Paula J. Crowley

Elongated fibrillar structure of a streptococcal adhesin assembled by the high-affinity association of α- and PPII-helices

Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein adhesin that interacts with salivary components within the salivary pellicle. AgI/II contributes to virulence and has been studied as an immunological and structural target, but a fundamental understanding of its underlying architecture has been lacking. Here we report a high-resolution (1.8 Å) crystal structure of the A3VP1 fragment of S. mutans AgI/II that demonstrates a unique fibrillar form (155 Å) through the interaction of two noncontiguous regions in the primary sequence. The A3 repeat of the alanine-rich domain adopts an extended α-helix that intertwines with the P1 repeat polyproline type II (PPII) helix to form a highly extended stalk-like structure heretofore unseen in prokaryotic or eukaryotic protein structures. Velocity sedimentation studies indicate that full-length AgI/II that contains three A/P repeats extends over 50 nanometers in length. Isothermal titration calorimetry revealed that the high-affinity association between the A3 and P1 helices is enthalpically driven. Two distinct binding sites on AgI/II to the host receptor salivary agglutinin (SAG) were identified by surface plasmon resonance (SPR). The current crystal structure reveals that AgI/II family proteins are extended fibrillar structures with the number of alanine- and proline-rich repeats determining their length.

Independent gene duplications of the YidC/Oxa/Alb3 family enabled a specialized cotranslational function

YidC/Oxa/Alb3 family proteins catalyze the insertion of integral membrane proteins in bacteria, mitochondria, and chloroplasts, respectively. Unlike gram-negative organisms, gram-positive bacteria express 2 paralogs of this family, YidC1/SpoIIIJ and YidC2/YgjG. In Streptococcus mutans, deletion of yidC2 results in a stress-sensitive phenotype similar to that of mutants lacking the signal recognition particle (SRP) protein translocation pathway, while deletion of yidC1 has a less severe phenotype. In contrast to eukaryotes and gram-negative bacteria, SRP-deficient mutants are viable in S. mutans; however, double SRP-yidC2 mutants are severely compromised. Thus, YidC2 may enable loss of the SRP by playing an independent but overlapping role in cotranslational protein insertion into the membrane. This is reminiscent of the situation in mitochondria that lack an SRP pathway and where Oxa1 facilitates cotranslational membrane protein insertion by binding directly to translation-active ribosomes. Here, we show that OXA1 complements a lack of yidC2 in S. mutans. YidC2 also functions reciprocally in oxa1-deficient Saccharomyces cerevisiae mutants and mediates the cotranslational insertion of mitochondrial translation products into the inner membrane. YidC2, like Oxa1, contains a positively charged C-terminal extension and associates with translating ribosomes. Our results are consistent with a gene-duplication event in gram-positive bacteria that enabled the specialization of a YidC isoform that mediates cotranslational activity independent of an SRP pathway.

Streptococcus mutans ffh, a gene encoding a homologue of the 54 kDa subunit of the signal recognition particle, is involved in resistance to acid stress

The ability of Streptococcus mutans, a bacterial pathogen associated with dental caries, to tolerate rapid drops in plaque pH (acidurance), is considered an important virulence factor. To study this trait, Tn917 mutants of S. mutans strain JH1005 which display acid sensitivity have been isolated and partially characterized. In this paper, the characterization of one of these mutants, AS17, is reported. Preliminary sequence analysis revealed that the transposon insertion in AS17 occurred in the intergenic region of a two-gene locus which has been named sat for secretion and acid tolerance. This locus displays a high degree of homology to the ylxM-ffh operon of Bacillus subtilis. The sat+ locus was cloned by complementation of a conditional Escherichia coli ffh mutant with an S. mutans genomic library. Sequencing of the complementing clone identified the intact ylxM and ffh genes as well as a partial ORF with homology to the proUlopuAC gene of B. subtilis which encodes the binding protein of the ProU/OpuA osmoregulated glycine betaine transport system. RNA dot blot experiments indicated steady-state levels of ffh mRNA in the mutant that were approximately eightfold lower compared to parental levels. This suggests a partial polar effect of the sat-1::Tn917 mutation on ffh expression. Upon acid shock (pH 5), wild-type ffh mRNA levels were found to increase approximately four- to eightfold compared to unstressed (pH 7.5) levels. Mutant levels remained unaltered under the same conditions. Experiments designed to investigate the origins of the acid-sensitivity of the mutant revealed a lack of an acid-adaptive/tolerance response. Assays of proton-extruding ATPase (H+/ATPase) specific activity measured with purified membranes derived from acid-shocked AS17 showed twofold lower levels compared to the parent strain. Also, AS17 was found to be unable to ferment sorbitol although it was able to grow in glucose and a variety of other sugar substrates. These findings suggest that Ffh may be involved in the maintenance of a functional membrane protein composition during adaptation of S. mutans to changing environmental conditions.

