Daniel P. Dulebohn

OspC-Independent Infection and Dissemination by Host-Adapted Borrelia burgdorferi

ABSTRACT Borrelia burgdorferi OspC is required for the spirochete to establish infection in a mammal by tick transmission or needle inoculation. After a brief essential period, the protein no longer is required and the gene can be shut off. Using a system in which spirochetes contain only an unstable wild-type copy of the ospC gene, we can obtain mice persistently infected with bacteria lacking OspC. We implanted pieces of infected mouse skin subcutaneously in naïve mice, using donors carrying wild-type or ospC mutant spirochetes, and found that both could infect mice by this method, with similar numbers of wild-type or ospC mutant spirochetes disseminated throughout the tissues of recipient mice. Recipient mouse immune responses to tissue transfer-mediated infection with wild-type or ospC mutant spirochetes were similar. These experiments demonstrate that mammalian host-adapted spirochetes can infect and disseminate in mice in the absence of OspC, thereby circumventing this hallmark of tick-derived or in vitro-grown spirochetes. We propose a model in which OspC is one of a succession of functionally equivalent, essential proteins that are synthesized at different stages of mammalian infection. In this model, another protein uniquely present on host-adapted spirochetes performs the same essential function initially fulfilled by OspC. The strict temporal control of B. burgdorferi outer surface protein gene expression may reflect immunological constraints rather than distinct functions.

Global repression of host-associated genes of the Lyme disease spirochete through post-transcriptional modulation of the alternative sigma factor RpoS.

Borrelia burgdorferi, the agent of Lyme disease, is a vector-borne pathogen that transits between Ixodes ticks and vertebrate hosts. During the natural infectious cycle, spirochetes must globally adjust their transcriptome to survive in these dissimilar environments. One way B. burgdorferi accomplishes this is through the use of alternative sigma factors to direct transcription of specific genes. RpoS, one of only three sigma factors in B. burgdorferi, controls expression of genes required during tick-transmission and infection of the mammalian host. How spirochetes switch between different sigma factors during the infectious cycle has remained elusive. Here we establish a role for a novel protein, BBD18, in the regulation of the virulence-associated sigma factor RpoS. Constitutive expression of BBD18 repressed transcription of RpoS-dependent genes to levels equivalent to those observed in an rpoS mutant. Consistent with the global loss of RpoS-dependent transcripts, we were unable to detect RpoS protein. However, constitutive expression of BBD18 did not diminish the amount of rpoS transcript, indicating post-transcriptional regulation of RpoS by BBD18. Interestingly, BBD18-mediated repression of RpoS is independent of both the rpoS promoter and the 5’ untranslated region, suggesting a mechanism of protein destabilization rather than translational control. We propose that BBD18 is a novel regulator of RpoS and its activity likely represents a first step in the transition from an RpoS-ON to an RpoS-OFF state, when spirochetes transition from the host to the tick vector.

Regulation of the Virulence Determinant OspC by bbd18 on Linear Plasmid lp17 of Borrelia burgdorferi

Persistent infection of a mammalian host by Borrelia burgdorferi, the spirochete that causes Lyme disease, requires specific downregulation of an immunogenic outer surface protein, OspC. Although OspC is an essential virulence factor needed by the spirochete to establish infection in the mammal, it represents a potent target for the host acquired immune response, and constitutive expression of OspC results in spirochete clearance. In this study, we demonstrate that a factor encoded on a linear plasmid of B. burgdorferi, lp17, can negatively regulate ospC transcription from the endogenous gene on the circular plasmid cp26 and from an ospC promoter-lacZ fusion on a shuttle vector. Furthermore, we have identified bbd18 as the gene on lp17 that is responsible for this effect. These data identify a novel component of ospC regulation and provide the basis for determining the molecular mechanisms of ospC repression in vivo.

Borrelia burgdorferi Linear Plasmid 38 Is Dispensable for Completion of the Mouse-Tick Infectious Cycle

ABSTRACT Borrelia burgdorferi, the causative agent of Lyme disease, exists in a complex enzootic cycle, transiting between its vector, Ixodes ticks, and a diverse range of vertebrate hosts. B. burgdorferi linear plasmid 38 (lp38) contains several genes that are differentially regulated in response to conditions mimicking the tick or mouse environments, suggesting that these plasmid-borne genes may encode proteins important for the B. burgdorferi infectious cycle. Some of these genes encode potential virulence factors, including hypothetical lipoproteins as well as a putative membrane transport system. To characterize the role of lp38 in the B. burgdorferi infectious cycle, we constructed a shuttle vector to selectively displace lp38 from the B. burgdorferi genome and analyzed the resulting clones to confirm the loss of lp38. We found that, in vitro, clones lacking lp38 were similar to isogenic wild-type bacteria, both in growth rate and in antigenic protein production. We analyzed these strains in an experimental mouse-tick infectious cycle, and our results demonstrate that clones lacking lp38 are fully infectious in a mouse, can efficiently colonize the tick vector, and are readily transmitted to a naive host.

