A novel cis antisense RNA AsfD promotes Salmonella enterica serovar Typhi motility and biofilm formation

Journal Pre-proof A novel cis antisense RNA AsfD promotes Salmonella enterica serovar Typhi motility and biofilm formation Long Chen, Liping Gu, Xinfeng Geng, Guoxin Xu, Xinxiang Huang, Xiaojue Zhu PII:

S0882-4010(19)31799-1

DOI:

https://doi.org/10.1016/j.micpath.2020.104044

Reference:

YMPAT 104044

To appear in:

Microbial Pathogenesis

Received Date: 14 October 2019 Revised Date:

19 January 2020

Accepted Date: 3 February 2020

Please cite this article as: Chen L, Gu L, Geng X, Xu G, Huang X, Zhu X, A novel cis antisense RNA AsfD promotes Salmonella enterica serovar Typhi motility and biofilm formation, Microbial Pathogenesis (2020), doi: https://doi.org/10.1016/j.micpath.2020.104044. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Long Chen: Conceptualization, Methodology, Writing - original draft, Writing - review & editing Liping Gu: Formal analysis Xinfeng Geng: Formal analysis Guoxin Xu: Formal analysis, Software Xinxiang Huang: Writing - review & editing, Supervision Xiaojue Zhu: Resources, Writing - review & editing

1

1

A novel cis antisense RNA AsfD promotes Salmonella enterica serovar Typhi motility and

2

biofilm formation

3

Long Chen1, Liping Gu1, Xinfeng Geng1, Guoxin Xu1, Xinxiang Huang2, Xiaojue Zhu1*

4

1

5

University, Zhangjiagang 215600, China

6

2

7

Zhenjiang, China

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* Correspondence and reprints: [email protected]

Department of Clinical Laboratory, The Affiliated Zhangjiagang Hospital of Soochow

Department of Biochemistry and Molecular Biology, School of Medicine, Jiangsu University,

9 10

Abstract

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Bacterial non-coding RNAs (ncRNAs) can participate in multiple biological processes, including

12

motility, biofilm formation, and virulence. Using high-throughput sequencing and transcriptome

13

analysis of Salmonella enterica serovar Typhi (S. Typhi), we identified a novel antisense RNA

14

located at the opposite strand of the flhDC operon. In this study, a northern blot and qRT-PCR

15

were used to confirm the expression of this newfound antisense RNA in S. Typhi. Moreover, 5′

16

RACE and 3′ RT-PCR were performed to reveal the molecular characteristics of the antisense

17

RNA, which was 2079 nt − 2179 nt in length, covered the entire flhDC operon sequence, and

18

termed AsfD. The level of AsfD expression was higher during the stationary phase of S. Typhi

19

and activated by the regulators, OmpR and Fis. When AsfD was overexpressed, the level of

20

flagellar gene flhDC transcription increased; moreover, the level of fliA and fljB expression, as

21

well as the motility and biofilm formation of S. Typhi were also enhanced. The results of this 1

2

22

study suggest that AsfD is likely to enhance the motility and biofilm formation of S. Typhi by

23

up-regulating flhDC expression.

24 25

Keywords: Salmonella enterica serovar Typhi; non-coding RNA; AsfD; flhDC; motility; biofilm

26 27

1. Introduction

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Salmonella enterica serovar Typhi (S. Typhi) is a Gram-negative enteropathogenic bacterium

29

that can infect humans and cause a severe systemic infection called typhoid fever, due to its

30

characteristic pathogenicity [1, 2]. After entering a host, S. Typhi passes through the acidic

31

stomach and duodenum, after which it invades the mucosa of the small intestine and survives in

32

macrophages and monocytes. During this process, S. Typhi takes advantage of various strategies

33

to overcome environmental stress through regulating gene expression, including increasing

34

motility and biofilm formation [3, 4].

