Functional Dissection of Streptococcus pyogenes M5 Protein: the Hypervariable Region is Essential for Virulence

The surface-localized M protein of Streptococcus pyogenes is a major virulence factor that inhibits phagocytosis, as determined ex vivo. Because little is known about the role of M protein in vivo we analyzed the contribution of different M protein regions to virulence, using the fibrinogen (Fg)-binding M5 protein and a mouse model of acute invasive infection. This model was suitable, because M5 is required for mouse virulence and binds mouse and human Fg equally well, as shown here. Mixed infection experiments with wild type bacteria demonstrated that mutants lacking the N-terminal hypervariable region (HVR) or the Fg-binding B-repeat region were strongly attenuated, while a mutant lacking the conserved C-repeats was only slightly attenuated. Because the HVR of M5 is not required for phagocytosis resistance, our data imply that this HVR plays a major but unknown role during acute infection. The B-repeat region is required for phagocytosis resistance and specifically binds Fg, suggesting that it promotes virulence by binding Fg. However, B-repeat mutants were attenuated even in Fg-deficient mice, implying that the B-repeats may have a second function, in addition to Fg-binding. These data demonstrate that two distinct M5 regions, including the HVR, are essential to virulence during the early stages of an infection. In particular, our data provide the first in vivo evidence that the HVR of an M protein plays a major role in virulence, focusing interest on the molecular role of this region

Interactions between streptococcal M proteins and human plasma proteins

Streptococcus pyogenes is a common human pathogen, which causes a variety of diseases. A major virulence factor of S. pyogenes is the surface-associated M protein, which mediates resistance to phagocytosis. Several studies indicate that M proteins inhibit complement deposition and thereby phagocytosis. It is often assumed that S. pyogenes lacking M protein activates complement via the alternative pathway and that M protein inhibits complement deposition via this pathway. However, recent evidence indicates that many S. pyogenes strains actually activate the classical pathway, and that the M proteins in these strains inhibit deposition via this pathway. For this purpose, the bacteria recruit the human complement inhibitor C4b-binding protein (C4BP), which specifically inhibits the classical pathway. The two first papers in this thesis are focused on the biological properties of M proteins that do not bind C4BP. These M proteins bind the human plasma protein fibrinogen (Fg). In the first paper, we studied the role of bound Fg under non-immune conditions, i.e. in the absence of opsonic antibodies, and found that bacteria-bound Fg inhibits complement deposition via the classical pathway, and hence phagocytosis. Thus, the available data indicate that M proteins may recruit either C4BP or Fg to inhibit complement deposition, which occurs via the classical pathway even under non-immune conditions. These findings allow us to propose a unifying mechanism by which M proteins recruit a human plasma protein to interfere with complement deposition and phagocytosis. The second paper analyzes the role of M protein under immune conditions, i.e. in the presence of anti-M antibodies. It is known that antibodies to the N-terminal hypervariable region (HVR) are opsonic, but the role of antibodies to other regions in M protein have remained unclear. We used the M5 protein to study this problem. In initial studies we showed that only the Fg-binding B-repeat region of this M protein is required for phagocytosis resistance under non-immune conditions. However, only antibodies to the HVR were able to opsonize M5 bacteria. This apparent paradox could be explained by the demonstration that Fg and albumin specifically inhibited binding of antibodies to the B- and C-repeat regions, respectively, in the M5 protein. Thus, non-opsonizing epitopes in M5 correspond to regions that are covered by human plasma proteins, Fg and albumin, that block binding of anti-M antibodies, while opsonic epitopes allow binding of antibodies. These data provide a molecular explanation for the difference between opsonic and non-opsonic epitopes in an M protein. The third part of this thesis is focused on the study of an M protein-derived peptide, which binds human serum IgA, and the use of this peptide to develop a one-step purification method to isolate IgA from serum

Enhanced Inflammasome Activity in Patients with Psoriasis Promotes Systemic Inflammation

