Co-fractionation-mass spectrometry to characterize native mitochondrial protein assemblies in mammalian neurons and brain.

Human mitochondrial (mt) protein assemblies are vital for neuronal and brain function, and their alteration contributes to many human disorders, e.g., neurodegenerative diseases resulting from abnormal protein-protein interactions (PPIs). Knowledge of the composition of mt protein complexes is, however, still limited. Affinity purification mass spectrometry (MS) and proximity-dependent biotinylation MS have defined protein partners of some mt proteins, but are too technically challenging and laborious to be practical for analyzing large numbers of samples at the proteome level, e.g., for the study of neuronal or brain-specific mt assemblies, as well as altered mtPPIs on a proteome-wide scale for a disease of interest in brain regions, disease tissues or neurons derived from patients. To address this challenge, we adapted a co-fractionation-MS platform to survey native mt assemblies in adult mouse brain and in human NTERA-2 embryonal carcinoma stem cells or differentiated neuronal-like cells. The workflow consists of orthogonal separations of mt extracts isolated from chemically cross-linked samples to stabilize PPIs, data-dependent acquisition MS to identify co-eluted mt protein profiles from collected fractions and a computational scoring pipeline to predict mtPPIs, followed by network partitioning to define complexes linked to mt functions as well as those essential for neuronal and brain physiological homeostasis. We developed an R/CRAN software package, Macromolecular Assemblies from Co-elution Profiles for automated scoring of co-fractionation-MS data to define complexes from mtPPI networks. Presently, the co-fractionation-MS procedure takes 1.5-3.5 d of proteomic sample preparation, 31 d of MS data acquisition and 8.5 d of data analyses to produce meaningful biological insights.

Sanitation enzymes: Exquisite surveillance of the noncanonical nucleotide pool to safeguard the genetic blueprint.

Reactive oxygen species (ROS) are common products of normal cellular metabolism, but their elevated levels can result in nucleotide modifications. These modified or noncanonical nucleotides often integrate into nascent DNA during replication, causing lesions that trigger DNA repair mechanisms such as the mismatch repair machinery and base excision repair. Four superfamilies of sanitization enzymes can effectively hydrolyze noncanonical nucleotides from the precursor pool and eliminate their unintended incorporation into DNA. Notably, we focus on the representative MTH1 NUDIX hydrolase, whose enzymatic activity is ostensibly nonessential under normal physiological conditions. Yet, the sanitization attributes of MTH1 are more prevalent when ROS levels are abnormally high in cancer cells, rendering MTH1 an interesting target for developing anticancer treatments. We discuss multiple MTH1 inhibitory strategies that have emerged in recent years, and the potential of NUDIX hydrolases as plausible targets for the development of anticancer therapeutics.

Insights into SACS pathological attributes in autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS)☆.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a rare early-onset neurodegenerative disease caused by mutations in the SACS gene, encoding Sacsin. Initial functional annotation of Sacsin was based on sequence homology, with subsequent experiments revealing the Sacsin requirement for regulating mitochondrial dynamics, along with its domains involved in promoting neurofilament assembly or resolving their bundling accumulations. ARSACS phenotypes associated with SACS loss-of-function are discussed, and how advancements in ARSACS disease models and quantitative omics approaches can improve our understanding of ARSACS pathological attributes. Lastly in the perspectives section, we address gene correction strategies for monogenic disorders such as ARSACS, along with their common delivery methods, representing a hopeful area for ARSACS therapeutics development.

A computational approach to rapidly design peptides that detect SARS-CoV-2 surface protein S.

The coronavirus disease 19 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) prompted the development of diagnostic and therapeutic frameworks for timely containment of this pandemic. Here, we utilized our non-conventional computational algorithm, InSiPS, to rapidly design and experimentally validate peptides that bind to SARS-CoV-2 spike (S) surface protein. We previously showed that this method can be used to develop peptides against yeast proteins, however, the applicability of this method to design peptides against other proteins has not been investigated. In the current study, we demonstrate that two sets of peptides developed using InSiPS method can detect purified SARS-CoV-2 S protein via ELISA and Surface Plasmon Resonance (SPR) approaches, suggesting the utility of our strategy in real time COVID-19 diagnostics. Mass spectrometry-based salivary peptidomics shortlist top SARS-CoV-2 peptides detected in COVID-19 patients' saliva, rendering them attractive SARS-CoV-2 diagnostic targets that, when subjected to our computational platform, can streamline the development of potent peptide diagnostics of SARS-CoV-2 variants of concern. Our approach can be rapidly implicated in diagnosing other communicable diseases of immediate threat.

