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The basic, intrinsically disordered regions of eukaryotic histones and their bacterial counterparts are presumed to act as signaling hubs to regulate the compaction of chromosomes or nucleoids and various DNA processes such as gene expression, recombination, and DNA replication. Posttranslational modifications (PTMs) on these regions are pivotal in regulating chromosomal or nucleoid compaction and DNA processes. However, the low sequence complexity and the presence of short lysine-rich repeats in the regions have hindered the accurate determination of types and locations of PTMs using conventional proteomic procedures. We described a limited proteolysis protocol using trypsin to analyze PTMs on mycobacterial DNA-binding protein 1 (MDP1), a nucleoid-associated protein in mycobacterial species that possesses an extended, lysine-rich, intrinsically disordered region in its C-terminal domain. This limited proteolysis approach successfully revealed significant methylation on many lysine residues in the C-terminal domain of MDP1 purified from Mycobacterium tuberculosis , which was lacking in the corresponding region of recombinant MDP1 expressed in Escherichia coli. • Limited proteolysis protocol for PTM analysis in bottom-up proteomics is described. • It unveils lysine-methylation of IDR region in DNA-binding protein 1. • Lysine-methylation occurs specifically on proteins purified from M. tuberculosis. • The methylation confers resistance to proteolytic digestion on the protein. [ABSTRACT FROM AUTHOR]

DNA is basically an intracellular molecule that stores genetic information and carries instructions for growth and reproduction in all cellular organisms. However, in some bacteria, DNA has additional roles outside the cells as extracellular DNA (eDNA), which is an essential component of biofilm formation and hence antibiotic tolerance. Mycobacteria include life-threating human pathogens, most of which are slow growers. However, little is known about the nature of pathogenic mycobacteria's eDNA. Here we found that eDNA is present in slow-growing mycobacterial pathogens, such as Mycobacterium tuberculosis, M. intracellulare, and M. avium at exponential growth phase. In contrast, eDNA is little in all tested rapid-growing mycobacteria. The physiological impact of disrupted eDNA on slow-growing mycobacteria include reduced pellicle formation, floating biofilm, and enhanced susceptibility to isoniazid and amikacin. Isolation and sequencing of eDNA revealed that it is identical to the genomic DNA in M. tuberculosis and M. intracellulare. In contrast, accumulation of phage DNA in eDNA of M. avium, suggests that the DNA released differs among mycobacterial species. Our data show important functions of eDNA necessary for biofilm formation and drug tolerance in slow-growing mycobacteria. [ABSTRACT FROM AUTHOR]

Tuberculosis remains a large health threat, despite the availability of the tuberculosis vaccine, BCG. As BCG efficacy gradually decreases from adolescence, BCG-Prime and antigen-booster may be an efficient strategy to confer vaccine efficacy. Mycobacterial DNA-binding protein 1 (MDP1, namely Rv2986c, hupB or HU) is a major Mycobacterium tuberculosis protein that induces vaccine-efficacy by co-administration with CpG DNA. To produce MDP1 for booster-vaccine use, we have created recombinant MDP1 produced in both Escherichia coli (eMDP1) and Mycolicibacterium smegmatis (mMDP1), an avirulent rapid-growing mycobacteria. We tested their immunogenicity by checking interferon (IFN)-gamma production by stimulated peripheral blood cells derived from BCG-vaccinated individuals. Similar to native M. tuberculosis MDP1, we observed that most lysin resides in the C-terminal half of mMDP1 are highly methylated. In contrast, eMDP1 had less post-translational modifications and IFN-gamma stimulation. mMDP1 stimulated the highest amount of IFN-gamma production among the examined native M. tuberculosis proteins including immunodominant MPT32 and Antigen 85 complex. MDP1-mediated IFN-gamma production was more strongly enhanced when combined with a new type of CpG DNA G9.1 than any other tested CpG DNAs. Taken together, these results suggest that the combination of mMDP1 and G9.1 possess high potential use for human booster vaccine against tuberculosis. [ABSTRACT FROM AUTHOR]

Background: Mycobacterium intracellulare is a major etiological agent of Mycobacterium avium-intracellulare pulmonary disease (MAC-PD). However, the characteristics of the virulence of M. intracellulare and the in vivo chemotherapeutic efficacy remain unclear. In this study, we examined the virulence of nine M. intracellulare strains with different clinical phenotypes and genotypes in C57BL/6 mice. Results: We classified three types of virulence phenotypes (high, intermediate, and low) based on the kinetics of the bacterial load, histological lung inflammation, and neutrophilic infiltration. High virulence strains showed more severe neutrophilic infiltration in the lungs than intermediate and low virulence strains, with 6.27-fold and 11.0-fold differences of the average percentage of neutrophils in bronchoalveolar lavage fluid, respectively. In particular, the high virulence strain M.i.198 showed the highest mortality in mice, which corresponded to the rapid progression of clinical disease. In mice infected with the drug-sensitive high virulence strain M019, clarithromycin-containing chemotherapy showed the highest efficacy. Monotherapy with rifampicin exacerbated lung inflammation with increased lymphocytic and neutrophilic infiltration into the lungs. Conclusions: The virulence phenotypes of clinical strains of M. intracellulare were diverse, with high virulence strains being associated with neutrophilic infiltration and disease progression in infected mice. These high virulence strains were proposed as a useful subject for in vivo chemotherapeutic experiments. [ABSTRACT FROM AUTHOR]

