Recombinant Salmonella enterica OmpX protein expression and its potential for serologically diagnosing Salmonella abortion in mares

Background and Aim: Salmonella abortion in mares is caused by Salmonella enterica subspecies enterica serovar abortus equi infection and is characterized by premature (abortion) or non-viable fetus birth. Although all horses are susceptible to infection, the condition is more often clinically manifested in pregnant mares, with most abortions recorded in young females. In addition, nonspecific clinical disease signs and poorly sensitive and effective bacteriological diagnostic methods hinder rapid and reliable infection diagnoses. Immunochemical methods such as enzyme-linked immunosorbent assay (ELISA) and immunochromatography assays can facilitate effective and rapid diagnoses. However, they require highly specific and active antigens and antibodies. This study aimed to generate a recombinant S. enterica outer membrane protein X (OmpX) and evaluate its suitability for serological diagnosis of Salmonella abortion in mares. Materials and Methods: Outer membrane protein X from the S. enterica antigen was synthesized de novo and expressed in Escherichia coli using the pET28 vector. Transformed E. coli cells were cultured under different conditions to detect recombinant OmpX (rOmpX) expression, and rOmpX purification and refolding were both conducted using metal affinity chromatography. Refolded and purified rOmpX was characterized by western blotting, liquid chromatography with tandem mass spectrometry, and ELISA. Results: After optimized rOmpX expression, a 23 kDa molecular weight protein was identified. Amino acid sequence analysis using Mascot program suggested that these peptides were the OmpX protein from S. enterica. High specificity and diagnostic efficiency were recorded when rOmpX was used in ELISA against 89 serum samples from aborted and contact mares. Conclusion: Recombinant outer membrane protein, in comparison to the O antigen, demonstrated better diagnostic characteristics against sera from mares who aborted and contact horses.


Introduction
Salmonella abortion in mares is an infectious disease of bacterial etiology and results in pregnancy termination and expulsion of dead or immature fetus from the uterus.In most mares, abortion occurs unexpectedly at 4-8 months.After abortion, increased body temperature and whitish mucus discharge from the vagina are observed.In a previous observational study (2-3 weeks), after abortion, animals were oppressed, refused to eat, and had a yellowish-brown vaginal discharge [1].Based on clinical disease signs (abortion) and pathoanatomical changes in aborted fetuses, Salmonella enterica subspecies enterica serovar abortus equi infection was cited as a contributory agent [2].However, a final diagnosis is often based on a bacteriological investigation of pathological material and specific pathogen detection [3].
The absence of specific clinical infections requires specific laboratory investigations.Furthermore, given often prolonged bacteriological analyses, several molecular methods have been developed, such as real-time polymerase chain reaction (PCR), and immunochemical methods such as enzyme-linked immunosorbent assay (ELISA) and immunochromatographic analysis, which are easy to perform and provide results within hours or even minutes [4][5][6][7].However, immunogenic bacterial membrane proteins are required to ensure immunochemical test specificity.Several S. enterica surface antigens have been genetically and immunologically investigated, Available at www.veterinaryworld.org/Vol.16/September-2023/3.pdf including flagellins, fliC and fljB, InvH and spvC, and the outer membrane protein X (OmpX) [8][9][10][11].Salmonella OMPs are important cell wall components found on peptidoglycan layers in Gram-negative bacteria and are major regulators of small molecule membrane permeability.As the main component of outer bacterial membranes, S. enterica OmpX is immunodominant and an attractive target for serum antibody interactions [12].
This study aimed to generate a recombinant OmpX (rOmpX) antigen from S. enterica and evaluate its suitability for serologically diagnosing Salmonella abortion in mares using ELISA.

Ethical approval
All animal experiments were conducted after obtaining permission from the Local Ethical Committee of S. Seifullin Kazakh Agrotechnical Research University (No.1, August 26, 2020), in accordance with the Guidelines for the maintenance and care of animals (Interstate standard, GOST 34088-2017).

Study period and location
The study was conducted from January 2022 to February 2023.Serum samples were collected from animals in private horse breeding farms and farmsteads in the Akmola, Karaganda, and Kostanay regions.The object of the study were mares that had previously experienced fetal abortion or had direct contact with them.

