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Chlamydial development.

Regulation of the growth cycle.

DNA structure

Histones Hc1 and Hc2

In chlamydial elementary bodies, the DNA is tightly condensed and is bound to two, basic, chlamydial histone-like proteins, Hc1 and Hc2. Hc1 is thought to have a major role in DNA condensation during EB formation, while Hc2 may be involved in stage specific gene expression [Pedersen et al., 1994; 1996]. DNA compaction at the stage of RB to EB conversion seems to be associated with a general silencing of gene expression [Kaul & Wenman, 1998]. There is some evidence that the chlamydial histone proteins may also promote endonuclease activity. Whether there is an interplay between endonucleolytic activity and Hc1-induced superhelicity of DNA remains to be explored. Since the basic histone proteins have a very high affinity for DNA, the question arises how does the observed decondensation of chlamydial DNA occur at the differentiation of elementary bodies to reticulate bodies despite the continued expression of histone. Grieshaber et al., 2004 found that a gene product of CT804, a gene homologous to the enzyme encoded by ychB in E. coli involved in a non-mevalonate pathway of isoprenoid biosynthesis, protects chlamydial nucleoid ghosts prepared by zwittergent extraction from disruption by DNAse.  Fosfidomycin, inhibits this CT804 rescue by blocking isoprenoid intermediates downstream of CT804. For the full story of this elegant work, see: editorial.

SW1-B and MDM2 domains

Chlamydiae are unique among bacteria in having an isolated protein from a mammalian host which is homologous with the mammalian SW1-B and MDM2 domains [Bennett-Lovsey et al., 2002]. Chlamydiae also have a further copy of the SW1-B domain fused to the C-terminus of a DNA topoisomerase. As parasitic bacteria Chlamydia are auxotrophic for foreign genetic material and they probably acquired the SW1-B domain from some mammalian host. The SW1-B domain and the MDM2 p53-binding domain of the MDM2 oncoprotein are distantly homologous but appear to share a common evolutionary origin and functional significance. MDM2 acts as a cellular inhibitor of the p53 tumour suppressor, binding the transactivation domain of p53 via a hydrophobic cleft, downregulating its ability to activate transcription. It is suggested that SW1-B too may interact with an amphipathic helical peptide through a cleft on its surface, perhaps playing a role in the chromatin condensation - decondensation of chlamydial DNA characteristic of chlamydiae but no other group of bacteria [Bennett-Lovsey et al., 2002]. DNA genomic transcriptomics studies [see below] indicate that a DNA topoisomerase fused to SW1-B domain [CT643], a DNA gyrase A paralogue [CT660] and histone like protein 2 [hct2] were all induced 24 hours post infection or later [Nicholson et al., 2003].

[Thanks to Grace Yu, MRC Protein Engineering Laboratory, Cambridge, for drawing MEW's attention to SW1-B].

Transcription model

C. trachomatis doubles its DNA content every 2 to 3 hours during replication, with synthesis beginning between 2 to 4 hours after infection. Three major temporal classes of gene transcript have been identified, notably  as a result of pioneering work by groups in Memphis and Aarhus:

• early transcripts; detected 2 or so hours after infection during the germination of EBs to RBs;
• mid-cycle transcripts; appearing between 6 and 12 hours after infection during the growth and multiplication of RBs, and
• late transcripts, which appear between 12 and 20 h after infection during the differentiation of RBs to EBs.

Collectively, early gene functions appear weighted toward initiation of macromolecular synthesis and the establishment of the intracellular niche by modification of the inclusion membrane. Late gene functions appear to be predominately those associated with the terminal differentiation of RBs back to EBs [Shaw et al., 2000].

