Time-resolved fluorimetry in the study of attachment of staphylococci and streptococci on substrate-bound fibronectin

Abstract The application of time-resolved fluorimetry was evaluated in the study of staphylococcal and streptococcal attachment to fibronectin-coated coverslips. The test system allowed the use of low bacterial concentrations (2 × 10⁵−10⁷ bacteria per ml), in contrast to the much higher concentrations of bacteria used in earlier assays. The bacteria attached much better to fibronectin-coated plastic surfaces than to albumin-coated ones, but there were differences between the individual strains. Soluble fibronectin inhibited the adsorption of staphylococci but enhanced streptococcal attachment to fibronectin-coated surfaces. Purified antibodies to fibronectin inhibited both staphylococcal and streptococcal adhesion in a dose-dependent way. Our results show that time-resolved fluorimetry is a very sensitive method for quantitating bacterial attachment.     

Pili of oral Streptococcus sanguinis bind to fibronectin and contribute to cell adhesion

Streptococcus sanguinis is a predominant bacterium in the human oral cavity and occasionally causes infective endocarditis. We identified a unique cell surface polymeric structure named pili in this species and investigated its functions in regard to its potential virulence. Pili of S. sanguinis strain SK36 were shown to be composed of three distinctive pilus proteins (PilA, PilB, and PilC), and a pili-deficient mutant demonstrated reduced bacterial adherence to HeLa and human oral epithelial cells. PilC showed a binding ability to fibronectin, suggesting that pili are involved in colonization by this species. In addition, ATCC10556, a standard S. sanguinis strain, was unable to produce pili due to defective pilus genes, which indicates a diversity of pilus expression among various S. sanguinis strains.     

Multilocus variable number tandem repeat analysis (MLVA) of Streptococcus pyogenes

We tested 21 polymorphic loci encoded by the genome of Streptococcus pyogenes (group A Streptococcus, GAS). Seven of them were chosen for the MLVA scheme. The primer pairs, designed for selected loci, detect from few to several alleles, and the method has a Simpson's Index of diversity of 0.957. To test the overall performance of the method, multiplex PCR reactions were carried out for over 700 GAS strains. Using the method we were able to detect differences between highly clonal strains that share the same emm, MLST and PFGE types. The most diverse strains were M4, M2, M3 and M28.We developed a typing method that can be employed to differentiate between GAS strains. The method has high resolution and measures diversity of the GAS core chromosome, on the contrary to methods such as PFGE.     

A novel fibronectin type III module binding motif identified on C-terminus of Leptospira immunoglobulin-like protein, LigB

Infection by pathogenic strains of Leptospira hinges on the pathogen’s ability to adhere to host cells via extracellular matrix such as fibronectin (Fn). Previously, the immunoglobulin-like domains of Leptospira Lig proteins were recognized as adhesins binding to N-terminal domain (NTD) and gelatin binding domain (GBD) of Fn. In this study, we identified another Fn-binding motif on the C-terminus of the Leptospira adhesin LigB (LigBCtv), residues 1708-1712 containing sequence LIPAD with a β-strand and nascent helical structure. This motif binds to 15th type III modules (15F₃) (K_D = 10.70 μM), and association (k_on = 600 M⁻¹ s⁻¹) and dissociation (k_off = 0.0129 s⁻¹) rate constants represents a slow binding kinetics in this interaction. Moreover, pretreatment of MDCK cells with LigB_1706-1716 blocked the binding of Leptospira by 39%, demonstrating a significant role of LigB_1706-1716 in cellular adhesion. These data indicate that the LIPAD residues (LigB_1708-1712) of the Leptospira interrogans LigB protein bind 15F₃ of Fn at a novel binding site, and this interaction contributes to adhesion to host cells.     

