Gene loss and cis-regulatory novelty shaped core histone gene evolution in the apiculate yeast Hanseniaspora uvarum.

Core histone genes display a remarkable diversity of cis-regulatory mechanisms despite their protein sequence conservation. However, the dynamics and significance of this regulatory turnover are not well understood. Here, we describe the evolutionary history of core histone gene regulation across 400 million years in budding yeasts. We find that canonical mode of core histone regulation-mediated by the trans-regulator Spt10-is ancient, likely emerging between 320 and 380 million years ago and is fixed in the majority of extant species. Unexpectedly, we uncovered the emergence of a novel core histone regulatory mode in the Hanseniaspora genus, from its fast-evolving lineage, which coincided with the loss of 1 copy of its paralogous core histone genes. We show that the ancestral Spt10 histone regulatory mode was replaced, via cis-regulatory changes in the histone control regions, by a derived Mcm1 histone regulatory mode and that this rewiring event occurred with no changes to the trans-regulator, Mcm1, itself. Finally, we studied the growth dynamics of the cell cycle and histone synthesis in genetically modified Hanseniaspora uvarum. We find that H. uvarum divides rapidly, with most cells completing a cell cycle within 60 minutes. Interestingly, we observed that the regulatory coupling between histone and DNA synthesis was lost in H. uvarum. Our results demonstrate that core histone gene regulation was fixed anciently in budding yeasts, however it has greatly diverged in the Hanseniaspora fast-evolving lineage.

In diverse conditions, intrinsic chromatin condensates have liquid-like material properties.

Nuclear DNA in eukaryotes is wrapped around histone proteins to form nucleosomes on a chromatin fiber. Dynamic folding of the chromatin fiber into loops and variations in the degree of chromatin compaction regulate essential processes such as transcription, recombination, and mitotic chromosome segregation. Our understanding of the physical properties that allow chromatin to be dynamically remodeled even in highly compacted states is limited. Previously, we reported that chromatin has an intrinsic capacity to phase separate and form dynamic liquid-like condensates, which can be regulated by cellular factors [B. A. Gibson et al., Cell 179, 470-484.e421 (2019)]. Recent contradictory reports claim that a specific set of solution conditions is required for fluidity in condensates that would otherwise be solid [J. C. Hansen, K. Maeshima, M. J. Hendzel, Epigenetics Chromatin 14, 50 (2021); H. Strickfaden et al., Cell 183, 1772-1784.e1713 (2020)]. We sought to resolve these discrepancies, as our ability to translate with confidence these biophysical observations to cells requires their precise characterization. Moreover, whether chromatin assemblies are dynamic or static affects how processes such as transcription, loop extrusion, and remodeling will engage them inside cells. Here, we show in diverse conditions and without specific buffering components that chromatin fragments form phase separated fluids in vitro. We also explore how sample preparation and imaging affect the experimental observation of chromatin condensate dynamics. Last, we describe how liquid-like in vitro behaviors can translate to the locally dynamic but globally constrained chromatin movement observed in cells.

TLR-9 Plays a Role in Mycobacterium leprae-Induced Innate Immune Activation of A549 Alveolar Epithelial Cells.

The respiratory tract is considered the main port of entry of Mycobacterium leprae, the causative agent of leprosy. However, the great majority of individuals exposed to the leprosy bacillus will never manifest the disease due to their capacity to develop protective immunity. Besides acting as a physical barrier, airway epithelium cells are recognized as key players by initiating a local innate immune response that orchestrates subsequent adaptive immunity to control airborne infections. However, to date, studies exploring the interaction of M. leprae with the respiratory epithelium have been scarce. In this work, the capacity of M. leprae to immune activate human alveolar epithelial cells was investigated, demonstrating that M. leprae-infected A549 cells secrete significantly increased IL-8 that is dependent on NF-κB activation. M. leprae was also able to induce IL-8 production in human primary nasal epithelial cells. M. leprae-treated A549 cells also showed higher expression levels of human ß-defensin-2 (hßD-2), MCP-1, MHC-II and the co-stimulatory molecule CD80. Furthermore, the TLR-9 antagonist inhibited both the secretion of IL-8 and NF-κB activation in response to M. leprae, indicating that bacterial DNA sensing by this Toll-like receptor constitutes an important innate immune pathway activated by the pathogen. Finally, evidence is presented suggesting that extracellular DNA molecules anchored to Hlp, a histone-like protein present on the M. leprae surface, constitute major TLR-9 ligands triggering this pathway. The ability of M. leprae to immune activate respiratory epithelial cells herein demonstrated may represent a very early event during infection that could possibly be essential to the generation of a protective response.

