Establishment Of Systemic Infection Research Articles

Association of host protein VARICOSE with HCPro within a multiprotein complex is crucial for RNA silencing suppression, translation, encapsidation and systemic spread of potato virus A infection.

In this study, we investigated the significance of a conserved five-amino acid motif ‘AELPR’ in the C-terminal region of helper component–proteinase (HCPro) for potato virus A (PVA; genus Potyvirus) infection. This motif is a putative interaction site for WD40 domain-containing proteins, including VARICOSE (VCS). We abolished the interaction site in HCPro by replacing glutamic acid (E) and arginine (R) with alanines (A) to generate HCProWD. These mutations partially eliminated HCPro-VCS co-localization in cells. We have earlier described potyvirus-induced RNA granules (PGs) in which HCPro and VCS co-localize and proposed that they have a role in RNA silencing suppression. We now demonstrate that the ability of HCProWD to induce PGs, introduce VCS into PGs, and suppress RNA silencing was impaired. Accordingly, PVA carrying HCProWD (PVAWD) infected Nicotiana benthamiana less efficiently than wild-type PVA (PVAWT) and HCProWD complemented the lack of HCPro in PVA gene expression only partially. HCPro was purified from PVA-infected leaves as part of high molecular weight (HMW) ribonucleoprotein (RNP) complexes. These complexes were more stable when associated with wild-type HCPro than with HCProWD. Moreover, VCS and two viral components of the HMW-complexes, viral protein genome-linked and cylindrical inclusion protein were specifically decreased in HCProWD-containing HMW-complexes. A VPg-mediated boost in translation of replication-deficient PVA (PVAΔGDD) was observed only if viral RNA expressed wild-type HCPro. The role of VCS-VPg-HCPro coordination in PVA translation was further supported by results from VCS silencing and overexpression experiments and by significantly elevated PVA-derived Renilla luciferase vs PVA RNA ratio upon VPg-VCS co-expression. Finally, we found that PVAWD was unable to form virus particles or to spread systemically in the infected plant. We highlight the role of HCPro-VCS containing multiprotein assemblies associated with PVA RNA in protecting it from degradation, ensuring efficient translation, formation of stable virions and establishment of systemic infection.

Cholecalciferol modulates the phenotype of differentiated monocyte-derived dendritic cells without altering HIV-1 transfer to CD4+ T cells.

Background Dendritic cells (DCs) play a crucial role during HIV-1 transmission due to their ability to transfer virions to susceptible CD4+ T cells, particularly in the lymph nodes during antigen presentation which favors the establishment of systemic infection. As mature dendritic cells (mDCs) exhibit a greater ability to transfer virions, compared to immature DCs (iDCs), maintenance of an iDC phenotype could decrease viral transmission. The immunomodulatory vitamin D (VitD) has been shown to reduce activation and maturation of DCs; hence, we hypothesized that it would reduce viral transference by DCs. Materials and methods We evaluated the effect of in vitro treatment with a precursor of VitD, cholecalciferol, on the activation/maturation phenotype of differentiated monocyte-derived DCs and their ability to transfer HIV-1 to autologous CD4+ T cells. Results Our findings show that although cholecalciferol decreases the activation of iDCs, it did not impact the maturation phenotype after LPS treatment nor iDCs' ability to transfer viral particles to target cells. Conclusion These findings suggest that despite cholecalciferol potentially modulates the phenotype of mucosal iDCs in vivo, such modulation might not impact the ability of these cells to transfer HIV-1 to target CD4+ T cells.

Proteomic analysis of the plasma membrane-movement tubule complex of cowpea mosaic virus.

Cowpea mosaic virus forms tubules constructed from the movement protein (MP) in plasmodesmata (PD) to achieve cell-to-cell movement of its virions. Similar tubules, delineated by the plasma membrane (PM), are formed protruding from the surface of infected protoplasts. These PM-tubule complexes were isolated from protoplasts by immunoprecipitation and analysed for their protein content by tandem mass spectrometry to identify host proteins with affinity for the movement tubule. Seven host proteins were abundantly present in the PM-tubule complex, including molecular chaperonins and an AAA protein. Members of both protein families have been implicated in establishment of systemic infection. The potential role of these proteins in tubule-guided cell-cell transport is discussed.

