This is the author s version of a work that was submitted/accepted for publication in the following source:

This is the author s version of a work that was submitted/accepted for publication in the following source: Roulis, Eileen, Polkinghorne, Adam, & Timms, Peter (2012) Chlamydia pneumoniae : modern insights into an ancient pathogen. Trends In Microbiology, 21(3), pp. 120-128. This file was downloaded from: http://eprints.qut.edu.au/56637/ c Copyright 2012 Elsevier. Notice: Changes introduced as a result of publishing processes such as copy-editing and formatting may not be reflected in this document. For a definitive version of this work, please refer to the published source: http://dx.doi.org/10.1016/j.tim.2012.10.009

Chlamydia pneumoniae: modern insights into an ancient pathogen Roulis, Eileen., 1 Polkinghorne, Adam., 1 Timms, Peter. 1 1 Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane 4059, Australia Corresponding Author: Timms, P. (p.timms@qut.edu.au) Keywords: Chlamydia pneumoniae, infection, genomics, cell biology, human, zoonosis 1

Chlamydia pneumoniae is an enigmatic human and animal pathogen. Originally discovered in association with acute human respiratory disease, it is now associated with a remarkably wide range of chronic diseases as well as having a cosmopolitan distribution within the animal kingdom. Molecular typing studies suggest that animal strains are ancestral to human strains and that C. pneumoniae crossed from animals to humans as the result of at least one relatively recent zoonotic event. Whole genome analyses appear to support this concept the human strains are highly conserved whilst the single animal strain that has been fully sequenced has a larger genome with several notable differences. When compared to the other, better known chlamydial species that is implicated in human infection, Chlamydia trachomatis, C. pneumoniae demonstrates pertinent differences in its cell biology, development and genome structure. Here we examine the characteristic facets of C. pneumoniae biology, offering insights into the diversity and evolution of this silent and ancient pathogen. The emergence of Chlamydia pneumoniae Chlamydia pneumoniae is a bacterial pathogen and member of the Chlamydiae, a diverse range of obligate intracellular bacteria that includes parasites of amoebae, fish, reptiles, mammals and humans[1]. The key feature shared by all members of this phylum is a biphasic development cycle, unique in the bacterial kingdom, which alternates between a highly condensed, non metabolic extracellular infectious form (the elementary body EB), and an intracellular, transcriptionally active, non infectious form (the reticulate body RB) [2]. While the related pathogen Chlamydia trachomatis is still considered the most clinically important chlamydial disease of humans, thanks to its leading role as an agent of genital 2

tract and ocular infections in humans worldwide [3], the role of C. pneumoniae infections and disease in humans is less clear. C. pneumoniae was initially isolated in 1965 from the eye of a child participating in a trachoma vaccine in Taiwan [4] and was first associated with respiratory disease as Chlamydia psittaci (TWAR) in 1985 when it was identified as the cause of a mild pneumonia epidemic in two geographically separated regions in Finland [5] and subsequently reclassified to C. pneumoniae in 1989 [6]. The precise incidence of C. pneumoniae in community acquired pneumonia (CAP) is unknown, however, it is believed to be around 10% [7]. Although clearly less common than other agents of CAP, studies into the relationship between this pathogen in respiratory diseases have suggested links between C. pneumoniae infection and asthma [8, 9], bronchitis and chronic obstructive pulmonary disease [10], in addition to CAP [11]. A single species with many pathologies Despite originally being identified as an acute respiratory pathogen, it is perhaps surprising to realise that the majority of C. pneumoniae research has focused on the role of this pathogen as a cause of persistent infections in human chronic disease. Initial efforts to do so were based on suspicions that C. pneumoniae may be linked to human cardiovascular disease [12, 13]. Subsequently, C. pneumoniae infection has also been implicated in a plethora of human pathologies, including Alzheimer s [14] and arthritis [15] as well as lung cancer [16]and diabetes [17]. It is pertinent to note that the links between C. pneumoniae and some of these diseases are tenuous, with minimal data to support the association. Not only has there been no 3

demonstration of causal relationship in a subset of these studies, infection is difficult to eradicate with antibiotic treatment and no statistically significant improvement of symptoms from any disease linked to C. pneumoniae infection, other than the respiratory diseases, has been demonstrated following antibiotic treatment [18 20]. The prevalence of C. pneumoniae in diseases with which it has a more concrete connection, such as pneumonia and atherosclerosis, vary widely between studies and detection methods [21]. Additionally, pneumonia caused by C. pneumoniae seems to be sporadic and occurs in discrete epidemics [5, 22, 23]. This contradication between prevalence and its enigmatic presentation in clinical surveys is further discussed in Box 1. Chlamydia pneumoniae in animals not just a human infection Alongside work attempting to piece together the relationship of C. pneumoniae infections and human disease, has been the realisation that this pathogen also infects both domesticated and wild animals. Chlamydiosis in the koala (Phascolarctos cinereus) was first described in 1974 [24] and was later revealed to be caused by two different strains of C. psittaci [25], with the type 1 strain reclassified as C. pneumoniae in 1994 [26]. Evidence for the presence of C. pneumoniae infection in animals other than the koala came with the discovery of C. pneumoniae in horses, frogs and reptiles [27]. As with human disease, animal C. pneumoniae infections manifest as vascular and respiratory pathologies, however conjunctival, genitourinary and systemic disease presentations are not uncommon. The demonstrated link of C. pneumoniae infection in chronic human and animal disease and the apparent genetic synteny of human strains has led to increasing scientific interest over recent years. The key questions, however, of how C. pneumoniae relates to these diseases 4

and why it has such a cosmopolitan host range and tissue presence still remain unanswered. Recent studies into the cell biology and genomics of C. pneumoniae have significantly advanced our understanding of this enigmatic bacterial pathogen as well as raising more questions on its origin and role in disease. In this review, we will present current knowledge on C. pneumoniae genetic diversity and cell biology, as well as highlighting key issues which warrant further investigation. Comparative genomics of Chlamydia pneumoniae: a potential origin in animals The family Chlamydiaceae were thought to have diverged from the environmental Chlamydiae some 700 million years ago, with the last common ancestor of the Chlamydiae having split from the planctobacterial phylum some 2 billion years ago [1]. The Chlamydiaceae have considerably smaller, highly conserved genomes when compared to the environmental and symbiotic Chlamydiae such as Parachlamydia acanthamoebae. 560 genes are conserved amongst all Chlamydiae, equating to approximately half the genome of a member of the Chlamydiaceae, while the remaining genes are genus and family specific [28]. Whole genome sequences of Chlamydia pneumoniae Whole genome sequences of four human C. pneumoniae strains have been available for over 10 years [29 31], and comparisons of these four strains reveal remarkable synteny with essentially 99% identity in their gene content and arrangement. Interestingly, of the 1073 genes in human C. pneumoniae, 186 genes are C. pneumoniae specific, having no homologs in any other organism, including other chlamydial species [30]. 5

