Accepted Manuscript Infection of human keratinocytes by Streptococcus dysgalactiae subspecies dysgalactiae isolated from milk of the bovine udderInfection of human keratinocytes by Streptococcus dysgalactiae subspecies dysgalactiae isolated from milk of the bovine udder Catarina Roma-Rodrigues, Cynthia Alves-Barroco, Luís R. Raposo, Mafalda N. Costa, Elvira Fortunato, Pedro Viana Baptista, Alexandra R. Fernandes, Ilda SantosSanches
PII:
S1286-4579(15)00253-1
DOI:
10.1016/j.micinf.2015.11.005
Reference:
MICINF 4351
To appear in:
Microbes and Infection
Received Date: 10 November 2015 Revised Date:
16 November 2015
Accepted Date: 21 November 2015
Please cite this article as: C. Roma-Rodrigues, C. Alves-Barroco, L.R Raposo, M.N Costa, E. Fortunato, P.V. Baptista, A.R Fernandes, I. Santos-Sanches, Infection of human keratinocytes by Streptococcus dysgalactiae subspecies dysgalactiae isolated from milk of the bovine udderInfection of human keratinocytes by Streptococcus dysgalactiae subspecies dysgalactiae isolated from milk of the bovine udder, Microbes and Infection (2015), doi: 10.1016/j.micinf.2015.11.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
RI PT
Infection of human keratinocytes by Streptococcus dysgalactiae subspecies dysgalactiae isolated from milk of the bovine udder
SC
Catarina Roma-Rodriguesa, Cynthia Alves-Barrocoa, Luís R Raposoa,b, Mafalda N Costa3, Elvira Fortunatoc, Pedro Viana Baptistaa, Alexandra R Fernandesa,b,*, Ilda Santos-Sanchesa,*
Affiliations:
M AN U
a – UCIBIO, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus de Caparica, 2829-516 Caparica, Portugal b – CQE, Centro de Quimica Estrutural, Complexo 1, Instituto Superior Técnico, Universidade de Lisboa, Avenida Rovisco Pais, 1049-001 Lisboa, Portugal
TE D
c – i3N/CENIMAT, Departamento de Ciência dos Materiais, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus de Caparica, 2829-516 Caparica, Portugal
Corresponding authors:
EP
Alexandra Fernandes
Email:
[email protected] AC C
Ilda Santos-Sanches
Email:
[email protected] Adress: Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, Campus de Caparica, 2829-516 Caparica, Portugal;
Phone: +351 212 948 530 Fax: +351 212 948 530
1
ACCEPTED MANUSCRIPT
Abstract (94/100) Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) are considered exclusive animal pathogens; however, a putative zoonotic upper limb cellulitis, a prosthetic joint infection and an infective endocarditis were described in humans. To unravel if bovine SDSD isolates are able to infect human cells, the adherence and internalization to human primary keratinocytes of two
RI PT
bovine SDSD strains isolated from milk collected from udder were analyzed. Bacterial adhesion assays and confocal microscopy indicate a high adherence and internalization of SDSD isolates
M AN U
SC
to human cells, suggesting for the first time the ability of bovine isolates to infect human cells.
