Information

8.10A: Chlamydiae - Biology


Chlamydiae are a bacterial phylum and class whose members are obligate intracellular pathogens.

Learning Objectives

  • Discuss the evidence that supports Chlamydiae as a unique bacterial evolutionary group

Key Points

  • Chlamydiae replicate inside the host cells and are termed intracellular.
  • Most intracellular chlamydiae are located in an inclusion body or vacuole.
  • Chlamydiae is a unique bacterial evolutionary group that separated from other bacteria approximately a billion years ago. It falls into the clade Planctobacteria in the larger clade Gracilicutes.
  • Chlamydia infection is a common sexually transmitted infection (STI) in humans caused by the bacterium Chlamydia trachomatis.

Key Terms

  • chlamydiae: Chlamydiae is a bacterial phylum and class whose members are obligate intracellular pathogens.
  • inclusion body: Inclusion bodies are nuclear or cytoplasmic aggregates of stainable substances, usually proteins.

Chlamydiae are a bacterial phylum and class whose members are obligate intracellular pathogens. Many chlamydiae coexist in an asymptomatic state within specific hosts. It is widely believed that these hosts provide a natural reservoir for these species. All known chlamydiae only grow by infecting eukaryotic host cells. They are as small or smaller than many viruses.

Chlamydiae replicate inside the host cells and are termed intracellular. Most intracellular chlamydiae are located in an inclusion body or vacuole. Outside of cells they survive only as an extracellular infectious form. Chlamydiae can only grow where their host cells grow. Therefore, chlamydiae cannot be propagated in bacterial culture media in the clinical laboratory. Chlamydiae are most successfully isolated while still inside their host cell.

Chlamydiae is a unique bacterial evolutionary group that separated from other bacteria approximately a billion years ago. Cavalier-Smith has postulated that the Chlamydiae fall into the clade Planctobacteria in the larger clade Gracilicutes. The species from this group can be distinguished from all other bacteria by the presence of conserved indels in a number of proteins such as RNA polymerase alpha subunit, Gyrase B, Elongation factor-Tu and Elongation factor-P, and by large numbers of signature proteins that are uniquely present in different chlamydiae species. Reports have varied as to whether Chlamydiae is related to Planctomycetales or Spirochaetes. However, genome sequencing indicates that 11% of the genes in Candidatus Protochlamydia amoebophila UWE25 and 4% in Chlamydiaceae are most similar to chloroplast, plant, and cyanobacterial genes. Phylogeny and shared presence of conserved indels in proteins such as RNA polymerase Beta subunit and lysyl-tRNA synthetase indicate that Verrucomicrobia are the closest free-living relatives of these parasitic organisms.

There are three described species of chlamydiae that commonly infect humans:

1. Chlamydia trachomatis, which causes the eye-disease trachoma and the sexually transmitted infection chlamydia.

2. Chlamydia pneumoniae, which causes a form of pneumonia.

3. Chlamydia psittaci, which causes psittacosis.

Chlamydia infection is a common sexually transmitted infection (STI) in humans caused by the bacterium Chlamydia trachomatis. The term Chlamydia infection can also refer to infection caused by any species belonging to the bacterial family Chlamydiaceae. C. trachomatis is found only in humans. Chlamydia is a major cause of blindness today, especially in developing countries.

Risk factors include a history of chlamydial or other sexually transmitted infection, new or multiple sexual partners, and inconsistent condom use. trachomatis infection can be effectively cured with antibiotics once it is detected. Current guidelines recommend: azithromycin, doxycycline, erythromycin, or ofloxacin. Agents recommended for pregnant women include erythromycin or amoxicillin.


Evolutionary Cell Biology of Division Mode in the Bacterial Planctomycetes- Verrucomicrobia- Chlamydiae Superphylum

Bacteria from the Planctomycetes, Verrucomicrobia, and Chlamydiae (PVC) superphylum are exceptions to the otherwise dominant mode of division by binary fission, which is based on the interaction between the FtsZ protein and the peptidoglycan (PG) biosynthesis machinery. Some PVC bacteria are deprived of the FtsZ protein and were also thought to lack PG. How these bacteria divide is still one of the major mysteries of microbiology. The presence of PG has recently been revealed in Planctomycetes and Chlamydiae, and proteins related to PG synthesis have been shown to be implicated in the division process in Chlamydiae, providing important insights into PVC mechanisms of division. Here, we review the historical lack of observation of PG in PVC bacteria, its recent detection in two phyla and its involvement in chlamydial cell division. Based on the detection of PG-related proteins in PVC proteomes, we consider the possible evolution of the diverse division mechanisms in these bacteria. We conclude by summarizing what is known and what remains to be understood about the evolutionary cell biology of PVC division modes.

