The cytokine interleukin-28B (IL-28B) has potential antiviral properties and regulatory roles in adaptive cellular immunity. A genome-wide association study identified a single nucleotide polymorphism near the IL-28B gene that strongly predicts response to hepatitis C treatment with interferon and ribavirin. In this study, we produced human serum albumin (HSA) fused to interleukin-28B (HSA-IL28B) in an attempt to determine the effects of albumin fusion on anti-Hepatitis C virus (HCV) activity and protein stability. HSA-IL28B was expressed at high levels in the yeast expression system we used and was easily purified. The biological activities of IL-28B were only retained when HSA was fused at the N-terminus. Compared with the native IL-28B, HSA-IL28B showed improved protein stability. HSA-IL28B inhibited HCV infection through the membrane receptors IL28R1and IL10R2. Additionally, we demonstrated that HSA-IL28B was able to induce interferon-stimulated genes, phosphorylate intracellular STAT1, and act in restricted cell types. Our findings highlight the potential clinical applications of the fusion protein during virus infection and for immune regulation. Interferons (IFNs) are cytokines produced naturally, or upon pathogen challenge. Currently, three types of IFNs  have been characterized, with type I IFN widely used to treat hepatitis C, leukemia, lymphomas, and recurrent melanoma. Hepatitis C virus (HCV) infects an estimated 170 million people worldwide Interleukin-28 (IL-28) has two isoforms, IL-28A and IL-28B, and are a part of the type III IFN family comprising IL-29, IL-28A and IL-28B . Recent genome-wide association studies have demonstrated that a genetic polymorphism in the il-28b gene was strongly associated with a sustained virological response during IFN treatment of chronic hepatitis C patients in vitro and in vivo for HCV, HIV, hepatitis B virus, herpes simplex virus and West Nile virus. Additionally, accumulating evidence suggests that type III IFNs have specific effects on the regulation of the immune system and inhibition of tumor cell growth.The type III IFNs transduce signals by binding to and stimulating a heterodimeric membrane receptor. This receptor is composed of a long, specific IL28 α chain (IL28R1) and a short, widely distributed IL10 β chain (IL10R2). Similar to type I IFNs, activation of IFN-λ receptors leads to phosphorylation of the Janus tyrosine kinase-signal transducer and activator of the transcription (JAK-STAT) pathway. Furthermore, phosphorylated STAT1 and STAT2, together with IFN regulatory factor 9 (IRF-9), form the IFN-stimulated gene factor 3 (ISGF3) complex. This regulates typical IFN-induced genes such as OAS and MxA. The therapeutic potential of type III IFNs to viral infection has been documented in vitro. However, development of an IL-28B molecule with sustained long-term effects has not been reported, but is urgently needed for treatment of HCV infections.Type III IFNs were first used in a clinical setting to treat hepatitis C. A pegylated IL-29 has been developed and is being used in phase 2 clinical trials. This particular molecule has shown some promising outcomes compared with pegylated IFN-α, with better tolerance and lower adverse effects observed for pegylated IL-29. Based on these results, we believe that type III IFNs can be applied as novel treatments for chronic hepatitis C. All type III IFNs were compared, and it was shown that IL-28B had potent antiviral activity, along with IL-29 and IL-28A Several technologies have been developed to enhance the pharmacokinetics of polypeptides, including chemical/covalent modification, micro-encapsulation and the use of protease-resistant variants. New approaches to improving pharmacokinetics of small proteins are based on binding to, or fusion with, circulating serum proteins such as human serum albumin (HSA). Albumin is the most abundant protein in the blood plasma; it is produced in the liver as a monomeric protein of 67 kDa In this study, we expressed and purified IL-28B that was fused to HSA (HSA-IL28B), and evaluated its efficacy against HCV.The PichiaPink™ Secreted Protein Expression Kit (Cat. No. A11151) and PichiaPink™ Media Kit (Cat. No. A11156) were purchased from Invitrogen . The human hepatoma cell line, Huh7.5.1, was a gift from Dr. Francis V. Chisari . The Huh7 cell line was from Apath Inc. HepG2, Caco-2, HeLa, HEK293T, A549, DU145, K562, MCF7 and U251 cells were obtained from ATCC . The HCV genotype 1b replicon-containing cell line (2−3+) was kindly provided by Dr. Stanley Lemon . T4 DNA ligase and all restriction enzymes were purchased from New England Biolabs . SP-Sepharose Fast Flow was from GE Healthcare . PEG-IFNα2b was from Schering Plough  and ribavirin was obtained from the Beijing YouAn Hospital. Recombinant IL28B protein was purchased from R&D Systems . Antibodies against STAT1, MX1, and ISG15 were obtained from Abcam, Cell Signaling Technology (phospho-STAT1-Tyr701), Thermo Scientific (HCV Core), and Sigma (β-actin). Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Jackson ImmunoResearch .Pichia codon bias and synthesized by Genscript . The coding region without a signal peptide (residues 18–196) was fused to the N- or C-terminus of HSA by overlap extension PCR using specific primers  were linearized with SpeI and introduced into PichiaPink Expression Strains by electroporation using a Micropulser  according to the manufacturer’s instructions. Expression of N-HSA-IL28B and IL28B-HSA-C was performed as previously described The human IL28B cDNA sequence (GenBank No. NM_172139) was optimized using  primers Table 1.To achieve high levels of protein expression, methanol concentration  and induction time  were optimized. Expression levels were analyzed by SDS-PAGE. Affi-Gel blue affinity gel (Blue Gel) that was covalently crosslinked with Blue F3GA dye was purchased from Bio-Rad and applied for the affinity purification of fusion proteins. Under optimal expression conditions, supernatant from yeast culture media was harvested by centrifugation and applied to a column containing Blue Gel pre-equilibrated with Buffer A . After loading, the gel was completely washed with Buffer A and the target protein eluted with Buffer B . The purified samples were analyzed by SDS-PAGE and MALDI-TOF mass spectrometry, then freeze-dried for subsequent biological assays.The production of firefly luciferase HCV reporter viruses (Jc1-Luc HCVcc) was performed as described elsewhere in vitro protease degradation assay of HSA-IL28B and native IL-28B were performed as described elsewhere To determine whether the HSA-IL28B fusion protein is more resistant to degradation by proteases, A single dose pharmacokinetic studies were conducted in CD-1 mice. Briefly, 12 normal CD-1 mice were randomly divided into two groups, each containing six animals. The mice were acclimatized to standard laboratory conditions of 12-h light/12-h dark cycle with free access to rodent chow and water. Mice were administered a single subcutaneous (s.c.) injection of 0.5 mg/kg of HSA-IL28B and native IL-28B. Blood samples were collected at 0, 30 min, 2 h, 6 h, 12 h, 24 h, 48 h, 72 h, 120 h and 144 h. Plasma concentrations of HSA-IL28B and native IL-28B were determined by ELISA.Serially diluted antibodies against IL28BR1 or IL10R2 (R&D Systems) were pre-incubated with HCVcc-infected Huh7.5.1 cells at 37°C for 2 h prior to HSA-IL28B (12×10–3 mg/L) treatment. These were removed by washing. A parallel IgG isotype treatment was used as a negative control. The cells were further cultured for 2 days in the presence of HSA-IL28B before luminescence was measured.Western blot assays were performed as previously described Data analyses were performed using the t-test for two independent samples. A P-value less than 0.05 was considered statistically significant. All experiments were performed in triplicate, and the values are presented as the mean ± SD.Pichia. Recombinant plasmids were transformed into electrocompetent PichiaPink yeast cells, and more than 100 clones screened by SDS-PAGE for expression levels of recombinant protein. The yeast clone with the highest expression level of N-HSA-IL28B was selected for further recombinant protein expression optimization. As shown in Pichia pastoris, Invitrogen).We expressed HSA-IL28B, with the HSA fused at either the N- or C-terminal of IL-28B, and designated it as N-HSA-IL28B or IL28B-HSA-C, respectively Fig. 1A.S3). Recombinant IL28B-HSA-C was purified and characterized in the same way as for N-HSA-IL28B (data not shown).As shown in We determined the anti-HCV activity of both HAS-IL28B forms using Jc1-Luc HCVcc. Fusion of HSA at the N-terminus of IL-28B resulted in a retention of IL-28B antiviral activity (IC50 of 3 ng/mL). However, activity was significantly lower compared with native human IL-28B (IC50 of 0.15 ng/mL) Fig. 2A The antiviral activity of native IL-28B gradually decreased over time in storage, declining to less than 50% activity after 4 days in storage. The activity of HSA-IL28B was completely retained during incubation and storage Click here for additional data file.Figure S2Peptide sequences from the human serum albumin (HSA) were identified by mass spectrometry (red).(TIF)Click here for additional data file.Figure S3Peptide sequences from the human IL28B were identified by mass spectrometry (red).(TIF)Click here for additional data file.


