# Evaluation and activity of new porphyrin-peptide cage-type conjugates for the photoinactivation of Mycobacterium abscessus

Matthéo Alcaraz, Sébastien Lyonnais, Chandramouli Ghosh, John Jairo

Aguilera-Correa, Sébastien Richeter, Sébastien Ulrich, Laurent Kremer

► To cite this version:

Matthéo Alcaraz, Sébastien Lyonnais, Chandramouli Ghosh, John Jairo Aguilera-Correa, Sébastien Richeter, et al.. Evaluation and activity of new porphyrin-peptide cage-type conjugates for the photoinactivation of Mycobacterium abscessus. Microbiology Spectrum, 2024, 12 (5), pp.e0000624. 10.1128/spectrum.00006-24 . hal-04603042

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# Evaluation and activity of new porphyrin-peptide cage-type conjugates for the photoinactivation of Mycobacterium abscessus

Matthéo Alcaraz, Sébastien Lyonnais, Chandramouli Ghosh, John Jairo

Aguilera-Correa, Sébastien Richeter, Sébastien Ulrich, Laurent Kremer

► To cite this version:

Matthéo Alcaraz, Sébastien Lyonnais, Chandramouli Ghosh, John Jairo Aguilera-Correa, Sébastien Richeter, et al.. Evaluation and activity of new porphyrin-peptide cage-type conjugates for the photoinactivation of Mycobacterium abscessus. Microbiology Spectrum, 2024, 12 (5), pp.e0000624. 10.1128/spectrum.00006-24 . hal-04603042

HAL Id: hal-04603042

https://hal.science/hal-04603042v1

Submitted on 6 Jun 2024

**HAL** is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
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<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Evaluation and activity of new porphyrin-peptide cage-type conjugates for the photoinactivation of *Mycobacterium abscessus*

Matthéo Alcaraz,¹ Sébastien Lyonnais,² Chandramouli Ghosh,³ John Jairo Aguilera-Correa,¹ Sébastien Richeter,⁴ Sébastien Ulrich,³ Laurent Kremer¹,⁵

**AUTHOR AFFILIATIONS** See affiliation list on p. 5.

**ABSTRACT** *Mycobacterium abscessus* is increasingly recognized as an emerging opportunistic pathogen causing severe lung diseases and cutaneous infections. However, treatment of *M. abscessus* infections remains particularly challenging, largely due to intrinsic resistance to a wide panel of antimicrobial agents. New therapeutic alternatives are urgently needed. Herein, we show that, upon limited irradiation with a blue-light source, newly developed porphyrin-peptide cage-type photosensitizers exert a strong bactericidal activity against smooth and rough variants of *M. abscessus* in planktonic cultures and in biofilms, at low concentrations. Atomic force microscopy unraveled important morphological alterations that include a wrinkled and irregular bacterial surface. The potential of these compounds for a photo-therapeutic use to treat *M. abscessus* skin infections requires further evaluations.

**IMPORTANCE** *Mycobacterium abscessus* causes persistent infections and is extremely difficult to eradicate. Despite intensive chemotherapy, treatment success rates remain very low. Thus, given the unsatisfactory performances of the current regimens, more effective therapeutic alternatives are needed. In this study, we evaluated the activity of newly described porphyrin-peptide cage-type conjugates in the context of photodynamic therapy. We show that upon light irradiation, these compounds were highly bactericidal against *M. abscessus* in vitro, thus qualifying these compounds for future studies dedicated to photo-therapeutic applications against *M. abscessus* skin infections.

**KEYWORDS** *Mycobacterium abscessus*, photodynamic inactivation, photosensitizer, therapeutic activity, atomic force microscopy

Mycobacterium abscessus is a rapidly growing mycobacterial species, often isolated in patients with cystic fibrosis (CF) or with other lung defects (1). In the context of lung infections, M. abscessus has emerged as an important opportunistic pathogen associated with significant mortality (2). It causes also cutaneous infections (3), which remains very difficult to treat, essentially due to its intrinsic resistance to many antibiotics, and requires prolonged periods of treatment, often associated with side effects. Thus, there is an unmet clinical need for new therapeutic approaches with better efficacy, while shortening the duration of the treatments.

In this context, photodynamic therapy (PDT), initially tested for the treatment of tumor cells, has appeared as a possible alternative to control infections. PDT relies on (organic) photosensitizers (PS) (4), which, following light irradiation, produces reactive oxygen species (ROS), leading the rapid photo-oxidation of various biomolecules, such as lipids, proteins, or nucleic acids, leading to rapid cell death. Porphyrins have been used as PS for PDT with antibiotic activity against several human pathogens (5).

**Editor** Gyanu Lamichhane, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

Address correspondence to Laurent Kremer, laurent.kremer@irim.cnrs.fr.

The authors declare no conflict of interest.

See the funding table on p. 6.

**Received** 16 January 2024

**Accepted** 24 March 2024

**Published** 15 April 2024

Copyright © 2024 Alcaraz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
<figure><img src="image_2.png" /></figure>

# Evaluation and activity of new porphyrin-peptide cage-type conjugates for the photoinactivation of *Mycobacterium abscessus*

Matthéo Alcaraz,¹ Sébastien Lyonnais,² Chandramouli Ghosh,³ John Jairo Aguilera-Correa,¹ Sébastien Richeter,⁴ Sébastien Ulrich,³ Laurent Kremer¹,⁵

**AUTHOR AFFILIATIONS** See affiliation list on p. 5.

**ABSTRACT** *Mycobacterium abscessus* is increasingly recognized as an emerging opportunistic pathogen causing severe lung diseases and cutaneous infections. However, treatment of *M. abscessus* infections remains particularly challenging, largely due to intrinsic resistance to a wide panel of antimicrobial agents. New therapeutic alternatives are urgently needed. Herein, we show that, upon limited irradiation with a blue-light source, newly developed porphyrin-peptide cage-type photosensitizers exert a strong bactericidal activity against smooth and rough variants of *M. abscessus* in planktonic cultures and in biofilms, at low concentrations. Atomic force microscopy unraveled important morphological alterations that include a wrinkled and irregular bacterial surface. The potential of these compounds for a photo-therapeutic use to treat *M. abscessus* skin infections requires further evaluations.

