Background
Cholera is a clinical-epidemiologic syndrome caused by ingestion of water contaminated with
Vibrio cholera serogroups O1 and O139. This disease is considered an important public health problem worldwide, though it mainly affects developing countries and alters the economies of these regions [
1]. From 1991 to 2001, the seventh pandemic of cholera affected Latin America, including Mexico. In October 2010, a cholera epidemic in Haiti resulted in over 180,000 cases in three months and spread rapidly to other countries, such as the Dominican Republic and Cuba [
2].
V. cholerae has the ability to survive in aquatic habitats of different characteristics, including wastewater. During the process of adaptation to conditions of extreme pH, salinity, temperature, and nutrient insufficiency as well as predation by heterotrophic protists and bacteriophages, the expression of different genes is activated. A viable but non-culturable state or biofilm is then induced, which contributes to adaptation by the bacterium for survival in different environmental conditions [
3]. Bacteriophages or phages (bacterial viruses) are mobile genetic elements that participate in horizontal gene transfer in bacteria, thereby contributing to their environmental adaptation and evolution. In addition, several bacterial virulence genes are present in phage genomes, and the mobile nature of phages can promote the emergence of new epidemic strains.
One of the main virulence factors of
V. cholerae is cholera toxin (CT), which is encoded by CTXØ, a lysogenic filamentous phage that has contributed to bacterial evolution through lysogenic conversion and genomic rearrangement [
4]. The
ctxAB genes present in the CTXØ genome of toxigenic
V. cholerae favor the conversion of nonpathogenic strains into toxigenic strains via CTXØ acquisition. The first vibrio phages were described in 1926 by d’Herelle, and in the 1950s, several distinct types of
V. cholerae phages were described [
5]. The use of bacteriophages as a tool for strain differentiation has contributed significantly to our understanding of cholera epidemiology [
6]. In addition, the first phage-typing scheme for
V. cholerae O1 was employed to study the spread of
V. cholerae strains of the El Tor biotype [
7]. Although, this phage-typing scheme has been used routinely for the classification of
V. cholerae O1 strains due to its limitations, new phage-typing schemes for O139 strain classification have been developed [
8]. Since 2007, more than 200 vibrio phages have been described; however, at present, only 17 genomes of
V. cholerae phages have been sequenced and annotated in the GenBank database.
In recent years, lytic phages have been proposed as important factors modulating populations of
V. cholerae serogroups O1 and O139 in the aquatic environment, thus affecting the seasonality and duration of cholera epidemics in endemic areas [
9]. In Bangladesh, which is considered an endemic cholera area, the prevalence of several predatory phages (JSF1 to JSF6) of
V. cholerae has been partially characterized. Fluctuations in and the presence of the most prevalent phage types have been correlated with temporal changes in the cyclical appearance of cholera, acting as factors that modulate the epidemic cycle in the short period as well as outbreak severity [
10]. In Mexico, conditions amenable to the survival of
V. cholerae Non-O1/Non-O139 in aquatic reservoirs have been reported for several years [
11]. However, the role of diverse phages in non-endemic cholera areas as elements that participate in the survival and occurrence of the bacterium during long interepidemic periods is not completely understood. In 2010, sporadic cholera cases were identified in Sinaloa State, México, and in 2013, an outbreak of 187 cases of cholera in Hidalgo State, México, was reported by the Secretaria de Salud de México (
www.epidemiologia.salud.gob.mx/dgae/boletin/intd_boletin.html;
www.sinave.gob.mx/). Although the phages involved in the epidemiology of cholera in Mexico have not yet been characterized, predation of
V. cholerae O1 by phages can be considered a key factor in understanding the long interepidemic periods of cholera in these regions. The main goal of this study was to isolate and characterize
V. cholerae phages from wastewater of the Endo Dam in Hidalgo State, México, and to assess their lytic activity against
V. cholerae O1 strains.
Discussion
Phages as biological entities are abundant and widely distributed in the world and have great relevance in the control of bacterial communities. Fluctuations in phage populations during the seasonal behavior of cholera and the surveillance of
V. cholerae in the aquatic environment are important factors that have been associated with cholera outbreaks [
9]. In endemic cholera regions,
V. cholerae phages have been detected in high frequency in different aquatic habitats, and these phages have been employed as strain markers for phage typing of
V. cholerae O1 and O139 [
8].
Thirteen bacterial isolates in wastewater samples collected from the Endhó Dam in Hidalgo State were identified as V. cholerae non-O1/O139, V. alginolyticus and A. veronii. Although toxigenic isolates of V. cholerae O1/O139 were not identified, the presence of non-O1/O139 V. cholerae strains is suggestive of the ability of these bacteria to survive for prolonged periods in sewage-polluted waters.