Auxotrophic and prototrophic conditional genetic networks reveal the rewiring of transcription factors in Escherichia coli.

Bacterial transcription factors (TFs) are widely studied in Escherichia coli. Yet it remains unclear how individual genes in the underlying pathways of TF machinery operate together during environmental challenge. Here, we address this by applying an unbiased, quantitative synthetic genetic interaction (GI) approach to measure pairwise GIs among all TF genes in E. coli under auxotrophic (rich medium) and prototrophic (minimal medium) static growth conditions. The resulting static and differential GI networks reveal condition-dependent GIs, widespread changes among TF genes in metabolism, and new roles for uncharacterized TFs (yjdC, yneJ, ydiP) as regulators of cell division, putrescine utilization pathway, and cold shock adaptation. Pan-bacterial conservation suggests TF genes with GIs are co-conserved in evolution. Together, our results illuminate the global organization of E. coli TFs, and remodeling of genetic backup systems for TFs under environmental change, which is essential for controlling the bacterial transcriptional regulatory circuits.

The conserved Tpk1 regulates non-homologous end joining double-strand break repair by phosphorylation of Nej1, a homolog of the human XLF.

The yeast cyclic AMP-dependent protein kinase A (PKA) is a ubiquitous serine-threonine kinase, encompassing three catalytic (Tpk1-3) and one regulatory (Bcy1) subunits. Evidence suggests PKA involvement in DNA damage checkpoint response, but how DNA repair pathways are regulated by PKA subunits remains inconclusive. Here, we report that deleting the tpk1 catalytic subunit reduces non-homologous end joining (NHEJ) efficiency, whereas tpk2-3 and bcy1 deletion does not. Epistatic analyses revealed that tpk1, as well as the DNA damage checkpoint kinase (dun1) and NHEJ factor (nej1), co-function in the same pathway, and parallel to the NHEJ factor yku80. Chromatin immunoprecipitation and resection data suggest that tpk1 deletion influences repair protein recruitments and DNA resection. Further, we show that Tpk1 phosphorylation of Nej1 at S298 (a Dun1 phosphosite) is indispensable for NHEJ repair and nuclear targeting of Nej1 and its binding partner Lif1. In mammalian cells, loss of PRKACB (human homolog of Tpk1) also reduced NHEJ efficiency, and similarly, PRKACB was found to phosphorylate XLF (a Nej1 human homolog) at S263, a corresponding residue of the yeast Nej1 S298. Together, our results uncover a new and conserved mechanism for Tpk1 and PRKACB in phosphorylating Nej1 (or XLF), which is critically required for NHEJ repair.

From fuzziness to precision medicine: on the rapidly evolving proteomics with implications in mitochondrial connectivity to rare human disease.

Mitochondrial (mt) dysfunction is linked to rare diseases (RDs) such as respiratory chain complex (RCC) deficiency, MELAS, and ARSACS. Yet, how altered mt protein networks contribute to these ailments remains understudied. In this perspective article, we identified 21 mt proteins from public repositories that associate with RCC deficiency, MELAS, or ARSACS, engaging in a relatively small number of protein-protein interactions (PPIs), underscoring the need for advanced proteomic and interactomic platforms to uncover the complete scope of mt connectivity to RDs. Accordingly, we discuss innovative untargeted label-free proteomics in identifying RD-specific mt or other macromolecular assemblies and mapping of protein networks in complex tissue, organoid, and stem cell-differentiated neurons. Furthermore, tag- and label-based proteomics, genealogical proteomics, and combinatorial affinity purification-mass spectrometry, along with advancements in detecting and integrating transient PPIs with single-cell proteomics and transcriptomics, collectively offer seminal follow-ups to enrich for RD-relevant networks, with implications in RD precision medicine.

Mitochondria under the spotlight: On the implications of mitochondrial dysfunction and its connectivity to neuropsychiatric disorders.