Functional amyloid formation by Streptococcus mutans.

Dental caries is a common infectious disease associated with acidogenic and aciduric bacteria, including Streptococcus mutans. Organisms that cause cavities form recalcitrant biofilms, generate acids from dietary sugars and tolerate acid end products. It has recently been recognized that micro-organisms can produce functional amyloids that are integral to biofilm development. We now show that the S. mutans cell-surface-localized adhesin P1 (antigen I/II, PAc) is an amyloid-forming protein. This conclusion is based on the defining properties of amyloids, including binding by the amyloidophilic dyes Congo red (CR) and Thioflavin T (ThT), visualization of amyloid fibres by transmission electron microscopy and the green birefringent properties of CR-stained protein aggregates when viewed under cross-polarized light. We provide evidence that amyloid is present in human dental plaque and is produced by both laboratory strains and clinical isolates of S. mutans. We provide further evidence that amyloid formation is not limited to P1, since bacterial colonies without this adhesin demonstrate residual green birefringence. However, S. mutans lacking sortase, the transpeptidase enzyme that mediates the covalent linkage of its substrates to the cell-wall peptidoglycan, including P1 and five other proteins, is not birefringent when stained with CR and does not form biofilms. Biofilm formation is inhibited when S. mutans is cultured in the presence of known inhibitors of amyloid fibrillization, including CR, Thioflavin S and epigallocatechin-3-gallate, which also inhibited ThT uptake by S. mutans extracellular proteins. Taken together, these results indicate that S. mutans is an amyloid-forming organism and suggest that amyloidogenesis contributes to biofilm formation by this oral microbe.

Membrane Composition Changes and Physiological Adaptation by Streptococcus mutans Signal Recognition Particle Pathway Mutants

Previously, we presented evidence that the oral cariogenic species Streptococcus mutans remains viable but physiologically impaired and sensitive to environmental stress when genes encoding the minimal conserved bacterial signal recognition particle (SRP) elements are inactivated. Two-dimensional gel electrophoresis of isolated membrane fractions from strain UA159 and three mutants (Deltaffh, DeltascRNA, and DeltaftsY) grown at pH 7.0 or pH 5.0 allowed us to obtain insight into the adaptation process and the identities of potential SRP substrates. Mutant membrane preparations contained increased amounts of the chaperones DnaK and GroES and ClpP protease but decreased amounts of transcription- and translation-related proteins, the beta subunit of ATPase, HPr, and several metabolic and glycolytic enzymes. Therefore, the acid sensitivity of SRP mutants might be caused in part by diminished ATPase activity, as well as the absence of an efficient mechanism for supplying ATP quickly at the site of proton elimination. Decreased amounts of LuxS were also observed in all mutant membranes. To further define physiological changes that occur upon disruption of the SRP pathway, we studied global gene expression in S. mutans UA159 (parent strain) and AH333 (Deltaffh mutant) using microarray analysis. Transcriptome analysis revealed up-regulation of 81 genes, including genes encoding chaperones, proteases, cell envelope biosynthetic enzymes, and DNA repair and replication enzymes, and down-regulation of 35 genes, including genes concerned with competence, ribosomal proteins, and enzymes involved in amino acid and protein biosynthesis. Quantitative real-time reverse transcription-PCR analysis of eight selected genes confirmed the microarray data. Consistent with a demonstrated defect in competence and the suggested impairment of LuxS-dependent quorum sensing, biofilm formation was significantly decreased in each SRP mutant.