In Vitro Anti-proliferative Activity and Mechanism of Action of Anemone nemorosa.

Anemone nemorosa is part of the Ranunculaceae genus Anemone (order Ranunculales) which comprises more than 150 species. Various parts of the plant have been used for the treatment of numerous medical conditions such as headaches, tertian agues, rheumatic gout, leprosy, lethargy, eye inflammation as well as malignant and corroding ulcers. The Anemone plants have been found to contain various medicinal compounds with anti-cancer, immunomodulatory, anti-inflammatory, anti-oxidant and anti-microbial activities. To date there has been no reported evidence of its use in the treatment of cancer. However, due to the reported abundance of saponins which usually exert anti-cancer activity via cell cycle arrest and the induction of apoptosis, we investigated the mode of cell death induced by an aqueous A. nemorosa extract by using HeLa cervical cancer cells. Cisplatin was used as a positive control. With a 50% inhibitory concentration (IC50) of 20.33 ± 2.480 µg/mL, treatment with A. nemorosa yielded a delay in the early mitosis phase of the cell cycle. Apoptosis was confirmed through fluorescent staining with annexin V-FITC. Apoptosis was more evident with A. nemorosa treatment compared to the positive control after 24 and 48 h. Tetramethylrhodamine ethyl ester staining showed a decrease in mitochondrial membrane potential at 24 and 48 h. The results obtained imply that A. nemorosa may have potential anti-proliferative properties.

Envolvimento do receptor TLR-9 na resposta imune do hospedeiro durante o curso da infecção pelo Mycobacterium leprae / Involvement of the TLR9 receptor in host immune responses during infection by Mycobacterium leprae

Mycobacterium leprae, agente etiológico da hanseníase, expressa em abundância uma proteína catiônica semelhante às histonas, denominada Hlp, presente tanto no envelope como no nucleóide bacteriano. O reconhecimento do DNA bacteriano, rico em motivos CpG não metilados, pelo receptor TLR-9 representa uma importante via para a ativação da resposta imune inata, a qual pode levar à eliminação do agente infeccioso ou mediar manifestações patológicas. Foi mostrado ainda que complexos DNA-histona são mais potentes agonistas de TLR-9 que DNA sozinho. Assim, o presente trabalho teve como objetivo investigar o envolvimento do receptor TLR-9 na ativação da resposta imune do hospedeiro durante o curso da infecção pelo M. leprae.Inicialmente foi analisada a participação do TLR-9 na ativação da resposta imune inata em células epiteliais alveolares da linhagem A549 após estímulo com M. leprae. M. leprae foi capaz de induzir aumento das quimiocinas IL-8 eMCP-1 e a transcrição gênica do peptídeo antimicrobiano HbetaD-2 nas células epiteliais. O aumento da expressão de CD80 na superfície celular também foi observada após estímulo com o bacilo. O complexo CpG-Hlp micobacteriano solúvel também induziu aumento na produção de IL-8 nas células A549. Foi observado que o aumento de IL-8 induzido pelo M. leprae ocorre de forma dependente da translocação nuclear do NF-capaB e que o antagonista sintético deTLR-9 afetou a secreção de IL-8 induzida pelo M. leprae. A adição de CpG aoM. smegmatis selvagem, mas não mutante para o gene hlp, aumentou a produção de IL-8 pelas células epiteliais. Em conjunto, esses dados sugerem que as células epiteliais respiratórias podem reconhecer M. leprae via TLR-9 e,assim, participar da resposta imune inata no sítio inicial da infecção. Uma vez que o aparecimento do eritema nodoso hansênico (ENH) está associado a liberação massiva de antígenos micobacterianos, foi investigado o envolvimento do TLR-9 na patogênese do ENH...

