Viewing entries in
Genetics and Genomics

Lineage structure of Streptococcus pneumoniae may be driven by immune selection on the groEL heat-shock protein.

Sci Rep. 2017 Aug 22;7(1):9023. doi: 10.1038/s41598-017-08990-z.

Lineage structure of Streptococcus pneumoniae may be driven by immune selection on the groEL heat-shock protein.

Lourenço J1, Watkins ER2, Obolski U2, Peacock SJ2, Morris C3, Maiden MCJ2, Gupta S2.

Author information

Abstract

Populations of Streptococcus pneumoniae (SP) are typically structured into groups of closely related organisms or lineages, but it is not clear whether they are maintained by selection or neutral processes. Here, we attempt to address this question by applying a machine learning technique to SP whole genomes. Our results indicate that lineages evolved through immune selection on the groEL chaperone protein. The groEL protein is part of the groESL operon and enables a large range of proteins to fold correctly within the physical environment of the nasopharynx, thereby explaining why lineage structure is so stable within SP despite high levels of genetic transfer. SP is also antigenically diverse, exhibiting a variety of distinct capsular serotypes. Associations exist between lineage and capsular serotype but these can be easily perturbed, such as by vaccination. Overall, our analyses indicate that the evolution of SP can be conceptualized as the rearrangement of modular functional units occurring on several different timescales under different pressures: some patterns have locked in early (such as the epistatic interactions between groESL and a constellation of other genes) and preserve the differentiation of lineages, while others (such as the associations between capsular serotype and lineage) remain in continuous flux.

PMID: 28831154 DOI: 10.1038/s41598-017-08990-z

The Transcriptome of Streptococcus pneumoniae Induced by Local and Global Changes in Supercoiling.

Front Microbiol. 2017 Jul 31;8:1447. doi: 10.3389/fmicb.2017.01447. eCollection 2017.

The Transcriptome of Streptococcus pneumoniae Induced by Local and Global Changes in Supercoiling.

de la Campa AG1,2, Ferrándiz MJ1, Martín-Galiano AJ1, García MT3, Tirado-Vélez JM1.

Author information

Abstract

The bacterial chromosome is compacted in a manner optimal for DNA transactions to occur. The degree of compaction results from the level of DNA-supercoiling and the presence of nucleoid-binding proteins. DNA-supercoiling is homeostatically maintained by the opposing activities of relaxing DNA topoisomerases and negative supercoil-inducing DNA gyrase. DNA-supercoiling acts as a general cis regulator of transcription, which can be superimposed upon other types of more specific trans regulatory mechanism. Transcriptomic studies on the human pathogen Streptococcus pneumoniae, which has a relatively small genome (∼2 Mb) and few nucleoid-binding proteins, have been performed under conditions of local and global changes in supercoiling. The response to local changes induced by fluoroquinolone antibiotics, which target DNA gyrase subunit A and/or topoisomerase IV, involves an increase in oxygen radicals which reduces cell viability, while the induction of global supercoiling changes by novobiocin (a DNA gyrase subunit B inhibitor), or by seconeolitsine (a topoisomerase I inhibitor), has revealed the existence of topological domains that specifically respond to such changes. The control of DNA-supercoiling in S. pneumoniae occurs mainly via the regulation of topoisomerase gene transcription: relaxation triggers the up-regulation of gyrase and the down-regulation of topoisomerases I and IV, while hypernegative supercoiling down-regulates the expression of topoisomerase I. Relaxation affects 13% of the genome, with the majority of the genes affected located in 15 domains. Hypernegative supercoiling affects 10% of the genome, with one quarter of the genes affected located in 12 domains. However, all the above domains overlap, suggesting that the chromosome is organized into topological domains with fixed locations. Based on its response to relaxation, the pneumococcal chromosome can be said to be organized into five types of domain: up-regulated, down-regulated, position-conserved non-regulated, position-variable non-regulated, and AT-rich. The AT content is higher in the up-regulated than in the down-regulated domains. Genes within the different domains share structural and functional characteristics. It would seem that a topology-driven selection pressure has defined the chromosomal location of the metabolism, virulence and competence genes, which suggests the existence of topological rules that aim to improve bacterial fitness.

