Comparative transcriptome analysis

revealed the role and mechanism of a

FeoC-like LuxR-type regulator in
intracellular survival of Aeromonas
hydrophila Zhe Zhang
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 Aquaculture 556 (2022) 738287

Contents lists available at ScienceDirect

Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture

Comparative transcriptome analysis revealed the role and mechanism of a

FeoC-like LuxR-type regulator in intracellular survival of
Aeromonas hydrophila
Zhe Zhang 1, Leilei Mao 1, Yingxue Qin *, Lingmin Zhao, Lixing Huang, Xiaojin Xu, Qingpi Yan *
Fisheries College, Key Laboratory of Health Mariculture for the East China, Ministry of Agriculture, Jimei University, Xiamen, China

A R T I C L E I N F O A B S T R A C T

Keywords: A. hydrophila is one of the most important pathogens in the aquaculture industry, and its drug-resistant strains
LuxR-type regulator continue to emerge due to the irregular use of drugs. Previous studies found A. hydrophila could survive in fish
FeoC regulator macrophages, so it was speculated that A. hydrophila may hide in host macrophages to escape the killing of drugs
Intracellular survival
and other antibacterial factors. The mechanism of A. hydrophila survival in fish macrophages is worthy of further
Macrophage
Aeromonas hydrophila
study. In this study, the expression of a FeoC-like LuxR-type regulator was stable silenced by shRNA to construct
the silencing strain feoC-RNAi. And the intracellular survival rate of feoC-RNAi decreased by about 67.5%
compared with that of the wild-type strain. Comprehensive transcriptome analysis suggested that this FeoC-like
LuxR-type regulator may regulate at least 1286 genes. According to GO and KEGG analysis of these 1286 DEGs, it
can be speculated that this regulator may affect A. hydrophila intracellular survival by regulating bacterial metal
ion metabolism, chemotaxis, type IV pilus biogenesis and binding, then further regulate the bacterial intracel­
lular survival. Further comparison on biological phenotype of A. hydrophila B11 and feoC-RNAi revealed this
FeoC-like LuxR-type regulator could regulate the expression of genes related to iron and magnesium metabolism
and greatly affect bacterial growth under the limited magnesium concentration, but has no effect on bacterial
growth under the limited iron concentration. And inhibited expression of this FeoC-like LuxR-type regulator led
to a decrease in chemotactic ability of A. hydrophila by about 60%, disappearance of pilus, and a decrease in
adhesion by about 50%. So a conclusion could be made that this FeoC-like LuxR-type regulator might play a vital
role in regulating the uptake of magnesium and the homeostasis of magnesium and iron, and chemotaxis, type IV
pilus biogenesis and adhesion of A. hydrophila, then further protect bacteria from the microbicidal actions and
keep survival in host macrophages.

1. Introduction A. hydrophila can survive in fish macrophages, which means that

A. hydrophila is an opportunist pathogen widely distributed
throughout the world, which not only causes aquatic animal diseases,
but also has high pathogenicity to other terrestrial animals including
human (Awan et al., 2018; Daskalov, 2006; Elwitigala et al., 2005; Lin
et al., 2017). As one of the most common pathogens in the aquaculture,
A. hydrophila has been regarded as a serious threat to the aquaculture
industry. At present, the measures to control the diseases caused by
A. hydrophila mainly relies on antibacterial drugs, and the irregular use
of antibiotics easily lead to the emergence of drug-resistant strains
(Kusdarwati and Rozi, 2017). Previous studies have shown that
A. hydrophila can keep host macrophage as a shelter to escape the killing
of drugs and other antibacterial factors, making the bacteria more
difficult to control (Qin et al., 2014; Wang et al., 2019; Zhang et al.,
2019; Zhang et al., 2018). These studies also suggested that A. hydrophila
may be a facultative intracellular bacterium.
It has been known that phagocytes have developed various antimi­
crobial defense mechanisms to eliminate pathogens, including the
oxidative burst, acidification of phagosomes, or fusion of phagosomes
with lysosomes (Kohler et al., 2002). At the same time, facultative
intracellular bacteria, in return, have developed strategies counteracting
the phagocytes defense to keep survive. Therefore, to elucidate the

* Corresponding authors.
E-mail addresses: yxqin@jmu.edu.cn (Y. Qin), yanqp@jmu.edu.cn (Q. Yan).
1
These authors contributed equally to this work and should be considered co-first authors

https://doi.org/10.1016/j.aquaculture.2022.738287
Received 7 February 2022; Received in revised form 14 April 2022; Accepted 20 April 2022
Available online 25 April 2022
0044-8486/© 2022 Elsevier B.V. All rights reserved.
Z. Zhang et al. Aquaculture 556 (2022) 738287

mechanism of intracellular survival of A. hydrophila will help to find Table 1

more effective treatments to control this bacterium. Strains and plasmids used in this study.
The genome analysis of the pathogenic A. hydrophila B11 isolated Strains or Characteristic(s) Source or
from diseased eel revealed that six LuxR family proteins were found in plasmids reference
this bacterium (Mao et al., 2020). Among them, LuxR05735 is the smallest Plasmids
one with only 243 bp, but it had the greatest impact on the intracellular pACYC184 (CmR TcR) provided by Prof.
survival of A. hydrophila B11 (Accession Number: MN327964) (Mao, Nie
2019). Furthermore, LuxR05735 is also annotated as FeoC-like tran­ pACYC184- pACYC184 with shRNA- pACYC184- This study
feoC-68 feoC− 68 (CmR)
scription regulator in databases. It means that LuxR05735 has both the pACYC184- pACYC184 with shRNA- pACYC184- This study
structure and function of LuxR-type family and FeoC transcriptional feoC− 78 feoC− 78 (CmR)
regulatory factors. FeoC is regarded as a member of Feo system which is pACYC184- pACYC184 with shRNA- pACYC184- This study
the first transport system commonly used by bacteria to acquire envi­ feoC− 175 feoC− 175(CmR)
Strains
ronment Fe2+ under anaerobic and low pH conditions, but the function
B11 A. hydrophila isolated from diseased Provided by Prof.
of FeoC is still controversial (Hung et al., 2012; Lau et al., 2016). LuxR Anguilla japonica (SmR) Guo
family proteins have been known to distribute in a variety of bacteria, feoC68-RNAi feoC of B11 was silenced by shRNA- feoC68 This study
which can regulate various physiological functions of bacteria (Zeng and (SmR, CmR)
Xie, 2011). Studies have also suggested that LuxR family proteins feoC78-RNAi feoC of B11was silenced by shRNA- feoC78 This study
(SmR, CmR)
involve in the adaptation of bacteria to the host environment, and have a feoC175-RNAi feoC of B11 was silenced by shRNA- feoC175 This study
regulatory effect on the intracellular viability of bacteria (Ahmer, 2004; (SmR, CmR)
Fang et al., 2013). At the same time, the host cells also evolve several E.coli DH5α deoR,recA1 endA1,hsdR17(rK− ,mK+),phoA, Takara
mechanisms to limit the survival and growth of bacteria in them, supE44,λ− ,thi-1,gyrA96,relA1
including generating reactive oxygen and nitrogen species, lowering the
pH of the compartment harboring and invading microbe, limiting
strain exhibited the best efficiency of gene silencing was designated as
pathogenic microbes access to metal ion and so on (Blanc-Potard and
feoC-RNAi and chosen for further studies.
Groisman, 2021; Gan et al., 2019; Nunez et al., 2018).As transition
metals such as iron, manganese, and zinc play a key role in nutrition for
both microbes and their hosts, intracellular bacteria must regulate the 2.3. Preparation of fish macrophages
uptake and metabolism of metal ion in the intracellular environment
and help bacteria resist cell killing, which may be the key factor in Healthy tilapia (Oreochromis spp) (1068.1 ± 40.2 g) from aquacul­
determining the survival of intracellular bacteria. What is worthy of ture farm was used to prepare fish macrophages according to the method
further study is how this regulator, which has both the LuxR and FeoC described by Isla et al. and Mccarthy et al (McCarthy et al., 2008).
characteristics, regulates the expression of A. hydrophila and further help Briefly, fish were anesthetized with 4-ethyl-amino-benzocaine, the head
bacteria survive in host macrophages. kidneys were removed and pooled under sterile conditions. The tissue
In this study, silencing strain feoC-RNAi was constructed and its was then pushed through a 100 mesh nylon mesh and suspended in L-15
intracellular survival ability was evaluated. Then, comparative tran­ medium (Biological Industries, Israel) with 100 IU streptomycin/peni­
scriptome was used to analyze the difference in expression between the cillin (S/P)/mL and 2% fetal bovine serum (FBS) (Thermo Fisher, USA).
wild-type strain and silencing strain. Finally, the biological phenotype The cell suspension was slowly added to a 34%/51% discontinuous
was verified to explore the role and mechanism of this FeoC-like LuxR- Percoll (Amersham Pharmacia Biotech, UK) separation solution with a
type regulator in the intracellular survival of A. hydrophila. syringe and centrifuged at 400 ×g for 30 min at 4 ◦ C. Cell bands were
collected in the middle of the 34%/51% interface and the cells were
2. Materials and methods washed twice and resuspended in L-15 medium containing 10% FBS,
100 IU S/P/mL. After that, the cells were incubated at 28 ◦ C for 4 h, non-
2.1. Bacterial strains and growth conditions adherent cells were removed by washing with L-15 medium and
adherent cells were collected by centrifugation. Then the cells were
The pathogenic A. hydrophila strain B11 was routinely grown in LB at resuspended in L-15 medium containing 10% FBS, 100 IU S/P/mL and
28 ◦ C, Escherichia coli was grown at 37 ◦ C in LB. The medium was sup­ adjusted to approximately 2 × 106 cells/mL.
plemented with the appropriate antibiotics at the following concentra­
tions: 50 μg/mL streptomycin (Sm) and 25 μg/mL chloromycetin (Cm). 2.4. Survival of A. hydrophila in fish macrophages in vitro
Table 1 listed the bacterial strains and plasmids used in this study.
Bacterial survival in fish macrophages was performed according to
2.2. Construction of feoC-RNAi the methods described by Larsen et al (Boesen et al., 2001) with some
modifications. Briefly, the assay was performed according to the
The gene of this FeoC-like LuxR-type regulator of A. hydrophila B11 following procedure: 1 mL of prepared fish macrophage suspension and
was stably silenced according to the protocol described before (Toku­ 1 mL of prepared bacteria suspension was added to a 6-well culture plate
naga et al., 2015; Zhang et al., 2019; Zhang et al., 2018). Briefly, three [multiplicity of infection (MOI) = 100 (100 bacteria per macrophage)]
short hairpin RNA sequences targeting different part of mRNA coding and incubated for 1 h at 28 ◦ C for cell phagocytosis. After that, the
regions were designed and synthesized by Shanghai Generay Biotech macrophages were collected and pooled in sterile tubes and centrifuged
Co., Ltd. (Shanghai, China) (Table 2). These annealed shRNA were at 100 ×g for 5 min at 28 ◦ C. Then the supernatant was carefully
ligated to the pACYC184 vector digested with SphI and BamHI (Takara, removed without disturbing the packed cells. After washing the packed
Kusatsu, Japan) usingT4 DNA ligase (Takara, Kusatsu, Japan). The re­ cells twice with cold PBS, the cells were resuspended in 2 mL PBS. The
combinant plasmids were identified and transformed into E.coli DH5α to cell suspension was treated with 2500 mg/mL gentamicin at 28 ◦ C for
amplification. And then, the recombinant plasmids were transformed 20 min to kill extracellular bacteria, and then washed twice with ice-
into A. hydrophila B11 by electroporation. The stable silencing strains cold PBS. After centrifugation to collect the cells, the supernatant was
were first screened by Chloramphenicol and Streptomycin, identified by removed and tested for sterility by plate count. The packed cells were
PCR and sequencing. Finally, the expression of this FeoC-like LuxR-type resuspended in fresh L-15 medium containing 10% FBS and 100 units S/
regulator in each silencing strains were evaluated by qRT-PCR and the P. The cell suspension was incubated at 28 ◦ C in 5% CO2, the cell

