The NH(2)-terminal region of Streptococcus pyogenes M5 protein confers protection against degradation by proteases and enhances mucosal colonization of mice.

BACKGROUND: The NH(2)-terminal sequence of the M protein from group A streptococci defines the serotype of the organism and contains epitopes that evoke bactericidal antibodies. METHODS: To identify additional roles for this region of the M protein, we constructed a mutant of M5 group A streptococci expressing an M protein with a deletion of amino acid residues 3-22 (DeltaNH(2)). RESULTS: M5 streptococci and the DeltaNH(2) mutant were resistant to phagocytosis and were similarly virulent in mice. However, DeltaNH(2) was significantly less hydrophobic, contained less lipoteichoic acid on its surface, and demonstrated reduced adherence to epithelial cells. These differences were abolished when organisms were grown in the presence of protease inhibitors. Treatment with cysteine proteases or with human saliva resulted in the release of M protein from the DeltaNH(2) mutant at a significantly greater rate than observed with the wild-type M5 strain. Compared with the parent strain, the DeltaNH(2) strain also showed a significant reduction in its ability to colonize the upper respiratory mucosa of mice. CONCLUSIONS: The NH(2) terminus of M5 protein has an important role in protecting the surface protein from proteolytic cleavage, thus preserving its function as an anchor for lipoteichoic acid, which is a primary mediator of adherence to epithelial cells and colonization.

Anti-adhesion therapy of bacterial diseases: prospects and problems.

The alarming increase in drug-resistant bacteria makes a search for novel means of fighting bacterial infections imperative. An attractive approach is the use of agents that interfere with the ability of the bacteria to adhere to tissues of the host, since such adhesion is one of the initial stages of the infectious process. The validity of this approach has been unequivocally demonstrated in experiments performed in a wide variety of animals, from mice to monkeys, and recently also in humans. Here we review various approaches to anti-adhesion therapy, including the use of receptor and adhesin analogs, dietary constituents, sublethal concentrations of antibiotics and adhesin-based vaccines. Because anti-adhesive agents are not bactericidal, the propagation and spread of resistant strains is much less likely to occur than as a result of exposure to bactericidal agents, such as antibiotics. Anti-adhesive drugs, once developed, may, therefore, serve as a new means to fight infectious diseases.

Pathways involved in the synergistic activation of macrophages by lipoteichoic acid and hemoglobin.

Lipoteichoic acid (LTA) is a Gram-positive cell surface molecule that is found in both a cell-bound form and cell-free form in the host during an infection. Hemoglobin (Hb) can synergize with LTA, a TLR2 ligand, to potently activate macrophage innate immune responses in a TLR2- and TLR4-dependent way. At low levels of LTA, the presence of Hb can result in a 200-fold increase in the secretion of IL-6 following macrophage activation. Six hours after activation, the macrophage genes that are most highly up-regulated by LTA plus Hb activation compared to LTA alone are cytokines, chemokines, receptors and interferon-regulated genes. Several of these genes exhibit a unique TLR4-dependent increase in mRNA levels that continued to rise more than eight hours after stimulation. This prolonged increase in mRNA levels could be the result of an extended period of NF-κB nuclear localization and the concurrent absence of the NF-κB inhibitor, IκBα, after stimulation with LTA plus Hb. Dynasore inhibition experiments indicate that an endocytosis-dependent pathway is required for the TLR4-dependent up-regulation of IL-6 secretion following activation with LTA plus Hb. In addition, interferon-ß mRNA is present after activation with LTA plus Hb, suggesting that the TRIF/TRAM-dependent pathway may be involved. Hb alone can elicit the TLR4-dependent secretion of TNF-α from macrophages, so it may be the TLR4 ligand. Hb also led to secretion of high mobility group box 1 protein (HMGB1), which synergized with LTA to increase secretion of IL-6. The activation of both the TLR2 and TLR4 pathways by LTA plus Hb leads to an enhanced innate immune response.