Mycobacterium leprae, etiological agent of leprosy, expresses in abundance acationic protein similar to histones, called histone-like protein (Hlp), present inthe envelope as well as in bacterial nucleoid.The recognition of bacterial DNArich in unmethylated CpG motifs by TLR-9 is an important pathway for activationof the innate immune response, which can lead to the elimination of theinfectious agent or mediate pathological manifestations. Moreover, studiesshowed that DNA-histone complexes are more potent agonists of TLR-9 thanDNA alone. This study aimed to investigate the involvement of TLR-9 in theactivation of the host immune response during the course of M. leprae infection.Initially, we analyzed the participation of TLR-9 activation on the innate immuneresponse in A549 alveolar epithelial cells after stimulation with M. leprae. It wasshown that M. leprae was able to induce the chemokines IL-8 and MCP-1, andgene transcription of antimicrobial peptide HbetaD-2 in epithelial cells. Theincrease of CD80 expression on the cell surface was also observed afterstimulation with bacillus. Soluble mycobacterial CpG-Hlp complex also inducedan increase in IL-8 in A549 cells. It was observed that the increase of IL-8,induced by M. leprae, occurs dependently nuclear translocation of NF-capaB andsynthetic TLR-9 antagonist affected IL-8 secretion induced by M. leprae. Theaddition of CpG to wild type M. smegmatis, but not to the mutant gene hlp,increased IL-8 production by epithelial cells. As a whole, these results suggestthat respiratory epithelial cells can recognize M. leprae via TLR-9 and thusparticipate in the innate immune response in the initial infection site. Since theappearance of erythema nodosum leprosum (ENL) is associated with themassive release of mycobacterial antigens, it was investigated the involvementof TLR-9 in the pathogenesis of ENL...

Mycobacterial laminin-binding histone-like protein mediates collagen-dependent cytoadherence

When grown in the presence of exogenous collagen I, Mycobacterium bovis BCG was shown to form clumps. Scanning electron microscopy examination of these clumps revealed the presence of collagen fibres cross-linking the bacilli. Since collagen is a major constituent of the eukaryotic extracellular matrices, we assayed BCG cytoadherence in the presence of exogenous collagen I. Collagen increased the interaction of the bacilli with A549 type II pneumocytes or U937 macrophages, suggesting that BCG is able to recruit collagen to facilitate its attachment to host cells. Using an affinity chromatography approach, we have isolated a BCG collagen-binding protein corresponding to the previously described mycobacterial laminin-binding histone-like protein (LBP/Hlp), a highly conserved protein associated with the mycobacterial cell wall. Moreover, Mycobacterium leprae LBP/Hlp, a well-characterized adhesin, was also able to bind collagen I. Finally, using recombinant fragments of M. leprae LBP/Hlp, we mapped the collagen-binding activity within the C-terminal domain of the adhesin. Since this protein was already shown to be involved in the recognition of laminin and heparan sulphate-containing proteoglycans, the present observations reinforce the adhesive activities of LBP/Hlp, which can be therefore considered as a multifaceted mycobacterial adhesin, playing an important role in both leprosy and tuberculosis pathogenesis.

Mycobacterial laminin-binding histone-like protein mediates collagen-dependent cytoadherence.