Regulatory Protein BBD18 of the Lyme Disease Spirochete: Essential Role During Tick Acquisition?

ABSTRACT The Lyme disease spirochete Borrelia burgdorferi senses and responds to environmental cues as it transits between the tick vector and vertebrate host. Failure to properly adapt can block transmission of the spirochete and persistence in either vector or host. We previously identified BBD18, a novel plasmid-encoded protein of B. burgdorferi, as a putative repressor of the host-essential factor OspC. In this study, we investigate the in vivo role of BBD18 as a regulatory protein, using an experimental mouse-tick model system that closely resembles the natural infectious cycle of B. burgdorferi. We show that spirochetes that have been engineered to constitutively produce BBD18 can colonize and persist in ticks but do not infect mice when introduced by either tick bite or needle inoculation. Conversely, spirochetes lacking BBD18 can persistently infect mice but are not acquired by feeding ticks. Through site-directed mutagenesis, we have demonstrated that abrogation of spirochete infection in mice by overexpression of BBD18 occurs only with bbd18 alleles that can suppress OspC synthesis. Finally, we demonstrate that BBD18-mediated regulation does not utilize a previously described ospC operator sequence required by B. burgdorferi for persistence in immunocompetent mice. These data lead us to conclude that BBD18 does not represent the putative repressor utilized by B. burgdorferi for the specific downregulation of OspC in the mammalian host. Rather, we suggest that BBD18 exhibits features more consistent with those of a global regulatory protein whose critical role occurs during spirochete acquisition by feeding ticks. IMPORTANCE Lyme disease, caused by Borrelia burgdorferi, is the most common arthropod-borne disease in North America. B. burgdorferi is transmitted to humans and other vertebrate hosts by ticks as they take a blood meal. Transmission between vectors and hosts requires the bacterium to sense changes in the environment and adapt. However, the mechanisms involved in this process are not well understood. By determining how B. burgdorferi cycles between two very different environments, we can potentially establish novel ways to interfere with transmission and limit infection of this vector-borne pathogen. We are studying a regulatory protein called BBD18 that we recently described. We found that too much BBD18 interferes with the spirochete’s ability to establish infection in mice, whereas too little BBD18 appears to prevent colonization in ticks. Our study provides new insight into key elements of the infectious cycle of the Lyme disease spirochete. Lyme disease, caused by Borrelia burgdorferi, is the most common arthropod-borne disease in North America. B. burgdorferi is transmitted to humans and other vertebrate hosts by ticks as they take a blood meal. Transmission between vectors and hosts requires the bacterium to sense changes in the environment and adapt. However, the mechanisms involved in this process are not well understood. By determining how B. burgdorferi cycles between two very different environments, we can potentially establish novel ways to interfere with transmission and limit infection of this vector-borne pathogen. We are studying a regulatory protein called BBD18 that we recently described. We found that too much BBD18 interferes with the spirochete’s ability to establish infection in mice, whereas too little BBD18 appears to prevent colonization in ticks. Our study provides new insight into key elements of the infectious cycle of the Lyme disease spirochete.

Borrelia burgdorferi Linear Plasmid 28-3 Confers a Selective Advantage in an Experimental Mouse-Tick Infection Model

ABSTRACT Borrelia burgdorferi, the bacterium that causes Lyme disease, has a unique segmented genome consisting of numerous linear and circular plasmids and a linear chromosome. Many of these genetic elements have been found to encode factors critical for B. burgdorferi to complete the infectious cycle. However, several plasmids remain poorly characterized, and their roles during infection with B. burgdorferi have not been elucidated. To more fully characterize the role of one of the four 28-kb linear plasmids, lp28-3, we generated strains specifically lacking lp28-3 and assayed the contribution of genes carried by lp28-3 to B. burgdorferi infection. We found that lp28-3 does not carry any genes that are strictly required for infection of a mouse or tick and that lp28-3-deficient spirochetes are competent at causing a disseminated infection. Interestingly, spirochetes containing lp28-3 were at a selective advantage compared to lp28-3-deficient spirochetes when coinjected into a mouse, and this advantage was reflected in the population of spirochetes acquired by feeding ticks. Our data demonstrate that genes carried by lp28-3, although not essential, contribute to the fitness of B. burgdorferi during infection.

