Transcriptional analysis of persistent Chlamydia pneumoniae infection in vitro

Chlamydia pneumoniae can cause acute respiratory infections including pneumonia. Repeated and persistent Chlamydia infections occur and persistent C. pneumoniae infection may have a role in the pathogenesis of atherosclerosis and coronary heart disease and may also contribute to the development of chronic inflammatory lung diseases like chronic obstructive pulmonary disease (COPD) and asthma. In this thesis in vitro models for persistent C. pneumonia infection were established in epithelial and monocyte/macrophage cell lines. Expression of host cell genes in the persistent C. pneumoniae infection model of epithelial cells was studied by microarray and RT-PCR. In the monocyte/macrophage infection model expression of selected C. pneumoniae genes were studied by RT-PCR and immunofluorescence microscopy. Chlamydia is able to modulate host cell gene expression and apoptosis of host cells, which may assist Chlamydia to evade the host cells' immune responses. This, in turn, may lead to extended survival of the organism inside epithelial cells and promote the development of persistent infection. To simulate persistent C. pneumoniae infection in vivo, we set up a persistent infection model exposing the HL cell cultures to IFN-gamma. When HL cell cultures were treated with moderate concentration of IFN-gamma, the replication of C. pneumoniae DNA was unaffected while differentiation into infectious elementary bodies (EB) was strongly inhibited. By transmission electron microscopy small atypical inclusions were identified in IFN-gamma treated cultures. No second cycle of infection was observed in cells exposed to IFN-gamma , whereas C. pneumoniae was able to undergo a second cycle of infection in unexposed HL cells. Although monocytic cells can naturally restrict chlamydial growth, IFN-gamma further reduced production of infectious C. pneumoniae in Mono Mac 6 cells. Under both studied conditions no second cycle of infection could be detected in monocytic cell line suggesting persistent infection in these cells. As a step toward understanding the role of host genes in the development and pathogenesis of persistent C. pneumoniae infection, modulation of host cell gene expression during IFN-gamma induced persistent infection was examined and compared to that seen during active C. pneumoniae infection or IFN-gamma treatment. Total RNA was collected at 6 to 150 h after infection of an epithelial cell line (HL) and analyzed by a cDNA array (available at that time) representing approximately 4000 human transcripts. In initial analysis 250 of the 4000 genes were identified as differentially expressed upon active and persistent chlamydial infection and IFN-gamma treatment. In persistent infection more potent up-regulation of many genes was observed in IFN-gamma induced persistent infection than in active infection or in IFN-gamma treated cell cultures. Also sustained up-regulation was observed for some genes. In addition, we could identify nine host cell genes whose transcription was specifically altered during the IFN-gamma induced persistent C. pneumoniae infection. Strongest up-regulation in persistent infection in relation to controls was identified for insulin like growth factor binding protein 6, interferon-stimulated protein 15 kDa, cyclin D1 and interleukin 7 receptor. These results suggest that during persistent infection, C. pneumoniae reprograms the host transcriptional machinery regulating a variety of cellular processes including adhesion, cell cycle regulation, growth and inflammatory response, all of which may play important roles in the pathogenesis of persistent C. pneumoniae infection. C. pneumoniae DNA can be detected in peripheral blood mononuclear cells indicating that the bacterium can also infect monocytic cells in vivo and thereby monocytes can assist the spread of infection from the lungs to other anatomical sites. Persistent infection established at these sites could promote inflammation and enhance pathology. Thus, the mononuclear cells are in a strategic position in the development of persistent infection. To investigate the intracellular replication and fate of C. pneumoniae in mononuclear cells we analyzed the transcription of 11 C. pneumoniae genes in Mono Mac 6 cells during infection by real time RT-PCR. Our results suggest that the transcriptional profile of the studied genes in monocytes is different from that seen in epithelial cells and that IFN-gamma has a less significant effect on