Effector Proteins Research Articles

Computational design of highly signalling-active membrane receptors through solvent-mediated allosteric networks.

Protein catalysis and allostery require the atomic-level orchestration and motion of residues and ligand, solvent and protein effector molecules. However, the ability to design protein activity through precise protein-solvent cooperative interactions has not yet been demonstrated. Here we report the design of 14 membrane receptors that catalyse G protein nucleotide exchange through diverse engineered allosteric pathways mediated by cooperative networks of intraprotein, protein-ligand and -solvent molecule interactions. Consistent with predictions, the designed protein activities correlated well with the level of plasticity of the networks at flexible transmembrane helical interfaces. Several designs displayed considerably enhanced thermostability and activity compared with related natural receptors. The most stable and active variant crystallized in an unforeseen signalling-active conformation, in excellent agreement with the design models. The allosteric network topologies of the best designs bear limited similarity to those of natural receptors and reveal an allosteric interaction space larger than previously inferred from natural proteins. The approach should prove useful for engineering proteins with novel complex protein binding, catalytic and signalling activities.

A bacterial type III effector hijacks plant ubiquitin proteases to evade degradation.

Gram-negative bacterial pathogens inject effector proteins inside plant cells using a type III secretion system. These effectors manipulate plant cellular functions and suppress the plant immune system in order to promote bacterial proliferation. Despite the fact that bacterial effectors are exogenous threatening proteins potentially exposed to the protein degradation systems inside plant cells, effectors are relative stable and able to perform their virulence functions. In this work, we found that RipE1, an effector protein secreted by the bacterial wilt pathogen, Ralstonia solanacearum, undergoes phosphorylation of specific residues inside plant cells, and this promotes its stability. Moreover, RipE1 associates with plant ubiquitin proteases, which contribute to RipE1 deubiquitination and stabilization. The absence of those specific phosphorylation sites or specific host ubiquitin proteases leads to a substantial decrease in RipE1 protein accumulation, indicating that RipE1 hijacks plant post-translational modification regulators in order to promote its own stability. These results suggest that effector stability or degradation in plant cells constitute another molecular event subject to co-evolution between plants and pathogens.

Multiscale footprints reveal the organization of cis-regulatory elements.

Cis-regulatory elements (CREs) control gene expression and are dynamic in their structure and function, reflecting changes in the composition of diverse effector proteins over time1. However, methods for measuring the organization of effector proteins at CREs across the genome are limited, hampering efforts to connect CRE structure to their function in cell fate and disease. Here we developed PRINT, a computational method that identifies footprints of DNA-protein interactions from bulk and single-cell chromatin accessibility data across multiple scales of protein size. Using these multiscale footprints, we created the seq2PRINT framework, which uses deep learning to allow precise inference of transcription factor and nucleosome binding and interprets regulatory logic at CREs. Applying seq2PRINT to single-cell chromatin accessibility data from human bone marrow, we observe sequential establishment and widening of CREs centred on pioneer factors across haematopoiesis. We further discover age-associated alterations in the structure of CREs in murine haematopoietic stem cells, including widespread reduction of nucleosome footprints and gain of de novo identified Ets composite motifs. Collectively, we establish a method for obtaining rich insights into DNA-binding protein dynamics from chromatin accessibility data, and reveal the architecture of regulatory elements across differentiation and ageing.

XopAE Effector from Xanthomonas phaseoli pv. manihotis Targets HSP20-Like p23 Cochaperone to Suppress Plant Basal Immunity.

Pathogenic bacteria use Type 3 effector proteins to manipulate host defenses and alter metabolism to favor their survival and spread. The non-model bacterial pathogen Xanthomonas phaseoli pv. manihotis (Xpm) causes devastating disease in cassava. The molecular role of Type 3 effector proteins from Xpm in causing disease is largely unknown. Here, we report that the XopAE effector from Xpm suppresses plant defense responses. Our results showed that XopAE is a suppressor of basal defenses such as callose deposition and the production of reactive oxygen species (ROS). XopAE targets a small heat shock protein (Mep23-1 cochaperone) in cassava and its homolog Atp23-1 in Arabidopsis. XopAE localizes to the nucleus and in scattered points throughout the cell border, while Mep23-1 shows a nucleocytoplasmic localization. Upon interaction, XopAE hijacks Mep23-1 to the scattered points throughout the cell border and they also interact in nucleus. Our results indicate that the interaction between XopAE and Mep23-1 is essential for suppressing basal plant defense. This study is one of the first to address the molecular mechanisms deployed by Xpm to cause disease in cassava, a non-model crop plant.

