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1.  Regulation of Chlamydial Infection by Host Autophagy and Vacuolar ATPase-Bearing Organelles ▿ 
Infection and Immunity  2011;79(10):4019-4028.
As arguably the most successful parasite, Chlamydia is an obligate intracellular bacterium replicating inside a vacuole of eukaryotic host cells. The chlamydial vacuole does not fuse with the defense cell organelle lysosome. We previously showed that chlamydial infection increases markers of autophagy, an innate antimicrobial activity requiring lysosomal function. However, the work presented here demonstrates that p62, an autophagy protein that is degraded in lysosomes, either remained unchanged or increased in chlamydia-infected human epithelial, mouse fibroblast, and mouse macrophage cell lines. In addition, the activities of three lysosomal enzymes analyzed were diminished in chlamydia-infected macrophages. Bafilomycin A1 (BafA), a specific inhibitor of vacuolar ATPase (vATPase) required for lysosomal function, increased the growth of the human pathogen Chlamydia trachomatis (L2) in wild-type murine fibroblasts and macrophages but inhibited growth in the autophagy-deficient ATG5−/− fibroblasts. BafA exhibited only slight inhibition or no effect on L2 growth in multiple human genital epithelial cell lines. In contrast to L2, the mouse pathogen Chlamydia muridarum (MoPn) was consistently inhibited by BafA in all cell lines examined, regardless of species origin and autophagy status. Finally, L2 but not MoPn grew more efficiently in the ATG5−/− cells than in wild-type cells. These results suggest that there are two types of vATPase-bearing organelles that regulate chlamydial infection: one supports chlamydial infection, while the other plays a defensive role through autophagy when cells are artificially infected with certain chlamydiae that have not been adapted to the host species.
PMCID: PMC3187247  PMID: 21807906
2.  The Conserved Tarp Actin Binding Domain Is Important for Chlamydial Invasion 
PLoS Pathogens  2010;6(7):e1000997.
The translocated actin recruiting phosphoprotein (Tarp) is conserved among all pathogenic chlamydial species. Previous reports identified single C. trachomatis Tarp actin binding and proline rich domains required for Tarp mediated actin nucleation. A peptide antiserum specific for the Tarp actin binding domain was generated and inhibited actin polymerization in vitro and C. trachomatis entry in vivo, indicating an essential role for Tarp in chlamydial pathogenesis. Sequence analysis of Tarp orthologs from additional chlamydial species and C. trachomatis serovars indicated multiple putative actin binding sites. In order to determine whether the identified actin binding domains are functionally conserved, GST-Tarp fusions from multiple chlamydial species were examined for their ability to bind and nucleate actin. Chlamydial Tarps harbored variable numbers of actin binding sites and promoted actin nucleation as determined by in vitro polymerization assays. Our findings indicate that Tarp mediated actin binding and nucleation is a conserved feature among diverse chlamydial species and this function plays a critical role in bacterial invasion of host cells.
Author Summary
Chlamydiae are bacterial obligate intracellular pathogens responsible for multiple human and veterinary diseases. The induction of cytoskeletal rearrangements to promote chlamydial internalization is partially mediated by a type III secreted effector protein called Tarp that is translocated upon contact with host cells and independently nucleates actin filament formation. Tarp from a C. trachomatis lymphogranuloma venereum (LGV) strain consists of a tyrosine-rich repeat domain, a proline-rich domain required for oligomerization, and a single actin binding domain. Oligomerization is required to bring multiple actin monomers together to initiate actin filament formation by a mechanism distinct from host actin nucleators. Here we have examined Tarp from several other strains of chlamydiae and find that certain of these contain up to four actin binding domains. Tarp fragments bearing multiple actin binding domains nucleate actin in in vitro assays even in the absence of the oligomerization domain. This suggests that Tarp from different chlamydial species may utilize hybrid mechanisms to induce actin nucleation. Determination of virulence determinants in chlamydiae is challenging due to the lack of tractable genetic systems. The direct introduction of anti-Tarp actin binding domain antibodies into the cytosol of host cells inhibited entry and thus demonstrates an essential role for Tarp in chlamydial pathogenesis.
PMCID: PMC2904776  PMID: 20657821
3.  Development of a Transformation System for Chlamydia trachomatis: Restoration of Glycogen Biosynthesis by Acquisition of a Plasmid Shuttle Vector 
PLoS Pathogens  2011;7(9):e1002258.
Chlamydia trachomatis remains one of the few major human pathogens for which there is no transformation system. C. trachomatis has a unique obligate intracellular developmental cycle. The extracellular infectious elementary body (EB) is an infectious, electron-dense structure that, following host cell infection, differentiates into a non-infectious replicative form known as a reticulate body (RB). Host cells infected by C. trachomatis that are treated with penicillin are not lysed because this antibiotic prevents the maturation of RBs into EBs. Instead the RBs fail to divide although DNA replication continues. We have exploited these observations to develop a transformation protocol based on expression of β-lactamase that utilizes rescue from the penicillin-induced phenotype. We constructed a vector which carries both the chlamydial endogenous plasmid and an E.coli plasmid origin of replication so that it can shuttle between these two bacterial recipients. The vector, when introduced into C. trachomatis L2 under selection conditions, cures the endogenous chlamydial plasmid. We have shown that foreign promoters operate in vivo in C. trachomatis and that active β-lactamase and chloramphenicol acetyl transferase are expressed. To demonstrate the technology we have isolated chlamydial transformants that express the green fluorescent protein (GFP). As proof of principle, we have shown that manipulation of chlamydial biochemistry is possible by transformation of a plasmid-free C. trachomatis recipient strain. The acquisition of the plasmid restores the ability of the plasmid-free C. trachomatis to synthesise and accumulate glycogen within inclusions. These findings pave the way for a comprehensive genetic study on chlamydial gene function that has hitherto not been possible. Application of this technology avoids the use of therapeutic antibiotics and therefore the procedures do not require high level containment and will allow the analysis of genome function by complementation.
Author Summary
C. trachomatis is a major human pathogen for which there is no means of genetically manipulating its DNA. It is an obligate intracellular bacterium which has a complex developmental cycle that takes place in a specialized host cell cytoplasmic vacuole known as an inclusion. We have constructed a shuttle vector based on the chlamydial plasmid and developed a new approach to select genetically modified bacteria. It uses rescue by selection of stable infectious, penicillin-resistant C. trachomatis from a pool of non-dividing, non-infectious C. trachomatis (induced by penicillin arrest of the developmental cycle). The transformed C. trachomatis is also cured of its endogenous plasmid by the selection of the transforming vector. The vector was modified to express the green fluorescent protein (GFP) in both Chlamydia and E. coli. We have genetically manipulated a plasmid-free recipient strain of C. trachomatis and shown restoration of the ability of this recipient strain to synthesize and accumulate glycogen within inclusions upon acquisition of the shuttle vector. The ability to transform and manipulate C. trachomatis using a complementation based vector is an important advance that opens up the field of Chlamydia research.
