Efficient genome editing in pathogenic mycobacteria using Streptococcus thermophilus CRISPR1-Cas9

The ability to genetically engineer pathogenic mycobacteria has increased significantly over the last decades due to the generation of new molecular tools. Recently, the application of the Streptococcus pyogenes and the Streptococcus thermophilus CRISPR‐Cas9 systems in mycobacteria has enabled gene editing and efficient CRISPR interference‐mediated transcriptional regulation. Here, we converted CRISPR interference into an efficient genome editing tool for mycobacteria. We demonstrate that the Streptococcus thermophilus CRISPR1-Cas9 (Sth1Cas9) is functional in Mycobacterium marinum and Mycobacterium tuberculosis, enabling highly efficient and precise DNA breaks and indel formation, without any off-target effects. In addition, with dual sgRNAs this system can be used to generate two indels simultaneously or to create specific deletions. The ability to use the power of the CRISPR-Cas9-mediated gene editing toolbox in M. tuberculosis with a single step will accelerate research into this deadly pathogen

Mycobacterium tuberculosis β-lactamase variant reduces sensitivity to ampicillin/avibactam in a zebrafish-Mycobacterium marinum model of tuberculosis

Abstract The β-lactamase of Mycobacterium tuberculosis, BlaC, hydrolyzes β-lactam antibiotics, hindering the use of these antibiotics for the treatment of tuberculosis. Inhibitors, such as avibactam, can reversibly inhibit the enzyme, allowing for the development of combination therapies using both antibiotic and inhibitor. However, laboratory evolution studies using Escherichia coli resulted in the discovery of single amino acid variants of BlaC that reduce the sensitivity for inhibitors or show higher catalytic efficiency against antibiotics. Here, we tested these BlaC variants under more physiological conditions using the M. marinum infection model of zebrafish, which recapitulates hallmark features of tuberculosis, including the intracellular persistence of mycobacteria in macrophages and the induction of granuloma formation. To this end, the M. tuberculosis blaC gene was integrated into the chromosome of a blaC frameshift mutant of M. marinum. Subsequently, the resulting strains were used to infect zebrafish embryos in order to test the combinatorial effect of ampicillin and avibactam. The results show that embryos infected with an M. marinum strain producing BlaC show lower infection levels after treatment than untreated embryos. Additionally, BlaC K234R showed higher infection levels after treatment than those infected with bacteria producing the wild-type enzyme, demonstrating that the zebrafish host is less sensitive to the combinatorial therapy of β-lactam antibiotic and inhibitor. These findings are of interest for future development of combination therapies to treat tuberculosis

The ESX-1 Substrate PPE68 Has a Key Function in ESX-1-Mediated Secretion in Mycobacterium marinum

Mycobacteria use specialized type VII secretion systems (T7SSs) to secrete proteins across their diderm cell envelope. One of the T7SS subtypes, named ESX-1, is a major virulence determinant in pathogenic species such as Mycobacterium tuberculosis and the fish pathogen Mycobacterium marinum. ESX-1 secretes a variety of substrates, called Esx, PE, PPE, and Esp proteins, at least some of which are folded heterodimers. Investigation into the functions of these substrates is problematic, because of the intricate network of codependent secretion between several ESX-1 substrates. Here, we describe the ESX-1 substrate PPE68 as essential for secretion of the highly immunogenic substrates EsxA and EspE via the ESX-1 system in M. marinum. While secreted PPE68 is processed on the cell surface, the majority of cell-associated PPE68 of M. marinum and M. tuberculosis is present in a cytosolic complex with its PE partner and the EspG1 chaperone. Interfering with the binding of EspG1 to PPE68 blocked its export and the secretion of EsxA and EspE. In contrast, esxA was not required for the secretion of PPE68, revealing a hierarchy in codependent secretion. Remarkably, the final 10 residues of PPE68, a negatively charged domain, seem essential for EspE secretion, but not for the secretion of EsxA and of PPE68 itself. This indicates that distinctive domains of PPE68 are involved in secretion of the different ESX-1 substrates. Based on these findings, we propose a mechanistic model for the central role of PPE68 in ESX-1-mediated secretion and substrate codependence. IMPORTANCE Pathogenic mycobacteria, such Mycobacterium tuberculosis and Mycobacterium marinum, use a type VII secretion system (T7SS) subtype, called ESX-1, to mediate intracellular survival via phagosomal rupture and subsequent translocation of the mycobacterium to the host cytosol. Identifying the ESX-1 substrate that is responsible for this process is problematic because of the intricate network of codependent secretion between ESX-1 substrates. Here, we show the central role of the ESX-1 substrate PPE68 for the secretion of ESX-1 substrates in Mycobacterium marinum. Unravelling the mechanism of codependent secretion will aid the functional understanding of T7SSs and will allow the analysis of the individual roles of ESX-1 substrates in the virulence caused by the significant human pathogen Mycobacterium tuberculosis

