Comment on "Electronic, vibrational and torsional couplings in N-methylpyrrole: Ground, first excited and cation states" [J. Chem. Phys. 154, 224305 (2021)].

Two-color (1 + 1') zero-electron-kinetic-energy (ZEKE) and photoionization efficiency (PIE) spectra are reported via different levels in the S1 ← S0 (Ã1A2←X̃1A1) one-photon transition of jet-cooled N-methylpyrrole. The laser radiation is produced using two dye lasers, one with an 1800 l/mm grating and one with 2400 l/mm. We report spectra where the excitation and ionization radiation are produced with both combinations of the dye lasers; these spectra differ markedly. This is attributed to Wood's anomalies with the 2400 l/mm grating: one aspect is a loss in light intensity over a range of wavelengths, attributed to a resonance anomaly. Another is the appearance of a "shadow" ZEKE spectrum and PIE curve at apparently higher ionization wavenumbers; under some conditions, a third ZEKE spectrum was observed-these latter observations arise from higher-order dispersion effects, likely caused by a Rayleigh anomaly. We comment on these observations and report more representative ZEKE and PIE spectra than those presented in a recent paper by our group [A. R. Davies, D. J. Kemp, and T. G. Wright, J. Chem. Phys. 154, 224305 (2021)] for four intermediate S1 levels.

Electronic, vibrational, and torsional couplings in N-methylpyrrole: Ground, first excited, and cation states.

The electronic spectrum associated with the S1 ← S0 (Ã1A2←X̃1A1) one-photon transition of jet-cooled N-methylpyrrole is investigated using laser-induced fluorescence (LIF) and (1 + 1) resonance-enhanced multiphoton ionization (REMPI) spectroscopy; in addition, the (2 + 2) REMPI spectrum is considered. Assignment of the observed bands is achieved using a combination of dispersed fluorescence (DF), two-dimensional LIF (2D-LIF), zero-electron-kinetic energy (ZEKE) spectroscopy, and quantum chemical calculations. The spectroscopic studies project the levels of the S1 state onto those of either the S0 state, in DF and 2D-LIF spectroscopy, or the ground state cation (D0 +) state, in ZEKE spectroscopy. The assignments of the spectra provide information on the vibrational, vibration-torsion (vibtor), and torsional levels in those states and those of the S1 levels. The spectra are indicative of vibronic (including torsional) interactions between the S1 state and other excited electronic states, deduced both in terms of the vibrational activity observed and shifts from expected vibrational wavenumbers in the S1 state, attributed to the resulting altered shape of the S1 surface. Many of the ZEKE spectra are consistent with the largely Rydberg nature of the S1 state near the Franck-Condon region; however, there is also some activity that is less straightforward to explain. Comments are made regarding the photodynamics of the S1 state.

Variations in Duschinsky rotations in m-fluorotoluene and m-chlorotoluene during excitation and ionization.

We investigate Duschinsky rotation/mixing between three vibrations for both m-fluorotoluene (mFT) and m-chlorotoluene (mClT), during electronic excitation and ionization. In the case of mFT, we investigate both the S1 → S0 electronic transition and the D0 + ← S1 ionization, by two-dimensional laser-induced fluorescence (2D-LIF) and zero-electron-kinetic energy (ZEKE) spectroscopy, respectively; for mClT, only the D0 + ← S1 ionization was investigated, by ZEKE spectroscopy. The Duschinsky mixings are different in the two molecules, owing to shifts in vibrational wavenumber and variations in the form of the fundamental vibrations between the different electronic states. There is a very unusual behavior for two of the mFT vibrations, where apparently different conclusions for the identity of two S1 vibrations arise from the 2D-LIF and ZEKE spectra. We compare the experimental observations to the calculated Duschinsky matrices, finding that these successfully pick up the key geometric changes associated with each electronic transition and so are successful in qualitatively explaining the vibrational activity in the spectra. Experimental values for a number of vibrations across the S0, S1, and D0 + states are reported and found to compare well to those calculated. Assignments are made for the observed vibration-torsion ("vibtor") bands, and the effect of vibrational motion on the torsional potential is briefly discussed.

Torsions, low-frequency vibrations, and vibration-torsion ("vibtor") levels in the m-chlorotoluene cation.