The mechanisms for the cytotoxicity of staphylococcal Panton- Valentine leukocidin ( PVL), a pore-forming toxin consisting of Luk S- PV and Luk F- PV, in human immune cells are still unclear. Because Luk S- PV binds to ganglioside GM1, a constituent of detergent-resistant membrane microdomains ( DRMs) of the plasma membrane, the role of DRMs in PVL cytotoxicity was examined in human polymorphonuclear neutrophils ( PMNs), monocytes, HL-60 cells, and THP-1 cells. PVL binding capacities in HL-60 and THP-1 cells were higher than those in PMNs and monocytes; however, the PVL concentration to obtain more than 80% cell lysis in HL-60 cells was 10 times higher than that in PMNs and PVL even at such concentration induced < 10% cell lysis in THP-1 cells. After incubation of PMNs with Luk S- PV, more than 90% of Luk S- PV bound to the detergent-soluble membranes. Subsequent incubation with Luk F- PV at 4 °C induced the accumulation of more than 70% of PVL components and 170- to 220-kDa complex formation in DRMs in an actin-independent manner. However, only 30% of PVL was found, and complex formation was under detectable level in DRMs in HL-60 cells. PVL did not accumulate in DRMs in THP-1 cells. Our observations strongly indicate that PVL accumulation in DRMs is essential for PVL cytotoxicity. [ABSTRACT FROM AUTHOR]

Methicillin-resistant Staphylococcus aureus (MRSA) is able to persist not only in hospitals (with a high level of antimicrobial agent use) but also in the community (with a low level of antimicrobial agent use). The former is called hospital-acquired MRSA (HA-MRSA) and the latter community-acquired MRSA (CA-MRSA). It is believed MRSA clones are generated from S. aureus through insertion of the staphylococcal cassette chromosome mec (SCC mec), and outbreaks occur as they spread. Several worldwide and regional clones have been identified, and their epidemiological, clinical, and genetic characteristics have been described. CA-MRSA is likely able to survive in the community because of suitable SCC mec types (type IV or V), a clone-specific colonization/infection nature, toxin profiles (including Pantone-Valentine leucocidin, PVL), and narrow drug resistance patterns. CA-MRSA infections are generally seen in healthy children or young athletes, with unexpected cases of diseases, and also in elderly inpatients, occasionally surprising clinicians used to HA-MRSA infections. CA-MRSA spreads within families and close-contact groups or even through public transport, demonstrating transmission cores. Re-infection (including multifocal infection) frequently occurs, if the cores are not sought out and properly eradicated. Recently, attention has been given to CA-MRSA (USA300), which originated in the US, and is growing as HA-MRSA and also as a worldwide clone. CA-MRSA infection in influenza season has increasingly been noted as well. MRSA is also found in farm and companion animals, and has occasionally transferred to humans. As such, the epidemiological, clinical, and genetic behavior of CA-MRSA, a growing threat, is focused on in this study. [ABSTRACT FROM AUTHOR]

When macrophages phagocytose chitin (N-acetyl-D-glucosamine polymer) microparticles, mitogen-activated protein kinases (MAPK) are immediately activated, followed by the release of Thi cytokines, but not IL-10. To determine whether phagocytosis and macrophage activation in response to chitin microparticles are dependent on membrane cholesterol, RAW264.7 macrophages were treated with methyl-β-cytodextrin (MBCD) and stimulated with chitin. These results were compared with the corresponding effects of bacterial components including heat-killed (HK) Mycobacterium bovis bacillus Calmette-Guèrin (BCG) and an ohgodeoxynucleotide (ODN) of bacterial DNA (CpG-ODN). The MBCD treatment did not alter chitin binding or the phagocytosis of chitin particles 20 mm after stimulation. At the same time, however, chitin-induced phosphorylation of cellular MAPK was accelerated and enhanced in an MBCD dose-dependent manner. The increased phosphorylation was also observed for chitin phagosome-associated p38 and ERK1/2. In contrast, CpG-ODN and HK-BCG induced activation of MAPK in MBCD-treated cells at levels comparable to, or only slightly more than, those of control cells. We also found that MBCD treatment enhanced the production of tumor necrosis factor-α (TNF-α) and the expression of cyclooxygenase-2 (COX-2) in response to chitin microparticles. In neither MBCD- nor saline-treated macrophages, did chitin particles induce detectable IL-10 mRNA synthesis. CpG-ODN induced TNF-α production, and COX-2 expression were less sensitive to MBCD treatment. Among the agonists studied, our results indicate that macrophage activation by chitin microparticles was most sensitive to cholesterol depletion, suggesting that membrane structures integrated by cholesterol are important for physiological regulation of chitin microparticle-induced cellular activation. [ABSTRACT FROM AUTHOR]

The article presents a study on the assembly of staphylococcal leukocidin into a pore-forming oligomer. Staphylococcal leukocidin (Luk) consists of LukS and LukF. It was found that LukS and LukF assembles into hetero-oligomeric pore complexes on the detergent-resistant membrane microdomains, lipid rafts of human polymorphonuclear leukocytes (HPMNLs). It was concluded that assembly of LukS and LukF into the pore-complex occurs in lipid rafts in HPMNLs.

