Biology subjects

Bourgeois, J.

Publications and source records attributed to Bourgeois, J..

8 recordsLinked to original sources

bioRxiv2026

The Alternative Polyadenylation Factor CFIm25 Orchestrates Macrophage Antibacterial Immunity by Amplifying TAB2-Mediated MAPK and NF-κB Signaling During Salmonella Infection

Macrophage antimicrobial programs are regulated not only by transcriptional networks but also by RNA processing mechanisms affecting signal transduction and effector responses. One such mechanism, alternative polyadenylation (APA), determines mRNA fate by changing the length of the 3' UTR. However, our understanding of the impact of APA on antibacterial functions and how we can manipulate it to influence infection outcomes remains limited. In this study, we identify the APA regulator CFIm25 (NUDT21) as a promoter of macrophage defense against Salmonella enterica serovar Typhimurium (STM). STM infection is known to drive macrophages toward an M2-like immunosuppressive state conducive to bacterial survival. Concurrent with this transition, the CFIm25 level is reduced, and the 3 UTRs of CFIm25 targets encoding key immune proteins, such as TAB2 and TBL1XR1, are lengthened, suggesting a role for APA changes in the response to STM. Overexpression of CFIm25 in infected macrophages blocks these Salmonella-induced 3 UTR changes, leading to greater mRNA and protein expression. Significantly, the increase in CFIm25 suppresses infection, thereby creating a more antimicrobial intracellular environment, improving macrophage survival, and reducing M2 properties that support bacterial replication. Specifically, CFIm25 enhances production of the antibacterial peptide LL-37, increases reactive oxygen species and nitric oxide levels, suppresses arginase activity and lactate production, and stimulates release of pro-inflammatory cytokines while inhibiting anti-inflammatory cytokines. Depletion studies show TAB2 mediates CFIm25s antibacterial effects by activating both MAPK and NF-kB pathways. Our findings highlight APA regulation as a potential target for boosting immune defenses and developing treatments for chronic bacterial infections.

immunology↗

bioRxiv2025

An Fc-silent OspA monoclonal antibody passively protects mice from tick and intradermal Borrelia burgdorferi challenge

The monoclonal antibody, LA-2, has played a pivotal role in the development of Outer surface protein A (OspA)-based vaccines for Lyme disease, a multisystem illness caused by the tick-borne spirochete, Borrelia burgdorferi sensu lato. Of particular significance was the demonstration more than three decades ago that LA-2 equivalent antibody titers, defined by a competitive-inhibition ELISA, serve as a reliable correlate of vaccine-induced protection across different species, including humans. In vitro characterization of LA-2 has identified both complement-dependent and -independent activities, although which of these attributes contribute to protection against B. burgdorferi remains unresolved. To address this issue, we generated and characterized an "Fc-silent" version of LA-2 IgG1 carrying so-called LALAPG substitutions (L234A, L235A, P329G). We demonstrate that LA-2 LALAPG retained OspA binding activity but was severely attenuated in in vitro complement deposition and complement-dependent borreliacidal assays. Nonetheless, LA-2 LALAPG was as effective as LA-2 at passively protecting C3H mice against nymphal tick-mediated B. burgdorferi challenge. LA-2 LALAPG was also equivalent to LA-2 in passively protecting BALB/c mice against intradermal B. burgdorferi challenge. In the intradermal challenge model, viable spirochetes were not recoverable 24 h after injection from skin biopsies of mice treated with LA-2 or LA-2 LALAPG, and an influx of pro-inflammatory cytokines and chemokines to the injection site was abrogated. Collectively, these results suggest that LA-2s primary mode of action involves direct physical interactions with the spirochete rather than complement-dependent killing. Elucidating these mechanisms may have implications for understanding the mechanistic correlates of OspA-based vaccine-induced immunity in humans.

