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OBJECTIVES: Klebsiella spp., an opportunistic infectious organism, is commensal in the nasal and oral cavities of humans. Recently, it has been reported that oral Klebsiella spp. ectopically colonize the intestinal tract and induce the accumulation of intestinal Th1 cells. For oral bacteria to colonize the intestinal tract, they need to compete for nutrients with indigenous intestinal bacteria. Therefore, we focused on mannose, a mucus-derived sugar, and the mannose phosphotransferase system (PTS) (ManXYZ), a mechanism for mannose uptake, in terms of their effects on intestinal colonization and immune responses to Klebsiella spp. METHODS: We generated a Klebsiella manXYZ-deficient strain and investigated whether the utilization of intestinal mucus-derived sugars is associated with the growth. Furthermore, we examine the virulence of this organism in the mouse intestinal tract, especially the ability to colonize the host using competition assay. RESULTS: We found that Klebsiella ManXYZ is a PTS that specifically takes up mannose and glucosamine. Through ManXYZ, mannose was used for bacterial growth and the upregulated production of extracellular polymeric substances. In vivo competition assays showed that mannose metabolism promoted intestinal colonization. However, ManXYZ was not involved in Th1 and Th17 induction in the intestinal tract. CONCLUSION: The fundamental roles of ManXYZ were to ensure that mannose, which is present in the host, is made available for bacterial growth, to promote the production of extracellular polymeric substances, thus facilitating bacterial adaptation to the host environment.
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Klebsiella , Manosa , Humanos , Animales , Ratones , Matriz Extracelular de Sustancias Poliméricas , Fosfotransferasas , Proliferación CelularRESUMEN
Rationale: The clinical implications of trehalose 6,6'-dimycolate (TDM) in nontuberculous mycobacterial pulmonary disease have not been studied. Objectives: To examine the presence of TDM in clinical isolates obtained from patients with Mycobacterium avium complex (MAC) pulmonary disease (PD) and its impact on disease severity and treatment outcomes. Methods: We analyzed clinical isolates from patients with diagnoses of MAC PD at Seoul National University Hospital between January 1, 2019, and December 31, 2021. The lipids were extracted from clinical isolates obtained at the time of diagnosis using mass spectrometry. Mass peaks between 300 and 3,500 m/z were obtained, and the peak patterns of the total lipids were analyzed. Results: TDM was identified in clinical isolates from 176 of 343 patients. Cavities were more prevalent in patients with TDM-negative isolates (19.8%) than in those with TDM-positive isolates (10.2%) (P = 0.015). The time to antibiotic treatment was shorter in patients with TDM-negative isolates (4 mo [interquartile range, 2-10 mo]) than in those with TDM-positive isolates (7 mo [interquartile range, 3-16 mo]) (P = 0.032). Patients with TDM-negative isolates had a significantly lower proportion of culture conversions (P = 0.012). TDM was associated with higher likelihood of culture conversion (adjusted hazard ratio, 2.29; P = 0.035). Conclusions: TDM-negative isolates were linked to a higher occurrence of cavities, earlier initiation of treatment, and worse treatment outcome in patients with MAC PD.
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Antibacterianos , Complejo Mycobacterium avium , Infección por Mycobacterium avium-intracellulare , Humanos , Masculino , Femenino , Infección por Mycobacterium avium-intracellulare/tratamiento farmacológico , Infección por Mycobacterium avium-intracellulare/microbiología , Infección por Mycobacterium avium-intracellulare/diagnóstico , Complejo Mycobacterium avium/aislamiento & purificación , Anciano , Persona de Mediana Edad , Antibacterianos/uso terapéutico , Estudios Retrospectivos , República de Corea , Enfermedades Pulmonares/microbiología , Enfermedades Pulmonares/tratamiento farmacológicoRESUMEN
Using anticancer drugs as examples, we examined the possibility of reusing residual drugs. The use of residual drugs is not widespread owing to concerns regarding bacterial contamination. We combined anticancer drugs and bacteria to investigate their effects on bacterial growth. The anticancer drugs carboplatin, paclitaxel, etoposide, irinotecan, methotrexate, and 5-fluorouracil (5-FU) were mixed with Staphylococcus aureus, Enterococcus faecalis, Serratia marcescens, and Escherichia coli. After a certain period, the bacteria were counted. Irinotecan showed no antibacterial activity, whereas 5-FU exhibited high antibacterial activity against the tested bacteria. The 5-FU also showed a minimum inhibitory concentration value in the range of 8-80 µg/mL, depending on the bacterial species. 5-FU dose-dependently inhibited S. aureus growth at more than 0.8 µg/mL. Because protein synthesis systems are reportedly antibiotic targets, we used a cell-free protein synthesis system to confirm the mechanism of the antibacterial activity of the anticancer agent. 5-FU and methotrexate had direct inhibitory effects on protein synthesis. It has been suggested that even if residual drugs are contaminated with bacteria, there will be no microbial growth, or the microbes will be killed by the drug. With careful monitoring, 5-FU can potentially be used for antimicrobial purposes.