**IMPORTANCE** *Mycobacterium abscessus* causes persistent infections and is extremely difficult to eradicate. Despite intensive chemotherapy, treatment success rates remain very low. Thus, given the unsatisfactory performances of the current regimens, more effective therapeutic alternatives are needed. In this study, we evaluated the activity of newly described porphyrin-peptide cage-type conjugates in the context of photodynamic therapy. We show that upon light irradiation, these compounds were highly bactericidal against *M. abscessus* in vitro, thus qualifying these compounds for future studies dedicated to photo-therapeutic applications against *M. abscessus* skin infections.

**KEYWORDS** *Mycobacterium abscessus*, photodynamic inactivation, photosensitizer, therapeutic activity, atomic force microscopy

Mycobacterium abscessus is a rapidly growing mycobacterial species, often isolated in patients with cystic fibrosis (CF) or with other lung defects (1). In the context of lung infections, M. abscessus has emerged as an important opportunistic pathogen associated with significant mortality (2). It causes also cutaneous infections (3), which remains very difficult to treat, essentially due to its intrinsic resistance to many antibiotics, and requires prolonged periods of treatment, often associated with side effects. Thus, there is an unmet clinical need for new therapeutic approaches with better efficacy, while shortening the duration of the treatments.

In this context, photodynamic therapy (PDT), initially tested for the treatment of tumor cells, has appeared as a possible alternative to control infections. PDT relies on (organic) photosensitizers (PS) (4), which, following light irradiation, produces reactive oxygen species (ROS), leading the rapid photo-oxidation of various biomolecules, such as lipids, proteins, or nucleic acids, leading to rapid cell death. Porphyrins have been used as PS for PDT with antibiotic activity against several human pathogens (5).

**Editor** Gyanu Lamichhane, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

Address correspondence to Laurent Kremer, laurent.kremer@irim.cnrs.fr.

The authors declare no conflict of interest.

See the funding table on p. 6.

**Received** 16 January 2024

**Accepted** 24 March 2024

**Published** 15 April 2024

Copyright © 2024 Alcaraz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

CAGE-Arg

M. abscessus S

M. abscessus R

<figure><img src="image_7.png" /></figure>

M. abscessus S

M. abscessus R

<figure><img src="image_8.png" /><figcaption>**FIG 1** Structures of the porphyrin-based PS used in this study and activity against *Escherichia coli* (B-D) and *M. abscessus* (E and F). (A) Structures of H2TMPyP, CAGE-H, and CAGE-Arg. (B-D) Cultures of *E. coli* were exposed 0.1 (light blue bars) and 1 µM (dark blue bars) of H2TMPyP (B), CAGE-Arg (C), or CAGE-H (D) for 30 min prior to irradiation with a blue light source for 5 or 10 min. Colony-forming unit (CFU) quantification was determined immediately after light irradiation. Untreated (black bars) and irradiated bacteria without PS (gray bars) were included as controls. Data are of three independent experiments in triplicate and were analyzed using one-way analysis of variance (ANOVA) test or with Kruskal-Wallis test. **P** ≤ 0.01; ****P ≤ 0.0001; ns, non-significant. (E and F) Cultures of *M. abscessus* S and R were exposed to 1 or 10 µM of CAGE-Arg (E) or CAGE-H (F) for 30 min prior to irradiation with a blue light source for 5 or 10 min. CFU quantification was determined immediately after light irradiation. Untreated (black columns) and irradiated bacteria without PS (gray columns) were included as controls. The error bars represent the standard deviation. Data are of three independent experiments in triplicate and were analyzed using one-way ANOVA test. **P** ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; ns, non-significant.</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>**FIG 1** Structures of the porphyrin-based PS used in this study and activity against *Escherichia coli* (B–D) and *M. abscessus* (E and F). (A) Structures of H2TMPyP, CAGE-H, and CAGE-Arg. (B–D) Cultures of *E. coli* were exposed 0.1 (light blue bars) and 1 µM (dark blue bars) of H2TMPyP (B), CAGE-Arg (C), or CAGE-H (D) for 30 min prior to irradiation with a blue light source for 5 or 10 min. Colony-forming unit (CFU) quantification was determined immediately after light irradiation. Untreated (black bars) and irradiated bacteria without PS (gray bars) were included as controls. Data are of three independent experiments in triplicate and were analyzed using one-way analysis of variance (ANOVA) test or with Kruskal-Wallis test. **P** ≤ 0.01; ****P ≤ 0.0001; ns, non-significant. (E and F) Cultures of *M. abscessus* S and R were exposed to 1 or 10 µM of CAGE-Arg (E) or CAGE-H (F) for 30 min prior to irradiation with a blue light source for 5 or 10 min. CFU quantification was determined immediately after light irradiation. Untreated (black columns) and irradiated bacteria without PS (gray columns) were included as controls. The error bars represent the standard deviation. Data are of three independent experiments in triplicate and were analyzed using one-way ANOVA test. **P** ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; ns, non-significant.</figcaption></figure>
In particular, cationic porphyrins promote strong electrostatic interactions with the polyanionic DNA backbone and membrane proteins (6, 7). The tetra-cationic N-methylated porphyrin (H2TMPyP) (Fig. 1A) has been reported to kill *M. abscessus* subsp. *massiliense* and *Mycobacterium fortuitum* (8), and there is current interest in exploring complex supramolecular and dynamic covalent assemblies as new antibiotics (9, 10). Herein, we explored porphyrin-peptide cage-type conjugates, precisely the positively charged CAGE-Arg and neutral CAGE-H (Fig. 1A) (11), as new architectures to inactivate *M. abscessus* in planktonic cultures and biofilms.