When grown in the presence of exogenous collagen I, Mycobacterium bovis BCG was shown to form clumps. Scanning electron microscopy examination of these clumps revealed the presence of collagen fibres cross-linking the bacilli. Since collagen is a major constituent of the eukaryotic extracellular matrices, we assayed BCG cytoadherence in the presence of exogenous collagen I. Collagen increased the interaction of the bacilli with A549 type II pneumocytes or U937 macrophages, suggesting that BCG is able to recruit collagen to facilitate its attachment to host cells. Using an affinity chromatography approach, we have isolated a BCG collagen-binding protein corresponding to the previously described mycobacterial laminin-binding histone-like protein (LBP/Hlp), a highly conserved protein associated with the mycobacterial cell wall. Moreover, Mycobacterium leprae LBP/Hlp, a well-characterized adhesin, was also able to bind collagen I. Finally, using recombinant fragments of M. leprae LBP/Hlp, we mapped the collagen-binding activity within the C-terminal domain of the adhesin. Since this protein was already shown to be involved in the recognition of laminin and heparan sulphate-containing proteoglycans, the present observations reinforce the adhesive activities of LBP/Hlp, which can be therefore considered as a multifaceted mycobacterial adhesin, playing an important role in both leprosy and tuberculosis pathogenesis.

A histone-like protein of mycobacteria possesses ferritin superfamily protein-like activity and protects against DNA damage by Fenton reaction.

Iron is an essential metal for living organisms but its level must be strictly controlled in cells, because ferrous ion induces toxicity by generating highly active reactive oxygen, hydroxyl radicals, through the Fenton reaction. In addition, ferric ion shows low solubility under physiological conditions. To overcome these obstacles living organisms possess Ferritin superfamily proteins that are distributed in all three domains of life: bacteria, archaea, and eukaryotes. These proteins minimize hydroxyl radical formation by ferroxidase activity that converts Fe(2+) into Fe(3+) and sequesters iron by storing it as a mineral inside a protein cage. In this study, we discovered that mycobacterial DNA-binding protein 1 (MDP1), a histone-like protein, has similar activity to ferritin superfamily proteins. MDP1 prevented the Fenton reaction and protects DNA by the ferroxidase activity. The K(m) values of the ferroxidase activity by MDP1 of Mycobacterium bovis bacillus Calmette-Guérin (BCG-3007c), Mycobacterium tuberculosis (Rv2986c), and Mycobacterium leprae (ML1683; ML-LBP) were 0.292, 0.252, and 0.129 mM, respectively. Furthermore, one MDP1 molecule directly captured 81.4±19.1 iron atoms, suggesting the role of this protein in iron storage. This study describes for the first time a ferroxidase-iron storage protein outside of the ferritin superfamily proteins and the protective role of this bacterial protein from DNA damage.

Chromatin condensing functions of the linker histone C-terminal domain are mediated by specific amino acid composition and intrinsic protein disorder.

Linker histones bind to the nucleosomes and linker DNA of chromatin fibers, causing changes in linker DNA structure and stabilization of higher order folded and oligomeric chromatin structures. Linker histones affect chromatin structure acting primarily through their approximately 100-residue C-terminal domain (CTD). We have previously shown that the ability of the linker histone H1 degrees to alter chromatin structure was localized to two discontinuous 24-/25-residue CTD regions (Lu, X., and Hansen, J. C. (2004) J. Biol. Chem. 279, 8701-8707). To determine the biochemical basis for these results, we have characterized chromatin model systems assembled with endogenous mouse somatic H1 isoforms or recombinant H1 degrees CTD mutants in which the primary sequence has been scrambled, the amino acid composition mutated, or the location of various CTD regions swapped. Our results indicate that specific amino acid composition plays a fundamental role in molecular recognition and function by the H1 CTD. Additionally, these experiments support a new molecular model for CTD function and provide a biochemical basis for the redundancy observed in H1 isoform knockout experiments in vivo.

Further biochemical characterization of Mycobacterium leprae laminin-binding proteins.

