Endoplasmic reticulum stress pathway-mediated apoptosis in macrophages contributes to the survival of Mycobacterium tuberculosis

Background

Apoptosis is thought to play a role in host defenses against intracellular pathogens, including Mycobacterium tuberculosis (Mtb), by preventing the release of intracellular components and the spread of mycobacterial infection. This study aims to investigate the role of endoplasmic reticulum (ER) stress mediated apoptosis in mycobacteria infected macrophages.

Methodology/Principal Findings

Here, we demonstrate that ER stress-induced apoptosis is associated with Mtb H37Rv-induced cell death of Raw264.7 murine macrophages. We have shown that Mtb H37Rv induced apoptosis are involved in activation of caspase-12, which resides on the cytoplasmic district of the ER. Mtb infection increase levels of other ER stress indicators in a time-dependent manner. Phosphorylation of eIF2α was decreased gradually after Mtb H37Rv infection signifying that Mtb H37Rv infection may affect eIF2α phosphorylation in an attempt to survive within macrophages. Interestingly, the survival of mycobacteria in macrophages was enhanced by silencing CHOP expression. In contrast, survival rate of mycobacteria was reduced by phosphorylation of the eIF2α. Futhermore, the levels of ROS, NO or CHOP expression were significantly increased by live Mtb H37Rv compared to heat-killed Mtb H37Rv indicating that live Mtb H37Rv could induce ER stress response.

Conclusion/Significance

These findings indicate that eIF2α/CHOP pathway may influence intracellular survival of Mtb H37Rv in macrophages and only live Mtb H37Rv can induce ER stress response. The data support the ER stress pathway plays an important role in the pathogenesis and persistence of mycobacteria.     

The Balance of Apoptotic and Necrotic Cell Death in Mycobacterium tuberculosis Infected Macrophages Is Not Dependent on Bacterial Virulence

Background

An important mechanism of Mycobacterium tuberculosis pathogenesis is the ability to control cell death pathways in infected macrophages: apoptotic cell death is bactericidal, whereas necrotic cell death may facilitate bacterial dissemination and transmission.

Methods

We examine M.tuberculosis control of spontaneous and chemically induced macrophage cell death using automated confocal fluorescence microscopy, image analysis, flow cytometry, plate-reader based vitality assays, and M.tuberculosis strains including H37Rv, and isogenic virulent and avirulent strains of the Beijing lineage isolate GC1237.

Results

We show that bacterial virulence influences the dynamics of caspase activation and the total level of cytotoxicity. We show that the powerful ability of M.tuberculosis to inhibit exogenously stimulated apoptosis is abrogated by loss of virulence. However, loss of virulence did not influence the balance of macrophage apoptosis and necrosis – both virulent and avirulent isogenic strains of GC1237 induced predominantly necrotic cell death compared to H37Rv which induced a higher relative level of apoptosis.

Conclusions

This reveals that macrophage necrosis and apoptosis are independently regulated during M. tuberculosis infection of macrophages. Virulence affects the level of host cell death and ability to inhibit apoptosis but other strain-specific characteristics influence the ultimate mode of host cell death and alter the balance of apoptosis and necrosis.     

Exosomes released from M. tuberculosis infected cells can suppress IFN-γ mediated activation of naïve macrophages

Background

Macrophages infected with Mycobacterium tuberculosis (M.tb) are known to be refractory to IFN-γ stimulation. Previous studies have shown that M.tb express components such as the 19-kDa lipoprotein and peptidoglycan that can bind to macrophage receptors including the Toll-like receptor 2 resulting in the loss in IFN-γresponsiveness. However, it is unclear whether this effect is limited to infected macrophages. We have previously shown that M.tb-infected macrophages release exosomes which are 30–100 nm membrane bound vesicles of endosomal origin that function in intercellular communication. These exosomes contain mycobacterial components including the 19-kDa lipoprotein and therefore we hypothesized that macrophages exposed to exosomes may show limited response to IFN-γ stimulation.