Neuropsychiatric disorders (NPDs) such as bipolar disorder (BD), schizophrenia (SZ) and mood disorder (MD) are hard to manage due to overlapping symptoms and lack of biomarkers. Risk alleles of BD/SZ/MD are emerging, with evidence suggesting mitochondrial (mt) dysfunction as a critical factor for disease onset and progression. Mood stabilizing treatments for these disorders are scarce, revealing the need for biomarker discovery and artificial intelligence approaches to design synthetically accessible novel therapeutics. Here, we show mt involvement in NPDs by associating 245 mt proteins to BD/SZ/MD, with 7 common players in these disease categories. Analysis of over 650 publications suggests that 245 NPD-linked mt proteins are associated with 800 other mt proteins, with mt impairment likely to rewire these interactions. High dosage of mood stabilizers is known to alleviate manic episodes, but which compounds target mt pathways is another gap in the field that we address through mood stabilizer-gene interaction analysis of 37 prescriptions and over-the-counter psychotropic treatments, which we have refined to 15 mood-stabilizing agents. We show 26 of the 245 NPD-linked mt proteins are uniquely or commonly targeted by one or more of these mood stabilizers. Further, induced pluripotent stem cell-derived patient neurons and three-dimensional human brain organoids as reliable BD/SZ/MD models are outlined, along with multiomics methods and machine learning-based decision making tools for biomarker discovery, which remains a bottleneck for precision psychiatry medicine.

HERG channel and cancer: A mechanistic review of carcinogenic processes and therapeutic potential.

The human ether-à-go-go related gene (HERG) encodes the alpha subunit of Kv11.1, which is a voltage-gated K+ channel protein mainly expressed in heart and brain tissue. HERG plays critical role in cardiac repolarization, and mutations in HERG can cause long QT syndrome. More recently, evidence has emerged that HERG channels are aberrantly expressed in many kinds of cancer cells and play important roles in cancer progression. HERG could therefore be a potential biomarker for cancer and a possible molecular target for anticancer drug design. HERG affects a number of cellular processes, including cell proliferation, apoptosis, angiogenesis and migration, any of which could be affected by dysregulation of HERG. This review provides an overview of available information on HERG channel as it relates to cancer, with focus on the mechanism by which HERG influences cancer progression. Molecular docking attempts suggest two possible protein-protein interactions of HERG with the ß1-integrin receptor and the transcription factor STAT-1 as novel HERG-directed therapeutic targeting which avoids possible cardiotoxicity. The role of epigenetics in regulating HERG channel expression and activity in cancer will also be discussed. Finally, given its inherent extracellular accessibility as an ion channel, we discuss regulatory roles of this molecule in cancer physiology and therapeutic potential. Future research should be directed to explore the possibilities of therapeutic interventions targeting HERG channels while minding possible complications.

Misconnecting the dots: altered mitochondrial protein-protein interactions and their role in neurodegenerative disorders.

Introduction: Mitochondria (mt) are protein-protein interaction (PPI) hubs in the cell where mt-localized and associated proteins interact in a fashion critical for cell fitness. Altered mtPPIs are linked to neurodegenerative disorders (NDs) and drivers of pathological associations to mediate ND progression. Mapping altered mtPPIs will reveal how mt dysfunction is linked to NDs.Areas covered: This review discusses how database sources reflect on the number of mt protein or interaction predictions, and serves as an update on mtPPIs in mt dynamics and homeostasis. Emphasis is given to mRNA expression profiles for mt proteins in human tissues, cellular models relevant to NDs, and altered mtPPIs in NDs such as Parkinson's disease (PD), Amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD).Expert opinion: We highlight the scarcity of biomarkers to improve diagnostic accuracy and tracking of ND progression, obstacles in recapitulating NDs using human cellular models to underpin the pathophysiological mechanisms of disease, and the shortage of mt protein interactome reference database(s) of neuronal cells. These bottlenecks are addressed by improvements in induced pluripotent stem cell creation and culturing, patient-derived 3D brain organoids to recapitulate structural arrangements of the brain, and cell sorting to elucidate mt proteome disparities between cell types.

A Gaussian process-based definition reveals new and bona fide genetic interactions compared to a multiplicative model in the Gram-negative Escherichia coli.

MOTIVATION: A digenic genetic interaction (GI) is observed when mutations in two genes within the same organism yield a phenotype that is different from the expected, given each mutation's individual effects. While multiplicative scoring is widely applied to define GIs, revealing underlying gene functions, it remains unclear if it is the most suitable choice for scoring GIs in Escherichia coli. Here, we assess many different definitions, including the multiplicative model, for mapping functional links between genes and pathways in E.coli. RESULTS: Using our published E.coli GI datasets, we show computationally that a machine learning Gaussian process (GP)-based definition better identifies functional associations among genes than a multiplicative model, which we have experimentally confirmed on a set of gene pairs. Overall, the GP definition improves the detection of GIs, biological reasoning of epistatic connectivity, as well as the quality of GI maps in E.coli, and, potentially, other microbes. AVAILABILITY AND IMPLEMENTATION: The source code and parameters used to generate the machine learning models in WEKA software were provided in the Supplementary information. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The transmembrane domain of the Staphylococcus aureus ESAT-6 component EssB mediates interaction with the integral membrane protein EsaA, facilitating partially regulated secretion in a heterologous host.

