Polymer Adhesin Domains in Gram-Positive Cell Surface Proteins

Surface proteins in Gram-positive bacteria are often involved in biofilm formation, host-cell interactions, and surface attachment. Here we review a protein module found in surface proteins that are often encoded on various mobile genetic elements like conjugative plasmids. This module binds to different types of polymers like DNA, lipoteichoic acid and glucans, and is here termed polymer adhesin domain. We analyze all proteins that contain a polymer adhesin domain and classify the proteins into distinct classes based on phylogenetic and protein domain analysis. Protein function and ligand binding show class specificity, information that will be useful in determining the function of the large number of so far uncharacterized proteins containing a polymer adhesin domain

Structural foundation for the role of enterococcal PrgB in conjugation, biofilm formation, and virulence

Type 4 Secretion Systems are a main driver for the spread of antibiotic resistance genes and virulence factors in bacteria. In Gram-positives, these secretion systems often rely on surface adhesins to enhance cellular aggregation and mating-pair formation. One of the best studied adhesins is PrgB from the conjugative plasmid pCF10 of Enterococcus faecalis, which has been shown to play major roles in conjugation, biofilm formation, and importantly also in bacterial virulence. Since prgB orthologs exist on a large number of conjugative plasmids in various different species, this makes PrgB a model protein for this widespread virulence factor. After characterizing the polymer adhesin domain of PrgB previously, we here report the structure for almost the entire remainder of PrgB, which reveals that PrgB contains four immunoglobulin (Ig)-like domains. Based on this new insight, we re-evaluate previously studied variants and present new in vivo data where specific domains or conserved residues have been removed. For the first time, we can show a decoupling of cellular aggregation from biofilm formation and conjugation in prgB mutant phenotypes. Based on the presented data, we propose a new functional model to explain how PrgB mediates its different functions. We hypothesize that the Ig-like domains act as a rigid stalk that presents the polymer adhesin domain at the right distance from the cell wall

Enterococcal PrgA Extends Far Outside the Cell and Provides Surface Exclusion to Protect against Unwanted Conjugation

Horizontal gene transfer between Gram-positive bacteria leads to a rapid spread of virulence factors and antibiotic resistance. This transfer is often facilitated via type 4 secretion systems (T4SS), which frequently are encoded on conjugative plasmids. However, donor cells that already contain a particular conjugative plasmid resist acquisition of a second copy of said plasmid. They utilize different mechanisms, including surface exclusion for this purpose. Enterococcus faecalis PrgA, encoded by the conjugative plasmid pCF10, is a surface protein that has been implicated to play a role in both virulence and surface exclusion, but the mechanism by which this is achieved has not been fully explained. Here, we report the structure of full-length PrgA, which shows that PrgA protrudes far out from the cell wall (approximately 40 nm), where it presents a protease domain. In vivo experiments show that PrgA provides a physical barrier to cellular adhesion, thereby reducing cellular aggregation. This function of PrgA contributes to surface exclusion, reducing the uptake of its cognate plasmid by approximately one order of magnitude. Using variants of PrgA with mutations in the catalytic site we show that the surface exclusion effect is dependent on the activity of the protease domain of PrgA. In silico analysis suggests that PrgA can interact with another enterococcal adhesin, PrgB, and that these two proteins have co-evolved. PrgB is a strong virulence factor, and PrgA is involved in post-translational processing of PrgB. Finally, competition mating experiments show that PrgA provides a significant fitness advantage to plasmid-carrying cells.

Gene structure of the eight aquaporin genes in <i>B</i>. <i>improvisus</i>.

<p>The <i>B</i>. <i>improvisus</i> aquaporin genes contain 4–6 exons. <i>AQP1</i> and <i>AQP2</i> are expressed as two alternative splice variants where exon 5 is excluded in one of the isoforms (indicated by bent arrows). Coding parts of exons are indicated in black, 5’ and 3’ UTR regions in grey and introns by thin lines. Exons, but not introns, are displayed to scale. For indication of intron lengths see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181192#pone.0181192.s002" target="_blank">S2 Fig</a>. The minimum lengths of the UTRs being part of the first or last coding exon are shown. An asterisk indicates that the UTR continues in an upstream or downstream non-coding exon not shown in the figure. Genomic data, cDNA clones and RNA-seq data were used to define the gene structure. In the case of the BIBL1 and BIBL2 mRNA, the sequence of the complete 3´end was determined by RACE.</p

Expression of aquaporins of <i>B</i>. <i>improvisus</i> in adults during exposure to various salinities.

<p>Adult individuals were incubated at three different salinities for 14 days (3, 20 and 33 PSU). For RNA preparation, soma, cirri and mantle of the adults were separated. For each salinity, the tissues (soma, cirri or mantle) from eighteen adults were pooled three-by-three to give six independent samples (n = 6). Quantitative PCR was used to determined aquaporin expression levels relative to actin. In case of cirri at 20 and 33 PSU only 5 independent samples were used in the qPCR due to very low RNA amounts obtained from one of the samples in each case. For some of the samples the expression was below the level of detection (N.D., not detected). Asterisks indicate significant levels (ANOVA): *** p<0.001, ** p<0.01, * p<0.05. Error bars show standard deviation.</p

Schematic overview of the <i>B</i>. <i>improvisus</i> aquaporins.

