Nonhemolysis of epidemic El Tor biotype strains of Vibrio cholerae is related to multiple functional deficiencies of hemolysin A

C6706 is a strain of V. cholerae El Tor biotype in the seventh pandemic with a typical hemolytic positive phenomenon and intact hlyA gene. We constructed a hlyA deletion strain from C6706, named CΔhlyA, and a hlyA complemented strain, CΔhlyA-C, by transformation of the recombinant plasmid pBAD33-hlyA carrying intact hlyA into CΔhlyA. The hemolysis tests showed that CΔhlyA lost the hemolysis phenomenon, but CΔhlyA-C regained hemolytic capacity (Fig. 1), showing that HlyA contributed mainly to hemolysis of the epidemic El Tor strain.

Furthermore, we randomly selected 85 toxigenic strains of O1 El Tor isolated from 1961 to 2007, and we performed hemolysis tests with these strains to confirm the hemolytic phenotype using strain C6706 as the positive control and CΔhlyA as the negative control. Among them, 71 hemolytic and 14 nonhemolytic strains were identified. The hlyA genes of these strains were then amplified and sequenced. Using the hlyA sequence of strain C6706 as the reference, a total of five hlyA sequence types were found and designed as ST.1 to ST.5, respectively (Table 1 and Fig. 2). The sequences that were identical to C6706 hlyA were designated ST.1 in this study. ST. 2 had a base variance at 1358 within the hlyA ORF and resulted in 453S instead of 453F in ST.1. The sequence type ST.3 had the same base variance as ST.1, with an additional base variance at 1797 that was a synonymous mutation. ST.4 had a 4-base deletion mutation at positions 1088–1091, which caused the termination code to appear in the middle of the original ORF and the generation of a truncated and mutant protein during translation. ST.5 had the same 4-base deletion and the same mutation in ST.2 at position 1358. The sequence mutations and number of strains with hemolytic and non-hemolytic phenomena in each hlyA sequence type are listed in Table 1.

It was noticed that all ST.2 strains were hemolytic, although an amino acid variation occurred when compared with the reference sequence ST.1, showing that the F453S variation did not affect the hemolytic activity of HlyA. In fact, in another study [19], ST.2 type hlyA was considered a wild sequence type, and this type of strain was also hemolytic. In the hlyA sequence type ST.3, in addition to F453S variation, another point mutation at 1979 (C → T) occurred but was a synonymous mutation. This strain was also hemolytic. Both strains (VC1279 and VC2568) in ST.4 and ST.5 were hemolysis negative, presenting a 4-base deletion of nucleotide 1088 to 1091 in the hlyA ORF. In addition, a point mutation was detected at nucleotide position 1358, causing the F453S mutation in strain VC2568, such as ST.2, but it did not affect the hemolytic activity of HlyA. Using the genome DNAs of strains VC1279 and VC2568 as templates, we cloned their hlyA genes into the expression plasmid pBAD33, and we generated the recombinant plasmids pBAD33-ST.4 and pBAD33-ST.5 and transferred them into strain CΔhlyA, respectively. Hemolysis tests showed that both complemental strains were still hemolytic-negative (Fig. 3). The recombinant plasmid pBAD33-hlyA was then transferred into strain VC1279 and VC2568, restoring the hemolytic phenotype (Fig. 3), which showed that the negative hemolytic phenotype resulted from the base deletion mutation in their hlyA genes.

Among the 37 strains with the ST.1 sequence type of hlyA, the hemolysis tests showed that 25 strains were hemolytic, but 12 were non-hemolytic. We then focused on these hemolytic-negative strains of ST.1 (ST.1^H− strains). HlyA is secreted extracellularly through T1SS in V. cholerae. The secretion of HlyA was detected by measuring the sheep blood cell hemolysis in the supernatant and cytoplasm of bacterial cells. Liquid cultures of all 12 ST.1^H− strains with an OD₆₀₀ of 0.6 were washed and disrupted by ultrasound, and the supernatants of the solution were obtained for the hemolysis tests, respectively. The positive control strain C6706 and negative control strain CΔhlyA were treated using the same protocol. Among the 12 ST.1^H− strains, seven showed positive hemolysis (Table 2 and Fig. 4), suggesting that in these seven strains, HlyA could be expressed but not secreted normally, resulting in hemolytic activity in the cytoplasm but non-hemolytic activity when tested with the cultured bacteria.

