Raman microspectroscopy reveals longterm ... - Wiley Online Library

Molecular Microbiology (2010) 77(3), 687–700 䊏

doi:10.1111/j.1365-2958.2010.07241.x First published online 11 June 2010

Raman microspectroscopy reveals long-term extracellular activity of chlamydiae mmi_7241 687..700

Susanne Haider,1 Michael Wagner,1* Markus C. Schmid,1 Barbara S. Sixt,1 Jan G. Christian,2,3 Georg Häcker,3 Peter Pichler,4 Karl Mechtler,5 Albert Müller,1 Christian Baranyi,1 Elena R. Toenshoff,1 Jacqueline Montanaro1 and Matthias Horn1 1 Department of Microbial Ecology, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria. 2 Institute for Medical Microbiology, Immunology, and Hygiene, Technical University Munich, Trogerstr. 30, 81675 Munich, Germany. 3 Institute for Medical Microbiology and Hygiene, Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Str. 11, 79104 Freiburg, Germany. 4 Christian Doppler Laboratory for Proteome Analysis, University of Vienna, Dr. Bohr-Gasse 9, 1030 Vienna, Austria. 5 IMP/IMBA, Dr. Bohr-Gasse 3, 1030 Vienna, Austria.

Summary The phylum Chlamydiae consists exclusively of obligate intracellular bacteria. Some of them are formidable pathogens of humans, while others occur as symbionts of amoebae. These genetically intractable bacteria possess a developmental cycle consisting of replicative reticulate bodies and infectious elementary bodies, which are believed to be physiologically inactive. Confocal Raman microspectroscopy was applied to differentiate between reticulate bodies and elementary bodies of Protochlamydia amoebophila and to demonstrate in situ the labelling of this amoeba symbiont after addition of isotope-labelled phenylalanine. Unexpectedly, uptake of this amino acid was also observed for both developmental stages for up to 3 weeks, if incubated extracellularly with labelled phenylalanine, and P. amoebophila remained infective during this period. Furthermore, P. amoebophila energizes its membrane and performs protein synthesis outside of its host. Importantly, amino acid uptake and protein synthesis after Accepted 21 May, 2010. *For correspondence. E-mail wagner@ microbial-ecology.net; Tel. (+43) 1 4277 54390; Fax (+43) 1 4277 54389.

© 2010 Blackwell Publishing Ltd

extended extracellular incubation could also be demonstrated for the human pathogen Chlamydia trachomatis, which synthesizes stress-related proteins under these conditions as shown by 2-D gel electrophoresis and MALDI-TOF/TOF mass spectrometry. These findings change our perception of chlamydial biology and reveal that host-free analyses possess a previously not recognized potential for direct experimental access to these elusive microorganisms.

Introduction Chlamydiae comprise many important human pathogens causing a variety of severe diseases such as pneumonia, trachoma (with over 90 million cases per year worldwide), and urogenital tract infections, which are a major cause of female infertility and make Chlamydia trachomatis the most frequently sexually transmitted bacterial pathogen (Schachter, 1999; WHO, 2001; 2008). However, the chlamydial host range is much broader and spans large parts of the animal kingdom. In particular, free-living amoebae harbour a wide variety of chlamydial symbionts (Fritsche et al., 2000; Horn et al., 2000; 2008; Corsaro et al., 2003; Schmitz-Esser et al., 2008) and some of these recently discovered intracellular bacteria can also infect human cells and are considered potential human pathogens (Greub and Raoult, 2002a; Horn, 2008). A hallmark of all chlamydiae is the intracellular developmental cycle, which consists of reticulate bodies (RBs), elementary bodies (EBs) and intermediate bodies (IBs) (Ward, 1988; Hatch, 1999; Greub and Raoult, 2002b; Abdelrahman and Belland, 2005). RBs represent the intracellular life stage; they are metabolically active and multiply inside host-derived vacuoles. The EB is a sporelike stage, which is considered to be metabolically inert, adapted for extracellular survival and infection of new host cells. IBs represent the transition stages between RBs and EBs. All chlamydiae possess reduced biosynthetic capabilities compared with free-living bacteria and thus must rely on the import of essential compounds such as nucleotides and amino acids from their host cells (Hatch, 1975; Tipples and McClarty, 1993; McClarty, 1994; Al-Younes et al., 2006). Because of this metabolic dependency on their hosts and their obligate intracellular developmental cycle, chlamydiae have been referred to as the

688 S. Haider et al. 䊏

ultimate auxotroph (Hatch, 1988). However, the interactions of chlamydiae with their host cells were up to now only inferred indirectly by comparative genomics (Stephens et al., 1998; Kalman et al., 1999; Stephens, 1999; Read et al., 2000; 2003; Horn et al., 2006), heterologous expression of transporters (Haferkamp et al., 2004; 2006; Schmitz-Esser et al., 2004; Trentmann et al., 2007) or by labelling studies with host-free chlamydial cells immediately after host cell lysis (Sarov and Becker, 1971; Hatch et al., 1982; 1985), but direct experimental confirmation of intracellular substrate uptake of chlamydial cells was not yet achieved. In this study we used Raman microspectroscopy to investigate the intra- and extracellular metabolic activity of Protochlamydia amoebophila, a model organism for symbiotic chlamydiae and the only non-pathogenic chlamydia for which a genome sequence is available (Horn et al., 2004). Furthermore, this technique was applied to study the host-free activity of the human pathogen C. trachomatis. Raman microspectroscopy is commonly used in chemistry for the identification of molecules and the characterization of materials (Baena and Lendl, 2004; De Gelder et al., 2007). Raman spectra provide fingerprints of the chemical composition of the analysed samples and can thus also be used for the characterization and differentiation of bacteria (Maquelin et al., 2002; Buijtels et al., 2008). The great potential of Raman microspectroscopy for biologists, however, lies in the observation that incorporation of heavy isotopes into cell compounds leads to pronounced changes (peak shifts) in the respective parts of the whole cell Raman spectrum (Huang et al., 2004; van Manen et al., 2005; 2008; Matthaus et al., 2008). This effect has recently been exploited by microbial ecologists to analyse the uptake and incorporation of labelled substrates into free-living bacteria on a single-cell level, without the need for cultivation (Huang et al., 2007). Here we used Raman microspectroscopy to differentiate between RBs and EBs of P. amoebophila and to proof that both developmental forms take up the 13C-labelled amino acid phenylalanine within the amoebal host. Furthermore, we demonstrate uptake of this amino acid and thus metabolic activity of P. amoebophila and C. trachomatis EBs in the absence of host cells and reveal extracellular protein biosynthesis of both chlamydial species by proteomic analyses.