2
Z. Zhang et al. Aquaculture 556 (2022) 738287

Table 2
Oligonucleotides used in producing shRNA for stable gene silencing.
Target gene shRNA sequence for stable gene silence

F:5’-GATCCGCCACTTCCACACCTCGGAAGTTCAAGAGACTTCCGAGGTGTGGAAGTGGCTTTTTTGCATG-3′
feoC-RNAi-68
R:5′ - CAAAAAAGCCACTTCCACACCTCGGAAGTCTCTTGAACTTCCGAGGTGTGGAAGTGGCG − 3’
F:5’- GATCCCGGTCCCATCGTCATGCTCATTCAAGAGATGAGCATGACGATGGGACCGTTTTTTGCATG − 3′
feoC-RNAi-78
R:5′ - CAAAAAACGGTCCCATCGTCATGCTCATCTCTTGAATGAGCATGACGATGGGACCGG − 3’
F:5’- GATCCGGCAAGAGCCTGGAGGTGATGTTCAAGAGACATCACCTCCAGGCTCTTGCCTTTTTTGCATG − 3′
feoC-RNAi-175
R:5′ - CAAAAAAGGCAAGAGCCTGGAGGTGATGTCTCTTGAACATCACCTCCAGGCTCTTGCCG − 3’

samples were taken out after incubation for 0 h and 1 h, then centrifuged
for 5 min at 100 ×g at 28 ◦ C. For the samples of 0 h and 1 h, the su­
pernatant was discarded, and 1 mL of sterile distilled water was added to
incubate for 30 min to lyse the cells. After that, the cell lysis was
centrifuged and the CFU of the precipitate was determined by plate
counting. Intracellular survival rate was defined as the number of CFU of
1 h cell lysate sample divided by the number of CFU of 0 h cell lysate
sample.
parameters were 95 ◦ C for 10 min, followed by 45 cycles of 95 ◦ C for 20
s, 56 ◦ C for 20 s, and 72 ◦ C for 20 s. Threshold cycling and dissociation
curves were determined using QuantStudioTM 6 Flex software to
confirm that only one PCR product was amplified and detected. The
relative expression of genes in qRT-PCR was calculated by Pair Wise
Fixed Reallocation Randomization Test (Pfaffl et al., 2002) using a
relative expression software tool (version 2, REST 2008). Primer se­
quences are listed in Table 3.

2.5. Transcriptomic analysis

2.8. Effect of iron and magnesium on the growth and related genes
expression of B11 and feoC-RNAi
Total RNA were extracted from B11 and feoC-RNAi strains from three
independent cultures. The RNA-seq libraries were constructed using
Iron chelate 2,2′ -bipyridine and magnesium chelate ammonium
TruSeq™ Stranded Total RNA Library Prep Kit (Illumina, San Diego, CA,
polyphosphate was added to LB medium respectively. And the concen­
USA). dUTP was used instead of dTTP in the dNTPs reagent for the
tration of iron chelate in LB was 0, 50, 100, 200, 300 and 400 μmol/L
second strand of cDNA to make the base in the second strand of cDNA
respectively. The concentration of magnesium chelate in LB was 10, 20,
contain A/U/C/G. Prior to PCR amplification, the second strand of the
30, 40, 50 mmol/L respectively. A. hydrophila B11 was inoculated into
cDNA was digested with the UNG enzyme such that the library con­
the medium for 36 h and the growth curves of bacteria in different
tained only the first strand of cDNA. After that, sequencing was carried
concentrations of chelate was draw. The lowest chelate concentration
out on the Illumina HiSeq4000 platforms at Majorbio Biotech Co., Ltd.
that completely inhibited bacterial growth was screen out.
(Shanghai, China).
According to the above screening results, the chelate that can
In order to ensure the accuracy of subsequent bioinformatics anal­
completely inhibited the growth of A. hydrophila B11 was added to the
ysis, the original sequencing data is first filtered to obtain high quality
LB medium. And different concentrations of Fe2+, Fe3+ and Mg2+ were
sequencing data (clean data). High quality sequences were aligned to the
added. Then, the medium with Fe2+ or Fe3+ concentration of 10− 3, 10− 4,
specified reference genome by Bowtie software. The differentially
10− 5, 10− 6 mol/L and the medium with Mg2+ concentration of 10− 2,
expressed genes were screened under the condition of P ≤ 0.05, |logFC|
10− 3, 10− 4, 10− 5 mol/L was prepared. A. hydrophila B11 and feoC-RNAi
≥ 1, which means that P ≤ 0.05 after correction, and the difference
was inoculated in these medium and cultured at 28 ◦ C, the values of
multiples more than 2 times were identified as significant differences.
Significant enrichment analysis of GO functions was performed on dif­
Table 3
ferential genes to illustrate the functional enrichment of differential
Primer sequences for PCR and qRT-PCR.
genes using the Blast2GO software, and the differences between samples
were clarified at the level of gene function. KOBAS software was used to Gene name Sequence
perform KEGG pathway enrichment analysis. AHA_2496F / 5′ - GGACAGCCATTTTCAGCC- 3’ / 5’
The transcriptome data were submitted to NCBI (Accession Number: AHA_2496R -CCTCTTTCACCACCACCG − 3’
SRR10681608, SRR10681622, SRR10681623, SRR10681612, AHA_3833F / 5′ - CCCAAGATCATTGCCCACC- 3’ / 5’
AHA_3833R -CTTCCAGAAACAGCACCCG- 3’
SRR10681613, SRR10681614). AHA_2558F / 5′ - CTGCTGGGGCTGGAAAG- 3′ / 5’-
AHA_2558R AACAGGTCGGGAATGGG- 3’
AHA_2559F 5’- TTTTGCGAGCGGGTGTTC- 3′ / 5’-
2.6. RNA extraction and reverse transcription
/AHA_2559R CCTGGCTGATGTGGGGAT- 3’
AHA_2831 F / 5’- CAGGGCAAACTGGTGGGG- 3′ / 5′ -
The RNA samples returned by the sequencing company was used for AHA_2831 R TGTCGAGCGGCGAGGAAC- 3’
qPCR verification to confirm the transcriptome data. The other RNA AHA_4206 F / 5′ - GTTCCCTTCCAAACACAT- 3′ / 5′ -
samples was extracted from freshly cultured bacteria and first-strand AHA_4206 R CAGTTCGCCTAAAAATTC- 3’
AHA_3869 F / 5′ - CGCCCTGCGTTCTTTCTT- 3′ / 5’-
cDNA synthesis were performed using EasyPure RNA Kit (TransGen
AHA_3869 R CATCCGGGTCAGCGTTTC- 3’
Biotech, China) and TransScript® ALL-in-one First-Strand cDNA Syn­ AHA_0691 F / 5′ - ACTGATGATTGCGGTAGCC- 3′ / 5’-
thesis SuperMix (TransGen Biotech, China) according to the manufac­ AHA_0691 R CATTGAGCGCCAGATTGTA- 3’
turer’s recommended protocol, respectively. AHA_3193 F / 5′ - CGGCTTCACTCGGCTTT- 3′ / 5’-
AHA_3193 R TCCTTGCGTCGCTCCTT- 3’
AHA_0686 F / 5’-GTTTTTGCGTTCAGGTTT- 3 / 5’-
2.7. qRT-PCR AHA_0686 R GTTCTGGGTCAGGTTGCT- 3
AHA_4123 F / 5’-GGGCTTTCGCCACCTCAT- 3 / 5’-
AHA_4123 R CCCCTCCATCGCCTTGTT- 3
qRT-PCR was performed using QuantStudio 6 Flex PCR system (ABI,
AHA_0937 F / 5’-GCTGGAATCCTTCATCG- 3 / 5’-
Garlsbad, CA, USA) with SYBR Green qPCR Mix (TransGen Biotech, AHA_0937 R CTCCCACTCCTCCTCGT- 3
China) according to the manufacturer’s instructions. 16S rRNA was used AHA_2529 F / 5’-GACCGAGACCCAGCAGAT- 3 / 5’-
as an internal control. The reaction was carried out in a 10 μL volume AHA_2529 R AGAGGGCGGGATAAAAGC- 3
mixture containing 0.2 μL PowerSYBR Green PCR Master Mix, 5 pmol L- AHA_1036 F / 5’-CAAAAACAAGGAGGGACG- 3 / 5’-
AHA_1036 R TGGAAGCGGTTGAAGGTA- 3
1 specific primers and approximately 50 ng cDNA. The cycling