35

Non-coding RNAs (ncRNAs), as nonnegligible and important regulatory factors, have been

36

reported to be involved in gene expression reprogramming process and post-transcriptional

37

regulation in both eukaryotes and prokaryotes. In bacteria, a growing number of studies have

38

confirmed that ncRNAs play important roles in regulatory networks of many cellular processes,

39

including acid resistance [5], quorum sensing [6], oxidative stress [7], and virulence genes [8, 9].

40

ncRNAs can be classified into small ncRNA and long ncRNAs (lncRNAs), which are

41

typically >200 nt-long. Most ncRNAs function by forming base pairs with target mRNAs.

42

According to the different locations and modes of action, these base-paired ncRNAs are primarily 2

3

43

divided into two categories: trans-encoded and cis-encoded ncRNA. Trans-encoded ncRNAs are

44

located in the intergenic region and form incomplete complementary base pairs with distant target

45

mRNAs. Cis-encoded ncRNAs, also known as antisense RNA, are located at the minus strands of

46

target genes and are fully complementary to the target mRNAs [10–12].

47

Using high-throughput sequencing and a transcriptome analysis of S. Typhi, we identified

48

several novel cis-encoded ncRNAs [11, 13–15]. A transcript encoded by the minus strand of the

49

flhDC operon attracted our attention. The flhDC operon is a two-gene operon that encodes

50

FlhD2C2, the master regulatory factor of flagellum that represents the structural and functional

51

basis of motility in Salmonella. As the only class-1 operon, the flhDC operon encoding the

52

regulator activates class-2 and class-3 flagellar gene transcription [16, 17]. The flhDC operon has

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also been reported to influence bacterial biofilm formation [18, 19].

54

In this study, we described the identification and characterization of a novel antisense RNA

55

encoded by the opposite strand of the flhDC operon, termed AsfD. We further demonstrate that

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AsfD may affect the motility and biofilm formation of S. Typhi by regulating flhDC expression.

57 58

2. Materials and methods

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2.1 Bacterial strains, plasmids, and growth conditions

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The strains and plasmids used in this study are listed in Table 1. Wild type S. Typhi GIFU10007

61

was used as the original strain for all strain generation. Unless otherwise noted, the strains were

62

grown at 37°C in Lysogeny broth (LB) medium. For the biofilm formation assay, bacteria were

63

grown in trypticase soy broth (TSB) medium. When necessary, ampicillin (100 µg/mL) or 3

4

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kanamycin (50 µg/mL) were added to the cultures. The transcription of AsfD was induced with

65

L-arabinose (w/v 0.2%).

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Table 1. Strains and plasmids used in this study. Strain or Plasmid

Relevant characteristics

Source

S. Typhi GIFU10007

wild type strain; Z66+

Gifu University

GIFU10007 carrying empty

This work

WT-pBAD pBAD-hisA empty plasmid WT- pBAD-AsfD

GIFU10007 carrying pBAD-AsfD

This work Laboratory

∆rpoS

GIFU10007 (∆rpoS) collection Laboratory

∆ompR

GIFU10007 (∆ompR) collection Laboratory

∆fis

GIFU10007 (∆fis) collection Laboratory

∆hfq

GIFU10007 (∆hfq) collection

E. coli DH 5α

E. coli host strain of T vector

Takara

pBAD-HisA

PlacO promoter; Ampr

Invitrogen

pBAD-AsfD

PlacO promoter, AsfD insert; Ampr

This work

pGEM-T vector

TA clone; Amprr

Promega

67 68

2.2 Plasmid and strain construction

4

5

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To construct the recombinant vector, pBAD-AsfD, a 2079-bp fragment spanning the entire flhDC

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operon sequence was amplified from the S. Typhi genome by PCR. The associated primers are

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presented in Table 2. The fragment containing the two restriction sites, Nco I and EcoR I, was

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cloned into a pBAD-HisA plasmid (Invitrogen). The wild type strain was transformed with the

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recombinant vector, pBAD-AsfD, and empty pBAD to be used as the AsfD over-expressing and

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control strains, respectively.