In parallel, hemolysis of these ST.1^H− strains was also tested by increasing the expression of hlyA genes through the introduction of plasmid pBAD33-hlyA into these strains. Eight strains became hemolytic in the hemolysis tests using the cultured bacterial cells (Table 2 and Fig. 5). Among them, three strains (VC1627, VC1554 and VC9) were previously non-hemolytic, but their cytoplasm and their hlyA overexpression transformants were hemolytic (Table 2 and Fig. 5a), suggesting that, for these wildtype strains, HlyA expression was too low, with normal or less efficient HlyA secretion. Five strains (VC1301, VC4979, VC4826, VC4983 and VC4732) for which the cytoplasm was still non-hemolytic became hemolytic when transformed with the hlyA overexpression plasmids (Table 2 and Fig. 5b), which might suggest that their hlyA genes were expressed at low levels in the cytoplasma, but the secretion of HlyA was normal.

Four ST.1^H− strains (VC3, VC136, VC2177 and VC4835) remained non-hemolytic when they were transferred with plasmid pBAD33-hlyA and tested with the cultured cells (Table 2 and Fig. 6). These strains were ultrasonically disrupted, and the hemolysis of their cytoplasm was tested. All of them were hemolytic, strongly suggesting an abnormal HlyA secretion; however, their hlyA transcription and expression might have been normal, since without the introduction of pBAD33-hlyA, the cytoplasm still possessed hemolytic activities.

We further determined the transcription levels of the hlyA gene in these four ST.1^H− wildtype strains (VC3, VC136, VC2177 and VC4835), using the recA gene of C6706 as the internal reference. The transcription levels of hlyA in this group were relatively high (Table 2). The hlyA transcription levels of the five ST.1^H− wildtype strains (VC1301, VC4979, VC4826, VC4983 and VC4732) were also determined, and they demonstrated much lower hlyA transcription levels than the previous group (Table 2). These data support that, in these five strains, expression of HlyA was reduced, but there was no deficiency in HlyA secretion. In contrast, four strains (VC3, VC136, VC2177 and VC4835) had normal expression but blocked secretion of HlyA.

The wildtype (WT) serogroup O1, El Tor biotype V. cholerae C6706 and its derivative mutants were grown in Luria–Bertani (LB) broth (pH 7.4) containing 1% NaCl (170 mM) at 37 °C unless specifically indicated. E. coli DH5αλpir and SM10λpir were cultured at 37 °C and used for cloning purposes. The culture media were supplemented with ampicillin (Amp, 100 mg/ml), streptomycin (Sm, 100 mg/ml) or chloramphenicol (Cm, 10 mg/ml) as required. All strains and plasmids used in this study are listed in Table 3.

In this study, 85 strains of O1 El Tor toxigenic strains isolated from different years (1961–2007) in China were randomly selected from the National Vibrio Collection Laboratory in National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention (China CDC), which is operated in our laboratory. Biotype identification of the serogroup O1 strains is performed in the local public health laboratories of the CDCs in different provinces before their submissions to the Collection Laboratory, and the strains were selected randomly for the biotype confirmation in this laboratory. The following criteria for biotype identification are conducted in all the public health laboratories in CDCs: the lysis by bacteriophage of classical IV, polymyxin B sensitivity and agglutination of chicken erythrocytes. The year of isolation and reconfirmed hemolytic phenotype of the strain in this study are shown in Additional file 1: Table S1.

