Abrogation of ospAB constitutively activates the Rrp2‐RpoN‐RpoS pathway (sigmaN‐sigmaS cascade) in Borrelia burgdorferi

Molecular mechanisms underlying the reciprocal regulation of the two major surface lipoproteins and virulence factors of Borrelia burgdorferi, OspA and OspC, are not fully understood. Herein, we report that inactivation of the ospAB operon resulted in overproduction of OspC and many other lipoproteins via the constitutive activation of the Rrp2‐RpoN‐RpoS pathway. Complementing the ospAB mutant with a wild‐type copy of ospA, but not an ospA variant that lacks the lipoprotein signal sequence, restored normal regulation of the Rrp2‐RpoN‐RpoS pathway; these results indicate that the phenotype was not caused by spurious mutations. Interestingly, while most of the ospAB mutant clones displayed a constitutive ospC expression phenotype, some ospAB mutant clones showed little or no ospC expression. Further analyses revealed that this OspC‐negative phenotype was independent of abrogation of ospAB. While activation of the Rrp2‐RpoN‐RpoS pathway was recently shown to downregulate ospA, our findings suggest that reduction of OspA can also activate this pathway. We postulate that the activation of the Rrp2‐RpoN‐RpoS pathway and downregulation of OspA form a positive feedback loop that allows spirochaetes to produce and maintain a constant high level of OspC and other lipoproteins during tick feeding, a strategy that is critical for spirochaetal transmission and mammalian infection.     

The BBA33 lipoprotein binds collagen and impacts Borrelia burgdorferi pathogenesis

Borrelia burgdorferi, the etiologic agent of Lyme disease, adapts to the mammalian hosts by differentially expressing several genes in the BosR and Rrp2‐RpoN‐RpoS dependent pathways, resulting in a distinct protein profile relative to that seen for survival in the Ixodes spp. tick. Previous studies indicate that a putative lipoprotein, BBA33, is produced in an RpoS‐dependent manner under conditions that mimic the mammalian component of the borrelial lifecycle. However, the significance and function for BBA33 is not known. Given its linkage to the BosR/Rrp2‐RpoN‐RpoS regulatory cascade, we hypothesized that BBA33 facilitates B. burgdorferi infection in the mammalian host. The deletion of bba33 eliminated B. burgdorferi infectivity in C3H mice, which was rescued by genetic complementation with intact bba33. With regard to function, a combinatorial peptide approach, coupled with subsequent in vitro binding assays, indicated that BBA33 binds to collagen type VI and, to a lesser extent, collagen type IV. Whole cell binding assays demonstrated BBA33‐dependent binding to human collagen type VI. Taken together, these results suggest that BBA33 interacts with collagenous structures and may function as an adhesin in a process that is required to prevent bacterial clearance.     

The BosR regulatory protein of Borrelia burgdorferi interfaces with the RpoS regulatory pathway and modulates both the oxidative stress response and pathogenic properties of the Lyme disease spirochete

Borrelia burgdorferi, the Lyme disease spirochete, adapts as it moves between the arthropod and mammalian hosts that it infects. We hypothesize that BosR serves as a global regulator in B. burgdorferi to modulate the oxidative stress response and adapt to mammalian hosts. To test this hypothesis, a bosR mutant in a low‐passage B. burgdorferi isolate was constructed. The resulting bosR::kan^R strain was altered when grown microaerobically or anaerobically suggesting that BosR is required for optimal replication under both growth conditions. The absence of BosR increased the sensitivity of B. burgdorferi to hydrogen peroxide and reduced the synthesis of Cdr and NapA, proteins important for cellular redox balance and the oxidative stress response, respectively, suggesting an important role for BosR in borrelial oxidative homeostasis. For the bosR mutant, the production of RpoS was abrogated and resulted in the loss of OspC and DbpA, suggesting that BosR interfaces with the Rrp2–RpoN–RpoS regulatory cascade. Consistent with the linkage to RpoS, cells lacking bosR were non‐infectious in the mouse model of infection. These results indicate that BosR is required for resistance to oxidative stressors and provides a regulatory response that is necessary for B. burgdorferi pathogenesis.     

