2011 the LuxS-Dependent Quorum Sensing System Regulates Early Biofilm Formation by Streptococcus...
Transcript of 2011 the LuxS-Dependent Quorum Sensing System Regulates Early Biofilm Formation by Streptococcus...
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The LuxS-dependent Quorum Sensing System Regulates Early Biofilm Formation1
by Streptococcus pneumoniae strain D392
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Jorge E. Vidal*1
, Herbert P. Ludewick1, Rebekah M. Kunkel
2, Dorothea Zhner
3and Keith P. Klugman
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91Hubert Department of Global Health, Rollins School of Public Health,
2Graduated Program in10
Population Biology, Ecology and Evolution and3Division of Infectious Diseases, School Medicine,11
Emory University12
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Running title: LuxS-mediated regulation ofS. pneumoniae early biofilms16
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*Corresponding author: Contact information, 1518 Clifton Rd NE Room 6007, Atlanta GA, 30322.20
Phone 404-712-8675, Fax: 404-712-8969. [email protected],21
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Abstract1
Streptococcus pneumoniae is the leading cause of mortality in children worldwide and form2
highly organized biofilms in the nasopharynx, lungs and middle ear mucosa. The luxS-controlled3
quorum sensing (QS) system has recently been implicated in virulence and persistence in the4
nasopharynx, but its role in biofilms had not been studied. Here, we show that this QS system plays a5
major role in controlling S. pneumoniae biofilm formation. Our results demonstrate that the luxS gene is6
encoded by invasive isolates and normal flora strains in a region that contains genes involved in division7
and cell wall biosynthesis. The luxS gene was maximally transcribed, as a monocistronic message, in the8
early-mid log phase of growth and this coincides with the appearance of early biofilms. Demonstrating9
the role of the LuxS system in regulating S. pneumoniae biofilms, at 24 h post-inoculation two different10
D39luxS mutants produced 80% less biofilm biomass than the wt strain D39. The complementing11
strains encoding either the luxS in a plasmid or integrated as a single copy into the genome restored12
biofilm levels to that of the wt. Moreover, a soluble factor secreted by wt strain D39 or purified AI-213
restored the biofilm phenotype of D39luxS. Our results also demonstrate that LuxS regulates, during14
the early-mid log phase of growth, levels of lytA transcript, an autolysin previously implicated in15
biofilms and also the transcript ofply, encoding the pneumococcal pneumolysin. In conclusion, the16
luxS-controlled QS system is a key regulator of early biofilm formation by S. pneumoniae strain D39.17
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Introduction1
Streptococcus pneumoniae (the pneumococcus) is a Gram positive pathogen that causes severe2
illnesses such as otitis media, pneumonia and meningitis, mainly in young children (
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antibiotic resistance amongst S. pneumoniae strains. For example, in vitro studies show that S.1
pneumoniaebiofilms, in comparison to their planktonic cultures, have an increased resistance profile to2
all the classes of oral antibiotics widely used to treat pneumococcal infections, namely betalactams,3
macrolides and fluoroquinolones (11, 13).4
Several molecular factors, including virulence factors, have been implicated in S. pneumoniae5
biofilm formation. Studies by Moscoso et al (2006) found that the amidases LytA, LytC and LytB, and6
adhesins such as CbpA, PcpA and PspA play some role in S. pneumoniae biofilms (32). Another study7
by Munoz-Elias et al (2008) identified 23 genes (genes encoding for adhesins, choline binding proteins8
and cell wall components) implicated in biofilm formation and colonization in a mouse model (34). Two9
other important virulence factors implicated in production of biofilms are PsrP (44) and the10
neuraminidase NanA (40). While it is clear that all these factors may play a role in the development of11
the biofilm structure, the specific mechanism by which S. pneumoniae biofilms are built remains to be12
elucidated.13
Biofilm formation requires a concerted mechanism regulated, in part, by numerous14
environmental signals (24). In S. intermedius, S. oralis, S. gordonii and S. mutans strains, regulation of15
biofilm formation has been linked to LuxS (1, 7, 31, 42), an enzyme that synthesizes the autoinducer 216
(AI-2) required for quorum sensing (QS). The phenomenon of QS is a cell-to-cell communication17
mechanism that utilizes molecules called autoinducers, to regulate gene expression in response to18
environmental changes and cell density (23).19
The first described QS mechanism implicated a 17-aa peptide [competence stimulating peptide20
(CSP)] secreted by S. pneumoniae that regulates their competence state (50). Besides this QS system, S.21
pneumoniae reference strains (e.g. D39 and TIGR4) encode a luxS gene and produce AI-2 (21, 48).22
Some evidence suggests that this LuxS-controlled QS system might be part of the regulatory network23
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controlling competence and LytA-dependent autolysis (43). In terms of pathogenicity, LuxS was1
implicated in virulence and persistence in the nasopharynx, by utilizing mouse models of pneumococcal2
infection (21, 48). The LuxS-generated signal has been implicated in differential expression of proteins3
in strain D39 (48) and regulation of genes involved in some metabolic processes, including the4
pneumolysin gene (ply) (21). In this work we demonstrate, for the first time, that LuxS plays a major5
role in controlling biofilm formation in S. pneumoniae strain D39. Two different D39-derivative luxS6
mutants were unable to produce early biofilms, while this phenotype was reversed by genetic7
complementation or physical complementation. Together, these results shed light on a new and8
important regulatory network of one of the most important human bacterial pathogens S. pneumoniae.9
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Material and Methods11
Strains and bacterial culture media. S. pneumoniae reference strains, and derivatives, utilized in this12
study are listed in Table 1. All other S. pneumoniae, invasive strains isolated from blood or13
cerebrospinal fluid (N=53) and normal flora strains (N=50), belong to our laboratory collection. These14
strains were isolated from different geographic region, including but not restricted to USA, Spain,15
Taiwan, Peru or Brazil. Identification and serotyping was performed by standard procedures (8). S.16
pneumoniae strains were cultured on blood agar plates (BAP) or Todd Hewitt broth containing 0.5%17
(w/v) yeast extract (THY]. When indicated, 2% maltose (w/v), ampicillin (100 g/ml), tetracycline (118
g/ml), erythromycin (0.5 g/ml) or spectinomycin (110 g/ml) was added to the culture medium.19
DNA extraction. Genomic DNA from S. pneumoniae Table 1 strains was purified from overnight20
cultures on blood agar plates (BAP) using the QIAamp DNA minikit (QIAGEN) following the21
manufacturers instructions. DNA-containing supernatant from invasive isolates and normal flora strains22
was extracted by the chelex method (10).23
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PCR reactions. Reactions were performed with genomic DNA (100 ng) or DNA-containing1
supernatant (3 l) as template, 1 M of the indicated pair of primers (Fig. 1A and Table 2), 1X Taq2
master mix (New England Biolabs) and DNA grade water. PCR reactions were run in a MyCycler3
Thermal Cycler System (Bio-Rad). Products were run in 2% agarose gels, stained with ethidium4