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Antineoplásicos , Staphylococcus aureus , Metotrexato/farmacología , Irinotecán/farmacología , Antibacterianos/farmacología , Bacterias , Antineoplásicos/farmacología , Fluorouracilo/farmacología , Escherichia coli , Pruebas de Sensibilidad MicrobianaRESUMEN
Scotochromogenic slow-growing mycobacteria were isolated from the sputum or bronchoalveolar lavage fluid of 12 patients in Japan. From a comparison of the whole-genome sequences, the representative strain IWGMT90018-18076T and the unknown strains obtained from the patients were found to represent a novel species related to the Mycobacterium gordonae complex. The average nucleotide identity values of IWGMT90018-18076T with Mycobacterium vicinigordonae, Mycobacterium paragordonae and M. gordonae were 86.7, 82.5 and 82.2â%, respectively. The genome size of the representative strain IWGMT90018-18076T was approximately 6.3 Mbp, and the genomic DNA G+C content was 67.1â%. The major fatty acid methyl esters were C16â:â0 (37.71â%), C18â:â1ω9c (29.5â%) and C16â:â1ω7c (10.32â%). In this study, we performed phylogenetic analyses, physiological and biochemical characteristic tests, drug susceptibility tests and fatty acid profiling of the clinical isolates. On the basis of the results obtained, we propose that the unknown clinical isolates represent a novel species, 'Mycobacterium kiyosense sp. nov,' with the type strain being IWGMT90018-18076T (=JCM 34837T =KCTC 49725T).
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Ácidos Grasos , Mycobacterium , Humanos , Ácidos Grasos/química , Filogenia , Análisis de Secuencia de ADN , ADN Bacteriano/genética , Composición de Base , ARN Ribosómico 16S/genética , Técnicas de Tipificación BacterianaRESUMEN
Mycobacterium infection gives rise to granulomas predominantly composed of inflammatory M1-like macrophages, with bacteria-permissive M2 macrophages also detected in deep granulomas. Our histological analysis of Mycobacterium bovis bacillus Calmette-Guerin-elicited granulomas in guinea pigs revealed that S100A9-expressing neutrophils bordered a unique M2 niche within the inner circle of concentrically multilayered granulomas. We evaluated the effect of S100A9 on macrophage M2 polarization based on guinea pig studies. S100A9-deficient mouse neutrophils abrogated M2 polarization, which was critically dependent on COX-2 signaling in neutrophils. Mechanistic evidence suggested that nuclear S100A9 interacts with C/EBPß, which cooperatively activates the Cox-2 promoter and amplifies prostaglandin E2 production, followed by M2 polarization in proximal macrophages. Because the M2 populations in guinea pig granulomas were abolished via treatment with celecoxib, a selective COX-2 inhibitor, we propose the S100A9/Cox-2 axis as a major pathway driving M2 niche formation in granulomas.
