Chihiro Aikawa

The small GTPases Rab9A and Rab23 function at distinct steps in autophagy during Group A Streptococcus infection

Autophagy 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.

Specific Behavior of Intracellular Streptococcus pyogenes That Has Undergone Autophagic Degradation Is Associated with Bacterial Streptolysin O and Host Small G Proteins Rab5 and Rab7

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 SLO-deficient 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.

Intraindividual variation in core microbiota in peri-implantitis and periodontitis

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.

CRISPR Regulation of Intraspecies Diversification by Limiting IS Transposition and Intercellular Recombination

Mobile genetic elements (MGEs) and genetic rearrangement are considered as major driving forces of bacterial diversification. Previous comparative genome analysis of Porphyromonas gingivalis, a pathogen related to periodontitis, implied such an important relationship. As a counterpart system to MGEs, clustered regularly interspaced short palindromic repeats (CRISPRs) in bacteria may be useful for genetic typing. We found that CRISPR typing could be a reasonable alternative to conventional methods for characterizing phylogenetic relationships among 60 highly diverse P. gingivalis isolates. Examination of genetic recombination along with multilocus sequence typing suggests the importance of such events between different isolates. MGEs appear to be strategically located at the breakpoint gaps of complicated genome rearrangements. Of these MGEs, insertion sequences (ISs) were found most frequently. CRISPR analysis identified 2,150 spacers that were clustered into 1,187 unique ones. Most of these spacers exhibited no significant nucleotide similarity to known sequences (97.6%: 1,158/1,187). Surprisingly, CRISPR spacers exhibiting high nucleotide similarity to regions of P. gingivalis genomes including ISs were predominant. The proportion of such spacers to all the unique spacers (1.6%: 19/1,187) was the highest among previous studies, suggesting novel functions for these CRISPRs. These results indicate that P. gingivalis is a bacterium with high intraspecies diversity caused by frequent insertion sequence (IS) transposition, whereas both the introduction of foreign DNA, primarily from other P. gingivalis cells, and IS transposition are limited by CRISPR interference. It is suggested that P. gingivalis CRISPRs could be an important source for understanding the role of CRISPRs in the development of bacterial diversity.

Complete Genome Sequence of the Serotype k Streptococcus mutans Strain LJ23

Streptococcus mutans is the major pathogen of dental caries and occasionally causes infective endocarditis. Here we report the complete genome sequence of serotype k S. mutans strain LJ23, which was recently isolated from the oral cavity of a Japanese patient.

Rab17‐mediated recycling endosomes contribute to autophagosome formation in response to Group A Streptococcus invasion

Autophagy plays a crucial role in host defence by facilitating the degradation of invading bacteria such as Group A Streptococcus (GAS). GAS‐containing autophagosome‐like vacuoles (GcAVs) form when GAS‐targeting autophagic membranes entrap invading bacteria. However, the membrane origin and the precise molecular mechanism that underlies GcAV formation remain unclear. In this study, we found that Rab17 mediates the supply of membrane from recycling endosomes (REs) to GcAVs. We showed that GcAVs contain the RE marker transferrin receptor (TfR). Colocalization analyses demonstrated that Rab17 colocalized effectively with GcAV. Rab17 and TfR were visible as punctate structures attached to GcAVs and the Rab17‐positive dots were recruited to the GAS‐capturing membrane. Overexpression of Rab17 increased the TfR‐positive GcAV content, whereas expression of the dominant‐negative Rab17 form (Rab17 N132I) caused a decrease, thereby suggesting the involvement of Rab17 in RE–GcAV fusion. The efficiency of GcAV formation was lower in Rab17 N132I‐overexpressing cells. Furthermore, knockdown of Rabex‐5, the upstream activator of Rab17, reduced the GcAV formation efficiency. These results suggest that Rab17 and Rab17‐mediated REs are involved in GcAV formation. This newly identified function of Rab17 in supplying membrane from REs to GcAVs demonstrates that RE functions as a primary membrane source during antibacterial autophagy.

