Ceramide-1-phosphate is a regulator of Golgi structure and is co-opted by the obligate intracellular bacterial pathogen Anaplasma phagocytophilum.

Many intracellular pathogens structurally disrupt the Golgi apparatus as an evolutionarily conserved promicrobial strategy. Yet, the host factors and signaling processes involved are often poorly understood, particularly for Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis. We found that A. phagocytophilum elevated cellular levels of the bioactive sphingolipid, ceramide-1-phosphate (C1P), to promote Golgi fragmentation that enables bacterial proliferation, conversion from its non-infectious to infectious form, and productive infection. A. phagocytophilum poorly infected mice deficient in ceramide kinase, the Golgi-localized enzyme responsible for C1P biosynthesis. C1P regulated Golgi morphology via activation of a PKCα/Cdc42/JNK signaling axis that culminates in phosphorylation of Golgi structural proteins, GRASP55 and GRASP65. siRNA-mediated depletion of Cdc42 blocked A. phagocytophilum from altering Golgi morphology, which impaired anterograde trafficking of trans-Golgi vesicles into and maturation of the pathogen-occupied vacuole. Cells overexpressing phosphorylation-resistant versions of GRASP55 and GRASP65 presented with suppressed C1P- and A. phagocytophilum-induced Golgi fragmentation and poorly supported infection by the bacterium. By studying A. phagocytophilum, we identify C1P as a regulator of Golgi structure and a host factor that is relevant to disease progression associated with Golgi fragmentation.IMPORTANCECeramide-1-phosphate (C1P), a bioactive sphingolipid that regulates diverse processes vital to mammalian physiology, is linked to disease states such as cancer, inflammation, and wound healing. By studying the obligate intracellular bacterium Anaplasma phagocytophilum, we discovered that C1P is a major regulator of Golgi morphology. A. phagocytophilum elevated C1P levels to induce signaling events that promote Golgi fragmentation and increase vesicular traffic into the pathogen-occupied vacuole that the bacterium parasitizes. As several intracellular microbial pathogens destabilize the Golgi to drive their infection cycles and changes in Golgi morphology is also linked to cancer and neurodegenerative disorder progression, this study identifies C1P as a potential broad-spectrum therapeutic target for infectious and non-infectious diseases.

Functional inhibition or genetic deletion of acid sphingomyelinase bacteriostatically inhibits Anaplasma phagocytophilum infection in vivo.

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.

Correction: Naimi, W.A., et al. Differential Susceptibility of Male versus Female Laboratory Mice to Anaplasma phagocytophilum Infection. Trop. Med. Infect. Dis. 2018, 3, 78.

The authors wish to make the following corrections to this paper [...].

Functional inhibition of acid sphingomyelinase disrupts infection by intracellular bacterial pathogens.

Intracellular bacteria that live in host cell-derived vacuoles are significant causes of human disease. Parasitism of low-density lipoprotein (LDL) cholesterol is essential for many vacuole-adapted bacteria. Acid sphingomyelinase (ASM) influences LDL cholesterol egress from the lysosome. Using functional inhibitors of ASM (FIASMAs), we show that ASM activity is key for infection cycles of vacuole-adapted bacteria that target cholesterol trafficking-Anaplasma phagocytophilum, Coxiella burnetii, Chlamydia trachomatis, and Chlamydia pneumoniae. Vacuole maturation, replication, and infectious progeny generation by A. phagocytophilum, which exclusively hijacks LDL cholesterol, are halted and C. burnetii, for which lysosomal cholesterol accumulation is bactericidal, is killed by FIASMAs. Infection cycles of Chlamydiae, which hijack LDL cholesterol and other lipid sources, are suppressed but less so than A. phagocytophilum or C. burnetii A. phagocytophilum fails to productively infect ASM-/- or FIASMA-treated mice. These findings establish the importance of ASM for infection by intracellular bacteria and identify FIASMAs as potential host-directed therapies for diseases caused by pathogens that manipulate LDL cholesterol.

Differential Susceptibility of Male Versus Female Laboratory Mice to Anaplasma phagocytophilum Infection.

Human granulocytic anaplasmosis (HGA) is a debilitating, non-specific febrile illness caused by the granulocytotropic obligate intracellular bacterium called Anaplasma phagocytophilum. Surveillance studies indicate a higher prevalence of HGA in male versus female patients. Whether this discrepancy correlates with differential susceptibility of males and females to A. phagocytophilum infection is unknown. Laboratory mice have long been used to study granulocytic anaplasmosis. Yet, sex as a biological variable (SABV) in this model has not been evaluated. In this paper, groups of male and female C57Bl/6 mice that had been infected with A. phagocytophilum were assessed for the bacterial DNA load in the peripheral blood, the percentage of neutrophils harboring bacterial inclusions called morulae, and splenomegaly. Infected male mice exhibited as much as a 1.85-fold increase in the number of infected neutrophils, which is up to a 1.88-fold increase in the A. phagocytophilum DNA load, and a significant increase in spleen size when compared to infected female mice. The propensity of male mice to develop a higher level of A. phagocytophilum infection is relevant for studies utilizing the mouse model. This stresses the importance of including SABV and aligns with the observed higher incidence of infection in male versus female patients.

