Biology subjects

Gruenheid, S.

Publications and source records attributed to Gruenheid, S..

15 recordsLinked to original sources

bioRxiv2026

Exploring the antibiotic-producing capabilities of Canadian High Arctic bacteria

Microbial communities in harsh environments such as the Canadian high Arctic, remain a relatively unexplored source of antimicrobial natural products. In this study, we developed a pipeline for screening and evaluating bacterial isolates cultured from 23 Canadian high Arctic soil, sediment, and active-layer permafrost samples to discover antibacterial compounds. The pipeline incorporated an undergraduate studentsourcing program and produced created a collection of over 350 bacterial isolates with preliminary antibacterial activity. Following dereplication, whole genome sequencing and secondary screening by co-culture assays, five isolates were prioritized to test crude extracts for antibacterial activity. Among bioactive crude extracts from these isolates, the Pseudomonas isolate with the most potent activity against S. aureus was analyzed. Using liquid chromatography bioactivity guided fractionation, we identified a potential bioactive compound known as koreenceine A, a compound previously only isolated from a Pseudomonas from temperate soil and involved in gene expression of other their microbial community. Here we expanded the known antibiotic activity of koreenceine A and confirmed that a {triangleup}kecBCD genetic knockout of the putative biosynthetic gene cluster significantly abrogated the antibacterial activity.

microbiology↗

bioRxiv2026

Repeated exposure to viral and bacterial danger signals in Parkin deficient mice induces inflammation but fails to trigger parkinsonism

Experimental models of Parkinson's disease (PD) traditionally involve direct perturbations of the functioning of dopamine (DA) neurons. However, PD is increasingly recognized as multifactorial in origin and involving both cell-autonomous vulnerability factors and non-cell-autonomous triggers including inflammatory signals deriving from bacterial or viral infections. Notably, genes associated with early-onset familial PD as PRKN (Parkin) and PINK1, are ubiquitously expressed and have been implicated in immune regulation, further supporting a role for inflammation in disease etiology. In the present study, we tested the hypothesis that alternate Polyinosinic:polycytidylic acid (Poly(I:C)), mimicking viral infection, and lipopolysaccharides (LPS), mimicking bacterial infection, may induce sufficient brain inflammation to impair the DA system in Parkin-deficient mice and recapitulate some of the systemic features of PD. We developed a protocol with alternating Poly(I:C) and LPS, inducing acute peripheral inflammation in both genotypes, with consistent body weight loss and an increase of fecal lipocalin-2. Modest levels of neuroinflammation were present six months after the treatment, with higher levels of the microglial marker Iba1 in the dorsal striatum and reduced levels of the astrocyte marker GFAP in the mesencephalon, potentially indicating reduced neuronal support in the longer term. Finally, we find that this alternating Poly(I:C)/LPS treatment does not induce dopaminergic denervation or motor dysfunctions, even in Parkin KO mice. Taken together, our findings suggest that repeated short-term exposure to pathogen-derived signals is insufficient to induce DA system impairment in young adult Parkin deficient mice. Instead, induction of PD-like pathology may require a convergence of factors, including a prolonged or recurrent inflammatory burden, aging, genetic susceptibility, and potentially additional environmental or cellular stressors, that together create a pathogenic "perfect storm". Thus, in its current form, the paradigm developed in the present study should not be regarded as a fully representative model of PD. Rather, it provides proof of concept that temporally alternating inflammatory stimuli can shape non-motor symptom-like phenotypes and underscores the need for future animal model research to incorporate interacting risk factors, disease duration, and age-dependent vulnerability.

neuroscience↗

bioRxiv2026

STING dampens the unfolded protein response to enable the presentation of self-antigens on MHC-I during inflammation.

