Early Steps Of Infection Research Articles

HIV-1 Vpr- and Reverse Transcription-Induced Apoptosis in Resting Peripheral Blood CD4 T Cells and Protection by Common Gamma-Chain Cytokines.

HIV-1 infection leads to the progressive depletion of the CD4 T cell compartment by various known and unknown mechanisms. In vivo, HIV-1 infects both activated and resting CD4 T cells, but in vitro, in the absence of any stimuli, resting CD4 T cells from peripheral blood are resistant to infection. This resistance is generally attributed to an intracellular environment that does not efficiently support processes such as reverse transcription (RT), resulting in abortive infection. Here, we show that in vitro HIV-1 infection of resting CD4 T cells induces substantial cell death, leading to abortive infection. In vivo, however, various microenvironmental stimuli in lymphoid and mucosal tissues provide support for HIV-1 replication. For example, common gamma-chain cytokines (CGCC), such as interleukin-7 (IL-7), render resting CD4 T cells permissible to HIV-1 infection without inducing T cell activation. Here, we find that CGCC primarily allow productive infection by preventing HIV-1 triggering of apoptosis, as evidenced by early release of cytochrome c and caspase 3/7 activation. Cell death is triggered both by products of reverse transcription and by virion-borne Vpr protein, and CGCC block both mechanisms. When HIV-1 RT efficiency was enhanced by SIVmac239 Vpx protein, cell death was still observed, indicating that the speed of reverse transcription and the efficiency of its completion contributed little to HIV-1-induced cell death in this system. These results show that a major restriction on HIV-1 infection in resting CD4 T cells resides in the capacity of these cells to survive the early steps of HIV-1 infection. A major consequence of HIV-1 infection is the destruction of CD4 T cells. Here, we show that delivery of virion-associated Vpr protein and the process of reverse transcription are each sufficient to trigger apoptosis of resting CD4 T cells isolated from peripheral blood. While these 2 mechanisms have been previously described in various cell types, we show for the first time their concerted effect in inducing resting CD4 T cell depletion. Importantly, we found that cytokines such as IL-7 and IL-4, which are particularly active in sites of HIV-1 replication, protect resting CD4 T cells from these cytopathic effects and, primarily through this protection, rather than through enhancement of specific replicative steps, they promote productive infection. This study provides important new insights for the understanding of the early steps of HIV-1 infection and T cell depletion.

ID: 26: PML-dependent inhibition of HIV-1 reverse-transcription by Daxx

PML, also known as TRIM19, belongs to the family of tripartite motif (TRIM) proteins. Like some other TRIM proteins, PML exerts a broad antiviral activity. PML is mainly expressed in the nucleus, where it forms dynamic structures known as PML nuclear bodies that recruit many proteins, such as Sp100 and Daxx. It was reported that HIV-1 infection induces the translocation of PML from the nucleus to the cytoplasm (Turelli et al., 2001). This paper also suggested that PML could interfere with HIV-1 infection but this was later contradicted (Berthoux et al., 2003). We sought to re-evaluate the implication of PML during HIV-1 infection. We show that infection by HIV-1 but also other retroviruses triggers the formation of PML cytoplasmic bodies, as early as 30 min post-infection. PML re-localization was blocked by reverse-transcription inhibitors and was not observed following infection with unrelated viruses, suggesting it is specifically triggered by retroviral reverse-transcription. Furthermore, we show that PML interferes with an early step of retroviral infection since PML knockdown dramatically increases RT efficiency. However, the effect of PML on retroviral infection was found to be indirect and to operate through the stabilization of one of its well-characterized partners, Daxx. In the presence of PML, Daxx inhibits an early step of reverse-transcription thereby interfering with HIV-1 infection. Like PML, Daxx is an interferon-inducible protein that provides intrinsic immunity against various viruses. Our study unravels for the first time its capacity to inhibit an early step of HIV-1 replication.