We found conditions where the rate of M.SssI mediated C-to-U deamination was at least 100-fold higher than the rate of m5C-to-T conversion. Although this difference in reactivities suggests that the enzyme could be used to identify C5-methylated cytosines in the epigenetically important CG dinucleotides, the rate of M.SssI mediated cytosine deamination is too low to become an enzymatic alternative to the bisulfite reaction. Amino acid replacements in the presumed SAM binding pocket of M.SssI (F17S and G19D) resulted in greatly reduced methyltransferase activity. The G19D variant showed cytosine deaminase activity in E. coli, at physiological SAM concentrations. Interestingly, the C-to-U deaminase activity was also detectable in an E. coli ung+ host proficient in uracil excision repair.The prokaryotic DNA(cytosine-5)methyltransferase M.SssI shares the specificity of eukaryotic DNA methyltransferases (CG) and is an important model and experimental tool in the study of eukaryotic DNA methylation. Previously, M.SssI was shown to be able to catalyze deamination of the target cytosine to uracil if the methyl donor S-adenosyl-methionine (SAM) was missing from the reaction. To test whether this side-activity of the enzyme can be used to distinguish between unmethylated and C5-methylated cytosines in CG dinucleotides, we re-investigated, using a sensitive genetic reversion assay, the cytosine deaminase activity of M.SssI. Confirming previous results we showed that M.SssI can deaminate cytosine to uracil in a slow reaction in the absence of SAM and that the rate of this reaction can be increased by the SAM analogue 5’-amino-5’-deoxyadenosine. We could not detect M.SssI-catalyzed deamination of C5-methylcytosine ( DNA (cytosine-5) methylation is catalyzed by C5-methyltransferases (C5-MTase), which transfer a methyl group from the methyl donor S-adenosyl-methionine (SAM) onto carbon 5 of cytosines in specific nucleotide sequences. Eukaryotic and prokaryotic C5-MTases share amino acid sequence similarity and are thought to function by the same catalytic mechanism . m5C) are chemically less stable than the other nucleobases. Cytosine deaminates, in a hydrolytic reaction, to uracil, and m5C deaminates to thymine. The rate of spontaneous C-to-U deamination in double-stranded DNA, under physiological conditions, was found to be 2.6 - 7 x 10-13/s , DH5α F- Φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17  phoA supE44 λ- thi-1 gyrA96 relA1[E. coli ER2357-kanS and DH10B-kanS carry the inactive kanamycin resistance gene of pUP41 (see below) integrated into the bacterial chromosome. To construct the strains, the 894 bp BstBI-DraI fragment of pUP41 containing the kanS allele was cloned between the BstBI and PmeI sites of the plasmid pMS26 [The following 14::IS10 , DH10B F ΔlacX74 , ER2357 id pMS26 , and subid pMS26 . R KnS) carries an inactive allele of the Tn5 kanamycin resistance gene, which can revert to KnR phenotype by a C-to-T mutation [Plasmid pUP41 (Apmutation . R) [sssIM allele cloned in pBHNS-MSssI was considered as wild-type for this work. Plasmids pBHNS-MSssI(F17S) and pBHNS-MSssI(G19D) encode mutant variants of M.SssI, and were created from pBHNS-MSssI by site-directed mutagenesis [R) [sssIM gene, M.SssI expression was under the control of the arabinose PBAD promoter and the AraC protein [Plasmid pBHNS-MSssI carries the gene of C-terminally His-tagged M.SssI  cloned iR) . The sssagenesis . Plasmidagenesis  containsesis [R)  characte protein . All M.S protein .E. coli for purification of M.SssI. Ampicillin (Ap), kanamycin (Kn) and chloramphenicol (Cm) were used at 100, 50 and 25 μg/ml concentration, respectively.Bacteria were routinely grown in LB medium  at 30 orCT GCT GGA ATT GGGTA TTT GAA GCT T) and AK234 (AC ATT GGT GCT CAA AGGAA GCT TTT GCT G) synthesized in this institute were used to introduce the F17S and G19D replacements, respectively. AK233 and AK234 represent the coding strand of the sssIM gene with the nucleotides corresponding to the mutations underlined.The deoxyoligonucleotides AK233  or pBHNS-MSssI(G19D) and grown in TB/Ap. At a cell density of OD600~0.5, 0.1% arabinose was added to induce MTase production, and growth was continued at 30°C for 4 - 6 hours. Cells from 400 ml culture were harvested, resuspended in a buffer containing 50 mM Tris-HCl pH 8.0, 1mM EDTA, 10mM β-mercaptoethanol, 5% glycerol and disrupted by sonication. Cell debris was removed by centrifugation, and the supernatant was loaded onto a heparin-agarose column. Proteins were eluted with a 0 - 1M NaCl gradient in a buffer containing 50 mM Tris-HCl pH 8.0 and 5% glycerol. Peak active fractions were pooled, diluted two-fold with Ni-agarose equilibration buffer  and loaded onto a Ni-agarose column  equilibrated with the same buffer. Proteins were eluted using a buffer containing 50 mM Tris-HCl pH 7.5, 0.5 M NaCl and 250 mM imidazole. M.SssI-containing fractions were concentrated by dialysis against storage buffer , and stored at -20°C. His-tagged wild-type and mutant M.SssI variants were purified from In some cases the heparin-agarose step was omitted and the diluted crude extract was loaded directly onto the Ni-agarose column. Purity of enzyme preparations varied between 60-80% as determined by SDS-polyacrylamide gel electrophoresis.M.SssI activity was routinely estimated by restriction protection assay. Samples from a serial dilution of M.SssI were incubated with 0.2 - 0.5 µg plasmid or lambda phage DNA in M.SssI reaction buffer  containing 160 μM SAM (New England Biolabs) at 30°C for one hour. After the reaction the DNA was purified by phenol/chloroform extraction and ethanol precipitation. The precipitated DNA was dissolved, digested with Hin6I restriction enzyme and analyzed by agarose gel electrophoresis. In some cases phenol/chloroform extraction was omitted and M.SssI was inactivated by heat treatment (60°C 20 min) before adding Hin6I. E. coli ER2357 ung or DH10B ung+ cells by electroporation. Appropriate dilutions of the bacterial suspension were spread on Ap and Kn plates to determine the number of ApR and KnR transformants. Plasmid pUP41 (70 - 110 ng) was incubated with purified M.SssI in M.SssI reaction buffer (see above) in 50 µl at 30°C for 4 h or as shown at the particular experiment. Under these conditions the concentration of double-stranded CG sites in the reaction was ~0.18 - 0.27 µM. M.SssI was used at concentrations indicated in the text. Some deamination reactions contained SAM, sinefungin (Ili Lilly or Sigma) or 5’-amino-5’-deoxyadenosine (Sigma) at concentrations indicated in the text. After the incubation, the reactions were stopped with 0.5% SDS, and the DNA was purified by phenol/chloroform extraction and ethanol precipitation. The precipitated DNA was dissolved in TE buffer , and was used to transform in vivo, in DH10B cells that also contained pSTC-MSssI and were grown in the presence of 0.1% arabinose, or in vitro using purified M.SssI and SAM. In either case complete methylation was verified by Hin6I digestion. For testing deamination of C5-methylcytosine, CG-specifically methylated pUP41 was prepared either in vivo was studied by two methods. In the simpler reversion test, E. coli ER2357-kanS ung and DH10B-kanS ung+ harboring one of the plasmids pBHNS-MSssI, pBHNS-MSssI(F17S) or pBHNS-MSssI(G19D) were grown in LB/Ap/0.2% glucose. The growth medium contained glucose to repress M.SssI expression. Cells from this culture were sedimented by centrifugation, washed in glucose-free LB, and used to inoculate fresh LB/Ap/0.1% arabinose medium. After 4 h growth at 30°C, frequency of KnR revertants was determined by spreading aliquots of serial dilutions on Kn and Ap plates. M.SssI-mediated cytosine deamination in vivo was determined by the fluctuation test. E. coli ER2357 ung and DH10B ung+ harboring pUP41 were transformed with pSTdC-MSssI, pSTdC-MSssI(F17S) or pSTdC-MSssI(G19D). ApR CmR transformants were grown in the presence of 0.2% glucose to repress M.SssI expression. Cells from overnight cultures were centrifuged, resuspended in glucose-free LB, and were used to inoculate 10 parallel 1 ml cultures in LB/Ap/Cm/0.1% arabinose. After 24 h growth at 30°C, the number of KnR and ApR colonies was determined as described above. The reversion rate was calculated by the on-line FALCOR program using the Ma-Sandri-Sarkar Maximum Likelihood Estimator method [The rate of C-to-U deamination r method . DNA cloning, PCR reactions, agarose gel electrophoresis of DNA and polyacrylamide gel electrophoresis of protein samples were done by standard methods . EnzymesR plasmid pUP41 carrying an inactive, mutant allele of the kanamycin resistance gene of the transposon Tn5. The mutant codon resulting in L94P substitution and kanamycin sensitivity is located within a SmaI restriction site CGGGCC. Conversion of the underlined cytosine to thymine reverts the amino acid substitution to wild-type Leu94 and restores kanamycin resistance. Because the underlined cytosine is in a CG dinucleotide, the substrate site for SssI DNA methyltransferase [To detect C-to-U deamination by M.SssI, we used a genetic reversion assay developed by Bhagwat and coworkers . This asnsferase , pUP41 cnsferase  and FiguE.coli ER2357 ung strain deficient in the repair of uracil containing DNA. The frequency of C-to-U conversions was derived from the ratio of the KnR and ApR transformants. Preliminary experiments testing the conditions of M.SssI-mediated cytosine deamination indicated that the number of KnR revertants reached maximal level at a ~two-fold excess of the enzyme over CG sites in the plasmid and after 4 h incubation at 30°C (not shown). The influence of the incubation temperature was not tested specifically for the deamination activity. Previously we found that the MTase activity of M.SssI was, in vitro as well as in vivo, higher at 30°C than at 37°C (unpublished results). Assuming that the deamination and methyltransferase activities of M.SssI have the same optimal temperature, 30°C was used throughout this work. In most experiments ~twofold MTase/CG site ratio and 4 h incubation time were used as standard conditions. Under these conditions, the reversion frequency varied between 10-4 and 10-5 for the plasmid incubated without the enzyme. M.SssI increased the reversion frequency ~10-fold. Addition of SAM lowered the reversion frequency back to the level of the untreated plasmid (+E. coli host DH10B indicating that reversion to KnR phenotype went through the C-to-U-to-T pathway (not shown). Plasmids were isolated from some of the KnR clones and the disappearance of the SmaI site and the concomitant appearance of a new MvaI site was verified by restriction digestion  in the SAM binding pocket of M.HpaII [Conserved block I characterized by the 5-MTases ,28,29. T M.HpaII . in vivo, we used the same strategy. In M.SssI, the amino acids corresponding to F38 and G40 of M.HpaII are F17 and G19 m5C sites [+ hosts. As expected, expression of the mutant MTases led to growth arrest in the McrBC+ host, whereas McrBC- cells continued growing after induction . To test the effect of the F17S and G19D replacements on the MTase activity, DH10B cells harboring pBHNS-MSssI or its mutant derivatives were grown to mid-log phase, then arabinose was added to induce M.SssI expression. Plasmid DNA was purified from the cultures after 4, 6 and 8 h induction, digested with the methylation sensitive restriction enzyme Hin6I and analyzed by agarose gel electrophoresis. After 4 h induction, pBHNS-MSssI encoding the WT MTase was completely resistant, whereas pBHNS-MSssI(F17S) and pBHNS-MSssI(G17D) were almost fully digestible. After 6 and 8 h growth partial digestion products appeared in the digests of the mutant plasmids . pBHNS-M5C sites , thus M.nduction . CompariT enzyme . Similarung and DH10B-kanS ung+. Due to the site specific mechanism of the technique used [sssIM alleles. These plasmids have much higher copy number than the pSTdC- plasmids and result in higher M.SssI concentration after arabinose induction. ER2357-kanS ung and DH10B-kanS ung+ harboring pBHNS-MSssI, pBHNS-MSssI(F17S) or pBHNS-MSssI(G19D) were induced at mid-log phase with arabinose, then after 4 h growth the frequency of KnR revertants was determined as described in Materials and Methods. The Ung- host not producing M.SssI showed a reversion frequency between 10-7 and 10-8, which can be considered to reflect the spontaneous C to U deamination rate under these conditions  and 5’-amino-5’-deoxyadenosine (AA) were previously shown to promote cytosine deamination by M.HpaII, HhaI, MspI as well as by M.SssI ,12. It wm5C-to-T deamination events, reversion frequency was determined using the Ung+ host DH10B. The reversion frequencies of the untreated plasmids were the same  in the absence of SAM using the same conditions as described for deamination of unmethylated pUP41. The only difference was that, to minimize the possibility of scoring C-to-U deaminations as the same . Under cthe same . This fin of ref .. The re at 37°C . To addr at 37°C   the quantitative results of this work and of previous studies [in vitro conditions , was ~2.7 x 10-4, which is ~10-fold higher than the frequency observed by Bandaru et al. after ~4 h incubation at 37°C [-4, but this value was determined after 16 h of incubation at 37°C [ Here we showed that M.SssI can catalyze deamination of cytosines located in CG dinucleotides in double-stranded DNA if the methyl donor SAM is omitted from the reaction . The dea studies ,10 are d-3, which is very close to the value obtained by the Jones group using 16h incubation [In our system the combined action of M.SssI and 250 µM 5’-amino-5’-deoxyadenosine resulted in an elevated reversion frequency of 3.3 x 10cubation . in vitro, we wished to test whether the enzyme can be used to deaminate cytosines in vivo, in the presence of SAM. To this end, we constructed two mutants, which carried the F17S or the G19D replacement in the presumed SAM binding pocket. The rationale of creating these mutations was to weaken binding of the methyl donor SAM to the MTase and thus mimic conditions of limiting SAM in vivo, at physiological SAM concentrations. This approach, previously applied to HpaII MTase [E. coli cells  are strictly conserved in C5-MTases and participate in forming the SAM binding pocket probably in all C5-MTases [The very low residual MTase activity of the mutant enzymes  and 3 an5-MTases ,28. For 5-MTases  and thisin vivo. This result raises the possibility of using G19D as a CG-specific targetable cytosine deaminase to induce C-to-T transitions at pre-determined CG sites in vivo. In the envisaged application, which has conceptually much in common with targeted DNA methylation [E. coli Ung+ hosts can be important for preventing repair of the pre-mutagenic U:G mismatch generated by the enzyme before conversion into a stable T:A mutant base pair.  The data reported here show that the G19D mutant of M.SssI can be used as a CG-specific cytosine deaminase hylation ,40, M.Sshylation ,42. The m5C-to-T conversion [ung- constructed in this study offers a directed evolution strategy to select M.SssI variants with increased cytosine deaminase activity. In the envisioned selection scheme a plasmid library carrying in vitro mutagenized sssIM gene variants would be introduced into this host strain and pools of KnR revertants would be selected after a period of growth. The plasmid preparation isolated from the KnR culture would be mutagenized again and transformed into ER2357 ung-. We expect that multiple rounds of mutagenesis coupled with successively shorter growth periods will gradually enrich M.SssI variants with higher cytosine deamination activity. This work was initiated on the assumption that cytosine and 5-methylcytosine would show different sensitivities to M.SssI-catalyzed deamination. This notion was based on the similar reaction mechanisms of C5-MTase mediated and bisulfite mediated C-to-U deamination. In particular, both reactions are thought to produce the unstable 5,6-dihydrocytosine intermediate, which readily undergoes hydrolytic deamination ,43. In snversion . This dinversion . We compnversion , the difin vitro conditions, M.SssI-catalyzed deamination of 5-methylcytosines was not detectable. Although the difference between the reactivities of C and m5C suggest that M.SssI could be used to determine the methylation status of cytosines in the epigenetically important CG sequence context, the slow rate of M.SssI-catalyzed C to U deamination makes this reaction, at present, impractical as an enzymatic alternative to the bisulfite reaction. We have shown that the G19D mutant of MSssI can catalyze C to U deamination in vivo, in E. coli, in the presence of SAM, even if the host is proficient in uracil excision repair. Thus, M.SssI(G19D) can function as a sequence-specific mutator that converts CG dinucleotides to TG. In summary, our results show that M.SssI can catalyze deamination of cytosines in CG dinucleotides in double-stranded DNA. Under the same Figure S1m5C to T change as a result of deamination.Detection of C to U and  Unmethylated or invivo M.SssI-methylated pUP41 was incubated with M.SssI in the absence of SAM and transformed into E. coli ung or ung+ host to detect KnR revertants . Plasmids isolated from KnR revertants were digested with SmaI or MvaI. Agarose gel electrophoresis of the digested plasmids.1) pUP41, untreated.invitro.2) pUP41 incubated with M.SssI invivo, and subsequently incubated with M.SssI invitro.3) pUP41 methylated by M.SssI M) Size marker .Deamination reactions did not contain SAM.CGGGCC) and appearance of a new MvaI site (CCWGG) indicates C to T change in the middle of the SmaI site (underlined). One of the new MvaI fragments (1249 bp), is marked by an arrow, and the disappearing SmaI (687 bp) fragment is marked by asterisk. Faint bands in samples 2 and 3 co-migrating with fragments of the untreated DNA (sample 1) probably indicate mixed plasmid population resulting from incomplete plasmid segregation. There are 2 SmaI sites and 13 MvaI sites in pUP41. Disappearance of a SmaI site ((TIF)Click here for additional data file.


Shigella flexneri IcsA (VirG) protein is a polarly distributed outer membrane protein that is a fundamental virulence factor which interacts with neural Wiskott-Aldrich syndrome protein (N-WASP). The activated N-WASP then activates the Arp2/3 complex which initiates de novo actin nucleation and polymerisation to form F-actin comet tails and allows bacterial cell-to-cell spreading. In a previous study, IcsA was found to have three N-WASP interacting regions (IRs): IR I (aa 185–312), IR II (aa 330–382) and IR III (aa 508–730). The aim of this study was to more clearly define N-WASP interacting regions II and III by site-directed mutagenesis of specific amino acids. Mutant IcsA proteins were expressed in both smooth lipopolysaccharide (S-LPS) and rough LPS (R-LPS) S. flexneri strains and characterised for IcsA production level, N-WASP recruitment and F-actin comet tail formation. We have successfully identified new amino acids involved in N-WASP recruitment within different N-WASP interacting regions, and report for the first time using co-expression of mutant IcsA proteins, that N-WASP activation involves interactions with different regions on different IcsA molecules as shown by Arp3 recruitment. In addition, our findings suggest that autochaperone (AC) mutant protein production was not rescued by another AC region provided in trans, differing to that reported for two other autotransporters, PrtS and BrkA autotransporters.The  Shigella flexneri is a human pathogen that causes bacillary dysentery by infecting and colonising the colonic epithelium S. flexneri, is polarly distributed at the outer membrane (OM). IcsA is required for inter- and intracellular spreading of S. flexneri within the host intestinal epithelium Shigella multiplies and interacts with the host actin regulatory protein neural Wiskott-Aldrich syndrome protein (N-WASP), which in turn activates the Arp2/3 complex that initiates actin polymerisation by recruiting the globular actin monomers to form the filamentous actin (F-actin) comet tails beta-barrel assembly machine) complex IcsA is a member of the autotransporter (AT) family (Type Va secretion system), the largest family of secreted proteins in Gram-negative bacteria In spite of the diversity in sequence, function and length, the passenger domains of most ATs possess a β-helical structure de novo actin polymerisation, leading to host cell motility and morphological changes 103–433 which contains glycine-rich