Decision letter: Spreading of a mycobacterial cell-surface lipid into host epithelial membranes promotes infectivity

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Several virulence lipids populate the outer cell wall of pathogenic mycobacteria. Phthiocerol dimycocerosate (PDIM), one of the most abundant outer membrane lipids, plays important roles in both defending against host antimicrobial programs and in evading these programs altogether. Immediately following infection, mycobacteria rely on PDIM to evade Myd88-dependent recruitment of microbicidal monocytes which can clear infection. To circumvent the limitations in using genetics to understand virulence lipids, we developed a chemical approach to track PDIM during Mycobacterium marinum infection of zebrafish. We found that PDIM's methyl-branched lipid tails enabled it to spread into host epithelial membranes to prevent immune activation. Additionally, PDIM’s affinity for cholesterol promoted this phenotype; treatment of zebrafish with statins, cholesterol synthesis inhibitors, decreased spreading and provided protection from infection. This work establishes that interactions between host and pathogen lipids influence mycobacterial infectivity and suggests the use of statins as tuberculosis preventive therapy by inhibiting PDIM spread. Introduction Mycobacterium tuberculosis, the causative pathogen of the pulmonary disease tuberculosis (TB), is estimated to have evolved within the confines of the human lung for millennia (Comas et al., 2013). A result of this co-evolution is a choreographed response of innate and adaptive immune cells culminating in the formation of granulomas, specialized structures that permit bacterial replication and ultimately promote transmission (Ramakrishnan, 2012). A key strategy used by mycobacteria throughout infection is to avoid and manipulate host immune pathways so as to afford the pathogen safe harbor in otherwise bactericidal myeloid cells (Cambier et al., 2014a; Urdahl, 2014). To better understand these host–pathogen interactions, we have taken advantage of the optically transparent zebrafish larva, a natural host of the pathogen M. marinum (Ramakrishnan, 2020; Takaki et al., 2013). Infection of the hindbrain ventricle (HBV), an epithelium-lined cavity, allows for the visualization and characterization of the cellular immune response (Davis and Ramakrishnan, 2009), a response that is comparable to that seen in the mouse lung following infection with M. tuberculosis (Srivastava et al., 2014). In both models, mycobacteria are initially phagocytosed by tissue-resident macrophages and are eventually transferred to monocytes which go on to form granulomas (Cambier et al., 2017; Cohen et al., 2018). In order to reach growth-permissive cells, mycobacteria must first evade prototypical anti-bacterial monocytes. In response to mucosal commensal pathogens, bactericidal monocytes are recruited downstream of toll-like receptor (TLR) signaling (Medzhitov, 2007). Screening of M. marinum genetic mutants found that the cell-surface lipid phthiocerol dimycocerosate (PDIM) is required to evade this antibacterial response (Cambier et al., 2014b). PDIM also promotes pathogenesis in other ways, such as being required for the relative impermeability of the mycobacterial cell wall (Camacho et al., 2001) and in promoting escape from phagolysosomes (Augenstreich et al., 2017; Barczak et al., 2017; Lerner et al., 2017; Quigley et al., 2017). However, the molecular details underlying PDIM’s myriad pathogenic functions remain unknown. To accomplish mechanistic studies of virulence lipids, we and others developed metabolic labeling strategies where unnatural metabolic precursors are fed to growing bacteria (Siegrist et al., 2015). The unnatural metabolite contains a bioorthogonal functional group that facilitates visualization of macromolecules in living bacteria (Sletten and Bertozzi, 2009). An example is the labeling of trehalose containing lipids with azide-functionalized trehalose (Swarts et al., 2012). Here, we have developed comparable chemical tools to monitor PDIM’s distribution during infection. We found that the first step in PDIM-mediated pathogenesis is to spread into epithelial cells in order to prevent the recruitment of microbicidal monocytes. Structure function analysis revealed that PDIM’s methyl-branched fatty acids increased lipid mobility and promoted spread. Spreading was also dependent on the lipid content of host