Zero-electron-kinetic-energy (ZEKE) spectra are presented for m-chlorotoluene (mClT), employing different low-lying torsional and vibration-torsional ("vibtor") levels of the S1 state as intermediates. The adiabatic ionization energy is determined to be 71 319 cm-1 ± 5 cm-1 (8.8424 ± 0.0006 eV). It is found that the activity in the ZEKE spectra varies greatly for different levels and is consistent with the assignments of the S1 levels of m-fluorotoluene (mFT) deduced in the recent fluorescence study of Stewart et al. [J. Chem. Phys. 150, 174303 (2019)] and the ZEKE study from Kemp et al. [J. Chem. Phys. 151, 084311 (2019)]. As with mFT, the intensities in the ZEKE spectra of mClT are consistent with a phase change in the torsional potential upon ionization, allowing a large number of torsions and vibtor levels to be observed for the cation. Vibration-induced modifications of the torsional potential are discussed. Calculated vibrational wavenumbers for the S0, S1, and D0 + states are also presented.

Identification of separate isoenergetic routes for vibrational energy flow in p-fluorotoluene.

A deceptively simple feature in the S1 ← S0 spectrum of p-fluorotoluene (pFT), 1013 cm-1 above the origin, is studied using both zero-electron-kinetic-energy (ZEKE) and two-dimensional laser-induced fluorescence (2D-LIF) spectroscopy. It is found to consist of a cornucopia of overlapped transitions to eigenstates that arise from numerous interacting levels. A significant variation in the activity is seen employing both the ZEKE and 2D-LIF techniques. Detailed insight into the complicated spectra can be achieved, owing to the large number of vibrational wavenumbers that have been previously determined for the S0, S1, and D0 + states, summarized herein. It is found that the activity is dominated by two overtones, which are individually interacting with other levels, so providing largely independent routes for vibrational energy flow at the same internal energy. Additionally, other weak features located 900-1050 cm-1 above the origin are examined.

Vibration-modified torsional potentials and vibration-torsion ("vibtor") levels in the m-fluorotoluene cation.

Zero-kinetic-energy (ZEKE) spectra are presented for m-fluorotoluene, employing different low-lying (<350 cm-1) intermediate torsional and vibration-torsional ("vibtor") levels of the S1 state. The adiabatic ionization energy (AIE) is found to be 71 997 ± 5 cm-1 (8.9265 ± 0.0006 eV). It is found that the activity in the ZEKE spectra varies greatly for different levels and is consistent with the assignments of the S1 levels deduced in the recent fluorescence study of Stewart et al. [J. Chem. Phys. 150, 174303 (2019)]. For cation torsional levels, the most intense band corresponds to changes in the torsional quantum number, in line with the known change in the phase of the torsional potential upon ionization. This leads to the observation of an unprecedented number of torsions and vibtor levels, with the pronounced vibtor activity involving out-of-plane vibrations. Interactions between levels involving torsions are discussed, with evidence presented, for the first time it is believed, for modification of a torsional potential induced by a vibration. Also, we discuss the possibility of distortion of the methyl group leading to a change from G6 molecular symmetry to Cs point group symmetry.

Complexity surrounding an apparently simple Fermi resonance in p-fluorotoluene revealed using two-dimensional laser-induced fluorescence (2D-LIF) spectroscopy.

Two-dimensional laser-induced fluorescence (2D-LIF) spectroscopy is a powerful tool allowing overlapped features in an electronic spectrum to be separated, and interactions between vibrations and torsions to be identified. Here the technique is employed to assign the 790-825 cm-1 region above the origin of the S1 ← S0 transition in para-fluorotoluene, which provides insight into the unusual time-resolved results of Davies and Reid [Phys. Rev. Lett. 109, 193004 (2012)]. The region is dominated by a pair of bands that arise from a Fermi resonance; however, the assignment is complicated by contributions from a number of overtones and combinations, including vibration-torsion ("vibtor") levels. The activity in the 2D-LIF spectra is compared to the recently reported zero-electron-kinetic-energy spectra [Tuttle et al., J. Chem. Phys. 146, 244310 (2017)] to arrive at a consistent picture of the energy levels in this region of the spectrum.

Effects of symmetry, methyl groups and serendipity on intramolecular vibrational energy dispersal.