Results and discussion Raman microspectroscopy-based identification of P. amoebophila RBs and EBs Identification of the two different life stages of chlamydiae is a fundamental prerequisite for investigating functional differences between them and for understanding the developmental cycle of these microorganisms. Tradition-

ally this differentiation is based on morphological criteria like the presence of condensed chromatin in EBs as observed by transmission electron microscopy (Rake, 1957; Ward, 1983; Matsumoto, 1988; Ward, 1988; Barry et al., 1992). To test whether this task can also be achieved by Raman microspectroscopy, RBs and EBs of P. amoebophila were physically separated by density gradient centrifugation (Friis, 1972; Howard et al., 1974; Knudsen et al., 1999) and subsequently aliquots of the respective fractions were analysed by transmission electron microscopy and Raman microspectroscopy respectively. However, for interpreting the results it should be noticed that this separation is not perfect (Yong et al., 1979; Caldwell et al., 1981) and that intermediate forms exist (Friis, 1972; Chi et al., 1987; Ward, 1988; Hatch, 1999). According to transmission electron microscopy 76% EBs, 16% IBs and 8% RBs were detected in the EB fraction (n = 530 cells), and 61% RBs, 34% IBs and 5% EBs were found in the RB fraction (n = 1106 cells). Raman spectra from cells from the EB fraction (n = 44) could be clustered in two groups. Ninety-five per cent of the spectra were highly similar with each other and were thus considered to represent EBs (including IBs with an EB-like chemical composition), while 5% of the spectra were clearly different and were thus assigned to the RBs (including IBs with a RB-like chemical composition). The same two groups of spectra were retrieved from the RB fraction (n = 93 cells), but in this case 76% were RB spectra while only 24% were EB spectra. Thus, the numbers of RBs and EBs determined in the two fractions were comparable but not identical between Raman and transmission electron microscopic analyses. The differences most likely reflect that IBs cannot be differentiated by Raman and that cells which are classified as IBs based on morphological criteria are, dependent on their chemical composition, either assigned to RBs or EBs by Raman. Further support for the reliability of the Raman-based RB/EB differentiation is provided by Raman-based analyses of P. amoebophila at different time points after infection of its amoebal host. After 1 h, 72 h or 96 h, 90% EBs and 10% RBs (n = 10), 20% EBs and 80% RBs (n = 10) and 64% EBs and 36% RBs (n = 11) were detected, respectively, and these numbers are fully consistent with the developmental cycle of P. amoebophila in Acanthamoeba sp. that is completed 96 h post infection (S. Haider, M. Wagner, A. Müller and M. Horn, manuscript in preparation). Raman spectra of RBs were clearly different from EB spectra (Fig. 1) and principal component analysis revealed that this discrimination is statistically significant and can be mainly attributed to three peaks at wavelengths of 746, 1585 and 1127 cm-1 (Fig. S1) of which the former two are absent and the latter is much less pronounced in EB spectra (Fig. 1). These peaks resemble three of the four peaks known to represent cytochrome c © 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 687–700

Intra- and extracellular activity of chlamydiae 689 A

B

Raman intensity

746 cm-1

1127 cm-1

1585 cm-1

RB

Fig. 1. Differentiation of protochlamydial RBs and EBs by Raman microspectroscopy. A. Comparison of mean Raman spectra of RBs (blue) and EBs (red). For each developmental stage 12 spectra were merged. The wave numbers of three peaks that are more pronounced in RB spectra than in EB spectra are indicated. B. Electron microscopic images of P. amoebophila RB (upper figure) and an EB (lower figure). Scale bars represent 200 nm.

EB 500

700

900 1100 1300 1500 Raman shift wave number (cm-1)

(Pätzold et al., 2008), but apparently do not originate from this compound as the fourth peak at 1311 cm-1, described for cytochrome c, does not vary significantly between RB and EB spectra. We did not find any unambiguous correlation between the three peaks and reported spectra of other biological molecules, but noticed that the peaks might represent nucleobases (De Gelder et al., 2007) and thus might reflect a higher DNA/RNA content in RBs. This is further supported by the observation of an increased RNA content in RBs compared with EBs, when P. amoebophila cell fractions were stained with the nucleic acid dye acridine orange (Fig. S2). Intracellular uptake of phenylalanine by P. amoebophila Raman microspectroscopy was subsequently used to test whether P. amoebophila takes up phenylalanine from its amoeba host during intracellular growth. Phenylalanine was selected as model substrate, because according to the annotation of the P. amoebophila genome the biosynthesis pathway for this amino acid is incomplete and the organism uses a proton/sodium neutral amino acid symporter for uptake of this essential amino acid from the host (Horn et al., 2004; 2006). However, as 62% of the genes of P. amoebophila lack homology to genes with recognized function, in silico genome analysis is insufficient to prove absence of a certain pathway and to infer the respective host-symbiont interaction. We investigated this interaction by combining EB and RB differentiation by Raman microspectroscopy with the recently documented capability of this technique to detect labelling of individual cells with stable isotope-tagged compounds (van Manen et al., 2005; 2008; Huang et al., 2007; Matthaus et al., 2008). Phenylalanine is ideally suited for such analyses as its Raman peak at 1003 cm-1, which is caused by the symmetric aromatic ring breathing mode (Wei et al., 2008), is easily detectable in EB and RB spectra of P. amoebophila and shows a pronounced shift to 967 cm-1 if the aromatic ring is fully 13C-labelled (Fig. 2). For the experiments, an

1700

unsynchronized amoeba culture containing P. amoebophila RBs and EBs as well as IBs was grown for different time periods in a chemically defined Acanthamoeba medium [DGM-21A (Schuster, 2002)] containing [13C9,15N]L-phenylalanine (13C-phenylalanine) as the only source of this amino acid. Subsequently, the amoeba host cells were lysed and the released symbionts were immediately monitored for uptake of the labelled amino acid by Raman microspectroscopy. After 264 h of incubation with labelled phenylalanine, the spectra of P. amoebophila showed a marked decrease of three peaks known to originate from unlabelled phenylalanine in bacterial biomass (Huang et al., 2004; Notingher et al., 2004), while three new peaks with a shifted wave number appeared representing the uptake/incorporation of labelled phenylalanine (Fig. 2). Almost identical peak shifts were observed when Raman spectra recorded with pure labelled or unlabelled phenylalanine were compared (Fig. S3). For all subsequent analyses, the most pronounced peak shift from 1003 cm-1 to 967 cm-1, representing the unlabelled and labelled aromatic ring (Wei et al., 2008), respectively, was used as indicator for phenylalanine uptake. Already after 24 h of incubation, the spectra of 50% of the P. amoebophila cells demonstrated phenylalanine uptake, while no peak shift was observed in the spectra of all cells in the corresponding control experiment with unlabelled phenylalanine (Fig. 3A). With longer incubation times with 13C-phenylalanine, the number of labelled cells increased, and after 120 h all chlamydial cells were labelled (Fig. 3A). In some cells the 12C-phenylalanine peak almost disappeared compared with spectra from unlabelled cells of the control experiment, unambiguously demonstrating that the observed peak shift was due to incorporation of the labelled amino acid into the biomass of the symbionts (Fig. S4). Interestingly, the Raman spectra of labelled P. amoebophila cells showed characteristics of RBs and EBs respectively (Fig. 3A). One explanation for this finding would be that the proposed spore-like and physiologically inert EBs (Ward, 1988; Hatch, 1999) were