Impact of bacteria and bacterial components on osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells

Adult mesenchymal stem cells (MSC) are present in several tissues, e.g. bone marrow, heart muscle, brain and subcutaneous adipose tissue. In invasive infections MSC get in contact with bacteria and bacterial components. Not much is known about how bacterial pathogens interact with MSC and how contact to bacteria influences MSC viability and differentiation potential. In this study we investigated the impact of three different wound infection relevant bacteria, Escherichia coli, Staphylococcus aureus, and Streptococcus pyogenes, and the cell wall components lipopolysaccharide (LPS; Gram-negative bacteria) and lipoteichoic acid (LTA; Gram-positive bacteria) on viability, proliferation, and osteogenic as well as adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (adMSC). We show that all three tested species were able to attach to and internalize into adMSC. The heat-inactivated Gram-negative E. coli as well as LPS were able to induce proliferation and osteogenic differentiation but reduce adipogenic differentiation of adMSC. Conspicuously, the heat-inactivated Gram-positive species showed the same effects on proliferation and adipogenic differentiation, while its cell wall component LTA exhibited no significant impact on adMSC. Therefore, our data demonstrate that osteogenic and adipogenic differentiation of adMSC is influenced in an oppositional fashion by bacterial antigens and that MSC-governed regeneration is not necessarily reduced under infectious conditions.     

A domain of the Leptospira LigB contributes to high affinity binding of fibronectin

Adhesion of pathogenic Leptospira spp. to mammalian cells is mediated by their adhesins interacting with host cell receptors. In a previous study, we have identified two potential fibronectin (Fn) binding sites in central variable region (LigBCen) and C-terminal variable region (LigBCtv) of LigB, an adhesin of pathogenic Leptospira spp. In this study, we have further localized the Fn-binding site on LigBCen and found a domain of LigB (LigBCen2) (amino acids 1014-1165) strongly bound to Fn. LigBCen2 bound to a 70kDa domain of Fn including N-terminal domain (NTD) and gelatin binding domain (GBD), but with a higher binding affinity to NTD (K(d)=272nM) than to GBD (K(d)=1200nM). Except Fn, LigBCen2 also bound laminin and fibrinogen. LigBCen2 could bind MDCK cells, and blocked the binding of Leptospira on MDCK cells by 45%. These results suggest that LigBCen2 contributed to high affinity binding on NTD or GBD of Fn, laminin, and fibrinogen and mediated Leptospira binding on host cells.     

Crystal structure of FtsA from Staphylococcus aureus

The bacterial cell-division protein FtsA anchors FtsZ to the cytoplasmic membrane. But how FtsA and FtsZ interact during membrane division remains obscure. We have solved 2.2 Å resolution crystal structure for FtsA from Staphylococcus aureus. In the crystals, SaFtsA molecules within the dimer units are twisted, in contrast to the straight filament of FtsA from Thermotoga maritima, and the half of S12–S13 hairpin regions are disordered. We confirmed that SaFtsZ and SaFtsA associate in vitro, and found that SaFtsZ GTPase activity is enhanced by interaction with SaFtsA.     

Characterization of cell membrane parameters of clinical isolates of Staphylococcus aureus with varied susceptibility to alpha-melanocyte stimulating hormone

Staphylococcus aureus (S. aureus), a major human pathogen of hospital and community acquired infections, is becoming resistant to almost all commercially available antibiotics. This has prompted development of antimicrobial peptides as therapeutic options. Alpha melanocyte stimulating hormone (α-MSH) is one such peptide known to possess antimicrobial properties. In the present study, we analyzed the antimicrobial activity of α-MSH against 75 clinical strains of S. aureus including both methicillin susceptible S. aureus (MSSA) and methicillin resistant S. aureus (MRSA) strains. Results of our previous study showed that membrane damage is the major mechanism of staphylocidal activity of α-MSH. In this context, we compared the various bacterial membrane parameters, viz., membrane fluidity, lipid composition, and surface charge of a few selected MSSA and MRSA strains that showed variable susceptibility to the melanocortin peptide. Our results showed that α-MSH killed both type of strains efficiently (≥70% killing in 84% clinical strains after exposure with 6μM of α-MSH for 1h). It was observed that compared to the α-MSH-susceptible strains, the α-MSH-non-susceptible strains had a different membrane order and phospholipid pattern. There was no consistent pattern of cell surface charge to distinguish α-MSH-susceptible strain from a non-susceptible strain. In conclusion, α-MSH possessed potential staphylocidal activity for both against MSSA and MRSA strains. S. aureus strains not susceptible to the peptide exhibited a rigid membrane and a higher amount of the cationic phospholipid as compared to the α-MSH-susceptible strains.     

