Development of an effective vaccine against human cytomegalovirus (HCMV) is a need of utmost medical importance. Generally, it is believed that a live attenuated vaccine would best provide protective immunity against this tenacious pathogen. Here, we propose a strategy for an HCMV vaccine that aims at the simultaneous activation of innate and adaptive immune responses. An HCMV strain expressing the host ligand ULBP2 for the NKG2D receptor was found to be susceptible to control by natural killer (NK) cells, and preserved the ability to stimulate HCMV-specific T cells. Infection with the ULBP2-expressing HCMV strain caused diminished cell surface levels of MHC class I molecules. While expression of the NKG2D ligand increased the cytolytic activity of NK cells, NKG2D engagement in CD8+ T cells provided co-stimulation and compensated for lower MHC class I expression. Altogether, our data indicate that triggering of both arms of the immune system is a promising approach applicable to the generation of a live attenuated HCMV vaccine. Human cytomegalovirus (CMV) is a major cause of morbidity and mortality in congenitally infected newborns and immunocompromised individuals, indicating an utmost need for a vaccine to protect these vulnerable groups. Recent experimental studies in animal models, including non-human primates, have shown that attenuated CMVs trigger a potent immune response and are attractive vaccine candidates. However, an effective CMV vaccine is still not available. Here, we demonstrate that rational engineering of a live attenuated human CMV vaccine candidate is feasible. We equipped a CMV strain with an immunostimulatory molecule that is a ligand for an activating receptor present on both Natural Killer cells and CD8+ T cells. Moreover, we deleted several immunoevasins involved in downregulation of MHC class I molecules and of a ligand for Natural Killer cells in order to elicit stronger immune responses. In vitro assays using human immune cells and a first assessment in a humanized mouse model in vivo suggest that the generated CMV strain is attenuated and has the ability to induce a virus-specific immune response. Our study proposes this novel approach for the development of a rationally engineered CMV vaccine. Human cytomegalovirus (HCMV) is a highly prevalent herpesvirus infecting most of the human population , 2. In iVaccination of adolescents, or ideally of all children at young age, would be the most effective strategy to reduce the incidence of congenital CMV infection . The immWith the exception of the well-established Oka vaccine that provides excellent protection against varicella-zoster virus, no attenuated vaccine against other herpesviruses has been approved. There are several difficulties that hamper the development of an effective live HCMV vaccine. The ability of HCMV to reactivate and to re-infect seropositive individuals indicates that immunity resulting from primary infection cannot completely prevent subsequent infections –25. AnotNumerous viral encoded immunoevasins prevent the development of full-blown CMV-specific immunity. Accordingly, genetically modified animal CMVs lacking immunoevasins exhibited outstanding vaccine properties in mouse and guinea pig models , 31. RecIt is highly desirable to test the suitability of similar approaches for a recombinant HCMV vaccine. However, the strategy successfully established for MCMV cannot simply be translated to HCMV. Divergence between rodents and primates started ~90 million years ago and resulted in some differences of the immune system of mouse and man . SimilarIn this study, we report on the construction and evaluation of a recombinant HCMV strain expressing the host ligand ULBP2 for the NKG2D receptor. We found that cells infected with the ULBP2-expressing strain increased the cytolytic activity of NK cells in an NKG2D-dependent manner, thereby preventing viral spread and containing the viral infection to few cells. Still, the strain exhibited a robust capacity to activate HCMV-specific T cells. Interestingly, the mechanisms steering T cell activation turned out to be different for the viral strains. Altogether, our data imply that the chosen strategy allows attenuation of an HCMV strain, while retaining its ability to stimulate innate NK cell and adaptive T cell immunity.To generate an HCMV strain for testing a novel vaccination approach that aims at activation of NK cells as well as T cells we used the bacterial artificial chromosome (BAC)-cloned strain TB40/E (herein referred as TB40)  that disMice reconstituted with human hematopoietic stem cells can serve as models for infection with several human-specific viruses , 43 and CMV-specific antibodies appear particularly important for prevention of congenital infection . We therFinally, we investigated whether CMV-specific T cells were primed in the humanized mice. When examined at day 14 p.i., CD8+ T cells specific for the HCMV epitopes NLVPMVATV and VLEETSVML  were found in blood of several animals . MoreoveTo investigate to which extent the two strains can establish infection in the humanized animals, viral loads were measured in several organs on day 18 p.i. using quantitative PCR (qPCR). Viral DNA was found only in spleens of infected animals, and not in the other organs analyzed. In spleen of most of the animals infected with the TB40 virus viral DNA was detected , whereasEncouraged by the observation that the ULBP2-TB40 strain could prime CD8 T cells in the humanized mice, we further investigated the activation of CMV-specific T cells by DC infected with this strain. It is well known that HCMV inhibits maturation of infected DC –53, and NK cells have the ability to modulate the outcome of T cell responses against CMV , 58. ThePrevious studies suggested that ULBP2 can co-stimulate CD8+ T cells via the NKG2D receptor . In ordeThe observed lower surface expression of HLA-I molecules in ULBP2-TB40 infected cells was expected to impair antigen presentation, leading to less CD8+ T cell activation than upon stimulation with TB40-infected cells. However, this was in contrast to the results presented in Figs in vitro assays using ex vivo-derived primary human immune cells and provide a first in vivo assessment in a humanized mouse model.In this report we propose a novel vaccination strategy that can be applied to the development of an HCMV vaccine. The ULBP2 ligand for the NKG2D receptor that is present on both NK and CD8+ T cells was expressed by an HCMV strain with the intention to stimulate innate as well as adaptive immunity. We investigated the properties of the resulting recombinant HCMV strain by An important goal in development of a live vaccine is attenuation. To this end, we endowed the HCMV strain with the ULBP2 gene, encoding a ligand for the activating NKG2D receptor of NK cells, and deleted the viral gene UL16, which normally counteracts the surface exposure of ULBP2 as well as of several other NKG2D ligands , 40. ThiAlthough attenuation is a central objective concerning safety of a live vaccine, too much attenuation could impair immunogenicity. Rapid control of the vaccine strain may for instance limit the production of viral antigens–below a level necessary for priming of the adaptive T cell response. To circumvent this problem, we chose an HCMV strain that lacked several of the MHC immunoevasins. Such strains modulate antigen presentation to a lesser extent than HCMV isolates expressing the full complement of immunoevasins , 74. In The seemingly neutral outcome of ULBP2 expression with respect to the frequency of activated CD8+ T cells Figs  and 4 caNCT01986010). The combination of the superior immunization approach presented here in this study with the safety features of a replication-deficient strain could lead to an HCMV vaccine that may even be safely applicable to immunocompromised patients. Additional preclinical studies are therefore warranted to further analyze the protective efficacy as well as the safety of such vaccination strategies against HCMV, particularly when improved humanized mouse models become available, ultimately paving the way for clinical trials.An excellent safety profile would increase the acceptance of a potential HCMV vaccine and will be an important goal, particularly when vaccination of immunocompromised patients is considered. Further attenuation can probably be achieved by deletion of additional genes for the numerous viral immunomodulatory functions, although it has to be examined that this does not go at the expense of reduced vaccination efficacy. Another option to increase safety is the application of replication- and spread-deficient vaccine strains. Studies in the mouse model revealed that such strains preserve the ability to stimulate CMV-specific T cell immunity , 83, andBuffy coat samples were obtained from the Institute for Transfusion Medicine at Hannover Medical School from voluntary healthy blood donors with known HCMV serological status. All materials and data were analyzed anonymously. All studies were performed in accordance with guidelines on human cell research and the approval of the Hannover Medical School Ethics Review Board. All animal research protocols were approved by the authorized Ethics Committee for Biomedical Research of the Clinical Hospital Rijeka and of the University of Rijeka  in accordance with the guidance of the European Parliament (Directive 2010/63/EU) and Croatian Federal Law about animal protection .PBMC were purified from buffy coat samples obtained from healthy blood donors using standard Biocoll (Biochrom) density gradient centrifugation according to a published protocol (Miltenyi Biotec). Cells were cryopreserved in fetal bovine serum (FBS) with 10% DMSO. Before NK cell isolation or prior to setting-up co-cultures, PBMC were thawed and cultured overnight with 500 IU/ml interleukin-2 (IL-2) (ImmunoTools) in X-VIVO 15 medium (Lonza) supplemented with 10% FBS and 5% human serum . Individual cell subsets  were isolated using human cell isolation kits (Miltenyi Biotec). NK cells were negatively selected, while CD8+ T cells and monocytes were positively selected. Purity of selection was analyzed by flow cytometry and was more than 94%. The pp65-specific T cell clone  was thawed 2 days before setting up assays in MEM-alpha medium (Lonza) supplemented with 120 IU/ml of IL-2 and 10% human serum. The CMV-CTL was tested for the absence of mycoplasma and its specificity was determined by intracellular cytokine staining and proliferation assays using pp65-peptide loaded cells as positive control. Human monocyte-derived dendritic cells (DC) were generated using a standard protocol . BrieflyFor the generation of the recombinant ULBP2-TB40 virus the ULBP2 ORF was amplified using primers 5´-GTCGGTACCGTCGCAGTCTTCGGTCTGACCACCGTAGAACGAGAGCTCCACCATGGCAGCAGCCGCCGCTACC-3´ and 5´-CCCGGATCCCTCTCCTCAGATGCCAGGGAGGATGAAG-3´ and an ULBP2 cDNA clone , and was cloned (via KpnI and