Search

Subversion of the plasminogen activation system is implicated in the virulence of group A streptococci (GAS). GAS displays receptors for the human zymogen plasminogen on the cell surface, one of which is the plasminogen-binding group A streptococcal M-like protein (PAM). The plasminogen binding domain of PAM is highly variable, and this variation has been linked to host selective immune pressure. Site-directed mutagenesis of full-length PAM protein from an invasive GAS isolate was undertaken to assess the contribution of residues in the a1 and a2 repeat domains to plasminogen binding function. Mutagenesis to alanine of key plasminogen binding lysine residues in the a1 and a2 repeats (Lys98 and Lys111) did not abrogate plasminogen binding by PAM nor did additional mutagenesis of Arg101 and His102 and Glu104, which have previously been implicated in plasminogen binding. Plasminogen binding was only abolished with the additional mutagenesis of Arg114 and His115 to alanine. Furthermore, mutagenesis of both arginine (Arg101 and Arg114) and histidine (His102 and His115) residues abolished interaction with plasminogen despite the presence of Lys98 and Lys111 in the binding repeats. This study shows for the first time that residues Arg101, Arg114, His102, and His115 in both the a1 and a2 repeat domains of PAM can mediate high affinity plasminogen binding. These data suggest that highly conserved arginine and histidine residues may compensate for variation elsewhere in the a1 and a2 plasminogen binding repeats, and may explain the maintenance of high affinity plasminogen binding by naturally occurring variants of PAM. [ABSTRACT FROM AUTHOR]

Zinc (Zn) and copper (Cu) are essential for optimal innate immune function, and nutritional deficiency in either metal leads to increased susceptibility to bacterial infection. Recently, the decreased survival of bacterial pathogens with impaired Cu and/or Zn detoxification systems in phagocytes and animal models of infection has been reported. Consequently, a model has emerged in which the host utilizes Cu and/or Zn intoxication to reduce the intracellular survival of pathogens. This review describes and assesses the potential role for Cu and Zn intoxication in innate immune function and their direct bactericidal function. [ABSTRACT FROM AUTHOR]

A recent analysis of group A Streptococcus (GAS) invasive infections in Australia has shown a predominance of M4 GAS, a serotype recently reported to lack the antiphagocytic hyaluronic acid (HA) capsule. Here, we use molecular genetics and bioinformatics techniques to characterize 17 clinical M4 isolates associated with invasive disease in children during this recent epidemiology. All M4 isolates lacked HA capsule, and whole genome sequence analysis of two isolates revealed the complete absence of the hasABC capsule biosynthesis operon. Conversely, M4isolates possess a functional HA-degrading hyaluronate lyase (HylA) enzyme that is rendered nonfunctional in other GAS through a point mutation. Transformation with a plasmid expressing hasABC restored partial encapsulation in wild-type (WT) M4 GAS, and full encapsulation in an isogenic M4 mutant lacking HylA. However, partial encapsulation reduced binding to human complement regulatory protein C4BP, did not enhance survival in whole human blood, and did not increase virulence of WT M4 GAS in a mouse model of systemic infection. Bioinformatics analysis found no hasABC homologs in closely related species, suggesting that this operon was a recent acquisition. These data showcase a mutually exclusive interaction of HA capsule and active HylA among strains of this leading human pathogen. [ABSTRACT FROM AUTHOR]

Mycoplasma hyopneumoniae colonizes the ciliated respiratory epithelium of swine, disrupting mucociliary function and inducing chronic inflammation. P97 and P102 family members are major surface proteins of M. hyopneumoniae and play key roles in colonizing cilia via interactions with glycosaminoglycans and mucin. The p102 paralog, mhp683, and homologs in strains from different geographic origins encode a 135-kDa pre-protein (P135) that is cleaved into three fragments identified here as P45683, P48683, and P50683. A peptide sequence (TTKF?QE) was identified surrounding both cleavage sites in Mhp683. N-terminal sequences of P48683 and P50683, determined by Edman degradation and mass spectrometry, confirmed cleavage after the phenylalanine residue. A similar proteolytic cleavage site was identified by mass spectrometry in another paralog of the P97/P102 family. Trypsin digestion and surface biotinylation studies showed that P45683, P48683, and P50683 reside on the M. hyopneumoniae cell surface. Binding assays of recombinant proteins F1683-F5683, spanning Mhp683, showed saturable and dose-dependent binding to biotinylated heparin that was inhibited by unlabeled heparin, fucoidan, and mucin. F1683-F5683 also bound porcine epithelial cilia, and antisera to F2683 and F5683 significantly inhibited cilium binding by M. hyopneumoniae cells. These data suggest that P45683, P48683, and P50683 each display cilium- and proteoglycan-binding sites. Mhp683 is the first characterized glycosaminoglycan-binding member of the P102 family. [ABSTRACT FROM AUTHOR]

