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Department of Molecular Biology1 and Microscopy Core Facility2 , Max-Planck-Institute for Infection Biology, Schumannstr. 21/22, 10117 Berlin, Germany
Correspondence
Thomas F. Meyer
meyer{at}mpiib-berlin.mpg.de
Received 30 November 2005
Accepted 23 March 2006
Abbreviations: EB, elementary body; Hyp, L-hydroxyproline; i.f.u., inclusion-forming unit; p.i., post-infection; RB, reticulate body.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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Because of the obligatory intracellular lifestyle of Chlamydia and its inability to synthesize amino acids, which are required for growth and multiplication, this pathogen appears to acquire these and other metabolic substrates from host pools (Grieshaber et al., 2002; Hatch, 1975; Kalman et al., 1999; McClarty, 1994; Tipples & McClarty, 1993). Any perturbation in the levels of these precursors inside host cells would affect chlamydial growth. Indeed, depletion of single amino acids or restriction of the amino acid content in the host-cell growth medium causes noticeable perturbation of chlamydial growth (Allan & Pearce, 1983; Allan et al., 1985; Coles et al., 1993; Harper et al., 2000; Karayiannis & Hobson, 1981; Kuo & Grayston, 1990). In our previous work (Al-Younes et al., 2004) we unexpectedly demonstrated that supplementation of cell cultures with increased amino acid levels caused various degrees of abnormality in C. trachomatis development. Of the 11 amino acids investigated, only three, leucine, isoleucine and methionine, markedly halted the inclusion development and resulted in a complete abrogation of chlamydial progeny infectivity, whereas phenylalanine led to a very strong reduction in the production of infectious C. trachomatis.
The objective of this study was to investigate and to compare the in vitro effects of individual amino acids, added in excess, on the development of C. pneumoniae and C. trachomatis. While some amino acids were found suppressive to their growth, others promoted the growth of both species. In addition, a differential response to increased concentrations of some amino acids was observed between the two species, which may represent a unique (novel) factor that could control the pathogenesis of both chlamydial species. It was noticeable that some amino acids led to a considerable suppression of C. trachomatis and C. pneumoniae growth, hinting at their potential in the prevention and treatment of chlamydial diseases.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Amino acids and antibodies. L-Amino acids and other chemicals were purchased from Sigma-Aldrich, Merck or Fluka. The amino acids used were tryptophan, lysine, valine, histidine, threonine, phenylalanine, methionine, isoleucine, leucine, glutamine, glutamate, alanine, aspartate, proline, asparagine, arginine, tyrosine, glycine, serine, and the proline analogue L-hydroxyproline. IM with exogenous free amino acids at appropriate concentrations was prepared as described previously (Al-Younes et al., 2004). The IMAGEN kit for the direct staining of Chlamydia was obtained from Dako.
Infection of cell cultures and supplementation with excess individual amino acids. HEp-2 cells were seeded in 6-well plates 1 day before infection. Next, host cells were infected with either C. trachomatis or C. pneumoniae, essentially as described previously (Al-Younes et al., 2001, 2004), using an m.o.i. of 0.5, unless otherwise indicated. Host cells were inoculated for 2 h with C. trachomatis at 35 °C and 5 % CO2 in a humidified tissue-culture incubator. Unlike infection with C. trachomatis, host cells were infected with C. pneumoniae with the aid of centrifugation (900 g at 35 °C for 1 h) and then incubated as described for C. trachomatis-infected cells for an additional 1 h. Inocula were then removed and infected cells were washed with IM. In a first group of experiments, infected cultures were supplemented immediately after the washing step with IM containing exogenous amino acids and incubated under standard conditions (35 °C and 5 % CO2 in a humidified tissue-culture incubator) until the end of the infection periods (44 h for C. trachomatis and 72 h for C. pneumoniae). In a second group of experiments and following the washing step, cells were provided with IM without additives and incubated under the conditions described above. IM was then aspirated from plates inoculated with C. trachomatis and C. pneumoniae at 19 and 31 h post-infection (p.i.), respectively. Next, cell monolayers were loaded with IM containing exogenous amino acids and incubated as described above until the end of the respective infection periods. As controls for both groups, infected cell monolayers were incubated under identical conditions in IM, but without exogenous supplements.
