Relationship Between Quorum Sensing and Secretion Systems

Abstract

Quorum sensing (QS) is a communication mechanism between bacteria that allows specific processes to be controlled, such as biofilm formation, virulence factor expression, production of secondary metabolites and stress adaptation mechanisms such as bacterial competition systems including secretion systems (SS). These SS have an important role in bacterial communication. SS are ubiquitous; they are present in both Gram-negative and Gram-positive bacteria and in Mycobacterium sp. To date, 8 types of SS have been described (T1SS, T2SS, T3SS, T4SS, T5SS, T6SS, T7SS and T9SS). They have global functions such as transport of proteases, lipases, adhesins, heme-binding proteins, amidases; and specific functions such as the synthesis of proteins in host cells, adaptation to the environment, the secretion of effectors to establish an infectious niche, transfer, absorption and release of DNA, translocation of effector proteins or DNA and autotransporter secretion. All of these functions can contribute to virulence and pathogenesis. In this review, we describe all types of secretion systems and discuss the ones that have been shown to be regulated by QS. Due to the large amount of information about this topic in some pathogens, we focus mainly on Pseudomonas aeruginosa and Vibrio sp.

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fmicb-10-01100 June 6, 2019 Time: 20:9 # 1
REVIEW
published: 07 June 2019
doi: 10.3389/fmicb.2019.01100
Edited by:
Tom Defoirdt,
Ghent University, Belgium
Reviewed by:
Romé Voulhoux,
UMR7255 Laboratoire d’Ingénierie
des Systèmes Macromoléculaires
(LISM), France
Jin Zhou,
Tsinghua University, China
*Correspondence:
Rodolfo García-Contreras
rgarc@bq.unam.mx
Maria Tomás
MA.del.Mar.Tomas.Carmona@
sergas.es
†These authors have contributed
equally to this work
Specialty section:
This article was submitted to
Microbial Physiology and Metabolism,
a section of the journal
Frontiers in Microbiology
Received: 20 February 2019
Accepted: 30 April 2019
Published: 07 June 2019
Citation:
Pena RT, Blasco L, Ambroa A,
González-Pedrajo B,
Fernández-García L, López M,
Bleriot I, Bou G, García-Contreras R,
Wood TK and Tomás M (2019)
Relationship Between Quorum
Sensing and Secretion Systems.
Front. Microbiol. 10:1100.
doi: 10.3389/fmicb.2019.01100
Relationship Between Quorum
Sensing and Secretion Systems
Rocio Trastoy Pena1†, Lucia Blasco1†, Antón Ambroa1, Bertha González-Pedrajo2,
Laura Fernández-García1, Maria López1, Ines Bleriot1, German Bou1,
Rodolfo García-Contreras3*†, Thomas Keith Wood4and Maria Tomás1*†
1Deapartamento de Microbiología y Parasitología, Complejo Hospitalario Universitario A Coruña (CHUAC), Instituto
de Investigación Biomédica (INIBIC), Universidad de A Coruña (UDC), A Coruña, Spain, 2Departamento de Genética
Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico, 3Departamento
de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City, Mexico,
4Department of Chemical Engineering, Pennsylvania State University, University Park, PA, United States
Quorum sensing (QS) is a communication mechanism between bacteria that allows
specific processes to be controlled, such as biofilm formation, virulence factor
expression, production of secondary metabolites and stress adaptation mechanisms
such as bacterial competition systems including secretion systems (SS). These SS have
an important role in bacterial communication. SS are ubiquitous; they are present in
both Gram-negative and Gram-positive bacteria and in Mycobacterium sp. To date, 8
types of SS have been described (T1SS, T2SS, T3SS, T4SS, T5SS, T6SS, T7SS, and
T9SS). They have global functions such as the transport of proteases, lipases, adhesins,
heme-binding proteins, and amidases, and specific functions such as the synthesis
of proteins in host cells, adaptation to the environment, the secretion of effectors to
establish an infectious niche, transfer, absorption and release of DNA, translocation
of effector proteins or DNA and autotransporter secretion. All of these functions can
contribute to virulence and pathogenesis. In this review, we describe the known types
of SS and discuss the ones that have been shown to be regulated by QS. Due to the
large amount of information about this topic in some pathogens, we focus mainly on
Pseudomonas aeruginosa and Vibrio spp.
Keywords: quorum, secretion, virulence, motility, competence
INTRODUCTION
Microorganisms coexist in competitive environments with other species, and they must develop
different survival strategies to compete for space, nutrients and ecological niches. Bacteria have
developed several molecular mechanisms that enable them to survive under stress conditions
in different environments. The general stress response (RpoS) (Battesti et al., 2011), tolerance
to reactive oxygen species (ROS) (Zhao and Drlica, 2014;Van den Bergh et al., 2017), energy
metabolism (cytochrome bd complex) (Korshunov and Imlay, 2010) and Tau metabolism (Javaux
et al., 2007), drug efflux pumps (Blanco et al., 2016), SOS response (Baharoglu and Mazel, 2014),
(p)ppGpp signaling under starvation conditions (Hauryliuk et al., 2015), toxin-antitoxin (TA)
systems (Wood et al., 2013) and quorum sensing (QS), which we will discuss in detail in this
review, are the main molecular mechanisms of tolerance and bacterial persistence (Harms et al.,
2016;Trastoy et al., 2018).
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Pena et al. Secretion Systems and Quorum Sensing
Quorum sensing acts by monitoring cell density through
chemical signals that allow communication between bacteria in
order to regulate the expression of genes involved in virulence,
competition, pathogenicity and resistance (Nealson et al., 1970;
Hawver et al., 2016;Paul et al., 2018). In general, QS systems
are species-dependent and contribute to processes such as cell
maintenance, biofilm formation and horizontal gene transfer. QS
also plays a role in other events involving the synchronization
of the whole population such as antibiotic production (Abisado
et al., 2018), natural competence (Shanker and Federle, 2017),
sporulation (Rai et al., 2015) and the expression of secretion
systems (SS). In this review, we will focus on the relationship
between QS networks and SS in two important bacterial
pathogens Pseudomonas aeruginosa and Vibrio spp.
