Staphylococcal Periscope proteins Aap, SasG, and Pls project noncanonical legume-like lectin adhesin domains from the bacterial surface.

Staphylococcus aureus and Staphylococcus epidermidis are frequently associated with medical device infections that involve establishment of a bacterial biofilm on the device surface. Staphylococcal surface proteins Aap, SasG, and Pls are members of the Periscope Protein class and have been implicated in biofilm formation and host colonization; they comprise a repetitive region ("B region") and an N-terminal host colonization domain within the "A region," predicted to be a lectin domain. Repetitive E-G5 domains (as found in Aap, SasG, and Pls) form elongated "stalks" that would vary in length with repeat number, resulting in projection of the N-terminal A domain variable distances from the bacterial cell surface. Here, we present the structures of the lectin domains within A regions of SasG, Aap, and Pls and a structure of the Aap lectin domain attached to contiguous E-G5 repeats, suggesting the lectin domains will sit at the tip of the variable length rod. We demonstrate that these isolated domains (Aap, SasG) are sufficient to bind to human host desquamated nasal epithelial cells. Previously, proteolytic cleavage or a deletion within the A domain had been reported to induce biofilm formation; the structures suggest a potential link between these observations. Intriguingly, while the Aap, SasG, and Pls lectin domains bind a metal ion, they lack the nonproline cis peptide bond thought to be key for carbohydrate binding by the lectin fold. This suggestion of noncanonical ligand binding should be a key consideration when investigating the host cell interactions of these bacterial surface proteins.

Model Systems to Study the Chronic, Polymicrobial Infections in Cystic Fibrosis: Current Approaches and Exploring Future Directions.

A recent workshop titled "Developing Models to Study Polymicrobial Infections," sponsored by the Dartmouth Cystic Fibrosis Center (DartCF), explored the development of new models to study the polymicrobial infections associated with the airways of persons with cystic fibrosis (CF). The workshop gathered 35+ investigators over two virtual sessions. Here, we present the findings of this workshop, summarize some of the challenges involved with developing such models, and suggest three frameworks to tackle this complex problem. The frameworks proposed here, we believe, could be generally useful in developing new model systems for other infectious diseases. Developing and validating new approaches to study the complex polymicrobial communities in the CF airway could open windows to new therapeutics to treat these recalcitrant infections, as well as uncovering organizing principles applicable to chronic polymicrobial infections more generally.

Culture-enriched metagenomic sequencing enables in-depth profiling of the cystic fibrosis lung microbiota.

Amplicon sequencing (for example, of the 16S rRNA gene) identifies the presence and relative abundance of microbial community members. However, metagenomic sequencing is needed to identify the genetic content and functional potential of a community. Metagenomics is challenging in samples dominated by host DNA, such as those from the skin, tissue and respiratory tract. Here, we combine advances in amplicon and metagenomic sequencing with culture-enriched molecular profiling to study the human microbiota. Using the cystic fibrosis lung as an example, we cultured an average of 82.13% of the operational taxonomic units representing 99.3% of the relative abundance identified in direct sequencing of sputum samples; importantly, culture enrichment identified 63.3% more operational taxonomic units than direct sequencing. We developed the PLate Coverage Algorithm (PLCA) to determine a representative subset of culture plates on which to conduct culture-enriched metagenomics, resulting in the recovery of greater taxonomic diversity-including of low-abundance taxa-with better metagenome-assembled genomes, longer contigs and better functional annotations when compared to culture-independent methods. The PLCA is also applied as a proof of principle to a previously published gut microbiota dataset. Culture-enriched molecular profiling can be used to better understand the role of the human microbiota in health and disease.

Streptococcus pneumoniae Colonization Is Required To Alter the Nasal Microbiota in Cigarette Smoke-Exposed Mice.

