The oral microbiota change dramatically with each part of the oral cavity, even within the same mouth. Nevertheless, the microbiota associated with peri-implantitis and periodontitis have been considered the same. To improve our knowledge of the different communities of complex oral microbiota, we compared the microbial features between peri-implantitis and periodontitis in 20 patients with both diseases. Although the clinical symptoms of peri-implantitis were similar to those of periodontitis, the core microbiota of the diseases differed. Correlation analysis revealed the specific microbial co-occurrence patterns and found some of the species were associated with the clinical parameters in a disease-specific manner. The proportion of Prevotella nigrescens was significantly higher in peri-implantitis than in periodontitis, while the proportions of Peptostreptococcaceae sp. and Desulfomicrobium orale were significantly higher in periodontitis than in peri-implantitis. The severity of the peri-implantitis was also species-associated, including with an uncultured Treponema sp. that correlated to 4 clinical parameters. These results indicate that peri-implantitis and periodontitis are both polymicrobial infections with different causative pathogens. Our study provides a framework for the ecologically different bacterial communities between peri-implantitis and periodontitis, and it will be useful for further studies to understand the complex microbiota and pathogenic mechanisms of oral polymicrobial diseases.

Streptococcus pyogenes, one of the major human pathogens, is a unique species since it has acquired diverse strain-specific virulence properties mainly through the acquisition of streptococcal prophages. In addition, S. pyogenes possesses clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems that can restrict horizontal gene transfer (HGT) including phage insertion. Therefore, it was of interest to examine the relationship between CRISPR and acquisition of prophages in S. pyogenes. Although two distinct CRISPR loci were found in S. pyogenes, some strains lacked CRISPR and these strains possess significantly more prophages than CRISPR harboring strains. We also found that the number of spacers of S. pyogenes CRISPR was less than for other streptococci. The demonstrated spacer contents, however, suggested that the CRISPR appear to limit phage insertions. In addition, we found a significant inverse correlation between the number of spacers and prophages in S. pyogenes. It was therefore suggested that S. pyogenes CRISPR have permitted phage insertion by lacking its own spacers. Interestingly, in two closely related S. pyogenes strains (SSI-1 and MGAS315), CRISPR activity appeared to be impaired following the insertion of phage genomes into the repeat sequences. Detailed analysis of this prophage insertion site suggested that MGAS315 is the ancestral strain of SSI-1. As a result of analysis of 35 additional streptococcal genomes, it was suggested that the influences of the CRISPR on the phage insertion vary among species even within the same genus. Our results suggested that limitations in CRISPR content could explain the characteristic acquisition of prophages and might contribute to strain-specific pathogenesis in S. pyogenes.

Autophagy is a cellular self-catabolic process wherein organelles, macromolecules, and invading microbes are sequestered in autophagosomes that fuse with lysosomes. In this study, we uncover the role of nitric oxide (NO) as a signaling molecule for autophagy induction via its downstream mediator, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP). We found that 8-nitro-cGMP-induced autophagy is mediated by Lys63-linked polyubiquitination and that endogenous 8-nitro-cGMP promotes autophagic exclusion of invading group A Streptococcus (GAS) from cells. 8-nitro-cGMP can modify Cys residues by S-guanylation of proteins. We showed that intracellular GAS is modified with S-guanylation extensively in autophagosomes-like vacuoles, suggesting the role of S-guanylation as a marker for selective autophagic degradation. This finding is supported by the fact that S-guanylated bacteria were selectively marked with polyubiquitin, a known molecular tag for selective transport to autophagosomes. These results collectively indicate that 8-nitro-cGMP plays a crucial role in cytoprotection during bacterial infections or inflammations via autophagy upregulation.

