Conservation of C4BP-binding sequence patterns in Streptococcus pyogenes M and Enn proteins.

Antigenically sequence variable M proteins of the major bacterial pathogen Streptococcus pyogenes (Strep A) are responsible for recruiting human C4b-binding protein (C4BP) to the bacterial surface, which enables Strep A to evade destruction by the immune system. The most sequence divergent portion of M proteins, the hypervariable region (HVR), is responsible for binding C4BP. Structural evidence points to the conservation of two C4BP-binding sequence patterns (M2 and M22) in the HVR of numerous M proteins, with this conservation applicable to vaccine immunogen design. These two patterns, however, only partially explain C4BP binding by Strep A. Here, we identified several M proteins that lack these patterns but still bind C4BP and determined the structures of two, M68 and M87 HVRs, in complex with a C4BP fragment. Mutagenesis of these M proteins led to the identification of amino acids that are crucial for C4BP binding, enabling formulation of new C4BP-binding patterns. Mutagenesis was also carried out on M2 and M22 proteins to refine or generate experimentally grounded C4BP-binding patterns. The M22 pattern was the most prevalent among M proteins, followed by the M87 and M2 patterns, while the M68 pattern was rare. These patterns, except for M68, were also evident in numerous M-like Enn proteins. Binding of C4BP via these patterns to Enn proteins was verified. We conclude that C4BP-binding patterns occur frequently in Strep A strains of differing M types, being present in their M or Enn proteins, or frequently both, providing further impetus for their use as vaccine immunogens.

Molecular characterization of the interaction between human IgG and the M-related proteins from Streptococcus pyogenes.

Group A Streptococcal M-related proteins (Mrps) are dimeric α-helical-coiled-coil cell membrane-bound surface proteins. During infection, Mrp recruit the fragment crystallizable region of human immunoglobulin G via their A-repeat regions to the bacterial surface, conferring upon the bacteria enhanced phagocytosis resistance and augmented growth in human blood. However, Mrps show a high degree of sequence diversity, and it is currently not known whether this diversity affects the Mrp-IgG interaction. Herein, we report that diverse Mrps all bind human IgG subclasses with nanomolar affinity, with differences in affinity which ranged from 3.7 to 11.1 nM for mixed IgG. Using surface plasmon resonance, we confirmed Mrps display preferential IgG-subclass binding. All Mrps were found to have a significantly weaker affinity for IgG3 (p < 0.05) compared to all other IgG subclasses. Furthermore, plasma pulldown assays analyzed via Western blotting revealed that all Mrp were able to bind IgG in the presence of other serum proteins at both 25 °C and 37 °C. Finally, we report that dimeric Mrps bind to IgG with a 1:1 stoichiometry, enhancing our understanding of this important host-pathogen interaction.

NLRP12 Senses the SARS-CoV-2 Membrane Protein and Promotes an Inflammatory Response.

COVID-19 is an acute respiratory disorder that is caused by SARS-CoV-2, in which excessive systemic inflammation is associated with adverse patient clinical outcomes. Here, we observed elevated expression levels of NLRP12 (nucleotide-binding leucine-rich repeat-containing receptor 12) in human peripheral monocytes and lung tissue during infection with SARS-CoV-2. Co-immunoprecipitation analysis revealed that NLRP12 directly interacted with the M protein through its leucine-rich repeat domain. Moreover, in vitro studies demonstrated that NLRP12 interacted with TRAF3 and promoted its ubiquitination and degradation, which counteracted the inhibitory effect of TRAF3 on the NF-κB/MAPK signaling pathway and promoted the production of inflammatory cytokines. Furthermore, an in vivo study revealed that NLRP12 knockout mice displayed attenuated tissue injury and ameliorated inflammatory responses in the lungs when infected with a SARS-CoV-2 M protein-reconstituted pseudovirus and mouse coronavirus. Taken together, these findings suggest that NLRP12 mediates the inflammatory responses during coronavirus infection.

A conserved 3D pattern in a Streptococcus pyogenes M protein immunogen elicits M-type crossreactivity.

