Biology subjects

Belew, A. T.

Publications and source records attributed to Belew, A. T..

10 recordsLinked to original sources

bioRxiv2026

Melanopsin regulates axonal translation underlying retinohypothalamic circuit assembly

Intrinsically photosensitive retinal ganglion cells (ipRGCs) influence visual system development via melanopsin before photoreceptor-mediated vision, but how melanopsin signaling contributes to ipRGC circuit assembly remains unknown. Here we show that melanopsin coordinates retinohypothalamic tract development by regulating local translation in developing ipRGC axons. Loss of melanopsin selectively disrupted local translation in axons without affecting somatic translation. The affected transcripts encoded cytoskeletal regulators, adhesion molecules, and trafficking proteins, and activity-dependent changes in translation were restricted to the period before eye-opening. Consistent with impaired axonal growth and synaptogenesis, Opn4 knockout mice showed reduced ipsilateral suprachiasmatic nucleus innervation and fewer retinohypothalamic synapses, while nanoscale synaptic molecular organization and microglial engulfment were unaffected. Reduced visual drive in Opn4 knockouts further altered developmental gene expression programs across the retina, suprachiasmatic nucleus, and lateral geniculate nucleus, with region-specific differences in expression timing. These findings identify melanopsin as a regulator of local axonal translation during early circuit development, linking sensory phototransduction to translational control mechanisms that guide retinohypothalamic tract assembly and postsynaptic target maturation.

neuroscience↗

bioRxiv2026

Identification of Pseudomonas aeruginosa genetic determinants that connect redox metabolism to alginate biosynthesis

Pseudomonas aeruginosa is a well-known human pathogen that contributes significantly to chronic infections, particularly in cystic fibrosis (CF) patients. During this chronic infection, P. aeruginosa undergoes a phenotype change, the inactivation of mucA, which leads to the production of exopolysaccharide alginate, known as mucoid, a key virulence factor associated with biofilm formation. This mucoid phenotype allows the bacterium to persist in the lungs of CF patients for the duration of their lives. Previously, we identified ebselen oxide (EbO) as an inhibitor that suppresses alginate production in P. aeruginosa. In the current study, we synthesized a series of structural analogs based on EbO and ebselen (Eb) and evaluated their ability to inhibit alginate production. These analogs did have similar or lower inhibitory activity than EbO. The mechanism by which EbO inhibits alginate production remains unclear. We employed RNA sequencing analysis of P. aeruginosa treated with inhibitors and identified several candidate genes potentially involved in this inhibitory pathway. Interestingly, we observed that a transposon and in-frame deletion mutants of the candidate genes were defective for alginate production. These findings suggest there are additional requirements for optimal alginate production in conditions that mimic the CF lung beyond the algD-A operon. IMPORTANCEWhen bacteria encounter the correct conditions, they can dedicate their energy toward a specific function to maximize the function. One example is the low calcium response in Yersinia pestis in which the bacteria arrest growth when grown at 37 {degrees}C in the absence of calcium because it uses all its energy for type III secretion. Another example is production of alginate by P. aeruginosa in the lungs of CF patients that can lead to occlusion of the airways. In both cases, the dedicated use of energy toward type III secretion for Y. pestis and alginate biosynthesis for P. aeruginosa reduces the ability of the bacteria to multiply. In the lab, suppressors can be easily identified that restore bacteria growth. The suppressor mutations are often located in the operons that are up-regulated and thereby prevent the execution of the energetically costly process. While these results indicate these processes are energetically costly, we still do not understand how the bacteria dedicate their energy toward these processes over other cellular processes such as growth. Previously, we identified ebselen oxide (EbO) as an inhibitor that suppresses alginate production in P. aeruginosa, but chemical analogues fail to improve the inhibitory activity. We used RNA sequencing analysis of P. aeruginosa treated with inhibitors and identified several candidate genes potentially involved in this inhibitory pathway. Interestingly, we observed that a transposon and in-frame deletion mutants of these genes involved in redox reactions were defective for alginate production. These findings suggest there proteins may shunt energy for optimal alginate production in conditions that mimic the CF lung beyond the algD-A operon. Results from P. aeruginosa alginate production may inform how other bacteria can similarly focus energy toward specific cellular processes.

