Interactions between the Re-Emerging Pathogen Corynebacterium diphtheriae and Host Cells

Abstract

Corynebacterium diphtheriae, the etiological agent of diphtheria, is a re-emerging pathogen, responsible for several thousand deaths per year. In addition to diphtheria, systemic infections, often by non-toxigenic strains, are increasingly observed. This indicates that besides the well-studied and highly potent diphtheria toxin, various other virulence factors may influence the progression of the infection. This review focuses on the known components of C. diphtheriae responsible for adhesion, invasion, inflammation, and cell death, as well as on the cellular signaling pathways activated upon infection.

Keywords: Shiga-like toxin; apoptosis; diphtheria; diphtheria toxin; mycolic acids; necrosis; pyroptosis.

Figure 1

C. diphtheriae infection. Infection pathway by respiratory droplets and pseudo-membrane formation (indicated in yellow) caused by colonization of the upper respiratory tract (figure created with BioRender.com).

Figure 2

Adhesion of C. diphtheriae: a multi-factorial process. C. diphtheriae can bind different epithelial cell types in a strain-specific manner. Several proteins involved in this process have been identified thus far, including adhesive pili and MSCRAMMS (Microbial Surface Components Recognizing Adhesive Matrix Molecules), which mediate attachment to fibrinogen or collagen. Deletion or disruption of single genes encoding one of these proteins results typically in only a minor loss of adhesion, indicating that a combination of independent adhesion mechanisms act together. In addition, C. diphtheriae can bind to human erythrocytes, which may support spreading of the bacteria via the bloodstream within the whole body [31] (figure created with BioRender.com).

Figure 3

Inflammatory response induced by C. diphtheriae. (a,b) Inflammatory response caused by non-invasive C. diphtheriae. Entering bacteria lead to recruitment of immune cells such as neutrophils and macrophages and removal of the pathogen. (c) Invasive C. diphtheriae remain undetected by the host immune system through unknown mechanisms, gain access to deeper tissues and blood vessels, and spread through the whole body by binding erythrocytes (hemagglutination) (figure created with BioRender.com).

Figure 4

C. diphtheriae recognition by macrophages. Binding of C. diphtheriae by TLR2 (1) leads on the one hand to upregulation of the C-type lectin receptor Mincle (2) and on the other hand to phagocytosis of the bacteria (3), resulting in phagosome–lysosome fusion, which is somehow delayed by C. diphtheriae (4). Furthermore, binding of C. diphtheriae to Mincle (5) triggers the production of pro-inflammatory cytokines (6), which was confirmed by reduced cytokine production in Clec4e-deficient cells (7). Additionally, in Myd88-deficient cells the cytokine production as well as the uptake of the bacteria was completely blocked (8). Further signs of inflammation caused by pathogenic corynebacteria are the activation of NFκ-B-signaling (9), resulting in upregulation of pro-inflammatory genes (10), and the production of nitric oxide (NO) (11). In the case of the infection of THP-1 cells, a cytotoxic effect of C. diphtheriae was detectable by LDH release (12). TLR-9 activation can be observed for non-toxigenic strains (13) [58] (figure created with BioRender.com).

Figure 5

Delivery and action of diphtheria toxin. The B-subunit of the toxin binds to the host receptor HB-EGF, leading to receptor-mediated endocytosis. Once in the endosome, acidification of the lumen induces pore formation, and the catalytic domain of the toxin is released into the cytoplasm. ADP ribosyltransferase activity of the catalytic domain inactivates elongation factor 2 (EF-2) and protein biosynthesis stops, inducing cell death by apoptosis (see below) (figure created with BioRender.com).

Figure 6

C. diphtheriae-induced necrosis and apoptosis in macrophages. Infection of human macrophage cell lines leads to induction of necrosis and apoptosis. Factors that are involved in these processes are Rbp from strain HCO4 and DIP0733 from CDC-E8392. The molecular mechanisms by which these proteins act is unclear thus far, and more detailed biochemical analyses are required to understand the cytotoxic activity. Necrosis is highly regulated by cellular processes that are characterized by a loss of cell membrane integrity, intracellular organelles, and cell swelling [84,85]. In contrast to apoptosis, necrosis represents a form of cell death that is optimally induced when caspases are inhibited [86,87,88]. Regulated or programmed necrosis eventually leads to cell lysis and release of cytoplasmic content into the extracellular region that often results in tissue damage and intensive inflammatory response. Apoptosis is characterized by nuclear chromatin condensation, cytoplasmic shrinking, dilated endoplasmic reticulum, and membrane blebbing [89]. Apoptosis is considered as controlled suicide of the cell, which, in contrast to necrosis, does not include the release of cell plasma and thus does not trigger an inflammatory reaction. (Receptor interacting protein kinase 1 and 2 (RIPK1, RIPK2), mixed lineage kinase domain-like (MLKL), reactive oxygen species (ROS), membrane targeted death ligand (tBID); figure created with BioRender.com).

Figure 7

C. diphtheria-induced pyroptosis in macrophages. Gasdermin D (GSDMD) serves as a specific substrate of caspase-1, -4, -5 (in humans), and -11 (in mice) and as an effector molecule for the lytic and highly inflammatory form of pyroptosis [95,96]. The pore-forming activity of the N-terminal cleavage product causes cell swelling and lysis to prevent intracellular pathogens from replicating, leading to the release of cytoplasmic content such as the inflammatory cytokines IL-1β and IL-18 into the extracellular space to recruit and activate immune cells to the site of infection [97] (figure created with BioRender.com).

Figure 8

Hypothetical C. diphtheriae-induced non-canonical and canonical inflammasome activation and pyroptosis. Inflammasomes are multimeric proteins that play a pivotal role in host defense against invading pathogens. Canonical inflammasomes such as NLRP3 respond to a wide range of PAMPs and DAMPs and their activation in macrophages requires two signals: (i) priming, which is provided by TLRs, NOD2, and TNFR1/2 mediated by MYD88, leading to NFκB-mediated expression of inflammasome genes (pro-IL-1β and NLRP3). Pro-IL-18 is constitutively expressed in the cell. (ii) PAMPs or DAMPs (phagosomal rupture) trigger NLRP3, ASC, and caspase-1 assembly to the inflammasome, which leads to proteolytic cleavage of pro-IL-1β and pro-IL-18 for secretion and the induction of pyroptosis [98]. The non-canonical inflammasome pathway is defined by its requirement of caspase-4/-5 in human macrophages. Thus far, this way of inflammasome activation has mainly been described to be triggered by lipopolysaccharide (LPS) of Gram-negative bacteria. Unpublished data by Ott and co-workers indicated that putatively secreted corynebacterial proteins bind an unknown receptor, leading to induction of an alternative NFκB-pathway and expression of inflammasome genes. Intracellular corynebacterial effector proteins seem to induce caspase-4/-5 inflammasome assembly, resulting in proteolytic cleavage of gasdermin D, pore assembly, and pyroptosis. In this case, there is no IL-1β secretion, but intracellular IL-1R2-bound pro-IL-1α to is processed to IL-1α by caspase-5, leading to passive efflux of IL-1α through GSDMD pores ([99], Ott et al., unpublished; figure created with BioRender.com).