KEYWORDS:

DNA supercoiling; DNA topoisomerases; fluoroquinolones; global transcription; interactome; novobiocin; seconeolitsine; topological domains

PMID: 28824578 PMCID: PMC5534458 DOI: 10.3389/fmicb.2017.01447

Clinical streptococcal isolates, distinct from Streptococcus pneumoniae, but containing the β-glucosyltransferase tts gene and expressing serotype 37 capsular polysaccharide.

PeerJ. 2017 Jul 18;5:e3571. doi: 10.7717/peerj.3571. eCollection 2017.

Clinical streptococcal isolates, distinct from Streptococcus pneumoniae, but containing the β-glucosyltransferase tts gene and expressing serotype 37 capsular polysaccharide.

Sheppard CL1, Kapatai G1, Broughton K1, Schaefer U2, Hannah M3, Litt DJ1, Fry NK1.

Author information

Abstract

The major virulence factor of the pneumococcus, and target for conjugate vaccines, is the polysaccharide capsule, which is usually encoded by the highly variable cps locus. Serotype 37 is an unusual pneumococcal type in which the single β-glucosyltransferase gene responsible for serotype capsule production (tts) is located outside of the capsular operon region. Using a previously described automated whole genome sequence (WGS)-based serotyping bioinformatics tool, PneumoCaT, we identified and investigated seven clinical isolates (three from blood cultures) of non-pneumococcal streptococci containing a highly homologous tts and included them in a study panel of 20 isolates which included a 11 further clinical isolates of S. pneumoniae serotype 37, a reference strain of serotype 37 and the S. pseudopneumoniae type strain BAA 960T. The seven non-pneumococcal isolates generated novel alleles at all pneumococcal MLST loci and gave low percentage similarity (<45%) to S. pneumoniae or S. pseudopneumoniae species by comparison of short sequence patterns in genomic data (k-mer analysis). The S. pseudopneumoniae BAA-960T isolate generated two novel alleles in the MLST and gave a high similarity (>99%) to the reference sequence for BAA-960T. Twelve isolates gave high similarity (>77%) to the Streptococcus pneumoniae 5652-06 serotype 19A reference genome sequence and had previously reported MLST alleles. Each of the seven clinical non-pneumococcal strains and all of the 12 S. pneumoniae possessed a β-glycosyltransferase gene (tts) with >95% similarity to the pneumococcal tts reference DNA sequence with 20-22 non-synonymous SNPs. All but two strains in which the tts gene was detected gave positive reactions for serotype 37 in slide agglutination tests with serotype 37 typing sera. Phylogenetic analysis using both SNP and MLST data showed distinct clades corresponding to strains identified as pneumococcus or non-pneumococcus by kmer WGS analysis. Extended k-mer database analysis and ribosomal MLST placed the non-pneumococcal isolates within the S. mitis group. Biochemical and bile solubility assays showed differences between the unusual isolates and S. pneumoniae. All isolates had detectable pneumolysin (ply) genes, but only those that identified as pneumococcus contained the genes for autolysin (lytA) or the ABC transporter lipoprotein A (piaA) with >80% coverage and >95% similarity. Here we report the existence of a novel group of strains distinct from S. pneumoniae, but which can express a pneumococcal serotype 37 capsular polysaccharide which can be associated with clinical disease.

KEYWORDS:

Microbial genomics; Microbial identification; Phylogenetics; Streptococci

PMID: 28729954 PMCID: PMC5518733 DOI: 10.7717/peerj.3571

Complete Genome Sequence of Streptococcus pneumoniae Virulent Phage MS1.

Genome Announc. 2017 Jul 13;5(28). pii: e00333-17. doi: 10.1128/genomeA.00333-17.

Complete Genome Sequence of Streptococcus pneumoniae Virulent Phage MS1.

Kot W1, Sabri M2, Gingras H3, Ouellette M3, Tremblay DM2,4,5, Moineau S6,4,5.

Author information

Abstract

The lytic Streptococcus pneumoniae phage MS1 was isolated from a throat swab of a patient with symptoms of upper respiratory tract infection. The genome of this siphophage has 56,075 bp, 42.3% G+C content, and 77 open reading frames, including queuosine biosynthesis genes. Phage MS1 is related to pneumococcal phage Dp-1.

Copyright © 2017 Kot et al.

