Cellular microbiology

), where they multiply by schizogony, undergoing development through ring, trophozoite and schizont stages that are responsible for malaria pathogenesis. Here, we report that a protein kinase-mediated signalling pathway involving host RBC PAK1 and MEK1, which do not have orthologues in the Plasmodium kinome, is selectively stimulated in Plasmodium falciparuminfected (versus uninfected) RBCs, as determined by the use of phospho-specific antibodies directed against the activated forms of these enzymes. Pharmacological interference with host MEK and PAK function using highly specific allosteric inhibitors in their known cellular IC 50 ranges results in parasite death. Furthermore, MEK inhibitors have parasiticidal effects in vitro on hepatocyte and erythrocyte stages of the rodent malaria parasite Plasmodium berghei, indicating conservation of this subversive strategy in malaria parasites. These findings have profound implications for the development of novel strategies for antimalarial chemotherapy.c mi_1582 836..845

The cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) pathway is central to signal transduction in many organisms. In pathogenic fungi such as Candida albicans, this signalling cascade has proven to be involved in several processes, such as virulence, indicating its potential importance in antifungal drug discovery. Candida glabrata is an upcoming pathogen of the same species, yet information regarding the role of cAMP-PKA signalling in virulence is largely lacking. To enable efficient monitoring of cAMP-PKA activity in this pathogen, we here present the usage of two FRET-based biosensors. Both variations in the activity of PKA and the quantity of cAMP can be detected in a time-resolved manner, as we exemplify by glucose-induced activation of the pathway. We also present information on how to adequately process and analyse the data in a mathematically correct and physiologically relevant manner. These sensors will be of great benefit for scientists interested in linking the...

The Cytolethal Distending Toxin (CDT) is a genotoxin produced by several pathogenic bacteria. It is generally admitted that CDT induces double-strand breaks (DSB) and cell cycle arrest in G2/M-phase, in an ATM-dependent manner. Most of these results were obtained at high dose (over 1 mg ml -1 ) of CDT and late after treatment (8-24 h). We provide here evidence that the Escherichia coli CDT (EcCDT) -at low dose (50 pg ml -1 or LD50) and early after treatment (3-6 h) -progressively induces DNA DSB, mostly in S-phase. DSB formation is related to the single-strand breaks induction by CDT, converted into DSB during the S-phase. We also show that homologous recombination is mobilized to these S-phase-associated DSB. This model unveils a new mechanism for CDT genotoxicity that may play a role in cells partly deficient in homologous recombination.

Plasmodium knowlesi is a simian malaria parasite primarily infecting macaque species in Southeast Asia. Although its capacity to infect humans has been recognized since the early part of the last century, it has recently become evident that human infections are widespread and potentially life threatening. Historically, P. knowlesi has proven to be a powerful tool in early studies of malaria parasites, providing key breakthroughs in understanding many aspects of Plasmodium biology. However, the necessity to grow the parasite either in macaques or in vitro using macaque blood restricted research to laboratories with access to these resources. The recent adaptation of P. knowlesi to grow and proliferate in vitro in human red blood cells (RBCs) is therefore a substantial step towards revitalizing and expanding research on P. knowlesi. Furthermore, the development of a highly efficient transfection system to genetically modify the parasite makes P. knowlesi an ideal model to study parasite biology. In this review we elaborate on the importance of P. knowlesi in earlier phases of malaria research and highlight the future potential of the newly available human adapted P. knowlesi parasite lines.

Leishmania species are recognized to be pathogenic parasites that infect almost 15 million people globally, and up to date there is no vaccine available to stop the spread of Leishmania infection. For Leishmania mexicana to lose its virulence, the parasite culture must undergo in vitro passaging, little is known about the process underlying the reduction of parasite virulence. Thus, the purpose of this work was to look into the expression of lipophosphoglycan (LPG), as a virulence factor, during L. mexicana in vitro culturing and assessing the parasite's capacity to infect target cells. Results illustrated the extreme downregulation in LPG expression on the surface of promastigotes. Since anti-LPG monoclonal antibody staining showed LPG expression was significantly less expressed in passage 20 (P20) compared to its expression in passage 1 (P1). In addition, this was confirmed by flow cytometry analysis, which also showed that LPG was highly expressed in P1 compared to P20. Moreover, an in vitro infectivity assay was performed using Balb/c and C57 macrophages. Both cells were infected with L. mexicana promastigotes from P1 and P20 for 24 hours. The results illustrated the ability of P1, but not P20, to transform into the non-flagellated amastigotes sage inside infected cells. Our results concluded the significant role of LPG protein in L. mexicana virulence and its ability to induce infection.

Leishmania hydrophilic acylated surface protein B (HASPB) family are only expressed in infective parasites (both extra-and intracellular stages) and, together with the peripheral membrane protein SHERP (small hydrophilic endoplasmic reticulum-associated protein), are essential for parasite differentiation (metacyclogenesis) in the sand fly vector. HASPB is a 'nonclassically' secreted protein, requiring N-terminal acylation for trafficking to and exposure on the plasma membrane. Here, we use live cell imaging methods to further explore this pathway to the membrane and flagellum. Unlike HASPB trafficking in transfected mammalian cells, we find no evidence for a phosphorylation-regulated recycling pathway in metacyclic parasites. Once at the plasma membrane, HASPB18-GFP (green fluorescent protein) can undergo bidirectional movement within the inner leaflet of the membrane and on the flagellum. Transfer of fluorescent protein between the flagellum and the plasma membrane is compromised, however, suggesting the presence of a diffusion barrier at the base of the Leishmania flagellum. Full-length HASPB is released from the metacyclic parasite surface on to macrophages during phagocytosis but while expression is maintained in intracellular amastigotes, HASPB cannot be detected on the external surface in these cells. Thus HASPB may be a dual function protein that is shed by the infective metacyclic but retained internally once Leishmania are taken up by macrophages.

