Rifampicin Or Capreomycin Induced Remodelling of the Mycobacterium

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Rifampicin or capreomycin induced remodelling of the Mycobacte- rium smegmatis mycolic acid layer is mitigated in synergistic combinations with cationic antimicrobial peptides

DeDe Kwun-Wai Man1,2, Tokuwa Kanno1, Giorgia Manzo1, Brian D. Robertson3, Jenny K.W. Lam2 and A. James Mason1*

1Institute of Pharmaceutical Science, School of Cancer & Pharmaceutical Sciences, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London, SE1 9NH, United Kingdom 2Department of Pharmacology & Pharmacy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong 3MRC Centre for Molecular Bacteriology and Infection, Department of Medicine, Imperial College London, London SW7 2AZ, United Kingdom KEYWORDS rifampicin, capreomycin, Mycobacteria, NMR metabolomics, synergy, tyloxapol

ABSTRACT: The mycobacterial cell wall affords natural resistance to antibiotics. Antimicrobial peptides (AMPs) modify the surface properties of mycobacteria and can act synergistically with antibiotics from differing classes. Here we investigate the response of Mycobacterium smegmatis to the presence of rifampicin or capreomycin, either alone or in combination with two synthetic, cationic, α-helical AMPs; distinguished by the presence (D-LAK120-HP13) or absence (D-LAK120-A) of a kink-inducing proline. Using a combination of high-resolution magic angle spinning (HR-MAS) NMR of bacteria, diphenylhexatriene (DPH) fluorescence anisotropy and laurdan emission spectroscopy we show that M. smegmatis responds to challenge with rifampicin or capreomycin by substantially altering its metabolism and, in particular, by remodelling the cell envelope. In NMR spectra of bacteria, reductions in intensity for mycolic acid lipid –(CH2)-, -CH3, R2CH-COOH, R2CH-OH and also -CH2–(CH=CH)- and -CH=CH- resonances were observed following challenge with rifampicin and capreomycin, while the latter also caused an increase in trehalose. These changes are consistent with a reduction of trehalose dimycolate and increase of trehalose monomycolate and are associated with an increase in rigidity of the mycolic acid layer observed following challenge by capreomycin but not rifampicin. Challenge with D-LAK120-A or D-LAK120-HP13 induced no or modest changes respectively in these metabolites and did not induce a significant increase in rigidity of the mycolic acid layer. Further, the response to rifampicin or capreomycin was significantly reduced when these were combined respectively with D-LAK120-HP13 and D-LAK120-A, suggesting a possible mechanism for the synergy of these combinations. The remodelling of the mycomembrane in M. smegmatis is therefore identified as an important countermeasure deployed against rifampicin or capreomycin, but this can be mitigated, and rifampicin or capreomycin efficacy potentiated, by combining with AMPs.

Introduction Mycobacterium spp. are responsible for a variety of diseases in- inner (6-7 nm) membranes, a layer of arabinogalactan-peptidogly- cluding tuberculosis, leprosy, pulmonary disease, lymphadenitis, can (6-7 nm) and a periplasm (14-17 nm) containing lipomannan skin and disseminated diseases. Although tuberculosis incidence is and lipoarabinomannan resulting in a cell envelope between 33 and falling globally at a rate of c 2% a year, it still carries the greatest 39 nm thick.4,5 The emergence of resistance to the limited number worldwide disease burden with 10.4 million people falling ill with of existing therapies for tuberculosis has focussed attention on the TB in 2016 and 1.7 million deaths.1 Infections due to non-tubercu- particular problems associated with finding drugs capable of lous mycobacteria are also increasingly recognised.2 Although the breaching the mycobacterial cell wall, since this may offer a means reduced global incidence of tuberculosis is welcome, first line ther- of either directly killing mycobacterial pathogens or of re-sensitiz- apies for tuberculosis are increasingly failing with 600,000 new ing them to existing first-line antibiotics. cases of tuberculosis resistant to the most effective first-line antibiotic, rifampicin. Of these, 490,000 cases were multi-drug resistant In this regard, antimicrobial peptides are of considerable interest (MDR) and a substantial proportion of these are extensively drug- since their mechanisms of action are often associated with either direct damage to bacterial plasma membranes and/or penetration resistant (XDR). While 95% of tuberculosis deaths occur in low- 6 and middle-income countries (LMICs), tuberculosis is also an in- within the bacterial cytoplasm to access intracellular targets , and creasing problem in the developed world where reactivation of la- these actions often result in rapid bacterial cell death – a property tent tuberculosis infections is of particular concern.3 Mycobacteria that may be desirable in reducing the incidence of resistance to AMPs. Antimicrobial peptides are therefore increasingly being are intrinsically resistant to many antibiotics that are effective 7,8 against other bacteria and this is thought to be due to the superior evaluated as anti-tuberculosis agents. protection offered by both mycobacterial outer (7-8 nm thick) and

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Figure 1. AMPs render Mycobacterium smegmatis permeable to fluorescein isothiocyanate (FITC) - dextran. Confocal microscope images of 1.1 x 108 CFU/ml M. smegmatis mc2 155 either unchallenged (A) or challenged with rifampicin (D), ½ MIC (B/C) or 2 x MIC (E/F)), D-LAK120-A (B/E) or D-LAK120-HP13 (C/F). In contrast to rifampicin, both peptides facilitate absorption or adsorption of the 150 kDa fluorescently labelled dextran. Indeed, not only have antimicrobial peptides been evaluated in iso- against mycobacteria is likely to be distinct from that which is ef- lation, combinations of cationic α-helical peptides with first line fective against Gram-negative bacteria. Consequently, our under- anti-mycobacterial agents have already been shown to be effec- standing of the structural features that promote anti-mycobacteria tive.9 All D-amino acid isomers of these peptides displayed im- activity is poorly developed. proved stability and enhanced mycobacterial selectivity.10 To address this gap in our understanding we have investigated in Our own research has focused on a series of highly cationic antimi- more detail how Mycobacterium smegmatis responds to challenge crobial peptides, comprising D-amino acids, rationally designed to with proline free (D-LAK120-A) and proline containing (D- adopt α-helix conformations within biological membranes.11 These LAK120-HP13) D-LAK peptides. We examined the activity of, AMPs have a detergent like ability enabling disruption of colonies and bacterial response to, combinations of each peptide with rifam- of Mycobacterium tuberculosis H37Ra11, are able to inhibit growth picin or capreomycin to investigate the mechanism of the observed, of MDR and XDR strains of M. tuberculosis when cultured in THP- albeit modest, synergy. Testing the hypothesis that the membrane 1 macrophage and potentiate the activity of first-line antibiotic iso- activity of the D-LAK peptides is key to both their anti-mycobac- niazid in vitro.12 The D-LAK peptides were designed to be cationic terial activity and their ability to potentiate the activity of rifam- and amphipathic, with the angle subtended by the positively picin and capreomycin, we incorporate high resolution magic angle charged lysine residues when the peptide adopts an idealised α-he- spinning (HR-MAS) NMR metabolomics with fluorescence spec- lix conformation, modified to enhance disruption of anionic model troscopy of M. smegmatis membrane-incorporated trans-1,6-diphe- membranes.11 Furthermore, the role of conformational flexibility nyl-1,3,5-hexatriene (DPH) and laurdan dyes which are sensitive to was investigated and proline residues were introduced to disrupt changes in membrane fluidity and order, respectively. These tech- the α-helix. The positioning of this proline kink was shown to be niques are able to distinguish between the two D-LAK peptides and important and could enhance activity against Gram-negative bacte- reveal that, when challenged with sub-inhibitory concentrations of ria while also mitigating haemolysis.11 Conformational flexibility rifampicin or capreomycin, M. smegmatis remodels its membrane. is a key property of potent antimicrobial peptides, such as pleuro- These observations suggest why and how antimicrobial peptides cidin, which is associated with greater penetration of bacterial may act and may be modified to improve their ability to potentiate membranes and the ability to reach intracellular targets.12,13 A tran- existing anti-mycobacterial treatments. scriptomic and NMR metabolomic approach to understanding the mechanism of action of D-LAK120-HP13 supports an ability to penetrate within Gram-negative bacteria.13 Interestingly however the proline containing D-LAK peptides did not always outperform their proline free analogues when tested against Mycobacterium tuberculosis H37Ra highlighting that the mechanism of action bioRxiv preprint doi: https://doi.org/10.1101/269324; this version posted February 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Materials and Methods FITC-labelled dextran (Sigma Aldrich, Cat. #46946) and D-AMPs

