interaction with R2 of H3. However, while the aromatic amino acids in the TTD of UHRF1 contribute to selectivity, the analogous residues in UHRF2 contribute to both selectivity and affinity. We also discovered that the PHD of UHRF2 contains a distinct asparagine in the H3R2 binding pocket that lowers the binding affinity of the PHD by reducing a potential electrostatic interaction with the H3 tail. Furthermore, we demonstrate the PHD and TTD of UHRF2 cooperate to interact with the H3 tail and that dual domain engagement with the H3 tail relies on specific amino acids. Lastly, our data indicate that the unique stretch region in the TTD of UHRF2 can decrease the melting temperature of the TTD-PHD and represents a disordered region. Thus, these subtle but important mechanistic differences are potential avenues for selectively targeting the histone binding interactions of UHRF1 and UHRF2 with small molecules.

Erratum: Author Correction: LepVax, a defined subunit vaccine that provides effective pre-exposure and post-exposure prophylaxis of M. leprae infection.

[This corrects the article DOI: 10.1038/s41541-018-0050-z.].

LepVax, a defined subunit vaccine that provides effective pre-exposure and post-exposure prophylaxis of M. leprae infection.

Sustained elimination of leprosy as a global health concern likely requires a vaccine. The current standard, BCG, confers only partial protection and precipitates paucibacillary (PB) disease in some instances. When injected into mice with the T helper 1 (Th1)-biasing adjuvant formulation Glucopyranosyl Lipid Adjuvant in stable emulsion (GLA-SE), a cocktail of three prioritized antigens (ML2055, ML2380 and ML2028) reduced M. leprae infection levels. Recognition and protective efficacy of a single chimeric fusion protein incorporating these antigens, LEP-F1, was confirmed in similar experiments. The impact of post-exposure immunization was then assessed in nine-banded armadillos that demonstrate a functional recapitulation of leprosy. Armadillos were infected with M. leprae 1 month before the initiation of post-exposure prophylaxis. While BCG precipitated motor nerve conduction abnormalities more rapidly and severely than observed for control infected armadillos, motor nerve injury in armadillos treated three times, at monthly intervals with LepVax was appreciably delayed. Biopsy of cutaneous nerves indicated that epidermal nerve fiber density was not significantly altered in M. leprae-infected animals although Remak Schwann cells of the cutaneous nerves in the distal leg were denser in the infected armadillos. Importantly, LepVax immunization did not exacerbate cutaneous nerve involvement due to M. leprae infection, indicating its safe use. There was no intraneural inflammation but a reduction of intra axonal edema suggested that LepVax treatment might restore some early sensory axonal function. These data indicate that post-exposure prophylaxis with LepVax not only appears safe but, unlike BCG, alleviates and delays the neurologic disruptions caused by M. leprae infection.

Nitazoxanide is active against Mycobacterium leprae.

Nitazoxanide (NTZ) is an anti-parasitic drug that also has activity against bacteria, including Mycobacterium tuberculosis. Our data using both radiorespirometry and live-dead staining in vitro demonstrate that NTZ similarly has bactericidal against M. leprae. Further, gavage of M. leprae-infected mice with NTZ at 25mg/kg provided anti-mycobacterial activity equivalent to rifampicin (RIF) at 10 mg/kg. This suggests that NTZ could be considered for leprosy treatment.

Regulation of HBV-specific CD8(+) T cell-mediated inflammation is diversified in different clinical presentations of HBV infection.

Chronic HBV infection is the leading cause of liver cirrhosis and hepatic cancer, but the individual responses toward HBV infection are highly variable, ranging from asymptomatic to chronic active hepatitis B inflammation. In this study, we hypothesized that the different individual responses to HBV infection was associated with differences in HBV-specific CD8(+) T cell-mediated inflammation and cytotoxicity. Blood samples were collected from subjects with asymptomatic HBV-infection, subjects undergoing active chronic HBV flares (active CHB), and subjects with HBV-infected hepatocellular carcinoma (HBV-HCC). By tetramer staining, we found that all three groups had similar frequencies of HBVspecific CD8(+) T cells. However, after HBV peptide stimulation, the HBV-specific CD8(+) T cells in asymptomatic subjects had significantly stronger interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and CD107a expression than those in active CHB and HBV-HCC patients. Examination of surface marker expression revealed that the PD-1(-)Tim-3(-) double-negative cell population was the main contributor to HBV-specific inflammation. In active CHB patients and HBV-HCC patients, however, the frequencies of activated PD-1(-)Tim-3(-) cells were significantly reduced. Moreover, the serum HBV DNA titer was not correlated with the frequencies of HBV-specific CD8(+) T cells but was inversely correlated with the frequencies of IFN-g-expressing and CD107a-express cells in response to HBV stimulation. Together, our data demonstrated that the status of HBVspecific CD8(+) T cell exhaustion was associated with different clinical outcomes of chronic HBV infection.

Current knowledge on Mycobacterium leprae transmission: a systematic literature review.