Recombinant protein plasmid construction, transformation, and expression
The OmpX reference amino acid sequence was obtained from the National Center for Biotechnology Information database (https://www.ncbi.nlm.nih.gov/protein/) (Accession No. NP_459810.1).Outer membrane protein X from S. enterica was codon optimized for E. coli using Vector NTI (https://vector-nti.software.informer.com/11.5/)and synthesized at the National Center for Biotechnology, Astana, Kazakhstan.
The synthesized DNA fragment was cloned into a pGEM-TEasy vector and transformed into chemo-competent E. coli DH5α cells.Positive colonies were selected using blue-white selection method and PCR screening of white colonies.Positive clones were selected and sequenced using an ABI PRISM® 310 Genetic Analyzer (Thermo Fisher Scientific, USA).The amino acid sequence encoding OmpX was cloned into the pET28 expression plasmid using EcoRI and XhoI restriction enzymes (Fermentas, Vilnius, Lithuania).The obtained expression vector was used to transform competent E. coli BL21 (DE3) cells, which were cultured on LB agar.After PCR screening, a single positive clone was inoculated into LB agar/ kanamycin (50 μg/mL) and cultured.To determine optimal cultivation conditions positive colonies were cultivated in LB/kanamycin medium to an optical density (600 nm) of 0.5, and different isopropyl-β-D-thiogalactopyranoside (IPTG) (0.1-1 mM) concentrations were added.Cells were incubated at 25°C and 37°C, and sampling was performed every 2 h.

Recombinant outer membrane protein purification and isolation
For protein isolation, cells were resuspended in 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, and 0.1 mM ethylenediaminetetraacetic acid buffer supplemented with phenylmethylsulfonyl fluoride (0.1 mM), and disrupted using a UP200S ultrasonicator (Hielscher Ultrasonics GmbH, Teltow, Germany).Lysates were pelleted by centrifugation at 4000×g at 4°C for 10 min and resuspended in buffer plus 1 M urea, incubated further at 25°C for 30-40 min, and reprecipitated by centrifugation.The pellet was then resuspended in 8 M urea buffer and incubation and pelleting procedures were repeated.Recombinant outer membrane protein was isolated from the supernatant using 1 mL HisTrap HP columns (Cytiva, Uppsala, Sweden).A linear imidazole gradient (20-500 mM) was used to elute rOmpX and fractions were subjected to electrophoresis on 12% polyacrylamide gels, according to the Laemmli method [13].Protein concentrations were determined using the Bradford assay.The rOmpX amino acid sequence was analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS), as described previously by Borovikov et al. [14].
ELISA sensitivity was determined on serum samples form aborted mares and confirmed using bacteriological and PCR methods in the laboratory of the National Veterinary Reference Center.For comparison, a commercial "Salmonella Latex Kit" (Liofilchem, Roseto d.Abruzzi, Italy) was used to detect specific antibodies.Specificity was determined in serum samples with the presence of antibodies against other pathogens (horse wash, infectious anemia of horses), which are found in the Republic of Kazakhstan.

Statistical analysis
Enzyme-linked immunosorbent assay optical density values were analyzed and processed using GraphPad Prism 9.4.1 software 9.4.1.(https://www.graphpad.com/updates/prism-941-release-notes).Serum samples were analyzed in triplicate.Statistical data analyses were performed using t-tests (Student's tests).The equality of the two populations was determined using F-tests.Data were represented by the mean ± standard error and significant differences were accepted at p < 0.05.

Recombinant outer membrane protein construction, expression, and purification
Codon-optimized OmpX from the S. enterica antigen was cloned into pET28.The hexahistidine tag and restriction sites are shown (Figure -1).
Escherichia coli BL21 cells transformed with pET28/OmpX were cultured in LB/kanamycin medium containing different IPTG concentrations.Increased rOmpX expression was observed in samples every 2 h.After optimizing cultivation conditions, maximum protein expression occurred at 37°C, 0.1-0.2mM IPTG, and a 6 h incubation period (Table-1).The recombinant protein was also localized to inclusion bodies (data not shown).
After HisTrap HP column chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis on eluted fractions indicated a protein of approximately 23 kDa molecular weight.The optimal imidazole elution concentration was 500 mM.The maximum protein concentration was ~950 μg/mL (Figure -2).sequence; QTTDYPTYKHDTSDYGFSYGAGLQFN peptides were identified.Amino acid sequence analysis using Mascot suggested that these peptides were the OmpX protein from S. enterica (Figure -3).To ensure correct protein folding after affinity chromatography, urea concentrations were consistently reduced and specific rOmpX reactivity was determined by western blotting.Finally, the protein reacted with a positive sera pool from mares who aborted, indicating correct protein folding (Figure -4).