Preliminary experiments showed that long periods of exposure to the blue light was associated with a decrease in the viability of *Escherichia coli* (not shown), prompting us to identify experimental conditions for short periods of irradiation to limit the toxic effect. *E. coli* Stellar cultures at a 600-nm optical density (OD₆₀₀) of 0.8 were washed twice with a 0.9% NaCl, dispensed in 96-well plates, and incubated either with the PS control (H2TMPyP) (12), CAGE-Arg, or CAGE-H at 0.1 µM or 1 µM for 30 min at 37°C in the dark. Then, plates were irradiated for 5 or 10 min using a led blue light source (EvoluChem Led Light HCK1012-01-12, 425 nm, 33 mW/cm²) kept at a 10 cm distance from the wells. Bacteria were diluted in a 10-fold bank dilution of phosphate buffered saline (PBS) and plated onto lysogeny broth (LB) agar to quantify CFU after overnight incubation at 37°C. Under these conditions, incubation with 0.1 µM H2TMPyP, followed by irradiation with a blue light for 5 min, was associated with a 2-Log reduction in the CFUs as compared to bacteria kept in the dark. This effect was further exacerbated by an additional 4-Log decrease in the CFUs with 1 µM H2TMPyP (Fig. 1B; Table S1). Similar results were observed when increasing the duration of the treatment to 10 min (Fig. 1B). This underscores the PDT activity of H2TMPyP against *E. coli*, validating our experimental setup. We next assessed and observed a dose-dependent activity of CAGE-Arg (Fig. 1C; Table S1) and CAGE-H (Fig. 1D; Table S1), although the neutral cage exerted a reduced activity as compared to the cationic cage. As for H2TMPyP, increasing the duration of photoactivation from 5 to 10 min did not improve the PDT activity of the compounds. In the absence of irradiation, the compounds did not affect bacterial viability (not shown). We evaluated the effect of CAGE-Arg and H2TMPyP, the most active compound against the planktonic state, on *E. coli* biofilm (Supplemental Materials and Methods, Fig. S1; Table S2). CAGE-Arg at 1 µM for 10 min provoked a 2-Log reduction in the CFUs (Fig. S1A), while H2TMPyP at 1 µM was associated with a 4-Log decrease in the CFUs at both time points (Fig. S1B).

We next tested the activity of CAGE-Arg and CAGE-H against the smooth (S) and the rough (R) variants of *M. abscessus* CIP104536T (13), characterized by the presence or absence of cell surface glycopeptidolipids, respectively, and differing in their virulence profile; the R strain exhibiting a more pathogenic behavior in mice (14), zebrafish (15), and CF patients (16). Cultures were grown in Middlebrook 7H9 supplemented with 10% OADC enrichment and tyloxapol 0.025% (7H9OADC/ty) to reach an OD₆₀₀nm = 1, washed twice with 0.9% NaCl to prevent scavengers of ROS in 7H9OADC/ty, and washed with a 0.9% NaCl solution containing 0.025% tyloxapol. Bacterial suspensions were sonicated for 3 min in a bath sonicator to remove clumps and dispensed in 96-well plates. PS were added at a concentration of 1 or 10 µM for 30 min in the dark at 37°C prior to irradiation for 5 or 10 min with the blue led light. Serial dilutions in PBS supplemented with 0.025% of tyloxapol were prepared, sonicated for 3 min, and plated onto LB agar, and CFU were counted after 3 days at 37°C. A robust PDT activity of CAGE-Arg (at 1 and 10 µM) was found against *M. abscessus* S and R (Fig. 1E; Table S3), while in general, neutral CAGE-H exhibited a weaker activity than cationic CAGE-Arg (Fig. 1F; Table S3) at comparable times of irradiation and concentrations. Overall, this indicates that CAGE-Arg and CAGE-H exert potent PDT activity upon photoactivation at 425 nm and that a 5-min irradiation is sufficient to kill *M. abscessus* in planktonic state.

*M. abscessus* can also be associated with biomaterial-related infections, where the main pathological mechanism involves biofilm formation, contributing also to persistence, rendering eradication even more challenging (17). We, thus, assessed the activity
In particular, cationic porphyrins promote strong electrostatic interactions with the polyanionic DNA backbone and membrane proteins (6, 7). The tetra-cationic N-methylated porphyrin (H2TMPyP) (Fig. 1A) has been reported to kill *M. abscessus* subsp. *massiliense* and *Mycobacterium fortuitum* (8), and there is current interest in exploring complex supramolecular and dynamic covalent assemblies as new antibiotics (9, 10). Herein, we explored porphyrin-peptide cage-type conjugates, precisely the positively charged CAGE-Arg and neutral CAGE-H (Fig. 1A) (11), as new architectures to inactivate *M. abscessus* in planktonic cultures and biofilms.

Preliminary experiments showed that long periods of exposure to the blue light was associated with a decrease in the viability of *Escherichia coli* (not shown), prompting us to identify experimental conditions for short periods of irradiation to limit the toxic effect. *E. coli* Stellar cultures at a 600-nm optical density (OD₆₀₀) of 0.8 were washed twice with a 0.9% NaCl, dispensed in 96-well plates, and incubated either with the PS control (H2TMPyP) (12), CAGE-Arg, or CAGE-H at 0.1 µM or 1 µM for 30 min at 37°C in the dark. Then, plates were irradiated for 5 or 10 min using a led blue light source (EvoluChem Led Light HCK1012-01-12, 425 nm, 33 mW/cm²) kept at a 10 cm distance from the wells. Bacteria were diluted in a 10-fold bank dilution of phosphate buffered saline (PBS) and plated onto lysogeny broth (LB) agar to quantify CFU after overnight incubation at 37°C. Under these conditions, incubation with 0.1 µM H2TMPyP, followed by irradiation with a blue light for 5 min, was associated with a 2-Log reduction in the CFUs as compared to bacteria kept in the dark. This effect was further exacerbated by an additional 4-Log decrease in the CFUs with 1 µM H2TMPyP (Fig. 1B; Table S1). Similar results were observed when increasing the duration of the treatment to 10 min (Fig. 1B). This underscores the PDT activity of H2TMPyP against *E. coli*, validating our experimental setup. We next assessed and observed a dose-dependent activity of CAGE-Arg (Fig. 1C; Table S1) and CAGE-H (Fig. 1D; Table S1), although the neutral cage exerted a reduced activity as compared to the cationic cage. As for H2TMPyP, increasing the duration of photoactivation from 5 to 10 min did not improve the PDT activity of the compounds. In the absence of irradiation, the compounds did not affect bacterial viability (not shown). We evaluated the effect of CAGE-Arg and H2TMPyP, the most active compound against the planktonic state, on *E. coli* biofilm (Supplemental Materials and Methods, Fig. S1; Table S2). CAGE-Arg at 1 µM for 10 min provoked a 2-Log reduction in the CFUs (Fig. S1A), while H2TMPyP at 1 µM was associated with a 4-Log decrease in the CFUs at both time points (Fig. S1B).