microbiology↗

bioRxiv2024

Interplay of human macrophage response and natural resistance of L. (V.) panamensis to pentavalent antimony

Macrophages are the principal host cells of Leishmania spp. in human infection and play a critical role in controlling infection and enabling parasite survival and persistence. Nevertheless, understanding of drug resistance in leishmaniasis has primarily focused on the parasite. This investigation provides evidence of the significant differential macrophage response to ex vivo infection with clinical strains of L. (V.) panamensis naturally resistant (zymodeme 2.3/zym 2.3) or sensitive (zymodeme 2.2/zym 2.2) to antimonial drug, and the distinct effect of this drug on the activation of macrophages. Transcriptome analysis of infected monocyte-derived macrophages from healthy donors revealed significant interferon responses and cytokine signaling by zym 2.3 strains compared to zym 2.2 strains. Furthermore, in the presence of antimony, macrophages infected with zym 2.3 strains, but not with zym 2.2 strains, significantly increased the expression of genes associated with M-CSF-generated macrophages (M-MO, anti-inflammatory). Notably, macrophages infected with zym 2.3 strains exhibited elevated expression of genes associated with homeostasis and microbicidal regulation, such as the IDO1/IL4I1-Kyn-AHR pathways and superoxide dismutase, and downregulation of transporters like ABC and AQP, compared to macrophages infected with zym 2.2 strains. Remarkably, the majority of these pathways remained upregulated even in the presence of the strong modulatory effect of antimonial drug. Together, these findings demonstrate that the initial and specific parasite-host interaction influences the ex vivo macrophage response to antimony. Identification of key pathways in macrophage responses associated with natural resistance to this antileishmanial, enhances understanding of host-response mechanisms in the outcome of Leishmania infection and response to treatment. IMPORTANCEDrug resistance and treatment failure are increasingly recognized in human leishmaniasis. Investigation of resistance has predominantly focused on parasite-mediated mechanisms. This study examines the role of host macrophages in natural resistance to antimonial drug. Our findings reveal distinct responses by macrophages infected with Leishmania (Viannia) panamensis strains that are naturally resistant to antimonial drug versus sensitive strains, both in the presence and absence of the drug. Distinctively, resistant parasites induced regulatory pathways that modulate inflammatory responses and alter host cell transporter expression, potentially contributing to parasite survival under antimony exposure. The host cell-parasite interaction in the context of drug resistant intracellular infections presents opportunities for innovative therapeutic strategies targeting host cell responses. Graphical abstractO_LIClinical strains of L. (V.) panamensis naturally resistant or sensitive to antimonial drug (SbV) induce different profiles of human macrophage activation. C_LIO_LIL. (V.) panamensis strains with natural SbV resistance induce a significant interferon response in macrophages, accompanied by overexpression of the IDO1/IL4I1-Kyn-AHR pathways and SOD2, associated with immune homeostasis and regulation of microbicidal activity. C_LIO_LIHomeostatic regulation by the IDO1/IL4I1-Kyn-AHR pathways, induced in macrophages by infection with naturally SbV-resistant strains of L. (V.) panamensis, prevails despite significant modulation of macrophage activation by antimony exposure. C_LIO_LIAntimony treatment promotes a more anti-inflammatory (M-MO) profile in macrophages infected with naturally resistant L. (V.) panamensis strains, while macrophages infected with sensitive strains maintain a more proinflammatory profile (GM-MO). C_LIO_LIInfection with the zym 2.3 strains in the presence of SbV leads to downregulation of specific macrophage transporter genes, supporting the capacity of these naturally SbV-resistant parasites to elicit macrophage responses that enable antimony resistance C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/620724v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@6a7397org.highwire.dtl.DTLVardef@cae4acorg.highwire.dtl.DTLVardef@6ae1d7org.highwire.dtl.DTLVardef@1110e5f_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

da_Tracker: Automated workflow for high throughput single cell and single phagosome tracking in infected cells.