Coiled coil-forming M proteins of the widespread and potentially deadly bacterial pathogen Streptococcus pyogenes (strep A) are immunodominant targets of opsonizing antibodies. However, antigenic sequence variability of M proteins into >220 M types, as defined by their hypervariable regions (HVRs), is considered to limit M proteins as vaccine immunogens because of type specificity in the antibody response. Surprisingly, a multi-HVR immunogen in clinical vaccine trials was shown to elicit M-type crossreactivity. The basis for this crossreactivity is unknown but may be due in part to antibody recognition of a 3D pattern conserved in many M protein HVRs that confers binding to human complement C4b-binding protein (C4BP). To test this hypothesis, we investigated whether a single M protein immunogen carrying the 3D pattern would elicit crossreactivity against other M types carrying the 3D pattern. We found that a 34-amino acid sequence of S. pyogenes M2 protein bearing the 3D pattern retained full C4BP-binding capacity when fused to a coiled coil-stabilizing sequence from the protein GCN4. We show that this immunogen, called M2G, elicited cross-reactive antibodies against a number of M types that carry the 3D pattern but not against those that lack the 3D pattern. We further show that the M2G antiserum-recognized M proteins displayed natively on the strep A surface and promoted the opsonophagocytic killing of strep A strains expressing these M proteins. As C4BP binding is a conserved virulence trait of strep A, we propose that targeting the 3D pattern may prove advantageous in vaccine design.

Early M-protein immune reconstitution after autologous haematopoietic stem cell transplantation is a good prognostic marker for patients with high-risk cytogenetic multiple myeloma.

The presence of transient abnormal protein banding (M-protein immune reconstitution) in serum immunofixation electrophoresis after autologous haematopoietic stem cell transplantation in patients with multiple myeloma has been reported. The purpose of this study was to investigate the impact of post-transplant M-protein immune reconstitution on the prognosis of patients with multiple myeloma. M-protein immune reconstitution was observed in 25.9% (75/290) of patients. The CR rate and MRD negativity were higher in the M-protein immune reconstitution group (85.3% vs. 69.3%, p = 0.013, 81.9% vs. 66.5%, p = 0.014). Although there were no significant differences between the groups, the overall median survival time was longer in the M-protein immune reconstruction group (80 vs. 72 m, p = 0.076; not reached vs. 105 m, p = 0.312). Among patients in the cytogenetic high-risk group, the occurrence of M-protein immune reconstitution predicted better PFS and OS (80 vs. 31 m, p = 0.010; not reached vs. 91 m, p = 0.026). Additionally, in revised-International Staging System stage III patients, PFS and OS were better in those who achieved M-protein immune reconstitution (80 vs. 20 m, p = 0.025; 57 vs. 32 m, p = 0.103). The better prognosis of M-protein immune reconstitution patients may be associated with the acquisition of a deeper response. In high-risk patients, early acquisition of M-protein immune reconstitution may suggest a better prognosis.

Cryoglobulinemic Vasculitis in Disguise: Cryofibrinogenemia as Variant of Monoclonal Gammopathy of Renal Significance.

Monoclonal gammopathy with cryoactivity (ie, cryoglobulins) that causes glomerulonephritis is considered within the spectrum of monoclonal gammopathy of renal significance. Cryofibrinogenemia (cryoactivity of coagulation factors) is very rarely associated with glomerulonephritis. We present a 39-year-old woman with a relapsing nephrotic syndrome. Laboratory investigation detected cryofibrinogen; the precipitate consisted of fibrinogen and a monoclonal immunoglobulin (M-protein; IgG-λ), and the latter was also detected in serum (4g/L). Initial conventional immunosuppressive therapy resulted in temporary renal remission. In view of the M-protein, subsequent therapy consisted of bortezomib/dexamethasone and high-dose melphalan followed by autologous hematopoietic stem cell transplantation, and resulted in a very good partial hematological response and temporary renal remission. However, after hematological and renal relapse, we performed unique experiments to clarify the role of the M-protein. Mixing patient serum with donor plasma resulted in cryoactivity, composed of M-protein+fibrinogen. Patient plasma deprived of M-protein did not have cryoactivity. Therefore, cryoactivity was dependent on the M-protein. We started lenalidomide, which resulted in very good partial hematological and renal remission. Thus, cryofibrinogenemia can be the consequence of an M-protein, which we suggest should be defined as monoclonal gammopathy of renal significance.

