Kenji M. Cunnion

Capsule Production and Growth Phase Influence Binding of Complement to Staphylococcus aureus

ABSTRACT Complement-mediated opsonization of bacteria by C3 binding is an important component of the host innate immune system. Little information is available concerning the interaction between complement proteins and capsule type 5 and 8 Staphylococcus aureusstrains, even though these isolates are responsible for ∼70% of human staphylococcal infections. To investigate the importance of an intact complement pathway in an experimental staphylococcal infection, control and C3-depleted mice were challenged intravenously with 107 CFU of a serotype 5 S. aureus isolate. Whereas only 8% of the control mice succumbed to the infection, 64% of the complemented-depleted animals died. In vitro parameters of C3 binding to two heavily encapsulated (CP++) strains, three encapsulated (CP+) strains, and an isogenic capsule-negative (CP−) mutant were examined. The alternative pathway contributed 90% of C3 binding in 20% serum at 30 min, whereas it accounted for only 13% of C3 binding in 2% serum. Stationary-phase organisms bound only 10% as much C3 as mid-log-phase organisms; this was only in part due to capsule. When the S. aureus strains were cultivated on solid medium, the CP++ isolates bound 50% less C3 than CP+ strains; a CP+ strain bound 42% less C3 than the CP− mutant. Both C3b and iC3b fragments of C3 bound to S. aureus cells, and about one-third of the bound C3 was shed from the staphylococcal surface as iC3b, regardless of the CP phenotype of the strain. Thus, the phase of growth and presence of capsule are critical to opsonization.

Availability of Complement Bound to Staphylococcus aureus To Interact with Membrane Complement Receptors Influences Efficiency of Phagocytosis

ABSTRACT Complement-mediated opsonization of encapsulated Staphylococcus aureus (CP+) of the predominant capsule types, 5 and 8, remains poorly understood. Our previous work showed that complement is important for mouse survival of CP+ type 5 bacteremia and that the type 5 capsule inhibits the binding of opsonic C3 fragments to the organism. The importance of complement-mediated opsonization of CP+ was tested by neutrophil phagocytosis assays. Complement-mediated opsonization of CP+ increased phagocytosis by 57% compared to opsonization in complement-inhibited serum. Agar-grown CP+, enhancing capsule expression, was phagocytosed only one-tenth as well as the capsule-negative organisms (CP−), supporting the belief that staphylococcal polysaccharide capsules impair phagocytosis. Despite relatively poor phagocytosis of CP+ compared to CP−, complement activation increased the phagocytosis of CP+ by 103%. Thus, complement in normal human serum may have an important role in opsonizing CP+, even when capsule expression is strong. The ability of bound C3 fragments to interact with complement receptor 1 (CD35) on the membrane of human erythrocytes was tested in an immune adherence assay. S. aureus capsule was able to mask C3 fragments on the organism from binding to complement receptor 1. The inhibition of C3 binding to CP+ and the masking of deposited C3 fragments caused by the presence of capsule was associated with markedly decreased phagocytosis. The addition of anti-capsule antibodies to normal human serum was found to markedly improve the recognition of deposited C3 fragments by complement receptor 1 even when the absolute number of C3 molecules bound to S. aureus was not increased.

Hypochlorite killing of community-associated methicillin-resistant Staphylococcus aureus.

We tested in vitro hypochlorite (bleach) killing of community-associated methicillin-resistant Staphylococcus aureus isolates to determine optimal concentration and duration. For all isolates maximal killing, >3-log decrease in colony forming units (CFU), was found after 5 minutes in 2.5 &mgr;L/mL bleach. We estimate that 2.5 &mgr;L/mL bleach is approximately one-half cup of bleach in one-quarter tub of water.

A comparison of antibacterial activity against Methicillin-Resistant Staphylococcus aureus and gram-negative organisms for antimicrobial compounds in a unique composite wound dressing.

