An lp17-encoded small non-coding RNA with a potential regulatory role in mammalian host adaptation by the Lyme disease spirochete

The bacterial agent of Lyme disease, Borrelia burgdorferi, relies on an intricate gene regulatory network to transit between the disparate Ixodes tick vector and mammalian host environments. We recently reported that a B. burgdorferi mutant lacking an intergenic region of lp17 displayed attenuated murine tissue colonization and pathogenesis due to altered antigen expression. In this study, a more detailed characterization of the putative regulatory factor encoded by the region was pursued through genetic complementation of the mutant with variants of the intergenic sequence. In cis complemented strains featuring mutations aimed at eliminating potential BBD07 protein translation were capable of full tissue colonization, suggesting that the region encodes an sRNA. In trans complementation resulted in elevated transcription levels and was found to completely abolish infectivity in both immunocompetent and immunodeficient mice. Quantitative analysis of transcription of the putative sRNA by wild type B. burgdorferi showed it to be highly induced during murine infection. Lastly, targeted deletion of this region resulted in significant changes to the transcriptome, including genes with potential roles in transmission and host adaptation. The findings reported herein strongly suggest that this lp17 intergenic region encodes for an sRNA with a critical role in the gene regulation required for adaptation and persistence of the pathogen in the mammalian host. Author Summary Lyme disease continues to emerge as a devastating infection that afflicts hundreds of thousands of people annually in the United States and abroad, highlighting the need for new approaches and targets for intervention. Successful development of these therapies relies heavily on an improved understanding of the biology of the causative agent, Borrelia burgdorferi. This is particularly true for the critical points in the life cycle of the pathogen where it must transition between ticks and mammals. Variation in the levels of bacterial gene expression is the lynchpin of this transition and is known to be driven partly by the activity of regulatory molecules known as small non-coding RNAs (sRNAs). In this work, we characterize one of these sRNAs by providing experimental evidence that the transcribed product does not code for a protein, by testing the effects of its overproduction on infectivity, and by interrogating whether its activity causes changes in expression levels of genes at the level of transcription. The findings of this study provide further evidence that regulatory sRNA activity is critical for transmission and optimal infectivity of B. burgdorferi and contribute to the recently growing effort to attribute specific roles to these important molecules in the context of Lyme disease.


Introduction
115 transcript from this same region was also detected in a pair of earlier studies investigating 116 Rrp2-, RpoN-, and RpoS-dependent genes, where its expression was shown to be highly 117 dependent on an intact alternative sigma factor pathway (38,39). 118 We recently demonstrated that an lp17 left-end deletion mutant lacking this region 119 displays attenuated murine tissue colonization and pathogenicity, which was ultimately 120 attributed to a 317 bp intergenic region encompassing the bbd07/ SR0726 locus (40). The 121 tissue colonization defect was not observed during infection of SCID mice, indicating a 122 potential role for this locus in avoidance of adaptive immunity. Further study provided 123 evidence that deletion of this region results in dysregulated antigen expression, 124 implicating the gene product as a regulatory factor.

125
In the current study, we aimed to test the hypothesis that the bbd07/SR0726 region 126 of lp17 encodes an intergenic regulatory sRNA that participates in the transcriptome and 127 proteome shift required for spirochetes to adapt to the mammalian host environment. To 128 do this, BBD07 protein production was first ruled out experimentally through non-native 129 bbd07/SR0726 complementation in the previously described lp17 left-end mutant (40).
130 Then, the effects of bbd07/SR0726 overexpression were studied in the context of murine 131 infection and antigen expression. These tests revealed that spirochetes expressing high 132 levels of sRNA bbd07/SR0726 cannot infect mice, and that this phenotype may be 133 independent of altered in vitro antigen expression. Next, the expression of this sRNA was 134 quantified under in vivo conditions, which confirmed its putative involvement in the 135 mammalian portion of the enzootic cycle. Finally, a targeted knockout clone of 136 bbd07/SR0726 was generated and used for RNA-seq analysis, which revealed that its 137 activity has effects on transcript levels in addition to the previously observed effects on 138 the in vivo-expressed antigenic proteome. This work represents a significant step forward 139 towards understanding the critical role that this sRNA has in host adaptation by the Lyme 140 disease pathogen.