immunology↗

bioRxiv2025

A Phage Ejectosome Protein Moonlights to Inhibit CBASS Anti-Viral Defense System

A family of prokaryotic antiviral systems collectively known as the cyclic oligonucleotide-based signaling system (CBASS) is found in many bacterial species. Phage defense in CBASS is mediated by the action of a specific effector activated by the cognate cyclic oligonucleotide, often resulting in abortive infection. In the prototypical Type II-A CBASS, phage defense is initiated by the production of cyclic GMP-AMP (cGAMP) by enzymes known as cGAS/DncV-like nucleotidyltransferases (CD-NTases) within the bacterial host, and subsequent activation of the CapV phospholipase by cGAMP leads to cell lysis, thereby limiting phage replication within the host population. Phage proteins that degrade cGAMP, sequester cGAMP, or prevent cGAMP synthesis have been identified in phages to evade CBASS protection. However, some phages are resistant to CBASS protection despite not encoding these proteins, suggesting that additional counter-defense mechanisms exist. To identify new anti-CBASS mechanisms in phages, we exploited bacterial host sensitivity to the antimicrobial compound sulfamethoxazole (SMX) as an indirect read-out for CBASS activity. Bacteria with active CBASS are more sensitive to SMX; therefore, we postulated that any viral proteins produced inside the bacterial host that target CBASS could increase bacterial SMX resistance. Using this screen, we identified that the Gp15 protein from the vibriophage ICP3 increases SMX resistance in Vibrio cholerae El Tor biotype that encodes an active CBASS. Gp15 is an essential virion protein involved in DNA ejection and conserved in many phages. Expression of Gp15 of another coliphage increases SMX resistance in Escherichia coli expressing an active CBASS, and it allows CBASS-sensitive phage to infect the bacterial host with CBASS, demonstrating an important role of Gp15 in phage defense evasion. Unlike other known anti-CBASS proteins, Gp15 does not degrade or sequester cGAMP, and it does not reduce the cellular level of cGAMP. Gp15 also does not interfere with CapV binding to cGAMP. Instead, Gp15 directly inhibits cGAMP-activated CapV serine hydrolase activity in a stoichiometric manner. Together, our study reveals a new mechanism for CBASS antagonism, in which a phage DNA ejection protein moonlights to inhibit a CBASS effector, thereby evading anti-phage defense.

microbiology↗

bioRxiv2024

Identifying essential genes in Schaalia odontolytica using a highly-saturated transposon library

The unique epibiotic-parasitic relationship between Nanosynbacter lyticus type strain TM7x, a member of the newly identified Candidate Phyla Radiation, now referred to as Patescibacteria, and its basibiont, Schaalia odontolytica strain XH001 (formerly Actinomyces odontolyticus), require more powerful genetic tools for deeper understanding of the genetic underpinnings that mediate their obligate relationship. Previous studies have mainly characterized the genomic landscape of XH001 during or post TM7x infection through comparative genomic or transcriptomic analyses followed by phenotypic analysis. Comprehensive genetic dissection of the pair is currently cumbersome due to the lack of robust genetic tools in TM7x. However, basic genetic tools are available for XH001 and this study expands the current genetic toolset by developing high-throughput transposon insertion sequencing (Tn-seq). Tn-seq was employed to screen for essential genes in XH001 under laboratory conditions. A highly saturated Tn-seq library was generated with nearly 660,000 unique insertion mutations, averaging one insertion every 2-3 nucleotides. 203 genes, 10.5% of the XH001 genome, were identified as putatively essential.

microbiology↗

bioRxiv2023

Comparative reservoir competence of Peromyscus leucopus and mouse models for Borrelia burgdorferi B31

