Reprogramming in Candida albicans Gene Expression Network under Butanol Stress Abrogates Hyphal Development.

Candida albicans is the causative agent of invasive fungal infections. Its hyphae-forming ability is regarded as one of the important virulence factors. To unravel the impact of butanol on Candida albicans, it was placed in O+ve complete human serum with butanol (1% v/v). The Candida transcriptome under butanol stress was then identified by mRNA sequencing. Studies including electron microscopy demonstrated the inhibition of hyphae formation in Candida under the influence of butanol, without any significant alteration in growth rate. The numbers of genes upregulated in the butanol in comparison to the serum alone were 1061 (20 min), 804 (45 min), and 537 (120 min). Candida cells exhibited the downregulation of six hypha-specific transcription factors and the induction of four repressor/regulator genes. Many of the hypha-specific genes exhibited repression in the medium with butanol. The genes related to adhesion also exhibited repression, whereas, among the heat-shock genes, three showed inductions in the presence of butanol. The fungal-specific genes exhibited induction as well as repression in the butanol-treated Candida cells. Furthermore, ten upregulated genes formed the core stress gene set in the presence of butanol. In the gene ontology analysis, enrichment of the processes related to non-coding RNA, ribosome biosynthesis, and metabolism was observed in the induced gene set. On the other side, a few GO biological process terms, including biofilm formation and filamentous growth, were enriched in the repressed gene set. Taken together, under butanol stress, Candida albicans is unable to extend hyphae and shows growth by budding. Many of the genes with perturbed expression may have fitness or virulence attributes and may provide prospective sites of antifungal targets against C. albicans.

Plasmodium berghei-Released Factor, PbTIP, Modulates the Host Innate Immune Responses.

The Plasmodium parasite has to cross various immunological barriers for successful infection. Parasites have evolved mechanisms to evade host immune responses, which hugely contributes to the successful infection and transmission by parasites. One way in which a parasite evades immune surveillance is by expressing molecular mimics of the host molecules in order to manipulate the host responses. In this study, we report a Plasmodium berghei hypothetical protein, PbTIP (PbANKA_124360.0), which is a Plasmodium homolog of the human T-cell immunomodulatory protein (TIP). The latter possesses immunomodulatory activities and suppressed the host immune responses in a mouse acute graft-versus-host disease (GvHD) model. The Plasmodium berghei protein, PbTIP, is expressed on the merozoite surface and exported to the host erythrocyte surface upon infection. It is shed in the blood circulation by the activity of an uncharacterized membrane protease(s). The shed PbTIP could be detected in the host serum during infection. Our results demonstrate that the shed PbTIP exhibits binding on the surface of macrophages and reduces their inflammatory cytokine response while upregulating the anti-inflammatory cytokines such as TGF-ß and IL-10. Such manipulated immune responses are observed in the later stage of malaria infection. PbTIP induced Th2-type gene transcript changes in macrophages, hinting toward its potential to regulate the host immune responses against the parasite. Therefore, this study highlights the role of a Plasmodium-released protein, PbTIP, in immune evasion using macrophages, which may represent the critical strategy of the parasite to successfully survive and thrive in its host. This study also indicates the human malaria parasite TIP as a potential diagnostic molecule that could be exploited in lateral flow-based immunochromatographic tests for malaria disease diagnosis.

Computational perspectives revealed prospective vaccine candidates from five structural proteins of novel SARS corona virus 2019 (SARS-CoV-2).