PMCID: PMC3178582  PMID: 21966270
4.  Fluorescence Lifetime Imaging Unravels C. trachomatis Metabolism and Its Crosstalk with the Host Cell 
PLoS Pathogens  2011;7(7):e1002108.
Chlamydia trachomatis is an obligate intracellular bacterium that alternates between two metabolically different developmental forms. We performed fluorescence lifetime imaging (FLIM) of the metabolic coenzymes, reduced nicotinamide adenine dinucleotides [NAD(P)H], by two-photon microscopy for separate analysis of host and pathogen metabolism during intracellular chlamydial infections. NAD(P)H autofluorescence was detected inside the chlamydial inclusion and showed enhanced signal intensity on the inclusion membrane as demonstrated by the co-localization with the 14-3-3β host cell protein. An increase of the fluorescence lifetime of protein-bound NAD(P)H [τ2-NAD(P)H] inside the chlamydial inclusion strongly correlated with enhanced metabolic activity of chlamydial reticulate bodies during the mid-phase of infection. Inhibition of host cell metabolism that resulted in aberrant intracellular chlamydial inclusion morphology completely abrogated the τ2-NAD(P)H increase inside the chlamydial inclusion. τ2-NAD(P)H also decreased inside chlamydial inclusions when the cells were treated with IFNγ reflecting the reduced metabolism of persistent chlamydiae. Furthermore, a significant increase in τ2-NAD(P)H and a decrease in the relative amount of free NAD(P)H inside the host cell nucleus indicated cellular starvation during intracellular chlamydial infection. Using FLIM analysis by two-photon microscopy we could visualize for the first time metabolic pathogen-host interactions during intracellular Chlamydia trachomatis infections with high spatial and temporal resolution in living cells. Our findings suggest that intracellular chlamydial metabolism is directly linked to cellular NAD(P)H signaling pathways that are involved in host cell survival and longevity.
Author Summary
Separate analysis of host and pathogen metabolic changes in intracellular C. trachomatis infections is arduous and has not been comprehensively realized so far. A more detailed understanding about the metabolic activity and needs of C. trachomatis and its specific interactions with the host cell would be the basis for the development of novel treatment strategies. We therefore applied fluorescence lifetime imaging (FLIM) of the metabolic coenzymes NAD(P)H using two-photon microscopy to directly visualize metabolic changes of host cells and pathogens in living cells. NAD(P)H fluorescence was detected both on the chlamydial inclusion membrane and inside the inclusion. Interestingly, changes in chlamydial growth and progeny induced by glucose starvation and IFNγ treatment were directly linked to significant changes of the NAD(P)H fluorescence lifetimes inside the inclusions. Furthermore, measurement of the NAD(P)H fluorescence lifetime in the host cell nucleus revealed that infected cells were programmed for starvation during the metabolically active phase of intracellular chlamydial growth. Our findings highlight for the first time a direct interaction between host and pathogen metabolism in intracellular bacterial infections that exceeds sole competition for nutrients. In conclusion, fluorescence lifetime imaging of NAD(P)H by two-photon microscopy enables real-time analysis of metabolic host-pathogen interactions in intracellular infections with high spatial and temporal resolution.
PMCID: PMC3136453  PMID: 21779161
5.  Treatment of Chlamydia trachomatis with a small molecule inhibitor of the Yersinia type III secretion system disrupts progression of the chlamydial developmental cycle 
Molecular microbiology  2006;61(6):1543-1555.
The obligate intracellular bacterium Chlamydia trachomatis possesses a biphasic developmental cycle that is manifested by differentiation of infectious, metabolically inert elementary bodies (EBs) to larger, metabolically active reticulate bodies (RBs). The cycle is completed by asynchronous differentiation of dividing RBs back to a population of dormant EBs that can initiate further rounds of infection upon lysis of the host cell. Chlamydiae express a type III secretion system (T3SS) which is presumably employed to establish and maintain the permissive intracellular niche by secretion of anti-host proteins. We hypothesize that T3SS activity is essential for chlamydial development and pathogenesis. However, the lack of a genetic system has confounded efforts to establish any role of the T3SS. We therefore employed the small molecule Yersinia T3SS inhibitor ′N′-(3,5-dibromo-2-hydroxybenzylidene)-4-nitrobenzohydrazide, designated compound 1 (C1), to examine the inter-dependence of the chlamydial T3SS and development. C1-treatment inhibited C. trachomatis but not T4SS-expressing Coxiella burnetii development in a dose-dependent manner. Although chlamydiae remained viable and metabolically active, they failed to divide significantly and RB to EB differentiation was inhibited. These effects occurred in the absence of host cell cytotoxicity and were reversible by washing out C1. We further demonstrate that secretion of T3S substrates is perturbed in C1-treated chlamydial cultures. We have therefore provided evidence that C1 can inhibit C. trachomatis development and T3SS activity and present a model in which progression of the C. trachomatis developmental cycle requires a fully functional T3SS.
PMCID: PMC1615999  PMID: 16968227
Chlamydia; type III secretion; Compound 1
6.  Use of cycloheximide to study independent lipid metabolism of Chlamydia trachomatis cultivated in mouse L cells grown in serum-free medium. 
Infection and Immunity  1981;31(2):668-673.
A system for measuring chlamydial lipid synthesis was developed with mouse L cells grown in serum-free modified Waymouth 752/l medium in a shaker culture. Host lipid synthesis was reduced approximately 90% when cells were incubated for 24 h in medium containing cycloheximide (2 micrograms/ml). Lipid metabolism was monitored by measuring the incorporation of [3H]isoleucine into the total lipid of normal and infected cells. The results suggested that lipid synthesis of Chlamydia trachomatis lymphogranuloma venereum (LGV-404L) was not inhibited by cycloheximide treatment when the chlamydiae were grown in L cells, whereas host lipid synthesis was inhibited. Chlamydial lipid metabolism began about 6 to 12 h after infection when the noninfectious reticulate body was found and continually increased until the beginning of the appearance of intracellular infectious elementary bodies at 24 to 30 h.
