NapA (Rv0430), a Novel Nucleoid-Associated Protein that Regulates a Virulence Operon in Mycobacterium tuberculosis in a Supercoiling-Dependent Manner

Comparison of Mycobacterium tuberculosis with Escherichia coli reveals a reduction in the diversity of DNA-managing proteins, such as DNA topoisomerases, although genome sizes are similar for the two species. The same is true for nucleoid-associated proteins (NAPs), important factors in bacterial chromosome compaction, chromosome remodeling, and regulation of gene expression. In a search for still uncharacterized NAPs, we found that M. tuberculosis protein Rv0430 has NAP-like features: it binds to DNA in a length- and supercoil-dependent fashion, prefers A/T-rich DNA sequences, protects DNA from damaging agents, and modulates DNA supercoiling. At a ratio of 1 dimer/40 bps of DNA, Rv0430 bridges distant DNA segments; at 1 dimer/20 bps, it coats DNA, forming inflexible rods. Rv0430 also stimulates the DNA relaxation activity of topoisomerase I. Remarkably, Rv0430 stimulates its own promoter in a supercoil-dependent manner. It is the first gene of an operon harboring two regulators of M. tuberculosis virulence (virR and sodC), and controls the expression of these downstream virulence regulators and therefore itself is a virulence regulator. The sensitivity of rv0430 expression to supercoiling is consistent with supercoiling being important for infection by M. tuberculosis. Thus, Rv0430 is a novel NAP, doubling up as a topology modulator of M. tuberculosis.     

Direct regulation of topoisomerase activity by a nucleoid-associated protein

The topological homeostasis of bacterial chromosomes is maintained by the balance between compaction and the topological organization of genomes. Two classes of proteins play major roles in chromosome organization: the nucleoid-associated proteins (NAPs) and topoisomerases. The NAPs bind DNA to compact the chromosome, whereas topoisomerases catalytically remove or introduce supercoils into the genome. We demonstrate that HU, a major NAP of Mycobacterium tuberculosis specifically stimulates the DNA relaxation ability of mycobacterial topoisomerase I (TopoI) at lower concentrations but interferes at higher concentrations. A direct physical interaction between M. tuberculosis HU (MtHU) and TopoI is necessary for enhancing enzyme activity both in vitro and in vivo. The interaction is between the amino terminal domain of MtHU and the carboxyl terminal domain of TopoI. Binding of MtHU did not affect the two catalytic trans-esterification steps but enhanced the DNA strand passage, requisite for the completion of DNA relaxation, a new mechanism for the regulation of topoisomerase activity. An interaction-deficient mutant of MtHU was compromised in enhancing the strand passage activity. The species-specific physical and functional cooperation between MtHU and TopoI may be the key to achieve the DNA relaxation levels needed to maintain the optimal superhelical density of mycobacterial genomes.     

Dual-targeting GroEL/ES chaperonin and protein tyrosine phosphatase B (PtpB) inhibitors: A polypharmacology strategy for treating Mycobacterium tuberculosis infections

Current treatments for Mycobacterium tuberculosis infections require long and complicated regimens that can lead to patient non-compliance, increasing incidences of antibiotic-resistant strains, and lack of efficacy against latent stages of disease. Thus, new therapeutics are needed to improve tuberculosis standard of care. One strategy is to target protein homeostasis pathways by inhibiting molecular chaperones such as GroEL/ES (HSP60/10) chaperonin systems. M. tuberculosis has two GroEL homologs: GroEL1 is not essential but is important for cytokine-dependent granuloma formation, while GroEL2 is essential for survival and likely functions as the canonical housekeeping chaperonin for folding proteins. Another strategy is to target the protein tyrosine phosphatase B (PtpB) virulence factor that M. tuberculosis secretes into host cells to help evade immune responses. In the present study, we have identified a series of GroEL/ES inhibitors that inhibit M. tuberculosis growth in liquid culture and biochemical function of PtpB in vitro. With further optimization, such dual-targeting GroEL/ES and PtpB inhibitors could be effective against all stages of tuberculosis – actively replicating bacteria, bacteria evading host cell immune responses, and granuloma formation in latent disease – which would be a significant advance to augment current therapeutics that primarily target actively replicating bacteria.     