repeats  is sufficient for N-WASP binding in vitroin vitro assays showed that IcsA53–508 is adequate to induce actin polymerisation i) S. flexneri IcsB and host autophagy protein, Atg5, binding region (aa 320–433) N-WASP is a key regulator of the actin cytoskeleton and functions as a link between signalling pathways and i mutants  with mutational alterations within the AC region have markedly reduced protein production in S. flexneri strain with native LPS O-antigen (LPS Oag)  and hence, were unable to form plaques, recruit N-WASP or form F-actin comet tail i mutants were expressed in S. flexneri strain without LPS Oag , IcsAi mutant production was restored to a level comparable to wild-type (WT) IcsA i production in S. flexneri R-LPS strains, we have identified that IcsAi633, IcsAi643, IcsAi677, but not IcsAi716, were able to recruit N-WASP and form F-actin comet tails From the 5 aa linker-insertion mutagenesis study, IcsAS. flexneri backgrounds. Using this approach, we have identified specific residues affecting N-WASP binding/interaction. In addition, we have also co-expressed IcsA mutant proteins with mutations at two N-WASP interaction regions, and showed for the first time that all three N-WASP interacting regions are essential for N-WASP and Arp3 recruitment, supporting a role for oligomeric IcsA in this interaction.In this study, we investigated the ability of IcsA mutants with point mutations within N-WASP IR II and III to recruit N-WASP and form F-actin comet tails in both S-LPS and R-LPS The anti-IcsA antiserum was produced under the National Health and Medical Research Council (NHMRC) Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and was approved by the University of Adelaide Animal Ethics Committee.The strains and plasmids used in this study are listed in S. flexneri strains were grown from a Congo Red positive colony as previously described −1), chloramphenicol , kanamycin , or tetracycline .All strains used in this study were routinely grown in Luria Bertani (LB) medium. E. coli K-12 DH5α was used for all cloning. DNA manipulation, PCR, transformation and electroporation into S. flexneri were performed as previously described Affinity purified rabbit polyclonal antibody to IcsA was produced as previously described 8 bacteria were centrifuged  and resuspended in 100 µl of 1x sample buffer The equivalent of 5×10Western immunoblotting was performed as described previously DpnI and transformed into XL 10-Gold ultracompetent cells (Stratagene). Mutations were confirmed by DNA sequencing.Single and multi site-directed mutagenesis were performed according to the QuikChange® Lightning Site-directed or QuikChange® Lightning Multi Site-directed protocol (Stratagene), respectively. Specific primers with the desired substitutions were designed for single site-directed mutagenesis  and degeWT, pSU23-IcsA::BIO, pSU23-IcsAi248 and pSU23-IcsA::BIO Y716G D717G were constructed by sub-cloning the PstI-SalI fragment of pIcsAWT, pIcsA::BIO, pIcsAi248 and pIcsA::BIO Y716G D717G (pMYRM322), encoding different IcsA proteins, respectively, into likewise digested pSU23.pSU23-IcsAet al. 8 bacteria mL−1 mid-exponential phase bacteria were diluted to 1∶1000 in DMEM, and 0.25 mL was added to each well. Trays were incubated in a CO2 (5%) incubator, and the trays were gently rocked every 15 min. At 120 min post-infection, the inoculum was carefully aspirated and 3 mL of the first overlay  FCS, 20 µg mL−1 gentamycin, 0.5% (w/v) agarose [Seakem ME]) was added to each well. The second overlay  FCS, 20 µg mL−1 gentamycin, 0.5% (w/v) agarose, 0.1% (w/v) Neutral Red solution [Gibco BRL]) was added at either 24 or 48 h post-infection and plaque formation observed 6–8 h later.Plaque assays were performed as described previously Indirect IF labelling of bacteria was performed as previously described 2 (5%). The infected monolayers were washed three times with D-PBS and incubated with MEM containing gentamycin for a further 1.5 h. Infected cells were then washed and fixed for 15 min in 3.7% (v/v) formalin, incubated with 50 mM NH4Cl in D-PBS for 10 min, and permeabilised with 0.1% (v/v) Triton X-100 for 5 min. The infected cells were blocked with 10% (v/v) FCS in PBS and incubated with the desired primary antibody at 37°C for 30 min. After washing in PBS, coverslips were incubated with Alexa Fluor 594-conjugated donkey anti-rabbit, Alexa Fluor 488-conjugated donkey anti-rabbit or Alexa Fluor 594-conjugated donkey anti-mouse secondary antibodies (Molecular Probes) (1∶100), where appropriate. F-actin was visualised by staining with Alexa Fluor 488-conjugated phalloidin (2 U mL−1), and 4′,6′-diamidino-2-phenylindole (DAPI) (10 µg mL−1) was used to counterstain bacteria and cellular nuclei.Infection of HeLa cell monolayers and IF staining were performed as previously described i mutants with insertion mutations within this region was unaffected but the mutants were defective in ABM when expressed in S. flexneri ΔicsA S-LPS i330 and IcsAi381), it was of interest to determine if altering specific residues would reproduce the same phenotype, thus, enabling the identification of specific amino acids involved in N-WASP recruitment within this region. Multi site-directed mutagenesis was performed on pIcsA::BIO (pMG55) to randomly substitute both amino acids, 330–331 and 381–382, respectively, by using degenerate primers listed in S. flexneri ΔicsA was assessed by Western immunoblotting with anti-IcsA antibody (data not shown). All of these IcsA mutant proteins were produced by S. flexneri at WT levels . The results are summarised in T330S G331G] and pMYRM539 [IcsA::BIO T330P G331G] both possess single mutations at residue 330 (in bold) but only mutant T330S G331G (both polar amino acids) was capable of recruiting N-WASP. However, it cannot be ruled out that the proline substitution at position 330 or 331 is causing a localised conformation change thereby affecting IcsA function. Furthermore, mutants with charged amino acids  recruited less N-WASP and formed less F-actin capping/tails  of those formed by S. flexneri expressing IcsA::BIO  that is positively charged. IcsA::BIO V382R (encoded by pMYRM590) mutant production by blotting , lane 3 blotting . IcsA::B plaques . These di mutants which had 5 aa insertion mutations within the IcsA AC region for their ability to recruit N-WASP, and identified that IcsAi716 was defective in N-WASP recruitment i mutants had extremely low protein production in the S-LPS background but WT comparable expression levels in the R-LPS background i716 mutant has a 5 aa insertion mutation after residue 716 which affects the Aro-X-Aro (aromatic residue-any-aromatic residue) motif, Y716-D717-Y718. This motif is commonly found in the β-domain of ATs and is thought to be a preferential binding site of SurA S. flexneri ΔicsA S-LPS and S. flexneri ΔicsA ΔrmlD R-LPS strains was determined by Western immunoblotting. The IcsA::BIO Y716*D717* mutant production was markedly reduced in the S-LPS strain, regardless of the amino acids substitutions  by performing site-directed mutagenesis on pIcsA::BIO to mutate Y716 to phenylalanine (F) or glycine (G), and D717 to glycine (G) using primer pairs shown in blotting  and IF mblotting . The Y71sA S-LPS , lane 2 csA::BIO , lane 5.i716 was defective in N-WASP recruitment and proposed that residues 716–717 could be an N-WASP binding/interaction site We have previously shown that IcsAS. flexneri ΔicsA S-LPS strain expressing IcsA::BIO Y716F recruited N-WASP and formed F-actin comet tails (S. flexneri ΔicsA S-LPS strain expressing IcsA::BIO Y716G recruited N-WASP (slightly less than IcsA::BIO) but had only F-actin capping formation and F-actin comet tails were not observed  , S. flexneri ΔicsA [IcsA::BIO Y716G] formed plaques that were 50% smaller (***P<0.001)  . These r plaques .S. flexneri (summary in icsA genes were cloned into vector pSU23 which has a similar copy number (intermediate) to pBR322. IcsA mutants with mutation at each N-WASP IR and defective in N-WASP recruitment when expressed individually in S. flexneri were chosen for co-expression/complementation analysis. pSU23 constructs that encode for IcsAWT, IcsA::BIO, IcsAi248 (N-WASP IR I) and IcsA::BIO Y716G D717G (N-WASP IR III) were constructed as detailed in Materials and Methods. As expected, all IcsA proteins were expressed at WT levels that were comparable to those expressed by the pBR322 constructs in both S-LPS and R-LPS backgrounds , IcsA::BIO V382R (N-WASP IR II) or IcsA::BIO Y716G D717G (N-WASP IR III) were subsequently electroporated into different S. flexneri strains (S-LPS and R-LPS) carrying the pSU23-IcsA constructs as shown in S. flexneri S-LPS strains that co-expressed IcsA proteins were assayed for N-WASP recruitment following infection of HeLa cells by IF microscopy, and the results are summarised in S. flexneri S-LPS strains that expressed either IcsAWT or the functionally equivalent IcsA::BIO were able to recruit N-WASP, regardless of the co-expressed IcsA protein. However, co-expression of two mutated IcsA proteins  did not result in N-WASP recruitment. As expected, S. flexneri strains that co-expressed a mutated IcsA protein with an empty vector did not recruit N-WASP. As the production of IcsA::BIO Y716G D717G was extremely low in the S-LPS background but restored to a WT comparable levels in the R-LPS background, various IcsA proteins were also co-expressed in S. flexneri R-LPS. Their ability to recruit N-WASP was examined and summarised in S. flexneri R-LPS strains which have the same IcsA co-expression combination in the S-LPS background, with the remarkable exception of the co-expressed combination IcsA::BIO V382R and IcsA::BIO Y716G D717G  that was able to recruit N-WASP  (MYRM697) was activated, Arp2/3 complex recruitment and F-actin comet tail formation were examined by IF microscopy with anti-Arp3 monoclonal antibody and Alexa Fluor 488-phalloidin staining, respectively. Arp3 recruitment was significantly detected at one pole of the bacterium which co-localised with N-WASP . We have quantitated the recruitment of N-WASP, Arp3 and F-actin comet tail/capping formation by scoring 20 infected HeLa cells (∼250–350 bacteria) when both N-WASP/Arp3 and N-WASP/F-actin tail/capping were observed respectively, in two independent experiments. MYRM697 