Smokers have nasal microbiota dysbiosis, with an increased frequency of colonizing bacterial pathogens. It is possible that cigarette smoke increases pathogen acquisition by perturbing the microbiota and decreasing colonization resistance. However, it is difficult to disentangle microbiota dysbiosis due to cigarette smoke exposure from microbiota changes caused by increased pathogen acquisition in human smokers. Using an experimental mouse model, we investigated the impact of cigarette smoke on the nasal microbiota in the absence and presence of nasal pneumococcal colonization. We observed that cigarette smoke exposure alone did not alter the nasal microbiota composition. The microbiota composition was also unchanged at 12 h following low-dose nasal pneumococcal inoculation, suggesting that the ability of the microbiota to resist initial nasal pneumococcal acquisition was not impaired in smoke-exposed mice. However, nasal microbiota dysbiosis occurred as a consequence of established high-dose nasal pneumococcal colonization at day 3 in smoke-exposed mice. Similar to clinical reports on human smokers, an enrichment of potentially pathogenic bacterial genera such as Fusobacterium, Gemella, and Neisseria was observed. Our findings suggest that cigarette smoke exposure predisposes to pneumococcal colonization independent of changes to the nasal microbiota and that microbiota dysbiosis observed in smokers may occur as a consequence of established pathogen colonization.

Investigating the Structural Compaction of Biomolecules Upon Transition to the Gas-Phase Using ESI-TWIMS-MS.

Collision cross-section (CCS) measurements obtained from ion mobility spectrometry-mass spectrometry (IMS-MS) analyses often provide useful information concerning a protein's size and shape and can be complemented by modeling procedures. However, there have been some concerns about the extent to which certain proteins maintain a native-like conformation during the gas-phase analysis, especially proteins with dynamic or extended regions. Here we have measured the CCSs of a range of biomolecules including non-globular proteins and RNAs of different sequence, size, and stability. Using traveling wave IMS-MS, we show that for the proteins studied, the measured CCS deviates significantly from predicted CCS values based upon currently available structures. The results presented indicate that these proteins collapse to different extents varying on their elongated structures upon transition into the gas-phase. Comparing two RNAs of similar mass but different solution structures, we show that these biomolecules may also be susceptible to gas-phase compaction. Together, the results suggest that caution is needed when predicting structural models based on CCS data for RNAs as well as proteins with non-globular folds. Graphical Abstract ᅟ.

The effects of inhaled aztreonam on the cystic fibrosis lung microbiome.

BACKGROUND: Aztreonam lysine for inhalation (AZLI) is an inhaled antibiotic used to treat chronic Pseudomonas aeruginosa infection in CF. AZLI improves lung function and quality of life, and reduces exacerbations-improvements attributed to its antipseudomonal activity. Given the extremely high aztreonam concentrations achieved in the lower airways by nebulization, we speculate this may extend its spectrum of activity to other organisms. As such, we sought to determine if AZLI affects the CF lung microbiome and whether community constituents can be used to predict treatment responsiveness. METHODS: Patients were included if they had chronic P. aeruginosa infection and repeated sputum samples collected before and after AZLI. Sputum DNA was extracted, and the V3-hypervariable region of the 16S ribosomal RNA (rRNA) gene amplified and sequenced. RESULTS: Twenty-four patients naïve to AZLI contributed 162 samples. The cohort had a median age of 37.1 years, and a  median FEV1 of 44% predicted. Fourteen patients were a priori defined as responders for achieving ≥3% FEV1 improvement following initiation. No significant changes in alpha diversity were noted following AZLI. Furthermore, beta diversity demonstrated clustering with respect to patients, but had no association with AZLI use. However, we did observe a decline in the relative abundance of several individual operational taxonomic units (OTUs) following AZLI initiation suggesting that specific sub-populations of organisms may be impacted. Patients with higher abundance of Staphylococcus and anaerobic organisms including Prevotella and Fusobacterium were less likely to respond to therapy. CONCLUSIONS: Results from our study suggest potential alternate/additional mechanisms by which AZLI functions. Moreover, our study suggests that the CF microbiota may be used as a biomarker to predict patient responsiveness to therapy suggesting the microbiome may be harnessed for the personalization of therapies.

The Evolving Cystic Fibrosis Microbiome: A Comparative Cohort Study Spanning 16 Years.