Structural and functional conservation of Mycobacterium tuberculosis GroEL paralogs suggests that GroEL1 Is a chaperonin

GroEL is a group I chaperonin that facilitates protein folding and prevents protein aggregation in the bacterial cytosol. Mycobacteria are unusual in encoding two or more copies of GroEL in their genome. While GroEL2 is essential for viability and likely functions as the general housekeeping chaperonin, GroEL1 is dispensable, but its structure and function remain unclear.Here, we present the 2.2-Å resolution crystal structure of a 23-kDa fragment of Mycobacterium tuberculosis GroEL1 consisting of an extended apical domain. Our X-ray structure of the GroEL1 apical domain closely resembles those of Escherichia coli GroEL and M. tuberculosis GroEL2, thus highlighting the remarkable structural conservation of bacterial chaperonins. Notably, in our structure, the proposed substrate-binding site of GroEL1 interacts with the N-terminal region of a symmetry-related neighboring GroEL1 molecule. The latter is consistent with the known GroEL apical domain function in substrate binding and is supported by results obtained from using peptide array technology. Taken together, these data show that the apical domains of M. tuberculosis GroEL paralogs are conserved in three-dimensional structure, suggesting that GroEL1, like GroEL2, is a chaperonin.     

NapM,a new nucleoid‐associated protein,broadly regulates gene expression and affects mycobacterial resistance to anti‐tuberculosis drugs

Nucleoid‐associated proteins (NAPs) play important roles in the global organization of bacterial chromosomes. However, potential NAPs and their functions are barely characterized in mycobacteria. In this study, NapM, an alkaline protein, functions as a new NAP. NapM is conserved in all of the sequenced mycobacterial genomes, and can recognize DNA in a length‐dependent but sequence‐independent manner. It prefers AT‐rich DNA and binds to the major groove. NapM possesses a clear DNA‐bridging function, and can protect DNA from DNase I digestion. NapM globally regulates the expression of more than 150 genes and the resistance of Mycobacterium smegmatis to two anti‐tuberculosis drugs, namely, rifampicin and ethambutol. An ABC transporter operon was found to be specifically responsible for the napM‐dependent ethambutol resistance of M. smegmatis. NapM also presents a similar regulation of anti‐tuberculosis drug resistance in M. tuberculosis. These results suggest that NapM is a new member of the mycobacterial NAP family. Our findings expand the range of identified NAPs and improve the understanding on the relationship between NAPs with antibiotic resistance in mycobacteria.     

Facilitated Oligomerization of Mycobacterial GroEL: Evidence for Phosphorylation-Mediated Oligomerization

