Anaplasma phagocytophilum Asp14 is an invasin that interacts with mammalian host cells via its C terminus to facilitate infection.

Anaplasma phagocytophilum, a member of the family Anaplasmataceae, is the tick-transmitted obligate intracellular bacterium that causes human granulocytic anaplasmosis. The life cycle of A. phagocytophilum is biphasic, transitioning between the noninfectious reticulate cell (RC) and infectious dense-cored (DC) forms. We analyzed the bacterium's DC surface proteome by selective biotinylation of surface proteins, NeutrAvidin affinity purification, and mass spectrometry. Transcriptional profiling of selected outer membrane protein candidates over the course of infection revealed that aph_0248 (designated asp14 [14-kDa A. phagocytophilum surface protein]) expression was upregulated the most during A. phagocytophilum cellular invasion. asp14 transcription was induced during transmission feeding of A. phagocytophilum-infected ticks on mice and was upregulated when the bacterium engaged its receptor, P-selectin glycoprotein ligand 1. Asp14 localized to the A. phagocytophilum surface and was expressed during in vivo infection. Treating DC organisms with Asp14 antiserum or preincubating mammalian host cells with glutathione S-transferase (GST)-Asp14 significantly inhibited infection of host cells. Moreover, preincubating host cells with GST-tagged forms of both Asp14 and outer membrane protein A, another A. phagocytophilum invasin, pronouncedly reduced infection relative to treatment with either protein alone. The Asp14 domain that is sufficient for cellular adherence and invasion lies within the C-terminal 12 to 24 amino acids and is conserved among other Anaplasma and Ehrlichia species. These results identify Asp14 as an A. phagocytophilum surface protein that is critical for infection, delineate its invasion domain, and demonstrate the potential of targeting Asp14 in concert with OmpA for protecting against infection by A. phagocytophilum and other Anaplasmataceae pathogens.

Anaplasma phagocytophilum outer membrane protein A interacts with sialylated glycoproteins to promote infection of mammalian host cells.

Anaplasma phagocytophilum is the tick-transmitted obligate intracellular bacterium that causes human granulocytic anaplasmosis (HGA). A. phagocytophilum binding to sialyl Lewis x (sLe(x)) and other sialylated glycans that decorate P selectin glycoprotein 1 (PSGL-1) and other glycoproteins is critical for infection of mammalian host cells. Here, we demonstrate the importance of A. phagocytophilum outer membrane protein A (OmpA) APH_0338 in infection of mammalian host cells. OmpA is transcriptionally induced during transmission feeding of A. phagocytophilum-infected ticks on mice and is upregulated during invasion of HL-60 cells. OmpA is presented on the pathogen's surface. Sera from HGA patients and experimentally infected mice recognize recombinant OmpA. Pretreatment of A. phagocytophilum organisms with OmpA antiserum reduces their abilities to infect HL-60 cells. The OmpA N-terminal region is predicted to contain the protein's extracellular domain. Glutathione S-transferase (GST)-tagged versions of OmpA and OmpA amino acids 19 to 74 (OmpA(19-74)) but not OmpA(75-205) bind to, and competitively inhibit A. phagocytophilum infection of, host cells. Pretreatment of host cells with sialidase or trypsin reduces or nearly eliminates, respectively, GST-OmpA adhesion. Therefore, OmpA interacts with sialylated glycoproteins. This study identifies the first A. phagocytophilum adhesin-receptor pair and delineates the region of OmpA that is critical for infection.

Postgenomic analyses reveal development of infectious Anaplasma phagocytophilum during transmission from ticks to mice.

Obligate intracellular bacteria of the Rickettsiales order have evolved to colonize both arthropod and mammalian hosts, but few details are known about the bacterial adaptations that occur during transmission from blood-feeding arthropods to mammals. Here we apply proteomics and transcriptome sequencing to Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, in Ixodes scapularis tick salivary glands, to detect proteins or genes expressed by the pathogen during transmission feeding by the tick. We detected expression of 139 genes, representing 11% of the open reading frames (ORFs) in the A. phagocytophilum genome. The predominant categories of proteins were ribosomal proteins, cell surface proteins, chaperones, and uncharacterized proteins. There was no evidence of DNA replication enzymes, suggesting that most of the A. phagocytophilum cells were no longer dividing. Instead, protein expression reflected conversion to the extracellular, infectious "dense-core" (DC) form. High expression of a DC-specific marker, APH_1235, further suggested this developmental transition in ticks. We showed that blocking APH_1235 with antibodies reduced A. phagocytophilum infection levels in mammalian cell culture. This work represents a starting point for clarifying essential proteins expressed by A. phagocytophilum during transmission from ticks to mammals and demonstrates that the abundantly expressed, DC-associated APH_1235 protein is important during in vivo infection by A. phagocytophilum.

The biology of Frankia sp. strains in the post-genome era.