Assessment of progeny infectivity, visualization of chlamydial infection, and host-cell metabolic activity. To explore the effects of excess individual amino acids on the production of infectious C. trachomatis L2 progeny, amino-acid-treated and -untreated HEp-2 cells were harvested 44 h p.i. from the 6-well plates, lysed and then serially diluted in IM. Next, dilutions were inoculated onto fresh HEp-2 cells seeded in 12-well plates and developed EBs in the lysates were allowed to adsorb to host cells for 2 h at 5 % CO2 and 35 °C. Monolayers were washed and incubated for 24 h in fresh IM under standard conditions. For the assessment of infectivity titre, the number of inclusion-forming units (i.f.u.) per millilitre was calculated. To assess the influence of amino acids on the yield of recoverable i.f.u. in treated cells infected with C. pneumoniae, infected cells were harvested 72 h after infection, homogenized, serially diluted and subpassaged onto HEp-2 cells grown on coverslips with the aid of centrifugation (900 g at 35 °C for 1 h). Next, cells were incubated under standard conditions for an additional 1 h, and inoculum was then removed. The cells were loaded with fresh MM and incubated in the humidified tissue-culture chamber. Two days after infection, chlamydial inclusions were visualized by immunostaining and counted to determine i.f.u. ml–1 (Al-Younes et al., 2001, 2004). The probability value (P) was determined by Student's t test, and the difference was considered significant when P was <0.05.
The developmental and growth features of the bacteria were examined by transmission electron microscopy or by staining cell cultures grown on coverslips with suitable antibodies (IMAGEN kit) followed by immunofluorescence microscopy (Al-Younes et al., 2001, 2004). The WST-1 colorimetric assay was used to estimate the effects of excess individual amino acids on the metabolic activity of host cells (Al-Younes et al., 2004).
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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The developmental cycles of C. trachomatis L2 and C. pneumoniae can be completed within approximately 44–48 h and 72–96 h, respectively (Grieshaber et al., 2002; Nicholson et al., 2003; Wolf et al., 2000). In the first group of experiments, amino acids were introduced approximately at the middle of the infection cycle, i.e. at 19 and 31 h p.i. for C. trachomatis and C. pneumoniae, respectively. At these time points, bacteria of both species exist at nearly equivalent developmental stages, at which intracellular bacteria are in the form of reticulate bodies (RBs) and are in the exponential phase of development (Grieshaber et al., 2002; Nicholson et al., 2003; Wolf et al., 2000). In the second group of experiments, amino acids were administered 2 h p.i. until the end of the infection course.
Influence of essential amino acids on the progress of C. trachomatis and C. pneumoniae infections
Single essential amino acids added at the middle of the infection had no or negligible effect on the size of C. trachomatis and C. pneumoniae inclusions (data not shown), except for Phe, Ile, Leu and Met, which evidently delayed maturation of both types of inclusions (Fig. 1A
). Interestingly, Met was less effective in arresting growth of C. pneumoniae inclusions, compared to its considerable effect on C. trachomatis inclusions (Fig. 1A). Next, titres of progeny infectivity of both species were determined in amino-acid-exposed cells and compared to those in untreated infected cells. Although they did not apparently modify the inclusion size, Trp, Lys and Val were able marginally to stimulate or reduce the formation of infectious elementary bodies (EBs) of both species, whilst His and Thr decreased progeny infectivity by about 40 % or more (Fig. 2A). Moreover, Leu, Ile, Phe and Met very strongly decreased progeny infectivity titres of C. trachomatis, to less than 1 % (Fig. 2A, closed bars), a finding that was fully reflected in their inhibitory effects on the growth of the inclusions (Fig. 1A, upper panels). Leu, Ile and Phe similarly suppressed the production of infectious progeny of C. pneumoniae. Interestingly, Met addition to C. pneumoniae-infected cells caused a moderate reduction in the recoverable i.f.u. (<50 %) (Fig. 2A, open bars), compared with more than 99 % reduction in C. trachomatis progeny infectivity (Fig. 2A, closed bars). In general, the effects of Leu, Ile, Met and Phe on the production of infectious chlamydial progeny of both species coincided with their influence on the inclusion size.