membranes. Administration of the cholesterol lowering drug, atorvastatin (Lipitor), led to a decrease in PDIM spreading, and subsequent resistance to mycobacterial infection. Our findings provide a mechanistic explanation for the association of statin use with a decrease in TB incidence (Lai et al., 2016) and support their use as a TB preventative therapy. Results Lipid removal and recoating of M. marinum PDIM lacks unique biosynthetic precursors to facilitate metabolic labeling (Onwueme et al., 2005). However, PDIM is removed following petroleum ether extraction (Moliva et al., 2019), a technique used to remove and add back mycomembrane lipids (Silva et al., 1985). Using this approach, we hypothesized that we could chemically install a biorthogonal handle onto extracted PDIM and use this modified lipid to elucidate the fundamental mechanisms underlying PDIM’s contribution to virulence. Similar to reports on M. tuberculosis and M. bovis, we validated that petroleum ether extraction did not affect the growth of M. marinum in culture (Figure 1—figure supplement 1A) and extracted lipids did not repopulate the mycomembrane following the first few days in culture (Figure 1—figure supplement 1B; Indrigo et al., 2003). Thus, this approach is well suited for loss of function studies of outer mycomembrane lipids in zebrafish. Extracted lipids could also be mixed with bacteria in petroleum ether followed by drying to recoat the bacterial surface (Figure 1A and Figure 1—figure supplement 1C). Evaluation by thin-layer chromatography and NMR demonstrated that the lipid composition of recoated bacteria was comparable to untreated bacteria (Figure 1—figure supplement 1D and Materials and methods). No protein was detected in the extracts (Figure 1—figure supplement 1E) suggesting minimal disruption of cell wall proteins. Following infection of zebrafish (Figure 1B) delipidated bacteria were attenuated for growth and this phenotype was rescued upon recoating (Figure 1C and D). Figure 1 with 1 supplement see all Download asset Open asset Lipid removal and recoating reveals that pre-infection PDIM reservoirs are required for M.marinum infection of zebrafish. (A) Model of lipid removal and recoating of M. marinum. (B) Model of zebrafish larva showing the hindbrain ventricle (HBV) injection site. (C) Representative images of the experiment in D (orange dots), wasabi (green) fluorescent protein expressing M. marinum in the HBV at 3 dpi are shown, scale bar = 50 μm. (D) Mean bacterial volume after HBV infection of wildtype fish with ~100 control, delipidated, or recoated M. marinum. (E) Model of lipid-swap experiment. (F) Mean bacterial volume at 3 dpi after HBV infection of wildtype fish with ~100 WT or ∆mmpL7 M. marinum treated as follows: non-extracted control (black), extracted and recoated with WT lipids (blue), or extracted and recoated with ∆mmpL7 lipids (orange). (G) Mean macrophage recruitment at 3 hpi of the HBV of wildtype or Myd88-depleted fish with ~100 ∆mmpL7 M. marinum as treated in F. (D), (F), and (G) representative of at least three separate experiments. Ordinary one-way ANOVA with (D) Sidak's multiple comparisons test for the comparison’s shown and (F) Tukey’s multiple comparisons test with selected adjusted P values shown. (G) Kruskal-Wallis ANOVA for unequal variances with Dunn’s multiple comparisons test with selected adjusted P values shown. Figure 1—source data 1 https://cdn.elifesciences.org/articles/60648/elife-60648-fig1-data1-v2.xlsx Download elife-60648-fig1-data1-v2.xlsx Pre-infection PDIM reservoirs are required for virulence M. marinum mutants in PDIM synthesis (∆mas) and localization to the mycomembrane (∆mmpL7) trigger TLR/Myd88-dependent immune responses (Cambier et al., 2014b). Myd88 signaling leads to the recruitment of activated monocytes that can clear bacteria in an inducible nitric oxide synthase-dependent fashion. PDIM-sufficient wildtype bacteria do not elicit this response and instead recruit a comparable number of permissive monocytes downstream of the chemokine CCL2 (Cambier et al., 2014b). However, since ∆mas and ∆mmpL7 M. marinum lack proteins required for PDIM’s synthesis or export, the associated phenotypes could be attributed to the missing proteins rather than to a lack of PDIM. While both of these mutants also lack the closely related phenolic glycolipid (Onwueme et al., 2005), results evaluating strains lacking only phenolic glycolipid ruled out this lipids role in mediating