DNA Aptamers Speci�c for Legionella Pneumophila: Systematic Evolution of Ligands by Exponential Enrichment in Whole Bacterial Cells

Legionella pneumophila is the major causative agent of Legionnaires’ disease and Pontiac fever, which pose major public health problems. Rapid detection of L. pneumophila is important for global control of these diseases. Aptamers, short oligonucleotides that bind to targets with high a�nity and speci�city, have great potential for use in pathogenic bacterium detection, diagnostics, and therapy. Here, we used a whole-cell SELEX (systematic evolution of ligands by exponential enrichment) method to isolate and characterize single-stranded DNA (ssDNA) aptamers against L. pneumophila. A total of 60 ssDNA sequences were identi�ed after 17 rounds of selection. Other bacterial species (Escherichia coli, Bacillus subtilis, Pseudomonas syringae, Staphylococcus aureus, Legionella quateirensis, and Legionella adelaidensis) were used for counterselection to enhance the speci�city of ssDNA aptamers against L. pneumophila. Four ssDNA aptamers showed strong a�nity and high selectivity for L. pneumophila, with K d values in the nanomolar range. Bioinformatic analysis of the most speci�c aptamers revealed predicted conserved secondary structures that might bind to L. pneumophila cell walls. In addition, the binding of these four �uorescently labeled aptamers to the surface of L. pneumophila was observed directly by �uorescence microscopy. This is the �rst study to use SELEX to target L. pneumophila whole cells. The aptamers identi�ed in this study could be used in the future to develop medical diagnostic tools and public environmental detection assays for L. pneumophila.


Introduction
Legionella species, the major causative agents of Legionnaires' disease and Pontiac fever, are commonly found in environmental samples (Xiong et al., 2016).They have been shown to induce lung infection and cause dysfunction in other organs, such as the heart and kidney, and in the central nervous system (Xiong et al., 2015).Legionella pneumophila speci cally has been identi ed as the primary cause of Legionnaires' disease.Early detection of L. pneumophila would allow for faster recovery and a reduction in the number of fatal cases.Additionally, monitoring the L. pneumophila population in the environment could reduce the likelihood of Legionnaires' disease outbreaks (Xiong et al., 2017).However, traditional culturing methods used for detecting L. pneumophila in samples rely on enrichment and selective plating followed by biochemical, serological, and molecular identi cation.These methods take 2 to 3 days to obtain a result and are labor intensive and tedious (Rajendhran and Gunasekaran, 2011).Recently, highly speci c detection techniques, such as ELISA, PCR, and real-time qPCR, have shortened detection time.
However, these methods depend on complex and technically di cult steps and are prohibitively expensive for routine testing (Woo et al., 2008).Biosensors have demonstrated a potential for rapid bacterial detection but rely on nanomaterials or complex platforms such as functional magnetic nanoparticles, electrochemical analysis systems, and eld-effect transistors (Gopinath et al., 2014).
Antibodies have also been applied for more than 40 years in assays for microbial detection but have many limitations despite their widespread application.For instance, the synthesis of antibodies requires a host animal, antibodies may be unstable at room temperature, and antibodies will not be obtained when target chemicals are toxic to the host organism.
Aptamers are single-stranded DNAs (ssDNAs) or RNAs that can bind to a wide range of non-nucleic acid targets with high a nity and speci city (Keefe et al., 2010).In detection and diagnostic assays, aptamers have several advantages over antibodies (Keefe et al., 2010).For instance, aptamers are obtained through an in vitro selection process, which is achieved at a lower cost and with less batch variation than in vivo antibody production.Due to their high stability at a range of temperatures and pHs, aptamers are also relatively easy to handle.In addition, aptamers can be easily modi ed with dyes or other functional groups to be labeled or immobilized on a substrate (Syed and Jamil, 2018).
Aptamer sequences are usually identi ed through the SELEX (systematic evolution of ligands by exponential enrichment) process, which starts with incubating the ssDNA or RNA library with the target, followed by separation and exponential ampli cation of the binding ssDNA or RNA (Torres-Chavolla and Alocilja, 2009).By repeating this process 8-20 times before cloning and sequencing the oligonucleotides that bind to the target, the most suitable aptamers can be identi ed (Dua et al., 2016).Aptamers can be selected for a wide variety of targets, from small molecules to whole cells (Bruno, 2015;Cao et al., 2009;Sypabekova et al., 2017).In recent years, aptamers have been selected against cells from different bacterial species, including Escherichia coli K88 (Peng et al., 2014) (Chen et al., 2007), and Campylobacter jejuni (Dwivedi et al., 2010).However, no speci c aptamers have been reported for L. pneumophila.
In this study, we isolated four sequences for DNA aptamers speci c to L. pneumophila using a whole bacterial cell SELEX process.The predicted secondary structures of selected aptamers were analyzed, and the binding of these aptamers to target cells was observed by uorescence microscopy.In addition, we characterized their a nity and selectivity for L. pneumophila cells.The results provide an important reference for pathogen detection using aptamers in the future.

