Evolved Resistance to a Novel Cationic Peptide Antibiotic Requires High Mutation Supply

Background and Objectives A key strategy for resolving the antibiotic resistance crisis is the development of new drugs with antimicrobial properties. The engineered cationic antimicrobial peptide WLBU2 (also known as PLG0206) is a promising broad-spectrum antimicrobial compound that has completed Phase I clinical studies. It has activity against Gram-negative and Gram-positive bacteria including infections associated with biofilm. No definitive mechanisms of resistance to WLBU2 have been identified. Methodology Here, we used experimental evolution under different levels of mutation supply and whole genome sequencing (WGS) to detect the genetic pathways and probable mechanisms of resistance to this peptide. We propagated populations of wild-type and hypermutator Pseudomonas aeruginosa in the presence of WLBU2 and performed WGS of evolved populations and clones. Results Populations that survived WLBU2 treatment acquired a minimum of two mutations, making the acquisition of resistance more difficult than for most antibiotics, which can be tolerated by mutation of a single target. Major targets of resistance to WLBU2 included the orfN and pmrB genes, previously described to confer resistance to other cationic peptides. More surprisingly, mutations that increase aggregation such as the wsp pathway were also selected despite the ability of WLBU2 to kill cells growing in a biofilm. Conclusions and implications The results show how experimental evolution and WGS can identify genetic targets and actions of new antimicrobial compounds and predict pathways to resistance of new antibiotics in clinical practice

Excess Dietary Sugar Alters Colonocyte Metabolism and Impairs the Proliferative Response to Damage

Background & Aims The colonic epithelium requires continuous renewal by crypt resident intestinal stem cells (ISCs) and transit-amplifying (TA) cells to maintain barrier integrity, especially after inflammatory damage. The diet of high-income countries contains increasing amounts of sugar, such as sucrose. ISCs and TA cells are sensitive to dietary metabolites, but whether excess sugar affects their function directly is unknown. Methods Here, we used a combination of 3-dimensional colonoids and a mouse model of colon damage/repair (dextran sodium sulfate colitis) to show the direct effect of sugar on the transcriptional, metabolic, and regenerative functions of crypt ISCs and TA cells. Results We show that high-sugar conditions directly limit murine and human colonoid development, which is associated with a reduction in the expression of proliferative genes, adenosine triphosphate levels, and the accumulation of pyruvate. Treatment of colonoids with dichloroacetate, which forces pyruvate into the tricarboxylic acid cycle, restored their growth. In concert, dextran sodium sulfate treatment of mice fed a high-sugar diet led to massive irreparable damage that was independent of the colonic microbiota and its metabolites. Analyses on crypt cells from high-sucrose–fed mice showed a reduction in the expression of ISC genes, impeded proliferative potential, and increased glycolytic potential without a commensurate increase in aerobic respiration. Conclusions Taken together, our results indicate that short-term, excess dietary sucrose can directly modulate intestinal crypt cell metabolism and inhibit ISC/TA cell regenerative proliferation. This knowledge may inform diets that better support the treatment of acute intestinal injury

Endosomal MR1 Trafficking Plays a Key Role in Presentation of Mycobacterium tuberculosis Ligands to MAIT Cells

Mucosal-Associated Invariant T (MAIT) cells, present in high frequency in airway and other mucosal tissues, have Th1 effector capacity positioning them to play a critical role in the early immune response to intracellular pathogens, including Mycobacterium tuberculosis (Mtb). MR1 is a highly conserved Class I-like molecule that presents vitamin B metabolites to MAIT cells. The mechanisms for loading these ubiquitous small molecules are likely to be tightly regulated to prevent inappropriate MAIT cell activation. To define the intracellular localization of MR1, we analyzed the distribution of an MR1-GFP fusion protein in antigen presenting cells. We found that MR1 localized to endosomes and was translocated to the cell surface upon addition of 6-formyl pterin (6-FP). To understand the mechanisms by which MR1 antigens are presented, we used a lentiviral shRNA screen to identify trafficking molecules that are required for the presentation of Mtb antigen to HLA-diverse T cells. We identified Stx18, VAMP4, and Rab6 as trafficking molecules regulating MR1-dependent MAIT cell recognition of Mtb-infected cells. Stx18 but not VAMP4 or Rab6 knockdown also resulted in decreased 6-FP-dependent surface translocation of MR1 suggesting distinct pathways for loading of exogenous ligands and intracellular mycobacterially-derived ligands. We postulate that endosome-mediated trafficking of MR1 allows for selective sampling of the intracellular environment.Career Development Award: (#IK2 CX000538); U.S. Department of Veterans Affairs Clinical Sciences Research and Development Program (MJH); U.S.Department of Veterans Affairs Biomedical Laboratory Research and Development Program (DML) Merit Award: (#I01 BX000533); American Lung Association: (RT-350058)