QS NETWORK
To explain the structure and functioning of the QS network,
we will focus on Gram-negative bacteria, in which the signaling
pathways are better described. In general terms, QS systems
are composed of synthase proteins that produce QS signals,
QS signals, and response regulators that bind QS signals and
reprogram gene expression (Ng and Bassler, 2009). N-acyl
homoserine lactones (AHLs) are the most common QS signals
in Gram-negative bacteria (Geske et al., 2008). Other QS signals
include autoinducer-2 (AI-2) in Vibrio harveyi (Surette et al.,
1999), PQS (Pseudomonas quinolone signal) (Pesci et al., 1999),
DSF (diffusible signaling factor) in Xanthomonas campestris
(Barber et al., 1997), indole in Escherichia coli (Lee and Lee, 2010),
and PAME (hydroxyl-palmitic acid methyl ester) in Ralstonia
solanacearum (Flavier et al., 1997). The LuxI/LuxR QS system of
Vibrio fischeri is the prototypical model system for Gram-negative
bacteria (Engebrecht et al., 1983;Engebrecht and Silverman,
1984). Homologs of luxI (which encode synthase proteins) and
luxR (which encode response regulators) are present in many
bacteria (Case et al., 2008). AHL signals are produced inside
the cell and most of them are transported freely to the local
environment. When the concentration of AHL reaches a certain
level outside of the cell, the molecule re-enters the cell (or binds
surface receptors) and binds/activates the LuxR-type receptor
to alter gene expression. AHL signals with small structural
differences are involved in the process of gene regulation (Fuqua
et al., 1994;Whiteley et al., 2017;Paul et al., 2018).
Pseudomonas aeruginosa possesses three well-known
QS systems: LasI/LasR, RhlI/RhlR, and PQS (Pseudomonas
quinolone signal)/PqsR (MvfR). The Las system consists
of LasI, a synthase protein which produces the AHL
N-(3-oxododecanoyl)-L-homoserine lactone (3O-C12-HSL), and
LasR, the transcriptional regulator (Seed et al., 1995;Stintzi et al.,
1998;Kariminik et al., 2017). Likewise, the RhlI/RhlR system
produces the N-hexanoyl-L-homoserine lactone (C4-HSL)
signal and the RhlR transcriptional regulator. Finally, the PQS
system comprises 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS
signal) and the PqsR (MvfR) receptor (Xiao et al., 2006;Jimenez
et al., 2012). In 2016, James and collaborators, analyzed the
role of a new binding receptor for PQS signals, i.e., MexG, an
inner membrane protein of the mexGHI-opmD operon and a
component of a resistance-nodulation-cell division (RND) efflux
pump (Hodgkinson et al., 2016).
Quorum quenching (QQ) enzymes have also been shown
to be important in the functioning of QS systems (Zhang and
Dong, 2004;Dong et al., 2007;Bzdrenga et al., 2017). Our
research group has recently described a new QQ enzyme (AidA)
which participates in the QS network in Acinetobacter baumannii
clinical strains (Lopez et al., 2017b, 2018).
SECRETION SYSTEMS
Bacterial pathogens secrete proteins through their cell
membranes in a fundamental process that enables them to
attack other microorganisms, evade the host immune system,
produce tissue damage and invade the host cells. Secreted
proteins can act as virulence factors that generate toxic products
to the host cells and may also facilitate adhesion to these cells.
Translocation of proteins across the phospholipid membranes
is carried out by several types of SS (Green and Mecsas, 2016).
SS play a significant role in bacterial communication. To date,
8 types of SS (T1SS, T2SS, T3SS, T4SS, T5SS, T6SS, T7SS, and
T9SS) have been made defined on their structure, composition
and activity (Figure 1). These differences can be attributed to the
differences between Gram-negative and Gram-positive bacteria
(Desvaux et al., 2009;Sato et al., 2010;Costa et al., 2015). The
characteristics of each type of SS are described in detail below.
T1SS
The type I secretion system is widely distributed in
Gram-negative bacteria such as P. aeruginosa,Salmonella
enterica,Neisseria meningitidis, and E. coli (Thomas et al., 2014).
The type I secretion system (T1SS), which has three structural
elements (ABC transporter protein, a membrane fusion protein
and an outer membrane factor), can transfer substrates across
both bacterial membranes in Gram negative bacteria in a
one-step process (Green and Mecsas, 2016). T1SS uses proteins
as substrates, e.g., proteases and lipases of different sizes and with
different functions; these proteins have a C-terminal uncleaved
secretion signal which is recognized by the ABC transporter
protein to form the translocation complex (Delepelaire, 2004;
Kanonenberg et al., 2013).
There are two systems described so far that regulate the
expression and secretion of substrates of T1SS, the Has system
of S. marcescens and P. aeruginosa, and the hemolysins of Vibrio
cholerae,N. meningitidis and in particular of uropathogenic E. coli
(Thomas et al., 2014).
T2SS
The type II secretion system (T2SS), which is conserved
in most Gram negative bacteria, is responsible for secreting
folded proteins from the periplasm. These proteins are first
transported through the IM by the general secretory (Sec) or
twin-arginine translocation (Tat) pathways, and then secreted
from the periplasm into the extracellular medium by the T2SS
(Nivaskumar and Francetic, 2014;Green and Mecsas, 2016).
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FIGURE 1 | Structure of secretion systems. Schematic representation of secretory systems: type I (T1SS), type II (T2SS), type V (T5SS), type IX (T9SS), type III
(T3SS), type IV (T4SS), type VI (T6SS), and type VII (T7SS). The type I pathway is exemplified by hemolysin A (HlyA) secretion in E. coli where TolC, HlyD, and HlyB
are the three components which constitute the channel to transport HlyA to extracellular space. Sec (general secretion route) and Tat (twin-arginine translocation
pathway) transfer the substrates of T2SS and T5SS across the inner membrane. Sec also participates in the transport of T9SS substrates across de inner
membrane. T9SS is also called Por Secretion System (PoSS). T4SS is represented by VirB/D system of Agrobacterium tumefaciens. The T7SS is based on a system
in Mycobacteria. Red squares represent the ATPases. HM, host membrane; EM, extracellular medium; OM, outer membrane; IM, inner membrane; MM,
mycomembrane. Substrates secreted by each secretory system are included in a circle in the top of the figure. Adapted from Tseng et al. (2009).
The Sec pathway consists of three structural parts: a
protein targeting component, a motor protein and a membrane
integrated conducting channel called SecYEG translocase. This
mechanism transports unfolded proteins with a hydrophobic
sequence at the N-terminus. Moreover, the secreted protein either
remains in the periplasm or is transported to the extracellular
space. The proteins may contain a SecB-specific signal sequence
for transport to the periplasm or the extracellular milieu;
however, if it has the signal recognition particle (SRP) signal it
can follow the SRP pathway and remain in the inner membrane
(Green and Mecsas, 2016;Tsirigotaki et al., 2017).