2 (D-LAK 120 A or D-LAK 120 HP13) or rifampicin at the respec- Mycobacterium smegmatis mc 155 was grown in Middlebrook tive MICs for 20 min. Following treatment, the bacterial cells were 7H9 broth (Difco, Detriot, Michigan, USA, Cat.# 271310) supple- centrifuged at 7500 rpm for 10 min and washed three times with mented with 10% (v/v) oleic acid-albumin-dextrose-catalase PBS. Cells were then fixed in 2% formaldehyde at R.T for 20 min. (OADC) enrichment (Becton Dickinson, Franklin Lakes, New Jer- After fixation, cells were washed twice and re-suspended in PBS. sey, USA, Cat.# 212240) and 0.5% (v/v) glycerol (Sigma Aldrich, 20 µl of sample were then allowed to air-dry on microscope slides Cat.#G-5516) in 50 ml Falcon tubes at 37°C without shaking. overnight. Samples were then imaged with a 65 x oil-immersion 0.025% (v/v) Tyloxapol surfactant (Sigma Aldrich, Cat.# T8761) objective lens using a Zeiss LSM-510 inverted confocal micro- was added as one of the treatments. Rifampicin (RIF; R3501) and scope (Carl Zeiss Inc.) FITC was excited with a 488 nm laser and capreomycin sulfate (CAP) from Streptomyces capreolus (C4142) detected with a 505 nm long-pass filter. were from Sigma Aldrich. D-AMPs D-LAK120-A 2 (KKLALALAKKWLALAKKLALALAKK-NH2) and D-LAK120- Transmission electron microscopy (TEM). M. smegmatis mc 155 7 HP13 (KKALAHALKKWLPALKKLAHALAKK-NH2) were sup- (1 × 10 CFU/ml) at mid-log phase (O.D. = 0.6) was treated with plied by China peptides Co. Ltd. (Shanghai, China). The purity of D-LAK120-A and D-LAK120-HP13 at their MICs respectively for synthesized peptides was above 80% and the peptides were used as 5 and 30 min. Samples were washed three times by PBS and cell supplied. Both peptides were amidated at the C-terminus. pellets were collected by centrifugation. Cell pellets were then fixed in 2.5% glutaraldehyde till further processing. Samples were Minimum inhibitory concentration (MIC) and Fractional inhibi- rinsed thrice with piperazine-N,N’-bis(ethanesulfonic acid); 1,4-pi- tory concentration (FIC) MIC of the anti-TB agents (rifampicin, D- 2 perazinediethanesulfonic acid (PIPES buffer) (Sigma Aldrich Cat.# AMPs and capreomycin) against M. smegmatis mc 155 was deter- P6757) followed by second fixation in 1% osmium tetroxide mined using broth micro-dilution assay in 96-well plates. Two-fold (OsO4) (Sigma Aldrich Cat.# 75632) for 1 h at R.T. 1st embedding serial dilutions were made in Middlebrook 7H9 broth supple- into agar was done to reduce sample loss. Pellets were then dehy- mented with oleic albumin dextrose catalase (OADC) and glycerol drated using ethanol (EtOH) (50, 70, 90% for 10 min each and in duplicate. An inoculum at an OD600 of 0.02 was prepared by di- 3 × 100% for 20 min). After dehydration, samples were infiltrated luting mid-log cultures and 100 μl was added in each well (~1 x 5 using 1:1 epoxy resin/ propylene oxide mixture overnight at 37 °C. 10 cfu). D-AMPs were prepared at 128 µM to 1 µM in Middle- The next day, pellets were infiltrated with fresh epoxy resin for 1h brook broth. RIF was serially diluted from 100 µg/ml to 0.195 at 37 °C followed by polymerization at 60 °C overnight in plastic µg/ml. Capreomycin was were prepared at 5 µg/ml to 0.004 µg/ml. moulds. Samples were viewed using Philips CM100 TEM 100 µl of mycobacterial suspension and anti-TB agent were added equipped with a TENGRA 2.3K X 2.3K camera. in each well to obtain the inoculum concentration of 5 x 104 CFU/ml. Growth control without drug and sterile medium were Anti-TB agent challenge assays. To evaluate the mechanism of ac- prepared in each assay. The plates were then incubated at 37°C for tion of anti-TB agents on bacterial growth, a ¾ MIC challenge as- 24h before adding 30 µl of 0.02% (w/v) resazurin dye and the col- say was conducted to observe the growth response of M. smegmatis our change was evaluated after incubation for another 24h. Resaz- mc2 155. 1% inoculum in the presence of ¾ MIC of each anti-TB urin assay is a colorimetric assay in which viable cells would re- agent, alone or in combination, was cultured in 10 ml Middlebrook duce blue resazurin (oxidized state) into fluorescent pink resorufin broth medium at 37°C. After 5 days, the bacteria were processed (reduced state). MIC of each anti-TB agent was determined as the and subjected to trans-1,6-diphenyl-1,3,5-hexatriene (DPH) fluo- lowest concentration that prevented growth, and therefore re- rescence assay, laurdan fluorescence emission and nuclear mag- mained blue. netic resonance (NMR) metabolomics analyses. Fractional inhibitory concentration (FIC) of the combination treat- Trans-1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence assay. ment of rifampicin or capreomycin and D-AMPs was determined M. smegmatis mc2 155 bacterial suspension was harvested and using a checkerboard assay.15 Synergistic interactions between fixed by 0.25% formaldehyde for 1 h at room temperature. After anti-TB agents in combination treatment was denoted by the Frac- fixing, the bacterial cells were centrifuged at 5000 rpm, 4 ̊C for 8 tional inhibitory concentration (FIC) index. FIC values of each min then washed by 5 ml phosphate buffered saline (PBS) once. combination were calculated from the following formula: Bacteria were re-suspended in PBS and stained by 2.5 µM DPH fluorescence probe. All tubes were wrapped with foil to prevent FIC index = FICA + FICB = [A] in combination/ MICA + [B] in combination/ light degradation of the dye and incubated at 37 ̊C for 30 min. Sam- MICB ples were added into the cuvette and excited at 358 nm using a Var- Where [A] is the lowest inhibitory concentration of D-AMPs in the ian Cary Eclipse Fluorescence Spectrophotometer. Signals were presence of RIF or CAP, MICA is the MIC of D-AMPs treated as a collected by single wavelength scanning at emission wavelength single agent and FICA is the FIC of D-LAK peptides; [B], MICB 430 nm. The spectral bandwidth of the emission monochromator and FICB are the corresponding values for RIF or CAP. Resembling and the measurement of emission spectra are set at 10 nm. Steady- a coordinate plane, the 96-well plate was set into x- and y-axis state fluorescence anisotropy, r, was calculated as which were designated for the addition of D-AMPs and RIF or CAP 퐼VV − 퐺퐼VH respectively. Two-fold serial dilutions of each anti-TB agent [x- 푟 = 퐼VV − 2퐺퐼VH axis: D-AMPs (4 µM to 0 µM); y-axis: RIF (100 µg/ml to 0 µg/ml); CAP (5 µg/ml to 0 µg/ml)] was added into the designated wells to where IVV and IVH are the parallel and perpendicular polarized flu- give combination treatments as follows: D-LAK120-A; RIF, D- orescence intensities measured with the vertically polarized excita- LAK120-A; CAP, D-LAK120-HP13; RIF and D-LAK120- tion light, IHV and IHH are the same fluorescence intensities meas- HP13;CAP. Mycobacterial suspension was prepared as described ured with the excitation light horizontally polarized, and G is the 5 above and 1 x 10 CFU/ml was seeded in each well. After 24h, the monochromator grating correction factor given by G = IHV/IHH. resazurin assay was performed and FIC was determined as the low- Laurdan fluorescence assay. Laurdan (6-dodecanolyl—N,N-dime- est combination concentrations that remained blue. thyl-2-napthtylamine) M. smegmatis mc2 155 bacterial suspension Confocal microscopy. M. smegmatis mc2 155 (1.1 × 108 CFU/ml) was harvested and fixed by 0.25% formaldehyde for 1 h at room mid-log phase (O.D. = 0.6) was treated with 250 µg/ml 150 kDa