The distinctive feature of the GroES-GroEL chaperonin system in mediating protein folding lies in its ability to exist in a tetradecameric state, form a central cavity, and encapsulate the substrate via the GroES lid. However, recombinant GroELs of Mycobacterium tuberculosis are unable to act as effective molecular chaperones when expressed in Escherichia coli. We demonstrate here that the inability of M. tuberculosis GroEL1 to act as a functional chaperone in E. coli can be alleviated by facilitated oligomerization. The results of directed evolution involving random DNA shuffling of the genes encoding M. tuberculosis GroEL homologues followed by selection for functional entities suggested that the loss of chaperoning ability of the recombinant mycobacterial GroEL1 and GroEL2 in E. coli might be due to their inability to form canonical tetradecamers. This was confirmed by the results of domain-swapping experiments that generated M. tuberculosis-E. coli chimeras bearing mutually exchanged equatorial domains, which revealed that E. coli GroEL loses its chaperonin activity due to alteration of its oligomerization capabilities and vice versa for M. tuberculosis GroEL1. Furthermore, studying the oligomerization status of native GroEL1 from cell lysates of M. tuberculosis revealed that it exists in multiple oligomeric forms, including single-ring and double-ring variants. Immunochemical and mass spectrometric studies of the native M. tuberculosis GroEL1 revealed that the tetradecameric form is phosphorylated on serine-393, while the heptameric form is not, indicating that the switch between the single- and double-ring variants is mediated by phosphorylation.GroEL, an essential chaperonin, is known to form a ring-shaped structure for sequestering substrate proteins from the crowded cellular milieu and is responsible for the occurrence of various cellular processes, such as de novo folding, transport, and macromolecular assembly, within a biologically relevant time scale (7, 26, 48, 53). In Escherichia coli, GroEL, along with its cofactor GroES, assists the folding of about 10 to 30% of cytosolic proteins, among which some are known to be essential for cell viability (15, 26, 27, 31). GroEL was originally identified as the host factor responsible for phage λ and T4 capsid protein assembly and was subsequently shown to be essential for cell viability (17, 20). E. coli groEL is found in an operonic arrangement with groES (groESL), and its expression is regulated by multiple promoter elements.GroEL function has been shown to be a complex interplay between its interaction with and encapsulation of substrate proteins, with concomitant conformational changes induced by ATP binding, hydrolysis, and GroES binding (24, 56, 62). E. coli GroEL exists as a homotetradecamer forming two isologous rings of seven identical subunits each. Crystallographic analyses have delineated the three-domain architecture of GroEL monomers and the GroES-GroEL interactions (4, 63). The central region of the GroEL polypeptide, spanning amino acid residues 191 to 376, constitutes the GroES and substrate polypeptide-binding apical domain. The equatorial ATPase domain spanning two extremities of the GroEL polypeptide, that is, residues 6 to 133 and 409 to 523, is responsible for the ATPase activity and the bulk of intersubunit interactions. The hinge-forming intermediate domain, spanning two regions on the polypeptide, namely, residues 134 to 190 and 377 to 408, connects the said two domains in the tertiary structure. The conformational changes resulting from ATP binding and hydrolysis at the equatorial domain are coupled to those occurring at the apical domain via this hinge region (4, 63).The usual size limit for the substrate proteins, as shown by both in vitro and in vivo studies, is around 57 kDa, although the cis cavity is reported to theoretically accommodate larger proteins, on the order of 104 kDa (10, 27, 35, 46). Productive in vivo folding of the proteins larger than the usual size limit, such as the 86-kDa maltose binding protein fusion and 82-kDa mitochondrial aconitase, has also been reported (9, 29). Since such large substrates are difficult to accommodate in the central cavity, it has been suggested that their productive folding might occur outside the cis cavity. These studies therefore indicate that the substrate recognition patterns of GroEL may be more diverse than initially thought.Recent genome annotation studies of various bacteria have revealed that a few bacterial genomes possess multiple copies of groEL genes (2, 18, 30). The Mycobacterium tuberculosis genome bears two copies of groEL genes (groELs). One of these, groEL1, is arranged in an operon, with the cognate cochaperonin groES being the first gene, while the second copy, groEL2, exists separately on the genome (13). Recombinant mycobacterial GroELs were shown to possess biochemical features that deviated significantly from the trademark properties of E. coli GroEL. The most striking feature of M. tuberculosis GroELs, however, was their oligomeric state, where contrary to expectations, in vitro they did not form the canonical tetradecameric assembly when purified from E. coli. The proteins instead existed as lower oligomers (dimers) irrespective of the presence or absence of cofactors, such as the cognate GroES or ATP (40, 41). Furthermore, they displayed weak ATPase activities and GroES independence in preventing aggregation of the denatured polypeptides.Evolutionary studies of M. tuberculosis groEL sequences have suggested rapid evolution of the groEL1 gene, yet without turning these into pseudogenes (21). The other hypothesis suggests that M. tuberculosis, being an organism that grows slowly, might require GroEL function that does not utilize ATP rapidly but, rather, with a slow turnover rate. Alternately, additional mechanisms might exist in M. tuberculosis which could mediate regulated oligomerization of M. tuberculosis chaperonins. Such regulation might help in the controlled utilization of ATP in nutrient-deprived M. tuberculosis, as observed for other chaperones, such as small heat shock proteins (23).In the present study, we have exploited the unusual oligomeric status of the recombinant M. tuberculosis GroELs to study the significance of oligomer formation for GroEL''s function as a molecular chaperone. Furthermore, we have explored the possibility of the existence of regulated oligomerization for native M. tuberculosis GroELs in their natural setting. We first show that M. tuberculosis groEL genes are not capable of complementing a conditional allele of E. coli groEL, namely, groEL44. The results of phenotypic and biochemical analyses of GroEL variants obtained by gene shuffling and domain swapping suggest that the impaired chaperoning ability of recombinant M. tuberculosis GroELs is a consequence of their inability to form higher-order oligomers in E. coli and that oligomerization is the prelude to the formation of an active GroEL chaperonin. Further, by immunochemical and mass spectrometric (MS) analysis of native mycobacterial GroELs, we show that M. tuberculosis GroEL1 exists in multiple oligomeric forms, viz., monomeric, dimeric, heptameric (single ring), and tetradecameric (double ring) forms, and that the switch between single-ring and double-ring variants is operated by phosphorylation on a serine residue. These observations suggest that the determinants of oligomerization for M. tuberculosis GroEL1 are distinct from those of its E. coli counterpart and that it does oligomerize in M. tuberculosis (its native environment), whereas it loses its oligomerization capability when expressed in E. coli. It could thus be possible that M. tuberculosis GroEL1 requires a certain native M. tuberculosis protein, probably a eukaryotic-like Ser-Thr protein kinase, to oligomerize properly, though the precise reason cannot be discerned by these observations.     