N-linked glycosylation of the M-protein variable region: glycoproteogenomics reveals a new layer of personalized complexity in multiple myeloma.

OBJECTIVES: Multiple myeloma (MM) is a plasma cell malignancy characterized by a monoclonal expansion of plasma cells that secrete a characteristic M-protein. This M-protein is crucial for diagnosis and monitoring of MM in the blood of patients. Recent evidence has emerged suggesting that N-glycosylation of the M-protein variable (Fab) region contributes to M-protein pathogenicity, and that it is a risk factor for disease progression of plasma cell disorders. Current methodologies lack the specificity to provide a site-specific glycoprofile of the Fab regions of M-proteins. Here, we introduce a novel glycoproteogenomics method that allows detailed M-protein glycoprofiling by integrating patient specific Fab region sequences (genomics) with glycoprofiling by glycoproteomics. METHODS: Glycoproteogenomics was used for the detailed analysis of de novo N-glycosylation sites of M-proteins. First, Genomic analysis of the M-protein variable region was used to identify de novo N-glycosylation sites. Subsequently glycopeptide analysis with LC-MS/MS was used for detailed analysis of the M-protein glycan sites. RESULTS: Genomic analysis uncovered a more than two-fold increase in the Fab Light Chain N-glycosylation of M-proteins of patients with Multiple Myeloma compared to Fab Light Chain N-glycosylation of polyclonal antibodies from healthy individuals. Subsequent glycoproteogenomics analysis of 41 patients enrolled in the IFM 2009 clinical trial revealed that the majority of the Fab N-glycosylation sites were fully occupied with complex type glycans, distinguishable from Fc region glycans due to high levels of sialylation, fucosylation and bisecting structures. CONCLUSIONS: Together, glycoproteogenomics is a powerful tool to study de novo Fab N-glycosylation in plasma cell dyscrasias.

Evaluating the HYDRASHIFT 2/4 Daratumumab assay: a powerful approach to assess treatment response in multiple myeloma.

OBJECTIVES: This study evaluates the HYDRASHIFT assay's effectiveness in mitigating daratumumab interference on serum protein tests during multiple myeloma (MM) treatment, aiming to ensure an accurate assessment of treatment response. METHODS: We analyzed 113 serum samples from 68 MM patients undergoing daratumumab treatment, employing both standard IF and the HYDRASHIFT assay. The assay's precision was determined through intra-day and inter-day variability assessments, while its specificity was verified using serum samples devoid of daratumumab. Comparative analysis of IF results, before and after the application of the HYDRASHIFT assay, facilitated the categorization of treatment responses in alignment with the International Myeloma Working Group's response criteria. RESULTS: The precision underscored the assay's consistent repeatability and reproducibility, successfully eliminating interference of daratumumab-induced Gκ bands. Specificity assessments demonstrated the assay's capability to distinguish daratumumab from both isatuximab and naturally occurring M-proteins. Of the analyzed cases, 91 exhibited successful migration of daratumumab-induced Gκ bands, thereby enhancing the accuracy of treatment response classification. The remaining 22 cases did not show a visible migration complex, likely due to the low concentration of daratumumab in the serum. These findings underscore the assay's critical role in distinguishing daratumumab from endogenous M-protein, particularly in samples with a single Gκ band on standard IF, where daratumumab and endogenous M-protein had co-migrated. CONCLUSIONS: The HYDRASHIFT assay demonstrates high precision, specificity, and utility in the accurate monitoring of treatment responses in MM patients receiving daratumumab. This assay represents a significant advancement in overcoming the diagnostic challenges posed by daratumumab interference.

M-protein diagnostics in multiple myeloma patients using ultra-sensitive targeted mass spectrometry and an off-the-shelf calibrator.