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Table 2. Oligonucleotides used in this study. Primer name

Sequence (5′′→3′′)

Primers used to construct strains CTACCATGGCATGGGTTTCAATCTC pBAD-AsfD PA TTCA GAGGAATTCAACAGAGGAGGGCGT pBAD-AsfD PB ATGCT Primers used for 5′′ RACE and 3′′RT-PCR GATTACGCCAAGCTTTCAACGAAG 5′RACE GSP AGATGGCAAACACACTGGG 3′RT PA

ACAGGGATGCCAAGACGTTC

3′RT PB1

CTCATTTAACGCAGGGCTGT

3′RT PB2

ATCGCCATATAGAAACGGTG

3′RT PB3

AATAATTTCCCCAACCAGCC

5

6

3′RT PB4

CGCTTAGTCGTGGCAGTGTT

3′RT PB5

GGCGCTCTATGGGTTGATTA

3′RT PB6

GGGCGCACCATACCTATAAA

3′RT PB7

CCAGTAAAGACACGACGATT

3′RT PB8

GCGATTGATTCACCGACACG

Primers used for Real-time PCR analyses 5s-qF

TTGTCTGGCGGCAGTAGC

5s-qR

TTTGATGCCTGGCAGTTC

AsfD qF

AATCCTGAGTCAAACGGGTG

AsfD qR

TCAACGAAGAGATGGCAAAC

flhDC-qF

AGCGTTTGATCGTCCAG

flhDC-qR

CGTCCACTTCATTGAGCA

fliA-qF

CGACCGATATGACGCTTTGC

fliA-qR

TTCCCGCCACTCATCGTAAG

fljB-qF

CAACCGCTAGTGATTTAGTTT

fljB-qR

CTGTCCCTGTAGTAGCCGTAC

Probe primers used for northern blot analysis P-AsfD PA

AATCCTGAGTCAAACGGGTG AATTGTAATACGACTCACTATAGG

P-AsfD PB GCG 76

6

7

77

2.3 RNA isolation

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Total RNA was isolated as described previously [20]. Briefly, bacteria were grown overnight in

79

LB broth at 37°C and 250 rpm. Overnight cultures were diluted 1:100 and grown at 37°C and

80

250 rpm. Total RNA from bacteria was extracted at OD600 = 0.8 using the RNeasy kit

81

(mini-column, Qiagen, Shanghai, China) according to the manufacturer’s instructions for the

82

detection of AsfD. For overexpression analysis, S. Typhi carrying pBAD or pBAD-AsfD

83

plasmids were grown in LB broth. L-arabinose was added in cultures (w/v 0.2%) at the log phase

84

(OD600 = 0.4) to induce pBAD plasmid. Total RNA was isolated with TRIzol Reagent

85

(Invitrogen) and then treatment with DNA removal kit (Invitrogen).

86

2.4 Northern blot analysis

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A northern blot analysis was performed as described previously [21]. To detect AsfD, a specific

88

probe was designed in the peak region of AsfD expression. The probe primers used in this study

89

are listed in Table 2. The RNA was labeled in an in vitro transcription reaction with

90

digoxigenin-11-UTP using a labeling mixture and an optimized transcription buffer. The total

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RNA sample (2 µg − 5 µg) was separated on 4% PAGE containing 8% urea and transferred to a

92

Hybond™ N+ membrane (300 mA 40 min). The membrane containing the RNA was washed twice

93

with SSC, and dried at 80°C for 2 h to fasten the RNA. Subsequently, the membrane was washed

94

three times with water and added to a hybridization solution containing a 45 ng/µL probe. The

95

membrane was exposed with a chemiluminescent analyzer.

96

2.5 5′′-rapid amplification of cDNA ends

7

8

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The 5′-rapid amplification of cDNA ends (5′RACE) was executed using a SMARTer 5′RACE Kit

98

(Takara). To obtain the RACE-Ready first-strand cDNA, reverse transcription was performed by

99

adding 1 µg of total RNA, Gene-Specific Primers (GSPs), and reverse transcriptase. A 5′RACE

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PCR reaction was performed using the cDNA as the template with a universal primer short (UPM

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short) and gene-specific primers (GSPs). The PCR products were inserted into a pUC19-based

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vector via in-fusion cloning. The AsfD transcription initiation site was determined by DNA

103

sequencing.