RATIONALE: The cystic fibrosis (CF) airways are infected with a diverse polymicrobial community. OBJECTIVES: Understanding how changes in the CF microbiome have occurred over time, similar to the observed changes in the prevalence of cultured pathogens, is key in understanding the microbiome's role in disease. METHODS: Drawing from a prospectively collected and maintained sputum biobank, we identified 45 patients with sputum samples collected between the ages of 18 and 21 years in three successive cohorts of adults transitioning to our CF clinic: A (1997-2000), B (2004-2007), and C (2010-2013). Patient demographics, clinical status, and medications were collected from detailed chart review. Microbial communities were assessed by Ilumina MiSeq sequencing of the variable 3 (V3) region of the 16S rDNA. RESULTS: The three cohorts were similar with respect to baseline demographics. There was a trend toward improved health and use of disease-modifying therapies in each successive cohort. Shannon diversity increased in the most recent cohort, suggesting an increase in the diversity of organisms between cohorts. Furthermore, the proportion of samples with Pseudomonas-dominated communities decreased over time, whereas Streptococcus increased. Although ß-diversity was associated with transition cohort, the greatest predictor of diversity remained lung function. Furthermore, core microbiome constituents were preserved across cohorts. CONCLUSIONS: Modest changes in the composition and structure of the microbiome of three successive cohorts of young adults with CF were observed, occurring in parallel with successive improvements in clinical status. Importantly, however, the core microbiome constituents were preserved across cohorts.

Longitudinal sampling of the lung microbiota in individuals with cystic fibrosis.

Cystic fibrosis (CF) manifests in the lungs resulting in chronic microbial infection. Most morbidity and mortality in CF is due to cycles of pulmonary exacerbations-episodes of acute inflammation in response to the lung microbiome-which are difficult to prevent and treat because their cause is not well understood. We hypothesized that longitudinal analyses of the bacterial component of the CF lung microbiome may elucidate causative agents within this community for pulmonary exacerbations. In this study, 6 participants were sampled thrice-weekly for up to one year. During sampling, sputum, and data (antibiotic usage, spirometry, and symptom scores) were collected. Time points were categorized based on relation to exacerbation as Stable, Intermediate, and Treatment. Retrospectively, a subset of were interrogated via 16S rRNA gene sequencing. When samples were examined categorically, a significant difference between the lung microbiota in Stable, Intermediate, and Treatment samples was observed in a subset of participants. However, when samples were examined longitudinally, no correlations between microbial composition and collected data (antibiotic usage, spirometry, and symptom scores) were observed upon exacerbation onset. In this study, we identified no universal indicator within the lung microbiome of exacerbation onset but instead showed that changes to the CF lung microbiome occur outside of acute pulmonary episodes and are patient-specific.

Reemergence of Lower-Airway Microbiota in Lung Transplant Patients with Cystic Fibrosis.

RATIONALE: Chronic lung infections are a hallmark of cystic fibrosis; they are responsible for progressive airway destruction and ultimately lead to respiratory death or the requirement for life-saving bilateral lung transplant. Furthermore, recurrent isolation of airway pathogens such as Pseudomonas aeruginosa in the allograft after transplant is associated with adverse outcomes, including bronchiolitis obliterans syndrome and acute infections. Little information exists on the impact of bilateral lung transplant on the lower-airway microbiota. OBJECTIVES: To compare, at a microbiome and single-pathogen level (P. aeruginosa), the bacterial communities in pre- and post-transplant samples. METHODS: We retrospectively accessed our biobank of sputum samples and sputum-derived bacterial pathogens for patients who had matched samples, including those who were clinically stable before transplant, those who had a pulmonary exacerbation before transplant, and those who had pulmonary exacerbation after transplant. We used 16S ribosomal RNA gene sequencing to characterize the lower-airway microbiome of 14 adult transplant patients with cystic fibrosis. Genotyping and phenotyping of P. aeruginosa isolates from 12 of these patients with matched isolates was performed. MEASUREMENTS AND MAIN RESULTS: Although α-diversity (richness and evenness) of patient microbiomes was similar before and after transplant, ß- diversity (core microbiome composition) measures stratified patients evenly into two groups with more similar and more dissimilar communities. P. aeruginosa strains isolated before transplant were found to reemerge in 11 of 12 patients; however, phenotypic variation was observed. CONCLUSIONS: These findings indicate that recolonization by P. aeruginosa after transplant is almost always strain specific, suggesting a within-host source. The polymicrobial colonization of the airways after transplant does not always reflect the pretransplant sputum microbiota.