OBJECTIVES: Minimal residual disease status in multiple myeloma is an important prognostic biomarker. Recently, personalized blood-based targeted mass spectrometry (MS-MRD) was shown to provide a sensitive and minimally invasive alternative to measure minimal residual disease. However, quantification of MS-MRD requires a unique calibrator for each patient. The use of patient-specific stable isotope labelled (SIL) peptides is relatively costly and time-consuming, thus hindering clinical implementation. Here, we introduce a simplification of MS-MRD by using an off-the-shelf calibrator. METHODS: SILuMAB-based MS-MRD was performed by spiking a monoclonal stable isotope labeled IgG, SILuMAB-K1, in the patient serum. The abundance of both M-protein-specific peptides and SILuMAB-specific peptides were monitored by mass spectrometry. The relative ratio between M-protein peptides and SILuMAB peptides allowed for M-protein quantification. We assessed linearity, sensitivity and reproducibility of SILuMAB-based MS-MRD in longitudinally collected sera from the IFM-2009 clinical trial. RESULTS: A linear dynamic range was achieved of over 5 log scales, allowing for M-protein quantification down to 0.001 g/L. The inter-assay CV of SILuMAB-based MS-MRD was on average 11 %. Excellent concordance between SIL- and SILuMAB-based MS-MRD was shown (R2>0.985). Additionally, signal intensity of spiked SILuMAB can be used for quality control purpose to assess system performance and incomplete SILuMAB digestion can be used as quality control for sample preparation. CONCLUSIONS: Compared to SIL peptides, SILuMAB-based MS-MRD improves the reproducibility, turn-around-times and cost-efficacy of MS-MRD without diminishing its sensitivity and specificity. Furthermore, SILuMAB can be used as a MS-MRD quality control tool to monitor sample preparation efficacy and assay performance.

An automated workflow based on data independent acquisition for practical and high-throughput personalized assay development and minimal residual disease monitoring in multiple myeloma patients.

OBJECTIVES: Minimal residual disease (MRD) status in multiple myeloma (MM) is an important prognostic biomarker. Personalized blood-based targeted mass spectrometry detecting M-proteins (MS-MRD) was shown to provide a sensitive and minimally invasive alternative to MRD-assessment in bone marrow. However, MS-MRD still comprises of manual steps that hamper upscaling of MS-MRD testing. Here, we introduce a proof-of-concept for a novel workflow using data independent acquisition-parallel accumulation and serial fragmentation (dia-PASEF) and automated data processing. METHODS: Using automated data processing of dia-PASEF measurements, we developed a workflow that identified unique targets from MM patient sera and personalized protein sequence databases. We generated patient-specific libraries linked to dia-PASEF methods and subsequently quantitated and reported M-protein concentrations in MM patient follow-up samples. Assay performance of parallel reaction monitoring (prm)-PASEF and dia-PASEF workflows were compared and we tested mixing patient intake sera for multiplexed target selection. RESULTS: No significant differences were observed in lowest detectable concentration, linearity, and slope coefficient when comparing prm-PASEF and dia-PASEF measurements of serial dilutions of patient sera. To improve assay development times, we tested multiplexing patient intake sera for target selection which resulted in the selection of identical clonotypic peptides for both simplex and multiplex dia-PASEF. Furthermore, assay development times improved up to 25× when measuring multiplexed samples for peptide selection compared to simplex. CONCLUSIONS: Dia-PASEF technology combined with automated data processing and multiplexed target selection facilitated the development of a faster MS-MRD workflow which benefits upscaling and is an important step towards the clinical implementation of MS-MRD.

Direct Mass Spectrometry-Based Detection and Antibody Sequencing of Monoclonal Gammopathy of Undetermined Significance from Patient Serum: A Case Study.

