Bacterial strains, cloning reagents and plasmids

Experiments were performed with the wild-type S. flexneri 5a strain M90T-Sm harbouring a streptomycin resistance mutation [27] and/or its derivative mutant strains ipaD [28] and mxiD [26]. Shigella strains were grown on trypticase soy broth (TSB) or trypticase soy agar in conditions indicated under the relevant sub-headings below.

The plasmids used for expression of bacterial proteins fused to the β-lactamase TEM-1 (referred to as Orf-bla chimeric proteins) are derived from pSU2718 (GenBank: M64731) with the following alterations: (i) modification of the multiple cloning site; (ii) upstream of the lac promoter, addition of the transcription terminator of trpA by mutagenesis PCR (resulting plasmid pSU2.1tt) and (iii) downstream of the lac promoter via KpnI/XbaI restriction sites, addition of the sequence coding for the mature β-lactamase TEM-1 isolated from pUC18, with 5’ insertion of a sequence coding for a six amino acid linker (resulting plasmid pSU2.1tt-bla). The sequences coding for the Shigella Orfs of interest were extracted from M90T pWR100 virulence plasmid DNA (using the annotations from [17]) and inserted in frame upstream of the linker via EcoRI/SmaI restriction sites (resulting plasmids pSU2.1tt-Orf-bla). A “consensus” Shine and Dalgarno (SD) sequence isolated from pQE-60 (see [29]) was part of the forward primer used for PCR amplification of each sequence inserted into pSU2.1tt and pSU2.1tt-bla. For each Orf, exchange of this “consensus” SD for the “endogenous” SD (endSD) sequence was performed either by mutagenesis PCR or by repeating extraction of the Orf of interest together with the 30 to 40 nucleotides upstream of its start codon in pWR100 sequence. Where indicated, mutagenesis PCR on the resulting pSU2.1tt-endSD-Orf-bla plasmids was performed to exchange the β-lactamase coding sequence for the c-Myc coding sequence (resulting plasmids pSU2.1tt-endSD-Orf-myc). The primers and plasmids used are listed in S1 and S2 Tables. NucleoSpin Plasmid miniprep kit (Macherey-Nagel), PCR clean up and gel extraction kits (Macherey-Nagel), PureLink HiPure midiprep kit (Invitrogen) were used for DNA preparation. Restriction enzymes, T4 DNA ligase, T4 polynucleotide kinase, Taq DNA polymerase and Phusion polymerase were purchased from ThermoFisher Scientific. Chemically competent Escherichia coli DH5α bacteria (Invitrogen) were used for all cloning steps and were grown at 37°C in 2xYeast Tryptone medium, supplemented with 30 μg/mL chloramphenicol when transformed with Orf-bla/myc-expressing plasmids.

Shigella strains were transformed with plasmids expressing Orf-bla or Orf-myc by electroporation and isolated on TSB agar supplemented with 0.01% Congo red and 10 μg/mL chloramphenicol after overnight incubation at 37°C. When indicated, WT Shigella strains expressing Orf-bla chimeric proteins were transformed with pUC18Δz-DsRed, a plasmid allowing constitutive expression of the fluorescent DsRed.T3 protein [30]. This plasmid was obtained by exchanging the insert of the pUC18Δz-TSAR plasmid for the DsRed coding sequence under the control of the rpsM promoter from the pTSAR1.3 plasmid, via SapI/HindIII restriction sites (both pUC18Δz-TSAR and pTSAR1.3 plasmids are described in [29], the former corresponding to the pFX4 plasmid with the coding sequence conferring resistance to zeocin instead of ampicillin). Shigella Orf-bla strains transformed with pUC18Δz-DsRed were isolated on TSB agar supplemented with 0.01% Congo red, 10 μg/mL chloramphenicol and 300 μg/mL zeocin after overnight incubation at 37°C.