Borrelia burgdorferi, a Lyme disease spirochete, causes a range of acute and chronic maladies in humans. However, a primary vertebrate reservoir in the United States, the white-footed deermouse Peromyscus leucopus, is reported not to have reduced fitness following infection. While laboratory strains of Mus musculus mice have successfully been leveraged to model acute human Lyme disease, the ability for these rodents to model B. burgdorferi-P. leucopus interactions remains understudied. Here we compared infection of P. leucopus with B. burgdorferi B31 with infection of the traditional B. burgdorferi murine models--C57BL/6J and C3H/HeN Mus musculus, which develop signs of inflammation akin to human disease. We find that B. burgdorferi were able to reach much higher burdens (10- to 30-times higher) in multiple M. musculus skin sites, and that the overall dynamics of infection differed between the two rodent species. We also found that P. leucopus remained transmissive to larval Ixodes scapularis for a far shorter period than either M. musculus strain. In line with these observations, we found that P. leucopus does launch a modest but sustained inflammatory response against B. burgdorferi in the skin, which we hypothesize leads to reduced bacterial viability and rodent-to-tick transmission in these hosts. Similarly, we also observe evidence of inflammation in infected P. leucopus hearts. These observations provide new insight into reservoir species and the B. burgdorferi enzootic cycle. ImportanceA Lyme disease-causing bacteria, Borrelia burgdorferi, must alternate between infecting a vertebrate host--usually rodents or birds--and ticks. In order to be successful in that endeavor the bacteria must avoid being killed by the vertebrate host before it can infect a new larval tick. In this work we examine how B. burgdorferi and one of its primary vertebrate reservoirs, Peromyscus leucopus, interact during an experimental infection. We find that B. burgdorferi appear to colonize its natural host less successfully than conventional laboratory mouse models which aligns with a sustained seemingly anti-bacterial response by P. leucopus against the microbe. These data enhance our understanding of P. leucopus host-pathogen interactions and could potentially serve as a foundation to uncover ways to disrupt the spread of B. burgdorferi in nature.

microbiology↗

bioRxiv2022

ztf-16 is a novel heterochronic modulator that opposes adult cell fate in dauer and continuous life histories in Caenorhabditis elegans

Animal development is a complex yet robust process that can withstand lengthy and variable interruptions. In Caenorhabditis elegans, adverse conditions can trigger entry into dauer diapause, a stress-resistant, developmentally arrested stage that occurs midway through larval development. Favorable conditions promote recovery from dauer and the completion of development. During larval development, hypodermal seam cells are multipotent and divide at each stage. At adulthood, seam cells differentiate and express the adult-specific COL-19 collagen. The larval-to-adult transition is controlled by a network of genes called the heterochronic pathway, including the LIN-29 transcription factor that directly activates col-19 expression, and the let-7 microRNA that indirectly promotes lin-29 expression. Notably, most heterochronic genes that oppose adult cell fate are dispensable after dauer. We performed a genetic screen for heterochronic genes that act after dauer and identified ztf-16, encoding a zinc finger transcription factor in the hunchback/Ikaros-like family. We found that ztf-16 opposes precocious expression of the adult cell fate marker col-19p::gfp equally during both life histories, making ztf-16(-) the first precocious heterochronic mutant to be unaffected by dauer. Our data indicate that ztf-16 regulates col-19p::gfp via a novel, lin-29-independent mechanism. Endogenous ztf-16b::gfp expression is regulated by let-7 and ztf-16 acts genetically downstream of let-7, but lin-29 is not required for col-19p::gfp expression in ztf-16 mutant larvae or adults. Taken together, this work illuminates a novel aspect of the heterochronic pathway relevant to both dauer and non-dauer development.

genetics↗

bioRxiv2021

Integration of the Salmonella Typhimurium methylome and transcriptome following environmental or metabolic perturbation reveals DNA methylation and transcriptional regulation are largely decoupled

