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ORIGINAL RESEARCH
published: 22 March 2017
doi: 10.3389/fmicb.2017.00434
Frontiers in Microbiology | www.frontiersin.org 1March 2017 | Volume 8 | Article 434
Edited by:
Kate Louise Seib,
Griffith University, Australia
Reviewed by:
Elena Del Tordello,
Valneva, France
Berenike Maier,
University of Cologne, Germany
Joseph P. Dillard,
University of Wisconsin-Madison, USA
*Correspondence:
Jesús Arenas
j.a.arenasbusto@uu.nl
†These authors have contributed
equally to this work.
Specialty section:
This article was submitted to
Infectious Diseases,
a section of the journal
Frontiers in Microbiology
Received: 12 December 2016
Accepted: 02 March 2017
Published: 22 March 2017
Citation:
Pérez-Ortega J, Rodríguez A, Ribes E,
Tommassen J and Arenas J (2017)
Interstrain Cooperation in
Meningococcal Biofilms: Role of
Autotransporters NalP and AutA.
Front. Microbiol. 8:434.
doi: 10.3389/fmicb.2017.00434
Interstrain Cooperation in
Meningococcal Biofilms: Role of
Autotransporters NalP and AutA
Jesús Pérez-Ortega †, Antonio Rodríguez †, Eduardo Ribes, Jan Tommassen and
Jesús Arenas*
Section Molecular Microbiology, Department of Biology, Utrecht University, Utrecht, Netherlands
Neisseria meningitidis (Nm) and Neisseria lactamica (Nl) are commensal bacteria
that live in the human nasopharynx, where they form microcolonies. In contrast
to Nl,Nm occasionally causes blood and/or meningitis infection with often fatal
consequences. Here, we studied interactions between neisserial strains during biofilm
formation. Fluorescent strains were engineered and analyzed for growth in single- and
dual-strain biofilms with confocal laser-scanning microscopy. Different strains of diverse
Neisseria species formed microcolonies of different sizes and morphologies. Pair-wise
combinations of two invasive Nm strains and one Nm carrier isolate showed that these
strains can coexist in spite of the fact that they produce toxins to combat congeners. This
lack of competition was even observed when the biofilms were formed under nutrient
limitation and can be explained by the observation that the separate microcolonies
within mixed biofilms are mostly lineage specific. However, these microcolonies showed
different levels of interaction. The coexistence of two strains was also observed in mixed
biofilms of Nm and Nl strains. Inactivation of the autotransporter NalP, which prevents
the release of the heparin-binding antigen NHBA and the α-peptide of IgA protease from
the cell surface, and/or the production of autotransporter AutA increased interactions
between microcolonies, as evidenced by close contacts between microcolonies on the
substratum. Qualitative and quantitative analysis revealed an altered spatial distribution of
each strain in mixed biofilms with consequences for the biomass, biofilm architecture and
bacterial viability depending on the synthesis of NalP and AutA, the expression of which is
prone to phase variation. Being in a consortium resulted in some cases in commensalism
and cooperative behavior, which promoted attachment to the substratum or increased
survival, possibly as result of the shared use of the biofilm matrix. We hypothesize that
Nm strains can cooperate during host colonization, but, possibly, the different capacities
of the microcolonies of each strain to resist the host’s defenses limits the long-term
coexistence of strains in the host.
Keywords: Neisseria, biofilms, bacterial interactions, AutA, NalP, NHBA, IgA protease
Pérez-Ortega et al. Interactions in Meningococcal Biofilms
INTRODUCTION
The genus Neisseria includes bacterial species that colonize
mucosal surfaces in humans, e.g., N. lactamica (Nl), and
the pathogenic N. meningitidis (Nm), which, like Nl, inhabits
the upper respiratory tract, and N. gonorrhoeae (Ng), which
infects the genitourinary tract. Nm forms microcolonies in the
nasopharynx (Sim et al., 2000). Such microbial communities are
similar to biofilms and offer protection against the host’s immune
response and other adverse conditions (Costerton et al., 1995).
Biofilms are defined as communities of microorganisms attached
to a surface and embedded in a self-produced extracellular
matrix (Costerton et al., 1995). Multiple factors influence
biofilm formation in Nm, such as the capsule, type IV pili,
surface-exposed proteins, and extracellular polymeric substances
of which extracellular DNA (eDNA) can be an important
component (Arenas and Tommassen, 2017). Depending on the
clonal complex (cc), the bacteria use two different strategies to
initiate biofilm formation, i.e., either dependent or independent
of eDNA (Lappann et al., 2010). In the former strategy, eDNA
is key to initial attachment and structure stabilization. These
processes are facilitated by cell-surface-exposed proteins that
attach the cells to eDNA via electrostatic interactions, a process
that may also occur in other bacterial species (Arenas et al., 2013a;
Arenas and Tommassen, 2017).
NalP, IgA protease, AutA, and AutB are autotransporters (AT)
involved in the initiation of biofilm formation (Arenas et al.,
2013a, 2015a, 2016). ATs are modular proteins constituted of a
translocator domain that is inserted into the outer membrane,
thereby allowing the transport of the fused passenger domain to
the cell surface. After translocation, the passenger may remain
attached to the cell or be released into the milieu after proteolytic
processing (Grijpstra et al., 2013). NalP (a.k.a. AspA) is a serine
protease whose autoproteolytic processing results in its release
from the cell surface (Turner et al., 2002). However, temporarily,
NalP remains attached at the cell surface by an N-terminal lipid
anchor, and in this position, it cleaves several ATs, including
IgA protease, and surface-exposed lipoproteins, including the
heparin-binding antigen NHBA (van Ulsen et al., 2003; Serruto
et al., 2010; Roussel-Jazédé et al., 2013). In addition, NalP cleaves
the complement factor C3, thus protecting Nm from complement
activation (Del Tordello et al., 2014). The passenger of IgA
protease consists of two separate domains, the protease domain
and the α-peptide, which are connected via a small γ-peptide
(Pohlner et al., 1987). The protease domain is released into the
extracellular milieu via autocatalytic processing (Pohlner et al.,
1987), but it may also be released connected to the α-peptide after
cleavage by NalP (van Ulsen et al., 2003; Roussel-Jazédé et al.,
2014).
The synthesis of NalP is prone to phase variation, which is
a stochastic turning on and off of gene expression (Saunders
et al., 2000). In the absence of NalP, the α-peptide of IgA protease
(αP) usually remains attached at the cell-surface (Roussel-Jazédé
et al., 2014). This αP contains nuclear localization signals, which
are positively charged, arginine-rich peptide segments (Pohlner
et al., 1995) that bind eDNA and therefore, when present at the
cell surface, they increase initiation of biofilm formation. NHBA
also contains an arginine-rich region, which is responsible for
binding heparin (Serruto et al., 2010) and presumably also for
the demonstrated DNA-binding capacity of the protein (Arenas
et al., 2013a). The binding of NHBA to eDNA is relevant for the
initiation of biofilm formation, at least in strains following an
eDNA-dependent strategy (Arenas et al., 2013a). NalP cleavage
releases a fragment of NHBA including the arginine-rich region,
which has been shown to increase endothelial permeability
by inducing the internalization of adherens junction proteins
(Casellato et al., 2014). However, this cleavage is not completely
effective since full-length NHBA molecules remain detectable at
the cell surface in the presence of NalP (Serruto et al., 2010).
This uncleaved form plays a role in the initiation of biofilm
formation, as deletion of the nhbA gene in a NalP+strain that
follows an eDNA-dependent strategy impairs biofilm formation
(Arenas et al., 2013a).
The autA gene is present in the genomes of various Neisseria
species. AutA is exposed at the cell surface where it binds
eDNA and interacts with AutA on neighboring cells. Its synthesis
induces autoaggregation (Arenas et al., 2015a), which has drastic
consequences for biofilm architecture. Expression of the autA
gene is prone to phase variation due to slipped-strand mispairing
at a tetranucleotide repeat within the coding region. In many
strains, however, the gene harbors a premature stop codon, an
insertion or a deletion that disrupts gene expression even if the
gene is in phase at the tetranucleotide repeat. Expression of
autB is also prone to phase variation and only very few strains
express the gene suggesting a negative selection pressure against
its expression (Arenas et al., 2016). Also AutB presumably binds
DNA.