Sequencing of the first animal C. pneumoniae strain, koala LPCoLN [32] provided a new perspective on C. pneumoniae infection. Whilst the core genome of the LPCoLN biovar shares high identity with all human strains, the chromosome itself is around 12Kb larger and contains several full length genes which are split or truncated in the human strains. Additionally, LPCoLN has several strain specific genes and an extrachromosomal plasmid which has not been described in human strains [33]. These characteristics provide support for at least one zoonotic transmission of C. pneumoniae from animal to human host at some time in the recent past this is further discussed in Box 2. Nucleotide metabolism C. pneumoniae has some key differences in respect to virulence and pathogenicity genes when compared to other chlamydial species. Perhaps the most important feature of the C. pneumoniae genome is the lack of a tryptophan recovery or biosynthesis pathway, including the trpabcr genes that are present in C. trachomatis, Chlamydia pecorum and Chlamydia caviae [34, 35]. The absence of these genes leaves C. pneumoniae completely reliant on the host tryptophan supply and therefore vulnerable to host immune defences as a result of interferon gamma (IFN ) attack. This immune mediator can eliminate microorganisms via tryptophan depletion through activation of indoleamine 2 3 dioxygenase and inducible nitric oxide synthase [36]. Regardless of the absence of these important synthesis genes, C. pneumoniae is able to survive and circumvent the host immune response, if not through an unknown synthesis pathway then very likely through a scavenger mechanism. One such mechanism may be found in C. pneumoniae gene Cpn1046, phha, which encodes an aromatic amino acid hydroxylase (Aro AAH), the substrates of which are all three aromatic amino acids (phenylalanine, tyrosine and tryptophan) leading to a potential C. pneumoniae 6

amino acid/tryptophan salvage pathway [37]. Additionally, C. pneumoniae encodes genes for purine and pyrimidine salvage as well as a complete biotin synthase pathway [30], which are not present in other chlamydial genomes, providing a mechanism for survival during nutrient withholding by the host. Presence of extrachromosomal elements Six of the nine Chlamydiaceae species have an extrachromosomal plasmid and of the five fully sequenced C. pneumoniae isolates, only one the LPCoLN strain possesses the extrachromosomal plasmid [32]. Chlamydial plasmids vary in size, however all contain eight open reading frames and a high degree of synteny across all species [33]. Additionally, AR39 a human respiratory strain carries the CPAR39 bacteriophage [29]. CPAR39 has been demonstrated to infect not only C. pneumoniae, but also Chlamydia abortus, C. pecorum and C. caviae [38] as well as being transmissible from the AR39 isolate to phage negative C. pneumoniae isolates such as CWL029 and Wein 2 [39]. Integration of a bacteriophage into a common C. pneumoniae ancestor is evidenced by the presence of a 350bp fragment similar to CPAR39 ORF4 within the genomes of all sequenced C. pneumoniae strains [39], with an additional remnant found immediately downstream of this fragment in LPCoLN, bearing 80% identity to the VP3 ORF of CPAR39 and Chp2 (C. psittaci phage 2) [33]. Membrane proteins Differences in the coding regions of membrane proteins make a significant contribution to diversity amongst all chlamydial genomes. The chlamydial major outer membrane protein gene (ompa) encodes a major component of the chlamydial EB membrane [40]. Sequence diversity across the four variable domains (VD) of ompa determines the serovariant or 7

antigenic identity of the infecting moiety, with the number of recognised serovariants varying amongst chlamydial species. Whilst C. pneumoniae is recognised as having a single ompa genotype [41] further studies have demonstrated some genetic variation in VD4 [42] as well as antigenic variation among different strains. The polymorphic membrane proteins (Pmps) are a family of autotransporter proteins found in all chlamydial species and share sequence similarity to the type V secretion system of gram negative bacteria [43]. The number of Pmps per species is variable [30, 44, 45], however all pmp genes tend to cluster in specific regions of the genome [46]. Whole genome comparisons of C. trachomatis serovar D and C. pneumoniae CWL029 demonstrated an additional 187 Kb of sequence in C. pneumoniae, 22% of this extra sequence encoded for an expanded number of Pmp homologs 21 in C. pneumoniae compared to 9 in C. trachomatis [30]. Two rare signature repetitive protein motifs are characteristic of Pmps in Chlamydia: GGA(I,L,V) and FXXN. Genomic analysis of Pmp families between chlamydial species, and in C. pneumoniae, suggests that these genes are subject to host immune pressure, are rapidly evolving and adapting to their specific hosts [33, 47]. Type 3 secretion and effectors An additional indicator of genetic diversity in Chlamydiae comes from the presence of a Type 3 secretion system (T3S). The T3S is believed to have originated as a flagellar protein system [28]. Organization of T3S structural genes is highly conserved across all organisms with a T3S system, possibly as a result of horizontal gene transfer [48]. The Chlamydiae are the only non proteobacterial phylum known to possess a T3S system [49] and genes encoding for the chlamydial T3S and its associated effectors are located in at least four clusters throughout the bacterial chromosome [50]. The location and arrangement of the 8

