Pilot examination of independent and interactive effects of maltreatment and neighborhood risk on child attachment.

Elucidating the influence of microsystem and exosystem factors on development is an important goal of developmental psychopathology. This study examined the effects of maltreatment and neighborhood risk on child-caregiver attachment. Maltreatment records, neighborhood risk indices, and Strange Situation data were collected from a diverse sample of 170 four-year-old children and their caregivers. Relative contributions of maltreatment, neighborhood risk, and their interaction on attachment insecurity and disorganization were explored via latent moderation. Maltreated children demonstrated higher rates of insecure attachment, but not attachment disorganization, independent of neighborhood risk. Controlling for maltreatment, preliminary results suggested no effects of neighborhood risk on attachment. Findings support prior research that has identified maltreatment as a salient risk to the formation of secure attachment relationships. However, results add heterogeneity to the limited research investigating effects of neighborhood on attachment. Overall, this study highlights the importance of examining multilevel ecological risk in relation to attachment relationship development.

2-Stage microfermentations.

Cell based factories can be engineered to produce a wide variety of products. Advances in DNA synthesis and genome editing have greatly simplified the design and construction of these factories. It has never been easier to generate hundreds or even thousands of cell factory strain variants for evaluation. These advances have amplified the need for standardized, higher throughput means of evaluating these designs. Toward this goal, we have previously reported the development of engineered E. coli strains and associated 2-stage production processes to simplify and standardize strain engineering, evaluation and scale up. This approach relies on decoupling growth (stage 1), from production, which occurs in stationary phase (stage 2). Phosphate depletion is used as the trigger to stop growth as well as induce heterologous expression. Here, we describe in detail the development of protocols for the evaluation of engineered E. coli strains in 2-stage microfermentations. These protocols are readily adaptable to the evaluation of strains producing a wide variety of protein as well as small molecule products. Additionally, by detailing the approach to protocol development, these methods are also adaptable to additional cellular hosts, as well as other 2-stage processes with various additional triggers.

Phase separation methods for protein purification: A meta-analysis of purification performance and cost-effectiveness.

Protein purifications based on phase separations (e.g., precipitation and liquid-liquid extraction) have seen little adoption in commercial protein drug production. To identify barriers, we analyzed the purification performance and economics of 290 phase separation purifications from 168 publications. First, we found that studies using Design of Experiments for optimization achieved significantly greater mean yield and host cell protein log10 removal values than those optimizing one factor at a time (11.5% and 53% increases, respectively). Second, by modeling each reported purification at scales from 10 to 10,000 kg product/year and comparing its cost-effectiveness versus chromatography, we found that cost-effectiveness depends strongly on scale: the fraction of phase separations predicted to be cost-effective at the 10, 100, and 1000 kg/year scales was 8%, 15%, and 43%, respectively. Total cost per unit product depends inversely on input purity, with phase separation being cheaper than chromatography at the 100 kg/year scale in 100% of cases where input purity was ≤ 1%, compared to about 25% of cases in the dataset as a whole. Finally, we identified a simple factor that strongly predicts phase separation process costs: the mass ratio of reagents versus purified product (the "direct materials usage rate"), which explains up to 58% of variation in cost per unit of purified product among all 290 reports, and up to 98% of variation within particular types of phase separation.

A telemedicine bridge clinic improves access and reduces cost for opioid use disorder care.

Objective: We evaluated the impact of a telemedicine bridge clinic on treatment outcomes and cost for patients with opioid use disorder. Telemedicine bridge clinics deliver low-barrier rapid assessment of patients with opioid use disorder via audio-only and audiovisual telemedicine to facilitate induction on medication therapy and connection to ongoing care. Methods: A pre-post analysis of UPMC Health Plan member claims was performed to evaluate the impact of this intervention on the trajectory of care for patients with continuous coverage before and after bridge clinic visit(s). Results: Analysis included 150 UPMC Health Plan members evaluated at the bridge clinic between April 2020 and October 2021. At least one buprenorphine prescription was filled within 30 days by 91% of patients; median proportion of days covered by buprenorphine was 73.3%, 54.4%, and 50.6% at 30, 90, and 180 days after an initial visit compared to median of no buprenorphine claims 30 days prior among the same patients. Patients had an 18% decline in unplanned care utilization 30 days after initial Bridge Clinic visit, with a 62% reduction in unplanned care cost per member per month (PMPM), 38% reduction in medical cost PMPM, and 10% reduction in total PMPM (medical + pharmacy cost) at 180 days. Primary care, outpatient behavioral health, and laboratory costs increased while emergency department, urgent care, and inpatient costs declined. Conclusion: Utilization of a telemedicine bridge clinic was associated with buprenorphine initiation, linkage to ongoing care with retention including medication treatment, reduced unplanned care cost, and overall savings.