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2.6 Quantitative real-time PCR

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Total RNA samples (4 µg) were used for cDNA synthesis with PrimeScript Reverse Transcriptase

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(Takara) and gene-specific reverse primers, according to the manufacturer’s protocol. A SYBR

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Premix Ex Taq II (Takara) was used for cDNA quantification. The primers used for quantitative

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real-time PCR are listed in Table 2. Then 5S rRNA was used as the internal reference. Each

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experiment was performed in triplicate.

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2.7 3′-reverse transcription PCR

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The forward primer for 3′ PA was designed in the peak area of AsfD expression. Reverse primers,

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3′ PB1 to 3′ PB8, were designed downstream of AsfD. These primers were used to search for the

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AsfD transcription termination site by reverse transcription and PCR. The primers used for

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3′-reverse transcription PCR are listed in Table 2.

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2.8 Western blot analysis

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A western blot analysis was conducted as previously described with some modifications [14].

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Briefly, WT-pBAD and WT-pBAD-AsfD were grown to an OD600 nm of 0.4. AsfD 8

9

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overexpression was induced by L-arabinose. Bacteria were harvested at four different points of

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induction (0 min, 15 min, 30 min, and 60 min). Protein extracts were prepared by sonication and

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subjected to 12% SDS-PAGE. FljB and DnaK were detected with anti-FljB antiserum (1:2000)

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[22] and anti-DnaK antibodies (Enzo Life Sciences diluted 1:5000), respectively, as the primary

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antibodies. Goat anti-rabbit and anti-mouse immunoglobulin G antibodies linked to horseradish

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peroxidase (diluted 1:5000), respectively, were used as the secondary antibodies. Signals were

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detected with ECL plus western blotting detection reagents (Thermo Scientific, USA).

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2.9 Bacterial motility assays

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WT-pBAD and WT-pBAD-AsfD were grown in LB until the OD600nm reached 0.4, and were then

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treated with L-arabinose (0.2% w/v) for 30 min to induce AsfD. Bacterial cultures (1 µL) were

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inoculated in a 0.3% agar LB plate containing 0.2% L-arabinose and incubated at 37°C for 12 h.

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Motility was determined by measuring the diameter of the circular zone formed by the moving

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bacteria. Each experiment was performed in triplicate.

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2.10 Biofilm formation assays

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Quantification of biofilm formation was conducted as previously described with some

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modifications [23]. The WT-pBAD and WT-pBAD-AsfD strains were grown in TSB until the

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OD600nm reached 0.4 and were then treated with L-arabinose (0.2% w/v) for 30 min. These strains

135

were inoculated into microtiter plates (Corning, USA) and incubated at 30°C without shaking for

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96 h. After incubation, the uncombined bacteria were eliminated and washed three times with PBS.

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Then, adherent bacteria were fixed by adding 200 µL methanol for 30 min. To observe the biofilm,

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250 µL crystal violet (0.5%) was added for 5 min after removing the methanol. The crystal violet 9

10

139

was wiped off and washed with water. Then, 275 µL of 30% v/v acetic acid was added into

140

microtiter plates for 10 min. The absorption of the eluted stain was measured at a 570 nm

141

wavelength to quantify the biomass of the biofilm.

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2.11 Statistical analysis

143

Data are presented as the means ± standard deviation. Statistical analysis was performed using a

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Student’s t-test. Differences were considered to be significant at P < 0.05.