© 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 687–700

690 S. Haider et al. 䊏 *Phe (967 cm-1)

Phe (1003 cm-1; aromatic ring breathing) *Phe (1532 cm-1)

Phe (1586 cm-1; C=C)

*Phe (599 cm-1)

Raman intensity

Phe (620 cm-1)

P. amoebophila + 13C Phe

P. amoebophila + 12C Phe 400

600

800

1000 1200 1400 Raman shift wave number (cm-1)

1600

1800

2000

Fig. 2. Peak shifts induced by labelled phenylalanine in the Raman spectrum of P. amoebophila. Mean Raman spectra of P. amoebophila after incubation of the amoebal host in medium for 264 h with unlabelled (lower spectrum) and labelled phenylalanine (upper spectrum) respectively. It should be noted that the peak at 1003 cm-1 does not completely disappear in fully labelled symbiont cells because labelled phenylalanine also has a minor peak at this wave number (Fig. S3). For each analysis 12 EB spectra, which do not contain the RB signature peaks shown in Fig. 1, were merged. For the analyses shown in Figs 3, 4 and S7, the most pronounced peak shift from 1003 cm-1 to 967 cm-1, representing the unlabelled and labelled aromatic ring (Wei et al., 2008), respectively, was used as indicator for phenylalanine uptake. It should be noted that because of the strongly reduced catabolic and anabolic pathways of chlamydiae, transfer of labelled C and N from phenylalanine to other chemical compounds in the cell is according to genome annotation very limited, explaining why many other compounds show no Raman peak shifts in the upper spectrum.

A

B *Phe

Phe

*Phe Phe *Phe Phe

7

1003 cm-1/967 cm-1 Peak ratio

6 5 4 3 2 1 P. amoebophila 24 48

72

96 120 264 24

48 72 Time (h)

UWC36 96 120 264 96

96

5a2 96

96

Fig. 3. Phenylalanine uptake by intracellular P. amoebophila (A) and other amoebal endosymbionts (B). Endosymbionts were incubated inside their host cells (using unsynchronized infected amoebal cultures) in medium containing labelled phenylalanine (*Phe). In parallel, control experiments with medium containing unlabelled phenylalanine were performed (Phe). The ratio of the 1003 cm-1 peak intensity (representing unlabelled phenylalanine) to the 967 cm-1 peak intensity (representing labelled phenylalanine) is indicated for individual cells. The lower the ratio the higher the labelling of the cells. Red diamonds represent EBs and blue diamonds RBs. Filled triangles represent the obligate Rickettsia-like endosymbiont UWC36, and filled circles the amoeba endosymbiont ‘Candidatus Amoebophilus strain 5a2’. The dashed line represents the threshold below which no values were observed for cells of the respective endosymbiont in the control experiment. The difference between ratios of labelled (n = 147) and unlabelled (n = 67) P. amoebophila cells is highly significant (Mann–Whitney U = 644, Z = 10.19, asymptotic 2-tailed significance P = 2.20E-24).


Intra- and extracellular activity of chlamydiae 691

Host-free uptake of phenylalanine and protein biosynthesis by P. amoebophila The observed putative intracellular activity of P. amoebophila EBs prompted us to investigate whether this sym-

A

B

7 1003 cm-1/967 cm-1 Peak ratio

metabolically active and took up phenylalanine from the amoeba host. Alternatively, the labelled EBs might have taken up phenylalanine as RBs or IBs and retained the label during conversion to EBs. Generally, more EBs than RBs were detected by Raman within the released P. amoebophila cells. This finding is consistent with corresponding transmission electron microscopy analyses (performed in a separate experiment with another unsynchronized amoebal culture infected with P. amoebophila), which showed that the experimental lysis step did not only destroy the host cells but also lysed some RBs, therefore increasing the relative abundance of EBs in the lysate. In detail, 46% RBs, 31% IBs and 23% EBs were detected by transmission electron microscopy within the amoebal host cells, while 20% RBs, 23% IBs and 57% EBs were counted after host cell lysis (Fig. S5). In addition to P. amoebophila, we also investigated two other bacterial symbionts of acanthamoebae, ‘Candidatus Amoebophilus asiaticus 5a2’ [Bacteroidetes; (Horn et al., 2001; Schmitz-Esser et al., 2008)] and the rickettsial symbiont UWC36 [Alphaproteobacteria; (Fritsche et al., 1999)], which are not members of the Chlamydiae. Interestingly, uptake of labelled phenylalanine was also observed for both symbionts within 96 h (Fig. 3B). Consequently, the capability to import phenylalanine from the amoeba host is not restricted to symbiotic chlamydiae but seems to be widespread among obligate intracellular bacteria thriving in these free-living protozoa. It is tempting to speculate that acanthamoebae as predators of bacteria and small eukaryotes (Rodriguez-Zaragoza, 1994) are generally well supplied with amino acids. The availability of these compounds in their host cells might have made de novo synthesis dispensable for the intracellular bacteria, and the respective biosynthetic pathways were thus lost from the genomes of these amoeba symbionts during genome streamlining and reduction. Particularly, the outsourcing of biosynthesis of aromatic amino acids including phenylalanine provides a selective advantage for the symbionts, as these amino acids are metabolically very costly to synthesize (Akashi and Gojobori, 2002). Consistent with this consideration, phenylalanine is also an important metabolic exchange product between other parasitic and mutualistic symbiotic partners like Listeria monocytogenes and macrophages (Eylert et al., 2008), Ignicoccus hospitalis and Nanoarchaeum equitans (Jahn et al., 2008) and the bacterial symbionts of various aphids and ants (Baumann et al., 1995; Zientz et al., 2004; Moran and Degnan, 2006).

