Partial Characterization of Novel Bacteriocin SF1 Produced by Shigella flexneri and Their Lethal Activity on Members of Gut Microbiota

A strain of Shigella flexneri producing bacteriocin was isolated from a patient with diarrhea. The main objective of this study was to isolate and partially characterize the bacteriocin. The producing microorganism was identified using biochemical, serological, and molecular methods. The lethal activity of the S. flexneri strain was studied using the drop method. This bacterial strain showed activity against different strains of E. coli and B. fragilis. Using immunological techniques, it was determined that S. flexneri belongs to serotype 2a, and by PCR, the presence of the ipaH plasmid was determined. By chromatographic techniques, it was determined that the bacteriocin is a peptide of high purity with a molecular weight of 66294.094 Da. The amino acid composition and sequence were determined by the Edman reaction, and a sequence of 619 amino acid residues was obtained. Only in five positions of this sequence, the amino acid glutamine changed to glutamic acid with respect to colicin U produced by S. boydii. From an ecological point of view, it could be assumed that SF1 bacteriocin contributes to eliminate some members of the normal microbiota of the human intestine, facilitating colonization and then producing the invasion process that characterizes the pathogenicity of Shigella.


Introduction
Bacteriocins are proteins or antimicrobial peptides produced by different bacterial species, which have a broad or narrow spectrum of lethal action [1,2]. Usually, these products exert their antagonistic role on other bacterial species by competing for the same ecological niche [3].
In vitro investigations related to the detection and characterization of bacteriocins showed that the biosynthesis is altered by various physical factors. Furthermore, it has been proposed that the production of bacteriocins can be induced by unfavorable conditions of bacterial growth or chemical agents such as mitomycin C [4][5][6].
Some members of the Enterobacteriaceae family have genetic determinants that encode bacteriocins, whose frequently are located in plasmids [2]. ese antibacterial products show a broad spectrum of action and have a variable molecular weight range between 25 and 80 kDa (colicins) or below 9 kDa (microcins) [7,8]. E. coli is noted for its colicin production, very similar to the products synthesized by S. sonnei, S. boydii, and S. dysenteriae [8]. A few studies show bacteriocinogenic activity produced by S. flexneri. Preliminary studies demonstrated for the first time the bacteriogenic activity of S. flexneri on E. coli and B. fragilis strains isolated from feces of healthy humans [9,10].
Shigella is the most common etiologic agent of dysenteric diarrhea. e species of this genus have the ability to invade and multiply in the human intestinal epithelium, causing an acute inflammatory response and tissue destruction. e infection usually spreads from person to person through the faecal-oral route, and a very small inoculum (10-100 bacterial cells) is enough to cause disease [8].
Previous studies demonstrated the presence of one strain of S. flexneri with antibacterial capacity. In addition, it was reported that the antibacterial product is a bacteriocin with antagonistic activity on E. coli and B. fragilis [9]. In order to deepen the knowledge about this interesting antibacterial substance, the main objective of this study was to purify and perform a partial characterization of the bacteriocin produced by the S. flexneri strain.

