Identification of in vivo-induced bacterial protein antigens during calf infection with Chlamydia psittaci.

International Journal of Medical Microbiology 305 (2015) 310–321

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Identification of in vivo-induced bacterial protein antigens during calf infection with Chlamydia psittaci Julia Kästner a , Hans Peter Saluz a,b,∗ , Frank Hänel a,∗∗ a Department of Cell and Molecular Biology, Leibniz-Institute for Natural Product Research and Infection Biology, Beutenbergstrasse 11a, D-07745 Jena, Germany b Friedrich Schiller University, D-07745 Jena, Germany

a r t i c l e

i n f o

Article history: Received 30 August 2014 Received in revised form 19 November 2014 Accepted 20 December 2014 Keywords: Obligate intracellular bacteria In vivo lung infection Immunogenic chlamydial proteins Virulence IVIAT Zoonosis

a b s t r a c t Chlamydia (C.) psittaci, the causative agent of ornithosis, is an obligate intracellular pathogen with a unique developmental cycle and a high potential for zoonotic transmission. Various mammalian hosts, such as cattle, horse, sheep and man that are in close contact with contaminated birds can get infected (referred to as psittacosis). Since little is known about long-term sequelae of chronic disease and the molecular mechanisms of chlamydial pathogenesis, a key step in understanding the in vivo situation is the identification of C. psittaci infection-associated proteins. For this, we investigated sera of infected calves. Using the immunoscreening approach In Vivo Induced Antigen Technology (IVIAT) including all relevant controls, we focused on C. psittaci proteins, which are induced in vivo during infection. Sera were pooled, extensively adsorbed against in vitro antigens to eliminate false positive results, and used to screen an inducible C. psittaci 02DC15 genomic expression library. Screening and control experiments revealed 19 immunogenic proteins, which are expressed during infection. They are involved in transport and oxidative stress response, heme and folate biosynthesis, DNA replication, recombination and repair, cell envelope, bacterial secretion systems and hypothetical proteins of so far unknown functions. Some of the proteins found may be considered as diagnostic markers or as candidates for the development of vaccines. © 2015 Elsevier GmbH. All rights reserved.

Introduction The obligate intracellular bacterium Chlamydia psittaci (C. psittaci) shows a unique life cycle consisting of infectious extracellular elementary bodies (EBs) and metabolically active noninfectious intracellular reticulate bodies (RBs). Primarily C. psittaci can cause respiratory disease in birds called ornithosis, psittacosis or parrot fever, but its transmission to mammalian hosts including cattle, pig, sheep, horse or even human through the respiratory route has been reported (Petrovay and Balla, 2008). Recently, Ostermann et al. (2013a) circumstantiated the potential for zoonotic transmission of a bovine C. psittaci isolate from one calf to another. This indicates the potential risk for man being

∗ Corresponding author at: Department of Cell and Molecular Biology, Leibniz Institute for Natural Product Research and Infection Biology, Beutenbergstrasse 11a, Jena D-07745, Germany. Tel.: +49 03641 532 1201; fax: +49 03641 532 2361. ∗∗ Corresponding author. Tel.: +49 0 3641 532 1145; fax: +49 0 3641 532 0806. E-mail addresses: [email protected] (H.P. Saluz), [email protected] (F. Hänel). http://dx.doi.org/10.1016/j.ijmm.2014.12.022 1438-4221/© 2015 Elsevier GmbH. All rights reserved.

in direct contact with infected animals of inhaling contaminated aerosols. C. psittaci colonizes mucosal epithelial cells and upon bacteremia symptoms in calf vary from clinically unapparent infections to acute respiratory illness or keratoconjunctivitis (Twomey et al., 2006), whereas in human influenza-like illness can appear up to fatal diseases (Crosse, 1990; Pandeli and Ernest, 2006; Petrovay and Balla, 2008). Intracellular pathogens evolved sophisticated strategies to escape from the host immune system and to ensure their survival in the host. Chlamydiae hide and replicate within an intracellular vacuolar compartment (inclusion) and were shown to secrete various effector proteins via type III secretion in the host to modulate cellular functions (Bastidas et al., 2013; Valdivia, 2008). Despite the above escape mechanism, the secreted proteins or surface proteins may be excellent targets for the host immune system (Roan and Starnbach, 2008). In addition, a non-infected cell adjacent to an infected one is still able to recognize and defend the chlamydial pathogen (Zhong, 2009). Both, humoral and cell mediated immunity (Knittler et al., 2014; Roan and Starnbach, 2008) are important for the protection against chlamydial infection and its clearance (Starnbach et al., 1994;

J. Kästner et al. / International Journal of Medical Microbiology 305 (2015) 310–321

Vanrompay et al., 1999; Zhang et al., 1989). Although progress was made in the characterization of the innate immune response against C. psittaci infection in mice and chicken embryos and its linkage to the adaptive response via the complement system as well as the T cell-mediated immune response (Knittler et al., 2014), further information is needed regarding the adaptive immune response of the host to suggest host–pathogen molecular interactions and the accompanied pathogenesis of C. psittaci infections. The attenuation of pathologies and the development of proper vaccines, which are not available at present, are of great interest. To understand chlamydial pathogenic mechanisms, we investigated proteins, which are expressed during infection in calves (Ostermann et al., 2013a; Reinhold et al., 2012). For this, corresponding sera were pooled and subjected to the In Vivo Induced Antigen Technology (IVIAT; see Fig. 1) (Rollins et al., 2005). The sera were adsorbed with antigens of the E. coli BL21 (DE3) expression host and C. psittaci grown in vitro to avoid false positive results of the screening approach. The IVIAT technique was already applied to various pathogenic gram-negative and gram-positive bacteria as well as parasites (Alam et al., 2013; Amerizadeh et al., 2013; Gu et al., 2009; Hang et al., 2003; Harris et al., 2006; John et al., 2005; Kim et al., 2003; Lowry et al., 2010; Rollins et al., 2008; Salim et al., 2005; Vigil et al., 2011; Yoo et al., 2007). However, the above technique has never been applied to obligate intracellular bacteria like Chlamydiae. Recently, a different screening approach using an expression library of C. abortus was reported by Forsbach-Birk et al. (2013). In the latter approach sera were not adsorbed with the in vitro grown pathogens thus obtaining a general immune response profile (Forsbach-Birk et al., 2013). In contrast, the results presented here focus on proteins, which are expressed during the infection process (Ostermann et al., 2013a; Reinhold et al., 2012). They comprise proteins involved in transport and oxidative stress response, DNA replication, recombination and repair, cofactor metabolism, cell envelope, bacterial secretion systems and hypothetical proteins. Materials and methods Bacteria, plasmids, and growth conditions C. psittaci 02DC15 was recovered from an aborted calf fetus in 2002 (Goellner et al., 2006) and its genome was sequenced (Schöfl et al., 2011). Propagation of C. psittaci, infection and immunofluorescence staining were performed according to Goellner et al. (2006). Genomic DNA from the bovine isolate C. psittaci 02DC15 was isolated according to Schöfl et al. (2011) and was used to establish an IPTG-inducible genomic expression library in Escherichia coli BL21 (DE3). E. coli DH5␣ (host strain for cloning), the expression host E. coli BL 21 (DE3) and the expression vector pET30a were achieved from Novagen® (Merck Chemicals Ltd., Nottingham, UK). All plasmid transformed strains were grown in Luria–Bertani (LB) media or on solid LB agar containing 30 ␮g/ml kanamycin (Kan; Serva, Heidelberg, Germany) at 30 ◦ C and/or 37 ◦ C as mentioned in the following section (referred to Inducible Chlamydia psittaci genomic expression library). For long term storage all bacteria were maintained in 15% glycerol (Carl Roth GmbH, Karlsruhe, Germany) at −80 ◦ C. DNA isolation and recombinant DNA methods Isolation of plasmid DNA and genomic C. psittaci DNA was performed using the QIAprep Spin Miniprep Kit or QIAamp DNA Mini Kit (QIAGEN GmbH, Hilden, Germany). To clone selected chlamydial genes or gene fragments, C. psittaci DNA was amplified by PCR

