Type III secretion a la ChlamydiaJan Peters1, David P. Wilson2, Garry Myers3, Peter Timms4 and Patrik M. Bavoil1

1 Department of Biomedical Sciences, University of Maryland, Baltimore, MD 21201, USA2 Faculty of Medicine, University of New South Wales, Sydney, NSW 2010, Australia3 The Institute for Genomic Research, Rockville, MD 20850, USA4 Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane 4059, Australia

Opinion TRENDS in Microbiology Vol.15 No.6

Type III secretion (T3S) is a mechanism that is central tothe biology of the Chlamydiaceae and many other patho-gens whose virulence depends on the translocationof toxic effector proteins to cytosolic targets withininfected eukaryotic cells. Biomathematical simulations,using a previously described model of contact-depend-ent, T3S-mediated chlamydial growth and late differen-tiation, suggest that chlamydiae contained in smallnon-fusogenic inclusions will persist. Here, we furtherdiscuss the model in the context of in vitro-persistent,stress-induced aberrantly enlarged forms and of recentstudies using small molecule inhibitors of T3S. A generalmechanism is emerging whereby both early- and mid-cycle T3S-mediated activities and late T3S inactivationupon detachment of chlamydiae from the inclusionmembrane are crucial for chlamydial intracellular devel-opment.

A family apart: the ChlamydiaceaeThe Chlamydiaceae and other members of the orderChlamydialesy are obligate intracellular bacteria thatinfect a broad spectrum of multicellular organisms in-cluding human, animal and insect species, in addition tounicellular organisms such as free-living amoeba. They arecharacterized by a unique biphasic developmental cyclethat initiates when the infectious, metabolically inertelementary body (EB) attaches to and enters a eukaryotichost cell. Post-internalization, the EB differentiates intothe non-infectious, but metabolically active reticulate body(RB), which replicates by binary fission for several gener-ations within a parasitophorous vacuole, termed theinclusion. Growth and multiplication of the RBs continuefor 18–36 h, depending on the strain. Upon an unidentifiedlate signal(s), RBs differentiate back to EBs through apoorly defined form, variably termed the initial or inter-mediate body (IB).

In addition to inducing their own internalization,chlamydiae interfere with host cell function includingsubversion of the cytoskeleton to facilitate intracellularredistribution of newly internalized EBs [1], early inhi-bition of apoptosis to ensure intracellular survival for theduration of the developmental cycle, and induction of celldeath [2] to release chlamydial progeny upon completion ofthe cycle. The endpoint of chlamydial intracellular devel-opment is the release of newly made EBs associated with

Corresponding author: Bavoil, P.M. (pbavoil@umaryland.edu).y The ‘compromise’ Chlamydiaceae taxonomy of Kalayoglu and Byrne [70] is used in

this article.Available online 7 May 2007.

www.sciencedirect.com 0966-842X/$ – see front matter . Published by Elsevier Ltd. doi:10.10

the death of the infected host cell, which is thought toinvolve an apoptosis-like mechanism [3–5].

RBs that are subjected to stress (e.g. tryptophanstarvation, exposure to antibiotics or phages) cease todivide, although they continue to replicate [6,7] and yieldaberrantly enlarged, multinucleated forms that phenoty-pically resemble stress-induced filamentous forms ofrod-shaped Gram-negative bacteria. Like other stressedbacteria, these aberrant RBs express elevated levels ofstress-response proteins [8] and do not resume normalgrowth until the stressor is removed. (In the case of Chla-mydia, RBs do not undergo late differentiation.) Althoughthese forms are often referred to as persistent chlamydiae,a link with clinically ‘persistent’ infection in humans is stillunproven. Hence, to avoid confusion and by analogy withGram-negative filamentous bacteria (sometimes termed‘maxicells’), stress-induced forms are referred to asmaxi-RBs (mRBs) in this review.

The genetic intractability ofChlamydia hasmade directfunctional analysis of suspected virulence factors prohibi-tively difficult. Moreover, experimental reproducibilityoften suffers from systematic contamination with ‘variant’chlamydiae and bacterial or eukaryotic debris becauseclonal isolation and purification of these obligate intra-cellular organisms is difficult. Because of these limitations,Chlamydia researchers have resorted to alternativeapproaches such as comparative analyses with bettercharacterized systems, and the use of surrogate systemswhenever possible. For example, comparative analysis ofhighly conserved genomes – a ‘poor-man’s’ genetic systemin Chlamydia research – has led to the identification oftryptophan synthase as a key determinant of organ trop-ism in Chlamydia trachomatis infection [9].