Mass spectrometry data collection

mxiD and ipaD strains were grown overnight at 30°C and sub-cultured 1/50 in TSB at 37°C for 6 hours. Sub-cultures were spun at 3,220 g, 4°C for 15 min and supernatants filtered with 0.45 μm syringe filters. Proteins contained in the filtered supernatants were precipitated overnight at 4°C with 10% (v/v) trichloroacetic acid. Protein pellets from centrifugation at 9,400 g, 4°C for 15 min were recovered in 400 μL Laemmli buffer 1X (Biorad), and pH re-equilibrated with a 2M sodium hydroxide solution. Proteins present in the lysates were separated using polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie G-250 staining (Merck). Each gel lane corresponding to a unique experimental condition was cut into a single piece, then de-stained, dehydrated and treated with trypsin (Promega) overnight at 37°C. The resulting peptides were desalted using C₁₈ ZipTip column (Merck Millipore) and subsequently dissolved in 0.1% formic acid prior to liquid chromatography mass spectrometry analysis (LC-MS/MS). MS was performed on an Orbitrap Fusion MS (Thermo Fisher Scientific, Waltham, MA, USA) with a TriVersa NanoMate (Advion, Ltd., Harlow, UK) source in LC chip coupling mode. The peptide lysates were separated on a UHPLC system (Ultimate 3000, Dionex/Thermo Fisher Scientific, Idstein, Germany). 5 μL samples were first loaded for 5 min on the pre-column (μ-precolumn, Acclaim PepMap, 75 μm inner diameter, 2 cm, C18, Thermo Scientific) at 4% mobile phase B (80% acetonitrile in nanopure water with 0.08% formic acid), 96% mobile phase A (nanopure water with 0.1% formic acid), then eluted from the analytical column (PepMap Acclaim C18 LC Column, 25 cm, 3 μm particle size, Thermo Scientific) over a 120 min linear gradient of mobile phase B (4–55% B). The mass spectrometer was set on top speed with a cycle time of 3 s, using the Orbitrap analyser for MS and MS/MS scans with higher energy collision dissociation (HCD) fragmentation at normalised collision energy of 28%. MS scans were measured at a resolution of 120,000 in the scan range of 400–1,600 m/z. MS ion count target was set to 4×10⁵ at an injection time of 60 ms. Ions for MS/MS scans were isolated in the quadrupole with an isolation window of 2 Daltons (Da) and were measured with a resolution of 15,000 in the scan range of 350–1,400 m/z in the Orbitrap. The dynamic exclusion duration was set to 30 s with a 10-ppm tolerance around the selected precursor and its isotopes. Automatic gain control target was set to 5×10⁴ with an injection time of 120 ms.

Proteome data analysis

Proteome Discoverer (v1.4, Thermo Scientific) was used for protein identification and the acquired MS/MS spectra were searched with SEQUEST HT algorithm against the Uniprot reviewed database of Shigella pWR100 proteins (database of 265 protein-coding sequences, containing pWR100 protein annotations from NCBI [17] and the S. flexneri chromosomal IpaH sequences, combined with the sequences of 150 common contaminants). Enzyme specificity was selected to trypsin with up to two missed cleavages allowed using 10-ppm peptide ion tolerance and 0.05 Da MS/MS tolerances. Oxidation (methionine) and carbamylation (lysine and arginine) were selected as variable modifications and carbamidomethylation (cysteine) as a static modification. Only peptides with a false discovery rate (FDR) <0.01 calculated by Percolator [31] and peptide rank = 1 were considered as identified. All peptides matching more than one protein were removed, because they could not be used to distinguish between the secretion of homologs (in particular peptides matching the constant carboxy-terminal domain of IpaH proteins). The only exceptions to this rule were OspE1/OspE2, IpaH1.4/IpaH2.5, IpaH1/IpaH6 and IpaH4/IpaH5 for which this filter would have removed nearly all peptides identified, as these proteins are almost identical (>98% identity at the amino acid level). Therefore, these protein pairs were treated as one entity and appear as a single entry in the MS data analysis. The abundance of each detected protein was obtained by averaging peak intensities of up to 3 of its most abundant peptides in each replicate [32,33]; abundance values obtained from at least three independent replicates were then used to derive the average abundance value. Proteins identified in the ipaD sample were sorted into hit tiers based on the following criteria: Tier 2 hits had either at least 1.5-fold higher abundance for peptides also identified in the mxiD sample (i.e. shared peptides), or no peptides identified in the mxiD sample for at least two identified in the ipaD sample; Tier 1 hits had either no peptides identified in the mxiD sample for one identified in the ipaD sample, or 1.5-fold more peptides detected in the ipaD sample with a relative abundance lower than 1.5-fold; Tier 0 hits were the proteins not falling into the previous categories.

Congo red induction, bacterial lysates and immunoblotting

Relevant Shigella strains were grown overnight at 30°C with the appropriate antibiotics and sub-cultured 1/50 in TSB at 37°C without antibiotics. When indicated, T3SA secretion of WT strains was induced in vitro after the bacterial sub-culture had reached an optical density at 600 nm (OD₆₀₀) of 0.1, through addition of 30 μg/mL of the T3SA inducer Congo Red (CR) for 4 hours. This protocol was essentially performed as in [34,35] except that a lower concentration of CR was used, as in [36]. Sub-cultures were spun at 9,000 g for 1 min and equivalent amounts of bacteria were resuspended in Laemmli buffer for pellet fractions (Biorad). For supernatant fractions, supernatants were filtered with 0.22 μm syringe filters and secreted proteins precipitated on ice for 30 min using 10% (v/v) trichloroacetic acid. Protein pellets resulting from spinning at 16,000 g for 30 min were resuspended in Laemmli buffer. Boiled lysates were analysed by SDS-PAGE and the following antibodies were used to detect proteins of interest: mouse anti-β-lactamase (Abcam ab12251 or Novus Biologicals NB120-12251; 1/1,000), rabbit anti-c-Myc (Santa Cruz sc789; 1/750), rabbit anti-RecA (Abcam ab63797; 1/500), rabbit anti-IpaH ([37,38]; 1/5,000), rabbit anti-Spa15 ([39,40]; 1/20,000), anti-mouse-HRP (GE Healthcare NXA931; 1/10,000), anti-rabbit-HRP (Cell Signalling 7074S; 1/7,000). Membranes were imaged using film detection or Amersham Imager 600 (GE Healthcare). When indicated, quantification of band intensity was performed using Image Studio^TM Lite software (LI-COR Biosciences).