Abstract: A practical and highly effective Th1 adjuvant should induce Th1 cytokines (IL-12, IL-18, and TNF-α) but not the Th2 cytokine IL-10, an inhibitor of Th1 responses. In this study, phagocytosis of N-acetyl-d-glucosamine polymer (chitin) particles by RAW 264.7 cells, a murine macrophage-like cell line, resulted in phosphorylation of MAPK (p38, Erk 1/2, and JNK), and production of relatively high levels of TNF-α and COX-2 with increased PGE2 release. Similar results were observed in response to oligonucleotides with CpG motifs, mycobacterial components and endotoxin. However, these bacterial components also induced a large amount of IL-10. Chitin particles, in contrast, failed to induce detectable levels of IL-10, although the production of high levels of PGE2 and TNF-α and the activation of MAPK’s are potentially positive signals for IL-10 production. Thus, our results indicate that chitin particles act as a unique Th1 adjuvant for macrophages without inducing increased production of IL-10. [Copyright &y& Elsevier]

Isoprenoids with reduced Z,E‐mixed prenyl groups are found in various organisms. To date, only polyprenol reductases (PR‐Dol) involved in dolichol biosynthesis have been identified as enzymes capable of reducing Z,E‐mixed prenyl groups. Although C35‐isoprenoids with reduced Z,E‐mixed prenyl groups are found in mycobacteria, Z,E‐mixed heptaprenyl reductase (HepR) remains unidentified. In the present study, the identification and functional analysis of HepR was performed. No PR‐Dol homolog gene was detected in the genome of Mycolicibacterium vanbaalenii. However, a homolog of geranylgeranyl reductase (GGR), which reacts with an all‐E prenyl group as a substrate, was encoded in the genome; thus, we analyzed it as a HepR candidate. In vitro enzymatic assay and in vivo gene suppression analysis identified the GGR homolog as HepR and revealed that HepR catalyzes the reduction of ω‐ and E‐ prenyl units in Z,E‐mixed heptaprenyl diphosphates, and C35‐isoprenoids are mainly biosynthesized using E,E,E‐geranylgeranyl diphosphate as a precursor. Thus, it was demonstrated that the Z,E‐mixed prenyl reductase family exists in the GGR homologs. To the best of our knowledge, this is the first identification of a new type of Z,E‐mixed prenyl reductase with no sequence homology to PR‐Dol. The substrate specificity of HepR significantly differed from that of GGR, suggesting that it is a new enzyme. HepR homologs are widely distributed in mycobacterial genomes, and lipid analysis suggests that many strains, including pathogenic species, produce HepR metabolites. The discovery of this new enzyme will promote further research on Z,E‐mixed isoprenoids. [ABSTRACT FROM AUTHOR]

The article cites a research study that analyzes the effects of Lactobacillus johnsonii Lal (LC1) on Helicobacter pylon colonization in the stomach. that Helicobacter pylori, a spiral-shaped, Gram-negative microaerophilic bacterium, colonization and gastritis in LC1-inoculated Mongolian gerbils were significantly less intense than those in the control animals.

In this study, we are constructing a database useful for understanding long-term seismic activity in Japan by investigating and analyzing high-quality historical diaries that include descriptions of the intensity of earthquakes and the resulting damage. We are also trying to determine the hypocenter of the described earthquakes in this historical database by comparing historical earthquakes to modern earthquakes of which hypocenters are analyzed for observed seismic intensity maps. In evaluating future earthquake occurrence risk in the Japanese archipelago, where earthquakes occur frequently, it is very important to understand long-term seismic activity. The historical diaries are essential to understanding daily seismic activity including small- and medium-scale earthquakes in the pre-modern era, which is hundreds of years before the development of modern earthquake observation instruments. The daily earthquake information between A.D. 1853 and 1856 were surveyed at 28 sites that are distributed widely in Japan and about 2,800 felt earthquake descriptions were registered in the historical database. The number of survey sites was 13 in our previous study (Nishiyama et al., 2018, EGU) and it was more than doubled to understand the whole image of seismic intensity distribution and to increase the number of events that can be covered by the historical database. To test the capability of earthquake detection through the present distribution of the survey sites, we used a modern earthquake record database provided by Japan Meteorological Agency (JMA). In the JMA database, we first found the nearby observation points of the 28 survey sites of the historical database. Then, we counted the number of earthquakes that have the seismic intensity of one and above at any of the 28 points. As a results, 5,847 (9.5%) events could be detected from the whole 61,352 events of the JMA database during A.D. 2000-2016. For only 2016, 273 (4.1%) events out of 6,712 events could be detected. To understand the seismic activity from the limited number of the survey sites of the historical database, we are trying to determine the hypocenter of the earthquake events recorded in the database. To do this, there is a challenge to determine the hypocenter only from the seismic intensity, as there is no seismic wave record. To check the possibility of this approach, we compared the events in 2016 to the events in 2000-2015 using the JMA database. For every one of the 273 events detected by the 28 observation points in 2016, we estimated the hypocenter of each event by finding the event that has the most similar earthquake intensity distribution in 2000-2015 and referring the hypocenter of the found similar event. As a result, 141 (51.6%) events showed less than 100-km difference between the locations of estimated and true epicenters. For the estimated magnitude, the average error was 19.1% and the standard deviation of the error was 16.1%. After refining the methodology, we will estimate the hypocenters of historical events. [ABSTRACT FROM AUTHOR]

