The exopolysaccharide matrix modulates the interaction between 3D architecture and virulence of a mixed-species oral biofilm.

Virulent biofilms are responsible for a range of infections, including oral diseases. All biofilms harbor a microbial-derived extracellular-matrix. The exopolysaccharides (EPS) formed on tooth-pellicle and bacterial surfaces provide binding sites for microorganisms; eventually the accumulated EPS enmeshes microbial cells. The metabolic activity of the bacteria within this matrix leads to acidification of the milieu. We explored the mechanisms through which the Streptococcus mutans-produced EPS-matrix modulates the three-dimensional (3D) architecture and the population shifts during morphogenesis of biofilms on a saliva-coated-apatitic surface using a mixed-bacterial species system. Concomitantly, we examined whether the matrix influences the development of pH-microenvironments within intact-biofilms using a novel 3D in situ pH-mapping technique. Data reveal that the production of the EPS-matrix helps to create spatial heterogeneities by forming an intricate network of exopolysaccharide-enmeshed bacterial-islets (microcolonies) through localized cell-to-matrix interactions. This complex 3D architecture creates compartmentalized acidic and EPS-rich microenvironments throughout the biofilm, which triggers the dominance of pathogenic S. mutans within a mixed-species system. The establishment of a 3D-matrix and EPS-enmeshed microcolonies were largely mediated by the S. mutans gtfB/gtfC genes, expression of which was enhanced in the presence of Actinomyces naeslundii and Streptococcus oralis. Acidic pockets were found only in the interiors of bacterial-islets that are protected by EPS, which impedes rapid neutralization by buffer (pH 7.0). As a result, regions of low pH (<5.5) were detected at specific locations along the surface of attachment. Resistance to chlorhexidine was enhanced in cells within EPS-microcolony complexes compared to those outside such structures within the biofilm. Our results illustrate the critical interaction between matrix architecture and pH heterogeneity in the 3D environment. The formation of structured acidic-microenvironments in close proximity to the apatite-surface is an essential factor associated with virulence in cariogenic-biofilms. These observations may have relevance beyond the mouth, as matrix is inherent to all biofilms.

Interactome analysis illustrates diverse gene regulatory processes associated with LIN28A in human iPS cell-derived neural progenitor cells.

A single protein can be multifaceted depending on the cellular contexts and interacting molecules. LIN28A is an RNA-binding protein that governs developmental timing, cellular proliferation, differentiation, stem cell pluripotency, and metabolism. In addition to its best-known roles in microRNA biogenesis, diverse molecular roles have been recognized. In the nervous system, LIN28A is known to play critical roles in proliferation and differentiation of neural progenitor cells (NPCs). We profiled the endogenous LIN28A-interacting proteins in NPCs differentiated from human induced pluripotent stem (iPS) cells using immunoprecipitation and liquid chromatography-tandem mass spectrometry. We identified over 500 LIN28A-interacting proteins, including 156 RNA-independent interactors. Functions of these proteins span a wide range of gene regulatory processes. Prompted by the interactome data, we revealed that LIN28A may impact the subcellular distribution of its interactors and stress granule formation upon oxidative stress. Overall, our analysis opens multiple avenues for elaborating molecular roles and characteristics of LIN28A.

Identification of sulfenylation patterns in trophozoite stage Plasmodium falciparum using a non-dimedone based probe.

Plasmodium falciparum causes the deadliest form of malaria. Adequate redox control is crucial for this protozoan parasite to overcome oxidative and nitrosative challenges, thus enabling its survival. Sulfenylation is an oxidative post-translational modification, which acts as a molecular on/off switch, regulating protein activity. To obtain a better understanding of which proteins are redox regulated in malaria parasites, we established an optimized affinity capture protocol coupled with mass spectrometry analysis for identification of in vivo sulfenylated proteins. The non-dimedone based probe BCN-Bio1 shows reaction rates over 100-times that of commonly used dimedone-based probes, allowing for a rapid trapping of sulfenylated proteins. Mass spectrometry analysis of BCN-Bio1 labeled proteins revealed the first insight into the Plasmodium falciparum trophozoite sulfenylome, identifying 102 proteins containing 152 sulfenylation sites. Comparison with Plasmodium proteins modified by S-glutathionylation and S-nitrosation showed a high overlap, suggesting a common core of proteins undergoing redox regulation by multiple mechanisms. Furthermore, parasite proteins which were identified as targets for sulfenylation were also identified as being sulfenylated in other organisms, especially proteins of the glycolytic cycle. This study suggests that a number of Plasmodium proteins are subject to redox regulation and it provides a basis for further investigations into the exact structural and biochemical basis of regulation, and a deeper understanding of cross-talk between post-translational modifications.

Human common salivary protein 1 (CSP-1) promotes binding of Streptococcus mutans to experimental salivary pellicle and glucans formed on hydroxyapatite surface.