Secretion assay

Relevant Shigella strains were grown overnight at 30°C with the appropriate antibiotics and sub-cultured 1/50 in TSB at 37°C for 4 hours without antibiotics. β-lactamase activity was measured by incubating 20 μL of the colorimetric β-lactamase substrate nitrocefin (500 μg/mL stock, EMD Millipore) with 100 μL of different dilutions of bacterial supernatants (after spinning sub-cultures at 9,000 g for 1 min) or cleared sonicated sub-cultures (after spinning sonicated sub-cultures at 10,600 g for 1 min). Sonication of sub-cultures was performed with a UP50H sonicator (Hielscher Ultrasound Technology) by continuous sonication over 30 seconds at 50% amplitude. β-lactamase-mediated hydrolysis of the nitrocefin substrate was assessed by measuring absorbance at 486 nm (A₄₈₆) over time using a plate reader (Infinite M200 Pro; TECAN). β-lactamase activity was calculated in equivalent Miller units (e.M.u.) and averaged over successive time points within the linear part of the curve: Activity [e.M.u.] = 1,000 x (A₄₈₆ [sample]—A₄₈₆ [TSB]) / (time(min) x volume(mL) x OD₆₀₀[subculture] x sample dilution).

Host cell infection for translocation assays

Jurkat T cells (clone E6-1, ATCC TIB-152) and HeLa cells were respectively cultured in RPMI1640 and DMEM 1 g/L glucose (Gibco), supplemented with 10% heat-inactivated foetal calf serum (HI-FCS) at 37°C with 5% CO₂. HeLa cells were seeded the day before the experiment at 0.5x10⁵ cells per cm². Prior to infection, cells were washed and put back in the incubator in assay medium (i.e. DMEM containing 2.5 mM probenecid (VWR), which is an anion transport inhibitor facilitating the retention of CCF2-AM within loaded cells) supplemented with 2 μM CCF2-AM (ThermoFisher Scientific). Jurkat T cells were seeded on the day of the experiment in 96-well plates round-bottom at 3-5x10⁵ cells per well in assay medium (i.e. RPMI1640 containing 2.5 mM probenecid) supplemented with 1 μM CCF2-AM. Relevant bacterial strains grown overnight at 30°C with the appropriate antibiotics were sub-cultured 1/50 in TSB at 37°C without antibiotics until early exponential phase was reached. Sub-cultures were adjusted in the appropriate assay medium to the indicated multiplicity of infection (MOI). CCF2-AM-loaded Jurkat or HeLa cells were infected by centrifugation of bacteria at a MOI of 50 onto the cells at 300 g, 37°C for 10 min. Alternatively, bacterial sub-cultures were coated with 10 μg/mL poly-L-lysine (Sigma) in PBS for 10 min, prior to infection of CCF2-AM-loaded HeLa cells for 15 min at room temperature at a MOI of 10. Infection was subsequently allowed to proceed for indicated times at 37°C with 5% CO₂. When indicated, host cells were incubated in assay medium supplemented with 50 μg/mL gentamycin to kill extracellular bacteria. Sample analyses of the translocation assay for both cell types are described in the next section.

Translocation assay data acquisition and analysis

Infected HeLa cells were washed three times in PBS containing 2.5 mM probenecid. Cells were then switched to the imaging medium composed of DMEM without Phenol Red (Gibco) containing 2.5 mM probenecid and 50 μg/mL gentamycin. Acquisitions were performed using an Axio Observer.Z1 microscope (Zeiss) equipped with a swept field confocal Opterra system (Bruker) and an Evolve 512 Delta EMCCD camera (Photometrics), using a 40x PlanAPOCHROMAT oil immersion/1.4 NA objective (Zeiss). Fluorescence images were sequentially acquired as follows: (i) excitation at 405 nm and detection with a 460/50 filter (Blue channel, cleaved CCF2-AM); (ii) excitation at 405 nm and detection with a 531/50 filter (FRET channel, uncleaved CCF2-AM); (iii) excitation at 488 nm and detection with a 535/50 filter (Green channel, total CCF2-AM); (iv) excitation at 561 nm and detection with a 630/75 filter (Red channel, DsRed bacteria). Confocal Z-stacks (15 Z-slices of 0.5 μm) were acquired using a 70 μm slit, with an exposition time of 50 ms. Post-acquisition image analysis and quantification were performed with Fiji 2.0.0 software [41]: regions of interest (ROI) were defined for invaded cells loaded with CCF2-AM (based on Red and Green channels) and for each ROI the total mean fluorescence of the 15 Z-slices in the Blue and FRET channels were used to calculate the cleaved/uncleaved CCF2-AM fluorescent ratio (Blue/FRET channels).