bioRxiv preprint doi: https://doi.org/10.1101/269324; this version posted February 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. temperature. The bacterial pellet was then fixed, washed and resus- based on previous published work.17 Principal component analysis pended as stated previously. The suspension was then stained by (PCA) and orthogonal partial least squares discriminant analysis 2.5 µM Laurdan fluorescence probe. All tubes were wrapped with (OPLS-DA) were done using MVAPACK18 and a software devel- foil to prevent light degradation of the dye and incubated at 37 ̊C oped in our previous study.17 The software involves the use of Py- for 1 h. Samples were added into the cuvette and excited at 350 nm thon programming language with NumPy and SciPy for calcula- using Varian Cary Eclipse Fluorescence Spectrophotometer. Sig- tions and visualization is performed by matplotlib. To minimize the nals were collected at emission wavelength range of 400 to 600 nm noise content, regions beyond 10.5 ppm, below 0 ppm and water (no. of scans = 20). General polarization (GP) value corresponding peak were excluded. These spectra were subjected to probabilistic 19 to the fluidity of membrane was calculation as below where I440 and quotient normalization (PQN) and autoscaled. Icoshift algorithm I490 are the emission intensities at corresponding wavelengths: was applied for further alignment and optimized bucketing algorithm with a 0.005 ppm bin size was employed for bucketing. 퐼440 − 퐼490 Cross-validation was performed where 85% of the samples were 퐺푃 푣푎푙푢푒 = used as a training set and the remaining 15% as a test set, ensuring 퐼440 + 퐼490 that the number of samples in the test set was proportional to the NMR metabolomics. M. smegmatis mc2 155 bacterial suspension total number of samples from each class, while at least one sample were pelleted by centrifugation at 5000 rpm, 4 ̊C for 8 min. Super- from each class was present in the test set. The F1-score and leave- natant was collected and filtered through 0.22 µm membrane filters one-out cross-validation was carried out on the samples in the train- to remove remaining bacterial cells. Bacterial pellet was washed ing set in order to choose the number of components for the model, twice and re-suspended in PBS. All supernatant samples were fro- with the additional constraint to use a maximum of 5 components. zen at -80 ̊C and pellets were snap frozen in liquid nitrogen before This double cross-validation was repeated 1000 times with ran- freeze drying using an Alpha 1-2 LD plus freeze dryer (Martin domly chosen samples in the training and test set to prevent bias Christ, Germany). After lyophilization, the supernatant was rehy- due to the choice of training or test set. This produces 7×1000 mod- drated by 10% D2O containing 2,2,3,3-D4-3-(Trimethylsilyl) pro- els (in the supplementary information, each of these models leads pionic acid sodium salt (TMSP-2,2,3,3-D4) to provide a deuterium to a point on the scores plot, but loadings and weights are presented lock signal with reference signal. Samples were then transferred to as averages over all these models). Reference Q2 value was gener- NMR tubes. 1H NMR spectra were recorded on a Bruker Avance II ated after the known or random class assignment process. It pro- 700 NMR spectrometer (Bruker BioSpin, Coventry, United King- vides a measure of goodness of fit after cross validation and gener- dom) equipped with a 5-mm helium-cooled quadruple resonance ally referred to be ‘good’ above 0.5.20,21 The Q2 value quoted is the cryoprobe with sample isolates tested (9 replicates) and kept at 4 ̊C. mean of all models and was compared between the genuine and 1D spectra were recorded under automation at 298K using a Carr- permutated class assignments in each case. Backscaled loadings Purcell-Meiboom-Gill pre-saturation (cpmgpr1) pulse sequence. plots were used to identify metabolites with high variance and Spectra were acquired with 64 transients, a spectrum length of 20.1 weight compared with the untreated control spectra after PQN nor- ppm and 65 k data points. For the freeze dried bacterial pellets, 40 malization. Heatmaps with Euclidian distance-hierarchical cluster µl of D2O was used for rehydration. The samples were introduced (HCL) analysis were generated by MultiExperiment Viewer (MeV) in the HRMAS probe on a 600 MHz Bruker Avance III spectrom- software. eter, keeping the temperature at 310 K. Spinning speed was 5 kHz. 1 Volcano plots and univariate analysis of metabolomics data. For H NMR spectra were collected with a Carr-Purcell-Meiboom-Gill volcano plots, significant results from PLS or OPLS-DA analysis pre-saturation (zgpr) pulse sequence, a spectrum width of 12.01 justified univariate analysis of peaks associated with predictive ppm and 16384 data points was used. A total of 128 scans were value in the multivariate models. Therefore, the integrals of as- used to obtain each of the NMR spectra. Free induction decay was signed NMR peaks were analysed using non-parametric univariate multiplied by an exponential function with line broadening of 0.3 methods. Volcano plots were compared the fold change in metab- Hz. To aid the assignment of metabolite resonance, correlation olite values between two conditions. Fold change was calculated as spectroscopy (COSY) (cosygpprqf) spectra were acquired for a the ratio between the treatment condition and the control (Treat- subset of samples. All peak positions were Fourier transformed and ment/Control). Mann-Whitney U test were used to compare means measured relative to the trimethylsilylpropanoic (TSP) acid-D4 ref- and associated p-values were False Discovery rate adjusted using erence peak set to 0.0 ppm. Phase correction of spectra was per- the Benjamini-Hochberg method (α = 0.05). Volcano plots were formed manually, and automatic baseline correction was applied. generated using custom scripts in Python with Numpy, Pandas, Data analysis and statistics. All microbial data presented in this Matplotlib and Seaborn packages. study were statistically analysed by GraphPad Prism (version 5.01 PLS regression - PLS Regression was performed in Python using for Windows, GraphPad Software, La Jolla California USA). One- the PLSRegression function in the scikit-learn software package. way ANOVA test was performed, followed by Dunnett's Multiple Initial model assessment determined that only one component was Comparison Test with a value of p ≤ 0.05 to establish statistical necessary for best model performance. Increased number of com- significance. Sigmodal dose-response (variable slope) was used for ponents led to a decrease in Q2. Monte Carlo cross-validation of fitting of growth inhibition calculations. All experiments were per- models was performed by randomly splitting data into 70/30 train- formed for at least 3 times. ing/test set splits. Model generation and assessment was repeated For metabolomics data, peak assignment was performed by com- 1000 times to avoid bias by sample separation and R2 and Q2 values paring with chemical shift values and multiplicities from J-resolved were calculated to assess model performance. The R2 metric NMR spectra to values from the BMRB,16 COSY spectra and Che- demonstrates how well the model describes the training data set nomix NMR suite software (Chenomx Inc., Edmonton, Canada) while Q2 is a metric of how well the model predicts the test set. with NMR metabolite templates. Multivariate data analysis was