We consider two key parameters that have been proposed to be important for vibrational energy delocalization, closely related to intramolecular vibrational redistribution (IVR), in molecules. These parameters are the symmetry of the molecule, and the presence of torsional (internal rotor) modes of a methyl group. We consider four para-disubstituted benzene molecules and examine their vibrational character. The molecules selected are para-difluorobenzene, para-chlorofluorobenzene, para-fluorotoluene, and para-xylene. This set of molecules allows the above parameters to be assessed in a systematic way. The probe we use is zero-electron-kinetic-energy (ZEKE) spectroscopy, which is employed in a resonant scheme, where the intermediate levels are selected vibrational levels of the S1 excited electronic state, with wavenumbers up to 1300 cm-1. We conclude that symmetry, and the presence of a methyl groups, do indeed have a profound effect on "restricted" IVR at low energies. This is underpinned by serendipitous coincidences in the energies of the levels, owing to small shifts in vibrational wavenumbers between molecules, so bringing levels into resonance. Additionally, methyl groups play an important role in opening up new routes for coupling between vibrations of different symmetry, and this is critical in the transition to "statistical" IVR at lower energies for molecules that contain them. Further, the presence of two methyl groups in the symmetrically-substituted p-xylene causes more widespread IVR than does the single methyl group in the asymmetrically-substituted p-fluorotoluene.

Identifying complex Fermi resonances in p-difluorobenzene using zero-electron-kinetic-energy (ZEKE) spectroscopy.

The vibrations of the ground state cation ( X̃2B2g) of para-difluorobenzene (pDFB) have been investigated using zero-electron-kinetic-energy (ZEKE) spectroscopy. A comprehensive set of ZEKE spectra were recorded via different vibrational levels of the S1 state (<00 + 1300 cm-1). The adiabatic ionization energy for pDFB was measured as 73 869 ± 5 cm-1. Use of different intermediate levels allows different cationic vibrational activity to be obtained via the modification of the Franck-Condon factors for the ionization step, allowing the wavenumbers of different vibrational levels in the cation to be established. In addition, assignment of the vibrational structure in the ZEKE spectra allowed interrogation of the assignments of the S1 ← S0 transition put forward by Knight and Kable [J. Chem. Phys. 89, 7139 (1988)]. Assignment of the vibrational structure has been aided by quantum chemical calculations. In this way, it was possible to assign seventeen of the thirty vibrational modes of the ground state pDFB+ cation. Evidence for complex Fermi resonances in the S1 state, i.e., those that involve more than two vibrations, was established. One of these was investigated using picosecond time-resolved photoelectron spectroscopy. In addition, we discuss the appearance of several symmetry-forbidden bands in the ZEKE spectra, attributing their appearance to a Rydberg state variation of an intrachannel vibronic coupling mechanism.

Vibrations of the p-chlorofluorobenzene cation.

The vibrations of the ground state cation (X[combining tilde]2B1) of para-chlorofluorobenzene (pClFB) have been investigated using zero-electron-kinetic-energy (ZEKE) spectroscopy. ZEKE spectra were recorded using different vibrational levels of the S1 state as intermediate levels, for which assignments were put forward in an earlier paper [W. D. Tuttle, A. M. Gardner, and T. G. Wright, Chem. Phys. Lett., 2017, 684, 339]. These different intermediate levels dramatically modify the Franck-Condon factors for the ionization step. The adiabatic ionization energy (AIE) for pClFB was measured as 72 919 ± 5 cm-1, and analysis of the vibrational structure in the ZEKE spectra allowed further interrogation of the assignments of the REMPI spectrum. Assignment of the vibrational structure has been achieved by comparison with corresponding spectra of related molecules, via quantum chemical calculations, and via shifts in bands between the spectra of the 35Cl and 37Cl isotopologues. In this way it was possible to assign twenty out of the thirty vibrational modes of the ground state pClFB+ cation. Additionally, evidence for Fermi resonances between some vibrational levels was found in the S1 state, but no large-scale intramolecular vibrational redistribution (IVR) was seen in the spectra here. Finally, we discuss trends in AIE shifts for benzenes with one or two halogen atoms or methyl substituents.

Complement inhibitors from scabies mites promote streptococcal growth--a novel mechanism in infected epidermis?

