Fibrinolytic-deficiencies predispose hosts to septicemia from a catheter-associated UTI.

Catheter-associated urinary tract infections (CAUTIs) are amongst the most common nosocomial infections worldwide and are difficult to treat partly due to development of multidrug-resistance from CAUTI-related pathogens. Importantly, CAUTI often leads to secondary bloodstream infections and death. A major challenge is to predict when patients will develop CAUTIs and which populations are at-risk for bloodstream infections. Catheter-induced inflammation promotes fibrinogen (Fg) and fibrin accumulation in the bladder which are exploited as a biofilm formation platform by CAUTI pathogens. Using our established mouse model of CAUTI, here we identified that host populations exhibiting either genetic or acquired fibrinolytic-deficiencies, inducing fibrin deposition in the catheterized bladder, are predisposed to severe CAUTI and septicemia by diverse uropathogens in mono- and poly-microbial infections. Furthermore, here we found that Enterococcus faecalis, a prevalent CAUTI pathogen, uses the secreted protease, SprE, to induce fibrin accumulation and create a niche ideal for growth, biofilm formation, and persistence during CAUTI.

Antimicrobial peptide-conjugated phage-mimicking nanoparticles exhibit potent bactericidal action against Streptococcus pyogenes in murine wound infection models.

Streptococcus pyogenes is a causative agent for strep throat, impetigo, and more invasive diseases. The main reason for the treatment failure of streptococcal infections is increased antibiotic resistance. In recent years, infectious diseases caused by pyogenic streptococci resistant to multiple antibiotics have been rising with a significant impact on public health and the veterinary industry. The development of antibiotic resistance and the resulting emergence of multidrug-resistant bacteria have become primary threats to the public health system, commonly leading to nosocomial infections. Many researchers have turned their focus to developing alternative classes of antibacterial agent based on various nanomaterials. We have developed an antibiotic-free nanoparticle system inspired by naturally occurring bacteriophages to fight antibiotic-resistant bacteria. Our phage-mimicking nanoparticles (PhaNPs) display structural mimicry of protein-turret distribution on the head structure of bacteriophages. By mimicking phages, we can take advantage of their evolutionary constant shape and high antibacterial activity while avoiding the immune reactions of the human body experienced by biologically derived phages. We describe the synthesis of hierarchically arranged core-shell nanoparticles, with a silica core conjugated with silver-coated gold nanospheres to which we have chemisorbed the synthetic antimicrobial peptide Syn-71 on the PhaNPs surface, and increased the rapidity of the antibacterial activity of the nanoparticles (PhaNP@Syn71). The antibacterial effect of the PhaNP@Syn71 was tested in vitro and in vivo in mouse wound infection models. In vitro, results showed a dose-dependent complete inhibition of bacterial growth (>99.99%). Cytocompatibility testing on HaCaT human skin keratinocytes showed minimal cytotoxicity of PhaNP@Syn71, being comparable to the vehicle cytotoxicity levels even at higher concentrations, thus proving that our design is biocompatible with human cells. There was a minimum cutoff dosage above which there was no evolution of resistance after prolonged exposure to sub-MIC dosages of PhaNP@Syn71. Application of PhaNP@Syn71 to a mouse wound infection model exhibited high biocompatibility in vivo while showing immediate stabilization of the wound size, and infection free wound healing. Our results suggest the robust utility of antimicrobial peptide-conjugated phage-mimicking nanoparticles as a highly effective antibacterial system that can combat bacterial infections consistently while avoiding the emergence of resistant bacterial strains.

Fibrinolytic-deficiencies predispose hosts to septicemia from a catheter-associated UTI.

Catheter-associated urinary tract infections (CAUTIs) are amongst the most common nosocomial infections worldwide and are difficult to treat due to multi-drug resistance development among the CAUTI-related pathogens. Importantly, CAUTI often leads to secondary bloodstream infections and death. A major challenge is to predict when patients will develop CAUTIs and which populations are at-risk for bloodstream infections. Catheter-induced inflammation promotes fibrinogen (Fg) and fibrin accumulation in the bladder which are exploited as a biofilm formation platform by CAUTI pathogens. Using our established mouse model of CAUTI, we identified that host populations exhibiting either genetic or acquired fibrinolytic-deficiencies, inducing fibrin deposition in the catheterized bladder, are predisposed to severe CAUTI and septicemia by diverse uropathogens in mono- and poly-microbial infections. Furthermore, we found that E. faecalis, a prevalent CAUTI pathogen, uses the secreted protease, SprE, to induce fibrin accumulation and create a niche ideal for growth, biofilm formation, and persistence during CAUTI.

Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension.

BACKGROUND: Epigenetic regulation of vascular remodeling in pulmonary hypertension (PH) is poorly understood. Transcription regulating, histone acetylation code alters chromatin accessibility to promote transcriptional activation. Our goal was to identify upstream mechanisms that disrupt epigenetic equilibrium in PH. METHODS: Human pulmonary artery smooth muscle cells (PASMCs), human idiopathic pulmonary arterial hypertension (iPAH):human PASMCs, iPAH lung tissue, failed donor lung tissue, human pulmonary microvascular endothelial cells, iPAH:PASMC and non-iPAH:PASMC RNA-seq databases, NanoString nCounter, and cleavage under targets and release using nuclease were utilized to investigate histone acetylation, hyperacetylation targets, protein and gene expression, sphingolipid activation, cell proliferation, and gene target identification. SPHK2 (sphingosine kinase 2) knockout was compared with control C57BL/6NJ mice after 3 weeks of hypoxia and assessed for indices of PH. RESULTS: We identified that Human PASMCs are vulnerable to the transcription-promoting epigenetic mediator histone acetylation resulting in alterations in transcription machinery and confirmed its pathological existence in PH:PASMC cells. We report that SPHK2 is elevated as much as 20-fold in iPAH lung tissue and is elevated in iPAH:PASMC cells. During PH pathogenesis, nuclear SPHK2 activates nuclear bioactive lipid S1P (sphingosine 1-phosphate) catalyzing enzyme and mediates transcription regulating histone H3K9 acetylation (acetyl histone H3 lysine 9 [Ac-H3K9]) through EMAP (endothelial monocyte activating polypeptide) II. In iPAH lungs, we identified a 4-fold elevation of the reversible epigenetic transcription modulator Ac-H3K9:H3 ratio. Loss of SPHK2 inhibited hypoxic-induced PH and Ac-H3K9 in mice. We discovered that pulmonary vascular endothelial cells are a priming factor of the EMAP II/SPHK2/S1P axis that alters the acetylome with a specificity for PASMC, through hyperacetylation of histone H3K9. Using cleavage under targets and release using nuclease, we further show that EMAP II-mediated SPHK2 has the potential to modify the local transcription machinery of pluripotency factor KLF4 (Krüppel-like factor 4) by hyperacetylating KLF4 Cis-regulatory elements while deletion and targeted inhibition of SPHK2 rescues transcription altering Ac-H3K9. CONCLUSIONS: SPHK2 expression and its activation of the reversible histone H3K9 acetylation in human pulmonary artery smooth muscle cell represent new therapeutic targets that could mitigate PH vascular remodeling.

Inactivation of the lysine binding sites of human plasminogen (hPg) reveals novel structural requirements for the tight hPg conformation, M-protein binding, and rapid activation.

