Christopher L. Joannou

Clinical, Microbial, and Biochemical Aspects of the Exfoliative Toxins Causing Staphylococcal Scalded-Skin Syndrome

SUMMARY The exfoliative (epidermolytic) toxins of Staphylococcus aureus are the causative agents of the staphylococcal scalded-skin syndrome (SSSS), a blistering skin disorder that predominantly affects children. Clinical features of SSSS vary along a spectrum, ranging from a few localized blisters to generalized exfoliation covering almost the entire body. The toxins act specifically at the zona granulosa of the epidermis to produce the characteristic exfoliation, although the mechanism by which this is achieved is still poorly understood. Despite the availability of antibiotics, SSSS carries a significant mortality rate, particularly among neonates with secondary complications of epidermal loss and among adults with underlying diseases. The aim of this article is to provide a comprehensive review of the literature spanning more than a century and to cover all aspects of the disease. The epidemiology, clinical features, potential complications, risk factors, susceptibility, diagnosis, differential diagnoses, investigations currently available, treatment options, and preventive measures are all discussed in detail. Recent crystallographic data on the toxins has provided us with a clearer and more defined approach to studying the disease. Understanding their mode of action has important implications in future treatment and prevention of SSSS and other diseases, and knowledge of their specific site of action may provide a useful tool for physiologists, dermatologists, and pharmacologists.

Nitrite and nitrosyl compounds in food preservation

Nitrite is consumed in the diet, through vegetables and drinking water. It is also added to meat products as a preservative. The potential risks of this practice are balanced against the unique protective effect against toxin-forming bacteria such as Clostridium botulinum. The chemistry of nitrite, and compounds derived from it, in food systems and bacterial cells are complex. It is known that the bactericidal species is not nitrite itself, but a compound or compounds derived from it during food preparation. Of a range of nitrosyl compounds tested, the anion of Roussins black salt [Fe4S3(NO)7]- was the most inhibitory to C. sporogenes. This compound is active against both anaerobic and aerobic food-spoilage bacteria, while some other compounds are selective, indicating multiple sites of action. There are numerous possible targets for inhibition in the bacterial cells, including respiratory chains, iron-sulfur proteins and other metalloproteins, membranes and the genetic apparatus.

Structure and Association of Human Lactoferrin Peptides with Escherichia coli Lipopolysaccharide

ABSTRACT An 11-amino-acid amphipathic synthetic peptide homologous to a helical region on helix 1 of human lactoferrin HLP-2 exhibited bactericidal activity against Escherichia coli serotype O111, whereas an analogue synthesized with Pro substituted for Met, HLP-6, had greatly reduced antimicrobial activity. The bactericidal activity of HLP-2 was 10-fold greater than that of HLP-6 in both buffer and growth medium by time-kill assays. These assays also showed a pronounced lag phase that was both concentration and time dependent and that was far greater for HLP-2 than for HLP-6. Both peptides, however, were shown to be equally efficient in destabilizing the outer membrane when the hydrophobic probe 1-N-phenylnaphthylamine was used and to have the same lipopolysaccharide (LPS) binding affinity, as shown by polymyxin B displacement. Circular dichroism (CD) spectroscopy was used to study the structure and the organization of the peptides in solution and upon interaction with E. coli LPS. In the presence of LPS, HLP-2 and HLP-6 were found to bind and adopt a β-strand conformation rather than an α-helix, as shown by nonimmobilized ligand interaction assay-CD spectroscopy. Furthermore, this assay was used to show that there is a time-dependent association of peptide that results in an ordered formation of peptide aggregates. The rate of interpeptide association was far greater in HLP-2 LPS than in HLP-6 LPS, which was consistent with the lag phase observed on the killing curves. These results allow us to propose a mechanism by which HLP-2 folds and self-assembles at the outer membrane surface before exerting its activity.

