Limits...
Dynamics of the type III secretion system activity of enteropathogenic Escherichia coli.

Mills E, Baruch K, Aviv G, Nitzan M, Rosenshine I - MBio (2013)

Bottom Line: We found that the two pathogens exhibit different translocation behaviors: in EPEC, a subpopulation that formed microcolonies carried out most of the translocation activity, while Salmonella executed protein translocation as planktonic bacteria.We next extended the study to determine the translocation dynamics of twenty EPEC effectors and found that all exhibited distinct levels of translocation efficiency.Further, we mapped the global effects of key TTSS-related components on TTSS activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Institute of Medical Research Israel-Canada, Faculty of Medicine,the Hebrew University of Jerusalem, Jerusalem, Israel.

ABSTRACT

Unlabelled: Type III secretion systems (TTSSs) are employed by pathogens to translocate host cells with effector proteins, which are crucial for virulence. The dynamics of effector translocation, behavior of the translocating bacteria, translocation temporal order, and relative amounts of each of the translocated effectors are all poorly characterized. To address these issues, we developed a microscopy-based assay that tracks effector translocation. We used this assay alongside a previously described real-time population-based translocation assay, focusing mainly on enteropathogenic Escherichia coli (EPEC) and partly comparing it to Salmonella. We found that the two pathogens exhibit different translocation behaviors: in EPEC, a subpopulation that formed microcolonies carried out most of the translocation activity, while Salmonella executed protein translocation as planktonic bacteria. We also noted variability in host cell susceptibility, with some cells highly resistant to translocation. We next extended the study to determine the translocation dynamics of twenty EPEC effectors and found that all exhibited distinct levels of translocation efficiency. Further, we mapped the global effects of key TTSS-related components on TTSS activity. Our results provide a comprehensive description of the dynamics of the TTSS activity of EPEC and new insights into the mechanisms that control the dynamics.

Importance: EPEC and the closely related enterohemorrhagic Escherichia coli (EHEC) represent a global public health problem. New strategies to combat EPEC and EHEC infections are needed, and development of such strategies requires better understanding of their virulence machinery. The TTSS is a critical virulence mechanism employed by these pathogens, and by others, including Salmonella. In this study, we aimed at elucidating new aspects of TTSS function. The results obtained provide a comprehensive description of the dynamics of TTSS activity of EPEC and new insights into the mechanisms that control these changes. This knowledge sets the stage for further analysis of the system and may accelerate the development of new ways to treat EPEC and EHEC infections. Further, the newly described microscopy-based assay can be readily adapted to study the dynamics of TTSS activity in other pathogens.

Show MeSH

Related in: MedlinePlus

Effector translocation in the absence of CesT or CesF or both. (A) Translocation of strains carrying effector-BlaM fusions genes was analyzed. The results of an experiment representative of three are shown. The mutants used are indicated. Each data point represents the average of quadruplicate results. The translocation dynamics of NleA, EspG, EspZ, and EspF, which represent four distinct behaviors, are presented. Translocation of EspZ and EspF by the ΔcesT ΔcesF double mutant was not tested. Results relating to the other effectors are shown in Fig. S4 in the supplemental material. Error bars are omitted for clarity. (B) The ratio of translocation by wild-type EPEC to that by the cesT and cesF mutants was determined. The average results of at least three experiments, each of which was conducted in duplicate or quadruplicate, are shown. The two dotted lines indicate a ratio of 1, indicating that translocation by a given mutant was similar to translocation by wild-type EPEC. The bars signify standard deviations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3735188&req=5

fig5: Effector translocation in the absence of CesT or CesF or both. (A) Translocation of strains carrying effector-BlaM fusions genes was analyzed. The results of an experiment representative of three are shown. The mutants used are indicated. Each data point represents the average of quadruplicate results. The translocation dynamics of NleA, EspG, EspZ, and EspF, which represent four distinct behaviors, are presented. Translocation of EspZ and EspF by the ΔcesT ΔcesF double mutant was not tested. Results relating to the other effectors are shown in Fig. S4 in the supplemental material. Error bars are omitted for clarity. (B) The ratio of translocation by wild-type EPEC to that by the cesT and cesF mutants was determined. The average results of at least three experiments, each of which was conducted in duplicate or quadruplicate, are shown. The two dotted lines indicate a ratio of 1, indicating that translocation by a given mutant was similar to translocation by wild-type EPEC. The bars signify standard deviations.

Mentions: CesT and CesF are chaperones required for efficient translocation of some effectors (6, 12–18). To examine their global effect on translocation dynamics, we inserted effector-BlaM fusion genes into the chromosomes of EPEC cesT and cesF mutants and compared the translocation by these mutants to that exhibited by wild-type EPEC (Fig. 5). As before, the Tir and group B effectors were analyzed by real-time translocation assays and group C effectors by STPT assays. Translocation of only one effector, EspF, was dependent on CesF (Fig. 5A). In the case of CesT, a more complex picture emerged: translocation of Map, EspH, EspJ, EspZ, NleG, NleH1, and NleH2 was reduced dramatically to less than 10% of that of the wild-type strain, while translocation of NleA, NleB1, NleB2, NleC, and Tir was reduced only partially (Fig. 5; see also Fig. S4 in the supplemental material). Translocation of EspG, NleD, and NleF was not dependent on CesT or CesF (Fig. 5). These results show that CesT has a wide range of impact on effector translocation, extending from no impact to strict CesT dependency.