which co-expressed IcsA::BIO V382R and IcsA::BIO Y716G D717G had an approximately 40–45% reduction in N-WASP (P***<0.001) and Arp3 recruitment (0.001<P**<0.01) (P***<0.001) (To investigate if the N-WASP recruited by **<0.01) , as well*<0.001) , compareS. flexneri, and is able to initiate F-actin comet tail formation through activation of the host N-WASP, leading to bacterial ABM and spreading within the host colonic epithelium. Although N-WASP is known to interact with IcsA and several IcsA regions have been reported to be involved in N-WASP recruitment IcsA (VirG) is one of the key virulence proteins of S. flexneri ΔicsA expressing IcsA::BIO G331W was capable of recruiting N-WASP and forming F-actin comet tail/capping but not as frequently as the positive control strain (IcsA::BIO). As a result, a reduction in plaque size (∼50%) was observed for IcsA::BIO G331W. In comparison, a single amino acid change at position 330 into a proline which is a non-polar amino acid , abolished N-WASP recruitment. Notably, pMYRM543 (T330G G331P) which possesses proline at position 331 was also defective in N-WASP recruitment. Although both pMYRM543 (IcsA::BIO T330G G331P) and IcsA::BIO G331W mutants have the polar and non-polar amino acids combination, a contradictory effect on function was obtained. Taken together, the results obtained suggest that the proline substitution at either position disrupts local secondary structure of IcsA and prevents N-WASP binding. Nevertheless, the results suggest that N-WASP interaction at this site does not rely on amino acid polarity, and the polar-polar combination is not essential. Indeed, the data suggest that the presence of specific amino acid residues at positions 330–331 is required for efficient N-WASP interaction and recruitment.Specific amino acids within the N-WASP IR II were randomly mutated . Screening of the resultant IcsA mutants suggested that for efficient N-WASP recruitment, a polar-polar combination is required for residues 330–331, while a non-polar amino acid is required for residue 382  and 3. HS. flexneri expressing the IcsA::BIO V382R mutant was incapable of recruiting N-WASP and forming plaques. In contrast, mutants T381M V382L [pMYRM547] and T381K V382M [pMYRM548] ((in bold), were able to recruit N-WASP and form plaques when expressed in S. flexneri, regardless of the amino acid substitution at residue 381. The data suggest that a non-polar residue is essential at position 382 and plays an important role in N-WASP recruitment. Collectively, site-directed mutagenesis within the N-WASP IR II revealed new N-WASP binding/interaction sites, and we have assigned them as N-WASP IR 2a (aa 330–331) and N-WASP IR 2b (aa 382), respectively.Consistent with the IcsA::BIO T381*V382* mutants that also have residue 382 substituted with polar arginine (R) [pMYRM544 and pMYRM549] , S. flexMYRM548] , which pi716 which had a 5 aa insertion mutation within the AC region (N-WASP IR III) was defective in N-WASP recruitment S. flexneri S-LPS and R-LPS backgrounds, and investigated for N-WASP recruitment. The IcsA::BIO Y716*D717* mutant protein production was markedly reduced in the S-LPS background but restored to a WT comparable levels in the R-LPS background  and IcsA::BIO Y716G (non-conserved mutation) were functional in both S-LPS and R-LPS backgrounds, except that only F-actin capping was observed for Y716G in the S-LPS background. This may be due to the lower levels of IcsA::BIO Y716G expression in the S-LPS background which affected N-WASP recruitment efficiency and/or activation, hence, resulting in F-actin capping formation only. Consistent with these results,  plaques , reflectIcsA::BIO D717G did not recruit N-WASP in the S-LPS background . Consequi AC mutant production In terms of IcsA biogenesis, we showed that the 716 aromatic residue is important for IcsA production but this was largely independent of LPS structure because the non-conservative mutation resulted in reduced IcsA::BIO Y716G protein production in both S-LPS and R-LPS backgrounds . This imet al.S. flexneri S-LPS co-expressing IcsAWT and IcsAi563 or IcsAi677 displayed reduced efficiency in ABM. We hypothesised that IcsA functions as a complex on the bacterial surface and different regions of IcsA  are involved in interacting with N-WASP. To investigate our hypothesis, we co-expressed two IcsA mutant proteins in both S-LPS and R-LPS backgrounds in different combinations, to complement one mutated N-WASP IR with another copy of IcsA that had mutation at another N-WASP IR  were expected to be similar as the compatible intermediate copy plasmids (pBR322 and pSU23) were used. In addition, May et al. i mutant did not affect the polar distribution and the amount of functional protein at the bacterial surface.Previous investigations on IcsA regions involved in N-WASP recruitment have been studied on individual N-WASP interacting regions (based on deletion and linker insertion mutations) -WASP IR  and 6. SWT or the functionally equivalent IcsA::BIO  production was severely affected in the S-LPS background. We have previously proposed that AC IcsA mutant could be degraded in the periplasm prior to translocation across the OM S. flexneri surface. IcsA proteins with different tags could be co-expressed in S-LPS S. flexneri in future to determine the IcsA::BIO Y716G D717G expression level. In addition, we have previously shown that the expression of individual AC IcsA mutants in both R-LPS S. flexneri and R-LPS E. coli was restored to WT comparable levels, which does not require complementation of an additional AC region to restore IcsA biogenesis Studies on other autotransporter AC regions (e.g. PrtS and BrkA) were performed on WT molecule. The function of N-WASP IR I (contains the GRRs) cannot be complemented by another copy of IcsA with mutation at another N-WASP IR (S. flexneri co-expressing both IcsAi248 (N-WASP IR I) and IcsAWT  was capable of recruiting N-WASP, suggesting that despite mutation in N-WASP IR I, the presence of another copy of functional IcsA that provides sufficient functional N-WASP IRs is able to complement the defect, and a negative dominance effect was not detected.The N-WASP interacting region complementation data ; Fig. 4 -WASP IR . HoweverS. flexneri IcsA protein where the nature of their interactions is poorly understood. However, it is still unclear which region of N-WASP  S. flexneri virulence IcsA protein and the host actin cytoskeleton regulatory N-WASP protein.Taken together, we have identified new amino acids within N-WASP IR II and III that are involved in N-WASP recruitment. In particular, the conserved Aro-X-Aro motif  that is located within the AC region could be a potential drug target site, by utilising the recently crystallised IcsA AC region structure. Furthermore, our N-WASP IR complementation data strongly suggest that IcsA functions as a multimer. We have shown for the first time that all three N-WASP IRs are involved in N-WASP recruitment and activation. Our findings have shed light on N-WASP activation by Figure S1Plaque formation by S. flexneri ΔicsA expressing IcsA::BIO T330*G331* and IcsA::BIO T381*V382* mutants. Confluent HeLa cell monolayers were infected with mid-exponential phase S. flexneri strains for 2 h, and plaques were observed 48 h post-infection as detailed in Materials and Methods. 30 plaques were measured from each experiment. Data are represented as mean ± SEM of three independent experiments. **, 0.001<P<0.01; ***, P<0.001 (determined by Student’s unpaired one-tailed t test).(TIF)Click here for additional data file.Figure S2Detection of surface IcsA::BIO, IcsA::BIO G331W or IcsA::BIO V382R proteins expressed by S-LPS S. flexneri. Mid-exponential phase S. flexneri ΔicsA strains expressing either IcsA::BIO, IcsA::BIO G331W, IcsA::BIO V382R or an empty vector alone, were formalin fixed and labelled with rabbit polyclonal anti-IcsA antibody and Alexa 488-conjugated goat anti-rabbit secondary antibody. Insert shows an enlargement for greater clarity. IF images were observed at 100×magnification. Scale bar = 10 µm.(TIF)Click here for additional data file.Figure S3Locations of Y716, D717 and Y718 residues mapped on the crystal structure of the IcsA AC region. Protein Data Bank accession no. 3ML3. Residue Y716 is illustrated in pink, residue D717 is yellow and residue Y718 is blue. Side chains of each residue, as well as the amino (N) and carboxyl (C) termini are shown.(TIF)Click here for additional data file.Figure S4Detection of surface IcsA::BIO Y716 or D717 proteins expressed by S-LPS and R-LPS S. flexneri. Mid-exponential phase S. flexneri ΔicsA (S-LPS) or S. flexneri ΔicsA ΔrmlD (R-LPS) strains expressing either IcsA::BIO, IcsA::BIO Y716F, IcsA::BIO Y716G, IcsA::BIO D717G or an empty vector, were formalin fixed and labelled with anti-IcsA antibody and Alexa 488-conjugated goat anti-rabbit secondary antibody. Insert shows an enlargement for greater clarity. IF images were observed at 100×magnification. Scale bar = 10 µm.(TIF)Click here for additional data file.Figure S5Detection of IcsA proteins encoded by pSU23-based plasmids in S-LPS and R-LPS S. flexneri. (A) Whole cell lysates from mid-exponential phase S. flexneri ΔicsA (S-LPS) or S. flexneri ΔicsA ΔrmlD (R-LPS) expressing IcsAWT, IcsAi248, IcsA::BIO IcsA::BIO Y716G D717G or an empty vector were prepared and analysed by Western immunoblotting using anti-IcsA antibody. Strain names are shown above each lane. The 120 kDa band corresponds to the full length IcsA; the 85 kDa band corresponds to the cleaved form (IcsA’). S = S-LPS; R = R-LPS. (B) Mid-exponential phase S. flexneri ΔicsA (S-LPS) or S. flexneri ΔicsA ΔrmlD (R-LPS) strains expressing either IcsA, IcsA::BIO, IcsAi248, IcsA::BIO Y716G D717G or an empty vector, were formalin fixed and labelled with rabbit polyclonal anti-IcsA antibody and Alexa 488-conjugated goat anti-rabbit secondary antibody. Insert shows an enlargement for greater clarity. IF images were observed at 100×magnification. Scale bar = 10 µm.(TIF)Click here for additional data file.Table S1Oligonucleotides used in this study.(TIF)Click here for additional data file.Table S2IcsA production and N-WASP recruitment by S. flexneri S-LPS and R-LPS straisn expressing various IcsA proteins.(TIF)Click here for additional data file.