Distribution of the four type VI secretion systems in Pseudomonas aeruginosa and classification of their core and accessory effectors

Bacterial type VI secretion systems (T6SSs) are puncturing molecular machines that transport effector proteins to kill microbes, manipulate eukaryotic cells, or facilitate nutrient uptake. How and why T6SS machines and effectors differ within a species is not fully understood. Here, we applied molecular population genetics to the T6SSs in a global population of the opportunistic pathogen Pseudomonas aeruginosa. We reveal varying occurrence of up to four distinct T6SS machines. Moreover, we define conserved core T6SS effectors, likely critical for the biology of P. aeruginosa, and accessory effectors that can exhibit mutual exclusivity between strains. By ancestral reconstruction, we observed dynamic changes in the gain and loss of effector genes in the species’ evolutionary history. Our work highlights the potential importance of T6SS intraspecific diversity in bacterial ecology and evolution.

Wheat Leaf Rust Effector Pt48115 Localized in the Chloroplasts and Suppressed Wheat Immunity.

Wheat leaf rust caused by Puccinia triticina (Pt) is a prevalent disease worldwide, seriously threatening wheat production. Pt acquires nutrients from host cells via haustoria and secretes effector proteins to modify and regulate the expression of host disease resistance genes, thereby facilitating pathogen growth and reproduction. The study of effector proteins is of great significance for clarifying the pathogenic mechanisms of Pt and effective control of leaf rust. Herein, we report a wheat leaf rust candidate effector protein Pt48115 that is highly expressed in the late stages of infection during wheat-Pt interaction. Pt48115 contains a signal peptide with a secretory function and a transit peptide that can translocate Pt48115 to the host chloroplasts. The amino acid sequence polymorphism analysis of Pt48115 in seven different leaf rust races showed that it was highly conserved. Pt48115 inhibited cell death induced by Bcl-2-associated X protein (BAX) from mice or infestans 1 (INF1) from Phytophthora infestans in Nicotiana benthamiana and by DC3000 in wheat, and its 145-175 amino acids of the C-terminal are critical for its function. Furthermore, Pt48115 inhibited callose deposition and reactive oxygen species accumulation in the wheat cultivar Thatcher, demonstrating that it is an effector that enhances Pt virulence by suppressing wheat defense responses. Our findings lay a foundation for future studies on the pathogenesis of Pt during wheat-fungus interaction.

Transient activation of YAP/TAZ confers resistance to morusin-induced apoptosis

BackgroundThe Hippo signaling pathway involves a kinase cascade that controls phosphorylation of the effector proteins YAP and TAZ, leading to regulation of cell growth, tissue homeostasis, and apoptosis. Morusin, a compound extracted from Morus alba, has shown potential in cancer therapy by targeting multiple signaling pathways, including the PI3K/Akt/mTOR, JAK/STAT, MAPK/ERK, and apoptosis pathways. This study explores the effects of morusin on YAP activation and its implications for apoptosis resistance.ResultsOur investigation revealed that morusin induces transient YAP activation, characterized by the dephosphorylation of YAP at S127 and nuclear localization, followed by gradual rephosphorylation in multiple cancer cells. Notably, this activation occurs independently of the canonical Hippo pathway and involves the LATS1/2, MINK1, and MAPK pathways during the YAP inactivation stage. Furthermore, morusin-induced stress granule formation was significantly impaired in YAP/TAZ-depleted cells, suggesting a role in apoptosis resistance. Additionally, the expression of constitutively active MINK1 maintained YAP activation and reduced apoptosis, indicating that prolonged YAP activation can enhance resistance to cell death.ConclusionsThese findings suggest that YAP/TAZ are crucial in resistance to morusin-induced apoptosis, and targeting YAP/TAZ could enhance the anti-cancer efficacy of morusin. Our study provides new insights into the molecular mechanisms of morusin, highlighting potential therapeutic strategies against cancer by disrupting apoptosis resistance.

