Biology subjects

Hogan, A. M.

Publications and source records attributed to Hogan, A. M..

7 recordsLinked to original sources

bioRxiv2025

Burkholderia Genes Involved in Extracellular Bioplastic Degradation Revealed by Tn-seq and CRISPR-Cas

Bioplastics represent promising alternatives to petroleum-based plastics, yet their biodegradation remains insufficiently understood. Identifying bacteria capable of degrading bioplastics extracellularly could enhance end-of-life management practices. To investigate Burkholderias capacities for the degradation of medium-chain-length polyhydroxyalkanoate (mcl-PHA), we screened a panel of Burkholderia strains and identified such capacity in strains of B. gladioli, B. multivorans, and B. vietnamiensis. To elucidate the genetic basis of this activity, we performed transposon mutagenesis followed by activity-based screening and Tn-seq on B. vietnamiensis LMG 16232. Disrupted genetic elements in transposon mutants with negative phenotypes were further investigated using a CRISPR-associated transposase (CAST) system. These included a lipase production gene cluster, encoding two putative triacylglycerol lipases and a chaperone, and genes coding for a a A24 family peptidase, a TetR/AcrR family transcriptional regulator and a type II secretion system (T2SS) protein. Complete loss or reduced extracellular mcl-PHA depolymerase activity was observed in the CAST mutants, validating their involvement in mcl-PHA degradation. Notably, only one of the two lipases encoded in the lipase production gene cluster was responsible for mcl-PHA degradation, suggesting that while lipases may show substrate promiscuity, lipase functional annotation does not necessarily imply mcl-PHA depolymerization. Docking experiments using the amino acid sequences of the two lipases supported these findings. Together, we identify a gene coding for an active mcl-PHA depolymerase in B. vietnamiensis and demonstrate the power of combining activity-based screening, Tn-seq, and CAST to rapidly establish gene-to-function links. ImportanceDue to their versatile metabolism, Burkholderia strains play critical roles in degradation of multiple compounds in the environment. Here we show that several Burkholderia species can extracellularly degrade medium-chain-length polyhydroxyalkanoates (mcl-PHAs), a promising class of bioplastics. By integrating transposon mutagenesis, Tn-seq, and CRISPR-associated transposase (CAST) technologies, we identify and validate key genetic determinants involved in mcl-PHA degradation in B. vietnamiensis. These genes encode a lipase, a secretion system component, and regulatory factors, underscoring the complexity and specificity of microbial bioplastic degradation pathways. These findings not only advance our understanding of PHA biodegradation but also identifies B. vietnamiensis as as a source of enzymes capable of degrading extracellular mcl-PHA.

microbiology↗

bioRxiv2025

Stromal cells modulate innate immune cell phenotype and function in colorectal cancer via the Sialic acid/Siglec axis