Relationship between expression of the family of M proteins and lipoteichoic acid to hydrophobicity and biofilm formation in Streptococcus pyogenes.

BACKGROUND: Hydrophobicity is an important attribute of bacteria that contributes to adhesion and biofilm formation. Hydrophobicity of Streptococcus pyogenes is primarily due to lipoteichoic acid (LTA) on the streptococcal surface but the mechanism(s) whereby LTA is retained on the surface is poorly understood. In this study, we sought to determine whether members of the M protein family consisting of Emm (M protein), Mrp (M-related protein), Enn (an M-like protein), and the streptococcal protective antigen (Spa) are involved in anchoring LTA in a manner that contributes to hydrophobicity of the streptococci and its ability to form biofilms. METHODOLOGY/PRINCIPAL FINDINGS: Isogenic mutants defective in expression of emm, mrp, enn, and/or spa genes of eight different serotypes and their parental strains were tested for differences in LTA bound to surface proteins, LTA released into the culture media, and membrane-bound LTA. The effect of these mutations on the ability of streptococci to form a hydrophobic surface and to generate biofilms was also investigated. A recombinant strain overexpressing Emm1 was also engineered and similarly tested. The serotypes tested ranged from those that express only a single M protein gene to those that express two or three members of the M protein family. Overexpression of Emm1 led to enhanced hydrophobicity and biofilm formation. Inactivation of emm in those serotypes expressing only a single emm gene reduced biofilm formation, and protein-bound LTA on the surface, but did not alter the levels of membrane-bound LTA. The results were more varied in those serotypes that express two to three members of the M protein family. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that the formation of complexes with members of the M protein family is a common mechanism for anchoring LTA on the surface in a manner that contributes to hydrophobicity and to biofilm formation in S. pyogenes, but these activities in some serotypes are dependent on a trypsin-sensitive protein(s) that remains to be identified. The need for interactions between LTA and M proteins may impose functional constraints that limit variations in the sequence of the M proteins, major virulence factors of S. pyogenes.

Inactivation of DltA modulates virulence factor expression in Streptococcus pyogenes.

BACKGROUND: D-alanylated lipoteichoic acid is a virtually ubiquitous component of gram-positive cell walls. Mutations in the dltABCD operon of numerous species exhibit pleiotropic effects, including reduced virulence, which has been attributed to increased binding of cationic antimicrobial peptides to the more negatively charged cell surface. In this study, we have further investigated the effects that mutating dltA has on virulence factor expression in Streptococcus pyogenes. METHODOLOGY/PRINCIPAL FINDINGS: Isogenic Delta dltA mutants had previously been created in two distinct M1T1 isolates of S. pyogenes. Immunoblots, flow cytometry, and immunofluorescence were used to quantitate M protein levels in these strains, as well as to assess their ability to bind complement. Bacteria were tested for their ability to interact with human PMN and to grow in whole human blood. Message levels for emm, sic, and various regulatory elements were assessed by quantitative RT-PCR. Cell walls of Delta dltA mutants contained much less M protein than cell walls of parent strains and this correlated with reduced levels of emm transcripts, increased deposition of complement, increased association of bacteria with polymorphonuclear leukocytes, and reduced bacterial growth in whole human blood. Transcription of at least one other gene of the mga regulon, sic, which encodes a protein that inactivates antimicrobial peptides, was also dramatically reduced in Delta dltA mutants. Concomitantly, ccpA and rofA were unaffected, while rgg and arcA were up-regulated. CONCLUSIONS/SIGNIFICANCE: This study has identified a novel mechanism for the reduced virulence of dltA mutants of Streptococcus pyogenes in which gene regulatory networks somehow sense and respond to the loss of DltA and lack of D-alanine esterification of lipoteichoic acid. The mechanism remains to be determined, but the data indicate that the status of D-alanine-lipoteichoic acid can significantly influence the expression of at least some streptococcal virulence factors and provide further impetus to targeting the dlt operon of gram-positive pathogens in the search for novel antimicrobial compounds.