three clusters of structural genes appears to be conserved in all species, however the arrangement of the effector genes in C. pneumoniae differs remarkably from C. trachomatis. The cluster containing the chlamydial protease like activity factor (CPAF) and cop structural genes is reversed in both read direction and arrangement, as is the cluster containing Tarp (Translocated actin recruiting protein) and crpa effectors. Additionally, C. pneumoniae demonstrates duplication and rearrangement of the Inc effector genes and possesses a single copy of the ca530 effector gene, which is absent from C. trachomatis [50]. Furthermore, the Inc family of T3S effectors is expanded in C. pneumoniae, with 92 Inc proteins compared with 55 for C. trachomatis and 79 in C. caviae [51]. Inc proteins are demonstrated to be involved in chlamydial host cell interactions at the inclusion membrane, and have been shown to interact with host Rab GTPases. As host Rabs are largely responsible for remodelling the Golgi apparatus, it stands to reason that differring interactions of Inc proteins with Rabs contributes to species specific host interactions and nutrient acquisition displayed by members of the Chlamydiae [52]. Host and tissue specificity is not always straightforward Studies seeking to establish a definitive link between sequence and tissue or host tropism in C. pneumoniae are limited. A striking example of genotypic diversity that may correlate with tissue tropism was described in the study relating the copy number of the tyrosine permease genes (tyrp) and either respiratory or cardiovascular pathology in humans [53]. As for host specificity, a study by Mitchell et al. (2010), identified a truncation in the AR39 hypothetical gene CP_1042 which was specific to marsupials, and the lack of the LPCoLN hypothetical gene CPK_ORF00678 in all human isolates [33]. Conversely, studies demonstrating the presence of multiple C. pneumoniae genotypes in human carotid arterial 9

tissues [42], and the similarity of Australian indigenous human C. pneumoniae isolates to the sequenced koala biovar [54], partially contradicts the notion of genetics specifying host or tissue tropism. Whilst whole genome comparisons between human and animal strains of C. pneumoniae have highlighted differences that may explain host adaptations for each of these strains, they have raised important questions concerning the evolution and acquisition of C. pneumoniae strains in humans from animals. A dissection of the unique biology of this intracellular pathogen Chlamydial infection is a complex process, involving both the direct effects of chlamydial proteins as well as Chlamydia driven mechanisms to exploit the host cell machinery. C. pneumoniae infection progresses similarly to other chlamydial species with attachment of the EB to the host cell resulting in internalisation of the pathogen in a host derived vacuole. Differentiation to RBs occurs in this vacuole and the bacterium replicates and divides in this privileged niche. Approximately 72 hours after infection with C. pneumoniae, redifferentiation of the RBs into EBs is complete and the EBs are released [55]. Much of our current knowledge of the process of chlamydial infection and its metabolism in humans has been elucidated from studies in C. trachomatis, however not all of these findings translate directly to C. pneumoniae infection. Transcriptionally, metabolically and morphologically, C. pneumoniae infection exhibits differences to other chlamydial species. In this next section, we examine the key differences in the biology of this enigmatic pathogen that may contribute to the range of diseases and lifestyles of C. pneumoniae. The Chlamydia pneumoniae development cycle 10

Growth and development rates vary amongst chlamydial species, and different strains within a species. In comparing C. pneumoniae and C. trachomatis, the time taken to complete a single development cycle in vitro ranges from 60 96h for C. pneumoniae [56], whilst C. trachomatis genital and lymphogranuloma venereum strains have a much shorter replication time of 36 48h [57]. A comparison of the development of HEp 2 cells infected with human AR39 and koala LPCoLN C. pneumoniae revealed startling differences in pathogen doubling time and inclusion morphology. The LPCoLN strain grew almost twice as fast as the AR39 strain, with larger fusogenic inclusions and by 24 hours, contained up to 45 times more LPCoLN genomes per host cell. The authors suggest that LPCoLN may have a more efficient attachment and uptake mechanism: the genetic differences between human and animal C. pneumoniae strains could translate to a strain specific phenomenon [56]. Alternately, the growth differences between AR39 and LPCoLN could support the zoonotic transmission of C. pneumoniae from animals to humans, with gene loss and slowed development a result of adaptation to the human host. Effect of phage infection on development, and propagation of infectious progeny Phage infection exhibits interesting effects on C. pneumoniae morphology, particularly on RBs and the inclusion. Phage infection causes the secreted proteins CPAF and IncA to accumulate within the inclusion and infected inclusions do not lyse. This indicates that while phage infection utilises C. pneumoniae protein synthesis to its own benefit, C. pneumoniae has the ability to encapsulate infected RBs and prevent premature lysis of the inclusion [58]. Release of the infectious EBs from the host cell has been demonstrated to proceed in two ways a lytic pathway and an extrusion pathway, where the inclusion itself protrudes from the host cell, compartmentalises around EBs which eventually detach propagating infection 11

and leaving the host cell intact [59]. C. pneumoniae has been shown to modulate host cell apoptosis to evade the host immune response, through interference with the TNF, NF B and mitochondrial apoptotic signalling pathways [60 62] so it stands to reason that extrusion of the EB, sparing the host cell, would allow for re infection and asymptomatic, chronic disease to continue. Gene transcription events in Chlamydia pneumoniae Gene transcription throughout the chlamydial replication cycle coincides with developmental events, response to host stimuli and growth requirements. Whilst a majority of genes involved in secretion, nutrient acquisition and energy utilisation are shared amongst chlamydial species, C. pneumoniae transcribes many energy and nutrient acquisition genes differently. Early gene expression involves the internalisation and EB to RB transition the inhibition of membrane proteins and upregulation of nutrient and transcriptional regulators (e.g. ycia and xerc) begins at an earlier point in the development cycle for C. pneumoniae [63, 64]. During the replicative (mid) phase of infection, the highest levels of expression relate to housekeeping genes involved in DNA/RNA synthesis, cell division and energy metabolism [65, 66]. Biotin synthesis and aromatic acid hydroxylase genes, specific to C. pneumoniae, are up regulated during this time and remain up regulated until the completion of the development cycle [37, 64]. Re differentiation of RBs back into EBs is an asynchronous process and begins with removal of the T3S apparatus from the inclusion [67]. In C. pneumoniae, late gene expression starts around 36h PI with peptidoglycan synthesis genes peaking (mura, murb, ftsk) as the bulk of RBs convert to EBs. Very late cycle genes, expressed from 60 72h post infection (PI), encode for a variety of EB mrna transcripts of unknown function, outer membrane protein and histone like genes 12