The ESX-1 Substrate PPE68 Has a Key Function in ESX-1-Mediated Secretion in Mycobacterium marinum

Mycobacteria use specialized type VII secretion systems (T7SSs) to secrete proteins across their diderm cell envelope. One of the T7SS subtypes, named ESX-1, is a major virulence determinant in pathogenic species such as Mycobacterium tuberculosis and the fish pathogen Mycobacterium marinum. ESX-1 secretes a variety of substrates, called Esx, PE, PPE, and Esp proteins, at least some of which are folded heterodimers. Investigation into the functions of these substrates is problematic, because of the intricate network of codependent secretion between several ESX-1 substrates. Here, we describe the ESX-1 substrate PPE68 as essential for secretion of the highly immunogenic substrates EsxA and EspE via the ESX-1 system in M. marinum. While secreted PPE68 is processed on the cell surface, the majority of cell-associated PPE68 of M. marinum and M. tuberculosis is present in a cytosolic complex with its PE partner and the EspG1 chaperone. Interfering with the binding of EspG1 to PPE68 blocked its export and the secretion of EsxA and EspE. In contrast, esxA was not required for the secretion of PPE68, revealing a hierarchy in codependent secretion. Remarkably, the final 10 residues of PPE68, a negatively charged domain, seem essential for EspE secretion, but not for the secretion of EsxA and of PPE68 itself. This indicates that distinctive domains of PPE68 are involved in secretion of the different ESX-1 substrates. Based on these findings, we propose a mechanistic model for the central role of PPE68 in ESX-1-mediated secretion and substrate codependence. IMPORTANCE Pathogenic mycobacteria, such Mycobacterium tuberculosis and Mycobacterium marinum, use a type VII secretion system (T7SS) subtype, called ESX-1, to mediate intracellular survival via phagosomal rupture and subsequent translocation of the mycobacterium to the host cytosol. Identifying the ESX-1 substrate that is responsible for this process is problematic because of the intricate network of codependent secretion between ESX-1 substrates. Here, we show the central role of the ESX-1 substrate PPE68 for the secretion of ESX-1 substrates in Mycobacterium marinum. Unravelling the mechanism of codependent secretion will aid the functional understanding of T7SSs and will allow the analysis of the individual roles of ESX-1 substrates in the virulence caused by the significant human pathogen Mycobacterium tuberculosis

An optimized retroviral toolbox for overexpression and genetic perturbation of primary lymphocytes

Genetic manipulation of primary lymphocytes is crucial for both clinical purposes and fundamental research. Despite their broad use, we encountered a paucity of data on systematic comparison and optimization of retroviral vectors, the workhorses of genetic modification of primary lymphocytes. Here, we report the construction and validation of a versatile range of retroviral expression vectors. These vectors can be used for the knockdown or overexpression of genes of interest in primary human and murine lymphocytes, in combination with a wide choice of selection and reporter strategies. By streamlining the vector backbone and insert design, these publicly available vectors allow easy interchangeability of the independent building blocks, such as different promoters, fluorescent proteins, surface markers and antibiotic resistance cassettes. We validated these vectors and tested the optimal promoters for in vitro and in vivo overexpression and knockdown of the murine T cell antigen receptor. By publicly sharing these vectors and the data on their optimization, we aim to facilitate genetic modification of primary lymphocytes for researchers entering this field

The ESX-5 System of Pathogenic Mycobacteria Is Involved In Capsule Integrity and Virulence through Its Substrate PPE10

Mycobacteria produce a capsule layer, which consists of glycan-like polysaccharides and a number of specific proteins. In this study, we show that, in slow-growing mycobacteria, the type VII secretion system ESX-5 plays a major role in the integrity and stability of the capsule. We have identified PPE10 as the ESX-5 substrate responsible for this effect. Mutants in esx-5 and ppe10 both have impaired capsule integrity as well as reduced surface hydrophobicity. Electron microscopy, immunoblot and flow cytometry analyses demonstrated reduced amounts of surface localized proteins and glycolipids, and morphological differences in the capsular layer. Since capsular proteins secreted by the ESX-1 system are important virulence factors, we tested the effect of the mutations that cause capsular defects on virulence mechanisms. Both esx-5 and ppe10 mutants of Mycobacterium marinum were shown to be impaired in ESX-1-dependent hemolysis. In agreement with this, the ppe10 and esx5 mutants showed reduced recruitment of ubiquitin in early macrophage infection and intermediate attenuation in zebrafish embryos. These results provide a pivotal role for the ESX-5 secretion system and its substrate PPE10, in the capsular integrity of pathogenic mycobacteria. These findings open up new roads for research on the mycobacterial capsule and its role in virulence and immune modulatio

