Ventajas del consumo de los alimentos orgánicos

membrane

periplasm

outer

membrane

medium

periplasmic

type IV

pili

proteins

(2-step)

I

one-step

secretion

Figure 1.5. T r a n s l o c a t i o n p a thways a cross the Gram n e g a t i v e c e 1 I env elope.

The d i a g r a m includes the one- and t w o - s t e p s e c r e t i o n pathways, and a s s e m b l y of proteins into the inner and outer memb r a n e s . IM = inner membrane, OM = outer membrane.

or c l e a v a g e of a classical signal sequence, a l t h o u g h it does a p p e a r to be directed by a C - t e r m i n a l s e cretion signal, as d i s c u s s e d later. The s e c r e t e d p rotein traverses both m e m b r a n e s at once, via a channel formed by several a c c e s s o r y proteins. The best c h a r a c t e r i s e d example is a l p h a - h a e m o i y s i n s e c r e t i o n by E. c o 1i , but a highly h o mologous s y s t e m is r e s p o n s i b l e for protease s e c r e t i o n by E r w i n i a species.

One d i g r e s s i o n from the c l a s s i f i c a t i o n of s e c r e t i o n m e c h a n i s m s into 1-step and 2 - s t e p is the e x i s t e n c e of several pro t e i n s w hich are S e c - d e p e n d e n t in their p a s s a g e a c r o s s the inner membrane, but require no other f a c t o r s for their t r a n s l o c a t i o n across the outer membrane. T hese a u t o s e c r e t o r y proteins include the E. c o 1 i e n t e r o t o x i n s , ST» (Yang et a!., 19921 and S T B (D r e f u s et al., 1992i.

1.7 H a e m o l y s i n secretion: a o n e - s t e p m e c h a n i s m

The h a e m o l y s i n secreted by u r o p a t h o g e n i c strains of E. coIi

is t r a n s l o c a t e d into the m e d i u m by a m e c h a n i s m w h i c h does not r e q u i r e a classical N - t e r m i n a l signal sequence, is i n d e p e n d e n t of the general e x p o r t (Seel pathway ( M a c K m a n et

al., 19871 a nd in w hich the s e c r e t e d pr o t e i n does not stop in the p e r i p l a s m (Mackman et al., 19861. Instead, s e c r e t i o n is d i r e c t e d by two spe c i f i c proteins, HlyB and HlyD, both ins e r t e d into the inner membrane, together with the outer m e m b r a n e p r o t e i n T o l C ( W a n d e r s m a n and D e l e p e l a i r e 19901.

The m e t a l l o p r o t e a s e s of Ech a r e also s e creted by a o n e - s t e p m e c h a n i s m (Barras et al., 19861, being s y n t h e s i s e d w i t h o u t a

signal sequence. B oth are s y n t h e s i s e d as inactive zymogens, a c t i v a t i o n involving c l e a v a g e of an N-terminal " p r o ­ s e quence", a p o s t - s e c r e t i o n event. The protease genes are found in an o p e r o n along w i t h h o m o l o g u e s of hlyB <prtD), h l y D (prtE) and t olC (prtF), as we 1 1 as inh, e n c o d i n g a p e r i p l a s m i c inhibitor, a s a f e t y m e c h a n i s m for the p r o d u c i n g cell ( D e l e p e l a i r e and U a n d e r s m a n 1989? Letoffe et a 1.,

1990). A similar operon is r e s p o n s i b l e for secr e t i o n of a l k a l i n e pr o t e a s e by P s e u d o m o n a s a e r u g i n o s a (Duong et aJ., 1992), e n c o d i n g A p r h o m o l o g u e s of PrtD, E and F, as well as 1 n h .

H o m o l o g u e s of HlyB and D have a lso been reported in the s e c r e t i o n of v arious m e m b e r s of the haem o l y s i n f a m i l y ( c y c l o l y s i n of B o r d e t e l l a pertussis, leukotoxin of

P a s t e u r e i J a haemoiytica, (Le t o f f e et ai., 1990), h a e m o l y s i n of V i b r i o cholerae, (Aim and M anning 1990)), as well as the a n t i m i c r o b i a l peptide, subtil in of B a c i l l u s su b t i l i s (Chung

et ai 1992), p r o t e a s e of S e r r a t i a marce s c e n s , the n o d u l a t i o n

p r o t e i n N o d O of R h i z o b i u m 1e g u m i n o s a r u m (Scheu et al., 1992), a n d the a n t i b i o t i c syri ng o m y c i n of P s e u d o m o n a s s y r i n g a e pv. s y r i n g a e ( Quigley et al., 1993).

1.7.1 A s s i g n i n g roles to the Hly p r o t e i n s and h o mologues

T o p o l o g i c a l s tudies c o m b i n i n g i m m u n o 1 oca 1i s a t i o n ? p r o t e a s e s e n s i t i v i t y (Delep e l a i r e and U a n d e r s m a n 1991); and a n a l y s i s of P h o A a n d Bla fusions (Wang et al., 1991; S c huleln et al., 1992) i n d i c a t e that HlyB and D (and their homologues) span the inner membrane, HlyB with six t r a n s m e m b r a n e s e g m e n t s and

H 1yD w ith one, a l t h o u g h d e p e n d i n g on the method used t h e r e is also e v i d e n c e for a s s o c i a t i o n with the outer m e mbrane, s u g g e s t i n g that t hey form part of a c o mplex that spans b oth m e m b r a n e s (Blight and H o lland 1990). Seq u e n c e h o m o l o g y (Blight and H o l l a n d 1990) indicates that the large c a r b o x y - terminal c y t o p l a s m i c d omain of H 1yB c o n t a i n s two n u c l e o t i d e b i n d i n g s i t e s ( H i g g i n s et al., 1986), p r o b a b l y the s o u r c e of e n e r g y for the t r a n s l o c a t i o n process. Indeed, r e c e n t i n v e s t i g a t i o n s ( K o r o n a k i s et al., 1993) have d e m o n s t r a t e d that H 1yB has A T P a s e activity, that it shows af f i n i t y for b o t h ATP a n d ADP, and that b i n d i n g of e ither results in a s i g n i f i c a n t c o n f o r m a t i o n a l c h a n g e in the soluble d omain. F i g u r e 1.6 shows a recent model based on these o b s e r v a t i o n s .

1.7.2 The s e a r c h for a s e c r e t i o n signal

M u t a t i o n a l a n a l y s i s and s e q u e n c e c o m p a r i s o n s (Koronakis et al., 1989; S t a n l e y et al., 1991; K e n n y et al., 1992) i n d i c a t e d that the secr e t i o n signal is located in the N- terminal r e g i o n and h i g h l i g h t e d the p o s s i b l e importance of three s e c o n d a r y s t r u c t u r a l features: an a m p h i p a t h i c helix, a c luster of c h a r g e d residues, and a w e a k l y h y d r o p h o b i c terminal sequence. This has been s u p p o r t e d by v a r i o u s st u d i e s in w h i c h p r o g r e s s i v e l y shorter C-te r m i n a l f r a g m e n t s w e r e f used to r e p o r t e r g enes (Kenny et el., 1991; D e l e p e l a i r e and W a n d e r s m a n 1990; L e t o f f e and W a n d e r s m a n 1992). T h e s e s t u d i e s iden t i f i e d a s hort s equence of 4 0 - 5 0 a m i n o a cids at the e x t r e m e C-terminus, c a p a b l e of d i r e c t i n g secretion. In m a n y of the f usions made, however, a m u c h larger section, e n c o m p a s s i n g a g l y c i n e - r i c h repeat motif, is