Novel adaptive immune systems in pristine Antarctic soils

Antarctic environments are dominated by microorganisms, which are vulnerable to viral infection. Although several studies have investigated the phylogenetic repertoire of bacteria and viruses in these poly-extreme environments with freezing temperatures, high ultra violet irradiation levels, low moisture availability and hyper-oligotrophy, the evolutionary mechanisms governing microbial immunity remain poorly understood. Using genome-resolved metagenomics, we test the hypothesis that Antarctic poly-extreme high-latitude microbiomes harbour diverse adaptive immune systems. Our analysis reveals the prevalence of prophages in bacterial genomes (Bacteroidota and Verrucomicrobiota), suggesting the significance of lysogenic infection strategies in Antarctic soils. Furthermore, we demonstrate the presence of diverse CRISPR-Cas arrays, including Class 1 arrays (Types I-B, I-C, and I-E), alongside systems exhibiting novel gene architecture among their effector cas genes. Notably, a Class 2 system featuring type V variants lacks CRISPR arrays, encodes Cas1 and Cas2 adaptation module genes. Phylogenetic analysis of Cas12 effector proteins hints at divergent evolutionary histories compared to classified type V effectors and indicates that TnpB is likely the ancestor of Cas12 nucleases. Our findings suggest substantial novelty in Antarctic cas sequences, likely driven by strong selective pressures. These results underscore the role of viral infection as a key evolutionary driver shaping polar microbiomes.

The flavonoid metabolic pathway genes Ac4CL1, Ac4CL3 and AcHCT1 positively regulate the kiwifruit immune response to Pseudomonas syringae pv. actinidiae.

Psa primarily utilises the type III secretion system (T3SS) to deliver effector proteins (T3Es) into host cells, thereby regulating host immune responses. However, the mechanism by which kiwifruit responds to T3SS remains unclear. To elucidate the molecular reaction of kiwifruit plants to Psa infection, M228 and mutant M228△hrcS strains were employed to inoculate Actinidia chinensis var. chinensis for performing comparative transcriptional and metabolomic analyses. Transcriptome analysis identified 973 differentially expressed genes (DEGs) related to flavonoid synthesis, pathogen interaction, and hormone signaling pathways during the critical period of Psa infection at 48h post-inoculation. In the subsequent metabolomic analysis, flavonoid-related differential metabolites were significantly enriched after the loss of T3SS.Through multi-omics analysis, 22 differentially expressed genes related to flavonoid biosynthesis were identified. Finally, it was discovered that the transient overexpression of 3 genes significantly enhanced kiwifruit resistance to Psa. qRT-PCR analysis indicated that Ac4CL1, Ac4CL3 and AcHCT1 promote host resistance to disease, while Ac4CL3 negatively regulates host resistance to Psa. These findings enrich the plant immune regulation network involved in the interaction between kiwifruit and Psa, providing functional genes and directions with potential application for breeding kiwifruit resistance to canker disease.

The Exocyst Subunits EqSec5 and EqSec6 Promote Powdery Mildew Fungus Growth and Pathogenicity.

The exocyst complex in eukaryotic cells modulates secretory vesicle transportation to promote exocytosis. The exocyst is also required for the hyphal growth and pathogenic development of several filamentous phytopathogens. Obligate biotrophic powdery mildew fungi cause considerable damage to many cash crops; however, the exocyst's roles in this group of fungi is not well studied. To verify the functions of the exocyst in powdery mildew fungus, we identified two exocyst subunits, EqSec5 and EqSec6, from Erysiphe quercicola, a powdery mildew fungus that infects the rubber tree Hevea brasiliensis. When GFP-fused EqSec5 and EqSec6 were introduced into E. quercicola and another phytopathogenic fungus, Magnaporthe oryzae, they primarily localized to the hyphal tip region. Inducing gene silencing of EqSec5 or EqSec6 caused growth and infection defects, and those defects could not be fully restored under the NADPH oxidase inhibitor treatment to the plant. The silenced strains also induced the host defense response including reactive oxygen species accumulation and callose deposition. The silencing of EqSec5 or EqSec6 also inhibited the secretion of the effector protein EqIsc1, interrupting plant salicylic acid biosynthesis. Yeast two-hybrid and gene overexpression assays suggested that EqSec5 and EqSec6 interact with each other and can complement each other's function during host infection. Overall, our study provides evidence that the exocyst in this powdery mildew fungus facilitates effector secretion, hyphal growth, and infection.

