Human age and skin physiology shape diversity and abundance of Archaea on skin

13 Pages·2017·1.02 MB·English

Human age and skin physiology shape diversity and abundance of Archaea on skin
-Supplementary Figures-

Christine Moissl-Eichinger 1,2,* , Alexander J. Probst3, Giovanni Birarda4,7, Anna Auerbach5, Kaisa
Koskinen1,2, Peter Wolf6, Hoi-Ying N. Holman7

1 Medical University of Graz, Department for Internal Medicine, Auenbruggerplatz 15, 8036 Graz, Austria
2 BioTechMed Graz, Krenngasse 37, 8010 Graz, Austria
3 Department of Earth and Planetary Science, University of California, Berkeley, 307 McCone Hall, Berkeley, CA 94720, USA
4 Elettra – Sincrotrone Trieste, Strada Statale 14 - km 163,5 in AREA Science Park, 34149 Basovizza, Trieste Italy
5 University of Regensburg, Department for Microbiology and Archaea Center, Universitaetsstr. 31, 93053 Regensburg, Germany
6 Medical University of Graz, Department for Dermatology, Auenbruggerplatz 8, 8036 Graz, Austria
7 Berkeley Synchrotron Infrared Structural Biology Program, Lawrence Berkeley National Laboratory, One Cyclotron Road, Berkeley,
California, United States of America
* Corresponding author.
Supplementary Figure S1: FTIR-FPA spectra of archaeal and bacterial reference strains. Based on their different membrane
architecture, archaea can be easily distinguished.
Supplementary Figure S2: Biplot of the Lipid/Protein ratio versus (A) the archaeal IR count and (B) the qPCR values
indicates trends that support the hypothesis that people with drier skin may host more archaea cells.

