FEMS Microbiology Reviews 29 (2005) 949–959
www.fems-microbiology.org
The chlamydial developmental cycle
Yasser M. AbdelRahman, Robert J. Belland
*
Department of Molecular Sciences, University of Tennessee Health Sciences Center, 858 Madison Avenue, Memphis, TN 38163, USA
Received 30 October 2004; received in revised form 9 March 2005; accepted 18 March 2005
First published online 30 April 2005
Abstract
Intracellular parasitism by bacterial pathogens is a complex, multi-factorial process that has been exploited successfully by a wide
variety of organisms. Members of the Order Chlamydiales are obligate intracellular bacteria that are transmitted as metabolically
inactive particles and must differentiate, replicate, and re-differentiate within the host cell to carry out their life cycle. Understanding
the developmental cycle has been greatly advanced by the availability of complete genome sequences, DNA microarrays, and
advanced cell biology techniques. Measuring transcriptional changes throughout the cycle has allowed investigators to determine
the nature of the temporal gene expression changes required for bacterial growth and development.
Ó 2005 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies.
Keywords: Chlamydia; Prokaryotic development; Microarray analysis
Contents
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7.
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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Elementary body . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Attachment and entry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Primary differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Carryover mRNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Early gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Reticulate body . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3. Inclusion modification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4. Type III secretion machinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.5. Cell division . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Secondary differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1. Late gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Alternate growth modes and persistence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Corresponding author. Tel.: +1 901 448 8544; fax: +1 901 448 3330.
E-mail address: [email protected] (R.J. Belland).
0168-6445/$22.00 Ó 2005 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies.
doi:10.1016/j.femsre.2005.03.002
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Y.M. AbdelRahman, R.J. Belland / FEMS Microbiology Reviews 29 (2005) 949–959
1. Introduction
The order Chlamydiales encompasses a large group
of bacteria characterized by their obligate growth in
eukaryotic cells. The last decade has seen a rapid expansion of the number of organisms within the order,
including organisms from numerous animal and environmental sources. This has led to the proposed division
of the family Chlamydiaceae to include two genera,
Chlamydia and Chlamydophila and the inclusion of
non-Chlamydiaceae such as Simkaniaceae, Waddliaceae
and Parachlamydiaceae [1]. The taxonomy of the
Chlamydiales is controversial at this time and many feel
the genera divisions are unnecessary. Since the taxonomic situation is changing rapidly, and is not the subject of this review, we have chosen to use the
terminology described in Schachter et al. [2] i.e. the
use of a single genus, Chlamydia.
Chlamydiaceae are the etiological agents of many
important human and animal diseases. In humans, the
genital serovars of Chlamydia trachomatis are the most
prevalent cause of sexually transmitted disease worldwide
[3], while the ocular serovars result in blinding trachoma
in developing countries [4]. Chlamydia pneumoniae is a
widespread respiratory pathogen [5] and chronic infections are associated with an enhanced risk of developing
atherosclerotic [6,7], cerebrovascular [8], and chronic lung
disease [9]. Pathogenic chlamydial isolates have been
characterized from a number of animal hosts including
birds, cats, rodents, cattle and pigs (e.g., [10]).
The Chlamydiales differ from the other main order of
intracellular bacteria, the Rickettsiales, in that growth is
associated with a biphasic developmental cycle. In fact,
given the diversity associated with host range and disease pathogenesis, the developmental cycle may be seen
as the major unifying feature of the group. The purpose
of this review is to discuss recent advances that have furthered our understanding of the developmental cycle.
For this purpose we have taken a generalist point of
view and include findings from one or more species that
elucidate relevant features of the developmental cycle,
given the caveat that most of these observations have
not been shown for all species members.
Chlamydia alternates between two morphological
forms, the elementary body (EB) and the reticulate body
(RB) [11]. EBs are extra-cellular, metabolically inert
forms, responsible for dissemination of infection by their
ability to attach to and invade susceptible cells. Upon
infection, EBs are internalized in membrane bound vacuoles termed inclusions. EBs differentiate into metabolically active forms, termed RBs, and undergo repeated
cycles of binary fission leading to secondary differentiation back to EBs. The host cell then lyses, releasing
EBs, that infect neighboring cells. Under stressful growth
conditions, imposed by immunological responses, antibiotics, or nutrient deprivation, the developmental cycle is
disrupted, resulting in the appearance of large, aberrant
RBs (reviewed in [12]). This altered growth scheme appears to be associated with continued expression of genes
associated with DNA replication but not with those
genes involved with bacterial cell division.