Infected Jurkat cells were washed in PBS containing 2.5 mM probenecid. They were then stained with the Live/Dead near-infrared dead cell stain kit (ThermoFisher Scientific) following the supplier guidelines except that 2.5 mM probenecid was maintained during all steps. Cells were finally resuspended in the flow cytometry buffer composed of ice-cold PBS containing 2.5 mM probenecid and 0.1% BSA. Samples were acquired on a BD FACSCanto II flow cytometer (BD Bioscience). Single cells were gated based on forward and side scatters and dead cells were excluded based on excitation with the 633 nm laser coupled to 750/60 nm emission filter. Cleaved and uncleaved CCF2-AM were detected within live cells with the 405 nm excitation laser and 450/50 and 510/50 nm emission filters, respectively. Data were analysed using Flow Jo 10.0.8 software.

Plaque assay

The Shigella single-mutant strains used in plaque assays were the non-spreading strain icsA [42] and orf86, orf13, orf131a and orf176 [43]. The tetracycline cassette flanked by flippase recognition target (FRT) sites present within each of the latter strains was removed upon transformation with the pCP20 plasmid that encodes the flippase recombinase (FLP), as described in [44]. Plaque assays were performed as described previously [45], using Caco-2/TC7 cells cultured to confluency for three weeks in DMEM 1 g/L glucose supplemented with non-essential amino acids, glutamine (all from Gibco) and 20% HI-FCS at 37°C with 10% CO₂. Following 2 hours of infection with 10⁶ bacteria at 37°C with 10% CO₂, an overlay of 0.5% agarose containing 50 μg/ml gentamycin in culture medium was applied to extensively washed infected monolayers. Three days later, ethanol fixation and Giemsa R solution (RAL Diagnostics) staining allowed for enumeration of plaques and measurement of their area using Fiji 2.0.0 software [41].

Data presentation and statistical analysis

Prism 6.0 (GraphPad Software, Inc.) was used for graphs and statistical analyses. Unpaired two-tailed Student’s t-test was used to compare two conditions unless indicated otherwise. Significant statistical differences are indicated by asterisks: *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001. Error bars represent standard deviation to the mean. Illustrator CS5 software (Adobe) was used for assembling figures.

Identification by mass spectrometry of proteins secreted by Shigella T3SA

Proteins found in the culture medium of ipaD and mxiD S. flexneri strains were recovered by trichloroacetic acid precipitation and analysed by MS. These MS data are retrievable using the dataset identifier PXD006086 from the ProteomeXchange Consortium via the PRIDE partner repository [46]. Detected peptides were identified by mining the pWR100 annotated proteins database supplemented with the five chromosomally-encoded IpaH proteins [17]. For each of the three biological replicates, an identified protein was classified as a hit tier 2, 1 or 0, based on the quantification of abundance and number of matching peptides detected in ipaD versus mxiD supernatants (tier 2 hits are the proteins identified as T3SA substrates with the highest confidence). Proteins classified as tier 2 hits in at least two biological replicates were categorised as high confidence predictions while intermediate confidence predictions were tier 2 or 1 hits in at least two biological replicates. Lastly, proteins classified as tier 2 hits in only one biological replicate were categorised as low confidence predictions.

This analysis of the MS results classified the chromosomally-encoded IpaH proteins and 30 out of 31 previously known or highly suspected T3SA substrates encoded on pWR100 as high or intermediate confidence predictions (Fig 2A and S3 Table). Only OspZ, the product of orf212, was not detected in the secreted fraction. OspZ shares homology with the enteropathogenic E. coli effector NleE and was previously found to be secreted by S. flexneri 2a strain 2457T [47]. However, expression of ospZ in the S. flexneri 5a strain M90T used herein has been reported to be among the lowest of all genes located on the virulence plasmid [48], which might explain why it was undetected in the secreted fraction. On the other hand, four proteins of unknown function and localisation, namely Orf86, Orf13, Orf48 and Orf176, were predicted at high or intermediate confidence to be T3SA substrates. By contrast, identification of the outer membrane protein MxiD as an intermediate confidence prediction is an experimental artefact. Indeed, most of the mxiD coding sequence is replaced with an antibiotic resistance cassette, precluding detection of several MxiD-matching peptides in the mxiD strain, but not in the ipaD strain (see [26] for description of the mxiD mutant). Besides, three proteins identified as low confidence predictions were discarded on the basis of their known cellular localisation: IcsA (VirG) and SepA are auto-transporters [49,50] and PhoN2 is a periplasmic enzyme [51]. Nevertheless, two proteins of unknown function and localisation named Orf131a and Orf182 were predicted at low confidence to be T3SA substrates. Furthermore, the MS analysis validated the T3SA-dependent secretion of OspI (Orf169b), whose effector function was recently uncovered [19]. Because mRNA expression of ospI was comparable to that of orf13 [48], and their peptide abundance in ipaD supernatant were similar (S3 Table), OspI was selected as a positive control for following experiments. Therefore, the proteins selected for further analysis were the novel putative T3SA substrates (i.e. Orf86, Orf48, Orf176, Orf13, Orf131a and Orf182) and the T3SA effector OspI.