Dynamics of the type III secretion system activity of enteropathogenic Escherichia coli.

Mills E, Baruch K, Aviv G, Nitzan M, Rosenshine I - MBio (2013)

Effector translocation in the absence of CesT or CesF or both. (A) Translocation of strains carrying effector-BlaM fusions genes was analyzed. The results of an experiment representative of three are shown. The mutants used are indicated. Each data point represents the average of quadruplicate results. The translocation dynamics of NleA, EspG, EspZ, and EspF, which represent four distinct behaviors, are presented. Translocation of EspZ and EspF by the ΔcesT ΔcesF double mutant was not tested. Results relating to the other effectors are shown in Fig. S4 in the supplemental material. Error bars are omitted for clarity. (B) The ratio of translocation by wild-type EPEC to that by the cesT and cesF mutants was determined. The average results of at least three experiments, each of which was conducted in duplicate or quadruplicate, are shown. The two dotted lines indicate a ratio of 1, indicating that translocation by a given mutant was similar to translocation by wild-type EPEC. The bars signify standard deviations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3735188&req=5

fig5: Effector translocation in the absence of CesT or CesF or both. (A) Translocation of strains carrying effector-BlaM fusions genes was analyzed. The results of an experiment representative of three are shown. The mutants used are indicated. Each data point represents the average of quadruplicate results. The translocation dynamics of NleA, EspG, EspZ, and EspF, which represent four distinct behaviors, are presented. Translocation of EspZ and EspF by the ΔcesT ΔcesF double mutant was not tested. Results relating to the other effectors are shown in Fig. S4 in the supplemental material. Error bars are omitted for clarity. (B) The ratio of translocation by wild-type EPEC to that by the cesT and cesF mutants was determined. The average results of at least three experiments, each of which was conducted in duplicate or quadruplicate, are shown. The two dotted lines indicate a ratio of 1, indicating that translocation by a given mutant was similar to translocation by wild-type EPEC. The bars signify standard deviations.
Mentions: CesT and CesF are chaperones required for efficient translocation of some effectors (6, 12–18). To examine their global effect on translocation dynamics, we inserted effector-BlaM fusion genes into the chromosomes of EPEC cesT and cesF mutants and compared the translocation by these mutants to that exhibited by wild-type EPEC (Fig. 5). As before, the Tir and group B effectors were analyzed by real-time translocation assays and group C effectors by STPT assays. Translocation of only one effector, EspF, was dependent on CesF (Fig. 5A). In the case of CesT, a more complex picture emerged: translocation of Map, EspH, EspJ, EspZ, NleG, NleH1, and NleH2 was reduced dramatically to less than 10% of that of the wild-type strain, while translocation of NleA, NleB1, NleB2, NleC, and Tir was reduced only partially (Fig. 5; see also Fig. S4 in the supplemental material). Translocation of EspG, NleD, and NleF was not dependent on CesT or CesF (Fig. 5). These results show that CesT has a wide range of impact on effector translocation, extending from no impact to strict CesT dependency.

Bottom Line: We found that the two pathogens exhibit different translocation behaviors: in EPEC, a subpopulation that formed microcolonies carried out most of the translocation activity, while Salmonella executed protein translocation as planktonic bacteria.We next extended the study to determine the translocation dynamics of twenty EPEC effectors and found that all exhibited distinct levels of translocation efficiency.Further, we mapped the global effects of key TTSS-related components on TTSS activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Institute of Medical Research Israel-Canada, Faculty of Medicine,the Hebrew University of Jerusalem, Jerusalem, Israel.

ABSTRACT

Unlabelled: Type III secretion systems (TTSSs) are employed by pathogens to translocate host cells with effector proteins, which are crucial for virulence. The dynamics of effector translocation, behavior of the translocating bacteria, translocation temporal order, and relative amounts of each of the translocated effectors are all poorly characterized. To address these issues, we developed a microscopy-based assay that tracks effector translocation. We used this assay alongside a previously described real-time population-based translocation assay, focusing mainly on enteropathogenic Escherichia coli (EPEC) and partly comparing it to Salmonella. We found that the two pathogens exhibit different translocation behaviors: in EPEC, a subpopulation that formed microcolonies carried out most of the translocation activity, while Salmonella executed protein translocation as planktonic bacteria. We also noted variability in host cell susceptibility, with some cells highly resistant to translocation. We next extended the study to determine the translocation dynamics of twenty EPEC effectors and found that all exhibited distinct levels of translocation efficiency. Further, we mapped the global effects of key TTSS-related components on TTSS activity. Our results provide a comprehensive description of the dynamics of the TTSS activity of EPEC and new insights into the mechanisms that control the dynamics.

Importance: EPEC and the closely related enterohemorrhagic Escherichia coli (EHEC) represent a global public health problem. New strategies to combat EPEC and EHEC infections are needed, and development of such strategies requires better understanding of their virulence machinery. The TTSS is a critical virulence mechanism employed by these pathogens, and by others, including Salmonella. In this study, we aimed at elucidating new aspects of TTSS function. The results obtained provide a comprehensive description of the dynamics of TTSS activity of EPEC and new insights into the mechanisms that control these changes. This knowledge sets the stage for further analysis of the system and may accelerate the development of new ways to treat EPEC and EHEC infections. Further, the newly described microscopy-based assay can be readily adapted to study the dynamics of TTSS activity in other pathogens.

Show MeSH
Related in: MedlinePlus