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Mycolic acids are critical for the survival and virulence of Mycobacterium tuberculosis, the causative agent of tuberculosis. Double bond formation in the merochain of mycolic acids remains poorly understood, though we have previously shown desA1, encoding an aerobic desaturase, is involved in mycolic acid desaturation. Here we show that a second desaturase encoded by desA2 is also involved in mycolate biosynthesis. DesA2 is essential for growth of the fast-growing Mycobacterium smegmatis in laboratory media. Conditional depletion of DesA2 led to a decrease in mycolic acid biosynthesis and loss of mycobacterial viability. Additionally, DesA2-depleted cells also accumulated fatty acids of chain lengths C19-C24. The complete loss of mycolate biosynthesis following DesA2 depletion, and the absence of any monoenoic derivatives (found to accumulate on depletion of DesA1) suggests an early role for DesA2 in the mycolic acid biosynthesis machinery, highlighting its potential as a drug target.
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Mycobacterium tuberculosis , Tuberculosis , Ácido Graso Desaturasas/genética , Humanos , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Ácidos MicólicosRESUMEN
Mycobacterium tuberculosis has a lipid-rich cell envelope that is remodeled throughout infection to enable adaptation within the host. Few transcriptional regulators have been characterized that coordinate synthesis of mycolic acids, the major cell wall lipids of mycobacteria. Here, we show that the mycolic acid desaturase regulator (MadR), a transcriptional repressor of the mycolate desaturase genes desA1 and desA2, controls mycolic acid desaturation and biosynthesis in response to cell envelope stress. A madR-null mutant of M. smegmatis exhibited traits of an impaired cell wall with an altered outer mycomembrane, accumulation of a desaturated α-mycolate, susceptibility to antimycobacterials, and cell surface disruption. Transcriptomic profiling showed that enriched lipid metabolism genes that were significantly down-regulated upon madR deletion included acyl-coenzyme A (aceyl-CoA) dehydrogenases, implicating it in the indirect control of ß-oxidation pathways. Electromobility shift assays and binding affinities suggest a unique acyl-CoA pool-sensing mechanism, whereby MadR is able to bind a range of acyl-CoAs, including those with unsaturated as well as saturated acyl chains. MadR repression of desA1/desA2 is relieved upon binding of saturated acyl-CoAs of chain length C16 to C24, while no impact is observed upon binding of shorter chain and unsaturated acyl-CoAs. We propose this mechanism of regulation as distinct to other mycolic acid and fatty acid synthesis regulators and place MadR as the key regulatory checkpoint that coordinates mycolic acid remodeling during infection in response to host-derived cell surface perturbation.
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Proteínas Bacterianas/metabolismo , Mycobacterium/metabolismo , Ácidos Micólicos/metabolismo , Racemasas y Epimerasas/metabolismo , Acilcoenzima A/metabolismo , Proteínas Bacterianas/fisiología , Pared Celular/metabolismo , Ácido Graso Desaturasas/metabolismo , Ácidos Grasos/metabolismo , Metabolismo de los Lípidos/fisiología , Infecciones por Mycobacterium , Mycobacterium tuberculosis/metabolismo , Racemasas y Epimerasas/fisiología , Factores de Transcripción/metabolismoRESUMEN
Eight spindle-shaped bacteria were isolated from clinical samples in Japan and investigated for their taxonomic position. Phylogenetic trees (based on 16S rRNA, rpoB, zinc protease, and gyrB gene sequence comparisons) showed distinct clustering of eight strains with the type strain of Fusobacterium nucleatum and its closely related species. In silico whole genome comparison analysis based on average nucleotide index based on BLAST (ANIb) and digital DNA-DNA hybridization (dDDH) data between our clinical isolates (PAGU 1795, PAGU 1796T, and PAGU 1797) and the type strain of the closely related species showed values of less than 92.4% and 49.5%, respectively. On the basis of its phylogenetic and genomic distinctiveness together with differential phenotypic properties and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) characteristic signal patterns, we propose Fusobacterium watanabei sp. nov., with the type strain PAGU 1796T (= GTC 21791T = CCUG 74246T).