PMCID: PMC351361  PMID: 7216466
7.  Inclusion Biogenesis and Reactivation of Persistent Chlamydia trachomatis Requires Host Cell Sphingolipid Biosynthesis 
PLoS Pathogens  2009;5(11):e1000664.
Chlamydiae are obligate intracellular pathogens that must coordinate the acquisition of host cell-derived biosynthetic constituents essential for bacterial survival. Purified chlamydiae contain several lipids that are typically found in eukaryotes, implying the translocation of host cell lipids to the chlamydial vacuole. Acquisition and incorporation of sphingomyelin occurs subsequent to transport from Golgi-derived exocytic vesicles, with possible intermediate transport through endosomal multivesicular bodies. Eukaryotic host cell-derived sphingomyelin is essential for intracellular growth of Chlamydia trachomatis, but the precise role of this lipid in development has not been delineated. The present study identifies specific phenotypic effects on inclusion membrane biogenesis and stability consequent to conditions of sphingomyelin deficiency. Culturing infected cells in the presence of inhibitors of serine palmitoyltransferase, the first enzyme in the biosynthetic pathway of host cell sphingomyelin, resulted in loss of inclusion membrane integrity with subsequent disruption in normal chlamydial inclusion development. Surprisingly, this was accompanied by premature redifferentiation to and release of infectious elementary bodies. Homotypic fusion of inclusions was also disrupted under conditions of sphingolipid deficiency. In addition, host cell sphingomyelin synthesis was essential for inclusion membrane stability and expansion that is vital to reactivation of persistent chlamydial infection. The present study implicates both the Golgi apparatus and multivesicular bodies as key sources of host-derived lipids, with multivesicular bodies being essential for normal inclusion development and reactivation of persistent C. trachomatis infection.
Author Summary
The genus Chlamydia is composed of a group of obligate intracellular bacterial pathogens that cause several human diseases of medical significance. C. trachomatis is the most commonly encountered sexually transmitted pathogen, as well as the leading cause of preventable blindness worldwide. The prevalence of chlamydial infections, and the extraordinary morbidity and health care costs associated with chronic persisting disease, justifies the research efforts in this area of microbial pathogenesis. Despite their clinical importance, the mechanisms by which these intracellular bacteria obtain nutrients essential to their growth remain enigmatic. Acquisition of sphingolipids, from the cells that chlamydiae infect, is essential for bacterial propagation. This study identifies a requirement for the lipid sphingomyelin from the infected host cell for bacterial replication during infection, and for long-term subsistence in persistent chlamydial infection. Blockage of sphingomyelin acquisition results in premature release of bacteria, a reduced bacterial number, and failure of the bacteria to cause a persisting infection. In this study, we have identified and subsequently disrupted specific sphingomyelin transport pathways, providing important implications on therapeutic intervention targeting this successful microbial pathogen.
PMCID: PMC2774160  PMID: 19936056
8.  Chlamydial Infection Induces Pathobiotype-Specific Protein Tyrosine Phosphorylation in Epithelial Cells  
Infection and Immunity  2005;73(4):1939-1946.
Members of the genus Chlamydia are strict obligate intracellular pathogens that exhibit marked differences in host range and tissue tropism despite sharing a remarkable level of genomic synteny. These pathobiotype differences among chlamydiae are also mirrored in their early interactions with cultured mammalian host cells. Chlamydial attachment and entry is known to trigger protein tyrosine phosphorylation. In this study, we examined the kinetics and pattern of protein tyrosine phosphorylation induced by infection with a comprehensive collection of chlamydial strains exhibiting diversity in host, tissue, and disease tropisms. We report new findings showing that protein tyrosine phosphorylation patterns induced by infection directly correlate with the pathobiotype of the infecting organism. Patterns of protein tyrosine phosphorylation were induced following early infection that unambiguously categorized chlamydial pathobiotypes into four distinct groups: (i) Chlamydia trachomatis trachoma biovars (serovars A to H), (ii) C. trachomatis lymphogranuloma venereum biovars (serovars L1 to L3), (iii) C. muridarum, and (iv) C. pneumoniae and C. caviae. Notably, chlamydia-infected murine and human epithelial cells exhibited the same protein tyrosine phosphorylation patterns; this is indirect evidence suggesting that the phosphorylated protein(s) is of chlamydial origin. If our hypothesis is correct, these heretofore-uncharacterized proteins may represent a novel class of bacterial molecules that influence pathogen-host range or tissue tropism.
PMCID: PMC1087445  PMID: 15784533
9.  Differential interaction with endocytic and exocytic pathways distinguish parasitophorous vacuoles of Coxiella burnetii and Chlamydia trachomatis. 
Infection and Immunity  1996;64(3):796-809.
Coxiella burnetii and Chlamydia trachomatis are bacterial obligate intracellular parasites that occupy distinct vacuolar niches within eucaryotic host cells. We have employed immunofluorescence, cytochemistry, fluorescent vital stains, and fluid-phase markers in conjunction with electron, confocal, and conventional microscopy to characterize the vacuolar environments of these pathogens. The acidic nature of the C. burnetii-containing vacuole was confirmed by its acquisition of the acidotropic base acridine orange (AO). The presence of the vacuolar-type (H+) ATPase (V-ATPase) within the Coxiella vacuolar membrane was demonstrated by indirect immunofluorescence, and growth of C. burnetii was inhibited by bafilomycin A1 (Baf A), a specific inhibitor of the V-ATPase. In contrast, AO did not accumulate in C. trachomatis inclusions nor was the V-ATPase found in the inclusion membrane. Moreover, chlamydial growth was not inhibited by Baf A or the lysosomotropic amines methylamine, ammonium chloride, and chloroquine. Vacuoles harboring C. burnetii incorporated the fluorescent fluid- phase markers, fluorescein isothiocyanate-dextran (FITC-dex) and Lucifer yellow (LY), indicating trafficking between that vacuole and the endocytic pathway. Neither FITC-dex nor LY was sequestered by chlamydial inclusions. The late endosomal-prelysosomal marker cation-independent mannose 6-phosphate receptor was not detectable in the vacuolar membranes encompassing either parasite. However, the lysosomal enzymes acid phosphatase and cathepsin D and the lysosomal glycoproteins LAMP-1 and LAMP-2 localized to the C. burnetii vacuole but not the chlamydial vacuole. Interaction of C. trachomatis inclusions with the Golgi-derived vesicles was demonstrated by the transport of sphingomyelin, endogenously synthesized from C6-NBD-ceramide, to the chlamydial inclusion and incorporation into the bacterial cell wall. Similar trafficking of C-NBD-ceramide was not evident in C. burnetii-infected cells. Collectively, the data indicate that C. trachomatis replicates within a nonacidified vacuole that is disconnected from endosome-lysosome trafficking but may receive lipid from exocytic vesicles derived from the trans-Golgi network. These observations are in sharp contrast to those for C. burnetii, which by all criteria resides in a typical phagolysosome.