6 5 4 3 2 1 P. amoebophila

C. trachomatis

12C

Phe 24h 48h 72h 96h 120h 8d 14d 21d 12C Phe 20h 20h Control +cmp Control Preincubation time with unlabelled phenylalanine

Fig. 4. Phenylalanine uptake by host-free P. amoebophila and C. trachomatis. (A) After lysis of amoeba or human host cells, chlamydial cells were incubated extracellularly for the indicated time periods in DGM-21A medium containing unlabelled phenylalanine. Subsequently, the medium was replaced by DGM-21A medium with 13C-labelled phenylalanine for 24 h (P. amoebophila) or 44 h [C. trachomatis, in the presence and absence of chloramphenicol (cmp)] before Raman spectra were recorded. In parallel, control experiments with medium containing unlabelled phenylalanine were performed (12C-Phe control). The lower the 1003 cm-1/967 cm-1 ratio the higher the labelling of EBs (red) and RBs (blue). The dashed line represents the threshold below which no values were observed for cells in the control experiment. The difference between ratios of cells exposed to labelled phenylalanine (n = 94 for P. amoebophila; n = 20 for C. trachomatis) and cells incubated with unlabelled phenylalanine (n = 21 for P. amoebophila, n = 19 for C. trachomatis) is highly significant (Mann–Whitney U = 0, Z = -7.665, asymptotic 2-tailed significance P = 1.79033E-14 for P. amoebophila and Mann–Whitney U = 11, Z = -5.307, asymptotic 2-tailed significance P = 1.115E-07 for C. trachomatis).

biont can be metabolically active outside of its host. For this purpose, chlamydial cells were released by lysis of the amoeba host cells, the absence of intact amoebae in the lysate was confirmed by light microscopy, and the host-free chlamydial cells were incubated in DGM-21A medium containing unlabelled phenylalanine. Live/dead staining of the released bacteria showed that immediately after host cell lysis 89% (SD ⫾ 5.7) of the chlamydiae were alive, and this number decreased to 15% (SD ⫾ 16.8) after 15 days of extracellular incubation. After incubation periods from 24 h to 21 days the medium was replaced by a medium containing 13C-phenylalanine and after another 24 h of incubation phenylalanine uptake was measured by Raman microspectroscopy. Interestingly, in all experiments all living host-free chlamydial cells became labelled and almost all of the active cells were EBs according to their Raman spectra (Fig. 4A), while cells that were dead according to the live/dead assay gave no analysable Raman spectra (data not shown). In contrast, many unlabelled cells were observed after 24 h



of incubation with labelled phenylalanine in the intracellular labelling experiment and complete labelling of the P. amoebophila population under these conditions took 120 h (Fig. 3A). This difference reflects that host-free cells have more direct access to the added amino acid and that the intracellular symbionts compete with the host for incorporation of the added amino acid. Inferred from the 1003 cm-1 to 967 cm-1 phenylalanine peak ratios, hostfree P. amoebophila cells take up within 24 h significant amounts of phenylalanine comparable to those measured for intracellular cells exposed to phenylalanine for much longer periods (Figs 3A and 4). The phenylalanine imported by P. amoebophila outside of its host is subsequently incorporated into protein as shown by 1-D protein gel electrophoretic analysis of protein extracts prepared from host-free cells of this organisms after exposure to [U-14C]L-phenylalanine (14C-phenylalanine) (Fig. S6). Thus, P. amoebophila is capable of host-free protein biosynthesis. Destruction of H+ and Na+ concentration gradients across the membrane of host-free P. amoebophila by addition of 10 mM of the ionophore carbonylcyanide m-chlorophenylhydrazone (CCCP) blocked the uptake of the labelled amino acid in almost all cells. Interestingly, this effect was reversible. RBs and EBs, which were treated with CCCP and subsequently washed to remove the ionophore, regained the capability to take up labelled phenylalanine (Fig. S7), demonstrating their ability to re-energize their membrane outside of their host by respiratory activity and/or H+/Na+ translocation via the F- and/or V-type ATPase encoded in the genome of P. amoebophila. To investigate whether extracellular P. amoebophila cells are still infectious after prolonged incubation periods, we performed co-incubation experiments with symbiontfree amoeba host cells. Extracellular P. amoebophila remained infective for amoebae over the complete experimental period of 21 days. This is much longer than those pathogenic chlamydiae like C. trachomatis require, which are spread by direct human-to-human transmission (Schachter, 1999), but comparable with other at least partially environmentally transmitted chlamydiae such as Simkania negevensis or the koala biovar of Chlamydophila pneumoniae (Rush and Timms, 1996; Kahane et al., 2004). Host-free uptake of phenylalanine and protein biosynthesis by C. trachomatis Unexpectedly, host-free activity of chlamydial cells is not restricted to chlamydial symbionts living in amoebae, but was also observed for the important human pathogen C. trachomatis. If C. trachomatis EBs were exposed for 44 h to 13C-phenylalanine after 20 h of host-free incubation

A kDa

1

2

B 3

4

1

2

3

4

70

35 27

Fig. 5. Host-free phenylalanine incorporation into proteins by C. trachomatis. After release from the host cells, C. trachomatis was incubated for 20 h in DGM-21A medium containing unlabelled phenylalanine. Subsequently, the medium was removed and DGM-21A medium with 14C-labelled phenylalanine (with or without chloramphenicol) was added for 44 h before total proteins and outer membrane proteins were extracted respectively. Protein extracts were analysed by SDS-PAGE and stained with Coomassie brilliant blue (A) or analysed for radioactivity with a phosphorimager (B). Lanes 1 and 2: Outer membrane and total proteins, respectively, from incubation without chloramphenicol. Lanes 3 and 4: Outer membrane and total proteins, respectively, from incubation with chloramphenicol.

