Variable number tandem repeat analysis of Mycobacterium bovis isolates from Gyeonggi-do, Korea

Bovine tuberculosis (TB) is a major zoonosis that's caused by Mycobacterium bovis (M. bovis). Being able to detect M. bovis is important to control bovine TB. We applied a molecular technique, the variable number tandem repeat (VNTR) typing method, to identify and distinguish the M. bovis isolates from Gyeonggi-do, Korea. From 2003 to 2004, 59 M. bovis clinical strains were isolated from dairy cattle in Gyeonggi-do, Korea, and these cattle had tuberculosis-like lesions. Twenty-four published MIRU-VNTR markers were applied to the M. bovis isolates and ten of them showed allelic diversity. The most discriminatory locus for the M. bovis isolates in Korea was QUB 3336 (h = 0.64). QUB 26 and MIRU 31 also showed high discriminative power (h = 0.35). The allelic diversity by the combination of all VNTR loci was 0.86. Six loci (MIRU 31, ETR-A and QUB-18, -26, -3232, -3336) displayed valuable allelic diversity. Twelve genotypes were identified from the 59 M. bovis isolates that originated from 20 cattle farms that were dispersed throughout the region of Gyenggi-do. Two genotypes [designation index (d.i.) = e, g] showed the highest prevalence (20% of the total farms). For the multiple outbreaks on three farms, two successive outbreaks were caused by the same genotype at two farms. Interestingly, the third outbreak at one farm was caused by both a new genotype and a previous genotype. In conclusion, this study suggests that MIRU-VNTR typing is useful to identify and distinguish the M. bovis isolates from Gyeonggi-do, Korea

An in vitro model of granuloma-like cell aggregates substantiates early host immune responses against Mycobacterium massiliense infection

Mycobacterium massiliense (M. mass), belonging to the M. abscessus complex, is a rapidly growing mycobacterium that is known to cause tuberculous-like lesions in humans. To better understand the interaction between host cells and M. mass, we used a recently developed in vitro model of early granuloma-like cell aggregates composed of human peripheral blood mononuclear cells (PBMCs). PBMCs formed granuloma-like, small and rounded cell aggregates when infected by live M. mass. Microscopic examination showed monocytes and macrophages surrounded by lymphocytes, which resembled cell aggregation induced by M. tuberculosis (M. tb). M. mass-infected PBMCs exhibited higher expression levels of HLA-DR, CD86 and CD80 on macrophages, and a significant decrease in the populations of CD4+ and CD8+ T cells. Interestingly, low doses of M. mass were sufficient to infect PBMCs, while active host cell death was gradually induced with highly increased bacterial loads, reflecting host destruction and dissemination of virulent rapid-growing mycobacteria (RGM). Collectively, this in vitro model of M. mass infection improves our understanding of the interplay of host immune cells with mycobacteria, and may be useful for developing therapeutics to control bacterial pathogenesis

<i>Mycobacterium massiliense</i> Induces Macrophage Extracellular Traps with Facilitating Bacterial Growth

<div><p>Human neutrophils have been known to release neutrophil extracellular traps (NETs), antimicrobial DNA structures capable of capturing and killing microbes. Recently, a similar phenomenon has been reported in macrophages infected with various pathogens. However, a role for macrophages extracellular traps (METs) in host defense responses against <i>Mycobacterium massiliense</i> (<i>M</i>. <i>mass</i>) has yet to be described. In this study, we show that <i>M</i>. <i>mass</i>, a rapid growing mycobacterium (RGM), also induces the release of METs from PMA-differentiated THP-1 cells. Intriguingly, this process is not dependent on NADPH oxidase activity, which regulates NET formation. Instead, <i>M</i>. <i>mass</i>-induced MET formation partially depends on calcium influx and requires phagocytosis of high bacterial load. The METs consist of a DNA backbone embedded with microbicidal proteins such as histone, MPO and elastase. Released METs entrap <i>M</i>. <i>mass</i> and prevent their dissemination, but do not have bactericidal activity. Instead, they result in enhanced bacterial growth. In this regard, METs were considered to provide interaction of <i>M</i>. <i>mass</i> with cells and an environment for bacterial aggregation, which may facilitate mycobacterial survival and growth. In conclusion, our results demonstrate METs as an innate defense response against <i>M</i>. <i>mass</i> infection, and suggest that extracellular traps play a multifaceted role in the interplay between host and bacteria.</p></div

<i>M</i>. <i>mass</i> R-induced MET formation is not dependent on NADPH oxidase activity, but does depend on calcium influx.