Melanin formation and assembly by fungi has largely been investigated mainly for its importance in pathogenesis, as well as to establish the functions and biosynthetic pathways of melanin formed during the process of successional wood decay. It is known that melanin formation varies based on fungal species, especially melanin produced by ascomycetes versus basidiomycetes, and that the mechanisms of melanin production by basidiomycetes are more complex and thus far not entirely elucidated. This study compares in vivo melanin formation by Oxyporus populinus in sugar maple and Fomes fomentarius in birch, and in vitro pigmentation by Trametes versicolor, Xylaria polymorpha and Inonotus hispidus in sugar maple and beech, with and without the influence of the melanin precursor, catechol. The results of this research indicate a bi-or multi-modal activity of melanin production and assembly by wood decay fungi, and identify possible variations in melanin formation mechanisms as influenced by fungal and wood species.

Melanin formation and assembly by fungi has largely been investigated mainly for its importance in pathogenesis, as well as to establish the functions and biosynthetic pathways of melanin formed during the process of successional wood decay. It is known that melanin formation varies based on fungal species, especially melanin produced by ascomycetes versus basidiomycetes, and that the mechanisms of melanin production by basidiomycetes are more complex and thus far not entirely elucidated. This study compares in vivo melanin formation by Oxyporus populinus in sugar maple and Fomes fomentarius in birch, and in vitro pigmentation by Trametes versicolor, Xylaria polymorpha and Inonotus hispidus in sugar maple and beech, with and without the influence of the melanin precursor, catechol. The results of this research indicate a bi-or multi-modal activity of melanin production and assembly by wood decay fungi, and identify possible variations in melanin formation mechanisms as influenced by fungal and wood species.

concerted efforts by the World Health Wuchereria bancrofti is a neglected vector-borne Organization to eradicate the disease, Nigeria parasitic worm that constitutes a serious public remains the hot spot of the disease in Africa and health problem in tropical countries. The parasite is ranked the third most endemic country in the world (Christiana ., 2021). is under-recognized especially in endemic et al regions Nigeria inclusive. Very often, infected individuals are asymptomatic for a long period, One of the major challenges confronting the making it very difficult to know. A 35-year-old Nigerian Lymphatic Filariasis Elimination male presented with secondary infertility with Programme is the lack of information on the true intermittent fever in Northwestern Nigeria. prevalence of the disease (Ibo et al., 2012) The Semen microscopy, culture, and sensitivity was worms are often under-recognized especially in carried out. The semen microscopy showed the the rural endemic areas with high infectivity presence of Wuchereria bancrofti while rates. Very often, infected individuals are Escherichia coli was isolated in the culture. A asymptomatic for a long period, making it very combined single doses of 400 mg/kg difficult to identify the geographical hot spots. Albendazole and Ivermectin were used for the worm. Escherichia coli was sensitive to Since the diagnosis of the filarial worm is based Ciprofloxacin 500 mg/kg. The fever resolved on the microscopic identification of the with normalization of semen. There is need for a microfilaria in blood smears (Arid et al., 2021), baseline data to assess and evaluate the progress we report for the first time the Wuchereria of mass drug administration in Nigeria towards bancrofti and Escherichia coli co-infection in eventual eradication of filariasis. In addition, semen: A Case Report of Male Infertility. awareness of the unusual presentation would also assist in the diagnosis and possible treatment Case presentation of infertility. As for the case study, a 36-year-old male patient's clinical samples were sent to the laboratory for Keyword: Neglected Tropical disease, semen microscopy, culture, and sensitivity as Filariasis, Wuchereria bancrofti, Escherichia described (Cheesbrough, 2004). In the coli, Infertility macroscopy examination, semen volume was 2 ml and appeared creamy-green. The viscosity

An overproduction of reactive oxygen species (ROS) mediated by NADPH oxidase 2 (NOX2) has been related to airway inflammation typical of influenza infection. Virus-induced oxidative stress may also control viral replication, but the mechanisms underlying ROS production, as well as their role in activating intracellular pathways and specific steps of viral life cycle under redox control have to be fully elucidated. In this study, we demonstrate that influenza A virus infection of lung epithelial cells causes a significant ROS increase that depends mainly on NOX4, which is upregulated at both mRNA and protein levels, while the expression of NOX2, the primary source of ROS in inflammatory cells, is downregulated. Inhibition of NOX4 activity through chemical inhibitors or RNA silencing blocks the ROS increase, prevents MAPK phosphorylation, and inhibits viral ribonucleoprotein (vRNP) nuclear export and viral release. Overall these data, obtained in cell lines and primary culture, describe a so far unrecognized role for NOX4-derived ROS in activating redox-regulated intracellular pathways during influenza virus infection and highlight their relevance in controlling specific steps of viral replication in epithelial cells. Pharmacological modulation of NOX4-mediated ROS production may open the way for new therapeutic approaches to fighting influenza by targeting cell and not the virus.

Giardia was single celled microscopic protozoan parasites that cause enteric disease in human. Because of the impact on socio-economic development, especially in developing countries, it was included in the "Neglected Disease Initiative" of the World Health Organization. This work conducted to descript morphology of Giardia spp in different hosts, which is first study of its type in Basrah, Iraq. This work started from September 2012 till June 2013, through this period collected 2393 stool samples from human and cows. 727 out of 2393 were positive. By using golden standard microscopic methods for diagnosis. A selective 30 positive stool samples for each hosts, measured the dimensions of stages of Giardia. There is different measurement in dimensions of both two phases of Giardia.