Bacterial Strains and Antimicrobial
Spectrum of Bacteriocin SF1. A bacteriocin-producer strain of S. flexneri was isolated from a patient of 31 years of age with dysenteric diarrhea in the Regional Hospital of Talca, Chile. e patient signed an informed consent for the use of the isolated strain. e bacterium was identified using microbiological and biochemical methods described in Bergey's Manual [11].
Furthermore, serological identification was performed by means of an agglutination test with polyvalent and monovalent serum for somatic O antigen (Denka Seiken, Japan) according to the manufacturer's instructions. e Scientific Ethics Committee of University of Talca approved this study.
As fragilis. ree strains were tested from each bacterial species. All target strains were grown in Tryptic Soy Broth (Merck, Darmstadt, Germany) at 37°C for 24 h until the early exponential growth phase (OD 0.4 at 600 nm in UV visible spectrophotometer Shimadzu, Japan) except for B. fragilis, which was grown on Agar Base Blood (Merck) supplemented with vitamin K1 and hemin in an anaerobic jar (Genbox Anaer Biomerieux, France). Subsequently, this culture was collected and diluted in distilled water until the same OD already mentioned was reached. After that, the target strains were grown on Mueller-Hinton agar (Merck, Germany), except B. fragilis, which was sown in the same anaerobic medium already mentioned and incubated in an anaerobic system. On the contrary, the bacteriocinogenic S. flexneri strain was cultivated overnight in the Tryptic Soy Broth and subsequently centrifuged to 10,000g for 20 min. e antibacterial activity of S. flexneri was determined using the drop method. Specifically, all the dishes with the target strains were dried for 10 min at 37°C, and then 5 μL of the S. flexneri supernatant was spotted on the lawn [9]. e dishes were incubated at 37°C for 5 h, and then the inhibitory zones were observed. in solution was determined spectrophotometrically at a wavelength of 260 nm. e purity was measured at 280 nm as previously described [12]. DNA integrity of the template was tested on agarose gel 1% (w/v) and stained with GelRed (Biotium Inc., USA). A PCR assay was performed in a final volume of 25 μL, with a reaction mixture containing 0.25 μg/ μL of template DNA, 50 pmol of each oligonucleotide sequence, 1 X PCR master mix, and DNAse-RNAse free distilled water.
e primers IpaH-F 5′-CCTTGACCGCCTTTCC-GATA-3′ and IpaH-R 5′-CAGCCACCCTCTGAGGTACT-3′ [13,14] were used. e amplifications were performed using a DNA Engine ermal Cycler (Laboratories BioRad, USA). PCR conditions were as follows: initial denaturation at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing of oligonucleotide sequences at 62°C for 1 min, and extension at 72°C for 2 min. is was followed by a final incubation for 10 min at 72°C and maintained at 4°C. In addition, a negative control without template DNA was used. PCR-amplified DNA fragments were separated by electrophoresis on 1.5% agarose gel. Furthermore, a wide-range molecular weight marker ladder 100 bp (Invitrogen, Waltham, Massachusetts, USA) was used as standard. e band was stained with GelRed (Biotium Inc). PCR products were visualized and images captured using a Gel Documentation System Doc 1000 (Laboratories BioRad).

Partial
Purification of Bacteriocin SF1. S. flexneri strain was grown in 500 mL BHI broth at 37°C for 24 h. Subsequently, the culture was centrifuged at 14,000g for 35 min at 4°C. e supernatant was treated by a progressive addition of ammonium sulfate to reach 30% saturation. e mixture was kept overnight at 4°C with constant stirring. en, it was centrifuged at 10,000g for 30 min at 4°C. e precipitate was suspended with 50 mM Tris-HCL (pH 8.0) buffer. e resulting supernatant was adjusted to 95% saturation with ammonium sulfate, as described above.
Both samples were centrifuged at 10,000g for 30 min at 4°C, and the final pellet containing the bacteriocin SF1 was suspended in a minimal volume of 50 mM Tris-HCL (pH 8.0). e obtained products were dialyzed separately in a MEMBRA-CEL ™ MC-18 (Viskase, USA) membrane at 4°C for 48 h in the same buffer used above. e resulting dialysate was loaded onto a column of ion exchange FPLC (fast performance liquid chromatography) using a Mono-Q ™ 5/ 50 GL (GE Healthcare, Sweden) column previously equilibrated with 50 mM Tris-HCL (pH 8.0) and eluted with the same buffer using a gradient of 0 to 1.0 M NaCl in Tris-HCl (pH 8.0). Two mL aliquots were collected and tested to determine antimicrobial activity. Active fractions were mixed, concentrated, and lyophilized. Subsequently, filtration chromatography was performed on FPLC gel using a Superose 12 HR column 10/30 (GE Healthcare Life Sciences, UK) equilibrated with 50 mM Tris-HCl (pH 8.0) and 0.2 M NaCl; the samples were eluted with the same buffer. Active aliquots were processed in HPLC (high-performance liquid chromatography) using a LiChroCART ® C18 column (250 × 4.0 mm) (Merck). e mobile phase was 0.1% (v/v) trifluoroacetic acid (TFA) and the solution of 80% (v/v) aqueous acetonitrile containing 0.1% (v/v) of TFA. Aliquots were assayed for detecting the bacteriocin activity. All those positive activity fractions were pooled, lyophilized, and suspended in Milli-Q ® , obtaining the partially purified bacteriocin [15].
For all assays, bacteriocinogenic activity related with processes of purification and partial characterization, E. coli (EC-7) strain which has a high sensitivity to the bacteriocin studied, was used. is E. coli strain was the one that showed the highest sensitivity to bacteriocin among all the studied strains.