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using Pfu DNA polymerase (Thermo Fisher Scientific GmbH, Dreieich, Germany) and specific oligonucleotides (Eurofins MWG GmbH, Ebersberg, Germany; Supplementary Table 1). For this, 20 ␮l reaction comprising 1 ␮l of DNA (50 ng), 5 pmol of each primer, 2 ␮l 10× Pfu buffer with MgSO4 pH 8.8, 0.4 ␮l of 10 ␮M dNTPs, 15 ␮l nuclease-free water, 1.25 u Pfu DNA polymerase was subjected to thermal cycling conditions as recommended (Thermo Fisher Scientific GmbH). The amplified genomic DNA was recovered from Agarose gel (NucleoSpin® Gel and PCR Clean-up; Macherey-Nagel, Düren, Germany) and, both the genomic DNA and the pET30a plasmid DNA were digested by the restriction endonucleases NdeI/XhoI or BamHI/XhoI (Thermo Fisher Scientific GmbH). Ligation was performed using T4 DNA ligase (Invitrogen, Darmstadt, Germany) as recommended by the manufacturer except that ligation reaction was incubated at 16 ◦ C for 16 h. Plasmid DNA was introduced into E. coli DH5␣ by electroporation at 1800 V and 250 ␮FD (Gene pulserTM and capacitance extender, Biorad, Germany) at room temperature using Gene Pulser 2-mm electrode spacing cuvettes (Bio-Rad, Munich, Germany). Subsequently, 1 ml of SOC medium (2% (w/v) tryptone, 0.5% (w/v) yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2 , 10 mM MgSO4 , 20 mM glucose, pH 7.0) was added and bacteria were grown for 1 h at 37 ◦ C before plating on LB-Kan-agar. Supplementary Table 1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijmm.2014.12.022. Western blot analysis For the expression analysis of the cloned chlamydial DNA in the host E. coli Western blot analysis was performed according to Laemmli (1970). Samples were boiled for 5 min in Laemmli buffer (Laemmli, 1970) and 20 ␮g of sample (expression in shaking flask; 20 ml culture) or 10 ␮l of IPTG-induced cells (expression in 96-well format; 400 ␮l culture concentrated 10× in Laemmli buffer) were separated using 13% or 4–20% precast gradient polyacrylamide gels (GE Healthcare, Munich, Germany) and transferred onto a 0.45 ␮m polyvinylidene diflouride (PVDF) membrane (GE Healthcare). Membranes were blocked with 5% skim milk (Carl Roth GmbH) in TBST (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1% (v/v) Tween® 20) for 1 h at room temperature, incubated over night at 4 ◦ C with monoclonal anti-hexahistidine tag antibody (Dianova, Hamburg, Germany), polyclonal anti-streptavidin antibody (Abcam plc, Cambridge, UK) or adsorbed bovine sera in 5% skim milk-TBST and after washing three times for 5 min with TBST at room temperature, the membranes were incubated with 1:10,000 horseradish peroxidase (HRP) conjugated protein G (GenScript, Piscataway, USA) for 1 h at room temperature. Proteins were visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific GmbH) as recommended. Adsorption of sera Sera from 7 male Holstein-Friesian calves that had been challenged intrabronchially with C. psittaci (Reinhold et al., 2012) were pooled (final volume 2.5 ml) and sequentially adsorbed against cell lysate of C. psittaci grown in BGM cells as well as whole cells and cell lysates from the expression host E. coli BL21 (DE3) as follows. E. coli BL21 (DE3) culture grown in LB-Kan media to an optical density at 595 nm (OD595nm ) of 5.8 were pelleted by centrifugation and pooled sera were incubated with whole cells twice at 4 ◦ C for 5 h or overnight on a rocking platform (KS 250basic; IKA® , Staufen, Germany) to eliminate anti-E. coli antibodies. Cell lysates of E. coli BL21 (DE3) were prepared by sonification on ice for 6 × 20 s at 30 kHz with 20 s break between each pulse (Sartorius Labsonic® M, Göttingen, Germany) as recommended (Sambrook and Russell, 2001) and immobilized on Protran® nitrocellulose

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Fig. 1. IVIAT applied to obligate intracellular bacteria. A protein expression library of C. psittaci genomic DNA was constructed in E. coli BL21(DE3) and induced with 1 mM isopropyl-␤-d-thiogalactoside (IPTG). Expressed proteins were probed with pooled sera of C. psittaci infected calves that previously had been serially adsorbed against E. coli BL21(DE3) harboring empty expression vector and C. psittaci grown in BGM cells. Bound antibodies were detected by incubation with HRP coupled protein-G and visualized by chemoluminescence. Insert DNA of positive clones was sequenced with plasmid specific forward primer. To confirm seroreactivity of in vivo induced proteins, subcloning of selected candidate genes was performed and again probed with the adsorbed sera.