Microsporidians are obligate intracellular parasites that have minimized their genome content and sub-cellular structures by reductive evolution. Here, we demonstrate that cristae-deficient mitochondria (mitosomes) of Trachipleistophora hominis are the functional site of iron-sulfur cluster (ISC) assembly, which we suggest is the essential task of these organelles. Cell fractionation, fluorescence imaging and immunoelectron microscopy demonstrate that mitosomes contain a complete pathway for [2Fe-2S] cluster biosynthesis that we biochemically reconstituted using purified mitosomal ISC proteins. The T. hominis cytosolic iron-sulfur protein assembly (CIA) pathway includes the essential Cfd1-Nbp35 scaffold complex that assembles a [4Fe-4S] cluster as shown by spectroscopic methods in vitro. Phylogenetic analyses reveal that the ISC and CIA pathways are predominantly bacterial, but their cytosolic and nuclear target Fe/S proteins are mainly archaeal. This mixed evolutionary history of F...

The biogenesis of the Salmonella-containing vacuole within mammalian cells has been intensively studied over recent years. However, the ability of Salmonella to sense and adapt to the intracellular environment of different types of host cells has received much less attention. To address this issue, we report the transcriptome of Salmonella enterica serovar Typhimurium SL1344 within epithelial cells and show comparisons with Salmonella gene expression inside macrophages. We report that S. Typhimurium expresses a characteristic intracellular transcriptomic signature in response to the environments it encounters within different cell types. The signature involves the upregulation of the mgtBC, pstACS and iro genes for magnesium, phosphate and iron uptake, and Salmonella pathogenicity island 2 (SPI2). Surprisingly, in addition to SPI2, the invasion-associated SPI1 pathogenicity island and the genes involved in flagellar biosynthesis were expressed inside epithelial cells at later stages of the infection, while they were constantly downregulated in macrophage-like cells. To our knowledge, this is the first report of the simultaneous transcription of all three Type Three Secretion Systems (T3SS) within an intracellular Salmonella population. We discovered that S. Typhimurium strain SL1344 was strongly cytotoxic to epithelial cells after 6 h of infection and hypothesize that the timedependent changes in Salmonella gene expression within epithelial cells reflects the bacterial response to host cells that have been injured by the infection process.

a technique enabling real-time imaging of bacterial proliferation and tissue responses in a live animal. Spatial and temporal control of the infection process was achieved by microinjecting GFP + -expressing uropathogenic Escherichia coli (UPEC) into tubules of exteriorized kidneys in live rats. GFP + was introduced in the clinical UPEC strain CFT073 as a single-copy chromosomal gene fusion. Within hours, bacterial colonization was accompanied by marked ischaemic effects, perivascular leakage, loss of tubular integrity and localized recruitment of immune cells. The pathophysiology was altered in response to an isogenic bacterial strain lacking the exotoxin haemolysin, revealing the subtle and temporal roles of bacterial virulence factors in vivo. Microdissection and RNA extraction of the injected nephron allowed molecular analysis of prokaryotic and eukaryotic gene expression. The techniques described here can be applied to study the integrated cell communication evoked by a variety of bacterial pathogens, assisting in the design of strategies to combat bacterial infections.

Coronavirus (SARS-CoV-2) is a respiratory infection virus that was first detected in Wuhan, China. The virus causes COVID-19 disease and the outbreak was recognised as a pandemic by the World Health Organization (WHO) in March 2020. SARS-CoV-2 virion was first imaged using cryo-electron microscopy by the Chinese Center for Disease Control and Prevention (CDC). Atomic Force Microscopy is a unique technique that can allow imaging of biomolecules under different conditions. In this work, we used Atomic Force Microscopy to characterize SARS-CoV-2 on tissue culture polystyrene (TCPS) and glass coverslip surfaces. We isolated SARS-CoV-2 and drop casted it on coverslip glass and tissue culture polystyrene surfaces. We analyzed height profiles, density, and aggregation behavior of the virion on glass and polystyrene surfaces. We observed the coffee ring effect on the drop casted samples and close packing of virions near the coffee rings on both surfaces with relatively higher virion distribution on the tissue culture polystyrene (TCPS) substrates. We compare virion agglomeration on the two types of surfaces. Finally, we applied ethanol disinfectant to virions on the surface to visualize the effect of ethanol and image the ultrastructure of SARS-CoV-2.

BackgroundJasmonates (JAs) mediate trade-off between responses to both biotic and abiotic stress and growth in plants. TheArabidopsis thalianaHISTONE DEACETYLASE 6 is part of the CORONATINE INSENSITIVE1 receptor complex, co-repressing the HDA6/COI1-dependent acetic acid-JA pathway that confers plant drought tolerance. The decrease in HDA6 binding to target DNA mirrors histone H4 acetylation (H4Ac) changes during JA-mediated drought response, and mutations in HDA6 also cause depletion in the constitutive repressive marker H3 lysine 27 trimethylation (H3K27me3). However, the genome-wide effect of HDA6 on H4Ac and much of the impact of JAs on histone modifications and chromatin remodelling remain elusive.ResultsWe performed high-throughput ChIP-Seq on the HDA6 mutant,axe1-5, and wild-type plants with or without methyl jasmonate (MeJA) treatment to assess changes in active H4ac and repressive H3K27me3 histone markers. Transcriptional regulation was investigated in parallel by microarray...

