M. Abdallah

Type VII secretion — mycobacteria show the way

Recent evidence shows that mycobacteria have developed novel and specialized secretion systems for the transport of extracellular proteins across their hydrophobic, and highly impermeable, cell wall. Strikingly, mycobacterial genomes encode up to five of these transport systems. Two of these systems, ESX-1 and ESX-5, are involved in virulence — they both affect the cell-to-cell migration of pathogenic mycobacteria. Here, we discuss this novel secretion pathway and consider variants that are present in various Gram-positive bacteria. Given the unique composition of this secretion system, and its general importance, we propose that, in line with the accepted nomenclature, it should be called type VII secretion.

ESX-1-mediated translocation to the cytosol controls virulence of mycobacteria

Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium leprae, are among the most potent human bacterial pathogens. The discovery of cytosolic mycobacteria challenged the paradigm that these pathogens exclusively localize within the phagosome of host cells. As yet the biological relevance of mycobacterial translocation to the cytosol remained unclear. In this current study we used electron microscopy techniques to establish a clear link between translocation and mycobacterial virulence. Pathogenic, patient‐derived mycobacteria species were found to translocate to the cytosol, while non‐pathogenic species did not. We were further able to link cytosolic translocation with pathogenicity by introducing the ESX‐1 (type VII) secretion system into the non‐virulent, exclusively phagolysosomal Mycobacterium bovis BCG. Furthermore, we show that translocation is dependent on the C‐terminus of the early‐secreted antigen ESAT‐6. The C‐terminal truncation of ESAT‐6 was shown to result in attenuation in mice, again linking translocation to virulence. Together, these data demonstrate the molecular mechanism facilitating translocation of mycobacteria. The ability to translocate from the phagolysosome to the cytosol is with this study proven to be biologically significant as it determines mycobacterial virulence.

A specific secretion system mediates PPE41 transport in pathogenic mycobacteria.

Mycobacterial genomes contain two unique gene families, the so‐called PE and PPE gene families, which are highly expanded in the pathogenic members of this genus. Here we report that one of the PPE proteins, i.e. PPE41, is secreted by pathogenic mycobacteria, both in culture and in infected macrophages. As PPE41 lacks a signal sequence a dedicated secretion system must be involved. A single gene was identified in Mycobacterium marinum that showed strongly reduced PPE41 secretion. This gene was located in a gene cluster whose predicted proteins encode components of an ESAT‐6‐like secretion system. This cluster, designated ESX‐5, is conserved in various pathogenic mycobacteria, but not in the saprophytic species Mycobacterium smegmatis. Therefore, different regions of this cluster were introduced in M. smegmatis. Only introduction of the complete ESX‐5 locus resulted in efficient secretion of heterologously expressed PPE41. This PPE secretion system is also involved in the virulence of pathogenic mycobacteria, as the ESX‐5 mutant of M. marinum was affected in spreading to uninfected macrophages.

PPE and PE_PGRS proteins of Mycobacterium marinum are transported via the type VII secretion system ESX-5

ESX‐5 is one of the five type VII secretion systems found in mycobacteria. These secretion systems are also known as ESAT‐6‐like secretion systems. Here, we have determined the secretome of ESX‐5 by a proteomic approach in two different strains of Mycobacterium marinum. Comparison of the secretion profile of wild‐type strains and their ESX‐5 mutants showed that a number of PE_PGRS and PPE‐MPTR proteins are dependent on ESX‐5 for transport. The PE and PPE protein families are unique to mycobacteria, are highly expanded in several pathogenic species, such as Mycobacterium tuberculosis and M. marinum, and certain family members are cell surface antigens associated with virulence. Using a monoclonal antibody directed against the PGRS domain we showed that nearly all PE_PGRS proteins that are recognized by this antibody are missing in the supernatant of ESX‐5 mutants. In addition to PE_PGRS and PPE proteins, the ESX‐5 secretion system is responsible for the secretion of a ESAT‐6‐like proteins. Together, these data show that ESX‐5 is probably a major secretion pathway for mycobacteria and that this system is responsible for the secretion of recently evolved PE_PGRS and PPE proteins.