Time-lapse microscopy has emerged as a crucial tool in cell biology, facilitating a deeper understanding of dynamic cellular processes. While existing tracking tools have proven effective in detecting and monitoring objects over time, the quantification of signals within these tracked objects often faces implementation constraints. In the context of infectious diseases, the quantification of signals at localized compartments within the cell and around intracellular pathogens can provide even deeper insight into the interactions between the pathogen and host cell organelles. Existing quantitative analysis at a single-phagosome level remains limited and dependent on manual tracking methods. We developed a near-fully automated workflow that performs with limited bias, high-throughput cell segmentation and quantitative tracking of both single cell and single bacterium/phagosome within multi-channel, z-stack, time-lapse confocal microscopy videos. We took advantage of the PyImageJ library to bring Fiji functionality into a Python environment and combined deep-learning-based segmentation from Cellpose with tracking algorithms from Trackmate. Our workflow provides a versatile toolkit of functions for measuring relevant signal parameters at the single-cell level (such as velocity or bacterial burden) and at the single-phagosome level (i.e. assessment of phagosome maturation over time). Its capabilities in both single-cell and single-phagosome quantification, its flexibility and open-source nature should assist studies that aim to decipher for example the pathogenicity of bacteria and the mechanism of virulence factors that could pave the way for the development of innovative therapeutic approaches.

cell biology↗

bioRxiv2023

Characterization of the Entner-Douderoff Pathway in Pseudomonas aeruginosa Catheter-associated Urinary Tract Infections

Pseudomonas aeruginosa is an opportunistic nosocomial pathogen responsible for catheter-associated urinary tract infections (CAUTI). In a murine model of P. aeruginosa CAUTI, we previously demonstrated that urea within urine suppresses quorum sensing and induces the Entner-Douderoff (E-D) pathway. The E-D pathway consists of the genes zwf, pgl, edd, and eda. Zwf and Pgl convert glucose-6-phosphate into 6-phosphogluconate. Edd hydrolyzes 6-phosphogluconate to 2-keto-3-deoxy-6-phosphogluconate (KDPG). Finally, Eda cleaves KDPG to glyceraldehyde-3-phosphate and pyruvate, which enters the citric acid cycle. Here, we generated in-frame E-D mutants in strain PA14 and assessed their growth phenotypes on chemically defined media. These E-D mutants have a growth defect when grown on glucose or gluconate as sole carbon source which are similar to results previously reported for PAO1 mutants lacking E-D genes. RNA-sequencing following short exposure to urine revealed minimal gene regulation differences compared to the wild type. In a murine CAUTI model, virulence testing of E-D mutants revealed that two mutants lacking zwf and pgl showed minor fitness defects. Infection with the {Delta}pgl strain exhibited a 20% increase in host survival, and the {Delta}zwf strain displayed decreased colonization of the catheter and kidneys. Consequently, our findings suggest that the E-D pathway in P. aeruginosa is dispensable in this model of CAUTI. ImportancePrior studies have shown that the Entner-Douderoff pathway is up-regulated when Pseudomonas aeruginosa is grown in urine. Pseudomonads use the Entner-Douderoff pathway to metabolize glucose instead of glycolysis which led us to ask whether this pathway is required for urinary tract infection. Here, single-deletion mutants of each gene in the pathway were tested for growth on chemically defined media with single-carbon sources as well as complex media. The effect of each mutant on global gene expression in laboratory media and urine was characterized. The virulence of these mutants in a murine model of catheter-associated urinary tract infection revealed that these mutants had similar levels of colonization indicating that glucose is not the primary carbon source utilized in the urinary tract.

microbiology↗

bioRxiv2021

Physiological Magnesium Concentrations Increase Fidelity of Diverse Reverse Transcriptases from HIV-1, HIV-2, and Foamy Virus, but not MuLV or AMV

