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. 2021 May 25:12:676192.
doi: 10.3389/fmicb.2021.676192. eCollection 2021.

The Borrelia burgdorferi Adenylate Cyclase, CyaB, Is Important for Virulence Factor Production and Mammalian Infection

Vanessa M Ante  1 , Lauren C Farris  1 , Elizabeth P Saputra  1 , Allie J Hall  2 , Nathaniel S O'Bier  3 , Adela S Oliva Chávez  4 , Richard T Marconi  3 , Meghan C Lybecker  2 , Jenny A Hyde  1
Affiliations

The Borrelia burgdorferi Adenylate Cyclase, CyaB, Is Important for Virulence Factor Production and Mammalian Infection

Vanessa M Ante et al. Front Microbiol. .

Abstract

Borrelia burgdorferi, the causative agent of Lyme disease, traverses through vastly distinct environments between the tick vector and the multiple phases of the mammalian infection that requires genetic adaptation for the progression of pathogenesis. Borrelial gene expression is highly responsive to changes in specific environmental signals that initiate the RpoS regulon for mammalian adaptation, but the mechanism(s) for direct detection of environmental cues has yet to be identified. Secondary messenger cyclic adenosine monophosphate (cAMP) produced by adenylate cyclase is responsive to environmental signals, such as carbon source and pH, in many bacterial pathogens to promote virulence by altering gene regulation. B. burgdorferi encodes a single non-toxin class IV adenylate cyclase (bb0723, cyaB). This study investigates cyaB expression along with its influence on borrelial virulence regulation and mammalian infectivity. Expression of cyaB was specifically induced with co-incubation of mammalian host cells that was not observed with cultivated tick cells suggesting that cyaB expression is influenced by cellular factor(s) unique to mammalian cell lines. The 3' end of cyaB also encodes a small RNA, SR0623, in the same orientation that overlaps with bb0722. The differential processing of cyaB and SR0623 transcripts may alter the ability to influence function in the form of virulence determinant regulation and infectivity. Two independent cyaB deletion B31 strains were generated in 5A4-NP1 and ML23 backgrounds and complemented with the cyaB ORF alone that truncates SR0623, cyaB with intact SR0623, or cyaB with a mutagenized full-length SR0623 to evaluate the influence on transcriptional and posttranscriptional regulation of borrelial virulence factors and infectivity. In the absence of cyaB, the expression and production of ospC was significantly reduced, while the protein levels for BosR and DbpA were substantially lower than parental strains. Infectivity studies with both independent cyaB mutants demonstrated an attenuated phenotype with reduced colonization of tissues during early disseminated infection. This work suggests that B. burgdorferi utilizes cyaB and potentially cAMP as a regulatory pathway to modulate borrelial gene expression and protein production to promote borrelial virulence and dissemination in the mammalian host.

Keywords: Borrelia burgdorferi spirochete; CyaB; Lyme disease; adenylate cyclase (AC); cyclic nucleotides; secondary messenger; small RNA (sRNA).

