Mohlopheni J. Marakalala

Syk Kinase-Coupled C-type Lectin Receptors Engage Protein Kinase C-δ to Elicit Card9 Adaptor-Mediated Innate Immunity

Summary C-type lectin receptors (CLRs) that couple with the kinase Syk are major pattern recognition receptors for the activation of innate immunity and host defense. CLRs recognize fungi and other forms of microbial or sterile danger, and they induce inflammatory responses through the adaptor protein Card9. The mechanisms relaying CLR proximal signals to the core Card9 module are unknown. Here we demonstrated that protein kinase C-δ (PKCδ) was activated upon Dectin-1-Syk signaling, mediated phosphorylation of Card9 at Thr231, and was responsible for Card9-Bcl10 complex assembly and canonical NF-κB control. Prkcd−/− dendritic cells, but not those lacking PKCα, PKCβ, or PKCθ, were defective in innate responses to Dectin-1, Dectin-2, or Mincle stimulation. Moreover, Candida albicans-induced cytokine production was blocked in Prkcd−/− cells, and Prkcd−/− mice were highly susceptible to fungal infection. Thus, PKCδ is an essential link between Syk activation and Card9 signaling for CLR-mediated innate immunity and host protection.

Differential Adaptation of Candida albicans In Vivo Modulates Immune Recognition by Dectin-1

The β-glucan receptor Dectin-1 is a member of the C-type lectin family and functions as an innate pattern recognition receptor in antifungal immunity. In both mouse and man, Dectin-1 has been found to play an essential role in controlling infections with Candida albicans, a normally commensal fungus in man which can cause superficial mucocutaneous infections as well as life-threatening invasive diseases. Here, using in vivo models of infection, we show that the requirement for Dectin-1 in the control of systemic Candida albicans infections is fungal strain-specific; a phenotype that only becomes apparent during infection and cannot be recapitulated in vitro. Transcript analysis revealed that this differential requirement for Dectin-1 is due to variable adaptation of C. albicans strains in vivo, and that this results in substantial differences in the composition and nature of their cell walls. In particular, we established that differences in the levels of cell-wall chitin influence the role of Dectin-1, and that these effects can be modulated by antifungal drug treatment. Our results therefore provide substantial new insights into the interaction between C. albicans and the immune system and have significant implications for our understanding of susceptibility and treatment of human infections with this pathogen.

Inflammatory signaling in human tuberculosis granulomas is spatially organized

Granulomas are the pathological hallmark of tuberculosis (TB). However, their function and mechanisms of formation remain poorly understood. To understand the role of granulomas in TB, we analyzed the proteomes of granulomas from subjects with tuberculosis in an unbiased manner. Using laser-capture microdissection, mass spectrometry and confocal microscopy, we generated detailed molecular maps of human granulomas. We found that the centers of granulomas have a pro-inflammatory environment that is characterized by the presence of antimicrobial peptides, reactive oxygen species and pro-inflammatory eicosanoids. Conversely, the tissue surrounding the caseum has a comparatively anti-inflammatory signature. These findings are consistent across a set of six human subjects and in rabbits. Although the balance between systemic pro- and anti-inflammatory signals is crucial to TB disease outcome, here we find that these signals are physically segregated within each granuloma. From the protein and lipid snapshots of human and rabbit lesions analyzed here, we hypothesize that the pathologic response to TB is shaped by the precise anatomical localization of these inflammatory pathways during the development of the granuloma.

The C-Type Lectin Receptor CLECSF8/CLEC4D Is a Key Component of Anti-Mycobacterial Immunity

Summary The interaction of microbes with pattern recognition receptors (PRRs) is essential for protective immunity. While many PRRs that recognize mycobacteria have been identified, none is essentially required for host defense in vivo. Here, we have identified the C-type lectin receptor CLECSF8 (CLEC4D, MCL) as a key molecule in anti-mycobacterial host defense. Clecsf8−/− mice exhibit higher bacterial burdens and increased mortality upon M. tuberculosis infection. Additionally, Clecsf8 deficiency is associated with exacerbated pulmonary inflammation, characterized by enhanced neutrophil recruitment. Clecsf8−/− mice show reduced mycobacterial uptake by pulmonary leukocytes, but infection with opsonized bacteria can restore this phagocytic defect as well as decrease bacterial burdens. Notably, a CLECSF8 polymorphism identified in humans is associated with an increased susceptibility to pulmonary tuberculosis. We conclude that CLECSF8 plays a non-redundant role in anti-mycobacterial immunity in mouse and in man.

Complement C3 Plays an Essential Role in the Control of Opportunistic Fungal Infections

ABSTRACT The innate recognition of fungal pathogens is a crucial first step in the induction of protective antifungal immunity. Complement is thought to be one key component in this process, facilitating fungal recognition and inducing early inflammation. However, the roles of the individual complement components have not been examined extensively. Here we have used mice lacking C3 to examine its role in immunity to opportunistic fungal pathogens and show that this complement component is essential for resistance to infections with Candida albicans and Candida glabrata. We demonstrate that the absence of C3 impairs fungal clearance but does not affect inflammatory responses. We also show that the presence of C3 contributes to mortality in mice challenged with very high doses of Saccharomyces cerevisiae, although these effects were found to be mouse strain dependent.