3
Z. Zhang et al.
OD600 nm were measured every hour, and growth curves were plotted.
According to the growth curve, the bacteria that grew in the medium
with Fe2+ or Fe3+ concentration of 10− 3, 10− 4 mol/L and Mg2+ con­
centration of 10− 2, 10− 3, 10− 4, 10− 5 mol/L were collected and evaluated
the expression of related genes by RT-PCR.
2.9. Chemotaxis of bacteira to the Surface Mucus of fish
Fish mucus was prepared according to the method described previ­
ously (Huang et al., 2015). Skin mucus of healthy tilapia (Oreochromis
spp.) was carefully collected by a soft scraper, then the skin mucus was
mixed with PBS and centrifuged at 20000 g for 30 min at 4 ◦ C. The su­
pernatant was collected and filtered by 0.45 μm and 0.22 μm filters,
respectively. The overnight cultured bacteria were centrifuged at 2000
rpm for 10 min at 4 ◦ C, and the collected bacteria were washed twice
with PBS. Then the bacteria were resuspended with PBS and the con­
centration was adjusted to OD600 = 1.0.
The capillary tubes with an inner diameter of 0.1 mm were sealed at
one end and filled with the fish skin mucus. A syringe was filled with the
bacterial suspension prepared above. Then, the unclosed end with fish
mucus of the capillary was dipped into the bacterial suspension in the
syringe, and incubated at 28 ◦ C for 1 h. During this process, the capillary
remains horizontal. Then the liquid in the capillary tubes was blown into
PBS in the eppendorf tubes, and was diluted to evaluate the number of
chemotactic bacteria by plate counting. Chemotactic index = chemo­
tactic cell of each strains / chemotactic cell of the wild-type strain.
2.10. Adhesion of bacteria to fish mucus
The bacteria adhesion ability was measured according to the method
of Huang et al. (Huang et al., 2015). Briefly, 20 μL of tilapia surface
mucus was evenly spread on a glass slide with a size of 22 mm × 22 mm,
and fix with methanol at room temperature for 20 min. Then 200 μL of
bacterial suspension (OD600 = 0.3) was evenly spread on the mucus-
coated glass slide and incubated at 28 ◦ C for 2 h, the PBS-coated glass
slide was used as a negative control. Then the slides were gently washed
5 times with PBS to remove unattached bacteria, and then the adhered
bacteria were fixed with 4% methanol for 30 min and stained with
crystal violet for 3 min. The number of adhered bacteria was counted
under microscope (Leica) (×1000). 20 fields of view of each sample
were randomly selected for counting.
2.11. Transmission electron micrographs
Formvar-coated grids were floated on 20 μL drops of bacterial sus­
pensions for 1 min. Excess sample was withdrawn by touching the edge
of the grid to a cut edge of Whatman filter paper. Then, the grids were
washed three times in sterile deionized water to remove the medium,
and the cells were negatively stained by floating the grids on a drop of
1% phosphotungstic acid. At last, the bacteria were observed with a
transmission electron microscope (Philips, Tecnai F20, Amsterdam,
Holland).
2.12. Data processing
Unless otherwise stated, all experiments were repeated at least three
times in triplicates. Data are presented as mean ± SD. Statistical analysis
was performed by one-way analysis of variance with Dunnett’s test using
SPSS 22.0 software (Chicago, IL, USA). P < 0.05 was considered statis­
tically significant.
4
Aquaculture 556 (2022) 738287
3. Results
3.1. feoC-RNAi construction and intracellular survival in fish
macrophages
The efficiency of feoC silencing in three silencing strains showed that
feoC78-RNAi exhibited the highest efficiency of gene silencing (Fig. 1A).
Furthermore, the wild-type strain and feoC78-RNAi displayed almost the
same growth curves (Fig. 1B). Therefore, feoC78-RNAi was selected for
further studies and designated as feoC-RNAi.
The intracellular survival rates of the wild-type and feoC-RNAi
(Fig. 1C) displayed that the intracellular survival rate of feoC-RNAi
decreased by about 67.5% compared with that of the wild-type strain.
This result indicates that the expression of the FeoC-like LuxR-type
regulator could greatly affect intracellular survival of A. hydrophila.
3.2. Transcriptomic analysis
The transcriptome of B11 and feoC-RNAi were compared and
analyzed. The gene expression profile was calculated and the changes in
the expression level met FDR < 0.05 & |log2FC| ≥ 1 were considered
statistically significant difference. According to this standard, there are
1286 differentially expressed genes (DEGs), of which 353 genes are
significantly upregulated and 933 genes are significantly downregulated
(Fig. 2). These data suggested that the FeoC-like LuxR-type regulator can
regulate the expression of a large number of genes and has an extremely
important function in A. hydrophila.
The functions of the 1286 DEGs were analyzed by GO and catego­
rized into different enriched functional groups, among which the
important functions included cell projection assembly, small molecule
metabolic process, binding, inorganic anion transmembrane transport
and so on (Fig. 3). The 1286 DEGs were also performed KEGG pathway
enrichment analysis and enriched in 59 KEGG pathways, including
bacterial chemotaxis, ABC transporters, two-component system, ribo­
some and so on (Fig. 4). Comprehensive transcriptome analysis sug­
gested that the FeoC-like LuxR-type regulator may regulate metal ion
metabolism, chemotaxis, type IV pilus biogenesis and binding of
A. hydrophila, then further regulate the bacterial intracellular survival.
According to the GO and KEGG analysis, the functional genes that may
be directly and closely related to the intracellular survival of bacteria
were screened out(Table 4).
3.3. Effects of the FeoC-like LuxR-type regulator expression on Fe and Mg
metabolism in A. hydrophila
qPCR verification confirmed the transcriptome data met the re­
quirements (Figure 5). And the trend of the effects of Fe2+ and Fe3+ on
bacterial growth and the expression of genes related to iron metabolism
almost the same (Figure 6 and 7). The growth curves of the wild-type
strain and the silent strain basically coincide. When the iron ion con­
centration is lower than 10− 4 mol/L, the wild-type strain and silent
strain only grew at a very low level. As the iron ion concentration de­
creases, the expression of the gene AHA2496 encoded a (Fe–S)-binding
protein is up-regulated or maintained the original level, while the
expression of gene AHA3833 encoded bacterioferritin (bfr) is signifi­
cantly down-regulated. These results suggested that the FeoC-like LuxR-
type regulator can regulate the expression of genes related to iron
metabolism, but has no effect on the growth of bacteria, indicating that
A. hydrophila may have other pathways to obtain iron, and those path­
ways are not regulated by the FeoC-like LuxR-type regulator.
The data showed that Mg2+ has a significant effect on the growth of
bacteria (Fig. 8A). Under different concentration of magnesium ion, the
OD600 of the maximum growth of the wild-type strain was all lower than
0.5, and the silent strain hardly grew. Therefore, the genes expression
could not evaluate in feoC-RNAi strain. And the expression of the genes
mgtE and yifB tended to be up-regulated with the decrease of Mg2+
Z. Zhang et al. Aquaculture 556 (2022) 738287

Fig. 1. feoC-RNAi construction and intracellular survival in fish macrophages. (A) The efficiency of feoC silencing. (B) The growth curves of wild-type strain and feoC-
RNAi strain. (C) The intracellular survival of the wild-type strain and silencing strain. The data are presented as the means ± SD; Values denoted by different letters
are significantly different. (P < 0.05).

Fig. 2. Volcano plot and statistics of differentially expressed genes. (A) Volcano plot (B) The number of up-regulated genes and down-regulated genes.

Fig. 3. Histogram presentation of clusters of GO classification of 1286 DEGs.

