ÝËÅÊÒÐÎÍÍÎ-ÌÈÊÐÎÑÊÎÏÈ×ÅÑÊÈÉ ÀÍÀËÈÇ ÏÐÎÍÈÊÍÎÂÅÍÈß SERRATIA GRIMESII Ò. Í. Åôðåìîâà
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2015 ÖÈÒÎËÎÃÈß Ò î ì 57, ¹ 10 ÝËÅÊÒÐÎÍÍÎ-ÌÈÊÐÎÑÊÎÏÈ×ÅÑÊÈÉ ÀÍÀËÈÇ ÏÐÎÍÈÊÍÎÂÅÍÈß ÓÑËÎÂÍÎ-ÏÀÒÎÃÅÍÍÛÕ ÁÀÊÒÅÐÈÉ SERRATIA GRIMESII  ÊËÅÒÊÈ HeLa © Å. Ñ. Áîæîêèíà, Ë. Â. Êåâåð, ß. Þ. Êîìèññàð÷èê, Ñ. Þ. Õàéòëèíà,1 Ò. Í. Åôðåìîâà Èíñòèòóò öèòîëîãèè ÐÀÍ, Ñàíêò-Ïåòåðáóðã, 194064; 1 ýëåêòðîííûé àäðåñ: [email protected] Å. Ñ. Áîæîêèíà è äð. Óñëîâíî-ïàòîãåííûå áàêòåðèè Serratia grimesii ñïîñîáíû ïðîíèêàòü â êëåòêè ýóêàðèîò, ãäå îíè îáíàðóæèâàþòñÿ â âàêóîëÿõ èëè ñâîáîäíî ëåæàùèìè â öèòîïëàçìå (Efremova et al., 2001; Bozhokina et al., 2011). Îäíàêî ýôôåêòèâíîñòü èíâàçèè ýòèõ áàêòåðèé íèçêà, è ìåõàíèçìû èíâàçèè, îïðåäåëÿåìûå íà÷àëüíûìè ñòàäèÿìè ïðîöåññà, íåèçâåñòíû.  íàñòîÿùåé ðàáîòå ýôôåêòèâíîñòü èíâàçèè áûëà ïîâûøåíà ââåäåíèåì â êóëüòóðàëüíóþ ñðåäó N-àöåòèëöèñòåèíà (Bozhokina et al., 2013), 24-÷àñîâàÿ èíêóáàöèÿ ñ êîòîðûì ïðåäøåñòâîâàëà èíôèöèðîâàíèþ êëåòîê HeLa M áàêòåðèÿìè.  ïðåïàðàòàõ êëåòîê, îáðàáîòàííûõ N-àöåòèëöèñòåèíîì, îáíàðóæåíî äâà âàðèàíòà ïðîíèêíîâåíèÿ áàêòåðèé.  áîëüøèíñòâå ñëó÷àåâ ïðîíèêíîâåíèå áàêòåðèé âíóòðü êëåòêè-õîçÿèíà ñîîòâåòñòâóåò îïèñàííîìó â ëèòåðàòóðå ìåõàíèçìó «çàñòåæêà-ìîëíèÿ», ïåðâûì ýòàïîì êîòîðîãî ÿâëÿåòñÿ âçàèìîäåéñòâèå áàêòåðèàëüíîãî èíâàçèíà ñ ïîâåðõíîñòíûì ðåöåïòîðîì êëåòêè-õîçÿèíà. Îäíàêî â ðÿäå ñëó÷àåâ áàêòåðèþ çàõâàòûâàþò âûðîñòû êëåòî÷íîé ïîâåðõíîñòè, âîçíèêàþùèå, ïî-âèäèìîìó, â îòâåò íà âíåäðåíèå â êëåòêó áàêòåðèàëüíûõ ýôôåêòîðîâ, êàê ýòî ïðîèñõîäèò â «òðèããåðíîì» ìåõàíèçìå èíâàçèè. Äëÿ òîãî ÷òîáû ïîäòâåðäèòü ñóùåñòâîâàíèå äâóõ ìåõàíèçìîâ èíâàçèè èëè âûÿâèòü ïðåèìóùåñòâåííûé ìåõàíèçì, íåîáõîäèìî äàëüíåéøåå èññëåäîâàíèå, âêëþ÷àþùåå â ñåáÿ âûÿâëåíèå áàêòåðèàëüíûõ è êëåòî÷íûõ ôàêòîðîâ, ó÷àñòâóþùèõ âî âçàèìîäåéñòâèè. Ýëåêòðîííî-ìèêðîñêîïè÷åñêèé àíàëèç ïðîíèêíîâåíèÿ óñëîâíî-ïàòîãåííûõ áàêòåðèé Ê ë þ ÷ å â û å ñ ë î â à: èíâàçèÿ áàêòåðèé, N-àöåòèëöèñòåèí, Serratia grimesii, ñòàäèè èíâàçèè. ñëåäîâàíèé. Îñîáåííî ýòî êàñàåòñÿ íà÷àëüíûõ ñòàäèé èíâàçèè áàêòåðèé. Èìåííî ýòè ñòàäèè îïðåäåëÿþò