BACKGROUND: Scabies is highly prevalent in socially disadvantaged communities such as indigenous populations and in developing countries. Generalized itching causes discomfort to the patient; however, serious complications can occur as a result of secondary bacterial pyoderma, commonly caused by Streptococcus pyogenes (GAS) or Staphylococcus aureus. In the tropics, skin damage due to scabies mite infestations has been postulated to be an important link in the pathogenesis of disease associated with acute rheumatic fever and heart disease, poststreptococcal glomerulonephritis and systemic sepsis. Treatment of scabies decreases the prevalence of infections by bacteria. This study aims to identify the molecular mechanisms underlying the link between scabies and GAS infections. METHODOLOGY/PRINCIPAL FINDINGS: GAS bacteria were pre-incubated with blood containing active complement, phagocytes and antibodies against the bacteria, and subsequently tested for viability by plate counts. Initial experiments were done with serum from an individual previously exposed to GAS with naturally acquired anti-GAS antibodies. The protocol was optimized for large-scale testing of low-opsonic whole blood from non-exposed human donors by supplementing with a standard dose of heat inactivated human sera previously exposed to GAS. This allowed an extension of the dataset to two additional donors and four proteins tested at a range of concentrations. Shown first is the effect of scabies mite complement inhibitors on human complement using ELISA-based complement activation assays. Six purified recombinant mite proteins tested at a concentration of 50 µg/ml blocked all three complement activation pathways. Further we demonstrate in human whole blood assays that each of four scabies mite complement inhibitors tested increased GAS survival rates by 2-15 fold. CONCLUSIONS/SIGNIFICANCE: We propose that local complement inhibition plays an important role in the development of pyoderma in scabies infested skin. This molecular link between scabies and bacterial infections may provide new avenues to develop alternative treatment options against this neglected disease.

Novel scabies mite serpins inhibit the three pathways of the human complement system.

Scabies is a parasitic infestation of the skin by the mite Sarcoptes scabiei that causes significant morbidity worldwide, in particular within socially disadvantaged populations. In order to identify mechanisms that enable the scabies mite to evade human immune defenses, we have studied molecules associated with proteolytic systems in the mite, including two novel scabies mite serine protease inhibitors (SMSs) of the serpin superfamily. Immunohistochemical studies revealed that within mite-infected human skin SMSB4 (54 kDa) and SMSB3 (47 kDa) were both localized in the mite gut and feces. Recombinant purified SMSB3 and SMSB4 did not inhibit mite serine and cysteine proteases, but did inhibit mammalian serine proteases, such as chymotrypsin, albeit inefficiently. Detailed functional analysis revealed that both serpins interfered with all three pathways of the human complement system at different stages of their activation. SMSB4 inhibited mostly the initial and progressing steps of the cascades, while SMSB3 showed the strongest effects at the C9 level in the terminal pathway. Additive effects of both serpins were shown at the C9 level in the lectin pathway. Both SMSs were able to interfere with complement factors without protease function. A range of binding assays showed direct binding between SMSB4 and seven complement proteins (C1, properdin, MBL, C4, C3, C6 and C8), while significant binding of SMSB3 occurred exclusively to complement factors without protease function (C4, C3, C8). Direct binding was observed between SMSB4 and the complement proteases C1s and C1r. However no complex formation was observed between either mite serpin and the complement serine proteases C1r, C1s, MASP-1, MASP-2 and MASP-3. No catalytic inhibition by either serpin was observed for any of these enzymes. In summary, the SMSs were acting at several levels mediating overall inhibition of the complement system and thus we propose that they may protect scabies mites from complement-mediated gut damage.

Increased allergic immune response to Sarcoptes scabiei antigens in crusted versus ordinary scabies.

Scabies, a parasitic skin infestation by the burrowing "itch" mite Sarcoptes scabiei, causes significant health problems for children and adults worldwide. Crusted scabies is a particularly severe form of scabies in which mites multiply into the millions, causing extensive skin crusting. The symptoms and signs of scabies suggest host immunity to the scabies mite, but the specific resistant response in humans remains largely uncharacterized. We used 4 scabies mite recombinant proteins with sequence homology to extensively studied house dust mite allergens to investigate a differential immune response between ordinary scabies and the debilitating crusted form of the disease. Subjects with either disease form showed serum IgE against recombinant S. scabiei cysteine and serine proteases and apolipoprotein, whereas naive subjects showed minimal IgE reactivity. Significantly (P < 0.05) greater serum IgE and IgG4 binding to mite apolipoprotein occurred in subjects with crusted scabies than in those with ordinary scabies. Both subject groups showed strong proliferative responses (peripheral blood mononuclear cells) to the scabies antigens, but the crusted scabies group showed increased secretion of the Th2 cytokines interleukin 5 (IL-5) and IL-13 and decreased Th1 cytokine gamma interferon (IFN-gamma) in response to the active cysteine protease. These data confirm that a nonprotective allergic response occurs in the crusted disease form and demonstrate that clinical severity is associated with differences in the type and magnitude of the antibody and cellular responses to scabies proteins. A quantitative IgE inhibition assay identified IgE immunoreactivity of scabies mite antigens distinct from that of house dust mite antigens, which is potentially important for specific scabies diagnosis and therapy.