The ESAT-6-like secretion system (ESS) of Staphylococcus aureus plays a significant role in persistent infections. EssB is a highly conserved bitopic ESS protein comprising a cytosolic N-terminus, single transmembrane helix and a C-terminus located on the trans-side of the membrane. Six systematic truncations covering various domains of EssB were constructed, followed by bacterial two-hybrid screening of their interaction with EsaA, another conserved integral membrane component of the ESS pathway. Results show that the transmembrane domain of EssB is critical for heterodimerization with EsaA. In vivo crosslinking followed by Western blot analysis revealed high molecular weight species when wild-type EssB and EsaA were crosslinked, but this band was not detected in the absence of the transmembrane domain of EssB. Heterologous overproduction of EssB, EsaA and five other components of the ESS pathway in Escherichia coli BL21(DE3), followed by fractionation experiments led to a remarkable increase in the periplasmic protein content, suggesting the assembly of partially regulated secretion mechanism. These data identify the transmembrane domain of EssB as indispensable for interaction with EsaA, thereby facilitating protein secretion across bacterial membranes in a fashion that requires other components of the ESS pathway.

The C-terminus of the ESAT6-like secretion system virulence factor EsxC mediates divalent cation-dependent homodimerization.

The human pathogen Staphylococcus aureus encodes the ESAT6-like Secretion System (ESS). The ESS pathway secretes pathogenic substrates such as EsxA, EsxB, EsxC, EsxD and EssD that mediate staphylococcal establishment in persistent abscess lesions. The biochemical behavior of these substrates is not fully understood. EsxC is species-specific lysine-rich homodimer that lacks recognizable topogenic sequence. Studies have shown that EsxC is required for the secretion of other substrates, thereby revealing its biomedical importance. Here, EsxC self-association was investigated in the presence of several metal ion chelators. Results show that EsxC homodimerization is abolished in the presence of EDTA and EGTA, suggesting a role for calcium in mediating EsxC self-association. Complementation experiments confirm that EsxC homodimerization is calcium-dependent. N- and C-terminal truncations of EsxC were constructed, followed by bacterial two-hybrid screening. Results show that EsxC self-association is mediated by its C-terminal domain. Affinity purification of recombinant EsxC to apparent homogeneity, followed by chemical crosslinking and SDS-PAGE led to the detection of the monomeric and dimeric forms of the protein. In contrast and when a purified EsxC variant lacking the C-terminus was subjected to similar conditions, only the monomeric form was observed. These in vivo and in vitro data highlight the contribution of the C-terminus of the virulence factor EsxC to self-association, and document a previously unreported role for calcium in mediating protein-protein interactions in this pathogenic secretion system.

Error-prone PCR mutagenesis and reverse bacterial two-hybrid screening identify a mutation in asparagine 53 of the Staphylococcus aureus ESAT6-like component EsxB that perturbs interaction with EsxD.

The ESAT6-like Secretion System (ESS) of the human pathogen Staphylococcus aureus secretes heterodimeric virulence effectors such as EsxB and EsxD. To gain insights into the nature of EsxB-EsxD interaction, randomly mutated esxB generated by error-prone PCR was co-transformed together with esxD as adenylate cyclase fusion constructs into cyclase-deficient Escherichia coli, followed by reverse bacterial two-hybrid screening. Three color species were observed: dark blue, light blue, and white (no EsxB-EsxD interaction). The esxB from white colonies was subjected to standard PCR to check for gene signal, followed by SDS-PAGE for variant stability assessment. The gene coding for a stable EsxB variant that perturbed interaction with EsxD was further subjected to DNA sequencing. A single point mutation in esxB at position 157 was identified, leading to an amino acid change from asparagine to aspartic acid at position 53 in the resulting protein. Structural modeling of EsxB reveals that N53 is surface exposed. Whereas N53S substitution by site-directed mutagenesis retained heterodimerization with EsxD, N53A substitution abrogated such interaction. In addition, N53D change in EsxB did not alter interaction with EssG, another soluble component of the ESS pathway, suggesting minimal impact of the N53D substitution on EsxB stability and solubility. Taken together, these data provide new insights into the nature of EsxB-EsxD interaction and offer a systematic approach for in vivo analysis of protein-protein interactions of pathogenic bacteria in non-pathogenic hosts.