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Fig 2. Mass spectrometry results.

(A) Group of proteins predicted to be T3SA substrates with low, medium and high confidence using the MS approach described in the text. Novel putative substrates and control substrate OspI selected for further analysis are highlighted in bold. (B) Genetic mapping of the ORFs selected for further investigation. Colours refer to the GC-content of the genes: red <40%; blue 40–50%; green >50%. Numbers refer to the coordinates on pWR100 sequence in kilobases (accession number AL391753).

https://doi.org/10.1371/journal.pone.0186920.g002

Features of mass spectrometry hits

The six T3SA substrate candidates are encoded by orf13, orf48, orf86, orf176, orf131a and orf182. Overall these genes are well conserved with more than 98% sequence identity with their orthologs in the four Shigella spp. (i.e. S. flexneri, boydii, sonnei and dysenteriae). The only exceptions were orf48 and orf86 for which no orthologs were found in S. sonnei. As represented in Fig 2B, orf13 and orf131a exhibit a GC-content below 40%, which is a hallmark of known T3SA substrates [17]. orf131a, also known as SPA-ORF10, is located at the end of the mxi-spa locus. Its protein product named uncharacterized 9.1 kDa protein in spaS 3'region (www.uniprot.org), which we called Orf131a for the sake of simplicity (as in [17]), exhibits 27% sequence identity with E. coli YmgB/AriR, which has been implicated in biofilm formation repression and acid-resistance [52]. orf13 is located 1031 bp upstream of the gene coding for the known T3SA substrate OspF [17] and its protein product Orf13 has no identified conserved domain or characterised homolog. Two of the MS hits encoded by genes with a higher GC-content, namely orf48 and orf86 (44.9% and 42.9%, respectively), share a domain predicted to interact with DNA (Clusters of Orthologous Group 4453, COG4453) and are related to the antitoxin component of type II toxin-antitoxin (TA) systems [53] (orf48 and orf86 being referred to as 0063 and 0112 in [53], based on another virulence plasmid annotation [18]). Plasmid-encoded TA systems are classically described as post-segregation killing systems ensuring the maintenance of low-copy plasmids by inducing death of plasmid-free daughter cells (reviewed in [54]). Recently, McVicker and colleagues reported that Orf48/0063, but not Orf86/0112, was part of a functional TA system contributing to the maintenance of S. flexneri large virulence plasmid under certain growth conditions [53]. Another MS hit, Orf176, encoded by a gene with a GC-content of 50.6%, displays a conserved domain related to predicted transcriptional regulators (COG3905) and shares 93% identity with the antitoxin-like YacA plasmid stabilisation protein in E. coli (Orf176 is referred to as YacA in [53]). A sequence coding for a protein similar to its cognate toxin YacB is found downstream of the orf176 gene on pWR100. However, presence of two supplementary nucleotides creates a premature stop codon, therefore probably inactivating the toxin of this putative yacA/yacB homolog [53]. Noteworthy, orf176 is intriguingly located between ipaH9.8 and ospG, which both encode T3SA substrates [17]. Lastly, orf182 displays a high GC-content (50.9%) and its protein product has a molecular weight higher than typical T3SA substrates. Orf182 harbours an ankyrin repeat domain that suggests a potential role in protein-protein interactions.

Validation of T3SA-dependent secretion of the novel T3SA substrates using a β-lactamase secretion assay

