A diversity of prokaryotes currently exhibit multicellularity with different generation mechanisms in a variety of contexts of ecology on Earth. In the present study, we report a new type of multicellular bacterium, HS-3, isolated from an underground stream. HS-3 self-organizes its filamentous cells into a layer-structured colony with the properties of a nematic liquid crystal. After maturation, the colony starts to form a semi-closed sphere accommodating clusters of coccobacillus daughter cells and selectively releases them upon contact with water. This is the first report that shows that a liquid-crystal status of cells can support the prokaryotic multicellular behavior. Importantly, the observed behavior of HS-3 suggests that the recurrent intermittent exposure of colonies to water flow in the cave might have been the ecological context that cultivated the evolutionary transition from unicellular to multicellular life. This is the new extant model that underpins theories regarding a role of ecological context in the emergence of multicellularity.

Vaccines against infectious diseases should elicit potent and long-lasting immunity, ideally even in those with age-related decline in immune response. Here we report a rational polysaccharide vaccine platform using probiotic Escherichia coli-derived membrane vesicles (MVs). First, we constructed a probiotic E. coli clone harboring the genetic locus responsible for biogenesis of serotype 14 pneumococcal capsular polysaccharides (CPS14) as a model antigen. CPS14 was found to be polymerized and mainly localized on the outer membrane of the E. coli cells. The glycine-induced MVs displayed the exogenous CPS14 at high density on the outermost surface, on which the CPS14 moiety was covalently tethered to a lipid A-core oligosaccharide anchor. In in vivo immunization experiments, CPS14⁺MVs, but not a mixture of free CPS14 and empty MVs, strongly elicited IgG class-switch recombination with a Th1/Th2-balanced IgG subclass distribution without any adjuvant. In addition, CPS14⁺MVs were structurally stable with heat treatment and immunization with the heat-treated MVs-elicited CPS14-specific antibody responses in mouse serum to levels comparable to those of non-treated CPS14⁺MVs. Notably, the immunogenicity of CPS14⁺MVs was significantly stronger than those of two currently licensed vaccines against pneumococci. The CPS14⁺MV-elicited humoral immune responses persisted for 1 year in both blood and lung. Furthermore, the CPS14⁺MV vaccine was widely efficacious in mice of different ages. Even in aged mice, vaccination resulted in robust production of CPS14-specific IgG that bound to the pneumococcal cell surface. Taken together, the present probiotic E. coli MVs-based vaccine platform offers a promising, generalizable solution against encapsulated pathogens.

The macrolide erythromycin (ERM) inhibits excessive neutrophil accumulation and bone resorption in inflammatory tissues. We previously reported that the expression of developmental endothelial locus-1 (DEL-1), an endogenous anti-inflammatory factor induced by ERM, is involved in ERM action. Furthermore, DEL-1 is involved in the induction of bone regeneration. Therefore, in this study, we investigated whether ERM exerts an osteoblastogenic effect by upregulating DEL-1 under inflammatory conditions. We performed in vitro cell-based mechanistic analyses and used a model of Porphyromonas gingivalis lipopolysaccharide (LPS)-induced periodontitis to evaluate how ERM restores osteoblast activity. In vitro, P. gingivalis LPS stimulation suppressed osteoblast differentiation and bone formation. However, ERM treatment combined with P. gingivalis LPS stimulation upregulated osteoblast differentiation-related factors and Del1, indicating that osteoblast differentiation was restored. Alveolar bone resorption and gene expression were evaluated in a periodontitis model, and the results confirmed that ERM treatment increased DEL-1 expression and suppressed bone loss by increasing the expression of osteoblast-associated factors. In conclusion, ERM restores bone metabolism homeostasis in inflammatory environments possibly via the induction of DEL-1.

Recruitment of plasminogen is an important infection strategy of the human pathogen Streptococcus pneumoniae to invade host tissues. In Streptococcus aureus, triosephosphate isomerase (TPI) has been reported to bind plasminogen. In this study, the TPI of S. pneumoniae (TpiA) was identified through proteomic analysis of bronchoalveolar lavage fluid from a murine pneumococcal pneumonia model. The binding kinetics of recombinant pneumococcal TpiA with plasminogen were characterized using surface plasmon resonance (SPR, Biacore), ligand blot analyses, and enzyme‐linked immunosorbent assay. Enhanced plasminogen activation and subsequent degradation by plasmin were also shown. Release of TpiA into the culture medium was observed to be dependent on autolysin. These findings suggest that S. pneumoniae releases TpiA via autolysis, which then binds to plasminogen and promotes its activation, thereby contributing to tissue invasion via degradation of the extracellular matrix.

Macrolides are used to treat various infectious diseases, including periodontitis. Furthermore, macrolides are known to have immunomodulatory effects; however, the underlying mechanism of their action remains unclear. DEL-1 has emerged as an important factor in homeostatic immunity and osteoclastogenesis. Specifically, DEL-1 is downregulated in periodontitis tissues. Therefore, in the present study, we investigated whether the osteoclastogenesis inhibitory effects of erythromycin (ERM) are mediated through upregulation of DEL-1 expression. We used a ligature-induced periodontitis model in C57BL/6Ncrl wild-type or DEL-1-deficient mice and in vitro cell-based mechanistic studies to investigate how ERM inhibits alveolar bone resorption. As a result of measuring alveolar bone resorption and gene expression in the tooth ligation model, ERM treatment reduced bone loss by increasing DEL-1 expression and decreasing the expression of osteoclast-related factors in wild-type mice. In DEL-1-deficient mice, ERM failed to suppress bone loss and gene expression of osteoclast-related factors. In addition, ERM treatment downregulated osteoclast differentiation and calcium resorption in in vitro experiments with mouse bone marrow-derived macrophages. In conclusion, ERM promotes the induction of DEL-1 in periodontal tissue, which may regulate osteoclastogenesis and decrease inflammatory bone resorption. These findings suggest that ERM may exert immunomodulatory effects in a DEL-1-dependent manner.

Pneumococcus is the main cause of bacterial pneumonia. Pneumococcal infection has been shown to cause elastase, an intracellular host defense factor, to leak from neutrophils. However, when neutrophil elastase (NE) leaks extracellularly, it can degrade host cell surface proteins such as epidermal growth factor receptor (EGFR) and potentially disrupt the alveolar epithelial barrier. In this study, we hypothesized that NE degrades the extracellular domain (ECD) of EGFR in alveolar epithelial cells and inhibits alveolar epithelial repair. Using SDS-PAGE, we showed that NE degraded the recombinant EGFR ECD and its ligand epidermal growth factor, and that the degradation of these proteins was counteracted by NE inhibitors. Furthermore, we confirmed the degradation by NE of EGFR expressed in alveolar epithelial cells in vitro. We showed that intracellular uptake of epidermal growth factor and EGFR signaling was downregulated in alveolar epithelial cells exposed to NE and found that cell proliferation was inhibited in these cells These negative effects of NE on cell proliferation were abolished by NE inhibitors. Finally, we confirmed the degradation of EGFR by NE in vivo. Fragments of EGFR ECD were detected in bronchoalveolar lavage fluid from pneumococcal pneumonia mice, and the percentage of cells positive for a cell proliferation marker Ki67 in lung tissue was reduced. In contrast, administration of an NE inhibitor decreased EGFR fragments in bronchoalveolar lavage fluid and increased the percentage of Ki67-positive cells. These findings suggest that degradation of EGFR by NE could inhibit the repair of alveolar epithelium and cause severe pneumonia.