in DGM-21A medium, a shifted phenylalanine band appeared in their Raman spectra indicating phenylalanine uptake (Fig. 4B). As the C. trachomatis cells analysed were harvested from human cells at a time point post infection at which almost exclusively mature EBs are present (Ward, 1988; Hatch, 1999) and as almost all host-free C. trachomatis cells took up labelled phenylalanine (Fig. 4B), the observed uptake proofs metabolic activity of mature EBs and can not be explained solely by host-free activity of IBs. Furthermore, C. trachomatis also incorporated added 14C-phenylalanine into proteins outside of the host and this activity (observed again for cell preparations consisting almost exclusively of EBs, although the presence of a few RBs or IBs cannot be excluded) included the synthesis of outer membrane proteins (Fig. 5). While uptake of phenylalanine outside of the host was not inhibited (but reduced) by chloramphenicol (Fig. 4B), addition of this inhibitor of protein synthesis during host-free incubation prevented the incorporation of phenylalanine into protein by C. trachomatis (Fig. 5). Combined radioactive and non-radioactive 2-D gel electrophoresis coupled with MALDI-TOF/TOF mass spectrometry allowed us to identify three proteins synthesized by C. trachomatis outside of its host as transcription termination factor Rho, Nudix phosphohydrolase, and as the outer membrane protein OmpH (Table 1 and Fig. S8). Interestingly, Rho and Nudix phosphohydrolases have been associated with stress response in other bacteria (Italiani et al., 2002; McLennan, 2006) and might thus be © 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 687–700


Outer membrane protein [C. trachomatis (strain L2/434/Bu)]

Transcription termination factor Rho [Chlamydia trachomatis (strain L2/434/Bu)] Major outer membrane protein [C. trachomatis (strain L2/434/Bu)]b Phosphohydrolase (MutT/Nudix family protein) [C. trachomatis (strain L2/434/Bu)]c

Protein

ompA

CTL0050

CTL0494

ompH

rho

CTL0752

CTL0140

Gene name

Locus tag

B0B7F8

B0B8Z7

B0B8Q7

B0B864

19.4 (17.3)

17.4

42.6 (40.3)

51.7

Mw (kDa)

4.81 (4.65)

5.04

5.06 (4.84)

6.84

pI

Theoreticala

14

17

30

52

Mw (kDa)

4.55

5

5.25

6.9

pI

Observed

129

702

164

115

Protein score

1

LQDDDYMEGLSETAAAELRK + Ox (M)

1

GNSASFNLVGLFGDNENHATVSDSK

1 1 1 1 1 1 1 1 1 1 1 1 1

1

VEELNVLCNAAEFTINKPK FFGTPDR EGPQEAAER LLNFPEIR EVTYFLAEVK ACFICHTDGK HEYSFGVIPIR TKHEYSFGVIPIR GHAEEKEGPQEAAER ELVEETGLGIVNFFPK IFVENYSFNDKEEIFVR IFVENYSFNDKEEIFVRK GEVHADPDEICDVQWLSFQEGLR LQDDDYMEGLSETAAAELR + Ox (M)

1

SATTVFDVTTLNPTIAGAGDVK

1

SGTRKEELLYHPGELEK

1

1

VLFENLTPLHPNER

GYVGQEFPLDLK

1

GLIVAPPR

Peptide sequence

Peptide charge

49.77

24.56 39.16 46.44 53.76 35.77 58.29 98.06 91.49 73.05 93.35 31.83 58.25 79.5

36.32

26.04

38.93

63.04

33.44

55.13

26.8

Peptide score

0.00027

0.085 0.0019 0.0017 0.00019 0.0039 8.60E-05 9.40E-09 1.60E-08 2.80E-06 1.60E-08 0.029 1.90E-05 2.00E-07

0.0044

0.16

0.0069

2.80E-05

0.025

0.00021

0.051

E-value for peptide match

a. Theoretical molecular weight (Mw) and isoelectric point (pI) values were calculated using the Compute pI/Mw tool at the ExPASy website (http://www.expasy.org/tools/). Theoretical Mw and pI of proteins with cleaved predicted signal peptides (SignalP 3.0) are indicated in brackets. b. The detection of OmpA in one of the excised protein spots representing newly synthesized (radioactive) proteins might not reflect host-free OmpA synthesis. OmpA, as component of the major outer membrane complex, is highly abundant in C. trachomatis and is detected not as a single but as multiple prominent spots in a 2-D gel (Shaw et al., 2002) (unusual migration patterns of membrane proteins are often found in 2-D gels). As the well described OmpA spots were not radioactively labelled on the 2-D gel of the host-free C. trachomatis cells, it is possible that the minor OmpA spot we excised just is at the same location as another spot of a newly synthesized protein, whose concentration was apparently too low for mass spectrometric detection. Alternatively, OmpA is synthesized without a host, but has a different location in the 2-D gel than OmpA synthesized within the host, e.g. because of differences in posttranslational modifications or proteolytic processing. c. Although the protein with the accession number B0B8Z7 is annotated as putative uncharacterized protein in public databases, it is described as a member of the Nudix hydrolase family of proteins at UniProt and contains a conserved Nudix motif [G[X5]E[X7]REUXEEXGU, where X is any amino acid and U is a bulky aliphatic residue (usually I, L or V)] (Bessman et al., 1996). The most closely related protein annotated as phosphohydrolase (MutT/Nudix family protein) is the homologous protein of C. trachomatis (strain A/HAR-13), which shares 98% sequence identity at the protein level with B0B8Z7. No experimental data exist for both of these proteins.

4

3

2

1

Spot number

UniProt accession number

Table 1. C. trachomatis proteins identified by MALDI-TOF/TOF.



DGM-21A medium

Hatch medium

B

E

F

C

D

G

H

NonEUB 338

Chls532

A

Fig. 6. Detection of C. trachomatis by fluorescence in situ hybridization (FISH) after host-free exposure in different media. Host-free cells of C. trachomatis were exposed to DGM-21A and Hatch medium, respectively, as described for the 14C-phenylalanine incubation experiments and analysed by FISH. (A and C) display FISH pictures for C. trachomatis cells incubated in DGM-21A medium stained with the Chlamydiales-specific probe Chls523 (A) and the nonsense probe NonEUB338 (C) respectively. The inset in (A) shows individual FISH-stained C. trachomatis cells at a higher magnification. Inset bar corresponds to 1 mm. (E and G) show the results for the same experiments with cells incubated in Hatch medium. All FISH pictures were recorded with identical confocal microscope settings. The figures in the right columns display the corresponding DIC pictures. Bar corresponds to 5 mm and applies to all panels (with the exception of the inset in A). While C. trachomatis cells gave bright FISH signals after incubation in DGM-21A medium, no FISH signals could be observed if the cells were exposed to Hatch medium. This difference most likely reflects that the cells in the Hatch medium reduced their ribosome content below the detection limit of standard FISH, a trait described for many different bacteria in response to unfavourable environmental conditions (Wagner et al., 2003). This explanation would also be consistent with the absence of host-free protein biosynthesis in Hatch medium (Fig. S9). However, lack of FISH signals in the Hatch-exposed cells could theoretically also be caused by a medium induced modification of the cell wall preventing probe penetration.

