Glen S. Tamura

Identification of novel adhesins from Group B streptococci by use of phage display reveals that C5a peptidase mediates fibronectin binding.

ABSTRACT Group B streptococci (GBS) are a major cause of pneumonia, sepsis, and meningitis in newborns and infants. GBS initiate infection of the lung by colonizing mucosal surfaces of the respiratory tract; adherence of the bacteria to host cells is presumed to be the initial step in and prerequisite for successful colonization (G. S. Tamura, J. M. Kuypers, S. Smith, H. Raff, and C. E. Rubens, Infect. Immun. 62:2450-2458, 1994). We have performed a genome-wide screen to identify novel genes of GBS that mediate adherence to fibronectin. A shotgun phage display library was constructed from chromosomal DNA of a serotype Ia GBS strain and affinity selected on immobilized fibronectin. DNA sequence analysis of different clones identified 19 genes with homology to known bacterial adhesin genes, virulence genes, genes involved in transport or metabolic processes, and genes with yet-unknown function. One of the isolated phagemid clones showed significant homology to the gene (scpB) for the GBS C5a peptidase, a surface-associated serine protease that specifically cleaves the complement component C5a, a chemotaxin for polymorphonuclear leukocytes. In this work we have demonstrated that affinity-purified recombinant ScpB and a peptide ScpB fragment (ScpB-PDF), similar to the peptide identified in the phagemid, bound fibronectin in a concentration-dependent manner. Adherence assays to fibronectin were performed, comparing an isogenic scpB mutant to the wild-type strain. Approximately 50% less binding was observed with the mutant than with the wild-type strain. The mutant phenotype could be fully restored by in trans complementation of the mutant with the cloned wild-type scpB gene, providing further evidence for the role of ScpB in fibronectin adherence. Our results suggest that C5a peptidase is a bifunctional protein, which enzymatically cleaves C5a and mediates adherence to fibronectin. Since binding of fibronectin has been implicated in attachment and invasion of eukaryotic cells by streptococci, our results may imply a second important role for this surface protein in the pathogenesis of GBS infections.

Group B streptococci adhere to a variant of fibronectin attached to a solid phase

Group B streptococci (GBS) are the leading cause of neonatal pneumonia and meningitis. Adherence of GBS to host tissues may play an important role in the pathogenesis of infection. The host molecules which mediate GBS adherence to host tissues are unknown. Many bacterial pathogens adhere to fibronectin, an important component of the extracellular matrix (ECM). Some pathogens adhere to both immobilized and soluble fibronectin, while others adhere to immobilized fibronectin, but not to soluble fibronectin. Previous data indicated that GBS do not adhere to soluble fibronectin. We studied the ability of GBS to adhere to immobilized fibronectin. Forty‐five per cent of the input inoculum of COH1, a virulent GBS isolate, adhered to fibronectin immobilized on polystyrene. COH1 did not adhere to the other ECM proteins tested (laminin, type I collagen, vitronectin, and tenascin). Nine out of nine GBS strains from human sources tested adhered specifically to fibronectin at levels varying from 4–60%. We considered the possibility that GBS were adherent to a contaminant in the fibronectin preparation. Properties of fibronectin, including the presence of an immunologic epitope of fibronectin and binding to collagen, were verified to be properties of the molecule to which GBS adhere. COH1 adhered to fibronectin captured by a monoclonal antibody to fibronectin (FN‐15), confirming that the molecule to which GBS adhere bears immunologic determinants of fibronectin. Adherence of COH1 to fibronectin was inhibited by collagen, confirming that the molecule to which GBS adhere binds to collagen. These data strongly suggest that GBS adhere to fibronectin, and not to a contaminant. Protein blot analysis revealed that GBS were adherent to a high‐molecular‐weight variant of non‐reduced fibronectin monomers and dimers. GBS did not adhere to reduced fibronectin monomers. We conclude that GBS adhere to a variant of plasma fibronectin when attached to a solid phase.

A Glutamine Transport Gene, glnQ, Is Required for Fibronectin Adherence and Virulence of Group B Streptococci