Genome-Wide Screen Reveals Valosin-Containing Protein Requirement for Coronavirus Exit from Endosomes.

Coronaviruses are RNA viruses with a large zoonotic reservoir and propensity for host switching, representing a real threat for public health, as evidenced by severe acute respiratory syndrome (SARS) and the emerging Middle East respiratory syndrome (MERS). Cellular factors required for their replication are poorly understood. Using genome-wide small interfering RNA (siRNA) screening, we identified 83 novel genes supporting infectious bronchitis virus (IBV) replication in human cells. Thirty of these hits can be placed in a network of interactions with viral proteins and are involved in RNA splicing, membrane trafficking, and ubiquitin conjugation. In addition, our screen reveals an unexpected role for valosin-containing protein (VCP/p97) in early steps of infection. Loss of VCP inhibits a previously uncharacterized degradation of the nucleocapsid N protein. This inhibition derives from virus accumulation in early endosomes, suggesting a role for VCP in the maturation of virus-loaded endosomes. The several host factors identified in this study may provide avenues for targeted therapeutics. Coronaviruses are RNA viruses representing a real threat for public health, as evidenced by SARS and the emerging MERS. However, cellular factors required for their replication are poorly understood. Using genome-wide siRNA screening, we identified novel genes supporting infectious bronchitis virus (IBV) replication in human cells. The several host factors identified in this study may provide directions for future research on targeted therapeutics.

Early inhibition of hepatocyte innate responses by hepatitis B virus

The outcome of hepatitis B virus (HBV) infection may be influenced by early interactions between the virus and hepatocyte innate immune responses. To date, the study of such interactions during the very early step of infection has not been adequately investigated. We used the HepaRG cell line, as well as primary human hepatocytes to analyze, within 24h of exposure to HBV, either delivered by a physiologic route or baculovirus vector (Bac-HBV), the early modulation of the expression of selected antiviral/pro-inflammatory cytokines and interferon stimulated genes. Experiments were also performed in the presence or absence of innate receptor agonists to investigate early HBV-induced blockade of innate responses. We show that hepatocytes themselves could detect HBV, and express innate genes when exposed to either HBV virions or Bac-HBV. Whereas Bac-HBV triggered a strong antiviral cytokine secretion followed by the clearance of replicative intermediates, a physiologic HBV exposure led to an abortive response. The early inhibition of innate response by HBV was mainly evidenced on Toll-like receptor 3 and RIG-I/MDA5 signaling pathways upon engagement with exogenous agonist, leading to a decreased expression of several pro-inflammatory and antiviral cytokine genes. Finally, we demonstrate that this early inhibition of dsRNA-mediated response is due to factor(s) present in the HBV inoculum, but not being HBsAg or HBeAg themselves, and does not require de novo viral protein synthesis and replication. Our data provide strong evidence that HBV viral particles themselves can readily inhibit host innate immune responses upon virion/cell interactions, and may explain, at least partially, the "stealthy" character of HBV.