bromide and photographed using a ChemiDoc XRS gel documentation System (Bio-Rad).5
RNA extraction. Total RNA was extracted as previously described (54, 55). Briefly, a cell suspension6
was prepared using 200 l of acetate solution (20 mM sodium acetate [pH=5], 1 mM EDTA, 0.5% of7
sodium duodecyl sulfate [SDS, Bio-Rad]) and added with 200 l of saturated phenol (Fisher scientific).8
This was incubated at 60C in a water bath with vigorous shaking for 5 min and centrifuged 17,000 x g9
at 4C for 5 min. Cold ethanol was added to that supernatant, mixed well by inverting the tube and10
centrifuged 17,000 x g at 4C for 5 min to obtain the RNA pellet. The pellet was resuspended in 100 l11
of DNase-free, RNase-free water and additionally treated with 2 U of DNase I (Promega) at 37C for 3012
min. RNA concentration was quantified and stored in 20 l aliquots at -80C.13
qRT-PCR analysis. Quantitative RT-PCR (qRT-PCR) was performed using the iScript One-Step RT-14
PCR kit with SYBR Green (Bio-Rad) and the CFX96 Real-Time PCR Detection System (Bio-Rad).15
qRT-PCR reactions were performed in triplicate with 20 ng of total RNA, 500 nM concentration of each16
primer (Table 2) and the following conditions; 1 cycle at 50C for 20 min, 1 cycle at 95C for 10 min17
and 40 cycles of 95C for 15s and 55C for 1 min. Melting curves were generated by a cycle of 95C for18
1 min, 55C for 1 min and 80 cycles of 55C with 0.5C increments. The relative quantitation of mRNA19
expression was normalized to the constitutive expression of the housekeeping 16SrRNA gene and20
calculated by the comparative CT(2-CT
) method (27).21
Preparation of D39 derivative luxS mutant SPJV05 and complementing strains.22
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To inactivate the luxS gene in strain D39, a DNA cassette containing the ermB gene (which1
confers resistance to erythromycin) flanked by 5 and 3 regions ofluxS was prepared. Briefly, the ermB2
was PCR amplified using primers Ery-L and Ery-R (Table 2) and cloned into pCR2.1 TOPO. Then, 53
(210 bp) or 3 (160 bp) sequences ofluxS were PCR amplified using primers Lux5-L and Lux5-R or4
Lux3-L and Lux3-R, respectively. Those PCR fragments were purified using Qiaquick gel extraction5
(Qiagen) and digested with KpnI and BamHI (5 luxS fragment) orXhoI and XbaI (3 luxS fragment).6
Digested fragments were again purified and ligated, using T4 DNA ligase (Promega), upstream or7
downstream respectively of pCR2.1 TOPO encoding the ermB to create plasmid pLuxS-ery-LuxS. The8
whole cassette luxS-ery-luxS (1.1 kb) was PCR amplified and then purified.9
The luxS-ery-luxS cassette was transformed into competent cells of wt strain D39 by standard10
procedures (18). This transformation reaction was incubated for 2 h at 37C, plated onto BAP containing11
erythromycin and incubated for 48 h at 37C in a 5% CO2 atmosphere. Colonies were screened by PCR12
using primers Lux5-L and Lux3-R and a transformant, named SPJV05, was obtained. The luxS mutation13
was also verified by sequencing. Another D39luxS strain EJ3, previously prepared and characterized14
(21), was kindly provided by Dr. Elizabeth Joyce from the Department of Microbiology and15
Immunology, Stanford University School of Medicine.16
To prepare complementing strains, the luxS wt gene was amplified using primers LuxReg-L and17
LuxReg-R or LuxP2-L and LuxP2-R (Table 2) and cloned into the Gram positive plasmid pReg696 (14)18
or the S. pneumoniae integrative vector pPP2 (15) to create pJVR6 and pJVPP9 (Table 1). Plasmid19
pJVR6 or pJVPP9 was extracted from ECJV10 or ECJV11, and used to transform competent cells of20
SPJV05 to create SPJV06 and SPJV07, respectively (Table 1). EJ3, resistant to spectinomycin, was only21
complemented with pJVPP9 or pPP2 (the empty vector).22
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Preparation of the inoculum for biofilm assays. An overnight BAP culture was used to prepare a cell1
suspension in THY broth to an OD600 of 0.05 and incubated at 37C in a 5% CO2 atmosphere until the2
culture reached an OD600 of0.2. An aliquot (7x105
CFU/ml) was inoculated in triplicate into either an3
8-well glass slide (Lab-Tek) or polystyrene-treated 96-well or polystyrene-treated 24-well microtiter4
plate (Corning) containing THY with no antibiotics and incubated at 37C with 5% CO2 for the5
indicated time.6
Quantification of biofilm biomass by crystal violet. Biofilms were washed three times with PBS and7
then allowed to dry for 15 min. Crystal violet (0.4%) was then added and incubated for 15 min. After8
washing, crystal violet-stained biofilms were further dried at room temperature for 15 min. To quantify9
biofilm biomass, crystal violet was removed by adding 33% acetic acid solution. The absorbance 63010
(A630) of solubilized dye was obtained in a Epoch microplate spectrophotometer (Biotek).11
Quantification of biofilm biomass by a fluorescence-based assay. Biofilms were fixed with 2%12
paraformaldehyde (Sigma) for 15 min and made permeable by adding 0.5% Triton X-100 (Roche) and13
incubating 5 min at room temperature. After washing three times with PBS, biofilms were blocked by14
adding 2% bovine serum albumin (BSA) and stained for 1 h at room temperature with a polyclonal anti-15
Streptococcus pneumoniae antibody (40 g/ml) coupled to fluorescein isothiocyanate [(FITC)16
ViroStat, Portland, ME)]. To quantify biofilm biomass, FITC-fluorescence readings (arbitrary units)17
were obtained using a VICTOR X3 Multilabel Plate Reader (Perkin-Elmer). Fluorescence arbitrary18
units of 24 h biofilms of the wt strain D39 were set as 100% of biofilm biomass and used to calculate the19
percentage of biofilm biomass of all other tested S. pneumoniae strains or different time-points.20
Physical complementation of the biofilm phenotype and AI-2 studies. To assess for physical21
complementation, a mixture of two strains were incubated in the same biofilm assay but only the22
biomass of one strain was quantified. Briefly, wt D39 or SPJV05 strain was transformed with plasmid23
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pMV158GFP, that encodes the gfp gene under the control of the inducible maltose promoter (36), to1
create SPJV01 and SPJV08 respectively (Table 1). Biofilm biomass of these fluorescent versions was2
similar to that of their respective parent strains (not shown). Then, a mixture of wt D39 and SPJV08, or3
wt D39 and SPJV01, was inoculated in the same biofilm bioassay. As controls, SPJV01 or SPJV08 was4
also inoculated in individual wells. After 6 h of incubation at 37C, biofilms were washed and5
fluorescence readings, or images, immediately obtained. For these experiments, arbitrary fluorescence6
units obtained from SPJV01 biofilms were set as 100% of biofilm biomass and biofilm biomass of all7
other experimental conditions was calculated.8
To physically separate those bacteria within the same biofilm assay, two chambers were created9
within the same well by installing a Transwell
filter device (Corning, NY USA). The Transwell
10
membrane (0.4 M) creates a physical barrier impermeable to bacteria, but allows passage of small11
molecules between the two chambers (top and bottom). The wt D39 strain was inoculated into the top12
chamber (Transwell filter), while SPJV08 was inoculated on the bottom chamber. To further confirm13
whether LuxS mediates this secreted QS signal, the luxS mutant strain SPJV05 was inoculated in the top14
chamber and SPJV08 in the bottom chamber. These biofilm-Transwell bioassays were incubated for 6 h15
at 37C in a 5% CO2 atmosphere and biofilms produced by SPJV08 in the bottom chamber were16
quantified and photographed as mentioned above.17