2. Elementary body
2.1. Morphology
The term ‘‘elementary body’’ (EB) refers to the small
(ca. 0.3 lm), round, electron dense, infectious form of
the organism (e.g., [13,14]). The term, as with other
developmental cycle terms, stems from viral nomenclature historically associated with chlamydial research.
The EB is metabolically inert and has been likened to
a ‘‘spore-like’’ form of the organism. The bacterial
nucleoid is highly compacted in EBs due to the condensation of nuclear material by the bacterial histone-like
proteins HctA and HctB [15,16]. The nucleoid has an
eccentric location in the cell body suggesting an association with the bacterial inner-membrane or cell wall.
Chlamydial EBs are unusual in that little or no peptidoglycan is present in the cell wall. Structural rigidity is
thought instead to be due to the highly cross-linked nature of the outer-membrane complex. Inter and intramolecular cystine bonds exist between the cysteine rich
proteins of the outer envelope including OmpA, OmcB,
and OmcA (reviewed in [17]). In addition, a hexagonally
arrayed protein layer (predominantly OmcB) at the inner surface of the outer-membrane complex is thought
to contribute to cellular stability in EBs.
Electron microscopic examination of EBs using
freeze fracture/freeze etch procedures demonstrated the
presence of hexagonally organized surface projections
arranged regularly with a center to center spacing of
approximately 50 nm [18–20]. These ‘‘supramolecular
structures’’ extend approximately 30 nm from the EB
surface and have a rotational symmetry corresponding
to a 9-subunit composition. Several authors have speculated that these spike-like projections correspond to
Type III secretion system (TTSS) ‘‘needle’’ structures,
similar to those seen in Salmonella enterica serovar
Typhimurium [21]. No conclusive evidence has been
shown to confirm these hypotheses but Fields et al.
[22] have used an indirect argument that strongly supports the presence of TTSS in EBs. They have shown
that inclusion membrane protein genes (inc) are
expressed early in the developmental cycle, much earlier
than the genes encoding structural components of the
TTSS. Furthermore, they showed that secreted Inc protein appeared in the inclusion membrane before TTSS
proteins were produced. Since Inc proteins were TTSS
substrates in heterologous Yersinia systems, they concluded that EBs must have pre-formed TTSS that func-
Y.M. AbdelRahman, R.J. Belland / FEMS Microbiology Reviews 29 (2005) 949–959
tion very rapidly upon the EB–host cell interaction. Further support for the presence and functioning of TTSS
in the initial stages of attachment and entry was provided in a subsequent report that will be discussed below
[23].
2.2. Attachment and entry
As might be expected for an obligate intracellular
parasite, chlamydiae have developed elaborate, and possibly redundant, mechanisms for host cell attachment
and entry (reviewed in [24]). The ability to efficiently infect numerous non-phagocytic cell types from diverse
animal species indicates that the bacterial adhesins involved must recognize conserved cellular receptors.
Despite the central importance of this interaction in bacterial pathogenesis, and the research effort directed towards this problem, no clear consensus exists as to the
nature of the adhesin/receptor molecules involved. Bacterial adhesin candidates include OmpA, OmcB, and
Hsp70 [24]. The polymorphic membrane protein (Pmp)
family of autotransporters has also been suggested to
play a role in attachment [25–27]. This family of related
proteins (21 members in C. pneumoniae) contains highly
repetitive motifs of 4 amino acids (GGAI and FxxN,
[27]) that are found in other bacterial adhesins such as
rOmpA from the obligate intracellular Rickettsia spp.
[25]. Wehrl et al., [28] demonstrated that PmpD of
C. pneumoniae was indeed processed as an autotransporter and that antisera raised to the N-terminal passenger domain blocked chlamydial infectivity.
Recent studies indicate the interaction of EBs with
host cells occurs in a two-stage process. The initial
attachment occurs through electrostatic interactions of
the bacteria with heparan sulfate containing glycosaminoglycans [29–32]. This interaction is reversible and has
been shown to occur through binding to the surface exposed OmpA protein [31], although clear differences can
be found between chlamydial species and serovars. A
second, irreversible, binding stage was demonstrated
using chemically mutagenized cell lines [33] but the
receptor responsible has yet to be identified. Davis
et al. [34] have shown that C. trachomatis serovar E is
closely associated with protein disulfide isomerase
(PDI), a component of the estrogen receptor complex,
during attachment and entry of polarized HEC-1B cells.