ìåõàíèçì âõîæäåíèÿ áàêòåðèé â ýóêàðèîòè÷åñêóþ êëåòêó. Ê ñîæàëåíèþ, ýòè ñòàäèè òðóäíî îáíàðóæèòü èç-çà èõ áûñòðîòå÷íîñòè. Òåì íå ìåíåå èíîãäà óäàåòñÿ âûÿâèòü ÿìêè èëè âäàâëèâàíèÿ áàêòåðèé â ïîâåðõíîñòü èíôèöèðîâàííîé êëåòêè èëè, íàïðîòèâ, ñïåöèôè÷åñêèå âûðîñòû êëåòî÷íîé ïîâåðõíîñòè. Òàêîãî ðîäà êàðòèíû ìîãóò óêàçûâàòü íà ðàçëè÷íûå ìåõàíèçìû, èñïîëüçóåìûå áàêòåðèÿìè äëÿ âõîäà â êëåòêó-õîçÿèíà. Ñïîñîáíîñòüþ ïðîíèêàòü â êëåòêè ýóêàðèîò îáëàäàþò íå òîëüêî ïàòîãåííûå, íî è óñëîâíî-ïàòîãåííûå áàêòåðèè, îäíàêî ìåõàíèçìû èõ ïàòîãåííîñòè èçó÷åíû î÷åíü ìàëî. Ðàíåå ìû ïîêàçàëè, ÷òî èíêóáàöèÿ òðàíñôîðìèðîâàííûõ êëåòîê ñ áàêòåðèÿìè Serratia grimesii, ïðîäóöåíòàìè ïðîòåàçû ÅÑÐ32/ãðèìåëèçèí (Khaitlina et al., 1991; Bozhokina et al., 2008), ïðèâîäèò ê ïîÿâëåíèþ áàêòåðèé âíóòðè êëåòêè-õîçÿèíà, ñíà÷àëà â âàêóîëÿõ, à çàòåì ñâîáîäíî ëåæàùèõ â öèòîïëàçìå (Efremova et al., 2001; Gamaley et al., 2006; Bozhokina et al., 2011). Ïðè ýòîì èíôèöèðîâàííûìè îêàçûâàþòñÿ òîëüêî îêîëî 10 % êëåòîê, èíêóáèðîâàííûõ ñ áàêòåðèÿìè, ÷òî ñîîòâåòñòâóåò ñòàòóñó óñëîâíîé ïàòîãåííîñòè ýòèõ áàêòåðèé (Grimont, Grimont, 2006), íî çàòðóäíÿåò âûÿâëåíèå ðàííèõ ñòàäèé èíâàçèè. Ïîâûøåíèå ÷óâñòâèòåëüíîñòè êëåòîê ýóêàðèîò ê áàêòåðèàëüíîé èíôåêöèè êàê â îðãàíèçìå, òàê è â ýêñïåðèìåíòàëüíûõ óñëîâèÿõ ìîæåò çàâèñåòü îò ôèçèîëîãè÷åñêîãî ñîñòîÿíèÿ êëåòêè-õîçÿèíà: ìåòàáîëèçìà ýóêà- Ïàòîãåííîñòü ìíîãèõ áàêòåðèé îñíîâàíà íà èõ ñïîñîáíîñòè ïðîíèêàòü â êëåòêè ýóêàðèîò è ïåðåäâèãàòüñÿ èç êëåòêè â êëåòêó, èñïîëüçóÿ àêòèíîâûå êîìïîíåíòû öèòîñêåëåòà è ñèãíàëüíûå ñèñòåìû êëåòêè-õîçÿèíà (Cossart, Sansonetti, 2004; Haglund, Welch 2011). Ïî ñîâðåìåííûì ïðåäñòàâëåíèÿì, ïàòîãåííûå áàêòåðèè ïîïàäàþò â êëåòêó-õîçÿèíà ñ ïîìîùüþ îäíîãî èç äâóõ ìåõàíèçìîâ.  ìåõàíèçìå «çàñòåæêà-ìîëíèÿ» (zipper mechanism) — ýòî ïðÿìàÿ èíòåðíàëèçàöèÿ áàêòåðèé â ðåçóëüòàòå âçàèìîäåéñòâèÿ áàêòåðèàëüíîãî àäãåçèíà ñ ïîâåðõíîñòíûì ðåöåïòîðîì êëåòêè-õîçÿèíà. Ýòî âçàèìîäåéñòâèå àêòèâèðóåò ñèãíàëüíûå ñèñòåìû êëåòêè-õîçÿèíà è ïðèâîäèò ê óìåðåííûì èçìåíåíèÿì öèòîñêåëåòà (Tiney, Portnoy, 1989; Pizarro-Cerda et al., 2012).  