Jason A. Carlyon

Canine and human anaplasmosis are tick-borne diseases caused by the obligate intracellular bacteria, Anaplasma phagocytophilum and Anaplasma platys , respectively. In veterinary medicine, peptides derived from the immunodominant P44 protein are used to detect antibodies in serological point-of-care tests for anaplasmosis. To inform efforts to develop improved chimeric diagnostic proteins for anaplasmosis, the structure of the predicted surface-exposed domain (residues 151–308) of P44 was determined at a resolution of 1.65 Å. This domain comprises six α-helices and six short β-strands connected by loop regions. A structural similarity search of the P44 _151–308 domain using the Distance Matrix Alignment server against the AlphaFold Database revealed that the domain represents a previously uncharacterized protein fold. The determined structure is consistent with AlphaFold v2 predictions, which further predict the core of P44 to be an eight-stranded β-barrel. Using P44 fragments, immunodominant epitopes were mapped within residues 151–308. Four chimeric genes (ADC1, ADC2, ADC3, and ADC4) were designed that incorporate the analogous immunodominant P44 domains from A. phagocytophilum and A. platys . The recombinant ADC chimerics were assessed for sensitivity and specificity by screening with serum from dogs diagnosed with anaplasmosis, Lyme disease, or ehrlichiosis, with sera from specific pathogen-free dogs serving as negative controls. Accuracy was further demonstrated by screening characterized serum samples from coyotes ( Canis latrans ; wild canids). The ADC1 chimeric provided 100% sensitivity and 98.04% specificity. The sensitivity and specificity of ADC1 support its potential use as a diagnostic Ag for anaplasmosis.

Anaplasma phagocytophilum is an obligate intracellular gram-negative bacterium and etiologic agent of human granulocytic anaplasmosis. A. phagocytophilum genomic sequencing has historically been performed via short-read platforms. Our optimized bacterial isolation protocol combined with Nanopore sequencing produced a single, closed 1,481,805 bp circular A. phagocytophilum strain NCH-1 chromosome.

Orientia tsutsugamushi is a mite-transmitted obligate intracellular bacterium that causes the potentially deadly zoonosis, scrub typhus. The absence of genetic tools for Orientia has limited studies of the microbe-host interactions that underlie scrub typhus. To address this gap, we developed a protocol for transforming and achieving allelic exchange in O. tsutsugamushi str. Ikeda. From evaluating multiple cell lines and antibiotics, we found that contact-inhibited EA.hy926 human endothelial-like cells best supported Orientia replication, and that chloramphenicol was an effective selection marker. We engineered a homologous recombination cassette encoding a codon-modified version of the O. tsutsugamushi ank13 gene (OTT_RS04140) (CM ank13 ) and its promoter alongside genes for mScarlet-I and chloramphenicol acetyltransferase under control of the O. tsutsugamushi tsa22 -up and tsa56 -down promoters, respectively. A PCR product encompassing the cassette and chromosomal flanking regions was transformed into O. tsutsugamushi via electroporation or CaCl ₂ , the latter of which better preserved bacterial and host cell viability. EA.hy926 cells inoculated with transformed O. tsutsugamushi were grown in glass-bottom plates in the presence of chloramphenicol and imaged by live-cell microscopy to identify cultures containing mScarlet-I-positive bacteria, which could be maintained in perpetuity. Chromosomal integration of the CM ank13 cassette and loss of wild-type ank13 were verified by PCR and nanopore sequencing. This report establishes platforms for genetically manipulating O. tsutsugamushi and building additional genetic tools to investigate this globally significant pathogen. IMPORTANCE: Orientia tsutsugamushi causes scrub typhus, a globally emerging rickettsiosis that can have a high mortality rate and has been a known human disease since the fourth century. Of the genera of obligate intracellular bacterial pathogens that cause human disease, Orientia is the only one for which genetic tools have not been developed. This has limited understanding of O. tsutsugamushi -host dynamics that drive the bacterium’s pathobiology and hinder the development of novel treatment or protection strategies against scrub typhus. Here, we successfully transformed and achieved allelic exchange in O. tsutsugamushi . Transgenic bacteria were selected via antibiotic resistance, validated by PCR and nanopore sequencing, and visualized by immunofluorescence and live-cell fluorescence imaging. Our report includes detailed descriptions of empirically determined host cell cultivation, multiplicity of infection, transformation, and selection conditions to provide a foundation on which other researchers can build. Overall, this work begins to establish a genetic toolbox for O. tsutsugamushi .