Psoriasis is linked to systemic inflammation and cardiovascular comorbidities, but studies of the underlying cellular mechanisms are lacking. The NLRP3 inflammasome is genetically associated with psoriasis, and its activation is increasingly linked with cardiovascular disease. In this study, we show that patients with psoriasis exhibited higher plasma levels of inflammasome-generated IL-1ß and IL-18, without any correlation to skin lesion severity. Increased constitutive expression of the inflammasome sensors NLRP3, NLRP1, and AIM2 was found in peripheral blood cells of the patients and also of those with mild disease, and this was accompanied by an increased caspase-1 reactivity in the myeloid blood subsets. TNF-a was found to activate selectively the NLRP3 inflammasome without the requirement for a priming signal. TNF-a was found to signal through the TNFR?caspase-8?caspase-1 alternative inflammasome pathway, which proceeds independently of pyroptosis. Patients who received anti-TNF therapy had normalized plasma IL-1ß and IL-18 levels as well as normalized caspase-1 reactivity. This was in contrast to the patients treated with methotrexate who exhibited persistent, increased caspase-1 reactivity. Thus, we show that the TNF-a-mediated activation of NLRP3 inflammasomes in patients with psoriasis may contribute to systemic inflammation. Anti-TNF therapy normalized inflammasome function, suggesting a mechanism for the cardiovascular risk?reducing effect.Funding agencies: Ingrid Asp Foundation, the Edvard Welander Foundation, the Swedish Psoriasis Association, and the Medical Research Council of Southeast Sweden.</p

Induction of a regulatory phenotype in human CD4(+) T cells by streptococcal M protein

Regulatory T cells (Tregs) participate in the control of the immune response. In the human system, an IL-10-secreting, T regulatory type 1 cell (Tr1)-like subset of Tregs can be induced by concurrent cross-linking of the TCR and CD46 on naive CD4(+) T cells. Because many viral and bacterial pathogens, including the major human pathogen Streptococcus pyogenes, bind to CD46, we asked whether this bacterium can directly induce Tr1-like cells through the streptococcal ligand for CD46, the M protein. The M5 and M22 proteins were found to induce T cells to develop into the IL-10-producing Tr1-like phenotype. Moreover, whole M5-expressing bacteria, but not isogenic M-negative bacteria, led to proliferation and IL-10 secretion by T cells. The interaction between the M5 protein and T cells was dependent on CD46 and the conserved C repeat region of M5. Supernatants derived from T cells stimulated with M proteins or M protein-expressing bacteria suppressed bystander T cell proliferation through IL-10 secretion. In addition, activation of CD46 through streptococcal M protein induced the expression of granzyme B, providing a second means for these cells to regulate an immune response. These findings suggest that binding to CD46 and exploiting its signaling pathway may represent a strategy employed by a number of important human pathogens to induce directly an immunosuppressive/regulatory phenotype in T cells

Isolation and detection of human IgA using a streptococcal IgA-binding peptide

Bacterial proteins that bind to the Fc part of IgG have found widespread use in immunology. A similar protein suitable for the isolation and detection of human IgA has not been described. Here, we show that a 50-residue synthetic peptide, designated streptococcal IgA-binding peptide (Sap) and derived from a streptococcal M protein, can be used for single-step affinity purification of human IgA. High affinity binding of IgA required the presence in Sap of a C-terminal cysteine residue, not present in the intact M protein. Passage of human serum through a Sap column caused depletion of >99% of the IgA, and elution of the column allowed quantitative recovery of highly purified IgA, for which the proportions of the IgA1 and IgA2 subclasses were the same as in whole serum. Moreover, immobilized Sap could be used for single-step purification of secretory IgA of both subclasses from human saliva, with a recovery of approximately 45%. The Sap peptide could also be used to specifically detect IgA bound to Ag. Together, these data indicate that Sap is a versatile Fc-binding reagent that may open new possibilities for the characterization of human IgA

The psoriasis-protective TYK2 I684S variant impairs IL-12 stimulated pSTAT4 response in skin-homing CD4+and CD8+memory T-cells

