Terahertz imaging of human skin pathologies using laser feedback interferometry with quantum cascade lasers.

Early detection of skin pathologies with current clinical diagnostic tools is challenging, particularly when there are no visible colour changes or morphological cues present on the skin. In this study, we present a terahertz (THz) imaging technology based on a narrow band quantum cascade laser (QCL) at 2.8 THz for human skin pathology detection with diffraction limited spatial resolution. THz imaging was conducted for three different groups of unstained human skin samples (benign naevus, dysplastic naevus, and melanoma) and compared to the corresponding traditional histopathologic stained images. The minimum thickness of dehydrated human skin that can provide THz contrast was determined to be 50 µm, which is approximately one half-wavelength of the THz wave used. The THz images from different types of 50 µm-thick skin samples were well correlated with the histological findings. The per-sample locations of pathology vs healthy skin can be separated from the density distribution of the corresponding pixels in the THz amplitude-phase map. The possible THz contrast mechanisms relating to the origin of image contrast in addition to water content were analyzed from these dehydrated samples. Our findings suggest that THz imaging could provide a feasible imaging modality for skin cancer detection that is beyond the visible.

A new family of cell surface located purine transporters in Microsporidia and related fungal endoparasites.

Plasma membrane-located transport proteins are key adaptations for obligate intracellular Microsporidia parasites, because they can use them to steal host metabolites the parasites need to grow and replicate. However, despite their importance, the functions and substrate specificities of most Microsporidia transporters are unknown. Here, we provide functional data for a family of transporters conserved in all microsporidian genomes and also in the genomes of related endoparasites. The universal retention among otherwise highly reduced genomes indicates an important role for these transporters for intracellular parasites. Using Trachipleistophora hominis, a Microsporidia isolated from an HIV/AIDS patient, as our experimental model, we show that the proteins are ATP and GTP transporters located on the surface of parasites during their intracellular growth and replication. Our work identifies a new route for the acquisition of essential energy and nucleotides for a major group of intracellular parasites that infect most animal species including humans.

Gas spectroscopy with integrated frequency monitoring through self-mixing in a terahertz quantum-cascade laser.

We demonstrate a gas spectroscopy technique, using self-mixing in a 3.4 terahertz quantum-cascade laser (QCL). All previous QCL spectroscopy techniques have required additional terahertz instrumentation (detectors, mixers, or spectrometers) for system pre-calibration or spectral analysis. By contrast, our system self-calibrates the laser frequency (i.e., with no external instrumentation) to a precision of 630 MHz (0.02%) by analyzing QCL voltage perturbations in response to optical feedback within a 0-800 mm round-trip delay line. We demonstrate methanol spectroscopy by introducing a gas cell into the feedback path and show that a limiting absorption coefficient of ∼1×10-4 cm-1 is resolvable.

Evolutionary conservation and in vitro reconstitution of microsporidian iron-sulfur cluster biosynthesis.

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 Fe/S-related proteins and pathways, and their strong conservation among highly reduced parasites, provides compelling evidence for the ancient chimeric ancestry of eukaryotes.

Microsporidia: Why Make Nucleotides if You Can Steal Them?

Microsporidia are strict obligate intracellular parasites that infect a wide range of eukaryotes including humans and economically important fish and insects. Surviving and flourishing inside another eukaryotic cell is a very specialised lifestyle that requires evolutionary innovation. Genome sequence analyses show that microsporidia have lost most of the genes needed for making primary metabolites, such as amino acids and nucleotides, and also that they have only a limited capacity for making adenosine triphosphate (ATP). Since microsporidia cannot grow and replicate without the enormous amounts of energy and nucleotide building blocks needed for protein, DNA, and RNA biosynthesis, they must have evolved ways of stealing these substrates from the infected host cell. Providing they can do this, genome analyses suggest that microsporidia have the enzyme repertoire needed to use and regenerate the imported nucleotides efficiently. Recent functional studies suggest that a critical innovation for adapting to intracellular life was the acquisition by lateral gene transfer of nucleotide transport (NTT) proteins that are now present in multiple copies in all microsporidian genomes. These proteins are expressed on the parasite surface and allow microsporidia to steal ATP and other purine nucleotides for energy and biosynthesis from their host. However, it remains unclear how other essential metabolites, such as pyrimidine nucleotides, are acquired. Transcriptomic and experimental studies suggest that microsporidia might manipulate host cell metabolism and cell biological processes to promote nucleotide synthesis and to maximise the potential for ATP and nucleotide import. In this review, we summarise recent genomic and functional data relating to how microsporidia exploit their hosts for energy and building blocks needed for growth and nucleic acid metabolism and we identify some remaining outstanding questions.