Orientia tsutsugamushi is a mite-transmitted obligate intracellular bacterium that causes the potentially deadly zoonosis, scrub typhus. The absence of genetic tools for Orientia have limited studies of the microbe-host interactions that underlie scrub typhus. To address this gap, we developed a protocol for transforming and achieving allelic exchange in O. tsutsugamushi str. Ikeda. From evaluating multiple cell lines and antibiotics, we found that contact-inhibited EA.hy926 human endothelial-like cells best supported Orientia replication and that chloramphenicol was an effective selection marker. We engineered a homologous recombination cassette encoding a codon-modified version of the O. tsutsugamushi ank13 gene (OTT_RS04140) (CM ank13 ) and its promoter alongside genes for mScarlet-I and chloramphenicol acetyltransferase under control of the O. tsutsugamushi tsa22 -up and tsa56 -down promoters, respectively. A PCR product encompassing the cassette and chromosomal flanking regions was transformed into O. tsutsugamushi via electroporation or CaCl ₂ , the latter of which better preserved bacterial and host cell viability. EA.hy926 cells inoculated with transformed O. tsutsugamushi were grown in glass-bottom plates in the presence of chloramphenicol and imaged by live-cell microscopy to identify cultures containing mScarlet-I positive bacteria, which could be maintained in perpetuity. Chromosomal integration of the CM ank13 cassette and loss of wild-type ank13 were verified by PCR and nanopore sequencing. This report establishes platforms for genetically manipulating O. tsutsugamushi and building additional genetic tools to investigate this globally significant pathogen.

Nuclear factor-κB (NF-κB) nuclear localization and gene accessibility is mediated by Ser22-phosphorylated lamin A/C (pSer22-lamin A/C). Here, we report that the intracellular bacterium Orientia tsutsugamushi impairs NF-κB nuclear accumulation by targeting lamin A using multiple ankyrin repeat (AR)-containing effectors (Anks). The Anks' immunomodulatory capability requires a conserved hydrophilic α-helical peptide that binds lamin A/pSer22-lamin A and lies between the AR and PRANC (pox proteins repeats of ankyrin C-terminal) domains. Orientia promotes pSer22-lamin A redistribution from the lamina to the nucleoplasm. This phenotype can be recapitulated in uninfected cells by ectopically expressing Anks that carry the lamin A-binding sequence. O. tsutsugamushi also alters chromatin accessibility at sites regulated by lamin A, NF-κB, and the NF-κB coactivator, adapter protein 1. These findings identify a mechanism by which a pathogen synergistically modulates lamin A and chromatin accessibility to counteract NF-κB, reinforce the regulatory link between lamin A and NF-κB, and indicate that pSer22-lamin A enhances chromatin accessibility to NF-κB and its coactivators.