We next tested the activity of CAGE-Arg and CAGE-H against the smooth (S) and the rough (R) variants of *M. abscessus* CIP104536T (13), characterized by the presence or absence of cell surface glycopeptidolipids, respectively, and differing in their virulence profile; the R strain exhibiting a more pathogenic behavior in mice (14), zebrafish (15), and CF patients (16). Cultures were grown in Middlebrook 7H9 supplemented with 10% OADC enrichment and tyloxapol 0.025% (7H9OADC/ty) to reach an OD₆₀₀nm = 1, washed twice with 0.9% NaCl to prevent scavengers of ROS in 7H9OADC/ty, and washed with a 0.9% NaCl solution containing 0.025% tyloxapol. Bacterial suspensions were sonicated for 3 min in a bath sonicator to remove clumps and dispensed in 96-well plates. PS were added at a concentration of 1 or 10 µM for 30 min in the dark at 37°C prior to irradiation for 5 or 10 min with the blue led light. Serial dilutions in PBS supplemented with 0.025% of tyloxapol were prepared, sonicated for 3 min, and plated onto LB agar, and CFU were counted after 3 days at 37°C. A robust PDT activity of CAGE-Arg (at 1 and 10 µM) was found against *M. abscessus* S and R (Fig. 1E; Table S3), while in general, neutral CAGE-H exhibited a weaker activity than cationic CAGE-Arg (Fig. 1F; Table S3) at comparable times of irradiation and concentrations. Overall, this indicates that CAGE-Arg and CAGE-H exert potent PDT activity upon photoactivation at 425 nm and that a 5-min irradiation is sufficient to kill *M. abscessus* in planktonic state.

*M. abscessus* can also be associated with biomaterial-related infections, where the main pathological mechanism involves biofilm formation, contributing also to persistence, rendering eradication even more challenging (17). We, thus, assessed the activity
<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /><figcaption>FIG 2 Quantitative imaging AFM analysis of *M. abscessus* treated in the presence of photoactivable CAGE-Arg. (A) Representative topographic (upper panel) and stiffness (lower panel) images of *M. abscessus* when submitted to four processing conditions: dark condition, light condition, CAGE-Arg (10 µM) treatment in dark condition, and CAGE-Arg (10 µM) treatment in light condition, respectively. (B) Bacterial height, length, and stiffness variations. Height and length were measured from topographic images using the calculated height channel at 80% of the force-distance curve (Dark, n = 23; Light, n = 22; Dark + CAGE-Arg, n = 21; Light + CAGE-Arg, n = 22). Stiffness measurements were extracted from the slope of the force-distance curves (Dark, n = 22; Light, n = 20; Dark + CAGE-Arg, n = 19; Light + CAGE-Arg, n = 22). P-values were calculated using an unpaired t-test. **P**≤0.01; ***P** ≤ 0.001; ****P ≤ 0.0001; ns, non-significant.</figcaption></figure>

of H2TMPyP on *M. abscessus* S biofilms growing in a leukocyte lysate medium, which promotes mycobacterial biofilm formation (18) and then focused on CAGE-Arg, since it displays a better killing activity than CAGE-H *in vitro*. H2TMPyP reduced 79.5% (0.688 Log₁₀, *P* = 0.0164) of mycobacterial viability at 10 µM after 5 min of irradiation inside the biofilm and this reduction was 79.7% (0.6932 Log₁₀, *P* = 0.0151) and 86.8% (0.8784 Log₁₀, *P* = 0.0005) at 1 and 10 µM, respectively, upon irradiation for 10 min (Fig. S2B; Table S4).
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>**FIG 2** Quantitative imaging AFM analysis of *M. abscessus* treated in the presence of photoactivable CAGE-Arg. (A) Representative topographic (upper panel) and stiffness (lower panel) images of *M. abscessus* when submitted to four processing conditions: dark condition, light condition, CAGE-Arg (10 µM) treatment in dark condition, and CAGE-Arg (10 µM) treatment in light condition, respectively. (B) Bacterial height, length, and stiffness variations. Height and length were measured from topographic images using the calculated height channel at 80% of the force-distance curve (Dark, n = 23; Light, n = 22; Dark + CAGE-Arg, n = 21; Light + CAGE-Arg, n = 22). Stiffness measurements were extracted from the slope of the force-distance curves (Dark, n = 22; Light, n = 20; Dark + CAGE-Arg, n = 19; Light + CAGE-Arg, n = 22). *P*-values were calculated using an unpaired *t*-test. \*\**$P \le 0.01$; \*\*\*\*$P \le 0.001$; \*\*\*\*\*$P \le 0.0001$; ns, non-significant.</figcaption></figure>

of H2TMPyP on *M. abscessus* S biofilms growing in a leukocyte lysate medium, which promotes mycobacterial biofilm formation (18) and then focused on CAGE-Arg, since it displays a better killing activity than CAGE-H *in vitro*. H2TMPyP reduced 79.5% (0.688 Log₁₀, *P* = 0.0164) of mycobacterial viability at 10 µM after 5 min of irradiation inside the biofilm and this reduction was 79.7% (0.6932 Log₁₀, *P* = 0.0151) and 86.8% (0.8784 Log₁₀, *P* = 0.0005) at 1 and 10 µM, respectively, upon irradiation for 10 min (Fig. S2B; Table S4).
Irradiation of CAGE-Arg reduced bacterial viability by 74.7% (0.5962 Log₁₀, *P* = 0.0115) and 89.1% (0.961 Log₁₀, *P* < 0.0001) at 1 and 10 µM after 5 min, while this reduction was 83.2% (0.7743 Log₁₀, *P* = 0.0003) and 95.4% (1.334 Log₁₀, *P* < 0.0001) at 1 and 10 µM, respectively, after 10 min of irradiation. This qualifies CAGE-Arg for a photo-therapeutic use to treat *M. abscessus* skin infections.

