Complexity and dynamics of host–fungal interactions

Pathologies attributable to fungal infections represent a growing concern in both developed and developing countries. Initially discovered as opportunistic pathogens of immunocompromised hosts, fungi such as Candida albicans are now being placed at the centre of a more complex and dynamic picture in which the outcome of an infection is the result of an intricate network of molecular interactions between the fungus, the host and the commensal microflora co-inhabiting various host niches, and especially the gastrointestinal (GI) tract. The complexity of the host-fungal interaction begins with the numerous pathogen-associated molecular patterns (PAMPs) present on the fungal cell wall that are recognized by multiple pathogen-recognition receptors (PRRs), expressed by several types of host cells. PAMP-PRR interactions elicit a variety of intracellular signalling pathways leading to a wide array of immune responses, some of which promote fungal clearance while others contribute to pathogenesis. The picture is further complicated by the fact that numerous commensal bacteria normally co-inhabiting the host's GI tract produce molecules that either directly modulate the survival and virulence of commensal fungi such as C. albicans or indirectly modulate the host's antifungal immune responses. On top of this complexity, this host-microbiome-fungal interaction exhibits features of a dynamic system, in which the same fungi can easily switch between different morphological forms presenting different PAMPs at different moments of time. Furthermore, fungal pathogens can rapidly accumulate genomic alterations that further modify their recognition by the immune system, their virulence and their resistance to antifungal compounds. Thus, based on available molecular data alone, it is currently difficult to construct a coherent model able to explain the balance between commensalism and virulence and to predict the outcome of a fungal infection. Here, we review current advances in our understanding of this complex and dynamic system and propose new avenues of investigation to assemble a more complete picture of the host-fungal interaction, integrating microbiological and immunological data under the lens of systems biology and evolutionary genomics.     

Cardiovascular applications of biomaterials and implants–an overview

This paper was originally commissioned by the UK Department of Health as a contribution to the work of its Biomaterials and Implants Research Advisory Group. This group was set up under the Chairmanship of Professor Sir Colin Berry with the following terms of reference: (i) to identify recent advances in the field of biomaterials; (ii) to consider the future contribution of biomaterials in improving human health; (Hi) to advise the Standing Group on Health Technology in areas where developments and assessment are needed; and (iv) to report to the Director of Research and Development (Professor Sir Michael Peckham) by October 1995. The cardiovascular field was one of several areas in biomaterials/implants considered by the Advisory Group. Amongst other areas considered were orthopaedics, dentistry, urology, wound repair and ophthalmology. Additionally, consideration was also given to such topics as chemical and biochemical sensors, drug release, hydrogels, membranes and artificial organs. The final report of the Advisory Group will be published at the end of this year. However, the Department has agreed that individual working papers such as the present one can be published independently in appropriate scientific journals.     

Quantification of host–microbe interactions by automated fluorescence microscopy

We describe an automated fluorescence microscopy-based assay that quantifies the invasion of mammalian cells by intracellular pathogens. Pathogens associated with host cell surfaces, intracellular pathogens and mammalian cells are directly counted based on their specific fluorescent labeling. Such approach utilizes automated image acquisition and processing, and is thus ideally suited for high-throughput analyses. This method was validated using Listeria monocytogenes as a model intracellular pathogen.     

Utilization of zebrafish for intravital study of eukaryotic pathogen–host interactions

Unique imaging tools and practical advantages have made zebrafish a popular model to investigate in vivo host–pathogen interactions. These studies have uncovered details of the mechanisms involved in several human infections. Until recently, studies using this versatile host were limited to viral and prokaryotic pathogens. Eukaryotic pathogens are a diverse group with a major impact on the human and fish populations. The relationships of eukaryote pathogens with their hosts are complex and many aspects remain obscure. The small and transparent zebrafish, with its conserved immune system and amenability to genetic manipulation, make it an exciting model for quantitative study of the core strategies of eukaryotic pathogens and their hosts. The only thing to do now is realize its potential for advancement of biomedical and aquaculture research.     