Clinical Insights into Pulmonary Exacerbations in Cystic Fibrosis from the Microbiome. What Are We Missing?

Pulmonary exacerbations account for much of the decrease in lung function and consequently most of the morbidity and mortality in patients with cystic fibrosis. These events are driven by an acute inflammatory response to infection. Recent technological advancements in molecular profiling techniques have allowed for a proliferation of microbiome studies of the lower airways of patients with cystic fibrosis. But these methods may not provide a comprehensive and unbiased measure of the lung microbiota in these patients and molecular profiles do not always translate to quantitative microbiology. Furthermore, these studies have not yet been able to provide much in the way of mechanistic insights into exacerbations or to guide patient therapy. We propose a model in which pulmonary exacerbations may be driven by an active subpopulation of the lung microbiota, which may represent only a small portion of the microbiota measured in a clinical sample. Methodology should be focused on the ultimate goal, which is to use the best available approaches to provide accurate quantitative measures of the microbiome to inform clinical decisions and provide rapid assessment of treatment efficacy. These strategies would be relevant to other chronic lung diseases such as chronic obstructive pulmonary disease and neutrophilic asthma.

Structural basis for DNA strand separation by a hexameric replicative helicase.

Hexameric helicases are processive DNA unwinding machines but how they engage with a replication fork during unwinding is unknown. Using electron microscopy and single particle analysis we determined structures of the intact hexameric helicase E1 from papillomavirus and two complexes of E1 bound to a DNA replication fork end-labelled with protein tags. By labelling a DNA replication fork with streptavidin (dsDNA end) and Fab (5' ssDNA) we located the positions of these labels on the helicase surface, showing that at least 10 bp of dsDNA enter the E1 helicase via a side tunnel. In the currently accepted 'steric exclusion' model for dsDNA unwinding, the active 3' ssDNA strand is pulled through a central tunnel of the helicase motor domain as the dsDNA strands are wedged apart outside the protein assembly. Our structural observations together with nuclease footprinting assays indicate otherwise: strand separation is taking place inside E1 in a chamber above the helicase domain and the 5' passive ssDNA strands exits the assembly through a separate tunnel opposite to the dsDNA entry point. Our data therefore suggest an alternative to the current general model for DNA unwinding by hexameric helicases.

The Evolution of the Scavenger Receptor Cysteine-Rich Domain of the Class A Scavenger Receptors.

The class A scavenger receptor (cA-SR) family is a group of five evolutionarily related innate immune receptors. The cA-SRs are known for their promiscuous ligand binding; as they have been shown to bind bacteria, such as Streptococcus pneumoniae and Escherichia coli, as well as different modified forms of low-density lipoprotein. Three of the five family members possess a scavenger receptor cysteine-rich (SRCR) domain while the remaining two receptors lack the domain. Previous work has suggested that the macrophage-associated receptor with collagenous structure (MARCO) shares a recent common ancestor with the non-SRCR-containing receptors; however, the origin of the SRCR domain within the cA-SRs remains unknown. We hypothesize that the SRCR domains of the cA-SRs have a common origin that predates teleost fish. Using the newly available sequence data from sea lamprey and ghost shark genome projects, we have shown that MARCO shares a common ancestor with the SRCR-containing proteins. In addition, we explored the evolutionary relationships within the SRCR domain by reconstructing the ancestral SRCR domains of the cA-SRs. We identified a motif that is highly conserved between the cA-SR SRCR domains and the ancestral SRCR domain that consist of WGTVCDD. We also show that the GRAEVYY motif, a functionally important motif within MARCO, is poorly conserved in the other cA-SRs and in the reconstructed ancestral domain. Further, we identified three sites within MARCO's SRCR domain, which are under positive selection. Two of these sites lie adjacent to the conserved WGTVCDD motif, and may indicate a potential biological function for these sites. Together, these findings indicate a common origin of the SRCR domain within the cA-SRs; however, different selective pressures between the proteins may have caused MARCOs SRCR domain to evolve to contain different functional motifs when compared to the other SRCR-containing cA-SRs.