evasion of TLRs (Cambier et al., 2014b). To test if the lipid content on the bacterial surface is responsible for these phenotypes, we performed a lipid-swap experiment. Wildtype and ∆mmpL7 M. marinum were either untreated (control) or extracted and recoated with their native lipids or the lipids from the other strain (Figure 1E). Petroleum ether extraction of wildtype bacteria removed both dimycocerosic acid (DIM) containing lipids, PDIM, and its metabolic precursor phthiodiolone dimycocerosate (PNDIM, Figure 1—figure supplement 1F) both of which were absent in ∆mmpL7 extracts (Figure 1—figure supplement 1G). Following infection, wildtype control and wildtype bacteria recoated with wildtype lipids grew normally whereas wildtype bacteria recoated with ∆mmpL7 lipids were attenuated for growth (Figure 1F). Conversely, ∆mmpL7 bacteria were attenuated for growth, as expected, unless they were recoated with wildtype lipids, in which case they grew at wildtype bacterial rates (Figure 1F). Using an antisense morpholino to knockdown Myd88 (Bates et al., 2007), we also found that the dependence on Myd88 to recruit monocytes to ∆mmpL7 bacteria was abolished with wildtype lipids (Figure 1G). Taken together these experiments highlight the strengths of this chemical approach. Not only does it recapitulate known phenotypes of PDIM genetic mutants, but it directly links the mutant phenotypes to the mycomembrane composition. Furthermore, our data suggest that the PDIM present on the surface of the bacterium from the onset of infection is required and sufficient to promote virulence, as mutants unable to replenish PDIM on their surfaces become infectious when they are recoated with wildtype lipids. Synthesis of a clickable, biologically active PDIM Given the pathogenic importance of the pre-infection mycomembrane lipid content, we hypothesized that labeling this pool of PDIM would shed light on its virulence mechanisms. Both PDIM and its biosynthetic precursor PNDIM are present in the mycomembrane. The only difference between these lipids is their diol backbones; PDIM has a methyl ether, while PNDIM has a ketone (Siméone et al., 2007). Either lipid can promote infection in mice (Siméone et al., 2007), suggesting chemical flexibility at this site with regards to virulence. Therefore, we converted the methyl ether of PDIM to an alkyl halide with trimethylsilyl iodide (Jung and Lyster, 1977). Subsequent addition of sodium azide provided azido-DIM (Figure 2A). Recoating of delipidated bacteria with lipids containing azido-DIM, followed by a copper-free click reaction with the cyclooctyne fluorophore DIBO-488 (Figure 2B) resulted in a ~100-fold increase in fluorescence (Figure 2C). Confocal microscopy revealed the fluorescence to be membrane-associated (Figure 2D and E), suggesting incorporation into the mycomembrane. Importantly, we found that adding back native DIMs or azido-DIM to DIM-depleted lipids prior to recoating and labeling (Figure 2—figure supplement 1) rescued DIM-depleted bacteria’s growth attenuation (Figure 2F). Thus, with this approach we can generate bacteria with chemically functionalized PDIM that retain their pathogenicity. Figure 2 with 1 supplement see all Download asset Open asset Synthesis and application of a chemically tractable, biologically active PDIM variant, azido-DIM. (A) Synthesis of azido-DIM. (B) Model of delipidation and recoating of bacteria with or without azido-DIM followed by treatment with an azide-reactive cyclooctyne, DIBO-488. (C) Flow cytometry analysis of M. marinum recoated with or without azido-DIM treated with DIBO-488. Image of (D) native lipid control or (E) azido-DIM recoated bacteria treated with DIBO-488, scale bar = 8 μm. (F) Mean bacterial volume 3 days following HBV infection of wildtype fish with ~100 delipidated M. marinum recoated with Native, DIM-depleted (DIM–), DIM– plus native DIMs (+DIMs), or DIM– plus azido-DIM (+Azido-DIM) lipids. Kruskal-Wallis ANOVA for unequal variances with Dunn’s multiple comparisons test with selected adjusted P values shown. (C), (F) representative of three separate experiments. Figure 2—source data 1 https://cdn.elifesciences.org/articles/60648/elife-60648-fig2-data1-v2.xlsx Download elife-60648-fig2-data1-v2.xlsx PDIM spreads into macrophage membranes To visualize PDIM’s distribution, we infected zebrafish with blue-fluorescent M. marinum that were recoated with azido-DIM followed by labeling with DIBO-488 (DIM-488). DIM-488 spread