Monoclonal gammopathy of undetermined significance (MGUS) is a plasma cell disorder characterized by the presence of a predominant monoclonal antibody (i.e., M-protein) in serum, without clinical symptoms. Here we present a case study in which we detect MGUS by liquid-chromatography coupled with mass spectrometry (LC-MS) profiling of IgG1 in human serum. We detected a Fab-glycosylated M-protein and determined the full heavy and light chain sequences by bottom-up proteomics techniques using multiple proteases, further validated by top-down LC-MS. Moreover, the composition and location of the Fab-glycan could be determined in CDR1 of the heavy chain. The outlined approach adds to an expanding mass spectrometry-based toolkit to characterize monoclonal gammopathies such as MGUS and multiple myeloma, with fine molecular detail. The ability to detect monoclonal gammopathies and determine M-protein sequences straight from blood samples by mass spectrometry provides new opportunities to understand the molecular mechanisms of such diseases.

Advances in minimal residual disease monitoring in multiple myeloma.

Multiple myeloma (MM) is characterized by the clonal expansion of plasma cells and the excretion of a monoclonal immunoglobulin (M-protein), or fragments thereof. This biomarker plays a key role in the diagnosis and monitoring of MM. Although there is currently no cure for MM, novel treatment modalities such as bispecific antibodies and CAR T-cell therapies have led to substantial improvement in survival. With the introduction of several classes of effective drugs, an increasing percentage of patients achieve a complete response. This poses new challenges to traditional electrophoretic and immunochemical M-protein diagnostics because these methods lack sensitivity to monitor minimal residual disease (MRD). In 2016, the International Myeloma Working Group (IMWG) expanded their disease response criteria with bone marrow-based MRD assessment using flow cytometry or next-generation sequencing in combination with imaging-based disease monitoring of extramedullary disease. MRD status is an important independent prognostic marker and its potential as a surrogate endpoint for progression-free survival is currently being studied. In addition, numerous clinical trials are investigating the added clinical value of MRD-guided therapy decisions in individual patients. Because of these novel clinical applications, repeated MRD evaluation is becoming common practice in clinical trials as well as in the management of patients outside clinical trials. In response to this, novel mass spectrometric methods that have been developed for blood-based MRD monitoring represent attractive minimally invasive alternatives to bone marrow-based MRD evaluation. This paves the way for dynamic MRD monitoring to allow the detection of early disease relapse, which may prove to be a crucial factor in facilitating future clinical implementation of MRD-guided therapy. This review provides an overview of state-of-the-art of MRD monitoring, describes new developments and applications of blood-based MRD monitoring, and suggests future directions for its successful integration into the clinical management of MM patients.

Dengue virus M and E proteins belonging to genotype II (Cosmopolitan) of serotype 2 are influenced by the nature of M residue 36.

Mosquito-borne dengue disease is caused by the dengue virus serotype-1 to serotype-4. The contemporary dengue outbreaks in the southwestern Indian ocean coincided with the widespread of dengue virus serotype 2 genotype II (Cosmopolitan), including epidemic viral strains DES-14 and RUN-18 isolated in Dar es Salaam (Tanzania) in 2014 and La Reunion Island (France) in 2018, respectively. Heterodimeric interaction between prM (intracellular precursor of surface structural M protein) and envelope E proteins is required during the initial stage of dengue virus assembly. Amino acid 127 of DES-14 prM protein (equivalent to M36) has been identified as an infrequent valine whereas RUN-18 has a common isoleucine. In the present study, we examined the effect of M-I36V mutation on the expression of a recombinant RUN-18 E protein co-expressed with prM in human epithelial A549 cells. The M ectodomain of dengue virus serotype 2 embeds a pro-apoptotic peptide referred as D2AMP. The impact of M-I36V mutation on the death-promoting capability of D2AMP was assessed in A549 cells. We showed that valine at position M36 affects expression of recombinant RUN-18 E protein and potentiates apoptosis-inducing activity of D2AMP. We propose that the nature of M residue 36 influences the virological characteristics of dengue 2 M and E proteins belonging to genotype II that contributes to global dengue burden.

Proteomic profiling identifies novel inflammation-related plasma proteins associated with ischemic stroke outcome.