By contrast, the Tat secretion pathway consists of 2–3 subunits,
TatA and TatB, which form a unique multifunctional protein in
Gram-positive bacteria, and TatC. This mechanism translocates
folded proteins with a twin-arginine motif. In Gram-positive
bacteria, most proteins are transported out of the cell, while in
Gram-negative bacteria the protein can remain in the periplasm
or it can be translocated to the extracellular space by the T2SS
(Patel et al., 2014;Green and Mecsas, 2016).
The T2SS, a complex structure composed of 15 proteins,
named general secretion pathway proteins (Gsp) in E. coli
(Korotkov et al., 2012), Eps in V. cholera (Abendroth et al.,
2009;Sloup et al., 2017) and Xcp in P. aeruginosa (Filloux et al.,
1998;Robert et al., 2005), has a wide range of substrates with
diverse functions, although all share one feature, an N-terminal
signal which enables them pass to the periplasm via the Sec
or Tat secretion mechanisms (Nivaskumar and Francetic, 2014;
Green and Mecsas, 2016).
The main function of the T2SS is to acquire nutrients
(Nivaskumar and Francetic, 2014). It is responsible for secreting
numerous exoproteins, most of which are hydrolytic enzymes
and other proteins such as toxins, adhesins and cytochromes
that have various roles in respiration, biofilm formation and
motility (Nivaskumar and Francetic, 2014). The T2SS has been
described in various environmental strains and also human
pathogens such as V. cholera (Overbye et al., 1993), P. aeruginosa,
Aeromonas sp. and enterotoxigenic Escherichia coli (ETEC)
(Nivaskumar and Francetic, 2014).
T3SS
The type III secretion system (T3SS) or injectisome, is
a double-membrane-embedded apparatus found in multiple
pathogenic Gram-negative bacteria such as Salmonella spp.,
Yersinia spp., enteropathogenic and enterohemorrhagic E. coli,
Shigella spp. and Pseudomonas spp. (Cornelis, 2006;Gaytan
et al., 2016;Deng et al., 2017). This complex nanomachine
promotes the transfer of virulence proteins called effectors from
the bacterial cytoplasm into the eukaryotic cell in a single step
(Galan and Waksman, 2018).
The T3SS is composed of approximately 25 proteins assembled
in three main structures: the basal body, a set of rings spanning
the two membranes of the bacterium; a hollow needle-shaped
component through which the semi-unfolded effectors are
transported (these first two structures are collectively called
“needle complex”); and the translocon, made up of a hydrophilic
protein that serves as a scaffold for forming a translocation
pore, constituted by two hydrophobic proteins, which is inserted
into the host cell membrane and through which effectors are
directly translocated. A unique set of effectors is delivered by each
pathogen, which subverts specific host-cell signaling pathways to
allow bacterial colonization (Izore et al., 2011;Notti and Stebbins,
2016;Deng et al., 2017).
The export apparatus associated with the basal body is formed
by five poly topic inner membrane proteins that are essential
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for substrate secretion. This protein complex, together with
a cytoplasmic sorting platform and the ATPase complex are
responsible for substrate recruitment and classification, and
for energizing the secretion process enabling chaperone-effector
dissociation and protein unfolding for initial entry into the
T3SS central channel that serves as the secretion pathway. These
components are highly conserved between different T3SS systems
and with the flagella, which is evolutionarily related to the
injectisome (Abby and Rocha, 2012;Galan and Waksman, 2018;
Lara-Tejero and Galan, 2019).
Several effectors of T3SS have been described such as ExoS,
ExoT, ExoU, and ExoY in P. aeruginosa; Tir and EspE in E. coli
and YopE, YopH, YopM, YopJ/P, YopO/YpkA, and YopT in
Yersinia sp. (Cornelis and Van Gijsegem, 2000).
T4SS
The type IV secretion system family is found in Gram-negative
and Gram-positive bacteria as well as in Archaea. T4SS is the
most cosmopolitan secretion system and differs from other SS as
it is able to transfer DNA in addition to proteins (Cascales and
Christie, 2003). More specifically, T4SS is capable of performing
contact-dependent secretion of effector molecules into eukaryotic
cells, conjugative transfer of mobile DNA elements and also
exchange of DNA without any contact with the outside of
the cell (Green and Mecsas, 2016;Grohmann et al., 2018).
T4SS can be divided on the basis of its functionality into
two subfamilies: conjugation systems and effector translocators.
Conjugation systems are responsible for the transfer of antibiotic
resistance genes and virulence determinants among bacteria.
The effector translocators introduce virulence factors into the
host cell (Christie, 2016). However, in Gram-negative bacteria
T4SS has been divided into two different subfamilies: IVA and
IVB. The E. coli conjugation apparatuses and VirB/D system
of Agrobacterium tumefaciens are the models used to study the
structure of type IVA of T4SS (Grohmann et al., 2018). The
VirB/D apparatus consists of 12 proteins which form a complex
envelope-spanning structure that facilitate the translocation
function. Two of these proteins, VirB2 and VirB5, make up
the pilus, while another three proteins act as ATPases, and
VirB1 is a lytic transglycosylase (Costa et al., 2015;Green and
Mecsas, 2016). The Legionella pneumophila Dot/Icm (Defective
for organelle trafficking/Intracellular multiplication) system is
the model used to study the IVB subfamily of T4SS (Nagai and
Kubori, 2011;Grohmann et al., 2018).
T5SS
The type V secretion system is unique because its substrates
transport themselves across the outer membrane. The substrates
use the Sec translocase to pass through the inner membrane
to the periplasm space. Various different types of T5SS
have been identified: autotransporters (T5aSS), two-partner
passenger-translocators (T5bSS), trimeric autotransporters
(T5cSS), hybrid autotransporters (T5dSS) and inverted
autotransporters (T5eSS) (Henderson et al., 2004;Leo et al.,
2012;Rojas-Lopez et al., 2017). In general, the T5SS transports
proteins across the asymmetric outer membrane (OM) that
contains lipopolysaccharides, through their own C-terminal
translocation domain that inserts into the OM as a β-barrel
to complete the secretion of the N-terminal passenger domain
via the barrel pore. Several periplasmic chaperones also
participate in transport through the OM, specifically the β-barrel
assembly machinery (BAM complex) and the translocation and
assembly module (TAM complex) facilitate protein secretion
(Rojas-Lopez et al., 2017).
