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.

Summary 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.

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.

Many intracellular pathogens hijack cholesterol. Inhibiting acid sphingomyelinase alters cholesterol traffic to target different intracellular bacteria, signifying a host-directed approach for treating infectious disease.

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.