Characterization of a pore-forming cytotoxin expressed by Salmonella enterica serovars typhi and paratyphi A

Abstract

Cytolysin A (ClyA) is a pore-forming cytotoxic protein encoded by the clyA gene that has been characterized so far only in Escherichia coli. Using DNA sequence analysis and PCR, we established that clyA is conserved in the human-specific typhoid Salmonella enterica serovars Typhi and Paratyphi A and that the entire clyA gene locus is absent in many other S. enterica serovars, including Typhimurium. The gene products, designated ClyA(STy) and ClyA(SPa), show >/=90% amino acid identity to E. coli cytolysin A, ClyA(EC), and they are immunogenically related. The Salmonella proteins showed a pore-forming activity and are hence functional homologues to ClyA(EC). The chromosomal clyA(STy) gene locus was expressed at detectable levels in the serovar Typhi strains S2369/96 and S1112/97. Furthermore, in the serovar Typhi vaccine strain Ty21a, expression of clyA(STy) reached phenotypic levels, as detected on blood agar plates. The hemolytic phenotype was abolished by the introduction of an in-frame deletion in the clyA(STy) chromosomal locus of Ty21a. Transcomplementation of the mutant with a cloned clyA(STy) gene restored the hemolytic phenotype. To our knowledge, Ty21a is the first reported phenotypically hemolytic Salmonella strain in which the genetic determinant has been identified.

FIG. 1.

PCR analysis of the osmC clyA DNA regions of various Salmonella isolates. PCR products were resolved by agarose gel electrophoresis following amplification using the primers stm1 and stm3, which amplify a 433- or a 420-bp fragment in strains lacking both clyA and osmC and a 2,650-bp fragment in clyA⁺ osmC⁺ strains (arrowheads on right and left, respectively).

FIG. 2.

(A) Schematic map of the gene arrangement in a 4.5-kb region containing the clyA locus in serovar Typhi S2369/96, serovar Typhimurium LT2, serovar Typhi JON42, and E. coli K-12. The regions were aligned using the clyA_STy gene sequence as a reference. Base pair coordinates are indicated relative to the arbitrarily chosen end point. Open reading frames are shown as shaded arrows, and the 48-bp unique sequence in serovar Typhimurium is shown as an open box. The 12 bp of the ΔclyA_STy allele in JON42 are shown with start and stop codons underlined. The approximate positions of recognition sequences for the restriction endonuclease HincII (H) and of sequences corresponding to the primers stm1, stm3, sal1, sal2, and sal6 (small arrows) are indicated. The extents of the clyA_STy DNA regions cloned in the constructs pMWK21 and pJON97 are shown as solid bars. ORF, open reading frame. (B) Alignment of nucleotide sequences flanking the osmC clyA DNA region of serovar Typhi S2369/96, serovar Paratyphi A ATCC9510, and serovar Typhimurium LT2. Start and stop codons are shown in boldface and are underlined. The 13 bp of the 48-bp “inserted fragment” that were absent in the serovar Havana strains SR15 and St 63/1992 (see Results) are shown in boldface. Positions are indicated relative to the clyA start codon (+1). (C) Alignment of clyA upstream sequences of serovar Typhi S2369/96 and the clyA promoter region of E. coli K-12. Promoter elements [(−35) and (−10)], the transcriptional start point (+1), the Shine-Dalgarno sequence (SD), the translational start codon (ATG), and binding sites for the CRP and FNR regulatory proteins (CRP/FNR), described for the clyA_EC locus (13, 25, 50), are indicated.

FIG. 3.