C. pneumoniae transcription in monocytes. Furthermore, our study shows that type III secretion system (T3SS) related genes are transcribed and that Chlamydia possesses a functional T3SS during infection in monocytes. Since C. pneumoniae infection in monocytes has been implicated to have reduced antibiotic susceptibility, this creates opportunities for novel therapeutics targeting T3SS in the management of chlamydial infection in monocytes.Geenien poikkeavasta ilmenemisestä mahdollisesti apua kroonisen keuhkoklamydian tunnistukseen ja hoitoon Keuhkoklamydia on Suomessa erittäin yleinen hengitystieinfektio. Krooninen keuhkoklamydiainfektio voi olla yksi tekijä, joka vaikuttaa sydän- ja verisuonitautien kehittymiseen, ja sillä saattaa olla rooli myös keuhkoahtaumataudin ja astman taudinkuvassa. Nämä krooniset taudit ovat merkittävä kansanterveysongelma, ja ne kuormittavat terveydenhuoltoa ja kansantaloutta. Vaikka keuhkoklamydiaan tehoavat useat antibioottilääkkeet, kroonisessa infektiossa se näyttää olevan vastustuskykyinen lääkehoidoille. Keinoa kroonisen keuhkoklamydiainfektion luotettavaan tunnistamiseen ei toistaiseksi ole. Nyt julkaistavassa väitöstutkimuksessa tunnistettiin useita keuhkoklamydian ja ihmisen geenejä, joista saattaa olla apua kroonisen infektion tunnistamisessa ja hoidossa. Lisäksi nyt tunnistetuilla, kroonisessa infektiossa poikkeavasti ilmenevillä geeneillä saattaa olla vaikutusta kroonisen keuhkoklamydiataudin kehittymiseen. Tutkimuksessa havaittiin, että krooninen keuhkoklamydiainfektio muuttaa ihmisen solun geenien ilmenemistä. Työssä tunnistettiin lisäksi keuhkoklamydian geeni, jonka tuotteella (RNA:lla tai valkuaisaineella) on potentiaalia biomerkkiaineeksi, jonka avulla krooninen keuhkoklamydiainfektio voitaisiin tunnistaa tulevaisuudessa. Tutkimuksessa havaittiin myös, että keuhkoklamydian tietty valkuaisaineiden eritysreitti on toiminnallinen myös monosyytti-infektion aikana. Havaittu eritysreitti on myös potentiaalinen lääkehoidon kohde. Tulevaisuudessa kroonisen keuhkoklamydiainfektion hoito voitaisiin ehkä kohdistaa monosyytteihin eli veressä kiertäviin valkosoluihin ja täten ehkäistä klamydiabakteerin leviämistä elimistössä ja mahdollista kroonisen taudin syntyä keuhkojen ulkopuolella. Geenien erilaisesta aktiivisuudesta mahdollinen apu tautidiagnostiikkaan Tässä työssä keuhkoklamydian ja ihmisen solun vuorovaikutuksia tutkittiin tätä varten pystytetyissä soluviljelymalleissa. Ihmisen solun geenien ilmenemistä peittosolujen kroonisessa keuhkoklamydiainfektiomallissa tutkittiin DNA-sirulla, joka sisälsi 4000 ihmisen geeniä. Tutkimuksessa havaittiin, että kroonisessa infektiossa monet solun geenit ilmenivät voimakkaammin ja usein pidempään verrattuna akuuttiin infektioon. Lisäksi tutkimuksessa tunnistettiin yhdeksän geeniä, joiden ilmeneminen liittyi erityisesti krooniseen keuhkoklamydiainfektioon. Keuhkoklamydian geenien ilmenemistä tutkittiin ihmisen monosyyttisoluissa geenimonistustekniikan avulla. Ensisijainen kiinnostuksen kohde oli tutkia geenejä, joiden tuottamat valkuaisaineet eritetään isäntäsoluun infektion aikana sekä sellaisia geenejä, jotka aikaisempien tutkimusten perusteella ilmenivät poikkeavasti peittosolujen kroonisessa infektiomallissa. Yhden näistä peittosoluissa poikkeavalla tavalla ilmenevistä geeneistä havaittiin ilmenevän samankaltaisesti myös monosyyteissä. Nämä tutkimustulokset antavat viitteitä siitä, että kyseistä geenituotetta voitaisiin tulevaisuudessa mahdollisesti käyttää kroonisen infektion tunnistamiseen. Estäjämolekyyleistä mahdollinen hoitokeino Tutkimuksessa osoitettiin ensimmäistä kertaa tiettyjen klamydiageenien ilmentyminen ja valkuaisaineiden erittyminen ihmisen monosyyttisoluihin infektion aikana. Yhden näistä tutkituista proteiineista on osoitettu aiemmin erittyvän keuhkoklamydian niin sanotun tyyppi III -eritysreitin avulla. Tätä eritysreittiä vastaan on olemassa estäjämolekyylejä, jotka pysäyttävät keuhkoklamydian kasvun peittosoluissa. Koska tutkimuksessa havaittiin, että tyyppi III -eritysreitti on toiminnallinen myös monosyyteissä, luo se mahdollisuuden, että tunnetuilla estäjämolekyyleillä voidaan tulevaisuudessa hoitaa kroonista keuhkoklamydiainfektiota ja estää bakteerin leviämistä elimistössä. Hyvin kannustavista tuloksista huolimatta lisätutkimuksia tarvitaan, jotta ymmärrettäisiin paremmin nyt tunnistettujen keuhkoklamydian ja ihmisen geenituotteiden mahdollinen merkitys kroonisen taudin kehittymisessä ja jotta kroonisen keuhkoklamydiainfektion tunnistamiseen ja hoitoon saataisiin uusia työkaluja

Ripulivirusten pikadiagnostiikka

English summaryPeer reviewe

Genotyping of Hepatitis C virus by nucleotide sequencing: A robust method for a diagnostic laboratory

Abstract Hepatitis C virus (HCV) is a globally significant blood-borne agent causing liver diseases, and it has infected over 170 million people worldwide. HCV is a diverse group of RNA viruses currently divided into genotypes 1–7 as well as subtypes. HCV infection can be treated with antiviral drugs, but the HCV genotype has to be determined for optimal selection of treatment strategy. The aim of this study was to set up a sequencing-based HCV genotyping method suitable for the workflow of a diagnostic laboratory. The established method is robust and stable, and it utilizes a one-step reverse transcription and PCR amplification of the 5’ untranslated region (5’UTR) and partial Core region of the HCV genome. Amplification products are sequenced using the standard Sanger method, and the genotype is determined by using a freely accessible web-based genotyping tool. The method was validated at the Helsinki University Hospital Laboratory using 238 previously genotyped serum samples. • A new one-step RT-PCR method for the amplification of the 5’ untranslated region and partial Core region of hepatitis C virus was established. • HCV genotype is determined using Sanger sequencing and a freely accessible, easy-to-use web-based genotyping tool. • The method is robust, reproducible and suitable for diagnostic laboratory workflow, and it requires no costly instrumentation or specialized sequence analysis skills.Peer reviewe

Development of a multiplex real-time PCR assay for detection of Mycoplasma pneumoniae, Chlamydia pneumoniae and mutations associated with macrolide resistance in Mycoplasma pneumoniae from respiratory clinical specimens

The aim of this study was to improve detection of Mycoplasma pneumoniae and Chlamydia pneumoniae in clinical specimens by developing a multiplex real-time PCR assay that includes identification of macrolide-resistant M. pneumoniae. Novel assays targeting a M. pneumoniae conserved hypothetical protein gene, M. pneumoniae 23S rRNA gene mutations associated with macrolide resistance and human beta-globin gene (an endogenous internal control) were designed and combined with a previously published C. pneumoniae PCR targeting ompA gene. The resulting quadraplex PCR was validated with a panel of clinical specimens supplemented with external quality assessment specimens, simulated specimens and various bacterial and viral strains. The obtained results were compared to those obtained by reference PCRs or confirmed by sequencing (typing of macrolide resistance). The novel multiplex PCR assay was in 100 % agreement with reference PCRs. Four M. pneumoniae strains with macrolide resistance-associated mutations were identified among 42 strains, which comprises 9.5 % of the study material. Amplification of an internal control excluded sample-derived inhibition possibly leading to false-negative reporting. In conclusion, we have developed a resources conserving multiplex real-time PCR assay for simultaneous detection of M. pneumoniae, C. pneumoniae and the most common mutations leading to macrolide resistance in M. pneumoniae. The assay is a widely useful tool for detection of these respiratory pathogens and will also shed light on the occurrence of macrolide resistance in M. pneumoniae.Peer reviewe

Transcriptional Expression of the ompA, cpaf, tarp, and tox Genes of Chlamydia trachomatis Clinical Isolates at Different Stages of the Developmental Cycle