Figure 2. AMPs disrupt membrane surface of M. smegmatis. Transmission electron micrographs of 1.1 x 108 CFU/ml M. smegmatis mc2 155 either unchallenged (A) or challenged with D-LAK120-A (B/C) or D-LAK120-HP13 (D/E) for 5 (B/D) or 30 (C/D) mins. r – membrane ruffling, cr – cytoplasmic retraction, m – mesosoma.

Transmission electron microscopy was exploited to visualize the Results effect of D-LAK peptides on the ultrastructural changes of M. Action of D-LAK peptides on M. smegmatis. The action of the two smegmatis (Fig. 2). Unchallenged cells have characteristic lipid in- D-LAK AMPs on M. smegmatis was first investigated to confirm clusions, a regular rod-shaped cell body with well-defined intact that, as suspected from their activity against M. tuberculosis, the cell membranes and homogeneous cytoplasm (Fig. 2A). The cell peptides acted on the mycobacterial cell wall. Confocal micro- envelope was distinctly visible with cytosol bound by the plasma scopic investigation of M. smegmatis mc2 155 challenged with sub- membrane, surrounded by an internal and then an outer electron or supra-MIC concentrations of D-LAK120-A, D-LAK120-HP13 dense layer.26,27 The cell content was slightly pulled away in some or rifampicin indicate that both peptides, but not rifampicin, cause control cells which is attributed to sample preparation process. the bacteria to stain positive with the FITC labelled 150 kDa dex- Membrane ruffling was observed after treating M. smegmatis mc2 tran (Fig. 1). FITC-dextran has been used in a number of studies 155 at 1 x MIC with D-LAK120-A (Fig. 2B) or D-LAK-120-HP13 with vesicles to probe the membrane permeabilization ability of di- (Fig. 2D) for 5 mins. Cytoplasmic retraction was observed as the verse antimicrobial peptides, and a series of labelled dextran con- inner cell membrane detaches from the outer cell wall. This phe- jugates are available to measure the size of pores that may be nomenon was reported when Escherichia coli and Bacillus subtilis formed by antimicrobial peptides.22-25 In the case of melittin, dex- were exposed to antimicrobial peptides.28 Similar findings have tran of 4 kDa was able to readily escape from loaded vesicles but been observed for exposure of Mycobacteria tuberculosis (Erdman much less of the 50 kDa analogue was released. This indicated a strain) to antimicrobial peptides; granulysin demonstrated inner pore with a defined diameter of 25-30 Å is formed.22 In contrast, cell membrane detachment as observed in osmotic lysis.29 The re- other peptides facilitate escape of dextrans across the full range of duction in membrane uniformity suggests a cell penetrating ability sizes tested but complete leakage is never achieved.24,25 These re- for the D-LAK peptides. Peptide treatment also induced notable in- sults have been interpreted as evidence for rather large but transient tracellular changes, with clumped cytosol and increased amount of lesions.24 At 150 kDa the dextran used in the present study is sub- mesosoma formation. Mesosome structures have been observed stantially larger than those used in previous studies of escape from previously in bacteria challenged by antimicrobial peptides.30,31 lipid vesicles and it is not possible from images with this resolution Formation of mesosomes was suggested as a repair mechanism by to determine whether the labelled dextran has penetrated within the bacteria to combat cell lysis.32 Prolonged treatment (30 min) by D- bacteria. It is may be that the same functionality that causes D-LAK LAK peptides resulted in large scale of cell lysis and presence of peptides to prevent aggregation of M. tuberculosis colonies also en- cell debris (Fig. 2C/E). This indicates that the fate of bacteria chal- ables the dextran to adsorb to the surface of M. smegmatis. bioRxiv preprint doi: https://doi.org/10.1101/269324; this version posted February 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. lenged with inhibitory concentrations of D-LAK peptides is dis- to determine changes in bacterial cytoplasmic membrane fluidity turbed cell membranes and cell wall disintegration, membrane rup- under environmental stress.33 ture and release of intracellular content. Table 1. Minimal inhibitory concentrations of D-LAK peptides, rifampicin and capreomycin against M. smegmatis mc2 155 in the presence or absence of tyloxapol.

MIC50 (µM) Tyloxapol No tyloxapol Rifampicin 19.9 ± 3.04 38.2 ± 5.83 Capreomycin 1.72 ± 0.43 1.34 ± 0.25 D-LAK120-A 0.82 ± 0.07 1.68 ± 0.43 D-LAK120-HP13 0.63 ± 0.03 1.80 ± 0.39

Activity of D-LAK peptides against M. smegmatis alone or in combination with rifampicin or capreomycin – All four antibiotics were tested in isolation against M. smegmatis mc2 155 both in the presence and absence of tyloxapol; a non-ionic liquid polymer which is often included when growing Mycobacteria in planktonic suspension to prevent clumping (Table 1). Notably both peptides and rifampicin were more potent when tyloxapol was present in the culture media but the activity of capreomycin was unchanged. Table 2. Fractional inhibitory concentrations of D-LAK peptides in combination with either rifampicin or capreomycin against M. smegmatis mc2 155 in the presence or absence of tyloxapol.

FIC Tyloxapol No tyloxapol Rifampicin/D-LAK120-A 1.58 2.50 Rifampicin/D-LAK120-HP13 1.64 0.81 Capreomycin/D-LAK120-A 0.45 0.71 Capreomycin/D-LAK120-HP13 0.53 0.97

Combinations of rifampicin or capreomycin were then tested with each of the D-LAK peptides (Table 2). In the absence of tyloxapol, the combinations of capreomycin and the D-LAK peptides are modestly synergistic. Effectively inhibition is achieved by D- LAK120-A and capreomycin when used in combination with approximately a third of each agent required to achieve the same effect as when each agent is used alone. The effect is more marked when the combination is used in the presence of tyloxapol with an FIC <0.5 which is widely accepted as a threshold for synergism. In these conditions approximately only one tenth of the amount of D- LAK120-A required for the same effect. A similar effect is seen Figure 3. Fluorescence spectroscopic perspective of M. smeg- when capreomycin is used in combination with D-LAK120-HP13 matis mc2 155 response to challenge with antibiotics. DPH fluo- but the synergistic effect is more modest in both the presence and rescence anisotropy (A), laurdan GP fluorescence (B) and correla- absence of tyloxapol. Rifampicin demonstrates no synergy with D- tion (R2 = 0.856) of DPH and laurdan (C). ** indicates p <0.05 LAK120A and these antibiotics may even be antagonistic when no with respect to 7H9O. tyloxapol is added to the growth media. In contrast, though modest, As a polyene hydrophobic dye DPH localises to the hydrophobic D-LAK120-HP13 may assist the activity of rifampicin and a little core of lipid bilayers. It aligns parallel to the acyl chains of lipids less than half as much rifampicin is required to achieve the same and hence its ability to re-orientate is dependent on the packing of inhibitory effect as when rifampicin is used alone. Analogous ex- its lipid neighbours. A high degree of orientation, and hence fluo- periments were performed with isoniazid but no synergism was de- rescence anisotropy, results from a more rigid lipid bilayer. Signif- tected with either peptide and hence these combinations were not icant increases (p < 0.05) in DPH fluorescence polarisation anisot- studied further in the present work. ropy (r) were observed for M. smegmatis mc2 155 challenged dur- DPH anisotropy and laurdan fluorescence indicate substantial ing growth with ¾ MIC or FIC capreomycin or its combinations changes in membrane physical properties when M. smegmatis is with D-LAK120A or D-LAK120-HP13 (Fig. 3A) or when grown cultured in the presence of capreomycin or tyloxapol – DPH fluo- in the presence of 0.025% tyloxapol; the latter producing a more rescence polarization or fluorescence anisotropy is commonly used modest increase. Small increases in fluorescence anisotropy were bioRxiv preprint doi: https://doi.org/10.1101/269324; this version posted February 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. observed when bacteria were challenged with the D-LAK peptides of individual anti-mycobacteria drugs, ¾ FIC of their combinations or with rifampicin, either alone or in combination with the D-LAK or 0.025% tyloxapol. The technique could determine significant peptides, but these were not significant when compared with un- differences, as determined by Q2 (Table 3), for each model in which challenged bacteria. Only challenge with capreomycin alone or in loadings are compared in a hierarchical clustered heatmap (Fig. combination with either D-LAK peptide, causes a substantial in- 4A). Volcano plots for individual comparisons allow the identifi- crease in membrane rigidity in M. smegmatis. cation of the magnitude and significance of changes in individual metabolites (Fig. 4B-F) while changes in key metabolites are com- Laurdan is a solvatochromic fluorescent dye which inserts into lipid pared across treatments to show the impact of each peptide, rifam- bilayers with the fatty acid chain embedded in the hydrophobic core picin, capreomycin and their combinations (Fig. 5). of the bilayer and the N,N-dimethyl-2-napththylamine in the more polar interfacial region. The dipole moment between the dimethyl- Table 3. Comparison of test and permutated Q2 scores for cross- amino and carbonyl moieties is sensitive to local changes in polar- validated OPLS-DA models comparing 1H HR-MAS spectra of un- ity which will shift the emission maximum. Calculating the differ- challenged M. tuberculosis mc2 155 with those obtained for bacte- ence in emission at 440 and 490 nm generates a generalised polar- ria in the indicated conditions. ization (GP) value from which membrane order can be inferred. As a lipid bilayer becomes disordered more water can penetrate the Q2 Q2 interfacial region, which becomes more polar, and the GP value decreases.34 (permutated) The GP value is therefore a measure of disorder in the lipid bilayer interfacial region although other factors may influence the polarity Tyloxapol (0.025%) 0.866 -0.377 of the environment in which the fluorophore is located and some caution is advisable when interpreting laurdan GP in biological sys- D-LAK120-A 0.769 -0.423 tems. Nevertheless, laurdan generalised polarization has been used in e.g. Bacillus subtilis to detect the effect of challenge with a syn- D-LAK120-HP13 0.729 -0.360 thetic cyclic hexapeptide cWFW35 and the organisation of the bac- 36 37 terial membrane by flotillins and MreB . Significant changes (p Rifampicin 0.581 -0.426 < 0.05) in laurdan GP were observed for M. smegmatis mc2 155 challenged during growth with ¾ MIC or FIC capreomycin or its Rifampicin/D-LAK120-A 0.430 -0.429 combinations with D-LAK120A or D-LAK120-HP13 (Fig. 3B). Again, a change in membrane properties was detected for bacteria Rifampicin/D-LAK120-HP13 0.763 -0.410 incubated with tyloxapol but a noticeable reduction in GP following challenge with rifampicin is non-significant. The change in GP for bacteria challenged with combinations of capreomycin is of a Capreomycin 0.881 -0.347 lower magnitude than that observed when the bacteria are challenged with capreomycin alone but the differences between these Capreomycin/D-LAK120-A 0.775 -0.367 three conditions are non-significant. Notably the GP values in each of conditions were reduced relative to the unchallenged bacteria, Capreomycin/D-LAK120-HP13 0.823 -0.344 which implies the local environment of the laurdan probe is becom- ing more polar, something that would normally be associated with Culturing M. smegmatis mc2 155 in the presence of 0.025% tylox- an increase in disorder in the interfacial region. Since the increase apol causes significant and substantial changes in several metabo- in DPH anisotropy for these conditions is associated with an in- lites and components of the mycobacterial membrane (Fig. 4B). An crease in membrane rigidity, a less ordered but more rigid mem- increase in the amount of glutamate in the cells is the greatest brane is perhaps surprising. The strong correlation between DPH change while there are highly significant reductions in lipid reso- and laurdan data (Fig. 3C) does however indicate the two tech- nances associated with mycolic acid including the saturated alkyl niques are reporting on the same event. The DPH dye would be chains –(CH2)n- and both R2CH-COOH and R2CH-OH. Notably, expected to reside deep within the hydrophobic core of the mem- resonances associated with unsaturated alkyl groups –CH=CH- or brane while the laurdan probe would be much closer to the aqueous -CH2-CH=CH- are unaffected (Fig. 4B, 5A-D). Mycobacterial my- surface. Consequently, it is possible that the result of M. smegmatis colic acids comprise a long branch mero chain of 40-60 carbons responding to the presence of capreomycin is to modify the hydro- and a short α branch of typically 24 carbons. The shorter α chain is phobic core of the membrane, reducing fluidity, while the interfa- completely saturated. Mero-mycolic acids in other Mycobacteria cial region becomes more disordered. While the precise locations contain cyclopropanated mycolic acids but these are uncommon in of both dyes in the Mycobacteria cell membranes are yet to be de- M. smegmatis where the major mycolic acids are a homologous, termined it can be concluded that the physical properties of the bac- αʹseries containing just a cis-alkene in the mero-chain.38-42 terial envelope are substantially altered in response to challenge with capreomycin but not with either rifampicin or the D-LAK pep- Since the fold changes in all four lipid resonances are similar, this tide. is consistent with a reduction in the number of saturated α chains, with mero-mycolic acids, containing unsaturated hydrocarbons, HR-MAS 1H NMR metabolomics reveals substantial changes in M. unaffected. Many of these differences are also observed when M. smegmatis membrane components - Cross-validated OPLS-DA smegmatis mc2 155 is challenged with either rifampicin or capreo- was used to identify significant changes in 1H HR-MAS spectra mycin (Fig. 4E/F). obtained for M. smegmatis mc2 155 when challenged with ¾ MIC