For AI-2 studies, D39 or SPJV05 was inoculated in triplicate in 96-well plates with or without18
different concentrations of chemically synthesized AI-2 (from 0.1 to 100 nM) (dihydroxypentanedione19
[DPD], Omm scientific) as utilized elsewhere (4, 9, 42) and incubated for 24 h. Biofilms were stained20
with fluorescence and biomass calculated as earlier mentioned. A concentration of 10 nM induced21
statistical different biofilm biomass when incubated along with D39 or SPJV05.22
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Statistical analyses. All data were analyzed using the non-parametric two tailed Students t test and1
using the Minitab 15 software.2
3
Results4
The luxS gene is encoded in the same chromosomal region in S. pneumoniae isolates. Bioinformatic5
analysis of genome sequenced S. pneumoniae strains indicated that the luxS gene is located in a region6
that contains genes involved in division and cell wall biosynthesis. Specifically, luxS is situated7
upstream (6.4 kb) of the gene cps4A (encoding a capsule polysaccharide biosynthesis protein) and 48
kb downstream thepbp2X gene, whose encoded protein is involved in cell wall biosynthesis and a target9
for -lactam antibiotics (30, 57), followed by mraY [encoding a phospho-N-acetylmuramoyl-10
pentapeptide-transferase (3 kb)] (29) and 294 bp downstream the clpL gene encoding a putative ATP-11
dependent Clp protease (Fig. 1A).12
To investigate the presence of the luxS gene among S. pneumoniae strains, PCR analyses were13
performed with DNA extracted from invasive isolates and strains isolated from the nasopharynx of14
healthy children. Those PCR analyses revealed that all surveyed isolates (N=103) encode the luxS gene15
(Fig. 1B). To further study the location of the luxS gene, primers were designed so that a series of16
overlapping PCR reactions mapped its chromosomal location. This overlapping PCR approach17
demonstrated that the luxS gene is always located downstream of an ORF annotated as Sp0341 (TIGR418
annotation), which encodes a protein of unknown function. The size of the obtained PCR products was19
always the same and so included, as predicted by bioinformatic analysis, an intergenic region of94 bp20
between Sp0341 and luxS gene (Figs. 1A and B).21
Overlapping PCR identified downstream of luxS, in all surveyed isolates, a gene annotated as22
clpL encoding a putative ATP-dependent protease (Fig. 1B). For some invasive isolates (N=23) or23
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normal flora strains (N=19) strains, the size of the PCR product correlated with that observed using, as1
template, DNA from the reference strain ATCC33400, which belongs to serotype 1 (1200 bp). All2
other strains allowed the amplification of a product (960 bp) similar to that of TIGR4 (serotype 4). The3
size of those PCR products could not be correlated with serotypes of the surveyed strain (not shown).4
Overlapping PCR detected genes mraY and pbp2X downstream ofluxS in 17 normal flora strains and5
22 invasive isolates (Fig. 1B). PCR analysis targeting mraY orpbp2X further clarified that these genes6
were not encoded in this position by the rest of the strains (not shown). Taken together, these results7
indicate that the luxS gene is encoded near the capsule locus (cps) in all surveyed S. pneumoniae8
isolates.9
Transcription of the luxS gene. In S. bovis and S. pyogenes, a homologous luxS gene is transcribed10
during early-mid log phase of growth as a monocistronic message (5, 46). Unlike S. bovis, whose11
direction of transcription is opposite to both its upstream and downstream genes (5), in S. pneumoniae12
the direction of transcription of luxS is the same as that of its upstream gene (Sp0341) and opposite to13
the downstream clpL gene (Fig. 1A).14
To evaluate whether luxS mRNA is cotranscribed with Sp0341 by S. pneumoniae strain D3915
qRT-PCR analyses were performed. These analyses showed that 4 h post inoculation, levels of luxS16
transcripts increased 20-fold with respect to the RNA from BAP (Fig. 2). While expression of Sp034117
mRNA also increased 16-fold, almost no change (0.5) was obtained for that of Sp0341-luxS. As18
expected, transcription of a housekeeping gene utilized as an internal control (gyrB) did not19
considerably change. Similar results were obtained by RT-PCR with S. pneumoniae strains TIGR4 and20
ATCC33400 (not shown). Overall, these results indicate that the luxS gene of S. pneumoniae is21
transcribed as a monicistronic message during the mid-log phase of growth.22
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Development of a new fluroscence-based assay to quantify S. pneumoniae biofilm biomass. Before1
addressing the role of LuxS in S. pneumoniae biofilms, we quantified the biofilm biomass produced by2
reference strains D39, TIGR4 and R6 a non encapsulated variant of strain D39. As can be seen in Fig.3
3A, the crystal violet assay demonstrated biofilm formation by all strains. TIGR4 biofilm biomass was4
low (A600
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formed by D39 is highly organized, forming compact layers of bacteria that create aggregates where the1
fluorescent signal is more intense (not shown). In contrast, fluorescence images of biofilms produced by2
SPJV05 or EJ3 clearly show few bacteria attached to the bottom of the wells (Fig. 4B). Indeed, both3
mutants appear to form small aggregates in the bottom of the well that do not progress to form a mature4
biofilm (Fig. 4B).5
To confirm the role ofluxS in S. pneumoniae biofilms, the complementing strains SPJV04 and6
SPJV06 were assessed for biofilm production. As shown in Fig. 4A, the luxS mutant complemented with7
either a copy of luxS integrated into bgaA or in a plasmid, SPJV04 and SPJV06 respectively, restored8
levels of biofilm biomass to that of the wt. Production of biofilm biomass by SPJV02, encoding the9
pPP2 empty vector, was similar to EJ3 (Fig. 4A), demonstrating that disruption ofbgaA did not alter the10
biofilm phenotype. Epifluorescence images of SPJV04 or SPJV06 biofilms show similar structures to11
the wt strain D39 (Fig. 4B).12
This biofilm phenotype is likely not due to growth rates or an autoaggregation defect.13
Comparative growth studies showed that D39 and its derivatives grew similarly in THY (not shown)14
Time-course studies also demonstrated that luxS mutants and complementing strains autoaggregate15
similar to strain D39 (data not shown).16
LuxS regulates early events in biofilm formation. To begin exploring the mechanism by which LuxS17
controls biofilm formation, a time-course study was conducted that evaluated early production of18
biofilms. Fig. 5A shows that 3 h post-inoculation 10% of biofilm biomass had already been produced19
by the wt strain. Biofilm biomass reached 25%, 45% and 80% after 4, 6 and 8 h of incubation20
respectively (Fig. 5A), while biofilm biomass 10 or 12 h post-inoculation was similar to those levels21
produced during a 24 h period (not shown). In contrast, biofilms produced by SPJV05 were undetectable22
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at early time points (i.e. 2 or 4 h post-inoculation) while 8 h post inoculation produced only 15% of1
biofilm biomass (Fig. 5A).2
As seen in Fig. 5B, small pneumococcus aggregates could be detected as soon as 2 h post-3
inoculation with the wt strain. Those D39 aggregates became more evident after 4 and 6 h of incubation4
and almost covered the entire surface after 8 h (Figs. 5C, 5D and 5E). Bacterial aggregates produced by5
SPJV05, however, were smaller and only covered 20% of the surface 8 h post inoculation (Fig. 5F). As6
expected, the complementing strain produced similar biofilm biomass than the wt 8 h post-inoculation7
(Fig. 5G).8
Evidence that the LuxS-mediated AI-2 regulates early biofilm formation.9