Lipoteichoic acid synergizes with glycosphingolipids to potently stimulate secretion of interleukin-6 from human blood cells.

In the present study, we found that lipoteichoic acid (LTA) synergizes with glycosphingolipids to stimulate human blood cells to secrete cytokines. We employed globoside, kerasin, and lactosylceramide as representative neutral glycosphingolipids and mixed gangliosides GM(2) and GM(3) as representative acidic glycosphingolipids. LTA and the glycosphingolipids enhanced cytokine secretion by human whole blood, peripheral blood mononuclear cells, and purified monocytes in a dose-dependent manner. The level of synergy ranged up to approximately 10-fold greater than the additive stimulation caused by LTA and glycosphingolipid alone. The greatest synergy was observed with GM(3). We also found that LTA synergizes with the synthetic bacterial lipopeptide mimic Pam3CysK4. In contrast, the glycosphingolipids suppressed the stimulation caused by Pam3CysK4. The stimulation of human cells requires the simultaneous presence of LTA and the glycosphingolipids and probably requires their physical interactions, as shown by dot blotting and nondenaturing polyacrylamide gel electrophoresis experiments. We hypothesize that the enhanced stimulation is due to heterooligomers that form between LTA and glycosphingolipids at the subcritical micelle concentrations used in these experiments. Previous studies showed that LTA also synergizes with hemoglobin. The data taken together suggest that LTA may be a pathogen-associated molecular pattern, although its full activity requires the presence of a synergistic partner(s).

Enhancement of macrophage stimulation by lipoteichoic acid and the costimulant hemoglobin is dependent on Toll-like receptors 2 and 4.

Macrophage stimulation by lipoteichoic acid (LTA) and hemoglobin (Hb) requires Toll-like receptors 2 and 4 (TLR2 and -4). There are two distinct temporal phases of interleukin-6 (IL-6) production. The first results in a slight enhancement of IL-6 secretion in response to LTA plus Hb compared to that with LTA alone and is TLR4 independent. The second requires TLR4 and accounts for most of the additional stimulation seen with LTA plus Hb.

Anti-phagocytic mechanisms of Streptococcus pyogenes: binding of fibrinogen to M-related protein.

A key attribute of invasive Streptococcus pyogenes is their ability to resist phagocytosis and multiply in human blood. M-related protein (Mrp) is a major anti-phagocytic factor but the mechanism whereby it helps streptococci to evade phagocytosis has not been demonstrated. We investigated phagocytosis resistance in a strain of serotype M4 by inactivating the mrp gene and also the emm, enn, sof and sfbX genes and by analysing the effect on streptococcal growth in blood and on complement deposition on the bacterial surface. Inactivation of enn4 and sfbX4 had little impact on growth in blood, but ablation of mrp4, emm4 or sof4 reduced streptococcal growth in human blood, confirming that Mrp and Emm are required for optimal resistance to phagocytosis and providing the first indication that Sof may be an anti-phagocytic factor. Moreover, antisera against Mrp4, Emm4 and Sof4 promoted the killing of S. pyogenes, but anti-SfbX serum had no effect. Growth of S. pyogenes in blood was dependent on the presence of fibrinogen and in the absence of fibrinogen there was a twofold increase in complement deposition. Inactivation of mrp4 resulted in a loss of fibrinogen-binding and caused a twofold increase in the binding of C3b that was inhibited by Mg-EGTA. Mrp contained two fibrinogen-binding sites, one of which is within a highly conserved region. These findings indicate that Mrp-fibrinogen interactions prevent surface deposition of complement via the classical pathway, thereby contributing to the ability of these streptococci to resist phagocytosis. This may be a common mechanism for evasion of phagocytosis because Mrp is expressed by approximately half of the clinical isolates of S. pyogenes.

Monocyte and macrophage activation by lipoteichoic Acid is independent of alanine and is potentiated by hemoglobin.