MFP1 is a thylakoid-associated,nucleoid-binding protein with a coiled-coil structure

Plastid DNA, like bacterial and mitochondrial DNA, is organized into protein–DNA complexes called nucleoids. Plastid nucleoids are believed to be associated with the inner envelope in developing plastids and the thylakoid membranes in mature chloroplasts, but the mechanism for this re-localization is unknown. Here, we present the further characterization of the coiled-coil DNA-binding protein MFP1 as a protein associated with nucleoids and with the thylakoid membranes in mature chloroplasts. MFP1 is located in plastids in both suspension culture cells and leaves and is attached to the thylakoid membranes with its C-terminal DNA-binding domain oriented towards the stroma. It has a major DNA-binding activity in mature Arabidopsis chloroplasts and binds to all tested chloroplast DNA fragments without detectable sequence specificity. Its expression is tightly correlated with the accumulation of thylakoid membranes. Importantly, it is associated in vivo with nucleoids, suggesting a function for MFP1 at the interface between chloroplast nucleoids and the developing thylakoid membrane system.     

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis ¹, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms ^2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) ^5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms ⁷.In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin ^8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere ⁹. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc²7000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen ⁹. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.     

Polyphosphate Deficiency in Mycobacterium tuberculosis Is Associated with Enhanced Drug Susceptibility and Impaired Growth in Guinea Pigs

Inorganic polyphosphate (polyP), a linear polymer of hundreds of phosphate residues linked by ATP-like phosphoanhydride bonds, is found in all organisms and performs a wide variety of functions. This study shows that polyP accumulation occurs in Mycobacterium tuberculosis upon exposure to various stress conditions. M. tuberculosis possesses a single homolog of ppk-1, and we have disrupted ppk-1 in the M. tuberculosis genome by allelic replacement. The mutant strain exhibited negligible levels of intracellular polyP, decreased expression of sigF and phoP, and reduced growth in the stationary phase and displayed a survival defect in response to nitrosative stress and in THP-1 macrophages compared to the wild-type strain. We report that reduction in polyP levels is associated with increased susceptibility of M. tuberculosis to certain TB drugs and impairs its ability to cause disease in guinea pigs. These results suggest that polyP contributes to persistence of M. tuberculosis in vitro and plays an important role in the physiology of bacteria residing within guinea pigs.     

Characterization of a Mycobacterium tuberculosis Nanocompartment and Its Potential Cargo Proteins

Mycobacterium tuberculosis has evolved various mechanisms by which the bacterium can maintain homeostasis under numerous environmental assaults generated by the host immune response. M. tuberculosis harbors enzymes involved in the oxidative stress response that aid in survival during the production of reactive oxygen species in activated macrophages. Previous studies have shown that a dye-decolorizing peroxidase (DyP) is encapsulated by a bacterial nanocompartment, encapsulin (Enc), whereby packaged DyP interacts with Enc via a unique C-terminal extension. M. tuberculosis also harbors an encapsulin homolog (CFP-29, Mt-Enc), within an operon with M. tuberculosis DyP (Mt-DyP), which contains a C-terminal extension. Together these observations suggest that Mt-DyP interacts with Mt-Enc. Furthermore, it has been suggested that DyPs may function as either a heme-dependent peroxidase or a deferrochelatase. Like Mt-DyP, M. tuberculosis iron storage ferritin protein, Mt-BfrB, and an M. tuberculosis protein involved in folate biosynthesis, 7,8-dihydroneopterin aldolase (Mt-FolB), have C-terminal tails that could also interact with Mt-Enc. For the first time, we show by co-purification and electron microscopy that mycobacteria via Mt-Enc can encapsulate Mt-DyP, Mt-BfrB, and Mt-FolB. Functional studies of free or encapsulated proteins demonstrate that they retain their enzymatic activity within the Mt-Enc nanocompartment. Mt-DyP, Mt-FolB, and Mt-BfrB all have antioxidant properties, suggesting that if these proteins are encapsulated by Mt-Enc, then this nanocage may play a role in the M. tuberculosis oxidative stress response. This report provides initial structural and biochemical clues regarding the molecular mechanisms that utilize compartmentalization by which the mycobacterial cell may aid in detoxification of the local environment to ensure long term survival.     