Isolation of a Membrane Protein Complex for Type VII Secretion in Staphylococcus aureus.

The ESAT6-like secretion system (ESS) of Staphylococcus aureus promotes effector protein transport across the bacterial envelope. Genes in the ESS cluster are required for S. aureus establishment of persistent abscess lesions and the modulation of immune responses during bloodstream infections. However, the biochemical functions of most of the ESS gene products, specifically the identity of secretion machine components, are unknown. Earlier work demonstrated that deletion of essB, which encodes a membrane protein, abolishes S. aureus ESS secretion. Loss-of-function mutations truncating the essB gene product cause dominant-negative phenotypes on ESS secretion, suggesting that EssB is a central component of the secretion machinery. To test this prediction, we purified native and affinity-tagged EssB from staphylococcal membranes via dodecyl-maltoside extraction, identifying a complex assembled from five proteins, EsaA, EssA, EssB, EssD, and EsxA. All five proteins are essential for secretion, as knockout mutations in the corresponding genes abolish ESS transport. Biochemical and bacterial two-hybrid analyses revealed a direct interaction between EssB and EsaA that, by engaging a mobile machine component, EsxA, may also recruit EssA and EssD.IMPORTANCE Type VII secretion systems support the lifestyle of Gram-positive bacteria, including important human pathogens such as Bacillus anthracis, Mycobacterium tuberculosis, and Staphylococcus aureus Genes encoding SpoIIIE-FtsK-like ATPases and WXG100-secreted products are conserved features of type VII secretion pathways; however, most of the genes in T7SS clusters are not conserved between different bacterial species. Here, we isolate a complex of proteins from the membranes of S. aureus that appears to represent the core secretion machinery, designated ESS. These results suggest that three membrane proteins, EsaA, EssB, and EssA, form a secretion complex that associates with EssC, the SpoIIIE-FtsK-like ATPase, and with EsxA, a mobile machine component and member of the WXG100 protein family.

EssE Promotes Staphylococcus aureus ESS-Dependent Protein Secretion To Modify Host Immune Responses during Infection.

Staphylococcus aureus, an invasive pathogen of humans and animals, requires a specialized ESS pathway to secrete proteins (EsxA, EsxB, EsxC, and EsxD) during infection. Expression of ess genes is required for S. aureus establishment of persistent abscess lesions following bloodstream infection; however, the mechanisms whereby effectors of the ESS pathway implement their virulence strategies were heretofore not known. Here, we show that EssE forms a complex with other members of the ESS secretion pathway and its substrates, promoting the secretion of EsxA, EsxB, EsxC, EsxD, and EssD. During bloodstream infection of mice, the S. aureus essE mutant displays defects in host cytokine responses, specifically in the production of interleukin-12 (IL-12) (p40/p70) and the suppression of RANTES (CCL5), activators of TH1 T cell responses and immune cell chemotaxis, respectively. Thus, essE-mediated secretion of protein effectors via the ESS pathway may enable S. aureus to manipulate host immune responses by modifying the production of cytokines. IMPORTANCE: Staphylococcus aureus and other firmicutes evolved a specialized ESS (EsxA/ESAT-6-like secretion system) pathway for the secretion of small subsets of proteins lacking canonical signal peptides. The molecular mechanisms for ESS-dependent secretion and their functional purpose are still unknown. We demonstrate here that S. aureus EssE functions as a membrane assembly platform for elements of the secretion machinery and their substrates. Furthermore, S. aureus EssE-mediated secretion contributes to the production or the suppression of specific cytokines during host infection, thereby modifying immune responses toward this pathogen.

Secretion of atypical protein substrates by the ESAT-6 secretion system of Staphylococcus aureus.

