Regulatory Role of fnr Gene in Growth and tolA Gene Expression in Salmonella Typhimurium

Salmonella enterica serovar Typhimurium (S.Typhimurium) is a broad host range pathogen that causes acute self-limiting gastroenteritis in humans, cattle, swine, poultry, other large vertebrates. It can cause bacteraemia and systemic infection in immunosuppressed hosts, occasionally in healthy adult humans and animals (Pegues, 2005). Bacteria adapt to the various adverse conditions in  the environment or in the host, which increases the persistency of infection (Chakroun et al., 2017). It causes non-typhoidal Salmonellosis (NTS) and infection occurs mainly through the ingestion of contaminated food or water. Bacteria survive in the acidic pH of the stomach and enter the intestine (HARRIS et al., 1972; Chopra et al., 1994). The organism generally targets and colonizes in the intestinal epithelium of the host and causes gastroenteritis (Finlay and Falkow, 1990). Salmonella pathogenicity island-1 (SPI-1) type III secretion system (T3SS) is important for the bacterial invasion of intestinal epithelial cells and its virulence (Miki et al., 2004). Bacterial adhesion and invasion to the epithelial cells occur more in anaerobic condition than in the aerobically grown cells (Lee and Falkow, 1990). The mechanisms or genes controlling the adaptation to the anaerobic conditions may play an important role in the virulence of this species (Fink et al., 2007; Fernández et al., 2018). The changes in the cellular process during the transition from aerobic to anaerobic environment have been studied in detail in E.coli. More than 70 genes, important for the adaptation to the anaerobic conditions are under the control of the DNA-binding protein FNR (Fumarate and Nitrate Reduction).
 
regulatory protein), a cytoplasmic sensor of oxygen. The active form of FNR contains one [4Fe-4S]²⁺ cluster per protein monomer and it gets converted to [2Fe-2S]²⁺, an inactive form along with other less defined iron species, following exposure to oxygen in both in vitro and in vivo condition (Park and Gunsalus, 1995; Khoroshilova et al., 1997; Crack et al., 2004). FNR binds to promoter sequences usually at position -41 relative to the start of transcription and may also bind at position -61,-71,-81, -91 (Wing et al., 1995). The sequence recognized by FNR is palindromic (TTGATN4ATCAA) and when bound to this sequence, it interacts with the RpoA subunit of RNA polymerase and increases the efficiency of transcription (Lombardo et al., 1991). A reduced level of oxygen in theintestine during the bacterial colonization forces the organism to opt for anaerobic respiration. Adaptation to anaerobic conditions is controlled either alone by FNR protein or by inducing/repressing various other regulators such as ArcA, RpoS, NarL, Fur, or SoxR (Spiro and Guest, 1990; Hassan and Sun, 1992; Gunsalus and Park, 1994). The  aim of our experiment was to generate fnr null mutant of S. Typhimurium through lambda red-recombinase mediated gene knock out, to assess the role of fnr gene through in vitro experiments and proteomic analysis of WT and Dfnr strains grown under anaerobic condition.

All experiments were carried out in the Division of Animal Biochemistry, ICAR-IVRI, Izzatnagar, Bareilly in the time period of 2017-18. For MALDI-TOF, the sample was sent to IISc, Bengaluru.
 
Bacterial strains and culture
 
Salmonella Typhimurium (S. Typhimurium) strain PM45 (poultry isolate) was used. The cultures were streaked on Hektoen Enteric (HE) agar (HiMedia). Isolated colonies were characterized by biochemical tests and further confirmed by amplification of invA, a salmonella specific gene (Park et al., 2008). DH5α strain of E.coli was obtained from Stratagene.
 
Vectors
 
A helper plasmid pKD46, donor plasmid pKD4 and pCP20 were procured from Addgene. Oligonucleotide Primers were Procured from Xceleris Labs limited, Ahmedabad, India and sequence details are given in Table 1.
 

 
Construction of fnr gene deletion mutant S.Typhimurium
 
fnr gene from S. Typhimurium was deleted by one step gene inactivation protocol (Datsenko and Wanner, 2000).
 
PCR reaction conditions
 
The confirmation of Dfnr strain was done in 50μL reaction volume containing 1x Taq DNA polymerase buffer, 1.5mM MgCl₂, 20 pmol of each primer (FNR outer primers), 200µM dNTPs, 1μL genomic DNA, 2U Taq DNA Polymerase (Thermo scientific) and nuclease free water to 50μL. After initial denaturation at 94°C for 5 min, the amplification was carried out for 34 cycles each of 94°C-30 s, 52°C-45 s, 72°C-2 min with a final extension of 10 min at 72°C. Further confirmation was done using fnr inner primers, it was carried out in 50μL reaction volume containing 1x Taq DNA polymerase buffer, 1.5mM MgCl₂, 20 pmol of each primer (FNR inner primers), 200µM dNTPs, 1μL genomic DNA, 2U Taq DNA Polymerase (Thermo scientific) and nuclease free water to 50μL. After initial denaturation at 94°C for 5 min, the amplification was carried out for 34 cycles each of 94°C-30 s, 52°C-30 s, 72°C-30 s with a final extension of 10 min at 72°C.
 