Here, we examine the genetic organization and proteinmachinery of one of the better-studied pathways of Chla-mydia sp., the type III secretion (T3S) system, which med-iates the translocation of bacterial toxins to the cytosol ofinfected cells in several important Gram-negative bacterialpathogens. Based on early ultra-structural observations,wepreviously proposed the T3S-mediated contact-dependenthypothesis, whereby chlamydiae replicate strictly incontact with the inclusion membrane while detachmentand coupled T3S inactivation constitute the signal for latedifferentiation. We have now expanded this hypothesisthrough biomathematical simulations that predict persist-ence of chlamydiae under conditions where multipleinclusions are formed in a single cell. This is discussed inthe context of the chlamydial response to stress and inhi-bition of T3S.

16/j.tim.2007.04.005

242 Opinion TRENDS in Microbiology Vol.15 No.6

Type III secretion of a different typeT3S, which facilitates the direct translocation of bacterialvirulence factors to the cytosol of the target eukaryotic cell[10] (vir-T3S), has been described in major human patho-gens such as those from the genera Yersinia, Salmonellaand Shigella, in Pseudomonas aeruginosa and pathogenicEscherichia coli, in the Chlamydiaceae and in bacteriathat infect plants [11,12]. Recently, vir-T3S genes havealso been identified in the genomes of environmentalChlamydia species: Candidatus ‘Protochlamydia amoe-bophila’ [13] and Simkania negevensis (G. Myers et al.,unpublished), both of which grow in amoeba [13,14]. Insome pathogens, such as Salmonella enterica serovarTyphimurium (S. Typhimurium) [15] and Yersinia enter-ocolitica [16], multiple vir-T3S systems encoded withinunlinked pathogenicity islands or plasmids have beendescribed. Although the function of each individual vir-T3S system is not always clear, it is thought that themultiple systems are functionally distinct. The SPI-1system of Salmonella, for example, is important for inva-sion of target cells [17] whereas the unlinked SPI-2 sys-tem is crucial for intracellular growth and survival[15,18].

Figure 1. Multiple T3S gene clusters in the Chlamydiales. T3S gene clusters of Chlamyd

amoebophila’ (Pam) [13] and Simkania negevensis (Sne) (G. Myers et al., unpublished)

Yersinia pestis (Ype) is shown for comparison. Gene names and ORF numbers from the

above and beneath each gene, respectively, when available. Not drawn to scale. Diagram

the figure.

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A genomic T3S pathogenicity ‘pathogenicity

archipelago’

Across the Chlamydiales, genes encoding the structuralproteins of the vir-T3S apparatus are found in three dis-tinct conserved genomic clusters (Figure 1), whereas genesencoding putative translocator proteins and flagellar-associated T3S (fla-T3S) proteins are at unlinked genomicsites in the Chlamydiaceae, and are apparently absent inthe Parachlamydiaceae. The molecular G + C content ofeach chlamydial T3S cluster is close to 40%, similar to therest of the genome, and there are no apparent vestiges ofrecent integration events such as insertion sequenceelements or repeats. This is in contrast to vir-T3S genesof other Gram-negative bacteria, whose clustering in chro-mosomal pathogenicity islands or on plasmids suggeststhat they have been recently acquired by horizontal genetransfer from a heterologous donor [19]. Figure 1 showsthat all Chlamydia sp. have conserved T3S clusters, bothin gene content and genomic location, with the exception oftwo clusters of C. trachomatis and Chlamydia muridarumthat are inverted relative to the origin (ori). A comparisonof chlamydial T3S gene order with that of the T3S plasmid(pCD1) of their closest phylogenetic relative, Yersinia

ia trachomatis (Ctr) [67], Chlamydia caviae (Cca) [68], Candidatus ‘Protochlamydia

are displayed using the cdsN cluster as a reference. The pCD1 T3S gene cluster of

TIGR Comprehensive Microbial Resource database (http://www.tigr.org/) are listed

s showing the plasmid genomic location of each cluster are shown at the bottom of

Opinion TRENDS in Microbiology Vol.15 No.6 243

pestis, reveals loose similarities (Figure 1). However, it isunclear whether T3S genes that appear to be missing inChlamydia relative to Yersinia are truly missing or simplydistantly related, and hence unannotated as T3S homologsin genome sequences. These differencesmight reflect a T3Sinjectisome that is functionally adapted to the develop-mental biology ofChlamydia and the need of this organismto survive host defenses on both sides of the eukaryoticplasma membrane.

What are chlamydial flagellar T3S genes for?

It is not currently known whether contemporary vir-T3Ssystems have evolved from an ancestral flagellar T3S (fla-T3S) system [20] or if both vir- and fla-T3S systems haveevolved independently from a common ancestor [21]. Chla-mydia represents a microcosm of this unresolved questionbecause, in addition to vir-T3S genes, all members of theChlamydiaceae examined to date have a subset of fla-T3Sgenes, even though they are non-motile organisms.