Virulence Factors of Erwinia amylovora: A Review.

Erwinia amylovora, a Gram negative bacteria of the Enterobacteriaceae family, is the causal agent of fire blight, a devastating plant disease affecting a wide range of host species within Rosaceae and a major global threat to commercial apple and pear production. Among the limited number of control options currently available, prophylactic application of antibiotics during the bloom period appears the most effective. Pathogen cells enter plants through the nectarthodes of flowers and other natural openings, such as wounds, and are capable of rapid movement within plants and the establishment of systemic infections. Many virulence determinants of E. amylovora have been characterized, including the Type III secretion system (T3SS), the exopolysaccharide (EPS) amylovoran, biofilm formation, and motility. To successfully establish an infection, E. amylovora uses a complex regulatory network to sense the relevant environmental signals and coordinate the expression of early and late stage virulence factors involving two component signal transduction systems, bis-(3′-5′)-cyclic di-GMP (c-di-GMP) and quorum sensing. The LPS biosynthetic gene cluster is one of the relatively few genetic differences observed between Rubus- and Spiraeoideae-infecting genotypes of E. amylovora. Other differential factors, such as the presence and composition of an integrative conjugative element associated with the Hrp T3SS (hrp genes encoding the T3SS apparatus), have been recently described. In the present review, we present the recent findings on virulence factors research, focusing on their role in bacterial pathogenesis and indicating other virulence factors that deserve future research to characterize them.

The Virulence Polysaccharide Vi Released by Salmonella Typhi Targets Membrane Prohibitin to Inhibit T-Cell Activation

T cells are critical to immunity against pathogenic Salmonella including Salmonella Typhi which causes systemic infection, typhoid, in humans. The strategies that this pathogen employs to keep T-cell mediated immune responses in check during establishment of systemic infection are not completely understood. Here, we show that the virulence polysaccharide Vi, which distinguishes S. Typhi from localized gastroenteritis-producing nontyphoidal Salmonella serovars, is a potent inhibitor of T-cell activation. Vi released by S. Typhi interacts with the membrane prohibitin complex and inhibits IL-2 secretion from T cells stimulated through the T-cell receptor (TCR) but does not affect PMA-activated interleukin 2 (IL-2) secretion. Treatment with Vi suppresses early activation events including TCR down-regulation, actin polymerization, and phosphorylation of ERK. Coadministration of Vi with anti-CD3 Ab reduces secretion of IL-2 and interferon γ in mice. Our findings reveal a mechanism by which S. Typhi may target T-cell immunity during establishment of typhoid.

Protective-antigen (PA) based anthrax vaccines confer protection against inhalation anthrax by precluding the establishment of a systemic infection

An intense effort has been launched to develop improved anthrax vaccines that confer rapid, long lasting protection preferably with an extended stability profile amenable for stockpiling. Protective antigen (PA)-based vaccines are most favored as immune responses directed against PA are singularly protective, although the actual protective mechanism remains to be unraveled. Herein we show that contrary to the prevailing view, an efficacious PA-based vaccine confers protection against inhalation anthrax by preventing the establishment of a toxin-releasing systemic infection. Equally importantly, antibodies measured by the in vitro lethal toxin neutralization activity assay (TNA) that is considered as a reliable correlate of protection, especially for PA protein-based vaccines adjuvanted with aluminum salts appear to be not absolutely essential for this protective immune response.

Artificial infection of Cirsium arvense with the rust pathogen Puccinia punctiformis by imitation of natural spore transfer by the weevil Ceratapion onopordi

Müller E, Jud P & Nentwig W (2011). Artificial infection of Cirsium arvense with the rust pathogen Puccinia punctiformis by imitation of natural spore transfer of the weevil Ceratapion onopordi. Weed Research51, 209–213.Summary Cirsium arvense is a noxious perennial weed. Because of the limited possibilities of chemical and physical control of this weed, many studies have been performed on biological methods. Successful control of C. arvense using the rust fungus Puccinia punctiformis needs the establishment of systemic infection, often connected with the weevil Ceratapion onopordi as a spore vector. To analyse whether an artificial infection of C. arvense with P. punctiformis through the plant stem is possible without the weevil, different infection tests were performed. Urediniospore solutions with different kinds of additives of weevil origin were applied. All treatments with infectious material led to an infection. Injection of pure urediniospores into the plant stem resulted in the highest infection rate (50%). Even though the infection‐via‐the‐stem‐method used in this study was successful in principle, this method is not practicable in the field. Therefore, further studies of infection techniques are needed, particularly to examine the spore stages that cause systemic infections.