Accelerated activation of the human plasminogen zymogen (hPg) to two-chain active plasmin (hPm) is achieved following conformational changes induced by ligand-binding at the lysine-binding sites (LBSs) in four of the five hPg kringle domains. In this manner, pattern D skin-trophic strains of Group A streptococci (GAS), through the expression of surface plasminogen-binding M-protein (PAM), immobilize surface hPg, thereby enabling rapid hPg activation by GAS-secreted streptokinase (SK). Consequently, GAS enhances virulence by digesting extracellular and tight cellular junctional barriers using hPm activity. Many studies have demonstrated the singular importance of the kringle-2 domain of hPg (K2hPg) to PAM-binding using hPg fragments. Recently, we showed, using full-length hPg, that K2hPg is critical for PAM binding. However, these studies did not eliminate any modulatory effects of the non-K2hPg LBS on this interaction. Moreover, we sought to establish the significance of the intramolecular interaction between Asp219 of the LBS of K2hPg and its serine protease domain binding partner, Lys708, to conformational changes in hPg. In the current study, selective inactivation of the LBS of K1hPg, K4hPg, and K5hPg revealed that the LBS of these kringle domains are dispensable for hPg binding to PAM. However, the attendant conformational change upon inactivation of K4hPg LBS increased the affinity of hPg for PAM by an order of magnitude. This finding suggests that the native hPg conformation encloses PAM-binding exosites or sterically hinders access to K2hPg. While simultaneous inactivation of the LBS of K1hPg, K4hPg, and K5hPg inhibited hPg/SK association alongside hPg activation, the replacement of Lys708 generated a slight conformational change that optimally accelerated hPg activation. Thus, we accentuate disparate functions of hPg LBS and conclude, using intact proteins, that K2hPg plays a central role in regulating hPg activation.

Generation and characterization of a plasminogen-binding group A streptococcal M-protein/streptokinase-sensitive mouse line.

BACKGROUND: Streptococcus pyogenes (GAS) is a human bacterial pathogen that generates various mild to severe diseases. Worldwide, there are approximately 700 million cases of GAS infections per year. In some strains of GAS, the surface-resident M-protein, plasminogen-binding group A streptococcal M-protein (PAM), binds directly to human host plasminogen (hPg), where it is activated to plasmin through a mechanism involving a Pg/bacterial streptokinase (SK) complex as well as endogenous activators. Binding to Pg and its activation are dictated by selected sequences within the human host Pg protein, making it difficult to generate animal models to study this pathogen. OBJECTIVES: To develop a murine model for studying GAS infection by minimally modifying mouse Pg to enhance the affinity to bacterial PAM and sensitivity to GAS-derived SK. METHODS: We used a targeting vector that contained a mouse albumin-promoter and mouse/human hybrid plasminogen cDNA targeted to the Rosa26 locus. Characterization of the mouse strain consisted of both gross and histological techniques and determination of the effects of the modified Pg protein through surface plasmon resonance measurements, Pg activation analyses, and mouse survival post-GAS infection. RESULTS: We generated a mouse line expressing a chimeric Pg protein consisting of 2 amino acid substitutions in the heavy chain of Pg and a complete replacement of the mouse Pg light chain with the human Pg light chain. CONCLUSION: This protein demonstrated an enhanced affinity for bacterial PAM and sensitivity to activation by the Pg-SK complex, making the murine host susceptible to the pathogenic effects of GAS.

Analysis and Characterization of Glutathione Peroxidases in an Environmental Microbiome and Isolated Bacterial Microorganisms.

Glutathione peroxidases (Gpx) are a group of antioxidant enzymes that protect cells or tissues against damage from reactive oxygen species (ROS). The Gpx proteins identified in mammals exhibit high catalytic activity toward glutathione (GSH). In contrast, a variety of non-mammalian Gpx proteins from diverse organisms, including fungi, plants, insects, and rodent parasites, show specificity for thioredoxin (TRX) rather than GSH and are designated as TRX-dependent peroxiredoxins. However, the study of the properties of Gpx in the environmental microbiome or isolated bacteria is limited. In this study, we analyzed the Gpx sequences, identified the characteristics of sequences and structures, and found that the environmental microbiome Gpx proteins should be classified as TRX-dependent, Gpx-like peroxiredoxins. This classification is based on the following three items of evidence: i) the conservation of the peroxidatic Cys residue; ii) the existence and conservation of the resolving Cys residue that forms the disulfide bond with the peroxidatic cysteine; and iii) the absence of dimeric and tetrameric interface domains. The conservation/divergence pattern of all known bacterial Gpx-like proteins in public databases shows that they share common characteristics with that from the environmental microbiome and are also TRX-dependent. Moreover, phylogenetic analysis shows that the bacterial Gpx-like proteins exhibit a star-like radiating phylogenetic structure forming a highly diverse genetic pool of TRX-dependent, Gpx-like peroxidases.

High-Resolution Single-Particle Cryo-EM Hydrated Structure of Streptococcus pyogenes Enolase Offers Insights into Its Function as a Plasminogen Receptor.

