The alveolins are a family of intermediate filament-like proteins that form cytoskeletal structures in both free-living and parasitic members of the alveolate kingdom. Despite their important functions, the alveolins' biochemical properties and organizing principles are still poorly understood. Here, we characterize four alveolins of Plasmodium falciparum, the deadliest malaria parasite, to understand how alveolin domains mediate protein-protein interactions and highly specific recruitment to substructures of the cytoskeleton. Unexpectedly, we uncover variable dependence on alveolin domains for each substructure rather than an overarching mechanism. While PfIMC1e requires 1f to be sequentially recruited to the basal complex, PfIMC1c and PfIMC1g do not require interactions with each other to localize properly to the inner membrane complex. Moreover, alveolin domains are not interchangeable-they contain unique signatures for specialized localization. Finally, we identify a region outside the alveolin domain of PfIMC1e that is important for basal complex recruitment. These results provide direct evidence that alveolin domains mediate both alveolin-alveolin interactions and compartment-specific localization.

The pathogenic fungus Cryptococcus neoformans must overcome iron limitation to cause disease in mammalian hosts. Previously, we reported a screen for insertion mutants with poor growth on haem as the sole iron source. In this study, we characterised one such mutant and found that the defective gene encoded a Vam6/Vps39/TRAP1 domain-containing protein required for robust growth on haem, an important iron source in host tissue. We designated this protein Vps3 based on reciprocal best matches with the corresponding protein in Saccharomyces cerevisiae. C. neoformans encodes a second Vam6/Vps39/TRAP1 domain-containing protein designated Vam6/Vlp1, and we found that this protein is also required for robust growth on haem as well as on inorganic iron sources. This protein is predicted to be a component of the homotypic fusion and vacuole protein sorting complex involved in endocytosis. Further characterisation of the vam6Δ and vps3Δ mutants revealed perturbed trafficking of iron acquisition functions (e.g., the high affinity iron permease Cft1) and impaired processing of the transcription factor Rim101, a regulator of haem and iron acquisition. The vps3Δ and vam6Δ mutants also had pleiotropic phenotypes including loss of virulence in a mouse model of cryptococcosis, reduced virulence factor elaboration and increased susceptibility to stress, indicating pleiotropic roles for Vps3 and Vam6 beyond haem use in C. neoformans. TAKE AWAYS: Two Vam6/Vps39/TRAP1-domain proteins, Vps3 and Vam6, support the growth of Cryptococcus neoformans on haem. Loss of Vps3 and Vam6 influences the trafficking and expression of iron uptake proteins. Loss of Vps3 or Vam6 eliminates the ability of C. neoformans to cause disease in a mouse model of cryptococcosis.

Hepatitis B virus (HBV) infection is a major health threat causing 880,000 deaths each year. Available therapies control viral replication but do not cure HBV, leaving patients at risk to develop hepatocellular carcinoma. Here, we show that HBV envelope proteins (HBs)-besides their integration into endosomal membranes-become embedded in the plasma membrane where they can be targeted by redirected T-cells. HBs was detected on the surface of HBV-infected cells, in livers of mice replicating HBV and in HBV-induced hepatocellular carcinoma. Staining with HBs-specific recombinant antibody MoMab recognising a conformational epitope indicated that membrane-associated HBs remains correctly folded in HBV-replicating cells in cell culture and in livers of HBV-transgenic mice in vivo. MoMab coated onto superparamagnetic iron oxide nanoparticles allowed to detect membrane-associated HBs after HBV infection by electron microscopy in distinct stretches of the hepatocyte plasma membrane. Last but not least, we demonstrate that HBs located on the cell surface allow therapeutic targeting of HBV-positive cells by T-cells either engrafted with a chimeric antigen receptor or redirected by bispecific, T-cell engager antibodies. TAKE AWAYS: HBs become translocated to the plasma membrane. Novel, recombinant antibody confirmed proper conformation of HBs on the membrane. HBs provide an interesting target by T-cell-based, potentially curative therapies.