Isolation,structural characterization,and immunological evaluation of a high-molecular-weight exopolysaccharide from Staphylococcus aureus

Colonization of implanted medical devices by coagulase-negative staphylococci such as Staphylococcus epidermidis is mediated by the bacterial polysaccharide intercellular adhesin (PIA), a polymer of beta-(1-->6)-linked glucosamine substituted with N-acetyl and O-succinyl constituents. The icaADBC locus containing the biosynthetic genes for production of PIA has been identified in both S. epidermidis and S. aureus. Whereas it is clear that PIA is a constituent that contributes to the virulence of S. epidermidis, it is less clear what role PIA plays in infection with S. aureus. Recently, identification of a novel polysaccharide antigen from S. aureus termed poly N-succinyl beta-(1-->6)-glucosamine (PNSG) has been reported. This polymer was composed of the same glycan backbone as PIA but was reported to contain a high proportion of N-succinylation rather than acetylation. We have isolated a glucosamine-containing exopolysaccharide from the constitutive over-producing MN8m strain of S. aureus in order to prepare polysaccharide-protein conjugate vaccines. In this report we demonstrate that MN8m produced a high-molecular-weight (>300,000 Da) polymer of beta-(1-->6)-linked glucosamine containing 45-60% N-acetyl, and a small amount of O-succinyl (approx 10% mole ratio to monosaccharide units). By detailed NMR analyses of polysaccharide preparations, we show that the previous identification of N-succinyl was an analytical artifact. The exopolysaccharide we have isolated is active in in vitro hemagglutination assays and is immunogenic in mice when coupled to a protein carrier. We therefore conclude that S. aureus strain MN8m produces a polymer that is chemically and biologically closely related to the PIA produced by S. epidermidis.     

Functional and structural characterization of Spl proteases from Staphylococcus aureus

Staphylococcus aureus is the major cause of nosocomial infections world-wide, with increasing prevalence of community-acquired diseases. The recent dramatic increase in multi-antibiotic resistance, including resistance to the last-resort drug, vancomycin, together with the lack of an effective vaccine highlight the need for better understanding of S.aureus pathogenicity. Comparative analysis of available bacterial genomes allows for the identification of previously uncharacterized S.aureus genes with potential roles in pathogenicity. A good example is a cluster of six serine protease-like (spl) genes encompassed in one operon, which encode for putative proteases with similarity to staphylococcal glutamylendopeptidase (V8 protease). Here, we describe an efficient expression system for the production of recombinant SplB and SplC proteases in Escherichia coli, together with structural and functional characterization of the purified enzymes. A unique mechanism of cytoplasm protection against activity of misdirected SplB was uncovered. Apparently, the co-translated signal peptide maintains protease latency until it is cleaved by the signal peptidase during protein secretion. Furthermore, the crystal structure of the SplC protease revealed a fold resembling that of the V8 protease and epidermolytic toxins. Arrangement of the active site cleft and substrate-binding pocket of SplC explains the mechanism of enzyme latency and suggests that some Spl proteases possess restricted substrate specificity similar to that of the V8 protease and epidermolytic toxins.     

Purification and biochemical characterization of Mur ligases from Staphylococcus aureus

The Mur ligases (MurC, MurD, MurE and MurF) catalyze the stepwise synthesis of the UDP-N-acetylmuramoyl-pentapeptide precursor of peptidoglycan. The murC, murD, murE and murF genes from Staphylococcus aureus, a major pathogen, were cloned and the corresponding proteins were overproduced in Escherichia coli and purified as His₆-tagged forms. Their biochemical properties were investigated and compared to those of the E. coli enzymes. Staphylococcal MurC accepted l-Ala, l-Ser and Gly as substrates, as the E. coli enzyme does, with a strong preference for l-Ala. S. aureus MurE was very specific for l-lysine and in particular did not accept meso-diaminopimelic acid as a substrate. This mirrors the E. coli MurE specificity, for which meso-diaminopimelic acid is the preferred substrate and l-lysine a very poor one. S. aureus MurF appeared less specific and accepted both forms (l-lysine and meso-diaminopimelic acid) of UDP-MurNAc-tripeptide, as the E. coli MurF does. The inverse and strict substrate specificities of the two MurE orthologues is thus responsible for the presence of exclusively meso-diaminopimelic acid and l-lysine at the third position of the peptide in the peptidoglycans of E. coli and S. aureus, respectively. The specific activities of the four Mur ligases were also determined in crude extracts of S. aureus and compared to cell requirements for peptidoglycan biosynthesis.     