From bacterial to human dihydrouridine synthase: automated structure determination.

The reduction of uridine to dihydrouridine at specific positions in tRNA is catalysed by dihydrouridine synthase (Dus) enzymes. Increased expression of human dihydrouridine synthase 2 (hDus2) has been linked to pulmonary carcinogenesis, while its knockdown decreased cancer cell line viability, suggesting that it may serve as a valuable target for therapeutic intervention. Here, the X-ray crystal structure of a construct of hDus2 encompassing the catalytic and tRNA-recognition domains (residues 1-340) determined at 1.9 Šresolution is presented. It is shown that the structure can be determined automatically by phenix.mr_rosetta starting from a bacterial Dus enzyme with only 18% sequence identity and a significantly divergent structure. The overall fold of the human Dus2 is similar to that of bacterial enzymes, but has a larger recognition domain and a unique three-stranded antiparallel ß-sheet insertion into the catalytic domain that packs next to the recognition domain, contributing to domain-domain interactions. The structure may inform the development of novel therapeutic approaches in the fight against lung cancer.

Major reorientation of tRNA substrates defines specificity of dihydrouridine synthases.

The reduction of specific uridines to dihydrouridine is one of the most common modifications in tRNA. Increased levels of the dihydrouridine modification are associated with cancer. Dihydrouridine synthases (Dus) from different subfamilies selectively reduce distinct uridines, located at spatially unique positions of folded tRNA, into dihydrouridine. Because the catalytic center of all Dus enzymes is conserved, it is unclear how the same protein fold can be reprogrammed to ensure that nucleotides exposed at spatially distinct faces of tRNA can be accommodated in the same active site. We show that the Escherichia coli DusC is specific toward U16 of tRNA. Unexpectedly, crystal structures of DusC complexes with tRNA(Phe) and tRNA(Trp) show that Dus subfamilies that selectively modify U16 or U20 in tRNA adopt identical folds but bind their respective tRNA substrates in an almost reverse orientation that differs by a 160° rotation. The tRNA docking orientation appears to be guided by subfamily-specific clusters of amino acids ("binding signatures") together with differences in the shape of the positively charged tRNA-binding surfaces. tRNA orientations are further constrained by positional differences between the C-terminal "recognition" domains. The exquisite substrate specificity of Dus enzymes is therefore controlled by a relatively simple mechanism involving major reorientation of the whole tRNA molecule. Such reprogramming of the enzymatic specificity appears to be a unique evolutionary solution for altering tRNA recognition by the same protein fold.

S-Adenosyl-S-carboxymethyl-L-homocysteine: a novel cofactor found in the putative tRNA-modifying enzyme CmoA.

Uridine at position 34 of bacterial transfer RNAs is commonly modified to uridine-5-oxyacetic acid (cmo(5)U) to increase the decoding capacity. The protein CmoA is involved in the formation of cmo(5)U and was annotated as an S-adenosyl-L-methionine-dependent (SAM-dependent) methyltransferase on the basis of its sequence homology to other SAM-containing enzymes. However, both the crystal structure of Escherichia coli CmoA at 1.73 Å resolution and mass spectrometry demonstrate that it contains a novel cofactor, S-adenosyl-S-carboxymethyl-L-homocysteine (SCM-SAH), in which the donor methyl group is substituted by a carboxymethyl group. The carboxyl moiety forms a salt-bridge interaction with Arg199 that is conserved in a large group of CmoA-related proteins but is not conserved in other SAM-containing enzymes. This raises the possibility that a number of enzymes that have previously been annotated as SAM-dependent are in fact SCM-SAH-dependent. Indeed, inspection of electron density for one such enzyme with known X-ray structure, PDB entry 1im8, suggests that the active site contains SCM-SAH and not SAM.

Ki-67 expression predicts radiotherapy failure in early glottic cancer.