Despite being in a golden age of bacterial epigenomics, little work has systematically examined the plasticity and functional impacts of the bacterial DNA methylome. Here, we leveraged SMRT sequencing to examine the m6A DNA methylome of two Salmonella enterica ser. Typhimurium strains: 14028s and a {Delta}metJ mutant with derepressed methionine metabolism, grown in Luria Broth or a media that simulates the intracellular environment. We find that the methylome is remarkably static--over 95% of adenosine bases retain their methylation status across conditions. Integration of methylation with transcriptomic data revealed limited correlation between changes in methylation and gene expression. Further, examining the transcriptome in {Delta}yhdJ bacteria, lacking the m6A methylase with the most dynamic methylation pattern in our dataset, revealed little evidence of YhdJ-mediated gene regulation. Curiously, despite G(m6A)TC motifs being particularly resistant to change across conditions, incorporating dam mutants into our analyses revealed two examples where changes in methylation and transcription may be linked across conditions. This includes the novel finding that the {Delta}metJ motility defect may be partially driven by hypermethylation of the chemotaxis gene tsr. Together, these data redefine the S. Typhimurium epigenome as a highly stable system that has rare, but important, roles in transcriptional regulation. Incorporating these lessons into future studies will be critical as we progress through the epigenomic era. ImportanceWhile recent breakthroughs have enabled intense study of bacterial DNA modifications, limitations in current work have potentiated a surprisingly untested narrative that DNA methylation is a common mechanism of the bacterial response to environmental conditions. Essentially, whether epigenetic regulation of bacterial transcription is a common, generalizable phenomenon is a critical unanswered question that we address here. We find that most DNA methylation is static in Salmonella enterica serovar Typhimurium, even when the bacteria are grown under dramatically different conditions that cause broad changes in the transcriptome. Further, even when the methylation of individual bases change, these changes generally do not correlate with changes in gene expression. Finally, we demonstrate methods by which data can be stratified in order to identify coupled changes in methylation and gene expression.

microbiology↗

bioRxiv2020

Identification of spacer and protospacer sequence requirements in the Vibrio cholerae Type I-E CRISPR/Cas System

The prokaryotic adaptive immune system CRISPR/Cas serves as defense against bacteriophage and invasive nucleic acid. A Type I-E CRISPR/Cas system has been detected in classical biotype isolates of Vibrio cholerae, the causative agent of the disease cholera. Experimental characterization of this system revealed a functional immune system that operates using a 5-TT-3 protospacer-adjacent motif (PAM) for interference. However, several designed spacers against the 5-TT-3 PAM do not interfere as expected, indicating further investigation of this system is necessary. In this study, we identified additional sequence requirements of a pyrimidine in the 5 position of the spacer and purine in the complementary position of the protospacer using 873 unique spacers and 2267 protospacers mined from CRISPR arrays in deposited sequences of V. cholerae. We present bioinformatic evidence that during acquisition the protospacer purine is captured in the prespacer and that a 5-RTT-3 PAM is necessary for spacer acquisition. Finally, we demonstrate experimentally that a 5-RTT-3 PAM is necessary for CRISPR interference by designing and manipulating spacer and cognate PAMs in a plasmid conjugation assay and discover functional consequences of base pairing with the 5 spacer pyrimidine in spacer efficacy. ImportanceBacterial CRISPR/Cas systems provide immunity by defending against phage and other invading elements. A thorough comprehension of the molecular mechanisms employed by these diverse systems will improve our understanding of bacteriophage-bacterial interactions and bacterial adaptation to foreign DNA. The Vibrio cholerae Type I-E system was previously identified in an extinct classical biotype and was partially characterized for its function. Here, using both bioinformatic and functional assays, we extend that initial study. We have found that the Type I-E system still exists in modern strains of V. cholerae. Furthermore, we defined additional sequence elements in both the CRISPR array and in target DNA that are required for immunity. CRISPR/Cas systems are now commonly used as precise and powerful genetic engineering tools. Knowledge of the sequences required for CRISPR/Cas immunity is a prerequisite for the effective design and experimental use of these systems. Our results greatly facilitate the effective use of one such system. Furthermore, we provide a publicly available script that assists in the detection and validation of CRISPR/Cas immunity requirements when such a system exists in any bacterial species.

microbiology↗