used by C. trachomatis to mitigate cell damage in a hostfree environment. Conclusions Collectively, these findings contradict the dogma that all chlamydial EBs are metabolically inactive outside of their host cells. This dogma is based on studies with host-free EBs of C. trachomatis and Chlamydophila psittaci, which showed that these cells were unable to incorporate radioactively labelled amino acids into protein (Hatch et al., 1985). Furthermore, host-free EBs of C. psittaci also did not take up ATP although this organism is an intracellular ATP parasite (Hatch et al., 1982). In contrast, host-free RBs of clinically relevant chlamydiae show a certain spectrum of metabolic activities including lysine uptake, but to the best of our knowledge such activities have only been reported for RBs, which were exposed to extracellular conditions for no longer than 2–4 h (Tamura, 1967; Weiss and Wilson, 1969; Hatch et al., 1982; 1985; Crenshaw et al., 1990). However, the strongly time-limited metabolic activity of extracellular RBs and the metabolic inactivity of host-free EBs observed in these studies might only reflect that the incubation medium used was not suitable to sustain the metabolic activity of chlamydial EBs outside of the host. Indeed, when we repeated our phenylalanine incorporation experiment with the host-free incubation medium used in one of the previous studies (Hatch et al., 1985), no host-free activity of C. trachomatis could be measured

(Fig. S9) and the C. trachomatis cells could, in contrast to the DGM-21A medium experiment, no longer be detected by fluorescence in situ hybridization (FISH) (Fig. 6). These results show that media composition is crucial for the host-free activity of C. trachomatis and that the use of the DGM-21A medium for our host-free incubation experiments was important. This finding should also be kept in mind for the interpretation of some other studies on the host-free activity of RBs and EBs of clinically relevant chlamydiae (Sarov and Becker, 1971; Hackstadt et al., 1985; Hatch, 1988; Crenshaw et al., 1990). Taken together our work shows that chlamydial metabolism is not exclusively confined to life in eukaryotic cells and that EBs of the amoeba symbiont P. amoebophila and the human pathogen C. trachomatis are metabolically active for extended time periods after being released from their host cell, if they are supplied with appropriate nutrients. It is tempting to speculate that the host-free synthesis of stress response proteins and the capability to energize their membrane outside of the host [which could be driven by exogenous substrates and/or endogenous storage compounds like starch and glycogen, which can be synthesized by P. amoebophila and C. trachomatis according to genome annotation (Stephens et al., 1998; Horn et al., 2006)] are important features of chlamydiae to survive in the extracellular environment after host cell lysis and to efficiently restart their metabolism upon infection of a new host cell. This newly discovered facet of chlamydial biology has important implications for our understanding of the developmental cycle of these organisms and opens © 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 687–700


new perspectives for the physiological characterization, as well as the development of host-free cultivation approaches for chlamydiae.

tance between individual symbiont cells on the CaF2 slide was larger than the spatial resolution limit of the Raman microspectrometer, enabling single-cell analyses.

Experimental procedures

Host-free phenylalanine uptake experiments

Separation of P. amoebophila RBs and EBs

Protochlamydia amoebophila was grown in A. castellanii Neff in peptone-yeast-glucose-medium (Visvesvara, 1999) at 20°C. Cells were harvested by centrifugation and resuspended in 1¥ Page’s saline. Amoebal cells were disrupted, and the chlamydial cells were harvested as described for the intracellular uptake experiments. Absence of intact amoebae in the lysate was confirmed by light microscopy. Chlamydial cells were then resuspended in 20 ml of medium DGM-21A supplemented with unlabelled phenylalanine and were incubated for 24 h, 48 h, 72 h, 96 h, 120 h, 8 days, 14 days and 21 days at 20°C. Then, the medium was replaced by DGM21A medium containing 13C-phenylalanine (5.45 mM final concentration) and cells were incubated for 24 h before cells were prepared for Raman measurements as described above. For some samples, the ionophore CCCP (Sigma) was added at a concentration of 5 or 10 mM during incubation. These samples were split in two aliquots; one aliquot was analysed by Raman microspectroscopy while the other was washed with 1¥ Page’s saline and then incubated again with 13 C-phenylalanine before Raman analysis. Chlamydia trachomatis serovar L2 was grown in T-REx 293 cells (Invitrogen) at 37°C for about 72 h prior to release by a bead-beating step. The obtained cell suspension was filtered using a 5 mm filter to remove host cell debris. Host-free C. trachomatis was incubated at 37°C for 20 h in 20 ml of medium DGM-21A supplemented with unlabelled phenylalanine. Then, the medium was replaced by DGM-21A medium containing 13 C-phenylalanine (5.45 mM final concentration) and cells were incubated for 44 h in the presence or absence of 50 mg ml-1 chloramphenicol before cells were prepared for Raman measurements as described above. Furthermore, C. trachomatis cells incubated without host cells in DGM21-A or Hatch medium (Hatch et al., 1985) were also analysed by FISH. For this purpose, cells were washed with 0.6% NaCl at the end of the incubation experiments and subsequently the cells were fixed with 3% paraformaldehyde for 3 h at 4°C using a standard protocol (Daims et al., 2005). FISH was performed according to Daims et al. (Daims et al., 2005) using Cy3labelled derivatives of the Chlamydiales-specific probe Chls523 (Poppert et al., 2002) and of the nonsense probe NonEUB338 (Wallner et al., 1993). Hybridized cells were analysed using the Zeiss LSM 510 confocal laser scanning microscope.

For physical separation of RBs and EBs, Acanthamoeba sp. UWC1 harbouring P. amoebophila UWE25 was grown in Trypticase soy broth with yeast extract (Visvesvara, 1999) at 20°C. Amoebae were harvested by centrifugation, washed in 1¥ Page’s saline (Page, 1988), resuspended in 6.5 ml of sucrose phosphate glutamic acid buffer (SPG; 75 g l-1 sucrose, 0.52 g l-1 KH2PO4, 1.53 g l-1 Na2HPO4*2H2O, 0.75 g l-1 glutamic acid) per 1 g wet weight and disrupted on ice by a dounce homogenizer (Wheaton). Released endosymbionts were harvested by centrifugation (5500 g, 20 min, 4°C), resuspended in 6 ml of SPG, filtered (1.2 mm pore size), and 1 ml of the suspension was laid onto 6.5 ml of 30% (v/v) gastrografin (Schering). After ultracentrifugation (40 000 g, 1 h, 4°C) pellets were resuspended in SPG and the suspension was homogenized by needle extrusion. One millilitre of sample was laid onto a gradient consisting of 3 ml of 30% (v/v) gastrografin and 3 ml of 50% (w/v) sucrose, and centrifuged (40 000 g, 2 h, 4°C). The pellet was resuspended in 0.75 ml of SPG, supplemented with 32.5 ml of DNase I (20 mg ml-1), 32.5 ml of RNase (20 mg ml-1; both from Roche Diagnostics GmbH) and 37.5 ml of MgCl2 (1 M) and incubated for 1 h at 37°C. The suspension was again homogenized by needle extrusion, and 1 ml of the sample was laid on top of a gradient consisting of 1.5 ml of 34% (v/v), 2 ml of 40% (v/v), 2 ml of 46% (v/v) and 2.5 ml of 52% (v/v) gastrografin. After centrifugation (40 000 g, 2 h, 4°C), bands that appeared at the 34%/40% (RB fraction) and 46%/52% (EB fraction) interfaces were collected, washed in SPG and resuspended in 100–500 ml of SPG. Aliquots were taken for Raman and TEM analyses.