Several fla-T3S genes, annotated as flhA, fliF and fliI(homologs of the contact-dependent secretion (cds) genescdsV, cdsJ and cdsN), are present in two genomic clustersin all genomes of the Chlamydiaceae. These genes are notfound in Protochlamydia or Simkania, suggesting thattheir selective acquisition by the Chlamydiaceae – orloss by the Parachlamydiaceae – might have had a rolein the transition of ancestral chlamydiae from unicellularto multicellular hosts. FlhA, an essential component of theflagellar export apparatus, is normally housed within theFliF basal-bodyMS (membrane and supramembrane) ring,where it also interacts with the flagellar ATPase FliI andits specific inhibitor protein FliH [22]. fliA encodes sigma-28, which is involved in transcriptional regulation of fla-gellar genes in other bacteria and is immediately down-stream of flhA in all chlamydial genomes. Althoughmicroarray and proteomic experiments have indicated thatthese genes are expressed at mid-cycle [23], their functionremains a mystery because they potentially encode only aportion of the flagellar basal body. Do these genes encode asimplified form of a flagellum that provides motility withinthe inclusion? If so, what other gene products compose theputative flagellum? Are the chlamydial flagellar proteinsable to interact with the vir-T3S injectisome? That is, dothey represent a reductive evolution solution to the needfor multifunctional T3S systems? Or do these genes havean entirely novel function that cannot be inferred fromsequence similarity with other systems?

The chlamydial T3S machineAll chlamydial genomes encode multiple conservedproteins of the vir-T3S injectisome, a molecular ‘nano-syringe’ made of �20–25 proteins, the translocator appar-atus and chaperone subclasses, which together arerequired for the assembly and functioning of the T3Spathway [24]. Conserved components of the chlamydialT3S machine are described in Table 1 and representedgraphically in Figure 2.

The chlamydial injectisome

The predicted lipoprotein, CdsJ, is predicted to span theperiplasmic space and associate with integral membrane

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proteins CdsR–V. CdsJ also probably interacts with theinner-membrane protein CdsD, which has also beendetected at the surface of C. trachomatis EBs (ORF664in Tanzer et al. [25]). The highly conserved N terminus ofCdsV displays seven predicted transmembrane domains,while the large C-terminal region is less conserved, morehydrophilic and is predicted to be localized in the cyto-plasm where it might interact with effector proteins,chaperones or other T3S apparatus proteins. The outer-membrane ring of the injectisome, which is necessary forthe T3S needle to cross the outermembrane, is composed ofCdsC, a homolog of YscC of Yersinia. In other systems, thering forms hexameric structures similar to the ‘rosette-like’structures observed by Matsumoto [26]. The innerdiameter of the chlamydial rosettes, estimated at4–5 nm, is similar to the inner diameter of the Yersiniaouter ring at 4.5 nm [27] and close to that of theSalmonellaouter ring at 7 nm [28].

The translocator proteins

The Chlamydia outer protein CopB and its paralog CopB2of C. trachomatis are homologs of the Yersinia T3S trans-locator protein YopB and, as such, are predicted to functionas the entry point for the T3S needle and to facilitatetranslocation of secreted effectors across the plasma mem-brane of the eukaryotic host cell. CopB is detectable in theinclusion membrane after infection, consistent with itspresumed function as a T3S translocator. By contrast,CopB2 is detected in the host cell cytosol [29], possiblyreflecting a function for chlamydial translocator proteinsdistinct from that in other species, where only one copy ofthe gene is present. Similar to enteric T3S, a single CopBhomolog is also found in Protochlamydia.

Effector proteinsIn contrast to apparatus components, T3S effectors dis-play little sequence homology, although they often dis-play common structural features and have similarenzymatic activities across bacterial genera. In othersystems, effectors harbor a variety of toxic effects rangingfrom cytoskeletal alterations, subversion of signal-trans-duction pathways and repression or activation of apop-tosis; they can also disrupt host transcriptionalregulation [12,30]. By analogy with other systems, effec-tor proteins are either secreted into the inclusion lumen(potentially to attack the host cell through receptors atthe surface of the inclusion membrane), are depositedin the inclusion membrane, or are translocated directly tothe cytosol of the host cell.

Attempts to identify T3S effector proteins of theChlamydiaceae have had varied success. Supportive evi-dence includes direct sequence or secondary structuresimilarity with T3S effectors of other species and, possibly,linkage to or co-precipitation with other T3S orthologs[29,31]. A more reliable indicator is usually the demon-stration of T3S-mediated secretion or translocation of acandidate effector by a surrogate host bacterium [29,32–35]. An extension of this strategy to the testing ofT3S-dependent secretion by Shigella flexneri of hybridproteins composed of a predicted T3S signal sequencefused to adenylate cyclase identified 24 new candidate

Table 1. Components of the chlamydial T3S machine and secreted effectors

Component InterProa accession

no.