Chlamydial attachment was previously shown to be dramatically enhanced in estrogen dominant primary human endometrial epithelial cells [35] and the receptor
complex is located at the apical surface of polarized
cells, an important consideration for entry from the
luminal surface of the genital epithelium. Furthermore,
the authors suggest that the disulfide isomerase activity
of PDI may play a role in the reduction of the highly
disulfide cross-linked EB OMC, a requirement for productive attachment and entry [36].
951
Clifton et al. [23] have recently shown that, immediately following the irreversible binding step, a TTSS exported protein is delivered into the host cell. This
protein, termed Tarp (Translocated actin-recruiting
phosphoprotein, CT456), is rapidly phosphorylated at
tyrosine residues and phosphorylation correlates spatially and temporally with actin recruitment (Fig. 1).
Further evidence for the direct involvement of Tarp
comes from expression studies with a Tarp-EGFP
fusion in which the product expressed in the cell cytoplasm is tyrosine-phosphorylated and recruits actin.
Tyrosine-phosphorylated Tarp is detected on the cytoplasmic face of the cell membrane while the EB is still
extracellular (i.e. prior to chromosome decondensation).
These results indicate that chlamydial EBs have a functional TTSS that delivers important signaling molecules
to the host cell prior to differentiation. The preformed
nature of the TTSS and early effector molecule(s) such
as Tarp, suggests that the expression of important proteins for attachment and entry, and their assembly into
functional complexes, must be part of the secondary differentiation process in which RBs are converted to EBs.
3. Primary differentiation
The chlamydial developmental cycle has a singular
enigmatic stage, the differentiation of the infecting EB
to a metabolically active RB. The process of differentiation can be blocked by the addition of antibiotic inhibitors of transcription or translation, suggesting that de
novo protein expression is required to begin intracellular
growth [37]. This finding is at odds with the generally
held understanding that the presence of the bacterial histone-like proteins HctA and HctB render the EB transcriptionally incompetent. This paradox has been at least
partially addressed by the findings of Greishaber et al.,
[38] Chlamydial histone–DNA interactions are disrupted upon germination by a small metabolite in the
non-mevalonate pathway (MEP) pathway of isoprenoid
biosynthesis. The metabolite is thought to be 2-C-methylerythritol 2,4-cyclodiphosphate and is involved in
functional antagonism of HctA. Findings showed that
an E. coli HctA expression system was rescued from
the lethal effects of HctA by ispE. ispE is an intermediate
enzyme in the MEP pathway, stepwise reconstruction of
this pathway showed convincing evidence that the aforementioned metabolite is the candidate to be the functional antagonist of HctA. Paradoxically, experimental
data adds another question, the E. coli orthologue of
ispE could not protect E. coli from the lethal effects of
HctA expression. The question arises whether it is special enzyme kinetics attributed to chlamydial IspE or
the transcriptional activity of the chlamydial ispE in
the presence of HctA, or another unknown function that
chlamydial ispE has that helps in the rescue from HctA
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Y.M. AbdelRahman, R.J. Belland / FEMS Microbiology Reviews 29 (2005) 949–959
Fig. 1. A schematic representation of the Chlamydial developmental cycle. The host cell cytoplasmic membrane (red line) is shown to depict the
interactions of Chlamydial EBs and the origin of the inclusion membrane. The major events in the developmental cycle are categorized as described in
the text to allow an overview of the processes and mechanisms associated with acute and persistent chlamdyial growth. TARP (Translocated ActinRecruiting Phosphoprotein, CT456), TTSS (Type III Secretion System), MEP (non-MEalonate Pathway), CPAF (Chlamydial Protease/proteasomelike Activity Factor).
lethality. Chlamydia possess another histone-like protein
HctB, it is still unknown what antagonizes its function
during the differentiation step [38].
3.1. Carryover mRNA
The chlamydial EB contains significant quantities of
mRNA and ribosomes, despite its lack of metabolic
activity. Following chromosome decondensation, the
genome rapidly becomes transcriptionally active. During primary differentiation the fate of the so called ‘‘carryover mRNA’’ presents an interesting problem. The
bulk of EB mRNA encodes late gene products, a reflection of the transcriptome during the final stages of the
RB to EB secondary differentiation. For example, transcript levels for the histone-like protein gene hctA are
high, much higher than those found for genes expressed
early in the cycle [39]. Despite the presence of high levels
of mRNA for hctA, there is no expression of the HctA
protein. Indeed the expression of HctA at the start of
the cycle would seem to block new transcriptional activity, in much the same way this occurs late in the cycle.