Figure 4. Capreomycin and rifampicin induce substantial changes in mycolic acid lipid components in M. smegmatis. Hierarchical clustered heatmap comparing loadings obtained from cross-validated OPLS-DA of 1H HR-MAS NMR spectra of M. smegmatis mc2 155 grown in the indicated conditions (A). Volcano plots are shown for individual comparisons of unchallenged bacteria and those challenged with 0.025% tyloxapol (B), D-LAK120-A (C), D-LAK120-HP13 (D), rifampicin (E) or capreomycin (F). Volcano plots are of PQN normal- ized data and allow comparison of fold changes and significance of each metabolite; blue – significant reductions, red – significant increases and grey – non-significant changes in the indicated metabolites.

Figure 5. Univariate analysis of relative metabolite levels in M. smegmatis mc2 155. Significant differences with respect to unchallenged bacteria, as determined by one-way ANOVA with Tukey post-hoc test, are indicated **. Other significant differences are described in the text. However, the magnitudes of the responses vary substantially and also formed of lipids based on mycolic acid, but these are consid- there are also qualitative differences, most notably since both ri- ered free to diffuse laterally. These lipids are trehalose dimycolate fampicin and capreomycin cause reductions in resonances associ- (TDM) and trehalose monomycolate (TMM). Interestingly, only ated with unsaturated alkyl chains. It is notable that the fold challenge with capreomycin induces any change in signal intensity changes in the differing lipid resonances are again similar. This is attributable to trehalose (Fig. 4F). The significant increase is of a consistent with a reduction in the number of intact mycolic acid similar magnitude to the decrease observed for the mycolic acid molecules rather than a shortening in the length of the mycolic acid resonances indicating that the ratio of mycolic acid to trehalose is alkyl chain. Since resonances for both saturated and unsaturated substantially altered in M. smegmatis following challenge with cap- hydrocarbons are reduced as well as those for the R2CH-OH and reomycin. This would be consistent with a shift in the balance of R2CH-COOH protons a general reduction in mycolic acid can be TDM and TMM, to favour the latter, in the outer leaflet of the my- inferred. comembrane. Mycolic acids are an important component of the mycomembrane. The scale of the reduction in the mycolic acid resonances caused The inner leaflet is formed of mycolic acid linked to arabinogalac- by capreomycin (Fig. 4F) is much greater than that caused by ri- tan which is in turn linked to peptidoglycan. The outer leaflet is fampicin (Fig. 4E) or even tyloxapol (Fig. 4B). When changes in

bioRxiv preprint doi: https://doi.org/10.1101/269324; this version posted February 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. mycolic acid lipid resonances are compared across the various chal- Increased membrane rigidity is associated with altered composi- lenges (Fig. 5A-D) it is clear capreomycin has a substantial impact tion of the mycomembrane - to better understand the contributions on those from both saturated and unsaturated hydrocarbons and that of the various changes in metabolite concentrations on the physical while significant (p < 0.05), the reductions in intensity due to ri- properties of the mycomembrane following challenge with capreo- fampicin are much more modest. Similarly, tyloxapol causes a mycin and/or D-LAK peptides or growth in the presence of 0.025% modest reduction in resonances from saturated hydrocarbons (Fig. tyloxapol, Spearman correlations between individual metabolites 5C/D) but has no significant effect on those from unsaturated hy- and the fluorescence anisotropy recorded for the same samples drocarbons (Fig. 5A/B). Unlike capreomycin, neither rifampicin were determined using partial least squares regression (Fig. 6, Fig. nor tyloxapol induce a significant increase in resonances attributa- S6). Significant (p < 0.0001) correlations were detected between ble to trehalose (Fig. 5F). Remodelling of the mycolic acid induced fluorescence anisotropy and each of the metabolites in which sub- by rifampicin and tyloxapol is therefore much subtler than that in- stantial changes resulted from challenge with capreomycin, D- duced by capreomycin and this is presumably the origin of the non- LAK120-HP13 or growth in the presence of 0.025% tyloxapol. The significant changes in membrane physical properties as detected strongest of these correlations were negative correlations with sat- above using fluorescence techniques. urated hydrocarbons (Fig. 6A, Fig. 6E) while weaker negative or

2 positive correlations were detected with, respectively, unsaturated The response of M. smegmatis mc 155 to challenge with ¾ MIC hydrocarbons (Fig. 6C/D) or trehalose (Fig. 6B). The strength of of the two D-LAK peptides is quantitatively and qualitatively dif- these correlations may reflect the impact of each component of the ferent to the response to either rifampicin or capreomycin. Alt- mycomembrane in determining its fluidity or this may reflect the hough the OPLS-DA models for challenge with either peptide in- observation that, while all three types of resonance are affected by dicate a significant response when all metabolites are considered challenge with capreomycin, D-LAK120-HP13 affects only lipid (Table 3; Fig. S3), the magnitude of changes in individual metabo- resonances and tyloxapol affects only saturated hydrocarbons in the lites is low and few changes pass a stringent significance threshold shorter α chain. Nevertheless, the increased rigidity of the my- (Fig. 4C/D). When comparing individual metabolites across the comembrane appears to be a direct result of the observed changes various conditions, both peptides trigger an increase in glutamate in its composition. in the bacteria (p <0.05), a similar effect is seen for capreomycin but not rifampicin (Fig. 4E). In contrast with capreomycin, neither Rifampicin but not capreomycin induce detectable changes in spent peptide triggered any change in trehalose (Fig. 5F). The proline media metabolite composition – Spent bacterial culture superna- containing, and proline free peptides can themselves be distin- tants were also analysed by OPLDS-DA (Fig. S7). Comparison of guished by the absence of any changes in lipid resonances in re- the models indicates that only those conditions where rifampicin is sponse to D-LAK120-A while significant (p < 0.05) reductions in present was any qualitative change in M. smegmatis metabolism lipid resonances are observed for both unsaturated and saturated detected (Fig. S7A). A binary comparison of spent media for M. hydrocarbons in response to D-LAK120-HP13 (Fig. 5A-D). smegmatis grown without challenge with fresh media reveals the