membrane (Whatman® , Germany) overnight at 4 ◦ C. Membranes were blocked for 1 h at room temperature with phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2 HPO4 , 2 mM KH2 PO4 , pH 7.4) containing 0.05% Tween® 20 (T; Carl Roth GmbH) and 5% non-fat milk (Carl Roth GmbH) and sera supplemented with 0.03% sodium azide (Carl Roth GmbH) were adsorbed at 4 ◦ C overnight on a rocking platform. BGM cells were infected with C. psittaci at multiplicity of infection (Moi) 5, incubated at 37 ◦ C and harvested by scraping 46 h post infection. Chlamydial lysate was prepared by incubation of the pelleted C. psittaci infected BGM cells in ice cold modified RIPA-buffer (50 mM Tris–HCl [pH 7.4], 150 mM NaCl, 1% Triton X 100) supplemented with 100× protease inhibitor cocktail (100 mM 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF)–HCl, 80 ␮M aprotinin, 5 mM bestatin, 1.5 mM E-64, 2 mM leupeptin, 1 mM pepstatin A [Thermo Fisher Scientific, Bonn, Germany]) to 1× working concentration for 30 min on ice and sonification at 4 ◦ C (amplitude 8, 3 × 30 s [Branson Sonifier Ultrasonic Desintegrator W-450, ULT Heinemann, Germany]). 5.5 mg–6.5 mg chlamydial lysate was coated on Protran® nitrocellulose, blocked as mentioned above and incubated in E. coli adsorbed sera at 4 ◦ C on a rocking platform for 3–4 h. The same adsorption processes were performed in parallel with pooled sera samples from a non-infected calf. Successful adsorption was controlled by incubation of sera samples from distinct adsorption steps with 100 ␮l of 1 ␮g/ml C. psittaci lysate or precleared E. coli BL21 (DE3) lysate by the enzyme-linked immunosorbent assay (ELISA) according to Gu et al. (2009) and by Western blot analysis as described (Reinhold et al., 2012), except that Western blots were developed by an enhanced chemiluminescence reaction (ECL; SuperSignal West Pico Chemiluminescent Substrate, Thermo Fisher Scientific GmbH) as recommended.

Inducible Chlamydia psittaci genomic expression library Genomic DNA from C. psittaci DC15 was isolated and purified from infected cells as previously described (Schöfl et al., 2011). The genomic expression library was generated by LGC Genomics (LGC Genomics GmbH, Berlin, Germany). Chlamydial DNA fragments between 0.5 kb and 1.5 kb were introduced into the EcoRV restriction site of the pET30a expression vector (pET Expression System 30, Novagen, Germany). E. coli DH10␣ was transformed with these constructs and the library was checked for proper insert sizes and insert frequency (>90%) by restriction analysis (LGC Genomics GmbH). Plasmid DNA from recombinants was recovered (Nucleobond® Xtra Midi/Maxi; Macherey-Nagel) and transformed into expression host E. coli BL21 (DE3) as described in the “DNA isolation and recombinant DNA methods” section. The control digest was performed with BamHI/XbaI (Thermo Fisher Scientific GmbH) for 1 h at 37 ◦ C and the sequencing analysis using pET30-specific forward primers revealed an insert rate of 75%. E. coli BL21 (DE3) transformed with the vector pET30a (E. coli BL21 (DE3)-pET30a) served as control. The expression library and controls were stored in 25% glycerol at −80 ◦ C until use. For immunoscreening the expression library was spread on LBKan plates and incubated at 30 ◦ C overnight resulting in 300–500 colonies. Therefore, dilution series of the expression library on LB-Kan plates were performed previously to determine optimal concentration (1:107 ). The agar plate was overlaid with a Protran® membrane and colonies adherent to the membrane were induced by placing on LB-Kan agar containing 1 mM isopropyl-␤d-thiogalactoside (IPTG; Carl Roth GmbH) for 4 h at 37 ◦ C. Colonies on the membrane were partially lysed by incubation in chloroform (Carl Roth GmbH) for 15 s or by exposure to chloroform vapor for


20 min in a glass jar. Filters were air dried at room temperature, blocked with PBS-T containing 5% non-fat milk for 1 h, washed with PBS-T and probed with adsorbed sera 1:1000 in PBS-T containing 5% non-fat milk at 4 ◦ C overnight on a tube roller (Stuart SRT1; Stuart Scientific, Staffordshire, UK). After washing the membranes three times with PBS-T at room temperature, detection of reactive colonies was carried out by incubation with the HRP-Protein G (Genscript, Piscataway, USA) 1:10,000 in PBS-T containing 5% non-fat milk at room temperature for 1 h at a shaking frequency of 15 cycles/min. Reactive colonies were visualized by ECL substrate as described in the “Adsorption of sera” section. To confirm results, putative positive clones were subjected to another two rounds of IVIAT screening. Therefore, individualized clones, stored in 96deep well plates (Macherey-Nagel), were inoculated in a second 96-deep well plate containing 300 ␮l LB-Kan media/well and incubated at 37 ◦ C for 16–18 h under shaking at 200 cycles/min. Aliquots of 1 ␮l of these cultures were spotted directly onto a nitrocellulose membrane placed onto LB-Kan agar and incubated for 1–1.5 h at 37 ◦ C. Induction and detection were carried out as mentioned above. Reactive clones were identified by eye by comparing with control E. coli BL21 (DE3)-pET30a and negative control sera. Clones represented by at least two positive dots in three screenings with the adsorbed sera were considered to be positive. Sequencing using a pET30-specific forward primer was performed by LGC Genomics. Identification of chlamydial proteins expressed during bovine infection All clones were sequenced and analyzed using the NCBI database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). With KEGG database chlamydial proteins were classified in different functional groups (http://www.genome.jp/kegg/pathway.html) and the subcellular localization of proteins was predicted using PSORTb v3.0 (http://psort.org/psortb/index.html). Results and discussion Within the framework of this study we aimed at the elucidation of C. psittaci proteins, which are synthesized during the infection of calves. Thereby, the infection-associated molecular processes are very complex including the expression of specific bacterial virulence genes and the corresponding response of the infected host to combat the pathogen (Knittler et al., 2014). A highly important reaction of the infected host concerns the production of specific antibodies against the pathogen molecules, which are accessible to the host immune system. For this reason we applied calf sera containing antibodies, which were produced against C. psittaci proteins during the infection process (Reinhold et al., 2012). Due to the fact that sera contain also antibodies against chlamydial proteins, which are always present at the surface of or within the pathogen, we enriched the antibodies against the infection-associated proteins by a pretreatment of the sera with lysates of BGM cells infected with C. psittaci. The enriched sera were directly used to screen a chlamydial genomic expression library. This process is known as IVIAT (see Fig. 1), a high throughput immunoscreening technique, which was previously successfully applied to various pathogens, e.g. Salmonella enterica (Alam et al., 2013; Harris et al., 2006), Streptococcus suis (Gu et al., 2009), Escherichia coli (John et al., 2005; Vigil et al., 2011) and others (Amerizadeh et al., 2013; Hang et al., 2003; Kim et al., 2003; Lowry et al., 2010; Rollins et al., 2008; Salim et al., 2005; Yoo et al., 2007). However, to our knowledge this is the first attempt where obligate intracellular bacteria were involved. Like almost all other techniques IVIAT has advantages and disadvantages. Compared to the two major works of Forsbach-Birk (Forsbach-Birk et al., 2013) and Wang (Wang