It has been demonstrated that the alpha2 chain of laminin-2 present on the surface of Schwann cells is involved in the process of attachment of Mycobacterium leprae to these cells. Searching for M. leprae laminin-binding molecules, in a previous study we isolated and characterized the cationic proteins histone-like protein (Hlp) and ribosomal proteins S4 and S5 as potential adhesins involved in M. leprae-Schwann cell interaction. Hlp was shown to bind alpha2-laminins and to greatly enhance the attachment of mycobacteria to ST88-14 Schwann cells. In the present study, we investigated the laminin-binding capacity of the ribosomal proteins S4 and S5. The genes coding for these proteins were PCR amplified and their recombinant products were shown to bind alpha2-laminins in overlay assays. However, when tested in ELISA-based assays and in adhesion assays with ST88-14 cells, in contrast to Hlp, S4 and S5 failed to bind laminin and act as adhesins. The laminin-binding property and adhesin capacity of two basic host-derived proteins were also tested, and only histones, but not cytochrome c, were able to increase bacterial attachment to ST88-14 cells. Our data suggest that the alanine/lysine-rich sequences shared by Hlp and eukaryotic H1 histones might be involved in the binding of these cationic proteins to laminin.

Further biochemical characterization of Mycobacterium leprae laminin-binding proteins

It has been demonstrated that the alpha2 chain of laminin-2 present on the surface of Schwann cells is involved in the process of attachment of Mycobacterium leprae to these cells. Searching for M. leprae laminin-binding molecules, in a previous study we isolated and characterized the cationic proteins histone-like protein (Hlp) and ribosomal proteins S4 and S5 as potential adhesins involved in M. leprae-Schwann cell interaction. Hlp was shown to bind alpha2-laminins and to greatly enhance the attachment of mycobacteria to ST88-14 Schwann cells. In the present study, we investigated the laminin-binding capacity of the ribosomal proteins S4 and S5. The genes coding for these proteins were PCR amplified and their recombinant products were shown to bind alpha2-laminins in overlay assays. However, when tested in ELISA-based assays and in adhesion assays with ST88-14 cells, in contrast to Hlp, S4 and S5 failed to bind laminin and act as adhesins. The laminin-binding property and adhesin capacity of two basic host-derived proteins were also tested, and only histones, but not cytochrome c, were able to increase bacterial attachment to ST88-14 cells. Our data suggest that the alanine/lysine-rich sequences shared by Hlp and eukaryotic H1 histones might be involved in the binding of these cationic proteins to laminin

Mycobacterium smegmatis laminin-binding glycoprotein shares epitopes with Mycobacterium tuberculosis heparin-binding haemagglutinin.

Mycobacterium tuberculosis, the causative agent of tuberculosis, produces a heparin-binding haemagglutinin adhesin (HBHA), which is involved in its epithelial adherence. To ascertain whether HBHA is also present in fast-growing mycobacteria, Mycobacterium smegmatis was studied using anti-HBHA monoclonal antibodies (mAbs). A cross-reactive protein was detected by immunoblotting of M. smegmatis whole-cell lysates. However, the M. tuberculosis HBHA-encoding gene failed to hybridize with M. smegmatis chromosomal DNA in Southern blot analyses. The M. smegmatis protein recognized by the anti-HBHA mAbs was purified by heparin-Sepharose chromatography, and its amino-terminal sequence was found to be identical to that of the previously described histone-like protein, indicating that M. smegmatis does not produce HBHA. Biochemical analysis of the M. smegmatis histone-like protein shows that it is glycosylated like HBHA. Immunoelectron microscopy demonstrated that the M. smegmatis protein is present on the mycobacterial surface, a cellular localization inconsistent with a histone-like function, but compatible with an adhesin activity. In vitro protein interaction assays showed that this glycoprotein binds to laminin, a major component of basement membranes. Therefore, the protein was called M. smegmatis laminin-binding protein (MS-LBP). MS-LBP does not appear to be involved in adherence in the absence of laminin but is responsible for the laminin-mediated mycobacterial adherence to human pneumocytes and macrophages. Homologous laminin-binding adhesins are also produced by virulent mycobacteria such as M. tuberculosis and Mycobacterium leprae, suggesting that this adherence mechanism may contribute to the pathogenesis of mycobacterial diseases.