Free-space terahertz radiation from a LT-GaAs-on-quartz large-area photoconductive emitter.

We report on large-area photoconductive terahertz (THz) emitters with a low-temperature-grown GaAs (LT-GaAs) active layer fabricated on quartz substrates using a lift-off transfer process. These devices are compared to the same LT-GaAs emitters when fabricated on the growth substrate. We find that the transferred devices show higher optical-to-THz conversion efficiencies and significantly larger breakdown fields, which we attribute to reduced parasitic current in the substrate. Through these improvements, we demonstrate a factor of ~8 increase in emitted THz field strength at the maximum operating voltage. In addition we find improved performance when these devices are used for photoconductive detection, which we explain through a combination of reduced parasitic substrate currents and reduced space-charge build-up in the device.

A bacterial encoded protein induces extreme multinucleation and cell-cell internalization in intestinal cells.

Despite extensive study, the molecular mechanisms that lead to multinucleation and cell enlargement (hypertrophy) remain poorly understood. Here, we show that a single bacterial virulence protein, EspF, from the human pathogen enteropathogenic E. coli induces extreme multi-nucleation in small intestinal epithelial cells. Ectopic expression of EspF induced cell-cell internalization events, presumably responsible for the enlarged multinucleated cells. These extreme phenotypes were dependent on a C-terminal polyproline-rich domain in EspF and not linked to the targeting of mitochondria or the nucleolus. The subversive functions of EspF may provide valuable insight into the molecular mechanisms that mediate cell fusion, multinucleation and cell hypertrophy.

The enteropathogenic E. coli (EPEC) Tir effector inhibits NF-κB activity by targeting TNFα receptor-associated factors.

Enteropathogenic Escherichia coli (EPEC) disease depends on the transfer of effector proteins into epithelia lining the human small intestine. EPEC E2348/69 has at least 20 effector genes of which six are located with the effector-delivery system genes on the Locus of Enterocyte Effacement (LEE) Pathogenicity Island. Our previous work implied that non-LEE-encoded (Nle) effectors possess functions that inhibit epithelial anti-microbial and inflammation-inducing responses by blocking NF-κB transcription factor activity. Indeed, screens by us and others have identified novel inhibitory mechanisms for NleC and NleH, with key co-operative functions for NleB1 and NleE1. Here, we demonstrate that the LEE-encoded Translocated-intimin receptor (Tir) effector has a potent and specific ability to inhibit NF-κB activation. Indeed, biochemical, imaging and immunoprecipitation studies reveal a novel inhibitory mechanism whereby Tir interaction with cytoplasm-located TNFα receptor-associated factor (TRAF) adaptor proteins induces their proteasomal-independent degradation. Infection studies support this Tir-TRAF relationship but reveal that Tir, like NleC and NleH, has a non-essential contribution in EPEC's NF-κB inhibitory capacity linked to Tir's activity being suppressed by undefined EPEC factors. Infections in a disease-relevant intestinal model confirm key NF-κB inhibitory roles for the NleB1/NleE1 effectors, with other studies providing insights on host targets. The work not only reveals a second Intimin-independent property for Tir and a novel EPEC effector-mediated NF-κB inhibitory mechanism but also lends itself to speculations on the evolution of EPEC's capacity to inhibit NF-κB function.

The EspF effector, a bacterial pathogen's Swiss army knife.