To confirm that a QS signal was responsible for the biofilm defect of the luxS mutants, the wt10
strain and SPJV08 (SPJV05 encoding the gfp gene under the control of the maltose promoter) were11
inoculated in the same well with or without physical contact. If a QS signal released by the wt could12
induce an increase on SPJV08 biofilm biomass, SPJV08 fluorescence would increase.13
When the wt D39 and SPJV08 were inoculated together with physical contact, biofilm biomass14
of SPJV08 was similar to levels of SPJV08 alone and statistically different to levels produced by wt D3915
encoding pMV158GFP (SPJV01) (Fig. 6A). Even when co-cultured with D39, Fig. 6B shows almost no16
bacterial aggregates of fluorescent SPJV08 6 h post-inoculation. We hypothesize that the wt strain was17
able to attach to the surface and produced early biofilms but unable to incorporate, once initial18
aggregates had already formed, cells of the luxS null mutant. As a control, the wt strain and SPJV0119
were inoculated together and incubated for 6 h. Biofilm biomass of SPJV01 was 40% demonstrating20
that the wt strain outcompeted SPJV01 to produce biofilms (Fig. 6A).21
To avoid the formation of biofilms by the wt strain D39 when co-inoculated with SPJV08 but22
allow the secretion of a secreted factor, a transwell device was used. This system contains a membrane23
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(0.45 M) that allowed the inoculation of the wt strain in the top and SPJV08 in the bottom while they1
were physically separated (Fig. 6A, inset). Given its pore size, the membrane allows the passage of2
small molecules. As shown in Fig. 6A, when the wt strain and SPJV08 where co-inoculated, SPJV083
was able to produce 25% of biofilm biomass of wt levels 6 h post-inoculation (Figs. 6A and 6C). Those4
biofilm levels were statistically different to levels produced by SPJV08 alone (Fig. 6A). In contrast,5
biofilm biomass of SPJV08 that had been inoculated in the bottom of the well, and SPJV05 in the6
transwell system, did not produce levels significantly different than SPJV08 alone (Fig. 6A).7
To confirm the role of secreted AI-2 in the biofilm phenotype, strain D39 or SPJV05 where8
inoculated along with chemically synthesized AI-2. As shown in Fig. 7, AI-2 allowed the production of9
more robust biofilm biomass than that produced by strains grown with no purified AI-2.10
Temporal expression of the luxS gene and evidences that LuxS regulates lytA mRNA and ply11
mRNA levels. Since we had demonstrated that LuxS regulates early biofilm formation, a time-course12
study was conducted to evaluate levels ofluxS mRNA expression during early-mid log phase of growth.13
The luxS transcript was found maximally expressed (28-fold increase) at 4 h post-inoculation (Fig.14
8A). At this time point, fold change ofluxS mRNA in some invasive isolates and normal flora ranged15
from 6 through 300-fold and expression could not be correlated to the subset of strains (not shown). A16
clear decline in levels ofluxS mRNA, in strain D39, was obtained 6 h (5-fold difference) and 8 h (3.8-17
fold difference) post-inoculation. As expected, gyrB mRNA levels did not significantly change after 2,18
4, 6 or 8 h of incubation (Fig. 8A). These results indicate that transcription of the luxS gene, and19
therefore its activity, is maximally expressed during the early-mid log phase of growth.20
Previous studies have demonstrated that LytA, the capsular polysaccharide, the neuraminidase21
NanA and choline binding proteins such as PspA play a role in S. pneumoniae biofilm formation (32,22
40). Increased levels of the pneumolysin (Ply) has also been detected in S. pneumoniae early biofilms23
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(2). Therefore, transcription of genes and potential LuxS-mediated regulation were evaluated. Our qRT-1
PCR analyses demonstrated that wt mRNA levels of lytA, ply, cps4A, nanA and pspA similarly2
increased during the early-mid log phase of growth and then decreased 6 h post inoculation (Fig. 8A and3
not shown). In contrast, lytA mRNA levels in the luxS mutant decreased 4 h post-inoculation (Fig. 8B)4
while levels of the ply transcript dramatically decreased 2, 4 and 6 h post inoculation (Fig. 8C).5
Transcription levels of nanA, csp4A and pspA genes remained similar to that of wt (not shown).6
Complementing stains showed at all time points similar level of transcripts to that of wt (not shown).7
8
Discussion9
S. pneumoniae strains usually colonize the nasopharynx of healthy children during the first10
months of life and either remain asymptomatic or go on to cause disease such as pneumonia, meningitis11
or otitis media (17, 37). It has been postulated that the pneumococcus resides in the human nasopharynx12
forming biofilms (33). Despite the increasing importance ofS. pneumoniae biofilms during the last few13
years, the regulatory network behind these structures has not been completely elucidated. The current14
study now demonstrates, for the first time, that the luxS-controlled quorum sensing (QS) system15
regulates S. pneumoniae early biofilm formation. This AI-2 mediated regulatory network appears to be16
specific for a subset of biofilm effectors since, in this research and elsewhere (21), LuxS was found to17
regulate a particular set of genes in planktonic cultures.18
Recent experimental evidence indicates that the LuxS QS system is implicated in persistence,19
virulence and dissemination ofS. pneumoniae (3, 21, 48). A previous study by Stroeher et al. (2003)20
demonstrated that a D39 derivative luxS null mutant was less able to spread to the lungs or the blood21
than the wt strain D39, suggesting that the QS signal might be important for dissemination within the22
host (48). This luxS mutant was also less virulent for mice than wt strain D39 (48). Another study by23
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Joyce et al. (2004) showed that this QS system is implicated in persistence in the nasopharynx of mice1
(21). Our results extend these observations by demonstrating that LuxS is absolutely required to2
establish early biofilm structures. While luxS mutants were unable to form early biofilms, the3
complementing strains fully restored the phenotype. In an attempt to verify that a secreted QS signal was4
controlling early biofilms, we utilized chemically synthesized AI-2, or AI-2 containing supernatants5
from wt D39, that demonstrated statistically significant more biofilm biomass when AI-2 was incubated6
along with the luxS mutant or wt (Fig. 7).7
Recent publications have also shown that the competence QS system (Com), which is regulated8
by the secreted competence-stimulating peptide (CSP), controls biofilm production by S. pneumoniae9
strains (38, 52). An investigation by Oggioni et al (2006) showed that supplementing strain D39 with10
exogenous CSP produces more biofilm biomass (38), and in a more recent publication, Trapetti et al11
(2011) demonstrated that a TIGR4 derivative comC mutant was unable to form biofilms while the12
phenotype could be restored by adding exogenous CSP (52). Specific CSP-regulated biofilm effectors13
and potential LuxS-Com synergism for biofilm development, if any, remain to be investigated.14
The current study shows that the luxS gene is encoded near the capsular locus in S. pneumoniae15
strains and transcribed as a monocistronic unit. Expression of the capsular polysaccharide has been16
implicated in virulence (35) and biofilm formation (32), however, the LuxS system does not seem to17
regulate capsule genes since our qRT-PCR studies showed that the luxS mutant contained similar levels18
ofcsp4A mRNA than the wt. Other proteins previously implicated in biofilms such as the neuraminidase19