Methodology/Principal Findings

Exosomes were isolated from resting as well as M.tb-infected RAW264.7 macrophages. Mouse bone marrow-derived macrophages (BMMØ) were treated with exosomes +/− IFN-γ. Cells were harvested and analyzed for suppression of IFN-γ responsive genes by flow cytometry and real time PCR. We found that exosomes derived from M.tb H37Rv-infected but not from uninfected macrophages inhibited IFN-γ induced MHC class II and CD64 expression on BMMØ. This inhibition was only partially dependent on the presence of lipoproteins but completely dependent on TLR2 and MyD88. The exosomes isolated from infected cells did not inhibit STAT1 Tyrosine phosphorylation but down-regulated IFN-γ induced expression of the class II major histocompatibity complex transactivator; a key regulator of class II MHC expression. Microarray studies showed that subsets of genes induced by IFN-γ were inhibited by exosomes from H37Rv-infeced cells including genes involved in antigen presentation. Moreover, this set of genes partially overlapped with the IFN-γ-induced genes inhibited by H37Rv infection.

Conclusions

Our study suggests that exosomes, as carriers of M.tb pathogen associated molecular patterns (PAMPs), may provide a mechanism by which M.tb may exert its suppression of a host immune response beyond the infected cell.     

Construction of a virtual Mycobacterium tuberculosis consensus genome and its application to data from a next generation sequencer

Background

Although Mycobacterium tuberculosis isolates are consisted of several different lineages and the epidemiology analyses are usually assessed relative to a particular reference genome, M. tuberculosis H37Rv, which might introduce some biased results. Those analyses are essentially based genome sequence information of M. tuberculosis and could be performed in sillico in theory, with whole genome sequence (WGS) data available in the databases and obtained by next generation sequencers (NGSs). As an approach to establish higher resolution methods for such analyses, whole genome sequences of the M. tuberculosis complexes (MTBCs) strains available on databases were aligned to construct virtual reference genome sequences called the consensus sequence (CS), and evaluated its feasibility in in sillico epidemiological analyses.

Results

The consensus sequence (CS) was successfully constructed and utilized to perform phylogenetic analysis, evaluation of read mapping efficacy, which is crucial for detecting single nucleotide polymorphisms (SNPs), and various MTBC typing methods virtually including spoligotyping, VNTR, Long sequence polymorphism and Beijing typing. SNPs detected based on CS, in comparison with H37Rv, were utilized in concatemer-based phylogenetic analysis to determine their reliability relative to a phylogenetic tree based on whole genome alignment as the gold standard. Statistical comparison of phylogenic trees based on CS with that of H37Rv indicated the former showed always better results that that of later. SNP detection and concatenation with CS was advantageous because the frequency of crucial SNPs distinguishing among strain lineages was higher than those of H37Rv. The number of SNPs detected was lower with the consensus than with the H37Rv sequence, resulting in a significant reduction in computational time. Performance of each virtual typing was satisfactory and accorded with those published when those are available.

Conclusions

These results indicated that virtual CS constructed from genome sequence data is an ideal approach as a reference for MTBC studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1368-9) contains supplementary material, which is available to authorized users.     

Variation among Genome Sequences of H37Rv Strains of Mycobacterium tuberculosis from Multiple Laboratories