BACKGROUND: The present pandemic COVID-19 is caused by SARS-CoV-2, a single-stranded positive-sense RNA virus from the Coronaviridae family. Due to a lack of antiviral drugs, vaccines against the virus are urgently required. METHODS: In this study, validated computational approaches were used to identify peptide-based epitopes from six structural proteins having antigenic properties. The Net-CTL 1.2 tool was used for the prediction of CD8+ T-cell epitopes, while the robust tools Bepi-Pred 2 and LBtope was employed for the identification of linear B-cell epitopes. Docking studies of the identified epitopes were performed using HADDOCK 2.4 and the structures were visualized by Discovery Studio and LigPlot+. Antigenicity, immunogenicity, conservancy, population coverage and allergenicity of the predicted epitopes were determined by the bioinformatics tools like VaxiJen v2.0 server, the Immune Epitope Database tools and AllerTOP v.2.0, AllergenFP 1.0 and ElliPro. RESULTS: The predicted T cell and linear B-cell epitopes were considered as prime vaccine targets in case they passed the requisite parameters like antigenicity, immunogenicity, conservancy, non-allergenicity and broad range of population coverage. Among the predicted CD8+ T cell epitopes, potential vaccine targets from surface glycoprotein were; YQPYRVVVL, PYRVVVLSF, GVYFASTEK, QLTPTWRVY, and those from ORF3a protein were LKKRWQLAL, HVTFFIYNK. Similarly, RFLYIIKLI, LTWICLLQF from membrane protein and three epitopes viz; SPRWYFYYL, TWLTYTGAI, KTFPPTEPK from nucleocapsid phosphoprotein were the superior vaccine targets observed in our study. The negative values of HADDOCK and Z scores obtained for the best cluster indicated the potential of the epitopes as suitable vaccine candidates. Analysis of the 3D and 2D interaction diagrams of best cluster produced by HADDOCK 2.4 displayed the binding interaction of leading T cell epitopes within the MHC-1 peptide binding clefts. On the other hand, among linear B cell epitopes the majority of potential vaccine targets were from nucleocapsid protein, viz; 59-HGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLS-105, 227-LNQLE SKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATK-266, 3-DNGPQNQRNAPRITFGGP-20, 29-GERSGARSKQRRPQGL-45. Two other prime vaccine targets, 370-NSASFSTFKCYGVSPTKLNDLCFTNV-395 and 260-AGAAAYYVGYLQPRT-274 were identified in the spike protein. The potential B-cell conformational epitopes were predicted on the basis of a higher protrusion index indicating greater solvent accessibility. These conformational epitopes were of various lengths and belonged to spike, ORF3a, membrane and nucleocapsid proteins. CONCLUSIONS: Taken together, eleven T cell epitopes, seven B cell linear epitopes and ten B cell conformational epitopes were identified from five structural proteins of SARS-CoV-2 using advanced computational tools. These potential vaccine candidates may provide important timely directives for an effective vaccine against SARS-CoV-2.

Small Colony Variants of Pseudomonas aeruginosa Display Heterogeneity in Inhibiting Aspergillus fumigatus Biofilm.

Pseudomonas aeruginosa and Aspergillus fumigatus are major microbes in cystic fibrosis (CF). We reported non-mucoid P. aeruginosa isolates more inhibitory to A. fumigatus than mucoid ones. Another CF P. aeruginosa phenotype, small colony variants (SCVs), is an unknown factor in intermicrobial competition with A. fumigatus. Clinical SCV isolates and reference CF non-mucoid isolate (Pa10, producing normal-sized colonies) were compared. Live cells of P. aeruginosa or filtrates from P. aeruginosa planktonic or biofilm cultures were co-incubated with A. fumigatus growing under conditions allowing biofilm formation or with preformed biofilm. Metabolic activity of A. fumigatus biofilm was then measured. When necessary, assays were done after adjustment for growth differences by adding fresh medium to the planktonic culture filtrate. Pyoverdine determinations were performed spectrophotometrically on the planktonic culture filtrates. In all experimental conditions (live cells and planktonic or biofilm culture filtrates of P. aeruginosa versus A. fumigatus biofilm formation or preformed biofilm), three SCV isolates were less inhibitory than Pa10, two equal or more inhibitory. Adjusting planktonic culture filtrates for growth differences showed SCV inhibition differences variably related to growth or deficient inhibitor production. Studies suggested the principal P. aeruginosa inhibitor to be pyoverdine. SCV isolates appear heterogeneous in their capacity to inhibit A. fumigatus biofilm. SCV isolates can be important in the CF microbiome, because they are capable of intermicrobial inhibition.