In vitro characterization of fnr gene deletion mutant of S.Typhimurium (Δfnr)
 
In vitro characterization of Dfnr was carried out in an anaerobic jar.
 
Growth competition assay
 
The WT and the Δfnr strains were grown anaerobically at 37°C in MOPS (morpholinepropanesulfonic acid)-buffered (100mM, pH 7.4) Luria Bertani (LB) broth supplemented with 20mM D-xylose (LB-MOPS-X). Anaerobic chamber (HiMedia) and anaerobic gas pack (BD BBL+ GasPak™ anaerobic and CO₂ indicators) were used. All solutions were pre-equilibrated for 48 hours in an anaerobic chamber. A single isolated colony of both WT and Δfnr were inoculated into LB-MOPS-X broth for 16 hrs in anaerobic chambers and later from these fresh cultures were again inoculated in the same media till saturation. After saturation, both cultures were mixed in equal volume (1.0 ml each) and grown in 100 ml of MOPS buffered LB medium and growth competition was monitored at different time intervals (20, 40 and 60 hrs) (Samhita et al., 2014). The numbers of Δfnr and WT strains were enumerated randomly by colony PCR.
 
Biofilm formation assay
 
Biofilm formation by Salmonella isolates was assessed using microtitre plate assay. The assay was performed in sterile 96-well flat-bottom polystyrene microplates filled with 180μL of LB-MOPS-X media. The negative control wells contained 200μL of media only. 20μL of overnight grown cultures of both WT and Dfnr were dispensed to the wells in triplicate. The inoculated plates were then incubated anaerobically at 37°C for 72 hours. After incubation, the contents of the plates were poured off and the wells were thoroughly washed thrice with PBS (pH 7.2). The adherent bacterial cells were then stained with 200 μL of 0.5% (w/v) crystal violet stain per well for 10 min. After staining, plates were washed thrice with sterile distilled water. The plates were air-dried and 250μL of 33% glacial acetic acid was added to each well and mixed properly with gentle shaking. The optical density (O.D.) of each well was measured at 590nm using an automated ELISA reader (Nair et al., 2015; Shukla and Rao, 2017).
 
Swimming motility assay
 
Assay was performed as per the standard protocol described (Monteiro et al., 2012). Both WT and Dfnr bacteria were grown overnight at 37°C on an HEA plate and stab inoculated on 0.3% LB without salt agar plate with a toothpick. The plates were incubated at 28°C for 7hrs in an anaerobic chamber. The diameters of migrating bacteria from the point of inoculation (turbid zone) were measured. The results shown are representative of at least three independent experiments.
 
Sample preparation for proteomic study
 
WT and Δfnr strains were grown in LB MOPS-Xylose broth in an anaerobic environment. Overnight grown cultures were pelleted by centrifugation at 4500 rpm for 15 minutes. Pellets were suspended in lysis solution (7M urea, 2M thiourea, 4% CHAPS, 20mM Tris) and the sample was sonicated and centrifuged for 30 minutes at 7000 rpm, supernatants were collected and stored. The protein concentrations of lysates were determined by the Lowry method (Lowry et al., 1951).
 
Sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE)
 
SDS-PAGE was carried out in midigel (Tarson, India) apparatus with 5% stacking and 12% separating gel as per protocol (Sambrook, 2001). Equal amounts (concentration) of samples were loaded with a 5x Laemmli sample loading buffer along with the prestained protein marker. Electrophoresis was carried out at 100 volts.
 
Peptide mass fingerprinting
 
Differentially expressed proteins were cut, sent for MALDI-TOF mass spectrometry and obtained mass to charge ratios of peptides were analyzed using Mascot search engine software (Peptide mass fingerprinting).
 
Real– time quantitative reverse transcription PCR (qRT-PCR)
 
This technique was used to analyze the expression level of TolA protein at the RNA level and to validate the result of SDS-PAGE and mass spectrometry.  Total RNA isolated from anaerobically grown overnight cultures of WT and Dfnr strains using Trizol reagent. Isolated RNA samples were treated with Dnase I and dissolved in nuclease free water. RT-PCR was performed as per the Thermo Scientific RevertAid First Strand cDNA Synthesis Kit protocol. qRT-PCR was carried out using Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific), AriaMx Real-Time PCR System (Agilent Technologies) and data were analyzed by Agilent Aria Software Setup 1.6. TolA RT and 16s rRNA primers used for qRT-PCR were given in Table 1 (Gupta et al., 2014; Behera et al., 2020). All the procedure was carried out according to the MIQE guidelines.
 
Statistical method
 
One-way ANOVA was carried out using SPSS version 20 to test whether absorbance was significantly variable among different groups.