Lipoteichoic acids (LTAs) are Gram-positive bacterial cell wall components that elicit mononuclear cell cytokine secretion. Cytokine-stimulating activity is thought to be dependent on retaining a high level of ester-linked D-alanine residues along the polyglycerol phosphate backbone. However, Streptococcus pyogenes LTA essentially devoid of D-alanine caused human and mouse cells to secrete as much IL-6 as LTA with a much higher D-alanine content. Furthermore, hemoglobin (Hb) markedly potentiates the stimulatory effect of various LTAs on mouse macrophages or human blood cells, regardless of their d-alanine content. LTA and Hb appear to form a molecular complex, based on the ability of each to affect the other's migration on native acrylamide gels, their comigration on these gels, and the ability of LTA to alter the absorption spectra of Hb. Because S. pyogenes is known to release LTA and secrete at least two potent hemolytic toxins, LTA-Hb interactions could occur during streptococcal infections and might result in a profound alteration of the local inflammatory response.

Defective expression of Tamm-Horsfall protein/uromodulin in COX-2-deficient mice increases their susceptibility to urinary tract infections.

Mice lacking a functional cyclooxygenase-2 (COX-2) gene develop abnormal kidneys that contain hypoplastic glomeruli and reduced proximal tubular mass, and they often die of renal failure. A comparison of kidney-specific gene expression between wild-type and COX-2-deficient mice by cDNA microarrays revealed that although more than 500 mRNAs were differentially expressed between the two strains of mice depending on their ages, the genes encoding pre-pro-epidermal growth factor (pre-pro-EGF) and Tamm-Horsfall protein (THP)/uromodulin were aberrantly expressed in the kidneys of COX-2 -/- mice at all stages of their development. Downregulation of EGF could potentially affect renal development, and THP/uromodulin gene has been implicated in abnormal kidney development and end-stage renal failure in humans. We assessed in detail mechanism of defective THP/uromodulin gene expression and its potential consequences in COX-2-deficient mice. Consistent with the microarray data, the steady-state levels of THP/uromodulin mRNA were severely reduced in the COX-2 -/- kidney. Furthermore, reduced expression of renal THP/uromodulin, as assessed by Western blot and immunohistological methods, was closely corroborated by a corresponding decline in the urinary secretion of THP/uromodulin in COX-2 -/- mice. Finally, we demonstrate that the bladders of COX-2 -/- mice, in contrast to those of the wild-type mice, are highly susceptible to colonization by uropathogenic Escherichia coli.

Mapping the fibrinogen-binding domain of serum opacity factor of group a streptococci.

Serum opacity factor (SOF) is a large, extracellular, and cell-bound protein of group A streptococci that has two known functions, opacification of serum and binding of fibronectin. Herein, we describe a new function of SOF, the binding of fibrinogen. Utilizing purified, truncated recombinant SOF proteins, the fibrinogen-binding domain was localized to a region in the C-terminus of SOF encompassing amino acid residues 844-1047. Western-blot analysis revealed that SOF bound primarily to the beta subunit of fibrinogen. A SOF-negative mutant bound 50% less fibrinogen than did its wild-type parent. Furthermore, fibrinogen blocked the binding of SOF to fibronectin. These data suggest that fibrinogen and fibronectin bind to the same domain within SOF. It remains to be determined whether the binding of fibrinogen to SOF contributes to the virulence of group A streptococci.

Serum opacity factor (SOF) of Streptococcus pyogenes evokes antibodies that opsonize homologous and heterologous SOF-positive serotypes of group A streptococci.

Serum opacity factor (SOF) is a protein expressed by Streptococcus pyogenes that opacifies mammalian serum. SOF is also a virulence factor of S. pyogenes, but it has not been previously shown to elicit a protective immune response. Herein, we report that SOF evokes bactericidal antibodies against S. pyogenes in humans, rabbits, and mice. Rabbit antiserum against purified recombinant SOF2 opsonized SOF-positive M type 2, 4, and 28 S. pyogenes in human blood but had no effect on SOF-negative M type 5 S. pyogenes. Furthermore, affinity-purified human antibodies against SOF2 also opsonized SOF-positive streptococci. A combination of antisera against M2 and SOF2 proteins was dramatically more effective in killing streptococci than either antiserum alone, indicating that antibodies against SOF2 enhance the opsonic efficiency of M protein antibodies. Mice tolerated an intravenous injection of 100 microg of SOF without overt signs of toxicity, and immunization with SOF protected mice against challenge infections with M type 2 S. pyogenes. These data indicate that SOF evokes opsonic antibodies that may protect against infections by SOF-positive serotypes of group A streptococci and suggest that different serotypes of SOF have common epitopes that may be useful vaccine candidates to protect against group A streptococcal infections.