PMID: 28705957 PMCID: PMC5511896 DOI: 10.1128/genomeA.00333-17

Development of PCRSeqTyping-a novel molecular assay for typing of Streptococcus pneumoniae.

Pneumonia (Nathan). 2017 May 25;9:8. doi: 10.1186/s41479-017-0032-3. eCollection 2017.

Development of PCRSeqTyping-a novel molecular assay for typing of Streptococcus pneumoniae.

Nagaraj G1, Ganaie F1, Govindan V1, Ravikumar KL1.

Author information

Abstract

BACKGROUND:

Precise serotyping of pneumococci is essential for vaccine development, to better understand the pathogenicity and trends of drug resistance. Currently used conventional and molecular methods of serotyping are expensive and time-consuming, with limited coverage of serotypes. An accurate and rapid serotyping method with complete coverage of serotypes is an urgent necessity. This study describes the development and application of a novel technology that addresses this need.

METHODS:

Polymerase chain reaction (PCR) was performed, targeting 1061 bp cpsB region, and the amplicon was subjected to sequencing. The sequence data was analyzed using the National Centre for Biotechnology Information database. For homologous strains, a second round of PCR, sequencing, and data analysis was performed targeting 10 group-specific genes located in the capsular polysaccharide region. Ninety-one pneumococcal reference strains were analyzed with PCRSeqTyping and compared with Quellung reaction using Pneumotest Kit (SSI, Denmark).

RESULTS:

A 100% correlation of PCRSeqTyping results was observed with Pneumotest results. Fifty-nine reference strains were uniquely identified in the first step of PCRSeqTyping. The remaining 32 homologous strains out of 91 were also uniquely identified in the second step.

CONCLUSION:

This study describes a PCRSeqTyping assay that is accurate and rapid, with high reproducibility. This assay is amenable for clinical testing and does not require culturing of the samples. It is a significant improvement over other methods because it covers all pneumococcal serotypes, and it has the potential for use in diagnostic laboratories and surveillance studies.

KEYWORDS:

Molecular serotyping; PCRSeqTyping; Streptococcus pneumoniae; cpsB sequencing

PMID: 28702310 PMCID: PMC5471960 DOI: 10.1186/s41479-017-0032-3

Phylogenetic analysis of emergent Streptococcus pneumoniae serotype 22F causing invasive pneumococcal disease using whole genome sequencing.

PLoS One. 2017 May 22;12(5):e0178040. doi: 10.1371/journal.pone.0178040. eCollection 2017.

Phylogenetic analysis of emergent Streptococcus pneumoniae serotype 22F causing invasive pneumococcal disease using whole genome sequencing.

Demczuk WHB1, Martin I1, Hoang L2, Van Caeseele P3, Lefebvre B4, Horsman G5, Haldane D6, Gubbay J7, Ratnam S8, German G9, Daley Bernier J10, Strudwick L11, McGeer A12, Zhanel GG13, Van Domselaar G1,14, Graham M1,14, Mulvey MR1.

Author information

Abstract

Since implementation of the 13-valent polyvalent conjugate vaccine (PCV13) in Canada during 2010, the proportion of PCV13 serotypes causing invasive pneumococcal disease (IPD) has declined from 55% (n = 1492) in 2010 to 31% (n = 764) in 2014. A concurrent increase of non-PCV13 serotypes has occurred and 22F has become the most prevalent serotype in Canada increasing from 7% (n = 183) to 11% (n = 283). Core single nucleotide variant phylogenetic analysis was performed on 137 Streptococcus pneumoniae serotype 22F isolates collected across Canada from 2005-2015. Six phylogenetic lineages (n = 117) were identified among a serotype 22F/ST433 clonal complex (CC), including a recently expanding erythromycin-resistant clone. Erythromycin-resistance was observed in 25 isolates possessing ermB, mef or a 23S rRNA A2061G point mutation; 2 penicillin-resistant isolates had recombinant pbp1a, pbp2a and/or pbp2x; 3 tetracycline-resistant isolates contained tetM; and 1 isolate was multidrug-resistant. Virulence factor analysis indicated a high level of homogeneity among the 22F/ST433 clonal complex strains. A group of 6 phylogenetic outlier strains had differing MLST, antimicrobial resistance and molecular profiles suggestive of capsule switching events. While capsule switch events among S. pneumoniae serotype 22F has been observed, increasing prevalence of S. pneumoniae serotype 22F can be attributed to an evolving homogenous clone expanding nationally through local transmission events.