Ectopic expression of cGAS in Salmonella typhimurium enhances STING-mediated IFN-β response in human macrophages and dendritic cells

Background Interferon (IFN)-β induction via activation of the stimulator of interferon genes (STING) pathway has shown promising results in tumor models. STING is activated by cyclic dinucleotides such as cyclic GMP–AMP dinucleotides with phosphodiester linkages 2′–5′ and 3′–5′ (cGAMPs), that are produced by cyclic GMP–AMP synthetase (cGAS). However, delivery of STING pathway agonists to the tumor site is a challenge. Bacterial vaccine strains have the ability to specifically colonize hypoxic tumor tissues and could therefore be modified to overcome this challenge. Combining high STING-mediated IFN-β levels with the immunostimulatory properties of Salmonella typhimurium could have potential to overcome the immune suppressive tumor microenvironment.Methods We have engineered S. typhimurium to produce cGAMP by expression of cGAS. The ability of cGAMP to induce IFN-β and its IFN-stimulating genes was addressed in infection assays of THP-I macrophages and human primary dendritic cells (DCs). Expression of catalytically inactive cGAS is used as a control. DC maturation and cytotoxic T-cell cytokine and cytotoxicity assays were conducted to assess the potential antitumor response in vitro. Finally, by making use of different S. typhimurium type III secretion (T3S) mutants, the mode of cGAMP transport was elucidated.Results Expression of cGAS in S. typhimurium results in a 87-fold stronger IFN-β response in THP-I macrophages. This effect was mediated by cGAMP production and is STING dependent. Interestingly, the needle-like structure of the T3S system was necessary for IFN-β induction in epithelial cells. DC activation included upregulation of maturation markers and induction of type I IFN response. Coculture of challenged DCs with cytotoxic T cells revealed an improved cGAMP-mediated IFN-γ response. In addition, coculture of cytotoxic T cells with challenged DCs led to improved immune-mediated tumor B-cell killing.Conclusion S. typhimurium can be engineered to produce cGAMPs that activate the STING pathway in vitro. Furthermore, they enhanced the cytotoxic T-cell response by improving IFN-γ release and tumor cell killing. Thus, the immune response triggered by S. typhimurium can be enhanced by ectopic cGAS expression. These data show the potential of S. typhimurium–cGAS in vitro and provides rationale for further research in vivo

Both the <i>esx-5</i> and <i>ppe10</i> mutants have reduced hemolytic activity and show reduced ubiquitin co-localization upon infection of host cells.

<p>A) Contact-dependent hemolysis of red blood cells (RBCs) by <i>M</i>. <i>marinum</i>. <i>M</i>. marinum E11 wild-type cells and isogenic transposon mutants disrupted in <i>espG</i>_<i>5</i>, <i>eccCb</i>_<i>1</i> and <i>ppe10</i> and the complemented <i>ppe10</i> mutant were grown in the presence or absence of Tween-80. Subsequently, washed cells were used for the hemolysis assay. B) Quantification of differentiated THP-1 cells that were infected with <i>M</i>. <i>marinum</i>. Only cells gated as positive for co-localization of the bacteria with ubiquitin were chosen for further analysis (full data and statistics in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005696#ppat.1005696.s006" target="_blank">S6 Fig</a>). The <i>ppe10</i>::<i>tn</i> mutant showed similar levels of ubiquitin co-localization as <i>eccCb</i>_<i>1</i>::<i>tn</i> indicating that this mutant has no cytosolic access in the early stages of infection. C) Images obtained by imaging flow cytometry of wild-type <i>M</i>. <i>marinum</i> and <i>ppe10</i>::<i>tn</i> three hours after infection of differentiated THP-1 cells. Bacteria express <i>mCherry</i> (Red), while ubiquitin is visualized by FK-2 antibody against poly-ubiquitin (Green). D) Super-resolution confocal microscopy images of wild-type <i>M</i>. <i>marinum</i> and <i>ppe10</i>::<i>tn</i> illustrating differential bacterial clustering and ubiquitin labeling of bacteria. Bacteria express <i>mCherry</i> (Red), while ubiquitin is visualized by FK-2 antibody against poly-ubiquitin (Green). A 3-dimensional view of these images can be found in supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005696#ppat.1005696.s009" target="_blank">S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005696#ppat.1005696.s010" target="_blank">S2</a> Movies.</p

The <i>M</i>. <i>marinum ppe10</i>::tn mutant is attenuated in zebrafish embryo infections.