The newly differentiating chlamydial cell therefore appears to have a mechanism to differentiate newly transcribed mRNA from carryover mRNA such that only
newly synthesized mRNAs are translationally competent. It is unclear at this point how the cell does this.
Speculative hypotheses involve; RNA binding protein(s)
that sequester the carryover mRNA from ribosomes,
carryover mRNA processing that renders the message
translationally inactive (i.e. removal of the 5 0 end,
including translational start signals). Carryover mRNA
is rapidly degraded in the bacterial cell and generally
drops below detectable levels by ca. 6 h post-infection
[39].
3.2. Early gene expression
Transcription begins within the differentiating RB almost immediately following internalization. New pro-
Y.M. AbdelRahman, R.J. Belland / FEMS Microbiology Reviews 29 (2005) 949–959
tein expression can be detected within 15 min PI using
intrinsic labeling procedures [40] and newly synthesized
RNA can be detected in ‘‘host-free’’ chlamydia by 1 h PI
[41]. Wichlan and Hatch [42] identified the first early
gene (euo, Early Upstream ORF) using ‘‘host-free’’ early
RNA to select clones from a genomic library. Transcription of euo was confirmed by Northern blotting and primer extension analysis. Euo appears to be specific to
chlamydia in that it is highly conserved within the genus
but has no homology to other bacterial proteins. Despite its early identification, the function of Euo has
not been clearly defined to date.
Following the sequencing of the serovar D genome, a
number of studies used RT-PCR to identify genes expressed early in the cycle. Shaw et al. [43], identified a
number of inclusion-associated protein genes (inc) as
early genes and proposed the generally accepted temporal classes of developmentally expressed genes as (i)
early (1–2 h PI), (ii) mid-cycle (3–18 h PI), (iii) late
(20–48 h PI). More recently, genome-wide DNA microarrays have been used to determine the transcriptome
throughout the cycle. Belland et al. [39] identified 29
genes expressed by C. trachomatis serovar D at 1 h PI
in HeLa 229 cells. Seven of these genes had been previously characterized as early genes, including the previously mentioned euo and inc genes [42,43]. Other genes
expressed at 1 h PI included genes that encode proteins
involved in (i) translocation of metabolites into the
bacterial cell (e.g., ADP/ATP translocase, nucleotide
phosphate transporter, oligopeptide permease and a
D-alanine/glycine permease), (ii) metabolite interconversions (e.g., malate dehydrogenase, nucleoside phosphohydrolase, and a methionine aminopeptidase), (iii)
inclusion modification (e.g., inc-like genes CT228 and
CT229, and the EEA1-like CT147), and (iv) unknown
functions (all genes were conserved among chlamydial
species). These studies suggest that genes expressed during primary differentiation serve two general purposes
i.e. establishing systems involved in nutrient acquisition
and modifying the parasitophorous vacuole (inclusion)
to prevent its entry into the endocytic pathway leading
to lysosomal fusion.
4. Reticulate body
4.1. Morphology
The ‘‘reticulate body’’ (RB) arises from the internalized EB following primary differentiation, as discussed
above. RBs are larger than EBs (ca. 1 lm) and the cytoplasm appears granular with diffuse, fibrillar nucleic
acids, in contrast with the highly condensed nucleic acid
content of the EB (reviewed in [44]). RBs are non-infectious and are bounded by an inner and outer-membrane,
resembling other, Gram-negative, eubacteria. The sur-
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face of RBs is covered with projections and rosettes that
extend from the bacterial surface through the inclusion
membrane (proposed TTSS needle structures), similar
to those found on EBs but at a higher density [19].
RBs undergo binary fission throughout the middle part
of the developmental cycle. In certain species, the RBs
tend to be closely associated with the inner face of the
inclusion membrane throughout the period of rapid
growth, but this may not apply to all species (i.e.
C. pneumoniae appears to completely fill the interior of
the inclusion and C. caviae tends to grow in an ‘‘articulated’’ form of the inclusion, without a large internal
space [44]).
