Characterization and genomic analysis of the Lyme disease spirochete bacteriophage ϕBB-1.

Lyme disease is a tick-borne infection caused by the spirochete Borrelia (Borreliella) burgdorferi. Borrelia species have highly fragmented genomes composed of a linear chromosome and a constellation of linear and circular plasmids some of which are required throughout the enzootic cycle. Included in this plasmid repertoire by almost all Lyme disease spirochetes are the 32-kb circular plasmid cp32 prophages that are capable of lytic replication to produce infectious virions called ϕBB-1.

Characterization and genomic analysis of the Lyme disease spirochete bacteriophage ϕBB-1.

Lyme disease is a tick-borne infection caused by the spirochete () . species have highly fragmented genomes composed of a linear chromosome and a constellation of linear and circular plasmids some of which are required throughout the enzootic cycle. Included in this plasmid repertoire by almost all Lyme disease spirochetes are the 32-kb circular plasmid cp32 prophages that are capable of lytic replication to produce infectious virions called ϕBB-1.

c-di-GMP regulates activity of the PlzA RNA chaperone from the Lyme disease spirochete.

PlzA is a c-di-GMP-binding protein crucial for adaptation of the Lyme disease spirochete Borrelia (Borreliella) burgdorferi during its enzootic life cycle. Unliganded apo-PlzA is important for vertebrate infection, while liganded holo-PlzA is important for survival in the tick; however, the biological function of PlzA has remained enigmatic. Here, we report that PlzA has RNA chaperone activity that is inhibited by c-di-GMP binding.

The glycerol-3-phosphate dehydrogenases GpsA and GlpD constitute the oxidoreductive metabolic linchpin for Lyme disease spirochete host infectivity and persistence in the tick.

We have identified GpsA, a predicted glycerol-3-phosphate dehydrogenase, as a virulence factor in the Lyme disease spirochete Borrelia (Borreliella) burgdorferi: GpsA is essential for murine infection and crucial for persistence of the spirochete in the tick. B. burgdorferi has a limited biosynthetic and metabolic capacity; the linchpin connecting central carbohydrate and lipid metabolism is at the interconversion of glycerol-3-phosphate and dihydroxyacetone phosphate, catalyzed by GpsA and another glycerol-3-phosphate dehydrogenase, GlpD.

Gene Regulation and Transcriptomics.

() , along with closely related species, is the etiologic agent of Lyme disease. The spirochete subsists in an enzootic cycle that encompasses acquisition from a vertebrate host to a tick vector and transmission from a tick vector to a vertebrate host. To adapt to its environment and persist in each phase of its enzootic cycle, wields three systems to regulate the expression of genes: the RpoN-RpoS alternative sigma factor cascade, the Hk1/Rrp1 two-component system and its product c-di-GMP, and the stringent response mediated by Rel and DksA.

Interactions Between Ticks and Lyme Disease Spirochetes.

sensu lato causes Lyme borreliosis in a variety of animals and humans. These atypical bacterial pathogens are maintained in a complex enzootic life cycle that primarily involves a vertebrate host and spp. ticks.

Characterization of 6S RNA in the Lyme disease spirochete.

6S RNA binds to RNA polymerase and regulates gene expression, contributing to bacterial adaptation to environmental stresses. In this study, we examined the role of 6S RNA in murine infectivity and tick persistence of the Lyme disease spirochete Borrelia (Borreliella) burgdorferi. B.

DksA Controls the Response of the Lyme Disease Spirochete to Starvation.

The pathogenic spirochete senses and responds to changes in the environment, including changes in nutrient availability, throughout its enzootic cycle in ticks and vertebrate hosts. This study examined the role of DnaK suppressor protein (DksA) in the transcriptional response of to starvation. Wild-type and mutant strains were subjected to starvation by shifting cultures grown in rich complete medium, Barbour-Stoenner-Kelly II (BSK II) medium, to a defined mammalian tissue culture medium, RPMI 1640, for 6 h under microaerobic conditions (5% CO, 3% O).

The Stringent Response-Regulated sRNA Transcriptome of .

The Lyme disease spirochete () must tolerate nutrient stress to persist in the tick phase of its enzootic life cycle. We previously found that the stringent response mediated by Rel globally regulates gene expression to facilitate persistence in the tick vector. Here, we show that Rel regulates the expression of a swath of small RNAs (sRNA), affecting 36% of previously identified sRNAs in .

RNase III Processing of rRNA in the Lyme Disease Spirochete Borrelia burgdorferi.

The rRNA genes of () are unusually organized; the spirochete has a single 16S rRNA gene that is more than 3 kb from a tandem pair of 23S-5S rRNA operons. We generated an null mutant in that exhibits a pleiotropic phenotype, including decreased growth rate and increased cell length. Here, we demonstrate that endoribonuclease III (RNase III) is, as expected, involved in processing the 23S rRNA in The 5' and 3' ends of the three rRNAs were determined in the wild type and mutants; the results suggest that RNase III in is required for the full maturation of the 23S rRNA but not for the 5S rRNA nor, curiously, for the 16S rRNA.

Genetic Transformation and Complementation.

The disciplines of Borrelia (Borreliella) burgdorferi microbiology and Lyme disease pathogenesis have come to depend on the genetic manipulation of the spirochete. Generating mutants in these recalcitrant bacteria, while not straightforward, is routinely accomplished in numerous laboratories, although there are several crucial caveats to consider. This chapter describes the design of basic molecular genetic experiments as well as the detailed methodologies to prepare and transform competent cells, select for and isolate transformants, and complement or genetically restore mutants.