Anaplasma phagocytophilum is an obligate intracellular bacterium that invades neutrophils to cause the potentially severe infection, human granulocytic anaplasmosis. The A. phagocytophilum invasin-host cell receptor interactions that facilitate bacterial uptake and hence disease are incompletely defined. Here, we show that EPHNCH_1611, which we refer to as A. phagocytophilum invasion protein B (AipB), mediates bacterial entry but not adhesion. The AipB host cell-interacting domain was narrowed down to amino acids 109 to 129 and is conserved among Anaplasma and Ehrlichia species. AipB _109-123 antisera reduced A. phagocytophilum infection of host cells by 28%. Combining anti-AipB _109-123 with antisera specific for the receptor binding domains of previously identified invasins AipA, Asp14, and OmpA reduced infection by 93%, suggesting a synergistic relationship among these surface proteins. Immunizing mice against AipB _109-123 alone yielded partial protection from A. phagocytophilum challenge that rivaled the protection achieved by immunizing against it together with other invasin binding domains. A yeast two-hybrid screen identified the N-terminal domain of CD18, the β2 integrin β-subunit found on the neutrophil surface, as an AipB binding partner. The interaction was validated by co-immunoprecipitation. siRNA knockdown of CD18 reduced permissiveness to A. phagocytophilum infection. Antibodies targeting the CD18 N-terminus inhibited A. phagocytophilum infection of, but not adherence to, HL-60 cells and human neutrophils. These results demonstrate that the AipB-CD18 interaction is critical for A. phagocytophilum infection in vitro and in vivo , further dissecting the multifaceted mechanism by which A. phagocytophilum invades host cells. IMPORTANCE: Anaplasma phagocytophilum causes human granulocytic anaplasmosis, an emerging tick-borne infection for which there is no vaccine and limited treatments. Although A. phagocytophilum absolutely relies on invading host neutrophils to survive and cause disease, the microbe-host cell interactions that predicate infection are inadequately defined. We found that the bacterium uses its outer surface protein AipB to engage CD18 as a critical invasion step. An AipB peptide that is conserved among its homologs in other Anaplasmataceae pathogens is responsible for binding CD18 and can be targeted by antibody to inhibit infection in vitro and in vivo . However, blocking of A. phagocytophilum infection is most effectively achieved by targeting this AipB peptide together with the functional domains of other invasins. AipB is the first rickettsial invasin identified to interact with a β2 integrin, is key for pathogenesis, and could be targeted to protect against diseases caused by A. phagocytophilum and other Anaplasmataceae bacteria.

Orientia tsutsugamushi is an obligate intracellular alphaproteobacterium and the causative agent of the potentially fatal rickettsiosis, scrub typhus. During infection, O. tsutsugamushi replicates exclusively in the eukaryotic cytosol near mitochondria and alters host metabolic pathways governed by mitochondria. We report that O. tsutsugamushi induces mitochondrial enzymatic impairment and structural abnormalities without altering mitochondrial abundance or the levels of proteins that maintain mitochondrial homeostasis. Confocal and structured illumination microscopy revealed a selective spatial association between O. tsutsugamushi and the mitochondrial membrane protein, voltage-dependent anion channel (VDAC) but not other mitochondrial proteins. Immunosignal for VDAC paralogs 1 and 3 colocalized with cytosolic O. tsutsugamushi organisms whereas VDAC2 did not. Additionally, the antibody specific for VDAC1 and VDAC3 detected proteins of the expected sizes in Orientia membrane fractions. These findings indicate that O. tsutsugamushi negatively impacts mitochondrial function without overt organelle loss and selectively associates with VDAC1/VDAC3.

Selenomonas sputigena is an understudied oral pathobiont associated with periodontitis and dental caries. We recently demonstrated that S. sputigena binds to gingival epithelial cells (GECs), where afterwards the bacterium causes robust in vitro pro‐inflammatory cytokine production and migration of monocytes and neutrophils. Here, we report that extracellular adherent S. sputigena are subsequently internalized by GECs. Fluorescently labeled intracellular S. sputigena were observed in two gingival keratinocyte cell lines and primary cells. Oxygen‐killed S. sputigena was found within GECs at a similar rate to living bacteria, suggesting active protein synthesis is not required for internalization. Host‐cell actin polymerization was essential for the internalization of S. sputigena . Electron microscopy revealed that intracellular S. sputigena is located within a single membrane vesicle tightly wrapped around the bacterium which is decorated with LAMP1, indicating that S. sputigena is ultimately trafficked to a lysosome‐like vesicle. Although S. sputigena mRNA was detectable within GECs, it rapidly decreased by 24 h post‐inoculation. This differed from bacterial morphology, which remained intact up to 48 h. Induction of CXCL8 expression was concurrent with intracellular S. sputigena morphological integrity, suggesting that, while the bacteria are likely dead, bacterial macromolecules perpetuate inflammation in GECs. Collectively, this study demonstrates S. sputigena can be taken into gingival keratinocytes, where the bacterium induces inflammatory responses, but S. sputigena is ultimately processed by lysosomal machinery and is cleared from the intracellular niche in GECs.