4.2. Gene expression
Microarray analyses of gene transcription during the
developmental cycle have been reported for C. trachomatis serovar L2 [45] and serovar D [39]. While a number of technical and experimental design differences were
used for the analyses, both reports indicate that by 6–8 h
PI the developing RBs were highly trancriptionally active. Belland et al. [39] classified temporal gene expression groups into early, mid-cycle and late categories
with a subgrouping of genes expressed at 1 h PI as
immediate-early. While this study focused on the immediate-early and late genes, the complete listing of transcriptional activities ([39] and the accompanying
supplementary Table 1) throughout the cycle indicated
that the period of intense transcriptional activity (16–
24 h PI) correlated with the rapid growth and division
of RBs. Furthermore, they reported that virtually every
gene in the organism is expressed at some point in the
cycle (exceptions being CT496.1 and the repressible trp
A and trp B genes), indicating that C. trachomatis has
virtually no facultative capacity, as might be expected
for an obligate intracellular organism with a ‘‘minimal’’
genome. Delayed expression (16 h PI) of genes involved
in certain aspects of cellular metabolism were seen,
including cell division and TTSS assembly (discussed
in more detail below).
Nicholson et al. [45] used a differential display procedure to classify gene expression data in relation to
expression values at 24 h PI, the metabolic and developmental midpoint of the cycle. Expression data were expressed as three developmental stages encompassing 7
temporal clusters, Stages I and II encompassing the
RB phase. Stage I genes were expressed by 6 h PI and
included the (i) early, (ii) constitutive, and (iii) mid-cycle
clusters, according to their expression patterns at later
times in the cycle. The largest group was the constitutive
cluster which comprised genes that were involved in
basic cellular functions including translation (and ribosome assembly), DNA replication, transport-related,
energy compound acquisition and membrane energetics.
Stage II genes were significantly transcribed by 18 h PI
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Y.M. AbdelRahman, R.J. Belland / FEMS Microbiology Reviews 29 (2005) 949–959
and contained a midlate I (remain at 18 h levels through
the remainder of the cycle) and midlate II (levels continue to increase through the remainder of the cycle)
clusters. The midlate I cluster consisted of genes involved in a number of cellular processes including cell
envelope biogenesis, energy metabolism, TTSS, protein
folding, and DNA replication and repair. The midlate
II cluster is comprised of genes that appear to have dual
transcriptional control systems e.g., the tandem promoters of the omp A gene which result in maximal transcription initiation rates at different times in the cycle (giving
an additive effect later in the cycle) [45]. Together these
stage-specific genes lead to the rapid growth of the RB
(Stage III, late genes are discussed below).
4.3. Inclusion modification
Once internalized, chlamydiae actively modify the
properties of the nascent vacuole. Modification of
the inclusion circumvents normal trafficking through
the host endocytic pathway (e.g., [46,47]), effectively dissociating it from late endosomes and lysosomes (reviewed
in [24]). The inability to detect endosomal and lysosomal
markers in the Chlamydial inclusion membrane demonstrates that the inclusion is non-fusogenic with endosomes or lysosomes [48–51]. Instead, through processes
that require the active participation of Chlamydia [37],
chlamydial inclusions intersect a subset of vesicles containing sphingomyelin [52] and cholesterol from the Golgi apparatus [53]. These processes occur early in the
developmental cycle and are necessary for successful replication of Chlamydia within a host cell. The molecular
mechanisms that Chlamydia utilize to control the biogenesis of vacuoles are not known. However, Chlamydial
gene expression is required [37], suggesting that Chlamydial proteins that are secreted into the host cell cytoplasm or incorporated into the inclusion membrane are
likely to be important mediators of these properties.
Within 2 h after entry into host cells, Chlamydial
inclusions are trafficked to the perinuclear region of
the host cell and remain in close proximity to the Golgi
apparatus, where they begin to fuse with a subset of host
vesicles containing sphingomyelin. Chlamydial migration from the cell periphery to the peri-Golgi region
resembles host cell vesicular trafficking. Chlamydia
moves toward the minus end of microtubules and aggregate at the microtubule-organizing center in a process
that is dependent on Chlamydial protein synthesis [54].