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et al., 2010) dealing with the human antibody response to chlamydial infections, we provide a rather fast and relatively cheap high-throughput approach, which also focuses on sets of chlamydial immunogenic protein determinants that are expressed in living organisms. However, similar to Rollins et al. (2005), we included an adsorbtion step in order to remove “antibodies that bind antigens expressed during in vitro cultivation”, while specific antibodies against in vivo-induced antigens were more or less sustained. Due to these absorption steps the overall signal intensity decreases, while the ratio of specific signals to the overall background signal improves, i.e. stronger specific signals are obtained. The calf infections were performed with the intracellular bacterium C. psittaci propagated in BGM cells. Therefore, the bacterial sample is unavoidable contaminated with some debris of the host cells, which also contribute to the immune response of the infected host. The first three adsorption steps were performed with lysed and non-lysed E. coli cells to eliminate anti-E. coli cross-reactions (E. coli expression system). Two additional adsorption steps were performed with a lysed C. psittaci isolate, which – as described above – was contaminated with some debris of BGM cells. Another positive effect of the system used is that screening is based on random fragments of the entire chlamydial genome (500–1500 bp in lengths) and not necessarily and exclusively on whole open reading frames. However, our approach requires several controls to guarantee e.g. the specificity of identified signals. In addition, one has to mention that due to the E. coli expression system, IVIAT is unable to provide complete data sets of all proteins which play a significant role during infection (similar to other techniques). Sera adsorption and identification of immunoreactive proteins For the identification of immunoreactive proteins it was important to pool the calf sera with the highest antibody titer to avoid the impact of individual immune responses (Rollins et al., 2005). The pooled sera were stepwise adsorbed against both, the expression host E. coli and C. psittaci grown in BGM cells. Fig. 2A and B shows the results of Western blotting experiments to demonstrate the degree of adsorption by the decreased reactivity of the pooled sera. The comparison of pooled sera with adsorbed sera in infected and non-infected calves (Fig. 2A) revealed a high titer of antibodies against E. coli thus indicating a high bacterial load in cattle. As shown in Fig. 3 different ELISA tests (Gu et al., 2009) were used to demonstrate the gradual depletion of antibodies against E. coli and C. psittaci grown in cell culture. For this, unadsorbed sera revealed a 5 fold higher reactivity against E. coli than sera where anti-E. coli antibodies were removed (Fig. 3A). Fig. 3B shows similar results obtained from experiments in which instead of E. coli, C. psittaci was used, i.e. also an approximately 5 fold higher reactivity of unadsorbed sera. Altogether, the serum was successfully eliminated of antibodies against in vitro grown E. coli and C. psittaci. Since the time point of harvest of C. psittaci infected cells was 46 h post infection, when the inclusions contain predominately infectious elementary bodies (EBs), the preadsorption of sera with lysate leads to a reduction of antibodies against EB proteins and maybe to a bias concerning the amount of anti-RB protein antibodies. Prior to screening we tested the expression library for IPTG inducible protein expression thus allowing us to define the experimental set up as described in “Materials and Methods”. In order to achieve at least 10 fold coverage (Clarke and Carbon, 1976) of the C. psittaci genome, it was necessary to screen approximately 33,000 out of 105,000 clones of the genomic expression library. To define positive clones, series of increasing exposure times, always using the same film material and developing conditions, allowed us to discriminate background signals from potentially immunoreactive clones. By this means the first screening revealed

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Fig. 2. Extensive adsorption of sera eliminated antibodies specific for E. coli and C. psittaci grown in vitro. Comparison of reactivity of unadsorbed and adsorbed sera from C. psittaci infected calves (referred to as +Cps infected calves) and sera from control calf (referred to as −Cps infected calf) against lysates from E. coli BL21(DE3) (BL21 pET) and lysates from ±C. psittaci infected BGM (BGM + Cps/BGM) and HEp-2 cells (HEp + Cps/HEp) by Western blot. (A) Lysates from E. coli BL21(DE3) or (B) lysates from ±Cps infected BGM and HEp-2 cells were run on 4–20% Tris–Glycin PAGE and blotted onto PVDF membranes. In (2A) membranes were probed with pooled sera from +Cps or −Cps infected calves, sera of +Cps or −Cps infected calves adsorbed with E. coli BL21(DE3) and completely adsorbed sera from +Cps or −Cps infected calves. In (2B) pooled sera (left) and completely adsorbed sera of +Cps infected calves (right) were used. Detection of ␤-actin with loading control antibody (antibodies-online GmbH, Aachen, Germany) is shown below. MW, Molecular weight marker; BSA, bovine serum albumin.

approximately 170 putative positive clones. For further screenings the C. psittaci major outer membrane protein (MOMP) was used as a positive control due to its massive immunogenicity and its potential to elicit neutralizing antibodies (Vanrompay et al., 1999; Zhang et al., 1989). For this the whole open reading frame (orf) of MOMP was cloned and expressed in pET30a. Several additional screenings involving the cloned MOMP as positive control and the E. coli containing expression vector as a negative control revealed 79 positive clones (Supplementary Table 2). These clones were subjected to sequence analysis. Thirty-two clones contained DNA fragments of one single gene. A number of 47 clones contained fragments of more than one gene. All these clones were used for Western blot analysis thus allowing to select 18 clones which corresponded precisely to the predicted C. psittaci peptides/proteins (data not shown) (Table 1). Three of the above 18 clones, i.e. trxA, sufB and CPS0B 1025 (encoding pGP6-D-like protein), were subcloned for screening because they contained multiple orfs. Figs. 4 and 5 show the representative colony immunoblots of the above selected 18 candidates expressing 19 different proteins. As expected, screening with the adsorbed sera of non-infected calf, used as a negative control, remained negative. In addition, E. coli containing the empty

pET30a vector also did not reveal any signal. All 19 proteins could be assigned to specific biological functions according to Kyoto Encyclopedia of Genes and Genomes (KEGG) database, Conserved Domain Database (CDD) and/or Clusters of Orthologous Groups of proteins (COG). Furthermore their subcellular locations were predicted using PSORTb v3.0 (Table 1). The presence of multiple transporters for nutrient import supports the above mentioned assumption regarding the bias of identified proteins (Saka et al., 2011). Furthermore Saka and colleagues claim that “the relative absence of the cytoplasmic accessory factors CdsQ and CdsN in the RB forms suggest that additional factors may substitute for these components, as has been suggested for flagellar T3S system. The CdsN homologue CT717 could potentially substitute for CdsN at this stage” (Saka et al., 2011). To this we found, the orthologous CPS0B 0972, which corresponds to CT717 of C. trachomatis. Our lysate preparation involved a modified RIPA-buffer, which is less stringent than the originally described RIPA buffer (Harlow and Lane, 1988) in order to retain the native conformation of the chlamydial proteins. However, we combined the use of the modified RIPA-buffer with moderate sonification (see “Materials and Methods”) to break down host cells and chlamydial outer membrane


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Table 1 Selection of C. psittaci genes identified by IVIAT. Functional categorya

Transport

Locus tag (gene name)b

Function of gene productc

Predicted cellular locationd

Identification number in figures

CPS0B 0063 (sufB) CPS0B 0164

FeS assembly protein SufB Extracellular solute-binding protein ABC transporter ATP-binding protein ABC transporter ATP-binding protein Thioredoxin

