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The intracellular parasite Toxoplasma gondii enhances its dissemination to distant organs by hijacking infected leukocytes via a Trojan Horse mechanism. Upon infecting dendritic cells (DCs), Toxoplasma induces a hypermigratory phenotype characterized by podosome dissolution and formation of F-actin stress fibers. We previously showed that these cytoskeletal changes depend on the effector protein Toxoplasma WAVE complex-interacting protein (TgWIP) secreted from parasites to infected leukocytes. Here, we identify the host adaptor proteins non-catalytic region of tyrosine kinase adaptor protein 1 and 2 (Nck1/2) and growth factor receptor-bound protein 2 (Grb2) as direct TgWIP interactors. TgWIP mainly uses two distinct proline-rich regions (PRRs) to interact with Nck1 and Grb2. Mutating these PRRs abrogates TgWIP binding to Nck1 and Grb2 and diminishes podosome dissolution and DC hypermotility. Furthermore, we show that TgWIP directly interacts with the actin nucleation-promoting factor WAVE regulatory complex (WRC) via a WRC-interacting receptor sequence (WIRS). Disrupting this interaction also influences actin cytoskeletal remodeling and DC hypermotility. Collectively, our data reveal that TgWIP directly interacts with multiple actin regulators, including Nck1, Grb2, and the WRC, to remodel the actin cytoskeleton of the host cells, elucidating a key mechanism that Toxoplasma exploits to enhance host cell migration and dissemination.IMPORTANCEThe parasite Toxoplasma gondii spreads throughout the body by hijacking immune cells and boosting their motility. This ability depends on secreted parasite proteins that manipulate the host cell’s actin cytoskeleton. One such effector, Toxoplasma gondii WAVE-interacting protein (TgWIP), induces dramatic changes in host cell shape and movement, but how it does this has remained unclear. Here, we show that TgWIP directly interacts with multiple host actin-regulatory proteins using distinct sequence motifs. Disrupting these interactions prevents cytoskeletal remodeling and impairs parasite-induced immune cell migration. Our study reveals that Toxoplasma uses defined motifs to co-opt host signaling hubs that control cell motility. Understanding how pathogens exploit the cytoskeleton not only sheds light on host-pathogen interactions but may also reveal broader principles of cell migration relevant to immunity, cancer, and development.

The intracellular parasite Toxoplasma gondii invades immune cells to spread through the circulatory system, eventually reaching the brains of humans and animals. It is not well understood how parasitized immune cells interact with blood vessel walls, a process that ultimately helps Toxoplasma colonize the brain tissue. We found that when Toxoplasma infects the cells lining the blood vessels (endothelium), these produce C-C motif chemokine ligand 5 (CCL5), a potent signaling and attractant molecule. CCL5 production was triggered by a parasite-derived secreted protein, GRA15. CCL5 activated and attracted infected immune cells. In mice, the levels of CCL5 increased quickly in the brain microvasculature after infection, helping the infected immune cells adhere to brain vessels. When the effect of CCL5 was pharmacologically blocked, fewer infected cells sequestered in the brain vessels, lowering the parasite loads. These findings reveal a mechanism through which Toxoplasma manipulates host cells to produce factors that facilitate its colonization of the brain.

IcsB-like k-FATs are found in the related Pseudomonadota and Thermodesulfobacteriota phyla, suggesting that they are a recent biochemical innovation. Like IcsB, new k-FATs are primarily found in proteobacterial species with a T3SS. This leaves open the possibility that they may play a role in the colonization of plants or animals. However, we characterized one k-FAT from an environmental bacterium that is unlikely to possess a T3SS. Additionally, measurable fatty acid acyltransferase activity was not detected in approximately 25% of the proteins tested. These results imply that the IcsB-like k-FAT family has undergone functional diversification and may have a more complex evolutionary origin than previously thought. In summary, this study describes the properties of the IcsB-like k-FAT family and presents yeast-based assays for characterizing new family members and unrelated proteins with similar fatty acid acyltransferase activity.

Macrophages are one of the key target cells of pathogenic Yersinia , where central immune response pathways, such as phagocytosis, gene expression, and inflammasome assembly, are suppressed by secreted bacterial effectors (Yops) in a highly coordinated fashion. Most studies analyzing cooperation between Yop proteins have utilized cell lines and mouse-derived macrophages, which strongly differ from human macrophages. This study employed primary human macrophages and analyzed cooperation between different Yersinia enterocolitica effector proteins on gene expression, histone phosphorylation, calcium signaling, and inflammasome assembly. We reveal synergistic, antagonistic, and individual roles of different Yersinia effector proteins. This work highlights how highly coordinated activities of a limited set of effectors can efficiently disarm macrophage immune responses and lead to a successful infection.

Chlamydia trachomatis is a human pathogen and a prevalent agent of sexually transmitted diseases. The ability to survive and propagate within a protected intracellular niche leads directly to pathology indicative of Chlamydia -mediated disease. The reduced chlamydial genome leads to comparatively limited biosynthetic capacity, thereby necessitating parasitism of metabolites and other resources from the infected host cell. Chlamydia relies heavily on type III secreted effectors to interface with and co-opt host pathways to acquire resources. We demonstrate herein that the plasma membranes of infected cells represent a potential reservoir of resources required for optimal intracellular growth. Chlamydiae employ at least one type III secreted effector protein, translocated membrane-associated effector A (TmeA), to redirect material to the vacuole by manipulating Arp2/3-dependent actin polymerization. This pathway represents a distinct mechanism by which Chlamydia acquires resources and provides evidence for TmeA function during intracellular development.

