Virulence Factors of Chlamydia Spp. Involving Human Infections

Abstract

Chlamydia spp. are the culprit of many human infections with severe complications, especially involving human eye, reproductive system, and lungs. The scope of the project is to delineate the virulence factors of the bacterium that facilitate invasion in human tissues, their mechanism of action, the ability to hide from immune system and the complications of infection. Chlamydia spp. are obligate intracellular pathogens that in their evolution, they use multiple mechanisms to enter host cell, to form the inclusion body, and to promote intracellular replication and survival. The T3SS effectors, the inclusion membrane proteins (Incs), are not only structural components of the membrane but also interfere with the host cell pathways. They also have an atypical mechanism of cell division. Description of the mechanisms of pathogenicity may lead to the development of new ways to face this major pathogen.

1. Introduction

The order Chlamydiales, family Chlamydiaceae comprises obligate intracellular bacteria, classified as Gram-negative bacteria due to the cell wall structure but are difficult to stain. The cell wall has no peptidoglycan but contains an outer lipopolysaccharide membrane. Instead of peptidoglycan it contains proteins which confer the same functional properties as peptidoglycan. Those proteins are rich in cysteine. Due to this unique cell wall structure, the microorganism can divide intracellularly and survive extracellularly. The shape is coccoid or rod-shaped. Both survive intracellularly in aerobic conditions and are not able to synthesize its own ATP or grow on an artificial medium [1, 2].

Chlamydiae are not metabolically active outside the host cell. This is a unique characteristic of Chlamydiae and in contrast with other intracellular bacteria [3]. The life cycle of Chlamydia is biphasic and is characterized by a succession between the infectious inactive elementary body which does not replicate and represents the dispersal form of the microorganism, and the noninfectious reticulate body which can replicate [1]. Upon contact with the host cell, the bacterium provokes endocytosis by injecting chlamydial proteins into the epithelial cell it attacks. Those injected proteins force the epithelial cells to endocytose particles they would never do otherwise [4]. Once inside the host cell, the interaction with glycogen drives the elementary body to germinate and take its reticulate form [4]. The microorganism survives intracellularly into a protective parasitophorous vacuole [1]. To do so, the microorganisms act on host cell cytoskeletal structures and endocytic pathways so as not to fuse the parasitophorous vacuole with the infected cell lysosomes [4]. In those vacuoles, the microbe uptake nutrients and energy and simultaneously alter host cell transcriptional pathways to prevent apoptosis and hide from host defense mechanisms [4]. Some proteins of the bacterium are secreted into the inclusion membrane. Those proteins are called inclusion membrane proteins and their role as virulence factors still needs to be elucidated [4]. The reticulate form has an incubation period of about 7–21 days in its host as it divides every 2–3 hours. When division is complete, in about 48 hours, it takes the elementary form and is released from the host cell via exocytosis to infect new cells [1].

Chlamydophila (Chlamydia) pneumoniae is a main culprit of community-acquired pneumonia, bronchitis, and adult-onset asthma. They are also linked in the literature to atherosclerosis and multiple sclerosis [2, 5]. Chlamydia psittaci causes infection in birds and can cause severe pneumonia in humans who inhale the feces of those birds [1].

Chlamydia trachomatis, depending on the disease they induce and different tissue tropisms, are divided into biovars. Depending on the humoral response they provoke are divided into serovars (genovars) [1, 4, 6]. Based on antigenic variation of the major outer membrane protein (MOMP), C. trachomatis is classified into 15 serovars [6]. The genital strains of C. trachomatis serovars D through K cause the sexually transmitted disease chlamydia (cervicitis in women and urethritis in men) [1, 6]. The microbe infects squamocolumnar or transitional epithelial cells. Ascending infection causes Pelvic Inflammatory Disease in women and epididymitis and reactive arthritis in men. In women, infection may lead to infertility, ectopic pregnancy, and chronic pelvic pain [6]. Serovars L1–L3 cause the invasive lymphoma granuloma venereum (LGV), also sexually transmitted infection [1, 6]. The ocular biovar that causes Trachoma includes the serovars A through C [1]. Trachoma is a state directly linked with blindness. The disease is transmitted via infected secretions of the genital urinary tract or through ocular discharge or contact with eye-seeking flies. The microorganism binds to the mucosal membranes of the cervix, rectum, urethra, throat, and conjunctiva [1].

Chlamydia gallinacea is an obligate intracellular bacterium and an opportunistic human pathogen. In human, it is known to cause pneumonia in poultry slaughterhouse workers [7].