Effect of Anaerobiasis or Hypoxia on Pseudomonas aeruginosa Inhibition of Aspergillus fumigatus Biofilm.

Pseudomonas aeruginosa (Pa) and Aspergillus fumigatus (Af) are the major bacterial and fungal pathogens in the airways of cystic fibrosis (CF) patients. This is likely related to their ability to form biofilms. Both microbes have been associated with CF disease progression. The interplay between these two pathogens has been studied under aerobic conditions, though accumulating data indicates that much of the CF airway is hypoxic or anaerobic. We studied the microbial interaction in these latter environments. Pa is an aggressor against Af forming biofilm or as established Af biofilm, whether Pa is cultivated in aerobic, hypoxic, or anaerobic conditions, or tested in aerobic or hypoxic conditions. Pa cells are generally more effective than planktonic or biofilm culture filtrates. Pa growth is less in anaerobic conditions, and filtrates less effective after anaerobic or hypoxic growth, or against hypoxic Af. These, and other comparisons shown, indicate that Pa would be less effective in such environments, as would be the case in a CF mucus plug. These observations would explain why Pa becomes established in CF airways before Af, and why Af may persist during disease progression.

Cytokines induce effector T-helper cells during invasive aspergillosis; what we have learned about T-helper cells?

Invasive aspergillosis caused by Aspergillus species (Aspergillus fumigatus, A. flavus, and A. terreus) is life-threatening infections in immunocompromised patients. Understanding the innate and adaptive immune response particularly T-helper cells (TH-cells) against these Aspergillus species and how the different sub-set of TH-cells are regulated by differentiating cytokines at primary target organ site like lung, kidney and brain is of great significance to human health. This review focuses on presentation of Aspergillus through Antigen presenting cells (APCs) to the naive CD4(+) T-cells in the host. The production of differentiating/effector cytokines that activate following TH-cells, e.g., TH1, TH2, TH9, and TH17 has been reported in association or alone in allergic or invasive aspergillosis. Chemokines (CXCL1, CXCL2, CCL1, and CCL20) and their receptors associated to these TH-cells have also been observed in invasive aspergillosis. Thus, further study of these TH-cells in invasive aspergillosis and other elements of adaptive immune response with Aspergillus species are required in order to have a better understanding of host response for safer and effective therapeutic outcome.

Cytokine milieu in renal cavities of immunocompetent mice in response to intravenous challenge of Aspergillus flavus leading to aspergillosis.

Investigations in mice have demonstrated that Aspergillus flavus is more virulent than all other Aspergillus species except A. tamari. However, there is a complete lack of information on the immune responses elicited by A. flavus in systemic model. This communication reports the progression of infection and cytokine profile in BALB/c mice in response to intravenous challenge of A. flavus. The pathogenesis of infection was evaluated morphologically and by the analysis of Colony Forming Units (CFUs) in kidney homogenates. The kinetics of regulated cytokines was determined in kidneys by cytokine-specific murine ELISA. During the initial phase of infection the rate of clearance of A. flavus was high, most likely through recruited neutrophils and the resident renal macrophages with concurrent significant release of pro-inflammatory cytokines (IFN-γ, TNF-α, IL-12/IL-23p40, IL-6) indicating antifungal innate immune response to be active at the site. However, at 24h PI there was a significant rise of IL-17 and IL-23 suggesting the activation of IL-17/IL-23 axis of inflammation resulting in rise of CFU. The lack of significant induction in the anti-inflammatory cytokines like IL-4 and IL-10 confirmed the absence of Th2 type of response. In the late phase, after 3days post-infection, there was a rise in the number of pathogen in the kidneys as determined by histopathology and CFU counts. The A. flavus hyphae were evident in the renal pelvis and ureter and we propose the production of blastoconidia by metamorphosed hyphae.