òðèããåðíîì ìåõàíèçìå (trigger mechanism) áàêòåðèè çàõâàòûâàþòñÿ âûðîñòàìè êëåòî÷íîé ïîâåðõíîñòè, îáðàçîâàíèå êîòîðûõ èíäóöèðóåòñÿ áåëêàìè, ââåäåííûìè áàêòåðèÿìè â êëåòêó-õîçÿèíà ÷åðåç àïïàðàò ñåêðåöèè III òèïà (Sansonetti, 1998; Carayol, Tran Van Nhieu, 2013; Valencia-Gallardo et al., 2015). Ýòè ïðîöåññû ñâÿçàíû ñ ìîðôîëîãè÷åñêèìè èçìåíåíèÿìè êëåòêè-õîçÿèíà è çíà÷èòåëüíûìè ïåðåñòðîéêàìè åå öèòîñêåëåòà. Íåñìîòðÿ íà òî ÷òî áèîõèìè÷åñêèå îñíîâû ýòèõ ïðîöåññîâ çà ïîñëåäíèå ãîäû èíòåíñèâíî èçó÷àëèñü è ïîëó÷åí ðÿä íîâûõ âàæíûõ ôàêòîâ (Pizarro-Cerd*a et al., 2012; Rutherford, Bassler, 2012; Navarro-Garcia et al., 2013; Lee et al., 2014; Valencia-Gallardo et al., 2015), èõ âèçóàëèçàöèÿ êàê íà ýëåêòðîííî-ìèêðîñêîïè÷åñêîì, òàê è íà ñâåòîîïòè÷åñêîì óðîâíå íåñîìíåííî òðåáóåò äàëüíåéøèõ èñ714 Ýëåêòðîííî-ìèêðîñêîïè÷åñêèé àíàëèç ïðîíèêíîâåíèÿ óñëîâíî-ïàòîãåííûõ áàêòåðèé 715 ðèîòè÷åñêîé êëåòêè, ñòàäèè êëåòî÷íîãî öèêëà, ïðîëèôåðàòèâíîé àêòèâíîñòè è ñòåïåíè èììîðòàëèçîâàííîñòè êëåòîê (Velge et al., 1995, 1997). Ïîêàçàíî òàêæå, ÷òî àíòèîêñèäàíòû îêàçûâàþò ñóùåñòâåííîå âëèÿíèå íà êóëüòèâèðóåìûå êëåòêè, âûçûâàÿ ïåðåñòðîéêè àêòèíîâîãî öèòîñêåëåòà (Åôðåìîâà è äð., 2004) è èçìåíÿÿ èõ ÷óâñòâèòåëüíîñòü ê áàêòåðèÿì (Ãàìàëåé è äð., 2006; Gamaley et al., 2006).  ÷àñòíîñòè, îáðàáîòêà êëåòîê N-àöåòèëöèñòåèíîì ïðèâîäèò ê 2—3-êðàòíîìó óâåëè÷åíèþ ýôôåêòèâíîñòè èíâàçèè (Bozhokina et al., 2013).  íàñòîÿùåé ðàáîòå ìû èñïîëüçîâàëè ýòîò ýôôåêò, ÷òîáû âûÿâèòü íà÷àëüíûå ñòàäèè ïðîíèêíîâåíèÿ áàêòåðèé S. grimesii â êëåòêè HeLa, îáðàáîòàííûå è íå îáðàáîòàííûå N-àöåòèëöèñòåèíîì. Ìàòåðèàë è ìåòîäèêà Êëåòêè êàðöèíîìû øåéêè ìàòêè ÷åëîâåêà HeLa M, ïîëó÷åííûå èç Ðîññèéñêîé êîëëåêöèè êëåòî÷íûõ êóëüòóð (Èíñòèòóò öèòîëîãèè ÐÀÍ, Ñàíêò-Ïåòåðáóðã), êóëüòèâèðîâàëè íà ñðåäå aÌÅÌ, ñîäåðæàùåé 10 % ýìáðèîíàëüíîé áû÷üåé ñûâîðîòêè è 1 % NEAA (non-essential amino acids), ïðè 37 °Ñ â àòìîñôåðå 5 % CO2 â òå÷åíèå 2 ñóò äî äîñòèæåíèÿ ~70%-íîãî ìîíîñëîÿ. Ïîñëå ýòîãî çàìåíÿëè êîíäèöèîíèðîâàííóþ ñðåäó ïîðöèåé ñâåæåé ñðåäû è äîáàâëÿëè 10 ìÌ N-àöåòèëöèñòåèí (NAC) (Sigma) íà 22—24 ÷. Áàêòåðèè S. grimesii âûðàùèâàëè íà ïèòàòåëüíîì áóëüîíå