Crystal Structure of the C-terminal Region of Streptococcus mutans Antigen I/II and Characterization of Salivary Agglutinin Adherence Domains

The Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein that adheres to salivary components and extracellular matrix molecules. Here we report the 2.5 Å resolution crystal structure of the complete C-terminal region of AgI/II. The C-terminal region is comprised of three major domains: C1, C2, and C3. Each domain adopts a DE-variant IgG fold, with two β-sheets whose A and F strands are linked through an intramolecular isopeptide bond. The adherence of the C-terminal AgI/II fragments to the putative tooth surface receptor salivary agglutinin (SAG), as monitored by surface plasmon resonance, indicated that the minimal region of binding was contained within the first and second DE-variant-IgG domains (C1 and C2) of the C terminus. The minimal C-terminal region that could inhibit S. mutans adherence to SAG was also confirmed to be within the C1 and C2 domains. Competition experiments demonstrated that the C- and N-terminal regions of AgI/II adhere to distinct sites on SAG. A cleft formed at the intersection between these C1 and C2 domains bound glucose molecules from the cryo-protectant solution, revealing a putative binding site for its highly glycosylated receptor SAG. Finally, electron microscopy images confirmed the elongated structure of AgI/II and enabled building a composite tertiary model that encompasses its two distinct binding regions.

YidC1 and YidC2 are functionally distinct proteins involved in protein secretion, biofilm formation and cariogenicity of Streptococcus mutans.

The cariogenic bacterium Streptococcus mutans has two paralogues of the YidC/Oxa1/Alb3 family of membrane protein insertases/chaperones. Disruption of yidC2 results in loss of genetic competence, decreased membrane-associated ATPase activity and stress sensitivity (acid, osmotic and oxidative). Elimination of yidC1 has less severe effects, with little observable effect on growth or stress sensitivity. To examine the respective roles of YidC1 and YidC2, a conditional expression system was developed allowing simultaneous elimination of both endogenous YidCs. The function of the YidC C-terminal tails was also investigated and a chimeric YidC1 protein appended with the C terminus of YidC2 enabled YidC1 to complement a ΔyidC2 mutant for stress tolerance, ATP hydrolysis activity and extracellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity. Elimination of yidC1 or yidC2 affected levels of extracellular proteins, including GtfB, GtfC and adhesin P1 (AgI/II, PAc), which were increased without YidC1 but decreased in the absence of YidC2. Both yidC1 and yidC2 were shown to contribute to S. mutans biofilm formation and to cariogenicity in a rat model. Collectively, these results provide evidence that YidC1 and YidC2 contribute to cell surface biogenesis and protein secretion in S. mutans and that differences in stress sensitivity between the ΔyidC1 and ΔyidC2 mutants stem from a functional difference in the C-termini of these two proteins.

Requirements for Surface Expression and Function of Adhesin P1 from Streptococcus mutans

ABSTRACT In this report, we define requirements for the successful translocation and functional maturation of the adhesin P1 of Streptococcus mutans. Conformational epitopes recognized by anti-P1 monoclonal antibodies (MAbs) were further characterized, thus facilitating the use of particular MAbs as tools to monitor the locations of various forms of the protein. We show that correct localization of P1 is dependent on structural features of the molecule itself, including a requisite A region-P region intramolecular interaction that occurs within the cell prior to secretion. P1 also was shown to be affected by several members of the protein-folding-secretion-turnover apparatus. It does not achieve a fully functional form in the absence of the trigger factor PPIase homolog RopA, and its translocation is delayed when DnaK levels are limited. In addition, dnaK message levels are differentially altered in the presence of P1 lacking the alanine-rich compared to the proline-rich repeat domains. Lastly, nonsecreted P1 lacking the P region accumulates within the cell in the absence of htrA, implying an intracellular HtrA protease function in the degradation and turnover of this particular internal-deletion polypeptide. However, the opposite effect is seen for full-length P1, suggesting a sensing mechanism and substrate-dependent alteration in HtrAs function and effect that is consistent with its known ability to switch between chaperone and protease, depending on environmental perturbations.