SummaryAutophagy mediates the degradation of cytoplasmic contents in the lysosome and plays a significant role in immunity. Here we identified the small GTPases Rab9A and Rab23 as novel autophagy regulators during Group A streptococcus (GAS) infection. Rab9A was recruited to GAS-containing autophagosome-like vacuoles (GcAVs) after autophagosomal maturation and its activity was required for GcAV enlargement and eventual lysosomal fusion. GcAV enlargement appeared to be related to homotypic fusion of GcAVs with Rab9A. Rab23 was recruited to GAS-capturing forming autophagosomes. Knockdown of Rab23 expression decreased both LC3-and Atg5-positive GAS formation and caused the accumulation of LC3-positive structures that did not associate with intracellular GAS. It was suggested, therefore, that Rab23 is required for GcAV formation and is involved in GAS targeting of autophagic vacuoles. Furthermore, knockdown of Rab9A or Rab23 expression impaired the degradation of intracellular GAS. Therefore, our data reveal that the Rab9A and Rab23 GTPases play crucial roles in autophagy of GAS. However, neither Rab9A nor Rab23 were localized to starvation-induced autophagosomes. Not only Rab9A but also Rab23 was dispensable for starvation-induced autophagosome formation. These findings demonstrate that specific Rab proteins function at distinct steps during autophagy in response to GAS infection.

Streptococcus pyogenes (group A streptococcus (GAS)) is a pathogen that invades non-phagocytic host cells, and causes a variety of acute infections such as pharyngitis. Our group previously reported that intracellular GAS is effectively degraded by the host-cell autophagic machinery, and that a cholesterol-dependent cytolysin, streptolysin O (SLO), is associated with bacterial escape from endosomes in epithelial cells. However, the details of both the intracellular behavior of GAS and the process leading to its autophagic degradation remain unknown. In this study, we found that two host small G proteins, Rab5 and Rab7, were associated with the pathway of autophagosome formation and the fate of intracellular GAS. Rab5 was involved in bacterial invasion and endosome fusion. Rab7 was clearly multifunctional, with roles in bacterial invasion, endosome maturation, and autophagosome formation. In addition, this study showed that the bacterial cytolysin SLO supported the escape of GAS into the cytoplasm from endosomes, and surprisingly, a SLOdeficient mutant of GAS was viable longer than the wild-type strain although it failed to escape the endosomes. This intracellular behavior of GAS is unique and distinct from that of other types of bacterial invaders. Our results provide a new picture of GAS infection and host-cell responses in epithelial cells. Streptococcus pyogenes (group A streptococcus; GAS)2 is the causative pathogen for a diverse collection of human diseases, such as pharyngitis, bacteremia, and necrotizing fasciitis (1). GAS strains produce a variety of pathogenic factors such as streptolysin O (SLO), superantigens, and DNase (2-4). Invasive GAS diseases occur in ϳ1/1000 cases, with associated mortality of 25% (5).Autophagy is defined as "self-eating," bulk degradation system for cytoplasmic components. During autophagy, double membrane structures are formed in the cytoplasm, in which cytoplasmic organelles and proteins are sequestered. These structures, called autophagosomes, subsequently fuse with lysosomes to degrade the components within them. This system is important for the physiological turnover of cytoplasmic components, and is involved in a number of clinical conditions and diseases (6, 7). Recently, we and others showed that GAS is captured and degraded by autophagy, not by the endosomelysosome system (8, 9). Some species of bacteria invade host non-phagocytic cells, such as epithelial cells, and are largely degraded by the endocytosis pathway (10 -12). However, some bacteria, including Listeria monocytogenes, Shigella flexneri, Mycobacterium tuberculosis, and Salmonella typhimurium, cannot be eliminated by endosomes, and replicate within the cytoplasm (13-15). The autophagic machinery is thought to act to remove these bacteria. However, it is still unclear under what circumstances the intracellular bacteria are sequestered by autophagosomes and what events in the infected cells lead to the degradation of bacteria by autophagy.A cholesterol-dependent cytolysin of GAS, SLO, is a homolog of listerio...