Group A Streptococcus (GAS) is a leading human pathogen producing a diverse array of infections from simple pharyngitis ("strep throat") to invasive conditions, including necrotizing fasciitis and toxic shock syndrome. The surface-anchored GAS M1 protein is a classical virulence factor that promotes phagocyte resistance and exaggerated inflammation by binding host fibrinogen (Fg) to form supramolecular networks. In this study, we used a virulent WT M1T1 GAS strain and its isogenic M1-deficient mutant to examine the role of M1-Fg binding in a proximal step in GAS infection-interaction with the pharyngeal epithelium. Expression of the M1 protein reduced GAS adherence to human pharyngeal keratinocytes by 2-fold, and this difference was increased to 4-fold in the presence of Fg. In stationary phase, surface M1 protein cleavage by the GAS cysteine protease SpeB eliminated Fg binding and relieved its inhibitory effect on GAS pharyngeal cell adherence. In a mouse model of GAS colonization of nasal-associated lymphoid tissue, M1 protein expression was associated with an average 6-fold decreased GAS recovery in isogenic strain competition assays. Thus, GAS M1 protein-Fg binding reduces GAS pharyngeal cell adherence and colonization in a fashion that is counterbalanced by SpeB. Inactivation of SpeB during the shift to invasive GAS disease allows M1-Fg binding, increasing pathogen phagocyte resistance and proinflammatory activities.

Objectives

To evaluate the diagnostic performance and predictive value of coronary computed tomography angiography (CCTA) on subsequent cardiac outcomes.

Background

CCTA has been suggested as an alternative method to invasive coronary angiography for detection of and ruling out coronary artery disease (CAD). However, the usefulness of CCTA findings in predicting patient outcome in routine clinical practice is still uncertain.

Materials and methods

A prospective, multicenter registry study of CCTA with a Visipaque injection 320 mg I/ml was carried out in symptomatic patients suspected of having CAD as part of their medical care. CCTA findings were used to guide patient management decisions. Patient cardiac outcomes were followed at 1, 6, and 12 months after the CCTA procedure for the occurrence of major adverse cardiac event (MACE) (cardiac death, nonfatal myocardial infarction, or unstable angina requiring hospitalization). All cardiac outcome events or deaths were adjudicated independently.

Results

Of 874 patients (mean age=59 years; 51% men) who received Visipaque, 857 were included in the efficacy analysis. Using cardiac outcomes as the endpoint, the sensitivity of CCTA was 96.1, 95.8, and 94.7%, specificity was 84.5, 86.6, and 87.0%, and negative predictive value more than 99.0% at 1, 6, and 12 months, respectively. At 12 months, the rate of MACE was 5.7% (10/174) in patients with a positive CCTA (one or more ≥50% stenosis) and 0.1% (1/683) in patients with a negative CCTA (99.9% MACE-free survival rate). The Cox proportional hazards analysis with CCTA outcome, age, sex, reasons for CCTA, and cardiac risk factors as covariates showed a hazard ratio of 87.6 for positive versus negative CCTA (P=0.0001).

Conclusion

CCTA is a highly accurate, noninvasive tool to detect or rule out subsequent cardiovascular events in patients with intermediate pretest probability of CAD or an uninterpretable/equivocal stress test. A positive CCTA finding contributed significantly toward the prediction of subsequent MACE whereas a negative CCTA carried excellent prognostic outcomes at 12 months.

Background

Although several studies have shown promise for noninvasive angiography by coronary computed tomographic angiography (CCTA), few prospective multicenter trials have been conducted. This study evaluated the diagnostic accuracy of Visipaque enhanced CCTA to detect obstructive coronary stenosis compared with quantitative coronary angiography (QCA).

Patients and methods

Three sites prospectively enrolled 77 patients (58.1% men, 54 years) with chest pain referred for invasive coronary angiography (ICA). Patients underwent CCTA (Lightspeed VCT/Visipaque 320) before ICA. CCTAs were graded on a 15-segment American Heart Association model by a CCTA core lab with blinded readers for the presence of obstructive stenosis (>50% or >70%); ICAs were independently graded for %stenosis by QCA, considered the reference standard. The efficacy of CCTA was assessed including all vessel segments for per-patient and per-vessel analyses.

Results

A total of 46 more than 50% stenoses in 27 (35%) patients, and 31 more than 70% stenoses in 20 (26%) patients, were identified by QCA. Per-patient and per-vessel efficacy of CCTA compared with QCA yielded sensitivities of 85% and specificities of 90 and 95%, respectively.

Conclusion

This study shows the high accuracy of CCTA to reliably detect more than 50% and more than 70% stenoses in low-probability to intermediate-probability chest pain patients being referred for ICA. The high negative predictive values observed (92-100%) indicate that CCTA is also an effective noninvasive alternative to exclude obstructive coronary stenosis.