Keywords: PVC superphylum budding cell division dcw cluster ftsZ peptidoglycan.


Evidence for horizontal gene transfer between Chlamydophila pneumoniae and Chlamydia phage

Chlamydia-infecting bacteriophages, members of the Microviridae family, specifically the Gokushovirinae subfamily, are small (4.5-5 kb) single-stranded circles with 8-10 open-reading frames similar to E. coli phage ϕX174. Using sequence information found in GenBank, we examined related genes in Chlamydophila pneumoniae and Chlamydia-infecting bacteriophages. The 5 completely sequenced C. pneumoniae strains contain a gene orthologous to a phage gene annotated as the putative replication initiation protein (PRIP, also called VP4), which is not found in any other members of the Chlamydiaceae family sequenced to date. The C. pneumoniae strain infecting koalas, LPCoLN, in addition contains another region orthologous to phage sequences derived from the minor capsid protein gene, VP3. Phylogenetically, the phage PRIP sequences are more diverse than the bacterial PRIP sequences nevertheless, the bacterial sequences and the phage sequences each cluster together in their own clade. Finally, we found evidence for another Microviridae phage-related gene, the major capsid protein gene, VP1 in a number of other bacterial species and 2 eukaryotes, the woodland strawberry and a nematode. Thus, we find considerable evidence for DNA sequences related to genes found in bacteriophages of the Microviridae family not only in a variety of prokaryotic but also eukaryotic species.

Keywords: gokushovirinae horizontal gene transfer microviridae putative replication initiation protein (PRIP).

Figures

Comparison among Chlamydia bacteriophages. (…

Comparison among Chlamydia bacteriophages. ( A ) Matrix derived from MUSCLE analysis of…

Phylogenetic Analysis of the 11…

Phylogenetic Analysis of the 11 PRIP Proteins. The evolutionary history was inferred by…