<p>A) <i>M</i>. <i>marinum</i> E11 wild type strain and the isogenic mutants <i>espG</i>_<i>5</i>::<i>tn</i>, <i>eccCb</i>_<i>1</i>::<i>tn</i> and <i>ppe10</i>::<i>tn</i>, as well as the complemented <i>ppe10</i>::<i>tn</i> strain (PPE10-C), were pre-cultured in liquid medium containing Tween-80. 50–100 CFUs of bacteria were injected in the bloodstream of zebrafish embryos at 28 hours post fertilization. Embryos were homogenized five days post infection and plated to establish the number of CFU per embryo. Three independent experiments of six embryos per group were performed and the data were pooled. B) Visualization of <i>M</i>. <i>marinum</i> infection of zebrafish embryos. Wild-type <i>M</i>. <i>marinum</i> or <i>ppe10</i>::<i>tn</i> containing the plasmid pSMT3::<i>mCherry</i> was injected in the bloodstream of zebrafish embryos as described above. After 5 days of infection the embryos were examined by brightfield (top) or fluorescence (bottom) microscopy. C) Quantification of fluorescence in infected zebrafish embryos. <i>M</i>. <i>marinum</i> wild type (closed symbols) and the <i>ppe10</i>::<i>tn</i> (open symbols) mutants expressing <i>mCherry</i> were injected in the bloodstream of zebrafish embryos 28 hours post fertilization. Images were acquired by fluorescence microscopy at day 5 (blue) and 7 (red) post infection and were analyzed for fluorescent intensity by dedicated software [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005696#ppat.1005696.ref043" target="_blank">43</a>]. Differences on day 5 and day 7 were analyzed by GraphPad Prism software using Mann-Whitney two-tailed test. * = <i>p</i><0.05, ** = <i>p</i><0.01, n.s. = not significant.</p

Surface labelling of capsular protein EspE and capsular glycans is dependent on ESX-5.

<p>A) Parental strains of wild-type <i>M</i>. <i>marinum</i> E11, or isogenic mutant strains <i>espG</i>_<i>5</i>::<i>tn</i>, <i>eccCb</i>_<i>1</i>::<i>tn</i> and <i>ppe10</i>::<i>tn</i> expressing pSMT3::<i>mCherry</i>, were grown in the presence or absence of Tween-80 and labeled with the α-EspE antibody and a FITC labeled secondary antibody. Subsequently, cells were analyzed by flow cytometry analysis. High levels of surface expression of EspE could be detected in the absence of Tween-80 in both wild-type <i>M</i>. <i>marinum</i> and the <i>espG</i>_<i>5</i>::<i>tn</i> mutant strain, while <i>ppe10</i>::<i>tn</i> showed an intermediate EspE surface labelling. When grown in the presence of Tween-80, surface labeling of EspE was almost completely lost and reduced to similar levels of the ESX-1 mutant strain <i>eccCb</i>_<i>1</i>::<i>tn</i>. B) Immuno-electron microscopy analysis of <i>M</i>. <i>tuberculosis</i> CDC1551, the <i>esx-5</i> mutant Mtb-<i>eccC</i>_<i>5</i>::<i>tn</i> and the mutant complemented with an integrative plasmid containing the complete <i>esx-5</i> region; pMV-<i>esx-5</i>. Cells were grown in liquid culture with 0.05% Tween-80 (lower row) or without Tween-80 (upper row) and labeled with a monoclonal antibody directed against PIM₆-LAM capsular glycolipids and a gold-labeled secondary antibody. C) Gold labeling of the different Mtb strains depicted in B was quantified by counting the number of gold particles per cell after growth in the presence or absence of Tween-80 (error bars indicate the standard deviation). D) The capsule morphology of wild-type <i>M</i>. <i>marinum</i> and <i>espG</i>_<i>5</i>::<i>tn</i> was analyzed after plunge freezing of bacteria grown in the absence of Tween-80 by cryo-electron microscopy. The black bar indicates the mycobacterial capsular layer. E) Reduced hydrophobicity of the <i>espG</i>_<i>5</i>::<i>tn</i> and <i>ppe10</i>::<i>tn</i> strains, measured as the OD of the aqueous phase after 1 OD of bacteria was incubated with 0.5% (v/v) Xylene in PBS for two hours. Statistical differences were calculated by GraphPad Prism software using one-way ANOVA and (Dunnett’s) multiple comparisons against a single control. ** = <i>p</i><0.01, n.s = not significant.</p