Biology subjects

Abukhalaf, M.

Publications and source records attributed to Abukhalaf, M..

7 recordsLinked to original sources

bioRxiv2024

Combined cellular and proteomics approach suggest differential processing of native and foreign vibrios in the sponge Halichondria panicea

Phagocytosis is a conserved cellular mechanism for food uptake, defense and general animal-microbe interactions in metazoans. How the discrimination and subsequent processing of different microbes in marine invertebrates is facilitated remains largely unknown. Thereto, we combined a recently developed phagocytic assay with proteomics analysis to compare the phagocytic activity of the sponge Halichondria panicea upon encounter with a native (i.e., H. panicea isolate; Hal 281) and a foreign (i.e., Nematostella vectensis isolate; NJ 1) Vibrio. The sponge cell fraction was recovered after Vibrio exposure of 30 and 60 min and used for cellular (fluorescence-activated cell sorting and microscopy) and proteomics analyses. While the number of phagocytic active cells was similar between the isolates (p = 0.19), the distribution of vibrios over cell types differed (p = 0.02) with the tendency for a higher accumulation of foreign vibrios in small phagocytic cells than the native vibrios. Initially, both vibrios elicited a mitochondrial proteomic response (e.g., TBXASI, CASP7), whereas after 60 min differentially abundant proteins were dominated by lysosomal proteins (e.g., AGA, FUCA, SCARB2) in response to the native compared to immune-related proteins (e.g., AQP9, H2A, SAMHDI) in response to the foreign vibrio. H. panicea may discriminate native and foreign vibrios post-cellular internalization into choanocytes, followed by differential cellular and molecular processing: Native vibrios are readily transferred to archeocytes and elicit a digestive response, whereas foreign vibrios further accumulate before being transferred and provoke an immune response. These findings provide a mechanism for immune specificity in sponges. ImportanceMetazoans recognize and discriminate between native (i.e., associated and/or commonly encountered) and foreign microbes. In marine invertebrates, the mechanisms of microbial discrimination and immune specificity are not well understood. Phagocytosis is a conserved cellular process from amoeba to humans that facilitates the ingestion and digestion of microbial cells and likely plays a role in this discrimination. To elucidate the molecular and cellular basis of immune specificity in marine invertebrates, we examined the differential phagocytic processing of native and foreign Vibrio isolates in a marine sponge. Our findings revealed that both vibrios provoke a generalist mitochondrial protein-mediated response, while native vibrios trigger a digestive response, and foreign vibrios elicit an immune response after being internalized into sponge cells. This study uncovers a mechanism for immune specificity in an early-divergent metazoan and provides a valuable model for studying the evolution of immunity and its role in animal-microbe interactions.

cell biology↗

bioRxiv2024

An ancient lysozyme in placozoans

Lysozymes are an essential part of immunity and nutrition in metazoans, degrading bacterial cell walls via the hydrolysis of peptidoglycan. Although various lysozymes have been reported for higher animals, the origin of animal lysozymes remains elusive as they seem to be lacking in all early branching phyla. In this study, we investigated a putative goose-type lysozyme (PLys, glycoside hydrolase family 23, GH23) of the placozoan Trichoplax sp. H2. We show that PLys is highly active and produced in gland cells of the ventral epithelium. PLys contains a protective and non-conserved cysteine-rich domain N-terminal of the conserved GH23 lysozyme domain. A truncation of this N-terminal domain in the maturation process of PLys leads to a drastic increase in enzymatic activity at the cost of stability. As the lysozyme is most active under acidic conditions, we investigated the pH trajectories during extracellular digestion in situ. Using a pH-senstive fluorescence reporter, we show that Trichoplax sp. H2 acidifies its temporary feeding grooves pulsatively during digestive events close to the optimum pH for PLys activity. To elucidate the evolutionary origin of the metazoan GH23 lysozyme family, we applied a structure-based phylogenetics approach to show that the metazoan g-type GH23 lysozymes originated from a horizontal gene transfer event from bacteria to an early pre-bilaterian ancestor. GH23 lysozymes have then been retained and expanded in many phyla, including Porifera, Cnidaria, Placozoa and chordates, acting as first animal lysozyme and a key component in the antibacterial arsenal since early animal evolution.