Comparison of the 5 C.…

Comparison of the 5 C. pneumoniae strains in the region containing the PRIP…


Perforin-2 Restricts Growth of Chlamydia trachomatis in Macrophages

Fig 1 Perforin-2 inhibits intracellular proliferation of C. trachomatis in macrophages. BV2 cells were transfected with either perforin-2-specific siRNA or scrambled siRNA as a negative control. Cultures were infected with C. trachomatis L2 at an MOI of 0.5 roughly 24 h posttransfection and examined or harvested 24 h postinfection. (A) Cultures were processed for analysis via indirect immunofluorescence assay by probing with anti- Chlamydia antibodies. The arrow indicates the enlarged area in panel A. Bar = 5 μm. (B) Whole-culture material from scrambled-siRNA- or perforin-2 (P-2) siRNA-treated cells was probed via immunoblot assay with anti-perforin-2 or anti-β-actin antibody as a loading control. (C) Data for progeny counts are represented as means ± standard deviations of triplicate samples, and a Student t test was used to assess the statistical significance of differences (** P < 0.002). (D) Data for invasion efficiency are presented as mean percentages of bacteria (n = 100 in triplicate cultures) internalized ± standard deviations. A Student t test indicated no significant difference (NS). Fig 2 Perforin-2 inhibits the growth of multiple Chlamydia serovars and species. BV2 (treated with scrambled siRNA or perforin-2 siRNA) and HeLa cells were infected with the same inocula containing C. muridarum or C. trachomatis serovar L2, D, or B. Cultures were fixed and stained to visualize chlamydial inclusions via indirect immunofluorescence assay (A to C) or harvested for enumeration of progeny chlamydiae (D). (A) Representative immunofluorescence micrographs of Chlamydia inclusions (red) within BV2 cells treated with perforin-2-specific siRNA. Host cell nuclei are also shown (blue). Bar = 5 μm. (B) Inclusion counts during primary infections are reported as the percentages of mature inclusions detected in BV2 cells compared to those in equivalently infected HeLa cells. (C) Areas of representative inclusions were measured and plotted with respective means and standard deviations shown. One-way ANOVA was used to compute the statistical significance of area measurement differences from those of HeLa control cells (****, P < 0.0001 ***, P < 0.0003 **, P < 0.0051). (D) Progeny inclusion counts are reported as the percentages of mature inclusions detected in BV2 cells compared to those in equivalently infected HeLa cells. Fig 3 Electron microscopic evidence of intact inclusions in perforin-2 siRNA-treated cells. BV2 cells were transfected with scrambled siRNA or perforin-2-specific siRNA and infected 24 h later with C. trachomatis L2 at an MOI of 1. HeLa cells were also infected as a control. Cultures were processed for TEM analysis at 24 h postinfection. All images were acquired at a magnification of ×7,900, and representative micrographs are shown for HeLa cells (A) or BV2 cells lacking (B) or containing (C) perforin-2. (C) Arrows indicate typical Chlamydia -containing vacuoles detected in scrambled-siRNA-treated cells. The black arrow indicates the enlarged area shown in panel D. Fig 4 Chlamydia infection induces perforin-2 expression in macrophages. Parallel BV2 cultures were mock infected (M) or infected with C. trachomatis (L2) at an MOI of 1, and whole-culture protein (A) or RNA (B) was harvested at 2, 6, 12, and 24 h postinfection. (A) Endogenous perforin-2 was detected in immunoblot assays with perforin-2-specific antibodies, and IDO and β-actin were visualized as controls. (B) Message levels were evaluated by quantitative PCR of cDNA. The perforin-2 message level was normalized to the GAPDH signal, and n-fold change is relative to a mock-treated control. Student's t test was used to evaluate the statistical significance of differences at each time point between triplicate mock- and Chlamydia -infected samples (*, P < 0.01 **, P < 0.001 ***, P < 0.0001). Fig 5 Perforin-2 expression in C. trachomatis -infected HeLa cells. (A) HeLa cells were mock treated, infected with C. trachomatis (L2) for 24 h, or treated with 10 U/ml IFN-γ or HK chlamydiae for 15 h. Immunoblot assays of whole-culture material were probed with antibodies specific for endogenous perforin-2 (P-2) or β-actin as a loading control. (B) HeLa cells were mock infected or infected at an MOI of 1 with C. trachomatis L2. Cultures were supplemented with 10 U/ml IFN-γ with or without 200 μg/ml chloramphenicol (Cm) at 24 h postinfection. Whole-culture protein was harvested 15 h later. Endogenous perforin-2 was detected in immunoblot assays, and IDO and β-actin were visualized as controls. (C) Quantitative PCR of cDNA was done to determine message levels in cultures treated as described for panel B. The perforin-2 (P2) message level was normalized to the parallel GAPDH signal, and n-fold change is compared to the appropriate mock-infected control. Data are presented as means ± standard deviations of triplicate samples (*, P < 0.01). Fig 6 Ectopic expression of perforin-2 in HeLa cells suppresses chlamydial growth. HeLa cells were transfected with plasmids encoding RFP or RFP–perforin-2 and infected 6 h later with C. trachomatis L2. An inactive version of perforin-2 carrying a K→Q mutation [RFP-P2 (K/Q)] was included as an additional negative control. At 24 h postinfection, parallel cultures were fixed and stained and inclusion areas were calculated (A) or cells were harvested for progeny IFU counting (B). One-way ANOVA was used to compute the statistical significance of area measurement differences from the RFP control (****, P < 0.0001) or progeny count differences from the RFP control (*, P < 0.01). wt, wild type. Fig 7 Perforin-2 localization during chlamydial infection. HeLa cells were transfected with RFP and an inactive version of perforin-2 carrying a K→Q mutation [RFP-P2 (K/Q)] 6 h prior to infection. Parallel cultures were also transfected with perforin-2–RFP (RFP-P2) 6 or 24 h (late) before infection. C. trachomatis L2 was used to infected cells at an MOI of 1, and cultures were fixed and stained with Chlamydia -specific antibodies at 24 h postinfection. RFP-containing proteins (red) are shown with chlamydiae (green), and the rim-like localization of perforin-2–RFP and perforin-2 (K/Q)–RFP is marked by arrows. Bar = 5 μm. MOMP, major outer membrane protein.