Background: Mycobacterium intracellulare is a representative etiological agent of emerging pulmonary M. avium-intracellulare complex disease in the industrialized countries worldwide. The recent genome sequencing of clinical strains isolated from pulmonary M. avium-intracellulare complex disease has provided insight into the genomic characteristics of pathogenic mycobacteria, especially for M. avium; however, the genomic characteristics of M. intracellulare remain to be elucidated. Results: In this study, we performed comparative genomic analysis of 55 M. intracellulare and related strains such as M. paraintracellulare (MP), M. indicus pranii (MIP) and M. yonogonense. Based on the average nucleotide identity, the clinical M. intracellulare strains were phylogenetically grouped in two clusters: (1) the typical M. intracellulare (TMI) group, including ATCC13950 and virulent M.i.27 and M.i.198 that we previously reported, and (2) the MP-MIP group. The alignment of the genomic regions was mostly preserved between groups. Plasmids were identified between groups and subgroups, including a plasmid common among some strains of the M.i.27 subgroup. Several genomic regions including those encoding factors involved in lipid metabolism (e.g., fadE3, fadE33), transporters (e.g., mce3), and type VII secretion system (genes of ESX-2 system) were shown to be hypermutated in the clinical strains. M. intracellulare was shown to be pan-genomic at the species and subspecies levels. The mce genes were specific to particular subspecies, suggesting that these genes may be helpful in discriminating virulence phenotypes between subspecies. Conclusions: Our data suggest that genomic diversity among M. intracellulare, M. paraintracellulare, M. indicus pranii and M. yonogonense remains at the subspecies or genovar levels and does not reach the species level. Genetic components such as mce genes revealed by the comparative genomic analysis could be the novel focus for further insight into the mechanism of human pathogenesis for M. intracellulare and related strains. [ABSTRACT FROM AUTHOR]

Accurate point-of-care testing (POCT) is critical for managing tuberculosis (TB). However, current antibody-based diagnosis shows low specificity and sensitivity. To find proper antigen candidates for TB diagnosis by antibodies, we assessed IgGs responsiveness to Mycobacterium tuberculosis proteins in pulmonary TB (PTB) patients. We employed major secreted proteins, such as Rv1860, Ag85C, PstS1, Rv2878c, Ag85B, and Rv1926c that were directly purified from M. tuberculosis. In the first screening, we found that IgG levels were significantly elevated in PTB patients only against Rv1860, PstS1, and Ag85B among tested antigens. However, recombinant PstS1 and Ag85B from Escherichia coli (E. coli) couldn't distinguish PTB patients and healthy controls (HC). Recombinant Rv1860 was not checked due to its little expression. Then, the 59 confirmed PTB patients from Soetomo General Academic Hospital, Surabaya, Indonesia, and 102 HC were tested to Rv1860 and Ag85B only due to the low yield of the PstS1 from M. tuberculosis. The ROC analysis using native Ag85B and Rv1860 showed an acceptable area under curve for diagnosis, which is 0.812 (95% CI 0.734–0.890, p < 0.0001) and 0.821 (95% CI 0.752–0.890, p < 0.0001). This study indicates that taking consideration of native protein structure is key in developing TB's POCT by antibody-based diagnosis. [ABSTRACT FROM AUTHOR]