Interactions between different neisserial strains in the
nasopharynx have been poorly studied so far. Previously, it
has been shown that a strain following an eDNA-independent
strategy of biofilm formation was outcompeted in vitro by strains
following the eDNA-dependent one (Lappann and Vogel, 2010),
whilst two strains both using an eDNA-dependent strategy could
coexist in a biofilm (Lappann et al., 2010; Lappann and Vogel,
2010). However, the mechanisms of such inter-strain competition
and interactions and the implications on biofilm structure remain
to be elucidated. It is noteworthy, in this respect, that Neisseria
strains synthesize a variety of toxins to compete congeners,
including the TpsA (Arenas et al., 2013b) and MafB (Arenas et al.,
2015b; Jamet et al., 2015) proteins, but their potential role in
dual-strain biofilms has never been studied. Here, we analyzed
the interactions between strains in dual-strain biofilms and used
different mutants to understand the mechanisms behind.
MATERIALS AND METHODS
Bacterial Strains and Growth Conditions
Bacterial strains used in this study are listed in Table 1 and Table
S1. Neisserial strains were grown overnight on GC agar base
medium (OXOID) supplemented with OxoidTM Vitox at 37◦C
in a CO2-enriched atmosphere provided by a candle jar. When
appropriate, erythromycin (7 µg ml−1), kanamycin (100 µg
ml−1), chloramphenicol (10 µg ml−1), rifampicin (50 µg ml−1)
or gentamicin (60 µg ml−1) was added to the medium. BB-1 and
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Pérez-Ortega et al. Interactions in Meningococcal Biofilms
TABLE 1 | Characteristics of strains HB-1, BB-1, and α14.
HB-1 BB-1 α14
Lineage cc32 cc11 cc53
Isolation Invasive Invasive carrier
Capsule − − −
Twitching motility + + −
NalP + + −
AutA −*−*+
DNase sensitivity biofilms + − +
*No full-length AutA can be produced even if the gene is in phase at the tetranucleotide
repeats because of the presence of a premature stop codon.
HB-1 are capsule-deficient derivatives of strains B16B6 (Frasch
and Chapman, 1972) and H44/76 (Holten, 1979), respectively,
which were isolated from patients with meningococcal disease
in the United States and Norway, respectively. Strain α14 was
isolated in Germany from a healthy carrier (Claus et al., 2005). A
rifampicin-resistant derivative of HB-11nalP was selected after
plating the strain on GC plates supplemented with rifampicin.
For liquid cultures and biofilm experiments, bacteria grown
on plates were resuspended in Tryptic Soy Broth (TSB)
(Scharlau) or RPMI (Gibco) to an optical density at 550 nm
(OD550) of 0.1 and incubated in polystyrene cell culture flasks
with shaking (120 rpm) until they reached the exponential
growth phase (OD550 of ∼1.0). To induce the expression of genes
from plasmids, 0.1 mM isopropyl-β-D-l-thiogalactopyranoside
(IPTG) was added to the medium.
Escherichia coli strain DH5αwas grown in lysogeny broth
(LB) with shaking or on solid LB medium at 37◦C. For plasmid
selection, the following antibiotics were included in the medium:
kanamycin (50 µg ml−1), chloramphenicol (25 µg ml−1) or
gentamicin (20 µg ml−1).
Plasmid Construction
All plasmids and PCR primers used in this study are listed
in Tables S1, S2, respectively. Regular PCR reactions were
performed by using Dream Taq-DNA polymerase (Thermo
Scientific), whilst PCR fragments generated for cloning were
obtained using the High Fidelity polymerase (Roche Diagnostics
GmbH) or Phusion DNA polymerase (Thermo Scientific). For
purification of PCR products, the Wizard R
SV Gel and PCR
Clean-Up System (Promega) were used. Plasmids were isolated
with the commercial E.Z.N.A. R
Plasmid Mini Kit I (Omega Bio-
Tek). PCR products and plasmids were both digested for 2 h with
appropriate restriction enzymes (Thermo Scientific) for which
cleavage sites were included in the primers, purified and ligated
using T4 DNA ligase (5 U/µl) (Thermo Scientific).
For the preparation of pIN plasmids, plasmids pCRT_hrtA
and phrtA_gm_rfp containing the hrtA region (Figure S1A)
were used. The gfp gene together with an upstream region
containing the lac promoter were amplified by PCR from
plasmid mut3.1 and inserted into plasmid pCRT_hrtA via MluI
and PpuMI digestion. Subsequently, a gentamicin-resistance
cassette amplified by PCR from phrtA_gm_rfp was inserted
via PpuMI, resulting in plasmid phrtA_gm_gfp. Fragments of
different length (221–273 bp) located upstream of the opaB
gene and containing the −35 and −10 boxes of the opaB
promoter (opaBP) were amplified by PCR from Ng strain
FA1090 using different primer pairs (Table S2) and used to
generate three promoter variants: opaBPM,opaBPL, and opaBPH
(Figure S1C). The sequence of opaBPMdiffers with respect to
opaBP in four nucleotides creating an NheI site located between
the ribosome-binding site (RBS) and the start codon (Figure
S1C). This fragment and a gfp gene, amplified by PCR from
plasmid phrtA_gm_gfp, were digested with NheI and ligated
together. The resulting ligation product was purified and inserted
into phrtA_gm_rfp via MluI and Van91I resulting in plasmid
pINM(Figure S1B). The fragment amplified for opaBPHwas
18 nucleotides smaller than that for opaBPM, replacing 52
nucleotides immediately upstream of the start codon by 34
nucleotides. This fragment was inserted into phrtA_gm_gfp and
phrtA_gm_rfp upstream of the gene that encodes the fluorescent
protein via MluI and SmaI. The resulting plasmids, pINHand
pINH−RED, respectively, contain a different sequence between the
-10 box and the start codon as compared with pINM(Figure S1C).
The fragment amplified for opaBPLwas 3 nucleotides larger than
opaBP, replacing 32 bp by 35 bp immediately upstream of the
start codon, and this was cloned into phrtA_gm_gfp via MluI and
SmaI. The resulting plasmid, pINL, contains a similar sequence
between the -10 box and start codon as pINHbut contains 21
additional bp in opaBP (Figure S1C). The correct insertion of the
fragments was confirmed by PCR and subsequent sequencing of
plasmids at the Macrogen sequencing service (Amsterdam).
Biofilm Formation
Biofilms were formed under static conditions in 24-wells plates
as previously described (Arenas et al., 2013a) with modifications.
Briefly, 5-h old cultures in TSB were adjusted to an OD550
of 1, and 500-µl samples were seeded per well on a round
glass cover slip. For mixed biofilms, 500-µl samples were
mixed 1:1, unless mentioned otherwise, and the mixture was
placed in the well. After 15 h of incubation, the medium was
removed from each well, and the biofilm was washed twice
with de-ionized water. Biofilms were chemically fixed with
0.1 mM PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4,
and 1.8 mM KH2PO4) containing 2% formaldehyde for 2 h
for microscopy analysis. In some experiments, bacteria were
harvested from cultures by centrifugation (4,500 g for 5 min)
and resuspended in the supernatant recovered from the culture
of another strain before initiation of biofilm formation. To
determine DNase sensitivity of biofilm formation, cultures were
treated with 100 mg ml−1of DNase I and biofilm formation
was determined using crystal violet as described (Arenas et al.,
2013a).
Microscopy, Image Analysis, and Films
Fixed, 15-h old biofilms were used for microscopy. All
microscopic observations and image acquisitions were
performed using a Zeiss LSM 700 confocal laser scanning
microscope (Carl Zeiss, Germany) equipped with detectors
and filter sets for monitoring fluorescence. Images were
obtained using a 20x/0.8 Plan-Apochromat, a 40x/1.30
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Pérez-Ortega et al. Interactions in Meningococcal Biofilms
Plan-Neofluar oil or a 63x/1.40 Plan-Apochromat oil
objective. Phenotypes were considered when at least
observed in three independent experiments performed in
duplicate. For the analysis of the structural parameters
of the biofilm (biomass, average thickness, roughness
coefficient, and surface to volume ratio), image stacks
at 0.4 µm z-intervals were acquired and analyzed with
the program COMSTAT (Heydorn et al., 2000) in the
image processing environment ImageJ (v1.48, NIH,
http://imagej.nih.gov/ij/).