OBJECTIVE:Antibiotic resistance is increasing among organisms that commonly cause wound infections. Therefore, it becomes increasingly desirable to prevent wound infections as systemic antibiotic treatment of established wound infections becomes more difficult, more expensive, and potentially more toxic. The ability to incorporate antimicrobial compounds into modern wound dressings provides an opportunity to prevent wound infections without the risk of systemic toxicity, thus diminishing morbidity, mortality, and cost to the healthcare system. DESIGN:In these studies, the authors tested 16 antimicrobial agents in a unique composite wound dressing (TheraGauze; Soluble Systems, LLC, Newport News, Virginia) against clinical methicillin-resistant Staphylococcus aureus isolates and Gram-negative organisms commonly associated with wound infections and antibiotic resistance. Disk diffusion susceptibility testing is used to quantify antimicrobial activity. RESULTS:Broad-spectrum antimicrobial activity was found for the following agents in the composite wound dressing: hydrogen peroxide, tobramycin, chlorhexidine digluconate, chlorhexidine gluconate, levofloxacin, and silver. CONCLUSION:These studies suggest that potent local antibacterial activity can be achieved with several antimicrobials in this wound dressing.

Hyperglycemia Inhibits Complement-Mediated Immunological Control of S. aureus in a Rat Model of Peritonitis

Hyperglycemia from diabetes is associated with increased risk of infection from S. aureus and increased severity of illness. Previous work in our laboratory demonstrated that elevated glucose (>6 mM) dramatically inhibited S. aureus-initiated complement-mediated immune effectors. Here we report in vivo studies evaluating the extent to which a hyperglycemic environment alters complement-mediated control of S. aureus infection in a rat peritonitis model. Rats were treated with streptozocin to induce diabetes or sham-treated and then inoculated i.p. with S. aureus. Rats were euthanized and had peritoneal lavage at 2 or 24 hours after infection to evaluate early and late complement-mediated effects. Hyperglycemia decreased the influx of IgG and complement components into the peritoneum in response to S. aureus infection and decreased anaphylatoxin generation. Hyperglycemia decreased C4-fragment and C3-fragment opsonization of S. aureus recovered in peritoneal fluids, compared with euglycemic or insulin-rescued rats. Hyperglycemic rats showed decreased phagocytosis efficiency compared with euglycemic rats, which correlated inversely with bacterial survival. These results suggest that hyperglycemia inhibited humoral effector recruitment, anaphylatoxin generation, and complement-mediated opsonization of S. aureus, suggesting that hyperglycemic inhibition of complement effectors may contribute to the increased risk and severity of S. aureus infections in diabetic patients.

Inhibition of Myeloperoxidase Activity in Cystic Fibrosis Sputum by Peptide Inhibitor of Complement C1 (PIC1)

Myeloperoxidase is the major peroxidase enzyme in neutrophil granules and implicated in contributing to inflammatory lung damage in cystic fibrosis. Free myeloperoxidase is present in cystic fibrosis lung fluid and generates hypochlorous acid. Here we report a new inhibitor of myeloperoxidase activity, Peptide Inhibitor of Complement C1 (PIC1). Using TMB as the oxidizing substrate, PIC1 inhibited myeloperoxidase activity in cystic fibrosis sputum soluble fractions by an average of a 3.4-fold decrease (P = 0.02). PIC1 also dose-dependently inhibited myeloperoxidase activity in a neutrophil lysate or purified myeloperoxidase by up to 28-fold (P < 0.001). PIC1 inhibited myeloperoxidase activity similarly, on a molar basis, as the specific myeloperoxidase inhibitor 4-Aminobenzoic acid hydrazide (ABAH) for various oxidizing substrates. PIC1 was able to protect the heme ring of myeloperoxidase from destruction by NaOCl, assayed by spectral analysis. PIC1 incubated with oxidized TMB reversed the oxidation state of TMB, as measured by absorbance at 450 nm, with a 20-fold reduction in oxidized TMB (P = 0.02). This result was consistent with an antioxidant mechanism for PIC1. In summary, PIC1 inhibits the peroxidase activity of myeloperoxidase in CF sputum likely via an antioxidant mechanism.

Peptide inhibitor of complement C1 modulates acute intravascular hemolysis of mismatched red blood cells in rats

Acute hemolytic transfusion reactions have a broad clinical presentation from mild and transitory signs and symptoms to shock, disseminated intravascular coagulation, renal failure, and death. We have recently developed a rat model of acute intravascular hemolysis showing that the classical complement pathway mediates antibody‐dependent hemolysis. The objective of this study was to evaluate the role of the classical pathway inhibitor peptide inhibitor of complement C1 (PIC1) in this animal model.