To test by an independent approach whether the T3SA substrate candidates identified by MS were indeed secreted by S. flexneri T3SA, we generated plasmids enabling expression of chimeric proteins composed of an amino-terminal T3SA substrate candidate and a carboxy-terminal β-lactamase (bla) moieties. The coding sequence for MS hits and the selected positive control were cloned individually into a low copy expression plasmid containing the coding sequence for the mature β-lactamase TEM-1 (i.e. lacking the signal peptide required for periplasm localization). For each β-lactamase construct, we chose to initiate translation of the chimeric proteins from the “endogenous” Shine and Dalgarno (SD) sequence encoded by the 30 to 40 nucleotides upstream of each of the selected ORFs in pWR100. Indeed we observed that this allowed an optimal expression when compared to the use of a constant “consensus” SD sequence (S2 Fig). The “endogenous” SD-based plasmids were introduced into ipaD strains and expression of the chimeric proteins was confirmed in bacterial lysates by immunoblotting using an antibody raised against the β-lactamase (Fig 3A). Most Orfs-bla yielded a single prominent band of the expected molecular weight. OspI-bla yielded a band corresponding to the predicted molecular weight of 53 kDa and a smaller band of approximately 45 kDa discussed below. Secretion of the Orf-bla proteins was assessed using an in vitro secretion assay: supernatants from the ipaD strains were incubated with nitrocefin, a colorimetric substrate of β-lactamase [55]. Absorbance at 486 nm was measured to assess hydrolysis of the substrate and thus quantify β-lactamase activity. Despite Orf182-bla being well expressed by ipaD bacteria (Fig 3A), very faint β-lactamase activity was detected in the supernatant of that strain (Fig 3B). This was not due to misfolding of the β-lactamase when fused to Orf182 because upon sonication-induced release of the cytosol, Orf182-bla processed nitrocefin similarly to OspI-bla and Orf48-bla (S3A Fig). Moreover, Orf182 was not secreted when fused to a smaller protein such as Myc while OspI-myc was (S3B Fig), ruling out the possibility that T3SA-mediated secretion of Orf182 was prevented due to the large size of the resulting β-lactamase chimeric protein. Altogether, this demonstrated that the low-confidence MS prediction Orf182 was a false positive from the MS analysis. Therefore, Orf182-bla was used as a non-secreted control in the following experiments. Of note, the additional lower molecular weight species in the strains expressing OspI-bla (Fig 3A) or OspI-myc (S3B Fig) displayed a similar size difference compared to the highest molecular weight species. This suggested that the lower molecular weight species in both lysates were due to an intrinsic feature of OspI rather than to the carboxy-terminal tag used. These lower molecular weight species were not secreted (S3B Fig), indicating that they did not contribute to the β-lactamase activity detected in ipaD supernatant (Fig 3B). Since the tag detected by immunoblotting is located in the carboxy-terminus of the chimeric proteins, these lower molecular species are likely amino-terminal truncations that might be produced by proteolytic digestion or from translation initiation at an internal start site, similarly to what was recently shown for an amino-terminal peptide of OspD1-bla [56].

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Fig 3. Experimental confirmation of secretion and translocation of MS hits.

(A) ipaD Shigella lysates were analysed by immunoblotting with anti-β-lactamase antibody. Load equivalent to a bacterial culture optical density at 600 nm (OD₆₀₀) of 0.2 for each lane. Chimeric proteins tested and their expected molecular weight (kiloDaltons, kDa): Orf182-bla (140), OspI-bla (53), Orf86-bla (40), Orf48-bla (41), Orf176-bla (40), Orf13-bla (51), Orf131a-bla (38). (B) Supernatants of ipaD strains expressing the Orf-bla chimeric proteins were incubated with nitrocefin. Enzymatic activity was calculated based on measurement of absorbance at 486 nm (A₄₈₆). e.M.u.: equivalent Miller unit. Data are from 3 independent experiments. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (unpaired two-tailed Student’s t-test compared to Orf182-bla). (C) CCF2-AM-loaded Jurkat T lymphocytes were infected with WT strains expressing Orf-bla chimeric proteins for 1 hour. Data are from 4 independent experiments. Translocated cells were detected by flow cytometry. *p<0.05; **p<0.01; ***p<0.001 (unpaired two-tailed Student’s t-test compared to Orf182-bla). (D-E) CCF2-AM-loaded HeLa cells were infected with WT-DsRed strains expressing Orf-bla chimeric proteins for 1 hour, followed by 1 hour of incubation with gentamycin. (D) Representative images of cells infected with WT Shigella strains expressing the indicated chimeric proteins, showing total CCF2-AM (Green channel), cleaved CCF2-AM (Blue channel) and uncleaved CCF2-AM (FRET channel). Dashed lines denote cells invaded by DsRed bacteria (Red channel) that were used for fluorescence quantification. (E) CCF2-AM cleavage was quantified within invaded cells based on fluorescence intensity of cleaved over uncleaved CCF2-AM within the defined regions of interest. Data are from 3 independent experiments, representing 50 to 100 invaded cells analysed per condition. ****p<0.0001 (unpaired two-tailed Welch’s t-test compared to Orf182-bla unless depicted otherwise).