5
Z. Zhang et al. Aquaculture 556 (2022) 738287

Fig. 4. The KEGG pathway enrichment of 1286 DEGs.

concentration in wild-type strain (Fig. 8B). These results suggested that

Table 4
the FeoC-like LuxR-type regulator could regulate the magnesium
The genes with significant difference in expression and closely related to the
metabolism of A. hydrophila, then further involved in bacterial growth
intracellular survival of A. hydrophila.
and intracellular survival.
Function Number in the Encoded protein (Gene)
transcriptome

Ion metabolism AHA_2496 (Fe–S)-binding protein 3.4. Effects of the FeoC-like LuxR-type regulator expression on
AHA_3833 Bacterioferritin (bfr) chemotaxis of A. hydrophila
AHA_2558 iron‑sulfur cluster binding
protein From the results of transcriptome analysis and qRT-PCR verification,
AHA_2559 Fe-S oxidoreductase
it can be considered that after the expression of the FeoC-like LuxR-type
AHA_2831 magnesium transporter (mgtE)
AHA_4206 Mg chelatase-like protein regulator was inhibited, a series of chemotaxis related genes were
(yifB) significantly down-regulated (Fig. 9A) in feoC-RNAi. Bacteria chemo­
Chemotaxis AHA_4123 methyl-accepting chemotaxis taxis assay also confirmed that the chemotactic ability of feoC-RNAi was
protein (mcp)
reduced by more than 60% (Fig. 9B). However, feoC-RNAi still had
AHA_0937 chemotaxis sensory transducer
family protein
flagella (Fig. 10C) and motility of feoC-RNAi was not reduced compared
AHA_2529 putative chemotaxis protein with the wild-type strain (Data not shown). These results suggested that
AHA_1036 chemotaxis protein (chA-1) the FeoC-like LuxR-type regulator can regulate the chemotaxis of
Type IV pilus biogenesis AHA_3869 type IV pilus assembly ATPase A. hydrophila, and then affect bacterial intracellular survival.
and binding (pilB)
AHA_0691 type IV pilus biogenesis protein
(pilY) 3.5. Effects of the FeoC-like LuxR-type regulator expression on pilus
AHA_0686 type IV pilus modification
biogenesis and adhesion of A. hydrophila
protein (pilV)
AHA_3193 type IV pilus biogenesis protein
(pilN) The results of transcriptome analysis and qPCR verification also
suggested that after the expression of the FeoC-like LuxR-type regulator
was inhibited, some genes related to type IV-A pilus biogenesis were

Fig. 5. Verification of differentially expressed genes related to Fe and Mg metabolism. (A) The expression of the genes related to Fe metabolism; (B) The expression of
the genes related to Mg metabolism.

6
Z. Zhang et al. Aquaculture 556 (2022) 738287

Fig. 6. Effects of Fe2+ on growth and the expression of genes related to iron metabolism of A. hydrophila.

Fig. 7. Effects of Fe3+ on the expression of genes related to iron metabolism and growth of A. hydrophila.

Fig. 8. Effects of Mg2+ on the expression of genes related to magnesium metabolism and growth of A. hydrophila.

significantly down-regulated (Fig. 10A). Electronic microscope obser­ 4. Discussion

vations showed that pili were distributed around the cells of the wild-
type strain (Fig. 10B), while no intact pilus was observed around the
cells of the foeC-RNAi (Fig. 10C). Adhesion assays revealed that the
adhesive ability of feoC-RNAi was reduced by about 50% (Fig. 10D, E,
F). The results indicated that the FeoC-like LuxR-type regulator can
regulate type IV-A pilus biogenesis and adhesion of A. hydrophila, and
then affected bacterial intracellular survival.
The key role of LuxR transcriptional regulator in bacteria has been
wildly recognized, and it has been confirmed that LuxR can regulate the
expression of a variety of genes, including those encoding Quorum
Sensing (QS), virulence factors, antibiotics biosynthesis, motility, bio­
film formation, and so on (Chen and Xie, 2011). The results in this study
also displayed that after the expression of this FeoC-like LuxR-type
regulator was inhibited, the intracellular survival of A. hydrophila was