(hctb) [64, 65]. Genes such as pmp, early citric acid cycle genes succ and sucd [64] and various hypothetical proteins are also highly up regulated at this time [68]. Interestingly, the purine metabolism gene (guab) that is present in some strains of C. pneumoniae, is upregulated in this very late phase. The guaab add cluster has been described as nonfunctional in C. pneumoniae, however the upregulation of this gene at this stage may suggest a possible energy storage mechanism for EBs [52, 64]. A schematic of selected genes involved in various cellular processes is described further in Figure 2. The persistent state Perhaps the most important aspect of C. pneumoniae biology that has been examined relative to our understanding of the relationship between this pathogen and human diseases, has been the observation that C. pneumoniae can enter a viable but culture negative state known as persistence [2, 69]. This state has not yet conclusively been demonstrated in vivo, but is believed to be a major contributor to the intractability of treating chlamydial infections as well as having an involvement in asymptomatic, chronic disease. The persistent state is characterised by enlarged, pleomorphic RBs termed aberrant bodies (AB). Persistence could be considered a suspended state of infection as ABs do not replicate within the inclusion, however chromosomal transcription and replication does occur [69]. Persistence in vitro has been induced by a variety of mechanisms including antibiotic treatment, nutrient/tryptophan starvation, iron restriction, IFN attack and phage infection [2]. Gene regulation in persistence 13

Studies of persistence in C. pneumoniae have shown that just as in infection, C. pneumoniae modulates host metabolic and apoptotic machinery up regulating genes involved with NF B and tyrosine kinase signalling pathways, or down regulating genes involved with host cytoskeletal modifications [70]. The method by which persistence is induced also plays a major role in determining gene regulation in C. pneumoniae. During antibiotic mediated persistence, DNA replication genes are expressed at normal levels, however cytokinetic (ftsk, ftsw) and heat shock proteins appear to be down regulated [65, 71]. On the other hand, in an iron induced persistent model, gene expression of ftsk remains normal [72]. In a model of IFN persistence, genes involved with glycolysis and peptidoglycan synthesis are up regulated, whilst transcription of several T3S genes is altered [64, 73]. Genes involved in the late stages of C. pneumoniae development, the outer membrane protein genes and hypothetical genes present in the EB, remain up regulated, probably to hasten differentiation and development upon removal of IFN [64]. Models of persistence also vary greatly depending on the type of cell infected as illustrated by the divergent effect of iron availability in epithelial and monocytic cells infected with C. pneumoniae, significantly decreasing the number of infected cells and number of inclusions in epithelial cells but not monocytes [36]. Selected genes and their expression profiles demonstrated during differentially induced persistence types are shown in Figure 2. Concluding remarks and future directions The field of C. pneumoniae research has been active since its description as an acute respiratory pathogen in humans in the late 1980 s, and whilst its discovery in chronic diseases such as atherosclerosis, asthma and Alzheimer s has important implications for 14

diagnosis and treatment of these diseases, a solid causal link with any of these pathologies has not yet been established. It is important to note the ubiquity of C. pneumoniae within tissues from the human body, and that once infected, C. pneumoniae easily disseminates from the respiratory system through the circulatory system. Findings of C. pneumoniae in the brain, heart, vasculature and joints seem to support this. C. pneumoniae infection is predominantly asymptomatic, and a single individual can be infected on multiple occasions throughout their life. Oftentimes C. pneumoniae is not tested for upon commencement of a disease, and indeed its association with chronic disease is made after the disease has significantly progressed. If C. pneumoniae infection were definitively linked to its associated chronic diseases, we would expect a much higher incidence of multiple morbidities in a larger percentage of the population so far this has not been demonstrated. Subsequent characterisation of a genetically distinct biovar of C. pneumoniae in animals, as well as the discovery of highly similar strains in human Indigenous Australians raises several questions [54]. What is the transmission route and are there potential reservoirs of C. pneumoniae in wildlife? There is evidence to suggest that C. pneumoniae can survive protracted periods of environmental exposure [74], though how infection could propagate from this state is undetermined. Secondly, how long ago did the initial zoonotic event take place how did the infection disseminate to infect such a large proportion of the human population, and is this high level of infection also present in the animal population? Finally, is there a reason for the apparent genetic clonality of the sequenced human C. pneumoniae strains? Could the clonality of human C. pneumoniae strains simply be a matter of sampling bias? Strains CWL029, AR39 are respiratory strains collected in the 1980 s from the USA, J138 a respiratory strain from Japan isolated in 1994 and TW183 was 15

collected from the eye of a child in Taiwan in 1965. Given that three of the four fully sequenced human strains are respiratory in origin, it is no surprise that the genomes appear to be so similar. Its finding in an ocular isolate from an asymptomatic child also reinforces the notion that C. pneumoniae is ubiquitous in the human population, and whilst it is described in acute and chronic diseases, it is predominantly a silent, asymptomatic infection in the majority of the population. An important question raised here is whether the clonality of the genome in human C. pneumoniae strains illustrates adaptation to the host and if so, how long did this adaptation take and can this be conclusively demonstrated? The apparent differences in the C. pneumoniae genome and biology when compared to C. trachomatis indicate that there may indeed be correlates to host and tissue tropism, however definitive answers are still forthcoming. Construction of a C. pneumoniae pangenome would allow for an in depth analysis of how the genetic diversity of C. pneumoniae strains translate to differences in biology and disease. This can only be achieved if more isolates, from a wider range of hosts, are fully sequenced. A summary of the more immediate questions that need to be answered are outlined in Box 3. As is the epitome of all infectious disease research, prevention is better than cure. Identification of diagnostic markers to characterise tissue tropism and drug targets specific to tissue and disease types would be a result of further whole genome analyses. Ideally, the development of systemic vaccines targeted to infection in both humans and animals would be realised, leading to control of chronic disease and transmission of infection in animals and humans. Research into the comparative genomics and biology of C. pneumoniae is ongoing, with the hopes that the secrets of this silent and ancient pathogen may be unlocked. 16