Intracellular phenylalanine uptake experiments Protochlamydia amoebophila, the rickettsial endosymbiont of Acanthamoeba sp. UWC36, and ‘Candidatus Amoebophilus asiaticus 5a2’ were grown in Acanthamoeba castellanii Neff using the defined medium DGM-21A (Schuster, 2002) supplemented with unlabelled or 13C-phenylalanine (Sigma; 5.45 mM final concentration) at 20°C. Cells were harvested by centrifugation (5400 g, 5 min) after 24, 48, 96, 120 and 264 h for P. amoebophila and 96 h for the other two endosymbionts and resuspended in 500 ml of 1¥ Page’s saline. Amoebal cells were disrupted by freezing (-20°C) and thawing (room temperature, RT). An equal volume of glass beads was added to the suspension, which was subsequently vortexed for 15 s. Then, the supernatant was centrifuged (300 g, 10 min, 20°C) to remove amoebal cell debris, and the supernatant was centrifuged again to harvest the chlamydial cells (20 800 g, 15 min). The pellet was washed with SPG and resuspended in 20 ml of SPG. For Raman measurements, 2 ml of the cell suspension were transferred to a CaF2 slide (Crystran), dried at RT, washed by dipping in ddH2O and dried again. Because of this procedure, the dis-

Live/dead staining Two microlitres of the chlamydial cell suspension were incubated for 1 h at RT with 1 mg ml-1 4′,6-diamidino-2phenylindole (DAPI; Lactan) and 10 mg ml-1 propidium iodide (PI; Molecular Probes). Subsequently, 10 ml of phosphatebuffered saline (NaCl 130 mM, Na2PO4 10 mM; pH 7.2–7.4) were added, the suspension was filtered onto a 0.2 mm filter (Millipore) and live (DAPI stained) as well as dead (PI stained) cells were counted by epifluorescence microscopy.



An aliquot of the stained cells was dried onto CaF2 slides and Raman spectra were recorded for live and dead cells after bleaching of the fluorescent dye using the protocol of Huang et al. (2007).

Transmission electron microscopy Density gradient purified cells were incubated in fixative solution (Lindsay et al., 1995) overnight at 4°C. After washing in 0.1 M cacodylate buffer, fixed cells were encapsulated into 12% gelatine in 0.1 M cacodylate buffer. Post-fixation in 0.5% osmium tetroxide for 2 h was followed by en bloc staining with 2% aqueous uranyl acetate for 1 h and a dehydration step in a graded ethanol series. Acetone incubation was used as an intermediate step before infiltration with low viscosity resin (Agar Scientific) overnight. Then, cells were embedded in fresh resin and polymerized at 60°C for 12 h. Ultra-thin sections were contrasted with uranyl acetate and lead citrate, and examined in the transmission electron microscope (Zeiss 902) at 80 kV accelerating voltage. To assess the relative proportion of the different developmental stages of P. amoebophila, 20 to 40 randomly collected images at 7000¥ original magnification were recorded for each sample. The developmental stages were distinguished by chromatin morphology and electron density (Rake, 1957; Barry et al., 1992). For the quantification of the proportion of the different developmental stages in the phenylalanine uptake experiments, amoebal cells infected with P. amoebophila were split into two fractions. One fraction was harvested by centrifugation (5400 g, 5 min) and fixed immediately in fixative solution (3 mM cacodylate buffer, 2.5% glutaraldehyde, 2% sucrose, pH 7.2) for 1 h at RT. In the second fraction, the amoebal cells were lysed as described for the uptake experiments and the released cells were incubated in fixative solution. After another centrifugation step (amoebal cells at 5400 g for 3 min; host-free cells at 20 800 g for 5 min), cells were washed in 0.1 M cacodylate buffer and fixed in 1% osmium tetroxide for 1 h. After dehydration in a graded ethanol series, the samples were transferred to acetone followed by acetonitrile incubation. Subsequently, the cells were infiltrated in 1 vol. of low viscosity resin and 1 vol. acetonitrile overnight. The next day, cells were embedded in resin and ultra-thin sections were produced as described above.

Infection experiments At 24 h, 48 h, 72 h, 96 h, 120 h, 8 days, 14 days and 21 days after host-free incubation, P. amoebophila was added to uninfected A. castellanii Neff grown in multi-well plates (Iwaki) and centrifuged at 130 g for 15 min. The cells were left at 20°C for 48 h and infection was visualized after DAPI staining by epifluorescence microscopy.

Confocal Raman microspectroscopy Raman microspectroscopy was performed using a LabRAM HR800 confocal Raman microscope (Horiba Jobin-Yvon). Excitation for Raman scattering was provided by a 532 nm Nd : YAG laser. For Raman spectral analysis of a chosen cell, the incident laser power was typically adjusted to 7 mW, to

avoid damaging of the sample while still maintaining spectral sensitivity. The pinhole of the Peltier cooled CCD detector was set to 250 mm enabling a spatial resolution in z-direction of approximately 2.5 mm. Single cells were selected randomly using a 100¥ objective with a numerical aperture of 0.9 (leading to a diameter of the laser spot of below 800 nm). Spectra were obtained from 8–40 individual cells per measurement. The signal acquisition time was 40–60 s per measurement with a spectral resolution of 1.5 cm-1. Initially, spectra were acquired from 381 to 2030 wave numbers (cm-1). Visual inspection of these spectra showed that the most informative range was between 400 and 1800 cm-1, and this was used for data analysis. Raman spectra were processed for baseline correction and normalization using the commercial Labspec software 5.25.15 (Jobin-Yvon). These data were exported to Microsoft Excel for further peak determinations and calculations. Calibration was periodically checked by recording the position of a known Raman line using a silicon Raman reference (520 cm-1). The wave number accuracy was estimated to be ⫾3 cm-1. Principal components analysis (PCA) was employed to reduce the dimensionality of the Raman data. Mann–Whitney U-test was performed to test for the significance of between group differences. All statistical analyses were performed using SPSS version 16.0 (SPSS, Chicago, IL).