A growing body of evidence supports the contribution of the long-lasting adaptive immune system in Parkinsons disease (PD). We showed that the PD-associated protein PINK1 negatively regulates the presentation of mitochondrial antigens (MitAP) on MHC-I molecules. In vivo evidence indicated that MitAP activation in mice, in the absence of PINK1, led to cytotoxic CD8+ T cell stimulation and severe motor impairments, reversible by L-DOPA. We show here that following TLR4 activation, MitAP is engaged through a pathway involving cGAS-STING, which acts as a rheostat to dampen the unfolded protein response (UPR). Without STING, the stress response is amplified, leading to a translational attenuation that inhibits the expression of XBP1s, a transcription factor required for MitAP. STING activity also regulates the repertoire of peptides displayed at the cell surface during inflammation, highlighting a potential role in immunosurveillance. These findings establish STING and the UPR as key immune regulators targetable for therapeutic intervention during autoimmune diseases and PD.

immunology↗

bioRxiv2026

Early life γδT cell activation enforces intestinal barrier integrity during intergenerational C. difficile colonization

Early postnatal life is a highly dynamic period in which the intestine is inundated with billions of microbes that not only battle for colonization, but also strongly influence immune system function and long-term health. This brief period of development provides a window of opportunity to imprint resilience against disease, yet the intercellular dialogue that dictates tissue protection are only beginning to be discovered. By performing a kinetic analysis of immune cell activation in the colon of mice from early life to adulthood, we identified transient activation of a microbiota-dependent type 3 immune response during the weaning period. This response was characterized by a selective increase in IL-17 production by fetal-derived {gamma}{delta} T cells that occurred in an IL-1 receptor-dependent, cell-intrinsic manner. The subsequent differentiation of IL-10 producing Ror{gamma}t+ T regulatory cells extinguished IL-17 production by {gamma}{delta} T cells and prevented immunopathology. Microbial gain-of-function approaches determined that colonic {gamma}{delta} T cell activation occurred in response to passive acquisition of C. difficile at birth. In turn, IL-17 limited C. difficile growth and intestinal barrier breach by the commensal microbiota. Collectively, our results reveal how an agile immune cell network meets the demands of a maturing microbiota to support gut health during early life. Highlights- Colonic {gamma}{delta}17 T cells are activated during early life in a microbiota-specific manner - Fetal-derived {gamma}{delta}17 T cells require IL-1 signals for activation - Passive acquisition of C.difficile induces {gamma}{delta}17 T cell activation - IL-17 limits C. difficile growth and systemic dissemination of commensals

immunology↗

bioRxiv2026

Myeloid PINK1 represses mtDNA release and immune signaling that impacts neuronal pathology in patient-derived idiopathic PD models

Parkinsons disease (PD) is a neurodegenerative disorder marked by the development of cardinal motor deficits preceded by a protracted prodromal period of non-motor symptoms often involving the gastrointestinal (GI) tract. There is an emerging consensus that both the peripheral immune system and local neuroinflammation play key roles in the etiology of PD. We previously demonstrated a critical function for the Parkinsons related proteins PINK1 and Parkin as repressors of the innate to adaptive immune response in cultured cells and mouse models of infection. However, it remained unclear whether these processes were conserved in patient-derived models, and precisely how immune signaling may ultimately drive the death of dopaminergic neurons. Here we show that GI infection of PINK1 knockout (KO) mice triggered acute neurodegeneration which was evident early in the enteric nervous system. Treating wild type enteric or dopaminergic neurons with conditioned medium from immune-stimulated PINK1 KO macrophages was sufficient to promote neuronal disruption in both mouse and human neurons in vitro. Within immune-activated macrophages, we reveal that loss of PINK1 led to an enhanced release of mitochondrial DNA (mtDNA) within mitochondrial derived vesicles, leading to the activation of cGAS/STING pathways. These changes were seen in both mouse/human in vitro models and in PD patient-derived primary macrophages. Notably, pharmacological modulation using a PINK1 activator with high therapeutic potential attenuated pro-inflammatory profiles elicited by the mtDNA-dependent STING/NF-{kappa}B pathway in idiopathic patient-derived macrophages. Ultimately, our study lays the foundation for understanding PINK1-related peripheral macrophage mechanisms in idiopathic PD and provides a target for further development to treat the disease at early stages. Graphical abstractPINK1-related immune mechanisms of Parkinsons disease and associations with early neurodegenerative events. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/694713v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@6f6488org.highwire.dtl.DTLVardef@13139aaorg.highwire.dtl.DTLVardef@c0d4d1org.highwire.dtl.DTLVardef@1d41e25_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

A single Citrobacter rodentium infection in Pink1 knockout and wild type mice leads to regional blood-brain-barrier perturbation and glial activation without dopamine neuron axon terminal loss

