Waddlia chondrophila
1. Classification
a. Higher order taxa
Domain: Bacteria
Phylum: Chalmydiae
Class: Chlamydiia
Order: Parachlamydiales
Family: Waddliaceae
Genus: Waddlia
Species: Waddlia chondrophila
2. Description and Significance
Waddlia chondrophila is an obligate intracellular bacterium related to Chlamydia and belongs to the Waddliaceae family [1], [2], [3]. It was first discovered in bovine abortion tissues and is found to grow rapidly within human macrophages [1], [2].
W. Chondrophila has been found to infect various cell lines such as endometrial cells, primary human macrophages, fibroblasts, peripheral blood mononuclear cells, and pneumocytes [3], [4]. Additionally, there is a connection between W. chondrophila and miscarriages in both humans and animals, genital infection, male infertility, and pneumonia [3], [4], [5]. Hosts with W. chondrophila genital infections do not show symptoms [3]. Doxycycline and azithromycin can be used to treat W. chondrophila infections, but W. chondrophila is resistant to β-lactams and fluoroquinolones [1].
Currently, there is a dearth of knowledge about the points of entry of W. chondrophila and the exact mechanism it uses to cause such effects. The natural host of W. chondrophila is unknown, but a mouse model has been used to investigate the spread of a W. chondrophila infection [3], [6].
3. Genome Structure
The genome of W. chondrophila consists of a 2,116,312 base pair circular chromosome with a guanine and cytosine (G+C) content of 43.8% and a 15,593 base pair plasmid with a G+C content of 37.6% [7]. There are 1,934 protein coding genes within the genome, with 13% being conserved. These conserved proteins show the most similarity to those in the Parachlamydiacae family, including the closely related Parachlamydia acanthamoebae. [7] Out of the remaining proteins, 65% had inferred protein families or function, while only 13% had no similarity to other known proteins [7].
Based on the average read depth, there are 11 plasmid copies per cell [7]. The plasmid encodes for 22 proteins. One of these proteins, also found in the Waddlia chromosome, is homologous to MazF, an endoribonuclease part of a toxin-antitoxin system [7]. Normally, MazF is accompanied with the antitoxin MazE [8]. It is currently unclear whether any genes in the W. chondrophila genome encode for the antitoxin, but the presence of this system may explain the ubiquitous nature of the plasmid [7].
4. Cell Structure
W. chondrophila is considered a Gram-negative bacterium, but differs from other Gram-negative bacteria in that the peptidoglycan layer is replaced by a proteinaceous layer filled with disulfide bonds [7]. After entering a cell, these disulphide-linked proteins are reduced, allowing the bacteria to swell in size and create a replicating body [7]. Controlling this process there are 11 novel beta-barrel proteins of the outer membrane protein (OMP) family. These proteins have a cysteine rich cluster, similar to other OMP proteins found in the Chlamydiaceae family [7].
5. Metabolic Processes
W. chondrophila is an obligate intracellular bacterium, meaning that it needs a host cell to properly grow [9]. However, compared to others in the Chlamydiales order, W. chondrophila has a higher level of host independence. This is completed by having anabolic capabilities not shared within the same order. As with many other pathogens, W. Chondrophila is capable of utilizing the host’s cell materials to grow, particularly by exchanging host ATP and bacterial ADP through a translocase [9].Through electron microscopy, it has been found that this pathogen gravitates to host cell mitochondria, which is consistent with its dependence on host ATP [9]. However, W. chondrophila has the ability to produce some energy, independent from the host, by using oxidative phosphorylation to create ATP [7].
W. chondrophila has the ability to produce NAD from metabolic intermediates such as nicotinamide [7]. Whereas other Chlamydiales rely solely on importing all nucleotides for replication, W. chondrophila is able to produce all pyrimidine derivatives, in addition to having a nucleotide import [7]. W. chondrophila is also able to produce many forms of lipids, such as glycerophospholipids, glycerolipids and sphingolipids, while others in the same order cannot [7].
Although W. chondrophila has a high level of independence, it still requires the host for some metabolic processes [7]. The bacterium is able to produce only 10 of the 20 amino acids. Many other Chlamydia have the ability to synthesize tryptophan but W. chondrophila cannot do so on its own [7]. W. chondrophila also lacks the ability to produce aminos acids such as tyrosine and phenylalanine as well. Instead, W. chondrophila relies on an amino acid transporter to create its required proteins [7].
