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Quantitative monitoring of the Chlamydia trachomatis developmental cycle using GFP-expressing bacteria, microscopy and flow cytometry.

ABSTRACT: Chlamydiae are obligate intracellular bacteria. These pathogens develop inside host cells through a biphasic cycle alternating between two morphologically distinct forms, the infectious elementary body and the replicative reticulate body. Recently, C. trachomatis strains stably expressing fluorescent proteins were obtained. The fluorochromes are expressed during the intracellular growth of the microbe, allowing bacterial visualization by fluorescence microscopy. Whether they are also present in the infectious form, the elementary body, to a detectable level has not been studied. Here, we show that a C. trachomatis strain transformed with a plasmid expressing the green fluorescent protein (GFP) accumulates sufficient quantities of the probe in elementary bodies for detection by microscopy and flow cytometry. Adhesion of single bacteria was detected. The precise kinetics of bacterial entry were determined by microscopy using automated procedures. We show that during the intracellular replication phase, GFP is a convenient read-out for bacterial growth with several advantages over current methods. In particular, infection rates within a non-homogenous cell population are easily quantified. Finally, in spite of their small size, individual elementary bodies are detected by flow cytometers, allowing for direct enumeration of a bacterial preparation. In conclusion, GFP-expressing chlamydiae are suitable to monitor, in a quantitative manner, progression throughout the developmental cycle. This will facilitate the identification of the developmental steps targeted by anti-chlamydial drugs or host factors.

SUBMITTER: Vromman F

PROVIDER: S-EPMC4049595 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Quantitative monitoring of the Chlamydia trachomatis developmental cycle using GFP-expressing bacteria, microscopy and flow cytometry.

Vromman François F Laverrière Marc M Perrinet Stéphanie S Dufour Alexandre A Subtil Agathe A

PloS one 20140609 6

Chlamydiae are obligate intracellular bacteria. These pathogens develop inside host cells through a biphasic cycle alternating between two morphologically distinct forms, the infectious elementary body and the replicative reticulate body. Recently, C. trachomatis strains stably expressing fluorescent proteins were obtained. The fluorochromes are expressed during the intracellular growth of the microbe, allowing bacterial visualization by fluorescence microscopy. Whether they are also present in ...[more]

PMID: 24911516

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Project description:BackgroundChlamydia 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 findingsAddition 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.ConclusionsWe 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.

Project description:Chlamydia trachomatis infects squamous and columnar epithelia at the mucosal surface. Research on gene expression patterns of C. trachomatis has predominantly focused on non-native host cells, with limited data on growth kinetics and gene expression of chlamydia in keratinocytes. Here, we investigated whether early, mid, and late chlamydial genes observed in HeLa cell line studies were co-ordinately regulated at the transcriptional level even in the keratinized cell line model and whether the expression was stage-specific during the developmental cycle. HaCaT cell lines were infected with chlamydia clinical isolates (US151and serovar E) and reference strain (L2 434). Expression of groEL-1, incB, pyk-F, tal, hctA, and omcB genes was conducted with comparative real-time PCR and transcriptional events during the chlamydial developmental cycle using transmission electron microscopy. The relative expression level of each gene and fold difference were calculated using the 2-ΔΔCT method. The expression of groEL-1 and pyk-F genes was highest at 2 hours post-infection (hpi) in the L2 434 and serovar E. The expression of incB gene increased at 2 hpi in L2 434 and serovar E but peaked at 12 hpi in serovar E. L2 434 and US151 had similar tal expression profiles. Increased expression of hctA and omcB genes were found at 2 and 36 hpi in L2 434. Both clinical isolates and reference strains presented the normal chlamydial replication cycle comprising elementary bodies and reticulate bodies within 36 hpi. We show different gene expression patterns between clinical isolates and reference strain during in vitro infection of keratinocytes, with reference strain-inducing consistent expression of genes. These findings confirm that keratinocytes are appropriate cell lines to interrogate cell differentiation, growth kinetics, and gene expression of C. trachomatis infection. Furthermore, more studies with different clinical isolates and genes are needed to better understand the Chlamydial pathogenesis in keratinocytes.

Project description:Chlamydia trachomatis is an obligate intracellular pathogen responsible for ocular and genital infections of significant public health importance. C. trachomatis undergoes a biphasic developmental cycle alternating between two distinct forms: the infectious elementary body (EB), and the replicative but non-infectious reticulate body (RB). The molecular basis for these developmental transitions and the metabolic properties of the EB and RB forms are poorly understood as these bacteria have traditionally been difficult to manipulate through classical genetic approaches. Using two-dimensional liquid chromatography - tandem mass spectrometry (LC/LC-MS/MS) we performed a large-scale, label-free quantitative proteomic analysis of C. trachomatis LGV-L2 EB and RB forms. Additionally, we carried out LC-MS/MS to analyse the membranes of the pathogen-containing vacuole ('inclusion'). We developed a label-free quantification approaches to measure protein abundance in a mixed-proteome background which we applied for EB and RB quantitative analysis. In this manner, we catalogued the relative distribution of > 54% of the predicted proteins in the C. trachomatis LGV-L2 proteome. Proteins required for central metabolism and glucose catabolism were predominant in the EB, whereas proteins associated with protein synthesis, ATP generation and nutrient transport were more abundant in the RB. These findings suggest that the EB is primed for a burst in metabolic activity upon entry, whereas the RB form is geared towards nutrient utilization, a rapid increase in cellular mass, and securing the resources for an impending transition back to the EB form. The most revealing difference between the two forms was the relative deficiency of cytoplasmic factors required for efficient type III secretion (T3S) in the RB stage at 18 h post infection, suggesting a reduced T3S capacity or a low frequency of active T3S apparatus assembled on a 'per organism' basis. Our results show that EB and RB proteomes are streamlined to fulfil their predicted biological functions: maximum infectivity for EBs and replicative capacity for RBs.

Dataset Information

Quantitative monitoring of the Chlamydia trachomatis developmental cycle using GFP-expressing bacteria, microscopy and flow cytometry.

Publications

Quantitative monitoring of the Chlamydia trachomatis developmental cycle using GFP-expressing bacteria, microscopy and flow cytometry.

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