UNLABELLED: Summary BACKGROUND: The transmission pathways of Mycobacterium leprae are not fully understood. Solid evidence exists for an increased risk for individuals living in close contact with leprosy patients but the existence of zoonotic leprosy, environmental reservoirs and trauma-related transmission has also been established. PURPOSE: To assess the current state of knowledge on M. leprae transmission, we conducted a systematic review of the peer-reviewed literature pertaining to this topic. METHOD: Major electronic bibliographic databases were searched for relevant peer-reviewed articles published up to January 2014. No restrictions on study types, participants and location were applied, and all outcomes demonstrated to contribute to the transmission of M. leprae were considered. Included studies were grouped by mode of transmission, namely (i) human-to-human via aerosols or direct contact; (ii) direct inoculation (e.g. injury); and (iii) transmission to humans from environmental or zoonotic reservoirs, and by insects. The importance of the different transmission pathways and the strength of the evidence were assessed considering the number of publications describing similar findings, the consistency of the findings and the methodological quality of the studies. RESULTS: A total of 79 relevant articles were retained out of 3,805 hits resulting from the application of the search strategy. Solid evidence for transmission among contacts exists, and for zoonotic leprosy in the southern States of the USA. Based on the extant evidence, skin-to-skin contact, aerosols/droplets and shedding of bacteria into the environment and subsequent infection, e.g. through dust or small wounds, all remain possible options. CONCLUSION: No study has unequivocally demonstrated the mechanisms by which M. leprae bacteria travel from one case of leprosy to another.

rBCG30-induced immunity and cross-protection against Mycobacterium leprae challenge are enhanced by boosting with the Mycobacterium tuberculosis 30-kilodalton antigen 85B.

Leprosy remains a major global health problem and typically occurs in regions in which tuberculosis is endemic. Vaccines are needed that protect against both infections and do so better than the suboptimal Mycobacterium bovis BCG vaccine. Here, we evaluated rBCG30, a vaccine previously demonstrated to induce protection superior to that of BCG against Mycobacterium tuberculosis and Mycobacterium bovis challenge in animal models, for efficacy against Mycobacterium leprae challenge in a murine model of leprosy. rBCG30 overexpresses the M. tuberculosis 30-kDa major secretory protein antigen 85B, which is 85% homologous with the M. leprae homolog (r30ML). Mice were sham immunized or immunized intradermally with BCG or rBCG30 and challenged 2.5 months later by injection of viable M. leprae into each hind footpad. After 7 months, vaccine efficacy was assessed by enumerating the M. leprae bacteria per footpad. Both BCG and rBCG30 induced significant protection against M. leprae challenge. In the one experiment in which a comparison between BCG and rBCG30 was feasible, rBCG30 induced significantly greater protection than did BCG. Immunization of mice with purified M. tuberculosis or M. leprae antigen 85B also induced protection against M. leprae challenge but less so than BCG or rBCG30. Notably, boosting rBCG30 with M. tuberculosis antigen 85B significantly enhanced r30ML-specific immune responses, substantially more so than boosting BCG, and significantly augmented protection against M. leprae challenge. Thus, rBCG30, a vaccine that induces improved protection against M. tuberculosis, induces cross-protection against M. leprae that is comparable or potentially superior to that induced by BCG, and boosting rBCG30 with antigen 85B further enhances immune responses and protective efficacy.

Drug resistance in patients with leprosy in the United States.

Molecular drug susceptibility testing was performed on 39 US patients with leprosy. Of these, 2 had dapsone-resistant Mycobacterium leprae and 1 of these patients also had rifampin-resistant M. leprae. Even though antileprosy drug resistance occurs in this leprosy population, resistance does not appear to be a major problem.

Molecular assays for determining Mycobacterium leprae viability in tissues of experimentally infected mice.

BACKGROUND: The inability of Mycobacterium leprae to grow on axenic media has necessitated specialized techniques in order to determine viability of this organism. The purpose of this study was to develop a simple and sensitive molecular assay for determining M. leprae viability directly from infected tissues. METHODOLOGY/PRINCIPLE FINDINGS: Two M. leprae-specific quantitative reverse transcription PCR (qRT-PCR) assays based on the expression levels of esxA, encoding the ESAT-6 protein, and hsp18, encoding the heat shock 18 kDa protein, were developed and tested using infected footpad (FP) tissues of both immunocompetent and immunocompromised (athymic nu/nu) mice. In addition, the ability of these assays to detect the effects of anti-leprosy drug treatment on M. leprae viability was determined using rifampin and rifapentine, each at 10 mg/kg for 1, 5, or 20 daily doses, in the athymic nu/nu FP model. Molecular enumeration (RLEP PCR) and viability determinations (qRT-PCR) were performed via Taqman methodology on DNA and RNA, respectively, purified from ethanol-fixed FP tissue and compared with conventional enumeration (microscopic counting of acid fast bacilli) and viability assays (radiorespirometry, viability staining) which utilized bacilli freshly harvested from the contralateral FP. Both molecular and conventional assays demonstrated growth and high viability of M. leprae in nu/nu FPs over a 4 month infection period. In contrast, viability was markedly decreased by 8 weeks in immunocompetent mice. Rifapentine significantly reduced bacterial viability after 5 treatments, whereas rifampin required up to 20 treatments for the same efficacy. Neither drug was effective after a single treatment. In addition, host gene expression was monitored with the same RNA preparations. CONCLUSIONS: hsp18 and esxA qRT-PCR are sensitive molecular indicators, reliably detecting viability of M. leprae in tissues without the need for bacterial isolation or immediate processing, making these assays applicable for in vivo drug screening and promising for clinical and field applications.

