Application of β-Lactamase Reporter Fusions as an Indicator of Effector Protein Secretion during Infections with the Obligate Intracellular Pathogen <i>Chlamydia trachomatis</i>

<div><p><i>Chlamydia</i> spp. utilize multiple secretion systems, including the type III secretion system (T3SS), to deploy host-interactive effector proteins into infected host cells. Elucidation of secreted proteins has traditionally required ectopic expression in a surrogate T3SS followed by immunolocalization of endogenous candidate effectors to confirm secretion by chlamydiae. The ability to transform <i>Chlamydia</i> and achieve stable expression of recombinant gene products has enabled a more direct assessment of secretion. We adapted TEM-1 β-lactamase as a reporter system for assessment of chlamydial protein secretion. We provide evidence that this system facilitates visualization of secretion in the context of infection. Specifically, our findings provide definitive evidence that <i>C</i>. <i>trachomatis</i> CT695 is secreted during infection. Follow-up indirect immunofluorescence studies confirmed CT695 secretion and indicate that this effector can be secreted at multiple points during the chlamydial developmental cycle. Our results indicate that the BlaM-fusion reporter assay will allow efficacious identification of novel secreted proteins. Moreover, this approach can easily be adapted to enable more sophisticated studies of the secretion process in <i>Chlamydia</i>.</p></div

Expression of fusion constructs in <i>C</i>. <i>trachomatis</i> L2.

<p>(A). Transcription of <i>blaM</i> fusions (grey bars) and <i>mCherry</i> (black bars) was measured by reverse transcription followed by quantitative real-time PCR. Levels were normalized to those of chlamydial <i>16s</i>. Total RNA was isolated 24 hpi from HeLa cells infected with transformed <i>C</i>. <i>trachomatis</i> L2 at an MOI of 1. (B). Total protein was isolated 24 hpi from HeLa cells infected with transformed <i>C</i>. <i>trachomatis</i> L2 at an MOI of 1, and samples were probed with BlaM- or chlamydial Hsp60-specific monoclonal antibodies. Size standards are indicated in kDa.</p

CT695 is secreted by EBs during invasion.

<p>(A). CT695-specific antibodies were used to probe lysates of <i>Chlamydia</i>-infected culture lysates (HeLa + L2) or lysates of purified <i>C</i>. <i>trachomatis</i> L2 EBs (EB). Lysates of corresponding uninfected HeLa cells (HeLa) were added as a negative control and all lysates were probed with α-actin as a loading control for whole-culture material. (B). Purified preparations of <i>C</i>. <i>trachomatis</i> L2 EBs were mock treated or treated with BSA and EDTA. Samples were subsequently centrifuged and material from cell-free supernatants (Sup) and chlamydiae-containing pellets (P) were probed in immunoblots with Hsp60, TarP, or CT695-specific antibodies. All proteins were visualized with HRP-conjugated secondary antibodies and subsequent chemiluminescence development. (C). HeLa cells were infected with CMPTX-labelled <i>C</i>. <i>trachomatis</i> L2 at an MOI of 10 and fixed paraformaldehyde fixed at 1 hpi. Cells were probed with TarP-, MOMP- or CT695-specific antibodies. Chlamydiae are shown in red while CT695, MOMP, and TarP localization was visualized with secondary antibodies conjugated to Alexa-488 (green). Merged channels of epifluorscence images are shown. Large arrow designates area of inset and small arrows signify other areas where CT695 appears as punctate signal adjacent to EBs. Scale bar = 5 μm.</p

A schematic of constructed plasmids using CT695 as an example.

<p>(A). pUCNmP. The <i>Neisseria meningitidis</i> promoter (NmP) was inserted into pUC19 upstream from BlaM. Insertion/Deletion PCR is used to insert any chlamydial sequence (<i>ct695</i> is shown) to create a translational fusion of the chlamydial gene (green) with the β-lactamase gene (blue). DNA elements can then be PCR amplified using primers NmP+BlaFus+AscI F and NmP+BlaFus+AscI R to generate a product flanked by AscI restriction sites. (B). pL2dest was created by replacement of the coding sequence for GFP/CAT of pGFP::SW2 with the mCherry gene. A chloramphenicol drug cassette flanked by AscI recognition sequences was introduced immediately downstream from mCherry coding sequence. (C). pCT659-BLA was created by ligation of AscI-digested PCR product into the AscI site in pL2dest. The resulting plasmid allows expression of CT695-Bla from the constitutive Nmp promoter.</p

Gene arrangement and relative expression of <i>ct694</i>, <i>ct695</i>, and <i>ct696</i>.