Complement Effectors of Inflammation in Cystic Fibrosis Lung Fluid Correlate with Clinical Measures of Disease

In cystic fibrosis (CF), lung damage is mediated by a cycle of obstruction, infection, and inflammation. Here we explored complement inflammatory effectors in CF lung fluid. In this study soluble fractions (sols) from sputum samples of 15 CF patients were assayed for complement effectors and analyzed with clinical measurements. The pro-inflammatory peptide C5a was increased 4.8-fold (P = 0.04) in CF sols compared with controls. Incubation of CF sols with P. aeruginosa or S. aureus increased C5a concentration 2.3-fold (P = 0.02). A peptide inhibitor of complement C1 (PIC1) completely blocked the increase in C5a concentration from P. aeruginosa in CF sol in vitro (P = 0.001). C5a concentration in CF sol correlated inversely with body mass index (BMI) percentile in children (r = -0.77, P = 0.04). C3a, which has anti-inflammatory effects, correlated positively with FEV1% predicted (rs = 0.63, P = 0.02). These results suggest that complement effectors may significantly impact inflammation in CF lung fluid.

Complement Activation Influences Staphylococcus aureus Adherence to Endothelial Cells

ABSTRACT The ability of Staphylococcus aureus to adhere to endothelial cells (EC) is a critical step in the development of metastatic infection. The role of complement in S. aureus binding to EC remains uninvestigated. Log-phase S. aureus, expressing minimal capsule, was incubated with serum under various conditions, washed, and then incubated at 37°C for 30 min with cultured human umbilical vein EC (ATCC CRL-1730). Adherence was scored visually after staining with acridine orange. Incubation in 10% heat-inactivated human serum increased adherence to endothelial cells by 488% compared to organisms incubated in buffer. Incubating S. aureus in complement-active normal human serum (NHS) decreased binding to EC by 58% compared to organisms incubated in heat-inactivated serum. The importance of active complement was confirmed by experiments using serum with added EDTA or cobra venom factor, a protein that depletes C3. The expression of capsule by S. aureus strongly interfered with adherence. It has been shown that an important protein for S. aureus adhesion to EC is fibronectin. S. aureus adherence to purified fibronectin increased by 511% after incubation in heat-inactivated serum, compared to that of organisms incubated in buffer. This decreased by 56% in complement-active serum, suggesting that inhibition of S. aureus adherence to EC is due, in part, to complement-mediated diminished binding to fibronectin. Interestingly, when EC were exposed to S. aureus-activated serum and then washed, binding by S. aureus was 234% higher than that of EC exposed to NHS. Thus, complement-activated EC have increased S. aureus binding, while complement on the bacterial surface markedly reduces adherence.

Complement Activation and STAT4 Expression Are Associated with Early Inflammation in Diabetic Wounds

Diabetic non-healing wounds are a major clinical problem. The mechanisms leading to poor wound healing in diabetes are multifactorial but unresolved inflammation may be a major contributing factor. The complement system (CS) is the most potent inflammatory cascade in humans and contributes to poor wound healing in animal models. Signal transducer and activator of transcription 4 (STAT4) is a transcription factor expressed in immune and adipose cells and contributes to upregulation of some inflammatory chemokines and cytokines. Persistent CS and STAT4 expression in diabetic wounds may thus contribute to chronic inflammation and delayed healing. The purpose of this study was to characterize CS and STAT4 in early diabetic wounds using db/db mice as a diabetic skin wound model. The CS was found to be activated early in the diabetic wounds as demonstrated by increased anaphylatoxin C5a in wound fluid and C3-fragment deposition by immunostaining. These changes were associated with a 76% increase in nucleated cells in the wounds of db/db mice vs. controls. The novel classical CS inhibitor, Peptide Inhibitor of Complement C1 (PIC1) reduced inflammation when added directly or saturated in an acellular skin scaffold, as reflected by reduced CS components and leukocyte infiltration. A significant increase in expression of STAT4 and the downstream macrophage chemokine CCL2 and its receptor CCR2 were also found in the early wounds of db/db mice compared to non-diabetic controls. These studies provide evidence for two new promising targets to reduce unresolved inflammation and to improve healing of diabetic skin wounds.