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3. Results

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3.1 Identification of non-coding RNA for AsfD in S. Typhi

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Using transcriptome sequencing of S. Typhi, we found a high-level transcription from the

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opposite strand of flhDC, which is likely a novel non-coding RNA (data not shown). To confirm

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the existence of this presumptive ncRNA, the total RNA of the wild type strains was extracted

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during the early-exponential grow phase (OD600nm ≈ 0.4) and subjected to northern blot analysis. A

152

specific DIG-labeled probe, which was base-paired with a high-level expression region, was

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designed and employed (Fig. 1A). The results of the northern blot analysis showed a hybridization

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band with a full-length of approximately 2000 nt (Fig. 1B).

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To determine the boundaries of this antisense RNA, 5′RACE and 3′RT-PCR were conducted.

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The 5′RACE results revealed that the 5′-end of the transcript was 590 bp downstream of the

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termination codon of the flhDC operon on the complementary strand. The 3′-end of the transcript

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was characterized at 558 bp − 657 bp upstream of the start codon of the flhDC operon using

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3′RT-PCR. Together, these results indicate that the antisense RNA was 2079 nt − 2179 nt in length. 10

11

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The gene structure and location of the antisense RNA are shown in Fig. 1A. The antisense RNA

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was transcribed from the complementary strand of the flhDC operon and covered the entire flhDC

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operon. From the entire sequence of the novel antisense RNA, no obvious ORF structure or SD

163

element was found. Thus, the novel antisense RNA of flhDC was termed AsfD.

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Fig. 1 Identification of AsfD in S. Typhi. (A) Genomic location and structure of asfD. The white

166

arrows represent the adjacent genes, otsA, yecG, flhDC, motA, and motBc. The gray arrow

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represents the location of asfD. The black bar indicates the specific probe used for the northern blot

168

analysis. +1, experimental determined transcription start site of asfD. (B) AsfD was detected by a

169

northern blot analysis using a specific DIG-labeled probe. Total RNA of the wild type strain was

170

extracted at an OD600nm of 0.4. Different quantities of total RNA (2, 3, and 5 µg) were obtained

171

for the northern blot analysis. The hybridization bands were compared with RNA Mark.

172 173

3.2 Expression and regulation of AsfD in S. Typhi

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To study the expression characteristics of AsfD, total RNA was extracted from the S. Typhi at an

175

OD600nm of 0.3, 0.8, 1.3, and 2.0, representing the bacterial growth phases from the exponential

176

phase to the stationary phase, and subjected to a northern blot analysis and qRT-PCR. Both of the

177

results showed that AsfD expression increased in conjunction with bacterial growth and achieved

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the highest levels during the stationary phase (Fig. 2A and B).

179

Furthermore, the flhDC operon was reported to be activated during the stationary phase under

180

high osmolarity stress and regulated by several regulators (such as, RpoS, OmpR, and Fis) [17, 24]. 11

12

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In consideration of the AsfD expression characteristics and the genome location between AsfD

182

and flhDC operons, we utilized qRT-PCR to investigate whether the regulation of flhDC depends

183

on AsfD. Compared with the wild type strains, the expression of AsfD and flhDC were both

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downregulated in ∆ompR and ∆fis, and upregulated in ∆rpoS, which indicated that the regulation

185

of AsfD by OmpR, Fis, and RpoS were consistent with the flhDC operon. At the transcriptional

186

level of both asfD and flhDC, there were no significant differences between the ∆hfq and wild

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type strains (Fig. 2C). The qRT-PCR results also indicated that the antisense RNA AsfD could

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act on flhDC without hfq.

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Fig. 2 Expression of AsfD at different growth phases of S. Typhi. (A) The results of the

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northern blot analysis. Total bacterial RNA was extracted at an OD600nm of 0.3, 0.8, 1.3, and 2.0,

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10 µg of each RNA was subjected to a northern blot analysis, separated on a 4% polyacrylamide

193

gel containing urea and transferred to a membrane. A 5S rRNA was used as the loading control. (B)

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The level of AsfD in S. Typhi was measured using qRT-PCR and normalized to 5S rRNA. (C)

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Comparison of the transcriptional levels of asfD and flhDC in wild type, ∆ompR, ∆fis, ∆rpoS, and

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∆hfq strains. The qRT-PCR results showed that both asfD and flhDC were downregulated by the

197

regulators, OmpR and Fis, and upregulated by RpoS. In comparing ∆hfq with wild type, there were

198

no significant differences in the transcriptional level of asfD and flhDC. ***, P < 0.001; ns, not

199

statistically significant.