BACKGROUND: The inflammatory response to cerebral ischemia is complex; however, most clinical studies of stroke outcome focus on a few selected proteins. We, therefore, aimed to profile a broad range of inflammation-related proteins to: identify proteins associated with ischemic stroke outcome that are independent of established clinical predictors; identify proteins subsets for outcome prediction; and perform sex and etiological subtype stratified analyses. METHODS: Acute-phase plasma levels of 65 inflammation-related proteins were measured in 534 ischemic stroke cases. Logistic regression was used to estimate associations to unfavorable 3-month functional outcome (modified Rankin Scale score > 2) and LASSO regressions to identify proteins with independent effects. RESULTS: Twenty proteins were associated with outcome in univariable models after correction for multiple testing (FDR < 0.05), and for 5 the association was independent of clinical variables, including stroke severity (TNFSF14 [LIGHT], OSM, SIRT2, STAMBP, and 4E-BP1). LASSO identified 9 proteins that could best separate favorable and unfavorable outcome with a predicted diagnostic accuracy (AUC) of 0.81; three associated with favorable (CCL25, TRAIL [TNFSF10], and Flt3L) and 6 with unfavorable outcome (CSF-1, EN-RAGE [S100A12], HGF, IL-6, OSM, and TNFSF14). Finally, we identified sex- and etiologic subtype-specific associations with the best discriminative ability achieved for cardioembolic, followed by cryptogenic stroke. CONCLUSIONS: We identified candidate blood-based protein biomarkers for post-stroke functional outcome involved in, e.g., NLRP3 inflammasome regulation and signaling pathways, such as TNF, JAK/STAT, MAPK, and NF-κB. These proteins warrant further study for stroke outcome prediction as well as investigations into the putative causal role for stroke outcome.

Nucleolar Protein Treacle Is Important for the Efficient Growth of Mumps Virus.

The nucleolus is the largest structure in the nucleus, and it plays roles in mediating cellular stress responses and regulating cell proliferation, as well as in ribosome biosynthesis. The nucleolus is composed of a variety of nucleolar factors that interact with each other in a complex manner to enable its function. Many viral proteins interact with nucleolar factors as well, affecting cellular morphology and function. Here, to investigate the association between mumps virus (MuV) infection and the nucleolus, we evaluated the necessity of nucleolar factors for MuV proliferation by performing a knockdown of these factors with small interfering (si)RNAs. Our results reveal that suppressing the expression of Treacle, which is required for ribosome biosynthesis, reduced the proliferative potential of MuV. Additionally, the one-step growth kinetics results indicate that Treacle knockdown did not affect the viral RNA and protein synthesis of MuV, but it did impair the production of infectious virus particles. Viral matrix protein (M) was considered a candidate Treacle interaction partner because it functions in the process of particle formation in the viral life cycle and is partially localized in the nucleolus. Our data confirm that MuV M can interact with Treacle and colocalize with it in the nucleolus. Furthermore, we found that viral infection induces relocalization of Treacle in the nucleus. Together, these findings suggest that interaction with Treacle in the nucleolus is important for the M protein to exert its functions late in the MuV life cycle. IMPORTANCE The nucleolus, which is the site of ribosome biosynthesis, is a target organelle for many viruses. It is increasingly evident that viruses can favor their own replication and multiplication by interacting with various nucleolar factors. In this study, we found that the nucleolar protein Treacle, known to function in the transcription and processing of pre-rRNA, is required for the efficient propagation of mumps virus (MuV). Specifically, our data indicate that Treacle is not involved in viral RNA or protein synthesis but is important in the processes leading to viral particle production in MuV infection. Additionally, we determined that MuV matrix protein (M), which functions mainly in viral particle assembly and budding, colocalized and interacted with Treacle. Furthermore, we found that Treacle is distributed throughout the nucleus in MuV-infected cells. Our research shows that the interaction between M and Treacle supports efficient viral growth in the late stage of MuV infection.

Expert-Level Immunofixation Electrophoresis Image Recognition based on Explainable and Generalizable Deep Learning.