LB, ñîäåðæàùåì 1 % ïåïòîíà, 0.5 % äðîææåâîãî ýêñòðàêòà è 1 % NaCl (pH 7.0), â òå÷åíèå 26 ÷ ïðè 37 °Ñ ñ àýðàöèåé. Çàòåì ñóñïåíçèþ áàêòåðèé â ðîñòîâîé ñðåäå öåíòðèôóãèðîâàëè ïðè 13 000 îá/ìèí â òå÷åíèå 5 ìèí. Îñàäîê ðåñóñïåíäèðîâàëè â ñðåäå aÌÅÌ áåç ñûâîðîòêè è äîáàâëÿëè ê êëåòêàì HeLa M, ïðåäâàðèòåëüíî ñìåíèâ ñðåäó, ñîäåðæàùóþ NAC, íà ñâåæóþ ñðåäó. Êëåòêè HeLa M èíêóáèðîâàëè ñ áàêòåðèÿìè 0.5—1.5 ÷ ïðè 37 °C. Ïîäãîòîâêó ïðåïàðàòîâ äëÿ ýëåêòðîííîé ìèêðîñêîïèè îñóùåñòâëÿëè ïî ñòàíäàðòíîé ìåòîäèêå (Ìèðîíîâ è äð., 1994). Êëåòêè ôèêñèðîâàëè íà ñòåêëàõ â 2.5%-íîì ðàñòâîðå ãëóòàðàëüäåãèäà íà ôîñôàòíîì áóôåðå (PBS) â òå÷åíèå 60 ìèí ïðè êîìíàòíîé òåìïåðàòóðå. Ïîñëå ýòîãî êëåòêè îòìûâàëè PBS è ïðîâîäèëè ïîñòôèêñàöèþ 1%-íûì ðàñòâîðîì îñìèåâîé êèñëîòû íà PBS â òå÷åíèå 30 ìèí ïðè êîìíàòíîé òåìïåðàòóðå. Äåãèäðàòàöèþ ïðîâîäèëè â ýòèëîâîì ñïèðòå âîçðàñòàþùèõ êîíöåíòðàöèé (îò 30 % äî àáñîëþòíîãî) è àáñîëþòíîì àöåòîíå.  ïðîöåññå äåãèäðàòàöèè êëåòêè ñíèìàëè ñî ñòåêîë ñ ïîìîùüþ ñêðåáêà è çàëèâàëè â ñìåñü ýïîíà è àðàëäèòà. Èç ïîëèìåðèçîâàííûõ áëîêîâ íà ìèêðîòîìå LKB III (Øâåöèÿ) ïîëó÷àëè óëüòðàòîíêèå ñðåçû, êîòîðûå êîíòðàñòèðîâàëè ïî 7 ìèí â öèòðàòå ñâèíöà è óðàíèë-àöåòàòå. Óëüòðàòîíêèå ñðåçû ïðîñìàòðèâàëè è ôîòîãðàôèðîâàëè â ýëåêòðîííîì ìèêðîñêîïå Carl Zeiss Libra 120 (Ãåðìàíèÿ). Ðåçóëüòàòû è îáñóæäåíèå Ýëåêòðîííî-ìèêðîñêîïè÷åñêèå êàðòèíû êëåòîê HeLa M è áàêòåðèé S. grimesii, ïîëó÷åííûå â íàñòîÿùåé ðàáîòå, íå îòëè÷àëèñü îò àíàëîãè÷íûõ êàðòèí, ìíîãîêðàòíî îïèñàííûõ â ëèòåðàòóðå (Erlandson, deHarve, 1971; Boatman et al., 1976; Balczon, 2001; Santos et al., 2008; Bozhokina et al., 2011). Ðèñ. 1. Óëüòðàòîíêèå ñðåçû êîíòðîëüíûõ êëåòîê HeLa M, íå îáðàáîòàííûõ (à) è ïðåäîáðàáîòàííûõ (á) N-àöåòèëöèñòåèíîì. Îáîçíà÷åíèÿ êî âñåì ðèñóíêàì: ì — ìèòîõîíäðèè, ìâ — ìèêðîâîðñèíêè, ìò — ìèêðîòðóáî÷êè, ïì — ïëàçìàòè÷åñêàÿ ìåìáðàíà, ýð — ýíäîïëàçìàòè÷åñêèé ðåòèêóëóì. Ñòðåëêàìè ïîêàçàí êîðòèêàëüíûé àêòèí. Ìàñøòàáíûé îòðåçîê — 1 ìêì. Ïðè àíàëèçå ýëåêòðîííî-ìèêðîñêîïè÷åñêèõ ôîòîãðàôèé îñíîâíîå âíèìàíèå óäåëÿëîñü ïîâåðõíîñòíûì ñòðóêòóðàì êëåòîê, èíôèöèðîâàííûõ áàêòåðèÿìè.  