200 201

3.3 Effects of AsfD overexpression on flhDC expression and S. Typhi motility 12

13

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To examine the effects of AsfD on its putative target mRNA, flhDC, we investigated the level of

203

flhDC mRNA using qRT-PCR after overexpressing a 2079-nt AsfD sequence in the

204

arabinose-inducible plasmid, WT-pBAD-AsfD. Compared with WT-pBAD, the relative level of

205

flhDC mRNA increased after inducing the overexpression of AsfD for 30 min (Fig. 3A). In

206

addition, the mRNA levels of the flagellum-related genes, fliA and fljB, which were regulated by

207

FlhD2C2, increased similarly. The results of the western blot analysis showed that the levels of the

208

FljB protein were substantially elevated after overexpressing AsfD, especially following treatment

209

with L-arabinose for 60 min (Fig. 3A and B). The flagellum-related genes, flhDC, fliA, and fljB

210

have all been reported to participate in Salmonella motility. Therefore, to determine the effects of

211

AsfD on the motility of S. Typhi, we performed motility assays on both WT-pBAD and

212

WT-pBAD-AsfD. The results of the motility assays indicated that the overexpression of AsfD

213

enhanced the motility of S. Typhi (Fig. 3C and D). Based on the above results, we concluded that

214

AsfD overexpression promotes S. Typhi motility by upregulating flhDC.

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Fig. 3 AsfD overexpression increases S. Typhi motility through upregulating flagellar gene

217

expression. (A) The mRNA level of flagellar genes (flhDC, fliA, and fljB) was determined by

218

qRT-PCR, using 5S rRNA as the internal reference. (B) The level of fljB protein expression was

219

determined by western blot. DnaK was used as the loading control. Error bars indicate standard

220

deviations. (C) Motility assays of the WT-pBAD and WT-pBAD-AsfDc strains. (D) A columnar

221

statistical analysis of the motility ring diameter. The Y-axis of this bar chart suggests the motility

222

ring diameters on swim-agar plates. 13

14

223 224

3.4 AsfD overexpression promotes S. Typhi biofilm formation

225

Since changes in motility can influence biofilm formation, we suspected that AsfD could influence

226

the biofilm formation of S. Typhi. The WT-pBAD and WT-pBAD-AsfD strains were subjected to

227

biofilm formation assays, which were performed with TSB in 96-well microtiter plates using a

228

crystal violet assay. The results showed that the biofilm formation ability of WT-pBAD-AsfD was

229

approximately 2.5 times that of the control strain, WT-pBAD, suggesting that AsfD could enhance

230

the biofilm formation of S. Typhi (Fig. 4).

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Figure 4. Biofilm biomasses influenced by AsfD overexpression

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(A) Biofilm formation of WT-pBAD and WT-pBAD-AsfD in 96-well microtiter plates prior to the

234

absorption measurement. (B) Columnar statistical analysis of the biofilm biomasses. The Y-axis of

235

this bar chart suggests the absorption of the eluted stain measured at a wavelength of 570 nm.

236 237

4. Discussion

238

lncRNAs are RNAs longer than 200 nt that are incapable of coding proteins because they lack an

239

ORF structure [25]. By performing a deep sequencing analysis, we discovered several novel

240

lncRNAs in Salmonella, which have been associated with several biological processes, including

241

the invasion of host cells and responses to stress [10, 12, 13, 14, 26]. By combining our previous

242

findings, we identified a novel cis-encoded lncRNA AsfD, which was complementary to the flhDC

243

operon. The full-length of AsfD is 2079 nt − 2179 nt. No obvious ORF structure or 14

15

244

Shine-Dalgarno element was found throughout the entire sequence (Fig. 1). Therefore, we

245

considered AsfD to be a novel lncRNA.