Glossary EB Elementary body. The infectious, extracellular non metabolic chlamydial form. RB Reticulate body. The non infectious, intracellular metabolic chlamydial form. AB Aberrant body. A viable, but culture negative chlamydial form implicated in chlamydial persistence. PI Post infection. T3S Type 3 secretion. A subset of structural genes and effectors believed to be involved in initial infection and modulation of the host cell response to chlamydial infection. IFN Interferon gamma. An inducible host response shown to play a role in clearance and persistence of chlamydial infection by modulation of host nutrient availability and immune responses. CWL029, AR39, J138, TW 183. Chlamydia pneumoniae strains isolated from human respiratory and ocular samples. LPCoLN. Chlamydia pneumoniae strain isolated from a koala respiratory sample. 17

Box 1 Chlamydia pneumoniae the enigmatic pathogen Atypical pneumonia which can result from a chlamydial infection has been reported in adults and infants since the 1930s. Early work struggled to characterise these infections as Chlamydia due to poor diagnostic tools, however they were eventually recognised and named C. psittaci TWAR [75, 76]. An epidemic of mild pneumonia affecting young adults was detected during a routine radiographic survey in Finland in 1978 [5]. This appears to be the first epidemic attributed to C. pneumoniae, which set the stage for what would be an explosion of research attributing infection with this pathogen to an array of diseases, and eventually, just as many hosts. During the late 1980's and 1990s, C. pneumoniae was frequently detected in association with a range of respiratory infections [10, 77, 78]. Whilst some diagnostic methodologies were clearly more reliable than others [21], these studies nevertheless showed that C. pneumoniae was a widespread pathogen in humans. Adding to this puzzle is the more recent realisation that the worldwide incidence of C. pneumoniae respiratory infections seem to have declined dramatically [79]. Epidemics of pneumonia caused by C. pneumoniae still occur, and they are most often reported within military institutions [22, 23, 80]. One explanation is that the mild presentation of C. pneumoniae respiratory disease results in it being overlooked by clinicians, however this does not account for the apparent decrease in detection of this pathogen in the community [81]. Alternately, this could mean that some level of immunity to C. pneumoniae has developed in humans. The connection between C. pneumoniae and atherosclerosis is perhaps less clear. While a causal link is still uncertain, there is no doubt that C. pneumoniae DNA can be reliably found 18

in coronary (and other) plaque [82 84], and animal models do suggest a causative or exacerbating link [85]. Standardisation of experimental methods and broader patient sampling will undoubtedly paint a more realistic picture of the global prevalence of human C. pneumoniae and its association with respiratory disease but also the myriad of chronic diseases that this enigmatic bacterial pathogen is associated with. 19

Box 2 Just how old is Chlamydia pneumoniae? C. pneumoniae is only a recently characterised bacterium. First described as a novel strain of C. psittaci in 1985, and subsequently renamed in 1989 [5, 6], its description as a cause of CAP in humans, as well as its links to other chronic diseases, appears to suggest it is a newly emerged pathogen. In animals, particularly koalas, strains of C. psittaci which may be identified as C. pneumoniae with today s genetic technology, were described as causative agents of disease as early as 1974 [24]. In fact, Cockram et al. (1981) went so far as to suggest that this strain of C. psittaci might have been a factor in the decline of the koala population, which was noted between 1885 and 1930 [86]. Given the relatively recent discovery of this bacterium in humans, what leads us to believe that C. pneumoniae is an ancient pathogen? A clue to the age of C. pneumoniae lies with its cosmopolitan distribution within the animal kingdom. C. pneumoniae has been described in horses and dogs from Europe, marsupials from Australia, reptiles from the American continents and frogs from Africa, Europe and Australia [27, 87]. Genotyping of C. pneumoniae from horses, koalas, bandicoots and frogs reveal infecting strains with significant differences to the described human strains. SNP analysis performed by Rattei et al. [88] demonstrated that animal strains of C. pneumoniae are evolutionarily basal to human strains, though advised some caution in the interpretation of these findings. In 2009, Myers et al [32] sequenced the whole genome of the koala C. pneumoniae biovar LPCoLN, and in comparing this strain to the four sequenced human strains, demonstrated several regions of apparent diversity between the koala and human strains with approximately 12kbp of extra sequence and several full length complements of human C. pneumoniae genes. Additionally, C. pneumoniae strains from the koala and 20

horse show the presence of an extrachromosomal plasmid [32], which are not present in any of the human C. pneumoniae isolates tested so far. Studies by Mitchell et al. (2010) extended these analyses, demonstrating the presence of two bacteriophage remnants in LPCoLN, which share a high degree of sequence similarity to the AR39 bacteriophage [33]. Additionally, two human isolates from Australian indigenous communities were demonstrated to share similarities with both the sequenced human C. pneumoniae and koala C. pneumoniae strains, with the accompanying notion that C. pneumoniae may have already been firmly established within the indigenous Australian human population prior to European settlement In 1788 [54]. Whilst the exact age of C. pneumoniae in animals cannot as yet be determined, these studies suggest an ancient lineage of C. pneumoniae, originally present in amphibians, having undergone at least two zoonotic events from amphibians to reptiles, reptiles to marsupials/mammals and reptiles to humans (described in Figure 1). 21

Box 3 Outstanding questions 1) What are the genetic relationships between animal C. pneumoniae strains? Do animal strains exhibit the same level of synteny as human C. pneumoniae strains? 2) Does C. pneumoniae genotype plus environment equal disease phenotype? Can this be demonstrated for all C. pneumoniae strains? 3) Are there differences in human and animal C. pneumoniae strains from different geographical regions? What is the biogeographical diversity of these strains? Do they also link to particular diseases? 4) Is C. pneumoniae more widespread in Australian fauna than currently believed? What implication does this have for the long term survival of vulnerable species where C. pneumoniae is endemic? 5) Is anthroponosis possible? Could human handlers of horses and reptiles transmit the infection to animals? How transmissible are the human strains to animals and vice versa? 22

Acknowledgments This work was partly supported by funding from an Australian Government NHMRC grant. We would like to thank Wilhelmina Huston Ph.D for reviewing the manuscript. 23