To determine the level of expression of fluorescent proteins
expressed by each pIN variant, cell were fixed by adding
formaldehyde (1% v/v) to an exponentially growing culture as
described (Arenas et al., 2008) and formalin-fixed cells were
visualized by fluorescence microscopy. An outline was drawn
around 20 formalin-fixed cells, and the mean fluorescence was
measured, along with several adjacent background readings.
Then, the total corrected cellular fluorescence (TCCF) =
integrated density – (area of selected cell ×mean fluorescence
of background readings), was calculated for each bacterium.
Statistical analyses were performed considering TCCF values
obtained from each construct.
To determine twitching motility in biofilms, time-lapse
videos of fluorescent bacteria in 0.5- and 2-h old biofilms of
strains following an eDNA-dependent and -independent strategy,
respectively, were recorded. Sixty images were taken at 0.1-
ms intervals. For the HB-11pilE mutant, biofilms were stained
with a LIVE/DEAD BacLight Bacterial Viability Kit (Molecular
Probes).
Preparation of Samples, SDS-Page and
Western Blotting
Liquid cultures grown to an OD550 of ∼2.0 were centrifuged
(4,500 g for 5 min), and the resulting cell pellet was resuspended
in H2O to an OD550 of 10. The spent media were centrifuged at
16,000 g for 15 min to remove residual cells, and proteins were
precipitated from the supernatant with 10% (w/v) trichloroacetic
acid in H2O. After centrifugation (16,000 g, 15 min), the
resulting pellets were washed with ice-cold acetone, air-dried
and resuspended in H2O. For analysis of protein production
in biofilms, 15-h old biofilms were disrupted by mechanical
forces, and bacterial cells were peletted (4,500 g for 5 min) and
resuspended in H2O to a final OD550 of 10. For analysis of
secreted proteins in biofilms, biofilms and planktonic cells were
mixed by pipetting and the suspensions were centrifuged at
16,000 g for 15 min to remove cells and debris, and proteins
were precipitated from the supernatants as described above. All
samples were resuspended in double-strength sample buffer for
electrophoresis and boiled for 10 min.
For SDS-PAGE, the Mini-PROTEAN R
Electrophoresis
System (Bio-Rad) was used. After electrophoresis, gels were
stained with Coomassie Brilliant blue. Western blot analysis
was performed as described (Arenas et al., 2015a). Blots were
developed with SuperSignal R
West Pico Chemiluminescent
Substrate (Thermo Scientific) for 1 min at room temperature,
and the image was acquired on a light-sensitive film (Fuji
Medical X-Ray Film) or in a bio-imaging system (BioRad). The
antiserum directed against GFP was purchased from Sigma-
Aldrich. The antisera directed against the translocator domain
of IgA protease (Roussel-Jazédé et al., 2014), the translocator
domain of NalP (Oomen et al., 2004), and the TPS domain
of TpsA of system 1 (van Ulsen et al., 2008) were previously
described. The antiserum directed against MafAMGI−3is from
our laboratory collection, and the monoclonal antibody SM1
directed against PilE was generously provided by John Heckels
(University of Southampton).
Quantification of Live Bacteria in the
Biofilm
Bacteria with different antibiotic resistance markers were used
to discriminate between two strains (indicated in Table S1).
The biofilm was washed twice with Hanks’ Balanced Salt
Solution Gibco R
(ThermoScientific). To disrupt the aggregates,
biofilms were incubated for 15 min at room temperature with a
solution containing 0.5 mg ml−1of DNase I (Sigma-Aldrich) and
0.02 mg ml−1of proteinase K (ThermoScientific) diluted in TSB.
Complete disruption of aggregates was confirmed by microscopy.
The bacterial suspensions were serially diluted and plated on GC
agar containing appropriate antibiotics, and the colony-forming
units (CFU) were quantified after 12 h of incubation.
Statistical Analysis
For statistical comparisons, data from at least three independent
experiments performed in duplicate were considered. To
determine the structural parameters of the biofilm, at least five
image stacks of each sample were obtained from representative
experiments. Data were analyzed using an unpaired statistical
t-test with GRAPH PAD v 6.0 (Graph Pad Software, Inc.).
RESULTS
Engineering Fluorescent Neisseriae
To facilitate the visualization of bacteria by fluorescence
microscopy and the discrimination between different strains or
species in mixed biofilms, we designed new plasmids to integrate
genes encoding fluorescent proteins into the chromosome. A
high rate of transformation (hrtA) region (Claus et al., 1998)
present in the chromosome of several Neisseria species with
>90% of sequence identity (Figure S1A) was exploited for this
purpose. In a plasmid containing this region, we introduced
(i) the promoter of the opaB gene (opaBP), which encodes
an abundant protein expressed in some Neisseria species and
previously used for protein production (Ramsey et al., 2012), (ii)
a gene encoding green or red fluorescent protein (GFP or RFP,
respectively), and (iii) a gentamicin-resistance cassette (Figure
S1B). This plasmid was designated pIN (plasmid for Integration
in Neisseria). Three different opaBP variants were tested in
the pIN backbone (Figure S1C), and they were called opaBPH,
opaBPM, and opaBPL. The pIN variants, which were called pINH,
pINM, and pINL, respectively, were used to transform Nm strain
HB-1 selecting for gentamicin-resistant recombinants. To test
protein expression levels, the intensity of fluorescence emission
was acquired for individual bacteria and analyzed with the image
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Pérez-Ortega et al. Interactions in Meningococcal Biofilms
processing program ImageJ. The results revealed large differences
in fluorescence intensity between the promoter variants in the
order opaBPH>opaBPM>opaBPL, in accordance with protein
production levels detected on Western blots (Figure S1D). The
fluorescence of pINH-derived transformants was visualized in
all our imaging devices and allowed for the discrimination of
fluorescent strains in mixed biofilms. Therefore, this plasmid was
used to generate fluorescent bacteria. Indeed, we could generate
fluorescent bacteria in strains of Nm,Nl, and Ng transformed
with this plasmid (Figure S1E).
Biofilm Structure of Fluorescent Neisseriae
To study the biofilm structure of various Nm and Nl strains,
biofilms of fluorescent bacteria were formed on glass and
visualized by confocal microscopy. All strains used were capsule
deficient, as capsule has been reported to inhibit biofilm
formation on abiotic surfaces (Yi et al., 2004; Lappann et al.,
2006). Figure 1A shows the structures of 15-h old biofilms
of various strains. Biofilms consisted of cell clusters, but the
size, dispersion and number of the clusters differed largely
between both species and between strains of the same species.
Nm strains generally formed smaller clusters than did the Nl
strains. Also, Nm strains of cc8 and cc11 formed much smaller
and less compact clusters with, together, a larger coverage of
the substratum than strains of cc32 and cc53 (Figure 1A). It
is noteworthy that strains of cc8 and cc11 use an eDNA-
independent strategy of biofilm formation in contrast to strains
of other cc (Lappann et al., 2010). Both Nl strains formed biofilms
that were sensitive to DNase I (data not shown). Thus, these
results confirmed that aggregation is a common feature during
neisserial biofilm formation.
eDNA-Binding Proteins Contribute to Cell
Aggregation in Single-Strain Biofilms
AutA and type IV pili are known to be involved in bacterial
aggregation during biofilm formation (Lappann et al., 2006;
Arenas et al., 2015a). Here, we explored the contribution in
this process of eDNA-binding proteins, which are cleaved from
the cell surface by the protease NalP. Figure 1B shows the
biofilm structure of mutants of Nm strains HB-1 and BB-
1 lacking nalP. The nalP mutants formed bigger and more
compact microcolonies than the corresponding wild types
(Figure 1A), although the difference was less pronounced in
BB-1, in accordance with the eDNA-independent strategy of
biofilm formation of this strain. The stronger aggregation of HB-
11nalP is not due to increased piliation, as Western blotting
assays showed a similar production of PilE, the major pilus
subunit, in the wild type and the nalP mutant (Figure S2A).
Since cc11 strain BB-1 produces a different type of pilin that is
not recognized by the antibodies (Cehovin et al., 2010) we could
not verify pilE expression in this strain and its nalP mutant.