1 2 bacteria consume glucose and produce tartrate and 2-hydroxyiso- HR-MAS H NMR data was also obtained for M. smegmatis mc butyrate (Fig. S7B). When challenged with rifampicin there is a 155 challenged with combinations of each D-LAK peptide and ei- slight increase in glucose and citrate consumption but substantially ther rifampicin or capreomycin. For synergistic combinations the reduced production of lactate, alanine, valine and pyruvate and amount of each antibiotic was substantially lower than that used for more modest reductions in production of malate and tartrate (Fig. each antibiotic alone. S7C). These changes were muted when rifampicin was applied in combination with D-LAK120-HP13 where increased consumption Modest synergism exists between D-LAK120-A and capreomycin of glutamate was also observed (Fig. S7D). In no other conditions but there is indifference or even antagonism between D-LAK120- were any changes in individual metabolites detected that passed the A and rifampicin. D-LAK120-A mitigates the reduction in satu- significance threshold. rated and unsaturated hydrocarbons and increase in trehalose due to capreomycin (p < 0.05) (Fig. 5A-D, Fig. S1A, Fig. S2A/B) but Discussion not the increase in glutamate (Fig 5E). The responses of M. smeg- 2 matis mc 155 to rifampicin or rifampicin/D-LAK120-A are indis- The combination of HR-MAS 1H NMR metabolomics and fluores- tinguishable (Fig. 5, Fig. S1C, Fig. S2). cent probes sensitive to changes in membrane fluidity and order reveals modest changes in the mycomembrane of M. smegmatis D-LAK120-HP13 has a mostly additive effect when used in com- mc2 155 in response to challenge with rifampicin but much more bination with capreomycin but experiences modest synergism with dramatic changes in response to challenge with capreomycin. The rifampicin. D-LAK120-HP13 has a weaker impact on metabolic D-LAK peptides cannot be distinguished based on any change in changes induced by capreomycin or rifampicin. Although the mag- the physical properties of the mycomembrane but changes in its nitude of reduction of lipid resonances in D-LAK120- composition reflect a distinct mechanism for the proline free and HP13/capreomycin combinations was consistently lower than that proline containing analogues. The combinations of both D-LAK observed when capreomycin was used alone (Fig. 5A-D, Fig. S1B, peptides with capreomycin are synergistic in the presence of tylox- Fig. S2A/B), the mitigation was in no cases significant. In contrast, apol but when M. smegmatis is cultured in the absence of tyloxapol although the magnitude of changes induced by rifampicin is less, a this is attenuated leaving only modest synergy and only with D- significant mitigation (p < 0.05) was observed for lipid -CH3 (Fig. LAK120-A and not D-LAK120-HP13; only the latter is modestly 5C) while for other metabolites the combination of D-LAK120- beneficial when combined with rifampicin. These findings may HP13 and rifampicin did not induce significant changes with re- provide a rationale for understanding the properties required for D- spect to the unchallenged bacteria (Fig. 5A/B/D). Notably the Vol- LAK peptides and related molecules to potentiate the activity of cano plot of the individual comparison indicates no metabolites first and second line treatments against mycobacterial infections were significantly changed when this combination was used (Fig. and understand whether there is more to the activity of either pep- S1D) in contrast to rifampicin (Fig. 4F) or indeed D-LAK120- tide than enhancing penetration of the mycomembrane. HP13 when used alone (Fig. 4D).

Figure 6. Increased membrane rigidity is associated with altered composition of the mycomembrane. Spearman correlations are shown between DPH anisotropy (r) and 1H HR-MAS NMR resonance intensities obtained for M. smegmatis mc2 155 grown without challenge or with 0.025% Tyloxapol or ¾ MIC of D-LAK120-A, D-LAK120-HP13, capreomycin, capreomycin + D-LAK120-A or capreomycin + D- LAK120-HP13. Data is shown for lipid –(CH2)n- (A), trehalose (B), lipid –CH=CH- (C), lipid –CH2-CH=CH- (D) and lipid -CH3 (E). Capreomycin acts by inhibiting protein synthesis but induces sub- there is a substantial shift in metabolism following capreomycin stantial remodelling of the mycomembrane – the antibiotic activity challenge resulting in greater consumption of fatty acids, depletion of capreomycin against both M. smegmatis and M. tuberculosis is of mycolic acid and a rigidification of the mycomembrane as tre- ascribed to its ability to inhibit protein translation by interfering halose monomycolate predominates over trehalose dimycolate. As with the function of ribosomes.43 Specifically capreomycin binds such, this response from mycobacteria to capreomycin challenge across the ribosomal subunit interface using tlyA- encoded methyl- affords a means of altering the barrier that other antimicrobials may ations in both 16S and 23S rRNAs.44 A transcriptomic approach to have to cross to reach their targets. understanding the mechanism of action of capreomycin confirmed the importance of this mechanism but additionally revealed sub- Rifampicin acts by inhibiting transcription and induces modest re- stantial changes in a variety of other gene classes including lipid modelling of the mycomembrane – the antibiotic activity of rifam- metabolism, cell wall and cell processes, and intermediary metab- picin is attributed to its ability to inhibit bacterial DNA-dependent olism and respiration in M. tuberculosis.45 Notably capreomycin RNA polymerase activity. M. tuberculosis is more susceptible to was shown to affect the glyoxylate shunt, an alternate pathway to rifampicin then M. smegmatis with the latter enjoying a higher level the TCA cycle, with up-regulation of icl (Rv0467 – isocitrate ly- of baseline resistance due to rifampin ADP ribosyltransferase, an ase), glcB (Rv1837c) and aceAa (Rv1915) presumably stimulating enzyme capable of inactivating rifampicin.46 However, exposure of a process where fatty acids become an important carbon source. rifampicin sensitive M. tuberculosis H37Rv to rifampicin induces Reduced expression of a block of genes associated with the electron changes in expression of genes in the same functional classes af- transport chain, coding for NADH dehydrogenase or NADH- fected by capreomycin challenge i.e. lipid metabolism, cell-wall ubiquinone oxidoreductase, as well as gdh, a probable NAD- and cell processes, and intermediary metabolism and respiration.47 dependent glutamate dehydrogenase. Though performed in M. Further, rifampicin, and isoniazid and streptomycin, trigger activa- smegmatis the present study is consistent with these findings; glu- tion of isocitrate lyases in M. tuberculosis48 suggesting the glyox- tamate is seen to accumulate following challenge with capreomycin ylate shunt is a general means of overcoming oxidative stress.49 An- (and also both D-LAK peptides) while the substantial reductions in timicrobial peptides including LL-37 and pleurocidin have been mycolic acid is consistent with fatty acids being diverted for use as shown to induce oxidative stress respectively in Escherichia coli a carbon source; notably, in contrast with rifampicin challenge, no and Candida albicans.50,51The modest changes in mycolic acid in- significant change in individual metabolite levels in spent culture duced by challenge with rifampicin and D-LAK120-HP13 may media was detected. Taken together the two studies indicate that therefore be indicative of oxidative stress and reflect penetration of bioRxiv preprint doi: https://doi.org/10.1101/269324; this version posted February 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. the bacterium; notably D-LAK120-A does not have this effect. The MIC for capreomycin is not reduced and may even be higher. It is response of M. smegmatis to rifampicin differs from that to capre- unlikely therefore that tyloxapol improves access for capreomycin omycin quantitatively in the HR-MAS study but also qualitatively though it may perform this function for either D-LAK peptide. Alt- in the study of spent culture composition where evidence for an hough the difference between the two peptides in terms of their in- alternate strategy to overcoming oxidative stress emerges. This teraction with capreomycin is small, D-LAK120-A consistently would be consistent with M. smegmatis altering its metabolism in outperforms D-LAK120-HP13. Both peptides seemingly have a response to rifampicin in a similar way to that used in response to similar effect on capreomycin activity but the poorer synergy be- capreomycin but being less reliant on metabolism of fatty acids. tween D-LAK120-HP13 and capreomycin may be related to the ability of both agents to trigger changes in the mycomembrane. Distinct membrane activity underpins synergistic combinations – Since both capreomycin and rifampicin trigger changes in the my- for antibiotics to have additive or synergistic effects when used in comembrane composition, albeit with very different magnitudes of combination they need to avoid both interactions that reduce the response, this argument seemingly runs counter to the idea that amount of each agent reaching their target(s) and triggering re- membrane interactions will determine the ability of such molecules sponses in the bacteria that counter the activity of the other agent. to interact synergistically against M. smegmatis. The answer to this For combinations of antibiotics to have synergistic effects, addi- may lie in the distinct physical properties of capreomycin and ri- tionally they need to act on different targets or have distinct effects fampicin. Capreomycin is a cationic molecule with a physiological on the same target such that the effect of each individual antibiotic net positive charge of four. Rifampicin is also cationic but its phys- is enhanced. The HR-MAS 1H NMR approach enables investiga- iological charge is only one. Both molecules must penetrate the tion of whether antibiotics do indeed differ in their effect on the bacteria to exert their main antibiotic effects and hence the D-LAK mycomembrane. This is achieved by obtaining a bacterial perspec- peptides, with a net positive charge of nine and a higher affinity for tive of the challenge and inferring differences in activity of each anionic components of the mycomembrane, may provide an im- antibiotic from differences in the response of the bacteria to each portant screen for interactions between the membrane and either challenge. Each of capreomycin, rifampicin, the D-LAK peptides capreomycin or rifampicin. Capreomycin, with the higher charge, and tyloxapol induce qualitatively and/or quantitatively different would benefit more from this screen and this may underlie the ob- responses from M. smegmatis but there is nevertheless considerable served synergy rather than any induced change in mycomembrane overlap. biophysical properties.