List of abbreviations
TE D
Keywords: adhesion; bovine; host; human keratinocytes; internalization; Streptococcus dysgalactiae subspecies dysgalactiae
CFUs – Colony Forming Units
DMEM – Dulbecco’s Modified Eagle Medium supplemented with 10% (v/v) Foetal Bovine Serum
EP
PBS – Phosphate Buffer Saline
SDSD – Streptococcus dysgalactiae subps. dysgalactiae
AC C
THB-5YE – Todd Hewitt Broth supplemented with 5% (w/v) yeast extract
2
ACCEPTED MANUSCRIPT
1. Introduction
2
Streptococcus dysgalactiae subspecies dysgalactiae (SDSD) are alpha-hemolytic or non-
3
hemolytic streptococci belonging to the Lancefield group C [1, 2]. They are considered as
4
exclusive animal pathogens, despite three sporadic cases of infection in humans being reported,
5
namely an ascending upper limb cellulitis in a woman that contacted raw fish [3], a prosthetic joint
6
infection after total knee arthroplasty [4], and very recently, an infective endocarditis in a male
7
patient [5]. Previous investigations in our group evidenced that SDSD strains recovered from
8
subclinical bovine mastitis carried and expressed phage-encoded virulence genes including
9
genes that code for the exotoxins SpeK, SpeC, SpeL and SpeM, genes coding for DNase I
M AN U
SC
RI PT
1
(Spd1), and streptodornase gene (sdn) of the strictly human pathogen Streptococcus pyogenes
11
[6, 7]. Despite these reported cases of human infections caused by SDSD, it has never been
12
reported that animal SDSD isolates, particularly bovine mastitis subclinical strains, are capable to
13
infect human cells. Nevertheless, the presence of S. pyogenes virulence factors in the genome of
14
bovine SDSD has lead to the hypothesis of SDSD dissemination to other hosts. Hence, here the
15
possibility of SDSD strains to adhere to and invade human cells was investigated. Adherence and
16
invasion of SDSD isolates in epithelial cells has been described to occur in mammary cell lines
17
and fish epithelial cell lines cultured in vitro [8-10]. The virulence of SDSD seems to be related to
18
cell surface properties, such as high hemagglutination and hydrophobic properties that determine
19
the main adhesion and invasive pathogenic mechanism of the species [8, 11-13].
EP
AC C
20
TE D
10
21
2. Materials and Methods
22
2.1. Bacterial species and culture conditions
3
ACCEPTED MANUSCRIPT Streptococcus dysgalactiae subsp. dysgalactiae strains VSD5 and VSD13 were isolated from
24
milk collected from the udder of cows with mastitis [6, 7]. Strain identified as GCS-Si was
25
responsible for an ascending upper limb cellulitis in a woman that was pricked by the fins and
26
scales of a raw fish [3]. S. pyogenes GAP58 is an invasive strain isolated from the blood a patient
27
and GAP8 is a strain isolated from the tonsils of an asymptomatic patient [14]. The
28
characterization of each strain based on 16S sequencing, emm subtype and MLST was
29
previously performed [7, 14]. Strains were cultivated in 20 mL Todd Hewitt broth supplemented
30
with 5 % (w/v) yeast extract (THB-5YE) in 100 mL Erlenmeyer-flasks and grown at 37ºC until
31
reach a standardized optical density at 600 nm (OD600) of 0.3 – 04 (5x107 – 1x108 cells/mL) .
32
For each strain, an aliquot of 1 mL of cell suspension was collected and cells were washed 3
33
times in fresh THB-5YE.
34
2.2. Human Cells and culture conditions
35
Human primary epidermal keratinocytes (ATCC-PCS-200-010, ATCC, Manassas, USA) were
36
grown in keratinocyte growth media according to ATCC (Manassas, USA) instructions. For
37
infection assays, cells were seeded in a 96-well culture plate at a density of 3 x 104 cells/well and
38
incubated for 24 h at 37 ºC, 5 % (v/v) CO2 and 99 % (v/v) relative humidity. For confocal
39
microscopy analysis 1x105 cells were placed over a Polylysine treated microscope slide and
40
incubated for 24 h at 37 ºC, 5 % (v/v) CO2 and 99 % (v/v) humidity.