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Schematic of the cyaB mutant and trans-complement strains. The cyaB deletion strain JH522 was generated through the insertion of an aadA antibiotic cassette to disrupt cyaB (bb0723) while keeping bb0722 intact. Chromosomal cyaB complementation strains were made through the introduction of an aacC1 antibiotic cassette. Complement strain VA200 contains the cyaB open reading frame (ORF) while truncating the sRNA SR0623, strain VA272 contains both the cyaB ORF and SR0623, and strain VA336 contains the cyaB ORF and a wobble mutation of SR0623. ORFs are indicated by arrows and sRNAs by wavy lines.
FIGURE 2
FIGURE 2
Verification of strains generated in this study. (A) Deletion of cyaB does not abolish transcription of neighboring genes bb0722 or bb0724. RT-PCR was used to verify the presence or absence of transcripts in the indicated B. burgdorferi strains. RNA was isolated and used to generate cDNA as describe in the Methods. The ± symbols indicate the cDNA reaction with or without reverse transcriptase. PCR reactions included cDNA and primers for cyaB (bb0723), bb0722, or bb0724. (B) sRNA-sequencing results are displayed in the coverage map (Popitsch et al., 2017). The + strand is shown in green and the − strand in blue. Northern blot analyses of total RNA fractionated on a 6% denaturing polyacrylamide gel, blotted to a nylon membrane, and hybridized with radioactive oligonucleotides. The black line represents the location of the oligonucleotide probes. The genomic context is indicated below the coverage plot. The predicted transcripts are denoted and marked with the appropriate band in the Northern blot. Northern blots are representative of three biological replicates. The following abbreviations are used to indicate strains: WT (P), cyaB– (M), c-cyaB (C), c-cyaB-SR0623 (S), and c-cyaB-SR0623w (W).
FIGURE 3
FIGURE 3
cyaB does not contribute to H2O2 resistance. WT and cyaB– strains were grown in BSK-glycerol to mid-log phase at 32°C 1% CO2, exposed to H2O2 in modified BSK media for 4 h at 32°C 1% CO2, and serial diluted for outgrowth to determine survival. Shown are the average and standard error of three independent biological replicates.
FIGURE 4
FIGURE 4
Deletion of cyaB reduces ospC expression. B. burgdorferi was grown in BSK-glycerol to mid-logarithmic phase at 32°C 1% CO2 for qRT-PCR analysis. (A) Comparison of the Ct values for the endogenous reference gene flaB. Relative transcript levels of virulence determinants for the (B) tick and (C) mammalian cycle. Fold changes are compared to WT. Shown are the averages and standard error of three biological replicates. Statistical analysis was performed using one-way ANOVA with Dunnett correction relative to WT, ***p < 0.001.
FIGURE 5
FIGURE 5
Deletion of cyaB reduces protein production of BosR, OspC, and DbpA. Borrelia burgdorferi was grown in BSK-glycerol to mid-logarithmic phase at 32°C 1% CO2. Protein was harvested and resolved on SDS–PAGE with approximately 4 × 107 B. burgdorferi in each lane. Immunoblots were prepared using the depicted antiserum. FlaB was used as a loading control. Displayed is the representative of three independent replicates. The following abbreviations are used to indicate strains: WT (P), cyaB– (M), c-cyaB (C), c-cyaB-SR0623 (S), and c-cyaB-SR0623w (W).
FIGURE 6
FIGURE 6
cyaB expression is induced with mammalian H4 cell co-culture. Borrelia burgdorferi was co-cultured with ISE6 tick cells or H4 mammalian cells. qRT-PCR was performed on samples collected at 3, 6, and 24 h co-incubation. flaB was used as an endogenous control. Shown is the average and standard error of four biological replicates. Statistical analysis was done using two-way ANOVA with Tukey correction, *p-value < 0.05.
FIGURE 7
FIGURE 7
Borrelial burden and tissue dissemination is reduced in mouse tissues infected with the cyaB mutant. C3H/HeN mice were infected with 105 Borrelia burgdorferi and tissues were harvested at 7, 14, and 21 dpi. qPCR was performed on (A) ears, (B) skin flanks, (C) joints, and (D) bladders to determine the number of borrelial genomes (recA) per 106 copies of mouse β-actin. Individual data points with at least n of 4 with lines representing average and standard error. Statistical analysis was done using two-way ANOVA with Dunnett correction relative to WT, **p-value < 0.01, ***p-value < 0.001, ****p-value < 0.0001.
FIGURE 8
FIGURE 8
Bioluminescent cyaB– has attenuated infection and dissemination. (A) Bioluminescent Borrelia burgdorferi is temporal and spatial tracked during infection of Balb/c mice with 105 WT, cyaB–, or c-cyaB-SR0623. Mice were imaged at 1 h and 1, 4, 7, 10, 14, and 21 dpi. The mouse in the first position of the image, indicated by (-), did not receive D-luciferin to serve as a background control. n of 5 for each infection group. (B) The bioluminescence of four mice was quantitated and averaged. Statistical analysis was performed using two-way ANOVA with Tukey correction relative to WT, ****p-value < 0.0001. (C) The percentage of tissues positive for B. burgdorferi at 21 dpi, grown in BSKII + NRS.
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