Tyloxapol improves the potency of rifampicin and D-LAK peptides Conclusion when used alone but the combination of D-LAK peptides and rifampicin is indifferent or even antagonistic in the presence of ty- The two D-LAK peptide analogues can be distinguished based on loxapol. Since both rifampicin and tyloxapol induce membrane re- the different response of M. smegmatis to AMP challenge and their modelling in M. smegmatis one might speculate that rifampicin and different abilities to enhance the activity of rifampicin and capreo- tyloxapol combine to produce substantial change in membrane that mycin; two anti-mycobacteria drugs with distinct mechanisms of inhibits the ability of D-LAK peptides to disrupt or penetrate mem- action but related in their requirements for efficacy and the bacte- brane. In the absence of tyloxapol, the change in membrane prop- rial response to their activity. In addition to distinguishing anti-my- erties induced by rifampicin are more modest and this may explain cobacterial drugs the combination of HR-MAS 1H NMR and fluo- why combinations of rifampicin with D-LAK120-A fare worse rescence spectroscopy reveals how bacterial responses to each than those containing D-LAK120-HP13 if the proline modification agent; remodelling of the mycomembrane may affect their ability enables this analogue to better penetrate the mycomembrane. to act in combination. In addition to consideration of the mode of action and physical properties of each antibiotic, understanding Both D-LAK peptides interact synergistically with capreomycin, such effects may be essential in designing effective antibiotic com- albeit modestly. The interaction is enhanced however when M. binations. smegmatis is cultured in the presence of 0.025% tyloxapol. While tyloxapol enhances the action of both peptides and rifampicin, the

ASSOCIATED CONTENT Supporting Information. Supplementary figures are available as described in the text. AUTHOR INFORMATION Corresponding Author * A. James Mason, King’s College London, Institute of Pharmaceutical Science, School of Cancer and Pharmaceutical Sciences, Franklin- Wilkins Building 150 Stamford Street, London, SE1 9NH, UK; Tel: + 44 207 848 4813; Fax: + 44 207 848 4800; E-mail: james.ma- [email protected] Author Contributions AJM and DM wrote the main manuscript text and prepared all figures. DM, AJM, BDR and JKWL designed experiments. BDR and JKWL provided materials. DM and TK performed and/or analysed NMR metabolomic experiments/data. DM conducted susceptibility testing and obtained electron microscopy and fluorescence microscopy images. All authors approved the manuscript.

Notes The authors declare no competing financial interest.