BACKGROUND: Immunofixation electrophoresis (IFE) is important for diagnosis of plasma cell disorders (PCDs). Manual analysis of IFE images is time-consuming and potentially subjective. An artificial intelligence (AI) system for automatic and accurate IFE image recognition is desirable. METHODS: In total, 12 703 expert-annotated IFE images (9182 from a new IFE imaging system and 3521 from an old one) were used to develop and test an AI system that was an ensemble of 3 deep neural networks. The model takes an IFE image as input and predicts the presence of 8 basic patterns (IgA-, IgA-, IgG-, IgG-, IgM-, IgM-, light chain and ) and their combinations. Score-based class activation maps (Score-CAMs) were used for visual explanation of the models prediction. RESULTS: The AI model achieved an average accuracy, sensitivity, and specificity of 99.82, 93.17, and 99.93, respectively, for detection of the 8 basic patterns, which outperformed 4 junior experts with 1 years experience and was comparable to a senior expert with 5 years experience. The Score-CAMs gave a reasonable visual explanation of the prediction by highlighting the target aligned regions in the bands and indicating potentially unreliable predictions. When trained with only the new system images, the models performance was still higher than junior experts on both the new and old IFE systems, with average accuracy of 99.91 and 99.81, respectively. CONCLUSIONS: Our AI system achieved human-level performance in automatic recognition of IFE images, with high explainability and generalizability. It has the potential to improve the efficiency and reliability of diagnosis of PCDs.

Effects of lipemia on capillary serum protein electrophoresis in native ultra-lipemic material and intravenous lipid emulsion added sera.

OBJECTIVES: This study aims to investigate the effect of natural ultralipemic material (NULM) and intravenous lipid emulsion (IVLE) on capillary serum protein electrophoresis (SPEP). METHODS: NULM material was prepared from leftover patients' lipemic serum sample (triglyceride concentration >2,000 mg/dL) pool by a refrigerated high-speed centrifuge, and IVLE Omegaven lipid emulsion (30%) was used. Serum pools for interference study were prepared from patient samples for which serum protein electrophoresis was studied as Normal SPEP and M Peak SPEP. For both types of lipemia (DULM and IVLE), five pools with triglyceride concentrations of ∼4.52 mmol/L, ∼7.91 mmol/L, ∼14.69 mmol/L, ∼21.47 mmol/L, and ∼28.25 mmol/L were prepared. SPEP was studied in each pool with Sebia Capillarys Minicap. A repeated measure ANOVA test was used to determine the difference between the pools, and interferograms were used to evaluate the interference effect. RESULTS: Interference was not detected in IVLE added Normal SPEP and M Peak SPEP pools, either % or concentrations of fractions. In NULM-added Normal SPEP and M Peak SPEP pools, significant positive interference in albumin % (p=0.002 and p<0.001 respectively) and significant negative interference in gamma% (p<0.001 and p=0.005 respectively) and M protein peak (p=0.002) fractions were detected. However, significant positive interference was seen only for albumin concentration fractions (p<0.001 for both pools). CONCLUSIONS: It is vital to use NULM instead of IVLE solutions in lipemia interference studies for all laboratory tests, including CZE SPEP. The fractions concentration values calculated with the total protein concentration should be used for evaluating SPEP results.

Quorum Sensing Regulation of a Major Facilitator Superfamily Transporter Affects Multiple Streptococcal Virulence Factors.

Cell-cell signaling mediated by Rgg-family transcription factors and their cognate pheromones is conserved in Firmicutes, including all streptococci. In Streptococcus pyogenes, or group A strep (GAS), one of these systems, the Rgg2/3 quorum sensing (QS) system, has been shown to regulate phenotypes, including cellular aggregation and biofilm formation, lysozyme resistance, and macrophage immunosuppression. Here, we show the abundance of several secreted virulence factors (streptolysin O, SpyCEP, and M protein) decreases upon induction of QS. The main mechanism underlying the changes in protein levels appears to be transcriptional, occurs downstream of the QS circuit, and is dysregulated by the deletion of an Rgg2/3 QS-regulated major facilitator superfamily (MFS) transporter. Additionally, we identify this MFS transporter as the factor responsible for a previously observed increase in aminoglycoside sensitivity in QS-induced cells. IMPORTANCE The production of virulence factors is a tightly regulated process in bacterial pathogens. Efforts to elucidate the mechanisms by which genes are regulated may advance the understanding of factors influencing pathogen behavior or cellular physiology. This work finds expression of a major facilitator superfamily (MFS) transporter, which is governed by a quorum sensing (QS) system, impacts the expression of multiple virulence factors and accounts for QS-dependent antibiotic susceptibility. Although the mechanism underlying this effect is not clear, MFS orthologs with high sequence similarity from S. pneumoniae and S. porcinus were unable to substitute indicating substrate specificity of the GAS MFS gene. These findings demonstrate novel associations between expression of a transmembrane transporter and virulence factor expression and aminoglycoside transport.

