Jukka Hytönen

Fluid- or Surface-Phase Human Salivary Scavenger Protein gp340 Exposes Different Bacterial Recognition Properties

ABSTRACT Salivary scavenger receptor cysteine-rich protein gp340 aggregates streptococci and other bacteria as part of the host innate defense system at mucosal surfaces. In this article, we have investigated the properties of fluid-phase gp340 and hydroxylapatite surface-adsorbed gp340 in aggregation and adherence, respectively, of viridans group streptococci (e.g., Streptococcus gordonii and Streptococcus mutans), non-viridans group streptococci (e.g., Streptococcus pyogenes and Streptococcus suis), and oral Actinomyces. Fluid-phase gp340 and surface-phase gp340 bioforms were differentially recognized by streptococci, which formed three phenotypic groupings according to their modes of interaction with gp340. Group I streptococci were aggregated by and adhered to gp340, and group II streptococci preferentially adhered to surface-bound gp340, while group III streptococci were preferentially aggregated by gp340. Each species of Streptococcus tested was found to contain strains representative of at least two of these gp340 interaction groupings. The gp340 interaction modes I to III and sugar specificities of gp340 binding strains coincided for several species. Many gp340 interactions were sialidase sensitive, and each of the interaction modes (I to III) for S. gordonii was correlated with a variant of sialic acid specificity. Adherence of S. gordonii DL1 (Challis) to surface-bound gp340 was dependent upon expression of the sialic acid binding adhesin Hsa. However, aggregation of cells by fluid-phase gp340 was independent of Hsa and involved SspA and SspB (antigen I/II family) polypeptides. Conversely, both gp340-mediated aggregation and adherence of S. mutans NG8 involved antigen I/II polypeptide. Deletion of the mga virulence regulator gene in S. pyogenes resulted in increased cell aggregation by gp340. These results suggest that salivary gp340 recognizes different bacterial receptors according to whether gp340 is present in the fluid phase or surface bound. This phase-associated differential recognition by gp340 of streptococcal species of different levels of virulence and diverse origins may mediate alternative host responses to commensal or pathogenic bacterial phenotypes.

The SpeB virulence factor of Streptococcus pyogenes, a multifunctional secreted and cell surface molecule with strepadhesin, laminin-binding and cysteine protease activity.

The interactions between pathogenic bacteria and the host need to be resolved at the molecular level in order to develop novel vaccines and drugs. We have previously identified strepadhesin, a novel glycoprotein‐binding activity in Streptococcus pyogenes, which is regulated by Mga, a regulator of streptococcal virulence factors. We have now identified the protein responsible for the strepadhesin activity and find that (i) strepadhesin activity is carried by SpeB, streptococcal pyrogenic exotoxin with cysteine protease activity; (ii) SpeB carries laminin‐binding activity of the bacteria; and (iii) SpeB is not only a secreted molecule but also occurs unexpectedly tightly bound to the bacterial cell surface. Thus, in contrast to the previous view of SpeB as mainly an extracellular protease, it is also present as a streptococcal surface molecule with binding activity to laminin and other glycoproteins.

Anti—Tumor Necrosis Factor—α Treatment Activates Borrelia burgdorferi Spirochetes 4 Weeks after Ceftriaxone Treatment in C3H/He Mice

BACKGROUND The effect of anti-tumor necrosis factor (TNF)-alpha treatment in Borrelia burgdorferi-infected and ceftriaxone-treated C3H/He mice was evaluated. METHODS Mice were infected with B. garinii A218 or B. burgdorferi sensu stricto N40. At 2 weeks of infection, one group was treated simultaneously with ceftriaxone and anti-TNF-alpha, whereas another received ceftriaxone at 2 weeks and anti-TNF-alpha 4 weeks later. One group received ceftriaxone treatment only. Infected and noninfected control groups were sham treated. RESULTS At 14 weeks of infection, B. burgdorferi could not be detected by cultivation or by polymerase chain reaction in tissue samples of any mouse treated with ceftriaxone only. However, spirochetes grew from the tissue samples of one-third of the mice treated with anti-TNF-alpha simultaneously or 4 weeks after ceftriaxone. These activated spirochetes showed ceftriaxone sensitivity rates, plasmid profiles, and virulence rates similar to those of bacteria used to infect the mice. All infected control mice and mice given anti-TNF-alpha only were culture positive. CONCLUSIONS This report shows that, after ceftriaxone treatment for 5 days, a portion of B. burgdorferi-infected mice still have live spirochetes in their body, which are activated by anti-TNF-alpha treatment.

Streptococcus pyogenes glycoprotein-binding strepadhesin activity is mediated by a surface-associated carbohydrate-degrading enzyme, pullulanase.