NanA (40) or choline binding protein PspA (32) were also found not to be regulated by this system.20
Expression of lytA, however, encoding the autolysin involved in cell wall degradation (20) and21
production of biofilms (32), was found to be regulated by LuxS during the mid log phase of growth (Fig.22
8B). Whereas our experiments showed that lytA transcripts were higher, in the wt, during the early-mid23
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phase (2-4 h) of growth, the mutant had reduced lytA mRNA levels 4 h post-inoculation. In line with1
these results, evidence indicating that LuxS may play a role in LytA-dependent autolysis in the2
stationary phase of growth (5-7 h post-inoculation) has been published (43). Therefore, LuxS appears to3
regulate mRNA levels oflytA in exponentially growing cultures that results in a defect of the autolysis4
phenotype during the stationary phase. A complete characterization of the biology of this phenomenon is5
under investigation in our labs.6
Evidence now directly links production of pneumococcal biofilms with LuxS regulation. For7
instance, a previous report found that a luxS mutant produced a different protein expression profile8
(cytosolic and membrane proteins) than the wt strain D39 (48). Joyce et al (2004) using microarrays9
detected 46 genes down-, or up-regulated by the LuxS system, only when they utilized RNA extracted10
from the wt strain D39 and the luxS mutant growing at early-mid log phase of growth (21). Since levels11
of the luxS transcript are also higher during early-mid log phase of growth (Fig. 8A), and luxS mutants is12
unable to produce biofilms (Fig. 4A), regulation of genes encoding proteins implicated in early biofilm13
formation, such as lytA, should be a main target for the LuxS-generated signal.14
Regulation of proteins present in early biofilms has been previously investigated (2). A particular15
association between early biofilm-produced proteins and the LuxS system was the discovery that levels16
of pneumolysin (Ply), a protein implicated in colonization and virulence in animal models (19, 39),17
increase during early stages of biofilm formation (2). Its encoded gene (ply) has also been detected by18
microarray analyses to be regulated by the LuxS system (21). Our studies now extend this observation19
by demonstrating, utilizing qRT-PCR, that LuxS dramatically impact levels ofply transcripts during the20
early-mid phase of growth (Fig. 8C). The contribution of the pneumolysin to S. pneumoniae-produced21
biofilms is unclear and, given that D39ply had reduced ability to colonize the mouse nasopharynx (39),22
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19
requires further elucidation. It may have a role in initial attachment since Ply is not secreted by1
pneumococcus, but rather located in the cell wall (41).2
Our study also introduced a new fluorescence-based biofilm assay, which is specific for S.3
pneumoniae strains. While other non-specific fluorescence methods have been used to visualize S.4
pneumoniae biofilms in vitro (2, 12, 32), our newly developed assay utilizes an anti-S. pneumoniae5
antibody coupled to fluorescein (FITC) that permits quantification of the biofilm biomass and direct6
visualization of those structures, in a single assay. This assay may also be useful for studies where a7
heterogeneous population of bacteria (i.e. normal flora strains) are present with or within S. pneumoniae8
biofilms, for example, in samples collected from children with pneumococcal diseases, animal studies9
and in vitro studies dissecting the contribution of other bacteria to pneumococcal biofilms.10
An important feature of our assay is that the permeabilization of the biofilm structure with Triton11
X-100 may allow those anti-S. pneumoniae antibodies to reach most pneumococci within the biofilm12
matrix. Fluorescence arbitrary units were consistently higher only when biofilms were made permeable13
before staining with the fluorescent antibody. Since S. pneumoniae biofilm structures have been14
calculated to be 25 M thick (32), it is possible that the crystal violet dye does not reach all15
pneumococcus cells within the matrix. The new fluorescent assay is more sensitive being able to both16
quantify and visualize early biofilm structures (bacterial aggregates) within 2 h post-inoculation (Fig. 3).17
In contrast, our experiments and those reported by Munoz-Elias et al (2008) (34) using crystal violet,18
detected S. pneumoniae biofilms 6-8 h post-inoculation.19
Utilizing this new assay to gain insights into the biofilm biology, pneumococcal biofilm20
formation evolved in three stages: (1) initial attachment between 2-4 h, (2) formation of bacterial21
aggregates between 4-6 h occupying 50% of the surface and (3) biofilm development. Similar stages22
have been previously described using a continuous-flow biofilm reactor, although stages in that study23
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20
were observed between 1 to 6 days (2). Once bacterial aggregates were produced (stage 2), biofilms1
continues exponentially growing even in the absence of planktonic cells (Kunkel et al, unpublished). In2
fact, with the only exception of csp4A whose mRNA levels remained similar, biofilm cells showed3
higher levels of nanA, ply, pspA and lytA transcripts 6 h post-inoculation than their counterpart4
planktonic cultures (not shown), clearly demonstrating an active metabolism within biofilm cells.5
Attempts to find out levels of these transcripts in a luxS background failed because the absence of6
biofilm cells at this time-point.7
In summary, we have demonstrated that the S. pneumoniae LuxS-controlled QS system regulates8
early biofilm formation. Biofilm structures might be important forS. pneumoniae strains to persist and9
possibly cause important diseases such as otitis media or pneumonia. Findings in the current study may10
have implications for developing new targets to reduce pneumococcal carriage and, therefore11
pneumococcal disease.12
13
Acknowledgment14
This research was supported in part by PHS Grant UL1 RR025008 from the Clinical and Translational15
Science Award program, NIH, National Center for Research Resources (JEV). We thank Dr. Elizabeth16
Joyce from Stanford University for providing us strain EJ3, Dr. Reinhold Brckner from the Department17
of Microbiology, University of Kaiserslautern, for providing pPP2, Dr. Finbarr Hayes from the18
University of Manchester UK for providing pReg6969 and Dr. Lesley McGee from the Centers for19
Disease Control and Prevention (CDC) and Dr. Carlos Grijalva from Vanderbilt University for20
providing some S. pneumoniae isolates. Also thanks to Dr. Manuel Espinoza from the Centro de21
Investigaciones Biologicas Madrid, Spain for his kind gift of plasmid pMV158GFP and Joshua Shak22
for critical reading and suggestions to this manuscript.23
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21
References1
1. Ahmed, N. A., F. C. Petersen, and A. A. Scheie. 2009. AI-2/LuxS is involved in increased2
biofilm formation by Streptococcus intermedius in the presence of antibiotics. Antimicrob Agents3
Chemother53:4258-63.4
2. Allegrucci, M., F. Z. Hu, K. Shen, J. Hayes, G. D. Ehrlich, J. C. Post, and K. Sauer. 2006.5
Phenotypic characterization ofStreptococcus pneumoniae biofilm development. J Bacteriol 188:2325-6
35.7
3. Armbruster, C. E., W. Hong, B. Pang, K. E. Dew, R. A. Juneau, M. S. Byrd, C. F. Love, N.8
D. Kock, and W. E. Swords. 2009. LuxS promotes biofilm maturation and persistence of nontypeable9
Haemophilus influenzae in vivo via modulation of lipooligosaccharides on the bacterial surface. Infect10
Immun 77:4081-91.11