PMCID: PMC173840  PMID: 8641784
10.  The trans-Golgi SNARE syntaxin 10 is required for optimal development of Chlamydia trachomatis 
Chlamydia trachomatis, an obligate intracellular pathogen, grows inside of a vacuole, termed the inclusion. Within the inclusion, the organisms differentiate from the infectious elementary body (EB) into the reticulate body (RB). The RB communicates with the host cell through the inclusion membrane to obtain the nutrients necessary to divide, thus expanding the chlamydial population. At late time points within the developmental cycle, the RBs respond to unknown molecular signals to redifferentiate into infectious EBs to perpetuate the infection cycle. One strategy for Chlamydia to obtain necessary nutrients and metabolites from the host is to intercept host vesicular trafficking pathways. In this study we demonstrate that a trans-Golgi soluble N-ethylmaleimide–sensitive factor attachment protein (SNARE), syntaxin 10, and/or syntaxin 10-associated Golgi elements colocalize with the chlamydial inclusion. We hypothesized that Chlamydia utilizes the molecular machinery of syntaxin 10 at the inclusion membrane to intercept specific vesicular trafficking pathways in order to create and maintain an optimal intra-inclusion environment. To test this hypothesis, we used siRNA knockdown of syntaxin 10 to examine the impact of the loss of syntaxin 10 on chlamydial growth and development. Our results demonstrate that loss of syntaxin 10 leads to defects in normal chlamydial maturation including: variable inclusion size with fewer chlamydial organisms per inclusion, fewer infectious progeny, and delayed or halted RB-EB differentiation. These defects in chlamydial development correlate with an overabundance of NBD-lipid retained by inclusions cultured in syntaxin 10 knockdown cells. Overall, loss of syntaxin 10 at the inclusion membrane negatively affects Chlamydia. Understanding host machinery involved in maintaining an optimal inclusion environment to support chlamydial growth and development is critical toward understanding the molecular signals involved in successful progression through the chlamydial developmental cycle.
PMCID: PMC4585193  PMID: 26442221
Chlamydia trachomatis; syntaxin 10; chlamydial development; lipid trafficking; trans-Golgi SNARE
11.  EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis 
PLoS Pathogens  2015;11(4):e1004846.
The obligate intracellular bacterium Chlamydia trachomatis invades into host cells to replicate inside a membrane-bound vacuole called inclusion. Multiple different host proteins are recruited to the inclusion and are functionally modulated to support chlamydial development. Invaded and replicating Chlamydia induces a long-lasting activation of the PI3 kinase signaling pathway that is required for efficient replication. We identified the cell surface tyrosine kinase EphrinA2 receptor (EphA2) as a chlamydial adherence and invasion receptor that induces PI3 kinase (PI3K) activation, promoting chlamydial replication. Interfering with binding of C. trachomatis serovar L2 (Ctr) to EphA2, downregulation of EphA2 expression or inhibition of EphA2 activity significantly reduced Ctr infection. Ctr interacts with and activates EphA2 on the cell surface resulting in Ctr and receptor internalization. During chlamydial replication, EphA2 remains active accumulating around the inclusion and interacts with the p85 regulatory subunit of PI3K to support the activation of the PI3K/Akt signaling pathway that is required for normal chlamydial development. Overexpression of full length EphA2, but not the mutant form lacking the intracellular cytoplasmic domain, enhanced PI3K activation and Ctr infection. Despite the depletion of EphA2 from the cell surface, Ctr infection induces upregulation of EphA2 through the activation of the ERK pathway, which keeps the infected cell in an apoptosis-resistant state. The significance of EphA2 as an entry and intracellular signaling receptor was also observed with the urogenital C. trachomatis-serovar D. Our findings provide the first evidence for a host cell surface receptor that is exploited for invasion as well as for receptor-mediated intracellular signaling to facilitate chlamydial replication. In addition, the engagement of a cell surface receptor at the inclusion membrane is a new mechanism by which Chlamydia subverts the host cell and induces apoptosis resistance.
Author Summary
Chlamydia trachomatis are major human pathogens causing ocular and sexually transmitted diseases with hundreds of millions of cases per year. Chlamydia replicate inside the host cell in a membrane bound vacuole called inclusion. The current concept on how Chlamydia communicates with the host cell during its replication is based on the identification of the host protein that interacts with Chlamydia. Here, we describe that C. trachomatis-serovar L2 and D use EphA2, a member of the largest class of human receptor tyrosine kinases, as an adherence and entry receptor that is endocytosed together with the bacteria. Cell surface EphA2 receptor is adopted by Chlamydia to function also at the inclusion to support growth and replication and to keep the infected cell in an apoptosis resistant state. Thus, we show that EphA2 is an undiscovered important surface and intracellular signaling receptor that is crucial for chlamydial infection and development.
PMCID: PMC4408118  PMID: 25906164
12.  Biosynthesis and disulfide cross-linking of outer membrane components during the growth cycle of Chlamydia trachomatis. 
Infection and Immunity  1987;55(1):162-168.
The synthesis and accumulation of Chlamydia trachomatis outer membrane proteins within infected HeLa 229 host cells were monitored by assessing the uptake of [35S]cysteine into chlamydial proteins during the 48-h growth cycle of a lymphogranuloma venereum strain, L2/434/Bu. Synthesis of the major outer membrane protein, a protein that accounts for about 60% of the outer membrane protein mass of elementary bodies (EB), was first detected between 12 and 18 h after infection. The uptake of [35S]cysteine into the 60,000-molecular-weight doublet (60K doublet) and 12.5K cysteine-rich proteins was not observed until 30 h after infection, when the intracellularly dividing reticulate bodies were beginning to transform into infectious EBs. By using a more sensitive immunoblotting method in conjunction with monoclonal antibodies specific for the 60K doublet proteins, synthesis of these proteins was detected even earlier, by 18 h after infection. These data suggest that the time and extent of synthesis of these outer membrane proteins are regulated by processes that coincide in time with the transformation of reticulate bodies into EBs. Additional studies were performed to determine the extent of disulfide cross-linking of outer membrane proteins during the growth cycle. Both the major outer membrane protein and the 12.5K protein became progressively cross-linked to about 60% during the last 24 h of the growth cycle, whereas the 60K doublet proteins were extensively cross-linked during most of the cycle. These data may indicate an intracellular cross-linking mechanism, possibly enzymatic, that exists in addition to an auto-oxidation mechanism that occurs upon host cell lysis and exposure to the extracellular environment.