BACKGROUND: Early-stage laryngeal squamous cell carcinoma is managed with radiotherapy or endoscopic surgery. Although cure rates are high, radiation failures often require total laryngectomy for salvage. Biomarkers that can predict tumour radioresistance may be useful in modifying the treatment approach for individual patients. METHODS: Retrospective patient chart review yielded 75 patients with T1-T2 glottic squamous cell carcinoma treated with radiation therapy at the London Health Sciences Centre. Pretreatment tumour biopsies were immunostained for B-cell lymphoma 2 (Bcl-2), Ki-67, and epidermal growth factor receptor (EGFR) to correlate biomarker expression with disease-free survival (DFS). RESULTS: Ki-67 expression was strongly associated with recurrence following radiation and independently predicted poor DFS (hazard ratio 4.86, 95% CI 1.58-15.00; p  =  .006). EGFR and Bcl-2 were not associated with a risk of recurrence. CONCLUSIONS: Ki-67 expression identified a subset of patients with increased risk of local recurrence after radiation therapy. Ki-67 expression can potentially guide improved personalized treatments for patients with early-stage glottic squamous cell carcinomas.

Crystallization and preliminary X-ray crystallographic analysis of the catalytic domain of human dihydrouridine synthase.

Dihydrouridine synthases catalyse the reduction of uridine to dihydrouridine in the D-loop and variable loop of tRNA. The human dihydrouridine synthase HsDus2L has been implicated in the development of pulmonary carcinogenesis. Here, the purification, crystallization and preliminary X-ray characterization of the HsDus2L catalytic domain are reported. The crystals belonged to space group P2(1) and contained a single molecule of HsDus2L in the asymmetric unit. A complete data set was collected to 1.9 Å resolution using synchrotron radiation.

A flexible brace maintains the assembly of a hexameric replicative helicase during DNA unwinding.

The mechanism of DNA translocation by papillomavirus E1 and polyomavirus LTag hexameric helicases involves consecutive remodelling of subunit-subunit interactions around the hexameric ring. Our biochemical analysis of E1 helicase demonstrates that a 26-residue C-terminal segment is critical for maintaining the hexameric assembly. As this segment was not resolved in previous crystallographic analysis of E1 and LTag hexameric helicases, we determined the solution structure of the intact hexameric E1 helicase by Small Angle X-ray Scattering. We find that the C-terminal segment is flexible and occupies a cleft between adjacent subunits in the ring. Electrostatic potential calculations indicate that the negatively charged C-terminus can bridge the positive electrostatic potentials of adjacent subunits. Our observations support a model in which the C-terminal peptide serves as a flexible 'brace' maintaining the oligomeric state during conformational changes associated with ATP hydrolysis. We argue that these interactions impart processivity to DNA unwinding. Sequence and disorder analysis suggest that this mechanism of hexamer stabilization would be conserved among papillomavirus E1 and polyomavirus LTag hexameric helicases.

Comparison of endoscopic laser resection versus radiation therapy for the treatment of early glottic carcinoma.

OBJECTIVE: Radiation therapy (RT) and transoral laser microsurgery (TLM) are established treatments for early glottic squamous cell carcinoma (SCC). Similar oncologic outcomes have been reported with both modalities, leading physicians to consider other factors when making clinical recommendations. One such factor is voice-related quality of life. This investigation sought to characterize differences in self-reported voice outcomes in patients undergoing RT or TLM for the treatment of Tis or T1a glottic SCC. METHODS: A retrospective cohort study was conducted of all individuals who received either RT or TLM for the treatment of Tis or T1a glottic SCC between 2004 and 2009 at the London Regional Cancer Program. The primary outcome measure was voice-related quality of life, as assessed by the Voice-Related Quality of Life questionnaire (V-RQOL). Secondary outcomes included local control, overall survival, and laryngectomy-free survival. RESULTS: Fifty-seven patients were eligible for this study; 34 received RT and 23 received TLM. Forty (70.2%) of the 57 patients completed the V-RQOL. No statistically significant difference in total V-RQOL score was observed between the RT and TLM cohorts (p  =  .228). There was, however, a trend toward higher scores (ie, less voice disability) in the physical function domain of the V-RQOL for the RT group (90.0%) compared to the TLM group (80.2%) (p  =  .05). No significant differences were observed in recurrence or overall survival between the two groups. CONCLUSION: Both oncologic outcomes and self-rated voice-related quality of life are similar in patients treated with RT and TLM for early glottic carcinoma.