The publication of the complete genome sequence for Mycobacterium tuberculosis H37Rv in 1998 has had a great impact on the research community. Nonetheless, it is suspected that genetic differences have arisen in stocks of H37Rv that are maintained in different laboratories. In order to assess the consistency of the genome sequences among H37Rv strains in use and the extent to which they have diverged from the original strain sequenced, we carried out whole-genome sequencing on six strains of H37Rv from different laboratories. Polymorphisms at 73 sites were observed, which were shared among the lab strains, though 72 of these were also shared with H37Ra and are likely to be due to sequencing errors in the original H37Rv reference sequence. An updated H37Rv genome sequence should be valuable to the tuberculosis research community as well as the broader microbial research community. In addition, several polymorphisms unique to individual strains and several shared polymorphisms were identified and shown to be consistent with the known provenance of these strains. Aside from nucleotide substitutions and insertion/deletions, multiple IS6110 transposition events were observed, supporting the theory that they play a significant role in plasticity of the M. tuberculosis genome. This genome-wide catalog of genetic differences can help explain any phenotypic differences that might be found, including a frameshift mutation in the mycocerosic acid synthase gene which causes two of the strains to be deficient in biosynthesis of the surface glycolipid phthiocerol dimycocerosate (PDIM). The resequencing of these six lab strains represents a fortuitous “in vitro evolution” experiment that demonstrates how the M. tuberculosis genome continues to evolve even in a controlled environment.Publication of the whole genome sequence of the H37Rv strain of Mycobacterium tuberculosis by Stewart Cole and colleagues in 1998 provided a breakthrough in tuberculosis (TB) research (8), leading to insights into the biology, metabolism, and evolution of this infectious pathogen. Large protein families related to fatty acid and polyketide biosynthesis, regulation (e.g., sigma factors and two-component sensor systems), drug efflux pumps and transporters, and the PE_PGRS proteins (a large duplicated family unique to the M. tuberculosis group of mycobacteria) were identified. In addition, transposons, prophage-like elements, and other repetitive and/or mobile genetic elements were identified (18). This genomic information has played an essential role in interpreting gene expression studies, modeling persistence, and identifying essential proteins as putative targets for drug discovery. However, to date the functions of only half of the genes (1,756/4,066) have been determined or predicted, and the rest remain annotated as “hypothetical proteins” (6).The H37Rv strain was initially selected for sequencing because it is a widely used laboratory strain that has retained its virulence. H37Rv was initially derived from a clinical isolate, H37, obtained from a patient with pulmonary tuberculosis in 1905. H37Rv falls in the T clade (5) and single-nucleotide polymorphism (SNP) cluster group SCG-6b (12). The virulence of H37Rv can be demonstrated in a number of animal models. For example, SCID mice infected with H37Rv typically have a mean time to death of 30 to 35 days, depending on the dose and route of inoculation (13).An avirulent strain, H37Ra, was also derived from H37 by culturing on solid egg medium and selecting for resistance to lysis (42). The strain was found not to cause disease in guinea pigs (43) or in mice (27). It has a colony morphology (smooth) different from that of H37Rv (rough) and several other phenotypic differences (14, 29). The H37Rv (ATCC 25618) and H37Ra (ATCC 25177) strains are maintained at the Trudeau Institute in New York (3), although unfortunately, the original H37 clinical isolate has been lost. Strain ATCC 27294 (TMC 102) is also frequently used as a representative of H37Rv in studies and treated equivalently in the literature. ATCC 25618 and ATCC 27294 were both isolated from the same patient in different years, and both are fully drug susceptible.The complete genome of H37Ra has been sequenced by Zheng et al. (48), who found 272 polymorphisms compared to the genome sequence determined by Cole et al. (8) for H37Rv. However, a subset of the polymorphic sites were found to match CDC1551, and upon resequencing of 85 such sites in H37Rv, 79 were determined to be errors in the H37Rv reference sequence. In addition, H37Ra has insertions of IS6110 at two novel sites and a loss of one, compared to the 16 sites in H37Rv. The 130 genuine H37Ra-specific polymorphisms found were divided into those in coding regions, those in upstream regulatory regions, and those in noncoding, nonregulatory intergenic regions in order to assess potential relevance to virulence. Polymorphisms in the promoter regions of sigC, nrdH (glutaredoxin-like electron transporter), and pabB (para-amino benzoate synthase), as well as nonsynonymous substitutions in mazG (regulator of stringent response), phoP (two-component sensor regulating biosynthesis of cell surface lipid antigens), pks12 (polyketide synthase involved in biosynthesis of mycoketides), and nrp (nonribosomal peptide synthetase potentially involved in phthiocerol dimycocerosate [PDIM] biosynthesis), were highlighted as possible causes of the loss of virulence. H37Ra does not synthesize a number of cell surface antigens, including sulfolipid-1, trehalose mycolates, and PDIM (7). The roles of mutations in phoP and sigM, both of which regulate expression of genes involved in biosynthesis of cell surface antigens, have been subsequently investigated, though neither seems to be singularly responsible for the avirulence of H37Ra (17, 35). Multiple mutations in PPE and PE_PGRS genes are also observed in H37Ra, and there has been speculation about the role of these genes in virulence (39). However, the RvD2 region (an 8-kb region present in H37Ra but deleted in H37Rv, including an IS6110 insertion element, mmpL14, and several hypothetical genes) is known not to be responsible for differences in virulence (25).Because of its importance as a model strain used in laboratory studies, it is essential to determine how consistent different stocks of H37Rv in different laboratories are with the reference genome sequence and with each other. Different stocks could accumulate independent polymorphisms over time, and such inconsistencies could potentially make results of studies obtained with H37Rv cultures from different labs difficult to compare, particularly if they affect virulence, drug tolerance, metabolism, cell wall constitution, etc. Furthermore, sequencing errors in the original genome sequence are possible. In order to evaluate differences among currently used variants of H37Rv, we resequenced the complete genomes of six extant H37Rv strains (two samples of ATCC 25618 and four of ATCC 27294) using Illumina sequencing technology. We compared differences among them and differences from the reference sequences for H37Rv and H37Ra available from GenBank. The results of this study identify a common set of 73 polymorphisms shared among all six sequenced strains relative to the H37Rv reference strain. Most (72) of these are shared with H37Ra and likely correspond to sequencing errors in the original H37Rv genome sequence. However, there are several sites where additional polymorphisms are shared among a subset of strains, and several strains have a small number of unique polymorphisms. Furthermore, examination of insertion sites of the IS6110 transposable element reveals several changes that have occurred among these strains. These results illustrate the ongoing evolution of this strain and divergence from the sequenced reference strain of H37Rv and highlight the importance of understanding the genetic differences unique to the stock used in each laboratory.     