Figure 1: The zoonotic radiation of C. pneumoniae. C. pneumoniae has recently been identified as a zoonotic pathogen. It has a wide range of host species and appears to have a globally cosmopolitan distribution. Host species include amphibians, reptiles and mammals, including humans [27]. It is believed that amphibians were the original host of C. pneumoniae, which passed directly to reptiles then, perhaps through an unknown intermediate host, to mammals [32]. Recent studies have demonstrated the wide genetic diversity of animal C. pneumoniae strains as compared to human strains, and suggest at least two separate zoonotic transmissions from animals to humans [33, 54]. It is unknown whether humans can transmit C. pneumoniae to animals. 24

Figure 2: Gene expression and chlamydial development A biphasic development cycle characterised by an infectious, non metabolic extracellular form (elementary body) and a non infectious, transcriptionally active intracellular form (reticulate body) is characteristic of the Chlamydiae. Gene expression for nutrient, energy, cell division and membrane proteins coincide with different phases of the development cycle. Gene expression for C. pneumoniae and C. trachomatis is similar in most respects, however several points of difference exist in the expression of energy and transcriptional regulatory genes these are indicated in bold text [63 65, 68, 89]. Additionally, C. pneumoniae expresses several energy, metabolic and cofactor genes which are species and strain specific these are indicated in bold underlined text [64]. Gene expression is altered during persistence, and the mode by which persistence is induced can have a divergent effect on regulation of various genes. The differences in persistent gene expression in C. pneumoniae as well as the inducer (IFN, Pen (penicillin) and Fe (iron limitation) are outlined in the shaded region [64, 71, 73, 90, 91]. 25

1 Horn, M., et al. (2004) Illuminating the evolutionary history of Chlamydiae. Science 304, 728 730 2 Hogan, R.J., et al. (2004) Chlamydial persistence: beyond the biphasic paradigm. Infect Immun 72, 1843 1855 3 World Health Organisation Department of HIV/AIDS (2001) Chlamydia. World Health Organisation http://www.who.int/docstore/hiv/grsti/003.htm 4 Grayston, J.T. (2000) Background and current knowledge of Chlamydia pneumoniae and atherosclerosis. J. Infect. Dis. 181, S402 S410 5 Saikku, P., et al. (1985) An Epidemic of Mild Pneumonia Due to an Unusual Strain of Chlamydia psittaci. J. Infect. Dis. 151, 832 839 6 Grayston, J.T., et al. (1989) Chlamydia pneumoniae SP Nov for Chlamydia SP strain TWAR. Int. J. Syst. Bacteriol. 39, 88 90 7 Teh, B., et al. (2012) Doxycycline vs. macrolides in combination therapy for treatment of community acquired pneumonia. Clin. Microbiol. Infect. 18, E71 E73 8 Hahn, D.L., et al. (2012) Chlamydia pneumoniae Specific IgE Is Prevalent in Asthma and Is Associated with Disease Severity. PLoS One 7 9 Zaitsu, M. (2007) The Development of Asthma In Wheezing Infants with Chlamydia pneumoniae Infection. J. Asthma. 44, 565 568 10 Hahn, D.L., et al. (2002) Chlamydia pneumoniae as a respiratory pathogen. Front. Biosci. 7, E66 E76 11 Kurz, H., et al. (2009) Role of Chlamydophila pneumoniae in Children Hospitalized for Community Acquired Pneumonia in Vienna, Austria. Pediatr. Pulmonol. 44, 873 876 12 Hasan, Z.N. (2011) Association of Chlamydia pneumoniae Serology and Ischemic Stroke. South.Med.J. 104, 319 321 13 Deniset, J.F., et al. (2010) Chlamydophila pneumoniae Infection Leads to Smooth Muscle Cell Proliferation and Thickening in the Coronary Artery without Contributions from a Host Immune Response. Am. J. Pathol. 176, 1028 1037 14 Dreses Werringloer, U., et al. (2009) Initial characterization of Chlamydophila (Chlamydia) pneumoniae cultured from the late onset Alzheimer brain. Int. J. Med. Microbiol. 299, 187 201 15 Carter, J.D. and Hudson, A.P. (2010) The evolving story of Chlamydia induced reactive arthritis. Curr. Opin. Rheumatol. 22, 424 430 16 Zhan, P., et al. (2011) Chlamydia pneumoniae infection and lung cancer risk: A meta analysis. Eur. J. Cancer 47, 742 747 17 Wang, C.M., et al. (2009) Acute Chlamydia pneumoniae Reinfection Accelerates the Development of Insulin Resistance and Diabetes in Obese C57BL/6 Mice. J. Infect. Dis. 200, 279 287 18 Sutherland, E.R., et al. (2010) A trial of clarithromycin for the treatment of suboptimally controlled asthma. J. Allergy Clin. Immunol. 126, 747 753 19 Deniset, J.F. and Pierce, G.N. (2010) Possibilities for therapeutic interventions in disrupting Chlamydophila pneumoniae involvement in atherosclerosis. Fundam. Clin. Pharmacol. 24, 607 617 20 Cannon, C.P., et al. (2005) Antibiotic treatment of Chlamydia pneumoniae after acute coronary syndrome. N. Engl. J. Med. 352, 1646 1654 21 Benitez, A.J., et al. (2012) Comparison of Real Time PCR and a Microimmunofluorescence Serological Assay for Detection of Chlamydophila pneumoniae Infection in an Outbreak Investigation. J. Clin. Microbiol. 50, 151 153 22 Oktem, I.M.A., et al. (2007) PCR and serology were effective for identifying Chlamydophila pneumoniae in a lower respiratory infection outbreak among military recruits. Jpn. J. Infect. Dis. 60, 97 101 23 Sevketbeyoglu, H., et al. (2012) Outbreak of Chlamydia pneumoniae infection in a military unit: The distribution of lobar and segmental infiltration. Nobel Med. 8, 26 31 24 Cockram, F.A. and Jackson, A.R.B. (1974) Isolation of a Chlamydia from cases of keratoconjunctivitis in Koalas. Aust. Vet. J. 50, 82 83 26