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Clasificación , Fusobacterium/clasificación , Fusobacterium/citología , Fusobacterium/genética , Genes Bacterianos , Fenotipo , Filogenia , Infecciones por Fusobacterium/genética , Variación Genética , Genoma , Humanos , JapónRESUMEN
A recent study proposed the novel classification of the family Mycobacteriaceae based on the genome analysis of core proteins in 150 Mycobacterium species. The results from these analyses supported the existence of five distinct monophyletic groups within the genus Mycobacterium. That is, Mycobacterium has been divided into two novel genera for rapid grower Mycobacteroides and Mycolicibacterium, and into three genera for slow grower Mycolicibacter, Mycolicibacillus, and an emended genus Mycobacterium, which include all the major human pathogens. Here, cryo-TEM examinations of 1,816 cells of 31 species (34 strains) belonging to the five novel genera were performed. The fundamental morphological properties of every single cell, such as cell diameter, cell length, cell perimeter, cell circularity, and aspect ratio were measured and compared between these genera. In 50 comparisons on the five parameters between any two genera, only five comparisons showed "non-significant" differences. That is, there are non-significant differences between slow grower genus Mycolicibacillus and genus Mycobacterium in average cell diameter (p = 0.15), between rapid grower genus Mycobacteroides and slow grower genus Mycobacterium in average cell length (p > 0.24), between genus Mycobacteroides and genus Mycobacterium (p > 0.68) and between genus Mycolicibacter and genus Mycolicibacillus (p > 0.11) in average cell perimeter, and between genus Mycolicibacterium and genus Mycobacterium in circularity (p > 0.73). The other 45 comparisons showed significant differences between the genera. Genus Mycobacteroides showed the longest average cell diameter, whereas the genus Mycolicibacter showed the shortest average diameter. Genus Mycolicibacterium showed the most extended average cell length, perimeter, and aspect ratio, whereas the genus Mycolicibacillus showed the shortest average cell length, perimeter, and aspect ratio. Genus Mycolicibacillus showed the highest average cell circularity, whereas genus Mycobacterium showed the lowest average cell circularity. These fundamental morphological data strongly support the new classification in the family Mycobacteriaceae, and this classification is rational and effective in the study of the members of the family Mycobacteriaceae. Because both the genus Mycolicibacterium and the genus Mycobacterium contain many species and showed larger significant standard deviations in every parameter, these genera may be divided into novel genera which show common genotype and phenotypes in morphology and pathogenicity.
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Two gram-negative, catalase-negative, oxidase-positive strains (PAGU 1467T and PAGU 1468) isolated from patients with infective endocarditis were investigated to determine their taxonomic status. 16S rRNA gene sequence analysis indicated that the two strains were members of the Bergeyella-Chryseobacterium-Riemerella branch of the family Flavobacteriaceae. Strains PAGU 1467T and PAGU 1468 were highly related to each other (98.8% 16S rRNA gene sequence similarity). Phylogenetically closely-related species to PAGU 1467T comprised Bergeyella zoohelcum (95.0% 16S rRNA gene sequence similarity), Riemerella anatipestifer (94.3%) and Cloacibacterium normanense (94.3%). The major fatty acids of the two isolates were iso-C15:0 , iso-C17:0 3-OH and iso-C15:0 3-OH. The presence of C16:0 3-OH and iso-C15:0 2-OH allowed these isolates to be distinguished from B. zoohelcum. Menaquinone MK-6 was the only respiratory quinone in these organisms; this is a consistent characteristic of the family Flavobacteriaceae. The guanine-plus-cytosine content of the genomic DNA was 42.0%, which is higher than that of other close phylogenetic relatives. On the basis of their phenotypic properties and genetic distinctiveness, isolates PAGU 1467T and PAGU 1468 were classified within the novel genus Spodiobacter, as Spodiobacter cordis gen. nov., sp. nov., which is also the type species. The type strain of S. cordis is PAGU 1467T ( = CCUG 65564T = NBRC 109998T ).
