Assessment of the hypothetical protein BB0616 in the murine infection of Borrelia burgdorferi

Abstract

bb0616 of Borrelia burgdorferi, the Lyme disease pathogen, encodes a hypothetical protein of unknown function. In this study, we showed that BB0616 was not surface-exposed or associated with the membrane through localization analyses using proteinase K digestion and cell partitioning assays. The expression of bb0616 was influenced by a reduced pH but not by growth phases, elevated temperatures, or carbon sources during in vitro cultivation. A transcriptional start site for bb0616 was identified by using 5' rapid amplification of cDNA ends, which led to the identification of a functional promoter in the 5' regulatory region upstream of bb0616. By analyzing a bb0616-deficient mutant and its isogenic complemented counterparts, we found that the infectivity potential of the mutant was significantly attenuated. The inactivation of bb0616 displayed no effect on borrelial growth in the medium or resistance to oxidative stress, but the mutant was significantly more susceptible to osmotic stress. In addition, the production of global virulence regulators such as BosR and RpoS as well as virulence-associated outer surface lipoproteins OspC and DbpA was reduced in the mutant. These phenotypes were fully restored when gene mutation was complemented with a wild-type copy of bb0616. Based on these findings, we concluded that the hypothetical protein BB0616 is required for the optimal infectivity of B. burgdorferi, potentially by impacting B. burgdorferi virulence gene expression as well as survival of the spirochete under stressful conditions.

Keywords: Borrelia burgdorferi; Lyme disease; RpoS; pathogenesis; regulation of gene expression; virulence regulation.

Fig 1

In silico analysis of B. burgdorferi BB0616. (A) Domain structure of BB0616. The numbers represent the relative positions of predicted domains including an acyl-CoA N-acyltransferase domain (amino acids 22–106) and two cation efflux protein domains (amino acids 128–215 and 270–375). (B) Alignment of B. burgdorferi BB0616 with its homologs. GNAT, Cetobacterium GNAT family N-acetyltransferase (WP_294093315.1); cation, Palaeococcus ferrophilus cation diffusion facilitator family transporter (WP_048151185.1). Multiple alignment was generated by using Clustal Omega.

Fig 2

Cellular localization of BB0616. (A) BB0616 is not surface-exposed. Spirochetes were incubated with proteinase K (PK) with or without Triton X-100 (TX100). The presence or absence of treated reagents was indicated using the plus (+) and minus (−) signs. Cell lysates were resolved by SDS-PAGE and analyzed by immunoblot. OspC and FlaB were utilized as internal controls. OspC, localized on the cell surface, is sensitive to proteinase K treatment; FlaB, a periplasmic protein, is resistant to proteinase K digestion. (B) Whole-cell fractionation using Triton X-114. Spirochetes were incubated with Triton X-114 overnight, and the PC, the aqueous (Aq) phase, and the detergent (Det) phase were isolated and analyzed by immunoblot. FlaB and outer-membrane-localized OspA were used as controls. (C) BB0616 is not associated with the membrane. Spirochetes were sonicated, incubated with Triton X-114, and phase-separated into Aq and Det fractions. The regulatory protein BosR and outer-membrane-localized OspC were used as controls. Specific antibodies, denoted as α-, are shown on the right, and the relevant molecular masses are indicated in kilodalton on the left. A representative image from two independent experiments is shown.

Fig 3

Expression of bb0616 in B. burgdorferi. (A) B. burgdorferi was cultivated in BSK-II with pH 7.6 at 37°C, and spirochetes were collected when bacterial growth reached the early logarithmic phase (E), mid-logarithmic phase (M), or stationary phase (S). (B) B. burgdorferi was cultivated in BSK-II with pH 7.6 at 23°C or 37°C, and spirochetes were collected at the stationary phase. (C) To test the effect of pH change on gene expression, B. burgdorferi was cultured at 37°C in BSK-II with pH 6.8 or pH 7.6, and spirochetes were collected when bacterial growth reached the stationary phase. (D) To test the effects of different carbon sources, B. burgdorferi was cultivated in BSK-II or BSK-glycerol (BSK-Gly) at 23°C or 37°C, and spirochetes were collected at the stationary phase. RNA was isolated from spirochetes, and gene expression was analyzed by qRT-PCR via relative quantification. Data were normalized by using B. burgdorferi flaB gene as an internal control. All data were collected from three independent experiments, and the bars represent the mean values ± standard deviation. Asterisks indicate statistical significance using ANOVA followed by the Tukey test (^*P < 0.05; ^***P < 0.00005).