Trade-offs, trade-ups, and high mutational parallelism underlie microbial adaptation during extreme cycles of feast and famine.

Microbes are evolutionarily robust organisms capable of rapid adaptation to complex stress, which enables them to colonize harsh environments. In nature, microbes are regularly challenged by starvation, which is a particularly complex stress because resource limitation often co-occurs with changes in pH, osmolarity, and toxin accumulation created by metabolic waste. Often overlooked are the additional complications introduced by eventual resource replenishment, as successful microbes must withstand rapid environmental shifts before swiftly capitalizing on replenished resources to avoid invasion by competing species. To understand how microbes navigate trade-offs between growth and survival, ultimately adapting to thrive in environments with extreme fluctuations, we experimentally evolved 16 Escherichia coli populations for 900 days in repeated feast/famine conditions with cycles of 100-day starvation before resource replenishment. Using longitudinal population-genomic analysis, we found that evolution in response to extreme feast/famine is characterized by narrow adaptive trajectories with high mutational parallelism and notable mutational order. Genetic reconstructions reveal that early mutations result in trade-offs for biofilm and motility but trade-ups for growth and survival, as these mutations conferred positively correlated advantages during both short-term and long-term culture. Our results demonstrate how microbes can navigate the adaptive landscapes of regularly fluctuating conditions and ultimately follow mutational trajectories that confer benefits across diverse environments.

A Novel Tiled Amplicon Sequencing Assay Targeting the Tomato Brown Rugose Fruit Virus (ToBRFV) Genome Reveals Widespread Distribution in Municipal Wastewater Treatment Systems in the Province of Ontario, Canada.

Tomato Brown Rugose Fruit Virus (ToBRFV) is a plant pathogen that infects important Solanaceae crop species and can dramatically reduce tomato crop yields. The ToBRFV has rapidly spread around the globe due to its ability to escape detection by antiviral host genes which confer resistance to other tobamoviruses in tomato plants. The development of robust and reproducible methods for detecting viruses in the environment aids in the tracking and reduction of pathogen transmission. We detected ToBRFV in municipal wastewater influent (WWI) samples, likely due to its presence in human waste, demonstrating a widespread distribution of ToBRFV in WWI throughout Ontario, Canada. To aid in global ToBRFV surveillance efforts, we developed a tiled amplicon approach to sequence and track the evolution of ToBRFV genomes in municipal WWI. Our assay recovers 95.7% of the 6393 bp ToBRFV RefSeq genome, omitting the terminal 5' and 3' ends. We demonstrate that our sequencing assay is a robust, sensitive, and highly specific method for recovering ToBRFV genomes. Our ToBRFV assay was developed using existing ARTIC Network resources, including primer design, sequencing library prep, and read analysis. Additionally, we adapted our lineage abundance estimation tool, Alcov, to estimate the abundance of ToBRFV clades in samples.

A qualitative assessment of emergency physicians' experiences with robust emergency department buprenorphine bridge programs.

OBJECTIVES: Emergency departments (EDs) are a critical point of entry into treatment for patients struggling with opioid use disorder (OUD). When initiated in the ED, buprenorphine is associated with increased addiction treatment engagement at 30 days when initiated. Despite this association, it has had slow adoption. The barriers to ED buprenorphine utilization are well documented; however, the benefits of prescribing buprenorphine for emergency physicians (EPs) have not been explored. This study utilized semistructured interviews to explore and understand how EPs perceive their experiences working in EDs that have successfully implemented ED bridge programs (EDBPs) for patients with OUD. METHODS: Semistructured interviews were conducted with EPs from four geographically diverse academic hospitals with established EDBPs. Interviews were recorded and transcribed, and emergent themes were identified using codebook thematic analysis. Analysis credibility and transparency were confirmed with peer debriefing. RESULTS: Twenty-two interviews were conducted across the four sites. Three key themes were constructed during the analyses: (1) provided EPs agency; (2) transformed EPs' emotions, attitudes, and behaviors related to treating patients with OUD; and (3) improved EPs' professional quality of life. CONCLUSIONS: Participants in this study reported several common themes related to participation in their hospital's BP. Overall our results suggest that physicians who participate in EDBPs may feel a renewed sense of fulfillment and purpose in their personal and professional lives. These positive changes may lead to increased job satisfaction in hospitals that have successfully launched EDBP.