Golgi-Resident GTPase Rab30 Promotes the Biogenesis of Pathogen-Containing Autophagosomes

Autophagy acts as a host-defense system against pathogenic microorganisms such as Group A Streptococcus (GAS). Autophagy is a membrane-mediated degradation system that is regulated by intracellular membrane trafficking regulators, including small GTPase Rab proteins. Here, we identified Rab30 as a novel regulator of GAS-containing autophagosome-like vacuoles (GcAVs). We found that Rab30, a Golgi-resident Rab, was recruited to GcAVs in response to autophagy induction by GAS infection in epithelial cells. Rab30 recruitment was dependent upon its GTPase activity. In addition, the knockdown of Rab30 expression significantly reduced GcAV formation efficiency and impaired intracellular GAS degradation. Rab30 normally functions to maintain the structural integrity of the Golgi complex, but GcAV formation occurred even when the Golgi apparatus was disrupted. Although Rab30 also colocalized with a starvation-induced autophagosome, Rab30 was not required for autophagosome formation during starvation. These results suggest that Rab30 mediates autophagy against GAS independently of its normal cellular role in the structural maintenance of the Golgi apparatus, and autophagosome biogenesis during bacterial infection involves specific Rab GTPases.

Comparative genome analysis and identification of competitive and cooperative interactions in a polymicrobial disease.

Polymicrobial diseases are caused by combinations of multiple bacteria, which can lead to not only mild but also life-threatening illnesses. Periodontitis represents a polymicrobial disease; Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia, called ‘the red complex’, have been recognized as the causative agents of periodontitis. Although molecular interactions among the three species could be responsible for progression of periodontitis, the relevant genetic mechanisms are unknown. In this study, we uncovered novel interactions in comparative genome analysis among the red complex species. Clustered regularly interspaced short palindromic repeats (CRISPRs) of T. forsythia might attack the restriction modification system of P. gingivalis, and possibly work as a defense system against DNA invasion from P. gingivalis. On the other hand, gene deficiencies were mutually compensated in metabolic pathways when the genes of all the three species were taken into account, suggesting that there are cooperative relationships among the three species. This notion was supported by the observation that each of the three species had its own virulence factors, which might facilitate persistence and manifestations of virulence of the three species. Here, we propose new mechanisms of bacterial symbiosis in periodontitis; these mechanisms consist of competitive and cooperative interactions. Our results might shed light on the pathogenesis of periodontitis and of other polymicrobial diseases.

Complete Genome Sequence of a Propionibacterium acnes Isolate from a Sarcoidosis Patient

ABSTRACT Propionibacterium acnes is a human skin commensal that resides preferentially within sebaceous follicles and is the only microorganism that has been isolated from sarcoid lesions. We report the complete genome sequence of P. acnes, which was isolated from a Japanese patient with sarcoidosis.

The STX6-VTI1B-VAMP3 complex facilitates xenophagy by regulating the fusion between recycling endosomes and autophagosomes

ABSTRACT Macroautophagy/autophagy plays a critical role in immunity by directly degrading invading pathogens such as Group A Streptococcus (GAS), through a process that has been named xenophagy. We previously demonstrated that autophagic vacuoles directed against GAS, termed GAS-containing autophagosome-like vacuoles (GcAVs), use recycling endosomes (REs) as a membrane source. However, the precise molecular mechanism that facilitates the fusion between GcAVs and REs remains unclear. Here, we demonstrate that STX6 (syntaxin 6) is recruited to GcAVs and forms a complex with VTI1B and VAMP3 to regulate the GcAV-RE fusion that is required for xenophagy. STX6 targets the GcAV membrane through its tyrosine-based sorting motif and transmembrane domain, and localizes to TFRC (transferrin receptor)-positive punctate structures on GcAVs through its H2 SNARE domain. Knockdown and knockout experiments revealed that STX6 is required for the fusion between GcAVs and REs to promote clearance of intracellular GAS by autophagy. Moreover, VAMP3 and VTI1B interact with STX6 and localize on the TFRC-positive puncta on GcAVs, and are also involved in the RE-GcAV fusion. Furthermore, knockout of RABGEF1 impairs the RE-GcAV fusion and STX6-VAMP3 interaction. These findings demonstrate that RABGEF1 mediates RE fusion with GcAVs through the STX6-VAMP3-VTI1B complex, and reveal the SNARE dynamics involved in autophagosome formation in response to bacterial infection.