The saliva proteome includes host defense factors and specific bacterial-binding proteins that modulate microbial growth and colonization of the tooth surface in the oral cavity. A multidimensional mass spectrometry approach identified the major host-derived salivary proteins that interacted with Streptococcus mutans (strain UA159), the primary microorganism associated with the pathogenesis of dental caries. Two abundant host proteins were found to tightly bind to S. mutans cells, common salivary protein-1 (CSP-1) and deleted in malignant brain tumor 1 (DMBT1, also known as salivary agglutinin or gp340). In contrast to gp340, limited functional information is available on CSP-1. The sequence of CSP-1 shares 38.1% similarity with rat CSP-1. Recombinant CSP-1 (rCSP-1) protein did not cause aggregation of S. mutans cells and was devoid of any significant biocidal activity (2.5 to 10 µg/mL). However, S. mutans cells exposed to rCSP-1 (10 µg/mL) in saliva displayed enhanced adherence to experimental salivary pellicle and to glucans in the pellicle formed on hydroxyapatite surfaces. Thus, our data demonstrate that the host salivary protein CSP-1 binds to S. mutans cells and may influence the initial colonization of this pathogenic bacterium onto the tooth surface.

Global site-specific analysis of glycoprotein N-glycan processing.

N-glycans contribute to the folding, stability and functions of the proteins they decorate. They are produced by transfer of the glycan precursor to the sequon Asn-X-Thr/Ser, followed by enzymatic trimming to a high-mannose-type core and sequential addition of monosaccharides to generate complex-type and hybrid glycans. This process, mediated by the concerted action of multiple enzymes, produces a mixture of related glycoforms at each glycosite, making analysis of glycosylation difficult. To address this analytical challenge, we developed a robust semiquantitative mass spectrometry (MS)-based method that determines the degree of glycan occupancy at each glycosite and the proportion of N-glycans processed from high-mannose type to complex type. It is applicable to virtually any glycoprotein, and a complete analysis can be conducted with 30 µg of protein. Here, we provide a detailed description of the method that includes procedures for (i) proteolytic digestion of glycoprotein(s) with specific and nonspecific proteases; (ii) denaturation of proteases by heating; (iii) sequential treatment of the glycopeptide mixture with two endoglycosidases, Endo H and PNGase F, to create unique mass signatures for the three glycosylation states; (iv) LC-MS/MS analysis; and (v) data analysis for identification and quantitation of peptides for the three glycosylation states. Full coverage of site-specific glycosylation of glycoproteins is achieved, with up to thousands of high-confidence spectra hits for each glycosite. The protocol can be performed by an experienced technician or student/postdoc with basic skills for proteomics experiments and takes ∼7 d to complete.

Differential processing of HIV envelope glycans on the virus and soluble recombinant trimer.

As the sole target of broadly neutralizing antibodies (bnAbs) to HIV, the envelope glycoprotein (Env) trimer is the focus of vaccination strategies designed to elicit protective bnAbs in humans. Because HIV Env is densely glycosylated with 75-90 N-glycans per trimer, most bnAbs use or accommodate them in their binding epitope, making the glycosylation of recombinant Env a key aspect of HIV vaccine design. Upon analysis of three HIV strains, we here find that site-specific glycosylation of Env from infectious virus closely matches Envs from corresponding recombinant membrane-bound trimers. However, viral Envs differ significantly from recombinant soluble, cleaved (SOSIP) Env trimers, strongly impacting antigenicity. These results provide a benchmark for virus Env glycosylation needed for the design of soluble Env trimers as part of an overall HIV vaccine strategy.

MudPIT: multidimensional protein identification technology.

Large-scale biology emerged out of the efforts to sequence genomes of important organisms. Based on resources created by whole genome sequencing, large-scale analyses of messenger RNA (mRNA) and protein expression are now possible. With the availability of large amounts of genomic sequence information, a convenient method for the identification and analysis of proteins based on proteolytic digestion into peptides emerged. Processes to fragment peptides using collision-activated dissociation (CAD) in tandem mass spectrometers and computer algorithms to match the tandem mass spectra of peptides to sequences in databases enable rapid identification of amino acid sequences, and hence proteins, present in mixtures. The inherent complexity of the peptide mixtures has necessitated improvements in methodology for mass spectrometry (MS) analysis of peptides.

Global site-specific N-glycosylation analysis of HIV envelope glycoprotein.

