The IpaC carboxyterminal effector domain mediates Src-dependent actin polymerization during Shigella invasion of epithelial cells

Shigella, the causative agent of bacillary dysentery, invades epithelial cells by locally reorganizing the actin cytoskeleton. Shigella invasion requires actin polymerization dependent on the Src tyrosine kinase and a functional bacterial type III secretion (T3S) apparatus. Using dynamic as well as immunofluorescence microscopy, we show that the T3S translocon component IpaC allows the recruitment of the Src kinase required for actin polymerization at bacterial entry sites during the initial stages of Shigella entry. Src recruitment occurred at bacterial-cell contact sites independent of actin polymerization at the onset of the invasive process and was still observed in Shigella strains mutated for translocated T3S effectors of invasion. A Shigella strain with a polar mutation that expressed low levels of the translocator components IpaB and IpaC was fully proficient for Src recruitment and bacterial invasion. In contrast, a Shigella strain mutated in the IpaC carboxyterminal effector domain that was proficient for T3S effector translocation did not induce Src recruitment. Consistent with a direct role for IpaC in Src activation, cell incubation with the IpaC last 72 carboxyterminal residues fused to the Iota toxin Ia (IaC) component that translocates into the cell cytosol upon binding to the Ib component led to Src-dependent ruffle formation. Strikingly, IaC also induced actin structures resembling bacterial entry foci that were enriched in activated Src and were inhibited by the Src inhibitor PP2. These results indicate that the IpaC effector domain determines Src-dependent actin polymerization and ruffle formation during bacterial invasion

Growth Modeling of the Maternal Cytokine Milieu throughout Normal Pregnancy: Macrophage-Derived Chemokine Decreases as Inflammation/Counterregulation Increases

Several recent studies have shown differences in the maternal immune milieu at different phases of pregnancy, but most studies have been cross-sectional or of relatively few time points. Levels of 42 cytokines were determined using a multiplex bead-based assay on archived serum from a cohort of pregnant women N=16 at median of 18 time points tested, from the first trimester through to parturition, per woman. Unconditional growth modeling was then used to determine time-dependent changes in levels of these cytokines. Macrophage-derived chemokine (MDC, aka CCL22) decreases as pregnancy progresses. IL-1β, IL-6, IL-8, IL-12p70, IL-13, IL-15, IP-10, and FLT3-ligand increase as a function of gestational weeks, and IFNα2, IL-1ra, IL-3, IL-9, IL-12p40, and soluble CD40 ligand increase as a function of trimester. As pregnancy normally progresses, a maternal shift away from a type 2-biased immune response and toward an inflammatory/counterregulatory response is observed

The Type III Secreted Protein BspR Regulates the Virulence Genes in Bordetella bronchiseptica

Bordetella bronchiseptica is closely related with B. pertussis and B. parapertussis, the causative agents of whooping cough. These pathogenic species share a number of virulence genes, including the gene locus for the type III secretion system (T3SS) that delivers effector proteins. To identify unknown type III effectors in Bordetella, secreted proteins in the bacterial culture supernatants of wild-type B. bronchiseptica and an isogenic T3SS-deficient mutant were compared with iTRAQ-based, quantitative proteomic analysis method. BB1639, annotated as a hypothetical protein, was identified as a novel type III secreted protein and was designated BspR (Bordetella secreted protein regulator). The virulence of a BspR mutant (ΔbspR) in B. bronchiseptica was significantly attenuated in a mouse infection model. BspR was also highly conserved in B. pertussis and B. parapertussis, suggesting that BspR is an essential virulence factor in these three Bordetella species. Interestingly, the BspR-deficient strain showed hyper-secretion of T3SS-related proteins. Furthermore, T3SS-dependent host cell cytotoxicity and hemolytic activity were also enhanced in the absence of BspR. By contrast, the expression of filamentous hemagglutinin, pertactin, and adenylate cyclase toxin was completely abolished in the BspR-deficient strain. Finally, we demonstrated that BspR is involved in the iron-responsive regulation of T3SS. Thus, Bordetella virulence factors are coordinately but inversely controlled by BspR, which functions as a regulator in response to iron starvation

Vesicular Stomatitis Virus Infection Promotes Immune Evasion by Preventing NKG2D-Ligand Surface Expression

Vesicular stomatitis virus (VSV) has recently gained attention for its oncolytic ability in cancer treatment. Initially, we hypothesized that VSV infection could increase immune recognition of cancer cells through induction of the immune stimulatory NKG2D-ligands. Here we show that VSV infection leads to a robust induction of MICA mRNA expression, however the subsequent surface expression is potently hindered. Thus, VSV lines up with human cytomegalovirus (HCMV) and adenovirus, which actively subvert the immune system by negatively affecting NKG2D-ligand surface expression. VSV infection caused an active suppression of NKG2D-ligand surface expression, affecting both endogenous and histone deacetylase (HDAC)-inhibitor induced MICA, MICB and ULBP-2 expression. The classical immune escape mechanism of VSV (i.e., the M protein blockade of nucleocytoplasmic mRNA transport) was not involved, as the VSV mutant strain, VSVΔM51, which possess a defective M protein, prevented MICA surface expression similarly to wild-type VSV. The VSV mediated down modulation of NKG2D-ligand expression did not involve apoptosis. Constitutive expression of MICA bypassed the escape mechanism, suggesting that VSV affect NKG2D-ligand expression at an early post-transcriptional level. Our results show that VSV possess an escape mechanism, which could affect the immune recognition of VSV infected cancer cells. This may also have implications for immune recognition of cancer cells after combined treatment with VSV and chemotherapeutic drugs