41
2.3. Bacterial internalization and adherence assays
42
The adhesion and internalization assays were based on previously described protocols [15, 16]
43
with few modifications. Bacteria were grown at 37 ºC in Todd Hewitt Broth supplemented with 5%
44
(w/v) yeast extract (THB-5YE) until the middle of the exponential phase. The infection was started
45
by adding 106 bacterial cells in Dulbecco’s Modified Eagle’s Medium supplemented with 10%
46
(v/v) Foetal Bovine Serum (DMEM) to 104 human primary epidermal keratinocytes (ATCC-PCS-
AC C
EP
TE D
M AN U
SC
RI PT
23
4
ACCEPTED MANUSCRIPT 200-010, ATCC, Manassas, USA). After 2 h of incubation at 37 ºC, 5% (v/v) CO2 and 99% (v/v)
48
relative humidity, cell monolayers were washed with Phosphate Buffer Saline (PBS) to remove
49
unbound bacteria. To obtain the number of bacteria that adhere and internalize to keratinocytes
50
[(Adh+Int)value] and the number of bacteria that internalize keratinocytes (Intvalue), infected
51
keratinocyte cell monolayers were incubated for 30 min with DMEM or DMEM supplemented with
52
Penicillin 100 units/mL, respectively. Keratinocytes were detached from the well, disrupted with
53
Triton X-100 0.01% (v/v) (Sigma), and the bacterial colony-forming units (CFUs) were calculated
54
using standard plate counting techniques. In parallel, the exact same procedure was performed in
55
keratinocyte free wells in order to obtain procedural negative control values. The values obtained
56
in the procedural control is subtracted from Intvalue and (Adh+Int)value. Intvalue was the averaged
57
CFUs counted for the keratinocytes infected cells treated with Penicillin minus the averaged
58
CFUs obtained in Intctrl. The (Adh+Int)value was the averaged CFUs counted for the keratinocytes
59
infected cells minus the averaged CFUs obtained in (Adh+Int)ctrl. The Adhvalue of each strain was
60
obtained by subtracting the Intvalue to the (Adh+Int)value. Every experiment was repeated at least 4
61
times.
62
2.4. Confocal laser scanning microscopy
63
For confocal laser scanning microscopy analysis, pelleted bacteria were resuspended in THB-
64
5YE supplemented with 0.2 mg/mL Hoechst 33258 (LifeTechnologies), incubated at 37 ºC for 45
65
min, washed with PBS and resuspended in DMEM. Keratinocytes in microscope slides were
66
washed 3 times with PBS and bacterial cells placed over the human cells. After 2 h of incubation
67
at 37 ºC, 5 % (v/v) CO2 and 99 % (v/v) relative humidity, cells were fixed with 2 % (w/v)
68
paraformaldehyde, lysed with 0.1 % (v/v) Triton X-100 and animal cells were stained with
69
AlexaFluor 488 Phalloidin (LifeTechnologies) according to the manufacturer’s instructions. After
70
washing 3 times with PBS, a drop of ProLong Diamond antifade mountant (LifeTechnologies) was
71
placed on top of the cells, covered with a cover slip and sealed. A confocal laser scanning
AC C
EP
TE D
M AN U
SC
RI PT
47
5
ACCEPTED MANUSCRIPT microscope (Carl Zeiss, LSM 700), using the 488 nm laser for visualization of cells stained with
73
AlexaFluor 488 Phalloidin, and the 405 nm laser for visualization of bacterial cells stained with
74
Hoechst 33258, and respective software (ZEN Black, 2011), was used for image acquisition. This
75
approach only used the bovine SDSD VSD13 and the human S. pyogenes GAP58. A control
76
sample was also prepared, consisting in keratinocytes stained for 2 h with 20 µg/mL Hoechst
77
33258.
RI PT
72
SC
78
3. Results
80
To unravel if bovine SDSD isolates were able to interact with human cells, human keratinocytes
81
were infected for 2 h with two bovine SDSD strains, VSD5 and VSD13, both isolated from milk
82
collected from udder [6, 7], and the adherence and internalization assessed. For comparative
83
purposes, adherence and internalization of the SDSD isolate that caused ascending upper limb
84
cellulitis (SDSD GCS-Si) [3], an invasive human isolate S. pyogenes GAP58 collected from the
85
blood of a patient, and a S. pyogenes GAP8 human isolate from the oropharynx of an
86
asymptomatic carrier [14] were also assessed. Primary keratinocytes were selected due to the
87
common colonization of non-intact skin by S. pyogenes [17] and the ascending upper limb
88
cellulitis caused by SDSD GCS-Si.