Effector proteins of Funneliformis mosseae BR221: unravelling plant-fungal interactions through reference-based transcriptome analysis, in vitro validation, and protein‒protein docking studies

BackgroundArbuscular mycorrhizal (AM) fungi form a highly adaptable and versatile group of fungi found in natural and man-managed ecosystems. Effector secreted by AM fungi influence symbiotic relationship by modifying host cells, suppressing host defense and promoting infection to derive nutrients from the host. Here, we conducted a reference-based transcriptome sequencing of Funneliformis mosseae BR221 to enhance understanding on the molecular machinery involved in the establishment of interaction between host and AM fungi.ResultsA total of 163 effector proteins were identified in F. mosseae isolate BR221, of these, 79.14% are extracellular effectors and 5.5% are predicted cytoplasmic effectors. In silico prediction using a pathogen-host interaction database suggested four of the 163 effectors could be crucial in establishing AM fungi-host interactions. Protein–protein docking analysis revealed interactions between these potential effectors and plant proteins known to be differentially expressed during mycorrhizal association, such as defensins, aquaporins, and PTO proteins. These interactions are multifaceted in modulating host physiological and defense mechanisms, including immune suppression, hydration, nutrient uptake, and oxidative stress modulation.ConclusionsThese findings of the current study provide a foundational understanding of fungal-host molecular interactions and open avenues for exploring pathways influenced by these effectors. By deepening our knowledge of these mechanisms, the use of AM fungi in biofertilizer formulations can be refined by selecting strains with specific effectors that enhance nutrient uptake, improve drought and disease resistance, and tailor the fungi’s symbiotic efficiency to different crops or environmental conditions, thus contributing to more targeted and sustainable agricultural practices.

The Toxoplasma rhoptry protein ROP55 is a major virulence factor that prevents lytic host cell death

Programmed-cell death is an antimicrobial defense mechanism that promotes clearance of intracellular pathogens. Toxoplasma counteracts host immune defenses by secreting effector proteins into host cells; however, how the parasite evades lytic cell death and the effectors involved remain poorly characterized. We identified ROP55, a rhoptry protein that promotes parasite survival by preventing lytic cell death in absence of IFN-γ stimulation. RNA-Seq analysis revealed that ROP55 acts as a repressor of host pro-inflammatory responses. In THP-1 monocytes ΔROP55 infection increased NF-κB p65 nuclear translocation, IL-1β production, and GSDMD cleavage compared to wild type or complemented parasites. ΔROP55 infection also induced RIPK3-dependent necroptosis in human and mouse primary macrophages. Moreover, ΔROP55 parasites were significantly impaired in virulence in female mice and prevented NF-κB activation and parasite clearance in mBMDM. These findings place ROP55 as a major virulence factor, dampening lytic cell death and enabling Toxoplasma to evade clearance from infected cells.

Molecular Characterization of Fusarium Isolates from Upland Cotton Roots in Uzbekistan and Whole-Genome Comparison with Isolates from the United States.

Fusarium oxysporum f. sp. vasinfectum (FOV) is a significant cotton (Gossypium spp.) pathogen causing vascular wilt, browning of the vascular tissues, and plant death in the most severe cases. This global disease is responsible for sizeable crop losses annually and is found in many cotton-producing regions, including the Republic of Uzbekistan and the United States. Specifically, FOV race 4 (FOV4) has been disrupting production for years. This study aimed to genetically characterize FOV4 isolates causing disease in the main cotton-producing region of Uzbekistan and compare them with FOV4 isolates from the United States. A field study conducted in the Bukhara region of the Republic of Uzbekistan in the spring of 2022 identified both FOV4 and new Fusarium isolates from Upland cotton exhibiting typical Fusarium wilt symptoms. Molecular markers were initially used to identify isolates of interest, and a phylogenetic analysis was performed using partial EF1-α sequences, followed by a comparative genomic analysis. We also report for the first time the isolation of F. solani and F. commune causing Fusarium wilt in Uzbekistan. Furthermore, we show that the FOV4 population within our sampling region of Uzbekistan may be dominated by a single biotype with an effector profile similar to that of FOV race 7. One of these effector proteins is also present in the F. commune isolate showing virulence to cotton. Whole-genome comparisons between FOV races can identify unique genetic markers for FOV4 and aid in the development of tools for breeding FOV-resistant cotton varieties.

CRISPR-Hybrid: A CRISPR-Mediated Intracellular Directed Evolution Platform for RNA Aptamers

Recent advances in gene editing and precise regulation of gene expression based on CRISPR technologies have provided powerful tools for the understanding and manipulation of gene functions. Fusing RNA aptamers to the sgRNA of CRISPR can recruit cognate RNA-binding protein (RBP) effectors to target genomic sites, and the expression of sgRNA containing different RNA aptamers permit simultaneous multiplexed and multifunctional gene regulations. Here, we report an intracellular directed evolution platform for RNA aptamers against intracellularly expressed RBPs. We optimize a bacterial CRISPR-hybrid system coupled with FACS, and identified high affinity RNA aptamers orthogonal to existing aptamer-RBP pairs. Application of orthogonal aptamer-RBP pairs in multiplexed CRISPR allows effective simultaneous transcriptional activation and repression of endogenous genes in mammalian cells.