Chitobiose utilization in Borrelia burgdorferi is dually regulated by RpoD and RpoS

Background  

Borrelia burgdorferi has limited biosynthetic capabilities and must scavenge N-acetylglucosamine (GlcNAc), an essential component of the microbial cell wall, from the surrounding environment. Spirochetes cultured in the absence of free GlcNAc exhibit biphasic growth; however, addition of chitobiose (a dimer of GlcNAc) substitutes for free GlcNAc resulting in a single exponential phase. We evaluated the effect of RpoS and RpoN, the only alternative sigma factors in B. burgdorferi, on biphasic growth and chitobiose utilization in the absence of free GlcNAc. In addition, we investigated the source of GlcNAc in the second exponential phase.     

The alternative sigma factors,rpoN1 and rpoN2 are required for mycophagous activity of Burkholderia gladioli strain NGJ1

Bacteria utilize RpoN, an alternative sigma factor (σ54) to grow in diverse habitats, including nitrogen-limiting conditions. Here, we report that a rice-associated mycophagous bacterium Burkholderia gladioli strain NGJ1 encodes two paralogues of rpoN viz. rpoN1 and rpoN2. Both of them are upregulated during 24 h of mycophagous interaction with Rhizoctonia solani, a polyphagous fungal pathogen. Disruption of either one of rpoNs renders the mutant NGJ1 bacterium defective in mycophagy, whereas ectopic expression of respective rpoN genes restores mycophagy in the complementing strains. NGJ1 requires rpoN1 and rpoN2 for efficient biocontrol to prevent R. solani to establish disease in rice and tomato. Further, we have identified 17 genes having RpoN regulatory motif in NGJ1, majority of them encode potential type III secretion system (T3SS) effectors, nitrogen assimilation, and cellular transport-related functions. Several of these RpoN regulated genes as well as certain previously reported T3SS apparatus (hrcC and hrcN) and effector (Bg_9562 and endo-β-1,3-glucanase) encoding genes are upregulated in NGJ1 but not in ΔrpoN1 or ΔrpoN2 mutant bacterium, during mycophagous interaction with R. solani. This highlights that RpoN1 and RpoN2 modulate T3SS, nitrogen assimilation as well as cellular transport systems in NGJ1 and thereby promote bacterial mycophagy.     

Transport activity of FhuA, FhuC, FhuD, and FhuB derivatives in a system free of polar effects, and stoichiometry of components involved in ferrichrome uptake

The Escherichia colifhu operon, composed of the fhuA, C, D, and B genes, is essential for the utilization of ferric siderophores of the hydroxamate type and for the uptake of the antibiotic albomycin. We have had difficulty studying the effects of missense mutations in individual plasmid-encoded transport genes because appropriate test strains were not found: all isolated chromosomal mutations in either one of the fhu genes (with a complete loss of function) negatively influenced the expression of other fhu genes in the operon. In order to analyze Fhu mutant proteins in a system free of polar effects, we constructed a plasmid-encoded gene cassette system by introducing unique restriction sites that allowed precise cloning of individual fhu genes. The fhu cassette operon expressed in a chromosomal fhu deletion mutant enabled us to evaluate the transport activity of mutated FhuA, FhuC, FhuD or FhuB derivatives. In addition, we found that transport across the outer membrane (via FhuA, TonB, ExbB, D) rather than transport across the cytoplasmic membrane (via FhuC, D, B) was rate limiting. The stoichiometry of the components involved in the uptake of iron(III) hydroxamates seems to be important for proper functioning. Received: 20 October 1997 / Accepted: 22 December 1997     

Acetyl-Phosphate Is Not a Global Regulatory Bridge between Virulence and Central Metabolism in Borrelia burgdorferi

