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Anaplasma phagocytophilum causes human granulocytic anaplasmosis, an emerging tick-borne infection for which there is no vaccine and limited treatments. Although A. phagocytophilum absolutely relies on invading host neutrophils to survive and cause disease, the microbe-host cell interactions that predicate infection are inadequately defined. We found that the bacterium uses its outer surface protein AipB to engage CD18 as a critical invasion step. An AipB peptide that is conserved among its homologs in other Anaplasmataceae pathogens is responsible for binding CD18 and can be targeted by antibody to inhibit infection in vitro and in vivo . However, blocking of A. phagocytophilum infection is most effectively achieved by targeting this AipB peptide together with the functional domains of other invasins. AipB is the first rickettsial invasin identified to interact with a β2 integrin, is key for pathogenesis, and could be targeted to protect against diseases caused by A. phagocytophilum and other Anaplasmataceae bacteria.

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.

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.

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.

Edwardsiella piscicida causes severe hemorrhagic septicemia in marine and freshwater fish worldwide, resulting in significant economic losses for the aquaculture industry (K. Y. Leung, Q. Wang, Z. Yang, and B. A. Siame, Virulence 10:555–567, 2019, https://doi.org/10.1080/21505594.2019.1621648 ). Our previous research identified a novel type III secretion system effector, EseQ, in E. piscicida whose function remains to be elucidated. In this work, we showed that EseQ binds to tubulin and GEF-H1 and destabilizes microtubules. GEF-H1 released from microtubules activates the RhoA-ROCK-MLCII signaling pathway, leading to stress fiber formation in epithelial cells. EseQ deforms the epithelial barrier and promotes E. piscicida ’s invasion in a stress fiber-dependent manner. This work contributes to the understanding of the mechanism by which E. piscicida invades host cells.

Life-threatening systemic infections often occur due to the translocation of pathogens across the gut barrier and into the bloodstream. While the microbial and host mechanisms permitting bacterial gut translocation are well characterized, these mechanisms are still unclear for fungal pathogens such as Candida albicans , a leading cause of nosocomial fungal bloodstream infections. In this study, we dissected the cellular mechanisms of translocation of C. albicans across intestinal epithelia in vitro and identified fungal genes associated with this process. We show that fungal translocation is a dynamic process initiated by invasion and followed by cellular damage and loss of epithelial integrity. A screen of >2,000 C. albicans deletion mutants identified genes required for cellular damage of and translocation across enterocytes. Correlation analysis suggests that hypha formation, barrier damage above a minimum threshold level, and a decreased epithelial integrity are required for efficient fungal translocation. Translocation occurs predominantly via a transcellular route, which is associated with fungus-induced necrotic epithelial damage, but not apoptotic cell death. The cytolytic peptide toxin of C. albicans , candidalysin, was found to be essential for damage of enterocytes and was a key factor in subsequent fungal translocation, suggesting that transcellular translocation of C. albicans through intestinal layers is mediated by candidalysin. However, fungal invasion and low-level translocation can also occur via non-transcellular routes in a candidalysin-independent manner. This is the first study showing translocation of a human-pathogenic fungus across the intestinal barrier being mediated by a peptide toxin. IMPORTANCE Candida albicans , usually a harmless fungus colonizing human mucosae, can cause lethal bloodstream infections when it manages to translocate across the intestinal epithelium. This can result from antibiotic treatment, immune dysfunction, or intestinal damage (e.g., during surgery). However, fungal processes may also contribute. In this study, we investigated the translocation process of C. albicans using in vitro cell culture models. Translocation occurs as a stepwise process starting with invasion, followed by epithelial damage and loss of epithelial integrity. The ability to secrete candidalysin, a peptide toxin deriving from the hyphal protein Ece1, is key: C. albicans hyphae, secreting candidalysin, take advantage of a necrotic weakened epithelium to translocate through the intestinal layer. Candida albicans , usually a harmless fungus colonizing human mucosae, can cause lethal bloodstream infections when it manages to translocate across the intestinal epithelium. This can result from antibiotic treatment, immune dysfunction, or intestinal damage (e.g., during surgery). However, fungal processes may also contribute. In this study, we investigated the translocation process of C. albicans using in vitro cell culture models. Translocation occurs as a stepwise process starting with invasion, followed by epithelial damage and loss of epithelial integrity. The ability to secrete candidalysin, a peptide toxin deriving from the hyphal protein Ece1, is key: C. albicans hyphae, secreting candidalysin, take advantage of a necrotic weakened epithelium to translocate through the intestinal layer.

Trichomonas vaginalis , the most common non-viral sexually transmitted parasite, relies on adherence to host epithelial cells to establish infection. Our previous work highlighted the importance of N6-methyladenine (6mA) DNA methylation in the regulation of transcription and three-dimensional chromatin structure. Now, our study integrates RNA-seq, MeDIP-seq, and assay for transposase-accessible chromatin sequencing data to reveal how 6mA and chromatin accessibility modulate gene expression during T. vaginalis interaction with human host cells. We identified over 3,600 differentially expressed genes upon parasite contact with prostate cells, including pathogenesis-related genes. Moreover, we identified transcriptionally active and repressive regions flanked by 6mA that remain largely stable during the process of host interaction. We mapped genome-wide chromatin accessibility and uncovered differentially accessible regions upon host cell contact associated with a subset of genes involved in adhesion. These results suggest that local chromatin accessibility has a major role in modulating gene expression of key virulence genes during host interaction.

Bacteria colonize surfaces in the environment and can also penetrate tissues and materials by entering micro- and nano-scale cracks and pores. has been observed within nanoscale channels in bone that are 2-3 times smaller than cell diameter. Inside the bone, bacteria are protected from host immunity and systemic antibiotics, potentially contributing to chronic and recurrent infections. The physical mechanisms that enable bacteria to enter channels smaller than the cell width are unclear. It has been proposed that bacteria traverse narrow passages through division, such that daughter cells form within small channels and proliferate in chains down the channel length. Here, we use microfluidics to test the idea that can traverse submicrometer channels through growth. We examined the net migration of growing cell chains within tapered nanochannels (width ~1.5-0.3 μm). We found that proliferation can facilitate migration, but only to cell deformations of 600 nm (65% cell width). Below 600 nm, mechanical confinement significantly slows or completely inhibits division in single cells. Interestingly, growth arrest occurs independent of the Z-ring assembly and is unrelated to the initial orientation of the division plane. Thus, our findings suggest that it is unlikely for to traverse nanoscale channels via division. Bacteria that colonize materials and tissues within the body can be difficult to remove, even with thorough cleaning and application of antibiotics. Recent studies show that bacteria not only colonize the surfaces of tissues in the body but can also squeeze into naturally occurring pores and channels and thereby gain protection from immune cells and antibiotics. Here, we ask how physical forces and cell growth might enable bacteria to enter small pores within materials. We use microfluidic devices to study the growth and migration of the human pathogenic bacteria, .