BamHI) between the MCMV major immediate-early promoter sequence (corresponding to nucleotides 182849 to 183094 of the MCMV Smith Strain (Genbank accession number NC004065.1)) and a kanamycin resistance (KanR) cassette flanked by FRT sites. The whole insert was amplified with primers 5´-GACACCGGGCTCCATGCTGACGTAGGTACCGACTGGGGTCAAAAGCCTTTAAACGGTACTTTCCCATAGC-3´ and 5´-CTTATAGCAGCGTGAACGTTGCACGTGGCCTTTGCGGTTATCCGTTCAGGAACACTTAACGGCTGA-3´, and inserted into the BAC-cloned genome of the HCMV strain TB40/E  by red-α+ L/D+ cell specific lysis—% CFSE+ L/D+ cell spontaneous lysis) / (100 - % CFSE+ L/D+ cell spontaneous lysis) × 100.HFF were infected with TB40 or ULBP2-TB40 as described above. One day p.i. the infection rate of the HFF was determined by flow cytometry following intranuclear staining with an Alexa Fluor 488-conjugated CMV IE1/2 antibody (Merck Millipore). Cells were cryopreserved and thawed at the day of establishing the cytotoxicity assay. Prior establishing co-cultures, cells were labeled with carboxyfluorescein succinimidyl ester  and co-cultured with freshly isolated human NK cells at effector to target ratios of 16:1, 8:1 and 4:1 in X-VIVO 15 medium supplemented with 500 IU/ml IL-2. After 4 h incubation at 37°C, specific lysis was determined by flow cytometry as measured by determination of dead cells using the Fixable Viability Kit (L/D) (BioLegend). To evaluate the spontaneous death of cells, samples containing target cells only were used. Cytolytic activity of NK cells expressed as percentage of specific lysis was determined by the following calculation: (% CFSE4 cells/well). NK cells were added at the ratio 1:0.25 (HFF:NK cells). After 3 days, the cultures were fixed with 80% acetone, and stained with the IE1/2 antibody. Infected cells per focus of infection and number of infectious foci were counted using fluorescence microscopy. Each sample was performed in quadruplicates.Focus expansion assays were established as previously described . Brieflyin vitro was performed using the NKG2D-specific antibody clone 1D11 and isotype control mouse IgG1 (BioLegend) (both at 10 μg/ml). In addition, human Fc Block  was added.Blocking of NKG2D of human NK cells 5 cells/well) isolated from HLA-A*02:01 HCMV-seropositive healthy donors were co-cultured with autologous monocyte-derived DC  and with γ-irradiated autologous CD14- feeder cells (2–3 × 105 cells/well). Co-cultures were set up in 96-well plates at the ratio 1:10 (DC:T cell) in X-VIVO 15 medium supplemented with 5% human serum and IL-2 (500 IU/ml), IL-7 and IL-15 . Every 2 days fresh medium containing cytokines was replenished. After 7 days, expanded cells were transferred to 48-well plates, and freshly prepared DC and feeder cells were added. After 2 weeks, the percentage of pp65-specific CD8+ T cells was determined by flow cytometry following staining with the PE-conjugated tetramer HLA-A*02.01-NLVPMVATV (MoBiTec). For intracellular cytokine staining, the expanded T cells, PBMC or the pp65-specific CTL clone were stimulated in 96-well V-bottom plates for 6 h with DC loaded with the pp65 peptide pool or the NLVPMVATV peptide or with uninfected/infected DC for PBMC and the pp65-specific T cell clone (at a ratio 1:10 [DC:T cells]). Protein transport inhibitor cocktail and a PE-Cy7-conjugated antibody against CD107a  were added at the beginning of the assay. For surface staining we used PerCp-Cy5.5-conjugated anti-human CD3 , PE-Texas Red-conjugated anti-human CD8  and in case of expanded T cells additionally the PE-conjugated tetramer HLA-A*02:01-NLVPMVATV. After fixation with 3% paraformaldehyde for 5 min at RT and permeabilization with 0.1% saponin for 20 min at RT, FITC-conjugated (for expanded T cells) or PE-conjugated anti-human IFNγ  and APC-conjugated anti-human TNFα Abs  were used for staining. One million of splenocytes isolated from humanized mice were incubated with 1 μg of the peptide 316-VLEETSVML-324 (HLA-A2 restricted immunodominant epitope from the CMV IE1 antigen) for 5 h at 37°C, with brefeldin A (eBioscience) added for the last 4 h of stimulation. For evaluation of IFNγ production by NK cells, 5 × 106 splenocytes were resuspended in 400 μl of RPMI medium supplemented with 10% FCS and IL2 (500 U/ml) and cultured for 5 h at 37°C in the presence of brefeldin A.For expansion of CMV-specific T cells, CD8+ T cells (10PrkdcscidIl2rgtm1WjlTg(HLA-A2.1)1Enge/SzJ mice expressing human HLA-A*02:01 (NSG-A2) were irradiated with 0.7 Gy and 4–6 h after irradiation injected with 1 × 105 CD34+ cells intrahepatically. 3 months post transfer engraftment of human cells was determined in blood. Humanization status ranged from 70–90% of human leukocytes (hCD45+ cells). Animals were randomly assigned to experimental groups with the humanization status being equal among the experimental groups. Mice were injected intraperitoneally with uninfected or TB40 or ULBP2-TB40 infected DC (3 × 105 cells/mouse) generated from monocytes of HLA-A*02:01 positive HCMV seronegative donors. Mice were daily treated with subcutaneous injections of recombinant human granulocyte colony-stimulating factor (G-CSF) . Mice were boosted after 7 days with freshly prepared, uninfected or infected DC (3 × 105 cells/mouse).Humanization of mice was performed at the Helmholtz Center for Infection Research . Briefly, fetal liver mononuclear cells were isolated over Ficoll-density gradients, and CD38- CD34+ cells were enriched using a MACS selection kit (Miltenyi Biotec). Newborn (3–5 days old) NOD.Cg-Analysis of CMV-specific IgM antibodies in serum of infected animals was performed using anti-CMV IgM Human in vitro ELISA kit (Abcam) according to the manufacturer’s instructions. Briefly, a 96-well plate was precoated with CMV antigens to bind cognate antibodies. Control and test samples were added to the wells in duplicates. Serum was isolated from blood using density gradient centrifugation applying the standard PBMC isolation protocol (Miltenyi Biotec) and stored at -20°C. The following controls were included: CMV IgM positive control, CMV IgM negative control and CMV IgM cut-off calibrator. Following washing, a horseradish peroxidase (HRP) labelled anti-human IgM conjugate was added to the wells and after incubation as according to the instructions of the manufacturer, the reaction was stopped by adding an acidic stop solution. Absorbance at 450 nm was measured immediately using a TriStar Version 1.04 microplate reader (BertholdTech).Sections from formalin fixed, paraffin-embedded spleens were stained with hematoxylin and eosin. Labelling of tissue sections was performed by an anti-human CD3 (F7.2.38) Ab (Dako) followed by biotinylated goat anti-mouse IgG (BD Pharmingen), streptavidin covalently coupled to horseradish peroxidase conjugate (Roche Applied Science) and DAB chromogene (Dako). Slides were examined with an Olympus BX51 microscope, images were acquired by an Olympus digital camera (DP71) and analyzed using Cell^B software.DNA was isolated from spleen, liver, bone-marrow, blood and salivary glands using the ReliaPrep Blood gDNA Miniprep Kit (Promega) according to the manufacturer’s protocol. Presence of HCMV DNA was determined by 7500 Fast Real-Time PCR system (Applied Biosystems) using a predefined two step PCR: 1 cycle at 95°C for 15 min followed by 40 cycles at 95°C for 15 s and 60°C for 1 min using Power SYBR Green PCR master mix and TaqMan Universal PCR mix (Applied Biosystems). To detect viral DNA primers specific for gB (gB_Fw 5’-AGG TCT TCA AGG AAC TCA GCA AGA and gB_Rv 5’-CGG CAA TCG GTT TGT TGT AAA) and a TaqMan gB probe with TAMRA quencher (6FAM—AAC CCG TCA GCC ATT CTC TCG GC—TAMRA) was used as previously described . For theFor flow cytometry analysis the following antibodies were used: anti-human Alexa Fluor 700-conjugated CD80 , Brilliant Violet 510 conjugated CD86 , PE-conjugated CD209 , PE-Cy7-conjugated CD14 , PE-CF594-conjugated HLA-DR , APC- -conjugated CD3 , Alexa Fluor780-conjugated CD8 , BV510-conjugated CD1a , APC-conjugated HLA-A2 , PE-conjugated ULBP2/5/6 , APC eBioscience), PE-Cy7–conjugated CD19 , PE-conjugated CD56 , APC-conjugated PD-1 , FITC-conjugated CD57 , FITC-CD45RA , PE-Cy7-CCR7 , PE-Cy7-CD62L  and FITC-CD45RO . As isotype controls we used PE‑conjugated mouse IgG2a (R&D Systems) and APC-conjugated mouse IgG2b (eBioscience). For dead cells exclusion Zombie NIR Fixable Viability Kit (BioLegend) was used. Intranuclear staining with an Alexa Fluor 488-conjugated IE1/2-specific antibody (Merck Millipore) after PFA fixation and permeabilization with 75% ice-cold ethanol was used to detect infected cells. For evaluation of the phenotype of CD8+ T cell we used anti-human APC-conjugated NKG2D . CMV-specific CD8+ T cells from blood and spleen of humanized mice were analyzed on days 14 and 18 p.i, respectively, using the PE-conjugated pp65-specific tetramer HLA-A*02.01-NLVPMVATV and the APC-conjugated IE1-specific tetramer HLA-A*02:01- VLEETSVML synthesized by the NIH tetramer core facility. Flow cytometry was performed using FACSVerse, FACSAria or LSRII (BD Biosciences), and data were analyzed using FlowJo software (Tree Star).Statistical analyses were performed with GraphPad Prism5 software and statistical tests used are indicated in the figure legends. No statistical method was used to predetermine sample size, and data analysis was not blinded. Exact sample sizes and number of independent experiments performed are indicated in each figure legend. No samples were excluded. All error bars represent SEM or range as noted in the figure legends. Differences were considered to be statistically significant for P values <0.05.S1 FigHFF were infected with 1 PFU per cells with TB40 (black) and ULBP2-TB40 (red) viruses. At indicated time points supernatants were harvested, and viral titers were determined by plaque assay. The values at day 0 represent the inocula.(PDF)Click here for additional data file.S2 FigFocus expansion assays were set up using TB40 or ULBP2-TB40 infected HFF and primary NK cells of 6 donors as described in (PDF)Click here for additional data file.S3 FigHumanized mice were treated as described in (PDF)Click here for additional data file.S4 FigImmunization was performed with TB40 or ULBP2-TB40-infected DC in humanized mice (as described in (PDF)Click here for additional data file.S5 FigHumanized mice were injected with infected DCs as described in (PDF)Click here for additional data file.S6 FigRepresentative dot plots depicting percentages of pp65-specific CD8+ T cells from one donor before expansion and after 14 days of co-culture with uninfected autologous DC or pp65-peptide loaded mature DC (pp65-mDC). Lower plots, staining with irrelevant tetramer as negative control (irrel-tet). The graph at right is compiled data from 4 donors. Horizontal bars are medians. Data are representative for one of two independent experiment performed.