Interaction of an uuter membrane protein of enterotoxigenic Escherichia coli with cell surface heparan sulfate proteoglycans.

We have previously shown that enterotoxigenic invasion protein A (Tia), a 25-kDa outer membrane protein encoded on an apparent pathogenicity island of enterotoxigenic Escherichia coli (ETEC) strain H10407, mediates attachment to and invasion into cultured human gastrointestinal epithelial cells. The epithelial cell receptor(s) for Tia has not been identified. Here we show that Tia interacts with cell surface heparan sulfate proteoglycans. Recombinant E. coli expressing Tia mediated invasion into wild-type epithelial cell lines but not invasion into proteoglycan-deficient cells. Furthermore, wild-type eukaryotic cells, but not proteoglycan-deficient eukaryotic cells, attached to immobilized polyhistidine-tagged recombinant Tia (rTia). Binding of epithelial cells to immobilized rTia was inhibited by exogenous heparan sulfate glycosaminoglycans but not by hyaluronic acid, dermatan sulfate, or chondroitin sulfate. Similarly, pretreatment of eukaryotic cells with heparinase I, but not pretreatment of eukaryotic cells with chrondroitinase ABC, inhibited attachment to rTia. In addition, we also observed heparin binding to both immobilized rTia and recombinant E. coli expressing Tia. Heparin binding was inhibited by a synthetic peptide representing a surface loop of Tia, as well as by antibodies directed against this peptide. Additional studies indicated that Tia, as a prokaryotic heparin binding protein, may also interact via sulfated proteoglycan molecular bridges with a number of mammalian heparan sulfate binding proteins. These findings suggest that the binding of Tia to host epithelial cells is mediated at least in part through heparan sulfate proteoglycans and that ETEC belongs on the growing list of pathogens that utilize these ubiquitous cell surface molecules as receptors.

Molecular mechanisms of adhesion, colonization, and invasion of group A streptococci.

The initial step in establishing a bacterial infection is adhesion of the organism to the epithelium of the host. Group A streptococci use multiple adhesins to attach to host cells and the types of adhesins expressed by a particular strain will determine its tissue specificity. Expression of adhesins is regulated in response to changing environmental and growth conditions. Thus, the array of adhesins expressed by a group A streptococcus will depend on the complement of its adhesin genes and on the environment. Expression of some adhesins may trigger internalization of the streptococci by host cells, which may enable the streptococci to evade antibiotics and to facilitate the penetration of deeper tissues. In this review, we present the different molecular mechanisms of adhesion utilized by group A streptococci and how these interactions lead to colonization and invasion.

Antibodies against a synthetic peptide of SagA neutralize the cytolytic activity of streptolysin S from group A streptococci.

Virtually all group A streptococci (GAS) produce streptolysin S (SLS), a cytolytic toxin that is responsible for the beta-hemolysis surrounding colonies of the organisms grown on blood agar. SLS is an important virulence determinant of GAS, and recent studies have identified a nine-gene locus that is responsible for synthesis and transport of the toxin. SLS is not immunogenic; thus, no neutralizing antibodies are evoked during the course of natural infection. In the present study, we show that a synthetic peptide containing amino acid residues 10 to 30 of the putative SLS (SagA) propeptide [SLS(10-30)] coupled to keyhole limpet hemocyanin evoked antibodies in rabbits that completely neutralized the hemolytic activity of the toxin in vitro. Inhibition of hemolysis was reversed by preincubation of the immune serum with soluble, unconjugated peptide, indicating the specificity of the antibodies. In addition, antibodies that were affinity purified over an SLS(10-30) peptide column completely inhibited SLS-mediated hemolysis. The SLS(10-30) antisera did not opsonize group A streptococci; however, when combined with type-specific M protein antisera, the SLS antibodies significantly enhanced phagocytosis mediated by M protein antibodies. Thus, we have shown for the first time that it is possible to raise neutralizing antibodies against one of the most potent bacterial cytolytic toxins known. Our data also provide convincing evidence that the sagA gene actually encodes the SLS peptide of GAS. The synthetic peptide may prove to be an important component of vaccines designed to prevent GAS infections.