The Impact of Mouse Passaging of Mycobacterium tuberculosis Strains prior to Virulence Testing in the Mouse and Guinea Pig Aerosol Models

Background

It has been hypothesized that the virulence of lab-passaged Mycobacterium tuberculosis and recombinant M. tuberculosis mutants might be reduced due to multiple in vitro passages, and that virulence might be augmented by passage of these strains through mice before quantitative virulence testing in the mouse or guinea pig aerosol models.

Methodology/Principal Findings

By testing three M. tuberculosis H37Rv samples, one deletion mutant, and one recent clinical isolate for survival by the quantitative organ CFU counting method in mouse or guinea pig aerosol or intravenous infection models, we could discern no increase in bacterial fitness as a result of passaging of M. tuberculosis strains in mice prior to quantitative virulence testing in two animal models. Surface lipid expression as assessed by neutral red staining and thin-layer chromatography for PDIM analysis also failed to identify virulence correlates.

Conclusions/Significance

These results indicate that animal passaging of M. tuberculosis strains prior to quantitative virulence testing in mouse or guinea pig models does not enhance or restore potency to strains that may have lost virulence due to in vitro passaging. It is critical to verify virulence of parental strains before genetic manipulations are undertaken and comparisons are made.     

Morphine suppresses IFN signaling pathway and enhances AIDS virus infection

Background

Opioids exert a profound influence on immunomodulation and enhance HIV infection and replication. However, the mechanism(s) of their action remains to be determined. We thus investigated the impact of morphine on the intracellular innate antiviral immunity.

Methodology/Principal Findings

Seven-day-cultured macrophages were infected with equal amounts of cell-free HIV Bal or SIV Delta_B670 for 2 h at 37°C after 24 h of treatment with or without morphine. Effect of morphine on HIV/SIV infection and replication was evaluated by HIV/SIV RT activity assay and indirect immunofluorescence for HIV p24 or SIV p28 antigen. The mRNA expression of cellular factors suppressed or induced by morphine treatment was analyzed by the real-time RT-PCR. We demonstrated that morphine treatment of human blood monocyte-derived macrophages significantly inhibited the expression of interferons (IFN-α, IFN-β and IFN-λ) and IFN-inducible genes (APOBEC3C/3F/3G and 3H). The further experiments showed that morphine suppressed the expression of several key elements (RIG-I and IRF-7) in IFN signaling pathway. In addition, morphine treatment induced the expression of suppressor of cytokine signaling protein-1, 2, 3 (SOCS-1, 2, 3) and protein inhibitors of activated STAT-1, 3, X, Y (PIAS-1, 3, X, Y), the key negative regulators of IFN signaling pathway.

Conclusions

These findings indicate that morphine impairs intracellular innate antiviral mechanism(s) in macrophages, contributing to cell susceptibility to AIDS virus infection.     

Interactions of attenuated Mycobacterium tuberculosis phoP mutant with human macrophages

Background

Mycobacterium tuberculosis phoP mutant SO2 derived from a clinical isolate was shown to be attenuated in mouse bone marrow-derived macrophages and in vivo mouse infection model and has demonstrated a high potential as attenuated vaccine candidate against tuberculosis.