HrcA is a negative regulator of the dnaK and groESL operons of Streptococcus pyogenes

The genome of Streptococcus pyogenes, an important human pathogen, encodes homologs of the principal bacterial heat shock proteins DnaK and GroES, -EL, as well as HrcA, a negative regulator of dnaK and groESL expression in other Gram-positive bacteria. Using nuclease protection assays to measure dnaK/groESL mRNA abundance and a "non-polar" insertion to disrupt hrcA, we demonstrate that heat shock triggers a 4- to 8-fold increase in dnaK and groESL-specific mRNAs within 5 min of the temperature shift and that HrcA is a negative regulator of S. pyogenes dnaK/groESL mRNA abundance in unstressed S. pyogenes. Although the loss of HrcA elevated dnaK and groESL mRNA levels under non-heat shock conditions, the relative abundance of these RNAs increased further in heat shocked S. pyogenes, suggesting an additional element contributing to their synthesis or stability.     

Identification and characterization of the chaperone-subunit complex-binding domain from the type 1 pilus assembly platform FimD

The outer membrane protein FimD represents the assembly platform of adhesive type 1 pili from Escherichia coli. FimD forms ring-shaped oligomers of 91.4 kDa subunits that recognize complexes between the pilus chaperone FimC and individual pilus subunits in the periplasm and mediate subunit translocation through the outer membrane. Here, we have identified a periplasmic domain of FimD (FimD(N)) comprising the N-terminal 139 residues of FimD. Purified FimD(N) is a monomeric, soluble protein that specifically recognizes complexes between FimC and individual type 1 pilus subunits, but does not bind the isolated chaperone, or isolated subunits. In addition, FimD(N) retains the ability of FimD to recognize different chaperone-subunit complexes with different affinities, and has the highest affinity towards the FimC-FimH complex. Overexpression of FimD(N) in the periplasm of wild-type E.coli cells diminished incorporation of FimH at the tip of type 1 pili, while pilus assembly itself was not affected. The identification of FimD(N) and its ternary complexes with FimC and individual pilus subunits opens the avenue to structural characterization of critical type 1 pilus assembly intermediates.     

The Fibrinogen-binding M1 Protein Reduces Pharyngeal Cell Adherence and Colonization Phenotypes of M1T1 Group A Streptococcus

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

Structural elucidation of the extracellular and cell-wall teichoic acids of Staphylococcus aureus MN8m, a biofilm forming strain

Extracellular teichoic acid, an essential constituent of the biofilm produced by Staphylococcus epidermidis strain RP62A, is also an important constituent of the extracellular matrix of another biofilm producing strain, Staphylococcus aureus MN8m. The structure of the extracellular and cell wall teichoic acids of the latter strain was studied by NMR spectroscopy and capillary electrophoresis-mass spectrometry. Both teichoic acids were found to be a mixture of two polymers, a (1-->5)-linked poly(ribitol phosphate), substituted at the 4-position of ribitol residues with beta-GlcNAc, and a (1-->3)-linked poly(glycerol phosphate), partially substituted with the D-Ala at 2-position of glycerol residue. Such mixture is unusual for S. aureus.     

Development of pooled suppression subtractive hybridization to analyze the pangenome of Staphylococcus aureus

We describe the development and application of a Pooled Suppression Subtractive Hybridization (PSSH) method to describe differences between the genomic content of a pool of clinical Staphylococcus aureus isolates and a sequenced reference strain. In comparative bacterial genomics, Suppression Subtractive Hybridization (SSH) is normally utilized to compare genomic features or expression profiles of one strain versus another, which limits its ability to analyze communities of isolates. However, a PSSH approach theoretically enables the user to characterize the entirety of gene content unique to a related group of isolates in a single reaction. These unique fragments may then be linked to individual isolates through standard PCR. This method was applied to examine the genomic diversity found in pools of S.aureus isolates associated with complicated bacteremia infections leading to endocarditis and osteomyelitis. Across four pools of 10 isolates each, four hundred and twenty seven fragments not found in or significantly divergent from the S. aureus NCTC 8325 reference genome were detected. These fragments could be linked to individual strains within its pool by PCR. This is the first use of PSSH to examine the S. aureus pangenome. We propose that PSSH is a powerful tool for researchers interested in rapidly comparing the genomic content of multiple unstudied isolates.     

