Biology subjects

Adler, B.

Publications and source records attributed to Adler, B..

6 recordsLinked to original sources

bioRxiv2025

Phage-based microbiome manipulation elucidates interactions within community context

Testing microbial interaction hypotheses remains a major challenge in microbiome research. Bacteriophages have the potential to probe organism-organism interactions within complex microbiomes, yet are rarely used for this purpose. Here, we isolated nine narrow host range phages that replicate in several ecologically important Variovorax species. As Variovorax CL14 degrades the plant root-stunting hormone auxin, we used phages to eliminate V. CL14 from a rhizosphere consortium and reestablished the stunted root phenotype. We used three of the phages to deplete another Variovorax, SCN45, from complex communities to test correlation network-based hypotheses of thiamine interdependencies. Genome-resolved metagenomics revealed that specific taxa decreased in relative abundance following Variovorax depletion and could be rescued by thiamine supplementation. Thus, we confirmed thiamine production as the mechanistic basis for interdependence. These experiments lay the foundation for research that employs wildtype and engineered phages to test interaction hypotheses and for targeted microbiome manipulation.

microbiology↗

bioRxiv2024

A dual role in virion attachment and entry makes the human cytomegalovirus gHgLgO trimer the central player in virion infectivity

Glycoproteins in the viral envelope of human cytomegalovirus (HCMV) orchestrate virion tethering, receptor recognition and fusion with cellular membranes. The glycoprotein gB acts as fusion protein. The gHgL complexes gHgLgO and gHgLpUL(128,130,131A) define the HCMV cell tropism. Studies with HCMV lacking gO had indicated that gO, independently of binding to its cellular receptor PDGFR, plays an important second role in infection. Here, we identified a gO mutation which abolished virus particle infectivity by preventing the interaction of gHgLgO with host cell heparan sulfate proteoglycans (HSPGs). We could not only show that gHgLgO - HSPG interactions are a genuine second role of gHgLgO, but also that gHgLgO is the main player in determining the infectivity of HCMV virus particles. This challenges long-accepted textbook knowledge on the role of gB and gMgN complexes in virion tethering. Additionally, it adds the gHgLgO complex to the antigens of interest for future HCMV vaccines or treatments.

microbiology↗

bioRxiv2023

Sequential membrane- and protein-bound organelles compartmentalize genomes during phage infection

Many eukaryotic viruses require membrane-bound compartments for replication, but no such organelles are known to be formed by prokaryotic viruses1-3. Bacteriophages of the Chimalliviridae family sequester their genomes within a phage-generated organelle, the phage nucleus, which is enclosed by a lattice of the viral protein ChmA4-10. Previously, we observed lipid membrane-bound vesicles in cells infected by Chimalliviridae, but due to the paucity of genetics tools for these viruses it was unknown if these vesicles represented unproductive, abortive infections or a bona fide stage in the phage life cycle. Using the recently-developed dRfxCas13d-based knockdown system CRISPRi-ART11 in combination with fluorescence microscopy and cryo-electron tomography, we show that inhibiting phage nucleus formation arrests infections at an early stage in which the injected phage genome is enclosed within a membrane-bound early phage infection (EPI) vesicle. We demonstrate that early phage genes are transcribed by the virion-associated RNA polymerase from the genome within the compartment, making the EPI vesicle the first known example of a lipid membrane-bound organelle that separates transcription from translation in prokaryotes. Further, we show that the phage nucleus is essential for the phage life cycle, with genome replication only beginning after the injected DNA is transferred from the EPI vesicle to the newly assembled phage nucleus. Our results show that Chimalliviridae require two sophisticated subcellular compartments of distinct compositions and functions that facilitate successive stages of the viral life cycle.

cell biology↗

bioRxiv2020

Anti-Leptospira immunoglobulin profiling in mice reveals strain specific IgG and persistent IgM responses associated with virulence and renal colonization