Methodology/Principal Findings

In this study, we analyze the adhesion and the intracellular growth and trafficking of SO2 in human macrophages. Our results indicate an enhanced adhesion to phagocitic cells and impaired intracellular replication of SO2 in both monocyte-derived macrophages and human cell line THP-1 in comparison with the wild type strain, consistent with murine model. Intracellular trafficking analysis in human THP-1 cells suggest that attenuation of SO2 within macrophages could be due to an impaired ability to block phagosome-lysosome fusion compared with the parental M. tuberculosis strain. No differences were found between SO2 and the wild-type strains in the release and mycobacterial susceptibility to nitric oxide (NO) produced by infected macrophages.

Conclusions/Significance

SO2 has enhanced ability to bind human macrophages and differs in intracellular trafficking as to wild-type M. tuberculosis. The altered lipid profile expression of the phoP mutant SO2 and its inability to secrete ESAT-6 is discussed.     

Application of Hyperbranched Rolling Circle Amplification for Direct Detection of Mycobacterium Tuberculosis in Clinical Sputum Specimens

Background

Global tuberculosis (TB) control is encumbered by the lack of a rapid and simple detection method for diagnosis, especially in low-resource areas. An isothermal amplification method, hyperbranched rolling circle amplification (HRCA), was optimized to detect Mycobacterium tuberculosis (Mtb) in clinical sputum specimens.

Methods

A clinical validation study was performed to assess the diagnostic accuracy of HRCA. In order to analyze the detection limit of HRCA under optimal conditions, the method was initially used to detect purified H37Rv strain DNA and culture suspensions. Next, three strains of Mycobacterium tuberculosis complex (MTC) and eight strains of non-tuberculosis mycobacterium (NTM) were analyzed in order to evaluate specificity. Sputum specimens from 136 patients with diagnosed pulmonary TB, 38 lung cancer patients, and 34 healthy donors were tested by HRCA to validate the clinical application of HRCA for the rapid detection of Mtb.

Results

The detection limit of HRCA for purified H37Rv DNA and culture suspensions was 740 aM and 200cfu/ml, respectively. The results of all MTC strains were positive in contrast to the NTM specimens which were all negative. The detection sensitivity for the 136 sputum specimens from TB patients was 77.2% (105/136), which was slightly lower than that of quantitative real-time PCR(79.4%, 108/136) and culture (80.9%,110/136). The sensitivity of all three methods was statistically higher than smear microscopy (44.9%, 61/136). The overall specificity of HRCA was 98.6% (71/72) which was similar to that of quantitative real-time PCR (qRT-PCR) and smear/culture methods (100%, 72/72).

Conclusions

Use of the HRCA assay for detection of Mtb within clinical sputum specimens was demonstrated to be highly sensitive and specific. Moreover, the performance of HRCA is simple and cost-effective compared with qRT-PCR and is less time consuming than culture. Therefore, HRCA is a promising TB diagnostic tool that can be used routinely in low-resource clinical settings.     

An incomplete TCA cycle increases survival of Salmonella Typhimurium during infection of resting and activated murine macrophages

Background

In comparison to the comprehensive analyses performed on virulence gene expression, regulation and action, the intracellular metabolism of Salmonella during infection is a relatively under-studied area. We investigated the role of the tricarboxylic acid (TCA) cycle in the intracellular replication of Salmonella Typhimurium in resting and activated macrophages, epithelial cells, and during infection of mice.

Methodology/Principal Findings

We constructed deletion mutations of 5 TCA cycle genes in S. Typhimurium including gltA, mdh, sdhCDAB, sucAB, and sucCD. We found that the mutants exhibited increased net intracellular replication in resting and activated murine macrophages compared to the wild-type. In contrast, an epithelial cell infection model showed that the S. Typhimurium ΔsucCD and ΔgltA strains had reduced net intracellular replication compared to the wild-type. The glyoxylate shunt was not responsible for the net increased replication of the TCA cycle mutants within resting macrophages. We also confirmed that, in a murine infection model, the S. Typhimurium ΔsucAB and ΔsucCD strains are attenuated for virulence.