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), compared to those receiving the same amino acids at the middle of infection (Fig. 1A). Again, C. pneumoniae was less responsive to the addition of Met; this may be compared to the striking adverse effect of Met on C. trachomatis inclusions (Fig. 1B). The effects of continuous exposure to essential amino acids on subsequent infectivity are shown in Fig. 2(B). In contrast to the limited adverse effects of Trp and Val on the yield of infectious progeny of C. trachomatis, these amino acids significantly stimulated the production of infectious C. pneumoniae EBs. Other tested amino acids exerted almost identical inhibitory effects on the subsequent infectivity of both species (Fig. 2B). Of these, Leu, Ile and Met completely inhibited the progeny infectivity of both species. Earlier work performed by other laboratories using amino acid limitation has provided detailed knowledge of amino acid requirements for the growth of chlamydiae (Allan & Pearce, 1983; Allan et al., 1985; Coles & Pearce, 1987; Coles et al., 1993; Harper et al., 2000; Karayiannis & Hobson, 1981; Kuo & Grayston, 1990). Reduction of amino acid concentrations in cell cultures disrupts the developmental cycle of C. trachomatis serovars L2 and E (Harper et al., 2000). Moreover, differences in amino acid requirements between four strains of Chlamydia (Chlamydophila) psittaci and 11 strains of C. trachomatis in McCoy cells have been revealed (Allan & Pearce, 1983). C. trachomatis strains show a need for the essential His, Val, Leu and Phe, and for the non-essential Gln. The same amino acids are required by C. psittaci, except for His. Interestingly, the amino acid requirements correlate with clinical syndromes caused by the different isolates. Trachoma serovars (C. trachomatis A, B and C) require Trp, whilst strains of oculogenital origin (C. trachomatis serovars D–I) show no need for Trp or Met. A lymphogranuloma venereum (LGV) strain exhibits a need for Met. Later, Kuo & Grayston (1990) demonstrated increased progeny infectivity titres of C. pneumoniae following depletion of 90–100 % of Lys or 70–90 % of Met. Overall, based on our and earlier published results, imbalances in the amino acid levels can effectively modulate chlamydial development.
The complete lack of recoverable infectious EBs of C. pneumoniae in infected cultures continuously treated with Met was unexpected, since inclusions of C. pneumoniae were relatively large compared to the very small inclusions of C. trachomatis that received identical treatment (Fig. 1B). This lack of subsequent infectivity was investigated by performing transmission electron microscopy, which revealed the presence of only the non-infectious RBs of C. pneumoniae (Fig. 3D). Of note was the observation that Met appeared not to halt the binary division of C. pneumoniae (Fig. 3D), while it induced complete inhibition of C. trachomatis proliferation (Fig. 3B).
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. The effect of different doses of Leu on subsequent infectivities of C. trachomatis and C. pneumoniae appeared to be similar. However, Ile and Met demonstrated clear differential activities against the two species. For instance, 0.1 mM Ile diminished the generation of infectious C. pneumoniae by about 80 %, compared to a 40 % reduction in C. trachomatis. In contrast, Met at concentrations ranging from 0.1 to 5 mM was less potent in decreasing C. pneumoniae progeny infectivity, compared to its effect on C. trachomatis.