A growing body of research suggests a link between immune system activation and the development of Parkinsons disease (PD). Previous work showed that repeated gastrointestinal infection with Citrobacter rodentium can induce PD-like motor dysfunction in Pink1 knockout (KO) mice, along with immune cell infiltration into the brain. To better understand mechanisms underlying immune-mediated brain attack in this model, we tested whether mild infections are sufficient to increase blood-brain barrier (BBB) permeability and trigger brain inflammation. Pink1 wild-type (WT) and KO mice were infected with C. rodentium, and gadolinium-enhanced magnetic resonance imaging (MRI) was performed at days 13 and 26 post-infection to assess BBB integrity. Quantitative MRI analysis revealed increased BBB permeability at day 26 in both WT and KO mice, particularly in the striatum, dentate gyrus, somatosensory cortex, and thalamus. Notably, this permeability was not associated with changes in tight junction protein expression or dopamine system markers in the striatum at either time point. However, persistent microglial activation was observed at day 26 post-infection, along with elevated levels of inflammatory mediators such as eotaxin, IFN-{gamma}, CXCL9, IL-17, and MIP-2 in the striatum. Additionally, serum levels of IL-17 and CXCL1 were increased in infected Pink1 KO mice. Flow cytometry revealed neutrophil infiltration in the brain at day 26 post-infection. Finally, a bulk RNA-seq transcriptome analysis revealed that gene sets related to synaptic function were particularly influenced by the infection and that inflammation-related genes were upregulated by the infection in the Pink1 KO mice. These findings support the hypothesis that even mild gastrointestinal infections can increase BBB permeability, disrupt brain homeostasis, and promote chronic neuroinflammation. In genetically susceptible individuals, such as those with Pink1 deficiency, this may represent a first hit that contributes to subsequent induction of PD pathology with aging. Author summaryWe hypothesize that immune system activation is linked to the development of Parkinsons disease (PD). Previous work revealed that repeated gastrointestinal infections with Citrobacter rodentium causes PD-like symptoms and immune cell invasion in the brain of Pink1 knockout (KO) mice. In the current study, we tested whether a single mild gut infection alters blood-brain barrier (BBB) permeability and causes brain inflammation. We infected Pink1 WT and KO mice with Citrobacter rodentium and used gadolinium-enhanced MRI to detect BBB permeability changes at 13- and 26-days post-infection. Results showed increased BBB permeability in specific brain regions at 26 days. While tight-junction and dopamine (DA)-related proteins remained unchanged, we observed altered expression of synaptic genes, chronic microglial activation, elevated inflammatory markers, and neutrophil infiltration in the brain. Our findings suggest that even mild gastrointestinal infections can increase BBB permeability, potentially enabling immune cell infiltration into the brain and exacerbating pathways implicated in the development of PD, particularly among individuals with genetic risk factors.

neuroscience↗

bioRxiv2024

LRRK2 G2019S mutation incites increased cell-intrinsic neutrophil effector functions and intestinal inflammation in a model of infectious colitis

Parkinsons Disease (PD) is a progressive, neurodegenerative disorder characterised by motor and non-motor symptoms. Emerging evidence suggests a link between PD and gastrointestinal dysfunction. Constipation is frequently observed years prior to development of motor dysfunction in PD, and people with inflammatory bowel disease (IBD) are more likely to develop PD. Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) account for approximately 1% of all PD cases and are associated with increased risk for IBD. Among them, LRRK2 Gly2019Ser (G2019S), located within the kinase domain, is the most common PD-associated mutation and increases kinase activity. It is unknown how LRRK2 mutation affects susceptibility to intestinal inflammation or pathogenesis of PD. Using single cell RNA sequencing (scRNAseq), we demonstrate that LRRK2 G2019S mutation promotes a dysregulated gene profile, especially within neutrophil, monocyte and {gamma}{delta} T cell populations, following Citrobacter rodentium infection in mice. Transcriptionally, LRRK2 G2019S neutrophils have a greater pro- inflammatory type I and II IFN response compared to those of WT mice. This is accompanied by an increase in neutrophil numbers in the lamina propria in LRRK2 G2019S mice. We also uncover cell-intrinsic functional defects in LRRK2 G2019S neutrophils, including increased chemotaxis, degranulation and neutrophil extracellular traps (NETosis) formation. Increased neutrophil infiltration is associated with an upregulation in Th17 immune responses, which may together contribute to the observed increase in colon pathology during infection. These findings increase our understanding of the role of PD-associated genes in immune cells and their contribution to immune dysregulation. Understanding the early perturbations driven by the LRRK2 G2019S mutation in gastrointestinal pathology may facilitate the development of biomarkers for early diagnosis and intervention in PD.