Put option is a contract to sell some underlying assets in the future with a certain price. On European put options, selling only can be exercised at maturity date. Behavior of European put options price can be modeled by using the Black-Scholes model which provide an analytical solution. Numerical approximation such as binomial tree, explicit and implicit finite difference methods also can be used to solve Black-Scholes model. Some numerical methods are applied and compared with the analytical solution to determine the best numerical method. The results show that numerical approximations using the binomial tree is more accurate than explicit and implicit finite difference method in pricing European put options. Moreover when the value of T is higher then the error obtained is also higher, while the error obtained is lower when the value of N is higher. The value of T and N cause the increase of the computation time. When the value of T is higher the computation time is lower, while computation time is higher if the value of N is higher. Overall, the lowest computation time is obtained by using an explicit finite difference method with an exceptional as the value of T is big and the value of N is small. The lowest computation time is obtained by using a binomial tree method.

Trichomonas vaginalis, the etiologic agent of the most common non-viral sexually transmitted infection worldwide. With an estimated annual prevalence of 276 million new cases, mixed infections with different parasite strains are expected. Although it is known that parasites interact with their host to enhance their own survival and transmission, evidence of mixed infections call into question the extent to which unicellular parasites communicate with each other. Here, we demonstrated that different T. vaginalis strains can communicate through the formation of cytoneme-like membranous cell connections. We showed that cytonemes formation of an adherent parasite strain (CDC1132) is affected in the presence of a different strain (G3 or B7RC2). Our findings provide evidence that this effect is contact-independent and that extracellular vesicles (EVs) are responsible, at least in part, of the communication among strains. We found that EVs isolated from G3, B7RC2, and CDC1132 strains conta...

Disruption of cell/ECM interactions resulting from uncontrolled pericellular proteolysis leads to detachment-induced cell apoptosis (anoikis), contributing to the morbid evolution of inflammatory vascular diseases. During cardiovascular infections, bacterial proteinases might induce vascular cells to enter a similar pathway. We focused on LasB, the predominant metalloproteinase secreted by the haematotropic pathogen Pseudomonas aeruginosa. While the exosecretome of the LasBdeficient pseudomonal strain PAO1lasBD had limited impact on human vascular cell adherence and viability, secretomes from the LasBexpressing reference strain, PAO1, or clinical isolates from patients with cardiac infection all induced anoikis, as did purified LasB. Immunofluorescence and/or immunoblotting analysis of heart valve myofibroblast cultures or whole tissue revealed an extensive, LasB-dependent degradation of ECM-associated fibronectin and vitronectin, that preceded cell de-adherence, whereas type I collagen showed limited degradation. Moreover, LasB produced a rapid endoproteolysis of the cell-associated urokinase receptor/uPAR, leaving a truncated receptor that is unable to support cell adherence and survival via interactions with vitronectin and integrins. Conversely, major myofibroblast integrins showed no or only minor alterations. Thus, among P. aeruginosa-secreted metalloproteinases, LasB can induce vascular cell anoikis through simultaneous proteolysis of ECM components and cell receptors, suggesting the uPAR-vitronectin axis as a major target in this process.

Rheumatoid arthritis (RA) is a systemic inflammatory disorder that can cause destructive joint disease, significant disability, and increased mortality. RA is the most frequent of all chronic inflammatory joint diseases, and its prevalence frequency in Pakistan is 1.6 per thousand people. Different cytokines and receptors were involved in the triggering of RA, including interleukin-6 (ILR-6), major histocompatibility complex (MHC) antigen human leukocyte (HLA-DR) receptor, and CD20. Several studies illustrated RA as an inherent immune response and triggered due to the “shared epitope.” Therefore, the involvement of all these receptors (IL-6, HLA-DR, and CD20) leads to the neurological, ocular, respiratory, cardiac, skin, and hematological manifestations that have been considered a potential therapeutic target for drug design. Various herbal, natural, and synthetic source inhibitors of interleukin-6 (IL-6), human leukocyte (HLA-DR), and CD20 were studied and reported previously. Repo...

Aims: To compare the gut microbiota dysbiotic pattern between the HIV-negative individual and HIV-positive patients with /or without first-line ARV and cotrimoxazole prophylaxis treatment through culture-dependent technique. And additionally to access the associations of gut microbiota at the genus level with sociodemographic and clinical factors. Study Design:  This was a cross-sectional study. Place and Duration of Study: Participants were selected from the South West region at the Buea Regional Hospital UPEC unit. The study spanned from August 2018 to April 2019. Methodology: We included 160 participants. Fecal and blood samples were collected from HIV-negative individuals (n=40), HIV-positive treatment naïve (n=40), HIV-positive + ARV (n=40) and HIV-positive + ARV + Cotrimoxazole prophylaxis (n=40). A self-structured questionnaire was administered to collect sociodemographic data. The stool samples were plated using three non-selective and ten selective media and colonies were i...

Agrobacterium has evolved sophisticated strategies to perceive and transduce plant-derived cues. Recent studies have found that numerous plant signals, including salicylic acid (SA), indole-3-acetic acid (IAA) and g-amino butyric acid (GABA), profoundly affect Agrobacterium-plant interactions. Here we determine and compare the transcriptome profiles of Agrobacterium in response to these three plant signals. Collectively, the transcription of 103, 115 and 95 genes was significantly altered by SA, IAA and GABA respectively. Both distinct cellular responses and overlapping signalling pathways were elicited by these three plant signals. Interestingly, these three plant compounds function additively to shut off the Agrobacterium virulence programme and activate the quorum-quenching machinery. Moreover, the repression of the virulence programme by SA and IAA and the inactivation of quorum-sensing signals by SA and GABA are regulated through independent pathways. Our data indicate that these plant signals, while cross-talk in plant signalling networks, also act as cross-kingdom signals and play redundant roles in tailoring Agrobacterium regulatory pathways, resulting in intensive signalling cross-talk in Agrobacterium. Our results support the notion that Agrobacterium has evolved the ability to hijack plant signals for its own benefit. The complex signalling interplay between Agrobacterium and its plant hosts reflects an exquisite co-evolutionary balance.