The genome of Chlamydial microorganisms has significant similarities that make it difficult to comprehend the way they provoke so diverse diseases [1]. Comparing the genome structure of C. trachomatis to Chlamydia pneumoniae may lead to better understanding of the ways those microorganisms act. The genome of C. trachomatis is described to be 1,042,519, while the respective of C. pneumoniae is 1,230,230 base pairs long. There are clear differences between the two: 186 genes on the C. pneumoniae genome are not present on the C. trachomatis genome, and seventy genes on the C. trachomatis genome are not represented on the C. pneumoniae genome [8]. The C. trachomatis genome is considered small. Nonetheless, there have been specified 900 coding sequences on the chromosome and the plasmid of the microbe. The over 200 open reading frames encode proteins their function still needs to be elucidated and are all responsible for the virulence, intracellular survival, and replication of the bacterium [9].

2. Virulence factors

Chlamydia can evade both intra- and extracellular host defense [1]. Further understanding of the virulence factors they possess, and elucidation of the mechanisms of action can provide essential tools for prevention and treatment.

2.1 The cell wall structure

The C. trachomatis LPS is a genus-specific antigen. It is a “rough” type molecule provoking weak macrophage activation. Lipid A portion is pentaacylated with more than C14 fatty acids. The cell wall of C. trachomatis inhibits phagolysosome fusion in phagocytes [10].

2.2 Type III secretion systems

Intracellular pathogens secrete contact-dependent protein products of conserved secretory genes [3]. Those proteins promote the viability and multiplication of the microbe within the host cell and are well-described virulence factors. Their role is to intercept host signaling pathways in favor of the intruder [3]. Type III secretion systems serve as a conduit to promote the delivery of pathogen-effector proteins into the cytoplasm of the host cell. Via this apparatus, the microorganism can inject proteins directly into the host cell and avoid lysosomes [10]. Chlamydiae have a unique life cycle where the elementary body is metabolically dormant and therefore expresses no contact secretion. This contrasts with other intracellular pathogens, where contact-dependent secretion begins before they have been internalized in the host cell [3]. In Chlamydiae contact secretion is triggered from within by the intracellular metabolically active reticulate body. Therefore, characterization and description of Chlamydiae contact-dependent secretion mechanisms are of great interest [3]. The product of scc1 gene has homology to chaperone proteins of other intracellular pathogens. Cds1 and cds2 locuses are acquired by horizontal transfer and thus are included in a classical pathogenicity island. They are expressed during the intracellular life cycle of the bacterium and are related to the translocation of the pathogen across the cytoplasmic membrane [3]. Effector protein IncA of C. trachomatis mediates inclusion fusion and is related to strain-dependent disease severity [10]. Another chlamydial phosphoprotein is delivered into host cell cytoplasm and promotes actin recruitment in favor of the pathogen. This so-called Translocated Actin Recruiting Phosphoprotein causes internalization of the microbe and is related to disease severity [10, 11].

2.3 Chlamydial proteins present in the cytosol of infected cells

Many chlamydial proteins have been described in literature to be present in the cytosol of infected cells, but their distinct role as virulence factors still needs to be elucidated. Those proteins are CPAF, cHtrA, CT621, CT622, CT311, CT795, C. trachomatis glycogen synthase (GlgA), the C. trachomatis outer membrane complex protein B, and Pgp3 [12]. Identification of these proteins can elucidate further the mechanism of pathogenic activity of the bacterium [12]. Bacterial antigens present in host cell cytosol are more immunogenic [4].

2.3.1 CPAF

CPAF factor responsible for chlamydial protease/proteasome-like activity is highly conserved [1]. It acts as a zymogen, which means it can self-activate and auto-process via vicinity-dependent homodimerization [1]. It is a secreted serine protease known to cleave a large amount of host proteins. Its role has been described in attacking certain host mechanisms to evade the immune system and to survive and replicate intracellularly. Their targets are the host transcriptional factors USF-1 23, RFX5 24, NF-κB, and HIF-1, the proapoptotic BH3-only proteins, the DNA repairing Poly-ADP-ribose polymerase, cyclin B1, cytoskeleton proteins involved in cell structure like keratin 8, keratin 18 and vimentin, and proteins involved in repairment of Golgi apparatus, proteins involved in cell adhesion like nectin-1 [1, 13]. The secreted CPAF into the cytoplasm of the infected cell degrades the transcription factors RFX5 and USF-1 that are responsible for MHC gene activation. In this way, the microorganism reduces immune recognition by affecting antigen presentation and suppressing IFNγ-inducible MHC class I expression [1, 13]. BH3-only proteins like Puma and Bim that act as intracellular stress sensor molecules via migration to the mitochondria induce apoptosis. The mechanism involves the activation of the multi-domain proapoptotic Bax and Bak to suppress the antiapoptotic function of BcL-2. CPAF degrades the BH3-only domain proteins and acts in favor of antiapoptotic activity. This mechanism still needs elucidation [1, 13]. Another distinct role of CPAF is cleavage of cytoskeletal proteins that lead to depolymerization of the cytoskeleton surrounding the inclusions. In this way the microbe uses the lack of ability of the infected host cells to maintain their cytoskeletal structure, to expand the chlamydial inclusions in favor of their rapid replication [1, 13]. The microbe uses Golgi-derived lipids like sphingomyelin and cholesterol via the chlamydial proteases. Cleavage of golgin-84 leads to recruitment of Golgi fragmentation to acquire nutrients [1, 13].