BackgroundThe immunosuppressive tumour microenvironment (TME) reduces immune response effectiveness in stromal-rich tumours, including CMS4 colorectal cancer (CRC). Mesenchymal stromal cells (MSCs), precursors to cancer-associated fibroblasts (CAFs), promote cancer progression by suppressing anti-tumour immune responses. Hypersialylation of glycans on tumours engages Siglec receptors on immune cells, driving immune dysfunction, but its role in stromal-mediated immunosuppression remains unclear. MethodsSialic acids and Siglec ligands were measured on CRC tissue, primary human CAFs, and tumour-conditioned-mesenchymal stromal cells (MSCTCS) and CAF using immunohistochemistry and flow cytometry. The effect of stromal cell sialylation on macrophages and NK cells was assessed in ex vivo primary stromal and immune cell co-cultures and expression of Siglec-10 and immune cell phenotype markers and function were measured by flow cytometry. Using an immunocompetent Balb/c CT26 mouse model, we induced tumours with/without conditioned stromal cells, with/without pre-treatment of stromal cells with sialyltransferase inhibitor (3FAX) or sialidase (E610). We assessed the effect of stromal cell sialylation on macrophages and NK cells in the tumour and secondary lymphoid tissues by flow cytometry. ResultsStromal cells, including CAFs, in CRC tumours are highly sialylated compared to epithelial cancer cells and are associated with high expression of ST6GalNAC6. Genetic knockdown of ST6GalNAC6 reduced the expression of stromal cell Siglec-10 ligands in MSCs. CAFs and MSCTCS induced Siglec-10 on macrophages and NK cells and impaired NK cell cytotoxicity. Sialidase treatment reduced Siglec-10 expression, restoring NK cell function. In vivo, desialylation of stromal cells increased macrophage activation (CD11b+CD80+) and reduced immunosuppressive marker expression (CD206, PD-L1, Siglec-G) in lymphoid tissues, indicating sustained systemic anti-tumour immunity. Intratumoural NK cells exhibited high Siglec-G expression and impaired cytotoxicity, and granzyme B expression significantly increased with sialidase treatment of stromal cells. In an inflammatory tumour model, inflammatory tumour-conditioned (iTCS) MSCs promoted metastasis and Siglec-G induction on NK cells and macrophages, both reversed by sialyltransferase inhibition, underscoring the effects of stromal modulation of innate immune cell function in inflammatory tumours. ConclusionStromal cell sialylation modulates innate immune suppression in CRC via the sialic acid/Siglec axis. Targeting stromal sialylation restores NK cytotoxicity and macrophage activation, offering a novel therapeutic strategy for immunosuppressive stromal-rich tumours. What is already known on this topicO_LIThe tumour microenvironment of consensus molecular subtype 4 (CMS4) colorectal cancer (CRC) is associated with high stromal burden, poor immune infiltration, poor response to anti-cancer therapies and thus poor patient prognosis. Immune checkpoint inhibitors (ICIs) have limited impact on stromal-rich CRC tumours, therefore highlighting the need to discover and target novel mechanisms of tumour immune evasion. C_LIO_LIEmerging studies have highlighted that stromal cells in CRC and pancreatic ductal adenocarcinoma (PDAC) are highly sialylated, expressing even higher levels of sialic acid on their cell surface than epithelial cancer cells. Targeting stromal cell sialylation has unveiled promising data in restoring the anti-tumour activity of T cells and macrophages. There is a need to explore the effects of targeting stromal cell sialylation on other immune cells of the TME and to evaluate the Siglec/sialic acid axis of stromal and immune cells in resistant CRC tumours. C_LI What this study addsO_LIWe reveal ST6GalNAC6 as a sialyltransferase enzyme that regulates the production of Siglec-10 ligands in CRC stromal cells. Overexpression of ST6GalNAC6 and Siglec-10 correlated with poor survival in CRC and mesenchymal CRC tumours. C_LIO_LIWe show for the first time an induction of Siglec-10 expression on macrophages and NK cells in stromal-immune co-culture experimental models with hypersialylated MSCs and CAFs in vitro and ex vivo. Targeting stromal cell sialylation increased NK cell cytotoxicity of CRC cells, indicating a direct functional role for stromal cell sialylation in immunosuppression. C_LIO_LIAn immunogenic mouse model of CRC was used to evaluate the potential therapeutic efficacy of targeting stromal cell sialylation in overcoming stromal cell-mediated immunosuppression in CRC. Sialic acid-targeting of stroma slowed tumour growth and reduced inflammation-driven metastasis. This was associated with greater infiltration and activation of macrophages and NK cells with stromal cell sialic acid depletion, highlighting stromal cell sialylation as a mechanism of innate immune cell suppression in stromal-rich CRC. C_LI How this study might affect Research, Practice or PolicyO_LIOur research provides insight into a novel mechanism of stromal cell-mediated immunosuppression of innate immune cells in CRC and may open up new avenues of research for targeting stromal cells in stromal-rich TMEs such as pancreatic, breast and ovarian cancers. C_LIO_LIOur research identifies a stromal cell effect of enhancing Siglec expression on tumour infiltrating innate immune cells as a novel immune checkpoint, which may be useful in identifying potential novel immunotherapeutic combinations in future. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/642985v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@cbafc3org.highwire.dtl.DTLVardef@80474forg.highwire.dtl.DTLVardef@1c9d050org.highwire.dtl.DTLVardef@8b6551_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Cell envelope structural and functional contributions to antibiotic resistance in Burkholderia cenocepacia