Genomic transcriptomic profiling

DNA microarray analysis has been used to analyse the developmental cycle of C. trachomatis l2/434/Bu [Nicholson et al., 2003] and C. trachomatis serovar D/UW3/Cx [Belland et al., 2003]. While it is unlikely that there are major differences in the developmental transcription profiles of C. trachomatis serovars D and L2, there were significant methodological and presentation differences between the two studies. Comparisons are inevitably invidious but this reviewer finds the study of Belland et al., 2003 to be the most informative, particularly because it looked at very early gene transcription one hour post infection. The Belland study, using a multiple array consisting of the 893 predicted open reading frames from the genomic sequence plus the eight open reading frames of the cryptic plasmid, is therefore chosen as the main focus here.

In this study: 

• Immediate-early genes: As early as one hour post infection 29 genes were transcriptionally active (immediate-early genes) at the stage of EB differentiation to RBs and expression of eight of these was confirmed by rtPCR. Prominent and expected among the immediate early genes were those encoding the inclusion proteins incD, E, F, & G, or putative inclusion proteins CT228 & CT 229; [see inclusion proteins], euo [Wichlan & Hatch, 1993] and the heat shock proteins groEL and groES.  Newly identified immediate early gene transcripts included genes translocating metabolites into the bacterial cell (ADP/ATP translocase, nucleotide phosphate transporter, oligopeptide permease, a D-alanine / glycine permease) and enzymes involved in metabolite interconversions (malate dehydrogenase, nucleoside phosphohydrolase, & methionine aminopeptidase). An active transcript of  particular interest was CT147, which encodes a 162 KDalton protein with strong functional homology to the human early endosomal antigen EEA1 involved in endosomal fusion and tethering. In chlamydial infected cells it is suggested that CT147 tethers chlamydial endosomes but  does not permit  their fusion with lysosomes, perhaps because CT147 lacks the Rab5 GTPase binding domain that in the human protein is involved with regulating endosomal fusion. CT147 is located in the membrane of the developing chlamydial endosome / inclusion, is post-translationally modified and may be the molecular basis of the important observation that the chlamydial envelope is capable of blocking fusion of chlamydial endosomes with lysosomes [Eissenberg et al., 1983; Scidmore et al., 2003].
  In the study of Nicholson et al., 2003 the earliest transcripts were measured at 6 hours post infection, when induction of two stage-specific genes were identified, incC and oppA, the latter encoding an oligopeptide binding protein. Interestingly, transcription of CT147 was not detected until very late in the growth cycle.
• Early genes. By 3 hours post infection a further 200 genes (early genes) were active. Virtually all genes were active during the mid portion of the cycle (16 - 24 hours) when RB were actively dividing, indicating that the full genomic capacity is utilised. Nicholson et al., 2003 point out that the majority of type III secretion pathway genes are markedly upregulated at 18 hours post infection apart from late stage transcription of lcrH and yscC. This is consistent with the concept that the type III secretion apparatus may be assembled relatively late in the developmental cycle to arm infectious EB for engagement with the target host cell.
• Late genes. A subset of 28 genes were only expressed late (40 hours) in the developmental cycle when the majority of RBs had differentiated to EBs. As expected these included the histone proteins HctA & HctB that mediate late DNA condensation into chromatin, and the cysteine-rich outer membrane proteins OmcA and OmcB which form a complex with OmpA in the outer membrane. Newly identified late transcripts included CT780 and CT783 encoding thioredoxin disulphide isomerases probably involved in the exchange of disulphide bonds among the cysteine-rich proteins of the outer membrane complex and the membrane  thiol-protease genes mtpA and mtpB with homology to adenoviral C5 proteases and which may play a proteolytic role in the formation of the outer membrane complex.

[Comment: The Belland study provides detailed and novel insights into transcriptional processes during chlamydial development. CT147 and mtpA and mtpB are highly conserved among chlamydiae, suggesting that their functions are important. These genes may have been derived from eukaryotic hosts by lateral gene transfer and then adapted to meet chlamydial requirements. The systematic use of microarrays as here provides a vital  insight into chlamydial development and pathobiology which may be useful for therapeutic and vaccine development and which would otherwise be frustrated by the lack of methods for manipulating chlamydial gene expression in vitro].

