Plasmid Interference in Regulation of the Type III Secretion System in Pseudomonas aeruginosa
Pseudomonas aeruginosa, an opportunistic pathogen, commonly colonizes patients with cystic fibrosis (1) and contributes to a sizeable proportion of antibiotic-resistant nosocomial infections (2). Acute P. aeruginosa infections are characterized by the expression of the type III secretion system (T3SS) comprised of a needle-like apparatus used to inject exoenzymes into neighboring cells (1, 3). These toxic effector proteins enable P. aeruginosa to spread throughout the host while evading immune responses such as phagocytosis (REF).
The T3SS is regulated through several pathways, one of which is the RsmAYZ system (3). This pathway is composed of …show more content…
Determine extent of plasmids’ effects on RsmAYZ system. I will examine how various plasmids affect the activity of several transcriptional reporters in P. aeruginosa as measured by a Miller (beta-galactosidase) assay. I will test the reporter strains PlacP1-lacZ to confirm that pJN105 does not affect transcription of Vfr, another gene involved in T3SS regulation. I will also examine the activity of transcriptional reporters for the exsD, rsmY, and rsmZ genes to confirm that these genes are reciprocally affected by the plasmid, as previously observed. PexsD-lacZ activity should decrease when carrying an empty vector and PrsmY-lacZ activity should increase, as previously observed. PrsmZ-lacZ activity should also increase with the addition of pJN105, as RsmY and RsmZ perform similar roles in the RsmAYZ pathway. The activity of PexsD-lacZ will be examined in the P. aeruginosa strain PAK to confirm that the effects observed are not unique to PA103. Additionally, as I believe that increased expression of RsmY and RsmZ is driving this interference pattern, II will look to see that RsmA regulation is not affected at the transcriptional level.
3. Identify element of plasmid responsible for interference. If multiple plasmids cause a change in the activity of the RsmAYZ system, it may be that a common DNA element is the source of the interference. In silico analyses of plasmids will be
Pseudomonas aeruginosa, an opportunistic pathogen, commonly colonizes patients with cystic fibrosis (1) and contributes to a sizeable proportion of antibiotic-resistant nosocomial infections (2). Acute P. aeruginosa infections are characterized by the expression of the type III secretion system (T3SS) comprised of a needle-like apparatus used to inject exoenzymes into neighboring cells (1, 3). These toxic effector proteins enable P. aeruginosa to spread throughout the host while evading immune responses such as phagocytosis (REF).
The T3SS is regulated through several pathways, one of which is the RsmAYZ system (3). This pathway is composed of …show more content…
Determine extent of plasmids’ effects on RsmAYZ system. I will examine how various plasmids affect the activity of several transcriptional reporters in P. aeruginosa as measured by a Miller (beta-galactosidase) assay. I will test the reporter strains PlacP1-lacZ to confirm that pJN105 does not affect transcription of Vfr, another gene involved in T3SS regulation. I will also examine the activity of transcriptional reporters for the exsD, rsmY, and rsmZ genes to confirm that these genes are reciprocally affected by the plasmid, as previously observed. PexsD-lacZ activity should decrease when carrying an empty vector and PrsmY-lacZ activity should increase, as previously observed. PrsmZ-lacZ activity should also increase with the addition of pJN105, as RsmY and RsmZ perform similar roles in the RsmAYZ pathway. The activity of PexsD-lacZ will be examined in the P. aeruginosa strain PAK to confirm that the effects observed are not unique to PA103. Additionally, as I believe that increased expression of RsmY and RsmZ is driving this interference pattern, II will look to see that RsmA regulation is not affected at the transcriptional level.
3. Identify element of plasmid responsible for interference. If multiple plasmids cause a change in the activity of the RsmAYZ system, it may be that a common DNA element is the source of the interference. In silico analyses of plasmids will be