Based on epidemiology studies, Chlamydia trachomatis has been proposed as a co-factor for human papillomavirus (HPV) in the development of cervical cancer. These two intracellular pathogens have been independently reported to induce the production of extra centrosomes, or centrosome amplification, which is a hallmark of cancer cells. We developed a cell culture model to systematically measure the individual and combined effects of Chlamydia and HPV on the centrosome in the same host cell. We found that C. trachomatis caused centrosome amplification in a greater proportion of cells than HPV and that the effects of the two pathogens on the centrosome were additive. Furthermore, centrosome amplification induced by Chlamydia, but not by HPV, strongly correlated with multinucleation and required progression through mitosis. Our results suggest that C. trachomatis and HPV induce centrosome amplification through different mechanisms, with the chlamydial effect being largely due to a failure in cytokinesis that also results in multinucleation. Our findings provide support for C. trachomatis as a co-factor for HPV in carcinogenesis and offer mechanistic insights into how two infectious agents may cooperate to promote cancer. TAKE AWAYS: • Chlamydia and HPV induce centrosome amplification in an additive manner. • Chlamydia-induced centrosome amplification is linked to host cell multinucleation. • Chlamydia-induced centrosome amplification requires cell cycle progression. • Chlamydia and HPV cause centrosome amplification through different mechanisms. • This study supports Chlamydia as a co-factor for HPV in carcinogenesis.

Canid herpesvirus 1 (CHV-1) is a Varicellovirus that causes self-limiting infections in adult dogs but morbidity and mortality in puppies. Using a multipronged approach, we discovered the CHV-1 entry pathway into Madin-Darby canine kidney (MDCK) epithelial cells. We found that CHV-1 triggered extensive host cell membrane lamellipodial ruffling and rapid internalisation of virions in large, uncoated vacuoles, suggestive of macropinocytosis. Treatment with inhibitors targeting key macropinocytosis factors, including inhibitors of Na+ /H+ exchangers, F-actin, myosin light-chain kinase, protein kinase C, p21-activated kinase, phosphatidylinositol-3-kinase and focal adhesion kinase, significantly reduced viral replication. Moreover, the effect was restricted to exposure to the inhibitors early in infection, confirming a role for the macropinocytic machinery during entry. The profile of inhibitors also suggested a role for signalling via integrins and receptor tyrosine kinases in viral entry. In contrast, inhibitors of clathrin, caveolin, microtubules and endosomal acidification did not affect CHV-1 entry into MDCK cells. We found that the virus colocalised with the fluid-phase uptake marker dextran; however, surprisingly, CHV-1 infection did not enhance the uptake of dextran. Thus, our results indicate that CHV-1 uses a macropinocytosis-like, pH-independent entry pathway into MDCK cells, which nevertheless is not based on stimulation of fluid uptake. TAKE AWAYS: CHV-1 enters epithelial cells via a macropinocytosis-like mechanism. CHV-1 induces extensive lamellipodial ruffling. CHV-1 entry into MDCK cells is pH-independent.

Zinc-dependent viral proteins rely on intracellular zinc homeostasis for successful completion of infectious life-cycle. Here, we report that the intracellular labile zinc levels were elevated at early stages of dengue virus (DENV) infection in hepatic cells and this increase in free zinc was abolished in cells infected with UV-inactivated virus or with a DENV replication inhibitor implicating a role for zinc homeostasis in viral RNA replication. This change in free zinc was mediated by zinc transporter, ZIP8, as siRNA-mediated knockdown of ZIP8 resulted in abrogation of increase in free zinc levels leading to significant reduction in DENV titers suggesting a crucial role for ZIP8 in early stages of DENV replication. Furthermore, elevated free zinc levels correlated with high copy numbers of dengue genome in peripheral blood leukocytes obtained from dengue patients compared to healthy controls suggesting a critical role for zinc homeostasis in dengue infection. TAKE AWAYS: Dengue virus utilises cellular zinc homeostasis during replication of its RNA. ZIP8 upregulates free zinc levels during dengue virus replication. Enhanced viremia associates with elevated intracellular free zinc in dengue.

Viruses of Archaea, arguably the most mysterious part of the virosphere due to their unique morphotypes and genome contents, exploit diverse mechanisms for releasing virus progeny from the host cell. These include virus release as a result of the enzymatic degradation of the cell wall or budding through it, common for viruses of Bacteria and Eukarya, as well as a unique mechanism of virus egress through small polygonal perforations on the cell surface. The process of the formation of these perforations includes the development of pyramidal structures on the membrane of the infected cell, which gradually grow by the expansion of their faces and eventually open like flower petals. This mechanism of virion release is operating exclusively in cells of hyperthermophilic hosts from the phylum Crenarchaeota, which are encased solely by a layer of surface proteins, S-layer. The review focuses on recent developments in understanding structural and biochemical details of all three types of egress mechanisms of archaeal viruses. TAKE AWAYS: Many archaeal viruses exit the host via polygonal perforations on the cell membrane. The molecular mechanism of exit via specific apertures is unique for archaeal viruses. Some enveloped archaeal viruses exploit the budding mechanism for egress.

