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Both Quorum Sensing (Qs)-I and Ii Systems Regulate Escherichia coli Flagellin Expression.

Byline: Yang Yang, Yun Liu, Mingxu Zhou and Guoqiang Zhu


For elucidating effect of quorum sensing (QS) systems I and II (QS-1 and QS-II) on Escherichia coli flagellin expression, E. coli F18ab strain 107/86 was modified to either express acyl-homoserine lactone (AHL) synthase (QS-I) or deleted for autoinducer 2 (AI-2) expression (QS-II). AHL expression and deletion of luxS (AI-2) both inhibited flagellin expression, as measured by motility assays, bacterial gene expression, and host responses to infection. The QS systems and flagellin were coordinately regulated, as deleting fliC caused decreased QS-II activity.

Key words

E.coli, Flagellin, Quorum sensing-I and II.


Porcine edema disease and porcine post-weaning diarrhea are two important diseases which bring pigs high morbidity and mortality. Shiga toxin-producing E. coli (STEC) is major pathogen of the diseases (Da et al., 2001; Frydendahl, 2002). Flagella participate in bacterial pathogenicity as important virulence factor. Besides providing motility, it also contributes to bacterial initial adhesion or colonization to host cells. In vivo, enteric bacteria could take advantage of flagella motility to compete with intestinal microbiota by exploiting inflammation (Stecher et al., 2004, 2008; Duan et al., 2012; Zhou et al., 2013).

Quorum sensing (QS) represents one crucial communication system, and was considered as a kind of bacterial-population based language between bacteria (Pacheco and Sperandio, 2009; Curtis and Sperandio, 2011). QS-I positive bacteria normally express acyl-homoserine lactone (AHL) synthase, whereas the QS-II system is regulated by LuxS and autoinducer 2 (AI-2) (Niu et al., 2013). We previously reported that QS-I expression in E. coli suppresses flagella expression (Yang et al., 2013). QS-II also regulates bacterial virulence strategies, including regulate motility by flhDC, type III secretion systems (Anand and Griffiths, 2003; Li et al., 2007; Han and Lu, 2009), as well as biofilm formation and bacterial pathogenicity (Sperandio et al., 2002; Clarke et al., 2006; Gonzalez et al., 2006).

Here we investigated the extent to which QS-II can also regulate flagellin expression in STEC and examined the potential for coordinate regulation between two QS systems.


Strains used in this study

Strains and plasmids used are listed in Table I. LB broth or LB agar plates were used for bacterial growth. Caco-2 cell line was cultivated in DMEM with 10 % FBS (37 AdegC, 5 % CO2). Human TNF and IL-8 immunoassay Kits (RandD Systems, Inc.) were purchased for relative experiments.

Construction of recombinant strains

The F18ab luxS gene in-frame deletion mutant (F18abIluxS) was constructed using I>>Red-based recombination system (Datsenko and Wanner, 2000). The luxS open reading frame (ORF) was amplified by primers LuxS-1/ LuxS-2 (Table II). Plasmid pBR-luxS was constructed and then transformed into F18abIluxS to obtain the complemented strain F18abIluxS/pluxS.

To over-express uvrY and csrB in F18ab, the primers uvrY-F/uvrY-R and csrB-F/csrB-R were used to PCR amplify uvrY and csrB, respectively. uvrY and csrB were cloned into pBR322 and transformed into F18ab E. coli.

AI-2 bioassays and motility assays

After grown to an OD600 of 1.3, supernatants of strains were collected. Bioluminescence was measured in luminescence mode by Tecan GPM reader (Han and Lu, 2009; Zhou et al., 2014). For motility assays, strains were seeded in the middle of motility agar plates. After appropriate growth time, motility halos were measured (Duan et al., 2013).

Table I.- Strains and plasmids used in this study.