The phosphatidylinositol derivative PI3P is a key second messenger that regulates multiple cellular processes, particularly membrane trafficking and autophagy. We report here that PikA, a T4SS substrate of R. rickettsii , functions as a PI-3 kinase that catalyzes the production of PI3P to promote autophagy influx. PikA achieves this by recruiting Beclin 1 through direct protein-protein interactions. The expression of the dual-specific PI phosphatase Myotubularin counteracted the effects of PikA and inhibited intracellular R . rickettsii replication. Our results reveal that the modulation of PI metabolism by a bacterial PI-3 kinase is critical for R . rickettsii virulence, and this pathway may provide potential target for the development of therapeutics against infections caused by this pathogen.

Bacterial pathogens have evolved intricate mechanisms to specifically avoid detection by the host autophagy pathway, which is a cell-autonomous innate immune pathway conserved in all eukaryotic organisms. The intracellular pathogen Legionella pneumophila has co-evolved with evolutionarily diverse protozoan hosts for over 100 million years. Thus, these bacteria have devised multiple strategies for evading host autophagy. In this study, we analyzed roughly 300 different Legionella effector proteins for their ability to disrupt autophagy in yeast. The Legionella effector protein Lem26 was found to specifically block autophagy in both yeast and mammalian cells. Biochemical studies revealed that this protein is tightly regulated and is activated upon binding to autophagosomal membranes, which stimulates Lem26 ADP-ribosyltransferase activity and results in the modification of critical autophagy proteins colocalized to these membranes. Thus, Lem26 has evolved the capacity to disrupt host autophagy by proximity labeling of host determinants on autophagosomal membranes, which represents a unique strategy for autophagy inhibition.

Alphaviruses are single stranded, positive sense RNA viruses that are often transmitted through mosquito vectors. With the increasing spread of mosquito populations throughout the world, these arboviruses represent a significant global health concern. Viruses such as Sindbis Virus (SINV), Chikungunya Virus (CHIKV) and Equine Encephalitis Viruses (EEV) are all alphaviruses. As viruses, these pathogens are dependent on the host cell environment for successful viral replication. It has been observed that viruses manipulate cellular metabolism and mitochondrial shape, activity, and dynamics to favor viral infection. This report looked to understand the metabolic changes present during Sindbis virus infection of hamster and human kidney cells. Cells were infected with increasing levels of SINV and at 24 hours post infection the mitochondria morphology was assessed with staining and mitochondrial activity was measured with a real-time Seahorse Bioanalyzer. The relative amount of mitochondrial staining intensity decreased with Sindbis virus infected cells. Both oxygen consumption rate and ATP production were decreased during SINV infection while non-mitochondrial respiration and extracellular acidification rate increased during infection. Collectively, the data indicates that SINV primarily utilizes non-mitochondrial metabolism to support viral infection within the first 24 hours. This understanding of viral preference for host cell metabolism may provide critical targets for antiviral therapies and help further define the nature of alphavirus infection.

The cycle inhibiting factors (Cifs) are a family of translocated effector proteins, found in diverse pathogenic bacteria, that interfere with the host cell cycle by catalyzing the deamidation of a specific glutamine residue (Gln40) in NEDD8 and the related protein ubiquitin. This modification prevents recycling of neddylated cullin-RING ligases, leading to stabilization of various cullin-RING ligase targets, and also prevents polyubiquitin chain formation. Here, we report the crystal structures of two Cif/NEDD8 complexes, revealing a conserved molecular interface that defines enzyme/substrate recognition. Mutation of residues forming the interface suggests that shape complementarity, rather than specific individual interactions, is a critical feature for complex formation. We show that Cifs from diverse bacteria bind NEDD8 in vitro and conclude that they will all interact with their substrates in the same way. The “occluding loop” in Cif gates access to Gln40 by forcing a conformational change in the C terminus of NEDD8. We used native PAGE to follow the activity of Cif from the human pathogen Yersinia pseudotuberculosis and selected variants, and the position of Gln40 in the active site has allowed us to propose a catalytic mechanism for these enzymes.

Summary Apoptosis is a critical process that intrinsically links organism survival to its ability to induce controlled death. Thus, functional apoptosis allows organisms to remove perceived threats to their survival by targeting those cells that it determines pose a direct risk. Central to this process are apoptotic caspases, enzymes that form a signalling cascade, converting danger signals via initiator caspases into activation of the executioner caspase, caspase‐3. This enzyme begins disassembly of the cell by activating DNA degrading enzymes and degrading the cellular architecture. Interaction of pathogenic bacteria with caspases, and in particular, caspase‐3, can therefore impact both host cell and bacterial survival. With roles outside cell death such as cell differentiation, control of signalling pathways and immunomodulation also being described for caspase‐3, bacterial interactions with caspase‐3 may be of far more significance in infection than previously recognized. In this review, we highlight the ways in which bacterial pathogens have evolved to subvert caspase‐3 both through effector proteins that directly interact with the enzyme or by modulating pathways that influence its activation and activity.