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. 2013 May;88(3):510-22.
doi: 10.1111/mmi.12202. Epub 2013 Mar 26.

Proteolysis of BB0323 results in two polypeptides that impact physiologic and infectious phenotypes in Borrelia burgdorferi

Toru Kariu  1 , Xiuli Yang, Carolyn B Marks, Xinyue Zhang, Utpal Pal
Affiliations

Proteolysis of BB0323 results in two polypeptides that impact physiologic and infectious phenotypes in Borrelia burgdorferi

Toru Kariu et al. Mol Microbiol. 2013 May.

Abstract

Borrelia burgdorferi gene product BB0323 is required for cell fission and pathogen persistence in vivo. Here, we show that BB0323, which is conserved among globally prevalent infectious strains, supports normal spirochaete growth and morphology even at early phases of cell division. We demonstrate that native BB0323 undergoes proteolytic processing at the C-terminus, at a site after the first 202 N-terminal amino acids. We further identified a periplasmic BB0323 binding protein in B. burgdorferi, annotated as BB0104, having serine protease activity responsible for the primary cleavage of BB0323 to produce discrete N- and C-terminal polypeptides. These two BB0323 polypeptides interact with each other, and either individually or as a complex, are associated with multiple functions in spirochaete biology and infectivity. While N-terminal BB0323 is adequate to support cell fission, the C-terminal LysM domain is dispensable for this process, despite its ability to bind B. burgdorferi peptidoglycan. However, the LysM domain or the precisely processed BB0323 product is essential for mammalian infection. As BB0323 is a membrane protein crucial for B. burgdorferi survival in vivo, exploring its function may suggest novel ways to interrupt infection while enhancing our understanding of the intricate spirochaete fission process.

