Journal: mBio

Article Title: Increased Production of Outer Membrane Vesicles by Salmonella Interferes with Complement-Mediated Innate Immune Attack

doi: 10.1128/mBio.00869-21

Figure Lengend Snippet: Serum resistance assay for Salmonella mutants grown under PhoPQ-activating conditions. (A) Survival of Salmonella deletion mutants in 25% NHS incubated at 37°C for 1 h. Deletion of Δ pgtE , Δ rck (used as positive controls), Δ phoP , and Δ pagC made the bacteria susceptible to complement-mediated killing. (B) A comparison of serum resistance between Salmonella Δ pagC , Δ rck , and Δ ompX single, double, and triple mutants. (C) Quantitative RT-PCR-based analysis of gene expression ratio of pagC , rck , and ompX (relative to housekeeping gene rpoB ) in wild-type Salmonella grown in LB or under PhoPQ-activating conditions (5.8L) showed the expression of pagC increased significantly under PhoPQ-activating conditions. (D) Resistance to serum-mediated attack was restored in single Salmonella mutants complemented with plasmids induced to express PagC, Rck, and OmpX in the corresponding mutants. Plasmids expressing Rck or PagC complemented best the triple mutant compared with mutants containing empty vector (pRS1). The data represent one of three separate, reproducible experiments, expressed as mean ± SEM. Statistical significance was calculated using one-way ANOVA multiple-comparison test and set at a P value of <0.05 (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; and ns, P > 0.05).

Article Snippet: For analyzing the sensitivity of Salmonella to serum-mediated killing, bacteria (∼1 × 10 5 CFU grown in 5.8L N-minimal media (optical density at 600 nm [OD 600 ], 0.5) were incubated with 25% normal human serum (NHS) (Astarte Biologics, Bothell, WA, USA), C3/C4-depleted human serum (Complement Technology Inc., TX, USA), or with normal mouse serum and Factor H mutant serum ( ) for 1 hour at 37°C.

Techniques: Incubation, Bacteria, Comparison, Quantitative RT-PCR, Gene Expression, Expressing, Mutagenesis, Plasmid Preparation

Journal: mBio

Article Title: Increased Production of Outer Membrane Vesicles by Salmonella Interferes with Complement-Mediated Innate Immune Attack

doi: 10.1128/mBio.00869-21

Figure Lengend Snippet: Serum protection assay by OMV produced under PhoPQ-activating conditions. (A) Survival of WT and Δ pagC Salmonella incubated with 25% NHS in the presence of sterile culture medium, OMV-depleted culture supernatants, or OMV-containing culture supernatants showed that bacteria incubated with OMV-containing culture supernatants from PagC-expressing bacteria (WT, Δ pagC + p pagC , and PhoP C ) were significantly more resistant to serum-mediated attack than bacteria incubated with sterile culture medium or culture supernatant from the Δ pagC mutant. Bacterial survival in sterile culture medium was comparable to the survival in OMV-depleted spent culture supernatants. (B) Survival of WT and Δ pagC Salmonella incubated with 25% NHS in the presence of purified OMVs at indicated concentrations (0 to 10 5 OMVs), isolated from bacteria grown under PhoPQ-activating (5.8L; solid line) or nonactivating (7.6H; dashed line) conditions. OMVs from bacteria expressing PagC (WT) grown under PhoPQ-activating conditions were more protective than OMVs from bacteria grown under nonactivating conditions. The data represent one of three separate, reproducible experiments, expressed as mean ± SEM. Statistical significance was calculated using Student’s t test and set at a P value of <0.05 (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; and ns, P > 0.05).

Article Snippet: For analyzing the sensitivity of Salmonella to serum-mediated killing, bacteria (∼1 × 10 5 CFU grown in 5.8L N-minimal media (optical density at 600 nm [OD 600 ], 0.5) were incubated with 25% normal human serum (NHS) (Astarte Biologics, Bothell, WA, USA), C3/C4-depleted human serum (Complement Technology Inc., TX, USA), or with normal mouse serum and Factor H mutant serum ( ) for 1 hour at 37°C.

Techniques: Produced, Incubation, Sterility, Bacteria, Expressing, Mutagenesis, Purification, Isolation

Journal: mBio

Article Title: Increased Production of Outer Membrane Vesicles by Salmonella Interferes with Complement-Mediated Innate Immune Attack