PMCID: PMC260295  PMID: 3793227
13.  Penicillin Induced Persistence in Chlamydia trachomatis: High Quality Time Lapse Video Analysis of the Developmental Cycle 
PLoS ONE  2009;4(11):e7723.
Chlamydia trachomatis is a major human pathogen with a unique obligate intracellular developmental cycle that takes place inside a modified cytoplasmic structure known as an inclusion. Following entry into a cell, the infectious elementary body (EB) differentiates into a non - infectious replicative form known as a reticulate body (RB). RBs divide by binary fission and at the end of the cycle they redifferentiate into EBs. Treatment of C.trachomatis with penicillin prevents maturation of RBs which survive and enlarge to become aberrant RBs within the inclusion in a non - infective persistent state. Persistently infected individuals may be a reservoir for chlamydial infection. The C.trachomatis genome encodes the enzymes for peptidoglycan (PG) biosynthesis but a PG sacculus has never been detected. This coupled to the action of penicillin is known as the chlamydial anomaly. We have applied video microscopy and quantitative DNA assays to the chlamydial developmental cycle to assess the effects of penicillin treatment and establish a framework for investigating penicillin induced chlamydial persistence.
Principal Findings
Addition of penicillin at the time of cell infection does not prevent uptake and the establishment of an inclusion. EB to RB transition occurs but bacterial cytokinesis is arrested by the second binary fission. RBs continue to enlarge but not divide in the presence of penicillin. The normal developmental cycle can be recovered by the removal of penicillin although the large, aberrant RBs do not revert to the normal smaller size but remain present to the completion of the developmental cycle. Chromosomal and plasmid DNA replication is unaffected by the addition of penicillin but the arrest of bacterial cytokinesis under these conditions results in RBs accumulating multiple copies of the genome.
We have applied video time lapse microscopy to the study of the chlamydial developmental cycle. Linked with accurate measures of genome replication this provides a defined framework to analyse the developmental cycle and to investigate and provide new insights into the effects of antibiotic treatments. Removal of penicillin allows recovery of the normal developmental cycle by 10–20 hrs and the process occurs by budding from aberrant RBs.
PMCID: PMC2769264  PMID: 19893744
14.  Interaction between a trachoma strain of Chlamydia trachomatis and mouse fibroblasts (McCoy cells) in the absence of centrifugation. 
Infection and Immunity  1981;31(2):584-591.
A system was devised for studying the interaction of a trachoma strain of Chlamydia trachomatis (G17) and mouse fibroblasts (McCoy cells) in the absence of centrifugation, which is usually employed to enhance the infection of cell cultures with non-lymphogranuloma venereum human strains of C. trachomatis. In this system, the conditions of infection more closely approached those encountered in natural infections, and the entry of G17 into host cells could be compared with the previously described entry of C. psittaci 6BC and a lymphogranuloma venereum strain (440L) of C. trachomatis. McCoy cells were infected by shaking at 37 degrees C with inocula suspended in 0.01 M phosphate buffer, pH 7.2, containing 0.2 M sucrose. The efficiency of infection (inclusion counts without centrifugation/inclusion counts with centrifugation) was 1.5% for monolayers and 7.5% for suspensions. When measured either by inclusion counts or by host cell-associated 14C-amino acid-labeled G17, association was proportional to G17 concentration and increased linearly for 60 min. Pretreatment of host cells with diethylaminoethyl-dextran (30 micrograms/ml, 30 min) raised the efficiency of infection to about 13% for both monolayers and suspensions. Host cells treated with cytochalasin B (2 x 10(-5) M, 90 min) or trypsin (50 micrograms/ml, 60 min) associated with G17 at undiminished rates. 14C-labeled G17 inactivated by heat (60 degrees C, 3 min) or ultraviolet light (1,800 ergs per mm2) associated with McCoy cells at the same rate as live G17. Comparison of these results with those previously reported for strains 6BC and 440L showed that strain G17 exhibited some, but not all, of the host cell association properties of the other two chlamydial strains.
PMCID: PMC351349  PMID: 7216462
15.  The cell-penetrating peptide, Pep-1, has activity against intracellular chlamydial growth but not extracellular forms of Chlamydia trachomatis 
In the course of studies to identify novel treatment strategies against the pathogenic bacterium, Chlamydia, we tested the carrier peptide, Pep-1, for activity against an intracellular infection.
Using a cell culture model of Chlamydia trachomatis infection, the effect of Pep-1 was measured by incubating the peptide with extracellular chlamydiae prior to infection, or by adding Pep-1 to the medium at varying times after infection, and assaying for inhibition of inclusion formation.
Pep-1 had a concentration-dependent effect on chlamydial growth with 100% inhibition of inclusion formation at 8 mg/L peptide. There was a window of susceptibility during the chlamydial developmental cycle with a maximal effect when treatment was begun within 12 h of infection. Pep-1 treatment caused a severe reduction in the production of infectious progeny even when started later, when the effect on inclusion formation was minimal. Furthermore, electron micrographs showed a paucity of progeny elementary bodies (EBs) in the inclusion. In contrast, pre-incubation of EBs with Pep-1 prior to infection did not affect inclusion formation. Taken together, these findings indicate that the antichlamydial effect was specific for the intracellular stage of chlamydial infection. By comparison, Pep-1 had no antimicrobial activity against Escherichia coli and Staphylococcus aureus or the obligate intracellular parasite, Toxoplasma gondii.
Pep-1 has antichlamydial activity by preventing intracellular chlamydial growth and replication but has no effect on extracellular chlamydiae.
PMCID: PMC2721699  PMID: 18957395
Chlamydia spp.; antimicrobial peptides; antimicrobial activity; antimicrobial agents
16.  Laboratory diagnosis of human chlamydial infections. 
Clinical Microbiology Reviews  1989;2(2):119-136.