àïèêàëüíîé îáëàñòè èññëåäóåìûõ êëåòîê ÷åòêî âûÿâëÿëèñü îñíîâíûå îðãàíîèäû è ñòðóêòóðíûå ýëåìåíòû: ïëàçìàòè÷åñêàÿ ìåìáðàíà (ðèñ. 1—4), ïðèìåìáðàííûé àêòèíîâûé ñëîé (ðèñ. 1, 2), ìèòîõîíäðèè (ðèñ. 1), ýíäîïëàçìàòè÷åñêèé ðåòèêóëóì (ðèñ. 1, 3) è ìèêðîòðóáî÷êè (ðèñ. 1, 3—6). Ïîëó÷åííûå ðåçóëüòàòû ïîêàçûâàþò, ÷òî äîáàâëåíèå NAC â ðîñòîâóþ ñðåäó ñïîñîáñòâóåò ïîÿâëåíèþ ìíîãî÷èñëåííûõ âûðîñòîâ (âîðñèíîê) (ðèñ. 1). Ìíîãî÷èñëåííûå âîðñèíêè íàáëþäàþòñÿ è â ïðèñóòñòâèè áàêòåðèé, åùå äî ñòàäèè ïåðâè÷íîãî êîíòàêòà (ðèñ. 2). Ïðè ñîâìåñòíîé èíêóáàöèè áàêòåðèé è êëåòîê HeLa M, âûðàùåííûõ â ñðåäå áåç äîáàâëåíèÿ àíòèîêñèäàíòà, ïîâåðõíîñòü êëåòîê ïðàêòè÷åñêè ëèøåíà âîðñèíîê (ðèñ. 2, à, á). Êëåòêè HeLa M, îáðàáîòàííûå NAC, îòëè÷àþòñÿ îò êîíòðîëüíûõ êëåòîê áîëüøèì êîëè÷åñòâîì âûðîñòîâ (ðèñ. 2, â, ã). Âîçìîæíî, èìåííî ýòî ñïîñîáñòâóåò áîëåå àêòèâíîìó ïðîöåññó çàõâàòà áàêòåðèé HeLa M è, òàêèì îáðàçîì, ïîâûøàåò èíòåíñèâíîñòü èíâàçèè áàêòåðèé, ÷òî ñîãëàñóåòñÿ ñ ðàíåå ïîëó÷åííûìè ðåçóëüòàòàìè êîëè÷åñòâåííîé îöåíêè èíâàçèè áàêòåðèé S. grimesii â êëåòêè, îáðàáîòàííûå NAC (Bozhokina et al., 2013). Âçàèìîäåéñòâèå áàêòåðèé ñ êëåòêàìè HeLa M íà÷èíàåòñÿ ñ îáðàçîâàíèÿ ïëîòíîãî êîíòàêòà (ðèñ. 3). Çàòåì ìîæíî îáíàðóæèòü äâà âàðèàíòà ïðîíèêíîâåíèÿ áàêòå- 716 Å. Ñ. Áîæîêèíà è äð. Ðèñ. 2. Íà÷àëüíàÿ ñòàäèÿ ñîâìåñòíîé èíêóáàöèè êëåòîê HeLa M ñ áàêòåðèÿìè Serratia grimesii. à, á — êîíòðîëüíûå êëåòêè; â, ã — êëåòêè HeLa Ì, ïðåäîáðàáîòàííûå N-àöåòèëöèñòåèíîì, ÿ — ÿäðî. ðèé â ýóêàðèîòè÷åñêóþ êëåòêó.  áîëüøèíñòâå ñëó÷àåâ áàêòåðèè êàê áû ïðîäàâëèâàþò ïîâåðõíîñòü ýóêàðèîòè÷åñêîé êëåòêè, à çàòåì ïðîíèêàþò âíóòðü (ðèñ. 4, à, á), ÷òî ñîîòâåòñòâóåò ìåõàíèçìó «çàñòåæêà-ìîëíèÿ». Òðèããåðíûé ìåõàíèçì ïðåäóñìàòðèâàåò ðåîðãàíèçàöèþ ïðèìåìáðàííîãî àêòèíà êëåòêè-õîçÿèíà â îòâåò íà âíåäðåíèå â íåå áàêòåðèàëüíûõ ýôôåêòîðîâ. Âîçíèêàþùèå ïðè ïîëèìåðèçàöèè àêòèíà âûðîñòû çàõâàòûâàþò áàêòåðèþ, êîòîðàÿ ïðè ñëèÿíèè âûðîñòîâ îêàçûâàåòñÿ âíóòðè êëåòî÷íîé âàêóîëè (ðèñ. 5, à, á). Êðîìå òîãî, ïîëó÷åííûå â äàííîé ðàáîòå ýëåêòðîííî-ìèêðîñêîïè÷åñêèå êàðòèíû äàþò îñíîâàíèå ñ÷èòàòü, ÷òî âîðñèíêè êëåòêè-õîçÿèíà ñïîñîáñòâóþò çàõâàòó