A review on host–pathogen interactions: classification and prediction

The research on host–pathogen interactions is an ever-emerging and evolving field. Every other day a new pathogen gets discovered, along with comes the challenge of its prevention and cure. As the intelligent human always vies for prevention, which is better than cure, understanding the mechanisms of host–pathogen interactions gets prior importance. There are many mechanisms involved from the pathogen as well as the host sides while an interaction happens. It is a vis-a-vis fight of the counter genes and proteins from both sides. Who wins depends on whether a host gets an infection or not. Moreover, a higher level of complexity arises when the pathogens evolve and become resistant to a host’s defense mechanisms. Such pathogens pose serious challenges for treatment. The entire human population is in danger of such long-lasting persistent infections. Some of these infections even increase the rate of mortality. Hence there is an immediate emergency to understand how the pathogens interact with their host for successful invasion. It may lead to discovery of appropriate preventive measures, and the development of rational therapeutic measures and medication against such infections and diseases. This review, a state-of-the-art updated scenario of host–pathogen interaction research, has been done by keeping in mind this urgency. It covers the biological and computational aspects of host–pathogen interactions, classification of the methods by which the pathogens interact with their hosts, different machine learning techniques for prediction of host–pathogen interactions, and future scopes of this research field.     

Avian metapneumovirus infection of chicken and turkey tracheal organ cultures: comparison of virus–host interactions

Avian metapneumovirus (aMPV) is a pathogen with worldwide distribution, which can cause high economic losses in infected poultry. aMPV mainly causes infection of the upper respiratory tract in both chickens and turkeys, although turkeys seem to be more susceptible. Little is known about virus–host interactions at epithelial surfaces after aMPV infection. Tracheal organ cultures (TOC) are a suitable model to investigate virus–host interaction in the respiratory epithelium. Therefore, we investigated virus replication rates and lesion development in chicken and turkey TOC after infection with a virulent aMPV subtype A strain. Aspects of the innate immune response, such as interferon-α and inducible nitric oxide synthase mRNA expression, as well as virus-induced apoptosis were determined. The aMPV-replication rate was higher in turkey (TTOC) compared to chicken TOC (CTOC) (P?<?0.05), providing circumstantial evidence that indeed turkeys may be more susceptible. The interferon-α response was down-regulated from 2 to 144 hours post infection in both species compared to virus-free controls (P?<?0.05); this was more significant for CTOC than TTOC. Inducible nitric oxide synthase expression was significantly up-regulated in aMPV-A-infected TTOC and CTOC compared to virus-free controls (P?<?0.05). However, the results suggest that NO may play a different role in aMPV pathogenesis between turkeys and chickens as indicated by differences in apoptosis rate and lesion development between species. Overall, our study reveals differences in innate immune response regulation and therefore may explain differences in aMPV – A replication rates between infected TTOC and CTOC, which subsequently lead to more severe clinical signs and a higher rate of secondary infections in turkeys.     

Microstructure,mechanical and corrosion properties of Mg–Dy–Gd–Zr alloys for medical applications

In previous investigations, a Mg–10Dy (wt.%) alloy with a good combination of corrosion resistance and cytocompatibility showed great potential for use as a biodegradable implant material. However, the mechanical properties of Mg–10Dy alloy are not satisfactory. In order to allow the tailoring of mechanical properties required for various medical applications, four Mg–10(Dy + Gd)–0.2Zr (wt.%) alloys were investigated with respect to microstructure, mechanical and corrosion properties. With the increase in Gd content, the number of second-phase particles increased in the as-cast alloys, and the age-hardening response increased at 200 °C. The yield strength increased, while the ductility reduced, especially for peak-aged alloys with the addition of Gd. Additionally, with increasing Gd content, the corrosion rate increased in the as-cast condition owing to the galvanic effect, but all the alloys had a similar corrosion rate (～0.5 mm year^?1) in solution-treated and aged condition.     

Characterization of dielectrophoresis-aligned nanofibrous silk fibroin–chitosan scaffold and its interactions with endothelial cells for tissue engineering applications