7
Z. Zhang et al.
Fig. 9. Effects of the FeoC-like LuxR-type regulator expression on chemotaxis
of A. hydrophila. (A) The results of qRT-PCR; (B) The chemotaxis index of B11
and feoC-RNAi strains.
decreased by about 67.5%, which meant luxR05735 played important role
on A. hydrophila survival in fish macrophages. The role of LuxR on
intracellular survival of other bacteria has also been revealed. Study on
the LuxR-type regulator VjbR in Brucella suggested that this regulator
directly modulated the expression of a subset of genes that not only
encompass the well-studied virulence factors known to control intra­
cellular trafficking and escape lysosome-mediated degradation, but also
involve electron transport chain components (Kleinman et al., 2017). In
this study, transcriptome analysis suggested that this FeoC-like LuxR-
type regulator may regulate the expression of at least 1286 genes. Ac­
cording to the GO and KEGG analysis of these 1286 DEGs, it is specu­
lated that this regulator may affect A. hydrophila intracellular survival by
Fig. 10. Effects of the FeoC-like LuxR-type regulator expression on pilus biogenesis and adhesion of A. hydrophila.
8
Aquaculture 556 (2022) 738287
regulating bacterial metal ion metabolism, chemotaxis, type IV pilus
biogenesis and binding.
Further studies showed that this FeoC-like LuxR-type regulator could
regulate the expression of some genes related to iron metabolism, but
not affect the growth of A. hydrophila under the limited Fe2+ or Fe3+
concentration. At the same time, restricted expression of this FeoC-like
LuxR-type regulator greatly affect the expression of mgtE and yifB and
growth of bacteria under the limited Mg2+ concentration. So it is spec­
ulated this FeoC-like LuxR-type regulator play a vital role in regulating
the uptake of magnesium and the homeostasis of magnesium and iron in
A. hydrophila to maintain growth and intracellular survival. The role of
LuxR-type regulator in the various physiological and biochemical
functions of bacteria has been extensively studied and elucidated.
However, the role of LuxR family regulators on regulating magnesium
and iron metabolism of bacteria is rarely mentioned. As a FeoC-like
regulator, the function of this regulator in bacteria remain enigmatic
(Brown et al., 2021; Smith et al., 2019). FeoC has been known to be one
of the three proteins that comprised FeoABC system, which is the most
widely distributed and dedicated Fe2+ uptake system across the pro­
karyotic domain (Brown et al., 2021) and has been shown to connected
most widely to the virulence of pathogenic prokaryotes (Hantke, 2003;
Lau et al., 2016). Gao et al. once provided indirect evidence that FeoC is
a negative regulator of the feoABC operon in Yersinia pestis (Gao et al.,
2008). But Fetherston et al. found a ΔfeoC mutant did not affect tran­
scription from the feo promoter under aerobic or micro aerobic condi­
tions (Fetherston et al., 2012). Duo to feoC conserved helis-turn-helix
(HTH) motif, it has been predicted to play a role as a transcriptional
regulator (Brown et al., 2021; Kammler et al., 1993; Lau et al., 2013).
However, there are no reports indicating that FeoC binds to DNA (Brown
et al., 2021). Therefore, the function of FeoC remains to be elucidated.
Z. Zhang et al.
Both iron and magnesium are essential element for bacteria survival
and growth and also critical for the pathogenesis (Blanc-Potard and
Groisman, 2021; Leon-Sicairos et al., 2015). Magnesium is the most
abundant divalent metal in living cells and is required for numerous
cellular activity (Moomaw and Maguire, 2008; Shin et al., 2014) and Fe
is needed in amounts 1000 to 10,000 times smaller than those of Mg in
the cell (Hantke, 1997). Moreover, study once suggested that Mg2+ and
ferrous iron shared a permease and ferrous iron can be taken up by the
magnesium transport system independently of Feo system, especially
when the medium contained a low concentration of magnesium
(Hantke, 1997). Based on these point of view, it could be explained the
results in this study. This regulator with both LuxR and FeoC charac­
teristics may not direct involve in ferrous iron uptake of A. hydrophila,
and this bacteria can obtain enough ferrous iron by other pathway,
including magnesium transporter system. So under the limited iron
concentration, foeC-RNAi could maintain the same growth trend as the
wild-type strain. While under the limited magnesium condition, the
growth of both wild-type strain and foeC-RNAi was inhibited, especially
feoC-RNAi hardly grew.
The results in this study also showed that restricted expression of this
FeoC-like LuxR-type regulator greatly affected the chemotaxis ability,
pilus biogenesis and adhesion of A. hydrophila, then further reduced the
ability of bacteria to survive in macrophages. The phagocytosis of
pathogenic bacteria by host phagocytes was once believed to be a pas­
sive process. Recent research has found that some pathogens can
actively choose host phagocytes as refuge and have evolved a number of
very effective strategies that permit intracellular survival within
phagocytes (Kaufmann, 2011; Roop II and Caswell, 2013). For example,
inhibition of phagosome-lysosome fusion, inhibition of phagosome
acidification, lysis of the phagosome and escape to enter the cytoplasm,
and so on (Qin et al., 2014). Weinstein et al. once reported flagellated
salmonella survived longer in macrophages than do nonflagellated
salmonellae (Weinstein et al., 1984). And some the mechanisms eluci­
dated by the authors may also be applicable to the results of this study.
For example: (i) Bacteria with pili may not trigger the full cascade of
early microbicidal events of macrophages. (ii) After invading macro­
phages, bacteria with pili or without pili may be sequestered in different
compartments of cells. (iii) Bacteria with pilus may consequently be
shielded from the microbicidal actions of the macrophages better than
are the bacteria without pili. (iv) The presence of pili may act as a cloak
to partially protect bacteria from the microbicidal enzymes, pH, and
other defenses of the macrophages. And during these processes,
chemotactic movement of A. hydrophila may help bacteria to find the
sites suitable for survival or escape the sites for microbicidal actions.
CRediT authorship contribution statement
Zhe Zhang: Investigation and Editing. Leilei Mao: Investigation and
Writing. Yingxue Qin: Investigation, Writing-Reviewing and Editing.
Lingmin Zhao: Investigation. Lixing Huang: Investigation. Xiaojin Xu:
Investigation. Qingpi Yan: Writing- Reviewing and Editing.
Declaration of Competing Interest
The authors declare that the research was conducted in the absence
of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Acknowledgements
This work was supported by grants from the Natural Science Foun­
dation of Fujian Province (Grant No. 2020J01663), the National Natural
Science Foundation of China (Grant No. 31502194), and Industry-Uni­
versity-Research cooperation project of Xiamen (Grant
No.3502Z20203049).
9
Aquaculture 556 (2022) 738287
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she had had a hand in that anyhow. It must be as Blount said, that
she was more of a tool herself than anything else. She probably
couldn’t help herself very well or didn’t know the lengths to which her
pretended “friends” were prepared to go. Her eyes seemed troubled,
sad. She seemed weaker, more futile, than at any time since he had
known her, and this, while it did not add particularly to his respect,
softened his personal animosity. He felt that under the circumstances
he might come to like her. He also thought that she might be made to
like him enough to help him. He had the emotional mastery of her, he
thought, and that was something. He had described the incident with
all the vividness of detail that he could, showing how he and Blount
had escaped death by a hair’s breadth. She seemed a little sick, and
shortly after left the table. Gregory had taken good care to make it
plain that the strangers in the other car had been informed as to the
exact details of the case, and had offered their services as witnesses
in case they were wanted.
“But we don’t propose to do anything about it,” he said genially,
“not now, anyhow,” and it was then that she seemed to become a
little sick or faint, and left him.
Whether owing to this conversation or the accident itself, or to
circumstances concerning which he knew nothing, there now
seemed to come a temporary lull in the activities of this group. The
Blue Trailer disappeared as an active daily fact in their lives. Mrs.
Skelton was called to the city on business for a few days, as well as
Mr. Diamondberg, the “cloak and suit man,” as Blount always called
him, who in all the time he had been there had never publicly joined
them. Mrs. Skelton came back later as cheerful and optimistic as
ever, but in the meanwhile there had been an approach on the part
of Imogene toward himself which seemed to promise a new order of
things. She was freer, more natural and more genial than she had
been hitherto. She was with him more, smiling, playful, and yet
concerned, he thought. Because of their conversation the morning
after the accident, he felt easier in her presence, more confidential,
as though he might be able to talk to her about all this soon and get
her to help him.
 They had two hours together on the second afternoon of the
absence of the others which brought them within sight of each
other’s point of view. It began after lunch, because Gregory had
some reports to examine and was staying here to do it. She came
over and stood beside him.
“What are you doing?” she asked.
“Oh, I’m looking up some facts,” he replied enigmatically, smiling
up at her. “Sit down.”
They fell into conversation first about a tennis match which was
being held here, and then about his work, which he described in part
after observing that she knew all about it, or ought to.
“Why do you always talk to me that way about everything in
connection with you?” she asked after a moment’s pause. “You have
such a queer way of speaking, as though I knew something I ought
not to know about your affairs.”
“Well, you do, don’t you?” he questioned grimly, staring at her.
“Now, there it is again! What do you mean by that?”
“Do you really need to have me explain to you?” he went on in a
hard cynical manner. “As though you didn’t know! I don’t suppose
you ever heard of the Union Bank of Penyank, for instance? Or Mr.
Swayne, its president? Or Mr. Riley, or Mr. Mears, the cashier?”
At the mention of these, as at the mention of the automobile
accident, there was something which seemed to click like a camera
shutter in her eyes, only this time there was no sign of pain, none
even of confusion. She seemed, except for a faint trace of color, to
be fairly calm and poised. She opened her mouth slightly, but more
in an attempted smile of tolerance than anything else.
“The Union Bank? Mr. Swayne? Mr. Tilney? What are you talking
about?” she persisted. “Who is Mr. Swayne, and where is the Union
Bank?”
“Really, now, Miss Carle,” he said with a kind of dogmatic fury, “if
you want me to have any regard of any kind for you in the future, quit
lying about this. You know well enough what I mean. You know who
Mr. Swayne is, all right, and why he left Eastridge. You also know Mr.
Diamondberg, although I heard you say you didn’t, and that right
after I had seen you walking with him out here on the dunes three
weeks ago. You don’t remember that, I suppose?” this as she
fluttered slightly.
She stared, completely shaken out of her composure, and a real
flush spread over her cheeks and neck. For the moment her
expression hardened the least bit, then gave way to one of mingled
weakness and confusion. She looked more or less guilty and
genuinely distrait.
“Why, Mr. Gregory,” she pleaded weakly, “how you talk! Positively,
I haven’t the slightest idea of what you mean, and I wish you
wouldn’t be so rough. I don’t think you know what you’re talking
about, or if you do you certainly don’t know anything about me. You
must have me mixed up with some one else, or with something that I
don’t know anything about.” She moved as if to leave.
“Now listen to me a minute,” he said sharply, “and don’t be so
ready to leave. You know who I am, and just what I’m doing. I’m
running an investigation bureau on my own account with which I
mean to break up the present city political ring, and I have a lot of
evidence which might cause Mr. Tilney and the mayor and some
others a lot of trouble this fall, and they know it, and that’s why you’re
out here. Mr. Tilney is connected with the mayor, and he used to be