Molecular Weight Determination of Bacteriocin SF1.
e molecular weight of the bacteriocin SF1 was determined by glycine SDS-PAGE with 5% stacking gel and 12% separating gel using the molecular weight standard Strep Tag II Perfect Protein (Merck) [16].
e gel was stained with Coomassie blue R-250 and washed at room temperature with a solution of acetic acid 5% to remove excess of stain.

Effect of Enzyme Action, pH, and Temperature on Bacteriocin SF1.
e bacteriocin SF1 was diluted 5 times in buffer with each enzyme (Sigma, USA) analyzed. e final enzyme concentration was 1 mg/mL. All enzymes were sterilized by filtration through a membrane filter of 0.22 μm (Merck, Germany). For assays, trypsin, α-chymotrypsin, and pepsin in a buffer Tris-HCl 20 mM pH 8.0; proteinase K in buffer 20 mM Tris-HCl pH 7.2; and papain in 50 mM phosphate buffer pH 7.0 were used. e enzyme activity was determined by incubating each sample at 37°C for 30 min, 1, and 4 h. Untreated bacteriocin samples were used as controls.
e bacteriocin activity was assessed at different pH values. e pH of the bacteriocin sterilized by filtration was adjusted using the following buffers: KCl-HCl (pH 2.0 and 3.0), acetate (pH 4.0 and 5.0), citrate (pH 6.0), and Tris-HCl (pH 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0). e bacteriocin was diluted twice in different buffers. e resulting mixtures were incubated at 37°C for 30 min. e assay previously described was then performed to detect bacteriocinogenic activity.
On the contrary, the bacteriocin was treated at −76, 4, 25, 37, 60, and 80°C for 30 min as well as 100 and 121°C for 15 min. After the treatment, the samples were diluted 1 : 2, 1 : 4, and 1 : 10 and kept at 4°C for 2 h. Later, tests were performed to determine the biological activity as described above.

Analysis of Antimicrobial Activity in Polyacrylamide Gel under Nondenaturing Conditions.
e bacteriocins SF1 was run in a polyacrylamide gel 7.5%. It was subsequently washed with distilled water and placed in a sterile Petri dish. en, it was covered with a thin mixture of 0.8% Brain Heart Infusion Agar (BHI) (Merck) and 10 4 CFU/mL E. coli EC-7. After, the plate was incubated for 5 h at 37°C for observing the inhibitory zone.

Amino Acid Composition and Sequence Analysis of Bacteriocin SF1.
e lyophilized partially purified bacteriocin SF1 was used to obtain the amino acid composition, and the sequence was determined by the Edman reaction in an automated sequencer PPSQ-31A (Shimadzu, Japan) [17].

Identification of the S. flexneri Strain.
Bacteriological identification of the S. flexneri strain was performed by biochemical and serological methods. e strain belongs to serotype 2a. On the contrary, the molecular identification showed the presence of plasmid 606 bp ipaH, confirming that the strain studied is S. flexneri.

Partial Purification of Bacteriocin SF1.
e specific activity of bacteriocin SF1 was increased 23 times during the purification process; sixty-nine percent of the antimicrobial activity was recovered (Table 1). e SDS-PAGE analysis showed a band of approximately 66 kDa in a triplicate assay (Figure 1(a)). e antibacterial activity analysis showed the presence of an inhibitory zone at the same level of the detected band (Figure 1(b)). e chromatogram of purified bacteriocin SF1 was obtained by HPLC (see Figure S1 in the Supplementary Material for analysis of purity of bacteriocin SF1).

Antimicrobial Spectrum of Bacteriocin SF1.
e antimicrobial spectrum of the bacteriocin SF1 was determined on different Gram positive and negative species. e bacteriocin was active only against the three target strains of E. coli and B. fragilis tested in this research ( Table 2).

Effect of Enzymes, pH, and Temperature on Bacteriocin
SF1. Only the enzymes proteinase K and papain inactivated the bacteriocin SF1. It was observed that the bacteriocin SF1 lost biological activity only when exposed to 100°C and 121°C. Moreover, the alkaline pH inhibited the antibacterial action (see Table S1 in the Supplementary Material for comprehensive analysis of enzymes activity, temperature, and pH on bacteriocin SF1).