Central to the pathogenesis of many bacterial pathogens is the ability to deliver effector proteins directly into the cells of their eukaryotic host. EspF is one of many effector proteins exclusive to the attaching and effacing pathogen family that includes enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli. Work in recent years has revealed EspF to be one of the most multifunctional effector proteins known, with defined roles in several host cellular processes, including disruption of the epithelial barrier, antiphagocytosis, microvillus effacement, host membrane remodelling, modulation of the cytoskeleton, targeting and disruption of the nucleolus, intermediate filament disruption, cell invasion, mitochondrial dysfunction, apoptosis, and inhibition of several important epithelial transporters. Surprisingly, despite this high number of functions, EspF is a relatively small effector protein, and recent work has begun to decipher the molecular events that underlie its multifunctionality. This review focuses on the activities of EspF within the host cell and discusses recent findings and molecular insights relating to the virulence functions of this fascinating bacterial effector.

The bacterial effectors EspG and EspG2 induce a destructive calpain activity that is kept in check by the co-delivered Tir effector.

Bacterial pathogens deliver multiple effector proteins into eukaryotic cells to subvert host cellular processes and an emerging theme is the cooperation between different effectors. Here, we reveal that a fine balance exists between effectors that are delivered by enteropathogenic E. coli (EPEC) which, if perturbed can have marked consequences on the outcome of the infection. We show that absence of the EPEC effector Tir confers onto the bacterium a potent ability to destroy polarized intestinal epithelia through extensive host cell detachment. This process was dependent on the EPEC effectors EspG and EspG2 through their activation of the host cysteine protease calpain. EspG and EspG2 are shown to activate calpain during EPEC infection, which increases significantly in the absence of Tir - leading to rapid host cell loss and necrosis. These findings reveal a new function for EspG and EspG2 and show that Tir, independent of its bacterial ligand Intimin, is essential for maintaining the integrity of the epithelium during EPEC infection by keeping the destructive activity of EspG and EspG2 in check.

The enteropathogenic Escherichia coli EspF effector molecule inhibits PI-3 kinase-mediated uptake independently of mitochondrial targeting.

Delivery of effector molecules into LMme(v) macrophages by enteropathogenic Escherichia coli, via its type three secretion system (T3SS), inhibits bacterial uptake by a phosphatidylinositol-3 (PI-3) kinase-dependent pathway. The T3SS system, encoded by the locus of enterocyte effacement (LEE) pathogenicity island, delivers LEE- and non-LEE-encoded effector proteins into host cells. Previous studies discounted essential roles for the LEE-encoded Map, EspF, Tir or Intimin proteins in this process but correlated it with loss of phosphorylation of the PI-3 kinase substrate, Akt (Celli et al., 2001, EMBO J 20: 1245-1258). Given the more recent finding that these bacterial proteins are multifunctional and can act together to subvert host cellular processes, we generated a quadruple deletion mutant (Map, Tir, EspF and Intimin deficient) to unearth any cooperativity in inhibiting uptake. The quadruple mutant was as defective as the T3SS-defective strain at preventing bacterial uptake with further studies revealing a surprising dependence on EspF but not Map, Tir or Intimin. Subversive activities previously associated with EspF are disruption of epithelial barrier function and programmed cell death, with the latter linked to EspF targeting mitochondria. Interestingly, the C-terminal domain possesses a polyproline motif associated with protein-protein interactions. We demonstrate that EspF-mediated inhibition of PI-3 kinase-dependent uptake: (i) is independent of mitochondrial targeting, (ii) requires the N-terminal domain with and (iii) the C-terminal domain is sufficient to disrupt barrier function but not inhibition of bacterial uptake. Moreover, loss of PI-3 kinase-dependent phosphorylation of Akt and gross changes in host phosphotyrosine protein profiles could not be linked to inhibition of the PI-3 kinase-dependent uptake process.

EPEC's weapons of mass subversion.

