TY - JOUR
T1 - Inclusion membrane growth and composition are altered by overexpression of specific inclusion membrane proteins in chlamydia trachomatis l2
AU - Olson-Wood, Macy G.
AU - Jorgenson, Lisa M.
AU - Ouellette, Scot P.
AU - Rucks, Elizabeth A.
N1 - Funding Information:
We thank M. Weber (University of Iowa, IA) for the kind gift of the C. trachomatis L2 CT483-FLAG strain (23), R. Suchland (University of Washington, WA) and D. Rockey (Oregon State University, OR) for the anti-CT223 antibody, and T. Hackstadt (NIAID, Rocky Mountain Laboratories, Hamilton, MT) for the anti-IncE and anti-IncG antibodies. We thank Tom Bargar and Nicholas Conoan of the Electron Microscopy Core Facility (EMCF) at the University of Nebraska Medical Center for technical assistance. The EMCF is supported by state funds from the Nebraska Research Initiative (NRI) and the University of Nebraska Foundation and institutionally by the Office of the Vice Chancellor for Research.
Funding Information:
This work was partially supported by UNMC startup funds for E.A.R. and S.P.O., NIH/ NIAID grant number R01AI114670-01A1 awarded to E.A.R., and NIH/NIAID grant number R01AI132406-01A1 awarded to E.A.R. and S.P.O. This work was also supported by a UNMC Program of Excellence Assistantship awarded to M.G.O.-W.
Publisher Copyright:
© 2021 Olson-Wood et al.
PY - 2021/7
Y1 - 2021/7
N2 - Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections. This obligate intracellular bacterium develops within a membranebound vacuole called an inclusion, which sequesters the chlamydiae from the host cytoplasm. Host-pathogen interactions at this interface are mediated by chlamydial inclusion membrane proteins (Incs). However, the specific functions of most Incs are poorly characterized. Previous work from our laboratories indicated that expressing an IncF fusion protein at high levels in C. trachomatis L2 negatively impacted inclusion expansion and progeny production. We hypothesize that some Incs function in the structure and organization of the inclusion membrane and that overexpression of those Incs will alter the composition of endogenous Incs within the inclusion membrane. Consequently, inclusion biogenesis and chlamydial development are negatively impacted. To investigate this, C. trachomatis L2 was transformed with inducible expression plasmids encoding IncF-, CT813-, or CT226-FLAG. Overexpression of IncF-FLAG or CT813-FLAG, but not CT226-FLAG, altered chlamydial development, as demonstrated by smaller inclusions, fewer progeny, and increased plasmid loss. The overexpression of CT813-FLAG reduced the detectable levels of endogenous IncE and IncG in the inclusion membrane. Notably, recruitment of sorting nexin-6, a eukaryotic protein binding partner of IncE, was also reduced after CT813 overexpression. Gene expression studies and ultrastructural analysis of chlamydial organisms demonstrated that chlamydial development was altered when CT813-FLAG was overexpressed. Overall, these data indicate that disrupting the expression of specific Incs changed the composition of Incs within the inclusion membrane and the recruitment of associated host cell proteins, which negatively impacted C. trachomatis development.
AB - Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections. This obligate intracellular bacterium develops within a membranebound vacuole called an inclusion, which sequesters the chlamydiae from the host cytoplasm. Host-pathogen interactions at this interface are mediated by chlamydial inclusion membrane proteins (Incs). However, the specific functions of most Incs are poorly characterized. Previous work from our laboratories indicated that expressing an IncF fusion protein at high levels in C. trachomatis L2 negatively impacted inclusion expansion and progeny production. We hypothesize that some Incs function in the structure and organization of the inclusion membrane and that overexpression of those Incs will alter the composition of endogenous Incs within the inclusion membrane. Consequently, inclusion biogenesis and chlamydial development are negatively impacted. To investigate this, C. trachomatis L2 was transformed with inducible expression plasmids encoding IncF-, CT813-, or CT226-FLAG. Overexpression of IncF-FLAG or CT813-FLAG, but not CT226-FLAG, altered chlamydial development, as demonstrated by smaller inclusions, fewer progeny, and increased plasmid loss. The overexpression of CT813-FLAG reduced the detectable levels of endogenous IncE and IncG in the inclusion membrane. Notably, recruitment of sorting nexin-6, a eukaryotic protein binding partner of IncE, was also reduced after CT813 overexpression. Gene expression studies and ultrastructural analysis of chlamydial organisms demonstrated that chlamydial development was altered when CT813-FLAG was overexpressed. Overall, these data indicate that disrupting the expression of specific Incs changed the composition of Incs within the inclusion membrane and the recruitment of associated host cell proteins, which negatively impacted C. trachomatis development.
KW - Chlamydia
KW - Chlamydia trachomatis
KW - Developmental cycle
KW - Inc
KW - Inclusion membrane
KW - Type III secretion
UR - http://www.scopus.com/inward/record.url?scp=85108386826&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85108386826&partnerID=8YFLogxK
U2 - 10.1128/IAI.00094-21
DO - 10.1128/IAI.00094-21
M3 - Article
C2 - 33875478
AN - SCOPUS:85108386826
VL - 89
JO - Infection and Immunity
JF - Infection and Immunity
SN - 0019-9567
IS - 7
M1 - e00094-21
ER -