https://doi.org/10.1371/journal.pone.0186920.g003

In comparison to Orf182-bla, significantly more enzymatic activity was detected in the supernatants of ipaD strains expressing the positive control OspI-bla or the five other Orf-bla chimeric proteins (Fig 3B). Differences in enzymatic activity between these ipaD strains could not be explained by variations in Orf-bla expression (Fig 3A) because no linear correlation was found between the β-lactamase activity detected in supernatants and the Orf-bla protein expression (R² = 0.005; see S4A Fig for protein quantification). Furthermore, the β-lactamase activity in ipaD supernatants was not due to bacterial lysis as the cytosolic protein RecA was not detected in secreted fractions (S3C Fig). As suggested by the MS analysis, Orf-bla secretion was T3SA-dependent because no enzymatic activity was detected in supernatants of T3SA-deficient mxiD strains expressing the chimeric proteins (S3D and S3E Fig). In addition, we assessed Orf-bla secretion by the WT strains through induction of the T3SA activity by the dye Congo Red (CR). Briefly, bacterial cultures at an optical density of 0.1 were supplemented or not with CR and allowed to grow for 4 hours until the late exponential-early stationary phase was reached. Because CR absorbs in the same wavelength as the cleaved nitrocefin, we assessed Orf-bla secretion by immunoblotting with the anti-β-lactamase antibody (S3F Fig). In the absence of CR, all Orf-bla but Orf182-bla were detected in WT supernatants. This is in agreement with a previous report indicating that in bacterial cultures grown beyond the late exponential phase, T3SA substrates such as the translocators IpaB and IpaC were significantly secreted in the absence of CR [57]. In the presence of CR, all Orf-bla but the Orf182-bla were present in the supernatants and in most cases more abundant compared to the basal condition. Taken together, these results support the notion that Orf86, Orf48, Orf176, Orf13 and Orf131a are secreted in a T3SA-dependent manner by S. flexneri.

Validation of T3SA-mediated translocation of the novel T3SA substrates into human cells using a β-lactamase translocation assay

Translocation of the novel T3SA substrates into host cells was then investigated by infecting human cell lines with WT S. flexneri strains expressing the different chimeric proteins (S5A Fig). As expected, no β-lactamase activity could be detected in the supernatants of these WT strains upon in vitro growth, except for OspI-bla that was secreted to a lower extent than in the ipaD strain (S5B Fig). Delivery of β-lactamase chimeric proteins into the host cell cytoplasm upon infection was assessed using an assay based on the cell-permeable fluorescence resonance energy transfer (FRET) β-lactamase substrate CCF2-AM [58]. Presence of the β-lactamase within the cytoplasm of cells loaded with this fluorescent dye results in the cleavage of CCF2-AM, which yields an easily detectable green-to-blue shift in the emission fluorescence wavelength [5,55,59]. We first investigated Orf-bla translocation into T lymphocytes by taking advantage of the accuracy of flow cytometry to measure translocation into the Jurkat T lymphocyte cell line, similarly to what was previously described using IpgD-bla-expressing WT bacteria [59]. The fluorescence intensity of cleaved versus uncleaved CCF2-AM within each cell was measured. Uninfected cells were used as the negative control to set the gate defining translocated host cells (S5C Fig). Using this gating strategy, we obtained the percentage of translocated Jurkat T cells, which was significantly higher for all T3SA substrate candidates compared to the non-translocated Orf182-bla (Fig 3C). The absence of cleaved CCF2-AM signal in Jurkat T cells infected with mxiD strains expressing a subset of Orf-bla confirmed that their translocation was T3SA-dependent (S5D and S5E Fig).

Next, we investigated whether translocation of the five novel substrates could be detected as well in human epithelial cells. To do this, we infected CCF2-AM-loaded HeLa cells with WT-DsRed Shigella strains expressing the different chimeric proteins. Infected cells were imaged using confocal microscopy (Fig 3D) and the fluorescence ratio between cleaved and uncleaved CCF2-AM was calculated for several invaded cells in each condition (Fig 3E). Cells invaded by Orf182-bla-expressing Shigella displayed a fluorescence ratio similar to that of cells invaded with WT bacteria devoid of Orf-bla (Fig 3E). The positive control OspI-bla and all the Orf-bla chimeric proteins induced cleavage of the CCF2-AM probe within invaded HeLa cells, as shown by the significantly higher fluorescence ratio compared to Orf182-bla-infected cells (Fig 3E). Intracellular bacterial lysis was excluded as a possible explanation for CCF2-AM cleavage as cells invaded with the WT strain expressing Orf182-bla did not display any significant substrate cleavage as compared to WT-invaded cells, while the β-lactamase was shown to be functional when fused to Orf182 (S3A Fig). In agreement with the ipaD secretion assay (Fig 3B), OspI-bla was more translocated into HeLa cells than the other Orf-bla chimeric proteins (Fig 3E). In contrast to what was previously described for the β-lactamase secretion assay (Fig 3B), translocation of Orf13-bla and Orf131a-bla into HeLa cells was significantly higher than that of the antitoxin-based Orf-bla (Fig 3E). These differences could not be explained by the variations in Orf-bla expression by WT Shigella (S5A Fig) as no significant linear correlation was found with CCF2-AM fluorescence ratio (R² = 0.24; see S4B Fig for protein quantification). Overall, the translocation assay into HeLa cells confirmed the results obtained with the Jurkat human T cell line, further asserting that Orf13, Orf131a, Orf86, Orf48 and Orf176 are T3SA substrates.