Leptospira interrogans is a pathogenic spirochete responsible for leptospirosis, a neglected, zoonotic reemerging disease. Humans are sensitive hosts and may develop severe disease. Some animal species, such as rats and mice can become asymptomatic renal carriers. More than 350 leptospiral serovars have been identified, classified on the basis of the antibody response directed against the lipopolysaccharide (LPS). Similarly to whole inactivated bacteria used as human vaccines, this response is believed to confer only short-term, serogroup-specific protection. The immune response of hosts against leptospires has not been thoroughly studied and correlates of protection would be required to test vaccine candidates. In this work, we studied the immunoglobulin (Ig) profiles in mice infected with L. interrogans over time to determine whether this humoral response confers long-term protection after homologous challenge six months post-infection. Groups of mice were injected intraperitoneally with 2x107 leptospires of one of three pathogenic serovars (Manilae, Copenhageni or Icterohaemorrhagiae), attenuated mutants or heat-killed bacteria. Leptospira-specific immunoglobulin (IgA, IgM, IgG and 4 subclasses) produced in the first weeks up to 6 months post-infection were measured by ELISA. Strikingly, we found sustained high levels of IgM in mice infected with the pathogenic Manilae and Copenhageni strains, both colonizing the kidney. In contrast, the Icterohaemorrhagiae strain did not lead to kidney colonization, even at high dose, and triggered a classical IgM response that peaked at day 8 post-infection and disappeared. The virulent Manilae and Copenhageni serovars elicited high levels and similar profiles of IgG subclasses in contrast to Icterohaemorrhagiae strains that stimulated weaker antibody responses. Inactivated heat-killed Manilae strains elicited very low responses. However, all mice pre-injected with leptospires challenged with high doses of homologous bacteria did not develop acute leptospirosis, and all antibody responses were boosted after challenge. Furthermore, we showed that 2 months post challenge, mice pre-infected with the M895 Manilae LPS mutant or heat-killed bacterin were completely protected against renal colonization. In conclusion, we observed a sustained IgM response potentially associated with chronic leptospiral renal infection. We also demonstrated in mice different profiles of protective antibody response after L. interrogans infection, depending on the serovar and virulence of strains. Author summaryLeptospira interrogans is a pathogenic spirochete responsible for leptospirosis, a neglected zoonotic reemerging disease. The immune response of hosts against these bacteria has not been thoroughly studied. Here, we studied over 6 months the immunoglobulin (Ig) profiles in mice infected with L. interrogans and determined whether this humoral response confers long-term protection after homologous challenge six months after primary infection. Groups of mice were infected intraperitoneally with 2x107 bacteria of one of three different pathogenic serovars (Manilae, Copenhageni and Icterohaemorrhagiae) and some corresponding attenuated avirulent mutants. We measured by ELISA each type of Leptospira-specific Ig (IgA, IgM, IgG and 4 subclasses) produced in the first weeks up to 6 months post-infection. We showed different profiles of antibody response after L. interrogans challenge in mice, depending on the serovar and virulence of strains. However, all infected mice, including the ones harboring low antibody levels, like mice vaccinated with an inactivated, heat-killed strain, were protected against leptospirosis after challenge. Notably, we also showed an unusual sustained IgM response associated with chronic leptospiral colonization. Altogether, this long-term immune protection is different from what is known in humans and warrants further investigation.

immunology↗

bioRxiv2020

Leptospiral LPS escapes mouse TLR4 internalization and TRIF?associated antimicrobial responses through O antigen and associated lipoproteins

Leptospirosis is a worldwide re-emerging zoonosis caused by pathogenic Leptospira spp. All vertebrate species can be infected; humans are sensitive hosts whereas other species, such as rodents, may become long-term renal carrier reservoirs. Upon infection, innate immune responses are initiated by recognition of Microbial Associated Molecular Patterns (MAMPs) by Pattern Recognition Receptors (PRRs). Among MAMPs, the lipopolysaccharide (LPS) is recognized by the Toll-Like-Receptor 4 (TLR4) and activates both the MyD88-dependent pathway at the plasma membrane and the TRIF-dependent pathway after TLR4 internalization. We previously showed that leptospiral LPS is not recognized by the human TLR4, whereas it signals through murine TLR4, which mediates mouse resistance to acute leptospirosis. However, leptospiral LPS has low endotoxicity in mouse cells and is an agonist of TLR2, the sensor for bacterial lipoproteins. Here, using confocal microscopy and flow cytometry, we showed that the LPS of L. interrogans did not induce internalization of TLR4 in mouse macrophages, unlike the LPS of Escherichia coli. Consequently, the LPS failed to induce the production of the TRIF-dependent nitric oxide and RANTES, both important antimicrobial responses. Using shorter O antigen LPS and repurified leptospiral LPS with reporter HEK cells, we further found this TLR4-TRIF escape to be dependent on both the co-purifying lipoproteins and the full-length O antigen. Furthermore, our data suggest that the O antigen could alter the binding of the leptospiral LPS to the co-receptor CD14 that is essential for TLR4-TRIF activation. Overall, we describe here a novel immune escape mechanism linked to leptospiral LPS. We hypothesize that the LPS, already known as a virulence factor, plays a major role in the innate immune evasion of the leptospires, thereby contributing to their stealthiness and chronicity in mice. Author summaryLeptospira interrogans is a bacterial pathogen, responsible for leptospirosis, a worldwide neglected reemerging disease. L. interrogans may cause an acute severe disease in humans, whereas rodents and other animals asymptomatically carry the leptospires in their kidneys. They can therefore excrete live bacteria in urine and contaminate the environment. Leptospires are stealth pathogens known to escape the innate immune defenses of their hosts. They are covered in lipopolysaccharide (LPS), a bacterial motif recognized in mammals through the Toll-like receptor 4 (TLR4), which triggers two different signaling pathways. We showed previously that pathogenic leptospires escape TLR4 recognition in humans. Here we show in mice that the leptospiral LPS triggers only one arm of the TLR4 pathway and escapes the other, hence avoiding production of antimicrobial compounds. Removing the lipoproteins that always co-purify with the leptospiral LPS, or using shorter LPS, restores the stimulation of both pathways. This suggests a novel escape mechanism linked to the LPS and involving lipoproteins that could be instrumental for leptospires to escape the mouse defense and allows for their chronic renal colonization.

