BASEHIT scores home run: elucidates pathogen-host interactions.

In a tour de force, Hart and colleagues recently used a technique known as BASEHIT (bacterial selection to elucidate host-microbe interactions in high throughput) to screen a yeast display library containing 3324 curated human exoproteins with 82 pathogen samples, focusing on vector-borne pathogens, to identify 1303 putative interactions.

PERK-mediated antioxidant response is key for pathogen persistence in ticks.

A crucial phase in the life cycle of tick-borne pathogens is the time spent colonizing and persisting within the arthropod. Tick immunity is emerging as a key force shaping how transmissible pathogens interact with the vector. How pathogens remain in the tick despite immunological pressure remains unknown. In persistently infected Ixodes scapularis, we found that Borrelia burgdorferi (causative agent of Lyme disease) and Anaplasma phagocytophilum (causative agent of granulocytic anaplasmosis) activate a cellular stress pathway mediated by the endoplasmic reticulum receptor PKR-like ER kinase (PERK) and the central regulatory molecule eIF2α. Disabling the PERK pathway through pharmacological inhibition and RNA interference (RNAi) significantly decreased microbial numbers. In vivo RNAi of the PERK pathway not only reduced the number of A. phagocytophilum and B. burgdorferi colonizing larvae after a bloodmeal but also significantly reduced the number of bacteria that survive the molt. An investigation into PERK pathway-regulated targets revealed that A. phagocytophilum and B. burgdorferi induce activity of the antioxidant response regulator, nuclear factor erythroid 2-related factor 2 (Nrf2). Tick cells deficient for nrf2 expression or PERK signaling showed accumulation of reactive oxygen and nitrogen species in addition to reduced microbial survival. Supplementation with antioxidants rescued the microbicidal phenotype caused by blocking the PERK pathway. Altogether, our study demonstrates that the Ixodes PERK pathway is activated by transmissible microbes and facilitates persistence in the arthropod by potentiating an Nrf2-regulated antioxidant environment. IMPORTANCE Recent advances demonstrate that the tick immune system recognizes and limits the pathogens they transmit. Innate immune mediators such as antimicrobial peptides and reactive oxygen/nitrogen species are produced and restrict microbial survival. It is currently unclear how pathogens remain in the tick, despite this immune assault. We found that an antioxidant response controlled by the PERK branch of the unfolded protein response is activated in ticks that are persistently infected with Borrelia burgdorferi (Lyme disease) or Anaplasma phagocytophilum (granulocytic anaplasmosis). The PERK pathway induces the antioxidant response transcription factor, Nrf2, which coordinates a gene network that ultimately neutralizes reactive oxygen and nitrogen species. Interfering with this signaling cascade in ticks causes a significant decline in pathogen numbers. Given that innate immune products can cause collateral damage to host tissues, we speculate that this is an arthropod-driven response aimed at minimizing damage to "self" that also inadvertently benefits the pathogen. Collectively, our findings shed light on the mechanistic push and pull between tick immunity and pathogen persistence within the arthropod vector.

An Anaplasma phagocytophilum T4SS effector, AteA, is essential for tick infection.

IMPORTANCE: Ticks are the number one vector of pathogens for livestock worldwide and for humans in the United States. The biology of tick transmission is an understudied area. Understanding this critical interaction could provide opportunities to affect the course of disease spread. In this study, we examined the zoonotic tick-borne agent Anaplasma phagocytophilum and identified a secreted protein, AteA, which is expressed in a tick-specific manner. These secreted proteins, termed effectors, are the first proteins to interact with the host environment. AteA is essential for survival in ticks and appears to interact with cortical actin. Most effector proteins are studied in the context of the mammalian host; however, understanding how this unique set of proteins affects tick transmission is critical to developing interventions.

PERK-mediated antioxidant response is key for pathogen persistence in ticks.