6. Growth and Replication
W. chondrophila uses a biphasic developmental cycle, also referred to as complete chlamydial replicative cycle [9]. The host cell engulfs the infectious form of W. chondrophila, known as the elementary body (EB), which migrates to the host’s vacuole. W. chondrophila can evade the endosomal pathway during the preliminary infection, preventing phagocytes from recognizing the infection when engulfed [10]. It migrates towards the ER and mitochondria through use of microtubules and filaments. [10] Then, the EB matures into the reticulate body (RB), the metabolically active form that replicates via binary fission [9]. Lastly, before the cycle repeats, the RB reverts to EB and proceeds to lyse the vacuole and host cell, allowing the infection of surrounding host cells. This cycle activates in bovine cells as early as 6-12 hours after infection [9].
7. Ecology
In 2012, forty well samples and thirty drinking water samples were analyzed in Catalonia, Spain and it was found that W. chondrophila was present in ten of the well samples. W. chondrophila’s presence in these well water sources may have contributed to the spread of this pathogen to bovine and human hosts [11].
8. Pathogenicity
W. chondrophila is capable of infecting a large array of living hosts, ranging from small microorganisms such as amoeba to larger organisms like humans and cattle [2]. When W. chondrophila infects a cell, cytoskeletal components are arranged around the inclusion and help with the acceptance and growth of the infection within the cell [6]. While the related bacterium Chlamydia trachomatis has an exceptional ability to inhibit apoptosis in infected cells, W. chondrophila does not protect infected cells from apoptosis, but rather promotes cell lysis [6]. Furthermore, C. trachomatis depends on the fragmentation of the Golgi apparatus during infection and replication while W. chondrophila does not cause or rely on such fragmentation to facilitate infection and replication [6]. Finally, unlike other members of Chlamydiae, who rely on this sphingomyelin transport, W. chondrophila does not transport sphingomyelin from the Golgi to the inclusion [6].
A) Role of W. chondrophila in Miscarriages and Host Cell Immune Response
W. chondrophila has a similar pathogenicity to other chlamydial abortifacients such as Chlamydia abortus [9]. W. chondrophila and Chlamydia abortus belong in the same order, Chlamydiales, so share many genomic and growth similarities [2]. The presence of anti-Waddlia antibodies in mothers has a correlation to early miscarriages, as high seropositivity of Waddlia is common in recurrent miscarriages [2] and some having traces in their placentas [9]. Women who had IgG Waddlia antibodies were known to have been directly involved with cattles or drink milk that may have been exposed to the pathogen. This suggests that W. chondrophila is a zoonotic pathogen, capable of transferring from animal to humans, and the miscarriages involved may have been a result of W. chondrophila indirectly increasing cytokine production or mimic fetal antigens [2].
W. chondrophila is abortifacient due to its ability to cause fetal deaths - either through miscarriage or stillbirth - in a matter of 2 weeks post-infection [9]. Although the mechanism of how the pathogen directly causes miscarriages is unclear, the host’s immune response appears to be related. Pro-inflammatory cytokines and chemokines are thought to be contributing factors in the onset of multiple inflammatory pathways, especially IL-1ß, and were expressed even with the UV killed pathogen [9]. This observation indicates that the cell has a surface recognition pathway, as opposed to intracellular recognition responses like with CXCL8 and TNF-, that is required for active infection. These findings were replicated using qPCR, fluorescent immunocytochemistry, and electron microscopy methods [9].
In the past, W. chondrophila was shown to be able to infect different types of cells, including some human macrophages. Cell lines of interest in investigating miscarriages include trophoblast cells (specialized placental cells). Their innate immune response is important in embryo and fetal health, especially in the protection from maternal pathogens [9].
B) W. chondrophila: Disease Progression in Relation to Genital Infection
Genital infection of W. chondrophila in mice has caused enlargement of the spleen, liver, lymph nodes, and inflammation of the wall of the uterus [3]. After W. chondrophila infection, liver cells exhibited an excess of neutrophils and macrophages, which accumulated into immune cell nodules or granulomas [3]. Multifocal granulomas replaced the white pulp – which contains lymphocytes – and the red pulp – which filters blood of antigens – of the spleen leading to an increase in the number of dead red and white pulp cells [3]. White blood cells and neutrophils are found in the endometrium of mice infected with W. chondrophila, as well as dead deciduomas, which are masses of tissue that form in the uterus after pregnancy [3].