(PDF)Click here for additional data file.S7 FigRepresentative dot plots of an experiment performed with cells from one donor indicating percentage of naive , central memory , effector memory  and terminally differentiated effector memory  CD8+ T cells before expansion and after 14 days of co-culture with TB40 or ULBP2-TB40 infected DC. Graphs display compiled data for four donors as percentages of (A) CM or EM CD8+ T cells and (B) CM or EM pp65-specific CD8+ T cells expanded with TB40 (black circles), ULBP2-TB40 (red circles) infected DC or pp65-peptide loaded DC (grey circles).  Percentage of PD-1+ CD8+ T cells (C) and PD1+ pp65-specific CD8+ T cells (D). Data obtained with cells from individual donors are connected by lines. Data are representative of one of two independent experiment performed. Statistical analysis was done with one-way ANOVA Friedman test followed by Dunn’s Multiple Comparison test. *, P < 0.05; ns, P > 0.05.(PDF)Click here for additional data file.S8 Fig(A) Percentage of IE1/2-positive DC 1 day after infection with 3 PFU per cell of TB40 (black circles) and ULBP2-TB40 (red circles). Compiled data for DC of four donors are given, horizontal bars represent medians.  Intracellular cytokine staining for IFNγ (B) and TNFα (C) expression of CD4 T cells (CD3+CD8-) upon co-cultivation of PBMC with autologous monocyte-derived DC that remained uninfected or were infected as indicated. Graphs are compiled data of experiments performed with cells of 4 HCMV-seropositive donors. Data generated with cells of individual donors are connected by lines and are representative of one of three independent experiment performed. Statistical analysis was done with one-way ANOVA Friedman test followed by Dunn’s Multiple Comparison test. *, P < 0.05; ns, P > 0.05.(PDF)Click here for additional data file.S9 Fig(A) Representative staining for CD80, CD86 and HLA-DR expression on TB40 or ULBP2-TB40 infected DC (IE1/2-positive cells) and on uninfected DC matured as described in Materials and Methods. Graphs at right provide compiled data for DC of 6 donors. Results obtained with DC of one donor are connected by lines. (B) Median fluorescence intensity (MFI) of CD80, CD86 and HLA-DR for TB40 (black circles) and ULBP2-TB40 (red circles) infected DC relative to MFI values of uninfected DC in the same cultures (IE1/2-negative bystander cells) (n = 6 donors). Horizontal bars represent medians. Representative experiment of two of seven independent experiments performed. Data were analyzed using one-way ANOVA Friedman test followed by Dunn’s Multiple Comparison test. *P < 0.05; ns, P > 0.05.(PDF)Click here for additional data file.S10 Figpp65-specific CD8+ T cells were obtained by co-culturing CD8+ T cells from HLA-A2+, HCMV seropositive donors with pp65-peptide loaded autologous DC as described in Materials and Methods. Representative plots of pp65-specific CD8+ T cells (derived from one donor) after co-culturing with autologous uninfected DC or DC infected with the indicated viruses show (A) IFNγ, TNFα and CD107a intracellular staining and (B) NKG2D surface expression. Similar data were obtained using cells derived from three different donors.(PDF)Click here for additional data file.S11 Fig(A) Histograms depict NKG2D expression on CD8+ T cells from one donor before expansion and after 2 weeks of co-culture with uninfected, TB40 or ULBP2-TB40 infected DC or pp65-peptides loaded mature DC (pp65-mDC). Black lines indicate staining with isotype antibody. (B) Compiled data depicting percentages of NKG2D positive CD8+ T cells from 4 donors before expansion and after expansion with the different DC. Data obtained with cells from one donor are connected by lines.(PDF)Click here for additional data file.


The amino acid sequences of the all gene segments from 2015 A(H1N1)pdm09 isolates identified several new mutations compared to the 2009 A(H1N1)pdm09 strains, which may have contributed towards enhanced virulence, compared to 2009 A(H1N1)pdm09 strains.In 2015, the swine derived A(H1N1)pdm09 pandemic strain outbreak became widespread throughout the different states of India. The reported cases and deaths in 2015 surpassed the previous years with more than 39000 laboratory confirmed cases and a death toll of more than 2500 people. There are relatively limited complete genetic sequences available for this virus from Asian countries. In this study, we describe the full genome analysis of influenza 2015 A(H1N1)pdm09 viruses isolated from West Bengal between January through December 2015. The phylogenetic analysis of the haemagglutinin sequence revealed clustering with globally circulating strains of genogroup 6B. This was further confirmed by the constructed concatenated tree using all eight complete gene segments of Kolkata A(H1N1)pdm09 isolates with the other strains from different timeline and lineages. A study from Massachusetts Institute of Technology (MIT) in 2015 reported novel mutations T200A and D225N in haemagglutinin gene of a 2014 Indian strain (A/India/6427/2014). However, in all the pandemic strains of 2014–2015 reported from India, so far including A(H1N1)pdm09 strains from Kolkata, D225N mutation was not observed, though the T200A mutation was found to be conserved. Neuraminidase gene of the analyzed strains did not show any oseltamivir resistant mutation H275Y suggesting continuation of Tamiflu Influenza A virus is the most diverse of all influenza viruses that remains a major public health threat in developing as well as developed countries . Being ain silico study from Massachusetts Institute of Technology (MIT), USA, the HA sequences of 2014 Indian H1N1 strains were predicted to have potential amino acid (aa) mutations in the receptor binding site (RBS) of A(H1N1)pdm09. It was proposed that these mutations may have caused the disease severity of 2015 outbreak [During the first quarter of 2015, a sudden increase in influenza A(H1N1)pdm09 activity was observed in India, though the usual influenza seasonal peak is observed in monsoon (June-September) . In an ioutbreak . Recentloutbreak .In order to understand the origin and the genetic variation of 2015 pandemic influenza viruses, a pilot study was initiated to analyze the major surface antigen HA, NA and M gene sequences of twenty-five 2015 A(H1N1)pdm09 isolates from Kolkata, India. In addition, all other gene segments  were analyzed for 5 out of these twenty-five isolates. In agreement with the reported HA sequences of 2015 A(H1N1)pdm09 isolates from India , the conBetween January and December 2015, 1869 nasopharyngeal and/or throat swab samples collected from the patients admitted to hospitals with severe acute respiratory illness for receiving medical care throughout the epidemic wave in West Bengal were referred to the Molecular Virology laboratory, National Institute of Cholera and Enteric Diseases (NICED), Kolkata, India, in cold chain for diagnosis.RNA extractions from clinical samples were performed using the QIAamp® viral RNA mini kit  according to the manufacturer's instructions. The RNA was tested by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR), following U.S. Centers for Disease Control and Prevention (CDC) recommended primer-probes and protocol  followedFull length genes of the eight segments of 2015 A(H1N1)pdm09 isolates were amplified by RT-PCR using Superscript III RT-PCR system  and the primer pairs recommended by the World Health Organization, USA . CompletThe nucleotide sequences were aligned and translated using ClustalW program implemented in MEGA 6 . The phyhttp://tree.bio.ed.ac.uk/software/figtree/). Due to sharing almost same genetic makeup (≈100% sequence identity) for HA, NA and M gene, we have shown only fifteen isolates out of twenty-five in the phylogenetic tree or figures showing amino acid variation.The coding regions of all segments for each five isolate were manually inspected, trimmed and then concatenated in the following sequence order of PB2, PB1, PA, HA, NP, NA, M and NS using the SequenceMatrix software . Global 18-344aa and HA2345-566aa) sequence of 2015 A(H1N1)pdm09 isolate from the present study, A/India/Kol-S4659/2015 (KX789441), was used for three-dimensional (3D) structure analysis. The 3D structure of the 2015 A(H1N1)pdm09 HA protein was generated by the coordinates of PDB ID: 3LZG using MODELLER software [The complete HA amino acid , Kolkata, India has been identified as a referral laboratory for H1N1 testing by the Department of Health and Family Welfare, Government of West Bengal. Written informed consent was taken by the hospital authorities from the patients or their guardians for diagnostic and therapeutic intervention. Samples were sent to NICED without any link with personal identifiers of the patients. The study also obtained necessary approval from the Institutional Ethics Committee of NICED (No. A-1/2016-IEC).During the outbreak of 2015, a total of 1869 acute phase nasopharyngeal and/or throat swab specimens from patients hospitalized with acute respiratory illness, were referred to the Division of Molecular Virology, NICED, Kolkata, India, from various hospitals in West Bengal. 438 cases (23.3%) were found to be positive for 2015 A(H1N1)pdm09 virus using qRT-PCR. The age-wise analysis suggests that the 2015 A(H1N1)pdm09 positive cases were distributed throughout all age groups with maximum positivity in 1 - >5 years (32%) age group and minimum positivity in 18 - <36 years group (20.5%) . Of 1869The aim of this pilot study was to assess the genetic variation of these 2015 A(H1N1)pdm09 strains. Therefore, twenty-five isolates were sequenced for HA, NA and M gene, of which 5 isolates were further characterized for all gene segments to decipher the exact clade and origin of 2015 outbreak strains. The phylogenetic tree of the concatenated genome of five 2015 A(H1N1)pdm09 isolates, along with the representative A(H1N1)pdm09 strains, is shown in To understand the genetic evolution of 2015 A(H1N1)pdm09 isolates, phylogenetic analysis of the twenty-five HA sequences, together with globally distributed A(H1N1)pdm09 strains representing all genogroups from different timeline and lineages, was undertaken. The HA sequences from the present study showed 97.2% amino acid sequence identity with the prototype A(H1N1)pdm09 strain A/California/04/2009 and 98.8–99.2% identity with the worldwide circulating A(H1N1)pdm09 strains of 2014–2015. In the phylogenetic tree, the isolates from the present study clustered with the strains of genogroup VI-B from 2013 to 2015 . The sig63-286) revealed at least eleven mutations in HA1 polypeptide. The characteristic genogroup 6 amino acid change at S203T was observed in all viruses examined in the present study [131–138 and 220-loop218–228 structure were found to be highly conserved among the strains, the amino acid variations of S185T and T197A were observed in the 190 helix184–195 regions of predicted antigenic sites of all HA sequences reported during 2014–2015 along with the viruses analyzed in this study. S84N, D97N and K163Q substitutions