THY-1 Cell Surface Antigen (CD90) Has an Important Role in the Initial Stage of Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) infects about 50% of the US population, is the leading infectious cause of birth defects, and is considered the most important infectious agent in transplant recipients. The virus infects many cell types in vivo and in vitro. While previous studies have identified several cellular proteins that may function at early steps of infection in a cell type dependent manner, the mechanism of virus entry is still poorly understood. Using a computational biology approach, correlating gene expression with virus infectivity in 54 cell lines, we identified THY-1 as a putative host determinant for HCMV infection in these cells. With a series of loss-of-function, gain-of-function and protein-protein interaction analyses, we found that THY-1 mediates HCMV infection at the entry step and is important for infection that occurs at a low m.o.i. THY-1 antibody that bound to the cell surface blocked HCMV during the initial 60 minutes of infection in a dose-dependent manner. Down-regulation of THY-1 with siRNA impaired infectivity occurred during the initial 60 minutes of inoculation. Both THY-1 antibody and siRNA inhibited HCMV-induced activation of the PI3-K/Akt pathway required for entry. Soluble THY-1 protein blocked HCMV infection during, but not after, virus internalization. Expression of exogenous THY-1 enhanced entry in cells expressing low levels of the protein. THY-1 interacted with HCMV gB and gH and may form a complex important for entry. However, since gB and gH have previously been shown to interact, it is uncertain if THY-1 directly binds to both of these proteins. Prior observations that THY-1 (a) interacts with αVβ3 integrin and recruits paxillin (implicated in HCMV entry), (b) regulates leukocyte extravasation (critical for HCMV viremia), and (c) is expressed on many cells targeted for HCMV infection including epithelial and endothelial cells, fibroblast, and CD34+/CD38- stem cells, all support a role for THY-1 as an HCMV entry mediator in a cell type dependent manner. THY-1 may function through a complex setting, that would include viral gB and gH, and other cellular factors, thus links virus entry with signaling in host cells that ultimately leads to virus infection.

Bunyaviruses: From Transmission by Arthropods to Virus Entry into the Mammalian Host First-Target Cells

The Bunyaviridae constitute a large family of animal RNA viruses distributed worldwide, most members of which are transmitted to vertebrate hosts by arthropods and can cause severe pathologies in humans and livestock. With an increasing number of outbreaks, arthropod-borne bunyaviruses (arbo-bunyaviruses) present a global threat to public health and agricultural productivity. Yet transmission, tropism, receptors and cell entry remain poorly characterized. The focus of this review is on the initial infection of mammalian hosts by arbo-bunyaviruses from cellular and molecular perspectives, with particular attention to the human host. We address current knowledge and advances regarding the identity of the first-target cells and the subsequent processes of entry and penetration into the cytosol. Aspects of the vector-to-host switch that influence the early steps of cell infection in mammalian skin, where incoming particles are introduced by infected arthropods, are also highlighted and discussed.

HIV-1 Resistance to the Capsid-Targeting Inhibitor PF74 Results in Altered Dependence on Host Factors Required for Virus Nuclear Entry.

During HIV-1 infection of cells, the viral capsid plays critical roles in reverse transcription and nuclear entry of the virus. The capsid-targeting small molecule PF74 inhibits HIV-1 at early stages of infection. HIV-1 resistance to PF74 is complex, requiring multiple amino acid substitutions in the viral CA protein. Here we report the identification and analysis of a novel PF74-resistant mutant encoding amino acid changes in both domains of CA, three of which are near the pocket where PF74 binds. Interestingly, the mutant virus retained partial PF74 binding, and its replication was stimulated by the compound. The mutant capsid structure was not significantly perturbed by binding of PF74; rather, the mutations inhibited capsid interactions with CPSF6 and Nup153 and altered HIV-1 dependence on these host factors and on TNPO3. Moreover, the replication of the mutant virus was markedly impaired in activated primary CD4(+) T cells and macrophages. Our results suggest that HIV-1 escapes a capsid-targeting small molecule inhibitor by altering the virus's dependence on host factors normally required for entry into the nucleus. They further imply that clinical resistance to inhibitors targeting the PF74 binding pocket is likely to be strongly limited by functional constraints on HIV-1 evolution. The HIV-1 capsid plays critical roles in early steps of infection and is an attractive target for therapy. Here we show that selection for resistance to a capsid-targeting small molecule inhibitor can result in viral dependence on the compound. The mutant virus was debilitated in primary T cells and macrophages--cellular targets of infection in vivo. The mutations also altered the virus's dependence on cellular factors that are normally required for HIV-1 entry into the nucleus. This work provides new information regarding mechanisms of HIV-1 resistance that should be useful in efforts to develop clinically useful drugs targeting the HIV-1 capsid.

Suramin inhibits chikungunya virus replication through multiple mechanisms

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes severe and often persistent arthritis. In recent years, millions of people have been infected with this virus for which registered antivirals are still lacking. Using our recently established in vitro assay, we discovered that the approved anti-parasitic drug suramin inhibits CHIKV RNA synthesis (IC50 of ∼5μM). The compound inhibited replication of various CHIKV isolates in cell culture with an EC50 of ∼80μM (CC50>5mM) and was also active against Sindbis virus and Semliki Forest virus. In vitro studies hinted that suramin interferes with (re)initiation of RNA synthesis, whereas time-of-addition studies suggested it to also interfere with a post-attachment early step in infection, possibly entry. CHIKV (nsP4) mutants resistant against favipiravir or ribavirin, which target the viral RNA polymerase, did not exhibit cross-resistance to suramin, suggesting a different mode of action. The assessment of the activity of a variety of suramin-related compounds in cell culture and the in vitro assay for RNA synthesis provided more insight into the moieties required for antiviral activity. The antiviral effect of suramin-containing liposomes was also analyzed. Its approved status makes it worthwhile to explore the use of suramin to prevent and/or treat CHIKV infections.

Inhibition of respiratory syncytial virus replication and virus-induced p38 kinase activity by berberine.

Respiratory syncytial virus (RSV) causes severe lower respiratory tract infection and poses a major public health threat worldwide. No effective vaccines or therapeutics are currently available; berberine, an isoquinoline alkaloid from various medicinal plants, has been shown to exert antiviral and several other biological effects. Recent studies have shown that p38 mitogen-activated protein kinase (MAPK) activity is implicated in infection by and replication of viruses such as RSV and the influenza virus. Because berberine has previously been implicated in modulating the activity of p38 MAPK, its effects on RSV infection and RSV-mediated p38 MAPK activation were examined. Replication of RSV in epithelial cells was significantly reduced by treatment with berberine. Berberine treatment caused decrease in viral protein and mRNA syntheses. Similar to previously reported findings, RSV infection caused phosphorylation of p38 MAPK at a very early time point of infection, and phosphorylation was dramatically reduced by berberine treatment. In addition, production of interleukin-6 mRNA upon RSV infection was significantly suppressed by treatment with berberine, suggesting the anti-inflammatory role of berberine during RSV infection. Taken together, we showed that berberine, a natural compound already proven to be safe for human consumption, suppresses the replication of RSV. In addition, the current study suggests that inhibition of RSV-mediated early p38 MAPK activation, which has been implicated as an early step in viral infection, as a potential molecular mechanism.

Hepatitis D Virus Infection of Mice Expressing Human Sodium Taurocholate Co-transporting Polypeptide.

Hepatitis D virus (HDV) is the smallest virus known to infect human. About 15 million people worldwide are infected by HDV among those 240 million infected by its helper hepatitis B virus (HBV). Viral hepatitis D is considered as one of the most severe forms of human viral hepatitis. No specific antivirals are currently available to treat HDV infection and antivirals against HBV do not ameliorate hepatitis D. Liver sodium taurocholate co-transporting polypeptide (NTCP) was recently identified as a common entry receptor for HDV and HBV in cell cultures. Here we show HDV can infect mice expressing human NTCP (hNTCP-Tg). Antibodies against critical regions of HBV envelope proteins blocked HDV infection in the hNTCP-Tg mice. The infection was acute yet HDV genome replication occurred efficiently, evident by the presence of antigenome RNA and edited RNA species specifying large delta antigen in the livers of infected mice. The resolution of HDV infection appears not dependent on adaptive immune response, but might be facilitated by innate immunity. Liver RNA-seq analyses of HDV infected hNTCP-Tg and type I interferon receptor 1 (IFNα/βR1) null hNTCP-Tg mice indicated that in addition to induction of type I IFN response, HDV infection was also associated with up-regulation of novel cellular genes that may modulate HDV infection. Our work has thus proved the concept that NTCP is a functional receptor for HDV infection in vivo and established a convenient small animal model for investigation of HDV pathogenesis and evaluation of antiviral therapeutics against the early steps of infection for this important human pathogen.

