Structure and Assembly of Group B Streptococcus Pilus 2b Backbone Protein

<div><p>Group B <i>Streptococcus</i> (GBS) is a major cause of invasive disease in infants. Like other Gram-positive bacteria, GBS uses a sortase C-catalyzed transpeptidation mechanism to generate cell surface pili from backbone and ancillary pilin precursor substrates. The three pilus types identified in GBS contain structural subunits that are highly immunogenic and are promising candidates for the development of a broadly-protective vaccine. Here we report the X-ray crystal structure of the backbone protein of pilus 2b (BP-2b) at 1.06Å resolution. The structure reveals a classical IgG-like fold typical of the pilin subunits of other Gram-positive bacteria. The crystallized portion of the protein (residues 185-468) encompasses domains D2 and D3 that together confer high stability to the protein due to the presence of an internal isopeptide bond within each domain. The D2+D3 region, lacking the N-terminal D1 domain, was as potent as the entire protein in conferring protection against GBS challenge in a well-established mouse model. By site-directed mutagenesis and complementation studies in GBS knock-out strains we identified the residues and motives essential for assembly of the BP-2b monomers into high-molecular weight complexes, thus providing new insights into pilus 2b polymerization.</p></div

Multiple structural alignment of BP-2b protein with other known structures using the DALI server.

<p>Hits are ranked by Z-Score with best hits at the top of the table.</p><p><i>PDB</i>: Protein Data Bank</p><p><i>rmsd</i>: root-mean-square deviation of Cα atoms of superimposed proteins in Angstroms</p><p><i>lali</i>: number of structurally equivalent positions</p><p><i>nres</i>: number of structurally equivalent aligned residues</p><p><i>%ide</i>: percentage of amino acid identity in aligned positions</p><p>Multiple structural alignment of BP-2b protein with other known structures using the DALI server.</p

Lys 175, Glu 423 and the sorting motif LPSTG are involved in BP-2b polymerization in GBS.

<p>Immunoblot analysis of total protein extracts from GBS mutant strain lacking the pilus 2b backbone protein gene (Δ<i>BP-2b</i>) complemented with plasmids expressing the wild-type BP-2b protein (WT) or BP-2b mutants carrying a deletion of the C-terminal sorting signal (BP-2b_ΔLPXTG), alanine substitutions of the putative pilin motif lysine (BP-2b_K175A, BP-2b_K118A BP-2b_K82A) or of the E-box E423 residue (BP-2b_E423A). Nitrocellulose membrane was probed with a mouse antiserum raised against the recombinant BP-2b protein (α-BP–2b).</p

Biochemical characterization of different BP-2b constructs.

<p><b>(A)</b> Time course of the trypsin-proteolysis reactions at 37°C of BP-2b full length and fragments, analyzed by SDS-PAGE. Different digestion patterns can be observed for the different constructs. Asterisks indicate the not-digested proteins. (<b>B)</b> Differential Scanning Fluorimetry (DSF) analysis of BP-2b proteins (D1+D2+D3, D2+D3 and single domains D1, D2, D3) in presence of Sypro orange showed different thermal stabilities. Graph shows the fluorescence intensity <i>vs</i>. the temperature for the unfolding different BP-2b constructs. (<b>C)</b> Correlation of BP-2b melting temperature with the concentration of Ca²⁺.</p

Structural comparisons of BP-2b_D2+D3 with other pilin backbone proteins.

<p>(<b>A)</b> BP-2b (blue cartoon) is shown overlaid onto: the pilus backbone protein RrgB (pdb 2x9x, red cartoon, left), the major pilin protein GBS80 (pdb 3pf2, green cartoon, middle), and on the major pilin protein BP-2a (pdb 2xtl, pink cartoon, right). (<b>B)</b> Domain architecture of GBS backbone proteins from pilus 1 (BP-1), pilus 2a (BP-2a) and pilus 2b (BP-2b). The proteins are comprised of a signal peptide (SP) at the N-terminus and a C-terminal LPXTG-like motif (in red) linked to the transmembrane domain (TM). BP-1 and BP-2b contain three domains, while BP-2a four domains. The pilin motif involved in pilus polymerization is located near the D1–D2 domain linker while the E-box is located close to the sorting signal. Residues involved in isopeptide bonds are indicated by black bars. Domains present in the crystal structures are included into the box outlined with dashed lines.</p

Isopeptide bonds of BP-2b.

<p>Domains D2 and D3 are colored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125875#pone.0125875.g001" target="_blank">Fig 1</a> in blue and orange, respectively. Isopeptide bonds between Asn330 and Lys 187 for D2, and between Asn462 and Lys358 in D3 are shown with blue and orange sticks, and 1σ 2<i>F</i>o-<i>F</i>c electron density map around this region is shown as blue mesh (carve = 1.1). The magenta colored regions in (A) and (B) show the location of hydrophobic residues surrounding the isopeptide bonds. Hydrogen-bonds between the isopeptide bonds and the nearby Asp (225, D2) and Glu (423, D3) are shown with black dashed lines.</p

Image_1_Signatures of disease outcome severity in the intestinal fungal and bacterial microbiome of COVID-19 patients.pdf

BackgroundCOVID-19, whose causative pathogen is the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), was declared a pandemic in March 2020. The gastrointestinal tract is one of the targets of this virus, and mounting evidence suggests that gastrointestinal symptoms may contribute to disease severity. The gut–lung axis is involved in the immune response to SARS-CoV-2; therefore, we investigated whether COVID-19 patients’ bacterial and fungal gut microbiome composition was linked to disease clinical outcome.MethodsIn May 2020, we collected stool samples and patient records from 24 hospitalized patients with laboratory-confirmed SARS-CoV-2 infection. Fungal and bacterial gut microbiome was characterized by amplicon sequencing on the MiSeq, Illumina’s integrated next generation sequencing instrument. A cohort of 201 age- and sex-matched healthy volunteers from the project PRJNA661289 was used as a control group for the bacterial gut microbiota analysis.ResultsWe observed that female COVID-19 patients had a lower gut bacterial microbiota richness than male patients, which was consistent with a different latency in hospital admittance time between the two groups. Both sexes in the COVID-19 patient study group displayed multiple positive associations with opportunistic bacterial pathogens such as Enterococcus, Streptococcus, and Actinomyces. Of note, the Candida genus dominated the gut mycobiota of COVID-19 patients, and adult patients showed a higher intestinal fungal diversity than elderly patients. We found that Saccharomycetales unassigned fungal genera were positively associated with bacterial short-chain fatty acid (SCFA) producers and negatively associated with the proinflammatory genus Bilophila in COVID-19 patients, and we observed that none of the patients who harbored it were admitted to the high-intensity unit.ConclusionsCOVID-19 was associated with opportunistic bacterial pathogens, and Candida was the dominant fungal taxon in the intestine. Together, we found an association between commensal SCFA-producers and a fungal genus that was present in the intestines of patients who did not experience the most severe outcome of the disease. We believe that this taxon could have played a role in the disease outcome, and that further studies should be conducted to understand the role of fungi in gastrointestinal and health protection.</p