Antibiotic activity is limited by the physical construction of the Gram-negative cell envelope. Species of the Burkholderia cepacia complex (Bcc) are known as intrinsically multidrug-resistant opportunistic pathogens with low permeability cell envelopes. Here, we re-examined a previously performed chemical-genetic screen of barcoded transposon mutants in B. cenocepacia K56-2, focusing on cell envelope structural and functional processes. We identified structures mechanistically important for resistance to singular and multiple antibiotic classes. For example, the polymeric O-antigen was important for resistance to cationic antibiotics, while defects in peptidoglycan precursor synthesis specifically increased susceptibility to cycloserine and revealed a new putative amino acid racemase. Susceptibility to novobiocin, avibactam, and the LpxC inhibitor, PF-04753299, was linked to the BpeAB-OprB efflux pump, suggesting these drugs are substrates for this pump in B. cenocepacia. Additionally, disruption of the periplasmic disulfide bond formation system caused pleiotropic defects on outer membrane integrity and {beta}-lactamase activity. Our findings highlight the layering of resistance mechanisms in the structure and function of the cell envelope. Consequently, we point out processes that can be targeted for developing antibiotic potentiators. ImportanceThe Gram-negative cell envelope is a double-layered physical barrier that protects cells from extracellular stressors, such as antibiotics. The Burkholderia cell envelope is known to contain additional modifications that reduce permeability. We investigated Burkholderia cell envelope factors contributing to antibiotic resistance from a genome-wide view by re-examining data from a transposon mutant library exposed to an antibiotic panel. We identified susceptible phenotypes for defects in structures and functions in the outer membrane, periplasm, and cytoplasm. Overall, we show that resistance linked to the cell envelope is multifaceted and provides new targets for the development of antibiotic potentiators.

microbiology↗

bioRxiv2022

Profiling cell envelope-antibiotic interactions reveals vulnerabilities to β-lactams in a multidrug-resistant bacterium

The cell envelope of the Gram-negative Burkholderia cepacia complex (Bcc) presents unique restrictions to antibiotic penetration. As a consequence, Bcc species are notorious for causing recalcitrant multidrug-resistant infections in immunocompromised individuals, such as those living with cystic fibrosis. To systematically identify cell envelope-associated resistance and susceptibility determinants at the genome level, we constructed a high-density, randomly-barcoded transposon mutant library in the clinical isolate B. cenocepacia K56-2 and exposed it to a panel of more than twenty cell envelope-targeting antibiotics. By quantifying relative mutant fitness with BarSeq, followed by validation with CRISPR-interference, we profiled over a hundred new functional associations and identified novel mediators of antibiotic susceptibility in the Bcc cell envelope. We revealed new connections between {beta}-lactam susceptibility, peptidoglycan synthesis, and blockages in undecaprenyl phosphate metabolism, which highlight a vulnerability in sharing this lipid intermediate. We then show that the clinically relevant synergy of the {beta}-lactam/{beta}-lactamase inhibitor combination ceftazidime/avibactam is primarily mediated by inhibition of the PenB carbapenemase. Importantly, we found that avibactam more strongly potentiates the activity of aztreonam and meropenem than ceftazidime in a panel of Bcc clinical isolates. Finally, we characterize for first time in the Bcc the iron and receptor-dependent activity of the novel siderophore-cephalosporin antibiotic, cefiderocol. Overall, our work has implications for antibiotic target prioritization, and for using additional combinations of {beta}-lactam/{beta}-lactamase inhibitors that can extend the utility of our current clinical arsenal of antibacterial therapies. Author SummaryWhile the Gram-negative cell envelope is a major barrier to antibiotic action, we have an incomplete picture of how each component contributes to antibiotic resistance. To answer this question from a genome-wide perspective in a model of antibiotic-resistant bacteria, we constructed a library of randomly-barcoded transposon mutants in Burkholderia cenocepacia and exposed it to a panel of diverse cell envelope-targeting antibiotics. We identified individual genes and whole pathways associated with antibiotic resistance, including several novel players. Focusing on clinically relevant antibiotics, our strategy dissected how the synergy of the important {beta}-lactam/{beta}-lactamase inhibitor combination ceftazidime/avibactam primarily depends upon inhibition of a single {beta}-lactamase. Additionally, we are the first to characterise the molecular basis of the antibiotic mechanism of cefiderocol in Burkholderia, a new and very potent siderophore-cephalosporin conjugate drug. Encouragingly, we found that cefiderocol susceptibility was greatest at physiological iron concentrations. Overall, we highlight several avenues that can be taken to enhance or develop new therapeutic strategies against Burkholderia infection.

microbiology↗

bioRxiv2021

New auranofin analogs with antibacterial properties against Burkholderia clinical isolates

Bacteria of the genus Burkholderia include pathogenic Burkholderia mallei, Burkholderia pseudomallei and the Burkholderia cepacia complex (Bcc). These Gram-negative pathogens have intrinsic drug resistance, which makes treatment of infections difficult. Bcc affects individuals with cystic fibrosis (CF) and the species B. cenocepacia is associated with one of the worst clinical outcomes. Following the repurposing of auranofin as an antibacterial against Gram-positive bacteria, we previously synthetized auranofin analogs with activity against Gram-negatives. In this work, we show that two auranofin analogs, MS-40S and MS-40, have antibiotic activity against Burkholderia clinical isolates. The compounds are bactericidal against B. cenocepacia and kill stationary-phase cells and persisters without selecting for multistep resistance. Caenorhabditis elegans and Galleria mellonella tolerated high concentrations of MS-40S and MS-40, demonstrating that these compounds have low toxicity in these model organisms. In summary, we show that MS-40 and MS-40S have the potential to be effective therapeutic options to treat Burkholderia infections.

microbiology↗

bioRxiv2021

Stromal cell sialylation suppresses T cells in inflammatory tumour microenvironments: A new tumour stromal cell immune checkpoint?

Immunosuppressive tumour microenvironments (TME) reduce the effectiveness of immune responses in cancer. Non-haematopoietic mesenchymal stromal cells (MSC), the precursor to cancer associated fibroblasts (CAFs), dictate tumour progression by enhancing immune cell suppression. Hyper-sialylation of glycans promotes immune evasion in cancer, but the role of sialyation in stromal cell-mediated immunosuppression is unknown. Here we study changes in sialyltransferase (ST) enzymes and associated surface expressed sialic acid in stromal cells following inflammatory and tumour secretome conditioning. We show that tumour conditioned stromal cells have increased levels of sialyltransferases, 2,3/6 linked sialic acid and siglec ligands. In tumour models of solid (colorectal cancer) and haematological (multiple myeloma) stromal rich tumours, stromal cell sialylation is associated with enhanced immunosuppression. Using datasets and patient samples, we confirm that targeting sialylation in tumour stromal cells reverses immune cell exhaustion. Targeting stromal cell sialylation may represent a novel immune checkpoint to reactivate anti-tumour immunity.

immunology↗

bioRxiv2019

A broad-host-range CRISPRi toolkit for silencing gene expression in Burkholderia

Genetic tools are critical to dissecting the mechanisms governing cellular processes, from fundamental physiology to pathogenesis. Members of the genus Burkholderia have potential for biotechnological applications but can also cause disease in humans with a debilitated immune system. The lack of suitable genetic tools to edit Burkholderia GC-rich genomes has hampered the exploration of useful capacities and the understanding of pathogenic features. To address this, we have developed CRISPR interference (CRISPRi) technology for gene silencing in Burkholderia, testing it in B. cenocepacia, B. multivorans and B. thailandensis. Tunable expression was provided by placing a codon-optimized dcas9 from Streptococcus pyogenes under control of a rhamnose-inducible promoter. As a proof of concept, the paaABCDE operon controlling genes necessary for phenylacetic acid degradation was targeted by plasmid-borne sgRNAs, resulting in near complete inhibition of growth on phenylacetic acid as the sole carbon source. This was supported by reductions in paaA mRNA expression. The utility of CRISPRi to probe other functions at the single cell level was demonstrated by knocking down phbC and fliF, which dramatically reduces polyhydroxybutyrate granule accumulation and motility, respectively. As a hallmark of the mini-CTX system is the broad host-range of integration, we putatively identified 67 genera of Proteobacteria that might be amenable to modification with our CRISPRi toolkit. Our CRISPRi tool kit provides a simple and rapid way to silence gene expression to produce an observable phenotype. Linking genes to functions with CRISPRi will facilitate genome editing with the goal of enhancing biotechnological capabilities while reducing Burkholderias pathogenic arsenal.\n\nAuthor contributionsSTC conceived the idea and design of the research; AMH designed and cloned the dCas9 constructs; AMH and ASMZ designed the sgRNAs, assessed knockdown phenotypes, processed data, and wrote and edited the manuscript; TJL performed RT-qPCR analysis and edited the manuscript; STC supervised the work and provided financial support.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC=\"FIGDIR/small/618413v3_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (22K):\norg.highwire.dtl.DTLVardef@b00aa5org.highwire.dtl.DTLVardef@18ae038org.highwire.dtl.DTLVardef@86af96org.highwire.dtl.DTLVardef@71bac4_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