Enteropathogenic and enterohaemorrhagic Escherichia coli are closely related enteric pathogens whose ability to cause disease in humans is linked with a capacity to deliver bacterial 'effector' proteins into host epithelia to alter cellular physiology. Although the essential role of the locus of enterocyte effacement (LEE) pathogenicity island, which encodes effector proteins and the delivery machinery, has been established, more recent studies are uncovering additional layers of complexity. This is illustrated by the emerging multifunctional nature of the effectors and their ability to work together in redundant, synergistic and antagonistic relationships. Furthermore, new virulence-associated factors are continually being uncovered that are encoded outside the LEE pathogenicity island, some of which are not injected into host cells.

Microbial infection causes the appearance of hemocytes with extreme spreading ability in monolayers of the tobacco hornworm Manduca sexta.

The ability to adhere to and spread on a surface is a common property of insect blood cells. Spreading on a glass surface by insect hemocytes is often used as a measure of immune fitness that can be inhibited by some insect pathogens and parasites. Here, we report that upon infection of the tobacco hornworm Manduca sexta with either a fungus (Beauveria bassiana) or a bacterium (Photorhabdus luminescens), a new type of hemocyte, not previously observed in healthy insects, was found in hemocyte monolayers. These cells have a distinctive morphology, characterised by extreme spreading ability. They achieve a diameter of up to 120 microm after 1 h on glass coverslips and are therefore extremely thin. These hyper-spreading cells first appear in fungal-infected insects prior to hyphal growth. Their numbers later fall to zero as the pathogen begins to proliferate. The same hyper-spreading cells are induced after a 24 h delay following an injection of laminarin, a source of the fungal cell wall polymer beta-1,3-glucans. Wounding, on the other hand, did not cause the appearance of hyper-spreading cells. Evidence is presented here that is consistent with these spreading cells having a role in the cellular immune response of nodule formation.

Modulation by eicosanoid biosynthesis inhibitors of immune responses by the insect Manduca sexta to the pathogenic fungus Metarhizium anisopliae.

Metarhizium anisopliae conidia (spores) reduced weight gain and caused death when injected into Manduca sexta larvae. When the fungus was co-injected with the eicosanoid biosynthesis inhibitor dexamethasone, larval weight gain was further reduced and mortality increased. These effects were reversed when dexamethasone was given together with the eicosanoid precursor arachidonic acid (AA). Similarly, treatment with other eicosanoid biosynthesis inhibitors (esculetin, phenidone, ibuprofen, and indomethacin) with differing modes of action enhanced the reduction in weight gain caused by mycosis. Injection of M. anisopliae conidia induced nodule formation in vivo; nodule numbers were reduced by dexamethasone, and restored by AA. Incubation of hemocytes with conidia caused microaggregation of hemocytes (indicative of nodule formation) in vitro and this was inhibited by dexamethasone, suggesting that dexamethasone acts directly on hemocytes, although inhibition was only partially reversed by AA. We suggest that the M. sexta immune response to fungal pathogens is normally modulated by physiological systems that include eicosanoid biosynthesis. This is the first demonstration that the virulence of a fungal entomopathogen can be enhanced by compromising the insect host's immune system.

Bacterial infection of a model insect: Photorhabdus luminescens and Manduca sexta.

Invertebrates, including insects, are being developed as model systems for the study of bacterial virulence. However, we understand little of the interaction between bacteria and specific invertebrate tissues or the immune system. To establish an infection model for Photorhabdus, which is released directly into the insect blood system by its nematode symbiont, we document the number and location of recoverable bacteria found during infection of Manduca sexta. After injection into the insect larva, P. luminescens multiplies in both the midgut and haemolymph, only later colonizing the fat body and the remaining tissues of the cadaver. Bacteria persist by suppressing haemocyte-mediated phagocytosis and culture supernatants grown in vitro, as well as plasma from infected insects, suppress phagocytosis of P. luminescens. Using GFP-labelled bacteria, we show that colonization of the gut begins at the anterior of the midgut and proceeds posteriorly. Within the midgut, P. luminescens occupies a specific niche between the extracellular matrix and basal membrane (lamina) of the folded midgut epithelium. Here, the bacteria express the gut-active Toxin complex A (Tca) and an RTX-like metalloprotease PrtA. This close association of the bacteria with the gut, and the production of toxins and protease, triggers a massive programmed cell death of the midgut epithelium.