In B. burgdorferi, the Rrp2-RpoN-RpoS signaling cascade is a distinctive system that coordinates the expression of virulence factors required for successful transition between its arthropod vector and mammalian hosts. Rrp2 (BB0763), an RpoN specific response regulator, is essential to activate this regulatory pathway. Previous investigations have attempted to identify the phosphate donor of Rrp2, including the cognate histidine kinase, Hk2 (BB0764), non-cognate histidine kinases such as Hk1, CheA1, and CheA2, and small molecular weight P-donors such as carbamoyl-phosphate and acetyl-phosphate (AcP). In a report by Xu et al., exogenous sodium acetate led to increased expression of RpoS and OspC and it was hypothesized this effect was due to increased levels of AcP via the enzyme AckA (BB0622). Genome analyses identified only one pathway that could generate AcP in B. burgdorferi: the acetate/mevalonate pathway that synthesizes the lipid, undecaprenyl phosphate (C₅₅-P, lipid I), which is essential for cell wall biogenesis. To assess the role of AcP in Rrp2–dependent regulation of RpoS and OspC, we used a unique selection strategy to generate mutants that lacked ackA (bb0622: acetate to AcP) or pta (bb0589: AcP to acetyl-CoA). These mutants have an absolute requirement for mevalonate and demonstrate that ackA and pta are required for cell viability. When the ΔackA or Δpta mutant was exposed to conditions (i.e., increased temperature or cell density) that up-regulate the expression of RpoS and OspC, normal induction of those proteins was observed. In addition, adding 20mM acetate or 20mM benzoate to the growth media of B. burgdorferi strain B31 ΔackA induced the expression of RpoS and OspC. These data suggest that AcP (generated by AckA) is not directly involved in modulating the Rrp2-RpoN-RpoS regulatory pathway and that exogenous acetate or benzoate are triggering an acid stress response in B. burgdorferi.     

PlzA is a bifunctional c-di-GMP biosensor that promotes tick and mammalian host-adaptation of Borrelia burgdorferi

In this study, we examined the relationship between c-di-GMP and its only known effector protein, PlzA, in Borrelia burgdorferi during the arthropod and mammalian phases of the enzootic cycle. Using a B. burgdorferi strain expressing a plzA point mutant (plzA-R145D) unable to bind c-di-GMP, we confirmed that the protective function of PlzA in ticks is c-di-GMP-dependent. Unlike ΔplzA spirochetes, which are severely attenuated in mice, the plzA-R145D strain was fully infectious, firmly establishing that PlzA serves a c-di-GMP-independent function in mammals. Contrary to prior reports, loss of PlzA did not affect expression of RpoS or RpoS-dependent genes, which are essential for transmission, mammalian host-adaptation and murine infection. To ascertain the nature of PlzA’s c-di-GMP-independent function(s), we employed infection models using (i) host-adapted mutant spirochetes for needle inoculation of immunocompetent mice and (ii) infection of scid mice with in vitro-grown organisms. Both approaches substantially restored ΔplzA infectivity, suggesting that PlzA enables B. burgdorferi to overcome an early bottleneck to infection. Furthermore, using a Borrelia strain expressing a heterologous, constitutively active diguanylate cyclase, we demonstrate that ‘ectopic’ production of c-di-GMP in mammals abrogates spirochete virulence and interferes with RpoS function at the post-translational level in a PlzA-dependent manner. Structural modeling and SAXS analysis of liganded- and unliganded-PlzA revealed marked conformational changes that underlie its biphasic functionality. This structural plasticity likely enables PlzA to serve as a c-di-GMP biosensor that in its respective liganded and unliganded states promote vector- and host-adaptation by the Lyme disease spirochete.     

The chitobiose transporter, <Emphasis Type="Italic">chbC</Emphasis>, is required for chitin utilization in <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis>

Background  

The bacterium Borrelia burgdorferi, the causative agent of Lyme disease, is a limited-genome organism that must obtain many of its biochemical building blocks, including N-acetylglucosamine (GlcNAc), from its tick or vertebrate host. GlcNAc can be imported into the cell as a monomer or dimer (chitobiose), and the annotation for several B. burgdorferi genes suggests that this organism may be able to degrade and utilize chitin, a polymer of GlcNAc. We investigated the ability of B. burgdorferi to utilize chitin in the absence of free GlcNAc, and we attempted to identify genes involved in the process. We also examined the role of RpoS, one of two alternative sigma factors present in B. burgdorferi, in the regulation of chitin utilization.     