The ESX-5 secretion system of Mycobacterium marinum modulates the macrophage response

The ESX-5 secretion system of pathogenic mycobacteria is responsible for the secretion of various PPE and PE-PGRS proteins. To better understand the role of ESX-5 effector proteins in virulence, we analyzed the interactions of Mycobacterium marinum ESX-5 mutant with human macrophages (Mφ). Both wild-type bacteria and the ESX-5 mutant were internalized and the ESX-5 mutation did not affect the escape of mycobacteria from phagolysosomes into the cytosol, as was shown by electron microscopy. However, the ESX-5 mutation strongly effected expression of surface Ags and cytokine secretion. Whereas wild-type M. marinum actively suppressed the induction of appreciable levels of IL-12p40, TNF-α, and IL-6, infection with the ESX-5 mutant resulted in strongly induced production of these proinflammatory cytokines. By contrast, infection with M. marinum wild-type strain resulted in a significant induction of IL-1β production as compared with the ESX-5 mutant. These results show that ESX-5 plays an essential role in the modulation of immune cytokine secretion by human Mφ. Subsequently, we show that an intact ESX-5 secretion system actively suppresses TLR signaling-dependent innate immune cytokine secretion. Together, our results show that ESX-5 substrates, directly or indirectly, strongly modulate the human Mφ response at various critical steps.

Mycobacterium marinum Strains Can Be Divided into Two Distinct Types Based on Genetic Diversity and Virulence

ABSTRACT Mycobacterium marinum causes a systemic tuberculosis-like disease in a large number of poikilothermic animals and is used as a model for mycobacterial pathogenesis. In the present study, we infected zebra fish (Danio rerio) with different strains of M. marinum to determine the variation in pathogenicity. Depending on the M. marinum isolate, the fish developed an acute or chronic disease. Acute disease was characterized by uncontrolled growth of the pathogen and death of all animals within 16 days, whereas chronic disease was characterized by granuloma formation in different organs and survival of the animals for at least 4 to 8 weeks. Genetic analysis of the isolates by amplified fragment length polymorphism showed that M. marinum strains could be divided in two clusters. Cluster I contained predominantly strains isolated from humans with fish tank granuloma, whereas the majority of the cluster II strains were isolated from poikilothermic species. Acute disease progression was noted only with strains belonging to cluster I, whereas all chronic-disease-causing isolates belonged to cluster II. This difference in virulence was also observed in vitro: cluster I isolate Mma20 was able to infect and survive more efficiently in the human macrophage THP-1 and the carp leukocyte CLC cell lines than was the cluster II isolate Mma11. We conclude that strain characteristics play an important role in the pathogenicity of M. marinum. In addition, the correlation between genetic variation and host origin suggests that cluster I isolates are more pathogenic for humans.

Conserved Pro-Glu (PE) and Pro-Pro-Glu (PPE) Protein Domains Target LipY Lipases of Pathogenic Mycobacteria to the Cell Surface via the ESX-5 Pathway

The type VII secretion system ESX-5 is a major pathway for export of PE and PPE proteins in pathogenic mycobacteria. These mycobacteria-specific protein families are characterized by conserved N-terminal domains of 100 and 180 amino acids, which contain the proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) motifs after which they are named. Here we investigated secretion of the triacylglycerol lipase LipY, which in fast-growing mycobacteria contains a signal sequence, but in slow-growing species appears to have replaced the signal peptide with a PE or PPE domain. Selected LipY homologues were expressed in wild-type Mycobacterium marinum and its corresponding ESX-5 mutant, and localization of the proteins was investigated by immunoblotting and electron microscopy. Our study shows that Mycobacterium tuberculosis PE-LipY (LipYtub) and M. marinum PPE-LipY (LipYmar) are both secreted to the bacterial surface in an ESX-5-dependent fashion. After transport, the PE/PPE domains are removed by proteolytic cleavage. In contrast, Mycobacterium gilvum LipY, which has a signal sequence, is not transported to the cell surface. Furthermore, we show that LipYtub and LipYmar require their respective PE and PPE domains for ESX-5-dependent secretion. The role of the PE domain in ESX-5 secretion was confirmed in a whole cell lipase assay, in which wild-type bacteria expressing full-length LipYtub, but not LipYtub lacking its PE domain, were shown to hydrolyze extracellular lipids. In conclusion, both PE and PPE domains contain a signal required for secretion of LipY by the ESX-5 system, and these domains are proteolytically removed upon translocation.