Previously, we found that Ureaplasma parvum internalised into HeLa cells and cytosolic accumulation of galectin-3. U. parvum induced the host cellular membrane damage and survived there. Here, we conducted vesicular trafficking inhibitory screening in yeast to identify U. parvum vacuolating factor (UpVF). U. parvum triggered endoplasmic reticulum (ER) stress and upregulated the unfolded protein response-related factors, including BiP, P-eIF2 and IRE1 in the host cells, but it blocked the induction of the downstream apoptotic factors. MicroRNA library screening of U. parvum-infected cells and UpVF-transfected cells identified miR-211 and miR-214 as the negative regulators of the apoptotic cascade under ER stress. Transient expression of UpVF induced HeLa cell death with intracellular vacuolization; however, some stable UpVF transformant survived. U. parvum-infected cervical cell lines showed resistance to actinomycin D, and UpVF stable transformant cell lines exhibited resistance to X-ray irradiation, as well as cisplatin and paclitaxel. UpVF expressing cervical cancer xenografts in nude mice also acquired resistance to cisplatin and paclitaxel. A mycoplasma expression vector based on Mycoplasma mycoides, Syn-MBA (multiple banded antigen)-UpVF, reduced HeLa cell survival compared with that of Syn-MBA after 72 hr of infection. These findings together suggest novel mechanisms for Ureaplasma infection and the possible implications for cervical cancer malignancy. TAKE AWAYS: • Ureaplasmal novel virulence factor, UpVF, was identified. • UpVF triggered ER stress but suppressed apoptotic cascade via miR-211 and -214. • UpVF conferred resistance to anticancer treatments both in vivo and in vitro. • Dual expression of MBA and UpVF in JCVI-syn3B showed host cell damage.

Biofilm plays an important role in fungal multidrug resistance (MDR). Our previous studies showed that BSC2 is involved in resistance to amphotericin B (AMB) through antioxidation in Saccharomyces cerevisiae. In this study, the overexpression of BSC2 and IRC23 induced strong MDR in S. cerevisiae. BSC2-overexpression affected cellular flocculation, cell surface hydrophobicity, biofilm formation and invasive growth. However, it failed to induce caspofungin (CAS) resistance and affect the invasive growth in FLO mutant strains (FLO11Δ, FLO1Δ, FLO8Δ and TUP1Δ). Furthermore, the overexpression of BSC2 compensated for chitin synthesis defects to maintain the cell wall integrity and significantly reduced the cell morphology abnormality induced by CAS. However, it could not repair the cell wall damage caused by CAS in the FLO mutant strains. Although BSC2 overexpression increased the level of mannose in the cell wall, DPM1 overexpression in both BY4741 and bsc2∆ could confer resistance to CAS and AMB. In addition, BSC2 overexpression significantly increased the mRNA expression of FLO11, FLO1, FLO8 and TUP1. BSC2 may function as a regulator of FLO genes and be involved in cell wall integrity in yeast. Taken together, our data demonstrate that BSC2 induces MDR in a FLO pathway-dependent manner via contributing to the formation of biofilms in S. cerevisiae. TAKE AWAYS: Overexpression of BSC2 induced strong MDR in S. cerevisiae. BSC2 affected cellular flocculation, CSH, biofilm formation and invasive growth. BSC2 could not repair the cell wall damage caused by CAS in the FLO mutants. BSC2 may function as a regulator of FLO genes to maintain cell wall integrity. BSC2 promotes biofilm formation in a FLO pathway-dependent manner to induce MDR.