89
Figure 1 shows that there is 1 internalized/adhered VSD5 cell per 10 keratinocytes. Also, data
90
show that 3 and 11 VSD13 cells are capable to internalize and adhere to a single keratinocyte,
91
respectively. These results suggest a higher adherence and internalization of VSD13 strains to
92
human keratinocytes. Also, higher adherence of the bovine isolate VSD13 and higher
93
internalization values of the bovine isolates are observed when compared to the two GAS strains.
AC C
EP
TE D
M AN U
79
6
ACCEPTED MANUSCRIPT The adherence and internalization of SDSD VSD13 was further confirmed by confocal
95
microscopy. Results suggest that only the nuclear DNA of the epithelial cells was stained, as can
96
be observed in the movie from additional file 1, which shows the X-Y images obtained along the
97
Z-plan for the control sample.
98
Confocal images analysis (Fig.2) corroborates adherence and internalization results. A higher
99
number of SDSD VSD13 (Fig.2, A) than S. pyogenes GAP58 cells (Fig.2, B) were observed to be
RI PT
94
interacting with the human keratinocytes. Two additional movies show with more detail the X-Y
101
images obtained along the Z plan, the additional file 2 shows the interaction of SDSD VSD13 with
102
keratinocytes and additional file 3 show the adherence and internalization of S. pyogenes GAP58.
M AN U
SC
100
103
4. Discussion
105
This work describes for the first time that SDSD bovine isolates collected from the milk of udder
106
are capable to adhere and internalize human cells. Remarkably, the adherence and
107
internalization values of bovine SDSD isolates are higher than those of human streptococci
108
isolates analyzed in this study.
109
Since the classification of S. dysgalactiae by Vandamme et al [2], that human S. dysgalactiae G
110
and C groups are classified as S. dysgalactiae subsp. equisimillis, while all strains of animal
111
origin are classified as S. dysgalactiae subsp. dysgalactiae [1, 2]. In this work, the ability of
112
bovine SDSD strains to adhere and internalize human primary keratinocyte cells is demonstrated,
113
raising the question if this division is adequate. In fact, the correct identification of S. dysgalactiae
114
species is paramount in clinical practice, since the information of the causative infection agent is
115
vital for the adequate diagnosis and treatment of streptococcal infections [18].
AC C
EP
TE D
104
116 7
ACCEPTED MANUSCRIPT
Acknowledgments
118
We thank the Fundação para a Ciência e a Tecnologia/Ministério da Educação e Ciência
119
(FCT/MEC) for financial support via PTDC/CVT-EPI/4651/2012, PTDC/BBB-NAN/1812/2012 and
120
UID/CTM/50025/2013.
RI PT
117
122
Conflict of interests
123
Authors declare no conflict of interests.
SC
121
M AN U
124 125
References
126
[1] Jensen A, Kilian M. Delineation of Streptococcus dysgalactiae, its subspecies, and its clinical
127
and phylogenetic relationship to Streptococcus pyogenes. J Clin Microbiol 2012;50:113-26.
TE D
128
[2] Vandamme P, Pot B, Falsen E, Kersters K, Devriese LA. Taxonomic study of lancefield
130
streptococcal groups C, G, and L (Streptococcus dysgalactiae) and proposal of S. dysgalactiae
131
subsp. equisimilis subsp. nov. Int J Syst Bacteriol 1996;46:774-81.
AC C
132
EP
129
133
[3] Koh TH, Sng LH, Yuen SM, Thomas CK, Tan PL, Tan SH, et al. Streptococcal cellulitis
134
following preparation of fresh raw seafood. Zoonoses Public Health 2009;56:206-8.