4. Armbruster, C. E., W. Hong, B. Pang, K. E. Weimer, R. A. Juneau, J. Turner, and W. E.12
Swords. 2010. Indirect pathogenicity of Haemophilus influenzae and Moraxella catarrhalis in13
polymicrobial otitis media occurs via interspecies quorum signaling. MBio 1(3). pii: e00102-10.14
5. Asanuma, N., T. Yoshii, and T. Hino. 2004. Molecular characterization and transcription of the15
luxS gene that encodes LuxS autoinducer 2 synthase in Streptococcus bovis. Curr Microbiol 49:366-71.16
6. Avery, O. T., C. M. Macleod, and M. McCarty. 1944. Studies on the Chemical Nature of the17
Substance Inducing Transformation of Pneumococcal Types : Induction of Transformation by a18
Desoxyribonucleic Acid Fraction Isolated from Pneumococcus Type Iii. J Exp Med 79:137-58.19
7. Blehert, D. S., R. J. Palmer, Jr., J. B. Xavier, J. S. Almeida, and P. E. Kolenbrander. 2003.20
Autoinducer 2 production by Streptococcus gordonii DL1 and the biofilm phenotype of a luxS mutant21
are influenced by nutritional conditions. J Bacteriol 185:4851-60.22
-
7/28/2019 2011 the LuxS-Dependent Quorum Sensing System Regulates Early Biofilm Formation by Streptococcus Pneumoni
22/42
22
8. da Gloria Carvalho, M., F. C. Pimenta, D. Jackson, A. Roundtree, Y. Ahmad, E. V. Millar,1
K. L. O'Brien, C. G. Whitney, A. L. Cohen, and B. W. Beall. 2010. Revisiting pneumococcal2
carriage by use of broth enrichment and PCR techniques for enhanced detection of carriage and3
serotypes. J Clin Microbiol 48:1611-8.4
9. De Keersmaecker, S. C., C. Varszegi, N. van Boxel, L. W. Habel, K. Metzger, R. Daniels, K.5
Marchal, D. De Vos, and J. Vanderleyden. 2005. Chemical synthesis of (S)-4,5-dihydroxy-2,3-6
pentanedione, a bacterial signal molecule precursor, and validation of its activity in Salmonella7
typhimurium. J Biol Chem 280:19563-8.8
10. de Lamballerie, X., C. Zandotti, C. Vignoli, C. Bollet, and P. de Micco. 1992. A one-step9
microbial DNA extraction method using "Chelex 100" suitable for gene amplification. Res Microbiol10
143:785-90.11
11. del Prado, G., V. Ruiz, P. Naves, V. Rodriguez-Cerrato, F. Soriano, and M. del Carmen12
Ponte. 2010. Biofilm formation by Streptococcus pneumoniae strains and effects of human serum13
albumin, ibuprofen, N-acetyl-l-cysteine, amoxicillin, erythromycin, and levofloxacin. Diagn Microbiol14
Infect Dis 67:311-8.15
12. Donlan, R. M., J. A. Piede, C. D. Heyes, L. Sanii, R. Murga, P. Edmonds, I. El-Sayed, and16
M. A. El-Sayed. 2004. Model system for growing and quantifying Streptococcus pneumoniaebiofilms17
in situ and in real time. Appl Environ Microbiol 70:4980-8.18
13. Garcia-Castillo, M., M. I. Morosini, A. Valverde, F. Almaraz, F. Baquero, R. Canton, and19
R. del Campo. 2007. Differences in biofilm development and antibiotic susceptibility among20
Streptococcus pneumoniae isolates from cystic fibrosis samples and blood cultures. J Antimicrob21
Chemother59:301-4.22
-
7/28/2019 2011 the LuxS-Dependent Quorum Sensing System Regulates Early Biofilm Formation by Streptococcus Pneumoni
23/42
23
14. Grady, R., and F. Hayes. 2003. Axe-Txe, a broad-spectrum proteic toxin-antitoxin system1
specified by a multidrug-resistant, clinical isolate ofEnterococcus faecium. Mol Microbiol 47:1419-32.2
15. Halfmann, A., R. Hakenbeck, and R. Bruckner. 2007. A new integrative reporter plasmid for3
Streptococcus pneumoniae. FEMS Microbiol Lett 268:217-24.4
16. Hall-Stoodley, L., F. Z. Hu, A. Gieseke, L. Nistico, D. Nguyen, J. Hayes, M. Forbes, D. P.5
Greenberg, B. Dice, A. Burrows, P. A. Wackym, P. Stoodley, J. C. Post, G. D. Ehrlich, and J. E.6
Kerschner. 2006. Direct detection of bacterial biofilms on the middle-ear mucosa of children with7
chronic otitis media. Jama 296:202-11.8
17. Hava, D. L., J. LeMieux, and A. Camilli. 2003. From nose to lung: the regulation behind9
Streptococcus pneumoniae virulence factors. Mol Microbiol 50:1103-10.10
18. Havarstein, L. S., G. Coomaraswamy, and D. A. Morrison. 1995. An unmodified11
heptadecapeptide pheromone induces competence for genetic transformation in Streptococcus12
pneumoniae. Proc Natl Acad Sci U S A 92:11140-4.13
19. Hirst, R. A., B. Gosai, A. Rutman, C. J. Guerin, P. Nicotera, P. W. Andrew, and C.14
O'Callaghan. 2008. Streptococcus pneumoniae deficient in pneumolysin or autolysin has reduced15
virulence in meningitis. J Infect Dis 197:744-51.16
20. Jedrzejas, M. J. 2001. Pneumococcal virulence factors: structure and function. Microbiol Mol17
Biol Rev 65:187-207.18
21. Joyce, E. A., A. Kawale, S. Censini, C. C. Kim, A. Covacci, and S. Falkow. 2004. LuxS is19
required for persistent pneumococcal carriage and expression of virulence and biosynthesis genes. Infect20
Immun 72:2964-75.21
-
7/28/2019 2011 the LuxS-Dependent Quorum Sensing System Regulates Early Biofilm Formation by Streptococcus Pneumoni
24/42
24
22. Kadioglu, A., J. N. Weiser, J. C. Paton, and P. W. Andrew. 2008. The role ofStreptococcus1
pneumoniae virulence factors in host respiratory colonization and disease. Nat Rev Microbiol 6:288-2
301.3
23. Kaper, J. B., and V. Sperandio. 2005. Bacterial cell-to-cell signaling in the gastrointestinal4
tract. Infect Immun 73:3197-209.5
24. Karatan, E., and P. Watnick. 2009. Signals, regulatory networks, and materials that build and6
break bacterial biofilms. Microbiol Mol Biol Rev 73:310-47.7
25. Klugman, K. P., S. A. Madhi, and W. C. Albrich. 2008. Novel approaches to the identification8
ofStreptococcus pneumoniae as the cause of community-acquired pneumonia. Clin Infect Dis 47 Suppl9
3:S202-6.10
26. Lanie, J. A., W. L. Ng, K. M. Kazmierczak, T. M. Andrzejewski, T. M. Davidsen, K. J.11
Wayne, H. Tettelin, J. I. Glass, and M. E. Winkler. 2007. Genome sequence of Avery's virulent12
serotype 2 strain D39 of Streptococcus pneumoniae and comparison with that of unencapsulated13
laboratory strain R6. J Bacteriol 189:38-51.14
27. Livak, K. J., and T. D. Schmittgen. 2001. Analysis of relative gene expression data using real-15
time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402-8.16
28. Lizcano, A., T. Chin, K. Sauer, E. I. Tuomanen, and C. J. Orihuela. 2010. Early biofilm17
formation on microtiter plates is not correlated with the invasive disease potential of Streptococcus18
pneumoniae. Microb Pathog 48:124-30.19
29. Massidda, O., D. Anderluzzi, L. Friedli, and G. Feger. 1998. Unconventional organization of20
the division and cell wall gene cluster ofStreptococcus pneumoniae. Microbiology 144 ( Pt 11):3069-21
78.22
-
7/28/2019 2011 the LuxS-Dependent Quorum Sensing System Regulates Early Biofilm Formation by Streptococcus Pneumoni
25/42
25
30. Maurer, P., B. Koch, I. Zerfass, J. Krauss, M. van der Linden, J. M. Frere, C. Contreras-1
Martel, and R. Hakenbeck. 2008. Penicillin-binding protein 2x ofStreptococcus pneumoniae: three2
new mutational pathways for remodelling an essential enzyme into a resistance determinant. J Mol Biol3
376:1403-16.4
31. Merritt, J., F. Qi, S. D. Goodman, M. H. Anderson, and W. Shi. 2003. Mutation of luxS5
affects biofilm formation in Streptococcus mutans. Infect Immun 71:1972-9.6
32. Moscoso, M., E. Garcia, and R. Lopez. 2006. Biofilm formation by Streptococcus pneumoniae:7
role of choline, extracellular DNA, and capsular polysaccharide in microbial accretion. J Bacteriol8
188:7785-95.9
33. Moscoso, M., E. Garcia, and R. Lopez. 2009. Pneumococcal biofilms. Int Microbiol 12:77-85.10
34. Munoz-Elias, E. J., J. Marcano, and A. Camilli. 2008. Isolation ofStreptococcus pneumoniae11
biofilm mutants and their characterization during nasopharyngeal colonization. Infect Immun 76:5049-12
61.13
35. Nelson, A. L., A. M. Roche, J. M. Gould, K. Chim, A. J. Ratner, and J. N. Weiser. 2007.14
Capsule enhances pneumococcal colonization by limiting mucus-mediated clearance. Infect Immun15
75:83-90.16