Ablation of the Tamm-Horsfall protein gene increases susceptibility of mice to bladder colonization by type 1-fimbriated Escherichia coli.

The adhesion of uropathogenic Escherichia coli to the urothelial surface of the bladder is a prerequisite for the establishment of bladder infections. This adhesion process relies on E. coli adhesins and their cognate urothelial receptors, and it also is influenced by an intricate array of defense mechanisms of the urinary system. In this study, we examined the in vivo role of Tamm-Horsfall protein (THP), the most abundant urinary protein, in innate urinary defense. We genetically ablated the mouse THP gene and found that THP deficiency predisposes mice to bladder infections by type 1-fimbriated E. coli. Inoculation of too few type 1-fimbriated E. coli to colonize wild-type mice caused significant bladder colonization in THP-knockout mice. In contrast, THP deficiency did not enhance the ability of P-fimbriated E. coli to colonize the bladder. Our results provide the first in vivo evidence indicating that under physiological conditions, the mannosylated THP can serve as an effective soluble "receptor," binding to the type 1-fimbriated E. coli and competitively inhibiting them from adhering to the uroplakin Ia receptors present on the urothelial surface. These results suggest that potential THP defects, either quantitative or qualitative, could predispose the urinary bladder to bacterial infections. The generation of THP-deficient mice established the role of THP as a first line of urinary defense and should help elucidate other potential functions of this major protein in urinary tract physiology and diseases.

Tamm-Horsfall protein is a critical renal defense factor protecting against calcium oxalate crystal formation.

BACKGROUND: The tubular fluid of the mammalian kidney is often supersaturated with mineral salts, but crystallization rarely occurs under normal conditions. The unique ability of the kidney to avoid harmful crystal formation has long been attributed to the inhibitory activity of the urinary macromolecules, although few in vivo studies have been carried out to examine this hypothesis. Here we examined the role of Tamm-Horsfall protein (THP), the principal urinary protein, in urinary defense against renal calcium crystal formation, using a THP knockout model that we recently developed. METHODS: Wild-type and THP knockout mice were examined for the spontaneous formation of renal calcium crystals using von Kossa staining. The susceptibility of these mice to experimentally induced renal crystal formation was evaluated by administering mice with ethylene glycol, a precursor of oxalate, and vitamin D(3), which increases calcium absorption. Renal calcium crystals were visualized by von Kossa stain, dark field microscopy with polarized light and scanning electron microscopy. RESULTS: Inactivating the THP gene in mouse embryonic stem cells results in spontaneous formation of calcium crystals in adult kidneys. Excessive intake of calcium and oxalate, precursors of the most common type of human renal stones, dramatically increases both the frequency and the severity of renal calcium crystal formation in THP-deficient, but not in wild-type mice. Under high calcium/oxalate conditions, the absence of THP triggers a marked, adaptive induction in renal epithelial cells of osteopontin (OPN), a potent inhibitor of bone mineralization and vascular calcification. Thus, OPN may serve as an inducible inhibitor of calcium crystallization, whereas THP can serve as a constitutive and apparently more effective inhibitor. CONCLUSION: These results provide the first in vivo evidence that THP is a critical urinary defense factor and suggest that its deficiency could be an important contributing factor in human nephrolithiasis, a condition afflicting tens of millions of people in the world annually.