1. Chlamydia trachomatis Infections

Stephen Jordan, David Nelson and William Geisler

Chlamydia trachomatis is an obligate intracellular human bacterial pathogen that infects epithelial cells of the eye, oropharynx, urogenital, and anorectal mucosa and is responsible for the clinical diseases of trachoma, chlamydia (urogenital, oropharyngeal, and anorectal), and lymphogranuloma venereum. C. trachomatis infections occur worldwide and infection rates are increasing. C. trachomatis can cause chronic asymptomatic infections and can lead to complications, such as irreversible reproductive tract morbidity (e.g. tubal factor infertility) from genital chlamydia, and blindness from trachoma. Clinical manifestations of C. trachomatis infections range broadly from asymptomatic infection (most common) or relatively mild signs or symptoms (e.g. urethral or vaginal discharge) to more severe disease (e.g. pelvic inflammatory disease, epididymitis). Diagnosis of C. trachomatis infection cannot be accurately made by clinical signs or symptoms and requires identification of the pathogen, usually by nucleic acid detection. All C. trachomatis infections should be treated with antibiotics. For genital infections, reinfection should be ruled out by repeat testing approximately 3 months after treatment. Trachoma management involves a combination of four interventions at the community and individual levels: surgery for turned-in eyelashes (trichiasis), antibiotics, facial cleanliness, and environmental improvements.

2. Chlamydia pneumoniae Infections

Lee Ann Campbell and David Hahn

Chlamydia pneumoniae is an obligate intracellular Chlamydia organism that is an established cause of human acute upper and lower respiratory conditions, primarily bronchitis and pneumonia. Seroepidemiological studies indicate that the majority of individuals worldwide have been infected by the time adulthood is reached, and that reinfection and/or chronic infection is common. The majority of acute infections are asymptomatic or only mildly symptomatic below the threshold that medical care is sought. The known propensity for other Chlamydia species to cause chronic infection associated with chronic immunopathologic diseases early on raised the question of whether chronic C. pneumoniae infection, either ongoing or 'hit and run', could be causally associated with a number of chronic inflammatory diseases of unknown etiology. Indeed, increasingly robust evidence implicates chronic C. pneumoniae infection as a potentially causal factor in chronic respiratory diseases (asthma, chronic bronchitis, chronic obstructive respiratory disease) and atherosclerotic cardiovascular diseases, cerebrovascular diseases, and lung cancers, among others. This chapter reviews historical aspects of the discovery of C. pneumoniae, its role in acute respiratory illnesses, and emerging evidence for the role of C. pneumoniae in the pathogenesis of selected chronic respiratory and vascular diseases

3. Chlamydia Adhesion and Invasion

Matthew D. Romero, Katja Mölleken, Johannes H. Hegemann and Rey A. Carabeo

Chlamydiae are obligate intracellular pathogens that require a specialized intracellular niche for their survival and replication. Hence, they possess an efficient means of gaining access to this niche. Adhesion and invasion of Chlamydia to host cells are the initiating processes of infection and pathogenesis, and thus it is important to characterize this process in detail. In vivo, the primary targets are the epithelial cells that line the mucosa of the ano-genital tract, conjunctiva, the respiratory tract, and the gut. These cells are non-phagocytic. In order for Chlamydia to invade these cells, they must induce their uptake by actively remodeling the cortical actin cytoskeleton and manipulating the endocytic machinery to facilitate the phagocytosis of infectious elementary bodies (EBs). Uptake is preceded by the stable adherence of EBs to the surface of epithelial cells. Stable adhesion is followed by the induction of signalling pathways that trigger a number of host cell processes, including remodeling of the cortical actin to facilitate uptake of the pathogen. Adhesion and the induction of signalling to the cytoskeletal remodeling machinery are shared by a number of chlamydial species, with C. trachomatis and C. pneumoniae being the best studied. Chlamydial adhesins stabilize the bacterium at the surface of the host cell via interaction with host cell surface receptors. This adhesion step does not need to be highly specific. It involves transient electrostatic interactions followed by more specific associations between chlamydial adhesins and specific receptors to trigger signalling within the host cell and initiate actin remodeling at the cell surface. Localized actin remodelling results in the formation of a variety of cell surface structures designed to internalize the invading bacteria. In this chapter, we will review the mechanisms of chlamydial adhesion and invasion, with a focus on bacterial and host factors that control these steps and that have been identified within the last decade.