A crucial phase in the lifecycle of tick-borne pathogens is the time spent colonizing and persisting within the arthropod. Tick immunity is emerging as a key force shaping how transmissible pathogens interact with the vector. How pathogens remain in the tick despite immunological pressure remains unknown. In persistently infected Ixodes scapularis , we found that Borrelia burgdorferi (Lyme disease) and Anaplasma phagocytophilum (granulocytic anaplasmosis) activate a cellular stress pathway mediated by the endoplasmic reticulum receptor PERK and the central regulatory molecule, eIF2α. Disabling the PERK pathway through pharmacological inhibition and RNAi significantly decreased microbial numbers. In vivo RNA interference of the PERK pathway not only reduced the number of A. phagocytophilum and B. burgdorferi colonizing larvae after a bloodmeal, but also significantly reduced the number of bacteria that survive the molt. An investigation into PERK pathway-regulated targets revealed that A. phagocytophilum and B. burgdorferi induce activity of the antioxidant response regulator, Nrf2. Tick cells deficient for nrf2 expression or PERK signaling showed accumulation of reactive oxygen and nitrogen species in addition to reduced microbial survival. Supplementation with antioxidants rescued the microbicidal phenotype caused by blocking the PERK pathway. Altogether, our study demonstrates that the Ixodes PERK pathway is activated by transmissible microbes and facilitates persistence in the arthropod by potentiating an Nrf2-regulated antioxidant environment.

An Anaplasma phagocytophilum T4SS effector, AteA, is essential for tick infection.

Pathogens must adapt to disparate environments in permissive host species, a feat that is especially pronounced for vector-borne microbes, which transition between vertebrate hosts and arthropod vectors to complete their lifecycles. Most knowledge about arthropod-vectored bacterial pathogens centers on their life in the mammalian host, where disease occurs. However, disease outbreaks are driven by the arthropod vectors. Adapting to the arthropod is critical for obligate intracellular rickettsial pathogens, as they depend on eukaryotic cells for survival. To manipulate the intracellular environment, these bacteria use Type IV Secretion Systems (T4SS) to deliver effectors into the host cell. To date, few rickettsial T4SS translocated effectors have been identified and have only been examined in the context of mammalian infection. We identified an effector from the tick-borne rickettsial pathogen Anaplasma phagocytophilum , HGE1_02492, as critical for survival in tick cells and acquisition by ticks in vivo . Conversely, HGE1_02492 was dispensable during mammalian cell culture and murine infection. We show HGE1_02492 is translocatable in a T4SS-dependent manner to the host cell cytosol. In eukaryotic cells, the HGE1_02492 localized with cortical actin filaments, which is dependent on multiple sub-domains of the protein. HGE1_02492 is the first arthropod-vector specific T4SS translocated effector identified from a rickettsial pathogen. Moreover, the subcellular target of HGE1_02492 suggests that A. phagocytophilum is manipulating actin to enable arthropod colonization. Based on these findings, we propose the name AteA for Anaplasma ( phagocytophilum ) tick effector A. Altogether, we show that A. phagocytophilum uses distinct strategies to cycle between mammals and arthropods. Importance: Ticks are the number one vector of pathogens for livestock worldwide and for humans in the US. The biology of tick transmission is an understudied area. Understanding this critical interaction could provide opportunities to affect the course of disease spread. In this study we examined the zoonotic tick-borne agent Anaplasma phagocytophilum and identified a secreted protein, AteA, that is expressed in a tick-specific manner. These secreted proteins, termed effectors, are the first proteins to interact with the host environment. AteA is essential for survival in ticks and appears to interact with cortical actin. Most effector proteins are studied in the context of the mammalian host; however, understanding how this unique set of proteins affect tick transmission is critical to developing interventions.

Combinatorial selection in amoebal hosts drives the evolution of the human pathogen Legionella pneumophila.

Virulence mechanisms typically evolve through the continual interaction of a pathogen with its host. In contrast, it is poorly understood how environmentally acquired pathogens are able to cause disease without prior interaction with humans. Here, we provide experimental evidence for the model that Legionella pathogenesis in humans results from the cumulative selective pressures of multiple amoebal hosts in the environment. Using transposon sequencing, we identify Legionella pneumophila genes required for growth in four diverse amoebae, defining universal virulence factors commonly required in all host cell types and amoeba-specific auxiliary genes that determine host range. By comparing genes that promote growth in amoebae and macrophages, we show that adaptation of L. pneumophila to each amoeba causes the accumulation of distinct virulence genes that collectively allow replication in macrophages and, in some cases, leads to redundancy in this host cell type. In contrast, some bacterial proteins that promote replication in amoebae restrict growth in macrophages. Thus, amoebae-imposed selection is a double-edged sword, having both positive and negative impacts on disease. Comparing the genome composition and host range of multiple Legionella species, we demonstrate that their distinct evolutionary trajectories in the environment have led to the convergent evolution of compensatory virulence mechanisms.