The autA gene is disrupted in HB-1 and BB-1 because of the
presence of a premature stop codon (Arenas et al., 2015a) and
can, therefore, not play a role in the differences between the NalP-
producing and non-producing strains. Furthermore, aggregation
was severely reduced when NalP was expressed in trans from
plasmid pEN300 in the nalP mutants (Figure 1B) demonstrating
that the increased aggregation of the nalP mutants is a direct
effect of the lack of NalP synthesis. Microscopic examination of
log-phase precultures grown under shaking conditions showed
the presence of only few very small aggregates in the nalP
mutant of HB-1 but not in the wild type (Figure S2B). The
size of these aggregates does not match those observed in
biofilms (Figure S2B). Probably, the abundance of eDNA at
the surface of the nalP mutant cells facilitates aggregation and
thereby microcolony formation. Such interactions may occur
already in liquid cultures, but they are disrupted by physical
forces during shaking. In conclusion, these results show that
microcolony formation occurs during biofilm formation and
that nalP expression influences this process, presumably by
cleaving eDNA-binding proteins from the cell surface. Thus,
these data expand the previously established role of NalP during
the initiation of biofilm formation by demonstrating its effect on
biofilm structuring.
Interbacterial Interactions in Dual-Strain
Biofilms
The high rates of Nm colonization (Sim et al., 2000) suggest an
intense traffic of strains within the nasopharynx. Consequently,
strains could interact with each other to compete or to benefit
during colonization. To study how different strains affect
each other during biofilm formation, we analyzed pairwise
combinations of three Nm strains in biofilm experiments. We
selected strains HB-1, BB-1, and α14 because different traits
relevant for biofilm formation. HB-1 and BB-1 were chosen
as representatives of strains following an eDNA-dependent
and -independent strategy of biofilm formation, respectively
(Arenas et al., 2013a). Strain α14 was chosen because it
produces AutA, which causes autoaggregation and thereby affects
biofilm architecture (Arenas et al., 2015a). HB-1 and BB-1
do not produce AutA (Arenas et al., 2015a). Other relevant
characteristics of these strains are listed in Table 1.
First, several relevant properties of these strains were further
analyzed. HB-1 and BB-1 clearly showed twitching motility in
biofilms (Videos 1,2, respectively, in Supplementary Material).
Strain α14, however, showed no twitching motility, similar
as a pilE mutant of HB-1 (Videos 3,4, respectively, in
Supplementary Material). Interestingly, Western blot analysis
revealed a considerable difference in the electrophoretic mobility
of the PilE proteins of HB-1 and α14 (Figure S2A), even though
the sequences of these proteins are identical according to the
available genome sequences (Schoen et al., 2008; Piet et al., 2011).
Additionally, the genome sequence of α14 showed that nalP is out
of phase; Western blotting confirmed that NalP is not synthesized
in this strain (Figure S2A). All relevant properties of the three
strains are listed in Table 1.
Each strain was grown independently in TSB, and, after
adjusting them to the same OD, they were mixed 1:1 for biofilm
formation. First, green- and red-fluorescent variants of strain
HB-1 were combined (Figure 2A). Both variants of the strain
formed separate clusters with hardly any intermixing, although
these separate clusters extensively interacted (Figure 2A). A
similar result was observed when green- and red-fluorescent
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Pérez-Ortega et al. Interactions in Meningococcal Biofilms
FIGURE 1 | Biofilm organization in various fluorescent Neisseria strains. (A) Biofilms of various strains of Nm and Nl. The clonal complex (cc) of the Nm strains
is indicated. (B) Influence of nalP inactivation on biofilm architecture. Wild-type phenotypes are shown in (A). HB-11nalP/NalP+and BB-11nalP/NalP+represent
nalP mutants overexpressing NalP from plasmid pEN300. All strains harbor an opaBPH-gfp or opaBPH-rfp inserted into hrtA locus. Representative pictures from at
least four experiments with two technical replicates are shown.
variants of α14 were mixed (Figure 2A). Presumably, these
separate clusters result from clonal outgrowth. In contrast, small
intermixed clusters were abundantly found when green- and
red-fluorescent variants of BB-1 were combined (Figure 2A),
suggesting that the less aggregative nature of this strain allows
for intermixing.
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Pérez-Ortega et al. Interactions in Meningococcal Biofilms
FIGURE 2 | Interbacterial interactions in dual-strain biofilms. (A) Biofilms constituted of two differently labeled derivatives of strain HB-1, α14 or BB-1. In the
enlargements of the BB-1 biofilms, the positions of three clusters are highlighted, one consisting exclusively of GFP-labeled cells, one consisting exclusively of
RFP-labeled cells, and one intermixed cluster consisting of both GFP- and RFP-labeled cells. (B) Combinations of α14 and BB-1 and several mutant derivatives.
(Continued)
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Pérez-Ortega et al. Interactions in Meningococcal Biofilms
FIGURE 2 | Continued
(C) Combinations of α14 or its autA mutant derivative with HB-1. (D) Combinations of HB-1 and BB-1 and several mutant derivatives. Strain names at the left are
shown in green or red, reflecting the expression of GFP or RFP, respectively. Designations AutA+and NalP+indicate expression of AutA and NalP from plasmids
pFPAutA and pEN300, respectively. For clarity, strain HB-31nhbA1iga (Table S1) is indicated here as HB-11nalP1nhbA1iga. However, in contrast to strain
HB-11nalP, the isogenic strain HB-3 contains markerless deletions of the nalP gene and the capsule locus, which was necessary to combine multiple mutations into
a single strain (Arenas et al., 2013a). Individual and combined fluorescence is displayed.
Next, strains of different genetic backgrounds were mixed.
Interestingly, strain BB-1, which forms only very small
clusters dispersed over the substratum in single-strain biofilms
(Figure 1A), formed large clusters, which coincided with those
of α14, in dual-strain biofilms (Figure 2B), indicating that these
strains can form intermixed microcolonies. In contrast, HB-1
and α14 formed separate but interacting clusters (Figure 2C),
whilst strains BB-1 and HB-1 attached to the substratum
without much apparent association between the clusters formed
(Figure 2D). Lack of intermixing without much association
between clusters was also observed in most cases when Nm
strains were combined with Nl strains (Figure S3). Only
associations of clusters of α14 with those of the Nl strains were
observed.
Next, we studied the possible role of AutA in the
formation of intermixed clusters of α14 and BB-1. Indeed,
intermixing was drastically reduced when an autA mutant
of α14 was used, and separate but interacting clusters of
the two strains were observed (Figure 2B). Unfortunately, the
introduction of plasmid pFPAutA in the autA mutant of α14
for complementation studies failed for unknown reasons (Arenas
et al., 2015a). To further investigate the possible role of AutA
in the formation of intermixed clusters with BB-1, the AutA-
encoding plasmid was introduced in another genetic background,
i.e., HB-1. The synthesis of AutA in HB-1, which was confirmed
on Western blots (Figure S4A), increased the interaction of its
clusters with those of BB-1, but it did not lead to intermixed
clusters (Figure 2D). Thus, additional traits of α14, such as
the absence of NalP or of twitching motility (Table 1), may
contribute to its ability to form intermixed clusters with BB-
1.
Considering that NalP inactivation increased aggregation
in single-strain biofilms (Figure 1B), we asked whether the
presence or absence of NalP may also affect the interaction
between different strains in mixed biofilms. Combinations of
strains HB-1 and BB-1 with their respective nalP mutant
derivatives generated biofilms consisting of separate clusters of
the individual strains, which, however, extensively interacted
with those of the other strain as evidenced by their co-localization
on the substratum (Figure 2D). The ectopic expression of
nalP from pEN300 in both nalP mutants drastically reduced
these interactions (Figure 2D). Furthermore, the combination
of BB-1 with a derivative of HB-1 lacking, besides nalP, also
the nhbA and iga genes showed only small clusters of the
HB-1 derivative that did not interact with those of BB-1
(Figure 2D), confirming that the increased aggregation observed
for the nalP mutant is mediated by the increased cell-surface
exposure of intact NHBA and αP in this strain. Interestingly,
inactivation of nalP in strain BB-1 interfered with the formation
of intermixed microcolonies with α14 and led to the formation of
separate clusters of the individual strains, which barely interacted
(Figure 2B). Probably, in this case, the increased interaction
between the BB-1 cells due to the inactivation of NalP prevents
intermixing with the α14 cells. In conclusion, Nm strains can
form microcolonies constituted of mixed lineages or separate
clusters of individual strains that variably interact depending
on the strains studied and on the synthesis of AutA and/or
NalP.
