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Interaction of Herpes Simplex Virus (HSV) glycoprotein D (gD) with the host cell surface during Chlamydia trachomatis/HSV co-infection stimulates chlamydiae to become persistent. During viral entry, gD interacts with one of 4 host co-receptors: HVEM (herpes virus entry mediator), nectin-1, nectin-2 and 3-O-sulfated heparan sulfate. HVEM and nectin-1 are high-affinity entry receptors for both HSV-1 and HSV-2. Nectin-2 mediates HSV-2 entry but is inactive for HSV-1, while 3-O-sulfated heparan sulfate facilitates HSV-1, but not HSV-2, entry. Western blot and RT-PCR analyses demonstrate that HeLa and HEC-1B cells express nectin-1 and nectin-2, but not HVEM. Because both HSV-1 and HSV-2 trigger persistence, these data suggest that nectin-1 is the most likely co-receptor involved. Co-infections with nectin-1 specific HSV-1 mutants stimulate chlamydial persistence, as evidenced by aberrant body (AB) formation and decreased production of elementary bodies (EBs). These data indicate that nectin-1 is involved in viral-induced chlamydial persistence. However, inhibition of signal transduction molecules associated with HSV attachment and entry does not rescue EB production during C. trachomatis/HSV-2 co-infection. HSV attachment also does not activate Cdc42 in HeLa cells, as would be expected with viral stimulated activation of nectin-1 signaling. Additionally, immunofluorescence assays confirm that HSV infection decreases nectin-1 expression. Together, these observations suggest that gD binding-induced loss of nectin-1 signaling negatively influences chlamydial growth. Chlamydial infection studies in nectin-1 knockdown (NKD) HeLa cell lines support this hypothesis. In NKD cells, chlamydial inclusions are smaller in size, contain ABs, and produce significantly fewer infectious EBs compared to C. trachomatis infection in control HeLa cells. Overall, the current study indicates that the actions of host molecule, nectin-1, are required for successful C. trachomatis development.

The bacterium Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infection worldwide. When left untreated, chlamydial infections can lead to severe complications, such as infertility. Lack in current prevention and management due to its asymptomatic course of infection highlight the need for an effective vaccine against chlamydia. There is no vaccine at present to protect against chlamydia, but research is ongoing. A research group at Örebro University has developed a protein antigen candidate. This project focused on the production of the candidate, here called Protein X, for preclinical trials. This included optimising production in Escherichia coli to maximise formation of soluble protein, optimising purification, buffer exchange and removal of His-tag. It was found that formation of soluble protein was favoured in lower expression temperatures. Furthermore, purification was performed on soluble and insoluble protein fractions using immobilised metal affinity chromatography. However, issues with inefficient binding to the resin and purity could not be solved and further optimisation is needed. Buffers were tested to find a suitable buffer for preclinical experiments, but the protein precipitated in all buffers. It was however found that protein from the insoluble fraction dissolved in pure water. Lastly, removal of the His-tag was performed with a non-enzymatic method that utilises nickel ions instead of expensive proteases. Efficient removal was however not achieved and enzymatic methods may be considered instead. In conclusion, this project highlighted issues in the production of Protein X and may guide the research group towards improving this process for efficient preclinical preparations.

Abstract Around 3% of pregnant women in Finland have genital Chlamydia trachomatis infection, which can be transmitted from mother to newborn at birth. The risk of transmission has been reported to be 10–70% in vaginal deliveries resulting in conjunctivitis in 10–30% of cases and lower respiratory tract infection in 0–20% of cases. Although usually benign, Chlamydia trachomatis infections in infancy may result in long-term consequences, including conjunctival and corneal scarring, chronic cough and abnormal lung function. Based on the transmission rates published in prior studies, chlamydial conjunctivitis should occur in approximately 200 infants and chlamydial lower respiratory tract infection in 100 infants each year in our country, but in clinical practice we rarely encounter or diagnose infants with Chlamydia trachomatis infections. To investigate the reason for this discrepancy and to improve the recognition of Chlamydia trachomatis-infected infants, we set out to study the risk of vertical