Conclusions/Significance

Our results suggest that disruption of the TCA cycle increases the ability of S. Typhimurium to survive within resting and activated murine macrophages. In contrast, epithelial cells are non-phagocytic cells and unlike macrophages cannot mount an oxidative and nitrosative defence response against pathogens; our results show that in HeLa cells the S. Typhimurium TCA cycle mutant strains show reduced or no change in intracellular levels compared to the wild-type [1]. The attenuation of the S. Typhimurium ΔsucAB and ΔsucCD mutants in mice, compared to their increased net intracellular replication in resting and activated macrophages suggest that Salmonella may encounter environments within the host where a complete TCA cycle is advantageous.     

Divergent effects of mycobacterial cell wall glycolipids on maturation and function of human monocyte-derived dendritic cells

Background

Mycobacterium tuberculosis (Mtb) is able to evade the immune defenses and may persist for years, decades and even lifelong in the infected host. Mtb cell wall components may contribute to such persistence by modulating several pivotal types of immune cells. Dendritic cells (DCs) are the most potent antigen-presenting cells and hence play a crucial role in the initial immune response to infections by connecting the innate with the adaptive immune system.

Principal Findings

We investigated the effects of two of the major mycobacterial cell wall-associated types of glycolipids, mannose-capped lipoarabinomannan (ManLAM) and phosphatidylinositol mannosides (PIMs) purified from the Mtb strains H37Rv and Mycobacterium bovis, on the maturation and cytokine profiles of immature human monocyte-derived DCs. ManLAM from Mtb H37Rv stimulated the release of pro-inflammatory cytokines TNF, IL-12, and IL-6 and expression of co-stimulatory (CD80, CD86) and antigen-presenting molecules (MHC class II). ManLAM from M. bovis also induced TNF, IL-12 and IL-6 but at significantly lower levels. Importantly, while ManLAM was found to augment LPS-induced DC maturation and pro-inflammatory cytokine production, addition of PIMs from both Mtb H37Rv and M. bovis strongly reduced this stimulatory effect.

Conclusions

These results indicate that the mycobacterial cell wall contains macromolecules of glycolipid nature which are able to induce strong and divergent effects on human DCs; i.e while ManLAM is immune-stimulatory, PIMs act as powerful inhibitors of DC cytokine responses. Thus PIMs may be important Mtb-associated virulence factors contributing to the pathogenesis of tuberculosis disease. These findings may also aid in the understanding of some earlier conflicting reports on the immunomodulatory effects exerted by different ManLAM preparations.     

Differential cell reaction upon Toll-like receptor 4 and 9 activation in human alveolar and lung interstitial macrophages

Background

Investigations on pulmonary macrophages (MΦ) mostly focus on alveolar MΦ (AM) as a well-defined cell population. Characteristics of MΦ in the interstitium, referred to as lung interstitial MΦ (IM), are rather ill-defined. In this study we therefore aimed to elucidate differences between AM and IM obtained from human lung tissue.

Methods

Human AM and IM were isolated from human non-tumor lung tissue from patients undergoing lung resection. Cell morphology was visualized using either light, electron or confocal microscopy. Phagocytic activity was analyzed by flow cytometry as well as confocal microscopy. Surface marker expression was measured by flow cytometry. Toll-like receptor (TLR) expression patterns as well as cytokine expression upon TLR4 or TLR9 stimulation were assessed by real time RT-PCR and cytokine protein production was measured using a fluorescent bead-based immunoassay.

Results

IM were found to be smaller and morphologically more heterogeneous than AM, whereas phagocytic activity was similar in both cell types. HLA-DR expression was markedly higher in IM compared to AM. Although analysis of TLR expression profiles revealed no differences between the two cell populations, AM and IM clearly varied in cell reaction upon activation. Both MΦ populations were markedly activated by LPS as well as DNA isolated from attenuated mycobacterial strains (M. bovis H37Ra and BCG). Whereas AM expressed higher amounts of inflammatory cytokines upon activation, IM were more efficient in producing immunoregulatory cytokines, such as IL10, IL1ra, and IL6.

Conclusion

AM appear to be more effective as a non-specific first line of defence against inhaled pathogens, whereas IM show a more pronounced regulatory function. These dissimilarities should be taken into consideration in future studies on the role of human lung MΦ in the inflammatory response.