What is the evidence that the putative Mycobacterium lepromatosis species causes diffuse lepromatous leprosy?

Han et al. have made a retrospective isolation of DNA from two patients with fatal Lucio's phenomenon. This DNA does have some molecular differences to M. leprae and may constitute a variant of M. leprae. However the experiments and data needed to confirm that this is a new leprosy-causing species have not yet been done. We have outlined the work that does need to be done. For the moment the assertion that 'M. lepromatosis' is a new leprosy-causing species is not proven.

Advances and hurdles on the way toward a leprosy vaccine.

Prevalence rates for leprosy have declined sharply over the past 20 y, with this decline generally attributed to the WHO multi-drug therapy (MDT) campaign to provide free-of-charge treatment to all diagnosed leprosy patients. The success of this program appears to have reached its nadir, however, as evidenced by the stalled decreases in both global prevalence and new case detection rates of leprosy. Mass BCG vaccination for the prevention of tuberculosis (TB) at national levels has had a positive effect on leprosy decline and is often overlooked as an important factor in current leprosy control programs. Because BCG provides incomplete protection against both TB and leprosy, newer more effective TB vaccines are being developed. The impact that application of these vaccines will have on current leprosy control programs is unclear. In this review, we assess the need for vaccines within leprosy control programs. We summarize and discuss leprosy vaccine strategies that have been deployed previously and discuss those strategies that are currently being developed to augment recent breakthroughs in leprosy control.

Probable zoonotic leprosy in the southern United States.

BACKGROUND: In the southern region of the United States, such as in Louisiana and Texas, there are autochthonous cases of leprosy among native-born Americans with no history of foreign exposure. In the same region, as well as in Mexico, wild armadillos are infected with Mycobacterium leprae. METHODS: Whole-genome resequencing of M. leprae from one wild armadillo and three U.S. patients with leprosy revealed that the infective strains were essentially identical. Comparative genomic analysis of these strains and M. leprae strains from Asia and Brazil identified 51 single-nucleotide polymorphisms and an 11-bp insertion-deletion. We genotyped these polymorphic sites, in combination with 10 variable-number tandem repeats, in M. leprae strains obtained from 33 wild armadillos from five southern states, 50 U.S. outpatients seen at a clinic in Louisiana, and 64 Venezuelan patients, as well as in four foreign reference strains. RESULTS: The M. leprae genotype of patients with foreign exposure generally reflected their country of origin or travel history. However, a unique M. leprae genotype (3I-2-v1) was found in 28 of the 33 wild armadillos and 25 of the 39 U.S. patients who resided in areas where exposure to armadillo-borne M. leprae was possible. This genotype has not been reported elsewhere in the world. CONCLUSIONS: Wild armadillos and many patients with leprosy in the southern United States are infected with the same strain of M. leprae. Armadillos are a large natural reservoir for M. leprae, and leprosy may be a zoonosis in the region. (Funded by the National Institute of Allergy and Infectious Diseases and others.).

Hansen's disease (leprosy) complicated by secondary mycobacterial infection.

A patient with Hansen's disease received corticosteroids for a type 1 leprosy reaction and subsequently developed a new cutaneous lesion at the original biopsy site from which Mycobacterium fortuitum was cultured. A review of the literature found only two other cases of coinfection with atypical mycobacteria and Mycobacterium leprae, although there are many reports of pulmonary tuberculosis in patients with leprosy. This case highlights the diagnostic difficulties encountered when a patient has two different mycobacterial infections of the skin. The published experience emphasizes that such coinfection is remarkably uncommon in leprosy, despite the frequent use of high doses of corticosteroids for leprosy reactions.

Characterisation of short tandem repeats for genotyping Mycobacterium leprae.

OBJECTIVE: Establish a typing system for Mycobacterium leprae based on polymorphic DNA structures known as short tandem repeats (STR). DESIGN: Assess 16 polymorphic STR for sensitivity, specificity and reproducibility in standard assays using reference strains of M. leprae. RESULTS: Primers for 16 STR loci were selected based on PCR product size and for their ability to sequence each STR locus from both directions. All primer pairs produced a visible PCR amplicon of appropriate size from PCR reactions containing 10 M. leprae cells. DNA sequences for each STR locus, except (AT) 15, was correctly identified as M. leprae-specific in replicate samples containing 1000 M. leprae using either the forward or reverse PCR primers. Twelve of 13 M. leprae STR loci were stable during passage in heavily infected armadillo tissues over a 5 year and 7 month infection cycle. CONCLUSIONS: Certain M. leprae STR provide suitable targets for strain typing with the potential for grouping M. leprae with shared genotypes that may prove useful for establishing linkages between leprosy cases within geographical regions.