ABSTRACT Group B streptococci (GBS) are a leading cause of neonatal sepsis and meningitis. GBS adhere to fibronectin when it is attached to a solid phase. We isolated a Tn917 transposon mutant, COH1-GT1, which shows decreased adherence to fibronectin. COH1-GT1 also shows decreased adherence to and invasion of respiratory epithelial cells in vitro and decreased virulence in vivo. COH1-GT1 contains a Tn917 insertion in a homolog of glnQ, a gene from Escherichia coli which is required for glutamine transport and codes for a cytoplasmic ATP-binding cassette protein. To confirm that the decreased fibronectin adherence of COH1-GT1 was due to the mutation in glnQ, we constructed COH1-GT2, a strain with a nonpolar site-directed mutation in glnQ. COH1-GT2 showed decreased binding to fibronectin. We also demonstrated that complementation of glnQ in trans restored fibronectin adherence to COH1-GT1. COH1-GT1 shows decreased uptake of radiolabeled glutamine and is resistant to the toxic glutamine analog γ-l-glutamylhydrazide, demonstrating that the glnQ gene is required for glutamine transport in GBS. glnQ lacks a signal sequence and is a cytoplasmic protein in E. coli and thus is unlikely to act as a fibronectin adhesin. glnQ is transcribed in an operon with a putative glutamine permease gene, glnP, which has a novel predicted structure containing three distinct domains linked in a single gene. The first two domains are putative glutamine binding domains with homology to the E. coli periplasmic glutamine binding gene glnH. The third is a putative permease domain with homology to the E. coli glutamine permease gene glnP. RT-PCR analysis demonstrated that glnP and glnQ are contained within a single transcript. Transcription of scpB, encoding the only known fibronectin-binding adhesin of GBS, is unaffected. We speculate that glnQ may regulate expression of fibronectin adhesins by affecting cytoplasmic glutamine levels and that regulation may be posttranscriptional.

Analysis of Restriction Fragment Length Polymorphisms of the Insertion Sequence IS1381 in Group B Streptococci

Group B streptococci (GBS) are typed by capsular polysaccharide type. IS1381, an insertion sequence previously described in Streptococcus pneumoniae, was cloned from GBS strain A909. The presence of multiple copies of IS1381 in A909 suggested that IS1381 analysis might be an effective subtyping tool. IS1381 was found by Southern blot analysis to be present in 18 (72%) of 25 of unrelated GBS isolates tested. IS1381 analysis allowed discrimination between strains that contain IS1381 with a discriminatory power >99%. Eight of 8 sets of epidemiologically related isolates containing IS1381 give identical or nearly identical patterns of IS1381 insertion. For 2 maternal/infant sets, a single additional insertion was seen in 1 strain, suggesting that an additional insertion occurred between maternal colonization and infection of the infant. Insertion patterns of IS1381 are an effective tool for subtyping GBS.

High-Affinity Interaction between Fibronectin and the Group B Streptococcal C5a Peptidase Is Unaffected by a Naturally Occurring Four-Amino-Acid Deletion That Eliminates Peptidase Activity

ABSTRACT The streptococcal C5a peptidase (ScpB) of group B streptococci (GBS) is found in virtually all clinical GBS isolates and is required for mucosal colonization in a neonatal mouse model. ScpB inhibits neutrophil chemotaxis by enzymatically cleaving the complement component C5a. We previously identified a second function of ScpB as a fibronectin (Fn) adhesin using phage display. However, phage display can identify low-affinity interactions. We therefore measured the affinity of both full-length recombinant ScpB (FL-ScpB) and the 110-amino-acid phage display fragment (Scp-PDF) for immobilized Fn using surface plasmon resonance. The affinity for Fn was very high for both FL-ScpB (equilibrium dissociation constant [KD] = 4.0 nM) and Scp-PDF (KD = 4.4 nM) and is consistent with a biologically significant role for the adhesin activity of ScpB. We also studied the Fn adhesin activity of a common natural variant of ScpB (ScpBΔ) that contains a 4-amino-acid deletion that eliminates peptidase activity. The integrity of scpB is otherwise maintained, suggesting that the Fn adhesin activity of ScpB may be responsible for its conservation in these strains. The affinities of both FL-ScpBΔ (KD = 2.4 nM) and ScpBΔ-PDF (KD = 1.4 nM) for Fn are unaffected by the deletion. Complementation in trans by both scpB and scpBΔ corrected the Fn-binding defect of an scpB deletion mutant GBS strain to an identical degree. The high affinity of ScpB for Fn and the maintenance of this affinity in ScpBΔ support our hypothesis that the Fn adhesin activity of scpB plays a role in virulence.

Comprehension on Family-Centered Rounds for Limited English Proficient Families

OBJECTIVE To describe communication with limited English proficient (LEP) families during family-centered rounds (FCR); to examine differences in family understanding of diagnosis and plan by English proficiency and provider and interpreter rounding behaviors. METHODS Forty-one English proficient (EP) and 40 LEP parents of pediatric inpatients participated in a prospective cohort study from January to October 2011. Eligible LEP families self-reported a preference for medical communication in Spanish, Somali, or Vietnamese. Rounds were observed; families were interviewed afterward. Parent- and provider-reported diagnosis and plan were compared and classified as correct, incorrect, or incomplete by 3 blinded investigators. Logistic regression adjusted for potential confounders. RESULTS Fifty percent of LEP rounding encounters involved interpreters filtering information conveyed to families; 43% involved initial medical discussions without families present (vs 12% for EP, P = .002). Providers more frequently provided a plain language summary for LEP families (88% vs 56%, P = .001). LEP and EP families had similar ability to correctly name the childs diagnosis (70% vs 83%, P = .17) and all plan elements (38% vs 39%, P = .88). Results were unchanged after adjusting for parental characteristics and hospital day. Among LEP families, naming the correct diagnosis was positively associated with experience with a hospitalized child (odds ratio 5.11, 95% confidence interval 1.04-24.9) and may be negatively associated with interpreter filtering (odds ratio 0.22, 95% confidence interval 0.05-1.13). CONCLUSIONS Having initial medical discussions without the family and information filtering are common for LEP patients; filtering may be associated with poorer diagnosis comprehension. Experience with a hospitalized child is associated with increased comprehension among LEP parents.