36. Nieto, C., and M. Espinosa. 2003. Construction of the mobilizable plasmid pMV158GFP, a17
derivative of pMV158 that carries the gene encoding the green fluorescent protein. Plasmid 49:281-5.18
37. Nobbs, A. H., R. J. Lamont, and H. F. Jenkinson. 2009. Streptococcus adherence and19
colonization. Microbiol Mol Biol Rev 73:407-50.20
38. Oggioni, M. R., C. Trappetti, A. Kadioglu, M. Cassone, F. Iannelli, S. Ricci, P. W. Andrew,21
and G. Pozzi. 2006. Switch from planktonic to sessile life: a major event in pneumococcal22
pathogenesis. Mol Microbiol 61:1196-210.23
-
7/28/2019 2011 the LuxS-Dependent Quorum Sensing System Regulates Early Biofilm Formation by Streptococcus Pneumoni
26/42
26
39. Ogunniyi, A. D., K. S. LeMessurier, R. M. Graham, J. M. Watt, D. E. Briles, U. H.1
Stroeher, and J. C. Paton. 2007. Contributions of pneumolysin, pneumococcal surface protein A2
(PspA), and PspC to pathogenicity ofStreptococcus pneumoniae D39 in a mouse model. Infect Immun3
75:1843-51.4
40. Parker, D., G. Soong, P. Planet, J. Brower, A. J. Ratner, and A. Prince. 2009. The NanA5
neuraminidase ofStreptococcus pneumoniae is involved in biofilm formation. Infect Immun 77:3722-6
30.7
41. Price, K. E., and A. Camilli. 2009. Pneumolysin localizes to the cell wall of Streptococcus8
pneumoniae. J Bacteriol 191:2163-8.9
42. Rickard, A. H., R. J. Palmer, Jr., D. S. Blehert, S. R. Campagna, M. F. Semmelhack, P. G.10
Egland, B. L. Bassler, and P. E. Kolenbrander. 2006. Autoinducer 2: a concentration-dependent11
signal for mutualistic bacterial biofilm growth. Mol Microbiol 60:1446-56.12
43. Romao, S., G. Memmi, M. R. Oggioni, and M. C. Trombe. 2006. LuxS impacts on LytA-13
dependent autolysis and on competence in Streptococcus pneumoniae. Microbiology 152:333-41.14
44. Sanchez, C. J., P. Shivshankar, K. Stol, S. Trakhtenbroit, P. M. Sullam, K. Sauer, P. W.15
Hermans, and C. J. Orihuela. 2010. The pneumococcal serine-rich repeat protein is an intra-species16
bacterial adhesin that promotes bacterial aggregation in vivo and in biofilms. PLoS Pathog 6.17
45. Sekhar, S., R. Kumar, and A. Chakraborti. 2009. Role of biofilm formation in the persistent18
colonization ofHaemophilus influenzae in children from northern India. J Med Microbiol 58:1428-32.19
46. Siller, M., R. P. Janapatla, Z. A. Pirzada, C. Hassler, D. Zinkl, and E. Charpentier. 2008.20
Functional analysis of the group A streptococcal luxS/AI-2 system in metabolism, adaptation to stress21
and interaction with host cells. BMC Microbiol 8:188.22
-
7/28/2019 2011 the LuxS-Dependent Quorum Sensing System Regulates Early Biofilm Formation by Streptococcus Pneumoni
27/42
27
47. Sloan, G. P., C. F. Love, N. Sukumar, M. Mishra, and R. Deora. 2007. TheBordetella Bps1
polysaccharide is critical for biofilm development in the mouse respiratory tract. J Bacteriol 189:8270-6.2
48. Stroeher, U. H., A. W. Paton, A. D. Ogunniyi, and J. C. Paton. 2003. Mutation of luxS of3
Streptococcus pneumoniae affects virulence in a mouse model. Infect Immun 71:3206-12.4
49. Tettelin, H., K. E. Nelson, I. T. Paulsen, J. A. Eisen, T. D. Read, S. Peterson, J. Heidelberg,5
R. T. DeBoy, D. H. Haft, R. J. Dodson, A. S. Durkin, M. Gwinn, J. F. Kolonay, W. C. Nelson, J. D.6
Peterson, L. A. Umayam, O. White, S. L. Salzberg, M. R. Lewis, D. Radune, E. Holtzapple, H.7
Khouri, A. M. Wolf, T. R. Utterback, C. L. Hansen, L. A. McDonald, T. V. Feldblyum, S.8
Angiuoli, T. Dickinson, E. K. Hickey, I. E. Holt, B. J. Loftus, F. Yang, H. O. Smith, J. C. Venter,9
B. A. Dougherty, D. A. Morrison, S. K. Hollingshead, and C. M. Fraser. 2001. Complete genome10
sequence of a virulent isolate ofStreptococcus pneumoniae. Science 293:498-506.11
50. Tomasz, A. 1965. Control of the competent state in Pneumococcus by a hormone-like cell12
product: an example for a new type of regulatory mechanism in bacteria. Nature 208:155-9.13
51. Tomasz, A. 2000. Streptococcus pneumoniae : molecular biology & mechanisms of disease.14
Mary Ann Liebert, Inc., Larchmont, NY.15
52. Trappetti, C., L. Gualdi, L. Di Meola, P. Jain, C. C. Korir, P. Edmonds, F. Iannelli, S.16
Ricci, G. Pozzi, and M. R. Oggioni. 2011. The impact of the competence quorum sensing system on17
Streptococcus pneumoniae biofilms varies depending on the experimental model. BMC Microbiol18
11:75.19
53. van der Poll, T., and S. M. Opal. 2009. Pathogenesis, treatment, and prevention of20
pneumococcal pneumonia. Lancet 374:1543-56.21
-
7/28/2019 2011 the LuxS-Dependent Quorum Sensing System Regulates Early Biofilm Formation by Streptococcus Pneumoni
28/42
28
54. Vidal, J. E., J. Chen, J. Li, and B. A. McClane. 2009. Use of an EZ-Tn5-based random1
mutagenesis system to identify a novel toxin regulatory locus in Clostridium perfringens strain 13. PLoS2
One 4:e6232.3
55. Vidal, J. E., K. Ohtani, T. Shimizu, and B. A. McClane. 2009. Contact with enterocyte-like4
Caco-2 cells induces rapid upregulation of toxin production by Clostridium perfringens type C isolates.5
Cell Microbiol 11:1306-28.6
56. Weimer, K. E., C. E. Armbruster, R. A. Juneau, W. Hong, B. Pang, and W. E. Swords. 7
2010. Coinfection with Haemophilus influenzae promotes pneumococcal biofilm formation during8
experimental otitis media and impedes the progression of pneumococcal disease. J Infect Dis 202:1068-9
75.10
57. Zerfass, I., R. Hakenbeck, and D. Denapaite. 2009. An important site in PBP2x of penicillin-11
resistant clinical isolates of Streptococcus pneumoniae: mutational analysis of Thr338. Antimicrob12
Agents Chemother53:1107-15.13
14
15
16
17
18
19
20
21
22
23
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Figure legends1
Fig. 1. Overlapping PCR approach to locate the luxS gene in S. pneumoniae isolates. A) Schematic2
representation of a 6.2 kb region that includes the quorum sensing luxS gene. Solid bars underneath the3
locus diagram depict the primers utilized for overlapping PCRs shown in panel B). B) DNA from a4
collection ofS. pneumoniae invasive isolates and normal flora strains (lanes 1-15), ATCC33400 (lane5
16) or TIGR4 (lane 17) was used as template in PCR reactions with primers indicated at right. Size of6
the PCR product, in bp, is indicated at left in all panels.7
Fig. 2. Monocistronic transcription of the luxS gene during the mid-log phase of growth.8
Quantitative RT-PCRs were performed with RNA extracted from D39 grown overnight in a blood agar9
plate (BAP) or THY broth 4 h post-inoculation. Primers amplified the ORF Sp0341, Sp0341 and the10
luxS gene (0341-luxS), the luxS gene or the gyrase B subunit gene. Average CT values were normalized11
to the housekeeping 16S rRNA gene and the fold differences were calculated using the comparative CT12
method (2C
T) (27). Values on the bars indicate the calculated fold change relative to the overnight13
BAP culture. Error bars represent the standard error of the mean calculated using data from three14
independent experiments.15
Fig. 3. Quantification of biofilm biomass ofS. pneumoniae strains. Panels A and B. An aliquot of the16
indicated strain, was inoculated in triplicate in 96-well plates and incubated for 24 h at 37C in a 5%17
CO2 atmosphere. In panels C and D, S. pneumoniae D39 was inoculated and incubated the indicated18
time. Biofilms were stained with (A and C) crystal violet or (B and D) with a polyclonal anti- S.19
pneumoniae antibody coupled to fluorescein. Biofilm biomass (arbitrary fluorescent units) of wt strain20
D39 24 h post-inoculation was adjusted to 100% and percentage of biomass of the other strains or time21
points was calculated.22
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Fig. 4. Biofilm formation by S. pneumoniae strain D39 is regulated by the luxS gene. A) The1
indicated strain was inoculated in 96-well plates and incubated for 24 h. Biofilms were stained by2