Cellular plasminogen (Pg) receptors (PgRs) are utilized to recruit Pg; stimulate its activation to the serine protease, plasmin (Pm); and sterically protect the surface Pm from inactivation by host inhibitors. One such PgR is the moonlighting enzyme, enolase, some of which leaves the cytoplasm and resides at the cell surface to potentially function as a PgR. Since microbes employ conscription of host Pg by PgRs as one virulence mechanism, we explored the structural basis of the ability of Streptococcus pyogenes enolase (Sen) to function in this manner. Employing single-particle cryo-electron microscopy (cryo-EM), recombinant Sen from S. pyogenes was modeled at 2.6 Å as a stable symmetrical doughnut-shaped homooctamer with point group 422 (D4) symmetry, with a monomeric subunit molecular weight of ∼49 kDa. Binding sites for hPg were reported in other studies to include an internal K252,255 and the COOH-terminal K434,435 residues of Sen. However, in native Sen, the latter are buried within the minor interfaces of the octamer and do not function as a Pg-binding epitope. Whereas Sen and hPg do not interact in solution, when Sen is bound to a surface, hPg interacts with Sen independently of K252,255,434,435. PgRs devoid of COOH-terminal lysine utilize lysine isosteres comprising a basic residue, "i", and an anionic residue at "i + 3" around one turn of an α-helix. We highlight a number of surface-exposed potential hPg-binding lysine isosteres and further conclude that while the octameric structure of Sen is critical for hPg binding, disruption of this octamer without dissociation exposes hPg-binding epitopes.

Spike protein receptor-binding domains from SARS-CoV-2 variants of interest bind human ACE2 more tightly than the prototype spike protein.

Several SARS-CoV-2 variants of interest (VOI) have emerged since this virus was first identified as the etiologic agent responsible for COVID-19. Some of these variants have demonstrated differences in both virulence and transmissibility, as well as in evasion of immune responses in hosts vaccinated against the original strain of SARS-CoV-2. There remains a lack of definitive evidence that identifies the genetic elements that are responsible for the differences in transmissibility among these variants. One factor affecting transmissibility is the initial binding of the surface spike protein (SP) of SARS-CoV-2 to human angiotensin converting enzyme-2 (hACE2), the widely accepted receptor for SP. This step in the viral replication process is mediated by the receptor binding domain (RBD) of SP that is located on the surface of the virus. This current study was conducted with the aim of assessing potential differences in binding affinity between recombinant hACE2 and the RBDs of emergent SARS-CoV-2 WHO VOIs. Mutations that affect the binding affinity of SP play a dominant initial role in the infectivity of the virus.

Streptolysin S targets the sodium-bicarbonate cotransporter NBCn1 to induce inflammation and cytotoxicity in human keratinocytes during Group A Streptococcal infection.

Group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram-positive human pathogen that employs several secreted and surface-bound virulence factors to manipulate its environment, allowing it to cause a variety of disease outcomes. One such virulence factor is Streptolysin S (SLS), a ribosomally-produced peptide toxin that undergoes extensive post-translational modifications. The activity of SLS has been studied for over 100 years owing to its rapid and potent ability to lyse red blood cells, and the toxin has been shown to play a major role in GAS virulence in vivo. We have previously demonstrated that SLS induces hemolysis by targeting the chloride-bicarbonate exchanger Band 3 in erythrocytes, indicating that SLS is capable of targeting host proteins to promote cell lysis. However, the possibility that SLS has additional protein targets in other cell types, such as keratinocytes, has not been explored. Here, we use bioinformatics analysis and chemical inhibition studies to demonstrate that SLS targets the electroneutral sodium-bicarbonate cotransporter NBCn1 in keratinocytes during GAS infection. SLS induces NF-κB activation and host cytotoxicity in human keratinocytes, and these processes can be mitigated by treating keratinocytes with the sodium-bicarbonate cotransport inhibitor S0859. Furthermore, treating keratinocytes with SLS disrupts the ability of host cells to regulate their intracellular pH, and this can be monitored in real time using the pH-sensitive dye pHrodo Red AM in live imaging studies. These results demonstrate that SLS is a multifunctional bacterial toxin that GAS uses in numerous context-dependent ways to promote host cell cytotoxicity and increase disease severity. Studies to elucidate additional host targets of SLS have the potential to impact the development of therapeutics for severe GAS infections.

