Figure 1.
Two-dimensional SDS-PAGE of isolated whole bacterial lysate derived from C. jejuni strain 108.
Proteins were visualized with silver. The encircled protein spot was picked and identified by mass spectrometry as a putative C. jejuni Hcp protein.
Figure 2.
Gene organization of the T6SS clusters in C. jejuni strain 108 and related species.
(A) The C. jejuni genes are designated according to the proposed nomenclature [5], [29] as ttsA-M; alternative gene names in V. cholerae are given in brackets for comparison. Functionally related group of genes are indicated in the same color. The lines and numbers below the colored genes indicate the PCR products and primers used for cloning and sequencing by primer walking. The same PCR primer sets were used to determine the gene organization in other Hcp-positive C. jejuni strains. (B) Comparison of the T6SS gene cluster organizations in C. jejuni strain 108, C. coli strain RM2228, H. hepaticus strain ATCC51449 and V. cholerae strain V52. Functionally related group of genes are indicated in the same color. (C) Diagram indicating the site of insertion of the C. jejuni 108 T6SS gene cluster relative to the CJIE3 element of C. jejuni RM1221.
Table 1.
Characteristics of C. jejuni T6SS components and their most related orthologs in several other species.
Figure 3.
PCR detection of CJIE3 and Hcp in different C. jejuni and C. coli strains.
Only CJIE3-positive strains are shown. C. jejuni strains used are 1 - 108, 2 - 202606, 3 - 209071, 4 - 205223, 5 - NCTC12502 (P3), 6 - RM1221, 7 - C09165, 8 - C019168, 9 - C626, 10 - C631, 11 -C10, 12 - 117, 13 - C356. C. coli strains used are: 14 - Han35, 15 - Han 153. Hcp-positive strains are marked with (+). The size of the PCR products is indicated in base pairs (bp). CJIE3 and hcp were not detected by PCR in the following C. jejuni strains: NCTC11168, NCTC81116, ATCC 33291, ATCC 49301, BAA527, BAA529, C013199, C011338, C017289, C011672, C011300, C013500, C012599, C012446, 5003, D3468, D3141, CCUG10950, D3226, 233.95, 308.95, 21.97, 386.96, 260.94, 41239B, 07479, 127955, 850312, 40707L, GB1, GB5, GB11, GB18, GB23, GB26, GB27, E98623, 480, 209071, 201191, 205224, 207251, 207252, 206470, 206710, 105713, 146719, 209755, 100756, 132960, 210388, 11271, 11279, 81176, A3004, C9, C12, C608, C618, C621, C627; and the C. coli strains: UA417, Han36, 2371, K1102/03, H1.
Figure 4.
Western blot demonstrating the presence of Hcp protein.
(A) Cellular (Cj) and culture supernatant (sup) fractions of C. jejuni strain 108 and its Hcp-, TssM- and RpoN-negative derivatives were separated by SDS-PAGE, blotted, and incubated with Hcp-specific antisera. Molecular mass markers are indicated in kilodalton (kDa). (B) Western blot of culture supernatants of the CJIE3-positive C. jejuni strains tested in Fig. 3, demonstrating secretion of Hcp by all T6SS-positive strains. Hcp-secreting strains are marked with (+). The apparent mass of the Hcp protein is indicated in kilodalton (kDa). The strains loaded in lanes 1 to 15 are listed in the legend of Fig. 3.
Figure 5.
Hemolysis assay depicting the T6SS-mediated hemolytic activity of C.
jejuni. (A) C. jejuni strain 108, its hcp-negative derivative 108ΔHcp, the complemented mutant strain 108ΔHcp/phcp, and the parent strain carrying phcp grown for 7-days on saponin plates and then for 16 hours in HI broth (7 p-16 h) were incubated (6 h) with red blood cells before hemolysis was determined. (B) Western blot confirming the successful restoration of Hcp secretion in 108ΔHcp after introduction of the complementation plasmid phcp. (C) Effect of the age of C. jejuni cultures in T6SS-induced hemolysis. Strain 108 and 108ΔHcp grown either on saponin plates for 3 days and then in HI broth for 8 h (3 p/8 h), or on saponin plates for 7 days and then in HI broth for 16 h (7 p/16 h) were incubated with red blood cells for 6 h. Then hemolysis was determined by measuring absorbance at 420 nm. Values are the mean ± SEM of at least three experiments.
Figure 6.
Effect of capsule expression on T6SS-mediated hemolysis.
(A) SDS-PAGE of C. jejuni strain 108 and its capsule-negative mutant 108ΔCPS stained for the presence of capsule polysaccharide with Alcian blue. Capsule expression was compared for equal amounts of C. jejuni grown on saponin plates for three (3 p) or seven (7 p) days and then in HI broth for the indicated times (8 h or 16 h). Molecular mass markers are indicated in kilodalton (kDa). (B) T6SS-mediated hemolysis (6 h) for C. jejuni strain 108 and 108ΔHcp in a capsule-negative background (ΔCPS). Hemolysis was determined by measuring absorbance at 420 nm. Values are the mean ± SEM of at least three experiments. (C) Hemolysis assay depicting the hemolytic activity (6 h) of C. jejuni strain 108 (WT), the capsule mutant 108ΔCPS, the double mutant 108ΔCPS/ΔHcp, and the Hcp secretion-defective strain 108ΔTssm, and of the T6SS-negative strain 81116 (WT) and its capsule mutant 81116ΔCPS. Strains used were grown for 3 days on saponin plates and then for 16 h in HI broth (3 p/16 h). The hemolysis results depicted in the left panel were quantified by measuring absorbance at 420 nm (right panel). (D) Effect of T6SS on J774A.1 macrophages. Macrophages were incubated with C. jejuni strain 108, 108ΔHcp, 108ΔCPS, 108ΔCPS/ΔHcp or S. Typhimurium SL1344. After 10 h of incubation, total cellular and released lactate dehydrogenase (LDH) were measured as indicator of cytotoxicity. Values are the mean ± SEM of at three independent experiments performed in triplicates. (E) Real-time RT-PCR results showing hcp transcript levels for C. jejuni strain 108 and 108ΔCPS at different age (3 p/8 h and 3 p/16 h). Note the strong relative increase in hcp transcript level in late exponential growth phase. Data are representative of three independent experiments with two independent preparations of RNA.