PLoS Pathogens最新文献

Binding phospholipid is a simple, yet flexible, strategy for anchorage of bacterial effectors at cell membrane to manipulate host signaling responses. Phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-biphosphate are the only two phospholipid species known to direct bacterial effectors to establish inner leaflet localization at the plasma membrane. Here, selectivity of phosphatidic acid (PA) by bacterial effectors for the plasma membrane anchorage and its molecular entity was identified. C-terminal BID domain of Bartonella T4SS effectors (Beps) directed the plasma membrane localization of Beps in host cells through binding with PA. A hydrophobic segment of the 'HOOK' subdomain from BID is inserted into the bilayer to enhance the interaction of positively charged residues with the lipid headgroups. Mutations of a conserved arginine facilitating the electrostatic interaction, a conserved glycine maintaining the stability of the PA binding groove, and hydrophobic residues determining membrane insertion, prevented the anchorage of Beps at the plasma membrane. Disassociation from plasma membrane to cytosol attenuated the BepC capacity to induce stress fiber formation and cell fragmentation in host cells. The substitution of alanine with aspartic acid at the -1 position preceding the conserved arginine residue hindered BepD anchoring at the plasma membrane, a vital prerequisite for its ability to elicit IL-10 secretion in host macrophages. In conclusion, our findings reveal the PA-binding properties of bacterial effectors to establish plasma membrane localization and will shed light on the intricate mechanisms employed by bacterial effectors within host cells.

Nucleases and 5' nucleotidase (5'-NT) play essential roles in cell biology and are often associated with bacterial virulence. In Mycoplasma spp., which have limited metabolic capacities and rely on nutrient availability, these enzymes are of significant importance for nucleotide salvage. This study explores the potential role of 2 membrane-associated lipoproteins, the major nuclease MnuA and 5'-NT, in Mycoplasma bovis mastitis. Mutants deficient in MnuA (mnuA::Tn) and in 5'-NT (0690::Tn) were identified through genome-wide transposon mutagenesis of M. bovis PG45 type strain and their fitness and virulence were assessed both in vitro, in axenic medium, and in vivo, using murine and cow mastitis models. The mnuA::Tn mutant demonstrated reduced nuclease activity, while 0690::Tn exhibited slow log-phase growth and impaired hydrolase activity towards nucleotides as well as deoxynucleotides (dAMP and dGMP). In comparison to the parent strain, the 0690::Tn mutant displayed markedly reduced fitness, as evidenced by a significant decrease or even absence in post-challenge mycoplasma counts in murine and cow mammary tissues, respectively. Moreover, the 0690::Tn mutant failed to induce mastitis in both experimental models. Conversely, the mnuA::Tn mutant induced inflammation in murine mammary glands, characterized by neutrophil infiltration and increased expression of major inflammatory genes. In cows, the mnuA::Tn was able to cause an increase in somatic cell counts in a manner comparable to the wild type, recruit neutrophils, and induce mastitis. Collectively, these findings provide complementary insights, revealing that disruption of 5'-NT significantly attenuated M. bovis pathogenicity, whereas a MnuA-deficient mutant retained the ability to cause mastitis.

Alphaviruses are mosquito borne RNA viruses that are a reemerging public health threat. Alphaviruses have a broad host range, and can cause diverse disease outcomes like arthritis, and encephalitis. The host ubiquitin proteasome system (UPS) plays critical roles in regulating cellular processes to control the infections with various viruses, including alphaviruses. Previous studies suggest alphaviruses hijack UPS for virus infection, but the molecular mechanisms remain poorly characterized. In addition, whether certain E3 ubiquitin ligases or deubiquitinases act as alphavirus restriction factors remains poorly understood. Here, we employed a cDNA expression screen to identify E3 ubiquitin ligase TRIM32 as a novel intrinsic restriction factor against alphavirus infection, including VEEV-TC83, SINV, and ONNV. Ectopic expression of TRIM32 reduces alphavirus infection, whereas depletion of TRIM32 with CRISPR-Cas9 increases infection. We demonstrate that TRIM32 inhibits alphaviruses through a mechanism that is independent of the TRIM32-STING-IFN axis. Combining reverse genetics and biochemical assays, we found that TRIM32 interferes with genome translation after membrane fusion, prior to replication of the incoming viral genome. Furthermore, our data indicate that the monoubiquitination of TRIM32 is important for its antiviral activity. Notably, we also show two TRIM32 pathogenic mutants R394H and D487N, related to Limb-girdle muscular dystrophy (LGMD), have a loss of antiviral activity against VEEV-TC83. Collectively, these results reveal that TRIM32 acts as a novel intrinsic restriction factor suppressing alphavirus infection and provides insights into the interaction between alphaviruses and the host UPS.