Cell Entry and Unusual Replication of SARS-CoV-2.

BACKGROUND: SARS-CoV-2 is the causative virus for the CoVID-19 pandemic that has frequently mutated to continue to infect and resist available vaccines. Emerging new variants of the virus have complicated notions of immunity conferred by vaccines versus immunity that results from infection. While we continue to progress from epidemic to endemic as a result of this collective immunity, the pandemic remains a morbid and mortal problem. OBJECTIVE: The SARS-CoV-2 virus has a very complex manner of replication. The spike protein, one of the four structural proteins of the encapsulated virus, is central to the ability of the virus to penetrate cells to replicate. The objective of this review is to summarize these complex features of viral replication. METHODS: A review of the recent literature was performed on the biology of SARS-CoV-2 infection from published work from PubMed and works reported to preprint servers, e.g., bioRxiv and medRxiv. RESULTS AND CONCLUSION: The complex molecular and cellular biology involved in SARS-CoV-2 replication and the origination of >30 proteins from a single open reading frame (ORF) have been summarized, as well as the structural biology of spike protein, a critical factor in the cellular entry of the virus, which is a necessary feature for it to replicate and cause disease.

Relationships Between Plasminogen-Binding M-Protein and Surface Enolase for Human Plasminogen Acquisition and Activation in Streptococcus pyogenes.

The proteolytic activity of human plasmin (hPm) is utilized by various cells to provide a surface protease that increases the potential of cells to migrate and disseminate. Skin-trophic Pattern D strains of Streptococcus pyogenes (GAS), e.g., GAS isolate AP53, contain a surface M-protein (PAM) that directly and strongly interacts (Kd ~ 1 nM) with human host plasminogen (hPg), after which it is activated to hPm by a specific coinherited bacterial activator, streptokinase (SK2b), or by host activators. Another ubiquitous class of hPg binding proteins on GAS cells includes "moonlighting" proteins, such as the glycolytic enzyme, enolase (Sen). However, the importance of Sen in hPg acquisition, especially when PAM is present, has not been fully developed. Sen forms a complex with hPg on different surfaces, but not in solution. Isogenic AP53 cells with a targeted deletion of PAM do not bind hPg, but the surface expression of Sen is also greatly diminished upon deletion of the PAM gene, thus confounding this approach for defining the role of Sen. However, cells with point deletions in PAM that negate hPg binding, but fully express PAM and Sen, show that hPg binds weakly to Sen on GAS cells. Despite this, Sen does not stimulate hPg activation by SK2b, but does stimulate tissue-type plasminogen activator-catalyzed activation of hPg. These data demonstrate that PAM plays the dominant role as a functional hPg receptor in GAS cells that also contain surface enolase.

Evolution of Streptococcus pyogenes has maximized the efficiency of the Sortase A cleavage motif for cell wall transpeptidation.

Trafficking of M-protein (Mprt) from the cytosol of Group A Streptococcus pyogenes (GAS) occurs via Sec translocase membrane channels that associate with Sortase A (SrtA), an enzyme that catalyzes cleavage of Mprt at the proximal C-terminal [-LPST355∗GEAA-] motif and subsequent transpeptidation of the Mprt-containing product to the cell wall (CW). These steps facilitate stable exposure of the N-terminus of Mprt to the extracellular milieu where it interacts with ligands. Previously, we found that inactivation of SrtA in GAS cells eliminated Mprt CW transpeptidation but effected little reduction in its cell surface exposure, indicating that the C-terminus of Mprt retained in the cytoplasmic membrane (CM) extends its N-terminus to the cell surface. Herein, we assessed the effects of mutating the Thr355 residue in the WT SrtA consensus sequence (LPST355∗GEAA-) in a specific Mprt, PAM. In vitro, we found that synthetic peptides with mutations (LPSX355GEAA) in the SrtA cleavage site displayed slower cleavage activities with rSrtA than the WT peptide. Aromatic residues at X had the lowest activities. Nonetheless, PAM/[Y355G] still transpeptidated the CW in vivo. However, when using isolated CMs from srtA-inactivated GAS cells, rapid cleavage of PAM/[LPSY355GEAA] occurred at E357∗ but transpeptidation did not take place. These results show that another CM-resident enzyme nonproductively cleaved PAM/[LPSYGE357∗AA]. However, SrtA associated with the translocon channel in vivo cleaved and transpeptidated PAM/[LPSX355∗GEAA] variants. These CM features allow diverse cleavage site variants to covalently attach to the CW despite the presence of other potent nonproductive CM proteases.