áàêòåðèé (ðèñ. 2, â, ã). Ñëåäóåò îòìåòèòü, ÷òî ïðèíöèïèàëüíîé ðàçíèöû ìåæäó âûðîñòàìè è âîðñèíêàìè íåò. Òå è äðóãèå ïðåäñòàâëÿþò ñîáîé àêòèíîâûå ôèáðèëëû, îêðóæåííûå ïëàçìàòè÷åñêîé ìåìáðàíîé. Ðàçíèöà ëèøü â òîì, ÷òî âîðñèíêè ñóùåñòâóþò äî êîíòàêòà êëåòêè-õîçÿèíà ñ áàêòåðèåé, à âûðîñòû îáðàçóþòñÿ â ðåçóëüòàòå êîíòàêòà áàêòåðèè ñ êëåòêîé-õîçÿèíîì. Êàêîå-òî âðåìÿ áàêòåðèè îñòàþòñÿ âíóòðè âàêóîëåé, ÷òî íàáëþäàåòñÿ ïðè âçàèìîäåéñòâèè áàêòåðèé ñ êëåòêàìè, êîòîðûå ðîñëè â ïðèñóòñòâèè àíòèîêñèäàíòà èëè â ñðåäå áåç àíòèîêñèäàíòà (ðèñ. 5, á; 6, a).  äàëüíåéøåì áàêòåðèè, êàê ïðàâèëî, ðàñòâîðÿþò ìåìáðàíó âàêóîëè, â êîòîðîé îíè íàõîäÿòñÿ, è âûõîäÿò â öèòîïëàçìó (ðèñ. 6, á), ÷òî ïðèâîäèò ê ïîñòåïåííîé ãèáåëè ïîâðåæäåííîé êëåòêè (Efremova et al., 2001). Íåçàâèñèìî îò ìåõàíèçìà ïðîíèêíîâåíèÿ, ïåðåìåùåíèå áàêòåðèé âíóòðè êëåòîê, ïî äàííûì ðÿäà èññëåäîâàíèé (Tilney, Portnoy, 1989; Theriot et al., 1992; Theriot, 2000), îñóùåñòâëÿåòñÿ ñ ïîìîùüþ àêòèíîâîãî «õâîñòà», ñôîðìèðîâàííîãî èç àêòèíà èíôèöèðîâàííîé êëåòêè. Îäíàêî àêòèíîâûå õâîñòû âûÿâëÿþòñÿ äîñòàòî÷íî ðåäêî (Åôðåìîâà è äð., 1998). Âìåñòå ñ òåì ìû äîâîëüíî ÷àñòî íàáëþäàëè âíóòðèêëåòî÷íûå áàêòåðèè, îêðóæåííûå áîëüøèì êîëè÷åñòâîì ìèêðîòðóáî÷åê (ðèñ. 6). Èç äàííûõ ëèòåðàòóðû èçâåñòíî, ÷òî ýòè îðãàíîèäû íå òîëüêî îñóùåñòâëÿþò òðàíñïîðò ðÿäà êëåòî÷íûõ îðãàíîèäîâ (Granger et al., 2014; Hancock, 2014), íî è ñïîñîáñòâóþò èíâàçèè íåêîòîðûõ áàêòåðèé (Yoshida et al., 2002; Dhakal, Mulvey, 2009; Haglund, Welch, 2011; Croinin, Backert, 2012). Ýòè íàáëþäåíèÿ óêàçûâàþò íà âîçìîæíîå ó÷àñòèå ìèêðîòðóáî÷åê â èíâàçèè è (èëè) âî âíóòðèêëåòî÷íîì òðàíñïîðòå áàêòåðèé S. grimesii. Ýëåêòðîííî-ìèêðîñêîïè÷åñêèé àíàëèç ïðîíèêíîâåíèÿ óñëîâíî-ïàòîãåííûõ áàêòåðèé Ðèñ. 3. Ïåðâîíà÷àëüíûå êîíòàêòû áàêòåðèé Serratia grimesii ñ êëåòêàìè HeLa Ì. Òàêèì îáðàçîì, ïðåäâàðèòåëüíàÿ îáðàáîòêà êëåòîê HeLa M N-àöåòèëöèñòåèíîì, èñïîëüçîâàííàÿ â íàñòîÿùåé ðàáîòå äëÿ óâåëè÷åíèÿ ýôôåêòèâíîñòè èíâàçèè, ïîçâîëèëà âûÿâèòü çíà÷èòåëüíîå êîëè÷åñòâî áàêòåðèé, íàõîäÿùèõñÿ íà ðàííèõ ñòàäèÿõ ïðîíèêíîâåíèÿ â êëåòêó-õîçÿèíà. Àíàëîãè÷íûå ñòàäèè áûëè âûÿâëåíû íà