Fluorescent image analysis of HIV-1 and HIV-2 uncoating kinetics in the presence of old world monkey TRIM5α.

Uncoating of Human Immunodeficiency Virus type 1 (HIV-1) and type 2 (HIV-2) conical cores is an important early step for establishment of infection. In Old World Monkey (OWM) cells, the TRIM5α cellular factor potently suppresses an early step of infection by HIV-1. Previously, biochemical studies using whole cell lysates of infected cells revealed that OWM TRIM5α accelerates the uncoating of HIV-1, leading to premature reverse transcription. In the present study, we re-evaluated uncoating kinetics of HIV-1 in the presence of OWM TRIM5α by using an in situ uncoating assay, which allowed us to differentiate productive HIV-1 entry from simple (non-productive) endocytosis. Results showed that the uncoating kinetics of HIV-1 was indeed accelerated in the presence of OWM TRIM5α. Furthermore, we adapted an in situ uncoating assay to HIV-2, which showed wide variations in TRIM5α sensitivity among different isolates. HIV-2 isolate GH123, whose infectivity was suppressed by cynomolgus monkey (CM) TRIM5α, showed accelerated uncoating in the presence of CM TRIM5α. In contrast, mutant HIV-2 ASA, whose infectivity was unaltered by CM TRIM5α, showed no change in uncoating kinetics in the presence of CM TRIM5α. These results confirmed and further extended the previous notion that accelerated uncoating is associated with restriction activity of TRIM5α against lentiviruses.

HPV16 E5 deregulates the autophagic process in human keratinocytes

Autophagy plays key roles during host defense against pathogens, but viruses have evolved strategies to block the process or to exploit it for replication and successful infection. The E5 oncoprotein of human papillomavirus type 16 (HPV16 E5) perturbs epithelial homeostasis down-regulating the expression of the keratinocyte growth factor receptor (KGFR/FGFR2b), whose signaling induces autophagy. Here we investigated the possible effects of 16E5 on autophagy in human keratinocytes expressing the viral protein. The 16E5 presence strongly inhibited the autophagic process, while forced expression and activation of KGFR counteracted this effect, demonstrating that the viral protein and the receptor exert opposite and interplaying roles not only on epithelial differentiation, but also in the control of autophagy. In W12 cells, silencing of the 16E5 gene in the context of the viral full length genome confirmed its role on autophagy inhibition. Finally, molecular approaches showed that the viral protein interferes with the transcriptional regulation of autophagy also through the impairment of p53 function, indicating that 16E5 uses parallel mechanisms for autophagy impairment. Overall our results further support the hypothesis that a transcriptional crosstalk among 16E5 and KGFR might be the crucial molecular driver of epithelial deregulation during early steps of HPV infection and transformation.

Role of EXT1 and Glycosaminoglycans in the Early Stage of Filovirus Entry.

