Involvement of the heat shock proteins DnaK/DnaJ in Yersinia T3S

• Yersinia  pseudotuberculosis  uses  a  type  III  secretion  system  (T3SS)  to  secrete  and  deliver  effectors  called Yops into target cells. These processes are highly regulated and the pathogen senses cell contact and respond accordingly by inducing Yop-effector expression.  A key component of the T3SS is the YscF needle present on the  surface of  the  pathogen. It has  been  suggested  that the  bacterium  can  switch  from  needle  export  to  Yop expression  and  secretion  and  that this  substrate  switch  is  important  for proper  regulation  during infection. YscU  is  an  essential  protein  regulating  the  substrate  switch  and  autoproteolysis  of  YscU  is  essential  for accurate  T3SS  regulation.  To  study  regulation  of  Yop  translocation  in  more  detail,  we  generated  mutants defective for expression of the heat shock proteins (HSPs) DnaJ and DnaK, since earlier studies had indicated a role of these proteins in regulation of effector translocation in Salmonella. The dnaJ mutant and the double dnaK/J  mutant  showed  significant  defects  in  Yop  translocation,  but  surprisingly  both  mutants  were  able  to secrete Yops in vitro much like the wild type.  However, both mutants showed a changed export pattern of the YscF  needle  with  a  pronounced  increased  export  of  the  YscF  needle  protein  after  incubation  in  calcium containing media. This phenotype was linked to defects in YscU autoproteolysis and in this respect the  hsp-mutants  were  identical  to  earlier  identified  autoprocessing  defective  mutants  in  YscU  (Single  amino  acid exchange mutants N263A and P264A). The hsp-mutants and the processing mutants accumulated full-length YscU,  which  surprisingly  was  associated  with  the  outer  membrane,  while  the  processed  form  of  YscU  was found  in  the  inner  membrane  fraction.  The  dnaJ  and  dnaK/J  mutants  were  strongly  affected  in  YscU autoproteolysis, which indicates a possible direct role for DnaJ in this process. Indeed a specific interaction between  YscU  and  DnaJ  could  be  found  suggesting  a  direct  role  of  the  HSPs  in  regulation  of  the  substrate switch in the T3SS.

Ämnesord

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

Nyckelord

Molecular biology

Molekylärbiologi

molekylärbiologi

Molecular Biology

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