Rab GTPases, key regulators of membrane trafficking
were seen to be recruited to Chlamydial inclusion membrane, recruitment was species specific, which may function to regulate trafficking or fusogenic properties of the
inclusion. The absence of Rab5 and the association of
Rab4 and Rab11 with the inclusion membrane demonstrate that inclusions are associated with markers or
endosomal domains that are characteristic of late steps
in the recycling pathway [55]. Recently it was postulated
that chlamydial inclusions intersect the cellular autophagic pathway to acquire nutrients [56]. Using markers for
the autophagy pathway (MAP-LC3, calreticulin), autophagic vesicles were proved to be in close proximity to
the inclusion, but direct fusion between the two vesicles
could not be established. The inclusion of autophagy
inhibitors (3-methyl adenine, several amino acids) in
the growth medium led to aberrant chlamydial growth
[56].
A group of chlamydial proteins (termed Inc) are
found in the inclusion membrane and are thought to
interact with host proteins. The Inc proteins share very
limited amino acid identity among themselves, but share
a common bilobular hydrophobicity motif that is
thought to span the inclusion membrane. Using the
characteristic hydropathy profile, in silico searching
for these proteins led to the prediction of 90 candidates
in C. pneumoniae J138 and 36 in C. trachomatis serovar
D [57]. Inc proteins, were originally identified in C. psittaci, and subsequently found in C. trachomatis [58].
IncA was experimentally proven to be an inclusion
membrane protein by intracellular reactivity of the protein following microinjection of fluorescent antibody
into infected cells [59]. IncD, IncE and IncF, and IncG
were identified in purified inclusion membrane preparations. RT-PCR analysis demonstrated that inc D, E, F,
and G, were transcribed within the first 2 h after internalization, making them candidates for chlamydial factors required for the modification of the nascent
inclusions [60,61].
C. trachomatis IncA plays a role in homotypic fusion
of chlamydial inclusions as microinjection of a-IncA
antibodies inhibits homotypic fusion [61]. C. psittaci
IncA however did not appear to influence homotypic fusion, rather over-expression of IncA in cells infected
with C. psittaci disrupted the development cycle [62].
IncA is also found within fibers that appear as chains
of vesicles extending away from the inclusion along distinct routes into the host cytoplasm [63]. The significance and composition of the IncA-laden fibers is
unknown. IncA was shown to be phosphorylated at serine and/or therionine residues in host cell by unknown
host kinase(s) [59]. Recently, it was shown that IncA
has a characteristic signature of the eukaryotic SNARE
superfamily, where 6 hepta-repeats (called the SNARE
complex) were present in a coiled-coil motif close to
the hydrophobic domain of the protein. SNAREs are
known to be involved in targeting vesicles. Interestingly,
expression of IncA in eukaryotic cells led to retention of
the protein inside the ER, and that over-expression of
the protein inhibits chlamydial development [64].
C. trachomatis IncG was shown to interact specifically with the eukaryotic protein 14-3-3 b using yeast
two-hybrid screening assays and this observation was
confirmed by detection of interaction in the inclusion
Y.M. AbdelRahman, R.J. Belland / FEMS Microbiology Reviews 29 (2005) 949–959
membrane by immunofluorescence microscopy of infected cells [65]. This was the first demonstration of
the interaction of the chlamydial inclusion with a host
protein, although its significance has not been determined. Genomic sequences indicated that C. trachomatis
inc G had no orthologs in C. caviae or C. pneumoniae, in
agreement with the lack of 14-3-3 b association with
bacterial inclusions by these organisms [65]. This raises
interesting questions related to the species-specific interactions with the host that may influence cellular tropism
and disease pathogenesis.
4.4. Type III secretion machinery
Genomic data reveals that all chlamydial species possess a complement of genes encoding type III secretion
system (TTSS) [66–68]. C. pneumoniae was shown to
possess at least 13 genes that are homologous with other
known TTSS systems. TTSS genes in chlamydia show
temporal regulation through out the developmental cycle [22,69,70]. In C. pneumoniae for example; (i) early
genes include yscC, yscS, yscL, yscJ and lcrH-2, (ii)
mid-cycle genes include lcr D, yscN, and yscR, and
(iii) late cycle genes include lcrE, sycE, lcrH-1, and yscT
[70]. C. pneumoniae may use TTSS to translocate different effectors into the host cell, depending on the phase of
the developmental cycle [70]. Several effector proteins
were proved to be substrates for TTSS in heterologous
secretion systems. It was demonstrated that IncA, IncB
and IncC hybrid proteins with cya reporter gene are secreted by TTSS of S. flexneri [71]. In another study to
identify bacterial secreted proteins, immunofluorescence
assays with antisera raised against Cpn0809 and
Cpn1020 showed signals that were distributed within
the host cell rather than inside the inclusions, implying
that these are proteins secreted by the bacteria to modify
host response [72]. The influence of chlamydial infection
on the cells apoptotic machinery is probably controlled
through secreted effectors. The question of whether
infection is pro or anti-apoptotic (or perhaps both at different points in the cycle) is controversial at this point
and the complexity of the arguments prevent their coverage here (readers are referred to a recent review [73]).