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, upper panels). On the other hand, Hyp and Ser were capable of causing a detectable decrease in C. pneumoniae inclusion size (Fig. 1C, lower panels). The amino acids Glu, Ala, Gln, Asp, Pro and Asn barely affected the generation of infectious EBs of the two species (Fig. 2C) when added at the middle of infection, causing either slight enhancement or reduction of progeny infectivity. Moreover, the response of both species to the addition of Arg or Tyr was similar, with a pronounced reduction of progeny infectivity (Fig. 2C). Major differences in the production of infectious EBs of the two species were seen when cells were exposed to Gly, Hyp and Ser, which had stronger adverse effects on subsequent C. trachomatis infectivity than on that of C. pneumoniae (Fig. 2C).
The addition of most non-essential amino acids 2 h p.i. generated C. trachomatis and C. pneumoniae inclusions that were the same size as inclusions in untreated cells (data not shown). Similar to their inhibitory effects on the inclusion growth observed when added 19 h p.i. (Fig. 1C, upper panels), Gly, Hyp and Ser, in particular, arrested the maturation of C. trachomatis inclusions (Fig. 1D, upper panels). In contrast, the last two amino acids, as well as Arg, markedly decreased the size of C. pneumoniae inclusions (Fig. 1D, lower panels; data for Arg not shown). Ser was less effective in inhibiting the growth of C. pneumoniae inclusions, compared to its dramatic effect on C. trachomatis inclusion size. The influence of continuous incubation with non-essential amino acids on the generation of infectious chlamydiae is depicted in Fig. 2(D). Glu, Gln and Asp enhanced the progeny infectivity of both species, especially that of C. pneumoniae. Other amino acids had more activity against the progeny infectivity of C. trachomatis than against C. pneumoniae infectivity; their activity against the latter was sometimes limited, as in the case of Pro and Asn. The only exception was Arg, which reduced the progeny infectivity of C. pneumoniae by more than 90 %, compared to a reduction of about 40 % in C. trachomatis progeny infectivity.
Based on the notion that genes that encode key enzymes involved in glucose formation are present in the chlamydial genome (Stephens et al., 1998), Iliffe-Lee & McClarty (2000) have been able to show that, in the absence of glucose, other gluconeogenic carbon sources, such as Glu, can support chlamydial growth. Evidence that some amino acids may be needed in greater amounts than normally present has come from the studies of Ojcius et al. (1998), which demonstrate the stimulation of Glu synthesis in cells infected with C. psittaci. Moreover, we showed here that excess Glu or Asp increased the progeny infectivity of C. trachomatis and C. pneumoniae. Taken together, optimal chlamydial growth may require relatively high levels of certain amino acids that could be utilized in chlamydial and/or host-cell functional metabolic pathways, such as the gluconeogenesis and energy-production pathways. The growth enhancement caused by these amino acids would be achieved if they were not antagonistic to other essential amino acids or could compete with amino acids whose depletion was capable of enhancing chlamydial growth. These hypotheses should be tested.
Our results indicate that the addition of certain essential and non-essential amino acids can modulate the growth of chlamydial inclusions and may drastically affect the production of infectious progeny by two strains representing C. trachomatis and C. pneumoniae. In some cases, the strains responded differentially to amino acid overload, and the time p.i. at which amino acids were added appeared to be critical for this differential response. Excess amino acids introduced at the middle of infection led to a similar response in both strains, except for Met, Gly, Hyp and, to a lesser extent, Ser. When present over the whole course of infection, most amino acids added in excess affected the chlamydial infections differentially. For example, the essential Trp and Val had marginal adverse effects on the generation of infectious C. trachomatis, but significant positive effects on C. pneumoniae progeny infectivity. The non-essential amino acids Glu, Gln and Asp also stimulated the progeny infectivity of C. pneumoniae more than that of C. trachomatis. Collectively, our data not only indicate that C. pneumoniae is less sensitive to certain amino acids, shown to be active against C. trachomatis, but also that C. pneumoniae may even benefit from the presence of certain amino acids at extremely high concentrations.