25 Girjes, A.A., et al. (1988) 2 distinct forms of Chlamydia psittaci associated with disease and infertility in Phascolarctos cinereus (Koala). Infect. Immun. 56, 1897 1900 26 Girjes, A.A., et al. (1994) Remarkable sequence relatedness in the DNA encoding the Major Outer Membrane Protein of Chlamydia psittaci (Koala Type I) and Chlamydia pneumoniae. Gene 138, 139 142 27 Bodetti, T.J., et al. (2002) Molecular evidence to support the expansion of the hostrange of Chlamydophila pneumoniae to include reptiles as well as humans, horses, koalas and amphibians. Syst. Appl. Microbiol. 25, 146 152 28 Collingro, A., et al. (2011) Unity in Variety The Pan Genome of the Chlamydiae. Mol. Biol. Evol. 28, 3253 3270 29 Read, T.D., et al. (2000) Genome sequences of Chlamydia trachomatis MoPn and Chlamydia pneumoniae AR39. Nucleic Acids Res. 28, 1397 1406 30 Kalman, S., et al. (1999) Comparative genomes of Chlamydia pneumoniae and C. trachomatis. Nature Genet. 21, 385 389 31 Shirai, M., et al. (2000) Comparison of whole genome sequences of Chlamydia pneumoniae J138 from Japan and CWL029 from USA. Nucleic Acids Res. 28, 2311 2314 32 Myers, G.S.A., et al. (2009) Evidence that Human Chlamydia pneumoniae Was Zoonotically Acquired. J. Bacteriol. 191, 7225 7233 33 Mitchell, C.M., et al. (2010) Comparison of koala LPCoLN and human strains of Chlamydia pneumoniae highlights extended genetic diversity in the species. BMC Genomics 11 34 Mojica, S., et al. (2011) Genome Sequence of the Obligate Intracellular Animal Pathogen Chlamydia pecorum E58. J. Bacteriol. 193, 3690 3690 35 Fehlner Gardiner, C., et al. (2002) Molecular basis defining human Chlamydia trachomatis tissue tropism A possible role for tryptophan synthase. J. Biol. Chem. 277, 26893 26903 36 Bellmann Weiler, R., et al. (2010) Divergent modulation of Chlamydia pneumoniae infection cycle in human monocytic and endothelial cells by iron, tryptophan availability and interferon gamma. Immunobiology 215, 842 848 37 Abromaitis, S., et al. (2009) Chlamydia pneumoniae encodes a functional aromatic amino acid hydroxylase. FEMS Immunol. Med. Microbiol. 55, 196 205 38 Everson, J.S., et al. (2003) Host range of chlamydiaphages phi CPAR39 and Chp3. J. Bacteriol. 185, 6490 6492 39 Rupp, J., et al. (2007) Prevalence, genetic conservation and transmissibility of the Chlamydia pneumoniae bacteriophage (phi Cpn1). FEMS Microbiol. Lett. 273, 45 49 40 Bavoil, P., et al. (1984) Role of disulphide bonding in outer membrane structure and permeability in Chlamydia trachomatis. Infect. Immun. 44, 479 485 41 Shirai, M., et al. (2000) Comparison of outer membrane protein genes omp and pmp in the whole genome sequences of Chlamydia pneumoniae isolates from Japan and the United States. J. Infect. Dis. 181, S524 S527 42 Cochrane, M., et al. (2005) Multiple genotypes of Chlamydia pneumoniae identified in human carotid plaque. Microbiology (UK) 151, 2285 2290 43 Henderson, I.R. and Lam, A.C. (2001) Polymorphic proteins of Chlamydia spp. autotransporters beyond the Proteobacteria. Trends Microbiol. 9, 573 578 44 Harley, R., et al. (2007) Molecular characterisation of 12 Chlamydophila felis polymorphic membrane protein genes. Vet. Microbiol. 124, 230 238 45 Tanzer, R.J., et al. (2001) Identification of polymorphic outer membrane proteins of Chlamydia psittaci 6BC. Infect. Immun. 69, 2428 2434 46 Wehrl, W., et al. (2004) From the inside out processing of the Chlamydial autotransporter PmpD and its role in bacterial adhesion and activation of human host cells. Mol. Microbiol. 51, 319 334 47 Voigt, A., et al. (2012) The Chlamydia psittaci Genome: A Comparative Analysis of Intracellular Pathogens. PLoS One 7 27