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Figures

Figure 1
Figure 1. BB0323 is highly conserved and expressed in B. burgdorferi sensu lato strains including during early growth phases
(A) Production of BB0323 in major infectious strains of B. burgdorferi sensu lato. Equal amounts of lysates prepared from B. burgdorferi isolates B31, 297, or N40, B. garinii isolate PBi, and B. afzelii isolate PKo were immunoblotted with BB0323 antisera generated against the B. burgdorferi B31 recombinant protein. Arrow denotes position of BB0323. Protein molecular weight markers are shown on the left. (B) BB0323 production in early and late growth phases of spirochetes in culture. Left panel denotes bb0323 transcripts that were measured using qRT-PCR and presented as copies of bb0323 transcripts per copy of flaB transcript. The bars represent the mean values, and the error bars represent the SEM values from three independent experiments. Differences between bb0323 transcript levels in early and late growth phases are significant (*P < 0.001). Right panels show protein levels in spirochetes at the indicated growth phases assessed using Ponceau S staining or by immunoblotting with BB0323 antiserum. Arrow denotes position of BB0323. Protein molecular weight markers are shown on the left. (C) Transmission electron microscopic analysis of spirochetes collected at different phases of cell growth. BB0323-associated cell fission defects (arrows) were evident at early and late growth phases of spirochetes in vitro.
Figure 2
Figure 2. BB0104 (BbHtrA) is a putative BB0323-processing protease
(A) Interaction of BbHtrA and BB0323 in a GST pull-down assay. GST or GST-fused BB0323 were incubated with glutathione-sepharose beads, washed, and incubated with BbHtrA. Bound proteins were detected by immunoblotting using anti-BbHtrA. (B) Proteolytic activity of BbHtrA produces N- and C-terminal polypeptides of BB0323. Recombinant BbHtrA, as wild-type protein or mutated version with the critical replacement of a serine residue with an alanine (BB0104S226A), was incubated with full-length BB0323 (top panel) or a control protein, BBA52 (bottom panel), at 37 °C for 0 to 4 h. Immunodetection of BbHtrA, BBA52, and N-terminal polypeptide of BB0323 was accomplished by Western blotting using polyclonal antibodies against the respective proteins. The C-terminal polypeptide of BB0323 is difficult to detect by Western blot analysis, likely due to poor immunogenicity but can be detectable by far-Western blotting, as detailed in Fig. 3C. (C) Detection of BbHtrA and BB0323 in the periplasmic fraction of B. burgdorferi cells. Periplasmic (Peri) or spheroplasmic (Sphero) proteins are extracted from cultured spirochetes (Bb) and immunoblotted using BbHtrA and BB0323 antibodies. The higher-molecular-weight band (arrow) could represent either cross-reacting antigens absent in the purified periplasmic fraction (Peri) from lower proteome diversity or a cell cylinder-specific HtrA precursor.
Figure 3
Figure 3. Specific interaction of N- and C-terminal polypeptides of BB0323
(A) Schematic representation of various truncated recombinant BB0323 proteins representing N- and C-terminal polypeptides. (B) Direct interaction of truncated recombinant BB0323 proteins representing N- and C-terminal polypeptides. N-terminal polypeptides were produced without GST tags (N1, N2, and N3). Recombinant glutathione S-transferase (GST) or GST-fused C-terminal polypeptide (GST-C) was immobilized on glutathione beads and incubated with various N-terminal polypeptides, as described in panel A. After thorough washing, bound proteins were recovered and assessed by immunoblotting using anti-BB0323 antiserum. For size comparison, N-terminal polypeptides N1, N2, and N3 were separated in the same gel. The arrowhead denotes a potential cross-reactive antigen. (C) Recombinant N-and C-terminal polypeptides recognize native version of C- and N-terminal BB0323 polypeptides in B. burgdorferi, as revealed by far-Western blot analyses. B. burgdorferi lysates from equal numbers of WT and bb0323 mutant spirochetes were separated by SDS-PAGE, blotted onto nitrocellulose membranes, and incubated with GST, GST-fused N2 (GST-N2), or GST-fused C-terminal polypeptide (GST-C). Bound proteins were detected by immunoblotting using anti-GST antibodies. Note that the GST-fused C-terminal polypeptide that encompasses only the LysM domain (LysM) failed to exhibit detectable interaction in the assay. Arrows denote positions of native BB0323 N- and C-terminal polypeptides.
Figure 4
Figure 4. BB0323 LysM domain binds to B. burgdorferi peptidoglycan
(A) Schematic representation of full-length BB0323 (BB0323), recombinant truncated BB0323 proteins with either a deletion of the LysM domain (BB0323ΔLysM) or with only the LysM motif (LysM). The proteins were purified as GST-fusion proteins in E. coli. (B) Immobilized peptidoglycan purified from B. burgdorferi binds recombinant LysM domain protein. Recombinant GST-tagged full-length BB0323, BB0323ΔLysM, LysM, or GST proteins were incubated in microtiter wells coated with peptidoglycan purified from B. burgdorferi. The wells were washed, and protein binding was quantified using anti-GST antibodies followed by HRP-conjugated secondary antibodies. The OD450 value of BB0323 was expressed as 1.0. The bars represent the mean values, and the error bars represent the SEM values from three independent experiments. The asterisk represents a significant decrease (P < 0.05) between BB0323ΔLysM binding to peptidoglycan compared to that of either LysM or BB0323. (C) Interaction of LysM domain with borrelial peptidoglycan in solution. Peptidoglycan extracted from B. burgdorferi cells was mixed with BB0323, BB0323ΔLysM, or LysM, centrifuged to separate pellet or supernatant (Sup) fractions, and analyzed by immunoblotting using anti-GST antibodies. Arrow denotes position of BB0323 proteins.
Figure 5
Figure 5. BB0323 LysM domain is not essential for normal B. burgdorferi growth in vitro
(A) Growth analysis of cultured spirochetes. Wild-type cells (WT), bb0323 deletion mutants (bb0323-), and isolates with deletion of the LysM domain (ΔLysM) or C-terminus of BB0323 (ΔC) were assessed for growth in vitro. Data represent results from three independent experiments. Differences in spirochete numbers between WT and ΔLysM or ΔC groups were not significant at any time point. Arrow indicates a significant difference (P< 0.05) between numbers of bb0323 deletion mutants compared to all other isolates. (B) BB0323 LysM domain is not essential for normal morphology and the fission process in B. burgdorferi cells. Complementation of bb0323 mutants with truncated bb0323 genes restored normal cell morphology. Arrows denote abnormal OM organization as well as cell fission (represented by non-separated cells) in cases of bb0323 mutants. WT, bb0323-, ΔLysM, and ΔC isolates were grown in culture and subjected to transmission electron microscopy.
Figure 6
Figure 6. BB0323 LysM domain is required for spirochete infection of murine hosts
Mice (3 animals/group) were infected with wild type (WT), bb0323ΔLysM (ΔLysM), or a bb0323 deletion mutant complemented with bb0323 (bb0323 Com). RNA samples isolated from each group of three mice were pooled together according to the tissue type, and spirochete levels were assessed by qRT-PCR analysis at day 21 by measuring copies of flaB transcripts normalized against mouse ß-actin levels. The entire animal infection experiment was independently repeated three times. The bars represent the mean values, and the error bars represent the SEM values from the three independent experiments. Asterisks indicate undetectable levels of bb0323ΔLysM (ΔLysM) mutants.
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