Characterization of Single- and Dual-Strain
Biofilms
To gain more insight into the implication of AutA and NalP
synthesis on biofilm architecture, the individual contribution
of each strain in mixed biofilms was analyzed with COMSTAT
software and statistically compared with results of single-
strain biofilms. Single-strain biofilms of HB-1, BB-1, and α14
differed considerably; the biomass, that reflects the amount
of both live and dead bacteria in the biofilms, was in
the order α14 >HB-1 >BB-1 (Figure 3A). Also other
biofilm parameters, such as thickness, surface/volume ratio,
and roughness, differed (Figure S5). To test bacterial viability,
the biofilms were disrupted and the numbers of CFU were
determined on selective GC plates. Viability was determined
at 15, 24, and 48 h after initiation of biofilm formation and
found to decrease drastically for HB-1 and α14 at 48 h (data
not shown). To better compare the differences in viability
between wild types and mutants of the three strains, results
are shown for 24-h old biofilms (Figure 3B), although similar
results were observed for 15-h old biofilms. In single-strain
biofilms, the numbers of CFU of strains HB-1 and BB-1 did
not differ but they were significantly different to those of α14
(Figures 3B).
Single-strain biofilms of the nalP mutants of BB-1 and HB-1
revealed higher biomasses compared to the corresponding
wild types, but the numbers of CFU were similar or
drastically decreased, respectively (Figures 3A,B). We did
not find differences in viability between the wild types
and the nalP mutants in liquid cultures (data not shown).
The discrepancy between biomass and numbers of CFU
indicates the presence of more dead cells in the biofilms
of the nalP mutants and could be explained by the more
compact clusters formed in the absence of NalP (Figure 1),
which could result in lower exposure of the biomass to
the nutrients. In summary, the pronounced aggregation
observed in nalP mutants (Figure 1B) correlates with increased
biomass and altered biofilm architecture but reduced bacterial
viability.
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Pérez-Ortega et al. Interactions in Meningococcal Biofilms
FIGURE 3 | Characteristics of single- and dual-strain biofilms. Biofilms
were formed of various combinations of Nm strains HB-1 and BB-1 and their
nalP mutant derivatives, and α14 and its autA mutant derivative and biomass
and CFU were determined. The strains present in the consortium are shown at
the bottom of each panel. The data shown are for the strain indicated in bold.
(A) The biomass of biofilms was calculated for each strain using COMSTAT.
The biomass for each strain in the consortium is shown separately. (B)
Numbers of CFU. The CFU for each strain in the consortium is shown. To allow
for strain discrimination in dual-strain biofilms, strains with different
antibiotic-resistance markers were used as indicated in Table S1. The CFU for
each strain were determined by plating on GC medium containing kanamycin,
rifampicin or gentamicin and overnight incubation. Results are means and
standard deviations of three independent experiments. Statistically significant
differences are marked with one (P<0.05), two (P<0.005) or three asterisks
(P<0.0005) (unpaired t-test).
In dual-strain biofilms, the biofilm biomass and structure
of HB-1 remained almost unaltered when in consortium with
BB-1 or BB-11nalP (Figure 3A and Figure S5 for additional
biofilm details). In contrast, the phenotypes of the HB-11nalP
mutant, such as increased biomass and reduced viability in
biofilms, appeared to be complemented when this strain was
grown with BB-1, but barely with BB-11nalP (Figures 3A,B
and Figure S5). This complementation was not caused by the
cleavage of the αP from the cell surface of HB-11nalP by
NalP secreted by BB-1 as revealed by Western blotting (Figure
S4B). Consistently, the phenotype of the HB-11nalP mutant
was not complemented when biofilms were formed in culture
supernatants from BB-1. Apparently, the interaction of BB-1
with clusters of HB-11nalP (Figure 2D) had an impact on the
architecture of HB-11nalP biofilms (Figure 3A and Figure S5)
and on the survival of the HB-11nalP bacteria within these
biofilms (Figure 3B).
The biomass and viability of BB-1 in biofilms increased
significantly when it was grown in a consortium with
HB-1 or HB-11nalP (Figures 3A,B). This shows that BB-
1 benefits when mixed with HB-1, but BB-1 benefited
less in consortium with HB-11nalP than with HB-1.
The biomass and viability of BB-11nalP were barely
affected when in consortium with HB-1 or HB-11nalP
(Figures 3A,B).
Biofilms of strain α14 had a lower biomass than those of its
autA mutant derivative (Figure 3A). However, the number of
CFU of the wild type in biofilms appeared higher (Figure 3B).
In consortium with BB-1, the biomass and number of CFU of
α14 and its 1autA mutant derivative were similar or decreased
(Figures 3A,B). The biofilm biomass and CFU of BB-1 were
increased in consortium with α14 or α141autA (Figure 3A) and
also other biofilm parameters were altered (Figure S5). Together
with the results of BB-1 grown in consortium with HB-1 or its
nalP mutant derivative, these data indicate that biofilm formation
of BB-1 increases when it is grown in consortium with other
strains, and the degree of the increase is influenced by the
synthesis of NalP or AutA in the partner strain.
Interbacterial Interactions under Growth
Limiting Conditions
Although Nm strains synthesize a variety of polymorphic toxins
against congeners (Arenas et al., 2013b, 2015b; Jamet et al.,
2015), the results in Figure 3B do not provide evidence of inter-
bacterial competition in dual-strain biofilms. The expression
of toxins in the biofilms was therefore evaluated in Western
blotting assays. TpsA1 was detected in considerably higher
amounts in the medium of disrupted biofilms than in liquid
cultures grown under shaking conditions (Figure S4C). Large
differences in the amounts of TpsA1 between the strains were
detected in the order BB-1 >HB-1 >α14 (Figure S4C). As
we have no suitable antisera available directed against MafB
proteins, we had recourse to an antiserum directed against
the MafA encoded by the Maf Genomic Island (MGI) 3,
where the corresponding gene is located in an operon with
amafB gene (Jamet et al., 2015). This antiserum detected
the MafAMGI−3protein in biofilms of BB-1 and α14 (Figure
S4D), indicating that also MafBMGI−3is expressed in biofilms
of these strains. MafAMGI−3was not detected in HB-1, where
the gene is disrupted by a premature stop codon. Notably,
MafAMGI−3was produced in higher abundance in biofilms than
in liquid cultures (Figure S4D). In conclusion, the apparent
absence of competition in the biofilm assays (Figure 3B) cannot
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Pérez-Ortega et al. Interactions in Meningococcal Biofilms
be explained by the lack of expression of TpsA or MafB
proteins.
Changing the ratio of HB-1 and BB-1 in dual-strain biofilms
to 1:0.2 or 0.2:1, did not result in the suppression of the growth
of either strain in the consortium (data not shown). Considering
that competition between strains is particularly important under
nutrient-limiting conditions, we also studied biofilm formation
in RPMI medium, a synthetic medium that contains a low
concentration of nutrient metals (Stork et al., 2010). In single-
strain biofilms, the autoaggregative character of HB-1 and α14
already observed in TSB medium (Figure 1A) was retained
(Figure 4A), while the biofilm biomass was reduced ∼3-fold
(compare Figure 3A and Figure 4B). Biofilms of BB-1 were
dispersed on the substratum and showed less compact clusters as
compared with those formed in TSB medium. Remarkably, the
biofilm biomass of BB-1 in the nutrient-limited RPMI medium
was increased ∼4-fold relative to that in TSB (compare Figure 3A
and Figure 4B). The three strains revealed a similar biomass
in single-strain biofilms (Figure 4B), but the number of CFU
in 24-h old biofilms varied by orders of magnitude in order
α14 >HB-1 >BB-1 (Figure 4B). In dual strain biofilms, BB-1
formed compact clusters that interacted with those of α14 or HB-
1 (Figure 4C). The biofilm biomass and CFU of BB-1 increased
when in combination with HB-1 and, to a lesser extent, with α14
(Figure 4B). The number of CFU of HB-1 increased significantly
in consortium with BB-1, whilst the CFU of α14 remained
FIGURE 4 | Biofilm formation under nutrient limitation. (A) Biofilms of various strains of Nm grown on RPMI medium. (B) Biomass (left panel) and CFU (right
panel) in biofilms were calculated for each strain in single- and dual-strain biofilms as in Figure 3. The strains present in the consortium are shown at the bottom of
each panel. The data shown are for the strain indicated in bold. Statistically significant differences are marked with one asterisk (P<0.05) or with three asterisks (P<
0.0005) (unpaired t-test). (C) Interbacterial interactions in dual-strain biofilms constituted of BB-1 and HB-1 or α14.