Filoviruses, including both Ebola virus (EBOV) and Marburg virus (MARV), can infect humans and other animals, causing hemorrhagic fever with a high mortality rate. Entry of these viruses into the host is mediated by a single filoviral glycoprotein (GP). GP is composed of two subunits: GP1, which is responsible for attachment and binding to receptor(s) on susceptible cells, and GP2, which mediates viral and cell membrane fusion. Although numerous host factors have been implicated in the entry process, the initial attachment receptor(s) has not been well defined. In this report, we demonstrate that exostosin 1 (EXT1), which is involved in biosynthesis of heparan sulfate (HS), plays a role in filovirus entry. Expression knockdown of EXT1 by small interfering RNAs (siRNAs) impairs GP-mediated pseudoviral entry and that of infectious EBOV and MARV in tissue cultured cells. Furthermore, HS, heparin, and other related glycosaminoglycans (GAGs), to different extents, can bind to and block GP-mediated viral entry and that of infectious filoviruses. These results strongly suggest that HS and other related GAGs are attachment receptors that are utilized by filoviruses for entry and infection. These GAGs may have therapeutic potential in treating EBOV- and MARV-infected patients. Infection by Ebola virus and Marburg virus can cause severe illness in humans, with a high mortality rate, and currently there is no FDA-approved vaccine or therapeutic treatment available. The ongoing 2014 outbreak in West Africa underscores a lack of our understanding in the infection and pathogenesis of these viruses and the urgency of drug discovery and development. In this study, we provide several pieces of evidence that demonstrate that heparan sulfate and other closely related glycosaminoglycans are the molecules that are used by filoviruses for initial attachment. Furthermore, we demonstrate that these glycosaminoglycans can block entry of and infection by filoviruses. Thus, this work provides mechanistic insights on the early step of filoviral infection and suggests a possible therapeutic option for diseases caused by filovirus infection.

Cocaine modulates HIV-1 integration in primary CD4+ T cells: implications in HIV-1 pathogenesis in drug-abusing patients.

Epidemiologic studies suggest that cocaine abuse worsens HIV-1 disease progression. Increased viral load has been suggested to play a key role for the accelerated HIV disease among cocaine-abusing patients. The goal of this study was to investigate whether cocaine enhances proviral DNA integration as a mechanism to increase viral load. We infected CD4(+) T cells that are the primary targets of HIV-1 in vivo and treated the cells with physiologically relevant concentrations of cocaine (1 µM-100 µM). Proviral DNA integration in the host genome was measured by nested qPCR. Our results illustrated that cocaine from 1 µM through 50 µM increased HIV-1 integration in CD4(+) T cells in a dose-dependent manner. As integration can be modulated by several early postentry steps of HIV-1 infection, we examined the direct effects of cocaine on viral integration by in vitro integration assays by use of HIV-1 PICs. Our data illustrated that cocaine directly increases viral DNA integration. Furthermore, our MS analysis showed that cocaine is able to enter CD4(+) T cells and localize to the nucleus-. In summary, our data provide strong evidence that cocaine can increase HIV-1 integration in CD4(+) T cells. Therefore, we hypothesize that increased HIV-1 integration is a novel mechanism by which cocaine enhances viral load and worsens disease progression in drug-abusing HIV-1 patients.

HPV16 early gene E5 specifically reduces miRNA-196a in cervical cancer cells.

High-risk human papillomavirus (HPV) type 16, which is responsible for greater than 50% of cervical cancer cases, is the most prevalent and lethal HPV type. However, the molecular mechanisms of cervical carcinogenesis remain elusive, particularly the early steps of HPV infection that may transform normal cervical epithelium into a pre-neoplastic state. Here, we report that a group of microRNAs (microRNAs) were aberrantly decreased in HPV16-positive normal cervical tissues, and these groups of microRNAs are further reduced in cervical carcinoma. Among these miRNAs, miR196a expression is the most reduced in HPV16-infected tissues. Interestingly, miR196a expression is low in HPV16-positive cervical cancer cell lines but high in HPV16-negative cervical cancer cell lines. Furthermore, we found that only HPV16 early gene E5 specifically down-regulated miRNA196a in the cervical cancer cell lines. In addition, HoxB8, a known miR196a target gene, is up-regulated in the HPV16 cervical carcinoma cell line but not in HPV18 cervical cancer cell lines. Various doses of miR196a affected cervical cancer cell proliferation and apoptosis. Altogether, these results suggested that HPV16 E5 specifically down-regulates miR196a upon infection of the human cervix and initiates the transformation of normal cervix cells to cervical carcinoma.

Porcine complement regulatory protein CD46 and heparan sulfates are the major factors for classical swine fever virus attachment in vitro.

