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Orientia tsutsugamushi is an obligate intracellular bacterium that causes scrub typhus, a potentially fatal rickettsiosis, and for which no genetic tools exist. Critical to addressing this technical gap is to identify promoters for driving expression of antibiotic resistance and fluorescence reporter genes in O. tsutsugamushi. Such promoters would need to be highly conserved among strains, expressed throughout infection, and exhibit strong activity. We examined the untranslated regions upstream of O. tsutsugamushi genes encoding outer membrane protein A (ompA), 22-kDa type-specific antigen (tsa22) and tsa56. The bacterium transcribed all three during infection of monocytic, endothelial and epithelial cells. Examination of the upstream noncoding regions revealed putative ribosome binding sites, one set of predicted -10 and -35 sequences for ompA and two sets of -10 and -35 sequences for tsa22 and tsa56. Comparison of these regions among geographically diverse O. tsutsugamushi patient isolates revealed nucleotide identities ranging from 84.8 to 100.0%. Upon examination of the candidates for the ability to drive green fluorescence protein expression in Escherichia coli, varying activities were observed with one of the tsa22 promoters being the strongest. Identification and validation of O. tsutsugamushi promoters is an initial key step toward genetically manipulating this important pathogen., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Anaplasma phagocytophilum infects neutrophils to cause granulocytic anaplasmosis. It poorly infects mice deficient in acid sphingomyelinase (ASM), a lysosomal enzyme critical for cholesterol efflux, and wild-type mice treated with desipramine that functionally inhibits ASM. Whether inhibition or genetic deletion of ASM is bacteriostatic or bactericidal for A. phagocytophilum and desipramine's ability to lower pathogen burden requires a competent immune system were unknown. Anaplasma phagocytophilum-infected severe combined immunodeficiency disorder (SCID) mice were administered desipramine or PBS, followed by the transfer of blood to naïve wild-type mice. Next, infected wild-type mice were given desipramine or PBS followed by transfer of blood to naïve SCID mice. Finally, wild-type or ASM-deficient mice were infected and blood transferred to naïve SCID mice. The percentage of infected neutrophils was significantly reduced in all desipramine-treated or ASM-deficient mice and in all recipients of blood from these mice. Infection was markedly lower in ASM-deficient and desipramine-treated wild-type mice versus desipramine-treated SCID mice. Yet, infection was never ablated. Thus, ASM activity contributes to optimal A. phagocytophilum infection in vivo, pharmacologic inhibition or genetic deletion of ASM impairs infection in a bacteriostatic and reversible manner and A. phagocytophilum is capable of co-opting ASM-independent lipid sources., (© The Author(s) 2020. Published by Oxford University Press on behalf of the American Society for Nutrition.)

The c-di-GMP network of Borrelia burgdorferi, a causative agent of Lyme disease, consists of Rrp1, a diguanylate cyclase/response regulator; Hpk1, a histidine kinase; PdeA and PdeB, c-di-GMP phosphodiesterases; and PlzA, a PilZ domain c-di-GMP receptor. Borrelia hermsii, a causative agent of tick-borne relapsing fever, possesses a putative c-di-GMP regulatory network that is uncharacterized. While B. burgdorferi requires c-di-GMP to survive within ticks, the associated effector mechanisms are poorly defined. Using site-directed mutagenesis, size exclusion chromatography, isothermal titration calorimetry and fluorescence resonance energy transfer, we investigate the interaction of c-di-GMP with the Borrelia PilZ domain-containing Plz proteins: B. burgdorferi PlzA and B. hermsii PlzC. The Plz proteins were determined to be monomeric in their apo and holo forms and to bind c-di-GMP with high affinity with a 1:1 stoichiometry. C-di-GMP binding induced structural rearrangements in PlzA and PlzC. C-di-GMP binding proved to be dependent on positive charge at R 145 of the PilZ domain motif, R 145 xxxR. Comparative sequence analyses led to the identification of Borrelia consensus sequences for the PilZ domain signature motifs. This study provides insight into c-di-GMP:Plz receptor interaction and identifies a possible switch mechanism that may regulate Plz protein effector functions., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

OspC is produced by all species of the Borrelia burgdorferi sensu lato complex and is required for infectivity in mammals. To test the hypothesis that the conserved C-terminal motif (C10) of OspC is required for function in vivo, a mutant B. burgdorferi strain (B31::ospCΔC10) was created in which ospC was replaced with an ospC gene lacking the C10 motif. The ability of the mutant to infect mice was investigated using tick transmission and needle inoculation. Infectivity was assessed by cultivation, qRT-PCR, and measurement of IgG antibody responses. B31::ospCΔC10 retained the ability to infect mice by both needle and tick challenge and was competent to survive in ticks after exposure to the blood meal. To determine whether recombinant OspC protein lacking the C-terminal 10 amino acid residues (rOspCΔC10) can bind plasminogen, the only known mammalian-derived ligand for OspC, binding analyses were performed. Deletion of the C10 motif resulted in a statistically significant decrease in plasminogen binding. Although deletion of the C10 motif influenced plasminogen binding, it can be concluded that the C10 motif is not required for OspC to carry out its critical in vivo functions in tick to mouse transmission., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)