A T5SS has been described in human pathogens
such as Bordetella pertussis and Haemophilus influenzae,
which have two-partner SS and uropathogenic E. coli,
which has chaperone-usher systems (Costa et al., 2015;
Green and Mecsas, 2016).
YadA of Yersinia enterocolitica and SadA of Salmonella are
T5SS type c (Leo et al., 2012). Intimin of E. coli and invasin of
enteropathogenic Yersinia spp. are type Ve SS (Leo et al., 2012).
A self-transporter (T5aSS) (Wilhelm et al., 2007) and three
T5bSS: LepA /LepB system (Kida et al., 2008), the CupB system
(Ruer et al., 2008) and PdtA/PdtB system (Faure et al., 2014),
have been reported in P. aeruginosa. In B. cenocepacia, four
T5SS (Holden et al., 2009) have been found, two with pertactin
domains and two with haemagglutinin autotransporters; this last
type is also present in S. maltophilia (Ryan et al., 2009).
T6SS
The type VI secretion system is widely represented in
Gram-negative bacteria (Coulthurst, 2013;Gallique et al., 2017b).
T6SS is an integrated secretion device within the membrane and
it transfers substrates, which are toxic effectors to eukaryotic
(Pukatzki et al., 2007) and prokaryotic cells (Russell et al., 2014).
It plays a crucial role in the pathogenesis and competition
among bacteria (Ho et al., 2014;Zoued et al., 2014;Costa
et al., 2015;Gallique et al., 2017a). The origin of T6SS is
related to bacteriophages (Leiman et al., 2009). T6SS is a huge
apparatus and consists of 13 core components organized into
atrans-membrane complex, a baseplate-like structure at the
cytoplasmic face of the inner membrane, and a sheathed inner
tube, which is the effector delivery module that is ejected to
the target cell. The tube-sheath complex is assembled from the
baseplate in the cytoplasm and the hollow tube is built from
hexamers of the hemolysin co-regulated protein (Hcp). The
sheath contracts and pushes the tube with the associated effectors
into targeted cells, using a puncturing mechanism similar to
the one used by the contractile tails of phages (Russell et al.,
2011, 2014;Cianfanelli et al., 2016;Green and Mecsas, 2016;
Galan and Waksman, 2018).
T7SS
Type VII secretory system has been described in some
Gram-positive bacteria such as Staphylococcus aureus and
in species of Mycobacterium and Corynebacterium. This SS
was reported for the first time in 2003 in Mycobacterium
tuberculosis and it was called ESX-1 (Stanley et al., 2003),
which is an important virulence factor in M. tuberculosis. To
date, five T7SS have been identified in Mycobacterium sp. but
the transport mechanisms across the mycobacterial membrane
are almost unknown (Costa et al., 2015;Ates et al., 2016;
Green and Mecsas, 2016).
Most of the substrates of T7SS belong to EscAB clan which
includes six protein families: Esx, PE, PPE, LXG, DUF2563,
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and DUF2580. ESAT-6 is a M. tuberculosis protein which
belongs to Esx family and which is secreted with EsxB (CFP-10)
(Ates et al., 2016).
T9SS
The type IX secretion system (T9SS) or Por secretion system
(PorSS) is the most recently discovered system (Lasica et al.,
2017). Its function is to transport molecules across the outer
membrane. Its substrates must include a Sec signal, which allows
transfer of proteins through the inner membrane with the aid of
the Sec system. The T9SS system has been described in almost
all members of the phylum Bacteroidetes, but it has mainly been
studied in oral pathogens such as Porphyromonas gingivalis and
Tannerella forsythia. In P. gigivalis, the T9SS system consists of 16
proteins with structural and functional activity, and another two
proteins involved in the regulation of the transport process (Sato
et al., 2010;Lasica et al., 2017).
REGULATION OF SECRETION SYSTEMS
BY QUORUM SENSING NETWORKS
(TABLE 1)
Pseudomonas aeruginosa
T1SS
Transcriptional studies in P. aeruginosa suggest that in this
bacterium T1SS is positively regulated by QS, since the
expression of its effector, the alkaline protease AprA, depends
on QS. In addition, the genes of the AprA inhibitor aprI
and the structural genes aprDEF also appear to be positively
regulated by QS (Hentzer et al., 2003;Schuster et al., 2003;
Wagner et al., 2003).
T2SS
Three T2SS systems, the Xcp, Hxc and Txc systems, have been
described in P. aeruginosa. The first of these, Xcp, secretes
the QS regulated virulence factors elastase A and B (LasA
and LasB) as well as the exotoxin A (ExoA) and it is itself
positively regulated by QS (Figure 2). Accordingly, recently it
was demonstrated by ChIPseq analysis that MvfR (the receptor
of the PQS autoinducer) is able to directly bind xcpQ-xcpP-xcpR
regions and this is related to their induction in the presence of
MvfR (Maura et al., 2016).
The second T2SS, Hxc, is regulated by the availability of
phosphate and secretes LapA a low-molecular weight alkaline
phosphatase (Wagner et al., 2003;Michel et al., 2007). Two
genes, xphA and xqhA, which encode the PaQa subunit of
the Xcp functional hybrid system, have been described. These
genes, which are located outside the xcp locus, are regulated by
environmental conditions but not by QS, in contrast to what
occurs with the rest of the Xcp system (Michel et al., 2007, 2011).
In contrast to the first two systems, the third system Txc has
just recently been described and so far only identified in a
region of genome plasticity of the strain PA7; it is regulated by
a two component system (TtsSR) and secretes the chitin binding
protein CpbE (Cadoret et al., 2014).
T3SS
Current evidence suggest that as in Vibrio spp., QS in
P. aeruginosa negatively regulates the expression of T3SS,
specifically the RhlI/RhlR system, as transcription of the
T3SS genes and secretion of ExoS increase significantly in
arhlI mutant and return to basal levels on the addition
of exogenous C4-HSL (Bleves et al., 2005;Kong et al.,
2009;Figure 2). In agreement, the expression of exoS is
also negatively regulated by QS, specifically by the RhlI/RhlR
system, as well as by the stationary phase sigma factor RpoS
(Hogardt et al., 2004).