were observed in Kolkata, as well as Southeast Asian strains of 2014–2015. No changes were observed in highly conserved residues at Y91, W150, H180, and Y192, which form the base of the receptor binding pocket (data not shown) [Potential changes in the antigenic site of HA polypeptide were analyzed to better understand the amino acid substitutions and antigenic evolution of 2015 A(H1N1)pdm09 strains. In respect to the California strain of 2009 , a significant amino acid change of Alanine to Threonine was observed at 5 aa upstream of the signal peptide of HA in Kolkata and other strains reported from this subcontinent . Analysint study ,23. In at shown) , was nott shown) . InsteadSignificant number of amino acid variations between the HA polypeptide of A/California/04/2009 strain and 2015 A(H1N1)pdm09 isolates from the present study suggested that the HA protein structure of 2015 A(H1N1)pdm09 isolates may have acquired major structural modifications in respect to earlier 2009 A(H1N1)pdm09 strains. A computational comparative study was carried out to predict the changes within different regions of HA polypeptide. Since the crystal structure of the whole HA protein of 2015 A(H1N1)pdm09 is not available, the structure of the full-length HA protein was generated by using MODELLER software  followedThe NA gene of 2015 A(H1N1)pdm09 isolates characterized in this study shared almost 99.9% amino acid identities between themselves and 97% with the 2009 A(H1N1)pdm09 strain A/California/04/2009. Furthermore, these strains showed almost 98% homology with the worldwide circulating A(H1N1)pdm09 strains of 2014–2015 and made distinct cluster along with the strains from south-eastern Asia in the phylogenetic tree .Generally H275Y and N295S mutations in the NA polypeptide have been associated with multiple drug resistance in Influenza A . Both thThe amino acid sequence of the NA gene of 2015 A(H1N1)pdm09 strains revealed large number of variations compared to A/California/04/2009 strain. It was reported earlier that the amino acids I223, H275, Q313 and I427 in NA protein are responsible for binding with Neuraminidase inhibitors (NAIs)  . Though Like NA, the M gene of A/California/04/2009 has the closest homology to the M gene of the Eurasian lineage of swine influenza viruses circulating during 2009 to 2011. The twenty-five strains from the present study showed almost 99% similarity with the recently circulating strains from south-eastern Asia and formed a different cluster in the phylogenetic tree . AnalyseDuring 2009 pandemic, several mutations were defined in A(H1N1)pdm09, namely NP—V100I, NS—I123V (NS1) and T48A (NEP), PA—P224S, PB1—I397M and I435T and PB2—V344M and I354L. Majority of these amino acid substitutions have persisted in the following years with accumulation of several new mutations. In circulating viruses of 2014–2015, a number of new mutations were observed in the NP (S498N), NS , PA , PB1 (G154D), and PB2  sequences, which were also observed in 2015 H1N1pdm2009 Kolkata strains. Additionally, few unique mutations like A22T in NP, M83I in NEP and T106A, E249K, R299/368K in PB2 gene segments were identified in 2015 A(H1N1)pdm09 strains from this study . The funDuring early 2015, a sudden increase in pandemic influenza activity was observed in almost all states of India and its surroundings. There have been reports of severe second pandemic wave in Asia and Europe during earlier pandemics too . The panUsing global isolates whose clade identities are known, the phylogenetic analysis of the concatenated genome showed that the initial introductions of the 2009 A(H1N1)pdm09 virus in the country belonged to two separate global clades namely III and IV, which further evolved during 2012–2015 to clade VI-A and VI-B . Though The influenza virus envelope protein HA, is the principal surface antigen and the most critical component of the influenza vaccines. Since 2009, HA genes of the A(H1N1)pdm09 lineage have gradually evolved  due to aThere are two classes of antiviral drugs available for preventing and treating influenza illness: Neuraminidase inhibitors (NAIs)  and M2 (matrix 2) ion channel blockers (adamantanes: amantadine and rimantadine) . PreviouRecent ferret model studies have suggested that highly infectious pandemic influenza viruses could emerge due to antigenic drift within the pandemic viruses or through reassortment with human seasonal influenza viruses . It has S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 FigIdentical residues to A/California/04/2009 strain are indicated by dots.(PDF)Click here for additional data file.


S306A mutant  is a homotrimer whose subunits contain the serine protease domain (PD) and the regulatory PDZ domain. In the inactive state, a tight interdomain interface limits penetration of both PDZ-activating ligands and PD substrates into their respective target sites. We successfully crystalized HtrA2V226K/S306A, whose active counterpart HtrA2V226K has had higher proteolytic activity, suggesting higher propensity to opening the PD-PDZ interface than that of the wild type HtrA2. Yet, the crystal structure revealed the HtrA2V226K/S306A architecture typical of the inactive protein. To get a consistent interpretation of crystallographic data in the light of kinetic results, we employed molecular dynamics (MD). V325D inactivating mutant was used as a reference. Our simulations demonstrated that upon binding of a specific peptide ligand NH2-GWTMFWV-COOH, the PDZ domains open more dynamically in the wild type protease compared to the V226K mutant, whereas the movement is not observed in the V325D mutant. The movement relies on a PDZ vs. PD rotation which opens the PD-PDZ interface in a lid-like (budding flower-like in trimer) fashion. The noncovalent hinges A and B are provided by two clusters of interfacing residues, harboring V325D and V226K in the C- and N-terminal PD barrels, respectively. The opening of the subunit interfaces progresses in a sequential manner during the 50 ns MD simulation. In the systems without the ligand only minor PDZ shifts relative to PD are observed, but the interface does not open. Further activation-associated events, e.g. PDZ-L3 positional swap seen in any active HtrA protein (vs. HtrA2), were not observed. In summary, this study provides hints on the mechanism of activation of wtHtrA2, the dynamics of the inactive HtrA2V325D, but does not allow to explain an increased activity of HtrA2V226K.HtrA2(Omi) protease controls protein quality in mitochondria and plays a major role in apoptosis. Its HtrA2 The human HtrA2 (high-temperature requirement A2) protease controls protein quality in mitochondria , 2. It iPostsynaptic density of 95 kDa, Disk large, Zonula occludens 1) domain(s). The protease domain (PD) of the chymotrypsin type consists of two perpendicularly arranged β-barrel lobes β1-β6 and β7-β12. The lobes are delimited by the N- and C-terminal α-helices, arranged parallel in space, in a C2-pseudosymmetrical manner. These secondary-structure elements are connected by loops, several of which are important for proteolytic activity [The HtrA proteases can be distinguished from other serine proteases by the presence of one or two C-terminal PDZ ..PostsynaE. coli DegP and DegQ proteases, which contain two PDZ domains, further oligomerize forming up to 24-mers. Regardless the oligomerization status, at low temperatures or in the absence of a substrate or activating peptides HtrAs adopt inactive conformations characterized by non-catalytic architecture of the triad and/or a restricted access to the active site. Hence, it is generally accepted that HtrA proteases are activated [The PDZ domains participate in the regulation of HtrA activity by recognizing and binding hydrophobic C-terminal sequences of substrates or regulatory peptides. The HtrA monomers form higher-order oligomers. The common structural unit is a pyramid-shaped trimer with PDs constituting the pyramid walls and their N-termini adding to a central core around the pyramid apex, while the PDZ domains protruding outward laterally at the pyramid base. Two bacterial HtrAs, the ctivated , 17–20 bHtrAs share ~60% sequence homology. A number of crystal structures of pro- and eukaryotic HtrAs have been solved to date , 20–34, 361YIGV364, the PDZ-specific carboxylate-binding loop β13-β14 [PD-(β14-α5)PDZ (hereafter hinge A) and (β5-LC-β6)PD-(β13/α7)PDZ (hinge B). With PDZ taken as a lid to PD, the hinges are located at the C- and N-barrels within the PD domain, respectively [Human HtrA2 has a single PDZ domain and in the crystal structure forms a pyramid-shaped homotrimer. The catalytic triad located in an interior (concave) of the trimer—a feature shared by the whole HtrA proteins family—comprises H198, D228, and S306  in a no β13-β14 . In HtrAectively . Accordiectively , HtrA2 aectively , 38. Morectively . HtrA2 aectively . Availabectively , 26. HowPD segment, e.g. V325D, without complete loss of protease activity. On the contrary, hinge B also retains major interactions upon HtrA2 activation, but may accommodate favorably selected mutations, e.g. V226K in LCPD, that increase the proteolytic activity of the protein compared to the wild type HtrA2 [Recently, we have investigated temperature-dependent (20–45°C) conformational changes in HtrA2 using single-tryptophan mutants at the PD/PDZ interface and monitoring fluorescence of this residue. Correlation of temperature dependence of fluorescence and catalytic rates in selected mutants allowed us to elucidate the role of particular amino acids in regulation of protease activity . We havepe HtrA2 .PDZ slightly closing distance to “own” LC and L2 and to LD* [In our most recent study we combined a temperature-dependent tryptophan-induced quenching (TrIQ) with selected mutation-activity analyses. TrIQ is a modern technique complementary to FRET and efficient at smaller distances (≤10Å) than the latter . We found to LD*  while sid to LD* , 38 movid to LD* , we haveS306A/V226K  revealed that S306/A/V226K mutant has the same (inactive) architecture, earlier found for HtrA2S306A [PD and α7PDZ onto the concave side of the trimer, thus destroying hinge A. Consequently, bringing inactive HtrA2 to this “canonical active architecture” would require an extensive rearrangement of L3, equivalent with a crack of hinge A and replacement it with a new (L2-L3)PD- (β13-α7)PDZ interactions, with simultaneous whole opening of the PD-PDZ interface. An alternative would mean an entirely different activated HtrA2 architecture than one typical of all active HtrAs resolved thus far.The aim of this study was to examine in details likely routes of HtrA2 activation, compatible with the above observations. We used the wild type HtrA2, the inactivating mutant in hinge A V325D) and the activating mutant in hinge B V226K) as models. Our successful crystallization and structure solution of HtrA2rA2S306A . SubsequK as moderA2S306A –30, 32, 5D and thE. coli