Cytoplasm Unknown

B 6

Cytoplasmic Membrane Cytoplasmic Membrane Cytoplasm

8

Cytoplasm

1

Cytoplasm Cytoplasm

9 2

CPS0B 0996

Oxygen-independent coproporphyrinogen III oxidase Dihydrofolate reductase Transcription-repair coupling factor Putative DNA helicase

Cytoplasm

7

CPS0B 0426 CPS0B 0689

Hypothetical protein Hypothetical protein

12 11

CPS0B 1065 CPS0B 1025

Hypothetical protein Virulence protein pGP6-D-like protein Major outer membrane porin Polymorphic outer membrane protein G family protein

Unknown Cytoplasmic Membrane Cytoplasm Cytoplasm Outer Membrane Outer Membrane (may have multiple locations) Unknown Cytoplasm

A 4

Cytoplasm

5

Cytoplasm

10

CPS0B 0593 CPS0B 0636 Posttranslational modification/ Energy production Cofactor metabolism Nucleotide excision repair Transcription/DNA replication, recombination, and repair Hypothetical proteins

Uncharacterized protein family (UPF0137)

CPS0B 0094 (trxA) CPS0B 0934 (hemN) CPS0B 1066 CPS0B 0932

Cell envelope

CPS0B 0059 CPS0B 0319 (pmp17G)

Bacterial secretion system

CPS0B 0598 CPS0B 1012

Flagellar assembly

CPS0B 0971 CPS0B 0972

Inclusion membrane protein A Type II/IV secretion system family protein Type III secretory flagellar biosynthesis Type III secretion system ATPase

14 C

9 D

3 13

a Published and predicted proteins were subjected to KEGG database (http://www.genome.jp/kegg/). If not assigned by KEGG database, proteins were classified according annotation from C. psittaci chromosome (accession # CP002806) or predicted using Cluster of Orthologous Groups. b ,c Results are based on accession number CP002806 (GenBank database, http://www.ncbi.nlm.nih.gov). d PSORTb v.3.0. (http://psort.org/psortb/index.html).

complexes (COMC) according to Manire and Tamura (Manire, 1966; Manire and Tamura, 1967; Tamura and Manire, 1967). The reason that some abundant membrane proteins, e.g. MOMP, Pmp17G and IncA, which are parts of EBs and RBs, were found in our study, could be due to their high abundancy. Supplementary Table 2 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijmm.2014.12.022. Transport and oxidative stress response Four proteins were identified, which belong to the ABC-type transporter system, i.e. the proteins encoded by CPS0B 0063 (SufB), CPS0B 0164 (extracellular solute-binding protein), CPS0B 0593 (ABC transporter ATP-binding protein), and CPS0B 0636 (ABC transporter ATP-binding protein). ABC transporters are a large family of proteins responsible for the transport of e.g. sugars, peptides etc. and also signal transduction (Garmory and Titball, 2004; Tam and Saier, 1993). Bacterial ABC transporters are known to be essential in cell viability, virulence, and pathogenicity (Klein and Lewinson, 2011; Tam and Saier, 1993). In numerous bacteria, including Chlamydia trachomatis and Chlamydia abortus, the immunogenic potential of periplasmic and inner membrane components of ABC transporters was previously proven (Forsbach-Birk et al., 2013; Garmory and Titball, 2004). Three of the above proteins belong to ABC transporters, the first of which, i.e. the extracellular solute binding protein, is known to be specific for the transport of dipeptides (Detmers et al., 2001; Hiles and Higgins, 1986), the second (ABC transporter ATP-binding protein) for the transport of polar amino acids (Hosie and Poole, 2001; Tam and Saier, 1993) and

the third (ABC transporter ATP-binding protein) for the transport of nitrate/sulfonate/bicarbonate, respectively (Kertesz, 1999; Tam and Saier, 1993). The fourth, SufB, is involved in the [Fe–S] cluster assembly (Outten et al., 2003). The extracellular solute-binding protein as well as the periplasmic permease SufB bind and transfer their respective incoming substrates (Young and Holland, 1999) to the inner membrane part of the ABC transporter (Garmory and Titball, 2004; Hiles and Higgins, 1986). In contrast, the identified ATPase components of both, polar amino acid transporter and inorganic ion transporter, are located at the inner membrane. The ATP-binding proteins provide the energy for the active transport by hydrolyzing ATP (Holland and Blight, 1999). The above four transporters ensure bacteria the uptake of important compounds, e.g. sulfur or amino acids, for cell survival and growth dependent on the ecological niche. The transporters responsible for the import of dipeptides allow bacteria the sensing/monitoring of the local environment. These dipeptides can also be used as a source of nitrogen or metabolic energy (Detmers et al., 2001). The polar amino acid transporter found in our study and elsewhere (Forsbach-Birk et al., 2013) has multiple functions in the carbon and nitrogen metabolism (Hosie and Poole, 2001; Hütter and Niederberger, 1983). The ABC transporter ATP-binding protein carries ions, like sulfonate, which are required by the bacteria for the synthesis of the amino acids cysteine and methionine or cofactors or as building block for [Fe–S] clusters (Kertesz, 1999). For the assembly of [Fe–S] clusters one of three bacterial sulfur transfer/mobilization mechanisms (Frazzon and Dean, 2003; Takahashi and Tokumoto, 2002) was evolved in Chlamydiaceae (Ellis et al., 2001), which is encoded i.e. by the suf operon (sufBCDS) of C. psittaci (Schöfl et al., 2011).

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Fig. 5. Colony immunoblot of the heterologous expressed C. psittaci (Cps) genes. Subclones were treated similarly as selected clones in Fig. 4 and described in “Materials and methods” section. Listed sera correspond to sera in Fig. 4. In addition, response of monoclonal anti-hexahistidine tag antibody (anti-(His)6 -tag mAB; Dianova, Hamburg, Germany) to the heterologous expressed Cps genes is shown in the last row. TrxA and pGP6-D-like protein are fusion proteins expressing Nterminal (His)6 -tag. MOMP and SufB are recombinant proteins without N-terminal (His)6 -tag. Numeration (#) is consistent with Table 1 and represents Cps protein designation or locus tag based on accession no. CP002806.

Fig. 3. Stepwise decrease of sera reactivity against E. coli and C. psittaci with increasing adsorption steps. Adsorption of sera as demonstrated by enzyme immunoassay reactivities of sera samples with lysates of in vitro grown E. coli and C. psittaci. ELISA plates were coated (A) with cleared lysats of E. coli BL21 (DE3) and (B) with whole cell extracts of C. psittaci-infected cells. OD values were corrected for background.