Rickettsia conorii is a Gram-negative, cytosolic intracellular bacterium that has classically been investigated in terms of endothelial cell infection. However, R. conorii and other human pathogenic Rickettsia species have evolved mechanisms to grow in various cell types, including macrophages, during mammalian infection. During infection of these phagocytes, R. conorii shifts the host cell's overall metabolism towards an anti-inflammatory M2 response, metabolically defined by an increase in host lipid metabolism and oxidative phosphorylation. Lipid metabolism has more recently been identified as a key regulator of host homeostasis through modulation of immune signalling and metabolism. Intracellular pathogens have adapted mechanisms of hijacking host metabolic pathways including host lipid catabolic pathways for various functions required for growth and survival. In the present study, we hypothesised that alterations of host lipid droplets initiated by lipid catabolic pathways during R. conorii infection is important for bacterial survival in macrophages. Herein, we determined that host lipid droplet modulation is initiated early during R. conorii infection, and these alterations rely on active bacteria and lipid catabolic pathways. We also find that these lipid catabolic pathways are essential for efficient bacterial survival. Unlike the mechanisms used by other intracellular pathogens, the catabolism of lipid droplets induced by R. conorii infection is independent of upstream host peroxisome proliferator-activated receptor-alpha (PPARα) signalling. Inhibition of PPARɣ signalling and lipid droplet accumulation in host cells cause a significant decrease in R. conorii survival suggesting a negative correlation with lipid droplet production and R. conorii survival. Together, these results strongly suggest that the modulation of lipid droplets in macrophage cells infected by R. conorii is an important and underappreciated aspect of the infection process. TAKE AWAYS: Host lipid droplets are differentially altered in early and replicative stages of THP-1 macrophage infection with R. conorii. Lipid droplet alterations are initiated in a bacterial-dependent manner and do not require host peroxisome proliferator-activated receptors α or ɣ activation. Pharmacological inhibition of host lipid catabolic processes during R. conorii infection indicates a requirement of lipid catabolism for bacterial survival and initiation of lipid droplet modulation. A significant increase in host lipid droplets during infection has a negative impact on R. conorii survival in THP-1 macrophages.

Predatory interactions among microbes are major evolutionary driving forces for biodiversity. The fungivorous amoeba Protostelium aurantium has a wide fungal food spectrum including foremost pathogenic members of the genus Candida. Here we show that upon phagocytic ingestion by the amoeba, Candida parapsilosis is confronted with an oxidative burst and undergoes lysis within minutes of processing in acidified phagolysosomes. On the fungal side, a functional genomic approach identified copper and redox homeostasis as primary targets of amoeba predation, with the highly expressed copper exporter gene CRP1 and the peroxiredoxin gene PRX1 contributing to survival when encountered with P. aurantium. The fungicidal activity was largely retained in intracellular vesicles of the amoebae. Following their isolation, the content of these vesicles induced immediate killing and lysis of C. parapsilosis in vitro. Proteomic analysis identified 56 vesicular proteins from P. aurantium. Although completely unknown proteins were dominant, many of them could be categorised as hydrolytic enzymes targeting the fungal cell wall, indicating that fungal cell wall structures are under selection pressure by predatory phagocytes in natural environments. TAKE AWAY: The amoeba Protostelium aurantium feeds on fungi, such as Candida parapsilosis. Ingested yeast cells are exposed to reactive oxygen species. A copper exporter and a peroxiredoxin contribute to fungal defence. Yeast cells undergo intracellular lysis. Lysis occurs via a cocktail of hydrolytic enzymes from intracellular vesicles.

Porphyromonas gingivalis is a major pathogen of human periodontitis and dysregulates innate immunity at the gingival epithelial surface. We previously reported that the bacterium specifically degrades junctional adhesion molecule 1 (JAM1), causing gingival epithelial barrier breakdown. However, the functions of other JAM family protein(s) in epithelial barrier dysregulation caused by P. gingivalis are not fully understood. The present results show that gingipains, Arg-specific or Lys-specific cysteine proteases produced by P. gingivalis, specifically degrade coxsackievirus and adenovirus receptor (CXADR), a JAM family protein, at R145 and K235 in gingival epithelial cells. In contrast, a gingipain-deficient P. gingivalis strain was found to be impaired in regard to degradation of CXADR. Furthermore, knockdown of CXADR in artificial gingival epithelium increased permeability to dextran 40 kDa, lipopolysaccharide and peptidoglycan, whereas overexpression of CXADR in a gingival epithelial tissue model prevented penetration by those agents following P. gingivalis infection. Together, these results suggest that P. gingivalis gingipains breach the stratified squamous epithelium barrier by degrading CXADR as well as JAM1, which allows for efficient transfer of bacterial virulence factors into subepithelial tissues. TAKEAWAYS: P. gingivalis, a periodontal pathogen, degraded coxsackievirus and adenovirus receptor (CXADR), a JAM family protein, in gingival epithelial tissues. P. gingivalis gingipains, cysteine proteases, degraded CXADR at R145 and K235. CXADR degradation by P. gingivalis caused increased permeability to lipopolysaccharide and peptidoglycan through gingival epithelial tissues.