strain BL21(DE3)  transformed with the pET-derived pDZ5 V226K plasmid [HtrA2V226K/S306A gene was used to overproduce mutant protein (amino acids 134–458) with His6-tag at the C-terminal end. The protein was purified by affinity chromatography on Ni-NTA according to the manufacturers' instructions . The concentration of HtrA2V226K/S306A was estimated using Amido Black as described before [V226K/S306A preparation was estimated at more than 95% by SDS-polyacrylamide gel electrophoresis. plasmid , carryind before . The pur2PO4 and 0,1 M NaH2PO4. The crystals were cryoprotected in 25% glycerol in mother liquor and cooled in liquid nitrogen. The diffraction data were collected using in house rotating anode copper source . Data were indexed and integrated with MOSFLM [free was utilized to monitor the refinement strategy. The data collection and refinement statistics are presented in Immediately prior to crystallization screening the buffer was exchanged to 5 mM Tris-HCl pH 8.0 containing 150 mM NaCl and 200 mM imidazole by gel filtration on Superdex s75 , the protein was concentrated to 18 mg/ml and screening was performed using sitting drop vapor-diffusion method. Crystals appeared after several days at room temperature in Crystal Screen 2 formulation 21 (Hampton Research). The initial conditions were optimized. The crystals used for measurements were obtained from 0,15 M MES pH 6.5 containing 2 M NaCl, 0,13 mM KHh MOSFLM . The folh MOSFLM . Data weh MOSFLM , 46. Molh MOSFLM  with alah MOSFLM . Water mh MOSFLM . ThroughS306A [S306A/V226K structure  have turned out to be negligible, the crystal structure of the former  PDZ-GWTMFWV complex (pdb code 2PZD) [As differences between the first HtrA2S306A  and the ode 1LCY ) was usefunction , while Lfunction . The obtfunction , 53. PDZde 2PZD)  because 3 symmetry typical of the 1LCY crystal. Nine constructed starting homotrimers were separately immersed in a rectangular TIP3P [3 size. To neutralize the negative charge on the protein system, 6, 9 and 12 Na+ ions were added to HtrA2V226K, HtrA2 and HtrA2V325D trimers (and to the matching S306A mutants), respectively. Each trimer was initially optimized using similar methodology as described for the monomers.Based on the above modeled structure the V226K and V325D mutants were constructed. Additionally their equivalents with the catalytic triad serine replaced by alanine were also built: S306A, V226K/S306A and V325D/S306A. Each of the latter three was modelled with and without the selective ligand GWTMFWV. All these models were optimized as described above. Homotrimers compatible with 1LCY crystal structure were made from optimized starting monomers by imposing the Car TIP3P  water boMD simulations for each of the 9 systems were carried out using the following parameters: 1 fs time step, constant pressure (1 atm) and periodic boundary conditions. SHAKE algorithm  was usedS306A (1LCY [282ARDLGLPQT290) and the linker (344RGEKKNSSSGISGSQ358) gaps in the sequence were preserved. Moreover, by trial-and-error we found crucial to truncate both the β8 inlet to and the β9 outlet from the L3 gap, each by extra 8 residues, 274IVSSAQRP281 and 291NVEYIQTD298, respectively. Otherwise both trunks would sterically hinder optimization of the 1LCY-like start structure to a target meeting the restraints deduced from TrIQ and having a more open PD-PDZ interface (see below). All above conditions held, an alternative start, compatible with the completely open (no PD-PDZ1 interface) structure of the active DegP (PDB file 3CS0) [α-Cα distance) in HtrA2 thermal activation [To this aim, the crystal structure of HtrA26A (1LCY ) monomerle 3CS0)  was usedle 3CS0) . As TrIQtivation , 41, we tivation , 53.V226K/S306A. Its overall architecture resembles that of HtrA2S306A [S306A . Minor changes are observed only around the mutated residue and on L3. The mutated K226 forms a hydrogen bond with E425, absent in the HtrA2S306A, while on L3 the unresolved gap reduces from 9 in HtrA2S306A [281RARDLG286, more firmly manifesting L3 to flap on a convex side of the trimer than in 1LCY. The additional hydrogen bond K226-E225 further stabilizes the interaction between PDZ and PD domains in hinge B region  [Since the gaps in L3 and the PD-PDZ linker, not defined in the crystal structure, were restored computationally see , all theA (1LCY)  (Table 3new contacts (using the 3.5 Å criterion) with PD, viz. those of W2 with F331(β12) and W6 with A197(LB). Based on the mere geometrical criterion, they appeared as modest as 7 contacts already present in the crystal structure of PDZ-ligand, viz. with β14, E376α5, β15 and α7 [Importantly, the ligand-binding and the apo structures have optimized similarly demonstrating the correctness of our approach to modeling the peptide in complex with the apo-HtrA2. The GWTMFWV peptide fits surprisingly well to the apoPD-PDZ interface. Notably, its insertion into 1LCY has introduced only 2 ,Y428)α7 . A minorα atoms, respectively. It is important to note that two of the four most dynamic structure elements, L3 (9 residues) and PD-PDZ-linker (15 residues) were those restored algorithmically [Within 50 ns MD simulations each system experienced mainly specific segmental/collective motions of PDZ and PD domains relative to each other , but rethmically . TherefoS306A and HtrA2V226K models, all monomers of HtrA2V226K/S306A, HtrA2V325D, HtrA2V325D/S306A models in the peptide containing trimers, as well as in the all apo HtrA2S306A, HtrA2V226K/S306A and HtrA2V325D/S306A trimers.The segmental motions observed in the simulated models classify into three basic types . The preS306A exhibit the second type of motion. With the MD progress two monomers within the former structure and one within the latter have performed a relatively consistent motion, illustrated in PD and α7PDZ, simultaneously passing near the N-front of α5PDZ. Some translation, hard to characterize with math rigor , accompany the rotations of these units. The rotations are equivalent to opening the PD-PDZ interface in a lid-like way. The two hinges (A and B) are made of clusters of hydrophobic residues. The rotation of up to 50°  have confirmed the above conclusions.The last type of motion, seen only in a single monomer of the ligand-bound HtrA2Our results demonstrate that the motions within the monomers in the trimer are not synchronous in the timescale of 50 ns . By seleγ(Ser)-Nε2(His) distance and to 3.3±0.4 Ǻ for the Nδ1(His)-Oδ1(Asp) distance  alike serve as a criterion of the catalytic competence of HtrA2. Neither any starting nor resultant structures have fitted these criteria. We speculate that substrate binding induces further rearrangements yielding a proteolytically competent conformation within the catalytic triad, though this is not reflected in our simulations where substrate has not been taken into account.The conformations of the catalytic triad residues were analyzed in the optimized starting and resultant product structures and have been found catalytically incompetent, as seen previously in the crystal structure. Typical distances in a catalytically ready triad in serine proteases of the HtrA type amount to 3.15±0.2 Ǻ for the Oistance  interface, in agreement with the MD results given here in Figs α-Cα distances given in the last column of In Figs S306A [S306A/V226K (this work) resolve to homotrimers having essentially identical architectures, characterized by a unique PD-PDZ arrangements not seen in the structures of other procaryotic and eukaryotic proteins of HtrA family. In contrast to the other family members, regardless in proteolytically more or less active forms, the catalytic and the PDZ domains in HtrA2 tightly pack against each other [The crystal structures of HtrA2S306A  and HtrAch other . A majorch other .S306A-ligand  distance in hinge B roughly intact, as concluded from their FRET measurements [PDZ carboxylate-binding loop, see above. In this work we neither pursue this issue nor the allosteric regulation of HtrA2 (caspase-dependent) via its N-terminus [In our 50 ns MD simulation, two subunits in the trimers of HtrA2-ligand and one in the trimer of HtrA2and Figs  Figs havand Figs . The rotd B Figs . These rurements . These Aurements –63. Theyurements  i.e. theE. coli HtrA(DegP) [Mycobacterium tuberculosis HtrA2 [E. coli DegS [Legionella falloni DegQ [Arabidopsis thaliana Deg1 [vs. PD domains. Both domains do not form an interface. In the human HtrA1 the PDZ domain is not resolved within the crystal structure due to its high flexibility [E. coli DegS [PD and α7PDZ(1), simultaneously being in contact with LD* of the proximal unit of the trimer (asterisk denotes a neighboring monomer). In fact, these commonalities apply to all three L3*-LD-L1-L2 “activation clusters” per trimer, mutually related by the C3-symmetry  , Mycobacis HtrA2 , E. colioli DegS , 26, 32,oni DegQ , plant Aana Deg1  and humaana Deg1 . We noteana Deg1 , 28) vs.xibility , howeverxibility  and E. coli DegS , 26, 32 symmetry  [15, 19,PDZ  are in HtrA2 PDZ cluster, while retaining hinge B, the (β5-LC-β6)PD-(β13-α7)PDZ cluster still conserved. Clearly, the exact structural changes involved in the activation mechanism of HtrA2 remain open to debate until the crystal structure in the proteolytically active form is solved.In summary, X-ray study combined with MD failed to explain higher proteolytic activity of the V226K mutant, because in the MD simulations wtHtrA2-ligand complex exhibits more pronounced PD-PDZ opening than HtrA2S1 FigThree most significant eigenvectors, modes 1–3, are represented as square displacements of sequential MD-time-averaged Cα coordinates. The secondary-structure elements of HtrA2 monomer (1LCY pdb entry) are indicated below the abscissa for reference. Clearly, the majority of segmental motions are explained in mode 1, as modes 2 and 3 contain only residual fluctuations of LB, L3 and the PD-PDZ linker. Unit A is in red, unit B green, unit C blue. The lower-bottom panel includes respective scree plots.(PDF)Click here for additional data file.S2 FigGeneral legend see (PDF)Click here for additional data file.S3 FigGeneral legend see (PDF)Click here for additional data file.S4 FigDirectional amplitudes (double-arrow modules ~ root squares of displacements in (PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S1 TableFactors 3 to 30 accumulate 48% to 85%, respectively, of total variance in Unit B, fair scree; and 73% to 93%, respectively, of total variance in Unit C, steep scree.(PDF)Click here for additional data file.S2 TableFactors 3 to 30 accumulate 67% to 91%, respectively, of total variance in Unit A, fair scree; and 81% to 96%, respectively, of total variance in Unit B, steep scree.(PDF)Click here for additional data file.S3 TableFactors 3 to 30 accumulate 49% to 88%, respectively, of total variance in Unit C, fair scree; and 69% to 91%, respectively, of total variance in Unit A, moderate scree.(PDF)Click here for additional data file.