The suf operon is known to be induced under oxidative stress and iron starvation (Frazzon and Dean, 2003; Outten et al., 2003). In Mycobacterium tuberculosis the SUF machinery is the only system of [Fe–S] cluster assembly and repair (Huet et al., 2005). By comparison with the E. coli SUF machinery, authors showed that the mycobacterial suf operon encodes SufB-, SufD-, SufC-, and SufS-like proteins (Huet et al., 2005). The SufB-encoding pps1 gene harbors an intein invading sequence and hence, the blockage of the protein splicing of SufB was suggested to fight tuberculosis (Huet et al., 2005). No transmembrane segments were identified for SufB and SufD. Only a single nucleotide binding domain within SufC indicates this protein being part of an incomplete transporter (Huet et al., 2005). In 2011, Kumar et al. (2011) investigated that the first gene of the SUF system (Rv1460), a probable transcriptional regulatory protein of the suf genes, is highly expressed in patients with pulmonar tuberculosis. In our study the 54 kDa antigen SufB was expressed during the infection process in the bovine lungs (Fig. 5). C. psittaci SufB harbors 37% (56% query coverage) and 55% identity (97% query coverage) with SufB from Mycobacterium tuberculosis and E. coli, respectively. The highest similarity of C. psittaci SufB with SufB from mycobacterial species is obtained with SufB from M. columbiense (49% identity, 97% query coverage) and M. avium

(39% identity, 97% query coverage). According to the analysis of M. tuberculosis genes involved in [Fe–S] cluster biosynthetic systems (Huet et al., 2005) C. psittaci harbors orthologous genes of the Isc (iron sulfur cluster) system and Nif (nitrogen fixation) system, found in E. coli, outside the suf locus. CPS0B 0438 and CPS0B 0968 encode cysteine desulfurases, the first of which is a NifS-like protein. CPS0B 0969 encodes a putative IscU- and NifU-like protein. If and how these proteins contribute to the chlamydial [Fe–S] cluster assembly or if the SUF machinery is the unique system used, has to be investigated. Furthermore, the E. coli glutamine transporter GlnQ homolog in Streptococci (99% query coverage; 65% identity), which seems to correspond to the polar amino acid transporter protein found in our study (88% query coverage; 37% identity), was involved in virulence (Tamura et al., 2002). Mutated glnQ showed a decreased adherence to fibronectin and respiratory epithelial cells A549 (Tamura et al., 2002). What is more, mutants were defective to invade A549 cells in vitro and neonatal rats in an intraperitoneal-injection model, confirming decreased virulence in vivo (Tamura et al., 2002). The authors speculated that in glnQ mutants the nutritional defect i.e. reduced cytoplasmic glutamine level is the cause for the decrease in fibronectin adherence (Tamura et al., 2002). In addition, metabolism and microbial pathogenesis are linked (Rohmer et al., 2011). Therefore, gene expression of virulence factors may be regulated through alterations in glutamine levels in vitro and in vivo (Tamura et al., 2002). Furthermore, mice infected with Streptococcus pneumonia mutated in one glutamine uptake system (D39gln0411/0412) showed significantly increased survival times indicating the requirement of the glutamine uptake system for virulence (Härtel et al., 2011) C. psittaci infection stimulate ATP synthesis and synthesis of glutamate in HeLa cells, both peaked around 24 h post infection (Ojcius et al., 1998). An increased concentration of glutamate in the bovine lung cells may therefore stimulate the bacterial glutamine transporter required for

Fig. 4. Serum IgG response of the selected clones by colony immunoblot. Representative secondary screening of clones that had been selected from primary screening using pooled and adsorbed sera of C. psittaci infected calves (Cps positive sera) and sera of control calf (Cps negative sera). E. coli expressing positive control MOMP (ultimate left) and E. coli control strain carrying an empty expression vector (ultimate right) are included. Numeration (#) is consistent with Table 1 and represents C. psittaci protein designation or locus tag based on accession no. CP002806. 1066a , clone with multiple genes expressing both, CPS0B 1066 and CPS0B 1065.


adherence and hence virulence (Ostermann et al., 2013a; Reinhold et al., 2012). Moreover, the activity of the glutamate transporter GadC in Francisella is required for the bacterium to neutralize the production of reactive oxygen species in the phagosome (Ramond et al., 2014). Hence a pathogenic linkage between the glutamate transport, the oxidative stress response and the phagosome exists thus guaranteeing the intracellular bacteria’s survival (Ramond et al., 2014). In addition to the ABC transporter proteins described above, we identified C. psittaci thioredoxin TrxA. As a common cellular defense strategy, the production of reactive oxygen species (ROS) is induced by the infection of the host cells by bacteria like Chlamydiae (Boncompain et al., 2010; Prusty et al., 2012). Reactive oxygen species are generated by the NADPH oxidase in phagocytes consisting of two membrane proteins and cytosolic regulatory proteins (e.g. Ras-related C3 botulinum toxin substrate, Rac) or as a byproduct in mitochondria or peroxisomes (Bedard and Krause, 2007). Alveolar macrophages were present in C. psittaci infected lungs indicating that the innate immune system was active (Reinhold et al., 2012). Furthermore, hypoxia in the lung tissue was observed (Ostermann et al., 2013b). This indicates that the ROS contributes to oxidative stress (Bedard and Krause, 2007). ROS can damage the DNA or [Fe–S] cluster (Imlay, 2003; Koháryová and Kolárová, 2008). To allow C. trachomatis to escape the immune defense, they inactivate the NADPH oxidase by the removal of Rac (Boncompain et al., 2010). Our study revealed that thioredoxin (trxA) is expressed during infection (Fig. 5). TrxA is known to be involved in the regulation of oxidative stress (Koháryová and Kolárová, 2008). It alters the redox state of proteins, scavenges reactive oxygen species (Koháryová and ´ 2001) and mediates redox Kolárová, 2008; Nordberg and Arner, dependent gene regulation (Zeller and Klug, 2006). Thioredoxin promotes virulence (Anwar et al., 2013; Bjur et al., 2006; Missall and Lodge, 2005; Rocha et al., 2007) i.e. by assisting the activity of the virulence associated Salmonella pathogenicity island (SPI2) type III secretion system (Negrea et al., 2009). Also for the chlamydial type III secretion system a similar redox-dependency of TrxA could be assumed (Betts-Hampikian and Fields, 2011; Negrea et al., 2009). Heme and folate biosynthesis C. psittaci is able to synthesize folate de novo (Adams et al., 2014) and encodes all genes required for heme biosynthesis. IVIAT revealed the oxygen-independent coproporphyrinogen III oxidase HemN. HemN belongs to a cascade of catalytic enzymes, which are encoded in a cluster of genes, involved in the biosynthesis of heme (Heinemann et al., 2008). Heme is a prosthetic group of e.g. peroxidases, catalases or cytochromes that mediate redox reactions. Furthermore, proteins containing heme are involved in many cellular processes that are coupled to the energy metabolism (Frankenberg et al., 2003). Heme biosynthesis includes the incorporation of iron into the protoporphyrin (Heinemann et al., 2008). Iron is an essential nutrient for virtually all organisms, not only for general cellular processes that base on its redox capacity, but also for virulence and replication (Wilks and Burkhard, 2007). The requirement for iron in C. psittaci may compete with the iron requirement of the calves for mounting an effective immune response (Beard, 2001; Reinhold et al., 2012). Recently, Engström and colleagues demonstrated in vitro a strong impact of the protoporphyrinogen oxidase (HemG) on the infectivity of C. trachomatis (Engström et al., 2013). In our studies we found HemN to be expressed during infection, which belongs to the heme biosynthesis pathway (Heinemann et al., 2008; Layer et al., 2010). In addition to the above described HemN, which is required for the cofactor heme biosynthesis, also the dihydrofolate reductase FolA was found via IVIAT, which is involved in the folate