Amino Acid Composition and Sequence Analysis of Bacteriocin SF1.
e bacteriocin SF1 obtained from S. flexneri strain showed a sequence of 619 amino acid residues (Figure 2). Its amino acid composition is shown in Table 3. Its molecular weight was 66294,094 Da.

Discussion
Species of the genus Shigella are among the bacterial pathogens most frequently isolated from patients with diarrhea. Five to fifteen percent of all diarrheal episodes worldwide can be attributed to an infection with Shigella, including 1.1 million fatal cases [18,19].
In this research, the novel bacteriocin produced by the S. flexneri strain was named SF1.
is substance was International Journal of Microbiology sensitive to treatment with proteolytic enzymes, particularly proteinase K and papain, showing their peptidic nature. According to the results, bacteriocin SF1 maintains antibacterial capacity at 80°C, but not at 100°C. e thermolability at 100 and 121°C of this antagonistic substance is also consistent with its chemical composition and corroborates data reported by other authors who have demonstrated that colicins produced by Shigella are generally heat labile [7,20]. By SDS-PAGE, a single band was obtained for bacteriocin SF1. is band was taken and used to detect the protein purity by HPLC. Also, it is important to note that the band in the gel in nondenaturing conditions was responsible for the antagonistic activity in the biological assay.
It was interesting to observe that the amino acid composition of bacteriocin SF1 is similar to that of colicin U produced by S. boydii [21]. e specific results of this research showed a small variation that affected only the proportion of glutamine (amino acid imide) and glutamic acid (amino acid) of bacteriocin SF1 in respect to colicin U. In comparison with bacteriocin SF1, the colicin U shows 38 residues of glutamic acid and 28 of glutamine. is result was confirmed by means of the amino acid sequence performed in this research. e amino acidic variation occurs only at five positions in the sequence in which glutamine are replaced by glutamic acid. e molecular weight calculated for the bacteriocin SF1 is 66294.094 Da, compared to the molecular weight of the colicin U which is 66289.1719 Da. Also, according to the results, it is possible to argue that the bacteriocin of S. flexneri could be the product of mutations, explaining the differences detected between colicin U and bacteriocin SF1.
In addition, the bacteriocinogenic S. flexneri strain might present a selective advantage during the colonization process and before the development of its invasive capacity.
us, the bacteriocinogenic activity of S. flexneri against E. coli and B. fragilis would allow understanding how a low infectious dose of S. flexneri is capable of displacing these members of the gut microbiota and prevailing in this ecological niche, facilitating colonization and later starting the invasiveness process [22]. erefore, the possible role of bacteriocin SF1 as a virulence factor should be studied, and further microbiological and molecular studies on the bacteriocin SF1 are necessary to understand in depth its ecological role.
ree strains were tested from each bacterial species. +: bacterial species sensitive to bacteriocin; −: bacterial species not sensitive to bacteriocin.

Conclusions
A novel bacteriocin of 619 amino acid residues and 66294,094 Da of molecular weight, produced by S. flexneri, named bacteriocin SF1 has been for the first time detected and partly purified. Bacteriocin SF1 shows lethal activity on E. coli and B. fragilis, important members of the normal microbiota of the human gut.

Data Availability
e experimental data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.
Acknowledgments is work was supported by a grant from Dirección de Investigación, Universidad de Talca (VAC 600500). Figure S1 shows the purity of bacteriocin SF1 by means of HPLC. Active aliquots of bacteriocin SF1 were processed in HPLC using a LiChroCART C18 reverse phase. HPLC: mobile phase A: 0.1% trifluoroacetic acid (TFA); mobile phase B: 80% aqueous acetonitrile solution containing 0.1% TFA; linear gradient 0-100% of B solution in 30 min flow rate 1 mL/min; temperature 35°C; active fraction: 34.6 min retention time. Table S1 shows the enzymes activity, temperature, and pH on bacteriocin SF1. Initially, from the untreated bacteriocin, the arbitrary units per mL (AU/mL) were calculated, estimating 25,600 AU/mL. e arbitrary units were calculated based on the reciprocal of the highest dilution with biological activity and multiplied by 100 (dilution factor). E. coli EC-7 was used as target strain of lethal action of the bacteriocin SF1. (Supplementary Materials)