246

The level of AsfD expression in S. Typhi was examined in different growth phases using a

247

northern blot and qRT-PCR. Moreover, we confirmed the expression characteristics of AsfD in S.

248

Typhi. The level of AsfD transcription increased gradually with growth and peaked during the

249

stationary phase (Fig. 2A and B). Furthermore, we utilized qRT-PCR to investigate the regulation

250

of RpoS, OmpR, Fis, and Hfq on AsfD and the flhDC operon. The results showed that the

251

regulators, OmpR and Fis, activated the transcription of AsfD and flhDC, whereas RpoS

252

suppressed their expression. In E. Coli Salmonella, most sRNAs binding to mRNA depend on the

253

chaperone Hfq [27]. In this study, the absence of Hfq did not influence the transcription of AsfD

254

and flhDC, which indicated that the cis-encoded ncRNA, AsfD, acting on flhDC did not require the

255

presence of Hfq (Fig. 2C).

256

In considering the AsfD expression characteristics, we suspected that the AsfD lncRNA was a

257

functional product in S. Typhi. Therefore, we first studied the influence of AsfD expression on the

258

S. Typhi phenotype by constructing WT-pBAD and WT-pBAD-AsfD strains. The results of the

259

motility assay suggested that AsfD may enhance the motility of S. Typhi by up-regulating the

260

flhDC operon (Fig. 3C and D). The flagella-associated genes are divided into three classes (class-1,

261

-2, and -3) [28–30]. The flhDC operon is the only class-1 operon which can regulate class-2

262

flagellar genes. fliA is a class-2 flagellar gene that can regulate class-3 flagellar genes, including

263

the flagellin genes, fliC and fljB. In addition, FljB is a phase 2 flagellin in S. Typhi GIFU10007

264

that functions as a key component of bacterial flagella [31]. FljB is also considered to be a PAMP 15

16

265

in host cells because it can bind to TLR5 and NLRC4 [32]. In the present study, AsfD

266

overexpression enhanced the expression of flhDC, fliA, and fljB (Fig. 3A and B). The results

267

showed that AsfD overexpression could enhance the motility of S. Typhi, likely by up-regulating

268

the target gene of the flhDC operon, as well as the class-2, class-3 flagellar genes.

269

Previous reports have indicated that motile bacteria have substantial advantages in the

270

adhesion of bacteria onto the surface during the early stages of biofilm formation compared to

271

non-motile bacteria [18]. Since AsfD was found to positively regulate S. Typhi motility, we

272

suspected that AsfD could also influence biofilm formation. To verify this theory, the biofilm

273

formation of the WT-pBAD and WT-pBAD-AsfD strains was determined by crystal violet

274

staining. The results showed that the overexpression of AsfD could promote bacterial biofilm

275

formation (Fig. 4A and B).

276

In summary, we demonstrated a novel antisense RNA-AsfD, which consisted of a 2079 nt −

277

2179 nt cis-encoded lncRNA encoded by the antisense strand of the flhDC operon. Moreover, the

278

expression of AsfD increases in conjunction with bacterial growth and peaks during the stationary

279

phase. Furthermore, AsfD is regulated by RpoS, OmpR, and Fis. Taken together, our findings

280

indicate that the overexpression of AsfD enhances the motility and biofilm formation of S. Typhi

281

by up-regulating the level of flhDC expression.

282 283

Acknowledgements

16

17

284

We would like to thank Takayuki Ezaki (Gifu University) for providing the bacterial strains. This

285

work was supported by the Zhang Jia Gang Science-Technology Supporting Plan of China

286

(ZKS1718, ZKS1824) and the National Natural Science Foundation of China (NSFC31670131).

287 288

References

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Highlights In this work, a novel antisense RNA located at the opposite strand of the flhDC operon was identified and termed AsfD AsfD can up-regulate the transcriptional levels of flhDC and other flagellar genes AsfD enhance the motility and biofilm formation of S. Typhi