Excess dietary sugar directly alters epithelial metabolism and macrophage polarization resulting in lethal colitis

The intestinal barrier is exposed to trillions of bacteria and dietary metabolites every day, requiring both continuous renewal of the epithelium and surveillance by resident immune cells. As the first line of cellular defense, intestinal epithelium must regenerate every 3-5 days via intestinal stem cells (ISCs) to maintain barrier integrity, especially after inflammatory damage. Macrophages are critical first responders when the barrier is breached and must play a balancing act between eradicating threats to the host while also maintaining an environment that promotes healing. Recent studies have explored how diet can alter not only the microbiome and corresponding immune responses, but also have direct effects on host cells that influence proliferative and inflammatory responses. Given the significant increase in consumption of processed foods over the last two hundred years, we were interested in how excess dietary sugar affects mouse models of colitis. We hypothesized that expansion of sucrose-consuming bacteria would drive a pro-inflammatory intestinal immune response, exacerbating disease. However, we found that sugar-induced exacerbation of colitis was lymphocyte-independent, rather, sugar reduced the number of M2 colonic macrophages and reduced the proliferative gene signature of ISCs. Using 3-dimensional colonoids, we demonstrated sugar directly inhibits colonoid development and alters metabolic pathways. Restoring metabolic flux of glycolytic metabolites by inhibiting pyruvate dehydrogenase kinase rescued sugar-impaired colonoid development. We validate these effects in v vivo using a mouse-model fed high-sucrose (HS) or high-fiber diets. Crypts isolated from HS-fed mice have an increased glycolytic response, yet also exhibit greater spare respiratory capacity, or unused oxidative potential, demonstrating inefficient glucose utilization for oxidative respiration. To recapitulate the rapid proliferative response of developing colonoids, we treated mice with dextran sodium sulfate (DSS) to induce colonic damage. Lineage tracing experiments showed reduced number and migration of daughter cells after DSS in mice fed HS. As a result, mice fed a HS diet failed to repair DSS-induced colonic damage, resulting in lethal intestinal pathology. Our results indicate that short-term, excess dietary sugar can directly inhibit epithelial proliferation in response to damage and may inform diets that better support the treatment of acute intestinal injury

Stx18, VAMP4, and Rab6 silencing alter TGN structure and intracellular MR1 localization.

<p>a) BEAS-2B cells treated with missense, Stx18, Rab6, or VAMP4 siRNA were transfected with pCI:MR1-GFP for 48 hours, then fixed and imaged. Shown are representative images of the cells used to enumerate MR1-GFP⁺ EC. TGN46 staining is shown at 1.5x magnification in greyscale below each image to demonstrate dispersion or contraction of the TGN. b) MR1-GFP⁺ EC were identified and quantified as previously described. On the left, each dot represents the number of MR1-GFP⁺ EC in a single cell, and the data shown is representative of 4 independent experiments. On the right, each dot represents the mean intensity of GFP signal in one individual MR1-GFP⁺ EC. The data represent all endosomes from the cells plotted on the left, and are representative of 4 independent experiments. The Mann-Whitney test was used to determine statistical significance, *p<0.01.</p

Cell surface translocation of MR1 in the presence of ligand.

<p>a) BEAS-2B or primary NHBE cells were transfected with pCI:MR1-GFP and incubated for 30 hours. Transfected cells were then incubated with 6-FP for 18 hours. For imaging, cells were fixed and surface stained with α-MR1 (26.5), then imaged. In NHBE cells, MR1 surface staining (red) in MR1-GFP-expressing cells (green) is denoted by the arrows in the enlarged insets. Images shown are representative of at least three independent experiments. b) BEAS-2B or NHBE cells were treated as described in a). Following 6-FP treatment cells were harvested and surface stained on ice with α-MR1 (26.5). After staining, cells were fixed and analyzed by flow cytometry. Data shown are representative of at least three independent experiments. Where indicated, cells were treated with 100ng/ml brefeldin A (BFA) for 2 hours prior to 6-FP addition. Shown is a representative histogram demonstrating BFA blockade of 6-FP-dependent surface stabilization and the geometric mean and SEM of from three independent experiments. c) BEAS-2B or NHBE cells were transfected with pCI:MR1-GFP and treated with 6-FP as described above. Where indicated, cells were treated with10ug/ml cycloheximide (CHX) for 2 hours prior to 6-FP addition. Cells were fixed, and where indicated, stained with α-β2M, and imaged. Shown are results from one of at least three independent experiments. The Mann-Whitney test was used to determine the statistical significance of CHX treatment. For all other statistical comparisons, a Student’s t-test was used, *p<0.01. d) MR1-GFP⁺ β2M⁺ EC were identified and quantified as described in the Materials and Methods. Each dot represents the number of endosomes in one cell for any of the conditions. Shown are results from one of at least three independent experiments, *p<0.01.</p

Identification of trafficking molecules involved in presentation of Mtb ligands on MR1.