Strain or plasmid###Description###Source or reference


E. coli F18ab 107/86###Wild-type:###Duan et al. (2012)

###O139:H1:F18ab, Stx2e; O139:H1:F18ab, Stx2e

E. coli F18ab/pyenI###107/86 carrying pyenI###Yang et al. (2013)

E. coli F18ab/pBR###107/86 carrying pBR322###Yang et al. (2013)

E. coli F18abfliC###fliC deletion mutant###Duan et al. (2012)

E. coli F18abfliC/pfliC###F18abfliC carrying pBR-fliC###Duan et al. (2012)

E. coli F18abluxS###luxS deletion mutant###This study

E. coli F18abluxS/pluxS###F18abluxS carrying pBR-luxS###This study

E. coli F18ab/pcsrB###107/86 carrying pcsrB###This study

E. coli F18ab/puvrY###107/86 carrying puvrY###This study

E. coli DH5a###AI-2 bioassay negative control###Takara Ltd.

Vibrio harveyi BB170###AI-2 bioassay reporter strain###Bassler et al. (1994); Yang et al. (2013)


pBR322###Expression vector, Ampr###Takara Ltd.

pBR-luxS###pBR322 carrying LuxS ORF###This study

pKD3###Cmr; Cm cassette template###Datsenko and Wanner (2000); Duan et al. (2012)

pKD46###Ampr, Red recombinase expression###Datsenko and Wanner (2000); Duan et al. (2012)

pCP20###Ampr,Cmr; Flp recombinase expression###Datsenko and Wanner (2000); Duan et al. (2012)

Table II.- Primers used in this study.

Primer###Sequence (5'-3')###Primer###Sequence (5'-3')



















Measurement of mRNA level

Tiangen RNA Extraction Kit (DP419) was employed in this study, through which total RNA from each strain was prepared (Han and Lu, 2009). Primers for flhD, fliC, csrA, barA, uvrY, pfs, and luxS genes were designed and listed in Table II. Gene gapA was chosen as the endogenous reference. Caco-2 (1x106 cells/well density) was plated in 6-well plates. 107 CFU of relative strains were injected into each well, and infected for 2 h. Then cells were dealt with Tiangen RNA Extraction Kit following the standard protocol. 2-IICT method was employed for data analysis.

ELISA assay

Monolayer of Caco-2 was prepared for infection of individual E. coli strains. After 2h incubation, supernatants were obtained by centrifugation (Duan et al., 2012). With commercial kits, expression levels of IL-8 and TNF were then measured.


All experiments were repeated at least 3 times. Data are presented as the mean + standard deviation. To evaluate statistical significance with p < 0.05 considered significant, the Student's t-test method was employed.


QS-1 and QS-II have differential impact on bacterial motility and affect E. coli-induced pro-inflammatory responses F18ab E. coli was transformed with the yenI gene from Y. enterocolitica to induce endogenous AHL production (QS-1) (Yang et al., 2013). Flagella involves in bacterial motility, adhesion, invasion. Furthermore, it could induce inflammation response in host cells. With the recombinant strain, function of AHL (QS-1 signals) upon flagella expression could be identified. Subsequently, expressing pyenI inhibited bacterial motility on swim agar plates (Fig. 1A). Reduced flagella expression was observed in AHL positive strain F18ab/pyenI, as well as decreased motility ability.

QS-II manipulates multiple genes expression through AI-2. In many pathogenic bacteria, AI-2 participates in regulating bacterial virulence strategies. To assess a similar role for QS-2 on motility, we deleted the luxS gene. F18abIluxS was deficient in AI-2 production (Fig. 1B) and was less motile than the WT strain (Fig. 1A). Consistent with their impaired motility, fliC expression was inhibited in both pyenI and in IluxS (Fig. 1C).

Bacterial flagellin can induce pro-inflammatory responses through TLR5. TLR5 stimulates pro-inflammatory genes in NF-KB and MAPK pathways (Vijay-Kumar et al., 2010; Salazar-Gonzalez and Navarro-Garcia, 2011). Researchers found that as one pathogen-associated molecular pattern (PAMP), flagellin also have TLR5-independent pro-inflammatory ability by Naip5 and Ipaf, members of the NLR family (Miao et al., 2006; Ren et al., 2006).