Toxoplasma gondii and play an essential role in the epidemiology of this parasite. The study aims at clarifying whether cats are able to develop specific antibodies against different clonal types of T. gondii and to determine by serotyping the T. gondii clonal types prevailing in cats as intermediate hosts in Germany.Cats are definitive hosts of T. gondii positive (n=21) and negative cat sera (n=52). To determine the clonal type-specific antibody response of cats in Germany, 86 field sera from T. gondii seropositive naturally infected cats were tested. In addition, we analyzed the antibody response in cats experimentally infected with non-canonical T. gondii types (n=7).To establish a peptide-microarray serotyping test, we identified 24 suitable peptides using serological T. gondii type the cats were infected with. When the array was applied to field sera from Germany, 98.8% (85/86) of naturally-infected cats recognized similar peptide patterns as T. gondii type II reference sera and showed the strongest reaction intensities with clonal type II-specific peptides. In addition, naturally infected cats recognized type II-specific peptides significantly more frequently than peptides of other type-specificities. Cats infected with non-canonical types showed the strongest reactivity with peptides presenting amino-acid sequences specific for both, type I and type III.Positive cat reference sera reacted predominantly with peptides harbouring amino acid sequences specific for the clonal T. gondii. Serotyping revealed for most seropositive field sera patterns resembling those observed after clonal type II-T. gondii infection. This finding is in accord with our previous results on the occurrence of T. gondii clonal types in oocysts shed by cats in Germany.Cats are able to mount a clonal type-specific antibody response against  Toxoplasma gondii is a zoonotic obligate intracellular parasite which causes toxoplasmosis in humans and animals. Felids are definitive hosts of this parasite and almost all warm-blooded mammals including humans and cats ) of ANOVA analyses. For the ROC based selection of peptides derived from polymorphic clonal type-specific regions, reactions of reference sera derived from cats infected with a homologous type of ormation ,66 peptiormation ; the higFrequency analysis  for serotyping data was performed with the R-package “vcd”, which was also used to visualize the results in mosaic plots as described previously ,30,68. Serum-peptide reactions  were cut-off normalized (CN) by subtracting the peptide-specific cut-off values from MSIV resulting in a value called CN-MSIV. This was done to achieve a better visualization of positive and negative serum-peptide reactions, e.g. positive reactions led to values ≥ 0 and negative results to values < 0. To perform multiple comparisons of CN-MSIV means between peptide groups or between single peptides, a Post-Hoc-Test (LSD [Least-Significant-Difference]) on ANOVA results was applied using the R package “agricolae” ,61. To analyse whether peptide patterns recognized by individual sera cluster in different groups, we performed explorative data analysis applying the artificial neural network-based Selforganizing Kohonen Network/Selforganizing Kohonen Maps (SOM) method -72. In tT. gondii negative (n=52), the reciprocal IFAT titres ranged from <25 to 100. Among the sera regarded as positive (n=114) reciprocal IFAT titres ranged from 200 to 51200 , the protein sequence revealed eight aa substitutions and six aa insertions in type II (ME49 strain), while seven aa substitutions were detected in type III GRA6 (NED strain). All peptides met most selection criteria S1. OnlyFor GRA7, 13 polymorphic peptide sequences were selected. All peptide sequences were located near the C-terminus. In comparison to GRA7 type I (RH strain), eight aa substitutions were observed in type II (BEVERLEY strain) and 15 aa substitutions in type III (NED strain) S1. All For the SAG2A protein, four polymorphic peptides from the C-terminal region were chosen. The selected peptide regions met the criteria of antigenicity, hydrophilicity, surface probability and presence of proline residues. However, the peptides failed to present an α-helical structure S1. PeptT. gondii serotyping individual peptides were first subjected to a ROC analysis. Those peptides for which ROC analysis revealed a diagnostic capacity were further analyzed by ANOVA and LSD-Post-Hoc-Test. In total, 101 peptides presenting single type-specific polymorphisms  as well as common polymorphisms for two of three clonal types simultaneously , were used initially. For the selection of peptides suitable for T. gondii serologically negative cats (n=52) and sera from experimentally-infected cats (n=21). In the individual ROC analyses, 40 of the 101 analyzed peptides yielded on AUC value ≥ 0.7 . The aa sequences of the 24 peptides showed the following specificities in detail: type I (n=4), type II (n=8), type III (n=7), type I/II (n=1) and type I/III (n=4). A total of 24 remaining peptides were recognized by sera with homologous specificity S4. Diagand 100% S4. The  [A]), followed by GRA5-I-41, and GRA6-I-216 . The lowest anti-clonal type I-specific reactivity was observed for peptide GRA6-I-173 ( [A]). The diagnostic sensitivity of individual peptides presenting clonal type I-specificity  ranged from 5.9% to 94.1% and the diagnostic specificity from 64.2% to 98.2%  were recognized by sera of clonal type I-infected cats (n=17) were significantly higher than the mean CN-MSIVs by which peptides with type II- or III-specific, i.e. heterologous aa sequences were recognized by the same sera (to 98.2% S4.  [B]). Positive clonal type I-specific peptide reactions were quantitatively overrepresented  among sera of cats infected with type I T. gondii. In addition, type I/II- and I/III-specific peptide reactions were also statistically significantly overrepresented as indicated by Pearson residuals > 3 . All type II- and III-specific positive peptide reactions were underrepresented  among sera from type I-infected cats ( [B]).The prevalence of positive and negative serum-peptide reactions within the group of type I-infected cats was analysed using a log-linear model. The resulting contingency tables and deviations from the hypothesis of independence were visualized by mosaic plots (T. gondii (n=3) revealed that peptides presenting type II-specific aa sequences  were recognized by significantly higher mean CN-MSIVs as compared to those peptides with heterologous specificity, i.e. clonal type I or III ( [A]), as compared to the remaining peptides used for serotyping . The lowest means of clonal type II-specific CN-MSIVs were observed in GRA7-II-225, SAG2A-II-93 and GRA1-II-159. Peptides specific for clonal type I or III were recognized by markedly lower intensity as compared to those peptides presenting clonal type II specificity  ( [A]). The diagnostic sensitivity of peptides with clonal type II specificity  ranged between 66.7% and 100% and their typing specificity ranged from 60% to 98.6% .Estimation of the prevalence of positive peptide reactions by log-linear modelling showed a statistically significant overrepresentation of clonal type II-  as well as clonal type I/II-specific peptide reactions . In contrast, reactions against peptides with clonal type I- or III-specific sequences were underrepresented  in cats infected with clonal type II T. gondii were observed in peptides with type I/III-specific aa sequences, followed by the CN-MSIVs observed in the group of clonal type III-specific peptides . The significantly highest CN-MSIVs of a serum from a cat infected with clonal type III  [A]) as compared to peptides with a homologous aa sequence specificity. Four peptides with heterologous specificity  were also recognized by the serum. However, the MSIVs recorded for these peptides were markedly lower ( [B]). Since only one reference serum specific for clonal III was available, it was not possible to establish a statistically significant log-linear model for the prevalence of positive or negative peptide reactions. Nevertheless, clonal type III- as well as I/III-specific positive peptide reactions were quantitatively overrepresented , whereas positive reactions from clonal type I-, II- and I/II-specific peptides were underrepresented (T. gondii seropositive cats from Germany (n=86) showed the strongest reactions (i.e. highest CN-MSIVs) with peptides of aa sequences homologous to clonal type II (II and I/II) as compared to reactions with peptides of clonal type I- or III-specific aa sequences . The highest CN-MSIVs were observed for peptide SAG2A-II-93 followed by SAG2A-II-135 and GRA7-II-225, which were both also recognized by significantly higher positive CN-MSVIs as compared to most remaining peptides, including GRA5-III-34 and GRA7-I/II-193 and GRA6-I/III-220  , followed by SAG2A-II-135 (n=60 [69.7%]), GRA7-II-225  and SAG2A-II-131 (SAG2A-II-93 was the peptide recognized by the highest number of [30.2%]) S4. [B]), whereas positive clonal type I- or III-specific peptide reactions were significantly underrepresented .Log-linear model analysis revealed that reactions with peptides displaying clonal type II-specific aa sequences  were statistically significantly overrepresented  . Peptides GRA3-I/III-28 and SAG2A-I/III-131 were recognized with the highest mean CN-MSIVs. Lower CN-MSIVs were observed in SAG2A-II-131, GRA3-I/III-189, GRA5-I-41 and GRA6-I/III-220; however, the differences in reaction intensities were not statistically significant when compared with those against GRA3-I/III-28 and SAG2A-I/III-131. The remaining peptides were recognized in average by significantly lower index values  , while 6 of 7 sera (86%) detected SAG2A-I/III-131and GRA3-I/III-28 S4.  [B]).Log-linear analysis of positive and negative peptide reactions among clonal type-specific peptides revealed that reactions with peptides containing sequences specific for both, type I and III, were statistically significantly overrepresented , whereas clonal type I- and III-specific peptide reactions were underrepresented . No significant differences were observed in the frequency of positive and negative reactions among clonal type II- and I/II-specific peptide cohorts   was performed.  [A], where sera from five cat groups  are clustered according to their similarity in peptide reactivity.The Y-map of SKN is presented in the T. gondii , clonal type I  or with natural infection  . Group N  consisted of six nodes (1-3 and 6-8) with 85 (98.8%) sera of naturally infected cats and of all three sera from cats infected with T. gondii type II. Three sera from cats infected with T. gondii clonal type I and two sera collected from cats infected by non-canonical T. gondii fell also into this group. The second group I  consisted of a single grid node (5) with 11 type-I-sera and one serum from a naturally infected cat. The third group A  consisted also of a single grid node (4) with five sera from cats infected with non-canonical T. gondii strains, three sera from type I-infected cats and one serum from a type I infected cat.XYF-SKN classification analysis of serum-peptide reactions predicted three cat groups consisting mainly of animals infected with non-canonical and clonal type III 5) ( [C]). Two of the remaining type I sera were grouped together with 15 sera obtained from naturally seropositive cats into grid node 1 . The remaining type I serum and four sera from naturally seropositive cats were placed in grid node 2 .Almost all sera from cats infected by clonal types I (n=11) and III (n=1) clustered in one unit (grid node 3 together with 8.1% (n=7) of the sera obtained from naturally seropositive cats sampled in Germany . The second type II-specific serum was grouped with 36.04% (n=31) of the sera obtained from naturally-seropositive cats sampled in Germany and two sera from cats infected with non-canonical T. gondii in grid node 6 . The third type II serum was sorted together with 27.9% (n=24) of T. gondii-positive cat field sera into grid node 7 .The serum of a type II infected cat clustered in grid node T. gondii clustered separately in grid node 4 together with one serum of a clonal type III-infected cat and three