Genome sequences of different chlamydial species, including C. trachomatis and C. pneumoniae, are now publicly available. Analysis of these sequences has revealed that chlamydiae generally possess incomplete pathways of amino acid biosynthesis (Stephens et al., 1998) and has also demonstrated some similarities and differences in the pathways of amino acid synthesis and transport. For example, C. trachomatis and C. pneumoniae encode genes for the same single amino acid transporters (e.g. glnPQ, braB and aaaT) as well as genes for the same di- and oligopeptide ATP binding cassette (ABC) transporters (dpp and opp operons). The most prominent difference between the two genomes is that C. pneumoniae possesses a truncated TrpA operon and, therefore, cannot synthesize Trp from its precursors, whereas some C. trachomatis serovars, including L2, can produce Trp from the intermediate precursor indole (Shaw et al., 2000). Another striking difference recently found is that C. pneumoniae, but not C. trachomatis, has the Arg repressor (ArgR) that controls a putative Arg transport system encoded by the glnPQ operon in response to changes in intracellular Arg levels (Schaumburg & Tan, 2006). Genome comparison between C. trachomatis and C. pneumoniae has revealed that C. trachomatis contains about 70 genes without homologues in C. pneumoniae, and that C. pneumoniae has about 200 genes not present in C. trachomatis (Kalman et al., 1999). Taken together, the already-confirmed differences in amino acid biosynthesis/transport pathways between chlamydial species and the presence of species-specific genes not yet characterized suggest that the differential response to amino acid overload, shown in the present work, is most likely due to differences in the genetic background of different chlamydiae.
In their elegant study, Coles & Pearce (1987) revealed that the growth inhibition of C. psittaci by the omission of single amino acids results from competition between residual amounts of omitted amino acids, still present in the cytoplasm, and other amino acids with which they share a common transport system. The concomitant omission of Val and Ile abrogates growth inhibition by Val omission, indicating that Ile is an antagonist of Val. It has also been shown that Trp is an antagonist of Phe, and that Ile plus Val are antagonists of Leu. Consistent with the findings of Coles & Pearce (1987), preliminary experiments performed in our laboratory showed that antagonism could be one mechanism that underlies the inhibitory effect on chlamydial growth of overload with some amino acids. For example, a complete recovery of growth was noticed when Val was simultaneously added to Ile-treated cells (unpublished results), suggesting that Ile is an antagonist of Val. However, ongoing work suggests the presence of at least one other inhibitory mechanism in addition to antagonism; for example, the effect of Gly cannot be abrogated by the addition of other amino acids. It is possible that the addition of some amino acids activates certain amino-acid-responsive mechanisms of chlamydial and/or host gene regulation which may negatively affect bacterial growth. Evidence of such a mechanism of transcriptional regulation in Chlamydia has recently been published by Schaumburg & Tan (2006), who indicated that the Arg transport system glnPQ could be transcriptionally regulated in vitro by the recombinant ArgR repressor in response to Arg levels. The challenge now is to investigate if some of the inhibitory amino acids studied here could function as regulatory elements of chlamydial gene transcription. Potential chlamydial amino-acid-responsive mechanisms can be identified by searching the substantial comparative genomic information now available and by establishing heterologous systems; unfortunately, chlamydiae cannot grow in cell-free systems, and it has been impossible until now to manipulate them genetically.
In conclusion, our data are consistent with previous studies on the influence of amino acid depletion on chlamydial growth and indicate that manipulation of the nutritional environment could significantly influence chlamydial development and pathogenesis. However, it is too early to conclude that the differential sensitivities of C. trachomatis and C. pneumoniae to excess amino acid concentrations are species-specific, because of the limited number of isolates examined. The present study, nevertheless, hints at the potential existence of such a phenomenon among chlamydial species. Eventually, abrogation of chlamydial growth by supplementation with certain amino acids could represent a novel approach for the curative treatment and prophylaxis of chlamydial infections.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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