4.5. Cell division
Cell division takes place during the RB stage. Genome sequencing indicated that chlamydia lack an identifiable ftsZ ortholog, which encodes a protein centrally
involved in bacterial cell division and found in all other
sequenced eubacteria. Another surprising finding in the
genomic analyses was the presence of a complete set of
genes for the synthesis of peptidoglycan [66,68,74].
Numerous studies had reported that Chlamydia lacked
peptidoglycan, with a single study reporting trace
amounts in EBs [75]. Similarly, attempts to identify pep-
955
tidoglycan in RBs were unsuccessful [76]. Somewhat surprisingly, penicillin and other b-lactams are inhibitory to
chlamydial growth (e.g., [77]) and probably target the
high molecular weight penicillin binding proteins. This
paradoxical situation has been referred to as the ‘‘chlamydial anomaly’’ [78]. RBs may synthesize small
amounts of peptidoglycan that play a role in bacterial
cell division, perhaps by substituting for the lack of FtsZ
in the formation of nascent division septa (e.g., [79]).
McCoy et al. [80] have shown that the chlamydial MurA
ortholog (UDP-N-acetylglucosamine enopyruvyl transferase), which catalyzes the first committed step in peptidoglycan biosynthesis, is functional in E. coli. The
chlamydial MurA was found to encode a fosfomycinresistant form of the enzyme and this resistance was imparted to E. coli expressing the chlamydial enzyme.
Expression studies indicated that chamydia are naturally
fosfomycin resistant and that murA is expressed at the
point in the developmental cycle immediately preceding
cell division [39,80], providing circumstantial evidence
for the involvement of peptidoglycan synthesis in cell
division.
5. Secondary differentiation
5.1. Late gene expression
Following the period of rapid cell division, RBs begin
to redifferentiate to EBs, here termed ‘‘secondary differentiation’’. The signal for this process is unknown and
efforts to identify quorum sensing type pathways are
not been supported by genomic analyses. Speculative
mechanisms have been proposed that involve the dissociation of dividing RBs from the inclusion membrane as
a trigger for secondary differentiation. The removal of
the TTSS system needle apparatus from the inner surface of the inclusion membrane has been suggested as
a critical event in this process [81]. This hypothetical
mechanism has many attractive features, including an
explanation for the asynchronous nature of secondary
differentiation, but much work remains to provide
experimental evidence supporting it.
Expression of a number of late-cycle genes occurs
during secondary differentiation (reviewed in [17]),
including genes that encode components of the outermembrane complex (e.g., OmcA and B) and proteins involved in the condensation of the chromosome (e.g.,
HctA and B). Microarray analyses have extended the
number of genes expressed during this stage of development. Belland et al. [39] identified 26 late genes including
a number of genes previously characterized as late genes
based on other molecular biological studies (e.g., omcAB, hctAB, ltuB, lcrH.1) and a number of new genes
that encode proteins with speculative and unknown
function. Several newly identified late genes encoded
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Y.M. AbdelRahman, R.J. Belland / FEMS Microbiology Reviews 29 (2005) 949–959
proteins with functions predicted to be important in the
formation of the highly disulfide-cross-linked outermembrane complex. These include two predicted thioredoxin disulfide isomerases (CT780 and CT783) and two
membrane thiol proteases (mtpA and mtpB). The membrane thiol proteases share homology with adenoviral
proteases that play a role in the maturation of viral particles [82]. Interestingly, expression of some late cycle
genes may be directed towards the arming of EBs with
proteins necessary during the early stages of the next
infectious cycle (e.g., the TTSS protein LcrH.1), hence
‘‘late’’ genes may encode ‘‘early’’ proteins.
Nicholson et al. [45] identified 70 late genes, classified
as Stage III in that they were not expressed until 24 h PI.
