The Borrelia burgdorferi RelA/SpoT Homolog and Stringent Response Regulate Survival in the Tick Vector and Global Gene Expression during Starvation.

Dan Drecktrah,Meghan Lybecker,Laura S Hall,Philipp Rescheneder,D Scott Samuels,Niko Popitsch,Jenifer Coburn

doi:10.1371/journal.ppat.1005160

Abstract

As the Lyme disease bacterium Borrelia burgdorferi traverses its enzootic cycle, alternating between a tick vector and a vertebrate host, the spirochete must adapt and persist in the tick midgut under prolonged nutrient stress between blood meals. In this study, we examined the role of the stringent response in tick persistence and in regulation of gene expression during nutrient limitation. Nutritionally starving B. burgdorferi in vitro increased the levels of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), collectively referred to as (p)ppGpp, products of the bifunctional synthetase/hydrolase RelBbu (RelA/SpoT homolog). Conversely, returning B. burgdorferi to a nutrient-rich medium decreased (p)ppGpp levels. B. burgdorferi survival in ticks between the larval and nymph blood meals, and during starvation in vitro, was dependent on RelBbu. Furthermore, normal morphological conversion from a flat-wave shape to a condensed round body (RB) form during starvation was dependent on RelBbu; rel Bbu mutants more frequently formed RBs, but their membranes were compromised. By differential RNA sequencing analyses, we found that RelBbu regulates an extensive transcriptome, both dependent and independent of nutrient stress. The RelBbu regulon includes the glp operon, which is important for glycerol utilization and persistence in the tick, virulence factors and the late phage operon of the 32-kb circular plasmid (cp32) family. In summary, our data suggest that RelBbu globally modulates transcription in response to nutrient stress by increasing (p)ppGpp levels to facilitate B. burgdorferi persistence in the tick.

Highlights

The Lyme disease spirochete Borrelia burgdorferi is maintained in an enzootic cycle involving ticks and vertebrates [1,2,3]
We demonstrated that the stringent response, governed by RelBbu, which synthesizes and hydrolyzes the alarmones guanosine tetraphosphate and guanosine pentaphosphate (collectively termed (p)ppGpp), is necessary for persistence in the tick
We assayed if B. burgdorferi increases (p) ppGpp levels during starvation by shifting in vitro cultures from the normal growth medium, Barbour-Stoenner-Kelly II medium containing 6% rabbit serum (BSK + RS), to a starvation medium (RPMI containing no serum)

Summary

Introduction

The Lyme disease spirochete Borrelia burgdorferi is maintained in an enzootic cycle involving ticks and vertebrates [1,2,3]. Several B. burgdorferi gene products important for persistence in the tick have been identified [13,16], including BptA, a lipoprotein [17]; Dps/NapA/BicA, a bacterioferritin homolog [18]; GlpD (glycerol-3-phosphate dehydrogenase), an enzyme involved in glycerol metabolism [19]; and proteins involved in cyclic-dimeric-GMP (c-di-GMP) metabolism: Rrp, a response regulator and diguanylate cyclase [20,21], Hk1, its cognate histidine kinase [22], PdeB, a phosphodiesterase [23], and PlzA, a c-di-GMP-binding protein [24] None of these tick persistence factors have been shown to be associated with the requisite adaptation to nutrient limitations

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Discussion

Conclusion