Chlamydiaceae are bacterial pathogens that cause diverse diseases in humans and animals. Despite their broad host and tissue tropism, all Chlamydia species share an obligate intracellular cycle of development and have evolved sophisticated mechanisms to interact with their eukaryotic host cells. Here, we have analysed interactions of the zoonotic pathogen Chlamydia psittaci with a human epithelial cell line. We found that C. psittaci recruits the ceramide transport protein (CERT) to its inclusion. Chemical inhibition and CRISPR/Cas9-mediated knockout of CERT showed that CERT is a crucial factor for C. psittaci infections thereby affecting different stages of the infection including inclusion growth and infectious progeny formation. Interestingly, the uptake of fluorescently labelled sphingolipids in bacteria inside the inclusion was accelerated in CERT-knockout cells indicating that C. psittaci can exploit CERT-independent sphingolipid uptake pathways. Moreover, the CERT-specific in...

Botulinum and tetanus neurotoxins are the most toxic substances known and form the growing family of clostridial neurotoxins (CNTs). They are composed of a metalloprotease light chain (L), linked via a disulfide bond to a heavy chain (H). H mediates binding to nerve terminals and the membrane translocation of L into the cytosol where their substrates, the three SNARE proteins, are localized. L translocation is accompanied by unfolding and L has to be reduced and reacquire the native fold to exert its neurotoxicity. The Thioredoxin reductase-Thioredoxin system is responsible for the reduction, but it is unknown whether the refolding of L is spontaneous or aided by host chaperones. Here we report that geldanamycin, a specific inhibitor of Hsp90, hampers the refolding of L after membrane translocation and completely prevents the cleavage of SNAREs. We also found that the effect of geldanamycin strongly synergises with that of PX-12, an inhibitor of thioredoxin, suggesting that the processes of L chain refolding and interchain disulphide reduction are strictly coupled. Indeed we found that Hsp90 and the Thioredoxin reductase-Thioredoxin system physically interact on synaptic vesicle where they orchestrate a chaperone-redox machinery which is exploited by CNTs to deliver their catalytic part in the cytosol.

The successful invasion of Plasmodium is an essential step in their life cycle. The parasite reticulocyte-binding protein homologues (RHs) and erythrocyte-binding like proteins are two families involved in the invasion leading to merozoite-red blood cell (RBC) junction formation. Ca(2+) signaling has been shown to play a critical role in the invasion. RHs have been linked to Ca(2+) signaling, which triggers the erythrocyte-binding like proteins release ahead of junction formation, consistent with RHs performing an initial sensing function in identifying suitable RBCs. RH5, the only essential RHs, is a highly promising vaccine candidate. RH5-basigin interaction is essential for merozoite invasion and also important in determining host tropism. Here, we show that RH5 has a distinct function from the other RHs. We show that RH5-Basigin interaction on its own triggers a Ca(2+) signal in the RBC resulting in changes in RBC cytoskeletal proteins phosphorylation and overall alterations in ...

Onchocerciasis is a disease caused by a microfilaria worm known as onchocerca volvulus usually transmitted to humans through the bite of an infected blackfly. It affects the skin and eye and the larva of the organism develops into adult filaria resulting in a large spectrum of manifestations, which increase the risk of blindness. This case report presents the case of a 28-year-old female patient with onchocerciasis, referral and follow-up. The study also discussed the clinical features, findings, and treatment options.

The host-virus interactions leading to cell infection with hepatitis C virus (HCV) are not fully understood. The tetraspanin CD-81 and human scavenger receptor SR-BI/Cla1 are major receptors mediating virus cell entry. However, HCV in patients' sera is associated with lipoproteins and infectious potential of the virus depends on lipoproteins associated to virus particles. We show here that lipoprotein lipase (LPL), targeting triglyceride-rich lipoproteins (TRL) to the liver, mediates binding and internalization of HCV to different types of cells, acting as a bridge between virusassociated lipoproteins and cell surface heparan sulfate proteoglycans (HSPG). The dimeric structure and catalytic activity of LPL are required for LPLmediated HCV uptake to cells. Unexpectedly, exogenous LPL significantly inhibits HCVcc infection in vitro. This effect is prevented by anti-LPL antibodies and by tetrahydrolipstatin (THL) a specific inhibitor of LPL enzymatic activity. In addition, we show that antibodies directed to apolipoprotein B (ApoB)-containing lipoproteins efficiently inhibits HCVcc infection. Our findings suggest that LPL mediates HCV cell entry by a mechanism similar to hepatic clearance of TRL from the circulation, promoting a non-productive virus uptake. These data provide new insight into mechanisms of HCV cell entry and suggest that LPL could modulate HCV infectivity in vivo.

Toll-like receptors (TLRs) are a class of membrane-spanning proteins of host cells. TLR2 and TLR4 are displayed on the surface of macrophages, neutrophils and dendritic cells and recognise structurally conserved microbial signatures defined as Pathogen associated molecular patterns (PAMPs). C3H mice are susceptible to tick-borne pathogens; Lyme disease causing Borrelia burgdorferi that manifests arthritis and carditis and Apicomplexan protozoan, Babesia microti (Bm) that causes significant parasitemia associated with erythrocytopenia and haemoglobinuria. B. burgdorferi lacks typical TLR4 ligand lipopolysaccharides (LPS) and Bm TLR ligand(s) remain unknown. Only Borrelia lipoproteins that signal through TLR2 are established as PAMPs of these pathogens for TLR2/ TLR4. Infection of C3H mice with each pathogen individually resulted in increase in the percentage of splenic B, T and FcR+ cells while their co-infection significantly diminished levels of these cells and caused increased B. burgdorferi burden in the specific organs. The most pronounced inflammatory arthritis was observed in co-infected C3H/HeJ mice. Parasitemia levels and kinetics of resolution of Bm in both mice strains were not significantly different. Transfected HEK293 cells showed pronounced signalling by B. burgdorferi through TLR2 and to some extent by TLR4 while Bm and infected erythrocytes did not show any response confirming our results in mice.