Cyanide bioremediation: the potential of engineered nitrilases.

The cyanide-degrading nitrilases are of notable interest for their potential to remediate cyanide contaminated waste streams, especially as generated in the gold mining, pharmaceutical, and electroplating industries. This review provides a brief overview of cyanide remediation in general but with a particular focus on the cyanide-degrading nitrilases. These are of special interest as the hydrolysis reaction does not require secondary substrates or cofactors, making these enzymes particularly good candidates for industrial remediation processes. The genetic approaches that have been used to date for engineering improved enzymes are described; however, recent structural insights provide a promising new approach.

Residue Y70 of the Nitrilase Cyanide Dihydratase from Bacillus pumilus Is Critical for Formation and Activity of the Spiral Oligomer.

Nitrilases pose attractive alternatives to the chemical hydrolysis of nitrile compounds. The activity of bacterial nitrilases towards substrate is intimately tied to the formation of large spiral-shaped oligomers. In the nitrilase CynD (cyanide dihydratase) from Bacillus pumilus, mutations in a predicted oligomeric surface region altered its oligomerization and reduced its activity. One mutant, CynD Y70C, retained uniform oligomer formation however it was inactive, unlike all other inactive mutants throughout that region all of which significantly perturbed oligomer formation. It was hypothesized that Y70 is playing an additional role necessary for CynD activity beyond influencing oligomerization. Here, we performed saturation mutagenesis at residue 70 and demonstrated that only tyrosine or phenylalanine is permissible for CynD activity. Furthermore, we show that other residues at this position are not only inactive, but have altered or disrupted oligomer conformations. These results suggest that Y70's essential role in activity is independent of its role in the formation of the spiral oligomer.

Probing C-terminal interactions of the Pseudomonas stutzeri cyanide-degrading CynD protein.

The cyanide dihydratases from Bacillus pumilus and Pseudomonas stutzeri share high amino acid sequence similarity throughout except for their highly divergent C-termini. However, deletion or exchange of the C-termini had different effects upon each enzyme. Here we extended previous studies and investigated how the C-terminus affects the activity and stability of three nitrilases, the cyanide dihydratases from B. pumilus (CynDpum) and P. stutzeri (CynDstut) and the cyanide hydratase from Neurospora crassa. Enzymes in which the C-terminal residues were deleted decreased in both activity and thermostability with increasing deletion lengths. However, CynDstut was more sensitive to such truncation than the other two enzymes. A domain of the P. stutzeri CynDstut C-terminus not found in the other enzymes, 306GERDST311, was shown to be necessary for functionality and explains the inactivity of the previously described CynDstut-pum hybrid. This suggests that the B. pumilus C-terminus, which lacks this motif, may have specific interactions elsewhere in the protein, preventing it from acting in trans on a heterologous CynD protein. We identify the dimerization interface A-surface region 195-206 (A2) from CynDpum as this interaction site. However, this A2 region did not rescue activity in C-terminally truncated CynDstutΔ302 or enhance the activity of full-length CynDstut and therefore does not act as a general stability motif.

Probing an Interfacial Surface in the Cyanide Dihydratase from Bacillus pumilus, A Spiral Forming Nitrilase.

Nitrilases are of significant interest both due to their potential for industrial production of valuable products as well as degradation of hazardous nitrile-containing wastes. All known functional members of the nitrilase superfamily have an underlying dimer structure. The true nitrilases expand upon this basic dimer and form large spiral or helical homo-oligomers. The formation of this larger structure is linked to both the activity and substrate specificity of these nitrilases. The sequences of the spiral nitrilases differ from the non-spiral forming homologs by the presence of two insertion regions. Homology modeling suggests that these regions are responsible for associating the nitrilase dimers into the oligomer. Here we used cysteine scanning across these two regions, in the spiral forming nitrilase cyanide dihydratase from Bacillus pumilus (CynD), to identify residues altering the oligomeric state or activity of the nitrilase. Several mutations were found to cause changes to the size of the oligomer as well as reduction in activity. Additionally one mutation, R67C, caused a partial defect in oligomerization with the accumulation of smaller oligomer variants. These results support the hypothesis that these insertion regions contribute to the unique quaternary structure of the spiral microbial nitrilases.