4. The Chlamydial Inclusion

Kevin Hybiske and Dagmar Heuer

A distinctive feature of Chlamydia species is that all of these bacterial species reside and replicate within an intracellular, host membrane-derived vacuole called the inclusion. Extensive modification of the inclusion membrane and lumen by Chlamydia is necessary to make it a hospitable home for the bacteria, which require nutrients and energy from the host cell. Given its central role in Chlamydia infection, the inclusion has been intensively studied for many decades. This chapter will summarize many of the important concepts that have been learned about the establishment and maintenance of the inclusion by Chlamydia . These include the mechanistic functions played by inclusion membrane proteins at the inclusion-host interface, and how Chlamydia scavenge and recruit critical nutrients and proteins from the host cell into the inclusion.

5. Interactions of the Chlamydial Inclusion with the Host Cell

Christine Sütterlin and Isabelle Derré

Bacteria of the Chlamydia genus are obligate intracellular pathogens that reside at all times within a membrane-bound cytoplasmic compartment called the chlamydial inclusion. This replicative niche is necessary for the chlamydial developmental cycle and depends on many interactions with the host cell. In this chapter, we will discuss how these bacteria orchestrate the intricate relationship of the inclusion with most of the cellular elements of their host cell. In the first section, we will focus on the cytoskeleton and summarize our knowledge on how and why the infection alters F-actin, intermediate filaments and microtubules. We will also discuss the interaction of the inclusion with the centrosome, including its potentially negative consequences for the host cell. In the second section, we will describe how interactions between the inclusion and the endoplasmic reticulum, the Golgi and mitochondria are established, and how they promote the infection. In the last section, we will focus on associations of the inclusion with lipid droplets and peroxisomes, which have both been found to translocate into the inclusion. Overall, this description shows that chlamydiae are master manipulators of their host cell.

6. Chlamydia and Cell-autonomous Defence: Apoptosis and Autophagy

Georg Häcker and Thomas Rudel

In this chapter, we will discuss two major mechanisms of cell-autonomous defence against chlamydial infection in mammalian cells: apoptosis and autophagy. Both have been implicated in chlamydial infection numerous times, and there is substantial evidence that Chlamydia has established ways to escape these two defence reactions. We will describe the molecular mechanisms of apoptosis and autophagy and review the literature concerning their roles during a chlamydial infection. We will also discuss how these mechanisms may be triggered during an infection to defend the host cell, and how they are subverted by the infecting bacteria. Chlamydia can inhibit apoptosis in infected cells: experimental stimuli that would normally induce apoptosis in human cells fail to do that in Chlamydia-infected cells. It is generally assumed that Chlamydia needs to inhibit apoptosis to keep its host cell alive, although this has not been experimentally substantiated. Autophagy can also be used as a defence mechanism against intracellular bacteria. Chlamydia uses at least two mechanisms to avoid recognition by the autophagy machinery. Both apoptosis and autophagy could defend a human cell against chlamydial infection, but the bacteria appear to have evolved mechanisms to escape these defence mechanisms.