Chlamydia trachomatis is a human pathogen that causes ocular disease (trachoma and inclusion conjunctivitis), genital disease (cervicitis, urethritis, salpingitis, and lymphogranuloma venereum), and respiratory disease (infant pneumonitis). Respiratory chlamydioses also occur with infection by avian strains of C. psittaci or infection by the newly described TWAR agent. Diagnosis of most acute C. trachomatis infections relies on detection of the infecting agent by cell culture, fluorescent antibody, immunoassay, cytopathologic, or nucleic acid hybridization methods. Individual non-culture tests for C. trachomatis are less sensitive and specific than the best chlamydial cell culture system but offer the advantages of reduced technology and simple transport of clinical specimens. Currently available nonculture tests for C. trachomatis perform adequately as screening tests in populations in which the prevalence of infection is greater than 10%. A negative culture or nonculture test for C. trachomatis does not, however, exclude infection. The predictive value of a positive nonculture test may be unsatisfactory when populations of low infection prevalence are tested. Tests that detect antibody responses to chlamydial infection have limited utility in diagnosis of acute chlamydial infection because of the high prevalence of persistent antibody in healthy adults and the cross-reactivity due to infection by the highly prevalent C. trachomatis and TWAR agents. Assays for changes in antibody titer to the chlamydial genus antigen are used for the diagnosis of respiratory chlamydioses. A single serum sample that is negative for chlamydial antibody excludes the diagnosis of lymphogranuloma venereum.
PMCID: PMC358106  PMID: 2650858
17.  Effect of interferon on the growth of Chlamydia trachomatis in mouse fibroblasts (L cells). 
Infection and Immunity  1983;39(1):362-370.
The effect of murine interferon on the growth of the lymphogranuloma venereum biotype of Chlamydia trachomatis (strain 440L) in murine fibroblasts (L cells) was examined. Treatment of infected cell cultures with interferon caused a reduction in the number of inclusion-bearing cells as seen by light and electron microscopy and a decrease in yields of chlamydiae as determined by infectivity assays. Interferon also inhibited cycloheximide-resistant (chlamydia-specific) protein synthesis in infected cells. The interferon effect was dose dependent, with 80 to 90% inhibition occurring at concentrations of greater than 200 IU/ml. The inhibitory effect was neutralized by anti-murine interferon globulin. Interferon did not inactivate extracellular chlamydiae, and both host cell RNA and protein synthesis were required for the development of the interferon-induced antichlamydial state. Inhibition of chlamydial growth by interferon was demonstrable in cells treated 18 h before infection or up to 4 h after infection. Cells infected after interferon was removed exhibited an antichlamydial activity decline which was complete by 30 h after interferon removal. We show that interferon treatment did not affect either entry of chlamydiae into host cells or chlamydial conversion to reticulate bodies but rather caused a reduction in the rate of reticulate body replication.
PMCID: PMC347947  PMID: 6185434
18.  A novel co-infection model with Toxoplasma and Chlamydia trachomatis highlights the importance of host cell manipulation for nutrient scavenging 
Cellular microbiology  2012;15(4):619-646.
Toxoplasmaand Chlamydia trachomatis are obligate intracellular pathogens that have evolved analogous strategies to replicate within mammalian cells. Both pathogens are known to extensively remodel the cytoskeleton, and to recruit endocytic and exocytic organelles to their respective vacuoles. However, how important these activities are for infectivity by either pathogen remains elusive. Here, we have developed a novel co-infection system to gain insights into the developmental cycles of Toxoplasma and C. trachomatis by infecting human cells with both pathogens, and examining their respective ability to replicate and scavenge nutrients. We hypothesize that the common strategies used by Toxoplasma and Chlamydia to achieve development results in direct competition of the two pathogens for the same pool of nutrients. We show that a single human cell can harbor Chlamydia and Toxoplasma. In co-infected cells, Toxoplasma is able to divert the content of host organelles, such as cholesterol. Consequently, the infectious cycle of Toxoplasma progresses unimpeded. In contrast, Chlamydia’s ability to scavenge selected nutrients is diminished, and the bacterium shifts to a stress-induced persistent growth. Parasite killing engenders an ordered return to normal chlamydial development. We demonstrate that C. trachomatis enters a stress-induced persistence phenotype as a direct result from being barred from its normal nutrient supplies as addition of excess nutrients, e.g., amino acids, leads to substantial recovery of Chlamydia growth and infectivity. Co-infection of C. trachomatis with slow growing strains of Toxoplasma or a mutant impaired in nutrient acquisition does not restrict chlamydial development. Conversely, Toxoplasma growth is halted in cells infected with the highly virulent Chlamydia psittaci. This study illustrates the key role that cellular remodeling plays in the exploitation of host intracellular resources by Toxoplasma and Chlamydia. It further highlights the delicate balance between success and failure of infection by intracellular pathogens in a co-infection system at the cellular level.
PMCID: PMC3625693  PMID: 23107293
19.  Persistent Infection of Mouse Fibroblasts (L Cells) with Chlamydia psittaci: Evidence for a Cryptic Chlamydial Form 
Infection and Immunity  1980;30(3):874-883.
When monolayers of mouse fibroblasts (L cells) were infected with enough Chlamydia psittaci (strain 6BC) to destroy most of the host cells, 1 in every 105 to 106 originally infected cells gave rise to a colony of L cells persistently infected with strain 6BC. In these populations, the density of L cells and 6BC fluctuated periodically and reciprocally as periods of host cell increase were followed by periods of parasite multiplication. Successive cycles of L-cell and 6BC reproduction were sustained indefinitely by periodic transfer to fresh medium. Isolation of L cells and 6BC from persistent infections provided no evidence that there had been any selection of variants better suited for coexistence. Persistently infected populations consisting mainly of inclusion-free L cells yielded only persistently infected clones, grew more slowly, and cloned less efficiently. They were also almost completely resistant to superinfection with high multiplicities of either 6BC or the lymphogranuloma venereum strain 440L of Chlamydia trachomatis. These properties of persistently infected L cells may be accounted for by assuming that all of the individuals in these populations are cryptically infected with 6BC and that cryptic infection slows the growth of the host cell and makes it immune to infection with exogenous chlamydiae. According to this hypothesis, the fluctuations in host and parasite density occur because some factor periodically sets off the conversion of cryptic chlamydial forms into reticulate bodies that multiply and differentiate into infectious elementary bodies in a conventional chlamydial developmental cycle.
PMCID: PMC551395  PMID: 6262243
20.  The trans-Golgi SNARE syntaxin 6 is recruited to the chlamydial inclusion membrane 
Microbiology  2011;157(Pt 3):830-838.
