Following mycobacterial entry into macrophages the ESX-1 type VII secretion system promotes phagosomal permeabilization and type I IFN production, key features of tuberculosis pathogenesis. The current model states that the secreted substrate ESAT-6 is required for membrane permeabilization and that a subsequent passive leakage of extracellular bacterial DNA into the host cell cytosol is sensed by the cyclic GMP-AMP synthase (cGAS) and stimulator of IFN genes (STING) pathway to induce type I IFN production. We employed a collection of Mycobacterium marinum ESX-1 transposon mutants in a macrophage infection model and show that permeabilization of the phagosomal membrane does not require ESAT-6 secretion. Moreover, loss of membrane integrity is insufficient to induce type I IFN production. Instead, type I IFN production requires intact ESX-1 function and correlates with release of mitochondrial and nuclear host DNA into the cytosol, indicating that ESX-1 affects host membrane integrity and DNA release via genetically separable mechanisms. These results suggest a revised model for major aspects of ESX-1–mediated host interactions and put focus on elucidating the mechanisms by which ESX-1 permeabilizes host membranes and induces the type I IFN response, questions of importance for our basic understanding of mycobacterial pathogenesis and innate immune sensing.

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are receptors believed to be important for regulation of cellular activation and inflammation. Several pathogenic microbes bind specific Siglecs via sialic acid-containing structures at the microbial surface, interactions that may result in modulation of host responses. Recently, it was shown that the group B Streptococcus (GBS) binds to human Siglec-5 (hSiglec-5), an inhibitory receptor expressed on macrophages and neutrophils, via the IgA-binding surface β protein, providing the first example of a protein/protein interaction between a pathogenic microbe and a Siglec. Here we show that the hSiglec-5-binding part of β resides in the N-terminal half of the protein, which also harbors the previously determined IgA-binding region. We constructed bacterial mutants expressing variants of the β protein with non-overlapping deletions in the N-terminal half of the protein. Using these mutants and recombinant β fragments, we showed that the hSiglec-5-binding site is located in the most N-terminal part of β (B6N region; amino acids 1–152) and that the hSiglec-5- and IgA-binding domains in β are completely separate. We showed with BIAcoreTM analysis that tandem variants of the hSiglec-5- and IgA-binding domains bind to their respective ligands with high affinity. Finally, we showed that the B6N region, but not the IgA-binding region of β, triggers recruitment of the tyrosine phosphatase SHP-2 to hSiglec-5 in U937 monocytes. Taken together, we have identified and isolated the first microbial non-sialic acid Siglec-binding region that can be used as a tool in studies of the β/hSiglec-5 interaction.

Human group IIA-secreted phospholipase A2 (sPLA2-IIA) is a bactericidal molecule important for the innate immune defense against Gram-positive bacteria. In this study, we analyzed its role in the host defense against Streptococcus pyogenes, a major human pathogen, and demonstrated that this bacterium has evolved a previously unidentified mechanism to resist killing by sPLA2-IIA. Analysis of a set of clinical isolates demonstrated that an ∼500-fold higher concentration of sPLA2-IIA was required to kill S. pyogenes compared with strains of the group B Streptococcus, which previously were shown to be sensitive to sPLA2-IIA, indicating that S. pyogenes exhibits a high degree of resistance to sPLA2-IIA. We found that an S. pyogenes mutant lacking sortase A, a transpeptidase responsible for anchoring LPXTG proteins to the cell wall in Gram-positive bacteria, was significantly more sensitive (∼30-fold) to sPLA2-IIA compared with the parental strain, indicating that one or more LPXTG surface proteins protect S. pyogenes against sPLA2-IIA. Importantly, using transgenic mice expressing human sPLA2-IIA, we showed that the sortase A-mediated sPLA2-IIA resistance mechanism in S. pyogenes also occurs in vivo. Moreover, in this mouse model, we also showed that human sPLA2-IIA is important for the defense against lethal S. pyogenes infection. Thus, we demonstrated a novel mechanism by which a pathogenic bacterium can evade the bactericidal action of sPLA2-IIA and we showed that sPLA2-IIA contributes to the host defense against S. pyogenes infection.

Group B streptococcus (GBS) is a leading neonatal pathogen and a growing cause of invasive disease in the elderly, with clinical manifestations such as pneumonia and sepsis. Despite its clinical importance, little is known about innate immunity against GBS in humans. Here, we analyze the role of human group IIA secreted phospholipase A2 (sPLA2-IIA), a bactericidal enzyme induced during acute inflammation, in innate immunity against GBS. We show that clinical GBS isolates are highly sensitive to killing by sPLA2-IIA but not by human antimicrobial peptides. Using transgenic mice that express human sPLA2-IIA, we demonstrate that this enzyme is crucial for host protection against systemic infection and lung challenge by GBS. We found that acute sera from humans diagnosed with invasive GBS disease contain increased levels of sPLA2-IIA compared with normal sera from healthy individuals, indicating that GBS induces an sPLA2-IIA response in blood during human infection. We demonstrate that clinically relevant GBS strains are rapidly killed in these acute sera. We also demonstrate that the bactericidal effect is entirely due to sPLA2-IIA, showing that sPLA2-IIA might represent an important component of humoral innate immunity against GBS. Our data provide experimental and clinical evidence that sPLA2-IIA protects humans against GBS infections.

The ability of macrophages to eradicate intracellular pathogens is normally greatly enhanced by IFNγ, a cytokine produced mainly after onset of adaptive immunity. However, adaptive immunity is unable to provide sterilizing immunity against mycobacteria, suggesting that mycobacteria have evolved virulence strategies to inhibit the bactericidal effect of IFNγ-signalling in macrophages. Still, the host-pathogen interactions and cellular mechanisms responsible for this feature have remained elusive. We demonstrate that the ESX-1 type VII secretion systems of Mycobacterium tuberculosis and Mycobacterium  marinum exploit type I IFN-signalling to promote an IL-12(low) /IL-10(high) regulatory macrophage phenotype characterized by secretion of IL-10, IL-27 and IL-6. This mechanism had no impact on intracellular growth in the absence of IFNγ but suppressed IFNγ-mediated autophagy and growth restriction, indicating that the regulatory phenotype extends to function. The IFNγ-refractory phenotype was partly mediated by IL-27-signalling, establishing functional relevance for this downstream cytokine. These findings identify a novel macrophage-modulating function for the ESX-1 secretion system that may contribute to suppress the efficacy of adaptive immunity and provide mechanistic insight into the antagonistic cross talk between type I IFNs and IFNγ in mycobacterial infection.