evolutionary biology↗

bioRxiv2024

An array of Zymoseptoria tritici effectors suppress plant immune responses

Zymoseptoria tritici is the most economically significant fungal pathogen of wheat in Europe. However, despite the importance of this pathogen, the molecular interactions between pathogen and host during infection are not well understood. Herein, we describe the use of two libraries of cloned Z. tritici effectors that were screened to identify effector candidates with putative pathogen associated molecular pattern (PAMP) triggered immunity (PTI)-suppressing activity. The effectors from each library were transiently expressed in Nicotiana benthamiana, and expressing leaves were treated with bacterial or fungal PAMPs to assess the effectors ability to suppress reactive oxygen species (ROS) production. From these screens, numerous effectors were identified with PTI-suppressing activity. In addition, some effectors were able to suppress cell death responses induced by other Z. tritici secreted proteins. We used structural prediction tools to predict the putative structures of all of the Z. tritici effectors, and used these predictions to examine whether there was enrichment of specific structural signatures among the PTI-suppressing effectors. From among the libraries, multiple members of the killer protein-like 4 (KP4) and killer protein-like 6 (KP6) effector families were identified as PTI-suppressors. This observation is intriguing, as these protein families were previously associated with antimicrobial activity rather than virulence or host manipulation. This data provides mechanistic insight into immune suppression by Z. tritici during infection, and suggests that similar to biotrophic pathogens, this fungus relies on a battery of secreted effectors to suppress host immunity during early phases of colonisation.

plant biology↗

bioRxiv2023

Changes in Protein Turn-over Rates Regulate Tryptophan and Glucosinolate Biosynthesis, IAA Transport and Photosynthesis in Arabidopsis Growth and Defense Transitions

An organism continuously experiences shifts in biological states necessitating extensive rearrangement of physiology and molecular order of the cell. Here we model transitions between optimal growth conditions (homeostasis), fully induced pattern triggered immunity (PTI) and back in Arabidopsis thaliana, chronologically measuring changes in protein synthesis and degradation rates, transcript, protein and phytohormone abundance of 99 targets using qPCR and LC-MS parallel reaction monitoring (PRM). Temporally changing synthesis and degradation rates were primary determinants of abundance, next to changes in mRNA levels, of tryptophan, glucosinolate (GS) biosynthesis and photosynthesis associated (PAP) proteins particularly in the earlier establisher phases but also in fully induced PTI. While transcripts returned to growth levels 3 to 16 hours post elicitation, protein levels remained at fully induced PTI levels up to 16 hours into the transitory phase back to optimal growth. A notable exception were polar auxin transporters PIN3 and PIN7 levels which decreased in PTI but quickly returned to initial homeostasis levels after transition, although global auxin levels only decreased by 20%. Tryptophan, GS and JA biosynthesis proteins all increased in abundance in the wild type and the myc234 mutant background linking induction of the tryptophan and GS biosynthesis pathways to flg22 treatment and PTI independent of MYC2 and homologs. PAPs abundance was depleted in fully induced PTI however not in the myc234 mutant linking this active immune response to these bHLH transcription factors. FERREDOXIN-NADP(+)-OXIDOREDUCTASE (FNR1) synthesis rates decreased while its degradation rate increased in the earlier PTI establisher phase. FNR1 is the penultimate protein in the photosynthetic electron transfer chain and imparts electrons onto NADP+ however in its absence electrons are used for oxygen photoreduction and H2O2 production, an active defense compound. Thus FNR1 may be a molecular switch that switches photosystem activity between growth and defense under post-transcriptional control. The myc234 mutation generally led to delayed changes in transcript and protein abundance and also abolished IAA depletion. Protein turn-over rates of a set of PAPs were affected in the mutant suggesting a possible positive role of the transcription factors in controlling post-transcriptional regulatory processes in PTI induction.

plant biology↗

bioRxiv2023

Closely related Bacteroides of the murine intestinal microbiota affect each other's growth positively or negatively