PMID: 28531208 PMCID: PMC5439729 DOI: 10.1371/journal.pone.0178040

An evolutionary model to predict the frequency of antibiotic resistance under seasonal antibiotic use, and an application to Streptococcus pneumoniae.

Proc Biol Sci. 2017 May 31;284(1855). pii: 20170679. doi: 10.1098/rspb.2017.0679.

An evolutionary model to predict the frequency of antibiotic resistance under seasonal antibiotic use, and an application to Streptococcus pneumoniae.

Blanquart F1, Lehtinen S2,3, Fraser C2,3.

Author information

Abstract

The frequency of resistance to antibiotics in Streptococcus pneumoniae has been stable over recent decades. For example, penicillin non-susceptibility in Europe has fluctuated between 12% and 16% without any major time trend. In spite of long-term stability, resistance fluctuates over short time scales, presumably in part due to seasonal fluctuations in antibiotic prescriptions. Here, we develop a model that describes the evolution of antibiotic resistance under selection by multiple antibiotics prescribed at seasonally changing rates. This model was inspired by, and fitted to, published data on monthly antibiotics prescriptions and frequency of resistance in two communities in Israel over 5 years. Seasonal fluctuations in antibiotic usage translate into small fluctuations of the frequency of resistance around the average value. We describe these dynamics using a perturbation approach that encapsulates all ecological and evolutionary forces into a generic model, whose parameters quantify a force stabilizing the frequency of resistance around the equilibrium and the sensitivity of the population to antibiotic selection. Fitting the model to the data revealed a strong stabilizing force, typically two to five times stronger than direct selection due to antibiotics. The strong stabilizing force explains that resistance fluctuates in phase with usage, as antibiotic selection alone would result in resistance fluctuating behind usage with a lag of three months when antibiotic use is seasonal. While most antibiotics selected for increased resistance, intriguingly, cephalosporins selected for decreased resistance to penicillins and macrolides, an effect consistent in the two communities. One extra monthly prescription of cephalosporins per 1000 children decreased the frequency of penicillin-resistant strains by 1.7%. This model emerges under minimal assumptions, quantifies the forces acting on resistance and explains up to 43% of the temporal variation in resistance.

© 2017 The Authors.

KEYWORDS:

adaptation; antimicrobial resistance; balancing selection; drug resistance; fluctuating selection; microbiology

PMID: 28566489 PMCID: PMC5454275 DOI: 10.1098/rspb.2017.0679

High-throughput CRISPRi phenotyping identifies new essential genes in Streptococcus pneumoniae.

Mol Syst Biol. 2017 May 10;13(5):931. doi: 10.15252/msb.20167449.

High-throughput CRISPRi phenotyping identifies new essential genes in Streptococcus pneumoniae.

Liu X1,2, Gallay C1, Kjos M1,3, Domenech A1, Slager J1, van Kessel SP1, Knoops K4, Sorg RA1, Zhang JR2, Veening JW5,6.

Author information

Abstract

Genome-wide screens have discovered a large set of essential genes in the opportunistic human pathogen Streptococcus pneumoniae However, the functions of many essential genes are still unknown, hampering vaccine development and drug discovery. Based on results from transposon sequencing (Tn-seq), we refined the list of essential genes in S. pneumoniae serotype 2 strain D39. Next, we created a knockdown library targeting 348 potentially essential genes by CRISPR interference (CRISPRi) and show a growth phenotype for 254 of them (73%). Using high-content microscopy screening, we searched for essential genes of unknown function with clear phenotypes in cell morphology upon CRISPRi-based depletion. We show that SPD_1416 and SPD_1417 (renamed to MurT and GatD, respectively) are essential for peptidoglycan synthesis, and that SPD_1198 and SPD_1197 (renamed to TarP and TarQ, respectively) are responsible for the polymerization of teichoic acid (TA) precursors. This knowledge enabled us to reconstruct the unique pneumococcal TA biosynthetic pathway. CRISPRi was also employed to unravel the role of the essential Clp-proteolytic system in regulation of competence development, and we show that ClpX is the essential ATPase responsible for ClpP-dependent repression of competence. The CRISPRi library provides a valuable tool for characterization of pneumococcal genes and pathways and revealed several promising antibiotic targets.

