Syed Z. Sultan

Analysis of a Borrelia burgdorferi phosphodiesterase demonstrates a role for cyclic-di-guanosine monophosphate in motility and virulence.

The genome of Borrelia burgdorferi encodes a set of genes putatively involved in cyclic‐dimeric guanosine monophosphate (cyclic‐di‐GMP) metabolism. Although BB0419 was shown to be a diguanylate cyclase, the extent to which bb0419 or any of the putative cyclic‐di‐GMP metabolizing genes impact B. burgdorferi motility and pathogenesis has not yet been reported. Here we identify and characterize a phosphodiesterase (BB0363). BB0363 specifically hydrolyzed cyclic‐di‐GMP with a Km of 0.054 µM, confirming it is a functional cyclic‐di‐GMP phosphodiesterase. A targeted mutation in bb0363 was constructed using a newly developed promoterless antibiotic cassette that does not affect downstream gene expression. The mutant cells exhibited an altered swimming pattern, indicating a function for cyclic‐di‐GMP in regulating B. burgdorferi motility. Furthermore, the bb0363 mutant cells were not infectious in mice, demonstrating an important role for cyclic‐di‐GMP in B. burgdorferi infection. The mutant cells were able to survive within Ixodes scapularis ticks after a blood meal from naïve mice; however, ticks infected with the mutant cells were not able to infect naïve mice. Both motility and infection phenotypes were restored upon genetic complementation. These results reveal an important connection between cyclic‐di‐GMP, B. burgdorferi motility and Lyme disease pathogenesis. A mechanism by which cyclic‐di‐GMP influences motility and infection is proposed.

Analysis of the HD-GYP Domain Cyclic Dimeric GMP Phosphodiesterase Reveals a Role in Motility and the Enzootic Life Cycle of Borrelia burgdorferi

ABSTRACT HD-GYP domain cyclic dimeric GMP (c-di-GMP) phosphodiesterases are implicated in motility and virulence in bacteria. Borrelia burgdorferi possesses a single set of c-di-GMP-metabolizing enzymes, including a putative HD-GYP domain protein, BB0374. Recently, we characterized the EAL domain phosphodiesterase PdeA. A mutation in pdeA resulted in cells that were defective in motility and virulence. Here we demonstrate that BB0374/PdeB specifically hydrolyzed c-di-GMP with a Km of 2.9 nM, confirming that it is a functional phosphodiesterase. Furthermore, by measuring phosphodiesterase enzyme activity in extracts from cells containing the pdeA pdeB double mutant, we demonstrate that no additional phosphodiesterases are present in B. burgdorferi. pdeB single mutant cells exhibit significantly increased flexing, indicating a role for c-di-GMP in motility. Constructing and analyzing a pilZ pdeB double mutant suggests that PilZ likely interacts with chemotaxis signaling. While virulence in needle-inoculated C3H/HeN mice did not appear to be altered significantly in pdeB mutant cells, these cells exhibited a reduced ability to survive in Ixodes scapularis ticks. Consequently, those ticks were unable to transmit the infection to naïve mice. All of these phenotypes were restored when the mutant was complemented. Identification of this role of pdeB increases our understanding of the c-di-GMP signaling network in motility regulation and the life cycle of B. burgdorferi.

Analysis of the Borrelia burgdorferi Cyclic-di-GMP-Binding Protein PlzA Reveals a Role in Motility and Virulence

ABSTRACT The cyclic-dimeric-GMP (c-di-GMP)-binding protein PilZ has been implicated in bacterial motility and pathogenesis. Although BB0733 (PlzA), the only PilZ domain-containing protein in Borrelia burgdorferi, was reported to bind c-di-GMP, neither its role in motility or virulence nor its affinity for c-di-GMP has been reported. We determined that PlzA specifically binds c-di-GMP with high affinity (dissociation constant [Kd ], 1.25 μM), consistent with Kd values reported for c-di-GMP-binding proteins from other bacteria. Inactivation of the monocistronically transcribed plzA resulted in an opaque/solid colony morphology, whereas the wild-type colonies were translucent. While the swimming pattern of mutant cells appeared normal, on swarm plates, mutant cells exhibited a significantly reduced swarm diameter, demonstrating a role of plzA in motility. Furthermore, the plzA mutant cells were significantly less infectious in experimental mice (as determined by 50% infectious dose [ID50]) relative to wild-type spirochetes. The mutant also had survival rates in fed ticks lower than those of the wild type. Consequently, plzA mutant cells failed to complete the mouse-tick-mouse infection cycle, indicating plzA is essential for the enzootic life cycle of B. burgdorferi. All of these defects were corrected when the mutant was complemented in cis. We propose that failure of plzA mutant cells to infect mice was due to altered motility; however, the possibility that an unidentified factor(s) contributed to interruption of the B. burgdorferi enzootic life cycle cannot yet be excluded.

