@misc{oai:ir.soken.ac.jp:00001036, author = {山口, ひとみ and ヤマグチ, ヒトミ and YAMAGUCHI, Hitomi}, month = {2016-02-17}, note = {Autophagy is an intracellular bulk degradation system，participating in intracellular protein turnover and in the biogenetic management in starvation conditions．The process involves dynamic membrane rearrangement．Initially，a cup-shaped isolation membrane surrounds a portion of the cytoplasm and organelles．The isolation membrane is sealed by fusion between the tips，forming a double membrane bound organelle，autophagosome，which is then fused with lysosomes. After the fusion，the inner membrane of autophagosomes and the sequestered cytoplasmic components are degraded in the lysosome lumen.
Nakagawa et al.（Nakagawa, et al., 2004）reported that Streptococcus pyogenes（also known as Group A streptococci，GAS）that invades the cytoplasm of host cells were effectively eliminated by autophagic machinery. GAS is a serious gram-positive human pathogen that causes a variety of infections including pharyngitis，impetigo, actute rheumatic fever and post streptococcal glomerular nephritis. These infections，as well as the manifestations of invasive disease include streptococcal toxic shock syndrome and necrotizing fasciitis. GAS has ability to efficiently invade various type of non-phagocytic cells by exploiting the cellular endocytic machinery. By secreting a cytolysin SLO (Streptolysin O), which binds cholesterol and permeabilize the membrane，GAS escapes from endosomal lumen to the cytoplasm. However，it is captured by membrane-bound compartments bearing LC3, which is a specific marker protein for autophagosomes （Kabeya，et al., 2000），and killed after fusion of the compartments with lysosomes．Formation of the membrane-bound compartments containing GAS is dependent on autophagy-related protein Atg5， a core component of autophagy （Mizushima，et al., 200l）．
Together with the presence of LC3，GAS is considered to be captured by autophagosome-related structures. Thus，the structure was named GAS-containing autophagosome-like vacuoles（GcAVs）and the specialized autophagy process was designated GAS autophagy．These results provide new insights into the mechanisms and roles of autophagy in highere eukaryotes．Although the starvation-induced canonical autophagy has been considered as non-selective，GcAVs induced by GAS invasion selectively engulf the bacteria．Furthermore，the GcAVs have diameters of about 10μm，which are about ten times as large as canonical autophagosomes.
In this study, I examined how such a large autophafagosome is formed. I have performed detailed analysis of membrane dynamics in GcAV formation at an ultrastructural level. Multiple isolation membranes emerged close by the cytoplasmic GAS．Electron microscopy serial sections suggested that fusion among the individual isolation membranes accounts for formation of large GcAVs．
Based on the morphological results，l have studied the GAS autophagy by using molecular biological methods．Firstly，I examined the involvement of a small GTPase Rab7．It has been shown that Rab7 is required for fusion between canonical autophagosomes and lusosomes. I showed that expression of GDP-bound form constitutive-inactive form Rab7，Rab7T22N， blocked GcAVs formation．It should be noted that Rab7 is not required for canonical　autophagosome formation per se．In the cells expressing Rab7T22N the isolation membranes were successfully formed near GAS，although they failed to form GcAVs．As mentioned above， the large GcAVs are formed by fusion among isolation membranes. Thus，it is possible that Rab7 is involved in these fusion events required for GcAV formation．Since Rab7 is unnecessary for canonical autophagosome formation，the finding demonstrates the existence of a distinct molecular basis for an autophagy system specialized for bacterial elimination．
After successful completion of the GcAVs，these structures underwent homotypic fusion， resulting in larger GcAVs. First GcAVs arose as chain－like structures，which reflect the original morphology of the streptococcal chain. Then，they fused with each other to form a bunch of grape-like structures, and then progressively formed into a single semi-spherical structure containing several streptococcal chains. The process may contribute to the effective sequestration and killing of the pathogen．
Finally，I examined the membrane supplier for the large GcAVs. In response to GAS infection and / or GcAV formation，Golgi stuck disappeared，implying that the massive membrane efflux happened. At this time, microtubules were intact in GAS-infected and GcAVs bearing cells.
Furthermore autophagy-related protein Atg9L l，a sole trans membrane protein transferred to GcAVs from the Golgi apparatus．Finally，the traffic of Golgi lipid marker，C6-NBD-ceramide to GcAVs occurred．Thus，I propose that the Golgi apparatus supplies membrane for GcAVs.
In summary，I have revealed distinct membrane dynamics and molecular mechanisms in the specialized autophagy used for killing intracellular GAS. Pathogens have been developing the means and ways to evade the host defense mechanism，while the host cells have also evolved to counter the strategies of pathogens，employing distinct molecular mechanism from canonical autophagy. This study is the first example of a specialized or variant autophagy that invoIves additional molecular mechanisms to establish a microbicidal process．, 総研大甲第1065号}, title = {Membrane dynamics and molecular mechanism of autophagic elimination of Group A Streptococcus}, year = {} }