Flagellin redundancy in Caulobacter crescentus and its implications for flagellar filament assembly.

Bacterial flagella play key roles in surface attachment and host-bacterial interactions as well as driving motility. Here, we have investigated the ability of Caulobacter crescentus to assemble its flagellar filament from six flagellins: FljJ, FljK, FljL, FljM, FljN, and FljO. Flagellin gene deletion combinations exhibited a range of phenotypes from no motility or impaired motility to full motility. Characterization of the mutant collection showed the following: (i) that there is no strict requirement for any one of the six flagellins to assemble a filament; (ii) that there is a correlation between slower swimming speeds and shorter filament lengths in ΔfljK ΔfljM mutants; (iii) that the flagellins FljM to FljO are less stable than FljJ to FljL; and (iv) that the flagellins FljK, FljL, FljM, FljN, and FljO alone are able to assemble a filament.

A highly unusual thioester bond in a pilus adhesin is required for efficient host cell interaction.

Many bacterial pathogens present adhesins at the tips of long macromolecular filaments known as pili that are often important virulence determinants. Very little is known about how pili presented by Gram-positive pathogens mediate host cell binding. The crystal structure of a pilus adhesin from the important human pathogen Streptococcus pyogenes reveals an internal thioester bond formed between the side chains of a cysteine and a glutamine residue. The presence of the thioester was verified using UV-visible spectroscopy and mass spectrometry. This unusual bond has only previously been observed in thioester domains of complement and complement-like proteins where it is used to form covalent attachment to target molecules. The structure also reveals two intramolecular isopeptide bonds, one of these formed through a Lys/Asp residue pair, which are strategically positioned to confer protein stability. Removal of the internal thioester by allele-replacement mutagenesis in S. pyogenes severely compromises bacterial adhesion to model host cells. Although current paradigms of bacterial/host cell interaction envisage strong non-covalent interactions, the present study suggests cell adhesion could also involve covalent bonds.

Roles of minor pilin subunits Spy0125 and Spy0130 in the serotype M1 Streptococcus pyogenes strain SF370.

Adhesive pili on the surface of the serotype M1 Streptococcus pyogenes strain SF370 are composed of a major backbone subunit (Spy0128) and two minor subunits (Spy0125 and Spy0130), joined covalently by a pilin polymerase (Spy0129). Previous studies using recombinant proteins showed that both minor subunits bind to human pharyngeal (Detroit) cells (A. G. Manetti et al., Mol. Microbiol. 64:968-983, 2007), suggesting both may act as pilus-presented adhesins. While confirming these binding properties, studies described here indicate that Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role as a wall linker. Pili were localized predominantly to cell wall fractions of the wild-type S. pyogenes parent strain and a spy0125 deletion mutant. In contrast, they were found almost exclusively in culture supernatants in both spy0130 and srtA deletion mutants, indicating that the housekeeping sortase (SrtA) attaches pili to the cell wall by using Spy0130 as a linker protein. Adhesion assays with antisera specific for individual subunits showed that only anti-rSpy0125 serum inhibited adhesion of wild-type S. pyogenes to human keratinocytes and tonsil epithelium to a significant extent. Spy0125 was localized to the tip of pili, based on a combination of mutant analysis and liquid chromatography-tandem mass spectrometry analysis of purified pili. Assays comparing parent and mutant strains confirmed its role as the adhesin. Unexpectedly, apparent spontaneous cleavage of a labile, proline-rich (8 of 14 residues) sequence separating the N-terminal approximately 1/3 and C-terminal approximately 2/3 of Spy0125 leads to loss of the N-terminal region, but analysis of internal spy0125 deletion mutants confirmed that this has no significant effect on adhesion.

Solution structure of the major (Spy0128) and minor (Spy0125 and Spy0130) pili subunits from Streptococcus pyogenes.