transmission of Chlamydia trachomatis in a population-based setting, to describe the typical features of Chlamydia trachomatis infections in infants and to evaluate the occurrence of Chlamydia trachomatis in both neonatal conjunctivitis and lower respiratory tract infections in infants. When studying the probability of vertical transmission of Chlamydia trachomatis a search through two national health registers for 1996–2011 yielded 206 children aged less than four years with a possible Chlamydia trachomatis infection. In a cohort of 933 823 births this represented an occurrence of 0.22 per 1000 live births (95% confidence interval 0.19–0.25). The risk of vertical transmission of Chlamydia trachomatis leading to a symptomatic infection in infancy was 0.8–1.8%. A review of patient charts to evaluate the typical features of Chlamydia trachomatis infections in infants (124/206) revealed that one-third of the infants with chlamydial conjunctivitis (33/124) had spontaneous bloody discharge from the infected eyes. Almost half of the infants with chlamydial lower respiratory tract infection (15/32) had wheezing, but the characteristic staccato cough was not recorded in any of them. The median diagnostic delay from the onset of symptoms was 13 (range 4–374) days for conjunctivitis and 25 (range 10–149) days for lower respiratory tract infection. One neglected child developed bilateral corneal scars due to untreated chlamydial conjunctivitis. To investigate the occurrence of Chlamydia trachomatis in neonatal conjunctivitis, 173 neonates with clinical conjunctivitis at child health clinics were examined prospectively during 2010–2015 and none of the 163 cases tested had chlamydial or gonococcal conjunctivitis (0%; 95% confidence interval 0%–2.2%). Viral conjunctivitis was diagnosed in 8/167 cases (4.8%; 95% confidence interval 2.1%–9.2%) and non-chlamydial bacterial conjunctivitis in 58/160 (36%; 95% confidence interval 29%–44%). To investigate the occurrence of Chlamydia trachomatis in lower respiratory tract infections, 228 infants aged less than six months with lower respiratory tract infection presenting at the paediatric emergency department of Oulu University Hospital were examined prospectively over a period of a complete epidemiological year. One infant (0.4%; 95% confidence interval 0.01%–2.4%) had lower respiratory tract infection caused by Chlamydia trachomatis and another was diagnosed with whooping cough (0.4%; 95% confidence interval 0.01%–2.4%). The majority of the infants with lower respiratory tract infection (203/228) had a respiratory viral infection. It may be concluded that the risk of mother-to-child transmission of Chlamydia trachomatis leading to a clinical illness in the infant in this era of nucleic acid-based diagnostics was less than 2%, which is significantly lower than in earlier studies. The population-based prevalence of neonatal chlamydial conjunctivitis in primary care was less than 2% and that of chlamydial lower respiratory tract infection in a hospital setting less than 2.5%. The long-term prognosis for Chlamydia trachomatis infections in infancy was good. Common respiratory viruses were detected in 5% of the neonatal conjunctivitis cases. / Tiivistelmä Noin 3 %:lla suomalaisista raskaana olevista naisista on klamydian (Chlamydia trachomatis) aiheuttama sukupuolitauti, joka voi tarttua äidistä lapseen synnytyksessä. Tartuntariskin on raportoitu olevan alatiesynnytyksessä noin 10–70 %. Noin 10–30 % tartunnan saaneista lapsista sairastuu silmätulehdukseen ja 0–20 % keuhkokuumeeseen. Vaikka imeväisten klamydiainfektiot ovat useimmiten lieviä tauteja, imeväisiällä sairastettu klamydiainfektio voi aiheuttaa silmän side- ja sarveiskalvon arpeutumista, pitkittynyttä yskää ja keuhkofunktion alenemaa. Aiempien tutkimusten perusteella arvioimme, että Suomessa sairastuu vuosittain noin 200 imeväistä klamydian aiheuttamaan silmätulehdukseen ja noin 100 imeväistä klamydiakeuhkokuumeeseen. Kliininen kokemuksemme on kuitenkin, että kohtaamme klamydiaa sairastavia imeväisiä varsin harvoin. Tämän ongelman ratkaisemiseksi ja klamydiaa sairastavien imeväisten paremmaksi tunnistamiseksi suunnittelimme tutkimuksen, jonka tarkoituksena on selvittää väestöpohjainen riski klamydian tarttumiselle äidistä vastasyntyneeseen, kuvata imeväisten klamydiainfektioiden tyypilliset piirteet sekä selvittää klamydian osuus imeväisten silmätulehduksissa ja alle kuuden kuukauden ikäisten imeväisten alahengitystieinfektioissa. Klamydian sairastaneet lapset poimittiin kahdesta suomalaisesta terveydenhuoltorekisteristä vuosina 1996–2011. Tuona aikana 206 lasta oli sairastanut mahdollisen klamydiainfektion, joten klamydian ilmaantuvuus oli 0,22/1000:tta elävänä syntynyttä kohti (95 % luottamusväli 0,19–0,25). Väestöpohjainen riski