To test whether these T3SA substrates contributed to Shigella capacity to multiply and disseminate within a cell monolayer, we used a plaque formation assay in a human colonic epithelial cell line. Both the number and the average size of plaques formed by relevant single-mutant strains were indistinguishable from the WT (S6 Fig). This suggested that Orf86, Orf176, Orf13 and Orf131a are not involved in the molecular events necessary for cellular spread in tissue culture cells. The role of Orf48 could not be assessed because as orf48-47 encode a fully functional TA system [53], the orf48 mutant strain displaying a complete deletion of the sequence coding for the antitoxin moiety of this TA module could not be obtained.

Investigation of the role of protein size in the β-lactamase secretion and translocation assays, using known antitoxins and chaperones

Strikingly, the novel T3SA substrates are relatively small proteins with four out of the five weighing approximately 10 kDa. To test whether these proteins were translocated because of their small size, we assessed the secretion of the 15 kDa Spa15 and IpgA T3SA effector chaperones [39,60]. In addition, since three newly identified T3SA substrates are related to antitoxin components of TA systems, we also investigated whether CcdA and MvpA (8 and 9 kDa, respectively), the antitoxins of two additional TA systems of pWR100 [61,62], could also be T3SA substrates. Indeed, such small proteins may have gone undetected by the mass spectrometer particularly if a small fraction of the total amount is secreted. The same strategy as for the confirmation of the T3SA-dependent secretion of the MS hits was employed to assess secretion into ipaD supernatants and translocation into host cells upon infection with WT strains. Orf182-bla was used as a negative control and the antitoxin-containing Orf48-bla was selected as a positive control displaying a molecular weight similar to that of proteins tested in this second set. Expression of this second set of Orf-bla chimeric proteins was confirmed within bacterial lysates (Figs 4A and S7A), secretion into ipaD and WT supernatants was assessed using the nitrocefin secretion assay (Figs 4B and S7B) and translocation into host cells investigated upon infection of Jurkat T cells (Fig 4C). The Orf-bla chimeric proteins based on the chaperones Spa15 and IpgA were neither significantly secreted into ipaD supernatants (Fig 4B) nor translocated into host cells upon infection with WT strains (Fig 4C). This was not due to inactivity of the β-lactamase when fused to these proteins because nitrocefin hydrolysis was detected upon sonication-induced bacterial lysis (S7C Fig). Interference of the β-lactamase with the secretion of Spa15 and IpgA is unlikely because secretion of Spa15-Myc and IpgA-Myc by ipaD strains was also undetected (S7D Fig). Interestingly, a study previously reported T3SA-mediated secretion of Spa15 at late time points [36]. A hypothesis explaining this discrepancy may be that the expression of chimeric proteins in our assays blocks secretion of Spa15. This was however invalidated by the failure to detect endogenous Spa15 in the supernatants of ipaD or CR-induced WT strains (S7E and S7F Fig). Since we studied a serotype 5a strain (M90T) while Spa15 secretion was previously reported in a serotype 2a strain (2457T) [36], an alternative hypothesis is that the behaviour of Spa15 might vary from strain to strain. Overall, the results obtained with T3SA effector chaperones support that the secretion and translocation of the novel T3SA-substrates is not an experimental artefact due to their small size.

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Fig 4. Investigation of secretion and translocation of antitoxins and chaperones.

(A) ipaD Shigella lysates were analysed by immunoblotting with anti-β-lactamase antibody. Load equivalent to a bacterial culture OD₆₀₀ of 0.2 for each lane. Chimeric proteins tested and their expected molecular weight (kDa): Orf182-bla (140), Orf48-bla (41), CcdA-bla (38), MvpA-bla (38), Spa15-bla (44), IpgA-bla (44). (B) Supernatants of ipaD strains expressing the Orf-bla chimeric proteins were incubated with nitrocefin. Enzymatic activity was calculated based on measurement of A_486nm. e.M.u.: equivalent Miller unit. Data are from 4 independent experiments. (C) CCF2-AM-loaded Jurkat T cells were infected with WT strains for 1 hour. Translocated cells were detected by flow cytometry. Data are from 3 independent experiments. (B-C) **p<0.01; ***p<0.001; ****p<0.0001 (unpaired two-tailed Student’s t-test compared to Orf182-bla).

https://doi.org/10.1371/journal.pone.0186920.g004

Similarly to the chimeric proteins based on the antitoxin or antitoxin-like proteins Orf86, Orf48 and Orf176, MvpA-bla was secreted by ipaD bacteria in a T3SA-dependent manner (Figs 4B and S7G) and translocated into host cells by WT strains (Fig 4C). On the contrary, CcdA-bla was faintly secreted by the ipaD strain when compared to Orf182-bla (Fig 4B). However, the β-lactamase activity detected in ipaD supernatant was not different from that in mxiD supernatant (S7G Fig) and CcdA-bla was not translocated into host cells by WT bacteria (Fig 4C). We eliminated the possibility that failure to detect CcdA-bla translocation was due to its low expression as it was comparable to that of Orf48-bla (Fig 4A), which is efficiently translocated into host cells (Fig 4C). Therefore, CcdA was not further considered a T3SA substrate.