The sequences of M proteins, the major surface-associated virulence factors of the widespread bacterial pathogen group A Streptococcus, are antigenically variable but have in common a strong propensity to form coiled coils. Paradoxically, these sequences are also replete with coiled-coil destabilizing residues. These features are evident in the irregular coiled-coil structure and thermal instability of M proteins. We present an explanation for this paradox through studies of the B repeats of the medically important M1 protein. The B repeats are required for interaction of M1 with fibrinogen (Fg) and consequent proinflammatory activation. The B repeats sample multiple conformations, including intrinsically disordered, dissociated, as well as two alternate coiled-coil conformations: a Fg-nonbinding register 1 and a Fg-binding register 2. Stabilization of M1 in the Fg-nonbinding register 1 resulted in attenuation of Fg binding as expected, but counterintuitively, so did stabilization in the Fg-binding register 2. Strikingly, these register-stabilized M1 proteins gained the ability to bind Fg when they were destabilized by a chaotrope. These results indicate that M1 stability is antithetical to Fg interaction and that M1 conformational dynamics, as specified by destabilizing residues, are essential for interaction. A "capture-and-collapse" model of association accounts for these observations, in which M1 captures Fg through a dynamic conformation and then collapses into a register 2-coiled coil as a result of stabilization provided by binding energy. Our results support the general conclusion that destabilizing residues are evolutionarily conserved in M proteins to enable functional interactions necessary for pathogenesis.

Recently, we showed that endothelial heparan sulfate facilitates entry of a bacterial pathogen into the central nervous system. Here, we show that normal bactericidal activity of neutrophils is influenced by the sulfation pattern of heparan sulfate. Inactivation of heparan sulfate uronyl 2-O-sulfotransferase (Hs2st) in neutrophils substantially reduced their bactericidal activity, and Hs2st deficiency rendered mice more susceptible to systemic infection with the pathogenic bacterium group B Streptococcus. Specifically, altered sulfation of heparan sulfate in mutant neutrophils affected formation of neutrophil extracellular traps while not influencing phagocytosis, production of reactive oxygen species, or secretion of granular proteases. Heparan sulfate proteoglycan(s) is present in neutrophil extracellular traps, modulates histone affinity, and modulates their microbial activity. Hs2st-deficient brain endothelial cells show enhanced binding to group B Streptococcus and are more susceptible to apoptosis, likely contributing to the observed increase in dissemination of group B Streptococcus into the brain of Hs2st-deficient mice following intravenous challenge. Taken together, our data provide strong evidence that heparan sulfate from both neutrophils and the endothelium plays important roles in modulating innate immunity.

Histones are essential elements of chromatin structure and gene regulation in eukaryotes. An unexpected attribute of these nuclear proteins is their antimicrobial activity. A framework for histone release and function in host defense in vivo was revealed with the discovery of neutrophil extracellular traps, a specialized cell death process in which DNA-based structures containing histones are extruded to ensnare and kill bacteria. Investigating the susceptibility of various Gram-positive pathogens to histones, we found high-level resistance by one leading human pathogen, group A Streptococcus (GAS). A screen of isogenic mutants revealed that the highly surface-expressed M1 protein, a classical GAS virulence factor, was required for high-level histone resistance. Biochemical and microscopic analyses revealed that the N-terminal domain of M1 protein binds and inactivates histones before they reach their cell wall target of action. This finding illustrates a new pathogenic function for this classic GAS virulence factor, and highlights a potential innate immune evasion strategy that may be employed by other bacterial pathogens.

Background

The impact of autosomal dominant polycystic kidney disease (ADPKD) on health-related quality of life (HRQoL) is not well understood due to a lack of instruments specific to the condition.

Study design

Content for a new self-administered patient-reported outcome (PRO) questionnaire to assess ADPKD-related HRQoL was developed through clinical expert and patient focus group discussions. The new PRO instrument was administered to study patients with ADPKD to evaluate its reliability and validity.

Setting & participants

1,674 adult patients with ADPKD participated in this research: 285 patients in focus groups to generate questionnaire content, 15 patients in debriefing interviews to refine the PRO questionnaire, and 1,374 patients to assess the performance and measurement properties of the PRO questionnaire.

Outcome

A new PRO questionnaire.

Results

The ADPKD Impact Scale (ADPKD-IS), consisting of 14 items representing 3 conceptual domains (physical, emotional, and fatigue) plus 4 additional questions, was developed. The instrument's reliability (regarding internal consistency and test-retest consistency) and validity (content and construct) were supported.

Limitations

Need for more responsiveness testing when more data from clinical use become available over time. Complex concepts such as ADPKD-related pain and impact on a patient's HRQoL need further evaluation.

Conclusions

The ADPKD-IS is a new patient-centric tool that reliably and validly provides a standardized method for assessing HRQoL and overall disease burden in patients with ADPKD.