Results
143 Mutations that disrupt potential BBD07 protein production do not perturb the 144 functionality of the gene product during murine infection.

145
The recent detection of a putative sRNA encoded within the intergenic space of 146 lp17 containing the discontinued bbd07 ORF annotation (NC_001849.1 [discontinued]), 147 coupled with numerous failed attempts to detect a BBD07 protein product, provided a 148 strong indication that the bbd07/SR0726 locus encodes an sRNA. However, more 149 conclusive results were needed to definitively identify the gene product as an sRNA. 150 To further rule out the possibility of BBD07 protein production, two in cis 151 complement strains were generated in an lp17 mutant strain lacking a region containing 152 bbd01-bbd07 that is incapable of murine heart tissue colonization (∆1-7; (40)). These two 153 genetically complemented strains harbored a bbd07/SR0726 copy containing either a 154 premature stop codon or a disrupted start codon, and were denoted Comp7Nstop c and 155 Comp7NΔstart c , respectively (  Figure 1B. 180 Sequencing was performed to verify the single base pair mutations in each construct, and 181 isogeneity to the parent clone was confirmed by multiplex PCR for native plasmid content 182 (data not shown) (43).

183
To determine if the altered bbd07/SR726 sequences were able to rescue the 184 mutant phenotype, the capacity for heart tissue colonization of immunocompetent mice 185 was selected for use as a readout. We reasoned that this was a reliable indicator of gene 186 functionality due to the fact that the ∆1-7 mutant has been previously shown to be unable 187 to colonize heart tissue in immunocompetent mice (40). Groups of five C3H mice each 198

202
215 Overexpression of ittB is deleterious to murine host infection.

216
Our laboratory recently reported that absence of the region encoding ittB affects 217 antigen expression in vivo, which could potentially explain the observed attenuation in 218 tissue colonization and pathogenesis (40). Thus, it was hypothesized that ittB 219 overexpression may lead to altered murine infectivity due to abnormal antigen production.
220 B. burgdorferi has been shown to maintain the pBSV2G shuttle vector at a higher copy 221 number than its native plasmids, and expression levels of genes complemented on this 222 vector can be elevated compared to wild type (42). Thus, in trans complemented strains 223 that harbored ittB gene copies on pBSV2G were generated in the ∆1-7 mutant 224 background to assess the effects of ittB overexpression. Complementation was 225 performed by transforming ∆1-7 cells with pBSV2G carrying either a wild-type copy of ittB 226 (Comp7N t ) or a copy containing an early stop codon (Comp7Nstop t ). An empty vector 227 control strain harboring pBSV2G without an ittB gene copy was also generated (CompE t ).
228 PCR verification of ittB presence or absence in these strains is illustrated in Figure 2A, 229 and isogeneity to the parent strain was confirmed via multiplex PCR (data not shown).

230
To determine the relative in vitro expression levels of ittB in the wild type and 231 Comp7N t , qRT-PCR analysis was performed using cDNA derived from triplicate cultures 232 of wild type, ∆1-7, and Comp7N t grown to late log-phase (1x10 8 spirochetes ml -1 ). As 233 shown in Figure 2B, a ~24-fold increase in ittB expression by Comp7N t spirochetes 234 compared to the wild type was observed. Wild-type expression of ittB was found to be 235 low, producing less than 2 ittB copies per 10 4 flaB copies. These results indicate that ittB 236 may be tightly regulated, and that residence of the ittB sequence on a high-copy vector 237 may disrupt this regulation that results in elevated in vitro transcription. To assess the effects of high copy ittB expression on murine infection, groups of 239 five C3H mice each were needle inoculated (5x10 3 total spirochetes) with wild type,  279 is highly elevated during in vitro cultivation (Fig. 2B), we predicted that any potential 280 resultant protein expression changes would be observable under the same conditions.