7. Protein Secretion in Chlamydia

Agathe Subtil and Richard D. Hayward

Protein secretion is fundamental to microbial physiology and underlies virulence-associated processes including adhesion, invasion, intracellular replication and the delivery of exotoxins. Protein secretion is key to the interaction with the environment, and is thus an essential feature of the biology of the Chlamydiaceae, which interact with a eukaryotic host throughout their developmental cycle. Here we review the mechanisms of secretion relevant to the pathogenesis of the Chlamydiaceae. The major secretion pathways are described, including the current knowledge of the structure, mechanism and function of the associated macromolecular machineries. Specifically, we review the role of the type 2, 3 and 5 secretion pathways in the interactions between these bacteria and their host cell, and we discuss the emerging role for vesicles released from the outer membrane of the intracellular reticulate bodies. We review how new genetic techniques and chemical inhibitors have advanced our understanding of the contribution of different secretory pathways to the infection process and allowed the definitive identification of secretion substrates. We also discuss how state-of-the-art imaging has contributed to further understanding the structure and mechanism of the associated macromolecular machineries. We consider the future opportunities for translating this knowledge into the development of vaccines, novel antimicrobials and diagnostic tools, and discuss the implications beyond the field of chlamydial research.

8. The Chlamydial Protease CPAF

Julie A. Brothwell, Christine Sütterlin, Thomas Rudel and Ming Tan

CPAF is a conserved chlamydial protease, but our understanding of its role in a Chlamydia infection is incomplete and evolving. CPAF is expressed as an inactive enzyme that requires secretion, homodimerization, and proteolytic processing for conversion into the active protease. It cleaves or degrades many chlamydial and host proteins, but it has been challenging to distinguish in vivo substrates that are true biological targets from in vitro substrates that do not undergo proteolysis during a Chlamydia infection. The subcellular localization of active CPAF in a Chlamydia-infected cell is also controversial because it is uncertain whether it is primarily localized in the chlamydial inclusion during infection or also present in the host cytoplasm. CPAF activity must be regulated to account for the observed differences between in vitro substrate susceptibility and in vivo proteolysis, but the control mechanisms have not been completely defined. Multiple functions have been proposed for CPAF. The strongest data indicate that CPAF modulates the host immune response by down-regulating the type I interferon response and by inhibiting neutrophil activation.In vivo, CPAF is necessary to maintain the Chlamydia infection in the genital tract of mice. CPAF is also a protective antigen that is being studied as a vaccine candidate. Thus, despite the controversy surrounding the true substrates of CPAF, CPAF remains an important virulence factor of Chlamydia.

9. Chlamydia Cell Division and Differentiation

George W. Liechti, Patrick H. Viollier, Gilbert Greub and Anthony T. Maurelli

In this chapter we review the current state of our knowledge regarding the molecular processes by which Chlamydia species divide and differentiate between their replicative and infectious forms. We will 1) give a general introduction to cell division in Gram-negative bacteria, 2) compare this process with the process of cell division in Chlamydia, 3) address the 'chlamydial anomaly' by discussing our collective knowledge of peptidoglycan synthesis in Chlamydia, 4) describe our current understanding of the localization and kinetics of peptidoglycan assembly, 5) compare binary fission and polarized division as models of Chlamydia replication, 6) review what is known about the elementary body (EB) to reticulate body (RB) transition, and 7) evaluate the current models for RB to EB differentiation. We will also describe similarities and differences observed regarding division, peptidoglycan and persistence among the different members of the Chlamydiae phylum.