The effect of treatment with CAGE-Arg on the morphology and mechanical proper-
ties of *M. abscessus* S was evaluated by atomic force microscopy (AFM). Bacteria were
exposed to 10 µM CAGE-Arg in 0.9% NaCl for 30 min, either kept in the dark or irradi-
ated for 10 min. The bacterial suspensions were next deposited on poly-L-lysine coated
glass slides and imaged with a NanoWizard 4XP Bio-AFM (Bruker Nano GmbH, Berlin,
Germany) in PBS at 20°C, using sharp nitride lever probes, cantilever C (SNL-C) probes
(Bruker Nano GmbH, mean cantilever spring constant kcant = 0.08 N/m). AFM analysis
was carried out using force-distance spectroscopy (QI mode, Bruker Nano GmbH), which
is based on force curves informing on the interaction between the AFM tip and the
sample. These curves give access to the sample's topography and stiffness simultane-
ously, upon a constant applied force (700 pN). Figure 2A shows common topography of
a typical *M. abscessus* S (dark condition), ~0.5 µm in diameter and length distribution
centered around 2 µm. The effective stiffness extracted from the force-distance curves
was found in the range of 5.10<sup>-2</sup> N/m, which fits with the typical range (10<sup>-2</sup> N/m to 10<sup>-1</sup>
N/m) reported for various bacteria immobilized on substrates in comparable conditions
(19-21). Irradiated bacteria (Fig. 2A) displayed similar morphology and no significant
changes neither in dimensions nor in cell stiffness. While exposure to CAGE-Arg in the
dark negatively impacted cell stiffness (Fig. 2B), pronounced changes were observed
when *M. abscessus* was exposed to CAGE-Arg and irradiated, resulting in a mixture of
intact and deflated cells harboring membrane wrinkles, protrusions, and extrusions (Fig.
2A). The treated cells appeared softer, indicating alteration of the bacterial membrane.
Such dramatic changes in cell surface shape and roughness and alterations in their
mechanical properties have been previously reported for bacteria treated with various
porphyrins, peptides, or lipopeptides attacking the cell wall (22-24). These results
provide evidence that combining light radiation and CAGE-Arg alter the membrane
integrity and the mechanical properties of *M. abscessus*.

In summary, we provide here evidence that these new porphyrin-peptide cage-type
conjugates, particularly CAGE-Arg, have potential for a photo-therapeutic use to treat *M.
abscessus* skin infections and await further evaluations.

ACKNOWLEDGMENTS

We acknowledge the Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation for funding the PhD of M.A., the French National Research Agency (ILlome-20-CE44-0019) and the "Equipe FRM EQU202103012588" funding to L.K., and MUSE, Université de Montpellier (ANR-16-IDEX-0006) for funding the post-doctoral fellowship of C.G.

The bio-AFM was funded by the REDSAIM program from Montpellier University.

AUTHOR AFFILIATIONS

<footnote n="1">¹Centre National de la Recherche Scientifique UMR 9004, Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier, Montpellier, France</footnote>

<footnote n="2">²CEMIPAI, UAR3725, CNRS, Université de Montpellier, Montpellier, France</footnote>

<footnote n="3">³Institut des Biomolécules Max Mousseron (IBMM), Université of Montpellier, CNRS, ENSCM, Montpellier, France</footnote>

<footnote n="4">⁴Institut Charles Gerhardt Montpellier (ICGM), Université de Montpellier, CNRS, ENSCM, Montpellier, France</footnote>

<footnote n="5">⁵INSERM, IRIM, Montpellier, France</footnote>
Irradiation of CAGE-Arg reduced bacterial viability by 74.7% (0.5962 Log₁₀, *P* = 0.0115) and 89.1% (0.961 Log₁₀, *P* < 0.0001) at 1 and 10 µM after 5 min, while this reduction was 83.2% (0.7743 Log₁₀, *P* = 0.0003) and 95.4% (1.334 Log₁₀, *P* < 0.0001) at 1 and 10 µM, respectively, after 10 min of irradiation. This qualifies CAGE-Arg for a photo-therapeutic use to treat *M. abscessus* skin infections.

The effect of treatment with CAGE-Arg on the morphology and mechanical proper-
ties of *M. abscessus* S was evaluated by atomic force microscopy (AFM). Bacteria were
exposed to 10 µM CAGE-Arg in 0.9% NaCl for 30 min, either kept in the dark or irradi-
ated for 10 min. The bacterial suspensions were next deposited on poly-L-lysine coated
glass slides and imaged with a NanoWizard 4XP Bio-AFM (Bruker Nano GmbH, Berlin,
Germany) in PBS at 20°C, using sharp nitride lever probes, cantilever C (SNL-C) probes
(Bruker Nano GmbH, mean cantilever spring constant kcant = 0.08 N/m). AFM analysis
was carried out using force-distance spectroscopy (QI mode, Bruker Nano GmbH), which
is based on force curves informing on the interaction between the AFM tip and the
sample. These curves give access to the sample's topography and stiffness simultane-
ously, upon a constant applied force (700 pN). Figure 2A shows common topography of
a typical *M. abscessus* S (dark condition), ~0.5 µm in diameter and length distribution
centered around 2 µm. The effective stiffness extracted from the force-distance curves
was found in the range of 5.10<sup>-2</sup> N/m, which fits with the typical range (10<sup>-2</sup> N/m to 10<sup>-1</sup>
N/m) reported for various bacteria immobilized on substrates in comparable conditions
(19-21). Irradiated bacteria (Fig. 2A) displayed similar morphology and no significant
changes neither in dimensions nor in cell stiffness. While exposure to CAGE-Arg in the
dark negatively impacted cell stiffness (Fig. 2B), pronounced changes were observed
when *M. abscessus* was exposed to CAGE-Arg and irradiated, resulting in a mixture of
intact and deflated cells harboring membrane wrinkles, protrusions, and extrusions (Fig.
2A). The treated cells appeared softer, indicating alteration of the bacterial membrane.
Such dramatic changes in cell surface shape and roughness and alterations in their
mechanical properties have been previously reported for bacteria treated with various
porphyrins, peptides, or lipopeptides attacking the cell wall (22-24). These results
provide evidence that combining light radiation and CAGE-Arg alter the membrane
integrity and the mechanical properties of *M. abscessus*.