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Endocarditis/microbiología , Flavobacteriaceae/clasificación , Flavobacteriaceae/aislamiento & purificación , Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano/genética , Ácidos Grasos/análisis , Flavobacteriaceae/genética , Humanos , Filogenia , ARN Ribosómico 16S/genéticaRESUMEN
Among non-tuberculous mycobacteria (NTM), the Mycobacterium simiae complex is one of the largest groups, consisting of 18 species of slow-growing mycobacteria. In 2009, a case of NTM-associated infectious skin disease was reported in Shiga Prefecture, Japan. The patient presented with scattered nodules on the chest, back and extremities, and an M. simiae-like organism was isolated from skin biopsy specimens obtained from one of these lesions. Based on several assessments, including multiple-gene analyses, biochemical characterization and drug susceptibility testing, we concluded that this isolate represented a novel species of NTM, and proposed the name 'Mycobacterium shigaense'. Since 2009, five more cases of NTM-associated infectious disease in which there was a suspected involvement of 'M. shigaense' have been reported. Interestingly, four of these six cases occurred in Shiga Prefecture. Here we performed multiple-gene phylogenetic analyses, physiological and biochemical characterization tests, drug susceptibility tests, and profiling of proteins, fatty acids and mycolic acids of eight clinical isolates from the six suspected 'M. shigaense' cases. The results confirmed that all of the clinical isolates were 'M. shigaense', a slow-growing, scotochromogenic species. Here M. shigaense is validly proposed as a new member of the M. simiae complex, with the type strain being UN-152T (=JCM 32072T=DSM 46748T).
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Infecciones por Mycobacterium/microbiología , Mycobacterium/clasificación , Filogenia , Enfermedades Cutáneas Bacterianas/microbiología , Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano/genética , Ácidos Grasos/química , Humanos , Japón , Mycobacterium/genética , Mycobacterium/aislamiento & purificación , Ácidos Micólicos/química , Micobacterias no Tuberculosas/clasificación , Micobacterias no Tuberculosas/genética , Micobacterias no Tuberculosas/aislamiento & purificación , Fosfolípidos/química , Pigmentación , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU), a WHO-defined neglected tropical disease. All Japanese BU causative isolates have shown distinct differences from the prototype and are categorized as M. ulcerans subspecies shinshuense. During repeated sub-culture, we found that some M. shinshuense colonies were non-pigmented whereas others were pigmented. Whole genome sequence analysis revealed that non-pigmented colonies did not harbor a giant plasmid, which encodes elements needed for mycolactone toxin biosynthesis. Moreover, mycolactone was not detected in sterile filtrates of non-pigmented colonies. Mice inoculated with suspensions of pigmented colonies died within 5 weeks whereas those infected with suspensions of non-pigmented colonies had significantly prolonged survival (>8 weeks). This study suggests that mycolactone is a critical M. shinshuense virulence factor and that the lack of a mycolactone-producing giant plasmid makes the strain non-pathogenic. We made an avirulent mycolactone-deletion mutant strain directly from the virulent original.
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Mycobacterium ulcerans/genética , Mycobacterium ulcerans/patogenicidad , Plásmidos , Animales , Úlcera de Buruli/microbiología , Úlcera de Buruli/patología , Cromosomas Bacterianos , Medios de Cultivo , Genes Bacterianos , Macrólidos/metabolismo , Ratones , Ratones Endogámicos BALB C , Mycobacterium ulcerans/crecimiento & desarrollo , Virulencia/genéticaRESUMEN
A previously undescribed rapidly growing, non-pigmented mycobacterium was identified based on biochemical and nucleic acid analyses, as well as growth characteristics. Seven isolates were cultured from samples collected from five thread-sail filefish (Stephanolepis cirrhifer) and two farmed black scraper (Thamnaconus modestus). Bacterial growth occurred at 15-35 °C on Middlebrook 7H11 agar. The bacteria were positive for catalase activity at 68 °C and urease activity, intermediate for iron uptake, and negative for Tween 80 hydrolysis, nitrate reduction, semi-quantitative catalase activity and arylsulfatase activity at day 3. No growth was observed on Middlebrook 7H11 agar supplemented with picric acid, and very little growth was observed in the presence of 5â% NaCl. α- and α'-mycolates were identified in the cell walls, and a unique profile of the fatty acid methyl esters and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiles of the protein and cell-wall lipids were acquired. Sequence analysis revealed that the seven isolates shared identical sequences for the 16S rRNA, rpoB, hsp65, recA and sodA genes. Phylogenetic analysis of the five gene sequences confirmed that the isolates were unique, but closely related to Mycobacterium chelonae. Antibiotic susceptibility testing revealed the minimum inhibitory concentration (MIC) of clarithromycin against this novel species was <0.25 µg ml-1, which was lower than that for Mycobacterium salmoniphilum. The hsp65 PCR restriction enzyme analysis pattern differed from those of M. chelonae and M. salmoniphilum. Based on these findings, the name Mycobacterium stephanolepidis sp. nov. is proposed for this novel species, with the type strain being NJB0901T (=JCM 31611T=KCTC 39843T).