Development and Evaluation of Detection Systems for Staphylococcal Exfoliative Toxin A Responsible for Scalded-Skin Syndrome

ABSTRACT Staphylococcal scalded-skin syndrome is usually diagnosed clinically by its characteristic exfoliating rash. Isolation ofStaphylococcus aureus from the patient further supports the diagnosis. Several detection systems have been developed to determine whether the isolated strain produces exfoliative toxin, but none are routinely available in hospital laboratories. In a novel approach, we used computer models to predict the structure of the exfoliative toxins based on other serine proteases and to identify surface epitopes for the production of antibodies that specifically bound the exfoliative toxin A (ETA) serotype. Several rapid immunologically based diagnostic tests for ETA were developed with these antibodies and compared with existing systems. Our results showed that Western blot analysis using these antibodies was in complete correlation with PCR, which has been validated against the “gold standard” mouse model. On the other hand, the double-antibody enzyme-linked immunosorbent assay (ELISA) and Ouchterlony immunodiffusion assay gave unacceptably high false-positive results due to interference by staphylococcal protein A. This problem was successfully overcome by the development of a F(ab′)2fragment ELISA, which was rapid and reproducible and was as sensitive and specific as PCR and Western blot analysis. The F(ab′)2fragment ELISA is superior to existing diagnostic systems because it is quantitative, which may be related to the severity of the condition, and can detect amounts of exfoliative toxin in the picogram range directly from serum. This is the first detection system with the potential to confirm the diagnosis of staphylococcal scalded-skin syndrome from a routine blood test within 3 h of presentation.

Difficulties in diagnosis and management of the staphylococcal scalded skin syndrome.

Staphylococcal scalded skin syndrome (SSSS) describes a collection of blistering skin disorders caused by the exfoliative toxins (ET) of Staphylococcus aureus. Infants and young children are particularly susceptible and, although mortality is low, SSSS carries significant morbidity, often causing great distress to parents. Rare cases reported in adults are usually fatal despite antibiotics, mostly because of underlying conditions such as renal failure, malignancy and immunocompromise. 3 SSSS continues to receive little attention from clinicians and researchers alike because of its relative rarity, ease of treatment and good prognosis in children. 2 However, the recent emergence of ETproducing methicillin-resistant S. aureus (ETMRSA) has heightened the urgency to reevaluate our current management of SSSS, with particular emphasis on alternative treatment strategies, before antibiotic resistance escalates. At present consensus regarding the management of SSSS is lacking, and no clear guidelines exist. 2 Diagnosis is usually limited to clinical presentation, investigations to confirm diagnosis are inadequate and treatment is often left to the discretion of the clinician. The clinical features of SSSS vary along a spectrum. Protective antitoxin antibodies in some children and most adults limit the lesions to a few localized blisters in mild forms, whereas lack of antibodies in generalized SSSS allows hematologic dissemination of ET to produce exfoliation that may cover the entire body surface. Initially SSSS patients often present with only a single or few superficial exfoliating lesions. 2 At this stage diagnosis is important because early antibiotic use treats any focal source of infection and can even halt progression of exfoliation, as has been shown in animal and human studies. This in turn could reduce the number of severe cases, along with the need for hospital admission, intravenous antibiotics and other invasive procedures. A host of differential diagnoses must be considered for any localized exfoliating rash. Distinction from streptococcal impetigo can often be achieved clinically because it tends to begin with small vesicles that rapidly evolve into characteristic irregular, golden, crusted plaques, whereas the lesions of localized SSSS take the form of superficial, fragile blisters that burst and leave a tender erythematous base that quickly dries to a thin shiny surface. Distinction of SSSS from viralor drug-induced erythema multiforme/toxic epidermal necrolysis (EM/TEN) is essential because this condition is often treated with steroids, which could encourage secondary infection in SSSS with potentially fatal outcome. 11 Misdiagnosing SSSS as EM/TEN (and we have seen several cases of this) further delays treatment and allows exfoliation to progress. 2, 4, 7, 11 Faced with such a patient, the investigations available to aid diagnosis are limited, particularly in primary care. Superficial swabs (skin lesions, nose, umbilicus, etc) serve little purpose because S. aureus is a common skin commensal, which can be isolated from up to 50% of EM/TEN lesions. Confirmation of SSSS currently requires isolating S. aureus from the patient and demonstrating that the strain produces ET. In the UK this service is provided by the Public Health Laboratory Service, London, but it takes several days and is therefore useful only retrospectively. Similarly blood cultures take time and are often sterile. 3 Histologic examination of a skin biopsy, therefore, is the most useful investigation because demonstration of midepidermal separation at the zona granulosa is virtually diagnostic of SSSS and excludes EM/TEN where dermoepidermal cleavage occurs. 3, 11 However, this investigation has practical difficulties, especially in children and in the primary care setting, and requires rapid microscopic evaluation by specialists. Fortunately SSSS usually presents with a localized lesion and spreads gradually. Therefore in primary care any localized exfoliating rash in an otherwise well child that resembles localized SSSS should be treated with oral antibiotics (Fig. 1). In many cases new exfoliating lesions appear for 24 to 36 h after starting treatment (because the disease is toxin-mediated) after Accepted for publication June 7, 2000. From the Division of Biomolecular Sciences, Kings College London, London, UK.

Meningococcal Transferrin-Binding Proteins A and B Show Cooperation in Their Binding Kinetics for Human Transferrin

ABSTRACT Neisseria meningitidis, a causative agent of bacterial meningitis and septicemia, obtains transferrin-bound iron by expressing two outer membrane-located transferrin-binding proteins, TbpA and TbpB. A novel system was developed to investigate the interaction between Tbps and human transferrin. Copurified TbpA-TbpB, recombined TbpA-TbpB, and individual TbpA and TbpB were reconstituted into liposomes and fused onto an HPA chip (BIAcore). All preparations formed stable monolayers, which, with the exception of TbpB, could be regenerated by removing bound transferrin. The ligand binding properties of these monolayers were characterized with surface plasmon resonance and shown to be specific for human transferrin. Kinetic data for diferric human transferrin binding showed that recombined TbpA-TbpB had Ka and Kd values similar to those of copurified TbpA-TbpB. Individual TbpA and TbpB also displayed Ka values similar to those of copurified TbpA-TbpB, but their Kd values were one order of magnitude higher. Chemical cross-linking studies revealed that TbpA and TbpB, in the absence of human transferrin, formed large complexes with TbpA as the predominant species. Upon human transferrin binding, a complex was formed with a molecular mass corresponding to that of a TbpB-human transferrin heterodimer as well as a higher-molecular-mass complex of this heterodimer cross-linked to TbpA. This indicates that TbpA and TbpB form a functional meningococcal receptor complex in which there is cooperativity in the human transferrin binding kinetics. However, iron loss from the diferric human transferrin-TbpA-TbpB complex was not greater than that from human transferrin alone, suggesting that additional meningococcal transport components are involved in the process of iron removal.

Expression and purification of functional recombinant meningococcal transferrin-binding protein A.

Pathogenic bacteria of the genus Neisseria have a siderophore-independent iron-uptake system reliant on a direct interaction between the bacterial cell and human transferrin (hTf), a serum protein. In the meningococcus, this uptake system is dependent on two surface-exposed, transferrin-binding proteins (Tbps), TbpA and TbpB. TbpA is highly conserved among meningococcal strains, and is thought to be a porin-like integral protein that functions as a gated channel for the passage of iron into the periplasm. TbpB is more variable in size, lipidated and fully surface-exposed. Given its location on the cell surface, its role in pathogenicity and interstrain sequence conservation, TbpA is currently being regarded for inclusion in a meningococcal vaccine effective against all serogroups. This requires gaining knowledge of the ligand-receptor interactions. In the present study we have optimized a procedure for obtaining purified, functionally active recombinant TbpA at a level and stability necessary for the initiation of such studies.

NAD-dependent glutamate dehydrogenase from Pseudomonas aeruginosa is a membrane-bound enzyme

Measurements of the deaminating activity of NAD-dependent glutamate dehydrogenase (NAD-GDH) in Pseudomonas aeruginosa strain 8602 (PAC 1) showed an initially constant rate that gave way to a 3.5-fold increased rate on prolonged incubation. Only the faster rate was observed when assay mixtures were preflushed with nitrogen or were treated with the detergent Triton X-100. Comparison of the intracellular distribution of NAD-GDH with marker enzymes showed it to be associated with the cytoplasmic membrane. The results suggest that NAD-GDH may be linked to oxygen through an electron-transport system.