Chlamydia trachomatis is an obligate intracellular pathogen that replicates within a parasitophorous vacuole termed an inclusion. The chlamydial inclusion is isolated from the endocytic pathway but fusogenic with Golgi-derived exocytic vesicles containing sphingomyelin and cholesterol. Sphingolipids are incorporated into the chlamydial cell wall and are considered essential for chlamydial development and viability. The mechanisms by which chlamydiae obtain eukaryotic lipids are poorly understood but require chlamydial protein synthesis and presumably modification of the inclusion membrane to initiate this interaction. A polarized cell model of chlamydial infection has demonstrated that chlamydiae preferentially intercept basolaterally directed, sphingomyelin-containing exocytic vesicles. Here we examine the localization and potential function of trans-Golgi and/or basolaterally associated soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in chlamydia-infected cells. The trans-Golgi SNARE protein syntaxin 6 is recruited to the chlamydial inclusion in a manner that requires chlamydial protein synthesis and is conserved among all chlamydial species examined. The localization of syntaxin 6 to the chlamydial inclusion requires a tyrosine motif or plasma membrane retrieval signal (YGRL). Thus in addition to expression of at least two inclusion membrane proteins that contain SNARE-like motifs, chlamydiae also actively recruit eukaryotic SNARE-family proteins.
PMCID: PMC3081085  PMID: 21109560
21.  Seroepidemiological and socioeconomic studies of genital chlamydial infection in Ethiopian women. 
Genitourinary Medicine  1992;68(4):221-227.
OBJECTIVE--To measure the prevalence of chlamydial genital infection in Ethiopian women attending gynaecological, obstetric and family planning clinics; to identify the epidemiological, social and economic factors affecting the prevalence of infection in a country where routine laboratory culture and serological tests for chlamydial species are unavailable; to determine the risk factors for genital chlamydial infection in those with serological evidence of other sexually transmitted diseases. SUBJECTS--1846 Ethiopian women, outpatient attenders at two teaching hospitals and a mother and child health centre in Addis Ababa, Ethiopia. SETTING--Gynaecological outpatient department, antenatal, postnatal and family planning clinics. METHODS--Sera were tested for type-specific anti-chlamydial antibodies using purified chlamydial antigens (C. trachomatis A-C (CTA-C), C. trachomatis D-K (CTD-K), Lymphogranuloma venereum (LGV1-3), and C. pneumoniae (CPn)), in a micro-immunofluorescence test. The genital chlamydia seropositivity was analysed against patient's age, clinic attended, ethnic group, religion, origin of residence, age at first marriage and first coitus, income, number of sexual partners, duration of sexual activity, marital status/profession, obstetric and contraceptive history, and seropositivity for other sexually transmitted diseases. RESULTS--Overall exposure to chlamydia species was found in 84%, genital chlamydial infection in 62%, and titres suggestive of recent or present genital infection in 42% of those studied. Genital chlamydial infection was highest (64%) in family planning and lowest (54%) in antenatal clinic attenders. Exposure to genital chlamydia species was influenced by ethnic group and religion. Those married and sexually active under 13 years of age had greater exposure (69%) to genital chlamydial infection than those first sexually active aged over 18 (46%). Prevalence of infection was highest in those with more than five sexual partners (78%) and in bargirls (84%). The lowest income groups had a higher prevalence (65%) of genital chlamydial infection than the wealthiest (48%). Multivariate analysis showed the most important factors to be age at first coitus, religion, prostitution and present age of the woman in that order. Risk for genital chlamydial infection was increased in those with seropositivity for syphilis, gonorrhoea, HSV-2 but not HBV infection. CONCLUSION/APPLICATION--Chlamydial genital infections are highly prevalent in both symptomatic and asymptomatic Ethiopian women. The high prevalence of infection reported reflects a complexity of socioeconomic factors: very early age at first marriage and first coitus, instability of first marriage, subsequent divorce and remarriage or drift into prostitution, all of which are influenced by ethnic group, religion and poverty--together with transmission from an infected group of prostitutes by promiscuous males to their wives, lack of diagnostic facilities and inadequate treatment of both symptomatic and asymptomatic men and women. The problem of chlamydial disease in Ethiopia needs to be addressed urgently in the context of control of STD.
PMCID: PMC1194877  PMID: 1398656
22.  Metabolic Features of Protochlamydia amoebophila Elementary Bodies – A Link between Activity and Infectivity in Chlamydiae 
PLoS Pathogens  2013;9(8):e1003553.
The Chlamydiae are a highly successful group of obligate intracellular bacteria, whose members are remarkably diverse, ranging from major pathogens of humans and animals to symbionts of ubiquitous protozoa. While their infective developmental stage, the elementary body (EB), has long been accepted to be completely metabolically inert, it has recently been shown to sustain some activities, including uptake of amino acids and protein biosynthesis. In the current study, we performed an in-depth characterization of the metabolic capabilities of EBs of the amoeba symbiont Protochlamydia amoebophila. A combined metabolomics approach, including fluorescence microscopy-based assays, isotope-ratio mass spectrometry (IRMS), ion cyclotron resonance Fourier transform mass spectrometry (ICR/FT-MS), and ultra-performance liquid chromatography mass spectrometry (UPLC-MS) was conducted, with a particular focus on the central carbon metabolism. In addition, the effect of nutrient deprivation on chlamydial infectivity was analyzed. Our investigations revealed that host-free P. amoebophila EBs maintain respiratory activity and metabolize D-glucose, including substrate uptake as well as host-free synthesis of labeled metabolites and release of labeled CO2 from 13C-labeled D-glucose. The pentose phosphate pathway was identified as major route of D-glucose catabolism and host-independent activity of the tricarboxylic acid (TCA) cycle was observed. Our data strongly suggest anabolic reactions in P. amoebophila EBs and demonstrate that under the applied conditions D-glucose availability is essential to sustain metabolic activity. Replacement of this substrate by L-glucose, a non-metabolizable sugar, led to a rapid decline in the number of infectious particles. Likewise, infectivity of Chlamydia trachomatis, a major human pathogen, also declined more rapidly in the absence of nutrients. Collectively, these findings demonstrate that D-glucose is utilized by P. amoebophila EBs and provide evidence that metabolic activity in the extracellular stage of chlamydiae is of major biological relevance as it is a critical factor affecting maintenance of infectivity.
