The InhA metalloproteases of Bacillus cereus contribute concomitantly to virulence

Abstract

The virulence of Bacillus cereus requires that bacteria have the capacity to colonize their host, degrade specific tissues, and circumvent the host immune system. To study this aspect of pathogenesis, we focused on three metalloproteases, InhA1, InhA2, and InhA3, which share more than 66% identity. The expression of these metalloprotease genes was assessed by transcriptional fusions with a lacZ reporter gene. The expression profiles suggest a complementary time course of InhA production. Indeed, the genes are simultaneously expressed but are oppositely controlled during stationary phase. We constructed single and multiple inhA mutants and assessed the bacterial locations of the proteins as well as their individual or additive roles in macrophage escape and toxicity, antibacterial-peptide cleavage, and virulence. InhA1, a major component of the spore exosporium, is the only InhA metalloprotease involved in bacterial escape from macrophages. A mutant lacking inhA1, inhA2, and inhA3 shows a strong decrease in the level of virulence for insects. Taken together, these results show that the InhA metalloproteases of B. cereus are important virulence factors that may allow the bacteria to counteract the host immune system.

FIG. 1.

Alignment of the B. thuringiensis 407 InhA1, InhA2, and InhA3 amino acid sequences. Numbers indicate positions in the amino acid sequence. Identical residues are shaded. The conserved zinc-binding domain (HEXXH) is boxed in gray. The putative cleavage site (AYAET) is boxed, and the lipobox-like (IIGC) signal in InhA2 is at positions 18 to 21.

FIG. 2.

Phylogenetic tree based on InhA protein sequence analysis. Protein sequences were from international databases and are designated as the strain name followed by the accession number for the NCBI or GenBank reference sequence. The evolutionary history was inferred using an improved neighbor-joining method. Numbers on branches are bootstrap values above 80%. The bar (0.05 unit) indicates the scale of evolutionary distance; the unit is the number of amino acid substitutions per site. Bt, B. thuringiensis; Bc, B. cereus; Ba, B. anthracis; Bw, B. weihenstephanensis.

FIG. 3.

Expression of the inhA1, inhA2, and inhA3 genes under various growth conditions. The specific β-galactosidase activities of strains 407 (A, B, and C), 407 ΔplcR (C), and 407 Δspo0A (C) harboring the transcriptional inhA1′-lacZ (pinhA1) (B), inhA2′-lacZ (pinhA2) (A), or inhA3′-lacZ (pinhA3) (A and C) fusion were measured when bacteria were grown in LB medium at 37°C (A and B) or in HCT medium at 30°C (C).

FIG. 4.

Bacterial localization of the InhA2 and InhA3 proteins. Strains 407 ΔinhA1 ΔinhA3 and 407 ΔinhA1 ΔinhA2 were grown on LB medium and were harvested at t2 and t4. Supernatant (Sn) and membrane (Mb) fractions were blotted with a rabbit polyclonal antibody recognizing the different InhA proteins, and the specific locations of InhA2 and InhA3 were determined.

FIG. 5.

inhA mutant phenotypes. Strains 407, 407 ΔinhA1, 407 ΔinhA2, 407 ΔinhA3, 407 ΔinhA1 ΔinhA2, 407 ΔinhA1 ΔinhA3, 407 ΔinhA1 ΔinhA2 ΔinhA3, 407 ΔinhA1 ΔinhA2 ΔinhA3/inhA3, 407 ΔplcR, and 407 Δplcr ΔinhA1 were tested for their capacities to cleave the antibacterial peptide cecropin (A), to induce macrophage membrane permeability at various dilutions (B), and to kill Galleria mellonella (C) or Bombyx mori (D) larvae. In panel A, the following strains were tested: 2, strain 407; 3, strain 407 ΔinhA1; 4, strain 407 ΔinhA2; 5, strain 407 ΔinhA3; 6, strain 407 ΔinhA1 ΔinhA2; 7, strain 407 ΔinhA1 ΔinhA3; 8, strain 407 ΔinhA1 ΔinhA2 ΔinhA3; 9, strain 407 ΔplcR; 10, strain 407 ΔplcR ΔinhA1. Disk 1 shows the result for cecropin alone. Additionally, purified InhA2 and InhA3 proteins were tested for their capacities to cleave cecropin (disks 11 and 12, respectively) and casein (disks 13 and 14, respectively).

FIG. 6.

Expression of the inhA1, inhA2, and inhA3 genes in vivo. The specific β-galactosidase activity of strain 407 harboring the transcriptional inhA1′-lacZ (pinhA1), inhA2′-lacZ (pinhA2), or inhA3′-lacZ (pinhA3) fusion was measured after injection into Galleria mellonella larvae.