2.3.2 Chlamydial HtrA

Chlamydial HtrA (cHtrA) is a hexamer with proteolytic activities. It is a periplasmic protein. It acts as a protease that acts on the endoplasmic reticulum of host cells and cleaves the transcription factors ATF6 and SREBP that are involved in cholesterol biosynthesis [14]. It also releases the sE-factor to activate stress response genes and is essential for the survival of the microorganism under high temperature [14]. HtrA is present in the chlamydial inclusion and is secreted in host cell cytosol, a unique property of chlamydial cells.

2.3.3 CT621 and CT622

Chlamydial cells use the type III secretion system to secrete CT621 and CT622 into the host cell cytoplasm [12, 15]. The presence of CT622 and CT621 in host cell cytoplasm should be involved in the pathogenetic mechanism of chlamydial infection, although their role needs to be further studied and elucidated. They seem to follow the same path but different kinetics in expression and secretion, meaning they play different roles in the survival and replication of the bacterium intracellularly [12].

2.3.4 CT311

Protein CT311 of C. trachomatis is secreted out of chlamydial inclusion into the cytosol of the infected cell and it enters host cell nucleus, thus it is a sufficient component for nuclear targeting [16]. The presence of this chlamydial component in the nucleus during the late stage of intracellular infection means that it can interact and modulate signal transduction pathways. This is an important tool to induce infection [16]. The most possible role of CT311 is alteration of host cell mechanisms to facilitate exiting of host cell and spreading [16].

2.3.5 CT795

The Chlamydia-specific protein CT795 is detected in the cytoplasm of infected host cells via a sec-dependent mechanism and not by a type III secretion pathway [4].

2.3.6 C. trachomatis glycogen synthase (GlgA)

In recent studies, chlamydial GlgA seems to appear in host cell cytosol and chlamydial inclusion lumen among all C. trachomatis serovars and is immunogenic in women urogenital infections. Its specific role in the pathogenicity of chlamydial infections still needs to be elucidated but seems to interfere with the accumulation of glycogen, within the inclusion lumen [17]. Glycogen is a nutritional source but also a TLR2 ligand. In Chlamydia muridarum infection plasmid dependent TLR2 activation is related to the promotion of infection and the chronic pathology of oviduct inflammation [18].

2.3.7 The C. trachomatis outer membrane complex protein B

The C. trachomatis outer membrane complex protein B (OmcB) is a well-described antigen as far as chlamydial infections are concerned. It is an abundant outer membrane protein, highly conserved among Chlamydia species that acts as an adhesin for chlamydial invasion into epithelial cells. It seems to provoke robust antigenic response via the release of the C-terminal region of the molecule (OmcBc) to host cell cytosol [4]. The fragment OmcBc is present in host cell cytosol. The fragment OmcBn stays in the chlamydial inclusions. OmcBc proved to be highly immunogenic in women infected by the microbe. OmcB is therefore a target for the development of diagnostic tools and vaccines [19].

2.4 Chlamydiae-induced antiapoptotic activity of infected host cells

Infected cells seem to express antiapoptotic mechanisms like inhibition of caspase 3 activation, blockade of mitochondrial cytochrome c release, inhibition of Bax/Bak NFκB activation. In this way the microbe detours apoptosis, a well-described mechanism of infected cells by intracellular pathogens. CPAF has been described to have an important role in this direction [13].