a bosom friend of your friend, Jack Swayne. And Diamondberg and
Mrs. Skelton are in his employ right now, and so are you. You think I
don’t know that Castleman and his friends were working with you
and Mrs. Skelton, and Diamondberg and these ‘brokers’ also, and
that Castleman tried to run into us the other night and kill me, and
that I’m being watched here all the time and spied on, but I am, and I
know it, and I’m not in the dark as to anything—not one thing—not
even you,” and he leered at her angrily.
“Now wait a moment,” he went on quickly as she opened her
mouth and started to say something. “You don’t look to me to be so
crafty and devilish as all this seems, or I wouldn’t be talking to you at
all, and your manner all along has been so different—you’ve
appeared so friendly and sympathetic, that I’ve thought at times that
maybe you didn’t know exactly what was going on. Now, however, I
see that you do. Your manner the other morning at breakfast made
me think that possibly you were not so bad as you seemed. But now
I see that you’ve been lying to me all along about all this, just as I
thought, only I must say that up to now I haven’t been willing to
believe it. This isn’t the first time an attempt has been made to get
people in this way, though. It’s an old political trick, only you’re trying
to work it once more, and I don’t propose that you shall work it on me
if I can help it. Plainly, you people wouldn’t hesitate to kill me, any
more than Tilney hesitated to ruin Crothers three years ago, or than
he would hesitate to ruin me or any other man or woman who got in
his path, but he hasn’t got me yet, and he’s not going to, and you
can tell him that for me. He’s a crook. He controls a bunch of crooks
—the mayor and all the people working with him—and if you’re in
with them, as I know you are, and know what you’re doing, you’re a
crook too.”
“Oh, oh, oh! Don’t!” she exclaimed. “Please don’t! This is too
terrible! To think that you should talk to me in this way!” but she
made no attempt to leave.
“Now I want to tell you something more, Miss Carle—if that’s your
real name—” Gregory went on as she was putting her hands to her
temples and exclaiming, and she winced again. “As I said before,
you don’t look to me to be as bad as you seem, and for that reason
I’m talking to you now. But just see how it is: Here I am, a young man
just starting out in the world really, and here you are trying to ruin
me. I was living here with my wife and my little two-year-old baby
peacefully enough until she had to go to the mountains because our
little boy was taken sick, and then you and Mrs. Skelton and
Diamondberg and Castleman and the ‘brokers’ and all the rest of the
crowd that are and have been around here watching and spying,
came and began to cause me trouble. Now I’m not helpless. And you
needn’t think I wasn’t warned before you came, because I was.
There are just as many influential men on my side of the fence right
now as there are on Tilney’s—will be—and he isn’t going to get away
with this thing as easily as he thinks. But just think of your part in all
this! Why should you want to ruin me or help these people? What
have I ever done to you? I can understand Tilney’s wanting to do it.
He thinks that I have facts which will injure him, and I have, and that
because I haven’t made any public statement the evidence is still in
my hands, and that if I am put out of the way or discredited the whole
thing will blow over and nothing will happen to him—but it won’t. Not
now any more. It can’t. This thing will go on just the same, whether I
am here or not. But that isn’t the point either. I was told two months
ago that you would come, not by Mrs. Skelton, but by friends of
mine, and that an attempt would be made on my life,” and at that she
opened her eyes wide and sat there apparently amazed, “and here
you are on schedule time and doing just as you were told, and
apparently you aren’t the least bit ashamed to do it. But don’t you
think it’s a pretty shabby game for you to play?” He stared at her
wearily and she at him, but now for the moment she said nothing,
just sat there.
“That big blue machine that was to have killed me the other night,”
he went on, stretching matters a little in so far as his own knowledge
was concerned, “was all arranged for long before you came down
here. I haven’t the slightest idea why you work for Tilney, but I know
now that that’s what you’re doing, and I’m sick of you and the whole
thing. You’re just a plain little crook, that’s all, and I’m through with
you and this whole thing, and I don’t want you to talk to me any
more. What’s more, I’m not going to leave this hotel, either, and you
can take that news to Tilney if you want to, or Mrs. Skelton or
whoever else is managing things here for him. I’ve kept a day-to-day
record of everything that’s happened so far, and I have witnesses,
and if anything more happens to me here I’m going to the
newspapers and expose the whole thing. If you had any sense of
decency left you wouldn’t be in on anything like this, but you haven’t
—you’re just a shabby little trickster, and that lets you out, and that’s
all I have to say.”
He stood up and made as if to walk off, while Miss Carle sat there,
seemingly dazed, then jumped up and called after him:
“Mr. Gregory! Please! Please! Mr. Gregory, I want to tell you
something!”
 He stopped and turned. She came hurriedly up to him.
“Don’t go,” she pleaded, “not just yet. Wait a minute. Please come
back. I want to talk to you.” And though he looked at her rather
determinedly, he followed her.
“Well?” he asked.
“You don’t understand how it is,” she pleaded, with a look of real
concern in her eyes. “And I can’t tell you either, just now, but I will
some time if you will let me. But I like you, and I really don’t want to
do you any harm. Really, I don’t. I don’t know anything about these
automobile things you’re telling about—truly I don’t. They’re all
terrible and horrible to me, and if they are trying to do anything like
that, I don’t know it, and I won’t have anything more to do with it—
really I won’t. Oh, it’s terrible!” and she clenched her hands. “I do
know Mr. Diamondberg now, I admit that, but I didn’t before I came
down here, and Mr. Swayne and Mr. Tilney. I did come here to see if
I could get you interested in me, but they didn’t tell me just why. They
told me—Mrs. Skelton did—that you, or some people whom you
represented, were trying to get evidence against some friends of
theirs—Mr. Tilney’s, I believe—who were absolutely innocent, that
you weren’t happy with your wife, and that if some one, any one,
were able to make you fall in love with her or just become very good
friends, she might be able to persuade you not to do it, you see.
There wasn’t any plan, so far as I know, to injure you bodily in any
way. They didn’t tell me that they wanted to injure you physically—
really they didn’t. That’s all news to me, and dreadful. All they said
was that they wanted to get some one to get you to stop—make it
worth your while in a money way, if I could. I didn’t think there was
anything so very wrong in that, seeing all they have done for me in
the past—Mr. Tilney, Mrs. Skelton and some others. But after I saw
you a little while I——” she paused and looked at him, then away, “I
didn’t think you were that kind of a man, you see, and so—well, it’s
different now. I don’t want to do anything to hurt you. Really I don’t. I
couldn’t—now.”
“So you admit now that you do know Mr. Tilney,” he commented
sourly, but not without a sense of triumph behind it all.
 “I just told you that,” she said.
She stopped, and Gregory stared at her suspiciously. That she
liked him was plain, and in a sense it was different from that of a
mere passing flirtation, and as for himself—well, he couldn’t help
liking her in a genial way. He was free to admit that to himself, in
spite of her trickery, and that she was attractive, and as yet she
personally had not done anything to him, certainly nothing that he
could prove. She seemed even now so young, although so
sophisticated and wise, and much about her face, its smoothness,
the delicate tracery of hair about her forehead, the drooping pout of
the upper lip, sharpened his interest and caused him to meditate.
“Well?” he inquired after a time.
“Oh, I wish you wouldn’t turn on me so and leave me,” she
pleaded. “I haven’t done anything to you, have I? Not yet, anyhow.”
“That’s just the point—not yet. There’s the whole story in a
nutshell.”
“Yes, but I promise you faithfully that I won’t, that I don’t intend to.
Really I don’t. You won’t believe me, but that’s true. And I won’t, I
give you my word,—truly. Why won’t you still be friends with me? I
can’t tell you any more about myself now than I have—not now—but
I will some time, and I wish you would still be friends with me. I
promise not to do anything to cause you trouble. I haven’t really,
have I? Have I?”
“How should I know?” he answered testily and roughly, the while
believing that this was a deliberate attempt on her part to interest
him in spite of himself, to get him not to leave yet. “It seems to me
you’ve done enough, being with these people. You’ve led me into
going about with them, for one thing. I would never have gone with
them on most of these trips except for you. Isn’t that enough? What
more do you want? And why can’t you tell me now,” he demanded,
feeling in a way the authority of a victor, “who these people are and
all about them? I’d like to know. It might be a help to me, if you really
wanted to do something for me. What are their plans, their game?”
 “I don’t know. I can’t tell you any more than I have, truly I can’t. If I
find out, maybe I will some time. I promise to. But not now. I can’t,
now. Can’t you trust me that much? Can’t you see that I like you,
when I tell you so much? I haven’t any plan to injure you personally,
truly I haven’t. I’m obliged to these people in one way and another,
but nothing that would make me go that far. Won’t you believe me?”
She opened her eyes very wide in injury. There was something new
in her expression, a luring, coaxing something.
“I haven’t any one who is really close to me any more,” she went
on, “not anybody I like. I suppose it’s all my own fault, but—” her
voice became very sweet.
In spite of his precautions and the knowledge that his wife was the
best and most suitable companion for him in the world, and that he
was permanently fixed through his affection for his child and the
helpful, hopeful mother of it, nevertheless he was moved by some
peculiarity of this girl’s temperament. What power had Tilney over
her, that he could use her in this way? Think of it—a beautiful girl like
this!
“What about Mrs. Skelton?” he demanded. “Who is she, anyhow?
And these three gardeners around here? What is it they want?”
(There were three gardeners of the grounds who whenever he and
Imogene had been alone together anywhere managed somehow to
be working near the scene—an arrival which caused him always
instanter to depart.) “And Diamondberg?”
She insisted that in so far as the gardeners were concerned she
knew absolutely nothing about them. If they were employed by Mrs.
Skelton or any one, it was without her knowledge. As for
Diamondberg, she explained that she had only met him since she
had come here, but that she really did not like him. For some reason
Mrs. Skelton had asked her to appear not to know him. Mrs. Skelton,
she persisted, had known her years before in Cincinnati, as she had
said, but more recently in the city. She had helped her to get various
positions, twice on the stage. Once she had worked for Mr. Swayne,
yes, for a year, but only as a clerk. She had never known anything
about him or his plans or schemes, never. When Gregory wanted to
know how it was that he was to be trapped by her, if at all, she
insisted that she did not believe that he was to be trapped. It was all
to have been as she said.
Gregory could not quite make out whether she was telling him the
exact truth, but it was near enough, and it seemed to him that she
could not be wholly lying. She seemed too frank and wishful. There
was something sensuously affectionate in her point of view and her
manner. He would know everything in the future, she insisted, if he
wanted to, but only not now—please not now. Then she asked about
his wife, where she was, when she was coming back.
“Do you love her very much?” she finally asked naïvely.
“Certainly I love her. Why do you ask? I’ve a two-year-old boy that
I’m crazy about.”
She looked at him thoughtfully, a little puzzled or uncertain, he
thought.
They agreed to be friends after a fashion before they were
through. He confessed that he liked her, but still that he did not trust