Recently, we showed that endothelial heparan sulfate facilitates entry of a bacterial pathogen into the central nervous system. Here, we show that normal bactericidal activity of neutrophils is influenced by the sulfation pattern of heparan sulfate. Inactivation of heparan sulfate uronyl 2-O-sulfotransferase (Hs2st) in neutrophils substantially reduced their bactericidal activity, and Hs2st deficiency rendered mice more susceptible to systemic infection with the pathogenic bacterium group B Streptococcus. Specifically, altered sulfation of heparan sulfate in mutant neutrophils affected formation of neutrophil extracellular traps while not influencing phagocytosis, production of reactive oxygen species, or secretion of granular proteases. Heparan sulfate proteoglycan(s) is present in neutrophil extracellular traps, modulates histone affinity, and modulates their microbial activity. Hs2st-deficient brain endothelial cells show enhanced binding to group B Streptococcus and are more susceptible to apoptosis, likely contributing to the observed increase in dissemination of group B Streptococcus into the brain of Hs2st-deficient mice following intravenous challenge. Taken together, our data provide strong evidence that heparan sulfate from both neutrophils and the endothelium plays important roles in modulating innate immunity.

Unlabelled

Inhibitory CD33-related Siglec receptors regulate immune cell activation upon engaging ubiquitous sialic acids (Sias) on host cell surface glycans. Through molecular mimicry, Sia-expressing pathogen group B Streptococcus binds inhibitory human Siglec-9 (hSiglec-9) to blunt neutrophil activation and promote bacterial survival. We unexpectedly discovered that hSiglec-9 also specifically binds high molecular weight hyaluronan (HMW-HA), another ubiquitous host glycan, through a region of its terminal Ig-like V-set domain distinct from the Sia-binding site. HMW-HA recognition by hSiglec-9 limited neutrophil extracellular trap (NET) formation, oxidative burst, and apoptosis, defining HMW-HA as a regulator of neutrophil activation. However, the pathogen group A Streptococcus (GAS) expresses a HMW-HA capsule that engages hSiglec-9, blocking NET formation and oxidative burst, thereby promoting bacterial survival. Thus, a single inhibitory lectin receptor detects two distinct glycan "self-associated molecular patterns" to maintain neutrophil homeostasis, and two leading human bacterial pathogens have independently evolved molecular mimicry to exploit this immunoregulatory mechanism.

Key message

HMW-HA is the first example of a non-sialic acid containing glycan to be recognized by CD33-related Siglecs. HMW-HA engagement of hSiglec-9 attenuates neutrophil activation. Group A Streptococcus exploits hSiglec-9 recognition via its polysaccharide HMW-HA capsule to subvert neutrophil killing.

Classification of streptococci is based upon expression of unique cell wall carbohydrate antigens. All serotypes of group A Streptococcus (GAS; Streptococcus pyogenes), a leading cause of infection-related mortality worldwide, express the group A carbohydrate (GAC). GAC, the classical Lancefield antigen, is comprised of a polyrhamnose backbone with N-acetylglucosamine (GlcNAc) side chains. The immunodominant GlcNAc epitope of GAC is the basis of all rapid diagnostic testing for GAS infection. We previously identified the 12-gene GAC biosynthesis gene cluster and determined that the glycosyltransferase GacI was required for addition of the GlcNAc side chain to the polyrhamnose core. Loss of the GAC GlcNAc epitope in serotype M1 GAS resulted in attenuated virulence in two animal infection models and increased GAS sensitivity to killing by whole human blood, serum, neutrophils, and antimicrobial peptides. Here, we report that the GAC biosynthesis gene cluster is ubiquitous among 520 GAS isolates from global sources, representing 105 GAS emm serotypes. Isogenic ΔgacI mutants were constructed in M2, M3, M4, M28, and M89 backgrounds and displayed an array of phenotypes in susceptibility to killing by whole human blood, baby rabbit serum, human platelet releasate, human neutrophils, and antimicrobial peptide LL-37. The contribution of the GlcNAc side chain to GAS survival in vivo also varied by strain, demonstrating that it is not a prerequisite for virulence in the murine infection model. Thus, the relative contribution of GAC to virulence in non-M1 serotypes appears to depend on the quorum of other virulence factors that each strain possesses.IMPORTANCE The Lancefield group A carbohydrate (GAC) is the species-defining antigen for group A Streptococcus (GAS), comprising ~50% of the cell wall of this major human pathogen. We previously showed that the GlcNAc side chain of GAC contributes to the innate immune resistance and animal virulence phenotypes of the globally disseminated strain of serotype M1 GAS. Here, we use isogenic mutagenesis to examine the role of GAC GlcNAc in five additional medically relevant GAS serotypes. Overall, the GlcNAc side chain of GAC contributes to the innate immune resistance of GAS, but the relative contribution varies among individual strains. Moreover, the GAC GlcNAc side chain is not a universal prerequisite for GAS virulence in the animal model.