The fact that the T3SS genes do not appear to be
repressed by QS in some global transcriptomic studies with
mutants may be explained by the presence of high calcium
concentrations in the media, or by the lack of resolution
of DNA microarrays (Hentzer et al., 2003;Schuster et al.,
2003). More striking is the fact that some QS inhibitors
like 6-gingerol and coumarin inhibit rather than increase the
expression of T3SS (Zhang et al., 2018). Nevertheless, these
studies were done in the presence of high calcium, and
QS-independent inhibition of T3SS has not been ruled out.
Moreover, a recent study in the PA01 strain, using a lasR
rhlR double mutant, demonstrated that it remains virulent in
a murine abscess model, despite that it does not produce QS-
dependent virulence factors and that the secretion of ExoT
and ExoS is fully functional in this mutant. Hence the authors
hypothesized that T3SS is the cause of the remaining virulence
(Soto-Aceves et al., 2019).
The P. aeruginosa QS network and its T3SS are also related
by the fact that VqsM, an AraC-family transcription factor, binds
to both the promoter region of lasI and the promoter of exsA,
which encodes a master regulator of the T3SS, regulating both
mechanisms (Liang et al., 2014;Figure 2).
T6SS
The T6SS is involved in iron transport, and a connection
has been observed between T6SS and QS through the TseF
protein, which is a substrate of T6SS and interacts with PQS
(Lin et al., 2017;Figure 2).
In P. aeruginosa, three loci which encode T6SS have been
found to be regulated by QS proteins (LasR and MvfR) (Lesic
et al., 2009). Expression of the second loci, H2-T6SS, is regulated
by the Las and Rhl QS systems in PAO1 strains (Sana et al.,
2012;Figure 2) and by the direct binding of MvfR in PA14
(Maura et al., 2016).
Vibrio sp.
T2SS
The formation of biofilms has multifactorial regulation in
V. cholerae as in other pathogens. The QS network controls
directly biofilm production which is related to type II secretion
system in V. cholerae (Teschler et al., 2015). Several proteins such
as RbmA, RbmC and Bap1, which are involved in the formation
of biofilms, are transported by T2SS. In addition, mutant strains
with inactivated T2SS have reduced biofilm formation (Johnson
et al., 2014;Teschler et al., 2015).
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TABLE 1 | Pathogens and QS elements related to secretion systems.
Type secretion
system
SS element QS regulation QS element Microorganisms References
T1SS Lip +Swr Serratia liquefaciens Riedel et al., 2001
T2SS Xcp +lasR/lasI rhLR/rhlI Pseudomonas aeruginosa Wagner et al., 2003;Michel
et al., 2007
DSF-type Xanthomonas species Qian et al., 2013
T3SS LEE operon +luxS Escherichia coli Sperandio et al., 1999
–Vibrio parahaemolyticus Vibrio
harveyi
Henke and Bassler, 2004
ExsA lasI Pseudomonas aeruginosa Liang et al., 2014
Yop-Ysc +Hfq Yersinia pseudotuberculosis
Yersinia pestis
Schiano et al., 2014
T4SS VirB/D +VjbR (LuxR-type QS) Brucella abortus Arocena et al., 2010;Li
et al., 2017
+luxI Roseobacter group Patzelt et al., 2013, 2016
T6SS Vibrio alginolyticus Yang et al., 2018
Hcp HapR and LuxO Vibrio cholerae Ishikawa et al., 2009;
Zheng et al., 2010;Kitaoka
et al., 2011;Leung et al.,
2011
Burkholderia thailandensis Majerczyk et al., 2016
AHL Pseudomonas fluorescens Gallique et al., 2017b
TseF PQS Pseudomonas spp. Lin et al., 2017
LasR and MvfR Pseudomonas aeruginosa Lesic et al., 2009
VipA,Hcp-1, VipB AbaR/AbaI Acinetobacter baumannii Lopez et al., 2017b
T3SS
In V. parahaemolyticus and V. harveyi (unlike in E. coli),
both the HAI-1 and AI-2 QS systems inhibit the expression
of T3SS genes (Henke and Bassler, 2004). QS also represses
T3SS during V. harveyi infections of gnotobiotic brine shrimp
(Ruwandeepika et al., 2015). Waters et al. (2010) have described
the regulatory pathway by which QS controls T3SS. At low cell
density when LuxR is repressed, which entails the derepression
of two promoters of the exsBA operon and the exsA operon,
ExsA activates the expression of genes that encode the structural
proteins of the type III secretion system. However, when the cell
density is high, LuxR directly represses transcription of the PB
promoter, preventing the production of ExsA and consequently
decreasing the expression of structural genes of T3SS (Waters
et al., 2010;Ball et al., 2017). OpaR inhibits the T3SS1 in
V. parahaemolyticus which is the most important factor in its
cytotoxicity (Gode-Potratz and McCarter, 2011).
T6SS
Several researchers have demonstrated the regulation of T6SS
by QS networks in Vibrio spp. We present the main findings
in this field here. In V. alginolyticus, activation of T6SS and
the QS network has been found to be coordinated by the
serine/threonine kinase PpkA cascade (Yang et al., 2018). PpkA2
is autophosphorylation and it transfers the phosphate group to
VstR. Phosphorylated VstR promotes the expression of both of
the T6SS in V. alginolyticus through the inhibition of LuxO
activity, which acts to impede the expression of LuxR, a promoter
of the T6SS. LuxR inhibits the expression of the first T6SS
(T6SS1) or promotes the expression of the second T6SS (T6SS2)
(Yang et al., 2018).
At low cell population density, LuxO is phosphorylated,
which activates the expression of specific small regulatory
RNAs (sRNAs) in conjunction with alternative sigma factor σ54
(Sheng et al., 2012). sRNAs inhibit the expression of LuxR with
the help of RNA chaperone Hfq (Liu et al., 2011). However, at
high cell population density, LuxO is dephosphorylated turning
off the transcription of the sRNAs and allowing the translation
of LuxR (Waters and Bassler, 2005;Milton, 2006). Sheng et al.
(2012) also demonstrated that the expression of the hcp T6SS
gene is growth phase-dependent and the QS regulators controls
the haemolysin co-regulated protein, which is one of the main
proteins of the T6SS functioning as an effector of the system
and/or an effector binding protein (Figure 2). The phosphatase
PppA also acts on the QS (modulating the transcription of LuxR)
and the expression and secretion of hcp1 and hcp2 (Sheng et al.,
2013). It is important to highlight that PppA permits the cross-
talk between the two T6SS in V. alginolyticus (Sheng et al., 2013).