Demonstration of Three Distinct High-Molecular-Weight Complexes between Plasminogen Activator Inhibitor Type 1 and Tissue-Type Plasminogen Activator.

BACKGROUND: Details of the molecular interaction between tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor type-1 (PAI-1) remain unknown. METHODS AND RESULTS: Three distinct forms of high-molecular-weight complexes are demonstrated. Two of the forms were detected by mass spectrometry. The high molecular mass detected by MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) was 107,029 Da, which corresponds to the sum of molecular masses of the intact tPA (65,320 Da) and the intact PAI-1 (42,416 Da). The lower molecular mass was 104,367 Da and is proposed to lack the C-terminal bait peptide of PAI-1 (calculated mass: 3,804 Da), which was detected as a 3,808 Da fragment. When the complex was analyzed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), only a single band was observed. However, after treatment by SDS and Triton X-100, two distinct forms of the complex with different mobilities were shown by SDS-PAGE. The higher molecular weight band demonstrated specific tPA activity on fibrin autography, whereas the lower molecular weight band did not. Peptide sequence analysis of these two bands, however, unexpectedly revealed the existence of the C-terminal cleavage peptide in both bands and its amount was less in the upper band. In the upper band, the sequences corresponding to the regions at the interface between two molecules in its Michaelis intermediate were diminished. Thus, these two bands corresponded to distinct nonacyl-enzyme complexes, wherein only the upper band liberated free tPA under the conditions employed. CONCLUSION: These data suggest that under physiological conditions a fraction of the tPA-PAI-1 population exists as nonacylated-enzyme inhibitor complex.

Fibrin is a critical regulator of neutrophil effector function at the oral mucosal barrier.

Tissue-specific cues are critical for homeostasis at mucosal barriers. Here, we report that the clotting factor fibrin is a critical regulator of neutrophil function at the oral mucosal barrier. We demonstrate that commensal microbiota trigger extravascular fibrin deposition in the oral mucosa. Fibrin engages neutrophils through the αMß2 integrin receptor and activates effector functions, including the production of reactive oxygen species and neutrophil extracellular trap formation. These immune-protective neutrophil functions become tissue damaging in the context of impaired plasmin-mediated fibrinolysis in mice and humans. Concordantly, genetic polymorphisms in PLG, encoding plasminogen, are associated with common forms of periodontal disease. Thus, fibrin is a critical regulator of neutrophil effector function, and fibrin-neutrophil engagement may be a pathogenic instigator for a prevalent mucosal disease.

Plasminogen Deficiency Significantly Reduces Vascular Wall Disease in a Murine Model of Type IIa Hypercholesterolemia.

The fibrinolytic system has been implicated in the genesis and progression of atherosclerosis. It has been reported that a plasminogen (Pg) deficiency (Plg-/-) exacerbates the progression of atherosclerosis in Apoe-/- mice. However, the manner in which Plg functions in a low-density lipoprotein-cholesterol (LDL-C)-driven model has not been evaluated. To characterize the effect of Pg in an LDL-C-driven model, mice with a triple deficiency of the LDL-receptor (LDLr), along with the active component (apobec1) of the apolipoprotein B editosome complex, and Pg (L-/-/A-/-/Plg-/-), were generated. Atherosclerotic plaque formation was severely retarded in the absence of Pg. In vitro studies demonstrated that LDL uptake by macrophages was enhanced by plasmin (Pm), whereas circulating levels of LDL were enhanced, relative to L-/-/A-/- mice, and VLDL synthesis was suppressed. These results indicated that clearance of lipoproteins in the absence of LDLr may be regulated by Pg/Pm. Conclusions: The results from this study indicate that Pg exacerbates atherosclerosis in an LDL-C model of atherosclerosis and also plays a role in lipoprotein modification and clearance. Therefore, controlling the Pg system on macrophages to prevent foam cell formation would be a novel therapeutic approach.