In endemic cholera areas, the presence of non-O1/O139 strains in the environment has been related to these bacteria serving as possible phage reservoirs with lytic activity against
V. cholerae O1/O139 [
9]. An abundant number of phages in wastewater treatment systems have been described, though little information regarding their population dynamics and their interaction with the microbial community has been published [
26]. In the present work, only one phage, named ØVC8, showed lytic activity again
V. cholerae O1 strains.
V. cholerae predation by lytic phages has been proposed to be an important factor involved in the cyclical occurrence and severity of cholera outbreaks in endemic areas [
10]. Thus, the presence of ØVC8, a lytic phage of
V. cholerae O1 strains, could be involved in the epidemiology of cholera in Mexico, possibly regulating the presence of
V. cholerae O1 strains for long periods; however, further studies are required to confirm this possibility.
The morphological characteristics of ØVC8 phage visualized by TEM showed a structure similar to
V. cholerae phages JSF3 and JSF6 of the
Podoviridae family, two phages that have been associated with the cyclic appearance of cholera in Bangladesh (Fig.
1). Considering the morphological classification of JSF3 and JSF6 phages, ØVC8 could be included in group III of the
V. cholerae phage C1 morphotype, which includes OXN-100P, 4996, I, and III [
5‐
27].
Sequencing of the ØVC8 genome revealed 48 putative ORFs distributed on both DNA strands and organized into packing, head-tail morphogenesis, metabolism, replication, and unknown function modules. ORFs 2 and 3 encode for proteins of the terminase family, which are implicated in translocation of the viral capsid DNA during the final stage of phage assembly. Terminases are the most conserved proteins among caudoviruses, and they have been identified in all podoviruses [
28]. Considering the presence of terminases as a potential marker of podoviruses, ORF3 of phage ØVC8 was analyzed by bioinformatics procedures. The results obtained allowed the identified terminases to be grouped in the same cluster with the terminases of phages VP2 and VP5 (Fig.
4). These data support that ØVC8 has a genomic organization that is similar to that of VP2 and VP5; therefore, ØVC8 could be included in the VP2-like subfamily proposed by Lavigne et al
. [
29].
During ØVC8 phage replication, a terminase protein is required for DNA packaging and for chromosomal end formation [
30]. ORF3 encodes a large terminase subunit that could participate in this process. Indeed, comparative analysis of the terminase protein coded by ORF3 suggests participation in the packing process via the “single-stranded cohesive ends” strategy, as has been described for lambda-like phages. Phages with these characteristics have a complementary sequence and generate protruding single strands called COS sites; these sites are highly conserved in the genome and are present in a region 1,000 bp upstream of the gene that encodes the small terminase subunit [
28]. The above findings led us to propose that the COS site of ØVC8 is located in a tandem sequence 299 bp upstream of the terminase small subunit (Fig.
3). The interaction of head-to-tail connecting proteins with one of the procapsid vertices of the mature phage promotes formation of an axial pore for DNA translocation in both directions [
31]. The presence of the head-to-tail connecting protein (ORF4) of ØVC8 suggests that this protein participates in this process during DNA packaging by translocating the chromosome into the procapsid and ejecting it during the infective stage of the phage. These results indicate that ORF4 is the portal protein of phage ØVC8.
In some podovirus phages such as P22 from
Salmonella sp., tail proteins have been described as molecules with the ability to recognize specific receptors during the initial stages of host infection [
32]. In the head-tail morphogenesis module of ØVC8, ORF14 encodes a tail protein that could be involved in the recognition and infection of
V. cholerae O1 strains. However, an Ig domain similar to that described for phage T4, which has been associated with functions of immune response and adhesion to eukaryotic cells, was also identified in ORF14 [
33]. This domain has been grouped into the classic Ig domain (I-Set), which is widely distributed among bacteria, as well as the fibronectin type 3 (FN3) and bacterial Ig-like domain (Big2) families. Bioinformatics analysis of ORF14 revealed that its Ig domain corresponds to the I-Set family [
34]. Recent studies have demonstrated that Ig-like domains are important in phage interaction with metazoan mucosal surfaces via specific adherence that might provide immunity independent of the host immune response [
35]. Thus, the Ig domain of ORF14 may be an important element in ØVC8 phage interaction with the human intestinal mucosa, which is associated with the lytic activity of the phage in preventing
V. cholerae O1 colonization.
Although ORF20 codes for a tail fiber protein, the presence of fibers in the ØVC8 phage was not observed by TEM. The tail fiber protein described for phage T7 consists of elongated homo-trimers that are responsible for the reversible initial recognition of a cell [
36]. These structures are commonly composed of six fibers that are attached to the phage capsid, which hinders TEM analysis, and these fibers can be only visualized when host interaction occurs [
37]. In our study, ØVC8 phage fibers were not observed, suggesting the possibility of a situation similar to that described for T7. In contrast, ORF21 of ØVC8 was annotated as a capsid protein, which encodes a BNR/Asp-box domain that has been described in the neuraminidase or sialidase family from bacteria and phages [
38]. Proteins with sialidase activity are important for the degradation of bacterial polysaccharides; expression of these enzymes is an attractive feature for phage therapy [
39]. Indeed, the presence of the BNR/Asp-box domain in ORF21 enables ØVC8 to be considered as a possible strategy for the treatment of cholera.
