Cellular Microbiology最新文献

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.

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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.

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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.

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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

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.

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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.