DksA controls the response of the Lyme disease spirochete Borrelia burgdorferi to starvation

Abstract

34The pathogenic spirochete Borrelia burgdorferi senses and responds to diverse environmental 35 challenges, including changes in nutrient availability, throughout its enzootic cycle in Ixodes 36 spp. ticks and vertebrate hosts. This study examined the role of DnaK suppressor protein (DksA) 37 in the transcriptional response of B. burgdorferi to starvation. Wild-type and dksA mutant B. 38 burgdorferi strains were subjected to starvation by shifting mid-logarithmic phase cultures 39 grown in BSK II medium to serum-free RPMI medium for 6 h under microaerobic conditions (5% 40 CO2, 3% O2). Microarray analyses of wild-type B. burgdorferi revealed that genes encoding 41 flagellar components, ribosomal proteins, and DNA replication machinery were downregulated 42 in response to starvation. DksA mediated transcriptomic responses to starvation in B. 43 burgdorferi as the dksA-deficient strain differentially expressed only 47 genes in response to 44 starvation compared to the 500 genes differentially expressed in wild-type strains. Consistent 45 with a role for DksA in the starvation response of B. burgdorferi, fewer CFUs were observed for 46 dksA mutant after prolonged starvation in RPMI medium compared to wild-type B. burgdorferi. 47 60 regulatory activity impacts B. burgdorferi metabolism, virulence gene expression, and the ability 61

DksA-dependent regulation of RpoS contributes toBorrelia burgdorferitick-borne transmission and mammalian infectivity

“…Additionally, DksA and (p)ppGpp control the expression of genes encoding products that facilitate glycerol and chitobiose utilization, along with genes encoding oligopeptide transporters, which are essential for the adaptation of B. burgdorferi during infection of I. scapularis (12-14). These observations suggest DksA and (p)ppGpp work synergistically to regulate transcription by RNA polymerase in B. burgdorferi (11) and are consistent with the generation of a stringent response due to the overlapping regulatory roles of DksA and (p)ppGpp in other organisms (15-18) where these regulators control the response to stationary phase growth and nutrient limiting conditions (19, 20). DksA derived from other bacteria such as Escherichia coli interacts with (p)ppGpp in vitro which can fundamentally change the direction and magnitude of gene regulation from specific gene promoters in E. coli using in vitro transcription reactions (21-24).…”

“…The DnaK suppressor protein (DksA) and the metabolite guanosine penta- and tetra phosphate ((p)ppGpp) produced by Rel bbu coordinate the stringent response of B. burgdorferi by regulating the transcription of genes in response to nutrient limitation (9-11). Furthermore, there is an extensive overlap in genes under the control of DksA and (p)ppGpp in B. burgdorferi (11). DksA and (p)ppGpp both control genes involved in protein synthesis, nutrient uptake pathways, as well as several lipoproteins.…”

“…B. burgdorferi requires a complex, nutrient-rich medium for growth and what constitutes nutrient limitation in B. burgdorferi is not well-defined. Previous attempts to study the stringent response were carried out by altering multiple environmental and metabolic factors simultaneously, making it difficult to interpret which key factors and metabolites played a role in the generation of a stringent response by B. burgdorferi (9, 11, 25-27). While limitation of nutrients including fatty acids, carbohydrates, or amino acids can activate the stringent response in bacteria, responses involving DksA and (p)ppGpp are also triggered by environmental changes, such as low pH and the presence of reactive nitrogen species (28-30).…”

“…For example, in E. coli , the adaptation to an acidic extracellular environment is aided by the regulation of the alternative sigma factor RpoS by DksA and DksA appears to be a key component of the response of E. coli to acid stress (31, 32). We recently observed dksA -deficient B. burgdorferi strains showed reduced expression of genes under the control of the alternative sigma factor RpoS (11). RpoS plays a gatekeeper function in the infectious cycle of B. burgdorferi by coordinating the expression of a substantial portion of genes during the transmission of B. burgdorferi from ticks to mammalian hosts.…”

“…In this study, we used both biochemical and genetic approaches to dissect the gene regulatory role played by DksA and its relationship with (p)ppGpp in B. burgdorferi . In our genetic approach, we assessed pH- and acetate-dependent responses of B. burgdorferi in the presence and absence of DksA and the (p)ppGpp synthase (Rel bbu ) because previous studies suggested DksA plays a much larger role in regulating RpoS (11, 25). To test the hypothesis that a synergy exists between DksA (p)ppGpp in regulating gene transcription, we used a newly developed in vitro transcription assay system using the B. burgdorferi RNA polymerase (58).…”