Immunocompromised individuals are at risk for developing lymphocryptovirus-associated lymphoproliferative diseases, such as Epstein Barr virus (EBV)-associated B cell lymphomas and post-transplant lymphoproliferative disorder (PTLD). We previously reported development of cynomolgus lymphocryptovirus (CyLCV)-associated PTLD in Mauritian cynomolgus macaques (MCMs) undergoing hematopoietic stem cell transplantation (HSCT), which mirrored EBV-PTLD in transplant patients. Here, we sought to develop a MCM model of lymphocryptovirus-associated lymphoproliferative disease in immunosuppressed MCMs without HSCT. Five simian immunodeficiency virus (SIV)-infected, CD8α+ cell-depleted MCMs received an infusion of autologous B-lymphoblastoid cells transformed with CyLCV, followed by varying degrees of immunosuppression. Four of five infused macaques developed masses coincident with increasing CyLCV plasma viremia, and necropsies confirmed the presence of multicentric lymphomas, which most commonly manifested in lymph nodes, gastrointestinal tract, adrenal glands, and pancreas. Affected tissues harbored neoplastic lymphocytes double-positive for CD20 and CyLCV EBNA2 antigen, large frequencies of proliferating B cells, and high levels of cell-associated CyLCV DNA. In addition, longitudinal 18F-fluorodeoxyglucose positron-emission tomography (18F-FDG PET) of one MCM successfully detected lymphoproliferative disease in the adrenal glands prior to clinical signs of disease. These data demonstrate successful induction of lymphocryptovirus-associated PTLD-like disease in 4 of 5 MCMs, and thus support the use of MCMs as a preclinical NHP model of EBV-associated lymphoproliferative disease that could be employed to test novel diagnostic and therapeutic modalities.

Babesia bovis, an apicomplexan intraerythrocytic protozoan parasite, causes serious economic loss to cattle industries around the world. Infection with this parasite leads to accumulation of infected red blood cells (iRBCs) in the brain microvasculature that results in severe clinical complications known as cerebral babesiosis. Throughout its growth within iRBCs, the parasite exports various proteins to the iRBCs that lead to the formation of protrusions known as "ridges" on the surface of iRBCs, which serve as sites for cytoadhesion to endothelial cells. Spherical body proteins (SBPs; proteins secreted from spherical bodies, which are organelles specific to Piroplasmida) are exported into iRBCs, and four proteins (SBP1-4) have been reported to date. In this study, we elucidated the function of SBP3 using an inducible gene knockdown (KD) system. Localization of SBP3 was assessed by immunofluorescence assay, and only partial colocalization was detected between SBP3 and SBP4 inside the iRBCs. In contrast, colocalization was observed with VESA-1, which is a major parasite ligand responsible for the cytoadhesion. Immunoelectron microscopy confirmed localization of SBP3 at the ridges. SBP3 KD was performed using the glmS system, and effective KD was confirmed by Western blotting, immunofluorescence assay, and RNA-seq analysis. The SBP3 KD parasites showed severe growth defect suggesting its importance for parasite survival in the iRBCs. VESA-1 on the surface of iRBCs was scarcely detected in SBP3 KD parasites, whereas SBP4 was still detected in the iRBCs. Moreover, abolition of ridges on the iRBCs and reduction of iRBCs cytoadhesion to the bovine brain endothelial cells were observed in SBP3 KD parasites. Immunoprecipitation followed by mass spectrometry analysis detected the host Band 3 multiprotein complex, suggesting an association of SBP3 with iRBC cytoskeleton proteins. Taken together, this study revealed the vital role of SBP3 in ridge formation and its significance in the pathogenesis of cerebral babesiosis.

Microsporidia MB is a promising candidate for developing a symbiont-based strategy for malaria control because it disrupts the capacity of An. arabiensis to transmit the Plasmodium parasite. The symbiont is predominantly localized in the reproductive organs and is transmitted vertically from mother to offspring and horizontally (sexually) during mating. Due to the contribution of both transmission routes, Microsporidia MB has the potential to spread through target vector populations and become established at high prevalence. Stable and efficient vertical transmission of Microsporidia MB is important for its sustainable use for malaria control, however, the vertical transmission efficiency of Microsporidia MB can vary. In this study, we investigate the mechanistic basis of Microsporidia MB vertical transmission in An. arabiensis. We show that vertical transmission occurs through the acquisition of Microsporidia MB by Anopheles cystocyte progenitors following the division of germline stem cells. We also show that Microsporidia MB replicates to increase infection intensity in the oocyte of developing eggs when mosquitoes take a blood meal suggesting that symbiont proliferation in the ovary is coordinated with egg development. The rate of Microsporidia MB transmission to developing eggs is on average higher than the recorded (mother to adult offspring) vertical transmission rate. This likely indicates that a significant proportion of An. arabiensis offspring lose their Microsporidia MB symbionts during development. The stability of germline stem cell infections, coordination of symbiont proliferation, and very high rate of transmission from germline stem cells to developing eggs indicate that Microsporidia MB has a highly specialized vertical transmission strategy in An. arabiensis, which may explain host specificity.

