Correction: Hypervirulent Mycobacterium tuberculosis strain triggers necrotic lung pathology associated with enhanced recruitment of neutrophils in resistant C57BL/6 mice.

[This corrects the article DOI: 10.1371/journal.pone.0173715.].

Hypervirulent Mycobacterium tuberculosis strain triggers necrotic lung pathology associated with enhanced recruitment of neutrophils in resistant C57BL/6 mice.

Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb) that in most cases induces irreversible necrosis of lung tissue as a result of excessive inflammatory reactions. The murine model of TB in resistant C57BL/6 mice infected with reference Mtb strains is widely used in TB studies; however, these mice do not show a necrotic pathology, which restricts their use in studies of irreversible tissue damage. Recently, we demonstrated that necrotic lung lesions could be induced in the C57BL/6 mice by highly virulent Mtb strains belonging to the modern Beijing sublineage. However, the pathogenic mechanisms leading to necrosis in this model were not elucidated. In this study, we investigated the dynamics of lung lesions in mice infected with highly virulent Beijing Mtb strain M299, compared with those infected with laboratory Mtb strain H37Rv. The data demonstrate that necrotic lung lesions in mice infected by the strain M299 were associated with enhanced recruitment of myeloid cells, especially neutrophils, and increased levels of proinflammatory cytokines, consistent with exacerbated inflammation. High levels of IFN-γ production contributed to the control of bacterial growth. Further progression to chronic disease was associated with a reduction in the levels of inflammatory mediators in the lungs, the accumulation of foamy macrophages and partial healing of the necrotic tissue by fibrosis. At a late stage of disease, degradation of foamy cells resulted in the liberation of accumulated lipids and persisting bacilli and further activation of inflammation, which promoted lung consolidation. Overall, our studies show that C57BL/6 mice infected with highly virulent Mtb strain may serve as a TB model reproducing an exacerbated inflammatory response in a resistant host to hypervirulent mycobacteria, leading to irreversible necrotic lung lesions.

Genetic diversity of the Mycobacterium tuberculosis Beijing family in Brazil and Mozambique and relation with infectivity and induction of necrosis in THP-1 cells.

INTRODUCTION: The success of the Mycobacterium tuberculosis Beijing (MtbB) lineage in different geographical regions has been attributed to high transmission, increased virulence, drug resistance and rapid adaptation to the host. In some countries of secondary MtbB dispersion like South Africa and Peru, rising prevalence of the Beijing strains is registered. However, in neighboring countries to affected regions such as Mozambique and Brazil, respectively, the prevalence of these strains is still low and this could be due to biological particularities of the circulating MtbB strains and/or differentiated host susceptibility. OBJECTIVE: To characterize genetically and phenotypically MtbB strains isolated in Brazil (n = 8) and Mozambique (n = 17). METHODS: This is a descriptive study of genotypes of the MtbB isolates, determined by spoligotyping, MIRU-VNTR typing, analysis of the IS6110 copy number in the NTF region and screening for mutations in mutT2, mutT4, rpoB, katG and pks 15/1 genes. Virulence-associated properties of the studied isolates were verified in the in vitro model of infection of human THP-1 cells. RESULTS: The genotypes defined by the 24VNTRs were distinct for all isolates included in this study and presented an HGDI of 0.997. The VNTR patterns with seven copies of MIRU26 and seven copies of QUB26, representative for the previously described MtbB genotype B0, dominant in Russia, were detected in 38.5% of the studied isolates. In addition, all isolates presented RD105 deletion and a 7 bp insertion in pks15/1 gene. Almost all tested strains belonged to the RD181 sublineage, with the exception of two strains from Mozambique of RD150 sublineage. Combined analysis of the NTF region integrity and mutations in mutT genes showed that 62.5% and 47% of isolates obtained in Brazil and Mozambique, respectively, were of the ancestral genotype. The virulence index of the ancient isolates, evaluated in the THP-1 cells, was significantly lower than that of the modern genotype group. CONCLUSIONS: These data demonstrate genotype particularities of the Beijing strains isolated in Brazil and Mozambique, two countries of low prevalence of the MtbB lineage in local Mtb populations. In contrast to the neighboring countries with high prevalence of the MtbB strains of modern sublineage, significant proportions of the isolates obtained in Brazil and Mozambique were presented by the strains of the ancient sublineage. Our data suggest that lower virulence of the ancient strains, compared with the modern strains, could be involved in the slow spread of the MtbB strains in some regions.

Pulmonary infection with hypervirulent Mycobacteria reveals a crucial role for the P2X7 receptor in aggressive forms of tuberculosis.