<p>a) A lentiviral shRNA library of trafficking molecules was screened by IFNγ ELISPOT assay with MR1, HLA-E, and HLA-B45 restricted T cell clones as described. Each bar represents separate shRNA wells and each well was analyzed individually in relation to the mean and characterized as a hit if it was at least 25% below the mean. The mean was normalized to 100% and is represented by red line. Results from shRNA knockdown of Rab2b, Rab7L1, or Stx18 from three independent experiments are shown. Stars indicate gene specific shRNA wells that met the candidate selection criteria. Candidates were selected if at least 2 of 5 independent shRNAs resulted in reduced T cell response. b) BEAS-2B cells were treated with missense or Stx18 siRNA, infected with Mtb (MOI:5), and used as APC in an IFNγ ELISPOT assay with 1x10⁴ MR1, HLA-E, or HLA-B45 restricted T cells clones per well. Shown are the results using 5 x 10³ APC that have been normalized to the response to the Missense siRNA treated cells (100%). **p<0.01. Error bars represent the mean and SEM from four independent experiments. qRT-PCR was performed on cDNA synthesized from mRNA isolated from missense or Stx18 siRNA silenced BEAS-2B cells that were uninfected or infected with Mtb (MOI:5). Error bars represent the mean and SEM for three independent experiments. Mtb colony forming units (CFU) from 1x10⁵ infected cells were determined by lysing and plating serial dilutions on 7H10 agar plates. Error bars represent the mean and SEM from 3 independent experiments. c) NHBE cells were treated with missense or Stx18 siRNA and infected with Mtb (MOI:10). IFNγ ELISPOT and qRT-PCR analyses were performed as described for BEAS-2B cells. Error bars represent the mean and SEM from two independent experiments. d) BEAS-2B cells were treated with missense, VAMP4, or Rab6 siRNA and infected with Mtb (MOI:5). IFNγ ELISPOT and qRT-PCR analyses were performed as described above. Error bars represent the mean and SEM from at least three independent experiments, *p<0.01 compared to missense. e) BEAS-2B cells were treated with missense, Sec22b, BNIP1, or Use1 siRNA and infected with Mtb (MOI:5). IFNγ ELISPOT and qRT-PCR analyses were performed as described above. Error bars represent the mean and SEM from at least two independent experiments, *p<0.01 compared to missense.</p

Mtb-derived ligands are loaded on MR1 using a pathway distinct from that of 6-FP.

<p>a) BEAS-2B cells treated with missense, Stx18, Rab6 or VAMP4 siRNA were transfected with pCI:MR1-GFP for 30 hours, then incubated or not with 6-FP for 18 hours. Cells were then harvested, surface stained with α-MR1 (26.5) and examined by flow cytometry. Cells were gated on expression of MR1-GFP based on an untransfected control. Shaded histograms represent the isotype control. Black solid histograms represent the cell surface expression of MR1 after treatment with missense control siRNA, while the dashed histograms represent the cell surface expression of MR1 in the after treatment with the indicated siRNA. Data is representative of three independent experiments. b) For Stx18, BEAS-2B cells were transfected with MR1-GFP and incubated for 24 hours, then transfected with Stx18-RFP and incubated for 16 hours. For VAMP4, BEAS-2B cells stably expressing MR1-GFP were transfected with VAMP4-RFP and incubated for 18 hours. Cells co-expressing MR1-GFP and Stx18 or VAMP4 were imaged live. Images are representative of at least three independent experiments. c) BEAS-2B cells were infected with Mtb (MOI:8) for 18 hours or incubated with supernatant from <i>M</i>. <i>smegmatis</i> cultures (Msm Sup). Where indicated, cells were pretreated with 6-FP for 2 hours prior to infection or addition of supernatant. Cells were then used as APC in an IFNγ ELISPOT assay with IFNγ production by an MR1-restricted T cell clone as a readout for antigen presentation. Data are representative of two independent experiments, *p<0.01, n.s. not significant.</p

MR1 candidates evaluated using siRNA gene knockdown.

<p>MR1 candidates evaluated using siRNA gene knockdown.</p