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Peces/microbiología , Mycobacterium/clasificación , Filogenia , Animales , Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano/genética , Ácidos Grasos/química , Genes Bacterianos , Japón , Mycobacterium/genética , Mycobacterium/aislamiento & purificación , Infecciones por Mycobacterium/microbiología , Mycobacterium chelonae , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
Mycobacterium abscessus (MAB) is a species of nontuberculous mycobacteria (NTM) and a major causative pathogen of pulmonary diseases especially in patients with cystic fibrosis. MAB infection is notoriously difficult to treat because of its intrinsic or inducible resistance to most antibiotics. The rough (R) morphotype of MAB, lacking cell surface glycopeptidolipids (GPLs), is associated with more severe and persistent infection than the smooth (S) type; however, the mechanisms underlying the R type's virulence and the relation with GPLs remain unclear. In this study, we found that R-type MAB is much more proapoptotic than the S type, as a result of GPL-mediated inhibition of macrophage apoptosis. Polar GPLs inhibited an apoptotic response (induced by proapoptotic stimuli) by suppressing ROS production and the cytochrome c release and by preserving mitochondrial transmembrane potential. Furthermore, GPLs were found to be targeted to mitochondria and interacted with cyclophilin D; their acetylation was essential for this interaction. Finally, GPLs inhibited the intracellular growth and bacterial spreading of R-type MAB among macrophages via apoptosis inhibition. These findings suggest that GPLs limit MAB virulence by inhibiting apoptosis and the spread of bacteria and therefore provide a novel insight into the mechanism underlying virulence of MAB.
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Ciclofilinas/metabolismo , Macrófagos/metabolismo , Mitocondrias/metabolismo , Mycobacterium abscessus/patogenicidad , Micobacterias no Tuberculosas/patogenicidad , Apoptosis , Peptidil-Prolil Isomerasa F , HumanosRESUMEN
Mesorhizobium loti is a member of rhizobia and establishes nitrogen-fixing symbioses with several Lotus species. Recently, we reported that M. loti MAFF 303099 bacterial cells and their lipopolysaccharide (LPS) preparations are involved in the beginning of the symbiotic process by inducing transient nitric oxide (NO) production in the roots of L. japonicus. We subsequently found that both the polysaccharide (PS) part and the lipid A moiety in LPS are responsible for the NO induction. In this study, we elucidated the chemical structure of M. loti O-polysaccharide (OPS) in PS. PS was prepared by mild acid hydrolysis of M. loti LPS followed by gel filtration chromatography. OPS was subjected to hydrazine treatment to obtain deacylated PS (dPS). Chemical composition analysis, ethylation analysis, and NMR spectra revealed the chemical structure of the M. loti OPS backbone in dPS to be â2)-α-l-6dTalp-(1 â 3)-α-l-6dTalp-(1 â 2)-α-l-Rhap-(1 â 2)-α-l-6dTalp-(1 â 3)-α-l-6dTalp-(1 â 3)-α-l-Rhap-(1â.
