Genetic variation of hemolysin co-regulated protein 1 affects the immunogenicity and pathogenicity of Burkholderia pseudomallei

Abstract

Hemolysin co-regulated protein 1 (Hcp1) is a component of the cluster 1 Type VI secretion system (T6SS1) that plays a key role during the intracellular lifecycle of Burkholderia pseudomallei. Hcp1 is recognized as a promising target antigen for developing melioidosis diagnostics and vaccines. While the gene encoding Hcp1 is retained across B. pseudomallei strains, variants of hcp1 have recently been identified. This study aimed to examine the prevalence of hcp1 variants in clinical isolates of B. pseudomallei, assess the antigenicity of the Hcp1 variants, and the ability of strains expressing these variants to stimulate multinucleated giant cell (MNGC) formation in comparison to strains expressing wild-type Hcp1 (Hcp1wt). Sequence analysis of 1,283 primary clinical isolates of B. pseudomallei demonstrated the presence of 8 hcp1 alleles encoding three types of Hcp1 proteins, including Hcp1wt (98.05%), Hcp1variant A (1.87%) and Hcp1variant B (0.08%). Compared to strains expressing Hcp1wt, those expressing the dominant variant, Hcp1variant A, stimulated lower levels of Hcp1variant A-specific antibody responses in melioidosis patients. Interestingly, when Hcp1variant A was expressed in B. pseudomallei K96243, this strain retained the ability to stimulate MNGC formation in A549 cells. In contrast, however, similar experiments with the Hcp1variant B demonstrated a decreased ability of B. pseudomallei to stimulate MNGC formation. Collectively, these results show that B. pseudomallei strains expressing variants of Hcp1 elicit variable antibody responses in melioidosis patients and differ in their ability to promote MNGC formation in cell culture.

Fig 1

(A) Amino acid alignment and (B) phylogenetic tree based on amino acid sequences of 9 Hcp1 types of B. pseudomallei recorded in the RefSeq: NCBI Reference Sequence Database on March 4^th, 2024, generated using the Maximum Likelihood method and JTT matrix-based model with 1,000 bootstraps. Bootstrap values are shown in red text. The branch lengths are presented under each branch.

Fig 2. Antigenic variation of recombinant Hcp1 proteins.

(A) Reactivity of mouse anti-polyhistidine (mAb-6×His), mouse monoclonal antibody against rHcp1^wt (mAb H1-3), mouse sera obtained from B. mallei rHcp1 immunization (anti-rHcp1) and mouse sera obtained from rHcp1^{variant A} immunization (anti-rHcp1^{variant A}) against rHcp1^wt (blue bar), rHcp1^{variant A} (red bar) and rHcp1^{variant B} (green bar). The experiment was performed in duplicate. The bar graph represents mean of OD values and error bar shows SD. (B) Reactivity of human sera from healthy donors, melioidosis patients infected with B. pseudomallei containing hcp1^wt, melioidosis patients infected with B. pseudomallei containing hcp1^{variant A} and melioidosis patient infected with B. pseudomallei containing hcp1^{variant B} against rHcp1^wt (blue dot), rHcp1^{variant A} (red square) and rHcp1^{variant B} (green triangle). The data points represent the mean of duplicate OD values. The black line represents the median of each group. The orange dotted line represents the cut-off value. * P ≤ 0.05, *** P ≤ 0.001. (C) IFN-γ secretion from PBMC of 10 healthy donors and 23 melioidosis patients infected with B. pseudomallei containing hcp1^wt after stimulated with PHA (purple diamond), rHcp1^wt (blue dot), rHcp1^{variant A} (red square) and rHcp1^{variant B} (green triangle). The data points present the mean of duplicate wells. The error bars represented 95% CI. (D) Heat map of IFN-γ secretion from PBMC of 10 healthy donors and 23 melioidosis patients infected with B. pseudomallei containing hcp1^wt after stimulated with PHA, rHcp1^wt, rHcp1^{variant A} and rHcp1^{variant B}. The data points present the mean of duplicate wells.

Fig 3. Multinucleated giant cell formation efficiency of clinical B. pseudomallei isolates with variant hcp1 genes in A549 cells.

(A) Multinucleated giant cell formation in A549 cells infected with strains K96243 (Hcp1^wt), DR1235 (Hcp1^{variant A}) and DR0089 (Hcp1^{variant B}) at MOI 50 for 10 h. The bar scale represents 20 μM length. MNGC formation efficiency was calculated by determining the percentage of a MNGC formation (B) and the average number of nuclei in each MNGC (C). The bar graphs show the mean of three independent experiments. The error bars represent the standard deviation (SD).

Fig 4. Multinucleated giant cells (MNGC) formation efficiency of genetically manipulated B. pseudomallei K96243 with variant hcp1 genes in A549 cells.

(A) MNGC of genetically manipulated B. pseudomallei isolates (strain K96243, K96243Δhcp1, K96243Δhcp1::hcp1^{allele 6} and K96243Δhcp1:: hcp1^{allele 7}) after infection at MOI 50 for 10 h. The bar scale represents 20 μM length. MNGC formation efficiency is presented by the percentage of MNGC formation (B) and average nuclei in MNGC (C). The bar graphs show the mean of three independent experiments. The error bars represent the standard deviation (SD).

Fig 5. Structural analysis of B. pseudomallei Hcp1^{variant B}.

The superposition of the T6SS tubes, comprising Hcp1 and sheath proteins, of B. pseudomallei and V. cholerae (5ojq) showed an overall structural similarity and identified intermolecular interfaces that may be impacted by mutations associated with Hcp1^{variant B}. X-ray structure of Hcp1^{variant B} (orange, yellow and green) was superposed onto Hcp1^wt (3wx6; light blue and grey). AlphaFold2 prediction of B. pseudomallei sheath protein (magenta) was superposed onto sheath (cyan) proteins in the cryo-EM model of T6SS tubes from V. cholerae (5ojq). (A) Two superposed subunits of B. pseudomallei Hcp1^{variant B} (orange and green) highlight a head-to-tail stacking of the Hcp1 hexamers in the T6SS tubes and a twist of the adjacent hexamers. (B) The superposed hexamer of B. pseudomallei Hcp1^{variant B} (orange and yellow) is formed by symmetry related subunits from the crystal structure and superposition highlights its strong structural conservation. (C) and (D) are zoom-ins of (A) and (B), respectively. (C) Four mutations and an insertion between the residues 90 and 97 are likely to have a strong effect on the conformation of the loops involved in the interactions between hexamers, while (D) the mutation Q14T affects the interaction of the Hcp1 hexamer with the sheath. Mutated residues in (C) and (D) are shown in sticks and the adjacent V. cholerae Hcp1 subunits in (B) are shown in light blue. (E) The amino acid sequence alignment of B. pseudomallei Hcp1^wt (3wx6) and B. pseudomallei Hcp1^{variant B} (8z7k) from, V. cholerae Hcp1 (5ojq) and P. aeruginosa Hcp1 (1y12). Highlighted regions indicate unmodelled amino acids in Hcp1^{variant B} crystal structure with mutations shown in red and conserved amino acids shown in pink.