Nod1 Signaling Overcomes Resistance of S. pneumoniae to Opsonophagocytic Killing

Abstract

Airway infection by the Gram-positive pathogen Streptococcus pneumoniae (Sp) leads to recruitment of neutrophils but limited bacterial killing by these cells. Co-colonization by Sp and a Gram-negative species, Haemophilus influenzae (Hi), provides sufficient stimulus to induce neutrophil and complement-mediated clearance of Sp from the mucosal surface in a murine model. Products from Hi, but not Sp, also promote killing of Sp by ex vivo neutrophil-enriched peritoneal exudate cells. Here we identify the stimulus from Hi as its peptidoglycan. Enhancement of opsonophagocytic killing was facilitated by signaling through nucleotide-binding oligomerization domain-1 (Nod1), which is involved in recognition of γ-D-glutamyl-meso-diaminopimelic acid (meso-DAP) contained in cell walls of Hi but not Sp. Neutrophils from mice treated with Hi or compounds containing meso-DAP, including synthetic peptidoglycan fragments, showed increased Sp killing in a Nod1-dependent manner. Moreover, Nod1^−/− mice showed reduced Hi-induced clearance of Sp during co-colonization. These observations offer insight into mechanisms of microbial competition and demonstrate the importance of Nod1 in neutrophil-mediated clearance of bacteria in vivo. Pathogens are generally studied in the laboratory one species at a time. Most exist, however, in complex environments where they must adapt not only to their host but also to other members of the microbial flora. Using a mouse model of co-colonization, we have shown that one bacterial species (Haemophilus influenzae) can take advantage of the innate immune response of its host to outcompete and eliminate another species (Streptococcus pneumoniae) that resides in the same microenvironment of the upper respiratory tract. The molecular mechanism for this effect involves recognition of a cell wall fragment found on H. influenzae, but not on S. pneumoniae. The response to this immunostimulatory fragment requires Nod1, a host molecule that transmits inflammatory signals in response to specific peptides of the bacterial cell wall. This Nod1-mediated inflammatory stimulation triggers an increase in the ability of a type of white blood cell (neutrophil) to engulf and then kill S. pneumoniae, effectively removing it from its niche on the mucosal surface of the host airway. Our study, therefore, provides a demonstration of the importance of Nod1 in neutrophil-mediated clearance of bacterial infection. In addition, we have described a mechanism for interspecies competition between microbes that occurs through selective stimulation of host innate immune responses.