Aligned three-dimensional nanofibrous silk fibroin–chitosan (eSFCS) scaffolds were fabricated using dielectrophoresis (DEP) by investigating the effects of alternating current frequency, the presence of ions, the SF:CS ratio and the post-DEP freezing temperature. Scaffolds were characterized with polarized light microscopy to analyze SF polymer chain alignment, atomic force microscopy (AFM) to measure the apparent elastic modulus, and scanning electron microscopy and AFM to analyze scaffold topography. The interaction of human umbilical vein endothelial cells (HUVECs) with eSFCS scaffolds was assessed using immunostaining to assess cell patterning and AFM to measure the apparent elastic modulus of the cells. The eSFCS (50:50) samples prepared at 10 MHz with NaCl had the highest percentage of aligned area as compared to other conditions. As DEP frequency increased from 100 kHz to 10 MHz, fibril sizes decreased significantly. eSFCS (50:50) scaffolds fabricated at 10 MHz in the presence of 5 mM NaCl had a fibril size of 77.96 ± 4.69 nm and an apparent elastic modulus of 39.9 ± 22.4 kPa. HUVECs on eSFCS scaffolds formed aligned and branched capillary-like vascular structures. The elastic modulus of HUVEC cultured on eSFCS was 6.36 ± 2.37 kPa. DEP is a potential tool for fabrication of SFCS scaffolds with aligned nanofibrous structures that can guide vasculature in tissue engineering and repair.     

Systems biology of host–mycobiota interactions: Dissecting Dectin-1 and Dectin-2 signalling in immune cells with DC-ATLAS

Modelling the networks sustaining the fruitful coexistence between fungi and their mammalian hosts is becoming increasingly important to control emerging fungal pathogens.     

The apolipoprotein L family of programmed cell death and immunity genes rapidly evolved in primates at discrete sites of host–pathogen interactions