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Mesorhizobium/metabolismo , Óxido Nítrico/biosíntesis , Antígenos O/química , Secuencia de Carbohidratos , Raíces de Plantas/metabolismoRESUMEN
Oral streptococci including mitis group streptococci are commensal residents and are also the first to colonize the oral cavity. However, various species of these oral streptococci have the potential to invade the host and occasionally lead to severe infectious disease such as cardiovascular diseases. Oral streptococci have close interactions with the host immune system including macrophages at the oral mucosal surface. One notable common trait of oral streptococcus including Streptococcus oralis (S. oralis) is the production of hydrogen peroxide (H2O2). Using a comprehensive microarray approach, we sought to understand the innate immune response profiling affected by H2O2 production from oral streptococci. We compared the gene expression patterns of macrophages infected with S. oralis wild type (WT) and streptococcal pyruvate oxidase knockout (SpxB-KO), a strain that does not produce H2O2. We found that H2O2 from S. oralis suppressed proinflammatory gene expression such as TNF-α, that is induced in response to infection, and activated the cellular stress genes such as Egr-1 in response to oxidative stress. A comparative gene ontology analysis of S. oralis WT and SpxB-KO strains revealed that during infection, down regulated genes were closely related to the processes involved in the host defense reaction and up regulated genes were related with the cellular stress responses. Using qPCR analysis, we also confirmed the same pattern of expression changes such as TNF-α, IL-6 and Egr-1. Furthermore, supernatant from SpxB-KO could not suppress the expression of TNF-α in macrophages stimulated with LPS. These findings suggested that H2O2 production from S. oralis leads to the suppression of inflammatory responses and NF-κB signaling pathways in macrophages as well as the induction of the oxidative stress response. We concluded that streptococcal H2O2 production has the beneficial effects of modulating the innate immune response, thereby stabilizing streptococcal colonization at the mucosal surface and even in the bloodstream leading to cardiovascular disease after invasion, in addition to the commensal role to compete other bacterial species as initial colonizer at oral cavity.
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Perfilación de la Expresión Génica/métodos , Peróxido de Hidrógeno/metabolismo , Macrófagos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Streptococcus oralis/metabolismo , Células 3T3 , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Western Blotting , Línea Celular , Análisis por Conglomerados , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Ontología de Genes , Interacciones Huésped-Patógeno , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Ratones , Ratones Endogámicos BALB C , Mutación , Piruvato Oxidasa/genética , Piruvato Oxidasa/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Streptococcus oralis/genética , Streptococcus oralis/fisiología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Mycolic acids are unique long chain fatty acids found in the cell walls of mycobacteria including the tubercle bacillus, Mycobacterium tuberculosis. The introduction of double bonds in mycolic acids remains poorly understood, however, genes encoding two potential aerobic desaturases have been proposed to be involved in this process. Here we show that one of these genes, desA1, is essential for growth of the saprophytic Mycobacterium smegmatis. Depletion of desA1 in a M. smegmatis conditional mutant led to reduction of mycolic acid biosynthesis and loss of viability. The DesA1-depleted cells exhibited two other phenotypes: using 14[C]-labelling, we detected the accumulation of minor mycolic acid-related species that migrated faster in a silver TLC plate. Spiral Time of Flight Mass Spectroscopic analysis suggested the presence of species with sizes corresponding to what were likely monoenoic derivatives of α-mycolic acids. Additionally, conditional depletion led to the presence of free fatty acyl species of lengths ~C26-C48 in the lysing cells. Cell viability could be rescued in the conditional mutant by Mycobacterium tuberculosis desA1, highlighting the potential of desA1 as a new drug target in pathogenic mycobacteria.
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Proteínas Bacterianas/metabolismo , Ácido Graso Desaturasas/metabolismo , Mycobacterium smegmatis/metabolismo , Ácidos Micólicos/metabolismo , Acetamidas/química , Acetamidas/farmacología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Radioisótopos de Carbono/química , Cromatografía en Capa Delgada , Ácido Graso Desaturasas/genética , Datos de Secuencia Molecular , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/crecimiento & desarrollo , Ácidos Micólicos/análisis , Alineación de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Porphyromonas strains, including Porphyromonas-like strains, have been isolated from oral and various other systemic infections. The characterization of such strains is a crucial issue, because such information contributes to both the taxonomy of anaerobic bacteria and the clinical aspects of infectious diseases. We previously isolated four Porphyromonas-like strains from intraoperative bronchial fluids of a patient with non-small cell lung cancer. This study aimed to characterize the genetic, biochemical and chemotaxonomic aspects of these isolates. Each strain only grew under anaerobic conditions and their colony morphology was convex, 0.1-1.0 mm in diameter, light gray, and slightly glistening colony, with no black or brown pigmentation on blood agar plates after five-day incubation. The pigmentation was helpful to differentiate the isolates from other Porphyromonas, as most of Porphyromonas species show the pigmentation. In the 16S rRNA gene phylogenetic analysis (98% sequence identity of isolates indicates the same species), the four isolates were closely related to one another (99.7-100.0%), but not related to Porphyromonas (P.) catoniae, the closest species (96.9%). In addition, the DNA-DNA hybridization data revealed less than 16% similarity values between a representative isolate and the P. catoniae, indicating that the strains were genetically independent. Biochemically, the isolates could be differentiated from closely related species, i.e., P. catoniae, P. gingivalis, P. gulae, and P. pogonae, with trypsin activity (negative only in the isolates) and leucine arylamidase activity (positive only in the isolates). We therefore propose a new species to include these isolates: Porphyromonas bronchialis sp. nov.