Classical swine fever virus (CSFV) is the causative agent of a severe multi-systemic disease of pigs. While several aspects of virus-host-interaction are known, the early steps of infection remain unclear. For the closely related bovine viral diarrhea virus (BVDV), a cellular receptor is known: bovine complement regulatory protein CD46. Given that these two pestiviruses are closely related, porcine CD46 is also a candidate receptor for CSFV. In addition to CD46, cell-culture-adapted CSFV strains have been shown to use heparan sulfates as an additional cellular factor. In the present study, the interaction of field-type and cell-culture-adapted CSFV with a permanent porcine cell line or primary macrophages was assessed using anti-porcine CD46 monoclonal antibodies and a heparan-sulfate-blocking compound, DSTP-27. The influence of receptor blocking was assessed using virus titration and quantitative PCR. Treatment of cells with monoclonal antibodies against porcine CD46 led to a reduction of viral growth in both cell types. The effect was most pronounced with field-type CSFV. The blocking could be enhanced by addition of DSTP-27, especially for cell-culture-adapted CSFV. The combined use of both blocking agents led to a significant reduction of viral growth but was also not able to abolish infection completely. The results obtained in this study showed that both porcine CD46 and heparan sulfates play a major role in the initial steps of CSFV infection. Additional receptors might also play a role for attachment and entry; however, their impact is obviously limited in vitro in comparison to CD46 and heparan sulfates.

Vesicles from different Trypanosoma cruzi strains trigger differential innate and chronic immune responses

Trypomastigote forms of Trypanosoma cruzi, the causative agent of Chagas Disease, shed extracellular vesicles (EVs) enriched with glycoproteins of the gp85/trans-sialidase (TS) superfamily and other α-galactosyl (α-Gal)-containing glycoconjugates, such as mucins. Here, purified vesicles from T. cruzi strains (Y, Colombiana, CL-14 and YuYu) were quantified according to size, intensity and concentration. Qualitative analysis revealed differences in their protein and α-galactosyl contents. Later, those polymorphisms were evaluated in the modulation of immune responses (innate and in the chronic phase) in C57BL/6 mice. EVs isolated from YuYu and CL-14 strains induced in macrophages higher levels of proinflammatory cytokines (TNF-α and IL-6) and nitric oxide via TLR2. In general, no differences were observed in MAPKs activation (p38, JNK and ERK 1/2) after EVs stimulation. In splenic cells derived from chronically infected mice, a different modulation pattern was observed, where Colombiana (followed by Y strain) EVs were more proinflammatory. This modulation was independent of the T. cruzi strain used in the mice infection. To test the functional importance of this modulation, the expression of intracellular cytokines after in vitro exposure was evaluated using EVs from YuYu and Colombiana strains. Both EVs induced cytokine production with the appearance of IL-10 in the chronically infected mice. A high frequency of IL-10 in CD4+ and CD8+ T lymphocytes was observed. A mixed profile of cytokine induction was observed in B cells with the production of TNF-α and IL-10. Finally, dendritic cells produced TNF-α after stimulation with EVs. Polymorphisms in the vesicles surface may be determinant in the immunopathologic events not only in the early steps of infection but also in the chronic phase.

Antiviral and Antioxidant Activities of Sulfated Galactomannans from Plants of Caatinga Biome.

Dengue represents a serious social and economic public health problem; then trying to contribute to improve its control, the objective of this research was to develop phytoterapics for dengue treatment using natural resources from Caatinga biome. Galactomannans isolated from Adenanthera pavonina L., Caesalpinia ferrea Mart., and Dimorphandra gardneriana Tull were chemically sulfated in order to evaluate the antioxidant, and antiviral activities and the role in the inhibition of virus DENV-2 in Vero cells. A positive correlation between the degree of sulfation, antioxidant and antiviral activities was observed. The sulfated galactomannans showed binding to the virus surface, indicating that they interact with DENV-2. The sulfated galactomannans from C. ferrea showed 96% inhibition of replication of DENV-2 followed by D. gardneriana (94%) and A. pavonina (77%) at 25 µg/mL and all sulfated galactomannans also showed antioxidant activity. This work is the first report of the antioxidant and antiviral effects of sulfated galactomannans against DENV-2. The results are very promising and suggest that these sulfated galactomannans from plants of Caatinga biome act in the early step of viral infection. Thus, sulfated galactomannans may act as an entry inhibitor of DENV-2.