The humoral response of a W. chondrophila genital infection was an IgG-mediated response consisting mostly of IgG2a subtypes, which is indicative of a skew towards a Th-1 immune response – a proinflammatory response to kill intracellular parasites [3].
C) Role of W. chondrophila in Pneumonia
W. chondrophila was tested as a potential agent that causes pneumonia among C57BL/6 mice that range from eight to twelve weeks old. Six groups of mice were given increasing concentrations of W. chondrophila such as 5 x 107, 1 x 108 and 2 x 108, 4 x108 bacteria into their nasal cavity to mimic the natural route of transmission pneumonia agents usually follow [5]. Three days after being infected, 50% of the mice population only retained 60% of their original weight and the bacterial concentration in each of the mice lungs grew ten times the amount compared to typical pneumonia-causing microbes, such as Chlamydia pneumoniae [5]. Moreover, the bacterial concentration in the lung was highest one to three days after initial infection (6 x 106 bacteria within each lung sample), but bacterial concentration decreased by the sixth day [5]. Thus, W. chondrophila replicates quickly once in a host and can spread from the lungs to other organs, like the kidney and the spleen [5]. Pneumonia cases caused by W. chondrophila induce the production of IL-6 and TNF, which are pro-inflammatory cytokines within the mice’s lungs [5]. Through this, W. chondrophila could be a potential pathogen that attributes to the growing pneumonia cases each year, and should be tested as a potential cause in pneumonia patients [5].
D) Diagnosis
The presence of W. chondrophila can be detected using PCR of the 16S rRNA gene [12]. Using this method, 32 children diagnosed with bronchiolitis were tested for the presence of W. chondrophila. Three were found to have W. chondrophila in their system [12]. This method is new, but has a promising future in diagnosis of these types of microbial infections [12].
9. Current Research
While much research has been done on the effects of W. chondrophila and its effects on the female reproductive system, little has been done in males. In recent years, W. chondrophila has been linked to reproductive disorders in men. Both serum and sperm samples were obtained after 2 to 5 days of sexual abstinence from 204 men of infertile couples [4]. Of these patients, 58.3% had W. chondrophila specific IgG antibodies, showing that these patients were previously exposed to the bacteria. Furthermore, the seroprevalence of men who are older than 30 were higher, suggesting that the risks of infection increases with age [4]. An epitope of spermatozoa that was found to be related to the one on the bacteria may have caused an autoimmune response [4].
There is currently a debate about whether W. chondrophila degrades host proteins or if the loss of host protein is due to extraction of the cell for testing [6]. Considering the relationship between W. chondrophila and C. trachomatis, further investigation on how W. chondrophila inhibits apoptosis in cells is currently being studied to understand the differences from the mechanism that C. trachomatis uses [6]. Differentiating between these two microbes would help with selecting the appropriate treatment method for infected individuals.
With W. chondrophila being a potential pathogen that causes pneumonia in mouse models, further research is necessary to understand how W. chondrophila responds to current drugs available to treat pneumonia and which virulence factors will help distinguish W. chondrophila from other pneumonia causing agents [5].
W. chondrophila has been detected in water sources, specifically well-water samples. However, more water sources can be tested, for example swimming pools or spas to see if these are a potential source for infection as well [11]. Understanding the environments W. chondrophila can inhabit can help prevent or reduce the risk of infection.
Current and future research attempts to investigate the newly developed real-time PCR technique to screen larger sample sizes and further characterize the role of Waddlia in prevalent human diseases [12].
10. Author Contributions
C.M.D. wrote the Introduction section; M.M.U. wrote the section on Genome Structure; M.M.U. wrote the section on Cell Structure; M.M.U. and M.E.L. wrote the section on Metabolic Processes; M.M.U. and M.E.L. wrote the section on Growth and Replication; A.Z. wrote the section on Ecology; C.M.D., S.M., A.Z., and M.E.L. wrote the section on Pathogenicity; C.M.D., S.M., M.E.L, and A.Z, wrote the section on Current Research; and C.M.D., S.M., M.M.U, A.Z., and M.E.L. edited the final article draft.