Burkholderia cenocepacia creates an intramacrophage replication niche in zebrafish embryos, followed by bacterial dissemination and establishment of systemic infection.

Bacteria belonging to the "Burkholderia cepacia complex" (Bcc) often cause fatal pulmonary infections in cystic fibrosis patients, yet little is know about the underlying molecular mechanisms. These Gram-negative bacteria can adopt an intracellular lifestyle, although their ability to replicate intracellularly has been difficult to demonstrate. Here we show that Bcc bacteria survive and multiply in macrophages of zebrafish embryos. Local dissemination by nonlytic release from infected cells was followed by bacteremia and extracellular replication. Burkholderia cenocepacia isolates belonging to the epidemic electrophoretic type 12 (ET12) lineage were highly virulent for the embryos; intravenous injection of <10 bacteria of strain K56-2 killed embryos within 3 days. However, small but significant differences between the clonal ET12 isolates K56-2, J2315, and BC7 were evident. In addition, the innate immune response in young embryos was sufficiently developed to control infection with other less virulent Bcc strains, such as Burkholderia vietnamiensis FC441 and Burkholderia stabilis LMG14294. A K56-2 cepR quorum-sensing regulator mutant was highly attenuated, and its ability to replicate and spread to neighboring cells was greatly reduced. Our data indicate that the zebrafish embryo is an excellent vertebrate model to dissect the molecular basis of intracellular replication and the early innate immune responses in this intricate host-pathogen interaction.

Characteristic infectivity of to mice depending on routes of infection and inherent fungal pathogenicity

Isolates of Sporothrix schenckii were examined for their infectivity in BALB/c mice. The mice were injected with yeast forms of S. schenckii isolates differing in clinical source (human cutaneous lesions and pulmonary lesions), and fungal growth was determined at intervals in the footpad and visceral organs. After subcutaneous injection of ≈10 colony forming units (cfu) of S. schenckii into the footpad, locally restricted fungal infection developed gradually. At the peak of the infection (3–4 weeks post-inoculation), viable fungal counts reached 102–106 cfu/footpad. Dissemination to other tissues and visceral organs was not observed. After intravenous or intraperitoneal injection of 106 cfu of yeast forms, three of four isolates from cutaneous sporotrichosis were unable to establish infection and were eliminated from blood and visceral organs. The development of systemic infection was observed only with S. schenckii isolates obtained from the human lung lesion. Thus, inherent properties of each clinical isolate and routes of infection were shown to be critical for the establishment of systemic infection in spite of the remarkably strong infectivity of S. schenckii to the cutaneous tissue.

Effects of viral silencing suppressors on tobacco ringspot virus infection in two Nicotiana species

This study investigated the effects of silencing suppressors derived from six different viruses (P1, P19, P25, HcPro, AC2 and 2b), expressed in transgenic Nicotiana tabacum and Nicotiana benthamiana plants, on the infection pattern of tobacco ringspot virus (TRSV) potato calico strain. In N. benthamiana, this virus produced an initial infection with severe systemic symptoms, but the infection was strongly reduced within a few weeks as the plant recovered from the infection. P25 and HcPro silencing suppressors effectively prevented recovery in this host, allowing continuous accumulation of the viral RNA as well as of the virus-specific small interfering RNAs, in the systemically infected leaves. In the P1-, P19-, AC2- or 2b-expressing transgenic N. benthamiana, the recovery was not complete. Susceptibility of N. tabacum to this virus was temperature sensitive. At lower temperatures, up to 25 degrees C, the plants became systemically infected, but at higher temperatures, the infections were limited to the inoculated leaves. In these preventative conditions, all silencing suppressor transgenes (except P25, which was expressed at very low levels) allowed the establishment of systemic infections. Very strong and consistent systemic infections were observed in HcPro- and AC2-expressing plants.