Confirmation of fnr gene deletion mutant (Δfnr)
 
Dfnr was confirmed by FNR outer primers which resulted in a product size of 120 bp size whereas WT gave a product of 825 bp (Fig 1) and with FNR inner primers WT gave 213 bp size whereas only primer dimers were seen in Δfnr, there was no PCR product (Fig 2). Primers were presented in Table 1.
 

 

 
Growth competition assay
 
At 20, 40 and 60 hrs interval, WT and Dfnr bacterial growth patterns were observed. WT strain outcompeted the growth of Δfnr strain at these time intervals. Strain abundance of Δfnr was found to be 38%, 16% and 16% at 20, 40 and 60 hrs respectively (Fig 3). In a growth competition assay, a considerable difference was observed in relative growth between WT and Δfnr strains. The strain abundance of Δfnr started decreasing from 38% to 16% from 20-60 hrs intervals. This indicates that fnr gene deletion reduced the ability to compete with WT strain in nutrient deficient conditions and thus resulted in some degree of growth attenuation.
 

 
fnr gene contribute to the biofilm formation
 
Biofilm formation ability was checked in 96-well plate as it is a measure of the persistence of bacteria in the environment and in the host by colonization through forming an exopolymer matrix (Grantcharova et al., 2010). The mean absorbance of wild strain (0.562±0.025) was significantly higher than that of Dfnr strain (0.346±0.018) (Fig 4). Statistical analysis: One way Anova, Post hock test Tukey’s, P*<0.05 P**<0.01, P***<0.001, N.S.-Not significant. Thus, fnr gene played important role in the attachment of bacteria and so it may affect the colonizing ability of bacteria in the intestinal epithelium, essential for bacterial pathogenesis (Grantcharova et al., 2010; Chelvam et al., 2014; Chakroun et al., 2018). fnr may also control the genes responsible for biofilm formation (Chakraborty et al., 2020).
 

 
Δfnr strain has reduced Swimming motility
 
The effect of the deletion of fnr gene on the motility of Salmonella Typhimurium was analyzed on 0.3% LB agar plate without salt. Δfnr had shown significant reduction (P<0.001) in motility as compared to WT strain. A swimming motility test, a marker of bacterial invasion through the flagellar movement was conducted (Iyoda et al., 2001). Motility of both WT and Δfnr strains was assessed by taking the diameter of the turbid zone around the stab inoculated region. The diameter of the migrating bacteria on semisolid media of Δfnr strain was profoundly less (2.032±0.0578) compared to the WT strain (2.967±0.152). It may indicate that fnr gene regulates flagellar genes and motility. It is important for the bacterial invasion to the host cells (Khoramian-Falsafi et al., 1990; Morimoto et al., 2017) (Fig 5). Statistical analysis: T-test, P*<0.05 P**<0.01, P***<0.001, N.S.-Not significant. Our findings are in correlation with the transcriptomic and proteomic analysis of Δfnr strain of S. Typhimurium under anaerobic condition, that flagellar genes are activated by fnr (Fink et al., 2007; Behera et al., 2020).
 

 
One dimension-PAGE analysis of WT and Dfnr showed differential expression of many proteins
 
An equal amount of proteins from both WT and Δfnr cell lysates (100µg) each was loaded onto SDS-PAGE and gel was stained with 0.25% Coomassie brilliant blue (CBB-R 250). There were significant changes in the expression of many proteins and differentially expressed protein in the 45 kDa region from WT lane was cut and sent for mass spectrometry analysis (MALDI-TOF) (Fig 6). Analyzed protein sequence through peptide mass fingerprinting and identified as TolA (Supplementary material, S.5). we are first to demonstrate that tolA gene is regulated by fnr under anaerobic condition.
 

 
Real –time quantitative reverse transcription PCR (qRT-PCR) demonstrate that Dfnr has reduced expression of tolA gene
 
Measured mRNA levels were normalized to the expression of housekeeping gene 16s rRNA. These normalized values are used for the calculation of fold expression. tolA gene was 27 fold down-regulated in Δfnr compared to WT. Which supports our proteomic study that fnr gene activates tolA gene under anaerobic condition. Further validation was done by qRT-PCR technique, tolA gene was 27 fold down-regulated Δfnr compare to WT strain at the RNA level (Fig 7). First time we are reporting that tolA gene is positively regulated by fnr gene in an anaerobic environment that isesponsible for bacterial growth and pathogenesis. TolA is an inner membrane integrity protein, important for bacterial virulence, maintenance of membrane integrity, LPS production and bacterial replication of Salmonella Typhimurium (Paterson et al., 2009; Masilamani et al., 2018).
 

In the current study, we reported that fnr gene plays an essential role in regulating the bacterial growth, motility and virulence of S. Typhimurium by controlling the expression of proteins like TolA and other proteins.

This study was funded by Department of Biotechnology (DBT,), New Delhi. Project number is BT/PR15993/NER/95/144/2015. We acknowledge Director, Indian Veterinary Research Institute, Izatnagar, Bareilly, India for providing facilities related to this work. We also aknowledge ICMR for providing fellowship (ICMR-JRF/SRF).