Tyrosine kinase 2 (TYK2) belongs to the Janus kinase (JAK) family of tyrosine kinases, which transmit signals from activated cytokine receptors. GWAS have consistently implicated TYK2 in psoriasis susceptibility. We performed an in-depth association analysis of TYK2 using GWAS and resequencing data. Strong genetic association of three nonsynonymous variants in the exonic regions of the TYK2 gene (rs34536443, rs12720356, and rs2304256) were found. rs12720356 encoding I684S is predicted to be deleterious based on its location in the pseudokinase domain. We analyzed PBMCs from 29 individuals representing the haplotypes containing each of the significantly associated signals. STAT4 phosphorylation was evaluated by phospho-flow cytometry after CD3/CD28 activation of cells followed by IL-12 stimulation. Individuals carrying the protective I684S variant manifested significantly reduced p-STAT4 levels in CD4 + CD25 + CD45RO + (mean Stimulation Index (S.I.) 48.08, n = 10) and CD8 + CD25 + CD45RO + cells (S.I. 55.71, n = 10), compared to controls homozygous for the ancestral haplotype (S.I. 68.19, n = 10 (p = 0.002) and 76.76 n = 10 (p = 0.0008) respectively). Reduced p-STAT4 levels were also observed in skin-homing, cutaneous lymphocyte associated antigen (CLA)-positive CD4 and CD8 cells from I684S carriers. No significant changes in p-STAT4 for the psoriasis-associated variant rs34536443 was found. These data establish the functional significance of the TYK2 I684S variant in psoriasis susceptibility.Funding Agencies|National Institutes of Health [R01AR042742, R01AR050511, R01AR054966, R01AR063611, R01AR065183, K01-AR072129, K01-AR064765]; GAIN award from the Foundation for the National Institutes of Health; Dermatology Foundation; National Psoriasis Foundation; Arthritis National Research Foundation; Ann Arbor Veterans Affairs Hospital; Dawn and Dudley Holmes Foundation; Babcock Memorial Trust; Ingrid Asp Foundation; Welander Foundation; Swedish Psoriasis Association; Medical Research Council</p

Accelerating translational research by clinically driven development of an informatics platform--a case study.

Translational medicine is becoming increasingly dependent upon data generated from health care, clinical research, and molecular investigations. This increasing rate of production and diversity in data has brought about several challenges, including the need to integrate fragmented databases, enable secondary use of patient clinical data from health care in clinical research, and to create information systems that clinicians and biomedical researchers can readily use. Our case study effectively integrates requirements from the clinical and biomedical researcher perspectives in a translational medicine setting. Our three principal achievements are (a) a design of a user-friendly web-based system for management and integration of clinical and molecular databases, while adhering to proper de-identification and security measures; (b) providing a real-world test of the system functionalities using clinical cohorts; and (c) system integration with a clinical decision support system to demonstrate system interoperability. We engaged two active clinical cohorts, 747 psoriasis patients and 2001 rheumatoid arthritis patients, to demonstrate efficient query possibilities across the data sources, enable cohort stratification, extract variation in antibody patterns, study biomarker predictors of treatment response in RA patients, and to explore metabolic profiles of psoriasis patients. Finally, we demonstrated system interoperability by enabling integration with an established clinical decision support system in health care. To assure the usefulness and usability of the system, we followed two approaches. First, we created a graphical user interface supporting all user interactions. Secondly we carried out a system performance evaluation study where we measured the average response time in seconds for active users, http errors, and kilobits per second received and sent. The maximum response time was found to be 0.12 seconds; no server or client errors of any kind were detected. In conclusion, the system can readily be used by clinicians and biomedical researchers in a translational medicine setting

Basic data elements and associated attributes per patient visit to the clinic, as in the Swedish Rheumatology Quality Registry.

<p>Basic data elements and associated attributes per patient visit to the clinic, as in the Swedish Rheumatology Quality Registry.</p