Sequence,biophysical, and structural analyses of the PstS lipoprotein (BB0215) from Borrelia burgdorferi reveal a likely binding component of an ABC‐type phosphate transporter

The Lyme disease agent Borrelia burgdorferi, which is transmitted via a tick vector, is dependent on its tick and mammalian hosts for a number of essential nutrients. Like other bacterial diderms, it must transport these biochemicals from the extracellular milieu across two membranes, ultimately to the B. burgdorferi cytoplasm. In the current study, we established that a gene cluster comprising genes bb0215 through bb0218 is cotranscribed and is therefore an operon. Sequence analysis of these proteins suggested that they are the components of an ABC‐type transporter responsible for translocating phosphate anions from the B. burgdorferi periplasm to the cytoplasm. Biophysical experiments established that the putative ligand‐binding protein of this system, BbPstS (BB0215), binds to phosphate in solution. We determined the high‐resolution (1.3 Å) crystal structure of the protein in the absence of phosphate, revealing that the protein's fold is similar to other phosphate‐binding proteins, and residues that are implicated in phosphate binding in other such proteins are conserved in BbPstS. Taken together, the gene products of bb0215‐0218 function as a phosphate transporter for B. burgdorferi.     

Identification and function of the RNA chaperone Hfq in the Lyme disease spirochete Borrelia burgdorferi

Hfq is a global regulatory RNA‐binding protein. We have identified and characterized an atypical Hfq required for gene regulation and infectivity in the Lyme disease spirochete Borrelia burgdorferi. Sequence analyses of the putative B. burgdorferi Hfq protein revealed only a modest level of similarity with the Hfq from Escherichia coli, although a few key residues are retained and the predicted tertiary structure is similar. Several lines of evidence suggest that the B. burgdorferi bb0268 gene encodes a functional Hfq homologue. First, the hfq_Bb gene (bb0268) restores the efficient translation of an rpoS::lacZ fusion in an E. coli hfq null mutant. Second, the Hfq from B. burgdorferi binds to the small RNA DsrA_Bb and the rpoS mRNA. Third, a B. burgdorferi hfq null mutant was generated and has a pleiotropic phenotype that includes increased cell length and decreased growth rate, as found in hfq mutants in other bacteria. The hfq_Bb mutant phenotype is complemented in trans with the hfq gene from either B. burgdorferi or, surprisingly, E. coli. This is the first example of a heterologous bacterial gene complementing a B. burgdorferi mutant. The alternative sigma factor RpoS and the outer membrane lipoprotein OspC, which are induced by increased temperature and required for mammalian infection, are not upregulated in the hfq mutant. Consequently, the hfq mutant is not infectious by needle inoculation in the murine model. These data suggest that Hfq plays a key role in the regulation of pathogenicity factors in B. burgdorferi and we hypothesize that the spirochete has a complex Hfq‐dependent sRNA network.     

The Lon-2 protease of Borrelia burgdorferi is critical for infection in the mammalian host

Borrelia burgdorferi encodes a functional homolog of canonical Lon protease termed Lon-2. To date, the contribution of Lon-2 to B. burgdorferi fitness and infection remains unexplored. Herein, we showed that expression of lon-2 was highly induced during animal infection, suggesting that Lon-2 is important for B. burgdorferi infection. We further generated a lon-2 deletion mutant. Compared with that of wild-type (WT) strain, the infectivity of the mutant was severely attenuated in a murine infection model. Although no growth defect was observed for the mutant in normal BSK-II medium, resistance of the lon-2 mutant to osmotic stress was markedly reduced. In addition, when exposed to tert-Butyl hydroperoxide, survival of the lon-2 mutant was impaired. In addition, we found that the protein levels of RpoS and RpoS-dependent OspC were decreased in the mutant. All these phenotypes were restored to WT or near-WT levels when lon-2 mutation was complemented in cis. Taken together, these results demonstrate that Lon-2 is critical for B. burgdorferi to establish infection and to cope with environmental stresses. This study provides a foundation for further uncovering the direct link between the dual roles of Lon-2 in protein quality control and bacterial pathogenesis.