Fig 4

Identification of the TSS and promoter elements for bb0616. (A) Schematic diagram (not drawn to scale) of the region surrounding bb0616. Primers used for 5′ RACE to determine the TSS are shown above the gene diagram. (B) Putative regulatory region of bb0616. The asterisk denotes the TSS, and the associated −35 and −10 elements and the RBS are underlined. The translational start codons (ATG) for bb0616 and bb0617 are in boldface; arrows indicate the direction of translation.

Fig 5

Mutation and complementation of bb0616. (A) Schematic representation (not drawn to scale) of the bb0616 locus in the WT strain 297, the mutant OY64 (Mut), and the relevant complemented strain OY443 (Com). Genes are shown as thick arrows circumscribing the respective gene numbers. PCR (B) and RT-PCR (C) analyses. The target gene names are indicated on the right. DNA sizes are indicated in bp on the left. Lane 1, WT 297; lane 2, the mutant; lane 3, the complemented strain. Arrows indicate the approximate position of the bb0616 promoter (P).

Fig 6

BB0616 is dispensable for the growth of B. burgdorferi. WT 297, the bb0616 mutant (Mut), and the complemented strains (Com) were inoculated into BSK-II and grown at 37°C (A) or 23°C (B). Spirochetes were enumerated using dark-field microscopy. Data are presented as the mean values ± standard deviation from three biological replicates.

Fig 7

bb0616 contributes to the infectivity of B. burgdorferi. Mice were injected intradermally with WT 297, the bb0616 mutant (Mut), or the complemented strains (Com) at a dose of 10,000 spirochetes per animal. At 4 weeks post-inoculation, mice were sacrificed, and the skin, heart, and joints were collected. DNA was isolated, and spirochete burdens were quantified by qPCR via absolute quantification. Data are presented as the average copy number of B. burgdorferi flaB per 10⁵ mouse β-actin gene copies, and the bars represent the mean values ± standard deviation. n = 8 mice for each strain. The asterisk indicates statistical significance using ANOVA followed by the Tukey test (^*P < 0.05; ^**P < 0.005; ^***P < 0.0005).

Fig 8

Contribution of BB0616 to B. burgdorferi stress response. WT 297, the bb0616 mutant (Mut), and the complemented strain (Com) were grown in BSK-II at 37°C. When growth reached the mid-logarithmic phase, spirochetes were exposed to varying amounts of H₂O₂ at 37°C for 4 h (A) or 1 M NaCl for 0, 15, 30, 60, or 90 min (B). Spirochete viability was determined by using flow cytometry, and percent survival was calculated. Data are presented as the mean values ± standard deviation from three biological replicates. Asterisks indicate statistical significance using ANOVA followed by the Tukey test (^***P < 0.0005; ^****P < 0.00005).

Fig 9

Productions of BosR, RpoS, OspC, and DbpA were decreased in the bb0616 mutant. B. burgdorferi was cultivated at 37°C in the BSK-II medium, and spirochetes were collected at the stationary phase. Protein levels were assessed via immunoblot. To ensure equal loading of spirochetes in each lane, immunoblotting for the flagellin protein FlaB was performed. (A) Specific antibodies, denoted as α, are indicated on the left. (B) Three independent Western blots were quantified by densitometry using ImageJ software. Results are presented as the mean fold changes (relative to protein levels in WT) ±standard deviation. The asterisks indicate statistical significance using ANOVA followed by the Tukey test (^***P < 0.0005; ^****P < 0.00005). WT, strain 297; Mut, the bb0616 mutant; Com, the complemented strain.