Transcriptional controls

Chlamydial growth necessitates the ordered and differential expression of the right genes at the right time for the morphological and functional events that are occurring. Relatively little is known about this process. It was once thought that the formation of elementary bodies from reticulate bodies might be an analogous process to that of spore formation in the common environmental bacterium, Bacillus subtilis. In this bacterium, spore formation is regulated by a complex series of regulatory genes. Transcription of these genes into messenger RNA is determined by a substantial number of sigma factors, which modulate the binding of RNA polymerase to specific gene targets, thereby providing a general mechanism for separately controlling the transcription of different clusters of developmentally regulated genes. Genomic sequencing has shown that C. trachomatis has three different sigma factors [Stephens et al., 1998] involved in the binding of RNA polymerase to achieve the DNA-based transcription of RNA. These are sigma²⁸; sigma⁵⁴ and sigma⁶⁶ and several predicted enhancer binding proteins, such as NifS. Chlamydial sigma⁶⁶ promoters are unlike those of E. coli sigma⁶⁶ . Moreover,  E. coli sigma⁷⁰ promoters are unable to transcribe most chlamydial genes [Wan et al., 2002]. A number of sigma54 promoters have been identified [Mathews & Timms, 2000; Wan et al., 2002] and a consensus sigma⁵⁴ promoter has been proposed [Wan et al., 2002]. Temporal expression of the 6 known sigma⁵⁴ controlled genes occurs in mid cycle closely following that of sigma⁵⁴ itself [Wan et al., 2002]. The sigma⁵⁴ regulated genes are in two distinct clusters, with rpoN, lpxA and nifS expressed at 8 to 10 hours post infection in C. trachomatis L2 and htrA, acpS & CT683 slightly later at 14 - 16 hours post infection. Two of these later genes, htrA and acpS,  may be associated with cell wall synthesis [Wan et al., 2002]. In other bacteria sigma⁵⁴ regulated genes are involved in a wide variety of processes [Melnick, 1993]. However sigma factors and promoter recognition alone are unlikely to be sufficient for all the intricacies of developmental regulation in chlamydiae [Timms & Mathews, 2002]. 

DNA binding proteins which may influence transcription, apart from the histone like proteins, include an integration host factor (IHF) which bound upstream of the cysteine rich protein (crp) operon (imcAB) to increase transcription [Zhong et al., 2001] and EUO (see below) a protein highly expressed early in the chlamydial growth cycle.

Chlamydial codon usage

The "genome hypothesis" notes that triplet nucleic acid codes used to encode the amino acid constituents of proteins tend to differ from organism to organism and also within different regions of the genome. This is particularly true for the nucleotide in position 3 of the triplet codon, and is true of chlamydiae also. The usual explanation for the unequal use of synonymous codons among microorganisms is that it is the result of the mutational biases and natural selection acting at the level of translation.  Romero et al., (2000) showed that the patterns of synonymous codon usage depends on whether the sequence is located on the leading or trailing replication strand of DNA. As with other bacteria, the most highly expressed genes in chlamydiae appear to be located on the leading strand. When genes located on the leading strand alone are compared, there is a difference in codon usage between lowly and highly expressed genes, with natural selection most likely to be active on the latter. It has been suggested that this invokes 'replicational - translational' selection which might be used by chlamydiae as another mechanism with which to control aspects of gene transcription.  Synonymous codon usage is also influenced by the hydropathy of the encoded protein and by the degree of amino Romero et al., 2000 considered that in C. trachomatis the pattern of synonymous codon choices was a complex equilibrium influenced by:

• strand specific mutational biases
• natural selection operating at the levels of replication, transcription and translation
• the hydropathy level of each protein and
• the levels of amino acid conservation.