Motility Is Crucial for the Infectious Life Cycle of Borrelia burgdorferi

ABSTRACT The Lyme disease spirochete, Borrelia burgdorferi, exists in a zoonotic cycle involving an arthropod tick and mammalian host. Dissemination of the organism within and between these hosts depends upon the spirochetes ability to traverse through complex tissues. Additionally, the spirochete outruns the host immune cells while migrating through the dermis, suggesting the importance of B. burgdorferi motility in evading host clearance. B. burgdorferis periplasmic flagellar filaments are composed primarily of a major protein, FlaB, and minor protein, FlaA. By constructing a flaB mutant that is nonmotile, we investigated for the first time the absolute requirement for motility in the mouse-tick life cycle of B. burgdorferi. We found that whereas wild-type cells are motile and have a flat-wave morphology, mutant cells were nonmotile and rod shaped. These mutants were unable to establish infection in C3H/HeN mice via either needle injection or tick bite. In addition, these mutants had decreased viability in fed ticks. Our studies provide substantial evidence that the periplasmic flagella, and consequently motility, are critical not only for optimal survival in ticks but also for infection of the mammalian host by the arthropod tick vector.

A Novel Gene Inactivation System Reveals Altered Periplasmic Flagellar Orientation in a Borrelia burgdorferi fliL Mutant

Motility and chemotaxis are essential components of pathogenesis for many infectious bacteria, including Borrelia burgdorferi, the causative agent of Lyme disease. Motility and chemotaxis genes comprise 5 to 6% of the genome of B. burgdorferi, yet the functions of most of those genes remain uncharacterized, mainly due to the paucity of a nonpolar gene inactivation system. In this communication, we describe the development of a novel gene inactivation methodology to target B. burgdorferi fliL, a putative periplasmic flagellar gene located in a large motility operon and transcribed by RNA polymerase containing σ(70). Although the morphology of nonpolar fliL mutant cells was indistinguishable from that of wild-type cells, the mutant exhibited a defective-motility phenotype. Cryo-electron tomography (cryo-ET) of intact organisms revealed that the periplasmic flagella in the fliL mutant were frequently tilted toward the cell pole instead of their normal orientation toward the cell body. These defects were corrected when the mutant was complemented in cis. Moreover, a comparative analysis of flagellar motors from the wild type and the mutant provides the first structural evidence that FliL is localized between the stator and rotor. Our results suggest that FliL is likely involved in coordinating or regulating the orientation of periplasmic flagella in B. burgdorferi.

Role of the histone-like nucleoid structuring protein in the regulation of rpoS and RpoS-dependent genes in Vibrio cholerae.

Production of the Zn-metalloprotease hemagglutinin (HA)/protease by Vibrio cholerae has been reported to enhance enterotoxicity in rabbit ileal loops and the reactogenicity of live cholera vaccine candidates. Expression of HA/protease requires the alternate sigma factor sigma(S) (RpoS), encoded by rpoS. The histone-like nucleoid structuring protein (H-NS) has been shown to repress rpoS expression in Escherichia coli. In V. cholerae strains of the classical biotype, H-NS has been reported to silence virulence gene expression. In this study we examined the role of H-NS in the expression of HA/protease and motility in an El Tor biotype strain by constructing a Deltahns mutant. The Deltahns mutant exhibited multiple phenotypes, such as production of cholera toxin in nonpermissive LB medium, reduced resistance to high osmolarity, enhanced resistance to low pH and hydrogen peroxide, and reduced motility. Depletion of H-NS by overexpression of a dominant-negative allele or by deletion of hns resulted in diminished expression of HA/protease. Epistasis analysis of HA/protease expression in Deltahns, DeltarpoS, and Deltahns DeltarpoS mutants, analysis of RpoS reporter fusions, quantitative reverse transcription-PCR measurements, and ectopic expression of RpoS in DeltarpoS and DeltarpoS Deltahns mutants showed that H-NS posttranscriptionally enhances RpoS expression. The Deltahns mutant exhibited a lower degree of motility and lower levels of expression of flaA, flaC, cheR-2, and motX mRNAs than the wild type. Comparison of the mRNA abundances of these genes in wild-type, Deltahns, DeltarpoS, and Deltahns DeltarpoS strains revealed that deletion of rpoS had a more severe negative effect on their expression. Interestingly, deletion of hns in the rpoS background resulted in higher expression levels of flaA, flaC, and motX, suggesting that H-NS represses the expression of these genes in the absence of sigma(S). Finally, we show that the cyclic AMP receptor protein and H-NS act along the same pathway to positively affect RpoS expression.