© 2017 The Authors. Published under the terms of the CC BY 4.0 license.

KEYWORDS:

DNA replication; bacterial cell wall; competence; gene essentiality; teichoic acid biosynthesis

PMID: 28490437 PMCID: PMC5448163

The accessory Sec system (SecY2A2) in Streptococcus pneumoniae is involved in export of pneumolysin toxin, adhesion and biofilm formation.

Microbes Infect. 2017 Apr 26. pii: S1286-4579(17)30064-3. doi: 10.1016/j.micinf.2017.04.003. [Epub ahead of print]

The accessory Sec system (SecY2A2) in Streptococcus pneumoniae is involved in export of pneumolysin toxin, adhesion and biofilm formation.

Bandara M1, Skehel JM2, Kadioglu A3, Collinson I4, Nobbs AH5, Blocker AJ6, Jenkinson HF7.

Author information

Abstract

In Streptococcus pneumoniae TIGR4, genes encoding a SecY2A2 accessory Sec system are present within a locus encoding a serine-rich repeat surface protein PsrP. Mutant strains deleted in secA2 or psrP were deficient in biofilm formation, while the ΔsecA2 mutant was reduced in binding to airway epithelial cells. Cell wall protein (CWP) fractions from the ΔsecA2 mutant, but not from the ΔpsrP mutant, were reduced in haemolytic (pneumolysin) activity. Contact-dependent pneumolysin (Ply) activity of wild type TIGR4 cells was ten-fold greater than that of ΔsecA2 mutant cells suggesting that Ply was not active at the ΔsecA2 cell surface. Ply protein was found to be present in the CWP fraction from the ΔsecA2 mutant, but showed aberrant electrophoretic migration indicative of protein modification. Proteomic analyses led to the discovery that the ΔsecA2 mutant CWP fraction was deficient in two glycosidases as well as other enzymes involved in carbohydrate metabolism. Taken collectively the results suggest that positioning of Ply into the cell wall compartment in active form, together with glycosyl hydrolases and adhesins, requires a functional accessory Sec system.

Copyright © 2017 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.

KEYWORDS:

O-glycosidase; Streptococcus pneumoniae; biofilms; export; pneumolysin; secA2

PMID: 28456649 DOI: 10.1016/j.micinf.2017.04.003

ComE, an Essential Response Regulator, Negatively Regulates the Expression of the Capsular Polysaccharide Locus and Attenuates the Bacterial Virulence in Streptococcus pneumoniae.

Front Microbiol. 2017 Mar 7;8:277. doi: 10.3389/fmicb.2017.00277. eCollection 2017.

ComE, an Essential Response Regulator, Negatively Regulates the Expression of the Capsular Polysaccharide Locus and Attenuates the Bacterial Virulence in Streptococcus pneumoniae.

Zheng Y1, Zhang X2, Wang X2, Wang L2, Zhang J2, Yin Y2.

Author information

Abstract

The capsular polysaccharide (CPS) of Streptococcus pneumoniae is the main virulence factors required for effective colonization and invasive disease. The capacity to regulate CPS production at the transcriptional level is critical for the survival of S. pneumoniae in different host niches, but little is known about the transcription regulators of cps locus. In the present study, we isolated and identified the response regulator ComE, the master competence switch in transformation of S. pneumoniae, as a transcriptional regulator of cps locus by DNA affinity chromatography-pulldown, MALDI-TOF mass spectrometry (MS) and electrophoretic mobility shift assay (EMSA). Our results showed that phosphorylated mimetic of ComE (ComED58E) bound specifically to the cps locus prompter in vitro, and phosphorylated ComE negatively impacted both cps locus transcription and CPS production attenuating the pneumococcal virulence in vivo. Compared with D39-WT strain, D39ΔcomE mutant exhibited much thicker capsule, attenuated nasopharyngeal colonization and enhanced virulence in both pneumonia and bacteremia models of Balb/c mice. Furthermore, it was demonstrated that CSP-ComD/E competence system involved in regulating negatively the CPS production during the progress of transformation in D39. Our CSP1 induction experiment results showed that the expression of ComE in D39-WT strain increased powerfully by 120% after 10 min of CSP1 induction, but the CPS production in D39-WT strain decreased sharply by 67.1% after 15 min of CSP1 induction. However, the CPS production in D39ΔcomE mutant was almost constant during the whole stage of induction. Additionally, we found that extracellular glucose concentration could affect both the expression of ComE and CPS production of D39 in vitro. Taken together, for the first time, we report that ComE, as a transcriptional regulator of cps locus, plays an important role in transcriptional regulation of cps locus and capsular production level.