Asunto(s)
Bronquios/microbiología , Carcinoma de Pulmón de Células no Pequeñas/microbiología , Neoplasias Pulmonares/microbiología , Porphyromonas/genética , Anciano , Líquidos Corporales/microbiología , Carcinoma de Pulmón de Células no Pequeñas/cirugía , ADN Bacteriano/genética , Ácidos Grasos/análisis , Fermentación , Humanos , Periodo Intraoperatorio , Neoplasias Pulmonares/cirugía , Masculino , Porphyromonas/química , Porphyromonas/aislamiento & purificación , ARN Ribosómico 16S/genética , Especificidad de la Especie , Tripsina/análisisRESUMEN
Mycobacterium smegmatis has been widely used as a mycobacterial infection model. Unlike the M. smegmatis mc(2)155 strain, M. smegmatis J15cs strain has the advantage of surviving for one week in murine macrophages. In our previous report, we clarified that the J15cs strain has deleted apolar glycopeptidolipids (GPLs) in the cell wall, which may affect its morphology and survival in host cells. In this study, the gene causing the GPL deletion in the J15cs strain was identified. The mps1-2 gene (MSMEG_0400-0402) correlated with GPL biosynthesis. The J15cs strain had 18 bps deleted in the mps1 gene compared to that of the mc(2)155 strain. The mps1-complemented J15cs mutant restored the expression of GPLs. Although the J15cs strain produces a rough and dry colony, the colony morphology of this mps1-complement was smooth like the mc(2)155 strain. The length in the mps1-complemented J15cs mutant was shortened by the expression of GPLs. In addition, the GPL-restored J15cs mutant did not survive as long as the parent J15cs strain in the murine macrophage cell line J774.1 cells. The results are direct evidence that the deletion of GPLs in the J15cs strain affects bacterial size, morphology, and survival in host cells.
Asunto(s)
Proteínas Bacterianas/fisiología , Glucolípidos/fisiología , Glicopéptidos/fisiología , Mycobacterium smegmatis/fisiología , Animales , Línea Celular , Genes Bacterianos , Prueba de Complementación Genética , Interacciones Huésped-Patógeno , Ratones , Viabilidad MicrobianaRESUMEN
Mycolic acids (MAs) are characteristic components of bacteria in the suborder Corynebacterineae, such as Mycobacterium. MAs are categorized into subclasses based on their functional bases (cyclopropane ring, methoxy, keto, and epoxy group). Since MAs have heterogeneity among bacterial species, analyzing of MAs are required in the chemotaxonomic field. However, their structural analysis is not easy because of their long carbon-chain lengths and several functional groups. In this study, total fatty acid (FA) methyl ester (ME) fraction of M. tuberculosis H37Rv was analyzed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOFMS) with a spiral ion trajectory (MALDI spiral-TOFMS). The distributions of carbon-chain length and their relative peak intensities were confirmed with those obtained by analysis of each subclass fraction which was separated from total FA ME fraction using thin-layer chromatography (TLC). The observed major peaks were reliably assigned as MAs owing to the high mass accuracy (error<3 ppm). The types of MA subclasses, their distributions of carbon-chain lengths, their relative peak intensities, and the ratio of even- and odd-numbered carbon-chain MAs for the total FA ME fraction were consistent with those of MA subclass fractions. To visualize whole MAs, contour maps of relative peak intensities for whole MAs were created. The contour maps indicated the MA subclasses and their distributions of carbon-chains with relative peak intensities at a glance. Our proposed method allows simple characterization in a short time and thus enables the analysis of large numbers of samples, and it would contribute to the chemotaxonomy.