<p>After staining THP-1 macrophages with TO-PRO-3, the MET formation (%) in each sample was determined by quantification of MET-positive cells to total cell count. (A) Differentiated THP-1 macrophages were stimulated with hydrogen peroxide (1 mM), <i>M</i>. <i>mass</i> R (MOI 5) with or without DPI (20 μM) for 24 hr. (B) Fluo-4-labeled THP-1 macrophages were untreated or pretreated with BAPTA (20 μM) or EGTA (1 mM) for 30 min and then infected with <i>M</i>. <i>mass</i> R (MOI 5). Ionomycin (1 μM) was used as positive control. Intracellular calcium transients of labeled cells were recorded in a microplate reader at 6 hr after infection. (C) Determination of MET formation (%] in each sample. MR: <i>M</i>. <i>mass</i> R. Data are representative of three independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001 compared to <i>M</i>. <i>mass</i> R-infected group by one-way ANOVA with Bonferroni’s post-test.</p

MET formation is dependent on phagocytosis and cell lysis.

<p>(A) CFUs of <i>M</i>. <i>mass</i> R recovered from infected macrophages with or without cytochalasin D (5 μM) for 24 hr. (B) MET formation (%) in the macrophages infected with <i>M</i>. <i>mass</i> R (MOI 10) after pretreatment with or without cytochalasin D. (C) CFUs recovered from macrophages infected with <i>M</i>. <i>mass</i> R at various MOI at 4 hr post infection. (D) Determination of MET formation (%) in each sample at 24 hr post infection. (E) Supernatants were collected from macrophages infected with <i>M</i>. <i>mass</i> R at various MOIs at 24 hr post infection. The level of cell lysis of each group was determined by LDH release assay. MR: <i>M</i>. <i>mass</i> R. Data are representative of three independent experiments. ns, non-significant; *, p<0.05; **, p<0.01; ***, p<0.001 by Student’s <i>t</i>-test (A) or one-way ANOVA with Bonferroni’s post-test (B-E).</p

<i>M</i>. <i>mass</i> R-induced METs induce release of mitochondrial as well as nuclear DNA, and contain Histone, MPO and Elastase.

<p>(A) PCR analysis was performed for nuclear (<i>β-actin</i>, <i>Gapdh</i>) and mitochondrial genes (<i>Atp6</i>, <i>Nds1</i>) using DNA isolated from the supernatants of <i>M</i>. <i>mass</i> R-infected THP-1 macrophages and uninfected control samples. Both nuclear and mitochondrial genes were detected in the infected samples. Data are representative of three independent experiments. (B) <i>M</i>. <i>mass</i> R-infected THP-1 macrophages were fixed and processed for histone 4, elastase and MPO staining. METs were stained by TO-PRO-3. Circles indicate co-localization of METs with each component. Data are representative of three independent experiments. H4: histone 4. Bar, 20μm.</p

Rough strain of <i>M</i>. <i>mass</i> strongly induces MET formation in differentiated THP-1 macrophages.

<p>(A) Differentiated THP-1 macrophages were stimulated with <i>M</i>. <i>mass</i> R (MOI 20) with or without DNase I (50 units/ml), <i>M</i>. <i>mass</i> CIP (MOI 20), LPS (10 μg/ml) or hydrogen peroxide (1 mM) for 24 hr, and then stained by TO-PRO-3 to examine METs. Bar, 50μm. (B) MET formation (%) of each sample was quantified by calculating percentages of MET-positive cells to total cells count. MR: <i>M</i>. <i>mass</i> R. Data are representative of three independent experiments with similar results. *, p<0.05; **, p<0.01 compared to <i>M</i>. <i>mass</i> R-infected group by one-way ANOVA with Bonferroni’s post-test.</p

Released METs have no bactericidal effects on <i>M</i>. <i>mass</i> R.

<p>To determine the bactericidal effect of METs on <i>M</i>. <i>mass</i> R, macrophages with METs-degrading DNase I 30 min before infection. (A) Cell-associated (intracellular or METs-bound), (B) Extracellular, (C) Total number of bacteria recovered from the samples infected by <i>M</i>. <i>mass</i> R (MOI 5) for 24 hr, with or without DNase (50 units/ml). (D) AFB staining of THP-1 macrophages infected by <i>M</i>. <i>mass</i> R with or without DNase for 24 hr. Bar, 20μm. Data are representative of three independent experiments with similar results. **, p<0.01 by Student’s <i>t</i>-test.</p

Microscopic examination of macrophage extracellular trap (MET) formation induced by <i>M</i>. <i>mass</i> infection.

<p>Differentiated THP-1 macrophages were infected with <i>M</i>. <i>mass</i> R or CIP at a MOI of 5 for 24 hr, and stained by DNA-binding dye, TO-PRO-3 (Red) to visualize extracellular trap-like structure. (A) Microscopic examination of extracellular trap-like structures in <i>M</i>. <i>mass</i> R-infected THP-1 macrophages. Bar, 200μm. (B, C) Microscopic examination (B) and TO-PRO staining (C) of extracellular trap-like structures induced by <i>M</i>. <i>mass</i> R infection. Bar, 50μm. (D, E) Fluorescence micrographs of CFSE-stained <i>M</i>. <i>mass</i> R (Green) entrapped by METs. Bar, 20μm. (F) Scanning electron microscopy (SEM) images of METs induced by <i>M</i>. <i>mass</i> CIP. Bar, 1μm. Arrow: Mycobacteria. Arrow heads: Extracellular trap-like structures.</p