Genetic Manipulation of Borrelia Spp.

The spirochetes Borrelia (Borreliella) burgdorferi and Borrelia hermsii, the etiologic agents of Lyme disease and relapsing fever, respectively, cycle in nature between an arthropod vector and a vertebrate host. They have extraordinarily unusual genomes that are highly segmented and predominantly linear. The genetic analyses of Lyme disease spirochetes have become increasingly more sophisticated, while the age of genetic investigation in the relapsing fever spirochetes is just dawning.

Interaction of the Lyme disease spirochete with its tick vector.

Borrelia burgdorferi, the causative agent of Lyme disease (along with closely related genospecies), is in the deeply branching spirochete phylum. The bacterium is maintained in nature in an enzootic cycle that involves transmission from a tick vector to a vertebrate host and acquisition from a vertebrate host to a tick vector. During its arthropod sojourn, B.

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

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.

Up-regulation of mRNA ventricular PRNP prion protein gene expression in air pollution highly exposed young urbanites: endoplasmic reticulum stress, glucose regulated protein 78, and nanosized particles.

Mexico City Metropolitan Area children and young adults exposed to high concentrations of air pollutants including fine and ultrafine particulate matter (PM) vs. clean air controls, exhibit myocardial inflammation and inflammasome activation with a differential right and left ventricular expression of key inflammatory genes and inflammasomes. We investigated the mRNA expression levels of the prion protein gene PRNP, which plays an important role in the protection against oxidative stress and metal toxicity, and the glucose regulated protein 78, a key protein in endoplasmic reticulum (ER) stress signaling, in ventricular autopsy samples from 30 children and young adults age 19.

An inverted repeat in the ospC operator is required for induction in Borrelia burgdorferi.

Borrelia burgdorferi, the spirochete that causes Lyme disease, differentially regulates synthesis of the outer membrane lipoprotein OspC to infect its host. OspC is required to establish infection but then repressed in the mammal to avoid clearance by the adaptive immune response. Inverted repeats (IR) upstream of the promoter have been implicated as an operator to regulate ospC expression.

Genetics of Borrelia burgdorferi.

The spirochetes in the Borrelia burgdorferi sensu lato genospecies group cycle in nature between tick vectors and vertebrate hosts. The current assemblage of B. burgdorferi sensu lato, of which three species cause Lyme disease in humans, originated from a rapid species radiation that occurred near the origin of the clade.

Borrelia burgdorferi malQ mutants utilize disaccharides and traverse the enzootic cycle.

Borrelia burgdorferi, the causative agent of Lyme disease, cycles in nature between a vertebrate host and a tick vector. We demonstrate that B. burgdorferi can utilize several sugars that may be available during persistence in the tick, including trehalose, N-acetylglucosamine (GlcNAc), and chitobiose.

Use of rpsL as a Counterselectable Marker in Borrelia burgdorferi.

We have demonstrated that rpsL, encoding the S12 protein of the small ribosomal subunit, can be used as a counterselectable marker in Borrelia burgdorferi, the causative agent of Lyme disease. Mutations in rpsL confer streptomycin resistance. Streptomycin susceptibility is dominant in an rpsL merodiploid, and streptomycin selects for the loss of wild-type rpsL carried in trans.

Ubiquitination of the bacterial inositol phosphatase, SopB, regulates its biological activity at the plasma membrane.

The Salmonella type III effector, SopB, is an inositol polyphosphate phosphatase that modulates host cell phospholipids at the plasma membrane and the nascent Salmonella-containing vacuole (SCV). Translocated SopB persists for many hours after infection and is ubiquitinated but the significance of this covalent modification has not been investigated. Here we identify by mass spectrometry six lysine residues of SopB that are mono-ubiquitinated.

Dynamic behavior of Salmonella-induced membrane tubules in epithelial cells.

Salmonella Typhimurium is a facultative intracellular pathogen that causes acute gastroenteritis in man. Intracellular Salmonella survive and replicate within a modified phagosome known as the Salmonella-containing vacuole (SCV). The onset of intracellular replication is accompanied by the appearance of membrane tubules, called Salmonella-induced filaments (Sifs), extending from the SCV.

Salmonella trafficking is defined by continuous dynamic interactions with the endolysosomal system.

Following invasion of non-phagocytic host cells, Salmonella enterica survives and replicates within a phagosome-like compartment known as the Salmonella-containing vacuole (SCV). It is now well established that SCV biogenesis, like phagosome biogenesis, involves sequential interactions with the endocytic pathway. However, Salmonella is believed to limit these interactions and, in particular, to avoid fusion of terminal lysosomes with the SCV.

The mechanism of Salmonella entry determines the vacuolar environment and intracellular gene expression.

Macrophages are an important intracellular niche for Salmonella particularly for systemic infection. The interaction of Salmonella with these cells is mediated by two type III secretion systems (TTSS), encoded on Salmonella pathogenicity islands 1 and 2 (SPI1, SPI2), which mediate distinct phases of the pathogen-host cell interaction. The SPI1 TTSS mediates invasion whereas the SPI2 TTSS is required for intramacrophage survival.

The Salmonella SPI1 effector SopB stimulates nitric oxide production long after invasion.

The ability of Salmonella enterica to invade and replicate within host cells depends on two type III secretion systems (TTSSs) encoded on pathogenicity islands 1 and 2 (SPI1 and SPI2). The current paradigm holds that these systems translocate two classes of effectors that operate sequentially and independently. In essence, the SPI1 TTSS mediates early events (i.