äidin sukupuoliklamydian tarttumiselle vastasyntyneeseen niin että lapselle aiheutuu oireinen infektio oli 0,8–1,8 %. Saatavilla olevien potilasasiakirjojen (124/206) perusteella kolmasosalla (33/124) imeväisistä, jotka sairastivat klamydian aiheuttamaa silmätulehdusta, oli oireena spontaani verinen kyynel- tai rähmäerite. Klamydiakeuhkokuumetta sairastavista puolella (15/32) esiintyi hengityksen vinkumista, mutta klamydiakeuhkokuumeelle tyypillistä hakkaavaa yskää (”staccato-yskä”) ei todettu yhdelläkään imeväisellä. Diagnostinen viive oli verrattain pitkä: 13 päivää (vaihteluväli 4–374) silmätulehduksessa ja 25 päivää (vaihteluväli 10–149) keuhkokuumeessa. Yhdelle laiminlyödylle lapselle kehittyi molemminpuoliset sarveiskalvoarvet hoitamattoman klamydiainfektion seurauksena. Vastasyntyneen silmätulehdustutkimukseen rekrytoitiin 173 alle 30 päivän ikäistä lasta Oulun kaupungin lastenneuvoloissa vuosina 2010–2015. Klamydian tai tippurin aiheuttamaa silmätulehdusta ei todettu yhdelläkään 163:sta tutkitusta vauvasta (0 %; 95 % luottamusväli 0 %–2,2 %). Viruksen aiheuttama silmätulehdus todettiin kahdeksalla vauvalla (4,8 %; 95 % luottamusväli 2,1 %–9,2 %) ja jonkin muun bakteerin kuin klamydian aiheuttama silmätulehdus 58:lla vauvalla (36 %; 95 % luottamusväli 29 %–44 %). Imeväisten alahengitystieinfektiotutkimukseen rekrytoitiin 228 alle kuuden kuukauden ikäistä imeväistä yliopistosairaalan lastenpäivystyksessä yhden epidemiologisen vuoden aikana. Klamydian aiheuttama hengitystieinfektio diagnosoitiin yhdellä imeväisellä (0,4 %; 95 % luottamusväli 0,01 %–2,4 %) ja hinkuyskä niin ikään yhdellä (0,4 %; 95 % luottamusväli 0,01 %–2,4 %). Valtaosalla (203/228) alahengitystieinfektio-oireisista imeväisistä oli viruksen aiheuttama infektio. Yhteenvetona voimme todeta, että klamydia tarttui äidistä lapseen alle 2 %:ssa synnytyksistä, mikä on huomattavasti harvinaisempaa kuin aiemmin on luultu. Klamydian aiheuttamien silmätulehdusten esiintyvyys oli alle 2 % ja alahengitystieinfektioiden alle 2,5 % alueemme lapsiväestössä. Klamydian aiheuttamat pitkäaikaishaitat olivat harvinaisia. Tavallisten hengitystievirusten osuus vastasyntyneiden silmätulehduksissa oli 5 %.

Chlamydia trachomatis is the most common sexually transmitted bacterial disease agent worldwide, and, though frequently asymptomatic, can cause extreme pathology including infertility. Chlamydial species exhibit a unique biphasic developmental cycle. Once attached to a cell surface, infectious elementary bodies (EB) are internalized within an inclusion, the membrane-bound structure in which EB transform to noninfectious, replicable reticulate bodies (RB). After multiple rounds of division, RB condense to form EB, which are released and can infect new host cells. In culture, exposure to stressors, such as beta-lactam antibiotics, induce chlamydiae to reversibly detour from normal development into a noninfectious, viable state termed persistence. Cell culture data suggest that persistent forms are resistant to azithromycin (AZM), a front-line antibiotic, and are able to alter the host transcriptome. Though persistence has been described in culture for over 50 years, whether or not it: i) occurs in vivo; and ii) influences chlamydial pathogenesis, transmission and therapy has remained unresolved. To address these questions, we developed an animal model of persistent chlamydial infection using amoxicillin (AMX) treatment. AMX exposure decreased shedding of infectious chlamydiae in C. muridarum-infected mice without affecting chlamydial viability, demonstrating the presence of persistent chlamydiae. Shedding of infectious EB resumed following AMX cessation. Shedding data and microarray analyses suggested that host immunity might limit chlamydia’s exit from persistence in our model. Thus, we hypothesized that cyclophosphamide (CTX) treatment would increase the magnitude of chlamydial shedding observed after AMX-treatment cessation. CTX treatment increased post-AMX shedding by more than 10-fold compared to AMX-only controls. To determine whether persistent chlamydiae are resistant to antibiotic eradication in vivo, we induced persistence by administering AMX and treated mice with various AZM dosing regimes. Persistently infected mice demonstrated increased treatment failure following AZM therapy compared to productively infected controls. These data suggest that persistent chlamydiae are refractory to treatment in vivo and provide an explanation for the observation that treatment fails in some patients. In addition to creating the first fully characterized, experimentally tractable, in vivo model of chlamydial persistence, these experiments provide evidence that persistent/stressed chlamydial forms may serve as a long-term reservoir of infectious organisms in vivo.