Rpo N (σ54) collaborates with QS in the regulation of T6SS
genes. It is involved in the regulation of the expression of
hcp and vgrG3 operons that encode T6SS secreted molecules,
but does not control the genes that encode the structural
and sheath components of T6SS (Ishikawa et al., 2009;
Dong and Mekalanos, 2012).
There are a few more studies in V. cholerae related to
this topic than other species. Two QS autoinducers, CAI-I
(cholerae autoinducer) and AI-2 (autoinducer-2), co-operate
to control the gene expression depending on the cell density
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FIGURE 2 | Secretion systems and QS network elements. The figure shows the relationship between QS networks and expression of secretion systems (blue
squares). The genes regulated by QS are in purple boxes. Each QS network is represented by a different color. Starting at the top right of the figure: The swr QS
system of S. liquefaciens controls the lipB genes of the T1SS (orange); Ax21 (QS effector) and QS system Rax regulate RaxABC TOSS (T1SS) in gram negative
bacteria (ochre); QS (RhlIR and LasIR) regulates expression of T2SS, T3SS, and T6SS in P. aeruginosa (brown); VqsM (an AraC family transcription factor) interacts
with the LasIR and ExsA promoters (a master regulator of T3SS) in P. aeruginosa (dark orange); T2SS is regulated by LuxS/LuxI/AI-2 QS in E. coli and indole
production by TnA (tryptophanase) regulates esp genes expression (T2SS) in this bacterium (blue); in Yersinia sp. the Hfq chaperone is connected with QS (AI-2) and
regulates the Yop-Ysc type III secretion system (T3SS) (green); in Xanthomonas sp. T2SS and T3SS are regulated by DSF (diffusible signal factor) which is a quorum
sensing signal (yellow); T4SS (virB operon) is regulated by VjbR (LuxR like protein) and LuxI in Brucella (turquoise) and Roseobacter (pink), respectively; a connection
between Acinetobacter baumanii QS (AbaI/AbaR, controlled by bile salts) and T6SS has been established (maroon); in Vibrio sp. there is a complex network which
relates QS (LuxO/HapR/TfoX) with T6SS (aquamarine); AhyRI (a QS network) in Aeromonas sp. and P. atrosepticum is involved in Hcp and VgrG secretion (sky blue)
and finally, iron is transported across the cell membrane accompanied by PQS, a quorum sensing signal in P. aeruginosa, and this process depend on Tse a
substrate of T6SS, which binds to OMVs (outer membrane vesicles) containing PQS- Fe3+.
(Ng and Bassler, 2009). Two enzymes are necessary for the
biosynthesis of these autoinducers: CqsA and LuxS, respectively
(Schauder et al., 2001;Miller et al., 2002;Chen et al., 2002;
Higgins et al., 2007). These signal molecules are detected by two
sensor kinases, LuxQ (sensor of CAI-I) and CqsS (sensor of AI-2).
Both pathways merge on LuxU, a phosphotranfer protein. At
low cell density (LCD), the two sensor kinases phosphorylate
LuxU due to the absence of their respective autoinducers.
There are two histidine kinases which also contribute to the
phosphorylation of LuxU: VpsS and CqsR (Jung et al., 2015).
Then, LuxU transfers the phosphorylate group to a DNA-binding
response regulator protein called LuxO. Phosphorylated LuxO
activates the expression of sRNA molecules (known as qrr1-
4) when the cell density is low thanks to the interaction with
the alternative sigma factor σ54 (Freeman et al., 2000;Lenz
et al., 2004). In conjunction with the RNA-binding protein Hfq,
LuxO represses the expression of HapR (Lenz et al., 2004), a
TetR-family global transcriptional regulator which acts on QstR
(Tsou et al., 2009;Shao and Bassler, 2014;Watve et al., 2015;
Figure 2). HapR is accumulated when the cell density is high
(Lenz et al., 2004) because LuxO is not phosphorylated and
transcription of the sRNAs is blocked. QstR is a master regulator
of the T6SS belonging to the LuxR-type family of regulators
(Jaskólska et al., 2018). QstR binds to the promoter region of the
T6SS cluster inducing the expression of the genes. The regulation
of the T6SS by cAMP-CRP pathway is not clear, but it is possible
that it influences T6SS genes through regulation of QS and
chitin-induced competency (Liang et al., 2007;Blokesch, 2012).
It is known that CRP positively regulates T6SS (Ishikawa
et al., 2009). Apart from the activation of QstR
via QS, it is also regulated by chitin and arabinose
(Lo Scrudato and Blokesch, 2012, 2013).
The expression of the three T6SS gene clusters in V. cholerae
requires TfoX, CytR, HapR, and QstR for the highest level of
expression (Watve et al., 2015). CytR and TfoX are required for
the expression of the T6SS genes but their regulatory effects are
only mediated by QstR (Figure 2).
Other Pathogens
T1SS
The swr QS system, which controls swarming motility, regulates
the Lip secretion system, a T1SS responsible for the secretion
of lipases, metalloproteases and S-layer proteins in Serratia
liquefaciens MG1 (Riedel et al., 2001). The swr QS system consists
of SwrI, which synthesizes C4-HSL, and SwrR, which regulates
gene transcription after binding the diffusible signal C4-HSL.
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QS-mediated regulation of lipB, which encodes the LipB exporter,
was demonstrated in swrI mutants with luxAB insertions, in
which the level of secreted proteins was lower (Riedel et al.,
2001;Figure 2). Other relationships between T1SS and QS
have also been observed. The rice pathogen recognition XA21
receptor recognizes a sulphated peptide (axYS22) derived from
the Ax21 protein (activator of XA21-mediated immunity) and
confers resistance to Xanthomonas oryzae strains. Ax21 may have
a key biological role because it is conserved in Xanthomonas
spp., Xylella fastidiosa, and Stenotrophomonas maltophilia. Ax21
requires RaxABC TOSS (type I secretion system) for secretion
and activity. The expression of rax genes which encode T1SS has
been demonstrated to be QS-dependent due to the cell-density
dependency (Han et al., 2011). These data indicate that Ax21
could have a role as a signaling molecule and a direct relationship
between the QS network and T1SS is established (Figure 2;
Lee et al., 2006).
T2SS
In Xanthomonas species, QS is mediated by the diffusible signal
factor (DSF). A proteomic analysis conducted in 2013 revealed 33
proteins that are controlled by DSF. Their putative functions are
associated with QS and include cellular processes, intermediary
metabolism, oxidative adaption, macromolecule metabolism,
cell-structure, protein catabolism, and hypothetical functions
(Qian et al., 2013). In this study, it was observed that three genes
encoding T2SS-dependent proteins and one gene which encodes
Ax21 (activator of XA21-mediated immunity)-like protein
are regulated by QS and are essential for virulence-associated
functions, including extracellular protease, cell motility,
antioxidative ability, extracellular polysaccharide biosynthesis
(EPS), colonization, and biofilm (Qian et al., 2013;Figure 2).