Safety and immunogenicity of an adjuvanted recombinant spike protein-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine, SpikeVet™, in selected Carnivora, Primates and Artiodactyla in Australian zoos.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect a broad range of animal species and has been associated with severe disease in some taxa. Few studies have evaluated optimal strategies to mitigate the risk to susceptible zoo animals. This study evaluated the safety and immunogenicity of a protein-based veterinary SARS-CoV-2 vaccine (SpikeVet™) in zoo animals. Two to three doses of SpikeVet™ were administered intramuscularly or subcutaneously 3-4 weeks apart to 354 zoo animals representing 38 species. SpikeVet™ was very well tolerated across all species. Minor adverse effects were observed in 1.69% of animals vaccinated, or 1.04% of vaccine doses administered. Preliminary immunogenicity analyses in representative carnivores (meerkats, lions) and an artiodactylid (domestic goat) showed SpikeVet™-immunized animals developed serum antibodies able to neutralize a range of SARS-CoV-2 variants, including the vaccine-homologous Wuhan and Mu variants, as well as vaccine-heterologous Omicron BA.2 and XBB.1 strains. Prior to vaccination, all eight lions were seropositive for Wuhan strain by surrogate viral neutralization testing, suggesting past infection with SARS-CoV-2 or cross-reactive antibodies generated by another closely related coronavirus. These results from a range of zoo species support the ongoing development of SpikeVet™ as a safe and effective veterinary SARS-CoV-2 vaccine.

Both Las17-binding sites on Arp2/3 complex are important for branching nucleation and assembly of functional endocytic actin networks in S. cerevisiae.

Arp2/3 complex nucleates branched actin filaments that drive membrane invagination during endocytosis and leading-edge protrusion in lamellipodia. Arp2/3 complex is maximally activated in vitro by binding of a WASP family protein to two sites-one on the Arp3 subunit and one spanning Arp2 and ARPC1-but the importance of each site in the regulation of force-producing actin networks is unclear. Here, we identify mutations in budding yeast Arp2/3 complex that decrease or block engagement of Las17, the budding yeast WASP, at each site. As in the mammalian system, both sites are required for maximal activation in vitro. Dimerization of Las17 partially restores activity of mutations at both CA-binding sites. Arp2/3 complexes defective at either site assemble force-producing actin networks in a bead motility assay, but their reduced activity hinders motility by decreasing actin assembly near the bead surface and by failing to suppress actin filament bundling within the networks. While even the most defective Las17-binding site mutants assembled actin filaments at endocytic sites, they showed significant internalization defects, potentially because they lack the proper architecture to drive plasma membrane remodeling. Together, our data indicate that both Las17-binding sites are important to assemble functional endocytic actin networks in budding yeast, but Arp2/3 complex retains some activity in vitro and in vivo even with a severe defect at either Las17-binding site.

Mitogenomic architecture and evolution of the soil ciliates Colpoda.

Colpoda are cosmopolitan unicellular eukaryotes primarily inhabiting soil and benefiting plant growth, but they remain one of the least understood taxa in genetics and genomics within the realm of ciliated protozoa. Here, we investigate the architecture of de novo assembled mitogenomes of six Colpoda species, using long-read sequencing and involving 36 newly isolated natural strains in total. The mitogenome sizes span from 43 to 63 kbp and typically contain 28-33 protein-coding genes. They possess a linear structure with variable telomeres and central repeats, with one Colpoda elliotti strain isolated from Tibet harboring the longest telomeres among all studied ciliates. Phylogenomic analyses reveal that Colpoda species started to diverge more than 326 million years ago, eventually evolving into two distinct groups. Collinearity analyses also reveal significant genomic divergences and a lack of long collinear blocks. One of the most notable features is the exceptionally high level of gene rearrangements between mitochondrial genomes of different Colpoda species, dominated by gene loss events. Population-level mitogenomic analysis on natural strains also demonstrates high sequence divergence, regardless of geographic distance, but the gene order remains highly conserved within species, offering a new species identification criterion for Colpoda species. Furthermore, we identified underlying heteroplasmic sites in the majority of strains of three Colpoda species, albeit without a discernible recombination signal to account for this heteroplasmy. This comprehensive study systematically unveils the mitogenomic structure and evolution of these ancient and ecologically significant Colpoda ciliates, thus laying the groundwork for a deeper understanding of the evolution of unicellular eukaryotes.IMPORTANCEColpoda, one of the most widespread ciliated protozoa in soil, are poorly understood in regard to their genetics and evolution. Our research revealed extreme mitochondrial gene rearrangements dominated by gene loss events, potentially leading to the streamlining of Colpoda mitogenomes. Surprisingly, while interspecific rearrangements abound, our population-level mitogenomic study revealed a conserved gene order within species, offering a potential new identification criterion. Phylogenomic analysis traced their lineage over 326 million years, revealing two distinct groups. Substantial genomic divergence might be associated with the lack of extended collinear blocks and relaxed purifying selection. This study systematically reveals Colpoda ciliate mitogenome structures and evolution, providing insights into the survival and evolution of these vital soil microorganisms.