Enzyme-linked immunosorbent assay analysis of pooled equine sera
Using rOmpX and a commercial S. enterica O antigen, 89 serum samples from mares who experienced abortions, and contact animals, were examined.For rOmpX-based ELISA, specific antibodies were established in eight samples and 12 samples for the O antigen.To determine standard deviation values, antibody titers were determined in six randomly selected serum samples from aborted and healthy mares at dilutions of 1:100-1:800.Enzyme-linked immunosorbent assay results showed antibodies against rOmpX at a titer of 1:800 (Figure -5).To establish average optical density values using different antigens, antibody titers were determined in all sera (Figure -6).Data analyses indicated the presence of antibodies to the OmpX antigen, and in some serum samples, antibody titers were higher when compared with the O antigen.Statistical analyses were performed using Student's t-tests (*p = 0.0001) and F-tests (*p = 0.0001).
Enzyme-linked immunosorbent assay sensitivity when compared with the "Salmonella Latex Kit" was 114% for rOmpX and 171% for the O antigen.Specificity was confirmed by the absence of interaction with sera containing antibodies to antigens of heterologous infections in diagnostic titers.

Discussion
Salmonella enterica abortus equi is responsible for Salmonella abortion in horses, which is characterized by premature birth (abortion) or non-viable fetus birth.After abortion, increased body temperature is associated with several complications, while metritis may also develop.Animals appear oppressed and refuse to feed, and complications such as arthritis,   bursitis, and subcutaneous tissue abscesses may be manifested.To expedite rapid infection diagnoses, immunochemical methods are advised to identify specific antibodies in sera from sick animals.However, the diagnostic efficiency of these tests can considerably vary because the production of native antigens is difficult to standardize.[15].
Hence, rOmpX from S. enterica was generated to serologically diagnose Salmonella abortion in mares, which can also be used to obtain specific monoclonal antibodies.This Salmonella antigen diagnostic approach has been used in several studies [16,17].Outer membrane protein X from S. enterica was obtained using a de novo method, codon optimized for E. coli expression, cloned into an expression plasmid, and transformed into competent E. coli cells.Bacterial expression has been widely used to generate virus, bacterial, and eukaryotic diagnostic antigens [18,19].When we compared rOmpX amino acid sequence with the reference sequence, a complete match was established.Thus, a rOmpX protein (molecular weight = 23 kDa) was obtained.Correct protein refolding was confirmed by western blotting and ELISA with positive control sera.
Recombinant outer membrane protein activity and specificity were determined by indirect ELISA using 89 sera samples from mares that aborted and direct contact horses.The presence of specific antibodies was established in eight samples.Furthermore, specific antibodies were detected by ELISA based on a commercial O antigen in 12 samples.Despite the fact that fewer positive samples were detected using rOmpX, antibody titers in some cases were higher than the O antigen.Thus, rOmpX showed higher specificity when compared with the O antigen, which may have been due to increased lipopolysaccharide antigen cross-reactions.Our results agreed with other studies [20,21] where recombinant antigens were successfully used to serologically diagnose salmonellosis in other animal and bird species.

Conclusion
We successfully generated a S. enterica rOmpX protein.The protein actively interacted with specific antibodies in positive sera from infected animals at high titers.When rOmpX was used in ELISA against serum samples from mares who aborted, activity and specificity were established.However, before rOmpX is recommended for the serological diagnosis of Salmonella abortion in horses, further research is required.It will be important to compare rOmpX effectiveness against different Salmonella serotype antigens, which may improve diagnostic methods for this harmful infection.

Figure- 3 :
Figure-3: Tandem mass spectrometry spectrum of the fragmented peptides of recombinant outer membrane protein.

Figure- 5 :
Figure-5: Titration histogram of six blood sera in enzymelinked immunosorbent assay using Salmonella enterica recombinant outer membrane protein and standard deviations.

Figure- 4 :
Figure-4: Polyacrylamide gel electrophoresis (a) and western blot (b) of recombinant outer membrane protein X with Salmonella enterica-positive serum.Lines 1-2: Recombinant outer membrane protein, MM=Molecular marker.b a

Figure- 6 :
Figure-6: Optical density of the enzyme-linked immunosorbent assay reaction of the recombinant outer membrane protein and Salmonella O antigen with mare sera.Each symbol represents a separate serum sample.
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