Zinc finger proteins (ZFPs) are a large diverse family of proteins with one or more zinc finger domains in which zinc is important in stabilising the domain. ZFPs can interact with DNA, RNA, lipids or even other proteins and therefore contribute to diverse cellular processes including transcriptional regulation, ubiquitin-mediated protein degradation, mRNA decay and stability. In this review, we provide the first comprehensive classification of ZFPs of the malaria parasite Plasmodium falciparum and provide a state of knowledge on the main ZFPs in the parasite, which include the C2H2, CCCH, RING finger and the PHD finger proteins. TAKE AWAYS: The Plasmodium falciparum genome encodes 170 putative Zinc finger proteins (ZFPs). The C2H2, CCCH, RING finger and PHD finger subfamilies of ZFPs are most represented. Known ZFP functions include the regulation of mRNA metabolism and proteostasis.

Proteases are powerful enzymes, which cleave peptide bonds, leading most of the time to irreversible fragmentation or degradation of their substrates. Therefore they control many critical cell fate decisions in eukaryotes. Bacterial pathogens exploit this power and deliver protease effectors through specialised secretion systems into host cells. Research over the past years revealed that the functions of protease effectors during infection are diverse, reflecting the lifestyles and adaptations to specific hosts; however, only a small number of peptidase families seem to have given rise to most of these protease virulence factors by the evolution of different substrate-binding specificities, intracellular activation and subcellular targeting mechanisms. Here, we review our current knowledge about the enzymology and function of protease effectors, which Gram-negative bacterial pathogens translocate via type III and IV secretion systems to irreversibly manipulate host processes. We highlight emerging concepts such as signalling by protease cleavage products and effector-triggered immunity, which host cells employ to detect and defend themselves against a protease attack. TAKE AWAY: Proteases irreversibly cleave proteins to control critical cell fate decisions. Gram-negative bacteria use type III and IV secretion systems to inject effectors. Protease effectors are integral weapons for the manipulation of host processes. Effectors evolved from few peptidase families to target diverse substrates. Effector-triggered immunity upon proteolytic attack emerges as host defence.

Lipid microdomains or lipid rafts are dynamic and tightly ordered regions of the plasma membrane. In mammalian cells, they are enriched in cholesterol, glycosphingolipids, Glycosylphosphatidylinositol-anchored and signalling-related proteins. Several studies have suggested that mammalian pattern recognition receptors are concentrated or recruited to lipid domains during host-pathogen association to enhance the effectiveness of host effector processes. However, pathogens have also evolved strategies to exploit these domains to invade cells and survive. In fungal organisms, a complex cell wall network usually mediates the first contact with the host cells. This cell wall may contain virulence factors that interfere with the host membrane microdomains dynamics, potentially impacting the infection outcome. Indeed, the microdomain disruption can dampen fungus-host cell adhesion, phagocytosis and cellular immune responses. Here, we provide an overview of regulatory strategies employed by pathogenic fungi to engage with and potentially subvert the lipid microdomains of host cells. TAKE AWAY: Lipid microdomains are ordered regions of the plasma membrane enriched in cholesterol, glycosphingolipids (GSL), GPI-anchored and signalling-related proteins. Pathogen recognition by host immune cells can involve lipid microdomain participation. During this process, these domains can coalesce in larger complexes recruiting receptors and signalling proteins, significantly increasing their signalling abilities. The antifungal innate immune response is mediated by the engagement of pathogen-associated molecular patterns to pattern recognition receptors (PRRs) at the plasma membrane of innate immune cells. Lipid microdomains can concentrate or recruit PRRs during host cell-fungi association through a multi-interactive mechanism. This association can enhance the effectiveness of host effector processes. However, virulence factors at the fungal cell surface and extracellular vesicles can re-assembly these domains, compromising the downstream signalling and favouring the disease development. Lipid microdomains are therefore very attractive targets for novel drugs to combat fungal infections.