Linker histones stabilize the intrinsic salt-dependent folding of nucleosomal arrays: mechanistic ramifications for higher-order chromatin folding.

Defined nucleosomal arrays reconstituted from core histone octamers and twelve 208 bp tandem repeats of Lytechinus 5S rDNA (208-12 nucleosomal arrays) possess the ability to form an unstable folded species in MgCl2 whose extent of compaction equals that of canonical higher-order 30 nm diameter chromatin structures [Schwarz, P. M., and Hansen, J. C. (1994) J. Biol. Chem. 269, 16284-16289]. To address the mechanistic functions of linker histones in chromatin condensation, purified histone H5 has been assembled with 208-12 nucleosomal arrays in 50 mM NaCl. Novel purification procedures subsequently were developed that yielded preparations of 208-12 chromatin model systems in which a majority of the sample contained both one histone octamer per 5S rDNA repeat and one molecule of histone H5 per histone octamer. The integrity of the purified 208-12 chromatin has been extensively characterized under low-salt conditions using analytical ultracentrifugation, quantitative agarose gel electrophoresis, electron cryomicroscopy, and nuclease digestion. Results indicate that histone H5 binding to 208-12 nucleosomal arrays constrains the entering and exiting linker DNA in a way that produces structures that are indistinguishable from native chicken erythrocyte chromatin. Folding experiments performed in NaC1 and MgC12 have shown that H5 binding markedly stabilizes both the intermediate and extensively folded states of nucleosomal arrays without fundamentally altering the intrinsic nucleosomal array folding pathway. These results provide new insight into the mechanism of chromatin folding by demonstrating for the first time that distinctly different macromolecular determinants are required for formation and stabilization of higher-order chromatin structures.

Reversible oligonucleosome self-association: dependence on divalent cations and core histone tail domains.

Regularly spaced nucleosomal arrays equilibrate between unfolded and highly folded conformations in <2 mM MgCl2, and self-associate above 2 mM MgCl2 [Schwarz, P. M., & Hansen, J. C. (1994) J. Biol. Chem. 269, 16284-16289]. Here we use analytical and differential sedimentation techniques to characterize the molecular mechanism and determinants of oligonucleosome self-association. Divalent cations induce self-association of intact nucleosomal arrays by binding to oligonucleosomal DNA and neutralizing its negative charge. Neither linker histones nor H2A/H2B dimers are required for Mg2+ - dependent self-association. However, divalent cations are unable to induce self-association of trypsinized nucleosomal arrays lacking their N- and C-terminal core histone tail domains. This suggests that the H3/H4 tail domains directly mediate oligonucleosome self-association through a non-Coulombic-based mechanism. Self-association occurs independently of whether the oligonucleosome monomers are folded or unfolded. The first step in the self-association pathway is strongly cooperative and produces a soluble association intermediate that sediments approximately 10 times faster than the oligonucleosome monomers. The size of the oligonucleosome polymers increases rapidly as a consequence of small increases in the divalent cation concentration, eventually producing polymeric species that sediment at >> 10 000 S. Importantly, all steps in the self-association pathway are freely reversible upon removal of the divalent cations. Taken together, these data indicate that short oligonucleosome fragments composed of only core histone octamers and DNA possess all of the structural features required to achieve chromosome-level DNA compaction. These findings provide a molecular basis for explaining many of the recently uncovered structural features of interphase and metaphase chromosomal fibers.

Modulation of chromatin folding by histone acetylation.