In vitro selection of aptamers
To select aptamers that bind to Legionella pneumophila, we applied a cell-SELEX strategy using a library of ssDNA sequences containing a 45-base random region anked by two de ned primer binding regions (5′-GCAATGGTACGGTACTTCC-N45-CAAAAGTGCACGCTACTTTGCTAA-3′; Table 1).A total of 17 rounds of selection were performed until the ssDNAs binding to the target cells dominated the DNA pool (Fig. 1).
The genus Legionella includes more than 57 species (including subspecies) and more than 70 serogroups, not all of which are associated with human disease.The species most frequently detected in diagnosed cases is L. pneumophila, consisting of 16 serogroups (Casini et al., 2017); L. pneumophila serogroup 1, responsible for the rst identi ed outbreak in 1976 in Philadelphia (USA), is the cause of 95% of infections in Europe and 85% of infections worldwide (Cloutman-Green et al., 2019).The phylogenetic diversity of this Philadelphia-1 strain has since expanded, now including the JR32 and Lp02 strains (Schmolders et al., 2017) commonly used for L. pneumophila research (Maita et al., 2016).In this study, JR32 and Lp02 were used as the target strains for aptamer selection.Live cells were used because the conformation of cell wall molecules, which represent the most likely aptamer targets, may change when cells die.Also, considering bacterial cell wall composition changes during growth in culture, we used stationary-phase bacterial cells in all experiments.Following incubation, the target-bound aptamers were eluted and enriched at each selection round by ampli cation using PCR.After each round of selection, the obtained ssDNAs were quanti ed by a spectrophotometer and analyzed by agarose gel electrophoresis (Fig. 1A).
With each SELEX round, the percentage of ssDNA binding to the target cells gradually increased.
However, there were slight drops in elution yield after the second and sixth rounds, most likely due to counterselection (Fig. 1B).During SELEX, four counterselection processes were performed to increase the selectivity of aptamers to the target cells using a mixture of other bacterial species (Escherichia coli, Bacillus subtilis, Pseudomonas syringae, Staphylococcus aureus, Legionella quateirensis, and Legionella adelaidensis) at the second, sixth, ninth, and thirteenth rounds of selection.After the 17 rounds of selection, ssDNA bound to target cells dominated the DNA pool with about 90% elution yield.A total of 60 cloned transformants (E. coli cells harboring a vector that contained the aptamer sequences) were sequenced to identify speci c aptamer candidates.From these transformants, we identi ed four unique aptamer sequences (S11, S25, S28, and S29) that were speci c to LP02 cells and JR32 cells (Table 2).

Sequencing and bioinformatics analysis
The sequences of the S11, S25, S28, and S29 L. pneumophila aptamers are listed in Table 2.The computed GC/AT ratio of all four aptamers varies from 1.63 to 0.96.Since the GC/AT ratio is one of the most basic sequence characteristics in terms of nucleotide composition, aptamer sequences can be modi ed and optimized in future studies by taking this ratio into account.To understand the impact of aptamer sequence structure on binding, we generated predicted secondary structures for the most e cient L. pneumophila-binding aptamers using the mfold algorithm.Aptamers S11 and S29 had similar predicted stem-loop region secondary structures, which contained GGGCA residues at the apical loops (Fig. 2).S25 and S29 also contained a nearly identical sequence, CAxCTGTA.In addition, S11, S25, and S28 all shared one conserved stem loop with TACTT residues, while S28 and S29 had an identical stem loop containing the sequence CAAAAGTG (Fig. 2).
For the aptamers S11, S25 and S28, mfold yielded four predicted secondary structures, while ve different alternative conformations were predicted for S29.However, the conserved loops in different aptamers were stable in different predicted secondary structures, which suggests that these features could play an important role in the interaction of aptamers with their ligands in the bacterial cell wall.

Assessment of selectivity
To determine that the selected aptamers were capable of binding L. pneumophila cells, the four L. pneumophila-binding DNA aptamers (S11, S25, S28, and S29) were examined using the same concentration of aptamers (250 nM) and visualized by uorescence microscopy.Other bacterial species (E.coli, B. subtilis, P. syringae, S. aureus, L. quateirensis, and L. adelaidensis) were used as negative controls.As shown in Fig. 3A, uorescently labeled L. pneumophila JR32 and Lp02 cells were observed with 6-carboxy uorescein (FAM)-labeled S29, while the negative control cells incubated with FAM-S29 did not show any de nite uorescence signal on cells.Using Multiscan Spectrum, the intensity of uorescence was detected.All four of the aptamers showed selective binding to L. pneumophila JR32 and Lp02, and they had very low binding capacity for other bacterial species, including L. quateirensis and L. adelaidensis cells (Fig. 3B).These results indicate that the four ssDNA aptamers (S11, S25, S28, and S29) showed a nity and high selectivity to L. pneumophila JR32 and Lp02.