Mycoplasma hyopneumoniae is the causative pathogen of porcine enzootic pneumonia, an economically significant disease that disrupts the mucociliary escalator in the swine respiratory tract. Expression of Mhp 107, a P97 paralog encoded by the gene mhp107, was confirmed using ESI-MS/MS. To investigate the function of Mhp107, three recombinant proteins, F1MhP107, F2MhP107, and F3MhP107, spanning the N-terminal, central, and C-terminal regions of Mhp107 were constructed. Colonization of swine by M. hyopneumoniae requires adherence of the bacterium to ciliated cells of the respiratory tract. Recent studies have identified a number of M. hyopneumoniae adhesins that bind heparin, fibronectin, and plasminogen. F1MhP107 was found to bind porcine heparin (KD ∼90 nM) in a dose-dependent and saturable manner, whereas F3Mhp107 bound fibronectin (KD ∼180 nM) at physiologically relevant concentrations. F1MhP107 also bound porcine plasminogen (KD = 24 nM) in a dose-dependent and physiologically relevant manner. Microspheres coated with F3MhP107 mediate adherence to porcine kidney epithelial-like (PK15) cells, and all three recombinant proteins (F1MhP107-F3MhP107) bound swine respiratory cilia. Together, these findings indicate that Mhp107 is a member of the multifunctional M. hyopneumoniaeadhesin family of surface proteins and contributes to both adherence to the host and pathogenesis. [ABSTRACT FROM AUTHOR]

The flesh-eating bacterium group A Streptococcus (GAS) binds and activates human plasminogen, promoting invasive disease. Streptococcal surface enolase (SEN), a glycolytic pathway enzyme, is an identified plasminogen receptor of GAS. Here we used mass spectrometry (MS) to confirm that GAS SEN is octameric, thereby validating in silico modeling based on the crystal structure of Streptococcuspneumoniae n-enolase. Site-directed mutagenesis of surface-located lysine residues (SEN[supK252 + 255A], SEN[supK344A], SEN[supK334A], SEN[supK344E], SEN[supK435L, and SEN[supΔ434-435]) was used to examine their roles in maintaining structural integrity, enzymatic function, and plasminogen binding. Structural integrity of the GAS SEN octamer was retained for all mutants except SEN[supK344E], as determined by circular dichroism spectroscopy and MS. However, ion mobility MS revealed distinct differences in the stability of several mutant octamers in comparison with wild type. Enzymatic analysis indicated that SEN[supK3444E] had lost a-enolase activity, which was also reduced in SEN[supK334A] and SEN[supΔ434-435]. Surface plasmon resonance demonstrated that the capacity to bind human plasminogen was abolished in SEN[supK252 + 255A], SEN[supK435L], and SEN[supΔ434-435]. The lysine residues at positions 252, 255,434, and 435 therefore play a concerted role in plasminogen acquisition. This study demonstrates the ability of combining in silico structural modeling with ion mobility-MS validation for undertaking functional studies on complex protein structures. [ABSTRACT FROM AUTHOR]

Serum opacity factor (SOF) is a unique multifunctional virulence determinant expressed at the surface of Streptococcus pyogenes and has been shown to elicit protective immunity against GAS infection in a murine challenge model. SOF consists of two distinct domains with different binding capacities: an N-terminal domain that binds apolipoprotein At and a C-terminal repeat domain that binds fibronectin and fibrinogen. The capacity of SOF to opacify serum by disrupting the structure of high density lipoproteins may preclude its use as a vaccine antigen in humans. This study generated mutant forms of recombinant SOF with reduced (100-fold) or abrogated opacity factor (OF) activity, for use as vaccine antigens. However, alterations introduced into the N-terminal SOF peptide (SOFΔFn) by mutagenesis to abrogate OF activity, abolish the capacity of SOF to protect against lethal systemic S. pyogenes challenge in a murine model. Mutant forms of purified SOFΔFn peptide were also used to assess the contribution of OF activity to the pathogenic processes of cell adhesion and cell invasion. Using latex beads coated with full-length SOF, SOFΔFn peptide, or a peptide encompassing the C-terminal repeats (FnBD), we demonstrate that adhesion to HEp-2 cells is mediated by both SOFΔFn and FnBD. The HEp-2 cell binding displayed by the N-terminal SOFΔFn peptide is independent of OF activity. We demonstrate that while the N terminus of SOF does not directly mediate intracellular uptake by epithelial cells, this domain enhances epithelial cell uptake mediated by full-length SOF, in comparison to the FnBD alone. [ABSTRACT FROM AUTHOR]