These F18ab E. coli strains did not activate the pro-inflammatory IL-8 or TNF responses of infected Caco-2 cells to a magnitude equal to that of infection by WT F18ab E. coli, as measured by both RT-PCR (Fig. 2A, B) or by ELISA (Fig. 2C, D). QS-1 expression inhibits F18 E. coli motility, whereas QS-II expression enhances motility by affecting fliC expression.

AI-1 and flagella expression influence AI-2

AI-2 activity was impaired in F18ab/pyenI, indicating that QS-1 influences AI-2 production (Fig. 1B). The phenomenon was consistent with the decreased mRNA level of both luxS and pfs in F18ab/pyenI (Fig. 3), which encode enzymes involved in AI-2 synthesis in E. coli (Zhu et al., 2007). Pfs, an important functional enzyme in AI-2 signal synthesis, encounter 25 % decrease of mRNA level under QS-1 influence, while flagella motility was reduced heavily in luxS mutant. FliC expression also regulated QS-II, as deleting fliC inhibited pfs and luxS expression (Fig. 3).

Coordinate regulation of flagella and QS expression

Bacteria utilize two-component systems for adaptation to environmental changes (Pernestig et al., 2001, 2003; Herren et al., 2006; Yang et al., 2014). The BarA/UvrY and the CsrA/CsrB two-component systems regulate flagella expression (Edwards et al., 2011). UvrY can activate barA expression through an auto-regulatory loop. Influence of CsrA upon csrB is regulated partly by barA, and a BarA-independent, UvrY-dependent mechanism also involves. CsrA indirectly induces csrB transcription. UvrY can directly activate csrB transcription, and also is included in up-regulation of CsrA. Increased transcription of uvrY has been observed when E. coli is sustained in an AHL-positive environment (Wei et al., 2001; Van Houdt et al., 2006).

In this study, expression of barA and uvrY were increased by 2.3- and 2.7-fold, respectively, in F18ab/pyenI (Fig. 4). Up-regulation of uvrY and barA can induce csrB transcription, which binds to CsrA and antagonizes its regulatory effects upon flhDC (Liu and Romeo, 1997; Mercante et al., 2009; Edwards et al., 2011). flhD expression did decreased 2.1-fold in pyenI (Fig. 4). Over-expressing uvrY and csrB caused a 1.7- and 2.3-fold reduction in fliC expression, respectively (Fig. 1C).


Overall, we show here that expressing QS-1 in E. coli inhibits flagella expression, whereas the LuxS QS-II system up-regulates flagella expression. These systems are coordinately regulated, as QS-I inhibited QS-II and flagellin expression positively regulated LuxS. We implicate the BarA/UvrY and the CsrA/CsrB two-component systems as possible regulators of this coordinated regulation. In the presence of cattle rumen AHLs, E. coli O157:H7 represses LEE gene expression and activates gad expression to improve acid tolerance (Sperandio, 2010; Sheng et al., 2013). Other aspects of E. coli biology are also regulated by AHLs, including cell division (Sitnikov et al., 1996) and antibiotic resistance (Rahmati et al., 2002). These data suggest that E. coli flagella expression is also regulated by AHLs.


This study was supported by grants from the 13th Five-Year National Key Development Program (2016YFD0501000), Natural Science Foundation of Jiangsu Province (BK20150442), the Chinese National Science Foundation Grants (Nos. 31502075, 31072136, 31270171 and 30771603), the Genetically Modified Organisms Technology Major Project of China (2014ZX08006-001B), Program for Chang Jiang Scholars and Innovative Research Team In University "PCSIRT" (IRT0978), 948 programme from Ministry of Agriculture of the People's Republic of China (grant No. 2011-G24) and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

Statement of conflict of interest

The authors or their institution do not have any relationships that may influence or bias the results and data presented in this manuscript. There is no conflict of interests regarding the publication of the manuscript.


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Author:Yang, Yang; Liu, Yun; Zhou, Mingxu; Zhu, Guoqiang
Publication:Pakistan Journal of Zoology
Article Type:Report
Date:Oct 31, 2018
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