Reverse transcriptases (RTs) are typically assayed in vitro using optimized Mg2+ concentrations ([~]5-10 mM) several-fold higher than physiological cellular free Mg2+ ([~]0.5 mM). Analysis of fidelity using lacZ-based -complementation assays showed that tested HIV RTs, including HIV-1 from subtype B (HXB2-derived), HIV-2, subtype A/E, and several drug-resistant HXB2 derivatives all showed significantly higher fidelity using physiological Mg2+. This also occurred with prototype foamy virus (PFV) RT. In contrast, Moloney murine leukemia virus (MuLV) and avian myeloblastosis virus (AMV) RTs demonstrated equivalent fidelity in both low and high Mg2+. In 0.5 mM Mg2+, all RTs demonstrated {approx} equal fidelity, except for PFV RT which showed higher fidelity. A Next Generation Sequencing (NGS) approach that used barcoding to accurately determine mutation rates and profiles was used to examine the types of mutations made by HIV-1 (subtype B, wild type) in low (0.5 mM) and high (6 mM) Mg2+ with DNA or RNA that coded for lacZ. Unlike the -complementation assay, which is dependent on LacZ activity, the NGS assay scores mutations at all positions and of every type. Consistent with -complementation assays, a [~]4-fold increase in mutations was observed in high Mg2+. These findings help explain why HIV RT displays lower fidelity in vitro (with high Mg2+ concentrations) than other RTs (e.g., MuLV and AMV), yet cellular fidelity for these viruses is comparable. Establishing in vitro conditions that accurately represent RTs activity in cells is pivotal to determining the contribution of RT and other factors to the mutation profile observed with HIV.

biochemistry↗

bioRxiv2020

Macrophage metallothioneins participate in the antileishmanial activity of antimonials

SYNOPSISO_ST_ABSBackgroundC_ST_ABSHost cell functions that participate in the pharmacokinetics and pharmacodynamics (PK/PD) of pentavalent antimonials for treatment of American cutaneous leishmaniasis (CL) are critical for drug efficacy. ObjectivesIn this study, we investigated whether macrophage mechanisms of xenobiotic detoxification contribute to drug-dependent elimination of intracellular Leishmania. MethodsTranscriptomes of primary macrophages from CL patients (n=6), exposed ex vivo to Leishmania infection and SbV were generated. Candidate genes were selected and validated using short harping RNA interference (shRNA) in THP-1 cells. ResultsStrong induction of metallothionein (MT) genes was observed upon Leishmania infection and exposure to SbV, with 7 MT genes (MT1 and MT2 family members) appearing within the top 20 up-regulated genes. Tandem knockdown (KD) of MT2-A and MT1-E, 1F, and 1X in THP-1 cells was achieved using a pan-MT shRNA., Intracellular parasite survival after SbV exposure was unaffected in tandem-KD cells, and this was a consequence of strong transcriptional upregulation of MTs by infection and SbV, overcoming the KD effect. Gene silencing of the metal transcription factor-1 (MTF-1) abrogated expression of MT1 and MT2-A genes. Upon exposure to SbV, intracellular survival of Leishmania in MTF-1KD cells was significantly enhanced (p [≤] 0.05). ConclusionsMTs are potent scavengers of heavy metals, and central elements of the mammalian cell machinery for xenobiotic detoxification. Results from this study highlight the participation of macrophage MTs in Sb-dependent parasite killing, revealing novel strategies for host-targeted optimization of antileishmanial drugs.

molecular biology↗

bioRxiv2020

Identification of zinc-dependent mechanisms used by Group B Streptococcus to overcome calprotectin-mediated stress

Nutritional immunity is an elegant host mechanism used to starve invading pathogens of necessary nutrient metals. Calprotectin, a metal binding protein, is produced abundantly by neutrophils and is found in high concentrations within inflammatory sites during infection. Group B Streptococcus (GBS) colonizes the gastrointestinal and female reproductive tracts and is commonly associated with severe invasive infections in newborns such as pneumonia, sepsis, and meningitis. Though GBS infections induce robust neutrophil recruitment and inflammation, the dynamics of GBS and calprotectin interactions remain unknown. Here we demonstrate that disease and colonizing isolate strains exhibit susceptibility to metal starvation by calprotectin. We constructed a mariner transposon (Krmit) mutant library in GBS and identified 258 genes that contribute to surviving calprotectin stress. Nearly 20% of all underrepresented mutants following treatment with calprotectin, are predicted metal transporters, including known zinc systems. As calprotectin binds zinc with picomolar affinity, we investigated the contribution of GBS zinc uptake to overcoming calprotectin-imposed starvation. Quantitative RT-PCR revealed a significant upregulation of genes encoding zinc-binding proteins, adcA, adcAII, and lmb, following calprotectin exposure, while growth in calprotectin revealed a significant defect for a global zinc acquisition mutant ({Delta}adcA{Delta}adcAII{Delta}lmb) compared to the GBS WT strain. Further, mice challenged with the {Delta}adcA{Delta}adcAII{Delta}lmb mutant exhibited decreased mortality and significantly reduced bacterial burden in the brain compared to mice infected with WT GBS; this difference was abrogated in calprotectin knockout mice. Collectively, these data suggest that GBS zinc transport machinery are important for combatting zinc-chelation by calprotectin and establishing invasive disease. ImportanceGBS asymptomatically colonizes the female reproductive tract but is a common causative agent of meningitis. GBS meningitis is characterized by extensive infiltration of neutrophils, carrying high concentrations of calprotectin, a metal chelator. To persist within inflammatory sites and cause invasive disease, GBS must circumvent host starvation attempts. Here, we identified global requirements for GBS survival during calprotectin challenge, including known and putative systems involved in metal ion transport. We characterized the role of zinc import in tolerating calprotectin stress in vitro, and in a mouse model of infection. We observed that a global zinc-uptake mutant was less virulent compared to the parental GBS strain and found calprotectin knockout mice to be equally susceptible to infection by WT and mutant strains. These findings suggest that calprotectin production at the site of infection results in a zinc-limited environment and reveals the importance of GBS metal homeostasis to invasive disease.