Accurate point-of-care testing (POCT) is critical for managing tuberculosis (TB). However, current antibody-based diagnosis shows low specificity and sensitivity. To find proper antigen candidates for TB diagnosis by antibodies, we assessed IgGs responsiveness to Mycobacterium tuberculosis proteins in pulmonary TB (PTB) patients. We employed major secreted proteins, such as Rv1860, Ag85C, PstS1, Rv2878c, Ag85B, and Rv1926c that were directly purified from M. tuberculosis. In the first screening, we found that IgG levels were significantly elevated in PTB patients only against Rv1860, PstS1, and Ag85B among tested antigens. However, recombinant PstS1 and Ag85B from Escherichia coli (E. coli) couldn't distinguish PTB patients and healthy controls (HC). Recombinant Rv1860 was not checked due to its little expression. Then, the 59 confirmed PTB patients from Soetomo General Academic Hospital, Surabaya, Indonesia, and 102 HC were tested to Rv1860 and Ag85B only due to the low yield of the PstS1 from M. tuberculosis. The ROC analysis using native Ag85B and Rv1860 showed an acceptable area under curve for diagnosis, which is 0.812 (95% CI 0.734–0.890, p < 0.0001) and 0.821 (95% CI 0.752–0.890, p < 0.0001). This study indicates that taking consideration of native protein structure is key in developing TB's POCT by antibody-based diagnosis. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is fatal in elephants, hence protecting elephants from TB is key not only in the conservation of this endangered animal, but also to prevent TB transmission from elephants to humans. Most human TB cases arise from long-term asymptomatic infections. Significant diagnostic challenges remain in the detection of both infection and disease development from latency in elephants due to their huge bodies. In this study, we assessed cryopreserved sera collected for over 16 years, from the first Japanese treatment case of elephant TB. Semi-quantification of IgG levels to 11 proteins showed high detection levels of 3 proteins, namely ESAT6/CFP10, MPB83 and Ag85B. The level of IgG specific to these 3 antigens was measured longitudinally, revealing high and stable ESAT6/CFP10 IgG levels regardless of onset or treatment. Ag85B-specifc IgG levels were largely responsive to onset or treatment, while those of MPB83 showed intermediate responses. These results suggest that ESAT6/CFP10 is immunodominant in both asymptomatic and symptomatic phases, making it useful in the detection of infection. On the other hand, Ag85B has the potential to be a marker for the prediction of disease onset and in the evaluation of treatment effectiveness in elephants. [ABSTRACT FROM AUTHOR]

Human intelectin-1 (hITLN-1) is a 120-kDa lectin recognizing galactofuranosyl residues found in cell walls of various microorganisms but not in mammalian tissues. Although mouse intelectin-1 (mITLN-1) has been identified previously, its biochemical properties and functional characteristics have not been studied. Therefore, we have compared structures and saccharide-binding specificities of hITLN-1 and mITLN-1 using recombinant proteins produced by mammalian cells. Recombinant hITLN-1 is a trimer, disulfide-linked through Cys-31 and Cys-48, and N-glycosylated at Asn-163. Despite 84.9% amino acid identity to hITLN-1, recombinant and intestinal mITLN-1 are unglycosylated 30-kDa monomers. Recombinant hITLN-1, as well as recombinant and intestinal mITLN-1 were purified by Ca2+-dependent adsorption to galactose-Sepharose. In competitive binding studies, hITLN-1 was eluted from galactose-Sepharose by 100 mM 2-deoxygalactose, a galactofuranosyl disaccharide, d-xylose, and both d- and l-ribose. In contrast, mITLN-1 was partially eluted by the galactofuranosyl disaccharide, and only minimally by the other saccharides indicating that the two intelectins have different saccharide-binding specificities. When the N- and C-terminal regions of hITLN-1 were replaced, respectively, with those of mITLN-1, galactose-Sepharose binding was associated with the C-terminal regions. Finally, hITLN-1 binding to galactose-Sepharose was not affected by the substitution of the Cys residues in the N-terminal region that are necessary for oligomer formation, nor was it affected by the removal of the N-linked oligosaccharide at Asn-163. Although both hITLN-1 and mITLN-1 recognize galactofuranosyl residues, our comparative studies, taken together, demonstrate that these intelectins have different quaternary structures and saccharide-binding specificities. [ABSTRACT FROM PUBLISHER]

The global incidence of the human nontuberculous mycobacteria (NTM) disease is rapidly increasing. However, knowledge of gene essentiality under optimal growth conditions and conditions relevant to the natural ecology of NTM, such as hypoxia, is lacking. In this study, we utilized transposon sequencing to comprehensively identify genes essential for growth in Mycobacterium intracellulare. Of 5126 genes of M. intracellulare ATCC13950, 506 genes were identified as essential genes, of which 280 and 158 genes were shared with essential genes of M. tuberculosis and M. marinum, respectively. The shared genes included target genes of existing antituberculous drugs including SQ109, which targets the trehalose monomycolate transporter MmpL3. From 175 genes showing decreased fitness as conditionally essential under hypoxia, preferential carbohydrate metabolism including gluconeogenesis, glyoxylate cycle and succinate production was suggested under hypoxia. Virulence-associated genes including proteasome system and mycothiol redox system were also identified as conditionally essential under hypoxia, which was further supported by the higher effective suppression of bacterial growth under hypoxia compared to aerobic conditions in the presence of these inhibitors. This study has comprehensively identified functions essential for growth of M. intracellulare under conditions relevant to the host environment. These findings provide critical functional genomic information for drug discovery. [ABSTRACT FROM AUTHOR]

Tuberculosis causes the highest mortality among all single infections. Asymptomatic tuberculosis, afflicting one third of the global human population, is the major source as 5–10% of asymptomatic cases develop active tuberculosis during their lifetime. Thus it is one of important issues to develop diagnostic tools for accurately detecting asymptomatic infection. Mycobacterial DNA-binding protein 1 (MDP1) is a major protein in persistent Mycobacterium tuberculosis and has potential for diagnostic use in detecting asymptomatic infection. However, a previous ELISA-based study revealed a specificity problem; IgGs against MDP1 were detected in both M. tuberculosis-infected and uninfected individuals. Although the tertiary structures of an antigen are known to influence antibody recognition, the MDP1 structural details have not yet been investigated. The N-terminal half of MDP1, homologous to bacterial histone-like protein HU, is predicted to be responsible for DNA-binding, while the C-terminal half is assumed as totally intrinsically disordered regions. To clarify the relationship between the MDP1 tertiary structure and IgG recognition, we refined the purification method, which allow us to obtain a recombinant protein with the predicted structure. Furthermore, we showed that an IgG-ELISA using MDP1 purified by our refined method is indeed useful in the detection of asymptomatic tuberculosis. [ABSTRACT FROM AUTHOR]