HIV-1 envelope glycoprotein (Env) is the sole target for broadly neutralizing antibodies (bnAbs) and the focus for design of an antibody-based HIV vaccine. The Env trimer is covered by ∼90N-linked glycans, which shield the underlying protein from immune surveillance. bNAbs to HIV develop during infection, with many showing dependence on glycans for binding to Env. The ability to routinely assess the glycan type at each glycosylation site may facilitate design of improved vaccine candidates. Here we present a general mass spectrometry-based proteomics strategy that uses specific endoglycosidases to introduce mass signatures that distinguish peptide glycosites that are unoccupied or occupied by high-mannose/hybrid or complex-type glycans. The method yields >95% sequence coverage for Env, provides semi-quantitative analysis of the glycosylation status at each glycosite. We find that most glycosites in recombinant Env trimers are fully occupied by glycans, varying in the proportion of high-mannose/hybrid and complex-type glycans.

Streptococcus mutans protein synthesis during mixed-species biofilm development by high-throughput quantitative proteomics.

Biofilms formed on tooth surfaces are comprised of mixed microbiota enmeshed in an extracellular matrix. Oral biofilms are constantly exposed to environmental changes, which influence the microbial composition, matrix formation and expression of virulence. Streptococcus mutans and sucrose are key modulators associated with the evolution of virulent-cariogenic biofilms. In this study, we used a high-throughput quantitative proteomics approach to examine how S. mutans produces relevant proteins that facilitate its establishment and optimal survival during mixed-species biofilms development induced by sucrose. Biofilms of S. mutans, alone or mixed with Actinomyces naeslundii and Streptococcus oralis, were initially formed onto saliva-coated hydroxyapatite surface under carbohydrate-limiting condition. Sucrose (1%, w/v) was then introduced to cause environmental changes, and to induce biofilm accumulation. Multidimensional protein identification technology (MudPIT) approach detected up to 60% of proteins encoded by S. mutans within biofilms. Specific proteins associated with exopolysaccharide matrix assembly, metabolic and stress adaptation processes were highly abundant as the biofilm transit from earlier to later developmental stages following sucrose introduction. Our results indicate that S. mutans within a mixed-species biofilm community increases the expression of specific genes associated with glucan synthesis and remodeling (gtfBC, dexA) and glucan-binding (gbpB) during this transition (P<0.05). Furthermore, S. mutans up-regulates specific adaptation mechanisms to cope with acidic environments (F1F0-ATPase system, fatty acid biosynthesis, branched chain amino acids metabolism), and molecular chaperones (GroEL). Interestingly, the protein levels and gene expression are in general augmented when S. mutans form mixed-species biofilms (vs. single-species biofilms) demonstrating fundamental differences in the matrix assembly, survival and biofilm maintenance in the presence of other organisms. Our data provide insights about how S. mutans optimizes its metabolism and adapts/survives within the mixed-species community in response to a dynamically changing environment. This reflects the intricate physiological processes linked to expression of virulence by this bacterium within complex biofilms.

Jmjd6 catalyses lysyl-hydroxylation of U2AF65, a protein associated with RNA splicing.

The finding that the metazoan hypoxic response is regulated by oxygen-dependent posttranslational hydroxylations, which regulate the activity and lifetime of hypoxia-inducible factor (HIF), has raised the question of whether other hydroxylases are involved in the regulation of gene expression. We reveal that the splicing factor U2 small nuclear ribonucleoprotein auxiliary factor 65-kilodalton subunit (U2AF65) undergoes posttranslational lysyl-5-hydroxylation catalyzed by the Fe(II) and 2-oxoglutarate-dependent dioxygenase Jumonji domain-6 protein (Jmjd6). Jmjd6 is a nuclear protein that has an important role in vertebrate development and is a human homolog of the HIF asparaginyl-hydroxylase. Jmjd6 is shown to change alternative RNA splicing of some, but not all, of the endogenous and reporter genes, supporting a specific role for Jmjd6 in the regulation of RNA splicing.

The proteomes of human parotid and submandibular/sublingual gland salivas collected as the ductal secretions.

Saliva is a body fluid with important functions in oral and general health. A consortium of three research groups catalogued the proteins in human saliva collected as the ductal secretions: 1166 identifications--914 in parotid and 917 in submandibular/sublingual saliva--were made. The results showed that a high proportion of proteins that are found in plasma and/or tears are also present in saliva along with unique components. The proteins identified are involved in numerous molecular processes ranging from structural functions to enzymatic/catalytic activities. As expected, the majority mapped to the extracellular and secretory compartments. An immunoblot approach was used to validate the presence in saliva of a subset of the proteins identified by mass spectrometric approaches. These experiments focused on novel constituents and proteins for which the peptide evidence was relatively weak. Ultimately, information derived from the work reported here and related published studies can be used to translate blood-based clinical laboratory tests into a format that utilizes saliva. Additionally, a catalogue of the salivary proteome of healthy individuals allows future analyses of salivary samples from individuals with oral and systemic diseases, with the goal of identifying biomarkers with diagnostic and/or prognostic value for these conditions; another possibility is the discovery of therapeutic targets.