Binding of the kringle-2 domain of human plasminogen to streptococcal PAM-type M-protein causes dissociation of PAM dimers.

The direct binding of human plasminogen (hPg), via its kringle-2 domain (K2hPg ), to streptococcal M-protein (PAM), largely contributes to the pathogenesis of Pattern D Group A Streptococcus pyogenes (GAS). However, the mechanism of complex formation is unknown. In a system consisting of a Class II PAM from Pattern D GAS isolate NS88.2 (PAMNS88.2 ), with one K2hPg binding a-repeat in its A-domain, we employed biophysical techniques to analyze the mechanism of the K2hPg /PAMNS88.2 interaction. We show that apo-PAMNS88.2 is a coiled-coil homodimer (M.Wt. ~80 kDa) at 4°C-25°C, and is monomeric (M.Wt. ~40 kDa) at 37°C, demonstrating a temperature-dependent dissociation of PAMNS88.2 over a narrow temperature range. PAMNS88.2 displayed a single tight binding site for K2hPg at 4°C, which progressively increased at 25°C through 37°C. We isolated the K2hPg /PAMNS88.2 complexes at 4°C, 25°C, and 37°C and found molecular weights of ~50 kDa at each temperature, corresponding to a 1:1 (m:m) K2hPg /PAMNS88.2  monomer complex. hPg activation experiments by streptokinase demonstrated that the hPg/PAMNS88.2  monomer complexes are fully functional. The data show that PAM dimers dissociate into functional monomers at physiological temperatures or when presented with the active hPg module (K2hPg ) showing that PAM is a functional monomer at 37°C.

Group A Streptococcus-Induced Activation of Human Plasminogen Is Required for Keratinocyte Wound Retraction and Rapid Clot Dissolution.

Invasive outcomes of Group A Streptococcus (GAS) infections that involve damage to skin and other tissues are initiated when these bacteria colonize and disseminate via an open wound to gain access to blood and deeper tissues. Two critical GAS virulence factors, Plasminogen-Associated M-Protein (PAM) and streptokinase (SK), work in concert to bind and activate host human plasminogen (hPg) in order to create a localized proteolytic environment that alters wound-site architecture. Using a wound scratch assay with immortalized epithelial cells, real-time live imaging (RTLI) was used to examine dynamic effects of hPg activation by a PAM-containing skin-trophic GAS isolate (AP53R+S-) during the course of infection. RTLI of these wound models revealed that retraction of the epithelial wound required both GAS and hPg. Isogenic AP53R+S- mutants lacking SK or PAM highly attenuated the time course of retraction of the keratinocyte wound. We also found that relocalization of integrin ß1 from the membrane to the cytoplasm occurred during the wound retraction event. We devised a combined in situ-based cellular model of fibrin clot-in epithelial wound to visualize the progress of GAS pathogenesis by RTLI. Our findings showed GAS AP53R+S- hierarchically dissolved the fibrin clot prior to the retraction of keratinocyte monolayers at the leading edge of the wound. Overall, our studies reveal that localized activation of hPg by AP53R+S- via SK and PAM during infection plays a critical role in dissemination of bacteria at the wound site through both rapid dissolution of the fibrin clot and retraction of the keratinocyte wound layer.

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.

Past and Present Behçet's Disease Animal Models.

Behçet's disease (BD) is presumably an autoinflammatory disease of unknown etiology for which several animal models have been described over the years. Agents and methods used for the development of these models have ranged from the herpes simplex type one virus (hsv-1) pathogen to the use of transgenic mice. Other models have also been used to investigate a possible autoimmune component. Each model possesses its own unique set of benefits and shortcomings, with no one model fully being able to recapitulate the disease phenotype. Here, we review the proposed models and provide commentary on their effectiveness and usefulness in studying the disease.