Apolipoprotein L1 (APOL1) is a human protein that confers immunity to Trypanosoma brucei infections but can be countered by a trypanosome-encoded antagonist SRA. APOL1 belongs to a family of programmed cell death genes whose proteins can initiate host apoptosis or autophagic death. We report here that all six members of the APOL gene family (APOL1-6) present in humans have rapidly evolved in simian primates. APOL6, furthermore, shows evidence of an adaptive sweep during recent human evolution. In each APOL gene tested, we found rapidly evolving codons in or adjacent to the SRA-interacting protein domain (SID), which is the domain of APOL1 that interacts with SRA. In APOL6, we also found a rapidly changing 13-amino-acid cluster in the membrane-addressing domain (MAD), which putatively functions as a pH sensor and regulator of cell death. We predict that APOL genes are antagonized by pathogens by at least two distinct mechanisms: SID antagonists, which include SRA, that interact with the SID of various APOL proteins, and MAD antagonists that interact with the MAD hinge base of APOL6. These antagonists either block or prematurely cause APOL-mediated programmed cell death of host cells to benefit the infecting pathogen. These putative interactions must occur inside host cells, in contrast to secreted APOL1 that trafficks to the trypanosome lysosome. Hence, the dynamic APOL gene family appears to be an important link between programmed cell death of host cells and immunity to pathogens.The apolipoprotein L (APOL) gene family is composed of six genes in humans, which are grouped within 619 kb on human chromosome 22 (see Fig. 1; Page et al. 2001). By far, the best-studied family member is APOL1, which encodes a trypanolytic factor in humans and gorillas, lysing pathogenic Trypanosoma brucei subspecies during bloodstream infections (Vanhamme et al. 2003). Most other primates lack APOL1 and do not have trypanolytic activity, except for certain species of baboons, mandrills, and mangabeys (Seed et al. 1990; Lugli et al. 2004). APOL1 is unique among APOL proteins because it can be secreted outside the cell, presumably due to its N-terminal signal peptide. Other APOL proteins do not have this signal peptide and have predicted localizations inside the cell (Page et al. 2001).Open in a separate windowFigure 1.Genome organization and coding exons of the human APOL gene family. (A) The genome organization of the six human APOL genes is shown. (B) Coding exons for human APOL genes and isoforms are shown. Differences between isoforms in noncoding regions are not shown.In addition to killing parasites, APOL proteins can initiate programmed cell death (PCD) of host cells. Each of the APOL genes putatively contains a BH3 protein domain, which is characteristic of the BH3-only family of pro-apoptosis genes (Liu et al. 2005; Vanhollebeke and Pays 2006). BH3-only proteins function as upstream activators of PCD, responding to stimuli such as cell detachment, cytokine withdrawal, or DNA damage before initiating cell death (Strasser 2005). The BH3 domain itself is a short peptide that binds to a groove on other PCD proteins, such as pro-survival Bcl-2 family members, which can bind BH3-only proteins under normal conditions to prevent cell death. Consistent with other BH3-only proteins, APOL6 was found to cause apoptosis when overexpressed in cancer cells (Liu et al. 2005).APOL1 was also recently shown to have an intracellular function, causing autophagic death of human cells (Wan et al. 2008). Autophagy is used by cells to “self-eat” during starvation and to recycle cellular contents. Cytosolic contents are gathered in an autophagosome, and trafficked via endosome fusion to the lysosome for degradation (Mizushima 2007). Although autophagy is generally considered to be a pro-survival process, autophagy proteins can cause PCD by a mechanism that is distinct from apoptosis (Degterev and Yuan 2008). The seemingly disparate roles of APOL1 in host autophagic death and trypanolysis are remarkably consistent in that they both utilize endosome trafficking to the lysosome.Further clues to APOL protein functions come from expression studies, genetic screens, and disease association studies. APOL genes have been implicated in schizophrenia (Mimmack et al. 2002), breast cancer (Dombkowski et al. 2006), cervical cancer (Ahn et al. 2004), and osteoarthritis (Okabe et al. 2007). However, the specific roles of APOL proteins in these diseases are not known. APOL genes are up-regulated by multiple pro-inflammatory signaling molecules, including interferon (IFN)-alpha (Hayashi et al. 2005), IFN-beta (Stojdl et al. 2003), IFN-gamma (Sana et al. 2005), and tumor necrosis factor alpha (TNF-alpha) (Monajemi et al. 2002). These regulations suggest that APOL proteins participate in the immune system, in addition to the role of secreted APOL1 in trypanosome immunity.Studies of how APOL1 kills trypanosomes have revealed the functions of individual protein domains. These functions may be shared with intracellular APOL proteins by virtue of shared domain architecture. APOL1 associates with a fraction of high-density lipoprotein (HDL) particles in the human bloodstream (it is this association for which the APOL genes were named) (Duchateau et al. 1997). Once introduced into hosts via insect vectors, T. brucei subspecies endocytose HDL particles, likely as a means to obtain both lipids and iron (Green et al. 2003; Vanhollebeke et al. 2008). As the HDL particles are trafficked from endocytic particles to the lysosome, the pH change from pH ∼7 to ∼5 induces a conformational change in the APOL1 membrane addressing domain (MAD). This causes a salt-bridge-linked hinge to open, which releases APOL1 from the HDL particle to insert in the lysosomal membrane (Perez-Morga et al. 2005). After insertion into the lysosomal membrane, APOL1 employs its pore-forming protein domain to create an anion-specific pore, which leads to lysosome swelling and ultimately pathogen lysis. The MAD and pore-forming domain are both required for killing (Perez-Morga et al. 2005).Two trypanosome subspecies have evolved a means to resist lysis by APOL1, causing the disease “sleeping sickness” in humans. The resistance mechanism of Trypanosoma brucei gambiense is unknown, but the mechanism of Trypanosoma brucei rhodesiense has been discovered (Xong et al. 1998). Trypanosoma brucei rhodesiense encodes the protein SRA that directly binds to APOL1 in the trypanosome lysosome to prevent lysis. SRA interacts with APOL1 using a coiled–coiled interaction at the APOL1 SRA-interacting domain (SID). The SID protein domain was shown to not be required for killing (Vanhamme et al. 2003), except in a recently reported mouse model (Molina-Portela et al. 2008). The role of the SID domain in trypanolysis thus remains somewhat ambiguous.We wondered whether the APOL1–SRA conflict was just one instance of similar, recurrent conflicts that APOL genes have encountered during primate evolution. Since proteins that directly interact with pathogen components often evolve under positive selection, especially at interaction interfaces, we sought to test the APOL gene family for evidence of rapid evolution. We present a detailed evolutionary analysis of the APOL gene family in primates and find evidence that indeed positive selection has occurred—in the entire gene family. We highlight particular protein regions that have evolved under positive selection, and provide strong evidence for additional sites of host–pathogen interaction. Overall, we show that the APOL gene family has been very dynamic during primate evolution, which likely reflects important roles for the family in immunity to pathogens.     