Characterization of an interplay between a Mycobacterium?tuberculosis MazF homolog,Rv1495 and its sole DNA topoisomerase I

The MazEF systems are thought to contribute to the capacity for long-term dormancy observed in the human pathogen, Mycobacterium tuberculosis. However, except for their functions as mRNA interferases, little is known regarding any additional cellular functions of these systems in the pathogen. In the present study, we observed a negative interplay between MazF protein Rv1495 and the sole M. tuberculosis DNA topoisomerase I (MtbTopA) with respect to protein functions. Through its C-terminal domain, MtbTopA physically interacted with and inhibited the mRNA cleavage activity of Rv1495. Rv1495, in turn, inhibited the DNA cleavage activity of MtbTopA as well as its function of relaxation of supercoiled DNA. An N-terminus fragment of Rv1495, designated Rv1495-N(29-56), lost mRNA cleavage activity, but retained a significant physical interaction and inhibitory effect on TopA proteins from both M. tuberculosis and M. smegmatis. This fragment, although less effective than the full-length protein, was able to inhibit mycobacterial growth when expressed through a recombinant plasmid in M. smegmatis. The Rv1495 physically interacted with the M. smegmatis TopA both in vitro and in vivo. Our findings imply that MazEF systems can affect bacterial survival by a novel mechanism that allows direct modulation of M. tuberculosis topoisomerase I.     

Mycobacterium tuberculosis Hip1 Modulates Macrophage Responses through Proteolysis of GroEL2

Mycobacterium tuberculosis (Mtb) employs multiple strategies to evade host immune responses and persist within macrophages. We have previously shown that the cell envelope-associated Mtb serine hydrolase, Hip1, prevents robust macrophage activation and dampens host pro-inflammatory responses, allowing Mtb to delay immune detection and accelerate disease progression. We now provide key mechanistic insights into the molecular and biochemical basis of Hip1 function. We establish that Hip1 is a serine protease with activity against protein and peptide substrates. Further, we show that the Mtb GroEL2 protein is a direct substrate of Hip1 protease activity. Cleavage of GroEL2 is specifically inhibited by serine protease inhibitors. We mapped the cleavage site within the N-terminus of GroEL2 and confirmed that this site is required for proteolysis of GroEL2 during Mtb growth. Interestingly, we discovered that Hip1-mediated cleavage of GroEL2 converts the protein from a multimeric to a monomeric form. Moreover, ectopic expression of cleaved GroEL2 monomers into the hip1 mutant complemented the hyperinflammatory phenotype of the hip1 mutant and restored wild type levels of cytokine responses in infected macrophages. Our studies point to Hip1-dependent proteolysis as a novel regulatory mechanism that helps Mtb respond rapidly to changing host immune environments during infection. These findings position Hip1 as an attractive target for inhibition for developing immunomodulatory therapeutics against Mtb.     

Nitazoxanide Stimulates Autophagy and Inhibits mTORC1 Signaling and Intracellular Proliferation of Mycobacterium tuberculosis

Tuberculosis, caused by Mycobacterium tuberculosis infection, is a major cause of morbidity and mortality in the world today. M. tuberculosis hijacks the phagosome-lysosome trafficking pathway to escape clearance from infected macrophages. There is increasing evidence that manipulation of autophagy, a regulated catabolic trafficking pathway, can enhance killing of M. tuberculosis. Therefore, pharmacological agents that induce autophagy could be important in combating tuberculosis. We report that the antiprotozoal drug nitazoxanide and its active metabolite tizoxanide strongly stimulate autophagy and inhibit signaling by mTORC1, a major negative regulator of autophagy. Analysis of 16 nitazoxanide analogues reveals similar strict structural requirements for activity in autophagosome induction, EGFP-LC3 processing and mTORC1 inhibition. Nitazoxanide can inhibit M. tuberculosis proliferation in vitro. Here we show that it inhibits M. tuberculosis proliferation more potently in infected human THP-1 cells and peripheral monocytes. We identify the human quinone oxidoreductase NQO1 as a nitazoxanide target and propose, based on experiments with cells expressing NQO1 or not, that NQO1 inhibition is partly responsible for mTORC1 inhibition and enhanced autophagy. The dual action of nitazoxanide on both the bacterium and the host cell response to infection may lead to improved tuberculosis treatment.     

