Orientia tsutsugamushi
is a genetically intractable obligate intracellular bacterium, causes scrub typhus, and has one of the largest known armamentariums of ankyrin repeat-containing effectors (Anks). Most have a C-terminal F-box presumed to interact with the SCF ubiquitin ligase complex primarily based on their ability to bind overexpressed Skp1.
Anaplasma phagocytophilum causes granulocytic anaplasmosis, a debilitating infection that can be fatal in the immunocompromised. It also afflicts animals, including dogs, horses, and sheep. No granulocytic anaplasmosis vaccine exists. Because A. phagocytophilum is an obligate intracellular bacterium, inhibiting microbial-host cell interactions that facilitate invasion can disrupt infection. The binding domains of A. phagocytophilum adhesins AipA, Asp14, and OmpA are essential for optimal bacterial entry into host cells, but their relevance to infection in vivo are undefined. In this study, C57BL/6J mice were immunized with a cocktail of keyhole limpet hemocyanin-conjugated peptides corresponding to the AipA, Asp14, and OmpA binding domains in alum followed by challenge with A. phagocytophilum. The bacterial peripheral blood burden was pronouncedly reduced in immunized mice compared to controls. Examination of pre- and post-challenge sera from these mice revealed that immunization elicited antibodies against AipA and Asp14 peptides, but not OmpA peptide. Nonetheless, pooled sera from pre- and post-challenge groups, but not from control groups inhibited A. phagocytophilum infection of HL-60 cells. Adhesin domain immunization also elicited IFNγ-producing CD8+ T cells. A follow up study confirmed that immunization against only the AipA or Asp14 binding domain was sufficient to reduce the bacterial peripheral blood load in mice following challenge and elicit antibodies that inhibit A. phagocytophilum cellular infection in vitro. These data demonstrate that AipA and Asp14 are critical for A. phagocytophilum to productively infect mice and immunization against their binding domains elicits a protective immune response.
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. A. phagocytophilum infected 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.
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