48 Gophna, U., et al. (2003) Bacterial type III secretion systems are ancient and evolved by multiple horizontal transfer events. Gene 312, 151 163 49 Troisfontaines, P. and Cornelis, G.R. (2005) Type III secretion: More systems than you think. Physiology 20, 326 339 50 Beeckman, D.S.A. and Vanrompay, D.C.G. (2010) Bacterial Secretion Systems with an Emphasis on the Chlamydial Type III Secretion System. Curr. Issues Mol. Biol. 12, 17 41 51 Lutter, E.I., et al. (2012) Evolution and Conservation of Predicted Inclusion Membrane Proteins in Chlamydiae. Compar. Funct. Genom. 10.1155/2012/362104 52 Saka, H.A. and Valdivia, R.H. (2010) Acquisition of nutrients by Chlamydiae: unique challenges of living in an intracellular compartment. Curr. Opin. Microbiol. 13, 4 10 53 Gieffers, J., et al. (2003) Genotypic differences in the Chlamydia pneumoniae tyrp locus related to vascular tropism and pathogenicity. J. Infect. Dis. 188, 1085 1093 54 Mitchell, C.M., et al. (2010) Chlamydia pneumoniae Is Genetically Diverse in Animals and Appears to Have Crossed the Host Barrier to Humans on (At Least) Two Occasions. PLoS Pathog. 6 55 Wolf, K., et al. (2000) Ultrastructural analysis of developmental events in Chlamydia pneumoniaeinfected cells. Infect. Immun. 68, 2379 2385 56 Mitchell, C.M., et al. (2009) In vitro characterisation of koala Chlamydia pneumoniae: Morphology, inclusion development and doubling time. Vet. Microbiol. 136, 91 99 57 Miyairi, I., et al. (2006) Different growth rates of Chlamydia trachomatis biovars reflect pathotype. J. Infect. Dis. 194, 350 357 58 Hoestgaard Jensen, K., et al. (2011) Influence of the Chlamydia pneumoniae AR39 bacteriophage phi CPAR39 on chlamydial inclusion morphology. FEMS Immunol. Med. Microbiol. 62, 148 156 59 Hybiske, K. and Stephens, R. (2007) Mechanisms of host cell exit by the intracellular bacterium Chlamydia. Proc Natl Acad Sci USA 104, 11430 11435 60 Karunakaran, K., et al. (2011) Evolutionary Conservation of Infection Induced Cell Death Inhibition among Chlamydiales. PLoS One 6 61 Sharma, M. and Rudel, T. (2009) Apoptosis resistance in Chlamydia infected cells: a fate worse than death? FEMS Immunol. Med. Microbiol. 55, 154 161 62 Wolf, K., et al. (2009) A protein secreted by the respiratory pathogen Chlamydia pneumoniae impairs IL 17 signalling via interaction with human Act1. Cell Microbiol. 11, 769 779 63 Nicholson, T.L., et al. (2003) Global stage specific gene regulation during the developmental cycle of Chlamydia trachomatis. J. Bacteriol. 185, 3179 3189 64 Maurer, A.P., et al. (2007) Gene expression profiles of Chlamydophila pneumoniae during the developmental cycle and iron depletion mediated persistence. PLoS Pathog. 3, 752 769 65 Albrecht, M., et al. (2011) The transcriptional landscape of Chlamydia pneumoniae. Genome Biol. 12 66 Saka, H.A., et al. (2011) Quantitative proteomics reveals metabolic and pathogenic properties of Chlamydia trachomatis developmental forms. Mol. Microbiol. 82, 1185 1203 67 Hoare, A., et al. (2008) Spatial constraints within the chlamydial host cell inclusion predict interrupted development and persistence. BMC Microbiol. 8 68 Miura, K., et al. (2008) Genome wide analysis of Chlamydophila pneumoniae gene expression at the late stage of infection. DNA Res. 15, 83 91 69 Schoborg, R.V. (2011) Chlamydia persistence a tool to dissect chlamydia host interactions. Microbes Infect. 13, 649 662 70 Eickhoff, M., et al. (2007) Host cell responses to Chlamydia pneumoniae in gamma interferoninduced persistence overlap those of productive infection and are linked to genes involved in apoptosis, cell cycle, and metabolism. Infect. Immun. 75, 2853 2863 71 Polkinghorne, A., et al. (2006) Differential expression of chlamydial signal transduction genes in normal and interferon gamma induced persistent Chlamydophila pneumoniae infections. Microbes Infect. 8, 61 72 28

72 Klos, A., et al. (2009) The transcript profile of persistent Chlamydophila (Chlamydia) pneumoniae in vitro depends on the means by which persistence is induced. FEMS Microbiol. Lett. 291, 120 126 73 Timms, P., et al. (2009) Differential transcriptional responses between the interferon gammainduction and iron limitation models of persistence for Chlamydia pneumoniae. J. Microbiol. Immunol. Infect. 42, 27 37 74 Matsuo, J., et al. (2010) Stability of Chlamydophila pneumoniae in a harsh environment without a requirement for acanthamoebae. Microbiol. Immunol. 54, 63 73 75 Smadel, J.E. (1943) Atypical pneumonia and psittacosis. J. Clin. Invest. 22, 57 65 76 Puolakkainen, M., et al. (1984) Chlamydial pneumonitis and its serodiagnosis in infants. J. Infect. Dis. 149, 598 604 77 Grayston, J.T., et al. (1990) A New Respiratory Tract Pathogen: Chlamydia pneumoniae Strain TWAR. J. Infect. Dis. 161, 618 625 78 Falck, G.ñ., et al. (1997) Prevalence of Chlamydia pneumoniae in healthy children and in children with respiratory tract infections. Pediatr. Infect. Dis. J. 16, 549 554 79 Kumar, S. and Hammerschlag, M.R. (2007) Acute respiratory infection due to Chlamydia pneumoniae: Current status of diagnostic methods. Clinical Infectious Diseases 44, 568 576 80 Coon, R.G., et al. (2011) Chlamydophila pneumoniae Infection Among Basic Underwater Demolition/SEAL (BUD/S) Candidates, Coronado, California, July 2008. Milit. Med. 176, 320 323 81 Senn, L., et al. (2011) Does Respiratory Infection Due to Chlamydia pneumoniae Still Exist? Clinical Infectious Diseases 53, 847 848 82 Edvinsson, M., et al. (2010) Presence of Chlamydophila pneumoniae DNA but not mrna in stenotic aortic heart valves. Int. J. Cardiol. 143, 57 62 83 Dabiri, H., et al. (2009) Detection of Chlamydia pneumoniae in Atherosclerotic Plaques of Patients in Tehran, Iran. Jpn. J. Infect. Dis. 62, 195 197 84 Borel, N., et al. (2008) Evidence for persistent Chlamydia pneumoniae infection of human coronary atheromas. Atherosclerosis 199, 154 161 85 Watson, C. and Alp, N.J. (2008) Role of Chlamydia pneumoniae in atherosclerosis. Clin. Sci. 114, 509 531 86 Cockram, F. and Jackson, A. (1981) Keratoconjunctivitis of the koala, Phascolarctos cinereus, caused by Chlamydia psittaci. J. Wildl. Dis. 17, 497 504 87 Blumer, C., et al. (2007) Chlamydiae in free ranging and captive frogs in Switzerland. Vet. Pathol. 44, 144 150 88 Rattei, T., et al. (2007) Genetic diversity of the obligate intracellular bacterium Chlamydophila pneumoniae by genome wide analysis of single nucleotide polymorphisms: evidence for highly clonal population structure. BMC Genomics 8 89 Albrecht, M., et al. (2010) Deep sequencing based discovery of the Chlamydia trachomatis transcriptome. Nucleic Acids Res. 38, 868 877 90 Kokab, A., et al. (2010) Analysis of modulated gene expression in a model of Interferon gammainduced persistence of Chlamydia trachomatis in HEp 2 cells. Microb. Path. 49, 217 225 91 Di Pietro, M., et al. (2012) Analysis of gene expression in penicillin G induced persistence of Chlamydia pneumoniae. J. Biol. Regul. Homeost. Agents. 26, 277 284 29