135 136
[4] Park MJ, Eun IS, Jung CY, Ko YC, Kim YJ, Kim CK, et al. Streptococcus dysgalactiae
137
subspecies dysgalactiae infection after total knee arthroplasty: a case report. Knee Surg Relat
138
Res 2012;24:120-3.
139
8
ACCEPTED MANUSCRIPT 140
[5] Jordal S, Glambek M, Oppegaard O, Kittang BR. New tricks from an old cow: infective
141
endocarditis caused by Streptococcus dysgalactiae subsp. dysgalactiae. J Clin Microbiol
142
2015;53:731-4.
143 [6] Rato MG, Bexiga R, Nunes SF, Vilela CL, Santos-Sanches I. Human group A streptococci
145
virulence genes in bovine group C streptococci. Emerg Infect Dis 2010;16:116-9.
RI PT
144
146
[7] Rato MG, Nerlich A, Bergmann R, Bexiga R, Nunes SF, Vilela CL, et al. Virulence gene pool
148
detected in bovine group C Streptococcus dysgalactiae subsp. dysgalactiae isolates by use of a
149
group A S. pyogenes virulence microarray. J Clin Microbiol 2011;49:2470-9.
M AN U
SC
147
150 151
[8] Abdelsalam M, Asheg A, Eissa AE. Streptococcus dysgalactiae: An emerging pathogen of
152
fishes and mammals. Int J Vet Sci Med 2013;1:1-6.
TE D
153
[9] Almeida RA, Oliver SP. Invasion of bovine mammary epithelial cells by Streptococcus
155
dysgalactiae. J Dairy Sci 1995;78:1310-7.
156
EP
154
[10] Calvinho LF, Oliver SP. Invasion and persistence of Streptococcus dysgalactiae within
158
bovine mammary epithelial cells. J Dairy Sci 1998;81:678-86.
159
AC C
157
160
[11] Abdelsalam M, Chen SC, Yoshida T. Surface properties of Streptococcus dysgalactiae
161
strains isolated from marine fish. Bull Eur Ass Fish Pathol 2009;29:16-24.
162
9
ACCEPTED MANUSCRIPT 163
[12] Calvinho LF, Almeida RA, Oliver SP. Influence of Streptococcus dysgalactiae surface
164
hydrophobicity on adherence to mammary epithelial cells and phagocytosis by mammary
165
macrophages. Zentralbl Veterinarmed B. 1996;43:257-66.
166 [13] Calvinho LF, Oliver SP. Characterization of mechanisms involved in uptake of Streptococcus
168
dysgalactiae by bovine mammary epithelial cells. Vet Microbiol 1998;63:261-74.
RI PT
167
169
[14] Pires R, Rolo D, Morais A, Brito-Avo A, Johansson C, Henriques-Normark B, et al.
171
Description of macrolide-resistant and potential virulent clones of Streptococcus pyogenes
172
causing asymptomatic colonization during 2000-2006 in the Lisbon area. Eur J Clin Microbiol
173
Infect Dis 2012;31:849-57.
174
M AN U
SC
170
[15] Agarwal V, Ahl J, Riesbeck K, Blom AM. An alternative role of C1q in bacterial infections:
176
facilitating Streptococcus pneumoniae adherence and invasion of host Cells. J Immunol
177
2013;191:4235-4245.
178
TE D
175
[16] Almeida RA, Dunlap JR, Oliver SP. Binding of host factors influences internalization and
180
intracellular trafficking of Streptococcus uberis in bovine mammary epithelial cells. Vet Med Int
181
2010;2010:Article ID 319192.
AC C
182
EP
179
183
[17] Chhatwal GS, McMillan DJ. Uncovering the mysteries of invasive streptococcal diseases.
184
Trends Mol Med 2005;11:152-5.
185 186
10
ACCEPTED MANUSCRIPT 187
[18] Nitsche-Schmitz DP, Chhatwal GS. Host-pathogen interactions in streptococcal immune
188
sequelae. Curr Top Microbiol Immunol 2013;368:155-71.