away from bacteria into infected macrophage membranes (Figure 3—figure supplement 1A). Real-time imaging revealed that the spreading was dynamic in nature, with DIM-488 moving relative to host cells (Video 1). To better visualize spreading of PDIM into macrophage membranes, we used the transgenic zebrafish line Tg(mfap4:tdTomato) whose macrophages express the fluorescent protein tdTomato (Walton et al., 2015). As early as 3 hr post-infection (hpi), DIM-488 had spread into infected macrophage membranes, directly adjacent to infecting bacteria (Figure 3A, arrows) and at more distal membrane sites (Figure 3A, arrow heads). These data suggest that lateral diffusion as well as propagation into discrete membrane compartments by PDIM is taking place. Spreading increased across macrophage membranes by 3 days post-infection (dpi) (Figure 3B). Similar spreading was seen when azido-DIM was conjugated to DIBO-647 (Figure 3—figure supplement 1B and C), suggesting that the lipid, not the fluorescent probe, was responsible for this phenotype. To quantify the extent of PDIM spreading, we imaged the entire HBV infection site and calculated the proportion of fluorophore labeled azido-DIM that no longer localized with bacteria (Figure 3—figure supplement 1D). Using this number as a proxy for lipid spread, we saw an increase in spreading as infection progressed (Figure 3C). Spreading also occurred following infection of THP-1 macrophages in culture (Figure 3—figure supplement 1E). Finally, PDIM spreading was not a result of homeostatic lipid turnover. DIM-488 labeled bacteria were fluorescent following 3 days in culture (Figure 3D), and signal remained localized to the cell wall (Figure 3E). Thus, PDIM spreading away from bacteria only occurs following interactions with host cells. Figure 3 with 1 supplement see all Download asset Open asset PDIM spreads into macrophage membranes. Images of M. marinum expressing a cytosolic blue-fluorescent protein recoated with DIBO-488 labeled azido-DIM (DIM-488) at (A) 3 hpi and (B) 3 dpi of ~100 M. marinum in the HBV of transgenic fish whose macrophages express a fluorescent protein. Scale bar = 10 μm. Arrows, DIM-488 spread in vicinity of infecting bacteria, arrowheads, DIM-488 spread throughout macrophage. (C) Mean percent DIM-488 not localized with bacteria following HBV infection of wildtype fish with ~100 M. marinum. Kruskal-Wallis ANOVA for unequal variances with Dunn’s multiple comparisons test with selected adjusted P values shown. (D) Flow cytometry analysis of M. marinum expressing a cytosolic blue-fluorescent protein recoated with DIM-488 following 0 or 3 days in culture. Representative of two separate experiments. (E) Representative images of bacteria from D, scale bar = 3 μm. (F) Mean percent fluorescent signal not localized with bacteria following HBV infection of wildtype fish with ~100 control or recoated M. marinum labeled with periodate-hydroxylamine chemistry. Two-tailed, unpaired t test. (G) Mean percent DIM-488 not localized with bacteria following HBV infection of wildtype fish with ~100 wildtype or ∆RD1 M. marinum. Two-tailed Mann Whitney test for 0 dpi and two-tailed, unpaired t test for one dpi (H) Mean (+/- SEM) percentage of discrete bacterial objects remaining following HBV infection of wildtype fish with ~100 wildtype or ∆mmpL7 M. marinum. Representative of two separate experiments. (C), (F) and (G) representative of three separate experiments. Figure 3—source data 1 https://cdn.elifesciences.org/articles/60648/elife-60648-fig3-data1-v2.xlsx Download elife-60648-fig3-data1-v2.xlsx Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg PDIM dynamics. Real-time video of M. marinum expressing blue-fluorescent protein recoated with DIBO-488 labeled azido-DIM at 3 hpi of the HBV with ~100 M. marinum. These results were consistent with a recent report that M. tuberculosis PDIM occupies cultured macrophage membranes (Augenstreich et al., 2019). Nevertheless, we wanted to rule out spreading as an artifact of our recoating method. We used an established pan-glycolipid labeling method previously used to track mycobacterial glycolipids through macrophage membranes (Beatty et al., 2000). Control and recoated M. marinum were treated with periodate and then reacted with a fluorescent hydroxylamine prior to infection (Beatty et al., 2000). We found equal spreading of the total pool of fluorophore-labeled glycolipids (Figure 3F and Figure 3—figure supplement 1F). Thus, recoating