The ongoing evolution and immune escape of SARS-CoV-2, alongside the potential threat of SARS-CoV-1 and other sarbecoviruses, underscore the urgent need for effective strategies against their infection and transmission. This study highlights the discovery of nanobodies from immunized alpacas, which demonstrate exceptionally broad and potent neutralizing capabilities against the recently emerged and more divergent SARS-CoV-2 Omicron subvariants including JD.1.1, JN.1, KP.3, KP.3.1.1, as well as SARS-CoV-1 and coronaviruses from bats and pangolins utilizing receptor ACE2. Among these, Tnb04-1 emerges as the most broad and potent, binding to a conserved hydrophobic pocket in the spike's receptor-binding domain, distinct from the ACE2 binding site. This interaction disrupts the formation of a proteinase K-resistant core, crucial for viral-cell fusion. Notably, intranasal administration of Tnb04-1 in Syrian hamsters effectively prevented respiratory infection and transmission of the authentic Omicron XBB.1.5 subvariant. Thus, Thb04-1 holds promise in combating respiratory acquisition and transmission of diverse sarbecoviruses.

Protein-energy malnutrition (PEM) is a risk factor for developing visceral leishmaniasis (VL). While nutrient deficiency can impair immunity, its mechanistic impact on protective adaptive immune responses following Leishmania infection remains unknown. To determine the potential negative impacts of malnutrition on anti-parasitic responses in chronic VL, we provided mice with a polynutrient-deficient diet (deficient protein, energy, zinc, and iron) that mimics moderate human malnutrition. The polynutrient-deficient diet resulted in growth stunting and reduced mass of visceral organs and following infection with Leishmania infantum, malnourished-mice harbored more parasites in the spleen and liver. Malnourished and infected mice also had fewer T lymphocytes, with reduced T cell production of IFN-γ required for parasite clearance and enhanced production of the immunosuppressive cytokine, IL-10. To determine if IL-10 was causative in disease progression in the malnourished mice, we treated infected mice with monoclonal antibody α-IL-10R. α-IL-10R treatment reduced the parasite number in malnourished mice, restored the number of T cells producing IFN-γ, and enhanced hepatic granuloma formation. Our results indicate that malnutrition increases VL susceptibility due to defective IFN-γ-mediated immunity attributable to increased IL-10 production.

The common rust disease of maize is caused by the obligate biotrophic fungus Puccinia sorghi. The maize Rp1-D allele imparts resistance against the P. sorghi IN2 isolate by initiating a defense response that includes a rapid localized programmed cell death process, the hypersensitive response (HR). In this study, to identify AvrRp1-D from P. sorghi IN2, we employed the isolation of haustoria, facilitated by a biotin-streptavidin interaction, as a powerful approach. This method proves particularly advantageous in cases where the genome information for the fungal pathogen is unavailable, enhancing our ability to explore and understand the molecular interactions between maize and P. sorghi. The haustorial transcriptome generated through this technique, in combination with bioinformatic analyses such as SignalP and TMHMM, enabled the identification of 251 candidate effectors. We ultimately identified two closely related genes, AvrRp1-D.1 and AvrRp1-D.2, which triggered an Rp1-D-dependent defense response in Nicotiana benthamiana. AvrRp1-D-induced Rp1-D-dependent HR was further confirmed in maize protoplasts. We demonstrated that AvrRp1-D.1 interacts directly and specifically with the leucine-rich repeat (LRR) domain of Rp1-D through yeast two-hybrid assay. We also provide evidence that, in the absence of Rp1-D, AvrRp1-D.1 plays a role in suppressing the plant immune response. Our research provides valuable insights into the molecular interactions driving resistance against common rust in maize.

Middle East respiratory syndrome coronavirus (MERS-CoV) and the pangolin MERS-like coronavirus MjHKU4r-CoV-1 employ dipeptidyl peptidase 4 (DPP4) as an entry receptor. MjHKU4r-CoV-1 could infect transgenic mice expressing human DPP4. To understand the mechanism of MjHKU4r-CoV-1 entry into cells, we determined the crystal structures of the receptor binding domain (RBD) of MjHKU4r-CoV-1 spike protein bound to human DPP4 (hDPP4) and Malayan pangolin DPP4 (MjDPP4), respectively. The overall hDPP4-binding mode of MjHKU4r-CoV-1 RBD is similar to that of MERS-CoV RBD. MjHKU4r-CoV-1 RBD shows higher binding affinity to hDPP4 compared to the bat MERS-like coronavirus Ty-BatCoV-HKU4. Via swapping residues between MjHKU4r-CoV-1 RBD and Ty-BatCoV-HKU4 RBD, we identified critical determinants on MjHKU4r-CoV-1 that are responsible for virus usage of hDPP4. Our study suggests that MjHKU4r-CoV-1 is more adapted to the human receptor compared to the bat HKU4 coronavirus and highlights the potential of virus emergence into the human population.