Progress in understanding symbiotic determinants involved in the N(2)-fixing actinorhizal plant symbioses has been slow but steady. Problems persist with studying the bacterial contributions to the symbiosis using traditional microbiological techniques. However, recent years have seen the emergence of several genomes from Frankia sp. strains and the development of techniques for manipulating plant gene expression. Approaches to understanding the bacterial side of the symbiosis have employed a range of techniques that reveal the proteomes and transcriptomes from both cultured and symbiotic frankiae. The picture beginning to emerge provides some perspective on the heterogeneity of frankial populations in both conditions. In general, frankial populations in root nodules seem to maintain a rather robust metabolism that includes nitrogen fixation and substantial biosynthesis and energy-generating pathways, along with a modified ammonium assimilation program. To date, particular bacterial genes have not been implicated in root nodule formation but some hypotheses are emerging with regard to how the plant and microorganism manage to coexist. In particular, frankiae seem to present a nonpathogenic presence to the plant that may have the effect of minimizing some plant defense responses. Future studies using high-throughput approaches will likely clarify the range of bacterial responses to symbiosis that will need to be understood in light of the more rapidly advancing work on the plant host.

Anaplasma phagocytophilum AptA modulates Erk1/2 signalling.

Anaplasma phagocytophilum causes human granulocytic anaplasmosis, one of the most common tick-borne diseases in North America. This unusual obligate intracellular pathogen selectively persists within polymorphonuclear leucocytes. In this study, using the yeast surrogate model we identified an A. phagocytophilum virulence protein, AptA (A. phagocytophilum toxin A), that activates mammalian Erk1/2 mitogen-activated protein kinase. This activation is important for A. phagocytophilum survival within human neutrophils. AptA interacts with the intermediate filament protein vimentin, which is essential for A. phagocytophilum-induced Erk1/2 activation and infection. A. phagocytophilum infection reorganizes vimentin around the bacterial inclusion, thereby contributing to intracellular survival. These observations reveal a major role for the bacterial protein, AptA, and the host protein, vimentin, in the activation of Erk1/2 during A. phagocytophilum infection.

Comparative secretome analysis suggests low plant cell wall degrading capacity in Frankia symbionts.

BACKGROUND: Frankia sp. strains, the nitrogen-fixing facultative endosymbionts of actinorhizal plants, have long been proposed to secrete hydrolytic enzymes such as cellulases, pectinases, and proteases that may contribute to plant root penetration and formation of symbiotic root nodules. These or other secreted proteins might logically be involved in the as yet unknown molecular interactions between Frankia and their host plants. We compared the genome-based secretomes of three Frankia strains representing diverse host specificities. Signal peptide detection algorithms were used to predict the individual secretomes of each strain, and the set of secreted proteins shared among the strains, termed the core Frankia secretome. Proteins in the core secretome may be involved in the actinorhizal symbiosis. RESULTS: The Frankia genomes have conserved Sec (general secretory) and Tat (twin arginine translocase) secretion systems. The potential secretome of each Frankia strain comprised 4-5% of the total proteome, a lower percentage than that found in the genomes of other actinobacteria, legume endosymbionts, and plant pathogens. Hydrolytic enzymes made up only a small fraction of the total number of predicted secreted proteins in each strain. Surprisingly, polysaccharide-degrading enzymes were few in number, especially in strain CcI3, with more esterolytic, lipolytic and proteolytic enzymes having signal peptides. A total of 161 orthologous proteins belong to the core Frankia secretome. Of these, 52 also lack homologs in closely related actinobacteria, and are termed "Frankia-specific." The genes encoding these conserved secreted proteins are often clustered near secretion machinery genes. CONCLUSION: The predicted secretomes of Frankia sp. are relatively small and include few hydrolases, which could reflect adaptation to a symbiotic lifestyle. There are no well-conserved secreted polysaccharide-degrading enzymes present in all three Frankia genomes, suggesting that plant cell wall polysaccharide degradation may not be crucial to root infection, or that this degradation varies among strains. We hypothesize that the relative lack of secreted polysaccharide-degrading enzymes in Frankia reflects a strategy used by these bacteria to avoid eliciting host defense responses. The esterases, lipases, and proteases found in the core Frankia secretome might facilitate hyphal penetration through the cell wall, release carbon sources, or modify chemical signals. The core secretome also includes extracellular solute-binding proteins and Frankia-specific hypothetical proteins that may enable the actinorhizal symbiosis.

Genome characteristics of facultatively symbiotic Frankia sp. strains reflect host range and host plant biogeography.

Soil bacteria that also form mutualistic symbioses in plants encounter two major levels of selection. One occurs during adaptation to and survival in soil, and the other occurs in concert with host plant speciation and adaptation. Actinobacteria from the genus Frankia are facultative symbionts that form N(2)-fixing root nodules on diverse and globally distributed angiosperms in the "actinorhizal" symbioses. Three closely related clades of Frankia sp. strains are recognized; members of each clade infect a subset of plants from among eight angiosperm families. We sequenced the genomes from three strains; their sizes varied from 5.43 Mbp for a narrow host range strain (Frankia sp. strain HFPCcI3) to 7.50 Mbp for a medium host range strain (Frankia alni strain ACN14a) to 9.04 Mbp for a broad host range strain (Frankia sp. strain EAN1pec.) This size divergence is the largest yet reported for such closely related soil bacteria (97.8%-98.9% identity of 16S rRNA genes). The extent of gene deletion, duplication, and acquisition is in concert with the biogeographic history of the symbioses and host plant speciation. Host plant isolation favored genome contraction, whereas host plant diversification favored genome expansion. The results support the idea that major genome expansions as well as reductions can occur in facultative symbiotic soil bacteria as they respond to new environments in the context of their symbioses.