BackgroundOrnithodoros turicata is a veterinary and medically important argasid tick that is recognized as a vector of the relapsing fever spirochete Borrelia turicatae and African swine fever virus. Historic collections of O. turicata have been recorded from Latin America to the southern United States. However, the geographic distribution of this vector is poorly understood in relation to environmental variables, their hosts, and consequently the pathogens they transmit.MethodologyLocalities of O. turicata were generated by performing literature searches, evaluating records from the United States National Tick Collection and the Symbiota Collections of Arthropods Network, and by conducting field studies. Maximum entropy species distribution modeling (Maxent) was used to predict the current distribution of O. turicata. Vertebrate host diversity and GIS analyses of their distributions were used to ascertain the area of shared occupancy of both the hosts and vector.Conclusions and SignificanceOur results predicted previously unrecognized regions of the United States with habitat that may maintain O. turicata and could guide future surveillance efforts for a tick capable of transmitting high–consequence pathogens to human and animal populations.

BackgroundWith the global distribution, morbidity, and mortality associated with tick and louse-borne relapsing fever spirochetes, it is important to understand the dynamics of vector colonization by the bacteria and transmission to the host. Tick-borne relapsing fever spirochetes are blood-borne pathogens transmitted through the saliva of soft ticks, yet little is known about the transmission capability of these pathogens during the relatively short bloodmeal. This study was therefore initiated to understand the transmission dynamics of the relapsing fever spirochete Borrelia turicatae from the vector Ornithodoros turicata, and the subsequent dissemination of the bacteria upon entry into murine blood.Methodology/Principal FindingsTo determine the minimum number of ticks required to transmit spirochetes, one to three infected O. turicata were allowed to feed to repletion on individual mice. Murine infection and dissemination of the spirochetes was evaluated by dark field microscopy of blood, quantitative PCR, and immunoblotting against B. turicatae protein lysates and a recombinant antigen, the Borrelia immunogenic protein A. Transmission frequencies were also determined by interrupting the bloodmeal 15 seconds after tick attachment. Scanning electron microscopy (SEM) was performed on infected salivary glands to detect spirochetes within acini lumen and excretory ducts. Furthermore, spirochete colonization and dissemination from the bite site was investigated by feeding infected O. turicata on the ears of mice, removing the attachment site after engorment, and evaluating murine infection.Conclusion/SignificanceOur findings demonstrated that three ticks provided a sufficient infectious dose to infect nearly all animals, and B. turicatae was transmitted within seconds of tick attachment. Spirochetes were also detected in acini lumen of salivary glands by SEM. Upon host entry, B. turicatae did not require colonization of the bite site to establish murine infection. These results suggest that once B. turicatae colonizes the salivary glands the spirochetes are preadapted for rapid entry into the mammal.

The Lyme disease bacterium Borrelia burgdorferi survives diverse environmental challenges as it cycles between its tick vectors and various vertebrate hosts. B. burgdorferi must withstand prolonged periods of starvation while it resides in unfed Ixodes ticks. In this study, the regulatory protein DksA is shown to play a pivotal role controlling the transcriptional responses of B. burgdorferi to starvation. The results suggest that DksA gene regulatory activity impacts B. burgdorferi metabolism, virulence gene expression, and the ability of this bacterium to complete its natural life cycle.

Borrelia (Borreliella) burgdorferi, 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, B. burgdorferi wields three systems to regulate the expression of genes: the RpoN-RpoS alternative sigma (σ) factor cascade, the Hk1/Rrp1 twocomponent system and its product c-di-GMP, and the stringent response mediated by RelBbu and DksA. These regulatory systems respond to enzootic phase-specific signals and are controlled or finetuned by transcription factors, including BosR and BadR, as well as small RNAs, including DsrABb and Bb6S RNA. In addition, several other DNA-binding and RNA-binding proteins have been identified, although their functions have not all been defined. Global changes in gene expression revealed by highthroughput transcriptomic studies have elucidated various regulons, albeit technical obstacles have mostly limited this experimental approach to cultivated spirochetes. Regardless, we know that the spirochete, which carries a relatively small genome, regulates the expression of a considerable number of genes required for the transitions between the tick vector and the vertebrate host as well as the adaptation to each. caister.com/cimb 223 Curr. Issues Mol. Biol. Vol. 42 Curr. Issues Mol. Biol. 42: 223-266. caister.com/cimb Gene Regulation and Transcriptomics Samuels et al.Figure 2. A model of the RpoN-RpoS σ factor cascade. During tick feeding and mammalian infection, environmental and host signals activate the RpoN-RpoS σ factor cascade. Activation of RpoN requires phosphorylation of Rrp2 and accumulation of BosR. Rrp2 is the sole prokaryotic enhancer-binding protein present in B. burgdorferi that is required for RpoN (σ 54 ) activation. Phosphorylation of Rrp2 not only is required for RpoN-RpoS activation, but also is indispensable for cell survival, presumably replication. Levels of BosR respond to environmental signals and accumulation of BosR activates rpoS at its RpoN-dependent promoter via an unknown mechanism. BadR represses rpoS transcription by directly binding near the RpoN-dependent promoter region. In addition to the major rpoS mRNA species transcribed from the RpoN-dependent promoter, a longer rpoS transcript is produced at low cell density from an RpoN-independent promoter located within the upstream flgJ gene. The sRNA DsrABb regulates the efficiency of long rpoS mRNA species translation in response to temperature. DDB18 can regulate RpoS (σ S ) levels at the post-transcriptional level. Accumulation of rpoS transcript leads to the production of OspC, DbpA, DbpB, BBK32, and other mammalian infection-associated proteins.