Author Summary
The Chlamydiae are a group of bacteria that strictly rely on eukaryotic host cells as a niche for intracellular growth. This group includes major pathogens of humans and animals as well as symbionts of protists. Unlike most other bacteria, chlamydiae alternate between two distinct developmental stages. Here we provide novel insights into the infective stage, the elementary body (EB), which has been described almost a century ago and is commonly referred to as an inert spore-like particle. Our analyses of EBs of the amoeba symbiont Protochlamydia amoebophila provide a detailed overview of their metabolism outside of, and independent from, their natural host cells. We demonstrated that these EBs are capable of respiration and are active in the major routes of central carbon metabolism, including glucose import, biosynthetic reactions, and catabolism for energy generation. Glucose starvation resulted in a rapid decline of metabolic activity in P. amoebophila EBs and a concomitant decrease in their potential to infect new host cells. The human pathogen Chlamydia trachomatis was also dependent on nutrient availability for extracellular survival. The extent of metabolic activity in chlamydial EBs and its consequences for infectivity challenge long-standing textbook knowledge and demonstrate that the infective stage is far more dependent on its environment than previously recognized.
PMCID: PMC3738481  PMID: 23950718
23.  In Vitro and In Vivo Functional Activity of Chlamydia MurA, a UDP-N-Acetylglucosamine Enolpyruvyl Transferase Involved in Peptidoglycan Synthesis and Fosfomycin Resistance 
Journal of Bacteriology  2003;185(4):1218-1228.
Organisms of Chlamydia spp. are obligate intracellular, gram-negative bacteria with a dimorphic developmental cycle that takes place entirely within a membrane-bound vacuole termed an inclusion. The chlamydial anomaly refers to the fact that cell wall-active antibiotics inhibit Chlamydia growth and peptidoglycan (PG) synthesis genes are present in the genome, yet there is no biochemical evidence for synthesis of PG. In this work, we undertook a genetics-based approach to reevaluate the chlamydial anomaly by characterizing MurA, a UDP-N-acetylglucosamine enolpyruvyl transferase that catalyzes the first committed step of PG synthesis. The murA gene from Chlamydia trachomatis serovar L2 was cloned and placed under the control of the arabinose-inducible, glucose-repressible ara promoter and transformed into Escherichia coli. After transduction of a lethal ΔmurA mutation into the strain, viability of the E. coli strain became dependent upon expression of the C. trachomatis murA. DNA sequence analysis of murA from C. trachomatis predicted a cysteine-to-aspartate change in a key residue within the active site of MurA. In E. coli, the same mutation has previously been shown to cause resistance to fosfomycin, a potent antibiotic that specifically targets MurA. In vitro activity of the chlamydial MurA was resistant to high levels of fosfomycin. Growth of C. trachomatis was also resistant to fosfomycin. Moreover, fosfomycin resistance was imparted to the E. coli strain expressing the chlamydial murA. Conversion of C. trachomatis elementary bodies to reticulate bodies and cell division are correlated with expression of murA mRNA. mRNA from murB, the second enzymatic reaction in the PG pathway, was also detected during C. trachomatis infection. Our findings, as well as work from other groups, suggest that a functional PG pathway exists in Chlamydia spp. We propose that chlamydial PG is essential for progression through the developmental cycle as well as for cell division. Elucidating the existence of PG in Chlamydia spp. is of significance for the development of novel antibiotics targeting the chlamydial cell wall.
PMCID: PMC142877  PMID: 12562791
24.  Response of Chlamydia trachomatis serovar E to iron restriction in vitro and evidence for iron-regulated chlamydial proteins. 
Infection and Immunity  1997;65(11):4539-4547.
Iron is a well-established mediator of virulence in several bacterial pathogens, yet little is known about the role of iron in infectious disease processes caused by obligate intracellular bacterial pathogens. In this study, the effect of iron limitation was examined for the sexually transmitted infectious agent Chlamydia trachomatis in an in vitro model of human genital infection using the intracellular iron-chelating reagent deferoxamine mesylate (Desferal). Iron restriction caused a significant reduction in infectivity of C. trachomatis elementary bodies (EB) harvested from Desferal-exposed polarized epithelial cells when compared to that of EB harvested from iron-sufficient control cell cultures. Replacement of the Desferal exposure medium with medium containing iron-saturated transferrin restored chlamydial infectivity, whereas replacement with growth medium alone had no effect. The following three prominent morphological features were observed by electron microscopic examination of chlamydia-infected cells exposed to Desferal: (i) inclusions containing chlamydiae greatly delayed in maturation, (ii) substantial blebbing within chlamydial inclusions, and (iii) electron-dense material surrounding inclusions. Protein analyses of highly purified EB by two-dimensional polyacrylamide gel electrophoresis revealed that there were at least 19 candidate iron-repressible proteins in C. trachomatis and at least one protein which was iron inducible. One putative iron-repressible protein was confirmed by Western blot (immunoblot) analysis to be the chlamydial heat shock protein 60 (hsp60). The enhanced production of this antigen by chlamydiae as a result of iron limitation is of particular importance since there is a well-documented association between chlamydial hsp60 and destructive immunopathological sequelae in infected patients.
PMCID: PMC175652  PMID: 9353031
25.  Chlamydia trachomatis Infection Leads to Defined Alterations to the Lipid Droplet Proteome in Epithelial Cells 
PLoS ONE  2015;10(4):e0124630.
The obligate intracellular bacterium Chlamydia trachomatis is a major human pathogen and a main cause of genital and ocular diseases. During its intracellular cycle, C. trachomatis replicates inside a membrane-bound vacuole termed an “inclusion”. Acquisition of lipids (and other nutrients) from the host cell is a critical step in chlamydial replication. Lipid droplets (LD) are ubiquitous, ER-derived neutral lipid-rich storage organelles surrounded by a phospholipids monolayer and associated proteins. Previous studies have shown that LDs accumulate at the periphery of, and eventually translocate into, the chlamydial inclusion. These observations point out to Chlamydia-mediated manipulation of LDs in infected cells, which may impact the function and thereby the protein composition of these organelles. By means of a label-free quantitative mass spectrometry approach we found that the LD proteome is modified in the context of C. trachomatis infection. We determined that LDs isolated from C. trachomatis-infected cells were enriched in proteins related to lipid metabolism, biosynthesis and LD-specific functions. Interestingly, consistent with the observation that LDs intimately associate with the inclusion, a subset of inclusion membrane proteins co-purified with LD protein extracts. Finally, genetic ablation of LDs negatively affected generation of C. trachomatis infectious progeny, consistent with a role for LD biogenesis in optimal chlamydial growth.
PMCID: PMC4409204  PMID: 25909443

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