2.5 Chlamydial cryptic plasmid

The removal of the chlamydial cryptic plasmid in the murine equivalent model of C. trachomatis, C. muridarum, has led to reduced bacterial load and upper genital tract and ocular pathologies [1]. The possible mechanisms include the action of two plasmid genes involved in the formation of the antigen Pgp3, a secreted protein component of the outer plasmid membrane, and Pgp4 which is a regulator of both plasmid and chromosomal genes [1]. The products of these genes are related to glycogen production and accumulation, a contributor to virulence [9, 18]. Pgp4 acts also as a transcriptional regulator of both plasmid and chromosomal virulence-associated genes [18]. Both pgp3 and pgp4 genes are essential for in vitro growth of the bacterium, which enforces the aspect that their products are virulence factors [18]. This aspect is enforced by the fact that pgp4 is expressed only three hours post-infection [10, 18]. In mice, pGP3 promotes ascending infection and tubal inflammation. The mechanism seems to be via neutralizing the host antimicrobial peptides, like human alpha-defensins, human neutrophil peptide 2, human beta-defensin 3, and cathelicidin LL-37. Host antimicrobial peptides are trapped by pGP3 to form stable complexes. In this way the progeny Elementary Bodies released can safely evade the next host cell [20]. The plasmid-encoded protein CPSIT_P7 of C. psittaci via Toll-like receptor 4 and TLR4/Mal/MyD88/NF-κB signaling axis, triggers the expression of interleukin-6, interleukin-8, and monocyte chemoattractant protein-1 [21].

2.6 Protein CT135

Protein CT135 is responsible for persistent urogenital infection in mice and prolonged time to clearance in vivo [1].

2.7 Silencing the NF-κB inflammatory pathway

Chlamydiae seem to suppress NF-κB activation in infected cells. This is in contrast with the production of proinflammatory cytokines that are induced during infection. It appears that the microbe activates MAP kinases in its favor to acquire the needed nutrients for survival. The mechanism seems to be cleavage of NF-κB p65 into the fragments p40 and p20 by a chlamydial protein called tail-specific protease. The overexpression of p40 leads to interaction of the fragment with the cytoplasmic inhibitor of NF-κB I-κBα and blockage of NF-κB pathways [13]. SINC protein of C. psittaci targets the inner nuclear membrane of infected host cells. As a result, it is involved in controlling of nuclear structure and gene silencing [7, 22].

2.8 Translocated actin recruiting protein (TarP)

Upon attachment of the elementary body to the host cell, TarP is injected into the host cell. This translocation is made via a chlamydial type 3 secretory system. TarP has three binding sites for vinculin [23]. Additionally, TarP is reported to interact with focal adhesion kinase (FAK) and thus be able to alter cell adhesion signaling [23]. In this way, Chlamydiae use extracellular matrix and cell-to-cell junction for their benefit to promote entry to the host cells [23].

2.9 Chlamydial polymorphic outer membrane proteins (Pmps)

Chlamydial polymorphic outer membrane proteins are surface proteins that serve as adhesins, but also have antigenic role. They are present in all chlamydial species. They act as autotransporters that is they can translocate. These proteins are considered virulence factors. Chlamydiae in contrast to other Gram-negative bacteria possess more autotransporter proteins. PmpB, C, D, and I of C. trachomatis are known to induce strong humoral immune responses in infected patients. Women patients suffering from pelvic inflammatory disease and proved positive for anti-PmpA specific antibodies had a significantly high infertility rate in comparison to women negative for anti-PmpA antibodies. C. pneumonia Pmp20 and Pmp21 induce IL-6 and monocyte chemoattractant protein-1 production in human umbilical vein endothelial cells (HUVECs) by activation of NF-kB. The production is dose-dependent and the two Pmps do not act synergistically. C. trachomatis PmpD induces strong and dose-dependent IL-8 production from the human monocyte cell line THP-1 [10, 24].

3. Conclusion

Chlamydia spp. are the culprit of many human infections with severe complications, especially involving human eye, reproductive system, and lungs. Many virulence factors of the bacterium have been described in literature. Those virulence factors facilitate invasion in human tissues and provide the microorganism with the ability to hide from immune system and use the host cell mechanisms in its favor. The cell wall structure, Type III secretion systems, chlamydial proteins present in the cytosol of infected cells, induction of antiapoptotic activity of infected host cells, chlamydial cryptic plasmid, protein CT135, silencing of the NF-κB inflammatory pathway, Translocated actin recruiting protein (TarP), chlamydial polymorphic outer membrane proteins (Pmps), are some of them. Elucidation of the mechanisms of pathogenicity may lead to the development of new ways to face this major pathogen.

Conflicts of interest

We declare no conflict of interest.

Funding

None.