Incorporation of SDS-PAGE

14

C-phenylalanine into proteins –

After release from their host cells, P. amoebophila and C. trachomatis were incubated for 20 h in DGM-21A (in the presence or absence of 50 mg ml-1 rifampicin or chloramphenicol for P. amoebophila and C. trachomatis, respectively) or Hatch medium (Hatch et al., 1985) with unlabelled phenylalanine. Subsequently, the media were replaced by the respective media containing 14C-labelled phenylalanine (Perkin Elmer, 10 mCi per incubation experiment) and incubated for 44 h at 20°C (P. amoebophila) or 37°C (C. trachomatis). Subsequently the cells were harvested and split into two fractions. From one fraction outer membrane proteins were extracted as described before (Hatch et al., 1985). For total protein extraction, cells of the other fraction were lysed with 100 ml of 0.2 N NaOH for 10 min at RT (or 37°C for Figs S6A and S9). After diluting the lysate with 400 ml of ddH2O (this step was not included in the analyses shown in Figs S6A and S9), it was concentrated with an Amicon Ultra 0.5 ml Ultracel – 10 K membrane filter column (Millipore) and washed twice with 500 ml of ddH2O (this washing step was not included in the analyses shown in Figs S6 and S9). Proteins from both fractions were denatured for 8 min at 95°C in Laemmli buffer (Laemmli, 1970) and were separated by SDSPAGE on 12.5% (w/v) gels, which were subsequently stained with colloidal Coomassie and analysed by phosphor-imaging using a Typhoon 8600 Imager (Molecular Dynamics) or by autoradiography film exposure.

Incorporation of 14C-phenylalanine into proteins – 2-D gel electrophoresis Chlamydia trachomatis cells after host-free incubation in the presence of 12C- or 14C-phenylalanine were resuspended in © 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 687–700


50 mM Tris/HCl (pH 7.5) containing 1% SDS and 1¥ EDTAfree protease inhibitor mix (Roche Diagnostics GmbH), incubated for 5 min at 90°C, and cooled down on ice for 5 min. Eight volumes lysis buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris, 65 mM DTT and 2% IPG buffer pH 3–10 NL (GE Healthcare) were added, and the sample was incubated for 1 h at 20°C at 600 r.p.m. Insoluble material was removed by centrifugation at 20 800 g for 20 min at 4°C. The protein concentration was determined using the 2-D Quant Kit (GE Healthcare) according to the manufacturer’s instructions. Equipment and chemicals used for 2-DE were purchased from GE Healthcare. IPG strips of wide pH range (pH 3–10 NL) were passively rehydrated with the protein containing lysis buffer. About 250 mg of protein was loaded per strip. The focusing on the IEF system Ettan™ IPGphor II™ was performed at 20°C according to the following programme: 1 h at 500 V, 1 h at 2000 V and 6 h at 10 000 V with the last step programmed in Vh. Strips were equilibrated for 15 min in 30% glycerol, 2% SDS, 6 M urea, 50 mM Tris/HCl (pH 8.8), 1% DTT and subsequently for 15 min in a buffer where 4% iodoacetamide replaced DTT. Electrophoresis was conducted at 20°C at 5 W per gel for 30 min, followed by 17 W per gel for 3–4 h using an Ettan™ Dalt six electrophoresis unit, 12.5% SDS-polyacrylamide gels, and PageRuler™ prestained protein ladder (Fermentas) as molecular weight marker. Coomassie stained gels were scanned and analysed using the Image Master 2-D Platinum software version 5.0 (GE Healthcare). Vacuum-dried gels containing 14C-labelled proteins were analysed by phosphor-imaging using a Typhoon 8600 Imager (Molecular Dynamics) (exposed for 8 days). Gel images and radioactive scans were overlaid according to radioactive marks applied to the dried gel before exposure.

Protein identification by mass spectrometry Protein spots were excised and in-gel digestion was performed as described (Shevchenko et al., 2006). Peptide samples were desalted and concentrated using C-18 NuTips (Glygen Corp.) according to the manufacturer’s instructions. Two microlitres of each sample were spotted twice onto an Opti-TOF MALDI target plate and covered with 1 ml of matrix solution (3 mg ml-1 a-cyano-4-hydroxycinnamic acid in 70% acetonitrile, 0.1% trifluoroacetic acid). Mass spectra were obtained on a 4800 MALDI TOF/TOF mass spectrometer (Applied Biosystems) acquiring 2000 shots in reflector positive mode from m/z 800–4000. Peaks detected on both spots of a sample that did not coincide with a list of ‘contaminant’ masses (peaks corresponding to matrix clusters, trypsin autolysis and frequent keratin peptides) were submitted to Mascot 2.2.04 for PMF (peptide mass fingerprint) analysis. The protein sequence database comprised all Swiss-Prot and TrEMBL sequences for C. trachomatis and all Swiss-Prot sequences for Homo sapiens extracted from the 2009-12-15 UniProt fasta release, as well as a set of frequent lab contaminant proteins. For protein hits scoring above the Mascot significance cut-off (P < 0.05) in PMF, tandem mass spectra were obtained acquiring 4000 shots for precursor masses that matched to peptides of the respective protein in PMF. MS/MS spectra were likewise searched with Mascot using the following parameters: trypsin, max 2 missed cleavages,

precursor mass tolerance 50 ppm, fragment mass tolerance 0.5 Da, fixed modification carbamidomethylation of cysteine, variable modification oxidation of methionine. Table 1 shows C. trachomatis proteins identified with at least two or more peptides in MS/MS and Mascot scores above the individual Mascot homology or identity thresholds for the respective peptide. A Mascot decoy search of the data did not reveal any decoy hits.

Acknowledgements We gratefully acknowledge Waltraud Klepal and the team of the Ultrastructure Laboratory (University of Vienna) and Kilian Stoecker for advice and assistance with electron microscopy and Raman microspectroscopy respectively. We thank Stephanie Füreder and David Hasenöhrl for help with the phosphorimager and Andreas Richter for valuable discussions. This work was funded by grants of the Austrian Science Fund FWF (Y277-B03) and the University of Vienna in the framework of the University Research Focus Symbiotic Interactions (FS573001).

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