Binding Forces of Streptococcus mutans P1 Adhesin

Streptococcus mutans is a Gram-positive oral bacterium that is a primary etiological agent associated with human dental caries. In the oral cavity, S. mutans adheres to immobilized salivary agglutinin (SAG) contained within the salivary pellicle on the tooth surface. Binding to SAG is mediated by cell surface P1, a multifunctional adhesin that is also capable of interacting with extracellular matrix proteins. This may be of particular importance outside of the oral cavity as S. mutans has been associated with infective endocarditis and detected in atherosclerotic plaque. Despite the biomedical importance of P1, its binding mechanisms are not completely understood. In this work, we use atomic force microscopy-based single-molecule and single-cell force spectroscopy to quantify the nanoscale forces driving P1-mediated adhesion. Single-molecule experiments show that full-length P1, as well as fragments containing only the P1 globular head or C-terminal region, binds to SAG with relatively weak forces (∼50 pN). In contrast, single-cell analyses reveal that adhesion of a single S. mutans cell to SAG is mediated by strong (∼500 pN) and long-range (up to 6000 nm) forces. This is likely due to the binding of multiple P1 adhesins to self-associated gp340 glycoproteins. Such a cooperative, long-range character of the S. mutans-SAG interaction would therefore dramatically increase the strength and duration of cell adhesion. We also demonstrate, at single-molecule and single-cell levels, the interaction of P1 with fibronectin and collagen, as well as with hydrophobic, but not hydrophilic, substrates. The binding mechanism (strong forces, cooperativity, broad specificity) of P1 provides a molecular basis for its multifunctional adhesion properties. Our methodology represents a valuable approach to probe the binding forces of bacterial adhesins and offers a tractable methodology to assess anti-adhesion therapy.

An intramolecular lock facilitates folding and stabilizes the tertiary structure of Streptococcus mutans adhesin P1

Significance Streptococcus mutans adhesin P1 is a target of protective immunity and a vaccine candidate. P1’s complex structure dictates its function and makes it of interest from a protein folding perspective as well. An interaction between N- and C-terminal sequences contributes to antigenicity, adherence behavior, and stability. This is now explained by the identification of a previously unidentified fold in which the N terminus forms a stabilizing scaffold at the base of P1’s helical stalk to physically lock it in place via interactions with the C terminus. Disruption of this intramolecular lock not only negatively affects stability, but also prevents proper folding of the purified full-length protein. The cariogenic bacterium Streptococcus mutans uses adhesin P1 to adhere to tooth surfaces, extracellular matrix components, and other bacteria. A composite model of P1 based on partial crystal structures revealed an unusual complex architecture in which the protein forms an elongated hybrid alpha/polyproline type II helical stalk by folding back on itself to display a globular head at the apex and a globular C-terminal region at the base. The structure of P1’s N terminus and the nature of its critical interaction with the C-terminal region remained unknown, however. We have cocrystallized a stable complex of recombinant N- and C-terminal fragments and here describe a previously unidentified topological fold in which these widely discontinuous domains are intimately associated. The structure reveals that the N terminus forms a stabilizing scaffold by wrapping behind the base of P1’s elongated stalk and physically “locking” it into place. The structure is stabilized through a highly favorable ΔGsolvation on complex formation, along with extensive hydrogen bonding. We confirm the functional relevance of this intramolecular interaction using differential scanning calorimetry and circular dichroism to show that disruption of the proper spacing of residues 989–1001 impedes folding and diminishes stability of the full-length molecule, including the stalk. Our findings clarify previously unexplained functional and antigenic properties of P1.