Pleomorphism is an evolutionary adaptation by which diverse microorganisms maximize their fitness by transitioning between morphologically distinct forms that perform disparate functions in response to the local microenvironment. Cell division is critical for morphotype transition in many pleomorphic bacterial systems. Anaplasma phagocytophilum , which causes the emerging disease granulocytic anaplasmosis, is a pleomorphic obligate intracellular bacterium that lives in a pathogen-modified vacuole except for when it is exocytically released for dissemination to naïve cells. This bacterium cycles between non-infectious, replicative reticulate cell (RC) and infectious, non-replicative dense-cored (DC) forms. Here, we establish that differential modes of A. phagocytophilum cell division drive morphotype development where RC bacteria divide symmetrically to expand the intravacuolar population after which they switch to sacrificial asymmetric division to produce DCs. A. phagocytophilum MreB is crucial for cell division, specifically septation, and thereby formation of both morphotypes. Inhibition of cell division prevents not only DC formation but also A. phagocytophilum vacuole maturation and infectious progeny release, which suggests that these pathogenic processes are coordinated. This study advances understanding of A. phagocytophilum growth and morphotype development and, thus, pathobiology. It also provides the first evidence linking cell division to morphotype development in the Anaplasmataceae . IMPORTANCE: Anaplasma phagocytophilum , an obligate intracellular bacterial pathogen that lives in a host cell-derived vacuole, causes human and veterinary diseases of global importance. In the pathogen-occupied vacuole, A. phagocytophilum transitions from a replicative, non-infectious morphotype to a non-replicative, infectious morphotype that is released to spread infection. We established that distinct modes of bacterial cell division drive not only A. phagocytophilum replication but also its differentiation to the infectious form and dissemination to naïve cells. How pleomorphism is regulated in most vacuole-adapted bacterial pathogens is poorly understood. Therefore, this study advances fundamental knowledge of vacuole-adapted pleomorphic bacteria pathobiology and could ultimately identify common novel antibiotic targets for treating the diseases they cause.

Infections by intracellular pathogens often cause insult to host cell DNA, which stimulates responses that ultimately eliminate the damaged cell and hence the microbial niche. p53 is an innate immunity mediator that responds to DNA damage and intracellular infection by transcriptionally activating pathways that arrest the cell cycle, repair DNA, and elicit apoptosis. How pathogens counter p53 are incompletely understood. Here, we demonstrate that the endotheliotropic obligate intracellular bacterium Orientia tsutsugamushi blocks transcription of TP53 to nearly deplete p53 levels. Contrary to the unrestricted proliferation expected based on the transcriptome of p53-deficient infected cells, Orientia arrests the cell cycle at S phase to promote bacterial replication. It protects host endothelial cells from DNA damage even if induced by etoposide and delays genotoxic-dependent apoptosis until late in infection once a high bacterial load has been achieved. TP53 downregulation, protection against genotoxicity, and inhibition of DNA damage-dependent apoptosis are executed by the Orientia nucleomodulatory effector, Ank13. Therefore, O. tsutsugamushi inhibits TP53 expression and genotoxicity to reconfigure the intracellular environment of its host cell into one that favors bacterial replication.