her—not yet. They were to go on as before, but only on condition
that nothing further happened to him which could be traced to her.
She frankly told him that she could not control the actions of the
others. They were their own masters, and, after a fashion, hers, but
in so far as she could she would protect him. She did not believe that
they intended to try much longer. In so far as she was concerned, he
might go away if he chose. She could see him anywhere, if he
would. She was not sure if that would make any difference in their
plans or not. Anyhow, she would not follow him if he did go unless he
wished it, but she would prefer that he did. Perhaps nothing more
would happen here. If she heard of anything she would tell him, or
try to, in time. But she could not say more than that now. After a
while, maybe, as soon as she could get out of here ... there were
certain things over which she had no control. She was very
enigmatic and secretive, and he took it to mean that she was
involved in some difficult situation and could not easily extricate
herself.
 “I wouldn’t take too much stock in her, at that,” Blount reflected
when Gregory had told him about it. “Just keep your eyes open,
that’s all. Don’t have anything to do with her in a compromising way.
She may be lying to you again. Once a crook, always a crook.” Such
was his philosophy.
Mrs. Skelton returned on the third day after his long conversation
with Imogene, and in spite of the fact that they had seemed to come
closer together than ever before, to have established a friendly semi-
defensive pact, still he sensed treachery. He could not make out
what it was. She seemed to be friendly, simple, gay, direct, even
wooing—and yet—what? He thought at one time that she might be
the unconscious psychologic victim of Mrs. Skelton or of some one
else; at other times, an absolutely unprincipled political philanderer.
While pretending to be “on the level,” as he phrased it, with him, she
was crossing his path in such odd ways, making him uncertain as to
whether, in spite of all she had said and was saying, she was still
engaged in trying to compromise him. The whole thing began to take
on the fascination of a game with the unconquerable lure of sex at
the bottom of it—steeled as he was against compromising himself in
any way.
Thus once, after a late card game, when he stepped out on a
small veranda or balcony which graced the end of the hall nearest
which his room was situated, and which commanded a splendid view
of the sea, he found her just outside his door, alone, diaphanously
attired, and very sympathetic and genial. Now that they were friends
and had had this talk, there was something in her manner which
always seemed to invite him on to a closer life with her without
danger to himself, as she seemed to say. She would shield him
against all, at her own expense. At the same time he was far—very
far—from yielding. More than once he had insisted that he did not
want to have anything to do with her in an affectional way, and yet
here she was on this occasion, and although there might or there
might not have been anything very alarming in that, he argued with
himself afterward, yet since he had told her, this could be made to
look as though she were trying to overpersuade him, to take him off
his guard. Any guest of the hotel might have done as much (her
room was somewhere near there), but Rule One, as laid down by
Blount, and as hitherto practised by him, was never, under any
circumstances which might be misinterpreted, to be alone with her.
And besides, when he withdrew, as he did at once, excusing himself
lightly and laughingly, he saw two men turning in at a cross corridor
just beyond, and one, seeing him turn back, said to the other, “It
must be on the other side, Jim.” Well, there might not have been
anything very significant in that, either. Any two men might
accidentally turn into a hall on an end balcony of which a maiden
was sitting in very diaphanous array, but still——
It was the same whenever he walked along the outer or sea wall at
night, listening to the thunder of the water against the rapp which
sustained the walk, and meditating on the night and the beauty of the
hotel and the shabbiness of politics. Imogene was always about him
when she might be with safety, as he saw it, but never under such
circumstances as could be made to seem that they were alone
together. Bullen, one of the two brokers, who seemed not a bad sort
after his kind, came out there one night with Mrs. Skelton and
Imogene, and seeing Gregory, engaged him in conversation and
then left Imogene to his care. Gregory, hating to appear asininely
suspicious under such circumstances, was genuinely troubled as to
what to do in such cases as these. Always now he was drawn to her,
painfully so, and yet—— He had told her more than once that he did
not wish to be alone with her in this way, and yet here she was, and
she was always insisting that she did not wish him to be with her if
he objected to it, and yet look at this! Her excuse always was that
she could not help it, that it was purely accidental or planned by
them without her knowledge. She could not avoid all accidents.
When he demanded to know why she did not leave, clear out of all of
this, she explained that without great injury to herself and Mrs.
Skelton she could not, and that besides he was safer with her there.
“What is this?” he asked on this occasion. “Another plan?” Feeling
her stop and pull back a little, he felt ashamed of himself. “Well, you
know what I’ve been telling you all along,” he added gruffly.
“Please don’t be so suspicious, Ed. Why do you always act so?
Can’t I even walk out here? I couldn’t avoid this to-night, truly I
couldn’t. Don’t you suppose I have to play a part too—for a time,
anyhow? What do you expect me to do—leave at once? I can’t, I tell
you. Won’t you believe me? Won’t you have a little faith in me?”
“Well, come on,” he returned crossly, as much irritated with himself
as any one. “Give me your arm. Give a dog a bad name, you know,”
and he walked her courteously but firmly in the direction of the
principal veranda, trying to be nice to her at the same time.
“I tell you, Imogene, I can’t and I won’t do this. You must find ways
of avoiding these things. If not, I’m not going to have anything to do
with you at all. You say you want me to be friends with you, if no
more. Very well. But how are we going to do it?” and after more
arguments of this kind they parted with considerable feeling, but not
altogether antagonistic, at that.
Yet by reason of all this finally, and very much to his personal
dissatisfaction, he found himself limited as to his walks and lounging
places almost as much as if he had been in prison. There was a little
pergola at one end of the lawn with benches and flowering vines
which had taken his fancy when he first came, and which he had
been accustomed to frequent as a splendid place to walk and
smoke, but not any more. He was too certain of being picked up
there, or of being joined by Mrs. Skelton and Imogene, only to be left
with Imogene, with possibly the three gardeners or a broker as
witnesses. He could not help thinking how ridiculous it all was.
He even took Imogene, he and Blount, in Blount’s car, and Mrs.
Skelton with them or not, as the case might be—it was all well
enough so long as Blount was along—to one place or another in the
immediate vicinity—never far, and always the two of them armed and
ready for any emergency or fray, as they said. It seemed a risky
thing to do, still they felt a little emboldened by their success so far,
and besides, Imogene was decidedly attractive to both of them. Now
that she had confessed her affection for Gregory she was most
alluring with him, and genial to Blount, teasing and petting him and
calling him the watchdog. Blount was always crowing over how well
he and Gregory were managing the affair. More than once he had
pointed out, even in her presence, that there was an element of sport
or fascinating drama in it, that she “couldn’t fool them,” all of which
was helping mightily to pass the time, even though his own and
Gregory’s life, or at least their reputation, might be at stake.
“Go on, go on, is my advice,” Blount kept saying now that he was
being amused. “Let her fall in love with you. Make her testify on your
behalf. Get a confession in black and white, if you can. It would be a
great thing in the campaign, if you were compelled to use it.” He was
a most practical and political soul, for all his geniality.
Gregory could not quite see himself doing that, however. He was
too fond of her. She was never quite so yielding, so close to him, as
now. When he and Blount were out with her, now, the two of them
ventured to rag her as to her part in all this, asking her whether the
other car were handy, whether the gardeners had been properly
lined up, and as to who was behind this tree or that house. “There’d
be no use in going if everything wasn’t just right,” they said. She took
it all in good part, even laughing and mocking them.
“Better look out! Here comes a spy now,” she would sometimes
exclaim at sight of a huckster driving a wagon or a farm-hand
pushing a wheelbarrow.
To both Blount and Gregory it was becoming a farce, and yet
between themselves they agreed that it had its charm. They were
probably tiring her backers and they would all quit soon. They hoped
so, anyhow.
But then one night, just as they had concluded that there might not
be so very much to this plot after all, that it was about all over, and
Mrs. Gregory was writing that she would soon be able to return, the
unexpected happened. They were returning from one of those
shorter outings which had succeeded the longer ones of an earlier
day, Blount and Gregory and Imogene, and true to his idea of
avoiding any routine procedure which might be seized upon by the
enemy as something to expect and therefore to be used, Blount
passed the main entrance and drove instead around to a side path
which led to a sunk-in porch flanked on either side by high box
hedges and sheltered furry pines. True also to their agreed plan of
never being separated on occasions like this, they both walked to the
door with Imogene, Blount locking his car so that it could not be
moved during his absence. On the steps of this side porch they
chaffered a little, bantering Imogene about another safe night, and
how hard it was on the gardeners to keep them up so late and
moving about in the dark in this fashion, when Imogene said she was
tired and would have to go. She laughed at them for their brashness.
“You two think you’re very smart, don’t you?” she smiled a little
wearily. “It would serve you right if something did happen to both of
you one of these days—you know so much.”
“Is that so?” chuckled Blount. “Well, don’t hold any midnight
conferences as to this. You’ll lose your beauty sleep if you do.”
To which Gregory added, “Yes, with all this hard work ahead of you
every day, Imogene, I should think you’d have to be careful.”
“Oh, hush, and go on,” she laughed, moving toward the door.
But they had not gone more than a hundred and fifty feet down the
shadowy side path before she came running after them, quite out of
breath.
“Oh dear!” she called sweetly as she neared them, and they
having heard her footsteps had turned. “I’m so sorry to trouble you,
but some one has locked that side door, and I can’t open it or make
them hear. Won’t one of you come and help me?” Then, as the two
of them turned, “That’s right. I forgot. You always work in pairs, don’t
you?”
Blount chortled. Gregory smiled also. They couldn’t help it. It was
so ridiculous at times—on occasions like this, for instance.
“Well, you see how it is,” Gregory teased, “the door may be very
tightly closed, and it might take the two of us to get it open.”
Seeing that Blount was really coming, he changed his mind. “I
guess I can get it open for her. Don’t bother this time. I’ll have to be
going in, anyhow,” he added. The thought came to him that he would
like to be with Imogene a little while—just a few moments.
Blount left them after a cautioning look and a cheery good night. In
all the time they had been together they had not done this, but this
time it seemed all right. Gregory had never felt quite so close to
Imogene as he did this evening. She had seemed so warm,
laughing, gay. The night had been sultry, but mellow. They had
tittered and jested over such trifling things, and now he felt that he
would like to be with her a while longer. She had become more or
less a part of his life, or seemingly so, such a genial companion. He
took her arm and tucked it under his own.
“It was nice over there at the Berkeley,” he commented, thinking of