A 28 days old male was admitted with high fever, hepatosplenomegaly and increased acute phase reactants with high ferritin values. An infection by CMV was detected, supporting the diagnosis of virus-induced macrophage activation syndrome (MAS). Familial hemophagocytic lymphohistiocytosis (fHLH) were excluded. Ganciclovir together with high dose dexamethasone were administered with good clinical response. One month later high-grade fever, without features of HLH, and bloody diarrhea, requiring total parenteral nutrition (PN), appeared. The colonoscopy showed inflammatory erosive colitis and increased cell apoptosis on histology without CMV infection. Polyendocrinopathy Enteropathy X-linked was excluded. The baby became steroid-dependent, in spite of tacrolimus and amino acid formula, with several bouts of fever accompanied by bloody diarrhea and severe stunting depending on PN. New colonoscopy showed severe inflammatory Crohn-like colitis. The histologic examination confirmed the presence of a chronic inflammatory infiltrate in the lamina propria and submucosa. No improvement with azathioprine, and with anti-TNF alpha therapy. Diverting ileostomy and afterward colectomy were necessary. However, recurrent fever and stunting persisted. An inborn error of immunity was again suspected: chronic granulomatous disease, IL10/IL10R and mevalonate kinase deficiencies were excluded. A novel mutation of x-linked inhibitor of apoptosis (XIAP) was found. He received allogenic haemopoietic stem cell transplantation (HSCT) at the age of 22 month with progressive good clinical response and weaning from PN. Conclusions: XIAP mutations are associated with a X-linked primary immunodeficiency which can manifest as primary intestinal severe inflammation, often refractory to immunomodulators and biologics. HSCT may offer a causal therapeutic approach.

The mouse pathogen Citrobacter rodentium is used to model infections with enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC). Pathogenesis is commonly modelled in mice developing mild disease (e.g., C57BL/6). However, little is known about host responses in mice exhibiting severe colitis (e.g., C3H/HeN), which arguably provide a more clinically relevant model for human paediatric enteric infection. Infection of C3H/HeN mice with C. rodentium results in rapid colonic colonisation, coinciding with induction of key inflammatory signatures and colonic crypt hyperplasia. Infection also induces dramatic changes to bioenergetics in intestinal epithelial cells, with transition from oxidative phosphorylation (OXPHOS) to aerobic glycolysis and higher abundance of SGLT4, LDHA, and MCT4. Concomitantly, mitochondrial proteins involved in the TCA cycle and OXPHOS were in lower abundance. Similar to observations in C57BL/6 mice, we detected simultaneous activation of cholesterol biogenesis, import, and efflux. Distinctly, however, the pattern recognition receptors NLRP3 and ALPK1 were specifically induced in C3H/HeN. Using cell-based assays revealed that C. rodentium activates the ALPK1/TIFA axis, which is dependent on the ADP-heptose biosynthesis pathway but independent of the Type III secretion system. This study reveals for the first time the unfolding intestinal epithelial cells' responses during severe infectious colitis, which resemble EPEC human infections.

In iron overload disorders a significant fraction of the total iron circulates in the plasma as low molecular weight complexes not bound to transferrin, known as non-transferrin-bound iron (NTBI). By catalyzing the formation of free radicals, NTBI accumulation results in oxidative stress and cellular damage, being a major cause of organ toxicity. NTBI is rapidly and preferentially cleared from circulation by the liver and the myocardium, the main disease targets in iron overload conditions. We have recently demonstrated that human peripheral blood T lymphocytes take up NTBI in vitro, with a pattern that resembles that of hepatocytes. Since T lymphocytes constitute a numerically important component of the circulating cell pool, these findings support a putative role for this cell type in the systemic protection against iron toxicity. Here we tested the hypothesis that the circulating peripheral blood T lymphocyte pool constitutes an important storage compartment for NTBI and is thus a modifier of NTBI deposition in target organs. First we show that NTBI uptake by human T lymphocytes increases the expression of the iron-storage protein ferritin and of the iron exporter ferroportin via an IRE-dependent mechanism. NTBI retention by T lymphocytes is shown to be critically controlled by the hepcidin-mediated modulation of ferroportin both in vitro and in vivo. Finally, the protective effect of T lymphocytes was tested by analyzing the patterns of iron accumulation in the T lymphocyte-deficient mouse model Foxn1 nu before and after reconstitution with T lymphocytes by adoptive transfer. The results confirmed a significant increase of liver and pancreas iron accumulation in T lymphocyte-deficient mice. NTBI accumulation in the liver and spleen was prevented by reconstitution with syngeneic T lymphocytes. Altogether, our results demonstrate that T lymphocytes are important components of a circulating "NTBI storage compartment" and show its physiological relevance as a modifier of tissue iron overload.

Human skin keratinocytes HaCat attacked by Staphylococcus aureus a-toxin showed a transient drop of cellular ATP levels whereas in toxin-perforated bovine mammary epithelial cells (BMEC), the ATP levels dropped more slowly. Morphologically, during the ATP level depletion, HaCat cell developed a spacious intracellular vacuole together with the transient influx of trypan blue. WST-1 signal, which tested the function of mitochondrial enzyme in viable cells, also decreased concomitantly. On the other hand, BMEC excluded trypan blue and vacuolation was not observed throughout the experiment. We conclude that mammary epithelial cells resist the toxin better than keratinocytes. This is the first report showing that a-toxin enhances transient membrane permeability to large molecules, temporary vacuole formation and the transient defect of mitochondrial enzyme in viable cells without cell lysis.