A B
FTIR-FPA vs Lipids / Proteins qPCR vs Lipids / Proteins
1.2 Linear Fit of Lipids / Proteins Linear Fit of Lipids / Proteins
1.1
95% LCL of Lipids / Proteins 95% LCL of Lipids / Proteins
95% UCL of Lipids / Proteins 95% UCL of Lipids / Proteins
1.0
0.9
0.9
ns (a.u.) s (a.u.) 0.8
ei n 0.7
ds / Prot 0.6 s / Protei 0.6
Lipi pid 0.5
Li
0.3 0.4
0.3
0.00 0.03 0.06 0.09
0 1 2 3 4 5
Archaea IR (%)
qPCR
Supplementary Figure S3: Display of the human skin archaeomes of different skin wipe samples as revealed in this study.
This chart is based on all archaeal sequences (reads) obtained via next generation sequencing of all 21 skin wipe samples.
Sample 14.m.28 is not shown, since no archaeal reads were obtained. Sample names, given in the middle of each chart,
refer to sample number, sex (w=female, m=male), and age (separated by "."). Thaumarchaeota are shown in red,
Euryarchaeota in green, Crenarchaeota are indicated in blue.
100%
90%
80%
a Thaum_Soil group I.1b
x 70%
a
t
al Thaum_SAGMCG-1
e 60%
a
h Thaum_MCG
c
r
f a 50% Thaum_Group C3
o
n Eury_Halobacteria
o 40%
ti
or Eury_Methanomicrobia
p
o 30%
Pr Eury_Methanobacteria
20% Eury_Thermococci
Eury_Thermoplasmata
10%
Cren_Thermoprotei
0%
2 4 4 5 5 6 6 2 2 9 4 7 8 9 3 2 2 7 2 7
01.w. 22.w. 23.m. 26.w. 27.m. 19.m. 25.m. 5.w.1 6.w.1 9.m.1 5.m.2 5.m.2 6.w.2 1.m.3 1.w.4 2.m.5 4.w.5 4.m.5 0.m.6 3.m.6
4 4 4 1 0 1 5 3 0 3 0 4 4
Sample
Supplementary Figure S4: Bubble plot of the 20 most abundant archaeal OTUs. Relative abundance is reflected by the size
of the square (see legend). Samples are grouped according to their age group (age group I: 1-11 years, II: 12-60 years, III:
>61 years), as indicated in the figure. Sample names, given in the middle of each chart, refer to sample number, sex
(f=female, m=male), and age (separated by ".").
Age group: I II III
Supplementary Figure S5: Inverse Simpson Index, reflecting the diversity and richness in the archaeal skin communities. No
significant differences were found with respect to age group (age group I: 1-11 years, II: 12-60 years, III: >61 years) and sex
(f=female, m=male). However, a trend towards a lower index was found in age group II.
Supplementary Figure S6: Shannon Index, reflecting the diversity of the archaeal skin communities. No significant
differences were found with respect to sex (f=female, m=male). However, significantly different diversities were retrieved
for the age groups (age group I: 1-11 years, II: 12-60 years, III: >61 years) , with the lowest shannon index in age group II.
Supplementary Figure S7: Redundancy analysis plots on OTU level, indicating how well samples can be separated according
to sex and age group. Age groups were found to be significantly associated with variation in the data matrix (p-values are
given in the graphs).
Female (f) vs. male (m) Age groups (I: 1-11; II: 12-60; III: 61-75)
p= 0.81 p= 0.028*
1 Supplementary Information: Methods
2 Human age and skin physiology shape diversity and abundance of Archaea on skin
3 Running title: Human skin archaeome
4
5 Christine Moissl-Eichinger 1,2,* , Alexander J. Probst3, Giovanni Birarda4,7, Anna Auerbach5, Kaisa
6 Koskinen1,2, Peter Wolf6, Hoi-Ying N. Holman7
7 1 Medical University of Graz, Department for Internal Medicine, Auenbruggerplatz 15, 8036 Graz,
8 Austria
9 2 BioTechMed Graz, Krenngasse 37, 8010 Graz, Austria
10 3 Department of Earth and Planetary Science, University of California, Berkeley, 307 McCone Hall,
11 Berkeley, CA 94720, USA
12 4 Elettra – Sincrotrone Trieste, Strada Statale 14 - km 163,5 in AREA Science Park, 34149 Basovizza,
13 Trieste Italy
14 5 University of Regensburg, Department for Microbiology and Archaea Center, Universitaetsstr. 31,
15 93053 Regensburg, Germany
16 6 Medical University of Graz, Department for Dermatology, Auenbruggerplatz 8, 8036 Graz, Austria
17 7 Berkeley Synchrotron Infrared Structural Biology Program, Lawrence Berkeley National Laboratory,
18 One Cyclotron Road, Berkeley, California, United States of America
19
20
21 DNA extraction from samples
22 DNA was extracted following the protocol established previously 1.In brief, sponges were extracted
23 for two minutes by sonication (Bandelin Sonorex Super 10 P); the sponges were squeezed and
24 obtained liquid was removed for further extraction. This procedure was repeated after adding
25 another 3 ml isotonic saline solution (0.9% NaCl (w/v), DNA-free). Both liquid fractions were pooled,
26 and volume was adjusted to 3 ml. One aliquot of the liquid was subjected to XS-buffer extraction as
27 described previously; the extraction procedure included a bead-beating step 1.
28
29 Amplification of archaeal 16S rRNA genes and next generation sequencing (NGS)
30 From all DNA extractions, covering the broad age-range of the volunteers, 21 samples (see
31 Supplementary Table S1) were selected for NGS using 454 pyrotag-technology. Besides archaeal
32 amplicons, bacterial amplicons were produced for two of the samples to verify that our skin
33 microbiome analysis procedure worked properly, that the retrieved samples were representative of
34 the human skin microbiome and to exclude biases in the sampling methodology (Supplementary
35 Table S6).
36 A(cid:373)pli(cid:272)o(cid:374)s were produ(cid:272)ed usi(cid:374)g (cid:271)ar(cid:272)oded pri(cid:373)er pairs (cid:1007)(cid:1008)(cid:1008)af/(cid:1013)(cid:1005)(cid:1009)ar (cid:894)Ar(cid:272)haea, (cid:1009)’-
37 ACGGGGYGCAGCAGGCGCGA-(cid:1007)’/ (cid:1009)’-GTGCTCCCCCGCCAATTCCT 2,3(cid:895) a(cid:374)d (cid:1007)(cid:1008)(cid:1005)f/(cid:1012)(cid:1004)(cid:1009)r (cid:894)Ba(cid:272)teria, (cid:1009)’-
38 CCTACGGGNGGCWGCAG-(cid:1007)’/ (cid:1009)’-GACTACHVGGGTATCTAATCC-(cid:1007)’ 4). For both runs, 35 PCR cycles were
39 performed, with annealing temperature 60°C and 55°C, respectively. Amplicons were verified by gel
40 electrophoresis, quantified via Qubit, and pooled for NGS sequencing (Roche 454 GS FLX). Library
41 production and sequencing was performed by Macrogen Corporation (Geumcheon District, South
42 Korea).
43
44 Processing of the reads, visualization and phylogenetic analysis
45 Raw reads (sff files) were processed using Qiime (version 1.9.0; 5) following the standard operating
46 procedures, which included chimera removal by usearch. Operational taxonomic units (OTUs) were