In summary, we provide here evidence that these new porphyrin-peptide cage-type
conjugates, particularly CAGE-Arg, have potential for a photo-therapeutic use to treat *M.
abscessus* skin infections and await further evaluations.

ACKNOWLEDGMENTS

We acknowledge the Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation for funding the PhD of M.A., the French National Research Agency (ILlome-20-CE44-0019) and the "Equipe FRM EQU202103012588" funding to L.K., and MUSE, Université de Montpellier (ANR-16-IDEX-0006) for funding the post-doctoral fellowship of C.G.

The bio-AFM was funded by the REDSAIM program from Montpellier University.

AUTHOR AFFILIATIONS

<footnote n="1">¹Centre National de la Recherche Scientifique UMR 9004, Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier, Montpellier, France</footnote>

<footnote n="2">²CEMIPAI, UAR3725, CNRS, Université de Montpellier, Montpellier, France</footnote>

<footnote n="3">³Institut des Biomolécules Max Mousseron (IBMM), Université of Montpellier, CNRS, ENSCM, Montpellier, France</footnote>

<footnote n="4">⁴Institut Charles Gerhardt Montpellier (ICGM), Université de Montpellier, CNRS, ENSCM, Montpellier, France</footnote>

<footnote n="5">⁵INSERM, IRIM, Montpellier, France</footnote>
## AUTHOR ORCIDs

Matthéo Alcaraz http://orcid.org/0000-0001-5308-9043

Sébastien Lyonnais http://orcid.org/0000-0002-3154-8568

John Jairo Aguilera-Correa http://orcid.org/0000-0002-4002-1944

Laurent Kremer http://orcid.org/0000-0002-6604-4458

## FUNDING

<table><thead><tr><td>Funder</td><td>Grant(s)</td><td>Author(s)</td></tr></thead><tbody><tr><td>Fondation pour la Recherche Médicale (FRM)</td><td>EQU202103012588</td><td>Laurent Kremer</td></tr><tr><td>Agence Nationale de la Recherche (ANR)</td><td>ILLome-20-CE44-0019</td><td>Laurent Kremer</td></tr><tr><td>Agence Nationale de la Recherche (ANR)</td><td>ANR-16-IDEX-0006</td><td>Sébastien Ulrich</td></tr><tr><td>Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation</td><td></td><td>Matthéo Alcaraz</td></tr></tbody></table>

## DATA AVAILABILITY

All data will be made available upon request to the corresponding author.

## ADDITIONAL FILES

The following material is available online.

## Supplemental Material

Supplemental material (c0af1847-d0fc-4883-803d-866bc8ecc495.docx). Fig. S1 and S2; Tables S1-S4.

## REFERENCES

1. Johansen MD, Herrmann J-L, Kremer L. 2020. Non-tuberculous mycobacteria and the rise of *Mycobacterium abscessus*. Nat Rev Microbiol 18:392–407. https://doi.org/10.1038/s41579-020-0331-1

2. Kwak N, Dalcolmo MP, Daley CL, Eather G, Gayoso R, Hasegawa N, Jhun BW, Koh W-J, Namkoong H, Park J, Thomson R, Ingen J, Zweijpfenning SMH, Yim J-J. 2019. *Mycobacterium abscessus* pulmonary disease: individual patient data meta-analysis. Eur Respir J 54:1801991. https://doi.org/10.1183/13993003.01991-2018

3. Sepulcri C, Vena A, Bassetti M. 2023. Skin and soft tissue infections due to rapidly growing mycobacteria. Curr Opin Infect Dis 36:74–80. https://doi.org/10.1097/QCO.00000000000000905

4. Nguyen V-N, Zhao Z, Tang BZ, Yoon J. 2022. Organic photosensitizers for antimicrobial phototherapy. Chem Soc Rev 51:3324–3340. https://doi.org/10.1039/d1cs00647a

5. Silva AF, Borges A, Giaouris E, Graton Mikcha JM, Simões M. 2018. Photodynamic inactivation as an emergent strategy against foodborne pathogenic bacteria in planktonic and sessile states. Crit Rev Microbiol 44:667–684. https://doi.org/10.1080/1040841X.2018.1491528

6. Oliveira VA, Iglesias BA, Auras BL, Neves A, Terenzi H. 2017. Photoactive meso-tetra(4-pyridyl)porphyrin-tetrakis-[chloro(2,2'bipyridine)platinum(II) derivatives recognize and cleave DNA upon irradiation. Dalton Trans 46:1660–1669. https://doi.org/10.1039/c6dt04634g

7. Ma H-M, Chen X, Zhang N, Han Y-Y, Wu D, Du B, Wei Q. 2009. Spectroscopic studies on the interaction of a water-soluble cationic porphyrin with proteins. Spectrochim Acta A Mol Biomol Spectrosc 72:465–469. https://doi.org/10.1016/j.saa.2008.10.019

8. Guterres KB, Rossi GG, Menezes LB, Anraku de Campos MM, Iglesias BA. 2019. Preliminary evaluation of the positively and negatively charge effects of tetra-substituted porphyrins on photoinactivation of rapidly growing mycobacteria. Tuberculosis (Edinb) 117:45–51. https://doi.org/10.1016/j.tube.2019.06.001

9. Gao S, Yan X, Xie G, Zhu M, Ju X, Stang PJ, Tian Y, Niu Z. 2019. Membrane intercalation-enhanced photodynamic inactivation of bacteria by a metallacycle and TAT-decorated virus coat protein. Proc Natl Acad Sci U S A 116:23437–23443. https://doi.org/10.1073/pnas.1911869116

10. Reuther JF, Dees JL, Kolesnichenko IV, Hernandez ET, Ukrainsev DV, Guduru R, Whiteley M, Anslyn EV. 2018. Dynamic covalent chemistry enables formation of antimicrobial peptide quaternary assemblies in a completely abiotic manner. Nature Chem 10:45–50. https://doi.org/10.1038/nchem.2847

11. Ghosh C, Ali LMA, Bessin Y, Clément S, Richeter S, Bettache N, Ulrich S. 2024. Self-assembled porphyrin-peptide cages for photodynamic therapy. Org Biomol Chem 22:1484–1494. https://doi.org/10.1039/d3ob01887c