immunology↗

bioRxiv2019

Influence of human cytomegalovirus glycoprotein O polymorphism on the inhibitory effect of soluble forms of trimer- and pentamer-specific entry receptors

Human cytomegalovirus (HCMV) envelope glycoprotein complexes, gH/gL/gO-trimer and gH/gL/UL128L-pentamer, are important for cell-free HCMV entry. While soluble Nrp2-Fc (sNrp2-Fc) interferes with epithelial/endothelial cell entry through UL128, soluble PDGFR-Fc (sPDGFR-Fc) interacts with gO thereby inhibiting infection of all cell types. Since gO is the most variable subunit we investigated the influence of gO polymorphism on the inhibitory capacities of sPDGFR-Fc and sNRP2-Fc.\n\nAccordingly, gO genotype 1c (GT1c) sequence was fully or partially replaced by gO GT2b, GT3, GT5 sequences in TB40-BAC4-luc background. All mutants were tested for fibroblast and epithelial cell infectivity, for virions gO and gH content, and for infection inhibition by sPDGFR-Fc and sNrp2-Fc.\n\nFull-length and partial gO GT swapping may strongly alter the virions gO and gH levels associated with enhanced epithelial cell infectivity. All gO GT mutants except recombinant gO GT1c/3 displayed a near-complete inhibition at 1.25 g/ml sPDGFR-Fc on epithelial cells (98% versus 91%) and all on fibroblasts ([≥] 99%). While gO GT replacement did not influence sNrp2-Fc inhibition at 1.25 g/ml on epithelial cells (96%-98%), it rendered mutants with low gO levels moderately accessible to fibroblasts inhibition (20%-40%). In contrast to the steep sPDGFR-Fc inhibition curves (slope >1.0), sNrp2-Fc dose-response curves on epithelial cells displayed slopes of ~1.0 suggesting functional differences between these entry inhibitors.\n\nOur findings suggest that targeting of gO-trimer rather than UL128-pentamer might be a promising target to inhibit infectivity independent of the cell type, gO polymorphism, and gO/gH content. However, intragenic gO recombination may lead to moderate resistence to sPDGFR-Fc inhibition.\n\nImportanceHuman cytomegalovirus (HCMV) is known for its broad cell tropism as reflected by the different organs and tissues affected by HCMV infection. Hence, inhibition of HCMV entry into distinct cell types could be considered as a promising therapeutic option to limit cell-free HCMV infection. Soluble forms of cellular entry receptor PDGFR rather than those of entry receptor neuropilin-2 inhibit infection of multiple cell types. sPDGFR specifically interacts with gO of the trimeric gH/gL/gO envelope glycoprotein complex. HCMV strains may differ with respect to the virions amount of trimer and the highly polymorphic gO sequence. In this study, we show that gO polymorphism rather than gO levels may affect the inhibitory capacity of sPDGFR. The finding that gO intragenic recombination may lead to moderate evasion from sPDGFR inhibition is of major value to the development of potential anti-HCMV therapeutic compounds based on sPDGFR.

microbiology↗