Live cell imaging of plant infection provides new insight into the biology of pathogenesis by the rice blast fungus Magnaporthe oryzae.

Magnaporthe oryzae is the causal agent of rice blast, one of the most serious diseases affecting rice cultivation around the world. During plant infection, M. oryzae forms a specialised infection structure called an appressorium. The appressorium forms in response to the hydrophobic leaf surface and relies on multiple signalling pathways, including a MAP kinase phosphorelay and cAMP-dependent signalling, integrated with cell cycle control and autophagic cell death of the conidium. Together, these pathways regulate appressorium morphogenesis.The appressorium generates enormous turgor, applied as mechanical force to breach the rice cuticle. Re-polarisation of the appressorium requires a turgor-dependent sensor kinase which senses when a critical threshold of turgor has been reached to initiate septin-dependent re-polarisation of the appressorium and plant infection. Invasive growth then requires differential expression and secretion of a large repertoire of effector proteins secreted by distinct secretory pathways depending on their destination, which is also governed by codon usage and tRNA thiolation. Cytoplasmic effectors require an unconventional Golgi-independent secretory pathway and evidence suggests that clathrin-mediated endocytosis is necessary for their delivery into plant cells. The blast fungus then develops a transpressorium, a specific invasion structure used to move from cell-to-cell using pit field sites containing plasmodesmata, to facilitate its spread in plant tissue. This is controlled by the same MAP kinase signalling pathway as appressorium development and requires septin-dependent hyphal constriction. Recent progress in understanding the mechanisms of rice infection by this devastating pathogen using live cell imaging procedures are presented.

Mechanistic determinants and dynamics of cA6 synthesis in type III CRISPR-Cas effector complexes.

Type III clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems (type III CRISPR-Cas systems) use guide RNAs to recognize RNA transcripts of foreign genetic elements, which triggers the generation of cyclic oligoadenylate (cOA) second messengers by the Cas10 subunit of the type III effector complex. In turn, cOAs bind and activate ancillary effector proteins to reinforce the host immune response. Type III systems utilize distinct cOAs, including cyclic tri- (cA3), tetra- (cA4) and hexa-adenylates (cA6). However, the molecular mechanisms dictating cOA product identity are poorly understood. Here we used cryoelectron microscopy to visualize the mechanism of cA6 biosynthesis by the Csm effector complex from Enterococcus italicus (EiCsm). We show that EiCsm synthesizes oligoadenylate nucleotides in 3'-5' direction using a set of conserved binding sites in the Cas10 Palm domains to determine the size of the nascent oligoadenylate chain. Our data also reveal that conformational dynamics induced by target RNA binding results in allosteric activation of Cas10 to trigger oligoadenylate synthesis. Mutations of a key structural element in Cas10 perturb cOA synthesis to favor cA3 and cA4 formation. Together, these results provide comprehensive insights into the dynamics of cOA synthesis in type III CRISPR-Cas systems and reveal key determinants of second messenger product selectivity, thereby illuminating potential avenues for their engineering.

Specialized killing across the domains of life by the type VI secretion systems of Pseudomonas aeruginosa.

Type VI secretion systems (T6SSs) are widespread bacterial protein secretion machines that inject toxic effector proteins into nearby cells, thus facilitating both bacterial competition and virulence. Pseudomonas aeruginosa encodes three evolutionarily distinct T6SSs that each export a unique repertoire of effectors. Owing to its genetic tractability, P. aeruginosa has served as a model organism for molecular studies of the T6SS. However, P. aeruginosa is also an opportunistic pathogen and ubiquitous environmental organism that thrives in a wide range of habitats. Consequently, studies of its T6SSs have provided insight into the role these systems play in the diverse lifestyles of this species. In this review, we discuss recent advances in understanding the regulation and toxin repertoire of each of the three P. aeruginosa T6SSs. We argue that these T6SSs serve distinct physiological functions; whereas one system is a dedicated defensive weapon for interbacterial antagonism, the other two T6SSs appear to function primarily during infection. We find support for this model in examining the signalling pathways that control the expression of each T6SS and co-ordinate the activity of these systems with other P. aeruginosa behaviours. Furthermore, we discuss the effector repertoires of each T6SS and connect the mechanisms by which these effectors kill target cells to the ecological conditions under which their respective systems are activated. Understanding the T6SSs of P. aeruginosa in the context of this organism's diverse lifestyles will provide insight into the physiological roles these secretion systems play in this remarkably adaptable bacterium.