Translation of chlamydial protein

EUO is one of the earliest proteins to be translated. The protein binds to the AT-rich region upstream of the crp operon (and probably other sites) to inhibit its transcription. A putative consensus sequence for EUO binding has been identified [Zhang et al., 1998]. EUO is a protease which digests the DNA-binding C-terminal portion of Hc1, leading to the decondensation of the characteristic nucleoid of the chlamydial elementary body necessary to initiate other gene transcriptions [Kaul et al., 1997]. EUO therefore plays a critical role in the initiation of the chlamydial developmental cycle. The following table summarises some of the other known key players in the chlamydial developmental cycle. For a general review of the chlamydial proteome see van Dahl et al., 2001; 2002.

Table 1. Molecular events during the chlamydial growth cycle.  Abbreviations used: EB elementary body; RB reticulate body; p.i. is post infection; MOMP  major outer membrane protein; CRP cysteine rich protein; HSP heat shock protein; euo early upstream open reading frame. 

Dynamic environmental responses & HrcA - CIRCE

Chlamydia have seven heat shock-related chaperone proteins (groEL, groES, grpE, danJ, dnaK, groEL' and groEL'').  These proteins are upregulated in response to heat or oxidative stress [See: heat stress proteins] or to gamma interferon and other products of cell mediated immunity [Byrne et al., 2002; See: hsp and cmi]. However a specific sigma factor for regulating heat shock proteins has not been identified [See: Control of gene expression]. However chlamydial HrcA has been demonstrated to be a regulator of chlamydial heat shock gene expression acting in conjunction with a cis-acting DNA element called CIRCE as a repressor-operator pair. HrcA repressed the in vitro transcription of a chlamydial heat shock promoter in a promoter specific manner [Wilson & Tan, 2002].  Gene expression in persistent infection is altered with respect to productive infection [Byrne et al., 2002;  Mathews et al., 1999; Molestina et al., 2002; ] and similar alterations are observed in the synovia of patients with reactive arthritis [Gerard et al., 2002; Villa-Real et al., 2002].

Iron is an essential nutrient for chlamydiae as for other bacteria. Iron limitation causes a substantial alteration in chlamydial protein expression but there is no obvious homologue for the E. coli ferric uptake regulator (Fur) protein, which is a selective repressor of transcription. However, the C. trachomatis genome sequence indicated 5 unassigned open reading frames that would encode proteins with limited sequence homology to Fur. In particular, the protein encoded by ORF CT296 was antigenically cross reactive with Fur. Moreover the chlamydial protein was able to complement Fur activity in a mutant strain of E. coli and also bound to a 19 bp consensus sequence found in promoters of iron-regulated genes in E. coli. This protein was renamed divalent cation-dependent regulator A, encoded by the gene dcrA, the first repressor described for chlamydiae [Wyllie & Raulston, 2001].

Many bacteria regulate their population growth through the use of autoinducers based on 'quorum sensing' elements. Once a threshold level or target population has been reached, various target genes are activated or repressed to bring population growth under control. No such sensing elements have yet been recognised in chlamydiae, although they would be valuable for constraining RB replication within the constrained environment of the inclusion [Timms & Mathews, 2002].

[MEW] June 2004

See also: Histone decondensation

NEXT: Control of gene expression

References

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Belland, R. J., Zhong, G., Crane, D. D., Hogan, D., Sturdevant, D., Sharma, J., Beatty, W. L. & Caldwell, H. D. (2003). Genomic transcriptional profiling of the developmental cycle of Chlamydia trachomatis. Proceedings of the National Academy of Sciences of the U S A. 100, 8478 - 8483.

Byrne, G. I., Ouellette, S. P., Wang, Z., Rao, J. P., Lu, L., Beatty, W. L. & Hudson, A. P. (2001). Chlamydia pneumoniae expresses genes required for DNA replication but not cytokinesis during persistent infection of HEp-2 cells. Infection and Immunity 69, 5423 - 5429. Full article  

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Zhang, L., Howe, M. M. & Hatch, T. P. (2000). Characterization of in vitro DNA binding sites of the EUO protein of Chlamydia psittaci. Infection and Immunity 68, 1337 - 1349. Full article    [Superb, thoughtful study of the binding sites of the early upstream open reading frame (EUO).]

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