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metabolism (Fan et al., 1992). FolA converts dihydrofolate in 5,6,7,8-tetrahydrofolate which is required for DNA synthesis, DNA replication and amino acid synthesis (Hartman, 1993). All investigated Chlamydia species, except C. pecorum, synthesize active folate (Adams et al., 2014; Sait et al., 2014). Folate was shown to play a significant role concerning virulence in various animals infected by Leishmania (Vickers and Beverley, 2011). The above influence on virulence of folate was also demonstrated for Francisella tularensis infections (Ulland et al., 2013). In addition, we found the putative folate metabolism gamma-glutamate ligase FolC2 (CPS0B 1065) to be expressed during infection. FolC2 is a homolog of archael CofE that belongs to the COG1478 family and is involved in the glutamylation of the archael cofactor F420 (Adams et al., 2014; de Crécy-Lagard et al., 2007). In Chlamydia FolC2 performs the glutamylation of folate (de Crécy-Lagard et al., 2007). For this reason we suppose that FolC2 is also involved in virulence of C. psittaci in calves (Ostermann et al., 2013a; Reinhold et al., 2012). Moreover, the FolC2 ortholog of C. muridarum was shown to be immunodominant upon infection of mice with live EB (Cruz-Fisher et al., 2011). DNA replication, recombination, and repair Two other proteins, the transcription-repair coupling factor (TRCF) and a putative DNA helicase, which were also found, are essential for basal bacterial DNA replication, DNA recombination, DNA repair (Lohman and Bjornson, 1996) and hence survival and proliferation of a cell (Jacob et al., 1963). The chlamydial TRCF senses DNA lesions on the actively transcribed strand and recruits the nucleotide excision repair machinery to this site (Deaconescu et al., 2006; Mellon and Hanawalt, 1989). The recognition of damaged DNA by TRCF and the involved repair mechanism likely promote infection as shown for the recombinational repair proteins AddAB in the recombinationcoupled DNA repair in Helicobacter pylori (Amundsen et al., 2008). The TRC factor could contribute to the chlamydial stress response i.e. the repair of DNA damage encountered during oxidative stress in infected calves (Ostermann et al., 2013a,b; Reinhold et al., 2012). The second immunoreactive protein is a putative DNA helicase. Both, this protein and TRCF belong to the superfamily II helicases and share the helicase superfamily C-terminal domain (Bork and Koonin, 1993). Since this domain is found in a wide variety of helicases and helicase-related proteins within eukaryotes and prokaryotes, both identified proteins may not be appropriate drug targets. However, structural comparisons of the chlamydial helicase with its eukaryotic counterparts could indicate whether it is a potential target for antibacterial drug discovery (Sanyal and Doig, 2012). We assume that the chlamydial helicase participates in DNA decondensation observed during the conversion of EBs to RBs in calves (Grieshaber et al., 2004; Reinhold et al., 2012; Smith and Peterson, 2005) which vice versa was attributed to histon-like proteins (Barry et al., 1992; Hackstadt et al., 1991). The mycobacterial DNA helicase XPB that belongs to the superfamily II helicases catalyzes not only the unwinding of DNA, but has also intrinsic strand annealing activity (Balasingham et al., 2012). Therefore, the activity of XPB is attributed to the pathogenesis of Mycobacterium (Balasingham et al., 2012). Proteins with unknown function/hypothetical proteins The C. psittaci genome comprises many hypothetical proteins with hitherto unknown functions. We found that three of them are induced in vivo (Figs. 4 and 5). The first is encoded by CPS0B 0689 and is predicted to be localized at the inner membrane. The second protein, which is encoded by CPS0B 0426, is predicted to

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be secreted by the chlamydial type III secretion system (Voigt et al., 2012). The third one, i.e. pGP6-D-like protein, belongs to the same uncharacterized protein family UPF0137 like the C. psittaci virulence plasmid-encoded protein pGP6-D. We predicted pathogenicity of the gene products of CPS0B 0689 (AEG87625) and CPS0B 0426 (AEG87390) in the infected calves by means of the software tool MP3 (http://metagenomics.iiserb.ac.in/mp3/index.php) that combines both, the support vector machine (SVM) and the hidden Markov model (HMM) (Gupta et al., 2014). The hypothetical protein encoded by CPS0B 0426 from C. psittaci corresponds to the orthologous gene product of TC 0381 from C. muridarum which was shown to be immunodominant in BALB/c mice (Cruz-Fisher et al., 2011). Cell envelope proteins Out of the 19 proteins which we identified to be expressed during infection, three were classified as membrane proteins, the major outer membrane protein (MOMP), the polymorphic membrane protein 17G (Pmp17G) and the inclusion membrane protein A (IncA). MOMP and Pmp are surface exposed proteins in the outer membrane (Caldwell et al., 1981; Byrne, 2010; Tan et al., 2009). IncA which harbors two transmembrane regions (Beeckman et al., 2008; Rockey et al., 2000) is exposed to the cytoplasmic face of the chlamydial inclusion (Rockey et al., 1997). Analysis of the humoral response against chlamydial infections in other large-scale studies uncovered MOMP, Pmps and IncA as immunodominant proteins within various Chlamydiae (Cruz-Fisher et al., 2011; Forsbach-Birk et al., 2013; Wang et al., 2010). The cysteine-rich MOMP (Bavoil et al., 1984) is the dominant surface protein (Caldwell et al., 1981) that functions as porin-like ion channel (Findlay et al., 2005; Wyllie et al., 1998). In addition, MOMP has been implicated to the attachment of C. trachomatis to the host cell (Su et al., 1988, 1990) and was shown to elicit neutralizing antibodies (e.g. Kari et al., 2009; Murdin et al., 1993; Peterson et al., 1991; Zhang et al., 1987). Pmps are characterized by a C-terminal autotransporter-like domain (Henderson and Lam, 2001), a central pmp middle domain and short repetitive peptide motifs, GGA (I, L, V) and FxxN, within the N-terminal part of the protein (Grimwood and Stephens, 1999; Voigt et al., 2012). These peptide motifs were shown to be essential for the adhesion of EBs to human cells (Becker and Hegemann, 2014; Mölleken et al., 2010). Although virulence and tissue tropism were assigned to Pmps (Gomes et al., 2006; Nunes et al., 2007), their functions are not fully understood (Byrne, 2010; Tanzer et al., 2001). Pmps are genetically highly variable thus eliciting various serologic responses (Abdelsamed et al., 2013; Tan et al., 2009; Taylor et al., 2011), which might offer the chlamydial species to adapt to specific niches and to evade the host immune system. Thus Pmps may be important virulence factors of C. psittaci in calves (Reinhold et al., 2012). IncA possesses a bilobed-hydrophobic secondary structure that is thought to enable the insertion of IncA into the chlamydial inclusion membrane (Mital et al., 2013; Rockey et al., 2000). Due to their exposure to the host cell cytosol they may mediate host–pathogen communication (Fields and Hackstadt, 2002). We suggest that the bovine anti-IncA antibodies might contribute to the destabilization of the chlamydial inclusion thus hindering the bacteria to propagate (Reinhold et al., 2012). MOMP, Pmps and IncA exhibit a high degree of variability thus evading host response and partly impact disease severity (Abdelsamed et al., 2013). Bacterial secretion and effector proteins Three immunogenic proteins detected, are parts of the type II and III secretion apparatuses, namely the cytoplasmic type