The tested sera in A-I were from 12 past tuberculosis (Past TB) patients, 8 active tuberculosis patients (Active TB), and 12 healthy control (Healthy) individuals (also for J). (J) IgG responses to rFull-MDP1 of the 10 healthy control and those to rFull-MDP1, rN-MDP1, and rC-MDP1 of 23 other past TB individuals. There are a number of errors in the caption for Fig 4, "ELISAs to detect human MDP1, CFP10, ESTA6, Ag85, and PPD-specific IgG antibodies.". [Extracted from the article]

Methicillin-resistant Staphylococcus aureus (MRSA) is a common multidrug-resistant (MDR) pathogen. We herein discussed MRSA and its infections in Krasnoyarsk, Siberian Russia between 2007 and 2011. The incidence of MRSA in 3,662 subjects was 22.0% and 2.9% for healthcare- and community-associated MRSA (HA- and CA-MRSA), respectively. The 15-day mortality rates for MRSA hospital- and community-acquired pneumonia (HAP and CAP) were 6.5% and 50%, respectively. MRSA CAP cases included pediatric deaths; of the MRSA pneumonia episodes available, ≥27.3% were associated with bacteremia. Most cases of HA-MRSA examined exhibited ST239/spa3(t037)/SCCmecIII.1.1.2 (designated as ST239Kras), while all CA-MRSA cases examined were ST8/spa1(t008)/SCCmecIV.3.1.1(IVc) (designated as ST8Kras). ST239Kras and ST8Kras strongly expressed cytolytic peptide (phenol-soluble modulin α, PSMα; and δ-hemolysin, Hld) genes, similar to CA-MRSA. ST239Kras pneumonia may have been attributed to a unique set of multiple virulence factors (MVFs): toxic shock syndrome toxin-1 (TSST-1), elevated PSMα/Hld expression, α-hemolysin, the staphylococcal enterotoxin SEK/SEQ, the immune evasion factor SCIN/SAK, and collagen adhesin. Regarding ST8Kras, SEA was included in MVFs, some of which were common to ST239Kras. The ST239Kras (strain OC3) genome contained: a completely unique phage, φSa7-like (W), with no att repetition; S. aureus pathogenicity island SaPI2R, the first TSST-1 gene-positive (tst+) SaPI in the ST239 lineage; and a super copy of IS256 (≥22 copies/genome). ST239Kras carried the Brazilian SCCmecIII.1.1.2 and United Kingdom-type tst. ST239Kras and ST8Kras were MDR, with the same levofloxacin resistance mutations; small, but transmissible chloramphenicol resistance plasmids spread widely enough to not be ignored. These results suggest that novel MDR and MVF+ HA- and CA-MRSA (ST239Kras and ST8Kras) emerged in Siberian Russia (Krasnoyarsk) associated with fatal pneumonia, and also with ST239Kras, a new (Siberian Russian) clade of the ST239 lineage, which was created through stepwise evolution during its potential transmission route of Brazil-Europe-Russia/Krasnoyarsk, thereby selective advantages from unique MVFs and the MDR. [ABSTRACT FROM AUTHOR]

The 2011 Tohoku earthquake generated a huge, destructive tsunami with coastal heights of up to 40 m recorded along northern Honshu. The Sanriku coast experienced similar tsunamis and damage from the 1896 and 1933 Sanriku earthquakes, whereas the only damaging tsunamis on both the Ibaraki and Chiba coasts in the previous century were from the 1960 and 2010 Chile earthquakes. We summarized 12 field surveys in which the height of the 2011 tsunami was recorded at 296 points, along with descriptions of the survey method, reliability, and accuracy. We then compared them with the above-mentioned tsunamis at locations for which specific measurements were given in previous reports. On the Sanriku coast, the 2011 tsunami heights are positively correlated with the previous Sanriku tsunamis, indicating that local variations resulting from the irregular coastline were more dominant factors than the earthquake location, type, or magnitude for near-field tsunamis. The correlations with the Chilean tsunami heights are less significant due to the differences between the local and trans-Pacific tsunamis. On the Ibaraki and Chiba coasts, the 2011 Tohoku and the two Chilean tsunami heights are positively correlated, showing the general decrease toward the south with small local variations such as large heights near peninsulas. [ABSTRACT FROM AUTHOR]