The transcriptional gene expression patterns of Chlamydia trachomatis have mainly been studied using reference strains propagated in cultured cells. Here, using five low-passage-number C. trachomatis clinical isolates that originated from asymptomatic or symptomatic female patients, the in vitro expression of the ompA, cpaf, tarp, and tox genes was studied with reverse transcriptase real-time PCR during the chlamydial developmental cycle. We observed dissimilarities in the gene expression patterns between the low-passage-number clinical isolates and the reference strains. The expression of ompA and the peak of the tox expression were observed earlier in the reference strains than in most of the clinical isolates. The expression of cpaf was high in the reference strains compared with the clinical isolates at the mid-phase (6–24 hours post infection) of the developmental cycle. All of the strains had a rather similar tarp expression profile. Four out of five clinical isolates exhibited slower growth kinetics compared with the reference strains. The use of low-passage-number C. trachomatis clinical isolates instead of reference strains in the studies might better reflect the situation in human infection

Transcriptional Expression of the ompA, cpaf, tarp, and tox Genes of Chlamydia trachomatis Clinical Isolates at Different Stages of the Developmental Cycle

The transcriptional gene expression patterns of Chlamydia trachomatis have mainly been studied using reference strains propagated in cultured cells. Here, using five low-passage-number C. trachomatis clinical isolates that originated from asymptomatic or symptomatic female patients, the in vitro expression of the ompA, cpaf, tarp, and tox genes was studied with reverse transcriptase real-time PCR during the chlamydial developmental cycle. We observed dissimilarities in the gene expression patterns between the low-passage-number clinical isolates and the reference strains. The expression of ompA and the peak of the tox expression were observed earlier in the reference strains than in most of the clinical isolates. The expression of cpaf was high in the reference strains compared with the clinical isolates at the mid-phase (6–24 hours post infection) of the developmental cycle. All of the strains had a rather similar tarp expression profile. Four out of five clinical isolates exhibited slower growth kinetics compared with the reference strains. The use of low-passage-number C. trachomatis clinical isolates instead of reference strains in the studies might better reflect the situation in human infection

JC polyomavirus DNA detection in clinical practice

Peer reviewe

Single-Molecule Sequencing Revealing the Presence of Distinct JC Polyomavirus Populations in Patients With Progressive Multifocal Leukoencephalopathy

Background. Progressive multifocal leukoencephalopathy (PML) is a fatal disease caused by reactivation of JC polyomavirus (JCPyV) in immunosuppressed individuals and lytic infection by neurotropic JCPyV in glial cells. The exact content of neurotropic mutations within individual JCPyV strains has not been studied to our knowledge. Methods. We exploited the capacity of single-molecule real-time sequencing technology to determine the sequence of complete JCPyV genomes in single reads. The method was used to precisely characterize individual neurotropic JCPyV strains of 3 patients with PML without the bias caused by assembly of short sequence reads. Results. In the cerebrospinal fluid sample of a 73-year-old woman with rapid PML onset, 3 distinct JCPyV populations could be identified. All viral populations were characterized by rearrangements within the noncoding regulatory region (NCCR) and 1 point mutation, S267L in the VP1 gene, suggestive of neurotropic strains. One patient with PML had a single neurotropic strain with rearranged NCCR, and 1 patient had a single strain with small NCCR alterations. Conclusions. We report here, for the first time, full characterization of individual neurotropic JCPyV strains in the cerebrospinal fluid of patients with PML. It remains to be established whether PML pathogenesis is driven by one or several neurotropic strains in an individual.Peer reviewe

Parachlamydia acanthamoebae Detected during a Pneumonia Outbreak in Southeastern Finland, in 2017–2018

Community-acquired pneumonia (CAP) is a common disease responsible for significant morbidity and mortality. However, the definite etiology of CAP often remains unresolved, suggesting that unknown agents of pneumonia remain to be identified. The recently discovered members of the order Chlamydiales, Chlamydia-related bacteria (CRB), are considered as possible emerging agents of CAP. Parachlamydia acanthamoebae is the most studied candidate. It survives and replicates inside free-living amoeba, which it might potentially use as a vehicle to infect animals and humans. A Mycoplasma pneumoniae outbreak was observed in Kymenlaakso region in Southeastern Finland during August 2017–January 2018. We determined the occurrence of Chlamydiales bacteria and their natural host, free-living amoeba in respiratory specimens collected during this outbreak with molecular methods. Altogether, 22/278 (7.9%) of the samples contained Chlamydiales DNA. By sequence analysis, majority of the CRBs detected were members of the Parachlamydiaceae family. Amoebal DNA was not detected within the sample material. Our study further proposes that Parachlamydiaceae could be a potential agent causing atypical CAP in children and adolescents