Notes related to predicted structure/function

CdsC IPR004846 Component of outer membrane ring (T2S/T3S system protein); forms hexameric structures similar to

‘rosette-like’ structures observed by Matsumoto [53–55].

CdsD IPR012843 Integral inner membrane ring protein; �400 residues longer than YscD; cytoplasmic N terminus

includes FHA domain, possibly a receptor for phosphorylated chaperone-effector complexes; detected

at the EB surface [25].

CdsJ IPR006182 Predicted lipoprotein; spans the periplasmic space.

CdsL IPR012842 ATPase inhibitor.

CdsN IPR005714 ATPase.

CdsQ IPR001543 Basal body protein; required for structural assembly; homolog of motor-switch protein of fla-T3S.

CdsR IPR005773 Integral IM protein with multiple transmembrane domains.

CdsS IPR006306 Integral IM protein with multiple transmembrane domains.

CdsT IPR002010 Integral IM protein with multiple transmembrane domains.

CdsU IPR006135 Integral IM protein; by analogy with fla-T3S, associates with CdsJ, CdsN ATPase, and its putative

negative regulator, CdsL [24].

CdsV IPR001712 Integral IM protein; belongs to the Flagellar/Hr/Invasion Protein Export Pore (FHIPEP) protein family;

highly conserved N terminus has 6–8 predicted transmembrane domains; large, less conserved,

hydrophilic C terminus, predicted in cytoplasm where it might interact with other T3S proteins.

FliF IPR006182 Fla-T3S protein; paralog of CdsJ; predicted basal body MS ring; interacts with FlhA.

FliI IPR005714 Fla-T3S protein; paralog of CdsN; ATPase; interacts with FlhA.

FlhA IPR001712 Fla-T3S protein; paralog of CdsV; predicted to be housed within the MS ring; interacts with FliI.

Scc1 (SycE1)b IPR010261 T3S chaperone.

SycE2 IPR010261 T3S chaperone.

SycE3 IPR010261 T3S chaperone.

Scc2 (SycD)b IPR005415 T3S chaperone.

Scc3 (SycD)b IPR005415 T3S chaperone.

SccB IPR013026 T3S chaperone? (Tetratricopeptide region)

CopB Not assigned Translocator protein [29].

CopB2 Not assigned Translocator protein? [29].

CopD Not assigned Translocator protein [34].

CopD2 Not assigned Translocator protein?

CopN IPR013401 Secreted effector; negative regulator of T3S [34,38].

Pkn5 IPR000719 Secreted effector; Ser/Thr protein kinase [34].

IncA IPR007285 Secreted effector; inclusion membrane protein [36].

IncB Not assigned Secreted effector; inclusion membrane protein [36].

IncC Not assigned Secreted effector; inclusion membrane protein [36,38].

Tarp IPR011443 Secreted effector [35].aInterPro can be accessed at http://www.ebi.ac.uk/interpro/.bScc, specific Chlamydia chaperone, an ortholog of syc, the syc gene product.

244 Opinion TRENDS in Microbiology Vol.15 No.6

effectors [36]. However, this method also identified severalproteins that are not known and/or not likely to besecreted, including orthologs of FliH (CPn0859), a predictedarginine decarboxylase (CPn1032) and a b-lactamase-likemetal-dependent hydrolase (CPn0879) (M. Pallen, personalcommunication). Notwithstanding the questionablereliability of surrogate T3S systems, several candidateeffectors have been identified, the most prominent of whichare briefly discussed in the next section.

Translocated actin-recruiting phosphoprotein (Tarp)

The Tir-like effector protein Tarp is translocated throughT3S by Yersinia pseudotuberculosis and is involved in therecruitment of actin to the C. trachomatis inclusion [35].Like Tir, the translocated receptor for enteropathogenicE. coli [37], Tarp might function as a receptor for an uni-dentified chlamydial intimin analog. Because the T3Smachine is functional early in the cell developmental cycle[38], it is conceivable that Tarp is ‘preloaded’ in the T3Sneedle of the EB so that it can mediate early cytoskeletalchanges during internalization [39]. In addition, Tarp-mediated actin recruitment might build a cytosolic ‘track’for Chlamydia-laden inclusions. Tarp is activated upontyrosine phosphorylation by an unidentified eukaryotickinase [40], as is the case for many other secreted virulence

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factors [41]. Likewise, Tir is phosphorylated by the proteinkinase Fyn [42], amember of the Src kinase family [41]. Theexistence of a Src consensusmotif in Tarp strongly supportsthehypothesis that it is also phosphorylated byaSrckinase.