The different morphological stages of Plasmodium falciparum show different physiological and biochemical characteristic. Lately there has been a renewed interest in the proteome of the parasite leading to considerable body of work towards identification of stage and sex specific proteins. In 2002 a whole chromosome shotgun sequencing of P falciparum clone 3D7 was completed. The chromosomes of P falciparum show gene clustering with coregulated gene groups and proteins. The Parasitophorous Vacuole Membrane and Gametocyte Exported Proteins are seen to be over expressed during early phase of sexual differentiation. The gametocyte proteome is in general specific to differing functionalities of the sexes. All ribosomal proteins are highly expressed in females. The sensitivity of this stage to antimalarials attacking respiration like primaquine and artemisinin based drugs is pronounced. Research on on NF54 isolate of P falciparum has revealed that of the total of 1289 proteins found by nan...

Mycoplasma have been shown to be involved in the alteration of several eukaryotic cell functions, such as cytokine production, gene expression and more. We have previously reported that infection of human myelomonocytic U937 cell line with live Mycoplasma fermentans (M. fermentans) inhibited tumour necrosis factor (TNF-alpha)-induced apoptosis. Mycoplasmal membrane lipoproteins are considered to be the most potent initiators of inflammatory reactions in mycoplasmal infections. The aim of this study was to clarify whether the inhibitory effect on TNFa-induced apoptosis is exerted by M. fermentans lipoproteins (LPMf). A significant reduction in TNFa-induced apoptosis was demonstrated by stimulation of U937 cells with M. fermentans total proteins, LPMf or MALP-2 (M. fermentans synthetic lipopeptide), but not with M. fermentans hydrophilic protein preparation (AqMf). To investigate the mechanism of M. fermentans antiapoptotic effect, the reduction of mitochondrial transmembrane potential (DYm) was measured. M. fermentans total proteins LPMf and MALP-2, but not AqMf, inhibited the reduction of DYm. In addition, M. fermentans total proteins LPMf and MALP-2, but not AqMf, downregulated the formation of active caspase-8. NF-kB was transactivated in cells treated with M. fermentans lipoproteins, and was essential for host cell survival, but not for the inhibition of TNFa-induced apoptosis by LPMf. Our results suggest that the inhibitory effect exerted by M. fermentans on TNFa-induced apopto-sis in U937 cells is due to the membrane lipoproteins of these bacteria.

The Oca (Oligomeric coiled-coil adhesin) family is a subgroup of the bacterial trimeric autotransporter adhesins, which includes structurally related proteins, such as YadA of Yersinia enterocolitica and NadA of Neisseria meningitidis. In this study, we searched in silico for novel members of this family in bacterial genomes and identified HadA (Haemophilus adhesin A), a trimeric autotransporter expressed only by Haemophilus influenzae biogroup aegyptius causing Brazilian purpuric fever (BPF), a fulminant septicemic disease of children. By comparative genomics and sequence analysis we predicted that the hadA gene is harboured on a mobile genetic element unique to BPF isolates. Biological analysis of HadA in the native background was limited because this organism is not amenable to genetic manipulation. Alternatively, we demonstrated that expression of HadA confers to a non-invasive Escherichia coli strain the ability to adhere to human cells and to extracellular matrix proteins and to induce in vitro bacterial aggregation and microcolony formation. Intriguingly, HadA is predicted to lack the typical N-terminal head domain of Oca proteins generally associated with cellular receptor binding. We propose here a structural model of the HadA coiled-coil stalk and show that the N-terminal region is still responsible of the binding activity and a KGD motif plays a role. Interestingly, HadA promotes bacterial entry into mammalian cells. Our results show a cytoskeleton re-arrangement and an involvement of clathrin in the HadA-mediated internalization. These data give new insights on the structure-function relationship of oligomeric coiled-coil adhesins and suggest a potential role of this protein in the pathogenesis of BPF.

We previously identified a Neisseria flavescens strain in the duodenum of celiac disease (CD) patients that induced immune inflammation in ex vivo duodenal mucosal explants and in CaCo-2 cells. We also found that vesicular trafficking was delayed after the CD-immunogenic P31-43 gliadin peptide-entered CaCo-2 cells and that Lactobacillus paracasei CBA L74 (L. paracasei-CBA) supernatant reduced peptide entry. In this study, we evaluated if metabolism and trafficking was altered in CD-N. flavescens-infected CaCo-2 cells and if any alteration could be mitigated by pretreating cells with L. paracasei-CBA supernatant, despite the presence of P31-43. We measured CaCo-2 bioenergetics by an extracellular flux analyser, N. flavescens and P31-43 intracellular trafficking by immunofluorescence, cellular stress by TBARS assay, and ATP by bioluminescence. We found that CD-N. flavescens colocalised more than control N. flavescens with early endocytic vesicles and more escaped autophagy thereby surviving longer in infected cells. P31-43 increased colocalisation of N. flavescens with early vesicles. Mitochondrial respiration was lower (P < .05) in CD-N. flavescens-infected cells versus not-treated CaCo-2 cells, whereas pretreatment with L. paracasei-CBA reduced CD-N. flavescens viability and improved cell bioenergetics and trafficking. In conclusion, CD-N. flavescens induces metabolic imbalance in CaCo-2 cells, and the L. paracasei-CBA probiotic could be used to correct CD-associated dysbiosis.