CheY3 of Borrelia burgdorferi Is the Key Response Regulator Essential for Chemotaxis and Forms a Long-Lived Phosphorylated Intermediate

Spirochetes have a unique cell structure: These bacteria have internal periplasmic flagella subterminally attached at each cell end. How spirochetes coordinate the rotation of the periplasmic flagella for chemotaxis is poorly understood. In other bacteria, modulation of flagellar rotation is essential for chemotaxis, and phosphorylation-dephosphorylation of the response regulator CheY plays a key role in regulating this rotary motion. The genome of the Lyme disease spirochete Borrelia burgdorferi contains multiple homologues of chemotaxis genes, including three copies of cheY, referred to as cheY1, cheY2, and cheY3. To investigate the function of these genes, we targeted them separately or in combination by allelic exchange mutagenesis. Whereas wild-type cells ran, paused (flexed), and reversed, cells of all single, double, and triple mutants that contained an inactivated cheY3 gene constantly ran. Capillary tube chemotaxis assays indicated that only those strains with a mutation in cheY3 were deficient in chemotaxis, and cheY3 complementation restored chemotactic ability. In vitro phosphorylation assays indicated that CheY3 was more efficiently phosphorylated by CheA2 than by CheA1, and the CheY3-P intermediate generated was considerably more stable than the CheY-P proteins found in most other bacteria. The results point toward CheY3 being the key response regulator essential for chemotaxis in B. burgdorferi. In addition, the stability of CheY3-P may be critical for coordination of the rotation of the periplasmic flagella.

Motor Rotation Is Essential for the Formation of the Periplasmic Flagellar Ribbon, Cellular Morphology, and Borrelia burgdorferi Persistence within Ixodes scapularis Tick and Murine Hosts

ABSTRACT Borrelia burgdorferi must migrate within and between its arthropod and mammalian hosts in order to complete its natural enzootic cycle. During tick feeding, the spirochete transmits from the tick to the host dermis, eventually colonizing and persisting within multiple, distant tissues. This dissemination modality suggests that flagellar motor rotation and, by extension, motility are crucial for infection. We recently reported that a nonmotile flaB mutant that lacks periplasmic flagella is rod shaped and unable to infect mice by needle or tick bite. However, those studies could not differentiate whether motor rotation or merely the possession of the periplasmic flagella was crucial for cellular morphology and host persistence. Here, we constructed and characterized a motB mutant that is nonmotile but retains its periplasmic flagella. Even though ΔmotB bacteria assembled flagella, part of the mutant cell is rod shaped. Cryoelectron tomography revealed that the flagellar ribbons are distorted in the mutant cells, indicating that motor rotation is essential for spirochetal flat-wave morphology. The ΔmotB cells are unable to infect mice, survive in the vector, or migrate out of the tick. Coinfection studies determined that the presence of these nonmotile ΔmotB cells has no effect on the clearance of wild-type spirochetes during murine infection and vice versa. Together, our data demonstrate that while flagellar motor rotation is necessary for spirochetal morphology and motility, the periplasmic flagella display no additional properties related to immune clearance and persistence within relevant hosts.

The cyclic-di-GMP signaling pathway in the Lyme disease spirochete, Borrelia burgdorferi

In nature, the Lyme disease spirochete Borrelia burgdorferi cycles between the unrelated environments of the Ixodes tick vector and mammalian host. In order to survive transmission between hosts, B. burgdorferi must be able to not only detect changes in its environment, but also rapidly and appropriately respond to these changes. One manner in which this obligate parasite regulates and adapts to its changing environment is through cyclic-di-GMP (c-di-GMP) signaling. c-di-GMP has been shown to be instrumental in orchestrating the adaptation of B. burgdorferi to the tick environment. B. burgdorferi possesses only one set of c-di-GMP-metabolizing genes (one diguanylate cyclase and two distinct phosphodiesterases) and one c-di-GMP-binding PilZ-domain protein designated as PlzA. While studies in the realm of c-di-GMP signaling in B. burgdorferi have exploded in the last few years, there are still many more questions than answers. Elucidation of the importance of c-di-GMP signaling to B. burgdorferi may lead to the identification of mechanisms that are critical for the survival of B. burgdorferi in the tick phase of the enzootic cycle as well as potentially delineate a role (if any) c-di-GMP may play in the transmission and virulence of B. burgdorferi during the enzootic cycle, thereby enabling the development of effective drugs for the prevention and/or treatment of Lyme disease.

Borrelia burgdorferi CheY2 is dispensable for chemotaxis or motility but crucial for the infectious life cycle of the spirochete

ABSTRACT The requirements for bacterial chemotaxis and motility range from dispensable to crucial for host colonization. Even though more than 50% of all sequenced prokaryotic genomes possess at least one chemotaxis signaling system, many of those genomes contain multiple copies of a chemotaxis gene. However, the functions of most of those additional genes are unknown. Most motile bacteria possess at least one CheY response regulator that is typically dedicated to the control of motility and which is usually essential for virulence. Borrelia burgdorferi appears to be notably different, in that it has three cheY genes, and our current studies on cheY2 suggests that it has varied effects on different aspects of the natural infection cycle. Mutants deficient in this protein exhibit normal motility and chemotaxis in vitro but show reduced virulence in mice. Specifically, the cheY2 mutants were severely attenuated in murine infection and dissemination to distant tissues after needle inoculation. Moreover, while ΔcheY2 spirochetes are able to survive normally in the Ixodes ticks, mice fed upon by the ΔcheY2-infected ticks did not develop a persistent infection in the murine host. Our data suggest that CheY2, despite resembling a typical response regulator, functions distinctively from most other chemotaxis CheY proteins. We propose that CheY2 serves as a regulator for a B. burgdorferi virulence determinant that is required for productive infection within vertebrate, but not tick, hosts.