Inc proteins

Chlamydial T3S effectors include inclusion membraneproteins IncA, IncB and IncC, whose Chlamydia pneumo-niae orthologs are secreted by the S. flexneri T3S system[33]. IncC of C. trachomatis is also demonstrably translo-cated into the cytosol of HeLa cells by the Y. enterocoliticaT3S system [38]. IncA is located on the outer face of theinclusion membrane towards the cytosol and is involved inthe homotypic fusion of multiple inclusions of C. tracho-matis [43,44] but not of sphingomyelin-containing vesicles[45]. IncA also forms long fibers extending from theinclusion that are used as cytosolic tracks mediating theformation of secondary inclusions [46]. Transfection of incAinto eukaryotic cells blocks normal chlamydial develop-ment in these cells [47,48].

CopN

A homolog of the Yersinia T3S regulator YopN, CopN istranslocated inaT3S-dependentmannerbyY. enterocolitica[29,32] andS. typhimurium (SPI-1) [34]. Demonstrated late

Figure 2. Diagram of the chlamydial T3S machine. The putative structure of the chlamydial injectisome is derived by comparison with the Yersinia vir-T3S and Salmonella

fla-T3S apparatus (http://www.genome.jp/kegg/) [24]. Protein names in bold text identify components for which a paralogous fla-T3S protein (in parentheses) is found in the

Chlamydiaceae.

Opinion TRENDS in Microbiology Vol.15 No.6 245

copN expression [49,50] is consistent with CopN beinginvolved both in T3S downregulation and physical shutoffof the injectisome as RBs start differentiating into IBs.Similarly,YopN is thought toblock theT3Schannel througha conformation-dependent interaction with its chaperoneand a cytoplasmic membrane site of the T3S injectisome[51]. Contactwith a susceptible cell (or removal of calcium invitro) is presumed todisrupt this interaction, allowingYopNsecretion and subsequent unblocking of the channel forother Yops. A direct comparison between YopN and CopNis not necessarily justified in view of the phylogenetic dis-tance and biological disparities between Chlamydia andYersinia. However, it is worth noting that similar rolesfor CopN and YopN are still possible because late expressedchlamydial proteins are likely to mediate early events inchlamydial pathogenesis.

The T3S contact-dependent development hypothesisChlamydia in the pre-omic era

Starting in 1973, nearly 20 years before the earliestdescription of the T3S system in Yersinia [52], Matsumotoand colleagues published electron micrographs showing

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rosette-like structures and projections at the surface ofChlamydia psittaci strainMn [26,53–55]. These and similarstructures have since been observed at the surface ofC. trachomatis [56], C. caviae [57], C. muridarum [58] andC. pneumoniae [59] EBs and have become known asMatsu-moto’s projections. In a few of the published micrographs,Matsumoto was able to demonstrate that the projec-tionswereanchored inthecytoplasmicmembrane,extendedthrough the outermembrane of the chlamydiae (Figure 3b),and that a cluster of hexagonally-arrayed projections deli-neated a zone of contact between the bacterium and theplasma-membrane-derived inclusion membrane (Figure 3).Based on their analogies with T3S systems, we previouslyproposed that Matsumoto’s projections were in fact T3Sinjectisomes [60]. This hypothesis, while still requiringimmunochemical, biochemical or genetic confirmation, isconsistent with physiological and structural properties ofT3S injectisomes. However, any model for chlamydial de-velopment that is built on the identity of the T3Smachineryand Matsumoto’s projections, including that discussed inthis article, must be preceded by the caution that it remainsan unverified hypothesis.

Figure 3. RB interaction with the chlamydial inclusion membrane. HeLa 229 cells infected with Chlamydia caviae GPIC were examined by (a) scanning and (b) transmission

electron microscopy. T3S projections (red arrows) are viewed from the cytosolic side of the infected cell extending across the inclusion membrane from underlying RBs (a),

or in cross-section of an RB bound to the inclusion membrane (b) with needle-like structures. In (b), the patch of projections delineates the area of contact between the RB

and the luminal face of the inclusion membrane. Micrographs courtesy of Akira Matsumoto.