Staphylococcus aureus encodes the specialized ESAT-6 Secretion System (ESS). EsxA and EsxB are secreted by the ESS pathway, and share sequence features of ESAT-6 and CFP-10 of the Type VII Secretion System (T7SS) of Mycobacterium tuberculosis. Unlike ESAT-6 and CFP-10, EsxA and EsxB do not interact. Instead, EsxB associates with a novel substrate, EsxD, and EsxA dimerizes with itself or EsxC (EsaC). Unlike EsxA and EsxB, EsxC and EsxD do not share obvious sequence features of WXG100 proteins nor PE/PPE and Esp families of proteins, all of which belong to the pfam EsxAB clan of mycobacterial T7SS. EsxD carries the C-terminal motif YxxxD/E that has been proposed to target T7 substrates for secretion in mycobacteria. Here, we find that deletion, but not amino acid substitutions, in this motif prevent secretion of EsxA and EsxC but not EsxB or EsxD. This is unlike the genetic inactivation of esxA, esxB, esxC or esxD that leads to loss of secretion of all four substrates. Thus, substrate secretion can be uncoupled by deleting the last six amino acids of EsxD. The physical association of EsxC and EsxD with canonical WXG100 proteins suggests that these proteins belong to the EsxAB clan.

Cell-free production of integral membrane aspartic acid proteases reveals zinc-dependent methyltransferase activity of the Pseudomonas aeruginosa prepilin peptidase PilD.

Integral membrane aspartic acid proteases are receiving growing recognition for their fundamental roles in cellular physiology of eukaryotes and prokaryotes, and may be medically important pharmaceutical targets. The Gram-negative Pseudomonas aeruginosa PilD and the archaeal Methanococcus voltae FlaK were synthesized in the presence of unilamellar liposomes in a cell-free translation system. Cosynthesis of PilD with its full-length substrate, PilA, or of FlaK with its full-length substrate, FlaB2, led to complete cleavage of the substrate signal peptides. Scaled-up synthesis of PilD, followed by solubilization in dodecyl-ß-d-maltoside and chromatography, led to a pure enzyme that retained both of its known biochemical activities: cleavage of the PilA signal peptide and S-adenosyl methionine-dependent methylation of the mature pilin. X-ray fluorescence scans show for the first time that PilD is a zinc-binding protein. Zinc is required for the N-terminal methylation of the mature pilin, but not for signal peptide cleavage. Taken together, our work identifies the P. aeruginosa prepilin peptidase PilD as a zinc-dependent N-methyltransferase and provides a new platform for large-scale synthesis of PilD and other integral membrane proteases important for basic microbial physiology and virulence.

The VirB5 protein localizes to the T-pilus tips in Agrobacterium tumefaciens.

The Agrobacterium tumefaciens VirB/D4 type IV secretion system (T4SS) mediates the transfer of single-stranded DNA and protein virulence factors into plant cells, and also determines the assembly of the T-pilus, which is believed to play a role in host recognition. The T-pilus is composed of the major component VirB2 and the minor component VirB5. Using immuno-electron microscopy we detected the major component VirB2 along the entire length of detached T-pili, but not on cell-bound T-pili or on the cell surface. In contrast, the minor T-pilus component VirB5 was detected on the tips of cell-bound T-pili as well as on the ends of detached T-pili and on the cell surface. To gain further insights into the role of VirB5 we introduced changes at its C terminus. C-terminal deletions of up to four amino acids and alanine replacements did not abolish T-pilus formation and incorporation of the VirB5 variants at the tip, although they did impact the length of T-pili. Also, these changes differentially affected the ability of the T4SS to transfer DNA into plant and bacterial recipients, suggesting differential effects on host-cell specificity. The data presented here suggest that VirB5 localizes at the T-pilus tip, and provide novel insights into its role during the type IV secretion process.

Role of endothelial cell adhesion molecules in the development of allergy in human fascioliasis.

The recent understanding of the key role of adhesion molecules in the pathogenesis of chest allergy in parasitic infections may provide a pharmacological target for the associated asthmatic symptoms. Circulating levels of the endothelial cell adhesion molecules (CAMs): sICAM-1, sVCAM-1 and sE-selectin in sera of 18 allergic asthmatic patients. 16 fascioliasis cases (acute & chronic), 24 fascioliasis cases with allergic chest manifestations and 10 apparently healthy control subjects were estimated by ELISA method. Also, IL-4 serum level was evaluated in all groups. Chest allergy in association with fascioliasis included mainly bronchial asthma, beside eosinophilic bronchitis, persistent wheezing and chronic cough. The study provided evidence that adhesion molecules expression is up regulated in acute and chronic fascioliasis cases with allergic chest manifestations. sVCAM-1 seemed to be an early indicator of asthma development in human fascioliasis. IL-4 cytokine was suggested to be responsible for the increased expression especially in the chronic phase of the disease, yet the role of other cytokines cannot be excluded.