Mycobacterial Secretion Systems ESX-1 and ESX-5 Play Distinct Roles in Host Cell Death and Inflammasome Activation

During infection of humans and animals, pathogenic mycobacteria manipulate the host cell causing severe diseases such as tuberculosis and leprosy. To understand the basis of mycobacterial pathogenicity, it is crucial to identify the molecular virulence mechanisms. In this study, we address the contribution of ESX-1 and ESX-5—two homologous type VII secretion systems of mycobacteria that secrete distinct sets of immune modulators—during the macrophage infection cycle. Using wild-type, ESX-1– and ESX-5–deficient mycobacterial strains, we demonstrate that these secretion systems differentially affect subcellular localization and macrophage cell responses. We show that in contrast to ESX-1, the effector proteins secreted by ESX-5 are not required for the translocation of Mycobacterium tuberculosis or Mycobacterium marinum to the cytosol of host cells. However, the M. marinum ESX-5 mutant does not induce inflammasome activation and IL-1β activation. The ESX-5 system also induces a caspase-independent cell death after translocation has taken place. Importantly, by means of inhibitory agents and small interfering RNA experiments, we reveal that cathepsin B is involved in both the induction of cell death and inflammasome activation upon infection with wild-type mycobacteria. These results reveal distinct roles for two different type VII secretion systems during infection and shed light on how virulent mycobacteria manipulate the host cell in various ways to replicate and spread.

Essential Role of the ESX-5 Secretion System in Outer Membrane Permeability of Pathogenic Mycobacteria

Mycobacteria possess different type VII secretion (T7S) systems to secrete proteins across their unusual cell envelope. One of these systems, ESX-5, is only present in slow-growing mycobacteria and responsible for the secretion of multiple substrates. However, the role of ESX-5 substrates in growth and/or virulence is largely unknown. In this study, we show that esx-5 is essential for growth of both Mycobacterium marinum and Mycobacterium bovis. Remarkably, this essentiality can be rescued by increasing the permeability of the outer membrane, either by altering its lipid composition or by the introduction of the heterologous porin MspA. Mutagenesis of the first nucleotide-binding domain of the membrane ATPase EccC5 prevented both ESX-5-dependent secretion and bacterial growth, but did not affect ESX-5 complex assembly. This suggests that the rescuing effect is not due to pores formed by the ESX-5 membrane complex, but caused by ESX-5 activity. Subsequent proteomic analysis to identify crucial ESX-5 substrates confirmed that all detectable PE and PPE proteins in the cell surface and cell envelope fractions were routed through ESX-5. Additionally, saturated transposon-directed insertion-site sequencing (TraDIS) was applied to both wild-type M. marinum cells and cells expressing mspA to identify genes that are not essential anymore in the presence of MspA. This analysis confirmed the importance of esx-5, but we could not identify essential ESX-5 substrates, indicating that multiple of these substrates are together responsible for the essentiality. Finally, examination of phenotypes on defined carbon sources revealed that an esx-5 mutant is strongly impaired in the uptake and utilization of hydrophobic carbon sources. Based on these data, we propose a model in which the ESX-5 system is responsible for the transport of cell envelope proteins that are required for nutrient uptake. These proteins might in this way compensate for the lack of MspA-like porins in slow-growing mycobacteria.

ESX-5-deficient Mycobacterium marinum is hypervirulent in adult zebrafish.

ESX‐5 is a mycobacterial type VII protein secretion system responsible for transport of numerous PE and PPE proteins. It is involved in the induction of host cell death and modulation of the cytokine response in vitro. In this work, we studied the effects of ESX‐5 in embryonic and adult zebrafish using Mycobacterium marinum. We found that ESX‐5‐deficient M. marinum was slightly attenuated in zebrafish embryos. Surprisingly, the same mutant showed highly increased virulence in adult zebrafish, characterized by increased bacterial loads and early onset of granuloma formation with rapid development of necrotic centres. This early onset of granuloma formation was accompanied by an increased expression of pro‐inflammatory cytokines and tissue remodelling genes in zebrafish infected with the ESX‐5 mutant. Experiments using RAG‐1‐deficient zebrafish showed that the increased virulence of the ESX‐5 mutant was not dependent on the adaptive immune system. Mixed infection experiments with wild‐type and ESX‐5 mutant bacteria showed that the latter had a specific advantage in adult zebrafish and outcompeted wild‐type bacteria. Together our experiments indicate that ESX‐5‐mediated protein secretion is used by M. marinum to establish a moderate and persistent infection.