Detection and Differentiation of Entomopathogenic Serratia spp. to Inform Reintroduction of the Critically Endangered Lord Howe Island Stick Insect Dryococelus australis.

Once rodents have been successfully eradicated from Lord Howe Island, Australia, the critically endangered Lord Howe Island stick insect (Dryococelus australis (Montrouzier)) may be reintroduced, a century after it was thought to have become extinct. In captive populations of D. australis, elevated mortalities have been associated with bacterial pathogens. To better define the infectious risk posed by entomopathogens to the reintroduction program, we investigated the bacteria isolated from captive D. australis kept at Melbourne Zoo and on Lord Howe Island and from environmental samples and free-living invertebrates collected on various parts of the island. At Melbourne Zoo, Serratia and Pseudomonas spp. were the bacteria most frequently isolated between 2013 and 2019. Serratia spp. were also the organisms most frequently isolated from insects sampled in April 2019 from the captive population on Lord Howe Island. In addition, Serratia spp. were isolated from a range of environmental samples collected on Lord Howe Island during March-April 2019. These environmental isolates had a broader range of biochemical and molecular characteristics than those obtained from the captive insect populations. A large proportion of these isolates were urease positive and had biochemical profiles previously not described for Serratia spp. This study highlights the need for better surveillance for potential pathogens in understudied regions and sites. We conclude that infections caused by Serratia spp. might pose a problem to the captive breeding program for D. australis but that the risk of introducing novel pathogens to Lord Howe Island through infected insects is low. Our study explores some of the potential risks involved in captive breeding and provides a valuable example of using pathogen surveillance to better inform an invertebrate conservation program.

Toxoplasma gondii Does Not Inhibit the Assisted Colonization of Eastern Barred Bandicoots (Perameles gunnii) to Phillip Island, Victoria, Australia.

Eastern barred bandicoots (Perameles gunnii) are thought to be highly susceptible to disease caused by infection with the protozoan parasite Toxoplasma gondii. This study followed a population of 67 P. gunnii introduced onto the Summerland Peninsula, Phillip Island, Australia, where the prevalence of T. gondii infection in the feral cat population was known to be very high. Prior to release, bandicoots were tested for serologic exposure to T. gondii using the modified agglutination test. A subset of bandicoots was tested on four occasions after release onto the peninsula. No seroconversion was detected at any time point. A subset of bandicoots was radiotracked after release and at two additional trapping sessions to help monitor survival. Toxoplasma gondii DNA was not detected by PCR in eight carcasses recovered for necropsy. Fourteen founder bandicoots (21% of founders) were known to be alive at 500 d post-release. A total of 29 unmarked bandicoots were trapped over the study period, confirming that the bandicoots were successfully reproducing on the island. Body weight, packed cell volume, and total plasma protein were used as measures of individual animal health; population health was inferred from these data. Body weight was significantly associated with trip number, with a general trend of increasing weight after release onto the island. This study showed that eastern barred bandicoots were able to establish a new population despite a probably high environmental load of T. gondii.

The use of buprenorphine to-go packs in the emergency department.