Single molecule Lipid Biosensors Mitigate Inhibition of Endogenous Effector Proteins.

Genetically encoded lipid biosensors uniquely provide real time, spatially resolved kinetic data for lipid dynamics in living cells. Despite clear strengths, these tools have significant drawbacks; most notably, lipid molecules bound to biosensors cannot engage with effectors, potentially inhibiting signaling. Here, we show that although PI 3-kinase (PI3K)-mediated activation of Akt is not significantly reduced in a cell population transfected with a PH-Akt1 PIP3/PI(3,4)P2 biosensor, single cells expressing PH-Akt at visible levels have reduced activation. Tagging endogenous AKT1 with neonGreen reveals its EGF-mediated translocation to the plasma membrane. Co-transfection with the PH-Akt1 or other PIP3 biosensors eliminates this translocation, despite robust recruitment of the biosensors. Inhibition is even observed with PI(3,4)P2-selective biosensor. However, expressing lipid biosensors at low levels, comparable with those of endogenous AKT, produced no such inhibition. Helpfully, these single-molecule biosensors revealed improved dynamic range and kinetic fidelity compared with over-expressed biosensor. This approach represents a non-invasive way to probe spatiotemporal dynamics of PI3K signaling in living cells.

New insights on the regulators and inhibitors of RhoA-ROCK signalling in Parkinson's disease.

A multifaceted and widely prevalent neurodegenerative disease, Parkinson's disease (PD) is typified by the loss of dopaminergic neurons in the midbrain. The discovery of novel treatment(s) that can reverse or halt the course of the disease progression along with identifying the most reliable biomarker(s) in PD remains the crucial concern. RhoA in its active state has been demonstrated to interact with three distinct domains located in the central coiled-coil region of ROCK. RhoA appears to activate effectors most frequently by breaking the intramolecular autoinhibitory connections, which releases functional domains from the effector protein. Additionally, RhoA is highly expressed in the nervous system and it acts as a central molecule for its several downstream effector proteins in multiple signalling pathways both in neurons and glial cells. Mitochondrial dysfunction, vesicle transport malfunction and aggregation of α-Synuclein, a presynaptic neuronal protein genetically and neuropathologically associated with PD. While the RhoA-ROCK signalling pathway appears to have a significant role in PD symptoms, suggesting it could be a promising target for therapeutic interventions. Thus, this review article addresses the potential involvement of the RhoA-ROCK signalling system in the pathophysiology of neurodegenerative illnesses, with an emphasis on its biology and function. We also provide an overview of the state of research on RhoA regulation and its downstream biological activities, focusing on the role of RhoA signalling in neurodegenerative illnesses and the potential benefits of RhoA inhibition as a treatment for neurodegeneration.

Anaplasma phagocytophilum AFAP targets the host nucleolus and inhibits induced apoptosis.

Anaplasma phagocytophilum, the etiologic agent of human granulocytic anaplasmosis (HGA), is an obligate intracellular Gram-negative bacterium. During infection, A. phagocytophilum transfers its type IV secretion system (T4SS) effector proteins into host cells to manipulate cellular processes. AFAP (an actin filament-associated Anaplasma phagocytophilum protein) was identified as a T4SS effector protein and found to interact with the host nucleolin, as described in a previous study. In this study, proteomic analysis was performed to extensively identify AFAP-interacting proteins in host cells and analyze the potential role of AFAP in modulating host cellular processes. A total of 586 host proteins were identified interacting with AFAP by data-independent acquisition mass spectrometry and annotated to 501 Gene Ontology (GO) terms, with the significantly over-represented ones related to ribosomes, nucleolus, DNA binding, and rRNA metabolic process. Given the role of the nucleolus in cellular stress response, the targeting of AFAP to the nucleolus, and the identification of dozens of AFAP-interacting proteins that were annotated to the GO term (GO:0072331, signal transduction by p53 class mediator), the role of AFAP in modulating host apoptosis was determined. AFAP was found capable of inhibiting induced apoptosis. Thus, the proteomic analysis of AFAP-interacting proteins and determination of AFAP with anti-apoptotic activity may help elucidate the role of this T4SS effector protein in HGA pathogenesis.