10. Chlamydia Gene Regulation

Christopher J. Rosario, Katelyn Soules, P. Scott Hefty and Ming Tan

A defining feature of the Chlamydia developmental cycle is the expression of chlamydial genes in three main temporal classes. Early genes are the only genes transcribed at the beginning of the intracellular infection, but the mechanism for their selective expression is unknown. The large majority of chlamydial genes are midcycle genes, which are transcribed during the midstage of the developmental cycle at the time of reticulate body (RB) growth and replication. Gene expression in midcycle is proposed to be activated by higher DNA supercoiling levels and to be regulated by a DNA gyrase and two DNA topoisomerases, which are enzymes that control DNA supercoiling. Late genes are up-regulated late in the developmental cycle, when RB to elementary body (EB) conversion takes place. There are two subsets of late genes that are respectively transcribed by σ⁶⁶RNA polymerase, which is the major chlamydial RNA polymerase, or an alternative RNA polymerase called σ²⁸RNA polymerase. However, both subsets of late genes are repressed by the transcription factor EUO, which is thus known as the master regulator of late gene expression. Transcription is then proposed to be silenced in EBs by two histone-like proteins, Hc1 and Hc2, which condense DNA. Chlamydia gene expression is also controlled by transcription factors that homeostatically regulate the levels of amino acids, nucleotides, metal ions and the major heat shock proteins. Regulators of chlamydial gene expression with less understood roles include a second alternative sigma factor, σ⁵⁴ , putative regulators of each of the three forms of chlamydial RNA polymerase, and the transcription factors, ChxR and GrgA. In addition, there are potentially many sRNAs that control gene expression by binding to target transcripts. Collectively, these mechanisms allow Chlamydia to regulate the expression of its genes in both a pre-programmed manner and in response to external signals.

11. Chlamydia Genetics

Colette E. O'Neill, Ian N. Clarke and Derek J. Fisher

Since the seminal publication in 2011 from Wang and colleagues on plasmid-based gene transfer and the introduction of competence in Chlamydia trachomatis, numerous significant advances have been made in the field of chlamydial genetics using their transformation technique. Collectively, we can now generate targeted insertions and gene deletions in the chromosome, transform four chlamydial species with a variety of different shuttle vectors, construct random chromosomal mutants using chemical and transposon mutagenesis methods, and even generate conditional gene knockdowns using CRISPRi technology. In this chapter, we seek to provide background on how the field of chlamydial genetics grew, where it currently rests, and the needs that remain. While the transformation problem has been overcome, the low transformation frequencies and obligatory intracellular lifestyle are a significant bottleneck. Consequently, mutant construction remains tedious and operon structure and gene essentiality complicate both mutant construction and analysis. Pitfalls that arise when new technologies are brought to bear in a field starved of experimental approaches long practiced for other bacteria are also discussed along with standardization of protocols. It is our hope that this approach will yield significant and reproducible insights into the molecular details underlying the pathogenesis of Chlamydia.

12. Chlamydia Genomics

Vítor Borges, Patrick Hyden, João Paulo Gomes and Thomas Rattei

The field of Chlamydia genomics has seen a rampant development, from the very first release of the whole genome sequence of a C. trachomatis strain to the current availability of hundreds of genomes from multiple Chlamydia species. While this scientific advance has revealed fascinating patterns of adaptation to the intracellular environment, it has also disclosed the main genetic mechanisms driving Chlamydia evolution as well as many genes of unknown function that lack orthologs in the microbial world. It has allowed advances not only in the understanding of fundamental aspects of the epidemiology and biology of these unique intracellular bacteria but also in deciphering the molecular basis of phenotypic differences, such as host and tissue tropism, invasiveness and ecological success. Improvements and decreased cost of next-generation sequencing are coupled with advances in mutagenesis and proteome and transcriptome profiling. There is a critical need for multidisciplinary efforts to gain insights into the biological role of specific genes, to identify the within-patient Chlamydia adaptive traits during infection, and to decipher the Chlamydia-host interactive players that may ultimately lead to the development of new therapies.

13. Chlamydia and Innate Immunity

Uma M. Nagarajan, Breanna J. Turman, Michael R. Knittler and Andreas Klos

As an obligate intracellular bacterium, Chlamydia is detected by intracellular and extracellular host proteins to mount an innate immune response. The extracellular innate detectors in body fluids include complement and antimicrobial peptides, while the intracellular detectors are the pathogen recognition receptors. Engagement of these receptors with Chlamydia lead to intracellular signalling resulting in cytokine/chemokine secretion. Innate immune cells, such as neutrophils, NK cells, and macrophages, come to the site of infection in response to the chemokine gradients and also produce cytokines such as IFN-g, which can reduce bacterial burden by activation of interferon-response genes and GTPases. The innate immune response to Chlamydia is ineffective at clearing the infection, a finding which has long suggested that the pathogen has evolved means, at the humoral, extra-cellular and cytosolic levels, to subvert this host response. However, the innate response during Chlamydia infection is also associated with tissue pathology. Therefore, a better understanding of the key mediators of chlamydial recognition and early host responses can provide us with potential targets to mitigate host pathology.