ABSTRACT The interactions between pathogenic bacteria and the host need to be resolved at the molecular level in order to develop novel antiadhesive drugs and vaccines. We have previously identified strepadhesin, a novel glycoprotein-binding activity in Streptococcus pyogenes binding to thyroglobulin, submaxillar mucin, fetuin, and asialofetuin. The activity is known to be regulated by Mga, a regulator of streptococcal virulence factors, and is carried by the surface-associated streptococcal cysteine protease, SpeB. In the present study, we focused on the high strepadhesin activity in an S. pyogenes strain (NZ131rgg) lacking SpeB expression. By extracting surface proteins from the bacteria, a new strepadhesin protein was identified, and mass spectrometric analysis and database search identified it as a putative pullulanase. The gene was cloned, and the recombinant pullulanase (PulA) exhibited pullulanase and starch hydrolyzing activity, as well as strepadhesin activity. Sequencing of the pulA gene revealed an open reading frame with 3,498 bp encoding a protein of 1,165 amino acids with a predicted molecular mass of 129 kDa. PulA exhibited properties typical for a gram-positive surface protein with a putative signal sequence and LPKTGE cell wall anchoring motif and contained the four highly conserved regions common to pullulanases. Mutant bacteria deficient in PulA expression showed diminished strepadhesin activity on bacterial dot blot assay and reduced adherence to thyroglobulin immobilized on microtiter plates. Thus, S. pyogenes strepadhesin activity is carried by a surface-bound pullulanase, which combines glycoprotein-binding and carbohydrate-degrading activities in the same molecule.

Leucine-rich Repeats of Bacterial Surface Proteins Serve as Common Pattern Recognition Motifs of Human Scavenger Receptor gp340

Scavenger receptors are innate immune molecules recognizing and inducing the clearance of non-host as well as modified host molecules. To recognize a wide pattern of invading microbes, many scavenger receptors bind to common pathogen-associated molecular patterns, such as lipopolysaccharides and lipoteichoic acids. Similarly, the gp340/DMBT1 protein, a member of the human scavenger receptor cysteine-rich protein family, displays a wide ligand repertoire. The peptide motif VEVLXXXXW derived from its scavenger receptor cysteine-rich domains is involved in some of these interactions, but most of the recognition mechanisms are unknown. In this study, we used mass spectrometry sequencing, gene inactivation, and recombinant proteins to identify Streptococcus pyogenes protein Spy0843 as a recognition receptor of gp340. Antibodies against Spy0843 are shown to protect against S. pyogenes infection, but no function or host receptor have been identified for the protein. Spy0843 belongs to the leucine-rich repeat (Lrr) family of eukaryotic and prokaryotic proteins. Experiments with truncated forms of the recombinant proteins confirmed that the Lrr region is needed in the binding of Spy0843 to gp340. The same motif of two other Lrr proteins, LrrG from the Gram-positive S. agalactiae and BspA from the Gram-negative Tannerella forsythia, also mediated binding to gp340. Moreover, inhibition of Spy0843 binding occurred with peptides containing the VEVLXXXXW motif, but also peptides devoid of the XXXXW motif inhibited binding of Lrr proteins. These results thus suggest that the conserved Lrr motif in bacterial proteins serves as a novel pattern recognition motif for unique core peptides of human scavenger receptor gp340.

Persistence of borrelial DNA in the joints of Borrelia burgdorferi-infected mice after ceftriaxone treatment.

Yrjänäinen H, Hytönen J, Hartiala P, Oksi J, Viljanen MK. Persistence of borrelial DNA in the joints of Borrelia burgdorferi‐infected mice after ceftriaxone treatment. APMIS 2010; 118: 665–73.

Deficiency of the Rgg Regulator Promotes H2O2 Resistance, AhpCF-Mediated H2O2 Decomposition, and Virulence in Streptococcus pyogenes

Streptococcus pyogenes (group A streptococcus [GAS]), a catalase-negative gram-positive bacterium, is aerotolerant and survives H2O2 exposures that kill many catalase-positive bacteria. The molecular basis of the H2O2 resistance is poorly known. Here, we demonstrate that serotype M49 GAS lacking the Rgg regulator is more resistant to H2O2 and also decomposes more H2O2 than the parental strain. Subgenomic transcriptional profiling and genome-integrated green fluorescent protein reporters showed that a bicistronic operon, a homolog of the Streptococcus mutans ahpCF operon, is transcriptionally up-regulated in the absence of Rgg. Phenotypic assays with ahpCF operon knockouts demonstrated that the gene products decompose H2O2 and protect GAS against peroxide stress. In a murine intraperitoneal-infection model, Rgg deficiency increased the virulence of GAS, although in an ahpCF-independent manner. Rgg-mediated repression of H2O2 resistance is divergent from the previously characterized peroxide resistance repressor PerR. Moreover, Rgg-mediated repression of H2O2 resistance is inducible by cellular stresses of diverse natures--ethanol, organic hydroperoxide, and H2O2. Rgg is thus identified as a novel sensoregulator of streptococcal H2O2 resistance with potential implications for the virulence of the catalase-negative GAS.