The purinergic P2X7 receptor (P2X7R) is a sensor of extracellular ATP, a damage-associated molecule that is released from necrotic cells and that induces pro-inflammatory cytokine production and cell death. To investigate whether the innate immune response to damage signals could contribute to the development of pulmonary necrotic lesions in severe forms of tuberculosis, disease progression was examined in C57BL/6 and P2X7R-/- mice that were intratracheally infected with highly virulent mycobacterial strains (Mycobacterium tuberculosis strain 1471 of the Beijing genotype family and Mycobacterium bovis strain MP287/03). The low-dose infection of C57BL/6 mice with bacteria of these strains caused the rapid development of extensive granulomatous pneumonia with necrotic areas, intense bacillus dissemination and anticipated animal death. In contrast, in P2X7R-/- mice, the lung pathology presented with moderate infiltrates of mononuclear leukocytes without visible signs of necrosis; the disease attenuation was accompanied by a delay in mortality. In vitro, the hypervirulent mycobacteria grew rapidly inside macrophages and induced death by a P2X7R-dependent mechanism that facilitated the release of bacilli. Furthermore, these bacteria were resistant to the protective mechanisms elicited in macrophages following extracellular ATP stimulation. Based on this study, we propose that the rapid intracellular growth of hypervirulent mycobacteria results in massive macrophage damage. The ATP released by damaged cells engages P2X7R and accelerates the necrotic death of infected macrophages and the release of bacilli. This vicious cycle exacerbates pneumonia and lung necrosis by promoting widespread cell destruction and bacillus dissemination. These findings suggest the use of drugs that have been designed to inhibit the P2X7R as a new therapeutic approach to treat the aggressive forms of tuberculosis.

Mycobacterium tuberculosis strains of the modern sublineage of the Beijing family are more likely to display increased virulence than strains of the ancient sublineage.

Strains of the Beijing genotype family of Mycobacterium tuberculosis are a cause of particular concern because of their increasing dissemination in the world and their association with drug resistance. Phylogenetically, this family includes distinct ancient and modern sublineages. The modern strains, contrary to the ancestral counterparts, demonstrated increasing prevalence in many world regions that suggest an enhanced bacterial pathogenicity. We therefore evaluated virulence of modern versus ancient Beijing strains with similar epidemiological and genotype characteristics. For this, we selected six strains that had very similar 24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing profiles and belonged to the region of difference 181 (RD181) subgroup but differed using markers (mutT2 and mutT4 genes and NTF locus) that discriminate between modern and ancient Beijing sublineages. The strains were isolated from native patients in Brazil and Mozambique, countries with a low prevalence of Beijing strains. The virulence levels of these strains were determined in models of pulmonary infection in mice and in vitro macrophage infection and compared with that of a strain from Russia, part of the epidemic and hypervirulent Beijing clone B0/W148, and of the laboratory strain H37Rv. The results showed that two of the three modern Beijing strains were highly pathogenic, exhibiting levels of virulence comparable with that of the epidemic Russian strain. In contrast, all isolates of the ancient sublineage displayed intermediate or low virulence. The data obtained demonstrate that the strains of the modern Beijing sublineage are more likely to exhibit highly virulent phenotypes than ancient strains and suggest that genetic alterations characteristic of the modern Beijing sublineage favor selection of highly virulent bacteria.

Emerging multidrug resistant Mycobacterium tuberculosis strains of the Beijing genotype circulating in Russia express a pattern of biological properties associated with enhanced virulence.

The epidemiologically important Mycobacterium tuberculosis Beijing genotype strains, highly endemic in East Asia, have become an emerging infection in certain geographic areas, including Russia, because of its increasing prevalence and association with multidrug resistance (MDR). The aim was to verify whether MDR Beijing strains circulating in the emerging regions present some biological particularities that could contribute to their success in causing disease in comparison with the sporadic strains from locations with low prevalence of the Beijing genotype. We evaluated virulence-associated characteristics of the MDR Beijing strains isolated in Russia and compared them with those of the drug-resistant and susceptible Beijing strains from Brazil and reference H37Rv strain. We found that Russian MDR strains demonstrated an increased bacterial fitness and growth in THP-1 macrophage-like cells, as well as a higher capacity to induce non-protective cytokine synthesis and necrotic macrophage death. By contrast, the biological properties of the strains isolated in Brazil largely resembled those of the H37Rv strain, with the exception of the drug-resistant isolates that presented significantly reduced fitness. The data demonstrate that the emerging MDR strains of the Beijing genotype circulating in Russia do express a pattern of properties associated with the enhanced virulence favouring its clonal dissemination in this region.

Emerging multidrug resistant Mycobacterium tuberculosis strains of the Beijing genotype circulating in Russia express a pattern of biological properties associated with enhanced virulence

The epidemiologically important Mycobacterium tuberculosis Beijing genotype strains, highly endemic in East Asia, have become an emerging infection in certain geographic areas, including Russia, because of its increasing prevalence and association with multidrug resistance (MDR). The aim was to verify whether MDR Beijing strains circulating in the emerging regions present some biological particularities that could contribute to their success in causing disease in comparison with the sporadic strains from locations with low prevalence of the Beijing genotype. We evaluated virulence-associated characteristics of the MDR Beijing strains isolated in Russia and compared them with those of the drug-resistant and susceptible Beijing strains from Brazil and reference H37Rv strain. We found that Russian MDR strains demonstrated an increased bacterial fitness and growth in THP-1 macrophage-like cells, as well as a higher capacity to induce non-protective cytokine synthesis and necrotic macrophage death. By contrast, the biological properties of the strains isolated in Brazil largely resembled those of the H37Rv strain, with the exception of the drug-resistant isolates that presented significantly reduced fitness. The data demonstrate that the emerging MDR strains of the Beijing genotype circulating in Russia do express a pattern of properties associated with the enhanced virulence favouring its clonal dissemination in this region.