an inn they had just left. “Beautiful grounds—and that music! It was
delightful, wasn’t it?” They had been dancing together.
“Oh, dear,” she sighed, “the summer will soon be over, and then I’ll
have to be going back, I suppose. I wish it would never end. I wish I
could stay here forever, just like this, if you were here.” She stopped
and looked at the treetops, taking a full breath and stretching out her
arms. “And do look at those fire-flies,” she added, “aren’t they
wonderful?” She hung back, watching the flashing fire-flies under the
trees.
“Why not sit down here a little while?” he proposed as they neared
the steps. “It isn’t late yet.”
“Do you really mean it?” she asked warmly.
“You see, I’m beginning to be so foolish as to want to trust you.
Isn’t that idiotic? Yes, I’m even going to risk fifteen minutes with you.”
“I wish you two would quit your teasing, just once,” she pleaded. “I
wish you would learn to trust me and leave Blount behind just once
in a while, seeing that I’ve told you so often that I mean to do nothing
to hurt you without telling you beforehand.”
Gregory looked at her, pleased. He was moved, a little sorry for
her, and a little sorrier for himself.
In spite of himself, his wife and baby, as he now saw, he had come
along a path he should not have, and with one whom he could not
conscientiously respect or revere. There was no future for them
together, as he well knew, now or at any other time. Still he lingered.
 “Well, here we are,” he said, “alone at last. Now you can do your
worst, and I have no one to protect me.”
“It would serve you right if I did, Mr. Smarty. But if I had suggested
that we sit down for a minute you would have believed that the wood
was full of spies. It’s too funny for words, the way you carry on. But
you’ll have to let me go upstairs to change my shoes, just the same.
They’ve been hurting me dreadfully, and I can’t stand them another
minute. If you want to, you can come up to the other balcony, or I’ll
come back here. I won’t be a minute. Do you mind?”
“Not at all,” he assented, thinking that the other balcony would not
be as open as this, much too private for him and her. “Certainly not.
Run along. But I’d rather you came back here. I want to smoke,
anyhow,” and he drew out his cigar and was about to make himself
comfortable when she came back.
“But you’ll have to get this door open for me,” she said. “I forgot
about that.”
“Oh, yes, that’s right.”
He approached it, looking first for the large key which always hung
on one side at this hour of the night, but not seeing it, looked at the
lock. The key was in it.
“I was trying before. I put it there,” she explained.
He laid hold of it, and to his surprise it came open without any
effort whatsoever, a thing which caused him to turn and look at her.
“I thought you said it wouldn’t open,” he said.
“Well, it wouldn’t before. I don’t know what makes it work now, but
it wouldn’t then. Perhaps some one has come out this way since.
Anyhow, I’ll run up and be down right away.” She hurried up the
broad flight of stairs which ascended leisurely from this entrance.
Gregory returned to his chair, amused but not conscious of
anything odd or out of the way about the matter. It might well have
been as she said. Doors were contrary at times, or some one might
have come down and pushed it open. Why always keep doubting?
Perhaps she really was in love with him, as she seemed to indicate,
or mightily infatuated, and would not permit any one to injure him
through her. It would seem so, really. After all, he kept saying to
himself, she was different now to what he had originally thought, and
what she had originally been, caught in a tangle of her own emotions
and compelled by him to do differently from what she had previously
planned. If he were not married as happily as he was, might not
something come of this? He wondered.
The black-green wall of the trees just beyond where he was sitting,
the yellow light filtering from the one bowl lamp which ornamented
the ceiling, the fireflies and the sawing katydids, all soothed and
entertained him. He was beginning to think that politics was not such
a bad business after all, his end of it at least, or being pursued even.
His work thus far had yielded him a fair salary, furnishing as it had
excellent copy for some of the newspapers and political
organizations—the best was being reserved for the last—and was
leading him into more interesting ways than the old newspaper days
had, and the future, outside of what had happened in the last few
weeks, looked promising enough. Soon he would be able to deal the
current administration a body blow. This might raise him to a high
position locally. He had not been so easily frustrated as they had
hoped, and this very attractive girl had fallen in love with him.
For a while he stared down the black-green path up which they
had come, and then fixed his eyes in lazy contemplation on one of
the groups of stars showing above the treetops. Suddenly—or was it
suddenly?—more a whisper or an idea—he seemed to become
aware of something that sounded, as he listened more keenly, like a
light footfall in the garden beyond the hedge. It was so very light, a
mere tickle of the grass or stirring of a twig. He pricked up his ears
and on the instant strained every muscle and braced himself, not
that he imagined anything very dreadful was going to happen, but—
were they up to their old tricks again? Was this the wonderful
gardeners again? Would they never stop? Removing the cigar from
his mouth and stilling the rocker in which he had been slowly moving
to and fro, he decided not to stir, not even to move his hands, so well
concealed was he from the bushes on either side by the
arrangement of the posts, one of which was to the left of him. In this
position he might see and not be seen. Did they know he was there?
How had they found out? Were they always watching yet? Was she
a part of it? He decided to get up and leave, but a moment later
thought it better to linger just a little, to wait and see. If he left and
she came back and did not find him there—could it be that there was
some new trick on foot?
While he was thus swiftly meditating, he was using his ears to their
utmost. Certainly there was a light footfall approaching along the
other side of the hedge to the left, two in fact, for no sooner was one
seemingly still, near at hand, than another was heard coming from
the same direction, as light and delicate as that of a cat—spies,
trappers, murderers, even, as he well knew. It was so amazing, this
prowling and stalking, so desperate and cruel, that it made him a
little sick. Perhaps, after all, he had better have kept Blount with him
—not have lingered in this fashion. He was about to leave, a nervous
thrill chasing up and down his spine, when he heard what he took to
be Imogene’s step on the stair. Then she was coming back, after all,
as she had said. She was not a part of this as he had feared—or
was she? Who could tell? But it would be foolish to leave now. She
would see that he was wholly suspicious again, and that stage had
somehow seemed to be passing between them. She had promised
on more than one occasion to protect him against these others, let
alone herself. Anyhow he could speak of these newcomers and then
leave. He would let her know that they were hanging about as usual,
always ready to take advantage of his good nature.
But now, her step having reached the bottom of the stair and
ceased, she did not come out. Instead, a light that was beside the
door, but out at this hour, was turned on, and glancing back he could
see her shadow, or thought he could, on the wall opposite, to the
right. She was doing something—what? There was a mirror below
the light. She might be giving her hair a last pat. She had probably
arrayed herself slightly differently for him to see. He waited. Still she
did not come. Then swiftly, a sense of something treacherous came
over him, a creeping sensation of being victimized and defeated. He
felt, over his taut nerves, this thrilling fear which seemed to almost
convey the words: Move! Hurry! Run! He could not sit still a moment
longer, but, as if under a great compulsion, leaped to his feet and
sprang to the door just as he thought he heard additional movements
and even whispers in the dark outside. What was it? Who? Now he
would see!
Inside he looked for her, and there she was, but how different!
When she had gone upstairs she had been arrayed in a light
summery dress, very smart and out-door-ish, but here she was
clothed in a soft clinging housedress such as one would never wear
outside the hotel. And instead of being adjusted with her customary
care, it was decidedly awry, as though she might have been in some
disturbing and unhappy contest. The collar was slightly torn and
pulled open, a sleeve ripped at the shoulder and wrist, the hang of
the skirt over the hips awry, and the skirt itself torn, a ragged slit over
the knee. Her face had been powdered to a dead white, or she
herself was overcome with fear and distress, and the hair above it
was disarranged, as though it had been shaken or pulled to one
side. Her whole appearance was that of one who had been assailed
in some evil manner and who had come out of the contest
disarranged as to her clothes and shaken as to her nerves.
Brief as his glance was, Gregory was amazed at the
transformation. He was so taken aback that he could not say
anything, but just what it all meant came to him in an intuitive flash.
To fly was his one thought, to get out of the vicinity of this, not to be
seen or taken near it. With one bound he was away and up the easy
stair three at a time, not pausing to so much as look back at her,
meeting her first wide half-frightened stare with one of astonishment,
anger and fear. Nor did he pause until he had reached his own door,
through which he fairly jumped, locking himself in as he did so. Once
inside, he stood there white and shaking, waiting for any sound
which might follow, any pursuit, but hearing none, going to his mirror
and mocking at himself for being such a fool as to be so easily
outwitted, taken in, after all his caution and sophisticated talk. Lord!
he sighed. Lord!
And after all her protests and promises, this very evening, too, he
thought. What a revelation of the unreliability and treachery of
human nature! So she had been lying to him all the time, leading him
on in the face of his almost boastful precautions and suspicions, and
to-night, almost at the close of the season, had all but succeeded in
trapping him! Then Tilney was not so easily to be fooled, after all. He
commanded greater loyalty and cunning in his employees than he
had ever dreamed. But what could he say to her, now that he knew
what she really was, if ever he saw her again? She would just laugh
at him, think him a fool, even though he had managed to escape.
Would he ever want to see her again? Never, he thought. But to
think that any one so young, so smooth, so seemingly affectionate,
could be so ruthless, so devilishly clever and cruel! She was much
more astute than either he or Blount had given her credit for.
After moving the bureau and chairs in front of the door, he called
up Blount and sat waiting for him to come.
Actually, as he saw it now, she had meant to stage a seeming
assault in which he would have been accused as the criminal and if
they had sufficient witnesses he might have had a hard time proving
otherwise. After all, he had been going about with her a great deal,
he and Blount, and after he had told himself that he would not.
Her witnesses were there, close upon him, in the dark. Even
though he might be able to prove his previous good character, still,
considering the suspicious fact that he had trifled with her and this
treacherous situation so long, would a jury or the public believe him?
A moment or two more, and she would have screamed out that he
was attacking her, and the whole hotel would have been aroused.
Her secret friends would have rushed forward and beaten him. Who
knows?—they might even have killed him! And their excuse would
have been that they were justified. Unquestionably she and her
friends would have produced a cloud of witnesses. But she hadn’t
screamed—there was a curious point as to that, even though she
had had ample time (and she had had) and it was expected of her
and intended that she should! Why hadn’t she? What had prevented
her? A strange, disturbing exculpating thought began to take root in
his mind, but on the instant also he did his best to crush it.
“No, no! I have had enough now,” he said to himself. “She did
intend to compromise me and that is all there is to it. And in what a