Weak Organic Acids Decrease Borrelia burgdorferi Cytoplasmic pH, Eliciting an Acid Stress Response and Impacting RpoN- and RpoS-Dependent Gene Expression

The spirochete Borrelia burgdorferi survives in its tick vector, Ixodes scapularis, or within various hosts. To transition between and survive in these distinct niches, B. burgdorferi changes its gene expression in response to environmental cues, both biochemical and physiological. Exposure of B. burgdorferi to weak monocarboxylic organic acids, including those detected in the blood meal of fed ticks, decreased the cytoplasmic pH of B. burgdorferi in vitro. A decrease in the cytoplasmic pH induced the expression of genes encoding enzymes that have been shown to restore pH homeostasis in other bacteria. These include putative coupled proton/cation exchangers, a putative Na+/H+ antiporter, a neutralizing buffer transporter, an amino acid deaminase and a proton exporting vacuolar-type VoV1 ATPase. Data presented in this report suggested that the acid stress response triggered the expression of RpoN- and RpoS-dependent genes including important virulence factors such as outer surface protein C (OspC), BBA66, and some BosR (Borrelia oxidative stress regulator)-dependent genes. Because the expression of virulence factors, like OspC, are so tightly connected by RpoS to general cellular stress responses and cell physiology, it is difficult to separate transmission-promoting conditions in what is clearly a multifactorial and complex regulatory web.

Long-Term Survival of Borrelia burgdorferi Lacking the Hibernation Promotion Factor Homolog in the Unfed Tick Vector

ABSTRACT Borrelia burgdorferi, a causative agent of Lyme borreliosis, is a zoonotic pathogen that survives in nutrient-limited environments within a tick, prior to transmission to its mammalian host. Survival under these prolonged nutrient-limited conditions is thought to be similar to survival during stationary phase, which is characterized by growth cessation and decreased protein production. Multiple ribosome-associated proteins are implicated in stationary-phase survival of Escherichia coli. These proteins include hibernation-promoting factor (HPF), which dimerizes ribosomes and prevents translation of mRNA. Bioinformatic analyses indicate that B. burgdorferi harbors an hpf homolog, the bb0449 gene. BB0449 protein secondary structure modeling also predicted HPF-like structure and function. However, BB0449 protein was not localized in the ribosome-associated protein fraction of in vitro-grown B. burgdorferi. In wild-type B. burgdorferi, bb0449 transcript and BB0449 protein levels are low during various growth phases. These results are inconsistent with patterns of synthesis of HPF-like proteins in other bacterial species. In addition, two independently derived bb0449 mutants successfully completed the mouse-tick infectious cycle, indicating that bb0449 is not required for prolonged survival in the nutrient-limited environment in the unfed tick or any other stage of infection by B. burgdorferi. We suggest either that BB0449 is associated with ribosomes under specific conditions not yet identified or that BB0449 of B. burgdorferi has a function other than ribosome conformation modulation.

Global Profiling of Lysine Acetylation in Borrelia burgdorferi B31 Reveals Its Role in Central Metabolism

The post-translational modification of proteins has been shown to be extremely important in prokaryotes. Using a highly sensitive mass spectrometry-based proteomics approach, we have characterized the acetylome of B. burgdorferi. As previously reported for other bacteria, a relatively low number (5%) of the potential genome-encoded proteins of B. burgdorferi were acetylated. Of these, the vast majority were involved in central metabolism and cellular information processing (transcription, translation, etc.). Interestingly, these critical cell functions were targeted during both ML (mid-log) and S (stationary) phases of growth. However, acetylation of target proteins in ML phase was limited to single lysine residues while these same proteins were acetylated at multiple sites during S phase. To determine the acetyl donor in B. burgdorferi, we used mutants that targeted the sole acetate metabolic/anabolic pathway in B. burgdorferi (lipid I synthesis). B. burgdorferi strains B31-A3, B31-A3 ΔackA (acetyl-P- and acetyl-CoA-) and B31-A3 Δpta (acetyl-P+ and acetyl-CoA-) were grown to S phase and the acetylation profiles were analyzed. While only two proteins were acetylated in the ΔackA mutant, 140 proteins were acetylated in the Δpta mutant suggesting that acetyl-P was the primary acetyl donor in B. burgdorferi. Using specific enzymatic assays, we were able to demonstrate that hyperacetylation of proteins in S phase appeared to play a role in decreasing the enzymatic activity of at least two glycolytic proteins. Currently, we hypothesize that acetylation is used to modulate enzyme activities during different stages of growth. This strategy would allow the bacteria to post-translationally stimulate the activity of key glycolytic enzymes by deacetylation rather than expending excessive energy synthesizing new proteins. This would be an appealing, low-energy strategy for a bacterium with limited metabolic capabilities. Future work focuses on identifying potential protein deacetylase(s) to complete our understanding of this important biological process.