immunology↗

bioRxiv2024

A rewiring of the earliest immune events leading to T-cell mediated disease following intestinal infection in a PINK1KO mouse model of Parkinsons disease

Parkinsons disease (PD) is characterized by a protracted period of non-motor symptoms, including gastrointestinal (GI) dysfunction, which can precede the development of the cardinal motor deficits by decades. This long prodrome of disease is highly suggestive of immune cell involvement in the initiation of disease, but currently the field lacks robust model systems to study such mechanisms. It has been hypothesized that pathology may be first initiated in the periphery due to environmental triggers, such as pathogens that enter the GI tract. We further speculate that the impact of such pathogens on the immune system could be exacerbated in genetically predisposed individuals. Our group has developed a GI-targeted pathogen-induced PD mouse model system in PINK1 KO mice with Gram-negative bacterial infections and found that T cells are a major player in driving PD-like motor symptoms at late stages following infection. Herein, we now map the initiating immune events at the site of infection at the earliest stages with the goal of shedding light on the earliest mechanisms triggering immune-mediated pathological processes relevant to PD. Using unbiased single cell sequencing, we demonstrate that myeloid cells are the earliest dysregulated immune cell type in PINK1 KO infected mice at 1-week post-infection, followed by a dysregulated T cell response shortly after, at 2 weeks post-infection. We find that these myeloid cells have an enhanced proinflammatory profile, are more mature, and develop enhanced capacity for antigen presentation. Using unbiased prediction analysis, our data suggest that cytotoxic T cells and myeloid cells are particularly poised for interacting with each other, and we identify possible direct cell-cell interaction pathways that might be implicated. Taken together, deciphering the earliest immune mechanisms in the periphery underpinning PD autoimmunity will be instrumental in the development of effective therapeutic targeting strategies before irrevocable neuronal damage ensues.

immunology↗

bioRxiv2024

Modeling gene-environment interactions in Parkinson's Disease: Helicobacter pylori infection of Pink1-/- mice induces CD8 T cell-dependent motor and cognitive dysfunction.

Parkinsons disease (PD) is a chronic neurodegenerative disorder characterized by progressive loss of motor function. Diagnosis occurs late: after motor symptom development downstream of the irreparable loss of a large proportion of the dopaminergic neurons in the substantia nigra of the brain. Understanding PD pathophysiology in its pre-motor prodromal phase is needed for earlier diagnosis and intervention. Genetic risk factors, environmental triggers, and dysregulated immunity have all been implicated in PD development. Here, we demonstrate in a mouse model deficient in the PD-associated gene Pink, that infection with the human PD-associated gastric bacterium Helicobacter pylori leads to development of motor and cognitive signs resembling prodromal features of PD. This was also associated with proliferation and activation of primary mitochondria-reactive CD8 T cells and infiltration of CD8 T cells into the brain. Development of the motor and cognitive phenotypes in the infected Pink1-/- mice was abrogated when CD8 T cells were depleted prior to infection. We anticipate that this new model, which integrates genetic PD susceptibility, a PD-relevant environmental trigger, and specific immune changes that are required for symptom development, will be a valuable tool for increasing our understanding of this complex disease.

immunology↗

bioRxiv2023

Characterizing the diversity of enteric neurons using Dopamine Transporter (DAT)-Cre reporter mice