II/IV secretion system family protein (CPS0B 1012), the type III secretory flagellar biosynthesis protein (CPS0B 0971) and the type III secretion system ATPase (CPS0B 0972). The cytoplasmic type II/IV secretion system family protein encoded by CPS0B 1012 belongs to the bacterial type II secretion system (Beeckman and Vanrompay, 2010). This two-step secretion process is responsible for the energy-dependent transport of lytic enzymes and/or virulence factors (Beeckman and Vanrompay, 2010; Hueck, 1998). It is a homolog of PulE from Klebsiella oxytoca which is essential for the secretion of the debranching enzyme pullulanase (Possot et al., 1992). The chlamydial type III secretory flagellar biosynthesis protein and the type III secretion system ATPase are orthologs of the flagella proteins FliH and FliI encoded by the “flagella genes” CPS0B 0971 and CPS0B 0972, respectively. They are paralogs of SctL/CdsL and SctN/CdsN of the chlamydial non-flagellar type III secretion system (injectisome) that evolved from the flagellum (Abby and Rocha, 2012; Beeckman and Vanrompay, 2010; Stone et al., 2008). Chlamydiae encode a complete type III secretion system and, in addition, a rudimentary basal body composed of the chlamydial FliI, FliH, FliN, FliF and FliA homologs (Betts-Hampikian and Fields, 2010). C. psittaci FliH is a putative effector protein of the type III secretion system (Voigt et al., 2012). Stone and co-workers observed interactions of the flagellar proteins with proteins of the type III secretion system (Stone et al., 2010). It was proposed that the flagellar proteins assist the chlamydial type III secretion by providing alternate components for the basal body of the secretion apparatus which is correlated with the developmental cycle (Beeckman and Vanrompay, 2010; Stone et al., 2010). Recently, effector proteins of C. psittaci that are secreted into the host cell cytosol via type III secretion system were predicted in silico (Voigt et al., 2012). In our study we found the above type III secretory flagellar biosynthesis protein, as well as IncA, Pmp17G, HemN, SufB, ABC transporter ATP-binding protein (CPS0B 0593), transcription-repair coupling factor (CPS0B 932) and the hypothetical protein (CPS0B 0426). Chlamydiae secrete effector proteins to manipulate its host, e.g. by phosphorylation and dephosphorylation of host cell proteins or by the induction of proteolysis to ensure efficient propagation and/or to evade the host immune defense (Betts et al., 2009; BettsHampikian and Fields, 2010; Zhong, 2009).

Conclusion While the obligate intracellular way of life essentially protects pathogens from the host defence mechanisms more or less, various proteins are highly immunogenic. For this reason we identified immunogenic proteins during the infection process of C. psittaci in calves. Our study revealed 19 chlamydial immunoreactive proteins, most of which have never been associated with chlamydial mechanisms with respect to host cell invasion, production and secretion of anti-host proteins, nutrient acquisition and host immune response. They represent members of protein groups involved in a broad spectrum of such infection-associated mechanisms, i.e. transport and oxidative stress response, heme and folate biosynthesis, DNA replication, recombination and repair, cell envelope and bacterial secretion, and some proteins with putative infection-linked functions. Some of the proteins found, like the ABC importers, folate biosynthesis-related proteins, the type III secretion and effector proteins, as well as Pmps are exclusively found in Chlamydiae and therefore, may represent ideal targets for the development of antibacterial vaccines, diagnostics or therapeutic approaches and finally, for the prevention of chronic chlamydial infections.


Acknowledgements We are grateful to Konrad Sachse, Petra Reinhold and Angela Berndt (Friedrich-Loeffler-Institute (Federal Research Institute for Animal Health), Institute of Molecular Pathogenesis, Jena) for providing the strain material and the sera samples. We thank Muslihudeen Abdul-Aziz for critical reading of the manuscript. This study was financially supported by the Federal Ministry of Education and Research (BMBF) of Germany under Grant no. 01KI1011D: Joint project: Zoonotic chlamydia: Interaction of zoonotic chlamydia with their host cells (Project 4).

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Risk factors associated with Chlamydia psittaci infection in psittacine birds.

Protection of sheep against Chlamydia psittaci infection with a subcellular vaccine containing the major outer membrane protein.

Growth of Chlamydia psittaci in macrophages.

Host-pathogen interactions in specific pathogen-free chickens following aerogenous infection with Chlamydia psittaci and Chlamydia abortus.

Infective endocarditis with glomerulonephritis associated with cat chlamydia (C. psittaci) infection.

Chlamydia psittaci inclusion membrane protein IncB associates with host protein Snapin.

Immunofluorescence of peritoneal phagocytes after infection of mice with L-cell-attenuated Chlamydia psittaci 6BC.

Immunoelectron microscopy of Chlamydia psittaci with monoclonal antibodies.

Interaction of Chlamydia psittaci reticulate bodies with mouse peritoneal macrophages.

Host-Microbe Protein Interactions during Bacterial Infection.

Insidious endocarditis caused by Chlamydia psittaci.

Immunity to Chlamydia psittaci with particular reference to sheep.

Hepatitis in a family infected by Chlamydia psittaci.

Multiple tandem promoters of the major outer membrane protein gene (omp1) of Chlamydia psittaci.

Chlamydia psittaci infection increases mortality of avian influenza virus H9N2 by suppressing host immune response.

Abortion in Japanese cows caused by Chlamydia psittaci.

Isolation of Chlamydia psittaci from pleural effusion in a dog.

Prevalence of Chlamydia psittaci and Other Chlamydia Species in Wild Birds in Poland.

Chlamydia psittaci in Eurasian Collared Doves (Streptopelia decaocto) in Italy.

Muramic acid is not detectable in Chlamydia psittaci or Chlamydia trachomatis by gas chromatography-mass spectrometry.

Pregnancy complicated by severe Chlamydia psittaci infection acquired from a goat flock: a case report.

Use of HeLa cell guanine nucleotides by Chlamydia psittaci.

Parasite-specified phagocytosis of Chlamydia psittaci and Chlamydia trachomatis by L and HeLa cells.

Cloning and sequence analysis of the major outer membrane protein gene of Chlamydia psittaci 6BC.