does not appreciably influence the spreading dynamics of mycomembrane lipids. These data demonstrate that the introduction of a chemically functionalized PDIM into the mycomembrane provides relevant information regarding PDIM’s host distribution during infection. We next wanted to understand how PDIM spreading might be promoting virulence. PDIM has been suggested to interact with the protein substrates of the type VII secretion system ESX-1, including EsxA (Barczak et al., 2017). PDIM and EsxA are both required for cytosolic escape from phagolysosomes (Osman et al., 2020; Quigley et al., 2017; van der Wel et al., 2007), where PDIM is suggested to enhance the pore-forming activity of EsxA through its ability to infiltrate macrophage membranes (Augenstreich et al., 2017). Thus, we hypothesized that PDIM’s localization may be dependent on EsxA’s pore forming ability. Region of difference-1 M. marinum mutants (∆RD1) which lack EsxA (Volkman et al., 2010), were recoated with DIM-488 prior to zebrafish infection. There was no difference in DIM-488 spreading kinetics between wildtype and ∆RD1 M. marinum (Figure 3G), suggesting that EsxA does not influence PDIM spreading. Besides playing a role in cytosolic escape, PDIM has also been shown to promote phagocytosis of extracellular bacteria (Astarie-Dequeker et al., 2009; Augenstreich et al., 2019). To determine the phagocytosis rate of wildtype or ∆mmpL7 M. marinum in vivo, we measured the number of discrete bacterial objects over time. As bacteria are phagocytosed by macrophages, individual bacteria can no longer be discerned by confocal microscopy and the number of objects decreases. There was no measurable difference in the rate of phagocytosis of wildtype or ∆mmpL7 bacteria (Figure 3H). PDIM spreads into epithelial membranes Given the discrepancy regarding PDIM’s role in promoting phagocytosis between the cultured macrophage and zebrafish models, we wondered if the activation state of responding immune cells in zebrafish larvae was influencing their phagocytic capacities. One clue to the timing of PDIM’s role in virulence was the kinetics of the myeloid response. Wildtype bacteria needed to reside within resident macrophages in order to recruit permissive monocytes. In contrast, PDIM-deficient bacteria recruited microbicidal monocytes independent of and concurrent to resident macrophages (Cambier et al., 2017). Therefore, we wondered if PDIM plays a critical role in evading immune detection prior to any of its documented roles in modulating macrophages. Upon closer examination, we observed DIM-488 deposits on zebrafish epithelium at 24 hpi (Figure 4—figure supplement 1). Imaging at 3 hpi we captured extracellular bacteria having spread DIM-488 in the vicinity of an infected macrophage (Figure 4A). To better visualize spreading on these cells, we injected bacteria intravenously into the transgenic zebrafish line Tg(flk1:mcherry), which has a red-fluorescent vascular endothelium (Wang et al., 2010). We found that the DIM-488 from bacteria contacting endothelium had spread away from the bacteria onto the surrounding tissue (Figure 4B, arrows). To confirm PDIM spreading into epithelial membranes, we infected human A549 epithelial cells whose plasma membranes were labeled with Alexa-fluor 594 wheat germ agglutinin. We observed DIM-488 spreading into labeled epithelial plasma membranes (Figure 4C, arrows). Figure 4 with 1 supplement see all Download asset Open asset PDIM spreads into epithelial membranes. (A) Image of M. marinum expressing a cytosolic blue-fluorescent protein recoated with DIBO-488 labeled azido-DIM (DIM-488) DIM-488 spread from bacteria to epithelial cells at 3 hpi of ~100 M. marinum in the scale bar = 10 μm. (B) Image of DIM-488 labeled M. marinum at 1 infection of transgenic fish whose endothelium express a red-fluorescent protein. Arrows, DIM-488 spread onto scale bar = μm. (C) Image of A549 epithelial cells whose plasma membranes are labeled with Alexa-fluor 594 wheat germ at one infection with DIM-488 labeled M. marinum at an of Arrows, DIM-488 spread into plasma scale bar = μm. (D) Image DIM-488 spread onto epithelial surfaces at 2 hpi of ~100 M. marinum in the scale bar = 10 μm. (E) Mean percent DIM-488 in macrophage or epithelial cells not localized with bacteria following HBV infection with ~100 M. marinum. Representative of two separate experiments. (F) Mean percent DIM-488 not localized with bacteria following HBV infection of or