sera from cats infected with T. gondii clonal type I . The remaining 5 of 7 sera collected from cats infected with non-canonical 8 only sera from naturally-seropositive cats (n=4) were present .In the grid node T. gondii is partially specific for the clonal type of the parasite [T. gondii in infected humans and animals [T. gondii and serotyping in cats, although the cat plays a major role as an important definitive host in the epidemiology of the T. gondii infection. Several studies conducted with human and murine sera have shown that the antibody response against parasite ,31. Base animals ,31,43,73T. gondii infection is a prerequisite for developing serotyping tools. Since sera from humans with a known clonal type of T. gondii infection are very rare, sera from infected mice were used to evaluate peptides that were possibly suitable for serotyping with human sera [T. gondii infections. The availability of sera from individuals with a known clonal type of man sera . FortunaT. gondii infection in individual cats. Since seroconversion to T. gondii is usually permanent, typing is not only possible in cats actually shedding oocysts  but alsT. gondii polymorphic peptides, i.e. peptides with clonal type specific aa sequences to identify molecules suitable for serotyping of T. gondii in cats. Sera from experimentally infected cats or cats, for which the type of the infecting T. gondii had been determined, as well as seronegative cats were used to select appropriate peptides, i.e. peptides with optimal specificity and sensitivity. From these 101 peptides, 54 had previously been characterized for T. gondii serotyping in humans [In the present study, we analyzed a total of 101 n humans . Twenty-n humans -77. T. gondii serotyping in humans and mice [T. gondii. In addition, these results show that resources available in both, human and veterinary medicine could be commonly used to improve sensitivity and specificity of serological tests as e.g. tests for serotyping. However, a large number of peptides showed a low clonal type-specificity in cats – although their aa sequences had indicated clonal type specificity – and were therefore not included into the final serotyping peptide panel. Similar results were obtained when peptides with polymorphic epitopes were validated with mouse sera [Interestingly, we found that peptides previously described as suitable for and mice ,30,78 couse sera . The lowuse sera .T. gondii infection in cats using sera obtained from these animals. Our experiments led to the detection of 24 peptides deemed suitable for typing canonical clonal types of T. gondii types the cats were infected with. We thus believe that this panel of polymorphic, clonal type-specific peptides can be used for epidemiological studies in cats in areas, where canonical types of T. gondii prevail, i.e. in Europe and North America. We applied the peptide panel to determine the distribution of the clonal types of T. gondii infection among naturally seropositive cats from Europe based on DNA from oocysts from naturally infected cats [There was no reference serum from cats infected with canonical clonal types that failed to recognize peptides presenting epitopes with a homologous specificity for those ted cats ,21. GenoT. gondii type II-infection, i.e. were assorted together with sera from in a single group (N) but into 6 different nodes suggesting slight differences in which peptides were recognized. Four peptides with type II specific aa sequences recognized by field sera were also recognized by cats with a confirmed or experimental T. gondii type II infection. However, in case of the peptide SAG2A-II-93 the reactions in cats with confirmed or experimental type II infection were only close to the cut-off while field sera reacted strongly with SAG2A-II-93. Differences in the expression of SAG2A between field and laboratory strains used for experimental infection in the present study, differences in the stage of infection between field and laboratory cats or host genetic factors could have contributed to this observation. Therefore, based on the serotyping results it is not unlikely that the majority of naturally seropositive cats from Germany were infected with type II T. gondii, which is in accord with genotyping results obtained with DNA from oocysts shed by cats [T. gondii serotyping results performed with human sera from Germany [Serotyping results in naturally seropositive cats revealed that clonal type II-specific peptides were recognized by the significantly highest MSIVs as compared to peptides with other sequence specificities. Moreover, type II-specific serum-peptide reactions were significantly overrepresented as compared to reactions with the remaining peptide groups. Explorative data analysis using XYF-SKN prediction revealed that 98.8% of sera (85/86) obtained from naturally seropositive cats clustered in one group and recognized similar peptide patterns as cats with a known  by cats ,21 and w Germany . T. gondii types (n=2) were assorted to these nodes. In addition, a few sera from cats experimentally infected with type I (3/16), type III (n=1/1) and atypical (5/7) strains clustered together in a single node in the XYF-SKN prediction, i.e. could not clearly separated from each other by serotyping. These results show the limitations of serotyping. As documented in Although in the XYF-SKN analysis naturally seropositive cats were mainly assorted to nodes containing sera from type II-infected cats, also a few sera from cats infected with clonal types I (n=3) or cats infected with non-canonical T. gondii can occur in cats under natural conditions [T. gondii coexist. Since infection in cats seem to be clearly age-dependent [Previous genotyping results suggested that mixed or superinfections with more than one canonical type of nditions ,35, but ependent , limitinT. gondii types from Africa and South America by using peptides derived from clonal type II- and I/III-specific polymorphic GRA6 protein regions. Most of these human sera recognized mainly a clonal type I/III GRA6 peptide [T. gondii from Brazil  and North America (TgBbUS1 and TgGoatUS6). Interestingly, these sera also recognized clonal type I/III-specific peptides with the significantly highest mean MSIVs and the number of positive type I/III peptides reactions were significantly overrepresented in these sera. Strong reactions with individual peptides specific for clonal types I, II or III were also observed. The strong IgG response against type II-, or type I/III-specific peptides in cats infected with non-canonical T. gondii may indicate that especially proteins with epitopes common in type II, or I/III are expressed by these non-canonical strains. Since sequences of proteins from these atypical T. gondii isolates are not available, it was not possible to test this hypothesis. Although cats infected with atypical T. gondii reacted strongly with peptides specific at the same time for type I and III (i.e. type I/III peptides) most of these cats showed peptide patterns different from cats infected with type I and type II  may cause an antibody response reactive with single peptides containing epitopes specific for canonical types of T. gondii. Therefore, serotyping results have to be analyzed with care to avoid misinterpretation. We therefore propose serotyping as an epidemiological tool for typing in areas where canonical T. gondii-infections prevail, but not as a tool for typing individual cats. Sousa et al. (2008)  reported peptide . In the  type II 6. This T. gondii clonal type and identified 24 peptides suitable for T. gondii serotyping in cats. Our results suggest that most T. gondii seropositive cats in Germany are infected with type II T. gondii. This finding is in accord with previous findings reporting a predominance of type II T. gondii in oocysts shed by cats in Germany [T. gondii serotyping in humans from Germany, which also suggested that type II infections prevailed [T. gondii serotyping in cats. In conclusion, in the present study we showed that cats mount an antibody response that is specific for the  Germany  and withrevailed . FurtherTable S1Toxoplasma gondii antigens.Polymorphic peptides based on protein sequences available for (XLS)Click here for additional data file.Table S2Toxoplasma gondii.Cat reference sera used to evaluate and select peptides for the serotyping of (XLS)Click here for additional data file.Table S3Corrected mean sample index values  listed for all peptides and sera.(XLS)Click here for additional data file.Table S4Diagnostic parameters of peptides used in this study.(XLS)Click here for additional data file.


In this study, we used a model polymicrobial infection including the opportunistic pathogen Aggregatibacter actinomycetemcomitans and the commensal Streptococcus gordonii to examine the importance of metabolite cross-feeding for establishing co-culture infections. Our results reveal that co-culture with S. gordonii enhances the pathogenesis of A. actinomycetemcomitans in a murine abscess model of infection. Interestingly, the ability of A. actinomycetemcomitans to utilize L-lactate as an energy source is essential for these co-culture benefits. Surprisingly, inactivation of L-lactate catabolism had no impact on mono-culture growth in vitro and in vivo suggesting that A. actinomycetemcomitans L-lactate catabolism is only critical for establishing co-culture infections. These results demonstrate that metabolite cross-feeding is critical for A. actinomycetemcomitans to persist in a polymicrobial infection with S. gordonii supporting the idea that the metabolic properties of commensal bacteria alter the course of pathogenesis in polymicrobial communities.Microbes within polymicrobial infections often display synergistic interactions resulting in enhanced pathogenesis; however, the molecular mechanisms governing these interactions are not well understood. Development of model systems that allow detailed mechanistic studies of polymicrobial synergy is a critical step towards a comprehensive understanding of these infections  Aggregatibacter actinomycetemcomitans and the common oral commensal Streptococcus gordonii to assess the impact of polymicrobial growth on pathogenesis. We found that the abilities of A. actinomycetemcomitans to persist and cause disease are enhanced during co-culture with S. gordonii. Remarkably, this enhanced persistence requires A. actinomycetemcomitans catabolism of L-lactate, the primary metabolite produced by S. gordonii. These data demonstrate that during co-culture growth, S. gordonii provides a carbon source for A. actinomycetemcomitans that is necessary for establishing a robust polymicrobial infection. This study also demonstrates that virulence of an opportunistic pathogen is impacted by members of the commensal flora.Many bacterial infections are not the result of colonization and persistence of a single pathogenic microbe in an infection site but instead the result of colonization by several. Although the importance of polymicrobial interactions and pathogenesis has been noted by many prominent microbiologists including Louis Pasteur, most studies of pathogenic microbes have focused on single organism infections. One of the primary reasons for this oversight is the lack of robust model systems for studying bacterial interactions in an infection site. Here, we use a model co-culture system composed of the opportunistic oral pathogen  Invading pathogens must also obtain carbon and energy from an infection site, and specific carbon sources are required for several pathogens to colonize and persist in the host Aggregatibacter actinomycetemcomitans and the common commensal Streptococcus gordonii to provide mechanistic insight into how specific carbon sources impact disease pathogenesis in polymicrobial infections The survival of pathogens in the human body has been rigorously studied for well over a century. The ability of bacteria to colonize, persist and thrive A. actinomycetemcomitans is a Gram-negative facultative anaerobic bacterium that inhabits the human oral cavity and is a proposed causative agent of localized aggressive periodontitis A. actinomycetemcomitans is found between the gums and tooth surface in the subgingival