Multiple Vaginal Exposures to Low Doses of R5 Simian‐Human Immunodeficiency Virus: Strategy to Study HIV Preclinical Interventions in Nonhuman Primates

A nonhuman-primate model of human immunodeficiency virus type 1 (HIV-1) infection that more closely emulates human heterosexual transmission by use of multiple exposures to low doses of virus is critical to better evaluate intervention strategies that include microbicides or vaccines. In this report, we describe such a system that uses female pig-tailed macaques exposed vaginally to a CCR5-using simian-human immunodeficiency virus (SHIV(SF162P3)) at weekly intervals. Results of dose-titration experiments indicated that 3 once-weekly exposures to 10 tissue culture infectious doses of SHIV(SF162P3) resulted in consistent transmission of virus and establishment of systemic infection. The efficacy of cellulose acetate phthalate (CAP) as a vaginal microbicide was evaluated by applying it to the vaginal vault of macaques (n = 4) 15 min before each weekly exposure to SHIV(SF162P3). One conclusion that can be drawn from the data derived from multiple exposures to virus is that CAP prevented infection in 12 of 13 possible chances for infection, over the course of 39 total exposures. Our findings provide a basis to refine monkey models for transmission of HIV-1, which may be relevant to preclinical evaluation for therapeutic interventions.

Use of Proteases to Improve the Insecticidal Activity of Baculoviruses

Abstract Basement membranes that surround the tissues of lepidopterous larvae act as potential barriers to baculovirus movement and establishment of systemic infection. Hence, one potential approach to improving the insecticidal activity of baculoviruses is to perforate or eliminate the basement membranes of their hosts, thereby facilitating the process of infection. Toward this end, we constructed six recombinant clones of Autographa californica nucleopolyhedrovirus (AcMNPV) that express three proteases that digest basement membrane proteins: rat stromelysin-1, human gelatinase A, and flesh fly ( Sarcophaga peregrina ) cathepsin L. Expression of these proteases was directed from either the ie-1 promoter (in AcIE1TV3.STR1, AcIE1TV3.GEL, and AcIE1TV3.ScathL) or the p6.9 promoter (in AcMLF9.STR1, AcMLF9.GEL, and AcMLF9.ScathL). Recombinant proteases were detected in the culture medium of cells infected with recombinant viruses by either zymography or azocoll assay. AcMLF9.STR1 and AcMLF9.ScathL caused premature cuticular melanization of 5th instar Heliothis virescens. Melanization and fragmentation of internal tissues were observed in half of the larvae infected with AcMLF9.ScathL and not at all in larvae infected with AcMLF9.STR1 or wild-type AcMNPV. Lethal-concentration bioassays revealed no significant differences in virulence toward H. virescens among the protease-expressing recombinants and wild-type AcMNPV. However, in survival-time bioassays, AcMLF9.ScathL killed H. virescens approximately 30% faster than AcMLF9.LqhIT2, a virus expressing an insect-selective scorpion neurotoxin from the p6.9 promoter. Larvae infected with AcMLF9.ScathL consumed approximately 26-fold less lettuce than wild-type virus-infected larvae. These results highlight the potential of improving baculovirus efficacy through the expression of proteases.

The establishment of systemic infection in leaves of oilseed rape by Leptosphaeria maculans

In a series of growth room experiments in which leaves of Brassica napus var. oleifera were inoculated with ascospores or pycnidiospores of Leptosphaeria maculans successful infections progressed through three consecutive phases. Initial establishment in the mesophyll was succeeded by a phase of intercellular exploration, when hyphal proliferation was highly variable and host cell necrosis always ensued, and then by a systemic phase when hyphae were consistently sparse. Host cells associated with the hyphal front were capable of autofluorescence, accumulation of vital stains and plasmolysis, indicating that they were viable and that the pathogen was biotrophic throughout this sequence. During either of the first two phases permanent fungistatic containment, involving the formation of vesicles by disintegration of the hyphae, often occurred. Localization at the first phase was symptomless; at the second it was signified by a lesion with a clearly defined margin.There was a negative correlation between biotrophic potential and necrotrophic potential of three pathogenic isolates, on both the moderately susceptible cultivar Primor and the resistant cultivar Jet Neuf. As leaves aged, a progressively larger proportion of infections failed to become systemic. With increasing inoculum load, symptomless localization of infection diminished, the phase of necrosis extended, and the probability of irreversible systemic development increased.