This work was supported by a joint HKU-KCL studentship for DM awarded to JKWL, the Wellcome Trust (Capital Award for the KCL Centre for Biomolecular Spectroscopy). This work was supported by the Francis Crick Institute through provision of access to the MRC Biomedical NMR Centre. The Francis Crick Institute receives its core funding from Cancer Research UK (FC001029), the UK Medical Research Council (FC001029), and the Wellcome Trust (FC001029). We are grateful to Drs Tom Frenkiel, Alain Oregioni and Andrew Atkinson for their assistance with NMR instrumentation. We also acknowledge the assistance of the Li Ka Shing Faculty of Medicine Faculty Core Facility, and the Electron Microscope Unit at the University of Hong Kong. REFERENCES [1] Tuberculosis Fact sheet. World health Organisation http://www.who.int/mediacentre/factsheets/fs104/en/ Retrieved 19/12/2017 [2] Johnson MM & Odell JA. (2014) Nontuberculous mycobacterial pulmonary infections. J. Thorac. Dis. 6: 210-220. [3] Tuberculosis in the UK: 2013 report. London, UK: Health Protection Agency; 2013. [4] Bansal-Mutalik R & Nikaido H. (2014) Mycobacterial outer membrane is a lipid bilayer and the inner membrane is unusually rich in diacyl phosphatidylinositol dimannosides. Proc. Natl, Acad. Sci. USA 111: 4958-4963. [5] Minnikin DE, Lee OY-C, Wu HHT, Nataraj V, Donoghue HD, Ridell M, Watanabe M, Alderwick L, Bhatt A & Besra GS. (2015) pathophysio- logical implications of cell envelope structure in Mycobacterium tuberculosis and related taxa, Tuberculosis - Expanding Knowledge, Dr. Wellman Ribón (Ed.), InTech, DOI: 10.5772/59585. Available from: https://www.intechopen.com/books/tuberculosis-expanding-knowledge/pathophysiolog- ical-implications-of-cell-envelope-structure-in-mycobacterium-tuberculosis-and-related [6] Gutsmann T. (2016) Interaction between antimicrobial peptides and mycobacteria. Biochimica et Biophysica Acta 1858: 1034–1043 [7] Padhi A, Sengupta M, Sengupta S, Roehm KH & Sonawane A. (2014) Antimicrobial peptides and proteins in mycobacterial therapy: Current status and future prospects. Tuberculosis 94: 363-373. [8] Silva JP, Appelberg R & Gama FM (2016) Antimicrobial peptides as novel anti-tuberculosis therapeutics. Biotechnology Advances 34: 924–940. [9] Khara JS, Wang Y, Ke Xi-Yu, Liu S, Newton SM, Langford PR, Yang YY & Ee PLR. (2014) Biomaterials 35: 2032-2038. [10] Khara JS, Priestman M, Uhía I, Hamilton MS, Krishnan N, Wang Y, Yang YY, Langford PR, Newton SM, Robertson BD & Ee PLR. (2016) Unnatural amino acid analogues of membrane-active helical peptides with anti-mycobacterial activity and improved stability. J. Antimicrob. Chemother. 71: 2181–2191. [11] Vermeer LS, Lan Y, Abbate V, Ruh E, Bui TT, Wilkinson L, Kanno T, Jumagulova E, Kozlowska J, Patel J, McIntyre CA, Yam WC, Siu G, Atkinson RA, Lam JKW, Bansal SS, Drake AF, Mitchell GH & Mason AJ. (2012) Conformational flexibility determines selectivity and antibacterial, antiplasmodial and anticancer potency of cationic α-helical peptides. J. Biol. Chem. 287: 34120-34133. [12] Lan Y, Lam JT, Siu GKH, Yam WC, Mason AJ & Lam JKW. (2014) Cationic amphipathic D-enantiomeric antimicrobial peptides with in vitro and ex vivo activity against drug-resistant Mycobacterium tuberculosis. Tuberculosis 94: 678-689. [13] Amos S-BTA, Vermeer LS, Kozlowska J, Davy M, Ferguson P, Bui TT, Drake AF, Lorenz CD & Mason AJ. (2016) Antimicrobial peptide potency is facilitated by greater conformational flexibility when binding to Gram-negative bacterial inner membranes. Scientific Reports 6: 37639. [14] Kozlowska J, Vermeer LS, Rogers GB, Rehnnuma N, Amos S-BTA, Koller G, McArthur M, Bruce KD & Mason AJ. (2014) Combined systems approaches reveal highly plastic responses to antimicrobial peptide challenge in Escherichia coli. PLoS Pathogens 10: e1004104. [15] Caleffi-Ferracioli KR, Maltempe FG, Siqueira VL & Cardoso RF. (2013) Fast detection of drug interaction in Mycobacterium tuberculosis by a checkerboard resazurin method. Tuberculosis 93: 660-663. [16] Ulrich EL, Akutsu H, Doreleijers JF, Harano Y, Ioannidis YE, Lin J, Livny M, Mading S, Maziuk D, Miller Z, Nakatani E, Schulte CF, Tolmie DE, Kent Wenger R, Yao H, Markley JL. (2008) BioMagResBank. Nucleic Acids Res 36: D402–D408. [17] Vermeer LS, Fruhwirth GO, Pandya P, Ng T & Mason AJ. (2012) NMR metabolomics of MTLn3E breast cancer cells identifies a role for CXCR4 in lipid and choline regulation. J. Proteome. Res. 11: 2996 –3003. [18] Worley B, Powers R. 2014. MVAPACK: a complete data handling package for NMR metabolomics. ACS Chem. Biol. 9:1138 –1144. [19] Dieterle F, Ross A, Schlotterbeck G & Senn H (2006) Probabilistic quotient normalization as robust method to account for dilution of complex biological mixtures. Application in 1H NMR metabonomics. Anal. Chem. 78: 4281–4290. [20] Szymanska E, Saccenti E, Smilde AK & Westerhuis JA (2012) Double-check: validation of diagnostic statistics for PLS-DA models in metabolomics studies. Metabolomics 8: S3–S16. [21] Westerhuis JA, Hoefsloot HCJ, Smit S, Vis DJ, Smilde AK, et al. (2008) Assessment of PLSDA cross validation. Metabolomics 4: 81–9. [22] Ladokhin AS, Selsted ME & White SH (1997) Sizing membrane pores in lipid vesicles by leakage of co-encapsulated markers: pore formation by melittin. Biophys. J. 72: 1762-1766. [23] Lee W & Lee DG. (2015) Fungicidal mechanisms of the antimicrobial peptide Bac8c. Biochimica et Biophysica Acta 1848: 673–679. J. Biol. Chem. 272: 24224-24233. [24] Hristova K, Selsted ME & White SH. (1997) Critical role of lipid composition in membrane permeabilization by rabbit neutrophil defensins. [25] Marcellini L, Borro M, Gentile G, Rinaldi AC, Stella L, Aimola P, Barra D & Mangoni ML, (2009) Esculentin-1b(1–18) – a membrane-active antimicrobial peptide that synergizes with antibiotics and modifies the expression level of a limited number of proteins in Escherichia coli. FEBS Journal 276: 5647–5664. 13 bioRxiv preprint doi: https://doi.org/10.1101/269324; this version posted February 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. [26] Etienne G, Laval F, Villeneuve C, Dinadayala P, Abouwarda A, Zerbib D, Galamba A & Daffé M. (2005) The cell envelope structure and properties of Mycobacterium smegmatis mc2155: is there a clue for the unique transformability of the strain? Microbiology 151: 2075-2086. [27] Hoffmann C, Leis A, Niederweis M, Plitzko PM & Engelhardt H. (2008) Disclosure of the mycobacterial outer membrane: Cryo-electron tomography and vitreous sections reveal the lipid bilayer structure. Proc. Natl. Acad. Sci. USA 105: 3963–3967. [28] Taute H, Bester MJ, Neitz AWH & Gaspar ARM. (2015) Investigation into the mechanism of action of the antimicrobial peptides Os and Os-C derived from a tick defensin. Peptides 71: 179-187. [29] Ernst WA, Thoma-Uszynski S, Teitelbaum R, Ko C, Hanson DA, Clayberger C, Krensky AM, Leippe M, Bloom BR, Ganz T & Modlin RL (2000) Granulysin, a T cell product, kills bacteria by altering membrane permeability. J. Immunol. 165; 7102-7108. [30] Hartmann M, Berditsch M, Hawecker J, Ardakani MF, Gerthsen D & Ulrich AS. (2010) Damage of the bacterial cell envelope by antimicrobial peptides gramicidin S and PGLa as revealed by transmission and scanning electron microscopy. Antimicrob. Agents Chemother. 54: 3132-3142. [31] Friedrich CL, Moyles D, Beveridge TJ & Hancock REW. (2000) Antibacterial action of structurally diverse cationic peptides on Gram-positive bacteria. Antimicrob. Agents Chemother. 44: 2086-2092. [32] van Breda SV, Buys A, Apostolides Z, Nardell EA & Stoltz AC (2015) The antimicrobial effect of colistin methanesulfonate on Mycobacterium tuberculosis in vitro. Tuberculosis 95: 440-446. [33] Mykytczuk NC, Trevors JT, Leduc LG & Ferroni GD. (2007) Fluorescence polarization in studies of bacterial cytoplasmic membrane fluidity under environmental stress. Prog. Biophys. Mol. Biol. 95: 60-82. [34] Munishkina LA & Fink AL. (2007) Fluorescence as a method to reveal structures and membrane-interactions of amyloidogenic proteins. Bio- chim Biophys Acta. 1768: 1862-1885. [35] Scheinpflug K, Wenzel M, Krylova O, Bandow JE, Dathe M & Strahl H. (2017) Antimicrobial peptide cWFW kills by combining lipid phase separation with autolysis. Scientific Reports 7: 44332. [36] Bach JN & Bramkamp M. (2013) Flotillins functionally organize the bacterial membrane. Molecular Microbiology 88: 1205–1217. [37] Strahl H, Bürmann F & Hamoen LW. (2013) The actin homologue MreB organizes the bacterial cell membrane. Nature Communications 5: 3442. [38] Verschoor JA, Baird MS & Grooten J. (2012) Towards understanding the functional diversity of cell wall mycolic acids of Mycobacterium tuberculosis. Progress in Lipid Research 51; 325-339. [39] Marrakchi H, Lanéelle M-A & Daffé M. (2014) Mycolic acids: structures, biosynthesis, and beyond. Chemistry & Biology 21: 67-85. [40] Krembel J, Etémadi A-H. (1966) Sur la structure d’un nouveau type d’acides mycoliques de Mycobacterium smegmatis. Tetrahedron 22: 1113– 1119. [41] Wong MYH & Gray GR. (1979) Structures of the homologous series of monoalkene mycolic acids from Mycobacterium smegmatis. J. Biol. Chem. 254: 5741–5744. [42] Wong MYH, Steck PA & Gray GR. (1979) The major mycolic acids of Mycobacterium smegmatis. J. Biol. Chem. 254: 5734–5740. [43] Maus CE, Plikaytis BB & Shinnick TM. (2005) Mutation of tlyA confers capreomycin resistance in Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 49: 571-577. [44] Johansen SK, Maus CE, Plikaytis BB & Douthwaite S. (2006) Capreomycin binds across the ribosomal subunit interface using tlyA-encoded 2ʹ-O-methylations in 16S and 23S rRNAs. Molecular Cell 23, 173–182. [45] Fu LM & Shinnick TM, (2007) Genome-wide exploration of the drug action of capreomycin on Mycobacterium tuberculosis using Affymetrix oligonucleotide GeneChips. J. Infection. 54: 277-284. [46] Alexander, DC, Jones JRW &Liu J. (2003) A Rifampin-hypersensitive mutant reveals differences between strains of Mycobacterium smegmatis and presence of a novel transposon, IS1623. Antimicrob. Agents Chemother. 47: 3208–3213. [47] de Knegt GJ, Bruning O, ten Kate MT, de Jong M, van Belkum A, Endtz HP, Breit TM, Bakker-Woudenberg IAJM & de Steenwinkel JEM. (2013) Rifampicin-induced transcriptome response in rifampicin-resistant Mycobacterium tuberculosis. Tuberculosis 93: 96-101 [48] Nandakumar M, Nathan C, & Rhee KY. (2014) Isocitrate lyase mediates broad antibiotic tolerance in Mycobacterium tuberculosis. Nature Communications 5: 4306 [49] Ahn,S, Jung J, Jang I-A, Madsen EL, & Park W. (2016) Role of glyoxylate shunt in oxidative stress response. J. Biol. Chem. 291: 11928–11938. [50] Choi H, Yang Z & Weisshaar JC. (2017) Oxidative stress induced in E. coli by the human antimicrobial peptide LL-37. PLoS Pathog. 13: e1006481. [51] Cho J & Lee DG. (2011) Oxidative stress by antimicrobial peptide pleurocidin triggers apoptosis in Candida albicans. Biochimie. 93:1873- 1879.