crevice 2 depending on tissue depth A. actinomycetemcomitans is able to catabolize A. actinomycetemcomitans has been proposed to primarily inhabit the aerobic A. actinomycetemcomitans, the subgingival crevice is home to a diverse bacterial population, including numerous oral streptococci Streptococcus mutans, are typically non-pathogenic and depending upon the human subject and method of sampling, comprise approximately 5% 2O2, acetate, and CO2, oral streptococci such as S. gordonii have been shown to influence the composition of oral biofilms S. gordonii-produced H2O2 influences interactions between A. actinomycetemcomitans and the host by inducing production of ApiA, a factor H binding protein that inhibits complement-mediated lysis In addition to A. actinomycetemcomitans preferentially catabolizes L-lactate over high energy carbon sources such as glucose and fructose in multiple strains, despite the fact that this bacterium grows more slowly with L-lactate A. actinomycetemcomitans resides in close association with oral streptococci in vivo benefit exists for A. actinomycetemcomitans L-lactate preference. To test this hypothesis, we investigated the importance of A. actinomycetemcomitans L-lactate catabolism during mono-culture and co-culture with S. gordonii in vitro and in a murine abscess model of infection. Our results reveal that co-culture with S. gordonii enhances colonization and pathogenesis of A. actinomycetemcomitans, and the ability to utilize L-lactate as an energy source is essential for these co-culture benefits. Surprisingly, inactivation of L-lactate catabolism had no impact on mono-culture growth in vitro and in vivo suggesting that A. actinomycetemcomitans L-lactate catabolism is only critical for establishing co-culture infections. Taken together, these results provide compelling mechanistic evidence that the metabolic properties of human commensals such as S. gordonii can alter the course of pathogenesis in polymicrobial communities.in vivo carbon sources for A. actinomycetemcomitans. However in contrast to glucose, L-lactate is also produced by the oral microbial flora, primarily oral streptococci A. actinomycetemcomitans to catabolize streptococcal-produced L-lactate has been demonstrated previously A. actinomycetemcomitans consumes streptococcal-produced L-lactate during co-culture. To assess the importance of A. actinomycetemcomitans L-lactate catabolism in polymicrobial communities in vitro, we examined the metabolic profile during catabolism of L-lactate and glucose under aerobic and anaerobic conditions. Aerobically, A. actinomycetemcomitans primarily produced lactate and acetate from glucose  than the wt (1.9 hr) and cell suspensions produced a metabolite profile that differed from the wt  and produced similar metabolites as the wt  indicating that S. gordonii does not receive a benefit, at least in regard to cell number, from co-culture with A. actinomycetemcomitans. As a control, in vivo growth of the A. actinomycetemcomitans apiA mutant, which is hypersusceptible to killing by innate immunity, was examined. As expected, the apiA mutant exhibited a >250-fold decrease in mono-culture in vivo survival, which was unchanged in the presence of S. gordonii .A. actinomycetemcomitans strains VT1169 Streptococcus gordonii strain Challis DL1.1 (ATCC 49818), S. gordonii spxB-Escherichia coli DH5α-λpir, and E. coli SM10-λpir were used in this study. A. actinomycetemcomitans and S. gordonii were routinely cultured using Tryptic Soy Broth + 0.5% Yeast Extract (TSBYE). For resting cell suspension A. actinomycetemcomitans metabolite analysis, a Chemically Defined Medium (CDM) 2 atmosphere shaking at 165 RPM, and anaerobic culture conditions were static growth at 37°C in an anaerobic chamber  with a 5% H2, 10% CO2 and 85% N2 atmosphere. E. coli strains were grown on Luria-Bertani (LB) medium at 37°C. Where applicable, antibiotics were used at the following concentrations: chloramphenicol, 2 µg/ml for A. actinomycetemcomitans and 20 µg/ml for E. coli; spectinomycin, 50 µg/ml for selection and 10 µg/ml for maintenance for A. actinomycetemcomitans and E. coli and 100 µg/ml for selection and maintenance for S. gordonii spxB-; kanamycin, 40 µg/ml for selection and 10 µg/ml for maintenance; naladixic acid, 25 µg/ml; streptomycin, 50 µg/ml for selection and 20 µg/ml for maintenance. For quantifying CFU/ml in co-culture assays, vancomycin (5 µg/ml) was added to agar plates to enumerate A. actinomycetemcomitans and streptomycin (100 µg/ml) was added to agar plates to enumerate S. gordonii.A. actinomycetemcomitans was isolated using DNeasy tissue kits (Qiagen), and plasmid isolations were performed using QIAprep spin miniprep kits (Qiagen). DNA fragments were purified using QIAquick mini-elute PCR purification kits (Qiagen), and PCR was performed using the Expand Long Template PCR system (Roche). DNA sequencing was performed by automated sequencing technology using the University of Texas Institute for Cell and Molecular Biology sequencing core facility.DNA and plasmid isolations were performed using standard methods apiA (AA2485) was carried out by double homologous recombination. For construction of the knockout construct, 856 bp and 842 bp DNA fragments flanking apiA were amplified and combined with the aphA gene (encoding kanamycin resistance) from pBBR1-MCS2 aphA was positioned between the upstream and downstream regions. Primers used were: Kan-5′ (ATGTCAGCTACTGGGCTATCTG) and Kan-3′ (ATTTCGAACCCCAGAGTCCCGC) for the 1074 bp aphA-containing fragment; ApiA-UF (CCGATAACAGTAAGATCTTCTAC) and ApiA-UR (CAGATAGCCCAGTAGCTGACATCCTTTTCGGCTTGAATTTATACC) for the upstream apiA fragment; and ApiA-DF (GCGGGACTCTGGGGTTCGAAATGCGGTCAGAATTTTAGGTGTTTT) and ApiA-DR (CGAAACCAACGAACTCTTTATTC) for the downstream apiA fragment. Underlined sequences indicate overlapping DNA sequences between the apiA fragments and aphA. The overlap extension product was TA-cloned into the pGEM-T Easy vector  and excised by EcoRI digest. The EcoRI fragment containing the overlap extension product was ligated into the unique EcoRI site within the λpir-dependent suicide vector pVT1461 E. coli DH5α-λpir then into E. coli SM10-λpir for conjugation into A. actinomycetemcomitans. Conjugation was performed as described A. actinomycetemcomitans and nalidixic acid to kill the E. coli donors. Kanamycin resistant, spectinomycin sensitive double recombinants were selected and verified by PCR. Enhanced susceptibility of the apiA mutant to serum was verified as described previously Allelic replacement of cydB gene was performed by single homologous recombination using a 543 bp internal piece of the cydB (AA2840) gene amplified using the primers cydB-KO5′ (AGATCTTTATGATTAATACTATCGCGCCGGA) and cydB-KO3′ (AGATCTCAAAACCATCTTTGAAAGATAACCAGA). Underlined sequences represent BglII restriction sites. The internal cydB fragment was digested with BglII and ligated into the A. actinomycetemcomitans suicide vector pMRKO-1 (see below) to generate pMRKO-cydB. pMRKO-cydB was transformed into E. coli SM10-λpir and conjugated into A. actinomycetemcomitans. A. actinomycetemcomitans recombinants were grown anaerobically on TSBYE agar containing spectinomycin and naladixic acid. Colonies were subcultured anaerobically on liquid medium at the same antibiotic concentrations and insertion into cydB was verified by PCR.Insertional mutagenesis of the GCGGCCGCCGATTTTCGTTCGTGAATACATGATAAGAAT) and 3′ Spec-cass-EcoRI (GAATTCCATATGCAAGGGTTTATTGTTTCG), digested with NotI-EcoRI and ligated into NotI-EcoRI digested pmCherry (Clontech) underlined sequences indicate NotI and EcoRI restriction sites. The 3105 bp region containing the pUC origin of replication, plac:mCherry and the spectinomycin resistance gene were PCR amplified using the primers: 5′pMcher-trunc (AGATCTGACCAAGTTTACTCATATATACTGA) and 3′ Spec-cass-EcoRI (GAATTCCATATGCAAGGGTTTATTGTTTCG). Underlined sequences indicate BglII and EcoRI restriction sites. This fragment was digested with BglII and EcoRI and ligated into the 2780 bp fragment from BglII-EcoRI digested pVT1461. The resulting plasmid  is a suicide vector for A. actinomycetemcomitans and contains oriT, mob, and tra genes from pVT1461 along with the pUC origin of replication, mCherry expressed from plac, and a spectinomycin resistance cassette.The spectinomycin resistance gene from pDMG4 A. actinomycetemcomitans was grown in CDM overnight either aerobically or anaerobically in the presence of 20 mM glucose or 40 mM L-lactate. Bacteria were then subcultured in 30 ml of medium and exponential phase cells (OD600 = 0.4) were collected by centrifugation  at 25°C. Cell pellets were resuspended in an equal volume of CDM lacking nucleotides, amino acids and any carbon source. Cells were incubated at 37°C aerobically or anaerobically depending on the test conditions for 1 hr. Cells were collected again by centrifugation as described above and resuspended to an OD600 of 2 in 3 ml of CDM without nucleotides, amino acids, pimelate and thioctic acid containing either 20 mM glucose or 40 mM lactate. Cells were incubated for 4 h at 37°C either aerobically or anaerobically. After incubation samples were stored at −20°C for HPLC analysis.D-lactate assays were performed as described A. actinomycetemcomitans and S. gordonii were grown overnight in CDM containing 3 mM glucose. 3 mM glucose was used to ensure that the medium was limited for catabolizable carbon. Cells were diluted 1∶50 in the same medium and allowed to grow to exponential phase (OD600 of 0.2). Cells were then diluted 1∶100 (2×106S. gordonii/ml and 1×107A. actinomycetemcomitans/ml) as mono-cultures or co-cultures in 3 ml CDM containing 3 mM glucose. Cultures were allowed to grow for 10 h aerobically or 12 h anaerobically, after which cells were serially diluted, plated on either TSBYE agar + vancomycin for A. actinomycetemcomitans enumeration or TSBYE agar + streptomycin for S. gordonii enumeration. Colonies were counted after incubation at 37°C for 48 h. An aliquot of the culture was also stored at −20°C for HPLC metabolite analysis.2SO4 elution buffer and a flow rate of 0.5 ml/minute. A Varian refractive index (RI) detector at 35°C was used for metabolite enumeration by comparison with acetate, ethanol, formate, glucose, L-lactate, D-lactate, pyruvate and succinate standards.Metabolite levels were quantified using a Varian HPLC with a Varian Metacarb 87H 300×6.5 mm column at 35°C. Samples were eluted using isocratic conditions with 0.025 N H7 CFU A. actinomycetemcomitans, S. gordonii or both. At 6 days post- infection, mice were euthanized and intact abscesses were harvested, weighed and placed into 2 ml of sterile PBS (or water for pH measurements). Tissues were homogenized, serially diluted and plated on Brain Heart Infusion (BHI) agar + 20 µg/ml Na2CO3 + vancomycin for A. actinomycetemcomitans enumeration or BHI agar + 20 µg/ml Na2CO3 + streptomycin for S. gordonii enumeration, to determine bacterial CFU/abscess. Experimental protocols involving mice were examined and approved by the Texas Tech University HSC Institutional Animal Care and Use Committee.Murine abscesses were generated essentially as described previously Text S1S. gordonii in mono- or co-culture with A. actinomycetemcomitans or A. actinomycetemcomitans lctD- in aerobic and anaerobic co-cultures. Strains were grown as mono- or co-cultures in 3 mM glucose aerobically or anaerobically for 10 or 12 h respectively, serially diluted, and plated on selective media to determine colony forming units per ml (CFU/ml). S. gordonii mono-cultures numbers are represented by black bars, co-culture numbers with A. actinomycetemcomitans are represented by white bars, and co-culture numbers with A. actinomycetemcomitans lctD- are represented by grey bars. Error bars represent 1 standard error of the mean, n = 3. Figure S2: Survival of S. gordonii and S. gordonii spxB- in a murine abscess model. A. Number of bacteria recovered from each abscess expressed as colony forming units per abscess (CFU/abscess). Wilcoxon signed-rank test value, p<0.03. B. Abscess weights 6 days post-inoculation. Error bars represent 1 standard error of the mean, n = 4.Figure S1: Growth of (DOC)Click here for additional data file.