A homogeneous oligonucleosome complex was prepared by reconstitution of highly hyperacetylated histone octamers onto a linear DNA template consisting of 12 tandemly arranged 208-base pair fragments of the 5 S rRNA gene from the sea urchin Lytechinus variegatus. The ionic strength-dependent folding of this oligonucleosome assembly was monitored by sedimentation velocity and electron microscopy. Both types of analysis indicate that under ionic conditions resembling those found in the physiological range and in the absence of histone H1, the acetylated oligonucleosome complexes remain in an extended conformation in contrast to their nonacetylated counterparts. The implications of this finding in the context of a multistate model of chromatin folding (Hansen, J. C., and Ausio, J. (1992) TIBS 197, 187-191) as well as its biological relevance are discussed.

Formation and stability of higher order chromatin structures. Contributions of the histone octamer.

Unique roles have been identified for the histone octamer in the formation and stabilization of higher order chromatin structures. Histone octamers were assembled onto 12 tandem repeats of Lytechinus 5 S rDNA, at either saturating or subsaturating ratios. The extent of oligonucleosome folding and intermolecular association in divalent salts was monitored using analytical and differential sedimentation techniques. Saturated oligonucleosomes (12 nucleosomes/DNA) sedimented at 29 S in very low salt buffer. In 1.0-2.0 mM MgCl2, saturated oligonucleosomes formed a maximally folded 55 S structure whose extent of compaction was equivalent to that of classical higher order 30-nm diameter chromatin structures. These results are in marked contrast to those obtained previously in NaCl, where the maximally folded oligonucleosome species sedimented at only approximately 40 S (Hansen, J. C., Ausio, J., Stanik, V. H., and van Holde, K. E. (1989) Biochemistry 28, 9129-9136). Mg(2+)-dependent formation of the 55 S conformation was inhibited by histone octamer depletion; the maximum sedimentation coefficient observed for rDNA molecules containing 10-11 nucleosomes in 2.0 mM MgCl2 was only 40 S. Above 2.0 mM MgCl2, the equilibrium was progressively shifted toward formation of large associated oligonucleosome species. The implications of these results to the mechanism of chromatin folding and its relationship to the biological activity of the chromatin fiber are discussed.

Class-specific immunoglobulins and antibodies to mycobacterial sonicates and autoantigens in leprosy patients and contacts.

A comprehensive analysis of the humoral immune response in leprosy patients and contacts was undertaken. Class-specific antibodies to four mycobacterial sonicates, three autoantigens and three haptens were estimated by ELISA. It was found that IgG levels varied more extensively than IgM or IgA and that total serum IgG was significantly higher in lepromatous bacterial index positive (LL+ve) and negative LL-ve) leprosy patients than in tuberculoid (TT/BT) patients and controls. The high levels of anti-mycobacterial antibodies found in untreated LL+ve patients were significantly reduced in LL-ve patients after effective chemotherapy. Considerable amount of anti-mycobacterial IgG was also detected in TT/BT patients. Each serum when assayed against sonicates of Mycobacterium leprae, Mycobacterium tuberculosis, ICRC bacilli and BCG gave a similar antibody profile suggesting that these antibodies were directed predominantly against cross-reactive antigens. Up to 75% of LL patients and 35% of TT/BT patients were found to be positive for antibodies to histone, collagen and fibronectin. However, antibodies to several haptens were not detected in any of the patients and controls studied. Taken together, these results suggested that the amount of IgG antibodies is directly correlated with the antigenic load in the system, and that there is no evidence for polyclonal activation. It may be speculated that the regulatory mechanism of antibody production is severely deranged in lepromatous leprosy patients.

Relationship between host histones and armadillo-derived Mycobacterium leprae.

A major protein previously recognized as being primarily associated with the cell walls of Mycobacterium leprae, major wall protein (MWP), is now identified as histoprotein H2b based on N-terminal amino-acid sequencing, electrophoretic comparisons, and several other properties. An avid association between several host/armadillo-derived histones and M. leprae was demonstrated. Since such armadillo-derived M. leprae are the basis of several ongoing vaccine trials, a simple procedure that permits the prompt solubilization and quantification of histones in M. leprae preparations is described. The quantity of histones associated with M. leprae is significant, ranging from 0.6 to 4.8 micrograms of histoprotein H2b per mg of bacteria.