<p>(A). Schematic arrangement of the <i>ct694</i>-<i>ct696</i>. The locus is flanked by phosphoglycerate kinase (<i>pgk)</i> and endonuclease III (<i>end3</i>) genes and contains 4 intergenic regions (IGS1-4) of 261, 48, 54, and 3 nucleotides, respectively. TransTermHP [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135295#pone.0135295.ref036" target="_blank">36</a>] predicts a Rho-independent transcriptional terminator between <i>pgk</i> and <i>ct694</i>. Arrows indicate relative positions of previously reported transcriptional start sites [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135295#pone.0135295.ref038" target="_blank">38</a>]. Amplicons used for expression analyses are represented schematically. 100 bp amplicons (solid lines) were generated for qRT-PCR wherease gene-spanning amplicons (double lines) were employed to test the possibility of polycistronic message. (B). Transcription of <i>ct694</i>, <i>ct695</i>, and <i>ct696</i> increases throughout chlamydial development. HeLa cells were infected with <i>C</i>. <i>trachomatis</i> L2 at an MOI of 0.5, and transcript levels were determined by quantitative real-time PCR at various time points throughout the chlamydial developmental life cycle. Expression levels were normalized against, and relative to, those for the constitutively expressed <i>rpoD</i>.</p

CT695 is secreted during late-cycle development and co-localizes with the chlamydial inclusion.

<p>HeLa cells were infected with <i>C</i>. <i>trachomatis</i> L2 at an MOI of 1 and paraformaldehyde fixed at 24 hpi. (A). CT695 or TarP localization were assessed using α-CT695 or α-TarP, respectively. Chlamydiae were detected using α-Hsp60. Individual and merged channels of epifluorscence images are shown with specific detection of <i>Chlamydia</i> (red) and CT695 or TarP (green). Arrows indicate apparent inclusion membrane localization and scale bar = 10 μm. (B) HeLa cells were infected with <i>C</i>. <i>trachomatis</i> L2 at an MOI of 1 and paraformaldehyde fixed at 24 hpi. CT695 was detected with α-CT695 (green) wherease the position of the chlamydial inclusion membrane was visualized via staining with antibodies specific for Syntaxin-6 (red). Scale bar = 5 μm.</p

Co-transcription of <i>ct694</i> and <i>ct695</i>.

<p>(A). The presence of transcripts containing multiple open reading frames was determined by reverse-transcription (RT) PCR with primers surrounding <i>ct694</i> and <i>ct695</i> or <i>ct695</i> and <i>ct696</i>. RNA was isolated from HeLa cells infected with <i>C</i>. <i>trachomatis</i> L2 at an MOI of 0.5 grown to various time points post infection. (B). The same samples were additionally analyzed by quantitative real-time PCR for increased sensitivity. Levels shown are relative to those detected 6 hpi. A Student’s T test with Welch’s correction was employed to assess statistical significance (*, P < 0.04).</p

Detection of BlaM fusions in the cytosol of the host cell.

<p>24 hpi, HeLa cells infected with transformed <i>C</i>. <i>trachomatis</i> L2 were examined for the presence of cytosolic BlaM with the GeneBLAzer Detection Kit. Samples were treated with CCF2-AM for 30 minutes, fixed with 4% paraformaldehyde, and examined by confocal microscopy. Chlamydial expression of CT694-, CT695-, and TarP-BlaM constructs produced significant blue-fluorescent signal in the cytosol of infected cells. Bar = 10 μm.</p

Assessing a Potential Role of Host Pannexin 1 during <i>Chlamydia trachomatis</i> Infection

<div><p>Pannexin 1 (Panx1) is a plasma membrane channel glycoprotein that plays a role in innate immune response through association with the inflammasome complex. Probenecid, a classic pharmacological agent for gout, has also been used historically in combination therapy with antibiotics to prevent cellular drug efflux and has been reported to inhibit Panx1. As the inflammasome has been implicated in the progression of <i>Chlamydia</i> infections, and with chlamydial infections at record levels in the US, we therefore investigated whether probenecid would have a direct effect on <i>Chlamydia trachomatis</i> development through inhibition of Panx1. We found chlamydial development to be inhibited in a dose-dependent, yet reversible manner in the presence of probenecid. Drug treatment induced an aberrant chlamydial morphology consistent with persistent bodies. Although Panx1 was shown to localize to the chlamydial inclusion, no difference was seen in chlamydial development during infection of cells derived from wild-type and Panx1 knockout mice. Therefore, probenecid may inhibit <i>C. trachomatis</i> growth by an as yet unresolved mechanism.</p></div

Absence of HRI reduces the translocation of <i>Listeria</i> into the cytosol.

<p>(<b>A</b>) Single-cell suspensions of splenocytes were prepared from <i>Hri</i> +/+ and -/- mice and either left uninfected or infected <i>in vitro</i> for 5 hrs with OVA-expressing <i>L. monocytogenes</i>. Histogram displays the levels of OVA surface staining on viable CD11b⁺-gated macrophages from a representative experiment and the relative median fluorescence intensities (MFI) of 3 separate experiments is shown in the right panel. The isotype control stained cells are shown in the grey filled plot. (<b>B</b>) Unstimulated peritoneal macrophages isolated from <i>Hri</i> +/+ and -/- mice were infected <i>in vitro</i> with GFP-expressing <i>L. monocytogenes</i> for 6 hr and then stained for actin. The fraction of <i>L. monocytogenes</i> (Lm) that were actin associated was determined by dividing the yellow and red-tailed green Lm by the total Lm (<i>Hri</i> +/+: N=418 Lm/168 macrophages; <i>Hri</i> -/-: N=542 Lm/167 macrophages). The fraction of cytosolic Lm that possessed actin tails was determined by dividing the red-tailed green Lm by the total number of yellow and red-tailed green Lm. (<i>P</i> values calculated using student <i>t</i> test.).</p