Iliopsoas abscesses (IPAs) from methicillin-resistant Staphylococcus aureus (MRSA) are rare; however, IPAs from community-associated MRSA (CA-MRSA) may be increasing. In Japan, we previously described an adolescent athlete case of Panton-Valentine leukocidin (PVL)-positive ST30 CA-MRSA (strain NN12). In this study, we describe an IPA and discitis case from a variant of the successful PVL-negative CA-MRSA clone (ST8 CA-MRSA/J) in Japan. The patient was a 62-year-old man with intractable eczema, who had been diagnosed with IPAs and discitis (L1-L2). CA-MRSA (strain NN55) was isolated from blood, pus, and joint fluid. The invasive infections seemed to have originated in his intractable eczema, and the characteristics of this case, systemic myalgia and marked thrombocytopenia, seemed to have been caused by an exotoxin. Molecular genetic analysis revealed that NN55 possessed genotype ST8/ spa606(t1767)/ agr1/CoaIII and SCC mecIV of a novel subtype (encoding new cell-wall-anchored surface protein/J [CWASP/J]), exhibited enhanced expression of the cytolytic peptide genes, psmα and hld, and was resistant to gentamicin (caused by aacA- aphD), similar to ST8 CA-MRSA/J; however, NN55 lacked pathogenicity island SaPIj50 [carrying tst, encoding toxic shock syndrome toxin-1 (TSST-1)] of ST8 CA-MRSA/J, suggesting a variant (ST8 CA-MRSA/Jv). Strains NN12 and NN55 both caused bacteremia, IPAs, and adjacent musculoskeletal infections, preceded by intractable skin infections, and possessed high potential for adherence and enhanced expression of psmα and hld. The data suggest the role of a combination of CA-MRSA adhesin/cytolytic peptides (not PVL or TSST-1) in the pathogenesis of IPAs (and perhaps of systemic myalgia and marked thrombocytopenia). [ABSTRACT FROM AUTHOR]

A 17-year-old female patient (a basketball player) suffered from recurrent pelvic abscesses from methicillin-resistant Staphylococcus aureus (MRSA). The first episode, from strain NN12, occurred in October 2004. Her cutaneous abscesses complicated into systemic progression to osteomyelitis and multifocal pelvic abscesses, adjacent to the sacroiliac joint. The second episode, abscesses at tissues adjacent to the sacroiliac joint from strain NN31A, occurred late in February 2005. The third episode, from strain NN31B, occurred on July 30, 2005, repeating the second episode. Three MRSA strains were identical in terms of genotypes (belonging to Panton-Valentine leukocidin [PVL]-positive ST30 community-acquired MRSA, CA-MRSA), pulsed-field gel electrophoresis patterns, and peptide cytolysin gene ( psmα) expression levels. The three MRSA strains exhibited superior THP-1 cell invasion ability over hospital-acquired MRSA (New York/Japan clone). The data suggest that PVL-positive ST30 CA-MRSA, with high levels of cell invasion and peptide cytolysins, causes recurrence of pelvic abscesses in a healthy adolescent. [ABSTRACT FROM AUTHOR]

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA), which often produces Panton-Valentine leukocidin (PVL), is an emerging threat in the community. In Japan, for example, PVL-positive ST8 CA-MRSA (USA 300), which originated from the United States, persisted in families for a year and caused severe invasive infection in a child. In this study, we describe a long-term familial infection cluster caused by novel PVL-positive CA-MRSA, which most probably originated from India. This MRSA persisted in related families for more than 2 years with colonization of, for example, the nares and cheek. At least 6 of 12 members (50%) developed deep cutaneous abscesses, including recurrent and multifocal abscesses, every 1.2 months on average. All MRSA isolates from colonization and abscesses were the same, albeit with a variant in pulsed-field gel electrophoresis analysis. The MRSA exhibited the genotype ST22/ spa113(t005)/SCC mecIVa/coagulase gene ( coa) novel type and strong hemolysis activity. Moreover, the MRSA exhibited high biofilm formation (which was markedly enhanced by sub-MICs of oxacillin). Some patients were treated with levofloxacin, with successful MRSA eradication even from the whole body surface sites; however, short-term patient follow-up was not sufficient to demonstrate eradication of the familial infection cluster. The data suggest that PVL-positive novel ST22 CA-MRSA emerged in Japan, causing a long-term familial infection cluster, and that the success of ST22 CA-MRSA as both a colonizer and a pathogen could result from the combination of its strong biofilm formation and other virulence factors. A long-term patient (or carrier) follow-up is needed in the community. [ABSTRACT FROM AUTHOR]

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has become a major concern worldwide. In the United States, ST8 CA-MRSA with SCC mecIVa (USA300) has been predominant, affecting the entire United States. In this study, we investigated Japanese ST8 CA-MRSA with new SCC mecIVl (designated ST8 CA-MRSA/J), which has emerged in Japan since 2003. Regarding community spread and infections, ST8 CA-MRSA/J spread in 16.2-34.4% as a major genotype in the community in Japan, and was associated with skin and soft tissue infections (SSTIs), colitis, and invasive infections (sepsis, epidural abscesses, and necrotizing pneumonia), including influenza prodrome cases and athlete infections, similar to USA300. It spread to even public transport and Hong Kong through a Japanese family. Regarding genetic diversity, ST8 CA-MRSA/J included ST and spa variants and was classified into at least three pulsed-field gel electrophoresis types, ST8 Jα to γ. Of those, ST8 Jβ was associated with severe invasive infections. As for genomics, ST8 CA-MRSA/J showed high similarities to USA300, but with marked diversity in accessory genes; e.g., ST8 CA-MRSA/J possessed enhanced cytolytic peptide genes of CA-MRSA, but lacked the Panton-Valentine leukocidin phage and arginine catabolic mobile element, unlike USA300. The unique features of ST8 CA-MRSA/J included a novel mosaic SaPI (designated SaPIj50) carrying the toxic shock syndrome toxin-1 gene with high expression; the evolution included salvage (through recombination) of hospital-acquired MRSA virulence. The data suggest that ST8 CA-MRSA/J has become a successful native clone in Japan, in association with not only SSTIs but also severe invasive infections (posing a threat), requiring attention. [ABSTRACT FROM AUTHOR]