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Pérez-Ortega et al. Interactions in Meningococcal Biofilms
unaltered. In conclusion, under low-nutrient conditions, the
biofilm architecture of the strains and the interactions with other
strains may deviate from those in TSB, but we did not find
evidence for competition.
DISCUSSION
What happens when two Nm strains colonize the nasopharynx
simultaneously? Do they generate mixed biofilms, do they
cooperate, or do they compete? Under conditions that are
limiting for resources, such as in the nasopharynx, strong
competition between Neisseria strains could be expected. This
supposition is based on different studies. First, both Nm and
Nl contain an arsenal of biological weapons, i.e., a variety
of secreted toxins of the TpsA and MafB families (Arenas
et al., 2013b, 2015b; Jamet et al., 2015). These toxins target
congeners to inhibit their growth. Up to date, the growth-
inhibitory activity of these systems has not been demonstrated
in mixed biofilms, although tpsA gene expression was reported
to be up-regulated in 48-h old biofilms (Neil and Apicella,
2009). Second, although colonization of the throat by different
strains has been demonstrated in carriage studies, it was very
rare and detected in only ∼1% of the carriers (Caugant et al.,
2007). Third, a recent study showed that a Nm strain that
uses an eDNA-independent strategy for biofilm formation was
outcompeted in a mixed biofilm by a strain following an eDNA-
dependent strategy (Lappann et al., 2010). In our assays, the
coexistence of two strains resulted in mutual or single benefits
in some combinations. This was obvious for strain BB-1, which
uses an eDNA-independent strategy of biofilm formation. When
BB-1 was in consortium with strains HB-1 or α14 or their
derivatives, which follow an eDNA-dependent strategy, this
strain benefited as evidenced by increased biofilm biomass and
CFU as compared to single-strain biofilms (Figures 3A,B). We
also detected an increase in the biomass of biofilms under
nutrient-limiting conditions when BB-1 was combined with HB-
1 (Figure 4B). These results indicate that BB-1 may acquire
a higher capacity to colonize the host when in consortium
with a strain following an eDNA-dependent strategy for biofilm
formation. Strains following the eDNA-independent strategy
have a reduced capacity to initiate biofilm formation. They
are often isolated from patients with meningococcal disease
rather than from carriers (Lappann et al., 2010), a phenotype
referred to as “spreaders” (Lappann and Vogel, 2010). In
contrast, strains following an eDNA-dependent strategy have a
high biofilm-forming ability; they are more often isolated from
carriers than spreaders and are called “settlers” (Lappann and
Vogel, 2010). Possibly, BB-1 uses the matrix dispersed on the
substratum by eDNA-dependent strains to generate biofilms. In
addition, BB-1 strongly associated with the clusters of NalP−
or AutA+strains, even forming intermixed microcolonies with
the AutA+strain α14. We presume that in this case the eDNA-
binding proteins (NHBA, αP or AutA) of the partner strains
anchor large quantities of eDNA and subsequently other matrix
components at their surface (Arenas and Tommassen, 2017),
thereby promoting the interaction with cells and microcolonies
of the same and other lineages. This would also explain the
pronounced aggregation of 1nalP mutants observed in single-
strain biofilms (Figure 1B) as result of extensive associations of
microcolonies. In any case, it appears that the capacity of BB-1
to colonize a host could be enhanced by public goods produced
by other Nm strains. Another example of profiting from being in
a consortium was strain HB-11nalP, whose survival in mature
biofilms increased by several orders of magnitude when in
consortium with BB-1 (Figure 3B). A possible explanation for
this effect is that BB-1, by interacting with microcolonies of HB-
11nalP, limits the aggregation of these microcolonies, whilst
extensive aggregation may limit nutrient supply. Thus, these
examples demonstrate that Nm strains can profit from growing
in dual-strain biofilms.
Contrary to our expectations, we did not find evidence
for strong competition in dual-strain biofilms even when
we explored biofilm formation under nutrient limitation.
Neisserial strains can coexist in a biofilm, but the level of
interaction between strains is apparently different. In most cases,
we observed biofilms constituted of separate clusters of the
participating strains rather than of intermixed clusters. Such
segregation was even observed between two differently labeled
derivatives of strains HB-1 and α14 (Figure 2A), suggesting that
these clusters were generated by clonal expansion of initially
attached bacteria without recruitment and incorporation of new
planktonic bacteria. This mechanism of microcolony expansion
was also observed in vitro or in animal models for other
mucosal pathogens, for example Vibrio cholerae (Nadell and
Bassler, 2011; Millet et al., 2014). This spatial distribution avoids
competition by contact-dependent growth-inhibition systems
between lineages (Nadell et al., 2016) and explains the coexistence
of microcolonies of different lineages within the same biofilm.
Our results are consistent with those of Lappann and Vogel
(2010) who reported that two Nm strains following the eDNA-
dependent strategy of biofilm formation form mixed biofilms
composed of separate clonal clusters. However, in contrast with
our results, these authors reported that two differently labeled
derivatives of the same strain completely intermix (Lappann
et al., 2006), whilst a strain following an eDNA-independent
strategy of biofilm formation was outcompeted by a strain
following the eDNA-dependent one (Lappann et al., 2010;
Lappann and Vogel, 2010). Differences in mixing behavior may
be due to differences in pilus-mediated motility (Lappann et al.,
2006; Oldewurtel et al., 2015). In our assays, HB-1 and BB-
1 showed twitching motility in biofilms but α14 did not. The
amino-acid sequences of PilE of α14 and HB-1 are identical,
but PilE of α14 showed a lower electrophoretic mobility on
gels as compared to that of HB-1 (Figure S2A), which could be
explained by differences in posttranslational modifications. In
any case, HB-1 and BB-1 were motile, and, whereas differently
labeled BB-1 derivatives did form intermixed clusters, those of
HB-1 did not. Possibly, the strongly aggregative nature of HB-1
prevents intermixing. The lack of inter-strain growth inhibition
in our experiments could not be attributed to a deficiency in
the production of toxins involved in inter-strain competition,
i.e., TpsA and Maf family proteins (Figures S4C,D). Although
we did not test all proteins involved in contact-dependent
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Pérez-Ortega et al. Interactions in Meningococcal Biofilms
growth inhibition, those proteins tested were produced even
in higher levels in biofilms as compared to planktonic cells
in accordance with previous reports (Neil and Apicella, 2009).
Thus, discrepancies with a previous study (Lappann and Vogel,
2010) could be attributed to the use of different strains or
methodologies.
What is the role of the large variety of growth-inhibition
systems in Nm if competition appears very limited? Interestingly,
genetically identical Burkholderia thailandensis strains expressing
different TpsA proteins formed clonal patches in biofilms,
indicating a contribution of TpsA in microcolony segregation
(Anderson et al., 2014). However, growth-inhibition systems in
Nm do not seem to have a similar role in Nm as differently labeled
lineages of the same strain expressing the same growth-inhibition
systems already formed clonal patches in biofilms of HB-1
and α-14. Contact-dependent growth-inhibition systems were
also proposed to have additional functions in self-recognition,
interaction, and interbacterial signaling. TpsA contributes to
biofilm development and structuration in Nm and other bacteria
at least in part by mediating cell-cell interactions (Neil and
Apicella, 2009; Anderson et al., 2012; Ruhe et al., 2015).
Furthermore, transmission of the toxic domain of TpsA to
immune kin cells has been shown to affect gene expression
and promote biofilm formation and other social behaviors
(Garcia et al., 2016). Possibly, these additional functions are
more important than the growth-inhibitory properties of the
systems.
Besides interactions between Nm strains, we observed that
Nm coexist and interact with Nl in biofilms. Nl form part
of the microbiome in the nasopharynx and colonize humans
early in life. To the best of our knowledge, the formation
of Nl biofilms has not been reported before. Probably, Nl
uses similar mechanisms as Nm.Nl biofilms were sensitive
to DNase I, and inspection of available genome sequences
shows that also this species carries nhbA,autA, and nalP
genes. However, it does not contain a gene for IgA protease.
Thus, one may speculate that the exposition of eDNA-binding
region of unprocessed NHBA at the cell surface explains the
aggregative phenotype of these strains and facilitates their
interaction.