246 Opinion TRENDS in Microbiology Vol.15 No.6

The size of the projection patch decreases during

development

Chlamydial surface projections are organized as a regularhexagonally arrayed patch located at one pole of bothdevelopmental forms of the bacterium. Matsumotoobserved that within a patch of C. psittaci strain Mn,the number of projections can vary from as low as 11 inthe smaller patch of the EBs to as many as 83 in the largerpatch of the RBs [54]. We previously hypothesized that thepatch of projections observed on individual chlamydiaerepresents the fixed, imprinted memory of the contact areabetween the chlamydial surface and the inclusion mem-brane and that ‘fixation’ might occur late upon generaloxidation of surface disulfide bonds [60]. Moreover, wespeculated that the decrease in the number of projectionsduring late differentiation signified that the contact areawas being reduced as the number of replicating chlamydiaeprogressively exceeded available space at the inclusionmembrane surface and were being physically ‘squeezedout’. An implication of this was that detachment from theinclusion membrane could represent the signal for latedifferentiation [60,61], echoing a previous suggestion byHackstadt [62]. The proposed identity of T3S injectisomesand surface projections added a new dimension to thishypothesis in that it implied that T3S has an essentialrole in sustaining replication and, conversely, that loss ofT3S activity through loss of contact could be the signal forlate differentiation. The presence of T3S projections on theEB further suggested a potential role for T3S, possiblythrough preloaded effectors, during the initial steps ofinfection.

T3S mediates intracellular development

The involvement of chlamydial T3S in growth anddevelopment is a departure from the role of T3S in otherpathogens where T3S is indispensable for infection butdispensable for growth. This hypothesis for modulation ofgrowth has been shown to be consistent with observationsand preliminary mathematical modeling analyses [63]

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(Boxes 1 and 2). We now revisit and extend the modelto encompass two alternate outcomes of chlamydialgrowth: the development of inclusions that persist invitro and that of multiple inclusions. Essential stepsand presumed effector involvement are shown inFigure 4.

The strength of the model is that it reconciles many oldand new observations and provides a unified concept ofchlamydial growth and differentiation across speciesboundaries. For instance, progressive detachment fromthe inclusion membrane is consistent with the perenniallyobserved lack of developmental synchronicity such thatEBs, IBs and RBs coexist in the late inclusion, even aftercareful synchronization of the initial infection step. Themodel, however, is also noteworthy in that it goes againstthe commonly held belief that chlamydiae are able toreplicate free in the lumen of the inclusion (i.e. out ofcontact with the inclusion membrane). This belief is basedon transmission electronmicroscopy (TEM) observations ofgrowing chlamydial inclusions that have accumulatedduring nearly 40 years. TEM reproducibly generateshigh-contrast images but also reproducibly introduces sys-tematic ‘displacement’ artifacts owing to the harsh dehy-dration conditions, subsequent embedding in a resin andthin sectioning.

In vitro persistence is a ‘consequence’ of the model

The phenomenon known as in vitro persistence, whichresults from exposing chlamydiae to stress, providesstrong indirect support for themodel and for the predictedpersistence of small inclusions (Box 3). StressedmRBs areaberrantly enlarged, typically reside within relativelysmall inclusions and are therefore characterized by thesame physical constraints as normal-sized, ‘persisting’RBs in multiple inclusions, albeit on a different spatialscale.

Whether RBs (or mRBs) are attached or detachedfrom the inclusion membrane is directly linked towhether T3S-mediated translocation of effectors to the

Box 2. Utility and limitations of biomathematical modeling

A specific limitation of biomathematical modeling is the quality and

quantity of available measurements used to inform the develop-

ment of model equations and parameters. In Chlamydia research,

this is a substantial hurdle because the organism presents an

unusual degree of experimental difficulty. For example, the number

of projections over developmental time has only been measured

once in a single Chlamydia species [54]. This result was obtained

long before T3S was discovered and laid nearly forgotten for

20 years. Matsumoto’s data have been used as an estimate of

projection numbers in the model, and the model is calibrated to

these data so that it produces normal developmental time courses

similar to observations. Future improved measurements will con-

tinuously test the model and allow its refinement. This in turn will

provide a quantitative framework against which other measure-

ments can be tested.

This model is also deterministic in nature, not accounting for

general stochasticity, especially for very small numbers of particles.

Future models could incorporate greater geometrical features so

that more specific predictions can be made. With any mathematical

model, care must be taken with the assumptions that underlie it and

they should be made explicit. If an important assumption used in a

model is incorrect, then all subsequent model outcomes will be

biased accordingly. The current model explicitly makes certain

assumptions (such as assuming that RBs replicate only in contact

with the inclusion membrane and that they detach from the

membrane once the number of projections decreases to a threshold

level). The mathematical model is adequate to explore the outcomes

of these assumptions and the model results are, by nature,

implications of this hypothesis. The model is not valid outside

these assumptions.

Mathematical modeling is an underexploited area of biology.