14. Adaptive Immunity to Chlamydia trachomatis Infection

Taylor B. Poston, Toni Darville and Raymond M. Johnson

Understanding mechanics of mucosal adaptive immunity may facilitate development of a Chlamydia trachomatis vaccine. This chapter details mechanisms involved in determining whether an individual's adaptive immune response (antigen-specific B and T cell response) to a chlamydial genital tract infection or urogenital vaccine results in asymptomatic clearance (protection) or disease (urethritis/salpingitis/infertility). Over 30 years of research have revealed that adaptive responses to C. trachomatis are more complex than the presence or absence of antigen-specific T cell production of interferon-gamma (IFN-ɣ) (Th1 immunity) and B cell production of neutralizing antibody against elementary bodies. Basic research is beginning to define adaptive immunity to Chlamydia infections as functioning in tissues rather than in lymph nodes and spleens. Central to local tissue immunity against the epithelial-tropic C. trachomatis serovars (A-K) are B cells, T cells, and highly specialized immune infrastructures localized immediately beneath the reproductive tract epithelium. These subepithelial immune structures, called memory lymphocyte clusters, harbour plasma B cells and tissue resident memory T cells that are likely to be critical to protective immunity. While this story is incomplete, we try to present a working narrative of how chlamydial-specific mucosal adaptive immunity may be relevant to vaccine development. We touch on trachoma adaptive immunity to highlight how the eye differs from the genital tract as that informs the possibility of a universal urogenital/trachoma vaccine. Adaptive immunity mechanisms in Chlamydia-associated reactive arthritis, C. trachomatis serovar L1-3 (causative agent of lymphogranuloma venereum), and other Chlamydiae (e.g.C. psittaci) are unique and cannot be extrapolated from C. trachomatis urogenital tract data.

15. Chlamydia Vaccines

Luis M. de la Maza, Sukumar Pal, Anja W. Olsen and Frank Follmann

Vaccination is the most cost effective and efficient way to control infectious diseases. Vaccines against several members of the Chlamydia genus are urgently needed, especially those against sexually transmitted Chlamydia trachomatis, where current measures have failed to control the global epidemic. Preclinical research has made significant progress over the last four decades, and vaccines are now moving into development and clinical testing. The first Phase I trial of a candidate vaccine for C. trachomatis sexually transmitted infections (STIs) has been completed and others are on the way. Here, we provide an overview of the status of Chlamydia vaccines against the principal pathogens in both humans and animals, focusing primarily on human C. trachomatis STIs. We discuss the approaches pursued following the trachoma clinical trials in the 1960's, including inactivated whole-cell, attenuated strain, and subunit vaccines. Novel adjuvants and delivery systems and the challenges of inducing adaptive immunity in the female genital tract are covered. Lastly, we envision the implementation of C. trachomatis STI vaccines by proposing how to move them through the clinical development phases I-IV.

16. Chlamydia Infections and the Microbiota

Simon Graspeunter, Jan Rupp, Peter Timms and Bonnie Quigley

The microbiota has become intensively studied in many human diseases. In this chapter, we highlight recent research about the human microbiota and its association with chlamydial infections. We introduce the vaginal microbiota in general, focusing on the microbiota composition with regard to genital Chlamydia trachomatis infection and provide current concepts about the exclusion of C. trachomatis by the most important members of the vaginal microbiota, the lactobacilli. We further provide evidence about how particular anaerobic members of the microbiota create a microenvironment supporting ongoing infection with C. trachomatis. The potential role of the microbiota in upper genital tract infections with C. trachomatis is further discussed. The limited data that are available for non-genital chlamydial infections, in particular C. trachomatis infection in the eye and C. pneumoniae infection in the lung, are also reviewed. This chapter finally discusses the recent advances in microbiota research linked to animal chlamydial diseases and provides future directions of microbiota research in chlamydial diseases.