The Pathogen-Occupied Vacuoles of Anaplasma phagocytophilum and Anaplasma marginale Interact with the Endoplasmic Reticulum.

The genus Anaplasma consists of tick-transmitted obligate intracellular bacteria that invade white or red blood cells to cause debilitating and potentially fatal infections. A. phagocytophilum, a human and veterinary pathogen, infects neutrophils to cause granulocytic anaplasmosis. A. marginale invades bovine erythrocytes. Evidence suggests that both species may also infect endothelial cells in vivo. In mammalian and arthropod host cells, A. phagocytophilum and A. marginale reside in host cell derived pathogen-occupied vacuoles (POVs). While it was recently demonstrated that the A. phagocytophilum-occupied vacuole (ApV) intercepts membrane traffic from the trans-Golgi network, it is unclear if it or the A. marginale-occupied vacuole (AmV) interacts with other secretory organelles. Here, we demonstrate that the ApV and AmV extensively interact with the host endoplasmic reticulum (ER) in endothelial, myeloid, and/or tick cells. ER lumen markers, calreticulin, and protein disulfide isomerase, and the ER membrane marker, derlin-1, were pronouncedly recruited to the peripheries of both POVs. ApV association with the ER initiated early and continued throughout the infection cycle. Both the ApV and AmV interacted with the rough ER and smooth ER. However, only derlin-1-positive rough ER derived vesicles were delivered into the ApV lumen where they localized with intravacuolar bacteria. Transmission electron microscopy identified multiple ER-POV membrane contact sites on the cytosolic faces of both species' vacuoles that corresponded to areas on the vacuoles' lumenal faces where intravacuolar Anaplasma organisms closely associated. A. phagocytophilum is known to hijack Rab10, a GTPase that regulates ER dynamics and morphology. Yet, ApV-ER interactions were unhindered in cells in which Rab10 had been knocked down, demonstrating that the GTPase is dispensable for the bacterium to parasitize the ER. These data establish the ApV and AmV as pathogen-host interfaces that directly engage the ER in vertebrate and invertebrate host cells and evidence the conservation of ER parasitism between two Anaplasma species.

Anaplasma phagocytophilum Rab10-dependent parasitism of the trans-Golgi network is critical for completion of the infection cycle.

Anaplasma phagocytophilum is an emerging human pathogen and obligate intracellular bacterium. It inhabits a host cell-derived vacuole and cycles between replicative reticulate cell (RC) and infectious dense-cored (DC) morphotypes. Host-pathogen interactions that are critical for RC-to-DC conversion are undefined. We previously reported that A. phagocytophilum recruits green fluorescent protein (GFP)-tagged Rab10, a GTPase that directs exocytic traffic from the sphingolipid-rich trans-Golgi network (TGN) to its vacuole in a guanine nucleotide-independent manner. Here, we demonstrate that endogenous Rab10-positive TGN vesicles are not only routed to but also delivered into the A. phagocytophilum-occupied vacuole (ApV). Consistent with this finding, A. phagocytophilum incorporates sphingolipids while intracellular and retains them when naturally released from host cells. TGN vesicle delivery into the ApV is Rab10 dependent, up-regulates expression of the DC-specific marker, APH1235, and is critical for the production of infectious progeny. The A. phagocytophilum surface protein, uridine monophosphate kinase, was identified as a guanine nucleotide-independent, Rab10-specific ligand. These data delineate why Rab10 is important for the A. phagocytophilum infection cycle and expand the understanding of the benefits that exploiting host cell membrane traffic affords intracellular bacterial pathogens.

Anaplasma phagocytophilum-Occupied Vacuole Interactions with the Host Cell Cytoskeleton.

Anaplasma phagocytophilum is an obligate intracellular bacterial pathogen of humans and animals. The A. phagocytophium-occupied vacuole (ApV) is a critical host-pathogen interface. Here, we report that the intermediate filaments, keratin and vimentin, assemble on the ApV early and remain associated with the ApV throughout infection. Microtubules localize to the ApV to a lesser extent. Vimentin, keratin-8, and keratin-18 but not tubulin expression is upregulated in A. phagocytophilum infected cells. SUMO-2/3 but not SUMO-1 colocalizes with vimentin filaments that surround ApVs. PolySUMOylation of vimentin by SUMO-2/3 but not SUMO-1 decreases vimentin solubility. Consistent with this, more vimentin exists in an insoluble state in A. phagocytophilum infected cells than in uninfected cells. Knocking down the SUMO-conjugating enzyme, Ubc9, abrogates vimentin assembly at the ApV but has no effect on the bacterial load. Bacterial protein synthesis is dispensable for maintaining vimentin and SUMO-2/3 at the ApV. Withaferin A, which inhibits soluble vimentin, reduces vimentin recruitment to the ApV, optimal ApV formation, and the bacterial load when administered prior to infection but is ineffective once vimentin has assembled on the ApV. Thus, A. phagocytophilum modulates cytoskeletal component expression and co-opts polySUMOylated vimentin to aid construction of its vacuolar niche and promote optimal survival.

Expanding the diversity of mycobacteriophages: insights into genome architecture and evolution.

Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists.