Biology subjects

Proulx, M. K.

Publications and source records attributed to Proulx, M. K..

9 recordsLinked to original sources

bioRxiv2026

CLN6 is a glycerophosphoglycerol epimerase in bis(monoacylglycero)phosphate biosynthesis

Lysosomal catabolism depends on bis(monoacylglycero)phosphate (BMP), a phospholipid with atypical S,S stereochemistry of unknown biosynthetic origin. CLN8 and CLN5 catalyze sequential acylation steps in BMP synthesis, and their loss of function causes Batten disease, a group of inherited neurodegenerative lysosomal storage disorders. Here we identify CLN6, a biochemically uncharacterized endoplasmic reticulum protein whose loss also causes Batten disease, as the calcium-dependent glycerophosphoglycerol epimerase that establishes BMP stereochemistry upstream of CLN8. CLN6 deficiency depletes BMP in cells, mice, and patient lysosomes, and reduces lysosomal hydrolase levels. BMP-precursor supplementation bypasses CLN6, restores BMP and increases hydrolase abundance in CLN6-deficient cells and mice. These findings place three Batten disease proteins in a single biosynthetic pathway and reveal BMP deficiency as a reversible cause of disrupted lysosomal hydrolase homeostasis.

biochemistry↗

bioRxiv2024

Raver1 links Ripk1 RNA splicing to caspase-8-mediated pyroptotic cell death, inflammation, and pathogen resistance

Multiple cell death and inflammatory signaling pathways converge on two critical factors: receptor interacting serine/threonine kinase 1 (RIPK1) and caspase-8. Careful regulation of these molecules is critical to control apoptosis, pyroptosis and inflammation. Here we discovered a pivotal role of Raver1 as an essential regulator of Ripk1 pre-mRNA splicing, expression, and functionality, and the subsequent caspase-8-dependent inflammatory cell death. Macrophages from Raver1-deficient mice exhibit altered splicing of Ripk1, accompanied by diminished cell death and reduced activation of caspase-8, Gasdermin D and E, caspase-1, as well as decreased interleukin-18 (IL-18) and IL-1{beta} production. These effects were triggered by Yersinia bacteria, or by restraining TAK1 or IKK{beta} in the presence of LPS, TNF family members, or IFN{gamma}. Consequently, animals lacking Raver1 showed heightened susceptibility to Yersinia infection. Raver1 and RIPK1 also controlled the expression and function of the C-type lectin receptor Mincle. Our study underscores the critical regulatory role of Raver1 in modulating innate immune responses and highlights its significance in directing in vivo and in vitro inflammatory processes. SignificanceCaspase-8 and the kinase RIPK1 are at focal points of several inflammation and cell death pathways. Thus, a careful regulation of their actions is needed. Our work identifies the RNA splicing factor Raver1 as a critical factor directing the splicing of Ripk1 in order to modulate RIPK1/caspase-8-driven pyroptosis, apoptosis and inflammation. Raver1 is central for macrophage responses to Yersinia bacteria, initiated after blockade of kinases TAK1 and IKK, measured as activation of RIPK1, caspase-8, Gasdermin D, caspase-3, IL-1{beta} and IL-18. Importantly, Raver1 is necessary for host resistance to Yersinia infection in vivo. We propose that Raver1 is key for correct tuning of RIPK1-caspase-8 dependent processes.

immunology↗

bioRxiv2024

Antibody-Fab and -Fc features promote Mycobacterium tuberculosis restriction.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a leading cause of death by an infectious disease globally, with no efficacious vaccine. Antibodies are implicated in Mtb control, but the mechanisms of antibody action remain poorly understood. We assembled a library of TB monoclonal antibodies (mAb) and screened for the ability to restrict Mtb in mice, identifying protective antibodies targeting known and novel antigens. To dissect the mechanism of mAb-mediated Mtb restriction, we optimized a protective lipoarabinomannan-specific mAb through Fc-swapping. In vivo analysis of these Fc-variants revealed a critical role for Fc-effector function in Mtb restriction. Restrictive Fc-variants altered distribution of Mtb across innate immune cells. Single-cell transcriptomics highlighted distinctly activated molecular circuitry within innate immune cell subpopulations, highlighting early activation of neutrophils as a key signature of mAb-mediated Mtb restriction. Therefore, improved antibody-mediated restriction of Mtb is associated with reorganization of the tissue-level immune response to infection and depends on the collaboration of antibody Fab and Fc.

immunology↗

bioRxiv2024

Chemical genetic interactions elucidate pathways controlling tuberculosis antibiotic efficacy during infection

Successful tuberculosis therapy requires treatment with an unwieldy multidrug combination for several months. Thus, there is a growing need to identify novel genetic vulnerabilities that can be leveraged to develop new, more effective antitubercular drugs. Consequently, recent efforts to optimize TB therapy have exploited Mtb chemical genetics to identify pathways influencing antibiotic efficacy, novel mechanisms of antibiotic action, and new targets for TB drug discovery. However, the influence of the complex host environment on these interactions remains largely unknown, leaving the therapeutic potential of the identified targets unclear. In this study, we leveraged a library of conditional mutants targeting 467 essential Mtb genes to characterize the chemical-genetic interactions (CGIs) with TB drugs directly in the mouse infection model. We found that these in vivo CGIs differ significantly from those identified in vitro. Both drug-specific and drug-agnostic effects were identified, and many were preserved during treatment with a multidrug combination, suggesting numerous strategies for enhancing therapy. This work also elucidated the complex effects of pyrazinamide (PZA), a drug that relies on aspects of the infection environment for efficacy. Specifically, our work supports the importance of coenzyme A synthesis inhibition during infection, as well as the antagonistic effect of iron limitation on PZA activity. In addition, we found that inhibition of thiamine and purine synthesis increases PZA efficacy, suggesting novel therapeutically exploitable metabolic dependencies. Our findings present a map of the unique in vivo CGIs, characterizing the mechanism of PZA activity in vivo and identifying novel targets for TB drug development. SignificanceThe inevitable rise of multi-drug-resistant tuberculosis underscores the urgent need for new TB drugs and novel drug targets while prioritizing synergistic drug combinations. Chemical-genetic interaction (CGI) studies have delineated bacterial pathways influencing antibiotic efficacy and uncovered druggable pathways that synergize with TB drugs. However, most studies are conducted in vitro, limiting our understanding of how the host environment influences drug-mutant interactions. Using an inducible mutant library targeting essential Mtb genes to characterize CGIs during infection, this study reveals that CGIs are both drug-specific and drug-agnostic and differ significantly from those observed in vitro. Synergistic CGIs comprised distinct metabolic pathways mediating antibiotic efficacy, revealing novel drug mechanisms of action, and defining potential drug targets that would synergize with frontline antitubercular drugs.

microbiology↗

bioRxiv2021

Chemical-genetic interaction mapping links carbon metabolism and cell wall structure to tuberculosis drug efficacy

Current chemotherapy against Mycobacterium tuberculosis (Mtb), an important human pathogen, requires a multidrug regimen lasting several months. While efforts have been made to optimize therapy by exploiting drug-drug synergies, testing new drug combinations in relevant host environments remains arduous. In particular, host environments profoundly affect the bacterial metabolic state and drug efficacy, limiting the accuracy of predictions based on in vitro assays alone. In this study, we utilize conditional Mtb knockdown mutants of essential genes as an experimentally-tractable surrogate for drug treatment, and probe the relationship between Mtb carbon metabolism and chemical-genetic interactions (CGI). We examined the anti-tubercular drugs isoniazid, rifampicin and moxifloxacin, and found that CGI are differentially responsive to the metabolic state, defining both environment-independent and -dependent interactions. Specifically, growth on the in vivo-relevant carbon source, cholesterol, reduced rifampicin efficacy by altering mycobacterial cell surface lipid composition. We report that a variety of perturbations in cell wall synthesis pathways restore rifampicin efficacy during growth on cholesterol, and that both environment-independent and cholesterol-dependent in vitro CGI could be leveraged to enhance bacterial clearance in the mouse infection model. Our findings present an atlas of novel chemical-genetic-environmental interactions that can be used to optimize drug-drug interactions as well as provide a framework for understanding in vitro correlates of in vivo efficacy. SignificanceEfforts to improve tuberculosis therapy include optimizing multi-drug regimens to take advantage of drug-drug synergies. However, the complex host environment has a profound effect on bacterial metabolic state and drug activity, making predictions of optimal drug combinations difficult. In this study, we leverage a newly developed library of conditional knockdown Mycobacterium tuberculosis mutants in which genetic depletion of essential genes mimics the effect of drug therapy. This tractable system allowed us to assess the effect of growth condition on predicted drug-drug interactions. We found that these interactions can be differentially sensitive to the metabolic state and select in vitro-defined interactions can be leveraged to accelerate bacterial killing during infection. These findings suggest new strategies for optimizing tuberculosis therapy.

microbiology↗

bioRxiv2020

Host-pathogen genetic interactions underlie tuberculosis susceptibility in genetically diverse mice

The outcome of an encounter with Mycobacterium tuberculosis depends on the pathogens ability to adapt to the variable immune pressures exerted by the host. Understanding this interplay has proven difficult, largely because experimentally tractable animal models do not recapitulate the heterogeneity of tuberculosis disease. We leveraged the genetically diverse Collaborative Cross (CC) mouse panel in conjunction with a library of Mtb mutants to associate bacterial genetic requirements with host genetics and immunity. We report that CC strains vary dramatically in their susceptibility to infection and produce qualitatively distinct immune states. Global analysis of Mtb mutant fitness across the CC panel revealed that many virulence pathways are only in specific host microenvironments, identifying the large fraction of the pathogens genome that has been maintained to ensure fitness in a diverse population. Both immunological and bacterial traits were associated with genetic variants distributed across the mouse genome, identifying the specific host-pathogen genetic interactions that influence pathogenesis.