<p>The length [number of amino acids (aa)] and main features of the aquaporins in <i>B</i>. <i>improvisus</i> are displayed. Included are also the splice forms for Aqp1 and Aqp2. The region spanning from transmembrane 1 to transmembrane 6 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181192#pone.0181192.g004" target="_blank">Fig 4</a>), is indicated in dark grey and the N- and C-termini in light grey. The sequences are roughly aligned according to the position of the NPA sites. The total number of amino acid residues for each aquaporin is indicated to the right.</p

mRNAexpression of aquaporins in <i>B</i>. <i>improvisus</i>.

<p>The mRNA expression of the <i>B</i>. <i>improvisus</i> aquaporins was determined by RNA-seq in an adult (A) and in cyprid larvae (B) cultivated at seawater salinity (≈ 30 PSU). Total RNA was prepared and sequenced by paired-end Illumina sequencing. Normalized expression levels for the aquaporins were estimated by mapping reads to the aquaporins ORFs using the program RSEM. The expression levels for the different AQP genes are shown as FPKM (Fragments Per Kilobase of transcript per Million mapped reads) for the adult and TMM-normalized FPKM for cyprids. The sample sizes were n = 1 for the adult and n = 4 for the cyprids, with 300 cyprids pooled in four independent replicates. Error bars for the cyprid batches in B shows standard deviation. AQP2 was significantly higher expressed in cyprids compared to all the other aquaporins (ANOVA, P<0.001; see supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181192#pone.0181192.s012" target="_blank">S1 Table</a> for significant changes for all pair-wise comparisons). Absolute expression values should not be compared between life stages due to lack of normalization between the life stages.</p

Analysis of aquaporins from the euryhaline barnacle <i>Balanus improvisus</i> reveals differential expression in response to changes in salinity

<div><p>Barnacles are sessile macro-invertebrates, found along rocky shores in coastal areas worldwide. The euryhaline bay barnacle <i>Balanus improvisus</i> (Darwin, 1854) (= <i>Amphibalanus improvisus</i>) can tolerate a wide range of salinities, but the molecular mechanisms underlying the osmoregulatory capacity of this truly brackish species are not well understood. Aquaporins are pore-forming integral membrane proteins that facilitate transport of water, small solutes and ions through cellular membranes, and that have been shown to be important for osmoregulation in many organisms. The knowledge of the function of aquaporins in crustaceans is, however, limited and nothing is known about them in barnacles. We here present the repertoire of aquaporins from a thecostracan crustacean, the barnacle <i>B</i>. <i>improvisus</i>, based on genome and transcriptome sequencing. Our analyses reveal that <i>B</i>. <i>improvisus</i> contains eight genes for aquaporins. Phylogenetic analysis showed that they represented members of the classical water aquaporins (Aqp1, Aqp2), the aquaglyceroporins (Glp1, Glp2), the unorthodox aquaporin (Aqp12) and the arthropod-specific big brain aquaporin (Bib). Interestingly, we also found two big brain-like proteins (BibL1 and BibL2) constituting a new group of aquaporins not yet described in arthropods. In addition, we found that the two water-specific aquaporins were expressed as C-terminal splice variants. Heterologous expression of some of the aquaporins followed by functional characterization showed that Aqp1 transported water and Glp2 water and glycerol, agreeing with the predictions of substrate specificity based on 3D modeling and phylogeny. To investigate a possible role for the <i>B</i>. <i>improvisus</i> aquaporins in osmoregulation, mRNA expression changes in adult barnacles were analysed after long-term acclimation to different salinities. The most pronounced expression difference was seen for AQP1 with a substantial (>100-fold) decrease in the mantle tissue in low salinity (3 PSU) compared to high salinity (33 PSU). Our study provides a base for future mechanistic studies on the role of aquaporins in osmoregulation.</p></div

Amino acids in the Ar/R constriction site of aquaporins in <i>B</i>. <i>improvisus</i>.

<p>The four amino acids in the Ar/R constriction motif in the <i>Balanus</i> aquaporins were identified based on protein alignments with the human water aquaporin AQP1 and the <i>E</i>. <i>coli</i> aquaglyceroporin Glpf, which have well characterized constriction regions. All human aquaporins are included for comparison. Positions and residues for human AQP1 and <i>E</i>. <i>coli</i> Glpf are indicated at the top. The names of the <i>B</i>. <i>improvisus</i> aquaporins are shown in bold.</p

Initial phylogenetic classification of the aquaporins in <i>B</i>. <i>improvisus</i>.

<p>A phylogenetic tree was constructed using aquaporin sequences from <i>B</i>. <i>improvisus</i> and other arthropods. In addition, the human aquaporins were included for reference. The analysis was done using the program PhyML 3.0 at the <a href="http://Phylogeny.fr" target="_blank">Phylogeny.fr</a> website, creating an unrooted tree. The four main subfamilies according to Stavang et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181192#pone.0181192.ref022" target="_blank">22</a>] are indicated to the right and different aquaporin subgroups to the left. Aqp8-type aquaammoniaporins are abbreviated to aquaammoniaporins. The <i>B</i>. <i>improvisus</i> aquaporins are marked with a dot. The numbers on the branches are aLRT SH-like support-values. The scalebar shows substitutions per site.</p