KEYWORDS:

ComE; Streptococcus pneumoniae; capsule; transcription regulation; virulence

PMID: 28326061 PMCID: PMC5339220 DOI: 10.3389/fmicb.2017.00277

A tetracycline inducible integrative expression system for Streptococcus pneumoniae.

FEMS Microbiol Lett. 2017 Feb 21. doi: 10.1093/femsle/fnx044. [Epub ahead of print]

A tetracycline inducible integrative expression system for Streptococcus pneumoniae.

Meiers M1, Laux A1, Eichinger D1, Sexauer A1, Marx P1, Bertram R2,3, Brückner R1.

Author information

Abstract

Inducible gene expression systems are very useful to analyze cellular processes. The ability to switch the expression state of genes of interest may even be crucial if essential traits or genetic instability are involved. An integrative plasmid, pTEX2, was designed using the (anhydro)tetracycline-inducible promoter Pxyl/tet from staphylococcal plasmid pRAB11 to control gene expression in Streptococcus pneumoniae. The system was evaluated by expressing genes of the two-component regulatory system ciaRH of S. pneumoniae. With full induction of Pxyl/tet, wild type levels of the response regulator CiaR were obtained, while the uninduced basal expression was low. Hyperactive variants of the kinase gene ciaH normally causing pronounced genetic instability could be handled without any problems upon cloning into pTEX2. Therefore, the expression system is well suited to express physiological levels of proteins in S. pneumoniae and also to aid regulatory studies.

© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

KEYWORDS:

Streptococcus pneumoniae; integrative plasmid; tetracycline inducible expression; two-component regulatory system CiaRH

PMID: 28333198 DOI: 10.1093/femsle/fnx044

Niacin-mediated Gene Expression and Role of NiaR as a Transcriptional Repressor of niaX, nadC, and pnuC in Streptococcus pneumoniae.

Front Cell Infect Microbiol. 2017 Mar 9;7:70. doi: 10.3389/fcimb.2017.00070. eCollection 2017.

Niacin-mediated Gene Expression and Role of NiaR as a Transcriptional Repressor of niaX, nadC, and pnuC in Streptococcus pneumoniae.

Afzal M1, Kuipers OP2, Shafeeq S3.

Author information

Abstract

NAD (Nicotinamide Adenine Dinucleotide) biosynthesis is vital for bacterial physiology and plays an important role in cellular metabolism. A naturally occurring vitamin B complex, niacin (nicotinic acid), is a precursor of coenzymes NAD and NADP. Here, we study the impact of niacin on global gene expression of Streptococcus pneumoniae D39 and elucidate the role of NiaR as a transcriptional regulator of niaX, nadC, and pnuC. Transcriptome comparison of the D39 wild-type grown in chemically defined medium (CDM) with 0 to 10 mM niacin revealed elevated expression of various genes, including niaX, nadC, pnuC, fba, rex, gapN, pncB, gap, adhE, and adhB2 that are putatively involved in the transport and utilization of niacin. Niacin-dependent expression of these genes is confirmed by promoter lacZ-fusion studies. Moreover, the role of transcriptional regulator NiaR in the regulation of these genes is explored by DNA microarray analysis. Our transcriptomic comparison of D39 ΔniaR to D39 wild-type revealed that the transcriptional regulator NiaR acts as a transcriptional repressor of niaX, pnuC, and nadC. NiaR-dependent regulation of niaX, nadC, and pnuC is further confirmed by promoter lacZ-fusion studies. The putative operator site of NiaR (5'-TACWRGTGTMTWKACASYTRWAW-3') in the promoter regions of niaX, nadC, and pnuC is predicted and further confirmed by promoter mutational experiments.

KEYWORDS:

NiaR; Pneumococcus; nadC; niaX; niacin; pnuC

PMID: 28337428 PMCID: PMC5343564 DOI: 10.3389/fcimb.2017.00070