In 2008 we isolated methicillin-resistant Staphylococcus aureus (MRSA) from an 11-month-old Japanese girl who lived in Saitama, Japan, and suffered from cellulitis of the lower thigh and sepsis. The MRSA (strain NN47) belonged to multilocus sequence type (ST) 8 and exhibited spa363 (t024), agr1, staphylococcal cassette chromosome mec (SCC mec) type IVa, and coagulase type III. It was positive for Panton–Valentine leukocidin (PVL) and the arginine catabolic mobile element (ACME). Pulsed-field gel electrophoresis (PFGE) demonstrated that the MRSA was the USA300 clone, which is the predominant community-acquired MRSA (CA-MRSA) in the US. Strain NN47 was divergent, in terms of the spa type and patterns of PFGE and plasmids, from the USA300-0114 type strain or USA300 strain NN36, previously isolated from a visitor (Indian girl) from the US. Strain NN47 was resistant to erythromycin, in addition to β-lactam agents (e.g., oxacillin). These data demonstrate the first emergence of the USA300 clone in Japanese children who have never been abroad and have had no contact with foreigners (and therefore, the first USA300 spread in Japan), and also emergence of multiple divergent strains of the USA300 clone in Japan. Because the USA300 clone is highly transmissible and virulent, surveillance of the USA300 clone is needed. [ABSTRACT FROM AUTHOR]

Background: Streptococcus pneumoniae and Haemophilus influenzae infections in children with influenza have been noted because of the severity of co-infection. In Japan, vaccination against S. pneumoniae and H. influenzae infections has been listed in the vaccine program in 2008, but the characteristics of the two organisms, colonizing at the initial stage of influenza infection, have not been investigated in detail. Methods: Nasopharyngeal swabs from children with influenza (flu+) ( n= 236; mean age, 6.2 years) were examined for bacterial pathogens, including S. pneumoniae and H. influenzae. They were then examined for serotypes, drug susceptibilities, and resistance genes (or gene mutations). As a reference, children with upper respiratory tract infection (URTI+, flu-; n = 189; mean age, 6.2 years) were also examined. Results: S. pneumoniae, β-streptococci (groups A, B, and G), methicillin-susceptible and -resistant S. aureus, Moraxella catarrhalis, and H. influenzae were isolated. For S. pneumoniae, nine serotypes were detected with prevalent types of 3, 6, 19 and 23. Penicillin resistance was detected in types 19 and 23, while resistance to macrolide and clindamycin was found in various types. For H. influenzae, only b serotype was detected, with marked ampicillin resistance. The majority was non-typeable. Very similar results were obtained even in URTI+ (flu-) cases. Conclusion: Multiple drug-resistant S. pneumoniae with major serotypes, for example, 19 and 23 and H. influenzae with serotype b were already present at the initial stage of influenza infection, similar to URTI+ flu- cases. They could be prevented by current vaccines, but drug-resistant non-typeable H. influenzae is troubling. [ABSTRACT FROM AUTHOR]

Intelectin is an extracellular animal lectin found in chordata. Although human and mouse intelectin-1 recognize galactofuranosyl residues included in cell walls of various microorganisms, the physiological function of mammalian intelectin had been unclear. In this study, we found that human intelectin-1 was a serum protein and bound to Mycobacterium bovis bacillus Calmette-Guérin (BCG). Human intelectin-1-binding to BCG was inhibited by Ca2+-depletion, galactofuranosyl disaccharide, ribose, or xylose, and was dependent on the trimeric structure of human intelectin-1. Although monomeric, mouse intelectin-1 bound to BCG, with its C-terminal region contributing to efficient binding. Human intelectin-1-transfected cells not only secreted intelectin-1 into culture supernatant but also expressed intelectin-1 on the cell surface. The cell surface intelectin-1 was not a glycosylphosphatidylinositol-anchored membrane protein. Intelectin-1-transfected cells captured BCG more than untransfected cells, and the BCG adherence was inhibited by an inhibitory saccharide of intelectin-1. Intelectin-1-preincubated cells took up BCG more than untreated cells, but the adhesion of intelectin-1-bound BCG was the same as that of untreated BCG. Mouse macrophages phagocytosed BCG more efficiently in medium containing mouse intelectin-1 than in control medium. These results indicate that intelectin is a host defense lectin that assists phagocytic clearance of microorganisms. [ABSTRACT FROM PUBLISHER]