Increased levels of circulating sphingosine-1-phosphate (S1P) have been reported in ulcerative colitis. The objective of this study was to examine the effect of S1P on colonic smooth muscle contractility and how is it affected by colitis.Colonic inflammation was induced by intrarectal administration of trinitrobenzene sulfonic acid. Five days later colon segments were isolated and used for contractility experiments and immunoblotting.S1P contracted control and inflamed colon segments and the contraction was significantly greater in inflamed colon segments. S1P-induced contraction was mediated by S1PR1 and S1PR2 in control and S1PR2 in inflamed colon segments. S1PR3 did not play a significant role in S1P-induced contractions in control or inflamed colon. S1PR1, S1PR2 and S1PR3 proteins were expressed in colon segments from both groups. The expression of S1PR1 and S1PR2 was significantly enhanced in control and inflamed colon segments, respectively. S1PR3 levels however were not significantly different between the two groups. Nifedipine significantly reduced S1P-induced contraction in control but not inflamed colon segments. Thapsigargin significantly reduced S1P-induced contraction of the inflamed colon. GF 109203X and Y-27632, alone abolished S1P-induced contraction of the control but not inflamed colon segments. Combination of GF 109203X, Y-27632 and thapsigargin abolished S1P-induced contraction of inflamed colon segments.S1P contracted control colon via S1PR1 and S1PR2 and inflamed colon exclusively via S1PR2. Calcium influx (control) or release (inflamed) and calcium sensitization are involved in S1P-induced contraction. Exacerbated response to S1P in colitic colon segments may explain altered colonic motility reported in patients and experimental models of inflammatory bowel disease. Inflammatory bowel diseases (IBD) such as ulcerative colitis and Crohn’s disease are characterized by chronic inflammation of unknown etiology. IBD involves a complex interaction between genetic and microbial factors and immune responses (recently reviewed in ). AbnormSphingosine-1-phosphate (S1P) is a bioactive sphingolipid that has been implicated in a variety of biological processes including cell proliferation, cell survival, cell migration and adaptive and immune responses . SphingoThe main objective of this project was to examine the effect of S1P on contraction of rat colon and how this is affected by experimentally induced colitis. The role of sphingosine-1-phosphate receptors (S1PRs) and potential downstream signal transduction pathways were also investigated.Sprague-Dawley rats weighing between 200–250 g were used in this study. The study was approved by Health Science Center, Kuwait University Ethics Committee for animal use. The study was conducted according to the laboratory’s animal care guidelines at Kuwait University, Kuwait in accordance with the international standards of animal care. Rats were maintained at 22°C on 12-hr light/dark cycle (7 am–7 pm) and water and food were available ad libitum. Rats were anesthetized with ketamine (10 mg/Kg) and xylazine (20 mg/Kg). Experimental colitis was induced by intrarectal administration of 2,4,6-trinitrobenzenesulfonic acid  dissolved in 0.5 ml of 50% ethanol via a catheter inserted approximately 8 cm from the anal margin and administered at a dose of 73.3 mg/Kg. Rats activity, type of stool and body weights were monitored daily post injection. Some animals died the day after TNBS injection. Dissecting the rats suggested perforation as a potential cause of death. About 5% of the animals died prior to the experimental endpoint. The animals were used five days after TNBS administration. In our experience maximal inflammatory response was obtained five days post TNBS treatment. Rats were sacrificed by concussion followed by exsanguination. Thereafter, the abdominal cavity was opened up and approximately 2 cm segments of the terminal colon was dissected and transferred into a petri-dish containing Krebs’ solution at room temperature for physiological experiments or at 4°C for immunoblotting experiments. The tissues were then cleaned and adherent tissues were removed. Colon segments were used immediately for physiological experiments or stored at -80°C for immunoblotting studies. Smaller colon segments were preserved in phosphate buffered saline containing 10% formalin and processed for histological evaluation of colonic inflammation as previously described [Rats were obtained from Charles River Research Models and Services. Seventy two male escribed .3, 25; KH2PO4, 1.2; MgSO4, 1.2; CaCl2, 2.5 and glucose, 11 mM. The solution was continuously gassed with a 5% CO2/95% O2 mixture. The preparations were allowed to equilibrate under a resting tension of 1.0 g for up to 60 min during which the bath fluid was changed at least once. Isometric contractions were recorded, through dynamometer UF1 transducers, on a Lectromed 4-channel polygraph (MultiTrace 4P). After the period of equilibration, KCl (80 mM) was added to the bath to test for tissue viability. The tissues were washed immediately after the peak contraction has been obtained. The tissues were washed repeatedly over the next 30 min period. The contraction to KCl was repeated every 30 min until two consecutive contractions did not differ by more than 5%. The last contraction to KCl was used as reference against which contractions to S1P were expressed. Thereafter, S1P  was added non-cumulatively to the bath and the contractions were recorded. Once the peak response to any concentration of S1P has been attained, the bath fluid was drained and replaced with fresh Krebs’ solution. At the end of the experiments, the tissues were washed, weighed, frozen and stored at -80°C until analyzed. S1P induced contraction was expressed as a percentage of KCl-induced contraction as mean KCl induced contraction was significantly not different between control and colitic colon segments . Similar experiments were performed using the selective S1PR1 receptor agonist, SEW2871. In experiments conducted with kinase inhibitors or receptor antagonists, the appropriate concentration of each antagonist was added to the organ baths and allowed to equilibrate with the tissues for 30 min before adding S1P. Only one antagonist was tested on any given preparation except when the antagonists were used in combination.After removing all adhering tissues, colon segments were set up longitudinally in Krebs’ solution at 37°C contained in a 25ml organ bath for isometric tension recording. The composition of the Krebs’ solution was as follows: NaCl, 119; KCl, 4.7; NaHCO2, 0.25 M sucrose, and 1x protease inhibitors . Homogenates were centrifuged at 3000 x g for 10 minutes at 4°C and supernatants were subjected to another centrifugation step at 100,000 x g for 1 hour at 4°C. The pellet was suspended in 50 mM Tris-HCl, pH 7.5 containing 1x protease inhibitors cocktail  and 0.1% sodium dodecyle sulfate. Protein concentration was measured using the BCA-protein determination kit  and samples were stored at -80°C for further analysis.Total membranes were prepared to determine the expression levels of S1PRs. Colon samples were homogenized on ice for 3 minutes in 20 mM Tris-HCl, pH 7.5 containing 2 mM MgClAfter boiling, samples from control and colitic colon segments (50 μg protein) were subjected to SDS-PAGE as previously described . The memThe following drugs were used in this study: S1P, SEW2871, W146, JTE-013 and CAY-10444, purchased from Cayman Chemical . Other drugs including nifedipine, thapsigargin, GF 109203X and Y-27632 were purchased from Tocris Bioscience . SEW2871, W146, JTE-013, nifedipine, CAY-10444 and GF 109203X were dissolved in dimethyl sulfoxide. Y-27632 was dissolved in water. S1P was dissolved in pre-warmed solution of bovine serum albumin (4 mg/ml) and incubated at 37°C for 30 min with vortexing in between. This was followed by sonication for 10 sec in a water bath sonicator.Data was analyzed using the Student’s t-test (Microsoft Excel), represented as mean ± standard error of the mean (SEM) and was considered statistically significant when P < 0.05.Rats treated with TNBS weighed significantly less than control rats on day 5 post TNBS injection . Colon segments from TNBS-treated rats were hypertrophic and weighted significantly (P < 0.05) more compared to colon segments from control rats .Macroscopically, colon segments from TNBS-treated rats were grayish thickened, ulcerated and filled with soft feces while colon segments from control rats were, on the other hand, pinkish and engorged with hard feces). Histologically, TNBS treatment resulted in mucosal ulcerations, goblet cells damage and depletion and inflammatory cell infiltration .S1P-induced concentration-dependent contractions of colon segments isolated from control rats .However, we were not able to attain the maximum response as this would involve a lot of S1P. We therefore chose to use 40 μM, a concentration that gave 30% contraction relative to KCl, for subsequent experiments. As shown in , S1P 40. S1PR1 sSEW2871 however failed to induce a contraction in colon segments from TNBS-treated rats . ConsistAs shown in , S1PR1, S1PR1 was expressed at significantly (P < 0.05) higher levels in colon segments from control rats relative to colon segments from TNBS-treated rats . S1PR2 pNifedipine (1 μM), a concentration that abolished KCl-induced contraction , signifiThe same concentration of nifedipine did not however affect S1P-induced contractions of colon segments from TNBS-treated rats . ThapsigThe objective of this study was to test the effects of S1P on colon contractility and test if these effects are modulated by colonic inflammation. The major findings of the study are: 1) S1P induced contractions in control and inflamed colon segments and these contractions were greater in inflamed colon segments. 2) S1P stimulated contractions are mediated by distinct S1PR isoforms in control and inflamed colon segments. 3) Calcium influx through L-type Calcium channels is required for S1P induced contraction in control but not inflamed colon segments. 4) Calcium release from the sarcoplasmic reticulum and calcium sensitization pathways all contributed to S1P induced contractions in control and inflamed colon segments however their contribution differed .i/o, G12/13 and Gq [i/o [S1P produces its effects by activating G-protein coupled S1P receptors (S1PRs). Of the five S1PRs that have been identified, S1PR1, S1PR2 and S1PR3 receptors are widely expressed in smooth muscles. One or more of these receptors have been reported to mediate the contractile effect of S1P in a variety of smooth muscle preparations. It has been reported that S1P-induced contractions were mediated via S1PR2 receptors in the urinary bladder , 31, 37,3 and Gq . S1PR1 o Gq [i/o . S1PR2, S1P-induced contractions are associated with increased intracellular concentration of calcium resulting from influx of extracellular calcium and/or release from intracellular stores. The contribution of extracellular or intracellular calcium varies between preparations and with the receptor type mediating the S1P response. S1P-induced contraction of porcine retinal arterioles involved influx of extracellular calcium through L-type voltage operated channels  with no In addition to increasing intracellular calcium concentration, contractile agonists also sensitize the contractile machinery to calcium. This process of sensitization involves PKC and RhoA/Rho-kinase pathways. According to Chiba et al (2010), S1P-induced contraction of murine bronchial smooth muscle was abolished by Y-27632, an inhibitor of Rho kinase indicating a role for Rho-kinase pathway of calcium sensitization in S1P-induced contraction of the bronchial smooth muscle. The Rho kinase pathway of calcium sensitization has also been implicated in S1P-induced contraction of guinea-pig trachea , urinaryIt was therefore concluded that S1P-induced contractions of the rat colon via S1PR1 and S1PR2 in segments from control rats but exclusively via S1PR2 in inflamed segments from TNBS-treated rats. The contractions involved multiple signaling pathways including influx of extracellular calcium through nifedipine-sensitive L-type voltage-gated channels, calcium release from intracellular stores and calcium sensitization through PKC and Rho-kinase pathways. Enhanced contractile response to S1P in inflamed colon accompanied with the reported increase in S1P levels may possibly contribute to the reported increase in colonic motility in patients and experimental models of inflammatory bowel disease.S1 File(XLSX)Click here for additional data file.


