Mutans streptococci are major etiological agents of dental caries, and several of their secreted products contribute to bacterial accumulation on teeth. Of these, Streptococcus mutans glucan binding protein B (GbpB) is a novel, immunologically dominant protein. Its biological function is unclear, although GbpB shares homology with a putative peptidoglycan hydrolase from S. agalactiae and S. pneumoniae, indicative of a role in murein biosynthesis. To determine the cellular function of GbpB, we used several approaches to inactivate the gene, analyze its expression, and identify interacting proteins. None of the transformants analyzed were true gbpB mutants, since they all contained both disrupted and wild-type gene copies, and expression of functional GbpB was always conserved. Thus, the inability to obtain viable gbpB null mutants supports the notion that gbpB is an essential gene. Northern blot and real-time PCR analyses suggested that induction of gbpB expression in response to stress was a strain-dependent phenomenon. Proteins that interacted with GbpB were identified in pull-down and coimmunoprecipitation assays, and these data suggest that GbpB interacts with ribosomal protein L7/L12, possibly as part of a protein complex involved in peptidoglycan synthesis and cell division.

Pyrin domain-only proteins (POPs) are recently evolved, primate-specific proteins demonstrated in vitro as negative regulators of inflammatory responses. However, their in vivo function is not understood. Of the four known POPs, only POP2 is reported to regulate NF-κB-dependent transcription and multiple inflammasomes. Here we use a transgenic mouse-expressing POP2 controlled by its endogenous human promotor to study the immunological functions of POP2. Despite having significantly reduced inflammatory cytokine responses to LPS and bacterial infection, POP2 transgenic mice are more resistant to bacterial infection than wild-type mice. In a pulmonary tularaemia model, POP2 enhances IFN-γ production, modulates neutrophil numbers, improves macrophage functions, increases bacterial control and diminishes lung pathology. Thus, unlike other POPs thought to diminish innate protection, POP2 reduces detrimental inflammation while preserving and enhancing protective immunity. Our findings suggest that POP2 acts as a high-order regulator balancing cellular function and inflammation with broad implications for inflammation-associated diseases and therapeutic intervention.

Pseudogenes are duplicated yet defunct copies of functional parent genes. However, some pseudogenes have gained or retained function. In this study we consider a functional role for the NLRP2-related, higher primate specific, processed pseudogene NLRP2P, which is closely related to Pyrin-only protein 2 (POP2/PYDC2), a regulator of NF-κB and the inflammasome. The NLRP2P open reading frame on chromosome X has features consistent with a processed pseudogene (retrotransposon), yet encodes a 45 amino acid, Pyrin-domain related protein. The open reading frame of NLRP2P shares 80% identity with POP2 and is under purifying selection across Old World primates. Although widely expressed, NLRP2P mRNA is upregulated by LPS in human monocytic cells. Functionally, NLRP2P impairs NF-κB p65 transactivation by reducing activating phosphorylation of RelA/p65. Reminiscent of POP2, NLRP2P reduces production of the NF-κB-dependent cytokines TNFα and IL-6 following TLR stimulation. In contrast to POP2, NLRP2P fails to inhibit the ASC-dependent NLRP3 inflammasome. In addition, beyond regulating cytokine production, NLRP2P has a potential role in cell cycle regulation and cell death. Collectively, our findings suggest that NLRP2P is a resurrected processed pseudogene that regulates NF-κB RelA/p65 activity and thus represents the newest member of the POP family, POP4.

We have previously demonstrated that immunization with inactivated Francisella tularensis, a Category A intracellular mucosal pathogen, combined with IgG2a anti-F. tularensis monoclonal antibody, enhances protection against subsequent F. tularensis challenge. To understand the mechanism(s) involved, we examined the binding, internalization, presentation, and in vivo trafficking of inactivated F. tularensis in the presence and absence of opsonizing monoclonal antibody. We found that when inactivated F. tularensis is combined with anti-F. tularensis monoclonal antibody, presentation to F. tularensis-specific T cells is enhanced, this enhancement is Fc receptor-dependent, and requires a physical linkage between the monoclonal antibody and the inactivated F. tularensis immunogen. This enhanced presentation is due, in part, to enhanced binding and internalization of inactivated F. tularensis by antigen presenting cells, and involves interactions with multiple Fc receptor types. Furthermore, targeting inactivated F. tularensis to Fc receptors enhances dendritic cell maturation and extends the time period over which antigen presenting cells stimulate T cells. In vivo trafficking studies reveal enhanced transport of inactivated F. tularensis immunogen to the Nasal Associated Lymphoid Tissue in the presence of monoclonal antibody, which is FcRn-dependent. In summary, these are the first comprehensive studies using a single vaccine protection model/immunogen to establish the array of mechanisms involved in enhanced immunity/protection mediated by an Fc receptor-targeted mucosal immunogen. These results demonstrate that multiple cellular/immune mechanisms contribute to Fc receptor-enhanced immunity.