A tractable experimental model for study of human and animal scabies.

BACKGROUND: Scabies is a parasitic skin infestation caused by the burrowing mite Sarcoptes scabiei. It is common worldwide and spreads rapidly under crowded conditions, such as those found in socially disadvantaged communities of Indigenous populations and in developing countries. Pruritic scabies lesions facilitate opportunistic bacterial infections, particularly Group A streptococci. Streptococcal infections cause significant sequelae and the increased community streptococcal burden has led to extreme levels of acute rheumatic fever and rheumatic heart disease in Australia's Indigenous communities. In addition, emerging resistance to currently available therapeutics emphasizes the need to identify potential targets for novel chemotherapeutic and/or immunological intervention. Scabies research has been severely limited by the availability of parasites, and scabies remains a truly neglected infectious disease. We report development of a tractable model for scabies in the pig, Sus domestica. METHODOLOGY/PRINCIPAL FINDINGS: Over five years and involving ten independent cohorts, we have developed a protocol for continuous passage of Sarcoptes scabiei var. suis. To increase intensity and duration of infestation without generating animal welfare issues we have optimised an immunosuppression regimen utilising daily oral treatment with 0.2 mg/kg dexamethasone. Only mild, controlled side effects are observed, and mange infection can be maintained indefinitely providing large mite numbers (> 6000 mites/g skin) for molecular-based research on scabies. In pilot experiments we explore whether any adaptation of the mite population is reflected in genetic changes. Phylogenetic analysis was performed comparing sets of genetic data obtained from pig mites collected from naturally infected pigs with data from pig mites collected from the most recent cohort. CONCLUSIONS/SIGNIFICANCE: A reliable pig/scabies animal model will facilitate in vivo studies on host immune responses to scabies including the relations to the associated bacterial pathogenesis and more detailed studies of molecular evolution and host adaptation. It is a most needed tool for the further investigation of this important and widespread parasitic disease.

Characterization of a serine protease homologous to house dust mite group 3 allergens from the scabies mite Sarcoptes scabiei.

The scabies mite, Sarcoptes scabiei var. hominis, infests human skin, causing allergic reactions and facilitating bacterial infection by Streptococcus sp., with serious consequences such as rheumatic fever and rheumatic heart disease. To identify a possible drug target or vaccine candidate protein, we searched for homologues of the group 3 allergen of house dust mites, which we subsequently identified in a cDNA library. The native protein, designated Sar s 3, was shown to be present in the mite gut and excreted in fecal pellets into mite burrows within the upper epidermis. The substrate specificity of proteolytically active recombinant rSar s 3 was elucidated by screening a bacteriophage library. A preference for substrates containing a RS(G/A) sequence at the P1-P2' positions was revealed. A series of peptides synthesized as internally quenched fluorescent substrates validated the phage display data and high performance liquid chromatography/mass spectrometry analysis of the preferred cleaved substrate and confirmed the predicted cleavage site. Searches of the human proteome using sequence data from the phage display allowed the in silico prediction of putative physiological substrates. Among these were numerous epidermal proteins, with filaggrin being a particularly likely candidate substrate. We showed that recombinant rSar s 3 cleaves human filaggrin in vitro and obtained immunohistological evidence that the filaggrin protein is ingested by the mite. This is the first report elucidating the substrate specificity of Sar s 3 and its potential role in scabies mite biology.

Scabies mite inactivated serine protease paralogs inhibit the human complement system.

Infestation of skin by the parasitic itch mite Sarcoptes scabiei afflicts 300 million people worldwide and there is a need for novel and efficient therapies. We have previously identified a multigene family of serine proteases comprising multiple catalytically inactive members (scabies mite-inactivated protease paralogs (SMIPPs)), which are secreted into the gut of S. scabiei. SMIPPs are located in the mite gut and in feces excreted into the upper epidermis. Scabies mites feed on epidermal protein, including host plasma; consequently, they are exposed to host defense mechanisms both internally and externally. We found that two recombinantly expressed SMIPPs inhibited all three pathways of the human complement system. Both SMIPPs exerted their inhibitory action due to binding of three molecules involved in the three different mechanisms which initiate complement: C1q, mannose-binding lectin, and properdin. Both SMIPPs bound to the stalk domains of C1q, possibly displacing or inhibiting C1r/C1s, which are associated with the same domain. Furthermore, we found that binding of both SMIPPs to properdin resulted in prevention of assembly of the alternative pathway convertases. However, the SMIPPs were not able to dissociate already formed convertases. Immunohistochemical staining demonstrated the presence of C1q in the gut of scabies mites in skin burrows. We propose that SMIPPs minimize complement-mediated gut damage and thus create a favorable environment for the scabies mites.