Th1 and Th2 cytokines in a self-healing primary pulmonary Aspergillus flavus infection in BALB/c mice.

Studies with non-immunocompromised mice have demonstrated that Aspergillus flavus is more virulent than almost all other Aspergillus species. However, the type of immune response this fungus induces in mammals has not been investigated thoroughly. The study was carried out to analyze the sequential pathogenesis of pulmonary A. flavus infection and the role of cytokines in host response in BALB/c mice. Two distinct phases were observed in mice: First, an intense rate of clearance of A. flavus occurred, most likely through recruited neutrophils and the resident alveolar macrophages with concurrent release of pro-inflammatory cytokines and second, fungal and cellular debris were cleaned by recruited monocytes, pro-inflammatory cytokine production rapidly decreased and infection self-healed. The pro-inflammatory cytokine IFN-γ demonstrated an upward trend up to 24h PI followed by a steady decline. The titers of TNF-α (a pro-inflammatory Th1 cytokine) were, however, inversely related to the titers of IL-10 an anti-inflammatory Th2 cytokine. The anti-inflammatory cytokine IL-4 showed slightly decreasing trend between 12 and 48 h PI, beyond that it again reached to the titers observed at 6h PI. The infected mice produced signs of Th1 type response with self healing capabilities.

Cytokine profile and cytotoxicity in response to acute intratracheal dose of Metarhizium anisopliae in BALB/c mice.

Metarhizium anisopliae has been considered neither pathogenic nor toxic to mammals. However, some recent reports demonstrate that it is capable of causing infections in mammals including humans. Therefore, there is an urgent need to investigate the nature of infections this fungus induces in these animals. We therefore investigated the immunological and cytotoxic responses to acute intratracheal dose (10(8) conidia/50 µl) of crude conidial suspension (CCS) of M. anisopliae (ARSEF 7487) in BALB/c mice. The secretion of proinflammatory cytokine IL-6 significantly increased at all the evaluated time points post infection. The titers of IFN-γ, TNF-α, IL-12/IL-23-total p40 and IL-12-p70 showed continuous increasing trends from 6 h post infection (h PI) onwards and increased significantly at 24 h PI. While IL-10 activity was almost constant throughout the observation period, the IL-4 activity first increased at 6 h PI, beyond which it continuously decreased at the evaluated periods of time. The lactate dehydrogenase (LDH) and total protein levels increased significantly at the evaluated h PI. Collectively, the cytokine data suggested that the activation of IL-23/Th17 pathways leading to uncontrolled Th1 type response (elevated production of TNF-α, IFN-γ and IL-6) in the early hours of infection contributed to neutrophilia and tissue damage. On the other hand significantly elevated levels of total protein, LDH and albumin indicated cytotoxicity.

Instability of CGG repeats in transgenic mice.

Dynamic mutation resulting in the expansion of CGG repeats in the untranslated region (UTR) of the first exon of the FMR1 gene in humans results in fragile X syndrome. Long stretches of CGG repeats that are known to be highly unstable in humans have so far failed to show similar intergenerational instability in transgenic mice. We generated transgenic lines that show a dramatic increase from 26 to >300 repeats in three generations. One of the salient features of our transgene is the inclusion of the origin of replication of simian virus-40 (SV40), which is known to exclude nucleosomes. Three founder mice in FVB/NJ background show expansion of CGG repeats present in the transgene, supporting a postzygotic mechanism for CGG expansion that is independent of a genomic imprinting effect. We discuss here the results of analyzing one of the lines established.

Genetic characterisation of Mus musculus and related forms from India.

The present study reports the biochemical genetic profile of commensal forms of Indian wild house mouse populations Mus musculus from ten localities as well as other species of genus Mus, M. platythrix, M. booduga and M. terricolor III. Extreme polymorphism seen by the presence of a large number of variants and novel alleles in these animals indicates that the Indian M. musculus holds considerable potential for development of useful models for biomedical research.