Adhesion of the serotype M1 Streptococcus pyogenes strain SF370 to human tonsil explants and cultured keratinocytes requires extended polymeric surface structures called pili. In this important human pathogen, pili are assembled from three protein subunits: Spy0125, Spy0128 and Spy0130 through the action of sortase enzymes. For this study, the structural properties of these pili proteins have been investigated in solution. Spy0125 and Spy0128 display characteristics of globular, folded proteins. Circular dichroism suggests a largely beta-sheet composition for Spy0128 and Spy0125; Spy0130 appears to contain little secondary structure. Each of the proteins adopts a monodisperse, monomeric state in solution as assessed by analytical ultracentrifugation. Further, small-angle X-ray scattering curves for Spy0125, Spy0128 and Spy0130 suggest each protein adopts an elongated shape, likely comprised of two domains, with similar maximal dimensions. Based on the scattering data, dummy atom models of each of the pili subunits have been reconstructed ab initio. This study provides the first insights into the structure of Streptococcus pyogenes minor pili subunits, and possible implications for protein function are discussed.

Drug information: from education to practice.

Drug information is a specialty area within the realm of clinical pharmacy that has evolved as technology and clinical practice have changed. Drug information specialists are trained individuals who have clinical knowledge and skills that allow them to provide clear, concise, and accurate recommendations regarding drug use. The constant changing culture of drug information and health care in general has prompted the need for continual growth and refinement of the standards that govern drug information practice. This article outlines specific standards to help ensure that the education and practice of drug information will continue to meet the needs of the health care community. This opinion paper is divided into two sections: Education and Training, and Practice Areas. The Education and Training section is organized to describe the role of drug information and that of the drug information specialist in the training of all pharmacy students and advanced trainees, as well as to describe the role of focused training for those individuals wishing to specialize in drug information. This article also affirms the recommendations for the standards-based approach to drug information education and specialty training. The Practice Areas section is organized to describe the role of the drug information specialist within various practice settings, to identify some of the challenges faced by the drug information specialist within those settings, and to provide recommendations for the different practice areas. The areas found within this section include academia, institutional health systems, managed care, industry, medical writing, and informatics.

Causes of hyperglycemia and hypoglycemia in adult inpatients.

PURPOSE: The underlying causes of hyperglycemia and hypoglycemia in adult medical and surgical inpatients were studied. METHODS: Hyperglycemic and hypoglycemic events occurring in adult medical and surgical patients admitted between February and July 2003 to a tertiary care hospital were identified prospectively from automated daily printouts of abnormal blood glucose levels generated by the hospital laboratory. Information on the causes of a random sample of events was ascertained within 24 hours through chart review and provider and patient interviews. Narratives were presented to an expert committee to assess the causes of each event and preventability. RESULTS: Eighteen of 24 hypoglycemic events and 26 of 26 hyperglycemic episodes were considered preventable. Failure to adjust antidiabetic drugs in response to decreases in oral intake and unexpected deviation from normal hospital routine were the most common factors contributing to hypoglycemia. Hyperglycemia was most often associated with an unwillingness of providers to take responsibility for diabetes management and the exclusive use of sliding-scale insulin regimens. CONCLUSION: Hyperglycemia and hypoglycemia in medical and surgical inpatients were mostly related to inadequate prescribing, monitoring, and communication practices.

Evaluation of seven i.v. drug compatibility references by using requests from a drug information center.

PURPOSE: The scope of coverage and clinical performance of i.v. drug compatibility references were evaluated for drug pairs. METHODS: Compatibility pairs were selected from a drug information center database of previous questions. Duplicate pairs, contrast media, and nondrug chemicals were excluded. Scope of coverage was defined as whether i.v. compatibility information was present for each pair and was reported as a percentage. Clinical performance included the agreement among the references on compatibility, the inclusion of specific concentration information for each drug in a pair, and the presence of references for the information provided. The analysis included one print reference (Handbook on Injectable Drugs, 14th edition) and six electronic i.v. drug compatibility references (King Guide to Parenteral Admixtures, Trissel's 2 Clinical Pharmaceutics Database, Micromedex's IV INDEX, Gold Standard's Clinical Pharmacology, Facts and Comparisons 4.0 IV Chek, and CompoundingToday.com). The manufacturers' online labeling was also consulted. RESULTS: A total of 97 unique drug pairs were analyzed. Four databases contained i.v. compatibility information on 76% of the drug pairs, one contained 62%, one contained 58%, one contained 56%, and the manufacturers' labeling contained 13% of the pairs. Seventy-nine percent of the pairs had agreement among the references as to whether the pair was compatible, incompatible, or variable. Compounding Today.com, Facts and Comparisons IV Chek, the Handbook on Injectable Drugs, IV INDEX, and Trissel's 2 reported concentrations and references for the information given on all pairs. CONCLUSION: The highest-performing references included in the evaluation used the compatibility information provided in Trissel's 2 database as their source of information. Other popular references identified fewer pairs, and the manufacturers' labeling rarely contained compatibility information.