Elevated non-esterified fatty acids, triglyceride, diacyiglycerol, and ceramide have all been associated with insulin resistance in muscle. We set out to investigate the role of intramyocellular lipid metabolites in the induction of insulin resistance in human primary myoblast cultures. Muscle cells were subjected to adenovirus-mediated expression of perilipin or incubated with fatty acids for 18 h, prior to insulin stimulation and measurement of lipid metabolites and rates of glycogen synthesis. Adenovirus-driven perilipin expression lead to significant accumulation of triacylglycerol in myoblasts, without any detectable effect on insulin sensitivity, as judged by the ability of insulin to stimulate glycogen synthesis. Similarly, incubation of cells with the monounsaturated fatty acid oleate resulted in triacylglycerol accumulation without inhibiting insulin action. By contrast, the saturated fatty acid palmitate induced insulin resistance. Palmitate treatment caused less accumulation of triacylglycerol than did oleate but also induced significant accumulation of both diacylglycerol and ceramide. Insulin resistance was also caused by cell-permeable analogues of ceramide, and palmitate-induced resistance was blocked in the presence of inhibitors of de novo ceramide synthesis. Oleate co-incubation completely prevented the insulin resistance induced by palmitate. Our data are consistent with ceramide being the agent responsible for insulin resistance caused by palmitate exposure. Furthermore, the triacylglycerol derived from oleate was able to exert a protective role in sequestering palmitate, thus preventing its conversion to ceramide. [ABSTRACT FROM AUTHOR]

Group A streptococci (GAS) display receptors for the human zymogen plasminogen on the cell surface, one of which is the plasminogen-binding group A streptococcal M protein (PAM). Characterization of PAM genes from 12 GAS isolates showed significant variation within the plasminogen-binding repeat motifs (a1/a2) of this protein. To determine the impact of sequence variation on protein function, recombinant proteins representing five naturally occurring variants of PAM, together with a recombinant M1 protein, were expressed and purified. Equilibrium dissociation constants for the interaction of PAM variants with biotinylated Gluplasminogen ranged from 1.58 to 4.99 nM. Effective concentrations of prototype PAM required for 50% inhibition of plasminogen binding to immobilized PAM variants ranged from 0.68 to 22.06 nM. These results suggest that although variation in the a1/a2 region of the PAM protein does affect the comparative affinity of PAM variants, the functional capacity to bind plasminogen is conserved. Additionally, a potential role for the al region of PAM in eliciting a protective immune response was investigated by using a mouse model for GAS infection. The al region of PAM was found to protect immunized mice challenged with a PAM-positive GAS strain. These data suggest a link between selective immune pressure against the plasminogen-binding repeats and the functional conservation of the binding domain in PAM variants. [ABSTRACT FROM AUTHOR]

L-Tryptophan is the least abundant essential amino acid in humans. Indoleamine 2,3-dioxgyenase (IDO) is a cytosolic heme protein which, together with the hepatic enzyme tryptophan 2,3-dioxygenase, catalyzes the first and rate-limiting step in the major pathway of tryptophan metabolism, the kynurenine pathway. The physiological role of IDO is not fully understood but is of great interest, because IDO is widely distributed in human tissues, can be up-regulated via cytokines such as interferon-γ, and can thereby modulate the levels of tryptophan, which is vital for cell growth. To identify which amino acid residues are important in substrate or heme binding in IDO, site-directed mutagenesis of conserved residues in the IDO gene was undertaken. Because it had been proposed that a histidine residue might be the proximal heme ligand in IDO, mutation to alanine of the three highly conserved histidines His[sup 16], His[sup 303], and His[sup 346] was conducted. Of these, only His[sup 346] was shown to be essential for heme binding, indicating that this histidine residue may be the proximal ligand and suggesting that neither His[sup 303] nor His[sup 16] act as the proximal ligand. Site-directed mutagenesis of Asp[sup 274] also compromised the ability of IDO to bind heme. This observation indicates that Asp[sup 274] may coordinate to heme directly as the distal ligand or is essential in maintaining the conformation of the heme pocket. [ABSTRACT FROM AUTHOR]