11. References
[1] Goy, G., and Greub, G. 2009. Antibiotic susceptibility of Waddlia chondrophila in Acanthamoeba castellanii amoebae. Antimicrobial agents and chemotherapy 53 (6): 2663–2666. https://doi.org/10.1128/AAC.00046-09
[2] Baud, D., Goy, G., Osterheld, M. C., Croxatto, A., Borel, N., Vial, Y., Pospischil, A., and Greub, G. 2014. Role of Waddlia chondrophila placental infection in miscarriage. Emerging infectious diseases 20 (3): 460–464. https://doi.org/10.3201/eid2003.131019
[3] Vasilevsky, S., Gyger, J., Piersigilli, A., Pilloux, L., Greub, G., Stojanov, M., and Baud, D. 2015. Waddlia chondrophila induces systemic infection, organ pathology, and elicits Th1-associated humoral immunity in a murine model of genital infection. Frontiers in Cellular and Infection Microbiology 5 (76). https://doi.org/10.3389/fcimb.2015.00076
[4] Baud, D., Vulliemoz, N., Zapata, M., Greub, G., Vouga, M., and Stojanov, M. 2020. Waddlia chondrophila and male infertility. Microorganisms 8 (1): 136. https://doi.org/10.3390/microorganisms8010136
[5] Ludovic, P., Didier, L., Christophe, B., Thierry R., and Gilbert, G. 2016. Mouse model of respiratory tract infection induced by Waddlia chondrophila. PLOS ONE 11 (3): 1-12. https://doi.org/10.1371/journal.pone.0150909
[6] Dille, S., Kleinschnitz, E. M., Kontchou, C. W., Nölke, T., and Häcker, G. 2015. In contrast to Chlamydia trachomatis, Waddlia chondrophila grows in human cells without inhibiting apoptosis, fragmenting the golgi apparatus, or diverting post-golgi sphingomyelin transport. Infection and immunity 83 (8): 3268–3280. https://doi.org/10.1128/IAI.00322-15
[7] Bertelli, C., Collyn, F., Croxatto, A., Rückert, C., Polkinghorne, A., Kebbi-Beghdadi, C., Goesmann, A., Vaughan, L., and Greub, G. 2010. The Waddlia genome: a window into chlamydial biology. PLOS ONE 5 (5): e10890. https://doi.org/10.1371/journal.pone.0010890
[8] Engelberg-kulka, H., Hazan, R., Amitai, S. 2005. mazEF: a chromosomal toxin-antitoxin module that triggers programmed cell death in bacteria. Journal of Cell Science 188: 4327-4332. http://doi.or/10.1242/jcs.02619
[9] Wheelhouse, N., Coyle, C., Barlow, P. G., Mitchell, S., Greub, G., Baszler, T., Rae, M. T., and Longbottom, D. 2014. Waddlia chondrophila infects and multiplies in ovine trophoblast cells stimulating an inflammatory immune response. PLOS ONE 9 (7): e102386. https://doi.org/10.1371/journal.pone.0102386
[10] Croxatto A., and Breub. G. 2010. Early intracellular trafficking of Waddlia chondrophila in human macrophages. Microbiology 156 (2): 340-355. https://doi.org/10.1099/mic.0.034546-0
[11] Codony, F., Fittipaldi, M., López, E., Morató, J., and Agustí, G. 2012. Well water as a possible source of Waddlia chondrophila infections. Microbes and Environments 27 (4): 529–532. https://doi.org/10.1264/jsme2.me12048
[12] Goy, G., Croxatto, A., Posfay-Barbe, K. M., Gervaix, A., and Greub, G. 2009. Development of a real-time PCR for the specific detection of Waddlia chondrophila in clinical samples. European Journal of Clinical Microbiology & Infectious Diseases 28:1483-1486. https://doi.org/10.1007/s10096-009-0804-7
Edited by Cloelia DeCioccio, Margarette Emin Lee, Shirley Mai, Madison Uyemura, Alexis Zeng, students of Jennifer Bhatnagar for BI 311 General Microbiology, 2015, Boston University.