To our knowledge, this is the first study that reports
cooperation between Nm strains in biofilms. Cooperative
behaviors are found in multispecies biofilms in nature and they
can be relevant in host-pathogen interactions (Røder et al.,
2016). For example, bacterial coaggregation is an important
cooperative interaction between oral bacteria of different species,
which facilitates the co-adhesion of bacteria to the tooth surface
(Rickard et al., 2003). Our results point in this direction. We
observed that a consortium of two strains can provide mutual or
single benefits and this may facilitate colonization. The variable
production of AutA and NalP proteins balances the spatial
distribution of the strains in the biofilm, as well as the biomass
and final architecture of the biofilm, which may be key to
protection against mechanical forces derived from mucus flow,
coughing or swallowing. Possibly, the long term capacity of
each microcolony to resist the host’s defenses explains the rare
coexistence of two Nm strains in one host.
AUTHOR CONTRIBUTIONS
JA conceived the study; AR, JP, JT, and JA designed the
experiments; AR, JP, ER, and JA performed the experiments; AR,
JP, JT, and JA analyzed the data and AR, JT, and JA wrote the
paper. All authors have read and approved the manuscript.
ACKNOWLEDGMENTS
AR, JP, and ER were supported by personal scholarships for
student mobility (European Commission). We would like to
thank Antonio Sorlózano (University of Granada) for supporting
the scholarship of AR, and Angel Miranda and Natalia Escobar
(Utrecht University) for technical assistance. We also would
like to thank Dr. Reindert Nijland (Wageningen University) for
kindly providing plasmid mut3.1.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: http://journal.frontiersin.org/article/10.3389/fmicb.
2017.00434/full#supplementary-material
Video 1 | https://figshare.com/s/f8826227b634bb63512a
Video 2 | https://figshare.com/s/d05f4d8add7e3957d0c2
Video 3 | https://figshare.com/s/2aa04f555f793c10d646
Video 4 | https://figshare.com/s/eb1abba727476892fc55
REFERENCES
Anderson, M. S., Garcia, E. C., and Cotter, P. A. (2012). The Burkholderia
bcpAIOB genes define unique classes of two-partner secretion and
contact dependent growth inhibition systems. PLoS Genet. 8:e1002877.
doi: 10.1371/journal.pgen.1002877
Anderson, M. S., Garcia, E. C., and Cotter, P. A. (2014). Kind discrimination
and competitive exclusion mediated by contact-dependent growth inhibition
systems shape biofilm community structure. PLoS Pathog. 10:e1004076.
doi: 10.1371/journal.ppat.1004076
Arenas, J., Abel, A., Sánchez, S., Marzoa, J., Berrón, S., van der Ley, P., et al. (2008).
A cross-reactive neisserial antigen encoded by the NMB0035 locus shows high
sequence conservation but variable surface accessibility. J. Med. Microbiol. 57,
80–87. doi: 10.1099/jmm.0.47172-0
Arenas, J., Cano, S., Nijland, R., van Dongen, V., Rutten, L., van der Ende,
A., et al. (2015a). The meningococcal autotransporter AutA is implicated in
autoaggregation and biofilm formation. Environ. Microbiol. 17, 1321–1337.
doi: 10.1111/1462-2920.12581
Arenas, J., de Maat, V., Catón, L., Krekorian, M., Herrero, J. C., Ferrara, F., et al.
(2015b). Fratricide activity of MafB protein of N. meningitidis strain B16B6.
BMC Microbiol. 15:156. doi: 10.1186/s12866-015-0493-6
Arenas, J., Nijland, R., Rodriguez, F. J., Bosma, T. N., and Tommassen, J. (2013a).
Involvement of three meningococcal surface-exposed proteins, the heparin-
binding protein NHBA, the α-peptide of IgA protease and the autotransporter
Frontiers in Microbiology | www.frontiersin.org 13 March 2017 | Volume 8 | Article 434
Pérez-Ortega et al. Interactions in Meningococcal Biofilms
protease NalP, in initiation of biofilm formation. Mol. Microbiol. 87, 254–268.
doi: 10.1111/mmi.12097
Arenas, J., Paganelli, F. L., Rodríguez-Castaño, P., Cano-Crespo, S., van der
Ende, A., van Putten, J. P., et al. (2016). Expression of the gene for
autotransporter AutB of Neisseria meningitidis affects biofilm formation
and epithelial transmigration. Front. Cell. Infect. Microbiol. 6:162.
doi: 10.3389/fcimb.2016.00162
Arenas, J., Schipper, K., van Ulsen, P., van der Ende, A., and Tommassen, J.
(2013b). Domain exchange at the 3’ end of the gene encoding the fratricide
meningococcal two-partner secretion protein A. BMC Genomics 14:622.
doi: 10.1186/1471-2164-14-622
Arenas, J., and Tommassen, J. (2017). Meningococcal biofilm formation: let’s stick
together. Trends Microbiol. 25, 113–124. doi: 10.1016/j.tim.2016.09.005
Casellato, A., Rossi Paccani, S., Barrile, R., Bossi, F., Ciucchi, L., Codolo, G., et al.
(2014). The C2 fragment of Neisseria meningitidis antigen NHBA increases
endothelial permeability by destabilizing adherens junctions. Cell. Microbiol.
16, 925–937. doi: 10.1111/cmi.12250
Caugant, D. A., Tzanakaki, G., and Kriz, P. (2007). Lessons from
meningococcal carriage studies. FEMS Microbiol. Rev. 31, 52–63.
doi: 10.1111/j.1574-6976.2006.00052.x
Cehovin, A., Winterbotham, M., Lucidarme, J., Borrow, R., Tang, C. M., Exley,
R. M., et al. (2010). Sequence conservation of pilus subunits in Neisseria
meningitidis. Vaccine 28, 4817–4826. doi: 10.1016/j.vaccine.2010.04.065
Claus, H., Frosch, M., and Vogel, U. (1998). Identification of a hotspot
for transformation of Neisseria meningitidis by shuttle mutagenesis
using signature-tagged transposons. Mol. Gen. Genet. 259, 363–371.
doi: 10.1007/s004380050823
Claus, H., Maiden, M. C., Wilson, D. J., McCarthy, N. D., Jolley, K. A., Urwin, R.,
et al. (2005). Genetic analysis of meningococci carried by children and young
adults. J. Infect. Dis. 191, 1263–1271. doi: 10.1086/428590
Costerton, J. W., Lewandowski, Z., Caldwell, D. E., Korber, D. R., and Lappin-
Scott, H. M. (1995). Microbial biofilms. Annu. Rev. Microbiol. 49, 711–745.
doi: 10.1146/annurev.mi.49.100195.003431
Del Tordello, E., Vacca, I., Ram, S., Rappuoli, R., and Serruto, D. (2014). Neisseria
meningitidis NalP cleaves human complement C3, facilitating degradation of
C3b and survival in human serum. Proc. Natl. Acad. Sci. U.S.A. 111, 427–432.
doi: 10.1073/pnas.1321556111
Frasch, C. E., and Chapman, S. S. (1972). Classification of Neisseria meningitidis
group B into distinct serotypes. II. Extraction of type-specific antigens for
serotyping by precipitin techniques. Infect. Immun. 6, 127–133.
Garcia, E. C., Perault, A. I., Marlatt, S. A., and Cotter, P. A. (2016).
Interbacterial signaling via Burkholderia contact-dependent growth
inhibition system proteins. Proc. Natl. Acad. Sci. U.S.A. 113, 8296–8301.
doi: 10.1073/pnas.1606323113
Grijpstra, J., Arenas, J., Rutten, L., and Tommassen, J. (2013). Autotransporter
secretion: varying on a theme. Res. Microbiol. 146, 562–582.
doi: 10.1016/j.resmic.2013.03.010
Heydorn, A., Nielsen, A. T., Hentzer, M., Sternberg, C., Givskov, M.,
Ersbøll, B. K., et al. (2000). Quantification of biofilm structures by
the novel computer program COMSTAT. Microbiology 146, 2395–2407.
doi: 10.1099/00221287-146-10-2395
Holten, E. (1979). Serotypes of Neisseria meningitidis isolated from patients in
Norway during the first six months of 1978. J. Clin. Microbiol. 9, 186–188.