Streptococcus suis is a zoonotic pathogen, causing meningitis and septicemia. We previously demonstrated that the gastrointestinal tract (GIT) is an entry site for zoonotic S. suis infection. Here we studied the contribution of Streptococcal adhesin Protein (SadP) to host-pathogen interaction at GIT level.in vitro.SadP expression in presence of Intestinal Epithelial Cells (IEC) was compared with expression of other virulence factors by measuring transcript levels using quantitative Real Time PCR (qRT-PCR). SadP variants were identified by phylogenetic analysis of complete DNA sequences. The interaction of SadP knockout and complementation mutants with IEC was tested sadP was significantly increased in presence of IEC. Sequence analysis of 116 invasive strains revealed five SadP sequence variants, correlating with genotype. SadP1, present in zoonotic isolates of clonal complex 1, contributed to binding to both human and porcine IEC and translocation across human IEC. Antibodies against the globotriaosylceramide Gb3/CD77 receptor significantly inhibited adhesion to human IEC.Expression of S. suis zoonotic potential.SadP is involved in the host-pathogen interaction in the GIT. Differences between SadP variants may determine different affinities to the Gb3/CD77 host-receptor, contributing to variation in adhesion capacity to host IEC and thus to  Streptococcus suis (SS) is an emerging zoonotic pathogen which can cause severe disease including meningitis and septic shock in human and pigs [S. suis which determines the serotype; the virulence and prevalence differ within and among serotypes [S. suis [S. suis strains are limited to certain sequence types (ST), as determined by multi locus sequence typing (MLST). S. suis serotype 2 (SS2) isolates belonging to MLST clonal complex 1 (SS2/CC1) are considered highly virulent and zoonotic [S. suis serotype 9 (SS9), part of MLST clonal complex 16 (SS9/CC16) and the main cause of porcine S. suis infections in Northern Europe, is rarely associated with human infection [and pigs . The caperotypes . In addi[S. suis , 4. Invanfection . TherefoS. suis zoonotic transmission through the S. suis translocation of the human gastrointestinal tract.Epidemiological and experimental studies indicated that SS2/CC1 infection is a foodborne disease in Southeast Asia in adult patients caused by the consumption of contaminated undercooked pork –9. So fain vitro, and translocated the gastrointestinal tract (GIT) in a piglet model after S. suis oral infection [S. suis isolates to adhere to human and porcine IEC correlated with serotype as SS9 isolates showed significantly less binding to human IEC than SS2 isolates, suggesting that CPS composition and structure mediate host species restriction. In addition, whilst the CPS was shown to prevent adhesion to host-cells in in vitro models [cps mutant strains of serotype 2 and 9 appeared to retain the host restriction of their encapsulated parental strains when interacting with human and porcine IEC [S. suis with IEC.We previously demonstrated that SS2/CC1 adhered to and translocated across intestinal epithelial cells (IEC) of both human and porcine origin nfection . The capo models , 10, unecine IEC . Thus, iStreptococcal adhesin P (SadP) is a cell-wall adhesin which can recognize galactosyl-α1-4galactose  [labiose)  present labiose) . Analysilabiose) . Gb3/CD7labiose) , 14. Furlabiose) .S. suis in the presence or absence of intestinal epithelial cells of well-characterized S. suis secreted or cell-associated factors selected on the basis of their functional role in bacterial interaction with the host epithelium [sadP was highest among eight virulence-associated factors. We hypothesized that SadP plays a dominant role in the interaction of S. suis with porcine and human intestinal epithelium, and we performed a comparative study of SadP mediated adherence of S. suis serotype 2 (belonging to CC1 and CC20) and 9 (belonging to CC16).First, we determined the relevance of SadP in adhesion to intestinal epithelial cells in comparison to other well-characterized virulence-associated factors. Therefore, we assessed transcription levels in ithelium . The difTable A in S1 File. All strains were grown in Todd-Hewitt Broth with 5% Yeast (THY) or on Columbia blood agar plates (Difco). E. coli BHB2600 [E. coli 50 mg/L spectinomycin (spc); for S. suis 200 mg/L kanamycin (kan) and 100 mg/L spc. Caco-2 cells  and IPEC-J2 cells  were grown as previously described [Bacterial isolates used are listed in  BHB2600  was cultescribed , 19.2. The co-culture of bacteria and IECs was stopped by adding ice-cold 95% ethanol/5% phenol, pelleted, and stored at -80°C [free™ (Life Technologies). Quantity and quality of the RNA were measured with NanoDrop 2000 (Thermo Scientific) and Bioanalyzer (Agilent technologies).Caco-2 and IPEC-J2 cells were cultivated in triplicate in T25 flasks until differentiation . Co-cultat -80°C . Total Rat -80°C  followedproS and gdh) as reference genes [Table B in S1 File and data were analyzed with LinRegPCR [Reverse transcription of the RNA was performed with ThermoScript rt-PCR (Invitrogen). Relative gene expression was determined by qRT-PCR using SYBR Green (Roche) using two housekeeping genes  of 116 Prodigal  predicteProdigal , and a pProdigal  with 100 Vietnam . ProdigaTable B in S1 File. sadP deletion was achieved by gene replacement with the Janus cassette [kan gene. PCR fragments containing the flanked region of sadP (~ 0.5 kb) and Janus cassette were digested with ApaI/BamHI or EcoRI/BamHI and then ligated. ΔsadP::Janus fragment was PCR amplified from the ligation using Phusion DNA Polymerase (Thermo Fisher) with external primers, and successively transformed into S. suis in presence of the competence-inducing peptide ComS13-21 as previously described [sadP::Janus was confirmed by PCR and sequencing.Primers used for mutagenesis are listed in cassette  carryingescribed . TransfosadP1 (Table A in S1 File). The plasmid was maintained into E. coli BHB2600 kindly provided by Dr. S.A.J. Zaat [sadP1, sadP2A, and sadP2B genes were amplified from genomic DNA of respective S. suis strains (Table B in S1 File) and cloned into pMX1 via EcoRI/BamHI sites. After ligation, the plasmid was directly introduced into 10ΔsadP1 in presence of ComS13-21 to construct 10ΔsadP1CsadP1, 10ΔsadP1CsadP2A and 10ΔsadP1CsadP2B complemented mutants.The pMX1 vector  was used.J. Zaat  and extrAdhesion and translocation assays were performed as previously described . For inht-test was performed to compare the percentage of adhesion to and translocation across IECs between 10ΔsadP1 mutant and its parental strain.Prism 6.0  was used to analyse the normalized qPCR fold-ratio to the control (no cells) by One-way ANOVA. Unpaired Student's sadP expression was upregulated in presence of human and porcine IEC as an indication of its importance in adhesion. Therefore, we compared sadP transcript levels in comparison with other well-characterized S. suis secreted or cell-associated factors selected on the basis of their functional role in bacterial interaction with the host epithelium [Table 1 [SS2/CC1 strain 10) and non-zoonotic (SS9/CC16 strain 8067) S. suis types (Table A in S1 File), following 4 hours of co-culture in cell culture media without IEC (control) and with human (Caco-2) and porcine (IPEC-J2) IECs. Transcript levels of sadP in both S. suis strains ranged from 4.5 to 8.5-fold (p<0.001) higher than bacteria grown without IEC . Interestingly, in S. suis grown in the presence of IECs transcript levels of cps were also higher (2.5 to 3.0-fold p<0.05 and p<0.001 respectively) compared to S. suis grown without IECs. Sly toxin transcript levels were 1.6 to 3.5-fold higher (p<0.05) in both strains when in contact with human IEC only. In contrast, transcript levels of ssnA in SS9/CC16 in presence of IEC were 1.4 fold lower compared to control (p<0.001). Transcript levels of genes coding for the other virulence factors were not significantly altered compared to the control without cells except for transcript levels of the protease ddp IV involved in cell invasion process [p<0.05 and IPEC-J2 p<0.001). The high levels of sadP transcripts compared to other virulence factors, suggested an important role of SadP during S. suis-IEC interaction among zoonotic and non-zoonotic isolates.We first used qPCR expression profiling during host-pathogen protein interactions to identify whether ithelium  as descr[Table 1 , 33–40.  