OBJECTIVE: Buprenorphine is an effective treatment for opioid use disorder (OUD). Patients in the emergency department (ED) can be initiated or continued on buprenorphine as a bridge to follow-up in the outpatient setting, but gaps in care may arise. The objective was to evaluate the impact of buprenorphine to-go packs as a continuing treatment option for patients presenting to the ED with OUD across a health system. METHODS: Adult patients discharged with a buprenorphine to-go pack from one of ten EDs within a major health system were included. The primary outcomes assessed within 30 days of ED discharge were: (1) return to a health system ED, and (2) fill history of buprenorphine in the state prescription drug monitoring program database. Data was analyzed using descriptive statistics in Microsoft Excel (Redmond, WA). RESULTS: A total of 124 patients received buprenorphine to-go packs. The sample was primarily male (79; 63.7%), white (89; 71.8%), on Medicaid (79; 63.7%), and had a mean age of 40.9 years. A total of 43 patients (34.7%) were initiated on buprenorphine for the first time, while 81 (65.3%) had received buprenorphine (prescription or to-go) previously. At 30 days post-visit, 76 (61.3%) had filled buprenorphine prescriptions, and 40 (32.3%) returned to an ED within the health system for opioid withdrawal (17; 42.5%), non-OUD-related reasons (22; 55%), or overdose (1; 2.5%). CONCLUSION: The implementation of a system-wide buprenorphine to-go supply at ED discharge is a feasible option to provide continuity of care to patients with OUD.

Lysinoalanine cross-linking is a conserved post-translational modification in the spirochete flagellar hook.

Spirochetes cause Lyme disease, leptospirosis, syphilis, and several other human illnesses. Unlike other bacteria, spirochete flagella are enclosed within the periplasmic space where the filaments distort and push the cell body by the action of the flagellar motors. We previously demonstrated that the oral pathogen Treponema denticola (Td) and Lyme disease pathogen Borreliella burgdorferi (Bb) form covalent lysinoalanine (Lal) cross-links between conserved cysteine and lysine residues of the FlgE protein that composes the flagellar hook. In Td, Lal is unnecessary for hook assembly but is required for motility, presumably due to the stabilizing effect of the cross-link. Herein, we extend these findings to other, representative spirochete species across the phylum. We confirm the presence of Lal cross-linked peptides in recombinant and in vivo-derived samples from Treponema spp., Borreliella spp., Brachyspira spp., and Leptospira spp. As was observed with Td, a mutant strain of Bb unable to form the cross-link has greatly impaired motility. FlgE from Leptospira spp. does not conserve the Lal-forming cysteine residue which is instead substituted by serine. Nevertheless, Leptospira interrogans FlgE also forms Lal, with several different Lal isoforms being detected between Ser-179 and Lys-145, Lys-148, and Lys-166, thereby highlighting species or order-specific differences within the phylum. Our data reveal that the Lal cross-link is a conserved and necessary posttranslational modification across the spirochete phylum and may thus represent an effective target for the development of spirochete-specific antimicrobials.

Contribution of the SOS response and the DNA repair systems to norfloxacin induced mutations in E. coli.

Antibiotic-resistant bacteria severely threaten human health. Besides spontaneous mutations generated by endogenous factors, the resistance might also originate from mutations induced by certain antibiotics, such as the fluoroquinolones. Such antibiotics increase the genome-wide mutation rate by introducing replication errors from the SOS response pathway or decreasing the efficiency of the DNA repair systems. However, the relative contributions of these molecular mechanisms remain unclear, hindering understanding of the generation of resistant pathogens. Here, using newly-accumulated mutations of wild-type and SOS-uninducible Escherichia coli strains, as well as those of the strains deficient for the mismatch repair (MMR) and the oxidative damage repair pathways, we find that the SOS response is the major mutagenesis contributor in mutation elevation, responsible for ~ 30-50% of the total base-pair substitution (BPS) mutation-rate elevation upon treatment with sublethal levels of norfloxacin (0 ~ 50 ng/mL). We further estimate the significance of the effects on other mutational features of these mechanisms (i.e., transversions, structural variations, and mutation spectrum) in E. coli using linear models. The SOS response plays a positive role in all three mutational features (mutation rates of BPSs, transversions, structural variations) and affects the mutational spectrum. The repair systems significantly reduce the BPS mutation rate and the transversion rate, regardless of whether antibiotics are present, while significantly increasing the structural variation rate in E. coli. Our results quantitatively disentangle the contributions of the SOS response and DNA repair systems in antibiotic-induced mutagenesis. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00185-y.

An integrative protocol for one-step PCR amplicon library construction and accurate demultiplexing of pooled sequencing data.