Determination of equilibrium dissociation constants
To evaluate aptamer binding in more detail, we determined the a nity of individual aptamers (S11, S25, S28, and S29) to L. pneumophila JR32 and Lp02 ).In our study, the representative strains JR32 and Lp02 (Maita et al., 2016) were used as target cells to develop speci c, high-a nity aptamers for pathogenic L. pneumophila.Notably, the binding a nity of each aptamer varied between these two strains (Fig. 3 and 4).These results may be due to experimental error during assessments of a nity and speci city for aptamers against JR32 and Lp02 cells.However, it is also possible that, although JR32 and Lp02 were isolated simultaneously, the two strains currently display distinct genomic features leading to differences in cell wall composition (Maita et al., 2016).
In this study, we determined that using uorescein-modi ed primers to amplify the aptamers makes the product more visible under ultraviolet light, which aids in product recovery.In addition, to avoid primer contamination during the SELEX process we used a gel extraction kit to purify and recover products.The uorescein group may help increase the recovery e ciency during puri cation and recovery steps.
Using the mfold algorithm, we generated predicted secondary structures for the most e cient L. pneumophila-binding aptamers (Fig. 2).We observed that in the constant region primer, S11, S25, and S28 share one conserved stem loop with TACTT residues.In-depth analysis revealed that the generations of these conserved secondary structures were due to site mutations in the constant primer regions of these aptamers during the PCR ampli cation process (Fig. 2).These results demonstrated that the conserved stem loop with TACTT residues could be the key secondary structures that allow these aptamers to bind the target cells.In future studies, we will test this hypothesis by truncation or sitedirected mutation assays.In addition, elucidation of the speci c binding targets on the cells themselves may also help us better understand this mechanism.
To our knowledge, this is the rst study to report the binding of aptamers to L. pneumophila.Importantly, some of the aptamers described here recognize pathogenic L. pneumophila strains with a nity and speci city suitable for potential use in clinical diagnosis, therapeutics, and environmental pathogen detection applications.

Conclusions
We applied the whole-cell SELEX (systematic evolution of ligands by exponential enrichment) method to isolate and characterize single-stranded DNA (ssDNA) aptamers against L. pneumophila.Four ssDNA aptamers were obtained and showed strong a nity and high selectivity for L. pneumophila, with K d values in the nanomolar range.Bioinformatic analysis of the most speci c aptamers revealed predicted conserved secondary structures that might bind to L. pneumophila cell walls.In addition, the binding of these four uorescently labeled aptamers to the surface of L. pneumophila was observed directly by uorescence microscopy.The aptamers identi ed in this study could be used in the future to develop medical diagnostic tools and public environmental detection assays for L. pneumophila.

Aptamer selection process (SELEX)
A whole-cell SELEX approach was used to select 6-carboxy uorescein (FAM)-labeled ssDNA aptamers with binding a nity and speci city to L. pneumophila.The method of Dwivedi et al. ( 2010) was used, with minor modi cations.To initiate the selection process, an 88-mer combinatorial ssDNA library (5′-the supernatant discarded.The cells then went through two rounds of washing and centrifugation, with the supernatants again discarded.The cells were resuspended in 40 μL of washing buffer.Imaging of the bacteria was performed with a microscope (ZEISS, Axio Scope.A1) under excitation of 488 nm and emission in the range of 502 to 554 nm.

Aptamer binding analysis
Aptamer K d was determined by measuring the uorescence intensity of each sample.The FAM-labeled ssDNA aptamers (in increasing concentrations of 12.5 , 31.25 , 62.5 125 , 250, and 500 nM) were incubated with the target strains (10 7 cells) for 60 min at 20℃.Cells were washed two times to remove unbound ssDNA from cells and resuspended in 100 μL of washing buffer.The K d values for each aptamer were determined by nonlinear regression analysis using Origin 9.5 software. In Binding a nity of four selected aptamers for Legionella pneumophila.
The 6-carboxy uorescein-labeled ssDNA aptamers (in increasing concentrations of 12.5 , 31.25 , 62.5 , 125 , 250, and 500 nM) were incubated with the target strains JR32 (A) (10 7 cells) and Lp02 (B) (10 7 cells) for 60 min at 20℃.The K d values for each aptamer were determined by nonlinear regression analysis using Origin 9.5 software.Data represent the mean SD of three independent experiments.
vitro selection of aptamers.A. The PCR products of the 9th round of selection were analyzed by agarose gel electrophoresis.Lane 1, DNA ladder; lane 2, ampli ed DNA products.Black arrow indicates the PCR product band.B. Progression of the selection process based on the portion of ssDNA bound to the target cells in the DNA pool.Numbers 1-17 represent the seventeen rounds of selection.Black arrows indicate the rounds of counter selection.

Table 2 .
List of four identi ed Legionella pneumophila binding ssDNA aptamers.