T3SS
The relationship between QS and T3SS in E. coli was
first demonstrated by Sperandio et al. (1999), who showed
that expression of the locus of enterocyte effacement (LEE)
operons that encode the T3SS is activated by QS in both
enterohemorrhagic (EHEC) and enteropathogenic (EPEC) E. coli
due to transcriptional control of the LEE operons by LuxS, which
directly activates the LEE1 and LEE2 operons and indirectly
activates (via the Ler regulator) the LEE3 and tir operons
(Figure 2). These researchers proposed that activation of the
T3SS by the AI-2 autoinducer synthesized by commensal E. coli
resident in the large intestine could explain the high infectivity
of E. coli O157: H7, which has an infectious dose of about 50
bacterial cells (Sperandio et al., 1999).
The major virulence factors of EHEC and EPEC are intimin
(T5eSS), Tir (the receptor for intimin) and the three secreted
proteins EspA, EspB and EspD. T3SS functions in the secretion of
the Tir and Esp proteins. The LuxR-type response regulator SdiA
negatively regulates the expression of EspD and intimin in the
same bacterium, indicating multifactorial regulation of the T3SS
by bacterial QS signals (Kanamaru et al., 2000).
Indole, which is produced by tryptophanase (TnA) in enteric
bacteria and reaches high concentrations in the gut, is another
signaling molecule that influences expression of T3SS in E. coli
(Lee et al., 2007, 2008). Indole increases the production and
secretion of the translocators EspA and EspB in EHEC O157:H7
(Hirakawa et al., 2009;Figure 2); hence, indole promotes the
development of attaching and effacing (A/E) lesions in HeLa cells.
The involvement of the RNA chaperone protein Hfq, which
also participates in QS, in T3SS expression was demonstrated
in Yersinia pseudotuberculosis and Yersinia pestis (Schiano
et al., 2014;Figure 2). Moreover, Schiano et al., 2014 have
demonstrated the regulation of T3SS by QS through virulence
regulators LcrF and YmoA in Y. pseudotuberculosis (Amy, 2018).
In Aeromonas hydrophila, an unique QS system, encoded in
ahyR/ahyI loci, has been described (Vilches et al., 2009;Garde
et al., 2010). Vilches et al. (2009) have used the A. hydrophila
AH-3 strain to study the T3SS regulation. AH-3: ahyI and AH-3:
ahyR mutants have reduced activity of the aopN-aopB promoter
(promoter of T3SS components) compared to the wild-type strain
(Figure 2). So they concluded that QS could be involved in the
positive regulation of the production of the T3SS component in
the AH-3 strain (Vilches et al., 2009).
T4SS
In Brucella abortus, there is a clear relationship between the QS
network and T4SS. For the virB operon, which encodes the T4SS
regulated by VjbR, a LuxR-type QS is responsible for the virulence
characteristics of B. abortus (Li et al., 2017). The virB operon is
responsible for establishing the replicative niche of the bacterium
once it enters the host cell. The T4SS in B. abortus, as in other
bacteria, translocates effector proteins into the host cell to avoid
the immune defense mechanisms, making it one of the two main
virulence factors for Brucella. Arocena et al. (2010) described
the binding site of VjbR to the virB operon (Li et al., 2017).
Otherwise, the conjugation process between two members of the
Roseobacter group mediated by T4SS, encoded in RepABC-type
plasmids, is controlled by the QS network. This was demonstrated
by construction of luxI mutant and the addition of external long
chain AHLs, which restored the phenotype (Patzelt et al., 2013,
2016;Figure 2).
T6SS
Quorum sensing has been reported to control expression of
T6SS toxin-immunity systems in Burkholderia thailandensis.
Moreover, a new role for T6SS in constraining the proliferation
of QS mutants has been described in B. thailandensis (Majerczyk
et al., 2016). Interestingly, it has been observed that T6SS
effectors function as cell-to-cell signals in a Pseudomonas
fluorescens MFE01 strain lacking the AHL QS pathway
(Gallique et al., 2017b).
In A. hydrophila, Hcp and VgrG- two of the “core” proteins
and also effectors of the T6SS- secretion have been suggested
to be regulated by the AhyRI QS regulon (Khajanchi et al.,
2009;Figure 2). Finally, our research group has described the
association between T6SS machinery and the activation of the QS
system by bile salts in A. baumannii clinical strains (Lopez et al.,
2017a;Figure 2).
T7SS
As with other bacteria, Mycobacterium spp regulate biofilm
formation by QS (Virmani et al., 2018). The second messenger
c-di-cGMP, an intracellular signaling molecule, coordinates
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biofilm production and QS signaling (Sharma et al., 2014).
Both M. tuberculosis (Kulka et al., 2012) and diverse species of
non-tuberculous mycobacteria (M. smegmatis, M. marinum, M.
fortuitum, M. chelonae, M. ulcerans, M. abscessus, M. avium, and
M. bovis) produce biofilm depending on certain environmental
conditions such as the availability of nutrients or the pH of the
medium (Hall-Stoodley et al., 1998;Bardouniotis et al., 2003;
Ojha et al., 2005;Marsollier et al., 2007;Johansen et al., 2009;
Rhoades et al., 2009).
In the recent work of Lai et al. (2018) it was demonstrated that
the espE, espF, espG, and espH genes, located in the T7SS ESX-1
operon, are crucial for sliding motility and biofilm formation in
M. marinum. Esp proteins, which regulate substrate transport,
are involved also in virulence. This paper clearly demonstrates
the role of M. marinum T7SS in the production of biofilm which,
as already mentioned, is related to QS (Lai et al., 2018).
The T7SS of S. aureus, a virulence factors export machinery,
plays a key role in the promotion of bacterial survival and
long-term persistence of subpopulations of staphylococci. The
expression of T7SS is regulated by the bacterial interaction with
host tissues (Lopez et al., 2017c) mediated by the secondary
sigma factor (σB) (Schulthess et al., 2012). Schulthess et al.