17. Recent Advances in Epidemiology, Pathology and Immunology of Veterinary Chlamydiae

Konrad Sachse and Nicole Borel

The veterinary chlamydiae comprise all currently known organisms of the family Chlamydiaceae, except Chlamydia trachomatis. C. abortus is an economically important infection in sheep and goats causing abortion, stillbirths and weak neonates. Moreover, it is a zoonotic pathogen, i.e. well known for being transmissible from animals to humans and posing a risk for pregnant women. C. psittaci, which causes avian chlamydiosis in birds, is the most important veterinary chlamydial agent in terms of economic impact and human health. Avian chlamydiosis has been recently extended by addition of the novel species C. avium, C. gallinacea and Candidatus C. ibidis. In addition, C. felis, causing conjunctivitis in cats, and C. caviae, a guinea pig pathogen, can both be zoonotic. The latter has been extensively used in experimental animal models. Similarly, C. muridarum infection models in mice have been in use for decades to investigate human genital chlamydial disease. C. pecorum has a broad host range, including livestock and wild ruminants, but also marsupials such as the koala in Australia. C. pneumoniae, originally thought to occur only in humans, has a broad host range, including horses, koalas, reptiles and amphibians. C. suis, a pathogen of pigs, is the only chlamydial species to date that can maintain stable tetracycline resistance.

18. Thinking Outside the Chlamydia Box

Alyce Taylor-Brown, Tamara Halter, Adam Polkinghorne and Matthias Horn

Chlamydiae have long been studied exclusively in the context of disease. Yet, accumulating evidence over nearly three decades shows that chlamydiae are ubiquitous in the environment, thriving as symbionts of unicellular eukaryotes such as amoeba and infecting a broad range of animal hosts. These chlamydiae share the characteristic chlamydial developmental cycle and other chlamydial hallmarks. Their discovery fundamentally changed our perspective on chlamydial diversity. Instead of a single genus, Chlamydia, including closely related pathogens, the chlamydiae comprise hundreds of families and genera. Investigating isolates and non-cultured representatives provided insights into features that are in common with or divergent from known Chlamydia species, and suggested that some of these chlamydiae may also be considered pathogens. Importantly, these studies have contributed to a better understanding of the biology of all chlamydiae, and they provide a framework for investigating the evolution of the chlamydial intracellular lifestyle and pathogenicity.

19. Animal Models of Chlamydia trachomatis Infection

Guangming Zhong, Alison Quayle, Ashok Aiyar and Tianyuan Zhang

Chlamydia trachomatis causes infections in the eye, urogenital, respiratory and gastrointestinal mucosa. Chronic infection can lead to significant disease; ocular infection can result in trachoma, a leading cause of preventable blindness, and genital infection can lead to the serious sequelae of ectopic pregnancy and infertility. These pathological sequelae have been proposed to result from the induction of a sustained inflammatory response, but the precise pathogenic mechanisms that lead to these diseases are not yet fully understood. Chlamydial biologists have used in vitro approaches that rely on biochemistry and/or cell biology, in conjunction with recently developed genetic tools, to identify bacterial factors required for interactions with host cells. Although some of the putative chlamydial virulence factors identified in vitro have been shown to promote chlamydial pathogenicity in animal models, it remains to be determined if this information is relevant to pathogenic mechanisms operating in humans. Furthermore, many putative virulence factors have not been evaluated in optimal animal tissue microenvironments despite the fact that various animal models with chlamydial infections have been developed. Here we describe the pathogenesis of C. trachomatis infections in humans and the unique features of the currently used model systems so that the most appropriate models can be chosen for addressing different aspects of C. trachomatis pathogenesis and evaluating vaccine candidates.