The V. cholerae deletion mutant was constructed with the reference strain C6706 by allelic gene exchange. Primers were designed based on the genome sequence of V. cholerae strain C6706. All primers used in this study were designed with SnapGene (GSL Biotech) and then blasted in primer-BLAST of PubMed (https://​www.​ncbi.​nlm.​nih.​gov/​tools/​primer-blast/​). To construct the hlyA gene deletion mutant of CΔhlyA from strain C6706, the upstream and downstream DNA fragments flanking the hlyA open reading frame (ORF) were amplified from C6706 genomic DNA using primer pairs hlyA-XhoI-F/middle-R and middle-F/hlyA-SpeI-R, respectively. The amplicons were mixed in equimolar concentrations and used as template to amplify the chromosomal fragment containing the hlyA deletion using the primer pair hlyA-XhoI-F/hlyA-SpeI-R. The resulting fragment was cloned into the suicide plasmid pWM91 to generate pWM91-ΔhlyA. pWM91-hlyA was constructed in E. coli DH5αλpir. pWM91-hlyA was extracted from DH5αλpir, transformed into strain SM10λpir, and mixed with strain C6706 for conjugation of pWM91-hlyA. Exconjugants were selected in LB medium containing Amp and Sm, and they were streaked on LB agar containing 15% (w/v) sucrose. Sucrose-resistant colonies were selected and tested for Amp sensitivity, and the hlyA deletion mutant of V. cholerae was confirmed by DNA sequencing.

To complement the tested hlyA genes in the hlyA mutants of CΔhlyA, plasmid pBAD33 was used to construct complementary plasmids carrying the inserted wildtype hlyA or its derivatives. The fragment containing hlyA was PCR-amplified from chromosomal DNA of C6706 with primers hlyA-XbaI-F/hlyA-KpnI-R, digested with the restriction enzymes XbaI/KpnI and inserted into pBAD33. The complementary plasmids were transformed into CΔhlyA or other mutants and induced with 0.01% arabinose for pBAD33. pBAD33 was also transformed into CΔhlyA as the negative control. The complemented strains were verified by hemolysis tests. All primers used in this study are listed in Additional file 1: Table S2.

Vibrio cholerae strains were grown in LB medium to an OD₆₀₀ of 0.6. Total RNA was extracted from the culture of C6706 and other test strains using the SuperScript™ III Reverse Transcriptase and DNA-free™ DNA Removal Kit (Thermo Fisher). The RNA samples were analyzed by quantitative real-time reverse transcription-PCR (qRT-PCR) using the One Step SYBR Primerscript RT-PCR Kit II (TaKaRa). Relative expression values (R) were calculated using the equation \( {\text{R}} = 2^{{ - \left( {\Delta Cq target - \Delta Cq reference} \right)}} \), where Cq is the fractional threshold cycle. recA of C6706 was used as an internal reference. Run the qRT-PCR as follows: Pre-incubation (1 cycle): 95 °C for 1 min; Amplification (40 cycles): Denaturation at 95 °C for 10 s, Annealing at 60 °C for 30 s (to collect fluorescence signals); Melting (1 cycle): 95 °C for 10 s, 65 °C for 30 s, and 97 °C for 1 s; Cooling (1 cycle): 37 °C for 30 s. A control mixture using total RNA as a template was performed for each reaction to exclude chromosomal DNA contamination. The primers used for these target genes, recA and hlyA are listed in Additional file 1: Table S2.

Vibrio cholerae strains were cultured in LB to an OD₆₀₀ of 0.6, and 20 ml liquid culture was centrifuged at 5000 r/min. The supernatant was discarded, and the cells were resuspended in 20 ml LB and then centrifuged for washing two times. For ultrasonic cell disruption, the sample tube of bacteria was placed in an ice bath. The ultrasonic breaking procedure was set as ultrasound for 5 s, with 5-s intervals, for a total of 5 min. After ultrasonic disruption, the sample cytoplasm was collected by centrifugation at 12,000 r/min and 4 °C, followed by resuspension in 20 ml LB.

Sheep blood was washed twice with sterilized isotonic sodium chloride solution at a threefold volume. During the third wash, the mixture was centrifuged at 2000 r/min for 10 min, and the sheep erythrocytes were obtained by discarding the supernatant of the mixture. Then, 1% sheep erythrocyte solution was prepared with sterilized isotonic sodium chloride solution, and 1 ml bacterial culture (OD₆₀₀ of 0.6) and 1 ml of 1% sheep erythrocyte solution were mixed in a test tube, incubated at 37 °C for 2 h in bacteriology incubator, and left at 4 °C overnight. Hemolysis was estimated by comparison with the positive and negative controls. For the hemolysis assay of the bacterial cytoplasm, the culture of the strain with an OD₆₀₀ of 0.6 was washed twice with sterilized isotonic sodium chlorides solution, resuspended in the same volume as the previous medium, and disrupted using the ultrasonic technique as described above.