Nuclear Pore Complex Protein Mediated Nuclear Localization of Dicer Protein in Human Cells

Human DICER1 protein cleaves double-stranded RNA into small sizes, a crucial step in production of single-stranded RNAs which are mediating factors of cytoplasmic RNA interference. Here, we clearly demonstrate that human DICER1 protein localizes not only to the cytoplasm but also to the nucleoplasm. We also find that human DICER1 protein associates with the NUP153 protein, one component of the nuclear pore complex. This association is detected predominantly in the cytoplasm but is also clearly distinguishable at the nuclear periphery. Additional characterization of the NUP153-DICER1 association suggests NUP153 plays a crucial role in the nuclear localization of the DICER1 protein

A Nuclear Localization of the Infectious Haematopoietic Necrosis Virus NV Protein Is Necessary for Optimal Viral Growth

The nonvirion (NV) protein of infectious hematopoietic necrosis virus (IHNV) has been previously reported to be essential for efficient growth and pathogenicity of IHNV. However, little is known about the mechanism by which the NV supports the viral growth. In this study, cellular localization of NV and its role in IHNV growth in host cells was investigated. Through transient transfection in RTG-2 cells of NV fused to green fluorescent protein (GFP), a nuclear localization of NV was demonstrated. Deletion analyses showed that the 32EGDL35 residues were essential for nuclear localization of NV protein, and fusion of these 4 amino acids to GFP directed its transport to the nucleus. We generated a recombinant IHNV, rIHNV-NV-ΔEGDL in which the 32EGDL35 was deleted from the NV. rIHNVs with wild-type NV (rIHNV-NV) or with the NV gene replaced with GFP (rIHNV-ΔNV-GFP) were used as controls. RTG-2 cells infected with rIHNV-ΔNV-GFP and rIHNV-NV-ΔEGDL yielded 12- and 5-fold less infectious virion, respectively, than wild type rIHNV-infected cells at 48 h post-infection (p.i.). While treatment with poly I∶C at 24 h p.i. did not inhibit replication of wild-type rIHNVs, replication rates of rIHNV-ΔNV-GFP and rIHNV-NV-ΔEGDL were inhibited by poly I∶C. In addition, both rIHNV-ΔNV and rIHNV-NV-ΔEGDL induced higher levels of expressions of both IFN1 and Mx1 than wild-type rIHNV. These data suggest that the IHNV NV may support the growth of IHNV through inhibition of the INF system and the amino acid residues of 32EGDL35 responsible for nuclear localization are important for the inhibitory activity of NV

Phagosomal Rupture by Mycobacterium tuberculosis Results in Toxicity and Host Cell Death

Survival within macrophages is a central feature of Mycobacterium tuberculosis pathogenesis. Despite significant advances in identifying new immunological parameters associated with mycobacterial disease, some basic questions on the intracellular fate of the causative agent of human tuberculosis in antigen-presenting cells are still under debate. To get novel insights into this matter, we used a single-cell fluorescence resonance energy transfer (FRET)-based method to investigate the potential cytosolic access of M. tuberculosis and the resulting cellular consequences in an unbiased, quantitative way. Analysis of thousands of THP-1 macrophages infected with selected wild-type or mutant strains of the M. tuberculosis complex unambiguously showed that M. tuberculosis induced a change in the FRET signal after 3 to 4 days of infection, indicating phagolysosomal rupture and cytosolic access. These effects were not seen for the strains M. tuberculosisΔRD1 or BCG, both lacking the ESX-1 secreted protein ESAT-6, which reportedly shows membrane-lysing properties. Complementation of these strains with the ESX-1 secretion system of M. tuberculosis restored the ability to cause phagolysosomal rupture. In addition, control experiments with the fish pathogen Mycobacterium marinum showed phagolysosomal translocation only for ESX-1 intact strains, further validating our experimental approach. Most importantly, for M. tuberculosis as well as for M. marinum we observed that phagolysosomal rupture was followed by necrotic cell death of the infected macrophages, whereas ESX-1 deletion- or truncation-mutants that remained enclosed within phagolysosomal compartments did not induce such cytotoxicity. Hence, we provide a novel mechanism how ESX-1 competent, virulent M. tuberculosis and M. marinum strains induce host cell death and thereby escape innate host defenses and favor their spread to new cells. In this respect, our results also open new research directions in relation with the extracellular localization of M. tuberculosis inside necrotic lesions that can now be tackled from a completely new perspective

Autoantibodies against type I IFNs in patients with life-threatening COVID-19

Interindividual clinical variability in the course of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is vast. We report that at least 101 of 987 patients with life-threatening coronavirus disease 2019 (COVID-19) pneumonia had neutralizing immunoglobulin G (IgG) autoantibodies (auto-Abs) against interferon-w (IFN-w) (13 patients), against the 13 types of IFN-a (36), or against both (52) at the onset of critical disease; a few also had auto-Abs against the other three type I IFNs. The auto-Abs neutralize the ability of the corresponding type I IFNs to block SARS-CoV-2 infection in vitro. These auto-Abs were not found in 663 individuals with asymptomatic or mild SARS-CoV-2 infection and were present in only 4 of 1227 healthy individuals. Patients with auto-Abs were aged 25 to 87 years and 95 of the 101 were men. A B cell autoimmune phenocopy of inborn errors of type I IFN immunity accounts for life-threatening COVID-19 pneumonia in at least 2.6% of women and 12.5% of men