(1995) J. The position of the peptides in the D3 domain is also indicated. represent the mean S.E. of at least three experiments. is usually a common and important human bacterial pathogen causing a wide spectrum of diseases ranging from uncomplicated throat and skin infections to life-threatening invasive conditions, such as necrotizing fasciitis, septicaemia, and toxic shock syndrome (13,C15). Based on antigenic differences in the M protein, a surface protein of gene shows a high degree of variation among different M1 strains, and nearly 300 alleles are known (25). SIC also binds to and interferes with the activity of several antibacterial proteins and peptides such as lysozyme, LL-37, members of the defensin family, and the chemokine MIG/CXCL9 (26,C29). Recently, SIC was also shown to inhibit an antibacterial peptide produced by by promoting growth in human plasma and bacterial dissemination in a mouse model of sepsis. EXPERIMENTAL PROCEDURES Bacteria, Growth Conditions, Plasma Sources, Analysis of Bacterial Multiplication, and SIC Production during Growth The strain AP1 (40/58), was from the World Health Business Collaborating Centre for Reference and Research on Streptococci (Prague, Czech Republic). ZT-12-037-01 The mutant strain SIC? has been described previously (29). Bacteria were cultivated in Todd-Hewitt broth (TH; Difco) at 37 C. Fresh frozen plasma from healthy individuals was obtained from the blood lender at Lund University Hospital (Lund, Sweden) and kept at ?80 C until use. Human plasma depleted of HK, PK, FXII, or FXI, respectively, was purchased from George King BioMedical, Inc. (Overland Park, KS). Bacterial cultivation in plasma was performed as follows. 10 l of an overnight bacterial culture in TH was added to 250 l plasma, and bacteria were left to grow at 37 C. At various time points, including time 0, growth was monitored by plating appropriate dilutions of the bacterial answer on TH agar plates. Plates were incubated overnight at 37 C, and the number of cfu were decided. The multiplication factor (MF) was calculated by dividing the number of cfu at the individual time points with the number of cfu at time point zero. Bacteria produced for 8 h in plasma (undiluted or diluted 1:1 in TH) were collected by centrifugation. Plasma supernatants were analyzed for SIC content by ELISA. Supernatants were also precipitated with 5% TCA for 30 min on ice followed by ZT-12-037-01 centrifugation at 15,000 (4 C for 20 ZT-12-037-01 min). Precipitated material was dissolved in SDS sample buffer and subjected to SDS-PAGE and Western blot analysis. The bacterial cells were washed with PBS, and bound proteins were eluted with 0.1 m glycine-HCl, pH 2.0. The pH of the eluted material was raised to 7.5 with the addition of 1 m Tris. Eluted proteins were TCA-precipitated and analyzed with SDS-PAGE and Western blot. Proteins, Antibodies, and Iodination Human HK was purchased from Kordia. The synthetic peptides based on sequences in domain name D3 of HK were described previously (31) and ZT-12-037-01 are shown in Fig. 2strain AP1 as described (24). Polyclonal antisera to protein SIC and NAT26 were raised in rabbits. HRP-conjugated goat anti-rabbit IgG was purchased from Pierce, and HRP-conjugated protein A was from Sigma. FITC-conjugated anti-rabbit IgG F(ab)2 was purchased from Sigma, and anti-NAT26 F(ab)2 fragments were prepared as described (9). Polyclonal anti-SIC IgG was affinity-purified using protein A-Sepharose (GE Healthcare), and the FabRICATOR? Kit (Genovis) was then utilized for preparation and purification of F(ab)2 antibody fragments. Proteins were radiolabeled with 125I using Rabbit polyclonal to SirT2.The silent information regulator (SIR2) family of genes are highly conserved from prokaryotes toeukaryotes and are involved in diverse processes, including transcriptional regulation, cell cycleprogression, DNA-damage repair and aging. In S. cerevisiae, Sir2p deacetylates histones in aNAD-dependent manner, which regulates silencing at the telomeric, rDNA and silent mating-typeloci. Sir2p is the founding member of a large family, designated sirtuins, which contain a conservedcatalytic domain. The human homologs, which include SIRT1-7, are divided into four mainbranches: SIRT1-3 are class I, SIRT4 is class II, SIRT5 is class III and SIRT6-7 are class IV. SIRTproteins may function via mono-ADP-ribosylation of proteins. SIRT2 contains a 323 amino acidcatalytic core domain with a NAD-binding domain and a large groove which is the likely site ofcatalysis iodobeads (Pierce) as described by the manufacturer. Binding of radiolabeled protein to bacteria was performed as described (32). ELISA Microtiter plates (Maxisorb, NUNC, Denmark) were coated with peptides spanning the HK domain name D3 (0.5 g/ml). After washing with PBST (PBS made up of 0.05% Tween 20), the plates were incubated with protein.