The current understanding of Stevens–Johnson syndrome and toxic epidermal necrolysis

Stevens–Johnson syndrome has long been considered to resemble erythema multiforme with mucosal involvement, but is now thought to form a single disease entity with toxic epidermal necrolysis. Although Stevens–Johnson syndrome is less severe, etiology, genetic susceptibility and pathomechanism are the same for Stevens–Johnson syndrome/toxic epidermal necrolysis. The condition is mainly caused by drugs, but also by infections and probably other risk factors not yet identified. Identification of the cause is important for the individual patient and in cases of drug-induced disease withdrawal of the inducing drug(s) has an impact on the patient’s prognosis. If an infectious cause is suspected, adequate anti-infective treatment is needed. Besides this, supportive management is crucial to improve the patient’s state, probably more than specific immunomodulating treatments. Despite all of the therapeutic efforts, mortality is high and increases with disease severity, patients’ age and underlying medical conditions. Survivors may suffer from long-term sequelae such as strictures of mucous membranes including severe eye problems.     

Coiled-coil irregularities of the M1 protein structure promote M1–fibrinogen interaction and influence group A Streptococcus host cell interactions and virulence

Group A Streptococcus (GAS) is a human pathogen causing a wide range of mild to severe and life-threatening diseases. The GAS M1 protein is a major virulence factor promoting GAS invasiveness and resistance to host innate immune clearance. M1 displays an irregular coiled-coil structure, including the B-repeats that bind fibrinogen. Previously, we found that B-repeat stabilisation generates an idealised version of M1 (M1*) characterised by decreased fibrinogen binding in vitro. To extend these findings based on a soluble truncated version of M1, we now studied the importance of the B-repeat coiled-coil irregularities in full length M1 and M1* expressed in live GAS and tested whether the modulation of M1–fibrinogen interactions would open up novel therapeutic approaches. We found that altering either the M1 structure on the GAS cell surface or removing its target host protein fibrinogen blunted GAS virulence. GAS expressing M1* showed an impaired ability to adhere to and to invade human endothelial cells, was more readily killed by whole blood or neutrophils and most importantly was less virulent in a murine necrotising fasciitis model. M1-mediated virulence of wild-type GAS was strictly dependent on the presence and concentration of fibrinogen complementing our finding that M1–fibrinogen interactions are crucial for GAS virulence. Consistently blocking M1–fibrinogen interactions by fragment D reduced GAS virulence in vitro and in vivo. This supports our conclusion that M1–fibrinogen interactions are crucial for GAS virulence and that interference may open up novel complementary treatment options for GAS infections caused by the leading invasive GAS strain M1.     

Selectivity of a translation-inhibitory factor,CpBV15β, in host mRNAs and subsequent alterations in host development and immunity

An endoparasitoid wasp, Cotesia plutellae, parasitizes young larvae of the diamondback moth, Plutella xylostella. Its symbiotic virus, C. plutellae bracovirus (CpBV), has been shown to play a crucial role in inducing physiological changes in the parasitized host. A viral gene, CpBV15β, exhibits a specific translational control against host mRNAs by sequestering a eukaryotic translation initiation factor, eIF4A. Inhibitory target mRNAs have high thermal stability (>≈9 kcal/mol) of their secondary structures in 5′UTR. To determine the specificity of translational control in terms of 5′UTR complexity, this study screened target/nontarget mRNAs of CpBV15β using a proteomics approach through an in vivo transient expression technique. A proteomics analysis of host plasma proteins showed that 12.9% (23/178) spots disappeared along with the expression of CpBV15β. A total of ten spots were chosen, in which five spots (‘target’) were disappeared by expression of CpBV15β and the other five (‘nontarget’) were insensitive to expression of CpBV15β, and further analyzed by a tandem mass spectroscopy. The predicted genes of target spots had much greater complexity (−12.3 to −25.2 kcal/mol) of their 5′UTR in terms of thermal stability compared to those (−3.70 to −9.00 kcal/mol) of nontarget spots. 5′UTRs of one target gene (arginine kinase:Px-AK) and one nontarget gene (imaginal disc growth factor:Px-IDGF) were cloned and used for in vitro translation (IVT) assay using rabbit reticulocyte lysate. IVT assay clearly showed that mRNA of Px-IDGF was translated in the presence of CpBV15β, but mRNA of Px-AK was not. Physiological significance of these two genes was compared in immune and development processes of P. xylostella by specific RNA interference (RNAi). Under these RNAi conditions, suppression of Px-AK exhibited much more significant adverse effects on larval immunity and larva-to-pupa metamorphosis compared to the effect of suppression of Px-IDGF. These results support the hypothesis that 5′UTR complexity is a molecular motif to discriminate host mRNAs by CpBV15β for its host translational control and suggest that this discrimination would be required for altering host physiology to accomplish a successful parasitism of the wasp host, C. plutellae.