High-throughput sequencing of amplicons has been widely used to precisely and efficiently identify species compositions and analyze community structures, greatly promoting biological studies involving large amounts of complex samples, especially those involving environmental and pathogen-monitoring ones. Commercial library preparation kits for amplicon sequencing, which generally require multiple steps, including adapter ligation and indexing, are expensive and time-consuming, especially for applications at a large scale. To overcome these limitations, a "one-step PCR approach" has been previously proposed for constructions of amplicon libraries using long fusion primers. However, efficient amplifications of target genes and accurate demultiplexing of pooled sequencing data remain to be addressed. To tackle these, we present an integrative protocol for one-step PCR amplicon library construction (OSPALC). High-quality reads have been generated by this approach to reliably identify species compositions of mock bacterial communities and environmental samples. With this protocol, the amplicon library is constructed through one regular PCR with long primers, and the total cost per DNA/cDNA sample decreases to just 7% of the typical cost via the multi-step PCR approach. Empirically tested primers and optimized PCR conditions to construct OSPALC libraries for 16S rDNA V4 regions are demonstrated as a case study. Tools to design primers targeting at any genomic regions are also presented. In principle, OSPALC can be readily applied to construct amplicon libraries of any target genes using DNA or RNA samples, and will facilitate research in numerous fields. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00182-1.

Evolutionary Genomics of Sister Species Differing in Effective Population Sizes and Recombination Rates.

Studies of closely related species with known ecological differences provide exceptional opportunities for understanding the genetic mechanisms of evolution. In this study, we compared population-genomics data between Daphnia pulex and Daphnia pulicaria, two reproductively compatible sister species experiencing ecological speciation, the first largely confined to intermittent ponds and the second to permanent lakes in the same geographic region. Daphnia pulicaria has lower genome-wide nucleotide diversity, a smaller effective population size, a higher incidence of private alleles, and a substantially more linkage disequilibrium than D. pulex. Positively selected genes in D. pulicaria are enriched in potentially aging-related categories such as cellular homeostasis, which may explain the extended life span in D. pulicaria. We also found that opsin-related genes, which may mediate photoperiodic responses, are under different selection pressures in these two species. Genes involved in mitochondrial functions, ribosomes, and responses to environmental stimuli are found to be under positive selection in both species. Additionally, we found that the two species have similar average evolutionary rates at the DNA-sequence level, although approximately 160 genes have significantly different rates in the two lineages. Our results provide insights into the physiological traits that differ within this regionally sympatric sister-species pair that occupies unique microhabitats.

A chemosensory-like histidine kinase is dispensable for chemotaxis in vitro but regulates the virulence of Borrelia burgdorferi through modulating the stability of RpoS.

As an enzootic pathogen, the Lyme disease bacterium Borrelia burgdorferi possesses multiple copies of chemotaxis proteins, including two chemotaxis histidine kinases (CHK), CheA1 and CheA2. Our previous study showed that CheA2 is a genuine CHK that is required for chemotaxis; however, the role of CheA1 remains mysterious. This report first compares the structural features that differentiate CheA1 and CheA2 and then provides evidence to show that CheA1 is an atypical CHK that controls the virulence of B. burgdorferi through modulating the stability of RpoS, a key transcriptional regulator of the spirochete. First, microscopic analyses using green-fluorescence-protein (GFP) tags reveal that CheA1 has a unique and dynamic cellular localization. Second, loss-of-function studies indicate that CheA1 is not required for chemotaxis in vitro despite sharing a high sequence and structural similarity to its counterparts from other bacteria. Third, mouse infection studies using needle inoculations show that a deletion mutant of CheA1 (cheA1mut) is able to establish systemic infection in immune-deficient mice but fails to do so in immune-competent mice albeit the mutant can survive at the inoculation site for up to 28 days. Tick and mouse infection studies further demonstrate that CheA1 is dispensable for tick colonization and acquisition but essential for tick transmission. Lastly, mechanistic studies combining immunoblotting, protein turnover, mutagenesis, and RNA-seq analyses reveal that depletion of CheA1 affects RpoS stability, leading to reduced expression of several RpoS-regulated virulence factors (i.e., OspC, BBK32, and DbpA), likely due to dysregulated clpX and lon protease expression. Bulk RNA-seq analysis of infected mouse skin tissues further show that cheA1mut fails to elicit mouse tnf-α, il-10, il-1ß, and ccl2 expression, four important cytokines for Lyme disease development and B. burgdorferi transmigration. Collectively, these results reveal a unique role and regulatory mechanism of CheA1 in modulating virulence factor expression and add new insights into understanding the regulatory network of B. burgdorferi.