Relapsing fever (RF) spirochetes colonize and are transmitted to mammals primarily by Ornithodoros ticks, and little is known regarding the pathogen's life cycle in the vector. To further understand vector colonization and transmission of RF spirochetes, Borrelia turicatae expressing a green fluorescent protein (GFP) marker (B. turicatae-gfp) was generated. The transformants were evaluated during the tick-mammal infectious cycle, from the third nymphal instar to adult stage. B. turicatae-gfp remained viable for at least 18 months in starved fourth-stage nymphal ticks, and the studies indicated that spirochete populations persistently colonized the tick midgut and salivary glands. Our generation of B. turicatae-gfp also revealed that within the salivary glands, spirochetes are localized in the ducts and lumen of acini, and after tick feeding, the tissues remained populated with spirochetes. The B. turicatae-gfp generated in this study is an important tool to further understand and define the mechanisms of vector colonization and transmission.IMPORTANCE In order to interrupt the infectious cycle of tick-borne relapsing fever spirochetes, it is important to enhance our understanding of vector colonization and transmission. Toward this, we generated a strain of Borrelia turicatae that constitutively produced the green fluorescent protein, and we evaluated fluorescing spirochetes during the entire infectious cycle. We determined that the midgut and salivary glands of Ornithodoros turicata ticks maintain the pathogens throughout the vector's life cycle and remain colonized with the spirochetes for at least 18 months. We also determined that the tick's salivary glands were not depleted after a transmission blood feeding. These findings set the framework to further understand the mechanisms of midgut and salivary gland colonization.

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

The relapsing fever spirochete Borrelia turicatae possesses a complex life cycle in its soft‐bodied tick vector, Ornithodoros turicata . Spirochetes enter the tick midgut during a blood meal, and, during the following weeks, spirochetes disseminate throughout O. turicata . A population persists in the salivary glands allowing for rapid transmission to the mammalian hosts during tick feeding. Little is known about the physiological environment within the salivary glands acini in which B. turicatae persists. In this study, we examined the salivary gland transcriptome of O. turicata ticks and detected the expression of 57 genes involved in oxidant metabolism or antioxidant defences. We confirmed the expression of five of the most highly expressed genes, including glutathione peroxidase ( gpx ), thioredoxin peroxidase ( tpx ), manganese superoxide dismutase ( sod‐1 ), copper‐zinc superoxide dismutase ( sod‐2 ), and catalase ( cat ) by reverse‐transcriptase droplet digital polymerase chain reaction (RT‐ddPCR). We also found distinct differences in the expression of these genes when comparing the salivary glands and midguts of unfed O. turicata ticks. Our results indicate that the salivary glands of unfed O. turicata nymphs are highly oxidative environments where reactive oxygen species (ROS) predominate, whereas midgut tissues comprise a primarily nitrosative environment where nitric oxide synthase is highly expressed. Additionally, B. turicatae was found to be hyperresistant to ROS compared with the Lyme disease spirochete Borrelia burgdorferi , suggesting it is uniquely adapted to the highly oxidative environment of O. turicata salivary gland acini.

The Lyme disease spirochete Borrelia burgdorferi exhibits dramatic changes in gene expression as it transits between its tick vector and vertebrate host. A major hurdle to understanding the mechanisms underlying gene regulation in B. burgdorferi has been the lack of a functional assay to test how gene regulatory proteins and sigma factors interact with RnA polymerase to direct transcription. to gain mechanistic insight into transcriptional control in B. burgdorferi, and address sigma factor function and specificity, we developed an in vitro transcription assay using the B. burgdorferi RnA polymerase holoenzyme. We established reaction conditions for maximal RnA polymerase activity by optimizing pH, temperature, and the requirement for divalent metals. Using this assay system, we analyzed the promoter specificity of the housekeeping sigma factor RpoD to promoters encoding previously identified RpoD consensus sequences in B. burgdorferi. Collectively, this study established an in vitro transcription assay that revealed RpoD-dependent promoter selectivity by RNA polymerase and the requirement of specific metal cofactors for maximal RNA polymerase activity. The establishment of this functional assay will facilitate molecular and biochemical studies on how gene regulatory proteins and sigma factors exert control of gene expression in B. burgdorferi required for the completion of its enzootic cycle. Borrelia burgdorferi is a highly fastidious host-associated bacterium in the phylum Spirochaetes 1,2. B. burgdorferi is adapted to a vector-host life cycle and possesses a condensed 1.3 Mb genome with limited metabolic capability 3,4. The genome lacks genes encoding components of the citric acid cycle, the electron transport chain, amino acid biosynthesis, and fatty acid biosynthesis pathways, wholly relying on the transport of sugars, fatty acids and amino acids from the environment for survival 5,6. B. burgdorferi growth occurs extracellularly in vertebrate tissues and in ticks following a blood meal, which poses additional nutritional constraints 7. In particular, the host competition for iron is hypothesized to have resulted in abstinence from iron utilization by B. burgdorferi 8-10. Instead, manganese is thought to largely replace iron as a metal cofactor for metabolic enzymes 9,11. B. burgdorferi regulates gene expression to adapt to environmental constraints faced in its enzootic cycle. B. burgdorferi responds to environmental changes in pH, temperature, nutrient availability, and manganese levels with dramatic shifts in transcription and growth 12-18. The transcriptional changes in B. burgdorferi during transmission from the arthropod vector Ixodes scapularis 19,20 are thought to be influenced by the mechanisms underlying environmental sensing and transcriptional responses. Mechanistic studies of how transcription factors regulate gene expression in B. burgdorferi have been hindered by the scarcity of biochemical tools. To understand the transcriptional mechanisms that support B. burgdorferi survival, we set out to ...