Dormant liver stage forms (hypnozoites) of the malaria parasite Plasmodium vivax present major hurdles to control and eradicate infection. Despite major research efforts, the molecular composition of hypnozoites remains ill defined. Here, we applied a combination of state-of-the-art technologies to generate the first transcriptome of hypnozoites. We developed a robust laser dissection microscopy protocol to isolate individual Plasmodium cynomolgi hypnozoites and schizonts from infected monkey hepatocytes and optimized RNA-seq analysis to obtain the first transcriptomes of these stages. Comparative transcriptomic analysis identified 120 transcripts as being differentially expressed in the hypnozoite stage relative to the dividing liver schizont, with 69 and 51 mRNAs being up- or down-regulated, respectively, in the hypnozoites. This lead to the identification of potential markers of commitment to and maintenance of the dormant state of the hypnozoite including three transcriptional r...

Bacterial toxins; the soluble antigens, emitted by various pathogenic microorganisms has a standing notoriety of being a toxin discharged over the span of pathogenesis. Toxins can balance the cell capacities by specifically focusing on various flagging pathways inside the host cell so as to tilt the equalization in microorganisms support. With the upgraded comprehension of the poison refinements strategies, quality cloning, protein arrangements, and three-dimensional crystallographic structures today, we can appreciate the interesting conduct of toxins, for example, restricting capacity to the explicit target cell and a few chemicals capacities. Bacterial pathogens have been causing different infections in people for quite a long time and even today these pathogenic microscopic organisms are coinciding and developing with us, regardless of having diverse means for their control in clinical practices (specifically antimicrobials, immunizations, and phage treatment). Toxins can be grouped into two general classes: exotoxin and endotoxin, and can be delivered by both Gram-positive and Gram-negative microbes. Exotoxins delivered by various microorganisms like botulinum, Clostridium, diphtheria poisons displaying protein exercises like ADP-ribosylation, phospholipase, adenylate cyclase, metalloprotease, deamidase, protease, and deoxyribonuclease action. The vast majority of these microscopic organisms apply their impeding impacts in the contaminated host through toxins. The elucidation of the cellular mechanism of action of the bacterial exotoxins remains a complex problem, but they appear to share a common mechanism of action such as binding to specific receptors on the plasma membranes of the sensitive cells, poreformation, internalization or translocation across the membrane barrier and direct secretion.

Inhalational anthrax is initiated by pulmonary exposure to Bacillus anthracis spores. Spore entry into lung epithelial cells is observed both in vitro and in vivo and evidence suggests it is important for bacterial dissemination and virulence. However the specific host receptor and spore factor that mediate the entry process were unknown. Here, we report that integrin a2b1 is a major receptor for spore entry. This is supported by results from blocking antibodies, siRNA knock-down, colocalization, and comparison of spore entry into cells that do or do not express a2. BclA, a major spore surface protein, is found to be essential for entry and a2b1-mediated entry is dependent on BclA. However, BclA does not appear to bind directly to a2. Furthermore, spore entry into a2-expressing cells is dramatically reduced in the absence of serum, suggesting that additional factors are involved. Finally, complement component C1q, also an a2b1 ligand, appears to act as a bridging molecule or a cofactor for BclA/a2b1-mediated spore entry and BclA binds to C1q in a dosedependent and saturable manner. These findings suggest a novel mechanism for pathogen entry into host cells as well as a new function for C1qintegrin interactions. The implications of these findings are discussed.

Helicobacter (H.) suis is the most prevalent non-H. pylori Helicobacter species colonizing the stomach of humans suffering from gastric disease. In the present study, we aimed to unravel the mechanism used by H. suis to induce gastric epithelial cell damage. H. suis lysate induced mainly apoptotic death of human gastric epithelial cells. Inhibition of g-glutamyl transpeptidase (GGT) activity present in H. suis lysate and incubation of AGS cells with purified native and recombinant H. suis GGT showed that this enzyme was partly responsible for the observed apoptosis. Supplementation of H. suis or H. pylori GGT-treated cells with glutathione strongly enhanced the harmful effect of both enzymes and resulted in the induction of oncosis/necrosis, demonstrating that H. suis and H. pylori GGT-mediated degradation of glutathione and the resulting formation of glutathione degradation products play a direct and active role in the induction of gastric epithelial cell death. This was preceded by an increase of extracellular H 2O2 concentrations, generated in a cell-independent manner and causing lipid peroxidation. In conclusion, H. suis and H. pylori GGTmediated generation of pro-oxidant glutathione degradation products brings on cell damage and causes apoptosis or necrosis, dependent on the amount of extracellular glutathione available as a GGT substrate.

Human papillomaviruses (HPVs) target PML nuclear bodies (NBs) during infectious entry and PML protein is important for efficient transcription of incoming viral genome. However, the transcriptional down regulation was shown to be promoter-independent in that heterologous promoters delivered by papillomavirus particles were also affected. To further investigate the role of PML protein in HPV entry, we used shRNA to knockdown PML protein in HaCaT keratinocytes. Confirming previous findings, PML knockdown in HaCaT cells reduced HPV16 transcript levels significantly following infectious entry without impairing binding and trafficking. However, when we quantified steady-state levels of pseudogenomes in interphase cells, we found strongly reduced genome levels compared to parental HaCaT cells. Since nuclear delivery was comparable in both cell lines, we conclude that viral pseudogenome must be removed after successful nuclear delivery. Transcriptome analysis by gene array revealed that PML knockdown in clonal HaCaT cells was associated with a constitutive interferon (IFN) response. Abrogation of JAK1/2 signaling prevented genome loss, however, did not restore viral transcription. In contrast, knockdown of PML protein in HeLa cells did not affect HPV genome delivery and transcription. HeLa cells are transformed by HPV18 oncogenes E6 and E7, which have been shown to interfere with the JAK/Stat signaling pathway. Our data imply that PML NBs protect incoming HPV genomes. Furthermore, they provide evidence that PML NBs are key regulators of the innate immune response in keratinocytes.