Interactions of the streptococcal C5a peptidase with human fibronectin

Group B Streptococci (GBS) is a leading cause of sepsis and meningitis in neonates and immunocompromised adults in western countries. GBS do not bind to fibronectin (Fn) in solution, but will bind to Fn adsorbed onto a solid surface. The reason for the specificity of this binding is unknown. Single molecule force spectroscopy was used to test the hypothesis that GBS, through streptococcal C5a peptidase (ScpB) molecules present on the surface of the bacteria, binds to a motif created by the juxtaposition of multiple adjacent Fn molecules. Atomic force microscopy (AFM) topographical images of adsorbed Fn deposited from various Fn coating concentrations were used to determine the Fn surface concentration. ScpB was tethered to an AFM tip with all surface modifications characterized by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry. At the lowest Fn coverages the probability of observing a ScpB-Fn binding event increased linearly with Fn surface coverage. As an Fn monolayer was reached the probability of a ScpB-Fn binding event occurring increased markedly ( approximately 50 fold), with a concomitant increase in the rupture force from 17 pN to 33 pN. These results are consistent with the hypothesis that ScpB binds to a motif created by the juxtaposition of multiple Fn molecules.

Where Do Medical Students Turn? The Role of the Assigned Mentor in the Fabric of Support During Medical School

Background: The University of Washington School of Medicine implemented an assigned mentoring program in 2002. The College Mentors are assigned at matriculation, advise students throughout medical school, and teach and evaluate students in the 2nd-year Introduction to Clinical Medicine course. Purpose: The purpose of the study was to determine from whom students report they would seek advice and support for academic, professional, personal, and research issues. Methods: A cross-sectional cohort survey asking students whom they would first contact about academic, personal, professional, and research issues was administered to three cohorts of students in 2007. Results: Students reported that they would contact their College Mentor first for general academic progress (49.6%), personal issues (36.2%), and professional issues (64.1%) but not for research issues. Conclusions: Students identified their College Mentor as a primary contact for academic, professional, and personal issues, suggesting that neither the mentors’ assigned status or evaluator role were barriers to the mentoring relationship.

Use of glnQ as a Counterselectable Marker for Creation of Allelic Exchange Mutations in Group B Streptococci

ABSTRACT Efficient allelic exchange mutagenesis in group B streptococci (GBS) has been hampered by the lack of a counterselectable marker system. Growth inhibition of GBS by the glutamine analog gamma-glutamyl hydrazide requires glnQ. We have used this phenomenon to create a counterselectable marker system for efficient selection of allelic exchange mutants in GBS.

Atomic force microscopy and surface plasmon resonance investigation of fibronectin interactions with group B streptococci

The interactions of fibronectin (Fn) with group B streptococci (GBS) were investigated using the atomic force microscope (AFM) and surface plasmon resonance (SPR) biosensing. Submonolayer amounts of Fn were immobilized onto the AFM tip by two different methods, using either a sulfosuccinimidyl-4-(N-maleimidomethyl) cycholhexane-1-carboxylate (SMCC) linker or a pyridyldithio poly(ethylene glycol) succinimidylpropionate (NHS-PEG-PDP) linker. Each step of both immobilization methods was characterized using x-ray photoelectron spectroscopy. Time-of-flight secondary ion mass spectrometry experiments indicated both methods produced Fn immobilized in a similar conformation. AFM force-distance curves from live GBS plated onto polystyrene exhibited several types of interactions between the Fn functionalized AFM tip and the surface of capsule-deficient GBS (no interactions, interactions with the cell wall, Fn unfolding, large specific unbinding events, and small specific unbinding events). From analysis of the force-distance curves that exhibited only a single specific unbinding event, the work of adhesion and rupture force for the SMCC immobilized Fn tips (11 131 pN nm and 213 pN) were larger than the corresponding values for the NHS-PEG-PDP immobilized Fn tips (8115 pN nm and 189 pN). The unbinding event occurred at distances approximately 100 nm further from the surface with the NHS-PEG-PDP immobilized Fn tip compared to SMCC immobilized Fn tip. The SPR experiments of soluble Fn with adsorbed serine protease C5a peptidase (Scp), the surface protein on GBS that binds Fn, showed that both low (millimolar) and high binding (nanomolar) affinity interactions were present. However, the low binding affinity interactions dominated the adsorption process and, with increasing Fn solution concentration, the amount of Scp bound to Fn via the high binding affinity interaction decreased. These data confirm that Scp binds only to adsorbed Fn at the Fn concentrations typically present in blood plasma.