Jamet, A., Jousset, A. B., Euphrasie, D., Mukorako, P., Boucharlat, A.,
Ducousso, A., et al. (2015). A new family of secreted toxins in pathogenic
Neisseria species. PLoS Pathog. 11:e1004592. doi: 10.1371/journal.ppat.10
04592
Lappann, M., Claus, H., van Alen, T., Harmsen, M., Elias, J., Molin,
S., et al. (2010). A dual role of extracellular DNA during biofilm
formation of Neisseria meningitidis.Mol. Microbiol. 75, 1355–1371.
doi: 10.1111/j.1365-2958.2010.07054.x
Lappann, M., Haagensen, J. A., Claus, H., Vogel, U., and Molin, S. (2006).
Meningococcal biofilm formation: structure, development and phenotypes
in a standardized continuous flow system. Mol. Microbiol. 62, 1292–1309.
doi: 10.1111/j.1365-2958.2006.05448.x
Lappann, M., and Vogel, U. (2010). Biofilm formation by the human
pathogen Neisseria meningitidis.Med. Microbiol. Immunol. 199, 173–183.
doi: 10.1007/s00430-010-0149-y
Millet, Y. A., Alvarez, D., Ringgaard, S., von Andrian, U. H., Davis, B. M., and
Waldor, M. K. (2014). Insights into Vibrio cholerae intestinal colonization
from monitoring fluorescently labeled bacteria. PLoS Pathog. 10:e1004405.
doi: 10.1371/journal.ppat.1004405
Nadell, C. D., and Bassler, B. L. (2011). A fitness trade-off between local
competition and dispersal in Vibrio cholerae biofilms. Proc. Natl. Acad. Sci.
U.S.A. 108, 14181–14185. doi: 10.1073/pnas.1111147108
Nadell, C. D., Drescher, K., and Foster, K. R. (2016). Spatial structure,
cooperation and competition in biofilms. Nat. Rev. Microbiol. 14, 589–600.
doi: 10.1038/nrmicro.2016.84
Neil, R. B., and Apicella, M. A. (2009). Role of HrpA in biofilm formation of
Neisseria meningitidis and regulation of the hrpBAS transcripts. Infect. Immun.
77, 2285–2293. doi: 10.1128/IAI.01502-08
Oldewurtel, E. R., Kouzel, N., Dewenter, L., Henseler, K., and Maier., B. (2015).
Differential interaction forces govern bacterial sorting in early biofilms. Elife
24:e10811. doi: 10.7554/eLife.10811
Oomen, C. J., van Ulsen, P., van Gelder, P., Feijen, M., Tommassen, J., and Gros,
P. (2004). Structure of the translocator domain of a bacterial autotransporter.
EMBO J. 23, 1257–1266. doi: 10.1038/sj.emboj.7600148
Piet, J. R., Huis in’t Veld, R. A., van Schaik, B. D., van Kampen, A. H., Baas, F., van
de Beek, D., et al. (2011). Genome sequence of Neisseria meningitidis serogroup
B strain H44/76. J. Bacteriol. 193, 2371–2372. doi: 10.1128/JB.01331-10
Pohlner, J., Halter, R., Beyreuther, K., and Meyer, T. F. (1987). Gene structure
and extracellular secretion of Neisseria gonorrhoeae IgA protease. Nature 325,
458–462. doi: 10.1038/325458a0
Pohlner, J., Langenberg, U., Wölk, U., Beck, S. C., and Meyer, T. F.
(1995). Uptake and nuclear transport of Neisseria IgA1 protease-
associated α-proteins in human cells. Mol. Microbiol. 17, 1073–1083.
doi: 10.1111/j.1365-2958.1995.mmi_17061073.x
Ramsey, M. E., Hackett, K. T., Kotha, C., and Dillard, J. P. (2012). New
complementation constructs for inducible and constitutive gene expression in
Neisseria gonorrhoeae and Neisseria meningitidis.Appl. Environ. Microbiol. 78,
3068–3078. doi: 10.1128/AEM.07871-11
Rickard, A. H., Gilbert, P., High, N. J., Kolenbrander, P. E., and Handley,
P. S. (2003). Bacterial coaggregation: an integral process in the
development of multi-species biofilms. Trends Microbiol. 11, 94–100.
doi: 10.1016/S0966-842X(02)00034-3
Røder, H. L., Sørensen, S. J., and Burmølle, M. (2016). Studying bacterial
multispecies biofilms: where to start? Trends Microbiol. 24, 503–513.
doi: 10.1016/j.tim.2016.02.019
Roussel-Jazédé, V., Arenas, J., Langereis, J. D., Tommassen, J., and van
Ulsen, P. (2014). Variable processing of the IgA protease autotransporter
at the cell surface of Neisseria meningitidis.Microbiology 160, 2421–2431.
doi: 10.1099/mic.0.082511-0
Roussel-Jazédé, V., Grijpstra, J., van Dam, V., Tommassen, J., and van Ulsen,
P. (2013). Lipidation of the autotransporter NalP of Neisseria meningitidis
is required for its function in the release of cell-surface-exposed proteins.
Microbiology 159, 286–295. doi: 10.1099/mic.0.063982-0
Ruhe, Z. C., Townsley, L., Wallace, A. B., King, A., Van der Woude, M. W., Low, D.
A., et al. (2015). CdiA promotes receptor-independent intercellular adhesion.
Mol. Microbiol. 98, 175–192. doi: 10.1111/mmi.13114
Saunders, N. J., Jeffries, A. C., Peden, J. F., Hood, D. W., Tettelin, H., Rappuoli, R.,
et al. (2000). Repeat-associated phase variable genes in the complete genome
sequence of Neisseria meningitidis strain MC58. Mol. Microbiol. 37, 207–215.
doi: 10.1046/j.1365-2958.2000.02000.x
Schoen, C., Blom, J., Claus, H., Schramm-Glück, A., Brandt, P., Müller, T., et al.
(2008). Whole-genome comparison of disease and carriage strains provides
insights into virulence evolution in Neisseria meningitidis. Proc. Natl. Acad. Sci.
U.S.A.105, 3473–3478. doi: 10.1073/pnas.0800151105
Serruto, D., Spadafina, T., Ciucchi, L., Lewis, L. A., Ram, S., Tontini, M.,
et al. (2010). Neisseria meningitidis GNA2132, a heparin-binding protein that
induces protective immunity in humans. Proc. Natl. Acad. Sci. U.S.A. 107,
3770–3775. doi: 10.1073/pnas.0915162107
Sim, R. J., Harrison, M. M., Moxon, E. R., and Tang, C. M. (2000). Underestimation
of meningococci in tonsillar tissue by nasopharyngeal swabbing. Lancet 356,
1653–1654. doi: 10.1016/S0140-6736(00)03162-7
Stork, M., Bos, M. P., Jongerius, I., de Kok, N., Schilders, I., Weynants, V.
E., et al. (2010). An outer membrane receptor of Neisseria meningitidis
Frontiers in Microbiology | www.frontiersin.org 14 March 2017 | Volume 8 | Article 434
Pérez-Ortega et al. Interactions in Meningococcal Biofilms
involved in zinc acquisition with vaccine potential. PLoS Pathog. 6:e1000969.
doi: 10.1371/journal.ppat.1000969
Turner, D. P., Wooldridge, K. G., and Ala’Aldeen, D. A. (2002). Autotransported
serine protease A of Neisseria meningitidis: an immunogenic, surface-exposed
outer membrane, and secreted protein. Infect. Immun. 70, 4447–4461.
doi: 10.1128/IAI.70.8.4447-4461.2002
van Ulsen, P., Rutten, L., Feller, M., Tommassen, J., and van der Ende,
A. (2008). Two-partner secretion systems of Neisseria meningitidis
associated with invasive clonal complexes. Infect. Immun. 76, 4649–4658.
doi: 10.1128/IAI.00393-08
van Ulsen, P., van Alphen, L., ten Hove, J., Fransen, F., van der
Ley, P., and Tommassen, J. (2003). A Neisserial autotransporter
NalP modulating the processing of other autotransporters.
Mol. Microbiol. 50, 1017–1030. doi: 10.1046/j.1365-2958.2003.
03773.x
Yi, K., Rasmussen, A. W., Gudlavalleti, S. K., Stephens, D. S., and Stojiljkovic,
I. (2004). Biofilm formation by Neisseria meningitidis.Infect. Immun. 72,
6132–6138. doi: 10.1128/IAI.72.10.6132-6138.2004
Conflict of Interest Statement: The authors declare that the research was
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be construed as a potential conflict of interest.
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