Bordetella pertussis isolates in Finland: Serotype and fimbrial expression

BackgroundBordetella pertussis causes whooping cough or pertussis in humans. It produces several virulence factors, of which the fimbriae are considered adhesins and elicit immune responses in the host. B. pertussis has three distinct serotypes Fim2, Fim3 or Fim2,3. Generally, B. pertussis Fim2 strains predominate in unvaccinated populations, whereas Fim3 strains are often isolated in vaccinated populations. In Finland, pertussis vaccination was introduced in 1952. The whole-cell vaccine contained two strains, 18530 (Fim3) since 1962 and strain 1772 (Fim2,3) added in 1976. After that the vaccine has remained the same until 2005 when the whole-cell vaccine was replaced by the acellular vaccine containing pertussis toxin and filamentous hemagglutinin. Our aims were to study serotypes of Finnish B. pertussis isolates from 1974 to 2006 in a population with > 90% vaccination coverage and fimbrial expression of the isolates during infection. Serotyping was done by agglutination and serotype-specific antibody responses were determined by blocking ELISA.ResultsAltogether, 1,109 isolates were serotyped. Before 1976, serotype distributions of Fim2, Fim3 and Fim2,3 were 67%, 19% and 10%, respectively. From 1976 to 1998, 94% of the isolates were Fim2 serotype. Since 1999, the frequency of Fim3 strains started to increase and reached 83% during a nationwide epidemic in 2003. A significant increase in level of serum IgG antibodies against purified fimbriae was observed between paired sera of 37 patients. The patients infected by Fim3 strains had antibodies which blocked the binding of monoclonal antibodies to Fim3 but not to Fim2. Moreover, about one third of the Fim2 strain infected patients developed antibodies capable of blocking of binding of both anti-Fim2 and Fim3 monoclonal antibodies.ConclusionDespite extensive vaccinations in Finland, B. pertussis Fim2 strains were the most common serotype. Emergence of Fim3 strains started in 1999 and coincided with nationwide epidemics. Results of serotype-specific antibody responses suggest that Fim2 strains could express Fim3 during infection, showing a difference in fimbrial expression between in vivo and in vitro.

Use of CFSE staining of borreliae in studies on the interaction between borreliae and human neutrophils

BackgroundSpecies of the tick-transmitted spirochete group Borrelia burgdorferi sensu lato (B. burgdorferi) cause Lyme borreliosis. Acute borrelial infection of the skin has unusual characteristics with only a mild local inflammatory response suggesting that the interaction between borreliae and the cells of the first-line defence might differ from that of other bacteria. It has been reported that human neutrophils phagocytose motile borreliae through an unconventional mechanism (tube phagocytosis) which is not observed with non-motile borreliae. Therefore, it would be of great interest to visualise the bacteria by a method not affecting motility and viability of borreliae to be able to study their interaction with the cells of the innate immunity. Carboxyfluorescein diacetate, succinimidyl ester (CFSE) labelling has been previously used for studying the adhesion of labelled bacteria to host cells and the uptake of labelled substrates by various cells using flow cytometry.ResultsIn this study, CFSE was shown to efficiently stain different genospecies of B. burgdorferi without affecting bacterial viability or motility. Use of CFSE staining allowed subsequent quantification of borreliae associated with human neutrophils with flow cytometry and confocal microscopy. As a result, no difference in association between different borrelial genospecies (Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii), or between borreliae and the pyogenic bacterium Streptococcus pyogenes, with neutrophils could be detected. Borrelial virulence, on the other hand, affected association with neutrophils, with significantly higher association of a non-virulent mutant B. burgdorferi sensu stricto strain compared to the parental virulent wild type strain.ConclusionThese results suggest that the flow cytometric assay using CFSE labelled borreliae is a valuable tool in the analysis of the interaction between borreliae and human neutrophils. The results also indicate a clear difference in the association with neutrophils between virulent and non-virulent borrelial strains.

Transcriptional response of human dendritic cells to Borrelia garinii--defective CD38 and CCR7 expression detected

Lyme borreliosis is a disease, which can affect several organs and cause a variety of symptoms. In some patients, the infection may become chronic, even after antibiotic therapy, and cause persisting damage. Dendritic cells (DC) are involved in the initiation of innate and adaptive immune responses. To study interactions between Borrelia garinii (Bg), one of the causative agents of Lyme borreliosis, and human DC, we used a cDNA microarray to compare the Bg‐induced DC transcriptional response with the response induced by LPS. The Bg‐induced response consisted of a smaller number of genes than the LPS‐induced response. The microarray showed that the ectoenzyme CD38, which has an important role in DC chemotaxis and migration to lymph nodes, was strongly up‐regulated by LPS but practically not at all by Bg. This finding was confirmed with quantitative RT‐PCR and with flow cytometry at the protein level. In addition, RT‐PCR showed that CCR7 expression was 11‐fold greater in LPS‐stimulated than in Bg‐stimulated cells. These findings suggest that Bg may affect crucial DC functions by blocking the up‐regulation of important molecules in DC migration to lymph nodes, thus affecting further immune responses in Lyme borreliosis infection.