Therapeutic Antiviral Effect of the Nucleic Acid Polymer REP 2055 against Persistent Duck Hepatitis B Virus Infection

Previous studies have demonstrated that nucleic acid polymers (NAPs) have both entry and post-entry inhibitory activity against duck hepatitis B virus (DHBV) infection. The inhibitory activity exhibited by NAPs prevented DHBV infection of primary duck hepatocytes in vitro and protected ducks from DHBV infection in vivo and did not result from direct activation of the immune response. In the current study treatment of primary human hepatocytes with NAP REP 2055 did not induce expression of the TNF, IL6, IL10, IFNA4 or IFNB1 genes, confirming the lack of direct immunostimulation by REP 2055. Ducks with persistent DHBV infection were treated with NAP 2055 to determine if the post-entry inhibitory activity exhibited by NAPs could provide a therapeutic effect against established DHBV infection in vivo. In all REP 2055-treated ducks, 28 days of treatment lead to initial rapid reductions in serum DHBsAg and DHBV DNA and increases in anti-DHBs antibodies. After treatment, 6/11 ducks experienced a sustained virologic response: DHBsAg and DHBV DNA remained at low or undetectable levels in the serum and no DHBsAg or DHBV core antigen positive hepatocytes and only trace amounts of DHBV total and covalently closed circular DNA (cccDNA) were detected in the liver at 9 or 16 weeks of follow-up. In the remaining 5/11 REP 2055-treated ducks, all markers of DHBV infection rapidly rebounded after treatment withdrawal: At 9 and 16 weeks of follow-up, levels of DHBsAg and DHBcAg and DHBV total and cccDNA in the liver had rebounded and matched levels observed in the control ducks treated with normal saline which remained persistently infected with DHBV. These data demonstrate that treatment with the NAP REP 2055 can lead to sustained control of persistent DHBV infection. These effects may be related to the unique ability of REP 2055 to block release of DHBsAg from infected hepatocytes.     

Streptomyces IHF uses multiple interfaces to bind DNA

BackgroundNucleoid associated proteins (NAPs) are essential for chromosome condensation in bacterial cells. Despite being a diverse group, NAPs share two common traits: they are small, oligomeric proteins and their oligomeric state is critical for DNA condensation. Streptomyces coelicolor IHF (sIHF) is an actinobacterial-specific nucleoid-associated protein that despite its name, shares neither sequence nor structural homology with the well-characterized Escherichia coli IHF. Like E. coli IHF, sIHF is needed for efficient nucleoid condensation, morphological development and antibiotic production in S. coelicolor.MethodsUsing a combination of crystallography, small-angle X-ray scattering, electron microscopy and structure-guided functional assays, we characterized how sIHF binds and remodels DNA.ResultsThe structure of sIHF bound to DNA revealed two DNA-binding elements on opposite surfaces of the helix bundle. Using structure-guided functional assays, we identified an additional surface that drives DNA binding in solution. Binding by each element is necessary for both normal development and antibiotic production in vivo, while in vitro, they act collectively to restrain negative supercoils.ConclusionsThe cleft defined by the N-terminal and the helix bundle of sIHF drives DNA binding, but the two additional surfaces identified on the crystal structure are necessary to stabilize binding, remodel DNA and maintain wild-type levels of antibiotic production. We propose a model describing how the multiple DNA-binding elements enable oligomerization-independent nucleoid condensation.General significanceThis work provides a new dimension to the mechanistic repertoire ascribed to bacterial NAPs and highlights the power of combining structural biology techniques to study sequence unspecific protein-DNA interactions.     

Autophagy induction by vitamin D inhibits both Mycobacterium tuberculosis and human immunodeficiency virus type 1

Low vitamin D levels in human immunodeficiency virus type-1 (HIV) infected persons are associated with more rapid disease progression and increased risk for Mycobacterium tuberculosis infection. We report that physiological concentrations of 1α,25-dihydroxycholecalciferol (1,25D3), the active form of vitamin D, inhibits M. tuberculosis and HIV replication in co-infected macrophages through human cathelicidin microbial peptide-dependent autophagy that requires phagosomal maturation. These findings provide a biological explanation for the importance of vitamin D sufficiency in HIV and M. tuberculosis-infected persons, and provide new insights into novel approaches to prevent and treat HIV infection and related opportunistic infections.