The enteric nervous system (ENS) comprises a complex network of neurons whereby a subset appears to be dopaminergic, although the characteristics, roles, and implications in disease are less understood. Most investigations relating to enteric dopamine (DA) neurons rely on immunoreactivity to tyrosine hydroxylase (TH) - a rate-limiting enzyme in the production of DA. However, TH immunoreactivity is likely to provide an incomplete picture given previous work has showed that some DA neurons contain little if any TH and its levels tend to be decreased in response to cellular stress. This study herein provides a comprehensive characterization of DA neurons in the gut using a well-accepted reporter mouse line, expressing a fluorescent protein (tdTomato) under control of the DA transporter (DAT) promoter. Our findings confirm a unique localization of DA neurons in the gut and unveil the discrete subtypes of DA neurons in this organ, which we characterized using both immunofluorescence and single-cell transcriptomics, as well as validated using in situ hybridization. We observed distinct subtypes of DAT-tdTomato neurons expressing co-transmitters and modulators across both plexuses; some of them likely co-releasing acetylcholine, while others were positive for a slew of canonical DA markers (TH, VMAT2 and GIRK2). Interestingly, we uncovered a seemingly novel population of DA neurons unique to the ENS which were ChAT/DAT-tdTomato-immunoreactive neurons and were characterised by the expression of Grp, Calcb and Sst. Given the clear heterogeneity of DAergic gut neurons, further investigation is warranted to define their functional signatures and discover any inherent vulnerabilities in disease. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/545271v3_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1e1830borg.highwire.dtl.DTLVardef@625579org.highwire.dtl.DTLVardef@1d9b59aorg.highwire.dtl.DTLVardef@219cab_HPS_FORMAT_FIGEXP M_FIG C_FIG Using a reporter mouse line, expressing a fluorescent protein under control of the dopamine transporter (DAT) promoter, discrete subtypes of dopaminergic neurons were unveiled across the ganglionated plexuses of the gut. A novel subpopulations of enteric DA neurons, expressing genes previously reported involved in dopamine signaling in the brain, exhibit a cholinergic phenotype.

neuroscience↗

bioRxiv2023

LRRK2 regulates the activation of the unfolded protein response and antigen presentation in macrophages during inflammation

While the contribution of inflammation in the pathological process leading to Parkinsons disease (PD) is well established, a growing body of evidence supports a role for the long-lasting adaptive immune system in the disease. We showed that, in inflammatory conditions, the PD-proteins PINK1 and Parkin negatively regulate the presentation of mitochondrial antigens on MHC I molecules, a process referred to as MitAP (Mitochondrial Antigen Presentation). In the absence of PINK1, over-activation of this pathway in antigen-presenting cells (APCs) engages autoimmune mechanisms leading to the establishment of cytotoxic CD8+ T cells. Co-culture of dopaminergic neurons (DNs) with these T cells led to neuronal cell death, suggesting that the MitAP in DNs made them susceptible to T cell-mediated destruction. In the present study, we used a pharmacological and genetic approach to characterize the MitAP pathway at the molecular level. We showed that this antigen presentation pathway is induced in APCs in response to inflammatory signals through the sequential activation of TLR4, cGAS-STING and the Unfolded Protein Response (UPR). A "UPR motif" present on a STING cytoplasmic domain was shown to specifically activate the UPR sensor IRE1. Remarkably, the PD-related protein LRRK2, acted with STING upstream of the UPR to regulate the transition from innate to adaptive immunity, thereby identifying this PD-related protein as a key player in the immune response during inflammation.

immunology↗

bioRxiv2022

Simulated colonic fluid replicates the in vivo growth capabilities of Citrobacter rodentium cpxRA mutants and uncovers additive effects of Cpx regulated genes on fitness

Citrobacter rodentium is an attaching and effacing (A/E) pathogen used to model enteropathogenic and enterohemorrhagic Escherichia coli infections in mice. During colonization, C. rodentium must adapt to stresses in the gastrointestinal tract such as antimicrobial peptides, pH changes, and bile salts. The Cpx envelope stress response (ESR) is a two-component system used by some bacteria to remediate stress by modulating gene expression and is necessary for C. rodentium pathogenesis in mice. Here, we utilized simulated colonic fluid (SCF) to mimic the gastrointestinal environment which we show strongly induces the Cpx ESR and highlights a fitness defect specific to the {Delta}cpxRA mutant. While investigating genes in the Cpx regulon that may contribute to C. rodentium pathogenesis, we found that the absence of the Cpx ESR resulted in higher expression of the LEE master regulator, ler, and the genes yebE, ygiB, bssR, and htpX relied on CpxRA for proper expression. We then determined that CpxRA and select gene mutants were essential for proper growth in SCF when in the presence of extraneous stressors and in competition. Although none of the Cpx-regulated gene mutants exhibited marked virulence phenotypes in vivo, the {Delta}cpxRA mutant had reduced colonization and attenuated virulence, as previously determined, which replicated the in vitro growth phenotypes specific to SCF. Overall, these results indicate that the {Delta}cpxRA virulence defect is not due to any single Cpx regulon gene examined. Instead, attenuation may be the result of defective growth in the colonic environment resulting from the collective impact of multiple Cpx-regulated genes.