Two clusters were identified in this stage, the late temporal cluster and the very late cluster, the latter consisting of
genes that show increased expression levels between 24
and 36 h PI. The late cluster included previously identified late genes (e.g., omcAB, hctAB, ltuB, lcrH.1), genes
potentially involved in chromosome condensation (e.g.,
CT643 and CT660 in addition to hctAB), and a large
number of genes (31) with unknown function and found
only in chlamydia. The very late temporal cluster consisted of six genes that encode chlamydia-specific proteins
of unknown function, including a gene (CT147) identified
as an immediate-early gene in the Belland et al. study.
6. Alternate growth modes and persistence
Many chlamydial diseases are associated with a long
term or chronic infectious state. In most cases it is difficult to establish whether chronic or recurrent infections
arise through the inability of the host to resolve the
infection or the occurrence of repeated infections with
similar species or genotypes. Despite the unresolved nature of the disease etiology, persistence models of chlamydial infection have been studied to provide insight
into the nature of chronic disease (reviewed in [12]). Persistence is defined as a long-term association between
Chlamydia and their host cell in which these organisms
remain in a viable but culture-negative state. The in vitro persistence systems often share altered Chlamydial
growth characteristics for example, many studies have
described enlarged, and pleomorphic RBs that neither
undergo binary fission, nor differentiate to EBs, but nevertheless continue to replicate their chromosomes. These
changes are generally reversible upon removal of the
growth inhibitory factor [12]. Persistent in vitro infections have been induced by penicillin treatment [83],
amino acid starvation [84], iron deficiency [85], IFN-c
exposure [86], monocyte infection [87], phage infection
[88], continuous culture [89]. This subject has been thoroughly reviewed in Hogan et al. [12].
IFN-c mediated inhibition of intracellular chamydial
replication occurs by depletion of the essential amino
acid tryptophan, via the induction of indoleamine-2,3dioxygenase (IDO) [90]. The effect of IFN-c on
Chlamydial infection could be reversed by addition of
tryptophan [91]. Belland et al. have studied the induction
of persistence with IFN-c and the subsequent reactivation, using microarray analysis. Persistent growth, characterized by large aberrant RBs, led to the up-regulation
of genes involved in tryptophan utilization, DNA repair
and recombination, phospholipid biosynthesis and
translation. Up-regulation of the repressible trp BA operon [92] confirms the previous observations that IFN-c
treatment reduces intracellular concentrations of tryptophan. In addition, a number of early genes were up-regulated, particularly the euo gene (30-fold increase) which
encodes a DNA-binding protein that has been shown to
bind to a late gene promoter region (i.e. omcAB [93]).
Down-regulation of genes involved in RB to EB differentiation (late genes such as hctAB and omcAB), proteolysis and peptide transport, and cell division were seen
during persistent growth. The transcriptional analyses
were consistent with the biological properties associated
with aberrant RBs in that cells were blocked in cytokinesis and the developmental cycle was arrested at a point
preceding late gene expression. Removal of IFN-c and
supplementation with added tryptophan led to a rapid
reactivation from persistent growth. During reactivation
the expression differences rapidly returned to control
levels, i.e. euo expression dropped 20-fold in 12 h. The
transcriptional changes in the presence of IFN-c that result in persistent growth appear to constitute a persistence stimulon. This coordinated biological response
appears to have evolved to allow the organism to rapidly
respond to immunological pressure in a manner that allows for a period of resistance followed by rapid recovery after the waning of the host response.
7. Concluding remarks
The chlamydial developmental cycle involves a complex interaction between the pathogen and its obligate
host. This unique, biphasic life cycle requires the coordinate application of biological functions that involve
interactions of protein complexes and membrane structures and is dependent on both physical and metabolic
factors. In particular, the temporal patterns of gene
expression drive the progression of the cellular infection
through the initial stages of primary differentiation, replication, and secondary differentiation. The use of whole
genome microarrays has allowed the identification of
genes involved in these processes. Functional characterization of the components of the process will require
continued molecular and cellular biological studies that
should lead to a greater understanding of the role of the
cycle in chamydial disease and stimulate new therapeutic
approaches to disease treatment.
Y.M. AbdelRahman, R.J. Belland / FEMS Microbiology Reviews 29 (2005) 949–959
Acknowledgments
The authors would like to apologize to authors of
publications that contributed to our understanding of
the developmental cycle that were omitted accidentally
or for space considerations. This work was supported
by NIH grant HL 71735-02.
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