12. Merchat M, Bertolini G, Giacomini P, Villanueva A, Jori G. 1996. Meso-substituted cationic porphyrins as efficient photosensitizers of Gram-positive and Gram-negative bacteria. J Photochem Photobiol B 32:153–157. https://doi.org/10.1016/1011-1344(95)07147-4

13. Ripoll F, Pasek S, Schenowitz C, Dossat C, Barbe V, Rottman M, Macheras E, Heym B, Herrmann J-L, Daffé M, Brosch R, Risler J-L, Gaillard J-L. 2009. Non mycobacterial virulence genes in the genome of the emerging pathogen *Mycobacterium abscessus*. PLoS One 4:e5660. https://doi.org/10.1371/journal.pone.0005660

14. Catherinot E, Clarissou J, Etienne G, Ripoll F, Emile J-F, Daffé M, Perronne C, Soudais C, Gaillard J-L, Rottman M. 2007. Hypervirulence of a rough variant of the *Mycobacterium abscessus* type strain. Infect Immun 75:1055–1058. https://doi.org/10.1128/IAI.00835-06

15. Bernut A, Viljoen A, Dupont C, Sapriel G, Blaise M, Bouchier C, Brosch R, de Chastellier C, Herrmann J-L, Kremer L. 2016. Insights into the smooth-to-rough transitioning in *Mycobacterium bolletii* unravels a functional Tyr residue conserved in all mycobacterial MmpL family members. Mol Microbiol 99:866–883. https://doi.org/10.1111/mmi.13283

16. Catherinot E, Roux A-L, Macheras E, Hubert D, Matmar M, Dannhoffer L, Chinet T, Morand P, Poyart C, Heym B, Rottman M, Gaillard J-L, Herrmann J-L. 2009. Acute respiratory failure involving an R variant of *Mycobacterium abscessus*. J Clin Microbiol 47:271–274. https://doi.org/10.1128/JCM.01478-08
## AUTHOR ORCIDs

Matthéo Alcaraz http://orcid.org/0000-0001-5308-9043

Sébastien Lyonnais http://orcid.org/0000-0002-3154-8568

John Jairo Aguilera-Correa http://orcid.org/0000-0002-4002-1944

Laurent Kremer http://orcid.org/0000-0002-6604-4458

## FUNDING

<table><thead><tr><td>Funder</td><td>Grant(s)</td><td>Author(s)</td></tr></thead><tbody><tr><td>Fondation pour la Recherche Médicale (FRM)</td><td>EQU202103012588</td><td>Laurent Kremer</td></tr><tr><td>Agence Nationale de la Recherche (ANR)</td><td>ILLome-20-CE44-0019</td><td>Laurent Kremer</td></tr><tr><td>Agence Nationale de la Recherche (ANR)</td><td>ANR-16-IDEX-0006</td><td>Sébastien Ulrich</td></tr><tr><td>Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation</td><td></td><td>Matthéo Alcaraz</td></tr></tbody></table>

## DATA AVAILABILITY

All data will be made available upon request to the corresponding author.

## ADDITIONAL FILES

The following material is available online.

## Supplemental Material

Supplemental material (c0af1847-d0fc-4883-803d-866bc8ecc495.docx). Fig. S1 and S2; Tables S1-S4.

## REFERENCES

1. Johansen MD, Herrmann J-L, Kremer L. 2020. Non-tuberculous mycobacteria and the rise of *Mycobacterium abscessus*. Nat Rev Microbiol 18:392-407. https://doi.org/10.1038/s41579-020-0331-1

2. Kwak N, Dalcolmo MP, Daley CL, Eather G, Gayoso R, Hasegawa N, Jhun BW, Koh W-J, Namkoong H, Park J, Thomson R, Ingen J, Zweijpfenning SMH, Yim J-J. 2019. *Mycobacterium abscessus* pulmonary disease: individual patient data meta-analysis. Eur Respir J 54:1801991. https://doi.org/10.1183/13993003.01991-2018

3. Sepulcri C, Vena A, Bassetti M. 2023. Skin and soft tissue infections due to rapidly growing mycobacteria. Curr Opin Infect Dis 36:74-80. https://doi.org/10.1097/QCO.00000000000000905

4. Nguyen V-N, Zhao Z, Tang BZ, Yoon J. 2022. Organic photosensitizers for antimicrobial phototherapy. Chem Soc Rev 51:3324-3340. https://doi.org/10.1039/d1cs00647a

5. Silva AF, Borges A, Giaouris E, Graton Mikcha JM, Simões M. 2018. Photodynamic inactivation as an emergent strategy against foodborne pathogenic bacteria in planktonic and sessile states. Crit Rev Microbiol 44:667-684. https://doi.org/10.1080/1040841X.2018.1491528

6. Oliveira VA, Iglesias BA, Auras BL, Neves A, Terenzi H. 2017. Photoactive meso-tetra(4-pyridyl)porphyrin-tetrakis-[chloro(2,2'bipyridine)platinum(II) derivatives recognize and cleave DNA upon irradiation. Dalton Trans 46:1660-1669. https://doi.org/10.1039/c6dt04634g

7. Ma H-M, Chen X, Zhang N, Han Y-Y, Wu D, Du B, Wei Q. 2009. Spectroscopic studies on the interaction of a water-soluble cationic porphyrin with proteins. Spectrochim Acta A Mol Biomol Spectrosc 72:465-469. https://doi.org/10.1016/j.saa.2008.10.019

8. Guterres KB, Rossi GG, Menezes LB, Anraku de Campos MM, Iglesias BA. 2019. Preliminary evaluation of the positively and negatively charge effects of tetra-substituted porphyrins on photoinactivation of rapidly growing mycobacteria. Tuberculosis (Edinb) 117:45-51. https://doi.org/10.1016/j.tube.2019.06.001

9. Gao S, Yan X, Xie G, Zhu M, Ju X, Stang PJ, Tian Y, Niu Z. 2019. Membrane intercalation-enhanced photodynamic inactivation of bacteria by a metallacycle and TAT-decorated virus coat protein. Proc Natl Acad Sci U S A 116:23437-23443. https://doi.org/10.1073/pnas.1911869116