microbiology↗

bioRxiv2021

Simulated colonic fluid replicates the in vivo growth capabilities of Citrobacter rodentium mutants and highlights a critical role for the Cpx envelope stress response in mediating stressors encountered in the gastrointestinal tract

Citrobacter rodentium is an attaching and effacing (A/E) pathogen used as a model for enteropathogenic and enterohemorrhagic Escherichia coli infections in mice. While in the host, C. rodentium must adapt to stresses in the gastrointestinal tract such as antimicrobial peptides, pH changes, and bile salts. The Cpx envelope stress response (ESR) is a two-component system used by some bacteria to remediate stress by modulating gene expression and is necessary for C. rodentium pathogenesis in mice. To investigate genes in the Cpx regulon that may contribute to C. rodentium pathogenesis, RNA-Seq, SILAC, and microarray data from previous research was mined and the genes yebE, ygiB, bssR, and htpX were confirmed to be strongly upregulated by the presence of CpxRA using lux reporter constructs. To determine the function of these genes in vivo, knockout mutants were tested in C57Bl/6J and C3H/HeJ mice. Although none of the mutants exhibited marked virulence phenotypes, the {Delta}cpxRA mutant had reduced colonization and attenuated virulence, as previously determined. We also found that the absence of the Cpx ESR resulted in higher expression of the LEE master regulator, ler. In addition, we determined that the {Delta}cpxRA mutant had a growth defect in medium simulating the colon, as did several of the mutants bearing deletions in Cpx-upregulated genes. Overall, these results indicate that the {Delta}cpxRA virulence defect is not due to any single Cpx regulon gene examined. Instead, attenuation may be the result of defective growth in the colonic environment resulting from the collective impact of multiple Cpx-regulated genes.

microbiology↗

bioRxiv2021

A Mediterranean-like fat blend protects against the development of severe colitis in the mucin-2 deficient murine model

The Mediterranean diet (MD) is a health-promoting, moderate fat diet (~ 40% energy). It is not known if the blend of fats found in the MD contribute to the beneficial protective effects. We compared the MD fat blend (high monounsaturated, 2:1 n-6:n-3 polyunsaturated and moderate saturated fat) to isocaloric diets composed with corn oil (CO, n-6 polyunsaturated-rich), olive oil (high monounsaturated-rich) or milk fat (MF, saturated-rich) on spontaneous colitis development in Muc2-/- mice. The MD resulted in lower clinical and histopathological scores, and induced tolerogenic CD103+CD11b+ dendritic, Th22 and IL-17+IL-22+ cells important for intestinal barrier repair. MD also reduced attendant insulin resistance and a shift to a higher health-promoting gut microbes including Lactobacillus B. animalis and Muribaculaceae, whereas CO showed higher prevalence of mucin-degraders (Akkermansia muciniphila) and colitis promoters (Enterobacteriaceae). Our findings suggest that the MD fat blend could be recommended as a maintenance diet for colitis.

immunology↗

bioRxiv2019

Identification and characterization of OmpT-like proteases in uropathogenic Escherichia coli clinical isolates

Bacterial colonization of the urogenital tract is limited by innate defenses, including the production of antimicrobial peptides (AMPs). Uropathogenic Escherichia coli (UPEC) resist AMP-killing to cause a range of urinary tract infections (UTIs) including asymptomatic bacteriuria, cystitis, pyelonephritis, and sepsis. UPEC strains have high genomic diversity and encode numerous virulence factors that differentiate them from non-UTI causing strains, including ompT. As OmpT homologues cleave and inactivate AMPs, we hypothesized that high OmpT protease activity-levels contribute to UPEC colonization during symptomatic UTIs. Therefore, we measured OmpT activity in 58 UPEC clinical isolates. While heterogeneous OmpT activities were observed, OmpT activity was significantly greater in UPEC strains isolated from patients with symptomatic infections. Unexpectedly, UPEC strains exhibiting the greatest protease activities harboured an additional ompT-like gene called arlC (ompTp). The presence of two OmpT-like proteases in some UPEC isolates led us to compare the substrate specificities of OmpT-like proteases found in E. coli. While all three cleaved AMPs, cleavage efficiency varied on the basis of AMP size and secondary structure. Our findings suggest the presence ArlC and OmpT in the same UPEC isolate may confer a fitness advantage by expanding the range of target substrates.

microbiology↗