Nontoxigenic Corynebacterium diphtheriae and Corynebacterium ulcerans cause invasive disease in humans and animals. Host sensing of corynebacteria is largely uncharacterized, albeit the recognition of lipoglycans by Toll-like receptor 2 (TLR2) appears to be important for macrophage activation by corynebacteria. The members of the order Corynebacterineae (e.g., mycobacteria, nocardia, and rhodococci) share a glycolipid-rich cell wall dominated by mycolic acids (termed corynomycolic acids in corynebacteria). The mycolic acid-containing cord factor of mycobacteria, trehalose dimycolate, activates the C-type lectin receptor (CLR) Mincle. Here, we show that glycolipid extracts from the cell walls of several pathogenic and nonpathogenic Corynebacterium strains directly bound to recombinant Mincle in vitro. Macrophages deficient in Mincle or its adapter protein Fc receptor gamma chain (FcR␥) produced severely reduced amounts of granulocyte colony-stimulating factor (G-CSF) and of nitric oxide (NO) upon challenge with corynebacterial glycolipids. Consistently, cell wall extracts of a particular C. diphtheriae strain (DSM43989) lacking mycolic acid esters neither bound Mincle nor activated macrophages. Furthermore, TLR2 but not TLR4 was critical for sensing of cell wall extracts and whole corynebacteria. The upregulation of Mincle expression upon encountering corynebacteria required TLR2. Thus, macrophage activation by the corynebacterial cell wall relies on TLR2-driven robust Mincle expression and the cooperative action of both receptors.KEYWORDS C-type lectin receptor, Mincle, Toll-like receptor, macrophage, Corynebacterium diphtheriae, cell wall lipids, mycolate, mycolic acid, corynomycolate D iphtheria caused by toxin-producing Corynebacterium diphtheriae is a severe, life-threatening infection, which has become rare in Europe due to the efficacy and coverage of toxoid immunization but still causes a considerable burden of disease globally. Corynebacterium ulcerans and Corynebacterium pseudotuberculosis can also harbor the tox gene encoding diphtheria toxin (1) and in addition may secrete the exotoxin phospholipase D, a virulence factor involved in caseous lymphadenitis of sheep and goats (2, 3). Rates of infections with nontoxigenic strains of C. diphtheriae, including bloodstream infections and endocarditis, appear to be increasing in Europe (4,5). Of the 90 species that comprise the genus Corynebacterium, many inhabit the human skin or mucosa as commensals (6), with C. striatum, C. tuberculostearicum, C. amycolatum, and C. jeikeium being typical examples of skin inhabitants, whereas C. urealyticum and C. glucuronolyticum are frequently found in the urogenital tract. All

Corynebacterium diphtheriae is the causative agent of diphtheria, a toxin mediated disease of upper respiratory tract, which can be fatal. As a member of the CMNR group, C. diphtheriae is closely related to members of the genera Mycobacterium, Nocardia and Rhodococcus. Almost all members of these genera comprise an outer membrane layer of mycolic acids, which is assumed to influence host-pathogen interactions. In this study, three different C. diphtheriae strains were investigated in respect to their interaction with phagocytic murine and human cells and the invertebrate infection model Caenorhabditis elegans. Our results indicate that C. diphtheriae is able to delay phagolysosome maturation after internalization in murine and human cell lines. This effect is independent of the presence of mycolic acids, as one of the strains lacked corynomycolates. In addition, analyses of NF-κB induction revealed a mycolate-independent mechanism and hint to detrimental effects of the different strains tested on the phagocytic cells. Bioinformatics analyses carried out to elucidate the reason for the lack of mycolates in one of the strains led to the identification of a new gene involved in mycomembrane formation in C. diphtheriae.