(2012) reported that the repression of esxA by σB is due to the
transcription of sarA induced by σB, which leads to a strong
repression of esxA. The activation of the esxA transcript, on
the other hand, is stimulated by arlR, the response regulator
of the ArlRS two-component system, SpoVG, a σ-dependent
element, and the Agr quorum detection system (Schulthess
et al., 2012). Agr QS system is composed by AIP (self-activating
peptide), the inducer ligand of AgrC which is the receptor of
the agr signal. In the case of the QS Agr system, the effector of
global gene regulation is an important regulatory RNA, RNAIII
(Novick and Geisinger, 2008).
T9SS
Moreover, an important relationship between T9SS and
biofilm formation has been observed in periodontopathogenic
pathogens such as Capnocytophaga ochracea,Porphyromonas
spp., Fusobacterium spp. and Prevotella spp. (Kita et al., 2016).
In the study by Kita et al. (2016), the participation of T9SS in
the formation of biofilm of C. ochracea is demonstrated. The
formation of biofilm of C. ochracea is crucial for the development
of dental plaque and the same happens with other periodontal
pathogens, in which it has also been seen that genes related to
T9SS are present. Therefore, the components of the T9SS could
be potential targets to inhibit the formation of biofilm and thus
avoid the formation of dental plaque (McBride and Zhu, 2013;
Kita et al., 2016). However, in depth analysis of the relationship
between T9SS and QS network in different pathogens is required.
DISCUSSION
To date, the T1SS, T2SS, T3SS, T4SS, T6SS, T7SS, and T9SS
SS have been found to have important relationships with
QS networks. The involvement of the T1SS system (Lip B
which is part of the Lip exporter) in the QS network (swr
quorum system) of S. liquefaciens MG1 has been investigated
(Riedel et al., 2001). In P. aeruginosa, two QS systems
(lasR/lasI and rhLR/rhlI) are linked to T2SS system by
microarrays and proteomic studies (Chapon-Herve et al., 1997;
Wagner et al., 2003;Michel et al., 2007), and DSF-type systems
are also linked to T2SS in Xanthomonas species through
proteome analysis (Qian et al., 2013). The QS signal AI-2 has
been associated with a T3SS system in E. coli (Sperandio et al.,
1999) and Vibrio spp. (Henke and Bassler, 2004). Moreover, this
T3SS system has been related to QS proteins in another two
pathogens, P. aeruginosa and Yersinia spp. (Liang et al., 2014;
Schiano et al., 2014).
Several T4SS (virB operon) are controlled by VjbR protein
which is a LuxR-type quorum-sensing regulator in B. abortus
(Arocena et al., 2010;Li et al., 2017). Moreover, in the Roseobacter
group, the conjugation of plasmids, which encode T4SS, is
QS-controlled and the QS system may detect a broad range of
long-chain AHLs at the cell surface (Patzelt et al., 2013, 2016).
There is a wealth of information relating the T6SS to QS in
pathogens such as Vibrio spp. For example, Hcp and VasH from
the T6SS system in V. cholerae are involved in QS (Ishikawa et al.,
2009;Zheng et al., 2010;Kitaoka et al., 2011;Leung et al., 2011;
Yang et al., 2018). For Pseudomonas spp,. there are numerous
works where the different T6SS are regulated by QS networks
(Lesic et al., 2009;Gallique et al., 2017b;Lin et al., 2017). In other
pathogens as Burkholderia thailandensis (Majerczyk et al., 2016),
and in A. baumannii, the relationship between QS and SS has
begun to be studied (Lopez et al., 2017a).
In M. marinum, the relationship between biofilm formation,
which is tightly connected with QS, and T7SS, has been
demonstrated (Lai et al., 2018). Also in S. aureus, the Agr QS
network has been related to T7SS (Schulthess et al., 2012). An
important relationship between T9SS and biofilm formation has
been observed in periodontopathogenic pathogens (Kita et al.,
2016). Finally, although the involvement of T5SS secretion system
in virulence, motility and competence is well-known, these
systems and their association with QS must be studied in greater
depth in order to clarify their roles.
Taking P. aeruginosa as a reference point, the positive effect
of QS in the expression of T1SS and T2SS could be related
to the fact that this organism secretes exoproducts that are
public goods (proteases and lipases); hence, it is better to
produce and secrete these compounds when a high cell density
is reached, since these products are costly and the benefits
associated to their production are higher at high cell densities.
Similarly, the T6SS, which is involved in killing competitors by
contact, will be more efficient at high cell densities since the
probability of finding target bacteria is higher. In contrast, the
T3SS appears to be negatively regulated by QS, and this may
be related to its role in an acute infection and its “inhibition
by QS” may be a way to facilitate the transition to a chronic
infection state. In addition to its well established role in
infections, T3SS has a broader ecological role suggested by
its role in killing biofilm associated Acanthamoeba castellanii
amoeba (Matz et al., 2008). Furthermore, it was recently
demonstrated that T3SS is susceptible of cheating by mutants
that do not produce it, allowing their establishment in infections
(Czechowska et al., 2014); hence, the selective forces that act over
T3SS are complex.
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Therefore, research into the relationship between QS and
SS must be further developed in order to better understand
human infections.
AUTHOR CONTRIBUTIONS
RTP, LB, AA, BG-P, LF-G, ML, IB, and GB developed the
redaction of the manuscript, figures and table. RG-C, TW, and
MT designed the review, assigned writing tasks to co-authors,
contributed to writing and proofread the final version.
FUNDING
This study was funded by grant PI16/01163 awarded to
MT within the State Plan for R+D+I 2013–2016 (National
Plan for Scientific Research, Technological Development and
Innovation 2008–2011) and co-financed by the ISCIII-Deputy
General Directorate for Evaluation and Promotion of
Research – European Regional Development Fund “A
way of making Europe” and Instituto de Salud Carlos III
FEDER, Spanish Network for the Research in Infectious
Diseases (REIPI, RD16/0016/0001 and RD16/0016/0006)
and by the Study Group on Mechanisms of Action
and Resistance to Antimicrobials, GEMARA (SEIMC,
http://www.seimc.org/). MT was financially supported
by the Miguel Servet Research Programme (SERGAS
and ISCIII). RTP and LF-G were financially supported
by, respectively, a post-speciality grant awarded by the
Fundación Novoa Santos (CHUAC-SERGAS, Galicia) and
predoctoral fellowship from the Xunta de Galicia (GAIN,
Axencia de Innovación). RG-C research is supported
by the grant PAPIIT-UNAM number IN214218. BG-P
was supported by grants PAPIIT-UNAM IN209617 and
CONACYT 284081.
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