Productive application of mathematics to biological systems

should be evaluated on a case-by-case basis, but the Chlamydia

system lends itself exquisitely to modeling because it is a ‘closed’

system (i.e. most of chlamydial biology occurs within the

physically restricted, measurable space of the infected cell). A

highly important asset of the chlamydial model is that ‘simulations’

that mimic real biological events can be produced easily; for

example, the cases of in vitro persistent aberrantly enlarged mRBs

and multiple inclusions are simulated by altering input parameters

and re-running the computerized model. Mathematical modeling

allows in silico experiments to be run and re-run with different

parameters at no real costs, whereas wet laboratory experiments

are expensive and time-consuming. Modeling can predict specific

relationships and threshold levels that are crucial for development

and can determine sensitivity relationships between outcomes and

experimental conditions. Experimentation can produce results that

modeling can attempt to describe and explain, and then modeling

can inform experimental design and provide experimentally

testable predictions. As has occurred in physics for many decades,

experimentation and modeling theory are highly compatible and

complementary disciplines, but their union in biological applica-

tions is currently underdeveloped and could be used considerably

more.

Box 1. A biomathematical model of chlamydial

development

Can mathematics succeed where genetics has so far failed? The T3S

contact-dependent hypothesis was analyzed through biomathema-

tical modeling [63]. Modeling the hypothesis formalizes its assump-

tions, quantifies it in alignment with experimental observations, and

produces testable predictions of its implications. Biomathematical

modeling can produce outcomes that are the logical conclusion of

several given assumptions; the outcomes might be beyond first-

level intuition but can be explained by the mechanistic model

components and in terms of the underlying assumptions in the

equations. The current hypothesized biological model can be

expressed mathematically by three differential equations, represent-

ing the rate of change in the number of RBs (R), IBs (I) and EBs (E)

over time along the developmental cycle (Figure I).

These equations take into account parameters that are either

known or confidently estimated, such as the volume of space

occupied by detached chlamydiae in an inclusion (V), the average

number of projections post-infection derived from Matsumoto’s

observations ( p(t)) and the doubling time of RBs during exponen-

tial growth (td). The model has been useful to verify the plausibility

of the T3S contact-dependent hypothesis and to predict the

testable implication of persistence according to constrained

geometry. When more experimental data become available, the

model can be further fine-tuned and more complexity can be

introduced.

Figure I. The ordinary differential equations of the biomathematical model that

describe the contact-dependent T3 hypothesis of chlamydial development.

Opinion TRENDS in Microbiology Vol.15 No.6 247

host cytosol is on or off. This leads to the hypothesis thatT3S turn-off on its own (i.e. with or without detachment)might be the signal for late differentiation and thatdetachment merely facilitates the process. Comparativetranscriptomics of normal versus IFN-g-induced in vitro-persistent C. trachomatis indicate that transcription ofT3S genes is unaffected [23], suggesting that mRBscontinue expressing and assembling T3S injectisomesat near-normal rates. The net result should be moreT3S injectisomes per mRB (compared to normal RBs),contributing to tether the mRB to the inclusion mem-brane. The T3S effector CopN is not expressed in thesecells and is only expressed late in normal RBs, coinci-dental with the expression of the cysteine-rich outermembrane protein OmcB (i.e. at the developmental stagewhere the first late developmental forms are usuallyobserved). A chicken-and-egg question then arises: doesCopN provoke RB detachment and coupled T3S shut-off,or is CopN expression a consequence thereof? Recent

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evidence for cytosolic targets of CopN in late infectedcells (J. Peters and P.M. Bavoil, unpublished) favors theformer mechanism, albeit tenuously.

Small chemical inhibitors of T3S inhibit development

Small chemical compounds that belong to a class ofacylated hydrazones of salicylaldehydes have beenrecently shown to specifically inhibit T3S and to alterdevelopment significantly, as demonstrated by a dramaticreduction in the infectious EB yield [64–66]. One suchcompound, INP0010, coincidentally inhibited T3S and de-velopment in C. pneumoniae-infected cells [66]. Two othercompounds, C1 [64] and INP0400 [65] were able to

Figure 4. Graphic representation of T3S-mediated chlamydial development. The diagram highlights the predicted dependence of key stages of chlamydial infection on a

functional T3S system from internalization to the onset of late differentiation. These include (i) the translocation of preloaded Tarp during internalization; (ii) the role of IncA,

mutant IncA* or downregulated IncA in inclusion fusogenicity; (iii) the increased area of contact (and consequent T3S activity) between stress-induced mRBs and the

inclusion membrane; and (iv) the coincidental expression and secretion of CopN during late differentiation. The diagram and time scales are approximate.

248 Opinion TRENDS in Microbiology Vol.15 No.6

block T3S-mediated secretion of IncA of C. trachomatis,resulting in the inhibition of homotypic vesicle fusion andformation of multiple small inclusions. INP0400 inhi-bition, but not C1 inhibition, was coupled with the detach-ment of RBs from the inclusion membrane. An importantquestion then becomes how to reconcile these findingswith our prediction that T3S inactivation provokes latedifferentiation.