microbiology↗

bioRxiv2020

A Trypanosoma cruzi Zinc Finger protein that controls expression of epimastigote specific genes and affects metacyclogenesis

SummaryTrypanosoma cruzi has three biochemically and morphologically distinct developmental stages that are programed to rapidly respond to environmental changes the parasite faces during its life cycle. Unlike other eukaryotes, Trypanosomatid genomes contain protein coding genes that are transcribed into polycistronic pre-mRNAs and control of gene expression relies on mechanisms acting at the post-transcriptional level. Transcriptome analyses comparing epimastigote, trypomastigote and intracellular amastigote stages revealed changes in gene expression that reflect the parasite adaptation to distinct environments. Several genes encoding RNA binding proteins (RBP), known to act as key post-transcriptional regulatory factors, were also differentially expressed. We characterized one T. cruzi RBP (TcZH3H12) that contains a zinc finger domain, and whose transcripts are upregulated in epimastigotes compared to trypomastigotes and amastigotes. TcZC3H12 knockout epimastigotes showed decreased growth rates and increased capacity to differentiate into metacyclic trypomastigotes. Comparative transcriptome analysis revealed a TcZC3H12-dependent expression of epimastigote specific genes encoding amino acid transporters and proteins associated with differentiation (PAD), among others. RNA immunoprecipitation assays showed that transcripts from the PAD family interact with TcZC3H12. Taken together, these findings suggest that TcZC3H12 positively regulates the expression of genes involved in epimastigote proliferation and also acts as a negative regulator of metacyclogenesis.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2018

Genetic insight into zinc and antimicrobial toxicity uncovers a glycerol phosphate modification on streptococcal rhamnose polysaccharides

Cell wall glycopolymers on the surface of Gram-positive bacteria are fundamental to bacterial physiology and infection biology. These structures have also gained interest as vaccine antigens, in particular for the human pathogens Group A Streptococcus (GAS) and Streptococcus mutans. Streptococcal cell wall glycopolymers are considered to be functional homologues of wall teichoic acids but surprisingly lack the biologically-relevant and characteristic anionic charge. Here we identify gacH, a gene of unknown function in the GAS Group A Carbohydrate (GAC) biosynthetic cluster, in two independent transposon library screens for its ability to confer resistance to zinc and susceptibility to the bactericidal enzyme human group IIA secreted phospholipase A2. To understand the underlying mechanism of these phenotypes, we determined the structure of the extracellular domain of GacH and discover that it represents a new family of glycerol phosphate (GroP) transferases. Importantly, we demonstrate the presence of GroP in both the GAC and the homologous Serotype c Carbohydrate (SCC) from S. mutans, which is conferred by gacH and sccH products, respectively. NMR analysis of GAC released from cell wall by non-destructive methods reveals that approximately 30% of the GAC GlcNAc side-chains are modified by GroP at the C6 hydroxyl group. This previously unrecognized structural modification impacts host-pathogen interaction and has implications for vaccine design. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/337519v4_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@6d7598org.highwire.dtl.DTLVardef@435695org.highwire.dtl.DTLVardef@e9515corg.highwire.dtl.DTLVardef@1dc78b7_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