Fibronectin aggregation and assembly: the unfolding of the second fibronectin type III domain

The mechanism of fibronectin (FN) assembly and the self-association sites are still unclear and contradictory, although the N-terminal 70-kDa region ((I)1-9) is commonly accepted as one of the assembly sites. We previously found that (I)1-9 binds to superfibronectin, which is an artificial FN aggregate induced by anastellin. In the present study, we found that (I)1-9 bound to the aggregate formed by anastellin and a small FN fragment, (III)1-2. An engineered disulfide bond in (III)2, which stabilizes folding, inhibited aggregation, but a disulfide bond in (III)1 did not. A gelatin precipitation assay showed that (I)1-9 did not interact with anastellin, (III)1, (III)2, (III)1-2, or several (III)1-2 mutants including (III)1-2KADA. (In contrast to previous studies, we found that the (III)1-2KADA mutant was identical in conformation to wild-type (III)1-2.) Because (I)1-9 only bound to the aggregate and the unfolding of (III)2 played a role in aggregation, we generated a (III)2 domain that was destabilized by deletion of the G strand. This mutant bound (I)1-9 as shown by the gelatin precipitation assay and fluorescence resonance energy transfer analysis, and it inhibited FN matrix assembly when added to cell culture. Next, we introduced disulfide mutations into full-length FN. Three disulfide locks in (III)2, (III)3, and (III)11 were required to dramatically reduce anastellin-induced aggregation. When we tested the disulfide mutants in cell culture, only the disulfide bond in (III)2 reduced the FN matrix. These results suggest that the unfolding of (III)2 is one of the key factors for FN aggregation and assembly.     

Extra domain A and type III connecting segment of fibronectin in assembly and cleavage

To determine the role of the extra domain A (EDA) and type III connecting segment (IIICS) of fibronectin in fiber assembly, topographical distribution and proteolytic cleavage, eight full-length human fibronectin cDNA variants (aa0, aa64, aa89, and aa120 variations in the IIICS with or without the EDA) tagged with the V5 epitope were cloned from human endothelial cells and were expressed in CHO-K1 cells. All eight variants were assembled on cell surfaces. However, only the EDA(+) variants, regardless of the type of the IIICS domain, formed extensive fibrous networks. In contrast, the EDA(-)/aa64 and EDA(-)/aa89 variants were present predominantly as a soluble form. Western analysis of both soluble and cell-associated fibronectin/V5 variants showed that aa64, aa89, and aa120 variants with or without the EDA domain produced the major 50- to 62-kDa C-terminal fragments, whereas the aa0 variants did not, suggesting that the IIICS domain provides proteolytic cleavage sites.     

The structure of the C5a receptor-blocking domain of chemotaxis inhibitory protein of Staphylococcus aureus is related to a group of immune evasive molecules

The chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) is a 121 residue excreted virulence factor. It acts by binding the C5a- (C5aR) and formylated peptide receptor (FPR) and thereby blocks specific phagocyte responses. Here, we report the solution structure of a CHIPS fragment consisting of residues 31-121 (CHIPS31-121). CHIPS31-121 has the same activity in blocking the C5aR compared to full-length CHIPS, but completely lacks FPR antagonism. CHIPS31-121 has a compact fold comprising an alpha-helix (residues 38-51) packed onto a four-stranded anti-parallel beta-sheet. Strands beta2 and beta3 are joined by a long loop with a relatively well-defined conformation. Comparison of CHIPS31-121 with known structures reveals striking homology with the C-terminal domain of staphylococcal superantigen-like proteins (SSLs) 5 and 7, and the staphyloccocal and streptococcal superantigens TSST-1 and SPE-C. Also, the recently reported structures of several domains of the staphylococcal extracellullar adherence protein (EAP) show a high degree of structural similarity with CHIPS. Most of the conserved residues in CHIPS and its structural homologues are present in the alpha-helix. A conserved arginine residue (R46 in CHIPS) appears to be involved in preservation of the structure. Site-directed mutagenesis of all positively charged residues in CHIPS31-121 reveals a major involvement of arginine 44 and lysine 95 in C5aR antagonism. The structure of CHIPS31-121 will be vital in the further unraveling of its precise mechanism of action. Its structural homology to S.aureus SSLs, superantigens, and EAP might help the design of future experiments towards an understanding of the relationship between structure and function of these proteins.