A major difference between the predicted late T3Sinactivation upon detachment and inhibition with smallchemical compounds is in the timing. In our model, T3Sinactivation occurs ‘naturally’ upon detachment (i.e. pre-sumably after all T3S effectors have been secreted).Chemical inhibition applied at the onset of infection,by contrast, might target T3S-mediated secretion of earlyand mid-cycle effectors, which are necessary for growth,further development and, presumably, further T3S. Theobservation that compound C1 added late (15 h post-in-fection) still inhibits C. trachomatis development [64]suggests that this inhibitor could alter secretion of a lateeffector. It is also intriguing that different inhibitors

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appear to have opposed activities on systems that arevery similar. For instance, INP0010 inhibits C. pneumo-niae T3S and has no effect on T3S of the closely relatedC. trachomatis but blocks that of the more-distant Yersi-nia pseudotuberculosis [66]. This is indicative of a highlevel of specificity that suggests that secretion of a specificeffector(s), or the effector itself, is the target of theinhibition. The contact-dependent hypothesis developedhere and recent results of small-inhibitor studies areoverall consistent with a model proposed by Wolf andcollaborators, whereby signals governing developmentare transduced back through the T3S apparatus [64] bya specific mid- to late-cycle effector. Failure to transducethese signals or failure to secrete the effector woulddownregulate late T3S expression and/or function result-ing in a developmental block. Although inhibitor studiesare still in their infancy and the actual targets of theseinhibitors are not known, they hold the potential forunraveling some of the most intricate aspects of chlamy-dial biology while providing new avenues for therapeuticintervention.

Box 3. Are small, non-fusogenic inclusions persistent?

The number of inclusions per infected cell varies between species and

within species. Multiple inclusions within a cell can arise from initial

infection by multiple EBs or by inclusion division. Conversely,

multiple inclusions within a cell can fuse to form a single late

inclusion. In Chlamydia trachomatis, inclusion fusogenicity is modu-

lated by the type III secreted protein IncA, whereby inclusions

containing mutant incA do not fuse [43]. Biomathematical simulations

indicate that varying the number (N) of inclusions per cell increases

EB progeny up to N � 5, and decreases thereafter (Figure I).

This is a logical result: as the number of inclusions increases, there

is increased RB surface area in contact with the inclusion membrane

and this facilitates greater growth overall until space becomes

restrictive (only once there are �5 inclusions). Increased numbers of

inclusions implies that the size of each inclusion decreases so that the

available luminal volume for detached RBs is restricted. Therefore,

the T3S contact-dependent model predicts that chlamydial species

that tend to produce multiple inclusions will be characterized by RBs

that do not detach from the inclusion membrane but ‘persist’ as RBs.

Although this has not been investigated systematically, it is well

known that chlamydial species that produce multiple or lobar

inclusions (e.g. C. pneumoniae and many veterinary Chlamydia

species) tend to grow ‘forever’ in vitro and/or are characterized by

inclusions that are tightly packed with RBs devoid of luminal space.

Non-fusogenic strains of C. trachomatis are also more frequently

associated with sub-clinical, ‘persistent’ infection than are their

fusogenic counterparts [69].

Figure I. Histogram of biomathematical simulations of the EB progeny at 48 hours post-infection as a function of the number of inclusions per infected cell.

Opinion TRENDS in Microbiology Vol.15 No.6 249

Concluding remarksThe presence of T3S genes in all Chlamydia speciesexamined to date suggests that T3S is essential to thesurvival of these bacteria. This is a fundamental differ-ence to other pathogens in which T3S can be optionallypresent or where it can be inactivated by mutationwithout penalty to the bacterium. A mathematicalmodel, based on the strict replication of chlamydiae inT3S-mediated contact with the inclusion membrane, pre-dicts that loss of contact and coupled T3S inactivationconstitutes the signal for late differentiation (Box 1).Biomathematical simulations suggest that chlamydiaecontained in multiple small inclusions will persist, asdo stress-induced aberrantly enlarged mRBs. Althoughthis simple model is applicable to Chlamydia acrossspecies boundaries, future refinements based on newfindings (e.g. differential activities of small-moleculeinhibitors of T3S in different species) will introducecomplexity, thus reflecting specific host–pathogen inter-actions that can be further simulated biomathematically.Notwithstanding predictable variations on the theme, the

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model and observations reported here suggest that thefundamental role of T3S in the success of Chlamydia as aparasite might be to modulate its efficient growth anddevelopment inside the host, a role that could overshadowits presumed role in virulence.

AcknowledgementsWe are grateful to Priscilla Wyrick for her critical reading of themanuscript. This work was partially supported by NIH R01 AI51472.

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