Streptococcus co-opts a conformational lock in human plasminogen to facilitate streptokinase cleavage and bacterial virulence.

Virulent strains of Streptococcus pyogenes (gram-positive group A Streptococcus pyogenes [GAS]) recruit host single-chain human plasminogen (hPg) to the cell surface-where in the case of Pattern D strains of GAS, hPg binds directly to the cells through a surface receptor, plasminogen-binding group A streptococcal M-protein (PAM). The coinherited Pattern D GAS-secreted streptokinase (SK2b) then accelerates cleavage of hPg at the R561-V562 peptide bond, resulting in the disulfide-linked two-chain protease, human plasmin (hPm). hPm localizes on the bacterial surface, assisting bacterial dissemination via proteolysis of host defense proteins. Studies using isolated domains from PAM and hPg revealed that the A-domain of PAM binds to the hPg kringle-2 module (K2hPg), but how this relates to the function of the full-length proteins is unclear. Herein, we use intact proteins to show that the lysine-binding site of K2hPg is a major determinant of the activation-resistant T-conformation of hPg. The binding of PAM to the lysine-binding site of K2hPg relaxes the conformation of hPg, leading to a greatly enhanced activation rate of hPg by SK2b. Domain swapping between hPg and mouse Pg emphasizes the importance of the Pg latent heavy chain (residues 1-561) in PAM binding and shows that while SK2b binds to both hPg and mouse Pg, the activation properties of streptokinase are strictly attributed to the serine protease domain (residues 562-791) of hPg. Overall, these data show that native hPg is locked in an activation-resistant conformation that is relaxed upon its direct binding to PAM, allowing hPm to form and provide GAS cells with a proteolytic surface.

Distinct Serotypes of Streptococcal M Proteins Mediate Fibrinogen-Dependent Platelet Activation and Proinflammatory Effects.

Sepsis is a life-threatening complication of infection that is characterized by a dysregulated inflammatory state and disturbed hemostasis. Platelets are the main regulators of hemostasis, and they also respond to inflammation. The human pathogen Streptococcus pyogenes can cause local infection that may progress to sepsis. There are more than 200 serotypes of S. pyogenes defined according to sequence variations in the M protein. The M1 serotype is among 10 serotypes that are predominant in invasive infection. M1 protein can be released from the surface and has previously been shown to generate platelet, neutrophil, and monocyte activation. The platelet-dependent proinflammatory effects of other serotypes of M protein associated with invasive infection (M3, M5, M28, M49, and M89) are now investigated using a combination of multiparameter flow cytometry, enzyme-linked immunosorbent assay (ELISA), aggregometry, and quantitative mass spectrometry. We demonstrate that only M1, M3, and M5 protein serotypes can bind fibrinogen in plasma and mediate fibrinogen- and IgG-dependent platelet activation and aggregation, release of granule proteins, upregulation of CD62P to the platelet surface, and complex formation with neutrophils and monocytes. Neutrophil and monocyte activation, determined as upregulation of surface CD11b, is also mediated by M1, M3, and M5 protein serotypes, while M28, M49, and M89 proteins failed to mediate activation of platelets or leukocytes. Collectively, our findings reveal novel aspects of the immunomodulatory role of fibrinogen acquisition and platelet activation during streptococcal infections.