An antimicrobial protein of the gut symbiont Bacteroides fragilis with a MACPF domain of host immune proteins

Abstract

Bacteroidales are the most abundant Gram-negative bacteria of the human intestinal microbiota comprising more than half of the bacteria in many individuals. Some of the factors that these bacteria use to establish and maintain themselves in this ecosystem are beginning to be identified. However, ecological competition, especially interference competition where one organism directly harms another, is largely unexplored. To begin to understand the relevance of this ecological principle as it applies to these abundant gut bacteria and factors that may promote such competition, we screened Bacteroides fragilis for the production of antimicrobial molecules. We found that the production of extracellularly secreted antimicrobial molecules is widespread in this species. The first identified molecule, described in this manuscript, contains a membrane attack complex/perforin (MACPF) domain present in host immune molecules that kill bacteria and virally infected cells by pore formation, and mutations affecting key residues of this domain abrogated its activity. This antimicrobial molecule, termed BSAP-1, is secreted from the cell in outer membrane vesicles and no additional proteins are required for its secretion, processing or immunity of the producing cell. This study provides the first insight into secreted molecules that promote competitive interference among Bacteroidales strains of the human gut.

Figure 1

Agar overlay spot assay showing growth inhibition of B. fragilis strains by wild type 638R and mutants. A. Each strain from Table 1 that was growth inhibited by 638R was tested for growth inhibition by transposon mutant M26 and deletion mutant ΔBF1646. Overlaid B. fragilis strains tested for growth inhibition listed vertically. B. BF638R-1646 provided in trans by plasmid pMCL139 restores inhibitory activity to ΔBF1646.

Figure 2

Region of the B. fragilis chromosome surrounding the transposon insertion of mutant M26. The genes in this region were analyzed in all 70 B. fragilis strains with genome sequences; 14 B. fragilis genomes were not included due to this region being incomplete or detectable on multiple contigs. These regions segregated into two different types, those similar to 638R (21 strains) and those similar to 9343 (34 strains), and one outlier. Genes encoding similar proteins are shown in the same color, with the shade distinguishing the genes encoding very similar 638R-like proteins from the 9343-like genes also encoding very similar proteins. The gene immediately upstream of the gene into which the transposon inserted (blue) encodes a putative outer membrane transporter that is 92.3–100% identical within the 638R-like or 9343-like genomes and 57.5–60.4% identical between these two groups. The gene upstream of that encodes a protein of unknown function (green) in which the 638R-like genes are 93.5 – 100% identical to each other, those the 9343-like are 90.3 – 100% identical to each other, whereas the % identity between groups is 71.9 – 86.0%. Although the genetic region of strain 2_1_16 is 9343-like, there is an additional gene, HMPREF0101_00590, encoding a protein of unknown function inserted between the blue and red genes. The gene shown in red encodes a putative nucleotide deaminase which is 94.6 – 100% among all B. fragilis strains.

Figure 3

Growth inhibitory activity of BSAP-1 does not require additional factors. A. Agar overlay spot assay showing that addition of BF638R_BF1646 (pMCL139), but not vector alone (pMCL140), to strain CM13 confers the growth inhibitory activity to this strain. B. Coomassie-stained gel of See-Blue Pus2 markers (Life Technologies, lane 1) or purified His-BSAP (lane 2). C. Agar overlay spot assay with two different purifications of His-BSAP-1 showing the potent ability of this protein to inhibit the growth of sensitive strains. Total amount of protein added to the plate in 5 μl volumes is shown in figure.

Figure 4

BSAP-1 inhibition of 12905 growth A. Addition of strain 12905 or 638R in 10-fold dilutions to plates containing His-BSAP-1 or PBS. Total cfu of 12905 added to plates is shown on the right and those recovered after overnight incubation is shown in Table 2. B. Broth co-culture experiments of strain 12905 with either wild type 638R or ΔBF1646. The cfus of sensitive strain 12905 are reported.

Figure 5

MACPF domain is necessary for BSAP-1 activity. A. Alignment of the MACPF domains of BSAP-1 and BT_3439 of B. thetaiotaomicron VPI-5482. A conserved MACPF motif is boxed with residues altered by site-directed mutagenesis highlighted in yellow. B. Western blot of supernatants of B. fragilis CM13 containing the gene for WT BSAP-1, or BSAP-1 site-directed mutant genes, probed with antiserum to BSAP-1. C. Coomassie-stained gel (left panel) or western immunoblot (right panel) showing the purification of His-tagged BSAP-1 or BSAP-1 site-directed mutants from BL21/DE3 induced at room temperature. The coomassie-stained gel shows the unconcentrated mutant proteins following purification whereas the western blot shows reactivity when the mutant protein samples were each concentrated 10-fold. The blot was probed with antiserum to BSAP-1. D. Analysis of the killing of strain 12905 in an agar overlay by purified BSAP-1 or concentrated BSAP-1 site-directed mutants E. Microscopic analysis of PI incorporation by strain 12905 exposed to BSAP-1 contained in culture supernatant or control supernatant. Fluorescence images are shown at the starting point and 30 minutes later.

Figure 6

BSAP-1 is secreted extracellularly in outer membrane vesicles. A. Western immunoblot of 638R pMCL139 whole cell lysate, supernatant, or OMV fraction probed with polyclonal antiserum to His-BSAP-1. B. Immunofluorescence analysis of OMV fraction of 638R pMCL139 (left) or ΔBF1646 (right) using antiserum to His-BSAP-1. C. Agar spot overlay assay showing growth inhibition of strain 12905 by purified OMVs from 638R pMCL139, but not ΔBF1646. OMVs were harvested at mid-log (OD₆₀₀ = 0.6) and 50 μl volumes containing 50 μg (ΔBF1646) or 52 μg (638R pMCL139) of total protein were added in 50 μl volumes to plates prior to overlay.