ïðåïàðàòàõ êëåòîê, íå îáðàáîòàííûõ N-àöåòèëöèñòåèíîì (íå ïðåäñòàâëåíî). Îäíàêî ïîëó÷åííûå ðåçóëüòàòû íå ïîçâîëÿþò îäíîçíà÷íî èäåíòèôèöèðîâàòü ìåõàíèçì âçàèìîäåéñòâèÿ áàêòåðèé ñ ýóêàðèîòè÷åñêîé êëåòêîé, ÷òî ñîãëàñóåòñÿ ñ àíàëèçîì äàííûõ, îïèñàííûõ â ëèòåðàòóðå (Croinin, Backert, 2012). Ñèòóàöèÿ îñëîæíÿåòñÿ åùå è òåì, ÷òî íåêîòîðûå áàêòåðèè èñïîëüçóþò äëÿ èíâàçèè îáà ìåõàíèçìà, õîòÿ îäèí èç íèõ, ïî-âèäèìîìó, ÿâëÿåòñÿ ïðåèìóùåñòâåííûì (Rosselin et al., 2010; Croinin, Backert, 2012; Grepinet et al., 2012; Velge et al., 2012; Boumart et al., 2014). Ïîýòîìó äëÿ ïîíèìàíèÿ ìåõàíèçìà èíâàçèè íåîáõîäèìî òàêæå âûÿâëåíèå áàêòåðèàëüíûõ è êëåòî÷íûõ ôàêòîðîâ, ó÷àñòâóþùèõ âî âçàèìîäåéñòâèè. Èçâåñòíî, ÷òî â ìåõàíèçìå «çàñòåæêà-ìîëíèÿ» áàêòåðèàëüíûé àäãåçèí àêòèâèðóåò èíâàçèþ, âçàèìîäåéñòâóÿ ñ Å-êàäãåðèíîì êëåòêè-õîçÿèíà (Lecuit et al., 2000; Hamon et al., 2006; Pizarro-Cerda et al., 2012), à êàñêàä ðåàêöèé, ïðèâîäÿùèõ ê çàõâàòó áàêòåðèè, ðåãóëèðóåòñÿ Src-êèíàçîé (Pizarro-Cerda et al., 2012). Ðàíåå ìû ïîêàçàëè, ÷òî ÷óâñòâèòåëüíîñòü êëåòîê HeLa Ì ê èíâàçèè áàêòåðèé S. grimesii êîððåëèðóåò ñ óñèëåíèåì ýêñïðåññèè ãåíîâ 717 Ðèñ. 4. Ïðîíèêíîâåíèå áàêòåðèé Serratia grimesii â êëåòêè HeLa Ì ïî ìåõàíèçìó «çàñòåæêà-ìîëíèÿ» ÷åðåç ðåöåïòîðîïîñðåäîâàííûé ýíäîöèòîç. à — íà÷àëüíûé ýòàï; á — ïîñëåäóþùèå ýòàïû âõîæäåíèÿ áàêòåðèé. êî — êëàòðèíîâîå îáðàìëåíèå, ýÿ — ýíäîöèòîçíàÿ ÿìêà. Å-êàäãåðèíà è b-êàòåíèíà è óâåëè÷åíèåì êîëè÷åñòâà Å-êàäãåðèíà â êëåòî÷íûõ ýêñòðàêòàõ (Bozhokina et al., 2013, 2015). Êðîìå òîãî, ïî íàøèì ïðåäâàðèòåëüíûì äàííûì, èíãèáèðîâàíèå Src-êèíàçû çíà÷èòåëüíî óìåíüøàåò ýôôåêòèâíîñòü èíâàçèè S. grimesii â êëåòêè HeLa Ì. Ñîïîñòàâëåíèå ðåçóëüòàòîâ, ïðåäñòàâëåííûõ â íàñòîÿùåé ðàáîòå, ñ ýòèìè äàííûìè ïîçâîëÿåò ïðåäïîëîæèòü, ÷òî ïðîíèêíîâåíèå áàêòåðèé S. grimesii â êëåòêè ýóêàðèîò ïðîèñõîäèò, ïî êðàéíåé ìåðå ÷àñòè÷íî, ñ ïîìîùüþ ìåõàíèçìà «çàñòåæêà-ìîëíèÿ», âêëþ÷àþùåãî â ñåáÿ Å-êàäãåðèí-êàòåíèíîâûé ïóòü. Ðàáîòà âûïîëíåíà ïðè ôèíàíñîâîé ïîääåðæêå Ðîññèéñêîãî ôîíäà ôóíäàìåíòàëüíûõ èññëåäîâàíèé (ïðîåêòû 14-04-00316 è 13-04-00101) è ïðîãðàììû ïðåçèäèóìà ÐÀÍ «Ìîëåêóëÿðíàÿ è êëåòî÷íàÿ áèîëîãèÿ» (¹ 2211). Ñïèñîê ëèòåðàòóðû Ãàìàëåé È. À., Åôðåìîâà Ò. Í., Êèðïè÷íèêîâà Ê. Ì., Êîìèññàð÷èê ß. Þ., Êåâåð, Ë. Â., Ïîëîçîâ Þ. Â., Õàéòëèíà Ñ. Þ. 2006. Óìåíüøåíèå ÷óâñòâèòåëüíîñòè òðàíñôîðìèðîâàííûõ êëåòîê 3Ò3-SV40, îáðàáîòàííûõ N-àöåòèëöèñòåèíîì, ê áàêòå- 718 Å. Ñ. Áîæîêèíà è äð. Ðèñ. 5. Ïðîíèêíîâåíèå áàêòåðèé Serratia grimesii â êëåòêè HeLa Ì ïî «òðèãåðíîìó» ìåõàíèçìó ñ ïîìîùüþ èíäóöèðîâàííûõ âûðîñòîâ êëåòî÷íîé ïîâåðõíîñòè, çàõâàòûâàþùèõ áàêòåðèþ. Ðèñ. 6. 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Ïîñòóïèëà 6 VII 2015 ENTRY OF FACULTATIVE PATHOGEN SERRATIA GRIMESII INTO HELA CELLS. ELECTRON MICROSCOPIC ANALYSIS E. S. Bozhokina, L. V. Kever, Ya. Yu. Komissarchik, S. Yu. Khaitlina,1 T. N. Efremova Institute of Cytology RAS, St. Petersburg, 194064; 1 e-mail: [email protected] Facultative pathogens Serratia grimesii are able to invade eukaryotic cells where they have been found in vacuoles and free in the cytoplasm (Efremova et al., 2001; Bozhokina et al., 2011). However, efficiency of this invasion is low, and the mechanisms of the invasion related to the initial steps of the process are not known. In the present study, we have increased the invasion efficiency by incubation of HeLa cells with N-acetylcysteine (NAC) preceding the infection. In the NAC-pretreated cells, two modes of S. grimesii to enter HeLa cells were observed. In the most cases, the penetration of S. grimesii into the cell was consistent with the «zipper mechanism», involving specific interaction of bacterial invasin with a host cell surface receptor. However, in some cases, bacteria were trapped by membrane ruffling probably produced by injected bacterial proteins that trigger the bacterial uptake process, as described in the «trigger mechanism». Further elucidation of bacterial and cellular factors involved in the bacteria-host cell interaction should clarify whether two different mechanisms or a predominant one operate during S. grimesii invasion. K e y w o r d s: bacterial invasion, N-acetylcysteine, Serratia grimesii, invasion steps.