DNA/RNA helicases are widely distributed proteins that are required for the ATP-dependent unwinding of double-stranded DNA, an essential step in replication, expression, recombination, and DNA repair. In the replication module of the ØVC8 genome, ORF34 encodes a DNA/RNA helicase of the SNF-2 family with a conserved domain in its amino-terminal region that is involved in chromatin structure remodeling [
40]. ORF31 codes for SSB, a protein that participates in replication, recombination and DNA repair processes [
41]. ORF29 encodes for a DNA polymerase I described in mitochondrial polymerase-g, prokaryotic DNA polymerase I, and diverse polymerases (T3, T5, and T7 phages) of the Pol A family [
42]. Therefore, the helicase of ORF34 identified in this study may participate in transcription and replication processes of the ØVC8 phage genome.
ORF28 exhibits sequence homology with integrases of
V. cholerae phages VP2 and VP5
. Integrases achieve viral genome integration into the host genome via site-specific recombination of DNA sequences of 30 to 40 bp, with the first located on the phage chromosome (attP) and the second on the bacterial chromosome (attB). These enzymes are classified into two major families based on their amino acid sequence homology and catalytic residues, either tyrosine or serine. However, bioinformatics analysis shows no tyrosine or serine residues in the sequence of ØVC8 integrase or the corresponding sequences of VP2 and VP5 phages. Suggesting that the sequence does not correspond to an integrase or is a non-functional protein. Conserved bifunctional-N-terminal primase/polymerase domains (N-Ter prim/pol) and other primase C-terminal-2 domains (PriCT-2) were identified in the ORF28 sequence of the ØVC8 genome. N-Ter prim/pol is a multifunctional enzymatic domain with ATPase, primase, DNA polymerase, and helicase activity [
43]. In contrast, the PriCT-2 domain belongs to the archaea-eukaryotic primase superfamily from the primase-polymerase clade (prim/pol-like) [
44]. N-Ter prim/pol and PriCT-2 are essential domains of multifunctional replication proteins of the phage replication machinery. Accordingly, we speculate that ORF28 has bifunctional DNA primase/polymerase activity involved in ØVC8 phage replication.
In the metabolism module of the ØVC8 genome, ORFs 25 and 27 encode for enzymes involved in metabolic pathways of amino acid synthesis; these proteins are auxiliary metabolic molecules that may provide additional support in host metabolism steps, allowing successful phage infection [
45]. HD-3, a conserved domain of ORF25, corresponds to proteins with a distinct combination of metal-chelating residues, nucleases and phosphodiesterase activities [
46]. These data suggest that ORF25 could participate in ØVC8 phage signaling and nucleotide metabolism. ORF27 encodes an adenylosuccinate synthetase that participates in purine biosynthesis by catalyzing the GTP-dependent conversion of inosine monophosphate to adenosine monophosphate [
47]. Notably, this enzyme is located at the same loci of VP2 (VP2p26) and VP5 (VP5_gp26) chromosomes, and the presence of this enzyme constitutes one of the main distinguishing characteristics of the proposed VP2-like subfamily [
29].
Recent studies have shown that some phages can employ alternative pathways of the classical holin-endolysin lysis system by employing the host cell secretion machinery to deliver their endolysins [
48]. Our observations showed that ØVC8 is a virulent phage with lytic activity against several
V. cholerae O1 strains; however, none of the identified genes of the ØVC8 genome appear to be involved in bacterial lysis. One possible explanation for the lytic activity of ØVC8 is that this phage uses a lysis pathway that differs from the classic system.
Comparative genome analysis of ØVC8, VP2, and VP5 showed similar genome sequences and genetic organization. The presence of an adenylosuccinate synthetase and the lack of a lysis cassette are unique traits of these three phages. However, the genome of ØVC8 shows five insertion/deletions that have not been identified in the VP2 and VP5 genomes; these insertion/deletions are located mainly in the unknown function region and in the replication module (Additional file
2: Figure S1). The effect of these insertion/deletions on the phenotypes of VP2 and VP5 remain unknown, largely because of a lack of data regarding the characteristics of these phages.
Mass spectrometric analysis of the structural proteins of phage ØVC8 showed that these proteins are distributed among the packing and structural modules (ORFs 17, 14, 8, and 4), indicating that ØVC8 requires these four structural proteins for prophage assembly and potentially for initial host recognition. Additionally, ORF8, which encodes a protein of 36.1 kDa, was identified as one of the most abundant structural proteins, suggesting that this is a protein with a high copy number that is presumably the major capsid protein.
Authors’ contribution
ASS and CEC conceived the study and designed the experiments. ASS performed the experiments and analyzed the data. UHU performed the data analysis. ANO performed the bacterial identification by biochemical and serological test. FMJ performed transmission electron microscopy experiments. ASS, CEC, and JXC, wrote the manuscript. CEC and JXC revised the manuscript. All authors read and approved the final manuscript.