genetics↗

bioRxiv2019

Functionally overlapping variants control TB susceptibility in Collaborative Cross mice

Host genetics plays an important role in determining the outcome of Mycobacterium tuberculosis (Mtb) infection. We previously found that Collaborative Cross mouse strains differ in their susceptibility to Mtb, and that the CC042/GeniUnc (CC042) strain suffered from a rapidly progressive disease and failed to produce the protective cytokine, IFN{gamma}, in the lung. Here, we used parallel genetic and immunological approaches to investigate the basis of CC042 susceptibility. Using a population derived from a CC001/Unc (CC001) x CC042 intercross, we mapped four QTL underlying Tuberculosis ImmunoPhenotypes (Tip1-4). These included 2 major effect QTL on Chromosome 7 (Tip1 and Tip2) that were associated with bacterial burden. Tip2, along with Tip3 (Chromosome 15) and Tip4 (Chromosome 16) also correlated with IFN{gamma} production following infection, whereas Tip1 appeared to control an IFN{gamma}-independent mechanism of bacterial control. Further immunological characterization revealed that CC042 animals recruited relatively few antigen-specific T cells to the lung and these T cells failed to express the Integrin alpha L (L; i.e., CD11a), which contributes to T cell activation and migration. These defects could be explained by a CC042 private variant in the Itgal gene, which encodes CD11a, and is found within the Tip2 interval. This 15bp deletion leads to aberrant mRNA splicing and is predicted to result in a truncated protein product. The ItgalCC042 genotype was associated with all measured disease traits, indicating that this variant is a major determinant of susceptibility in CC042. The combined effect of functionally distinct Tip variants likely explains the profound susceptibility of CC042 and highlights the multigenic nature of TB control in the Collaborative Cross.\n\nImportance statementThe variable outcome of Mycobacterium tuberculosis infection observed natural populations is difficult to model in genetically homogenous small animal models. The newly-developed Collaborative Cross (CC) represents a reproducible panel of genetically-diverse mice that display a broad range of phenotypic responses to infection. We explored the genetic basis of this variation, focusing on a CC line that is highly susceptible to M. tuberculosis infection. This study identified multiple quantitative trait loci associated with bacterial control and cytokine production, including one that is caused by a novel loss-of-function mutation in the Itgal gene that is necessary for T cell recruitment to the infected lung. These studies verify the multigenic control of mycobacterial disease in the CC panel, identify genetic loci controlling diverse aspects of pathogenesis, and highlight the utility of the CC resource.

microbiology↗

bioRxiv2018

New inhibitors of Mycobacterium tuberculosis identified using systems chemical biology

New antibiotics are needed to combat rising resistance, with new Mycobacterium tuberculosis (Mtb) drugs of highest priority. Conventional whole-cell and biochemical antibiotic screens have failed. We developed a novel strategy termed PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets) in which we screen compounds against pools of strains depleted for essential bacterial targets. We engineered strains targeting 474 Mtb essential genes and screened pools of 100-150 strains against activity-enriched and unbiased compounds libraries, measuring > 8.5-million chemical-genetic interactions. Primary screens identified >10-fold more hits than screening wild-type Mtb alone, with chemical-genetic interactions providing immediate, direct target insight. We identified > 40 novel compounds targeting DNA gyrase, cell wall, tryptophan, folate biosynthesis, and RNA polymerase, as well as inhibitors of a novel target EfpA. Chemical optimization yielded EfpA inhibitors with potent wild-type activity, thus demonstrating PROSPECTs ability to yield inhibitors against novel targets which would have eluded conventional drug discovery.

microbiology↗

bioRxiv2018

Gain of Function Analysis Reveals Non-Redundant Roles for the Yersinia pestis Type III Secretion System Effectors YopJ, YopT, and YpkA

Virulence of Yersinia pestis in mammals requires the type III secretion system, which delivers seven effector proteins into the cytoplasm of host cells to undermine immune responses. All seven of these effectors are conserved across Y. pestis strains, but three - YopJ, YopT, and YpkA - are apparently dispensable for virulence. Some degree of functional redundancy between effector proteins would explain both observations. Here, we use a combinatorial genetic approach to define the minimal subset of effectors required for full virulence in mice following subcutaneous infection. We found that a Y. pestis strain lacking YopJ, YopT, and YpkA is attenuated for virulence in mice, and that addition of any one of these effectors to this strain increases lethality significantly. YopJ, YopT, and YpkA likely contribute to virulence via distinct mechanisms. YopJ is uniquely able to cause macrophage cell death in vitro and to suppress accumulation of inflammatory cells to foci of bacterial growth in deep tissue, whereas YopT and YpkA cannot. The synthetic phenotypes that emerge when YopJ, YopT, and YpkA are removed in combination provide evidence that each enhances Y. pestis virulence, and that YopT and YpkA act through a mechanism distinct from that of YopJ.

microbiology↗