Structural mechanisms of inactivation in scabies mite serine protease paralogues.

The scabies mite (Sarcoptes scabiei) is a parasite responsible for major morbidity in disadvantaged communities and immuno-compromised patients worldwide. In addition to the physical discomfort caused by the disease, scabies infestations facilitate infection by Streptococcal species via skin lesions, resulting in a high prevalence of rheumatic fever/heart disease in affected communities. The scabies mite produces 33 proteins that are closely related to those in the dust mite group 3 allergen and belong to the S1-like protease family (chymotrypsin-like). However, all but one of these molecules contain mutations in the conserved active-site catalytic triad that are predicted to render them catalytically inactive. These molecules are thus termed scabies mite inactivated protease paralogues (SMIPPs). The precise function of SMIPPs is unclear; however, it has been suggested that these proteins might function by binding and protecting target substrates from cleavage by host immune proteases, thus preventing the host from mounting an effective immune challenge. In order to begin to understand the structural basis for SMIPP function, we solved the crystal structures of SMIPP-S-I1 and SMIPP-S-D1 at 1.85 A and 2.0 A resolution, respectively. Both structures adopt the characteristic serine protease fold, albeit with large structural variations over much of the molecule. In both structures, mutations in the catalytic triad together with occlusion of the S1 subsite by a conserved Tyr200 residue is predicted to block substrate ingress. Accordingly, we show that both proteases lack catalytic function. Attempts to restore function (via site-directed mutagenesis of catalytic residues as well as Tyr200) were unsuccessful. Taken together, these data suggest that SMIPPs have lost the ability to bind substrates in a classical "canonical" fashion, and instead have evolved alternative functions in the lifecycle of the scabies mite.

Scabies mite inactivated serine protease paralogues are present both internally in the mite gut and externally in feces.

The scabies mite, Sarcoptes scabiei, is the causative agent of scabies, a disease that is common among disadvantaged populations and facilitates streptococcal infections with serious sequelae. Previously, we encountered large families of genes encoding paralogues of house dust mite protease allergens with their catalytic sites inactivated by mutation (scabies mite inactivated protease paralogues [SMIPPs]). We postulated that SMIPPs have evolved as an adaptation to the parasitic lifestyle of the scabies mite, functioning as competitive inhibitors of proteases involved in the host-parasite interaction. To propose testable hypotheses for their functions, it is essential to know their locations in the mite. Here we show by immunohistochemistry that SMIPPs exist in two compartments: 1) internal to the mite in the gut and 2) external to the mite after excretion from the gut in scybala (fecal pellets). SMIPPs may well function in both of these compartments to evade host proteases.

A cluster of ring stage-specific genes linked to a locus implicated in cytoadherence in Plasmodium falciparum codes for PEXEL-negative and PEXEL-positive proteins exported into the host cell.

Blood stages of Plasmodium falciparum export proteins into their erythrocyte host, thereby inducing extensive host cell modifications that become apparent after the first half of the asexual development cycle (ring stage). This is responsible for a major part of parasite virulence. Export of many parasite proteins depends on a sequence motif termed Plasmodium export element (PEXEL) or vacuolar transport signal (VTS). This motif has allowed the prediction of the Plasmodium exportome. Using published genome sequence, we redetermined the boundaries of a previously studied region linked to P. falciparum virulence, reducing the number of candidate genes in this region to 13. Among these, we identified a cluster of four ring stage-specific genes, one of which is known to encode an exported protein. We demonstrate that all four genes code for proteins exported into the host cell, although only two genes contain an obvious PEXEL/VTS motif. We propose that the systematic analysis of ring stage-specific genes will reveal a cohort of exported proteins not present in the currently predicted exportome. Moreover, this provides further evidence that host cell remodeling is a major task of this developmental stage. Biochemical and photobleaching studies using these proteins reveal new properties of the parasite-induced membrane compartments in the host cell. This has important implications for the biogenesis and connectivity of these structures.