The role of Syk/CARD9-coupled C-type lectin receptors in immunity to Mycobacterium tuberculosis infections.

There is increasing interest in understanding the mechanisms underlying the interactions that occur between Mycobacterium tuberculosis and host innate immune cells. These cells express pattern recognition receptors (PRRs) which recognise mycobacterial pathogen-associated molecular patterns (PAMPs) and which can influence the host immune response to the infection. Although many of the PRRs appear to be redundant in the control of M. tuberculosis infection in vivo, recent discoveries have revealed a key, nonredundant, role of the Syk/CARD9 signalling pathway in antimycobacterial immunity. Here we review these discoveries, as well as recent data investigating the role of the Syk/CARD9-coupled PRRs that have been implicated in mycobacterial recognition, including Dectin-1 and Mincle.

The Syk/CARD9-coupled receptor Dectin-1 is not required for host resistance to Mycobacterium tuberculosis in mice

There is interest in identifying the pattern recognition receptors involved in initiating protective or non-protective host responses to Mycobacterium tuberculosis (Mtb). Here we explored the role of the Syk/CARD9-coupled receptor, Dectin-1, using an aerosol model of Mtb infection in wild-type and Dectin-1 deficient mice. We observed a reduction in pulmonary bacilli burdens in the Dectin-1 deficient animals, but this did not correlate with significant changes in pulmonary pathology, cytokine levels or ability of these animals to survive the infection. Thus Dectin-1 makes a minor contribution to susceptibility to Mtb infections in mice.

Mycobacterium tuberculosis FtsX extracellular domain activates the peptidoglycan hydrolase, RipC

Significance During growth and division, bacterial cells partition the cell wall to daughter cells, but the “inside-out” signals that regulate peptidoglycan (PG) hydrolysis are not well understood. Our studies of mycobacterial proteins achieve in vitro reconstitution of the regulation of the PG hydrolase, RipC, by the FtsX extracellular domain (ECD), a ubiquitous transmembrane component of the divisome. RipC control is remarkable for the enormous conformational change encompassing the N- and C-terminal domains. The FtsX–ECD crystal structure shows not only the RipC binding site—which we confirm in vitro and in vivo—but also the flexibility of the site that provides a potential basis to control RipC affinity. Our results expand and sharpen the FtsX paradigm for regulation of PG hydrolysis. Bacterial growth and cell division are coordinated with hydrolysis of the peptidoglycan (PG) layer of the cell wall, but the mechanisms of regulation of extracellular PG hydrolases are not well understood. Here we report the biochemical, structural, and genetic analysis of the Mycobacterium tuberculosis homolog of the transmembrane PG-hydrolase regulator, FtsX. The purified FtsX extracellular domain binds the PG peptidase Rv2190c/RipC N-terminal segment, causing a conformational change that activates the enzyme. Deletion of ftsEX and ripC caused similar phenotypes in Mycobacterium smegmatis, as expected for genes in a single pathway. The crystal structure of the FtsX extracellular domain reveals an unprecedented fold containing two lobes connected by a flexible hinge. Mutations in the hydrophobic cleft between the lobes reduce RipC binding in vitro and inhibit FtsX function in M. smegmatis. These studies suggest how FtsX recognizes RipC and support a model in which a conformational change in FtsX links the cell division apparatus with PG hydrolysis.

Dectin-1: a role in antifungal defense and consequences of genetic polymorphisms in humans

The clinical relevance of fungal infections has increased dramatically in recent decades as a consequence of the rise of immunocompromised populations, and efforts to understand the underlying mechanisms of protective immunity have attracted renewed interest. Here we review Dectin-1, a pattern recognition receptor involved in antifungal immunity, and discuss recent discoveries of polymorphisms in the gene encoding this receptor which result in human disease.

Conjugates of plumbagin and phenyl-2-amino-1-thioglucoside inhibit MshB, a deacetylase involved in the biosynthesis of mycothiol

N-Acetylglucosaminylinositol (GlcNAc-Ins)-deacetylase (MshB) and mycothiol-S-conjugate amidase (Mca), structurally related amidases present in mycobacteria and other Actinomycetes, are involved in the biosynthesis of mycothiol and in the detoxification of xenobiotics as their mycothiol-S-conjugates, respectively. With substrate analogs of GlcNAc-Ins, MshB showed a marked preference for inositol as the aglycon present in GlcNAc-Ins. The inhibition of MshB and Mca by 10 thioglycosides, 7 cyclohexyl-2-deoxy-2-C-alkylglucosides, and 4 redox cyclers was evaluated. The latter contained plumbagin tethered via 2 to 5 methylene carbons and an amide linkage to phenyl-2-deoxy-2-amino-1-thio-alpha-d-glucopyranoside. These proved to be the most potent amongst the 21 compounds tested as inhibitors of MshB. Their inhibitory potency varied with the length of the spacer, with the compound with longest spacer being the most effective.