189
AC C
EP
TE D
M AN U
SC
RI PT
190
11
ACCEPTED MANUSCRIPT
Figure Legends:
192
Figure 1: Infection potential of bovine Streptococcus dysgalactiae subspecies
193
dysgalactiae strains of human keratinocytes. Adhesion (open bars) and internalization (filled
194
bars) in human primary epithelial keratinocytes of bovine isolates Streptococcus dysgalactiae
195
subsp. dysgalactiae (SDSD) VSD5 and VSD13, and human isolates SDSD GCS-Si,
196
Streptococcus pyogenes (SPYO) GAP58 and GAP8. Represented values are the average value
197
with SD. Represented values were corrected by subtracting values of procedural control
198
(keratinocyte-free wells). Results were only considered when procedural control values were
199
significantly lower (p-value < 0.05) than adherence and internalization values. *;**;***,**** P value
200
< 0.05.
M AN U
SC
RI PT
191
201
Figure 2: Infection of human keratinocytes by bovine Streptococcus dysgalactiae subsp.
203
dysgalactiae. Representative confocal laser scanning ortho-image of Z-stacks of 2 h infected
204
human keratinocytes (green, stained with AlexaFluor 488 Phaloidin, LifeTechnologies) with
205
Streptococcus (blue, stained with Hoechst 33258, LifeTechnologies). The centered panel is the
206
X-Y view of the image, corresponding to the blue line in the upper and right panels. The upper
207
panel, with a green outline, is the X-Z cross section of the green line in the centered image. The
208
image at the right, with a red outline, is the Y-Z cross section of the red line in the X-Y view. A)
209
Human keratinocytes infected with S. dysgalactiae subps. dysgalactiae VSD13 collected from the
210
milk of bovine udder. C) Human keratinocytes infected with the invasive human isolate S.
211
pyogenes GAP58. Red arrows point to internalized bacteria; yellow arrows point to adhered
212
bacteria.
AC C
EP
TE D
202
213
12
ACCEPTED MANUSCRIPT
Supporting information
215
Additional file 1
216
File format: .MOV
217
Title of data: Control sample.
218
Description of data: Representative movie obtained using confocal laser scanning microscopy,
219
of the X-Y images obtained along the Z-plane of human keratinocytes stained for 2 h with 20
220
µg/mL Hoechst 33258 (blue, LifeTechnologies), fixed with 2 % paraformaldehyde and stained
221
with AlexaFluor 488 Phaloidin (green, LifeTechnologies).
M AN U
SC
RI PT
214
222
Additional file 2
224
File format: MOV
225
Title of data: Infection of human keratinocytes by Streptococcus dysgalactiae subsp.
226
dysgalactiae VSD13 collected from the milk of the bovine udder.
227
Description of data: Representative movie obtained using confocal laser scanning microscopy,
228
of the X-Y images obtained along the Z-plane, of human keratinocytes (green, stained with
229
AlexaFluor 488 Phaloidin, LifeTechnologies) infected for 2 h with S. dysgalactiae subps.
230
dysgalactiae VSD13 (blue, stained with Hoechst 33258, LifeTechnologies).
AC C
EP
TE D
223
231 232
Additional file 3
233
File format: MOV
13
ACCEPTED MANUSCRIPT Title of data: Infection of human keratinocytes by human Streptococcus pyogenes GAP58.
235
Description of data: Representative movie obtained using confocal laser scanning microscopy,
236
of the X-Y images obtained along the Z-plane, of human keratinocytes (green, stained with
237
AlexaFluor 488 Phaloidin, LifeTechnologies) infected for 2 h with S. pyogenes GAP58 (blue,
238
stained with Hoechst 33258, LifeTechnologies).
239
SC
240
RI PT
234
AC C
EP
TE D
M AN U
241
14
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT