N-acetylglucosaminylation of serine-aspartate repeat proteins promotes Staphylococcus aureus bloodstream infection.

Staphylococcus aureus secretes products that convert host fibrinogen to fibrin and promote its agglutination with fibrin fibrils, thereby shielding bacteria from immune defenses. The agglutination reaction involves ClfA (clumping factor A), a surface protein with serine-aspartate (SD) repeats that captures fibrin fibrils and fibrinogen. Pathogenic staphylococci express several different SD proteins that are modified by two glycosyltransferases, SdgA and SdgB. Here, we characterized three genes of S. aureus, aggA, aggB (sdgA), and aggC (sdgB), and show that aggA and aggC contribute to staphylococcal agglutination with fibrin fibrils in human plasma. We demonstrate that aggB (sdgA) and aggC (sdgB) are involved in GlcNAc modification of the ClfA SD repeats. However, only sdgB is essential for GlcNAc modification, and an sdgB mutant is defective in the pathogenesis of sepsis in mice. Thus, GlcNAc modification of proteins promotes S. aureus replication in the bloodstream of mammalian hosts.

Apolipoprotein A1 and C-terminal fragment of α-1 antichymotrypsin are candidate plasma biomarkers associated with acute renal allograft rejection.

BACKGROUND: Current diagnostic methods of renal allograft rejection are neither sensitive nor specific. Needle biopsies are invasive and associated with patient morbidity. Thus, it is desirable to develop noninvasive tests to predict and diagnose rejection. METHODS: Using a case-control approach, surface-enhanced laser desorption/ionization time-of-flight mass spectrometry was used to identify plasma proteins associated with renal allograft rejection. From each rejection patient (n=16), two plasma samples (one near the biopsy date and the other at a time postbiopsy) were compared. Biopsy-confirmed nonrejection patients (n=48) were further analyzed as controls. Antibody-based quantitative enzyme-linked immunosorbent assay was performed to validate candidate biomarker apolipoprotein A1 (Apo A1) in a subset of the original and a second cohort of biopsy-confirmed rejection (n=40) and nonrejection (n=70) patients. RESULTS: Twenty-two proteins/peptides showed significant differences between rejection and postrejection samples. Peptides 5191 Da and 4467 Da detected rejection with 100% sensitivity and 94% specificity. The 4467 Da peptide was identified as the C-terminal fragment of α-1 antichymotrypsin and a 28 kDa protein was determined as Apo A1. Both protein levels were significantly lower at rejection compared with postrejection. Protein levels of nonrejection patients were similar to the postrejection samples. Apo A1 enzyme-linked immunosorbent assay results showed significantly lower Apo A1 levels (P=0.001 for the original and P=4.14E-11 for the second cohort) at the time of rejection compared with nonrejection which coincides with the SELDI findings. CONCLUSIONS: Together α-1 antichymotrypsin, Apo A1, and the unidentified 5191 Da peptide provide a plasma molecular profile, and this is associated with acute cellular renal allograft rejection.

Proteomic analysis reveals innate immune activity in intestinal transplant dysfunction.

BACKGROUND: Many patients with intestinal failure require intestinal transplantation (ITx) to survive. Acute cellular rejection poses a challenge in ITx because its biologic components are incompletely understood. New methodologies for its integrative and longitudinal analysis are needed. METHODS: In this study, we characterized episodes of acute cellular rejection in ITx recipients using a noninvasive proteomic analysis. Ostomy effluent was obtained from all patients undergoing ITx at University of California, Los Angeles from July 2008 to September 2009 during surveillance endoscopies in the first 8 weeks post-ITx. Effluent was analyzed using 17-plex Luminex technology and matrix-assisted laser desorption/ionization proteomics. RESULTS: Of 56 ostomy effluent samples from 17 ITx recipients, 14% developed biopsy-proven rejection at a median of 25 days post-ITx. Six had mild rejection, two were indeterminate for rejection, and no graft loss was seen in the first 3 months posttransplantation. Effluent levels of five innate immune cytokines were elevated in the posttransplantation phase: granulocyte colony-stimulating factor, interleukin-8, tissue necrosis factor-α, interleukin-1ß, and interferon-γ. Proteomic analysis revealed 17 protein features differentially seen in rejection, two identified as human neutrophil peptide 1 and 2. This was confirmed by the presence of human neutrophil peptide-positive lamina propria neutrophils in biopsy tissue samples. CONCLUSIONS: Proteomic and cytokine analysis of ostomy effluents suggests an early and unappreciated role of innate immune activation during rejection.

ProteinChip array-based amyloid beta assays.

Amyloid-beta (A beta) fragments are found in plaques of patients with Alzheimers. Three secretases cleave the amyloid precursor protein, producing multiple A beta fragments that accumulate in the brain and fluids of patients with Alzheimers. A beta peptides are difficult to detect using standard methods because of their small size and multiple isoforms. However, multiple peptide fragments can be detected using a single ProteinChip Array-Based assay. Specific antibodies recognizing various amyloid epitopes are immobilized on a ProteinChip Array. Crude samples, such as tissue lysates, serum, cerebral spinal fluid (CSF), or cell culture media, are applied to the antibody-coated arrays. A beta peptides are specifically retained by the antibody, whereas other sample components are removed by washing. The multiple peptide fragments are detected by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), which can easily resolve the different fragments because of the corresponding changes in peptide mass.

DNA affinity capture and protein profiling by SELDI-TOF mass spectrometry: effect of DNA methylation.

We report here that surface enhanced laser desorption/ionization-time of flight (SELDI-TOF) mass spectrometry, as performed on a Ciphergen Biosystems ProteinChip System, can be used in conjunction with DNA affinity capture (DACA) to study specific DNA-protein binding. Using DNA molecules bound to a surface, sequence-specific interactions can be detected as demonstrated by a mutation affecting the binding profile for TBP, a transcription factor. Also, a comparison between methylated and unmethylated promoter-containing DNA fragments shows numerous binding profile differences over a mass range extending to >60 kDa. The binding of several proteins is inhibited by methylation of the DNA.