281
To assess the effects of ittB overexpression on overall antigen production, cultures 282 of wild type, ∆1-7, Comp7N c , Comp7N t , and CompE t spirochetes were grown in triplicate 283 under the same conditions used to quantify ittB transcript levels in the Comp7N t strain.
284 Protein lysates (10 9 total cells) of each strain were subjected to Western blot analysis 285 using murine immune sera harvested from mice that had been infected with wild type B.
286 burgdorferi for 28 days. Surprisingly, no notable differences could be observed between 287 the in vitro antigenic profiles of the strains tested (Fig. 3). This result is suggestive that 288 the non-infectious phenotype exhibited by Comp7N t may not be due to dysregulated 289 antigen expression. 303 bladder, or joints, respectively (Fig. 4). These values range from a 100-to 400-fold 304 increase in ittB transcription levels relative to that observed for in vitro grown wild-type 305 spirochetes (~2 copies of ittB per 10 4 copies of flab, see Fig. 2B), supporting the 306 hypothesis that ittB transcription is upregulated during mammalian infection. Despite ittB 307 transcription being ~100-fold higher in heart-resident spirochetes than those grown in 308 vitro, ittB expression levels in these spirochetes were significantly lower than those 309 colonizing joint tissue (p≤0.05). Differences in ittB transcription either between heart and 310 bladder or bladder and joint did not reach significance. 338 infection, heart, bladder, joint, and ear tissue were harvested and cultured for detection 339 of spirochete growth by dark field microscopy as described before. As was previously 340 observed for Δ1-7, none of the heart tissue cultures from ΔittB-infected mice were positive 341 for spirochete growth, further demonstrating that ittB is required for heart tissue 342 colonization in mice (Fig. 6)  In this study, we attempted to further characterize the bbd07/SR0726 locus of lp17 386 by determining whether the functional product encoded by this intergenic region is an 387 RNA or protein, and assessing any potential regulatory effects resulting from its targeted     552 PBS. Lysis was performed by adding 30μL of 4X Laemmli loading dye containing 10% β-553 mercaptoethanol, and then heating the samples to 80°C for 10 minutes following at least 554 one freeze-thaw cycle. Lysates (10 9 cells) were electrophoresed on a precast 4-15% 555 polyacrylamide gradient gel (Bio-Rad). Western blotting was performed as described 556 previously (52), using sera harvested from C3H mice infected with 5x10 3 spirochetes of 557 the indicated strain for 28 days. Purified polyclonal antibodies (Rockland) were used in 558 anti-FlaB Western blots.

560
Cultures were grown in triplicate under standard culture conditions to mid-log 561 phase (5x10 7 spirochetes mL -1 ), then temperature shifted by 1:100 dilution into room 562 temperature BSK-II. Cultures were allowed to grow to mid log phase, at which point they 563 were subcultured again 1:100 into warm BSK-II. These final cultures were then allowed 564 to grow under standard conditions to late log-phase (1x10 8 spirochetes mL -1 ). Cultures 565 were pooled, and RNA was extracted using a hot phenol method described previously Fisher's exact test was used to determine significant differences in the ability to 577 recover recombinant strains by culturing of tissues compared with wild 578 type B. burgdorferi. Student's t-test was performed to determine significant differences in 579 spirochete burden in heart tissue samples from qPCR analyses, where the average 580 burden in heart tissues from mice infected with a given recombinant strain was compared 581 to that from mice infected with the wild type. Student's t-test was also used to determine 582 significant differences in the average in vitro ittB transcription levels between a given 583 recombinant strain and the wild type by qRT-PCR. One-way ANOVA followed by all 584 pairwise multiple comparison (Holm-Sidak) was used to determine significantly different 585 levels of average in vivo ittB transcription by the wild type between each tissue tested.