doi: 10.1128/mBio.00869-21

Figure Lengend Snippet: Blocking of complement activation by OMVs from PagC-expressing bacteria. (A) Survival of WT Salmonella incubated with 25% NHS treated with 5 mM EGTA or EDTA, in the presence or absence of 1 × 10 9 purified OMVs from WT or Δ pagC mutant. OMVs from PagC-expressing WT bacteria block the alternative pathway of the complement more efficiently than the OMVs from the Δ pagC mutant. Statistical significance was calculated using Student’s t test. (B) Schematic representation of C3 and its cleavage products C3b and iC3b (molecular weight [MW] of the α and β chain and the cleaved or uncleaved products is indicated in the brackets). (C) WT OMVs (10 8 to 10 10 OMVs) incubated with 5% NHS, when immunoblotted with anti-C3 antibody, showed bands for C3b inactivation at 68 kDa (iC3bα′) and 46 kDa (iC3bα″) in OMV pellet and supernatant fractions, respectively. No C3b inactivation bands were found when Δ pagC OMVs were incubated with NHS, suggesting that WT OMVs bind to C3b and inactivate C3b into iC3b in a dose-dependent manner. The Western blot image was spliced to remove an empty lane in between pellet and supernatant fractions. The panel on the right represents relative protein band densities (WT versus Δ pagC OMVs) as analyzed by NIH ImageJ software from 3 separate, reproducible experiments, expressed as mean ± SEM. Statistical significance was calculated using Student’s t test. (D) WT or Δ pagC mutant incubated with C3/C4-depleted human serum (gray bars) survived significantly better than the bacteria incubated with NHS (black bars). Presence of OMV-containing culture supernatant (+ OMV) contributed to bacterial survival when incubated with NHS, compared with bacteria incubated in the presence of OMV-depleted culture supernatants (− OMV). (E) Incubation of the Δ pagC Δ rck Δ ompX Δ pgtE mutant with NHS in the presence of OMV-containing culture supernatant prevented C3b deposition on bacterial cell surfaces. OMVs from the quadruple mutant expressing PagC prevented C3 deposition most significantly. The bar graph below the blot represents relative protein band densities as analyzed by NIH ImageJ software from 3 separate, reproducible experiments, expressed as mean ± SEM. For iC3bα′, relative protein densities were calculated for bacteria incubated in sterile culture medium (−) with no OMVs versus bacteria incubated with OMV-containing spent culture supernatant. Statistical significance was calculated using the one-way ANOVA multiple-comparison test. The data represent one of three separate, reproducible experiments, expressed as mean ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; and ns, P > 0.05).

Article Snippet: For analyzing the sensitivity of Salmonella to serum-mediated killing, bacteria (∼1 × 10 5 CFU grown in 5.8L N-minimal media (optical density at 600 nm [OD 600 ], 0.5) were incubated with 25% normal human serum (NHS) (Astarte Biologics, Bothell, WA, USA), C3/C4-depleted human serum (Complement Technology Inc., TX, USA), or with normal mouse serum and Factor H mutant serum ( ) for 1 hour at 37°C.

Techniques: Blocking Assay, Activation Assay, Expressing, Bacteria, Incubation, Purification, Mutagenesis, Molecular Weight, Western Blot, Software, Sterility, Comparison

Journal: Blood Advances

Article Title: A fully recombinant human IgG1 Fc multimer (GL-2045) inhibits complement-mediated cytotoxicity and induces iC3b

doi: 10.1182/bloodadvances.2016001917

Figure Lengend Snippet: GL-2045 sequesters C1q and prevents deposition of C1q, C4b, and MAC on the surface of Ab-opsonized cells. (A) The ability of testing compounds binding to plate-coated C1q was measured by ELISA. (B) GL-2045 blocks RTX-mediated C1q, C4b, and MAC deposition on Ramos cells in a CDC assay. GL-2045, HAGG, or IVIG was incubated in NHS and added to RTX-opsonized Ramos cells. Incubations were carried out for 15 minutes for C1q and C4b deposition or 30 minutes for MAC formation. The cells were then stained with FITC-conjugated anti-C1q, anti-C4b, or anti-C5b-9 mAbs to examine C1q, C4b, or MAC deposition. Data are shown as percent of C1q-, C4b-, or MAC-positive cells within the total cell population. (C) GL-2045 inhibits purified C1q deposition on RTX-opsonized cells. GL-2045, HAGG, or IVIG was preincubated with purified C1q (20 μg/mL) for 10 minutes and the resultant mixture was incubated with RTX-opsonized SUDHL4 cells at 37°C for 15 minutes. C1q deposition was detected with FITC anti-C1q Ab and evaluated by flow cytometry. Data are shown as percentage of C1q-positive cells within the total cell population. (D) GL-2045 inhibits the activation of the classical complement pathway in a plate-based assay. HAGG was coated on a plate to activate the classical pathway. Test compounds were added to the wells and incubated with 1.5% NHS at 37°C for 30 minutes for C1q deposition or 1 hour for MAC deposition. C1q and MAC depositions were determined by ELISA. The results are shown as the least squares mean ± SE estimated by ANCOVA. Natural log variance stabilizing transformation and Tukey procedure for multiple comparisons adjustment were used to test the differences. *P < .05, **P < .01, ***P < .001 compared with no-treatment control.

Article Snippet: Normal human serum (NHS) and rituximab (RTX) were purchased from Cedarlane Labs (Burlington, NC) and from Genentech Inc, respectively.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, CDC Assay, Incubation, Staining, Purification, Flow Cytometry, Activation Assay, Transformation Assay, Control