Expression of cloned serovar Typhi, serovar Paratyphi A, and E. coli ClyA determinants and subcellular localization of ClyA_STy in serovar Typhi. Immunoreactive bands were visualized using the ECL⁺ blotting detection system (see Materials and Methods). (A) Immunoblot analysis, using a monoclonal ClyA_EC antibody (final dilution, 1:5,000), of expression of cloned clyA_STy (constructs pJON97 and pMWK21), clyA_SPa (construct pJON80), and clyA_EC (construct pYMZ80) gene loci in E. coli YMZ19 (clyA::kan) and MC1061 (clyA⁺) and serovar Typhi Ty21a. Approximately 5 × 10⁷ cells of each strain were used for the extracts applied on the gel. (B) Subcellular fractionation of serovar Typhi Ty21a/pJON97 (lanes 2 to 5) and Ty21a/pGEM-T Easy (lanes 6 to 9) in membrane (m), cytosolic (c), periplasmic (p), and supernatant (s) fractions. Above, immunoblot analysis using a monoclonal antibody raised against ClyA_EC (final dilution 1:5,000) is shown. The position of the band corresponding to the 30-kDa protein of the molecular-mass marker (mw; lane 1) is indicated by an asterisk, and the position of the immunoreactive band representing the ClyA protein is indicated by an arrowhead. Below, immunoblot analysis of the same subcellular fractions (corresponding to lanes 2 to 9 in the upper blot) using a polyclonal antiserum against β-lactamase is shown. The position of the immunoreactive band representing β-lactamase is indicated by an arrowhead. An amount of the subcellular fraction equal to approximately 3 μg of protein was applied in each lane of the gel.

FIG. 4.

Pore-forming activity of ClyA_STy evidenced by osmotic protection using different sugar solutions in combination with suspensions of the serovar Typhimurium LT2 strains GT3258/pJON97 (clyA_STy⁺) (shaded bars) and GT3258/pGEM-T Easy (vector control) (solid bars). Cytolytic activity was determined as described in Materials and Methods. The bacteria were grown on LB agar plates at 37°C for 16 to 17 h before being harvested. Approximately 10⁸ bacterial cells were used for the samples applied in each well of the microtiter plate (final density, 10⁹ bacterial cells ml⁻¹). The activity of GT3258/pJON97 in 1× PBS was arbitrarily set to 1.0. Osmotic protectants (lane 1, 1× PBS; lane 2, maltose; lane 3, raffinose; lane 4, PEG1000; lane 5, PEG1500; and lane 6, dextran 4) were added at a final concentration of 30 mM.

FIG. 5.

Phenotypic expression of ClyA cytotoxin by serovar Typhi Ty21a and loss of lytic phenotype upon inactivation of the clyA_STy gene locus. (A) Western immunoblotting detection of ClyA_STy protein content in serovar Typhi strains using a polyclonal ClyA_EC antiserum (I) or a monoclonal antibody raised against ClyA_EC (final dilution, 1:1,000) (II) and the ECL⁺ Western blotting detection system for visualization of immunoreactive bands (see Materials and Methods). The following approximate numbers of bacterial cells were used for the extracts loaded in the lanes. For the Salmonella strains, 3 × 10⁸ cells were used for the polyclonal antiserum and 1 × 10⁸ cells were used for the monoclonal antibody. For YMZ19/pJON97 (clyA::kan clyA_STy⁺) and YMZ19/pGEM-T Easy (clyA::kan; vector), approximately 8 × 10⁶ bacterial cells were used. The positions of reactive bands corresponding to ClyA are indicated with arrowheads on the left. The phenotypes of the strains on blood agar plates containing horse erythrocytes are indicated at the bottom (III) and were scored after 16 h of growth at 37°C (+, small lysis zones around individual colonies; −, no lysis). (B) Effect of inactivation of the clyA_STy locus in serovar Typhi Ty21a. Shown are the phenotypes of Ty21a and the clyA_STy in-frame deletion mutant JON42 on a blood agar plate containing horse erythrocytes after incubation at 37°C for 17 h.

FIG. 6.

H-NS-dependent repression of clyA_STy in the E. coli K-12 strains YMZ19 (clyA hns⁺) (shaded bars) and YMZ16 (clyA hns) (solid bars) carrying different constructs. The bacteria were grown on LB agar plates at 37°C for 16 to 17 h before being harvested. Shown is the relative cytolytic activity as determined by quantification of the release of hemoglobin from erythrocytes (see Materials and Methods). Approximately 4.0 × 10⁷ bacterial cells were used for the samples applied in each well of the microtiter plate, giving a final density of 4 × 10⁸ cells per ml. The activity of YMZ19/pJON97 was arbitrarily set to 1.0. The error bars indicate standard errors of the mean from two separate experiments.