treated fish with ~100 M. marinum. Two-tailed Mann Whitney test for 0 dpi and two-tailed, unpaired t test for one Representative of three separate experiments. Figure data 1 Download To the timing of PDIM spread into epithelial and macrophage membranes we imaged infected zebrafish at 2 hpi of the prior to macrophage and at and hpi by which the of bacteria reside within macrophages. any spreading onto macrophages, DIM-488 had spread onto epithelial cells by 2 hpi (Figure and Figure where bacteria are found within macrophages (Figure spreading is seen on macrophage membranes. These data suggest that mycobacteria spread PDIM onto epithelial cells prior to interactions with macrophages, and that this spread PDIM within these epithelial cells after bacteria are phagocytosed by macrophages. macrophages from zebrafish larva using et al., we that DIM-488 spreading still occurs in the of macrophages (Figure These data demonstrate that PDIM spreads onto epithelial cells independent of and prior to macrophage PDIM’s mobility promotes spread into epithelial cell membranes We to understand PDIM’s that its ability to spread into host membranes. is well established that increased membrane a by the mobility of individual membrane lipids, promotes membrane et al., studies from our found that the mycomembrane of Mycobacterium is by using fluorescence after of labeled trehalose et al., 2017). We this for M. marinum Using the metabolic (Swarts et al., followed by reaction with DIBO-488 to track we found following of with only of the labeled lipids being (Figure and Figure supplement 1A). we PDIM, we found DIM-488 to be with a of 3 and of the signal being (Figure and Figure supplement Thus, PDIM lipids are more than lipids. Figure with 2 see all Download asset Open asset PDIM’s mobility promotes spread into epithelial cell membranes. (A) Representative images of DIM-488 and labeled M. scale bar = 2 μm. (B) after of DIM-488 or labeled M. the signal from = 10 cells. (C) Mean which is the following of data in to a with a (D) Mean percent DIM-488 or in macrophage or epithelial cells not localized with bacteria following HBV infection with ~100 M. marinum. (E) Mean percent DIM-488 or not localized with bacteria 24 hr following HBV infection of or treated fish with ~100 M. marinum. (F) after of or plus DIM-488 labeled M. the signal from = cells. (G) Mean which is the following of data in to a with a (H) Mean percent DIM-488 in macrophage or epithelial cells not localized with bacteria 2 hr following HBV infection with ~100 M. marinum treated as in F. Images of or treated DIM-488 labeled M. marinum at 2 hpi of the HBV with ~100 bacteria, scale bar = μm. Mean percent DIM-488 not localized with bacteria 24 hr following infection of treated fish or A549 epithelial cells with or DIM-488 labeled M. marinum. (C), and two-tailed, unpaired t test. and (H) one-way ANOVA with Tukey’s multiple comparisons test with selected adjusted P values shown. and representative of three separate experiments. Figure data 1 Download increased membrane and lipid mobility promote membrane then not be to spread into host cells as as PDIM we found that to spread onto epithelial cells at 2 hpi (Figure when bacteria are within macrophages at 24 hpi was spreading detected (Figure and Figure supplement 1C). in the of macrophages, where bacteria have a with epithelial cells, spreading was (Figure Thus, lipid mobility with the ability to spread into host epithelial membranes. To determine if is a between mobility and spreading, we used to that decreased PDIM’s after chemical (Figure and Figure supplement However, with a more for membrane-associated proteins et al., resulted in an in the of DIM-488 (Figure and Figure supplement treatment decreased DIM-488 spread onto epithelial cells at 2 hpi (Figure and DIM-488 did not spread into epithelial membranes following a 24 hr infection of zebrafish (Figure did it spread onto A549 epithelial cells after 24 hr (Figure these results suggest that PDIM’s mobility promotes spreading into epithelial membranes. PDIM’s methyl-branched acids promote mobility and spreading To test if PDIM’s mobility promotes its spreading, we to advantage of our ability to PDIM’s Lipid is known to lipid mobility and membrane and lipids form closely to more while lipids do not as and more on otherwise lipids have also been shown to increase membrane et al., et al., 2014). Therefore, we hypothesized that PDIM’s methyl-branched acids enhance its To test