The Type III Secretion System (T3SS) is a Determinant for Rice-Endophyte Colonization by Non-Photosynthetic Bradyrhizobium.

Plant associations by bradyrhizobia have been detected not only in leguminous plants, but also in non-leguminous species including rice. Bradyrhizobium sp. SUTN9-2 was isolated from Aeschynomene americana L., which is a leguminous weed found in the rice fields of Thailand. This strain promoted the highest total rice (Oryza sativa L. cultivar Pathum Thani 1) dry weight among the endophytic bradyrhizobial strains tested, and was, thus, employed for the further characterization of rice-Bradyrhizobium interactions. Some known bacterial genes involved in bacteria-plant interactions were selected. The expression of the type III secretion component (rhcJ), type IV secretion component (virD4), and pectinesterase (peces) genes of the bacterium were up-regulated when the rice root exudate was added to the culture. When SUTN9-2 was inoculated into rice seedlings, the peces, rhcJ, virD4, and exopolysaccharide production (fliP) genes were strongly expressed in the bacterium 6–24 h after the inoculation. The gene for glutathione-S-transferase (gst) was slightly expressed 12 h after the inoculation. In order to determine whether type III secretion system (T3SS) is involved in bradyrhizobial infections in rice plants, wild-type SUTN9-2 and T3SS mutant strains were inoculated into the original host plant (A. americana) and a rice plant (cultivar Pathum Thani 1). The ability of T3SS mutants to invade rice tissues was weaker than that of the wild-type strain; however, their phenotypes in A. americana were not changed by T3SS mutations. These results suggest that T3SS is one of the important determinants modulating rice infection; however, type IV secretion system and peces may also be responsible for the early steps of rice infection.

Mycobacterium marinum MgtC plays a role in phagocytosis but is dispensable for intracellular multiplication.

MgtC is a virulence factor involved in intramacrophage growth that has been reported in several intracellular pathogens, including Mycobacterium tuberculosis and Salmonella enterica serovar Typhimurium. MgtC participates also in adaptation to Mg2+ deprivation. Herein, we have constructed a mgtC mutant in Mycobacterium marinum to further investigate the role of MgtC in mycobacteria. We show that the M. marinum mgtC gene (Mma mgtC) is strongly induced upon Mg2+ deprivation and is required for optimal growth in Mg2+-deprived medium. The behaviour of the Mma mgtC mutant has been investigated in the Danio rerio infection model using a transgenic reporter zebrafish line that specifically labels neutrophils. Although the mgtC mutant is not attenuated in the zebrafish embryo model based on survival curves, our results indicate that phagocytosis by neutrophils is enhanced with the mgtC mutant compared to the wild-type strain following subcutaneous injection. Increased phagocytosis of the mutant strain is also observed ex vivo with the murine J774 macrophage cell line. On the other hand, no difference was found between the mgtC mutant and the wild-type strain in bacterial adhesion to macrophages and in the internalization into epithelial cells. Unlike the role reported for MgtC in other intracellular pathogens, Mma MgtC does not contribute significantly to intramacrophage replication. Taken together, these results indicate an unanticipated function of Mma MgtC at early step of infection within phagocytic cells. Hence, our results indicate that although the MgtC function is conserved among pathogens regarding adaptation to Mg2+ deprivation, its role towards phagocytic cells can differ, possibly in relation with the specific pathogen's lifestyles.