The mammalian intestine is a unique ecosystem for thousands of bacterial species and strains. How naturally coexisting bacteria of the microbiota interact with each other is not yet fully understood. Here, we isolated formerly coexisting, closely related strains of the genus Bacteroides from the intestines of healthy, wild-derived mice. The effect of one strain on another strains growth was tested in 169 pairs in vitro. We find a vast diversity of growth promoting and growth inhibiting activities. A strong positive effect was observed between two strains with differing metabolisms. Growth inhibition among a subset of strains was associated with the known bacterial toxin bacteroidetocin B. Across all strains, we observed growth promotion more often than growth inhibition. The effects were independent of two strains belonging to the same or different species. In some cases, one species differed in its effect on another according to host origin. These findings on obligate host-associated bacteria demonstrate that closely related and naturally coexisting strains have the potential to affect each others growth positively or negatively. These results have implications for our basic understanding of host-associated microbes and the design of synthetic microbial communities.

microbiology↗

bioRxiv2021

LC-MS Based Draft Map of the Arabidopsis thaliana Nuclear Proteome and Protein Import in Pattern Triggered Immunity

Despite its central role as the ark of genetic information and gene expression the plant nucleus is surprisingly understudied. We isolated nuclei from the Arabidopsis thaliana dark grown cell culture left untreated and treated with flg22 and nlp20, two elicitors of pattern triggered immunity (PTI) in plants, respectively. An LC-MS based discovery proteomics approach was used to measure the nuclear proteome fractions. An enrichment score based on the relative abundance of cytoplasmic, mitochondrial and golgi markers in the nuclear protein fraction allowed us to curate the nuclear proteome producing high quality catalogs of around 3,000 nuclear proteins under untreated and both PTI conditions. The measurements also covered low abundant proteins including more than 100 transcription factors and transcriptional co-activators. Protein import into the nucleus in plant immunity is known. Here we sought to gain a broader impression of this phenomenon employing our proteomics data and found 157 and 73 proteins to be putatively imported into the nucleus upon stimulus with flg22 and nlp20, respectively. Furthermore, the abundance of 93 proteins changed significantly in the nucleus following elicitation of immunity. These results suggest promiscuous ribosome assembly and retrograde signaling from the mitochondrion to the nucleus including Prohibitins and Cytochrome C, in the two forms of PTI.

plant biology↗

bioRxiv2020

Reshaping of the Arabidopsis thaliana proteome landscape and co-regulation of proteins in development and immunity

Proteome remodeling is a fundamental adaptive response and proteins in complex and functionally related proteins are often co-expressed. Using a deep sampling strategy we define Arabidopsis thaliana tissue core proteomes at around 10,000 proteins per tissue and absolutely quantify (copy numbers per cell) nearly 16,000 proteins throughout the plant lifecycle. A proteome wide survey of global post translational modification revealed amino acid exchanges pointing to potential conservation of translational infidelity in eukaryotes. Correlation analysis of protein abundance uncovered potentially new tissue and age specific roles of entire signaling modules regulating transcription in photosynthesis, seed development and senescence and abscission. Among others, the data suggest a potential function of RD26 and other NAC transcription factors in seed development related to desiccation tolerance as well as a possible function of Cysteine-rich Receptor-like Kinases (CRKs) as ROS sensors in senescence. All of the components of ribosome biogenesis factor (RBF) complexes were co-expressed tissue and age specifically indicating functional promiscuity in the assembly of these little described protein complexes in Arabidopsis. Treatment of seedlings with flg22 for 16 hours allowed us to characterize proteome architecture in basal immunity in detail. The results were complemented with parallel reaction monitoring (PRM) targeted proteomics, phytohormone, amino acid and transcript measurements. We obtained strong evidence of suppression of jasmonate (JA) and JA-Ile levels by deconjugation and hydroxylation via IAA-ALA RESISTANT3 (IAR3) and JASMONATE-INDUCED OXYGENASE 2 (JOX2) under the control of JASMONATE INSENSITIVE 1 (MYC2). This previously unknown regulatory switch is another part of the puzzle of the as yet understudied role of JA in pattern triggered immunity. The extensive coverage of the Arabidopsis proteome in various biological scenarios presents a rich resource to plant biologists that we make available to the community.

plant biology↗