process , which wFig 2). We designated the variants SadP1 (CC1), SadP2A (CC20), SadP2B (CC16), SadP3 (CC13) and SadP4 (CC27/29). An expanded search including an additional 375 strains from Vietnam and the UK [S. suis . At the amino acid (AA) level, SadP1 shares a similarity of 79% and 77% with SadP2A and SadP2B respectively. SadP2A and SadP2B exhibit a 99% similarity in AA-sequence and differ by the absence of the LPXTG cell-anchor motif in SadP2B, which suggests SadP2B might be secreted instead of anchored to the cell wall . The partial crystal structure of the galabiose-binding domain of the SadP1 adhesin (from 139–323 AA) has recently been resolved (PDB: 5BOA) [Fig 3B).To determine the contribution of SadP to the bacteria-IEC interaction, we investigated whether genetic variation of SadP occurred among zoonotic and non-zoonotic isolates with different serotypes and genotypes. We detected five genetic variants of SadP in 111 out of 116 strains covering CC1, CC13, CC16, CC20, and CC27/29. The clustering of the five genetic variants occurred independent of the serotype but according to their clonal complex (B: 5BOA) . The preFig 2). We deleted sadP1 in SS2/CC1 strain 10 and its unencapsulated mutant 10Δcps2 (10ΔsadP1 and 10Δcps2ΔsadP1 respectively). Adhesion was expressed as the total number of IEC associated bacteria, including both adherent and intracellular bacteria, proportional to the total number of bacteria added to the IEC infection model, since we have previously shown that under the test conditions the number of invasive bacteria is very low (from 0.04% to 0.01% of the starting inocolum) and can be neglected [sadP1 showed a two-fold reduction in adhesion to Caco-2 as well as to IPEC-J2 cells compared to the parental strain indicating that SadP1 contributes to the adhesion to both human and porcine IEC . The double knockout 10Δcps2ΔsadP1 also showed a two-fold reduction in adhesion compared to parental 10Δcps2 strain . However, no significant reduction in adhesion was observed when we tested 10Δcps2ΔsadP1 in contact with IPEC-J2 cells. Finally, we created sadP knockouts in SS9/CC16 strain 8067 (8067ΔsadP2B) and SS2/CC20 strain 2001171 (2001171ΔsadP2A) to test the adhesion properties of SadP2A and SadP2B in their natural genetic background. Adhesion capacity of both 2001171ΔsadP2A and 8067ΔsadP2B was similar compared to their parental strains when brought into contact with Caco-2 cells. In contrast, 8067ΔsadP2B adhered significantly less (1.6-fold) to the IPEC-J2 cells than the parental strain 8067 WT (p<0.01) indicating that SadP2B in SS9/CC16 strain 8067 contributed to the adhesion to porcine IEC .Next, we evaluated the contribution of SadP variants to adhesion to human and porcine IEC. We focused on SadP1 and two subvariants of SadP2 (SadP2A and SadP2B) as these variants were present in the most common zoonotic SS2/CC1 (SadP1) and SS2/CC20 (SadP2A) and non-zoonotic SS9/CC16 (SadP2B) strains  in the 10ΔsadP1 with SS2/CC1 background. Growth kinetics did not differ significantly between the wild-type (WT) strain, 10ΔsadP1 and complementation mutants, and transcripts of each sadP variant were detected by qPCR (data not shown). Adhesion to Caco-2 cells was increased by expression of any of the three sadP variants in 10ΔsadP1, although not to WT levels . Adhesion of 10ΔsadP1CsadP1 to Caco-2 cells was 1.2-fold higher than that of 10ΔsadP1 (p = 0.057), but adhesion to IPEC-J2 cells was similar to that of the knockout. However, adhesion to Caco-2 cells of 10ΔsadP1CsadP2A was 1.4-fold higher (p = 0.02) and 10ΔsadP1CsadP2B was 1.3-fold higher (p = 0.004) than that of 10ΔsadP1. Only 10ΔsadP1CsadP2A was able to restore adhesion to IPEC-J2 cells, which was 3-fold higher than that of the 10ΔsadP1 (p = 0.003) .Complementation mutants were subsequently created by sub-cloning variants of with three different sadP (cps2 was compared with the unencapsulated mutant lacking SadP1 function (10Δcps2ΔsadP1) since this mutant showed dramatically reduced adhesion  compared to its parental unencapsulated strain (10Δcps2). The adhesion of 10Δcps2 to human IEC was inhibited by anti-Gb3/CD77 in a dose-dependent manner while no significant inhibition effect could be observed with the double mutant 10Δcps2ΔsadP1 .To further investigate if adhesion is indeed mediated through binding of SadP1 to Gb3/CD77, we performed an inhibition assay with anti-Gb3/CD77. The inhibition of adhesion by strain 10ΔsadP1 and 10Δcps2ΔsadP1 mutants and their parental strains for their translocation capacity across polarized Caco-2 cells [cps2 and its isogenic 10Δcps2ΔsadP1 mutant  .To test whether SadP1 might have an effect on the translocation across the intestinal epithelium, we tested the 10Δ-2 cells . We obseSimilar to other streptococcal surface proteins involved in a wide range of physiological functions , SadP reTranscript levels of SadP were highly up-regulated compared to transcript levels of other virulence factors in zoonotic and non-zoonotic isolates when interacting with human and porcine IEC, although SadP transcript levels varied between strains. Thus, we hypothesized that SadP is involved in the interaction with host IEC. We identified five main SadP genetic variants among 116 zoonotic and non-zoonotic isolates, which clustered according to genotype. We focused on a comparison of three SadP variants present in the most virulent genotypes circulating in the Netherlands: CC1, CC16 and CC20. In particular, SadP1 was associated to zoonotic and virulent isolates with CC1 genetic background.S. suis strains carrying different SadP variants. The SadP1 deletion mutant of SS2/CC1 encapsulated strain 10 showed significantly lower adhesion to human as well as to porcine IEC than the parent strain. Previously it has been demonstrated that the expression of the cps could change in response to environmental stimuli in vivo [cps2), in which bacterial adhesins could have better access to bind specific host-cell receptors. The double 10Δcps2ΔsadP1 mutant still showed a significant reduction in adhesion to human IEC compared to its parental mutant but did not show a significant reduction in binding to porcine IEC. These results suggest that SadP1 strongly promotes the bacterial adhesion to the human intestinal epithelium independent of CPS expression, and contributes to the zoonotic potential of SS2/CC1 strains that mostly carry the SadP1 variant. Although the complemented SadP1 failed the full restoration of adhesion activity of the wild-type phenotype, its adhesion capacity with human IEC was significantly increased compared to the deletion mutant ΔsadP1. The somewhat lower adhesion of the complemented SadP1 may have been due to a low sadP1 gene expression or due to a suboptimal folding and assembly of SadP1 protein on the bacterial cell wall in our complementation system thus interfering with a correct docking of the SadP adhesin onto the galabiose moiety of the Gb3/CD77 receptor.We observed differences in the ability of SadP to adhere to IEC between  in vivo , 48 and  in vivo , 10. TheS. suis translocation across the intestinal mucosa after adhesion. However, SadP1 deletion does not entirely abolish the bacterial adhesion capacity, similar to observations from other investigators when studying S. suis Fhb-mediated adhesion to epithelial and endothelial cells originating from other organ systems [S. suis [Interestingly, both encapsulated and unencapsulated SadP1 mutants were also impaired in their translocation across human IEC, supporting the hypothesis that direct adhesion and interaction of bacteria with specific host-cell receptors, potentially leading to the opening of the cellular tight junctions, is required for bacterial translocation . Thus, w systems , indicat[S. suis .sadP2B in an SS9/CC16 strain resulted in a reduction of adhesion to porcine IEC, deletion of sadP2A in an SS2/CC20 strain had no effect on adhesion to both human and pig IEC. However, complementation with SadP2A, but not with SadP2B, restored the adhesion capacity to human (p< 0.05) and particularly to porcine IEC (p< 0.01) of ΔsadP1 in a SS2/CC1 strain. Thus, in the SS2/CC20 strain, other factors than SadP may contribute to adhesion masking the effect of SadP2A deletion, while the LPXTG cell-anchor motif present in SadP2A, but absent in SadP2B, might be a prerequisite for SadP mediated adhesion of CC1 to porcine IEC.SadP1 and SadP2 notably differ in AA composition (77% similarity), while SadP2A and SadP2B share 99% similarity and differ only for the lack of LPXTG cell-anchor motif in SadP2B. The lack of this motif might suggest that SadP2B will be secreted instead of being anchored to the cell wall. We have previously shown that the zoonotic SS2/CC20 clone emerged from the non-zoonotic SS9/CC16 clone and the sequence similarity (99%) of the SadP2 variants present in CC20 and CC16 isolates is consistent with this observation . While dThese results suggest that SadP2 variant is involved in the adhesion of IEC, but with a different degree of “host-specificity’ than SadP1, since it seems to mediate a major effect in the binding with porcine IEC.233→Ala233, Glu249→Gln249, Asn285→Asp285) in the binding pocket of the galabiose. These three AA are critical for the binding specificity with α-D-Gal of Gb2, and these substitutions might result in differences in binding affinity to different isoforms and forms of Gbs receptors among the SadP variants. This observation would be analogous to Stx variants, associated with different clinical outcomes of infections, which bind various isoforms and forms of Gbs receptors with different affinity as determined by variation in fatty acid composition [S. suis disease incidence in South-East Asia [We demonstrated that SadP1 contributes to the adhesion to human IEC by binding of Gb3/CD77 confirming previous functional studies on recombinant galabiose-binding N-terminal domain of SadP recognizing Gb3/CD77 as its natural glycolipid host-cell receptor , 47. Recposition . Similarposition , 54. Gbsposition , 14, 51.position , 51. Witposition . It is tS. suis SadP1 contributes to the adhesion and translocation across host IEC. Different SadP variants might contribute to differences in host restriction between S. suis clones, due to differences in affinity and specificity of SadP-Gbs receptors interactions. Thus, components that interfere with bacterial binding to these receptors may have potential to reduce the burden of S. suis infection in both pigs and humans.Our findings contribute to a better understanding of specific host-pathogen interactions, showing that S1 Fileet al. [S. suis ΔsadP2A and ΔsadP2B deletion mutants to human and porcine IEC. Percentage of adhesion of strain SS2/CC20 2001171 WT and its 2001171ΔsadP2A mutant; SS9/CC16 8067 WT strain and its isogenic mutant ΔsadP2B (8067ΔsadP2B) to human (Caco-2) and porcine (IPEC-J2) intestinal cells. Three independent experiments were performed in triplicate and the combined together. Unpaired Student's t-test was used to compare each ΔsadP2A and ΔsadP2B mutants with its own parental strains . Table A: List of S. suis strains used in this study. Table B: List of primers used in this study.Fig A: Phylogenetic analysis of SadP variants including strains from Vietnam and the UK. The five identified SadP variants cluster together even when 375 strains from Weinert et al.  were inc(DOCX)Click here for additional data file.