fluorescence and biomass of wt D39 adjusted as 100% to calculate all others. In all panels, error bars3
represent the standard error of the mean calculated using data from, at least, four independent4
experiments. Asterisks (*) indicate statistical significance (p0.05), calculated using a non-parametric t-5
test, in comparison with wt D39. B) Biofilms were imaged using an inverted fluorescence microscope.6
Bar at right bottom panel is also valid for all panels.7
Fig. 5. Time-course study of biofilm formation. Strain D39 or SPJV05, was inoculated in 96-well8
plates and incubated for 1-8 or 24 h at 37C in a 5% CO2 atmosphere. A) Biofilms were stained with an9
anti-S. pneumoniae antibody coupled to fluorescein and quantified using a fluorometer. Biofilm biomass10
of wt strain D39 24 h post-inoculation was set to 100% and all others calculated. Error bars represent the11
standard error of the mean calculated using data from three independent experiments. Asterisk (*)12
indicates statistical significance (p0.05), calculated using non-parametric t-test, in comparison with wt13
D39 8 h post-inoculation. Biofilms formed by the wt D39 after 2 h (B), 4 h (C), 6 h or 8 h (E) or SPJV0514
(F) 8 h post-inoculation or complementing SPJV06 8 h post-inoculation were imaged using an inverted15
fluorescence microscope. Bar at left bottom panel is valid for all panels.16
Fig. 6. Physical complementation of the biofilm phenotype of the luxS mutant. A) SPJV01, SPJV0817
or a mixture of the indicated strains were inoculated in 24-well microplates and incubated for 6 h.18
Transwell filters were installed in some wells (inset) and SPJV08 was inoculated in the bottom and D3919
or SPJV05 in the top. These Transwell-biofilm bioassays were also incubated for 6 h and biofilms20
produced by SPJV08 on the bottom was quantified. * or#
indicates statistical significance (p0.05),21
calculated using non-parametric t-test, in comparison with wt D39 or SPJV05 respectively. Panel (B)22
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31
shows the fluorescence image of SPJV08 inoculated along with wt D39 and incubated for 6 h.1
Fluorescent biofilms formed by SPJV08 on the bottom of the Transwell-biofilm bioassay, when strain2
wt D39 (C) or SPJV05 (D) had been inoculated in the top chamber and incubated for 6 h.3
Fig. 7. Exogenous AI-2 increases the biofilm biomass of wt and luxS mutant. StrainD39 or SPJV054
was inoculated in 96 well plates and incubated for 24 h at 37C in a 5% CO2 atmosphere. Where5
indicated AI-2 (10 M) was added to the wells. Biofilms were stained by fluorescence and biomass of6
D39 wt set as 100% to calculate all others. * or # indicates statistical significance (p0.05), calculated7
using non-parametric t-test, in comparison with wt D39 or SJPV05, respectively.8
Fig. 8. Regulation of lytA mRNA and ply mRNA transcripts by luxS. Strain D39 or SPJV05 was9
inoculated in THY broth and incubated for 2, 4, 6 or 8 h at 37C. RNA was extracted from these THY10
cultures or from an overnight culture in BAP. A) RNA from D39 wt was utilized as template in qRT-11
PCR reactions with primers that amplified the lytA, luxS, ply orgyrB gene. Average CT values were12
normalized to the 16S rRNA gene and the fold differences, relative to BAP, were calculated using the13
comparative CT method (2CT) (27). Values of each time point indicate the calculated fold change14
relative to the overnight BAP culture. (B and C) RNA from D39 or SPJV05 was used as template qRT-15
PCR reactions with primers targeting the lytA gene (B) orply gene (C). Average CT values were16
normalized to the 16S rRNA gene and the fold differences, relative to D39 wt, were calculated as17
mentioned. Panel show representative figures of three independent biological replicates.18
19
20
21
22
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Table 1. Strains and plasmids used in this study.1
Strain Description Reference or source
D39 Avery strain, clinical isolate capsular serotype 2 (6, 26)
R6 D39-derivative unencapsulated laboratory strain (26)
TIGR4 Invasive clinical isolate, capsular serotype 4 (49)
SPJV01 D39 encoding pMV158GFP, TetR This study
EJ3 D39-derivative luxS null mutant, SpecR (21)
SPJV02 EJ3 encoding pPP2, SpecR, TetR This study
SPJV04 EJ3 encoding pJVPP9, SpecR, TetR This study
SPJV05 D39-derivative luxS null mutant, EryR This study
SPJV06 SPJV05 encoding pJVR6, EryR, SpecR This study
SPJV07 SPJV05 encoding pJVPP9, EryR, TetR This study
SPJV08 SPJV05 encoding pMV158GFP, EryR, TetR This study
E. coli TOP10 cells Cloning host Invitrogen
ECJV10 E. coli TOP10 encoding pJVR6 This study
ECJV11 E. coli TOP10 encoding pJVPP9 This study
Plasmids
pCR2.1 TOPO Cloning vector Invitrogen
pReg696 Low copy plasmid for Gram positives (14)
pPP2 Integrative plasmid forS. pneumoniae strains (15)
pJVPP9 pPP2 encoding the luxS wt gene from strain D39 This study
pJVR6 pReg696 encoding the luxS wt gene from strain D39 This study
pLux-ery-Lux pCR2.1TOPO encoding the luxS-ery-luxS cassette This study
pMV158GFP S. pneumoniae mobilizable plasmid encoding the greenfluorescent protein gene
(36)
3
4
5
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Table 2. Primers used in this study.1
2
Primers Target gene(s)* Sequence** PCR product size (bp)
JVS1L pbp2X-mraY AGTAAGTCAACAAAGTCCTTATCC 600
JVS2R TATTGCTGAATTGGCTACTAAATA
JVS3L mraY-clpL GGTAAATGAAAGCCTTACTAGAAC 482
JVS4R TGTTAAGTTTCGACCTAGTTTTG
JVS5L clpL-luxS CTAAGGAAGACCTTTCTAAGATTG 953
JVS6L ACATCATCTCCAATTATGATATTC
JVS7L luxS-Sp0341 CTATCACAGCTACAGAAAATCCT 306
JVS8R AAAACTTTCGACAATAACTTCTTT
lytA-L lytA AGTTTAAGCATGATATTGAGAAC 272
lytA-R TTCGTTGAAATAGTACCACTTAT
luxS-L luxS ACATCATCTCCAATTATGATATTC 257
luxS-R GACATCTTCCCAAGTAGTAGTTTC
0341-L Sp0341 (560) TATGTCCAATATGTACCACGAC 386
0341-R TGAAGTCAAGAACTGTTTGATAGT
gyrB-L gyrB AATAGTTGGAGATACGGATAAAAC 227
gyrB-R TATATTCAACGTAACTAGCAATCC
16SrRNA-L rpsP AACCAAGTAACTTTGAAAGAAGAC 126
16SrRNA-R AAATTTAGAATCGTGGAATTTTT
LuxP2-L luxS TTGGTACCGAGAGGTTTTCTCTCTGTCTCA 554
LuxP2-R TTTCTAGATTAAATCACATGACGTTCAAAG
Ery-L ermB AAAAATTTGTAATTAAGAAGGAGT 795
Ery-R CCAAATTTACAAAAGCGACTCA
Lux5-L luxS (30) TTGGTACCGAACTTGACCACACCATTGTC 208
Lux5-R TTGGATCCATGGTGAACAGTCAATCATGC
Lux3-L luxS (275) TTCTCGAGACGTCACACCAGTGCTAAAAT 159
Lux3-R TTTCTAGAATGAGTCTTGCCCATTCTTTA
LuxReg-L luxS TTGGATCCGAGAGGTTTTCTCTCTGTCTCA 554
LuxReg-R TTGAGCTCTTAAATCACATGACGTTCAAAG
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Fig.1
A
1 3 4 5 7 8 9 10 11 12 132 6 14 15B 16 17
JVS78380
luxS300
JVS56
960
1200
JVS34480
JVS12600
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.
20
ssion
mBAP
+19.3
10
15
mRNAexpre
extractedfr
.
0
5
Foldchangei
relativetoRN
+2.06
lux
Sp034
0341-lux
gyr
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Fig.3
A B
0.60100
0.2
0.4
iofilmsCV
A6
iofilmbiom
as
40
60
80
D39
TIGR4
R6
%
0
20
D39
TIGR4
R6
C
0.6
0.8
CVA630
0.2
0.4
Biofilm
Time (h)
D100
120
mass
40
60
80
%Biofilm
bio
Time (h)
2 4 6 8 10 12
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Fig.4
A
biomass
80
100
120
%Biofilm
20
40
60
**
*
0
D39
EJ3
SP
JV
04
SP
JV
02
SP
JV
05
SP
JV
06
SPJV04D39 EJ3
100m
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Fig.5
A
)84.27.28
8 14.73.5SPJV05 *
Time(h
253.5
24.69.3
9.43.6
1.870.62
3
4
5
39
20 40 60 80
% Biofilm biomass
E F G
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Fig.6
SPJV08+SPJV05
answell
*
A
SPJV08+D39
SPJV08 **
*SPJV01+D39T
20 40 60 80 100
% Biofilm biomass
B C D
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Fig.7
D39+AI-2
SPJV05
SPJV05+AI-2
**
*
#
20 40 60 80 100 120 140
% Biofilm biomass
D39
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Fig.8
A40
50 luxSly tA
gyrB
ply
RNAexpression
oBAP
50.9
31.328.9
0
10
20
o
ldchangeinm
relative
-2.7-0.82
-3.4
10.4
22.3
13.8 13.8
28.0
5.02
22.3