Crystal structure of Streptococcus pyogenes sortase A: implications for sortase mechanism.

Sortases are a family of Gram-positive bacterial transpeptidases that anchor secreted proteins to bacterial cell surfaces. These include many proteins that play critical roles in the virulence of Gram-positive bacterial pathogens such that sortases are attractive targets for development of novel antimicrobial agents. All Gram-positive pathogens express a "housekeeping" sortase that recognizes the majority of secreted proteins containing an LPXTG wall-sorting motif and covalently attaches these to bacterial cell wall peptidoglycan. Many Gram-positive pathogens also express additional sortases that link a small number of proteins, often with variant wall-sorting motifs, to either other surface proteins or peptidoglycan. To better understand the mechanisms of catalysis and substrate recognition by the housekeeping sortase produced by the important human pathogen Streptococcus pyogenes, the crystal structure of this protein has been solved and its transpeptidase activity established in vitro. The structure reveals a novel arrangement of key catalytic residues in the active site of a sortase, the first that is consistent with kinetic analysis. The structure also provides a complete description of residue positions surrounding the active site, overcoming the limitation of localized disorder in previous structures of sortase A-type proteins. Modification of the active site Cys through oxidation to its sulfenic acid form or by an alkylating reagent supports a role for a reactive thiol/thiolate in the catalytic mechanism. These new insights into sortase structure and function could have important consequences for inhibitor design.

Pili mediate specific adhesion of Streptococcus pyogenes to human tonsil and skin.

Very little is known about the biological functions of pili that have recently been found to be expressed by important Gram-positive pathogens such as Corynebacterium diphtheriae, Streptococcus agalacticae, S. pneumoniae and S. pyogenes. Using various ex vivo tissue and cellular models, here we show that pili mediate adhesion of serotype M1 S. pyogenes strain SF370 to both human tonsil epithelium and primary human keratinocytes, which represent the two main sites of infection by this human-specific pathogen. Mutants lacking minor pilus subunits retained the ability to express cell-surface pili, but these were functionally defective. In contrast to above, pili were not required for S. pyogenes adhesion to either immortalized HEp-2 or A549 cells, highlighting an important limitation of these extensively used adhesion/invasion models. Adhering bacteria were internalized very effectively by both HEp-2 and A549 cells, but not by tonsil epithelium or primary keratinocytes. While pili acted as the primary adhesin, the surface M1 protein clearly enhanced adhesion to tonsil, but surprisingly, had the opposite effect on adhesion to keratinocytes. These studies provide clear evidence that S. pyogenes pili display an adhesive specificity for clinically relevant human tissues and are likely to play a critical role in the initial stages of infection.

Evaluation of drug interaction software to identify alerts for transplant medications.

BACKGROUND: The ability of computerized physician order entry (CPOE) systems to identify clinically significant drug interactions is dependent upon the integrity of the drug information populating the software. A CPOE system with incomplete or inaccurate drug information will fail to identify clinically important drug interactions and, therefore, fail to reduce preventable adverse drug events (pADEs). OBJECTIVE: To evaluate, from the prescribers' perspective, the ability of a common drug interaction database to identify clinically important drug interactions involving drugs used in transplantation. METHODS: The clinical significance of drug interactions involving 5 transplant drugs was evaluated by an expert panel to determine whether alerts should be generated for physicians not involved in the transplant at the time of order entry. Drug interactions included in the analysis were generated from the expert panel, a common drug interaction database, and 2 standard drug interaction references. Responses on the clinical significance were used to calculate the sensitivity, specificity, and positive and negative predictive values for each severity setting of a common electronic drug interaction database. RESULTS: Overall, the database failed to identify approximately 70% of interactions considered significant by the expert panel. Of the alerts that were generated, >85% were considered clinically significant. The database was most deficient in identifying interactions resulting from additive toxicity. CONCLUSIONS: To expect a decrease in pADEs caused by drug interactions, the information used to populate CPOE systems must be validated. Establishing consistency and integrity of this information may be a future role for pharmacists.