10. Reuther JF, Dees JL, Kolesnichenko IV, Hernandez ET, Ukrainsev DV, Guduru R, Whiteley M, Anslyn EV. 2018. Dynamic covalent chemistry enables formation of antimicrobial peptide quaternary assemblies in a completely abiotic manner. Nature Chem 10:45-50. https://doi.org/10.1038/nchem.2847

11. Ghosh C, Ali LMA, Bessin Y, Clément S, Richeter S, Bettache N, Ulrich S. 2024. Self-assembled porphyrin-peptide cages for photodynamic therapy. Org Biomol Chem 22:1484-1494. https://doi.org/10.1039/d3ob01887c

12. Merchat M, Bertolini G, Giacomini P, Villanueva A, Jori G. 1996. Meso-substituted cationic porphyrins as efficient photosensitizers of Gram-positive and Gram-negative bacteria. J Photochem Photobiol B 32:153-157. https://doi.org/10.1016/1011-1344(95)07147-4

13. Ripoll F, Pasek S, Schenowitz C, Dossat C, Barbe V, Rottman M, Macheras E, Heym B, Herrmann J-L, Daffé M, Brosch R, Risler J-L, Gaillard J-L. 2009. Non mycobacterial virulence genes in the genome of the emerging pathogen *Mycobacterium abscessus*. PLoS One 4:e5660. https://doi.org/10.1371/journal.pone.0005660

14. Catherinot E, Clarissou J, Etienne G, Ripoll F, Emile J-F, Daffé M, Perronne C, Soudais C, Gaillard J-L, Rottman M. 2007. Hypervirulence of a rough variant of the *Mycobacterium abscessus* type strain. Infect Immun 75:1055-1058. https://doi.org/10.1128/IAI.00835-06

15. Bernut A, Viljoen A, Dupont C, Sapriel G, Blaise M, Bouchier C, Brosch R, de Chastellier C, Herrmann J-L, Kremer L. 2016. Insights into the smooth-to-rough transitioning in *Mycobacterium bolletii* unravels a functional Tyr residue conserved in all mycobacterial MmpL family members. Mol Microbiol 99:866-883. https://doi.org/10.1111/mmi.13283

16. Catherinot E, Roux A-L, Macheras E, Hubert D, Matmar M, Dannhoffer L, Chinet T, Morand P, Poyart C, Heym B, Rottman M, Gaillard J-L, Herrmann J-L. 2009. Acute respiratory failure involving an R variant of *Mycobacterium abscessus*. J Clin Microbiol 47:271-274. https://doi.org/10.1128/JCM.01478-08
17. Clary G, Sasindran SJ, Nesbitt N, Mason L, Cole S, Azad A, McCoy K, Schlesinger LS, Hall-Stoodley L. 2018. *Mycobacterium abscessus* smooth and rough morphotypes form antimicrobial-tolerant biofilm phenotypes but are killed by acetic acid. Antimicrob Agents Chemother 62:e01782-17. https://doi.org/10.1128/AAC.01782-17

18. Chakraborty P, Kumar A. 2019. The extracellular matrix of mycobacterial biofilms: could we shorten the treatment of mycobacterial infections? *Microb Cell* 6:105-122. https://doi.org/10.15698/mic2019.02.667

19. Dufrêne YF. 2002. Atomic force microscopy, a powerful tool in microbiology. *J Bacteriol* 184:5205-5213. https://doi.org/10.1128/JB.184.19.5205-5213.2002

20. Whitehead KA, Rogers D, Colligon J, Wright C, Verran J. 2006. Use of the atomic force microscope to determine the effect of substratum surface topography on the ease of bacterial removal. *Colloids Surf B Biointerfaces* 51:44-53. https://doi.org/10.1016/j.colsurfb.2006.05.003

21. Arnoldi M, Fritz M, Bäuerlein E, Radmacher M, Sackmann E, Boulbitch A. 2000. Bacterial Turgor pressure can be measured by atomic force

microscopy. Phys Rev E Stat Phys Plasmas Fluids Relat Interdiscip Top 62:1034-1044. https://doi.org/10.1103/physreve.62.1034

22. Nasompag S, Siritongsuk P, Thammawithan S, Srichaiyapol O, Prangkio P, Camesano TA, Sinthuvanich C, Patramanon R. 2021. AFM study of nanoscale membrane perturbation induced by antimicrobial lipopeptide $C_{14}$ KYR. *Membranes* (Basel) 11:495. https://doi.org/10.3390/membranes11070495

23. Rossi GG, Guterres KB, da Silveira CH, Moreira KS, Burgo TAL, Iglesias BA, de Campos MMA. 2020. Peripheral tetra-cationic Pt(II) porphyrins photo-inactivating rapidly growing mycobacteria: first application in mycobacteriology. *Microb Pathog* 148:104455. https://doi.org/10.1016/j.micpath.2020.104455

24. Yang C-H, Chen Y-C, Peng S-Y, Tsai AP-Y, Lee TJ-F, Yen J-H, Liou J-W. 2018. An engineered arginine-rich α-helical antimicrobial peptide exhibits broad-spectrum bactericidal activity against pathogenic bacteria and reduces bacterial infections in mice. *Sci Rep* 8:14602. https://doi.org/10.1038/s41598-018-32981-3
17. Clary G, Sasindran SJ, Nesbitt N, Mason L, Cole S, Azad A, McCoy K, Schlesinger LS, Hall-Stoodley L. 2018. *Mycobacterium abscessus* smooth and rough morphotypes form antimicrobial-tolerant biofilm phenotypes but are killed by acetic acid. Antimicrob Agents Chemother 62:e01782-17. https://doi.org/10.1128/AAC.01782-17

18. Chakraborty P, Kumar A. 2019. The extracellular matrix of mycobacterial biofilms: could we shorten the treatment of mycobacterial infections? Microb Cell 6:105-122. https://doi.org/10.15698/mic2019.02.667

19. Dufrêne YF. 2002. Atomic force microscopy, a powerful tool in microbiology. J Bacteriol 184:5205-5213. https://doi.org/10.1128/JB.184.19.5205-5213.2002

20. Whitehead KA, Rogers D, Colligon J, Wright C, Verran J. 2006. Use of the atomic force microscope to determine the effect of substratum surface topography on the ease of bacterial removal. Colloids Surf B Biointerfaces 51:44-53. https://doi.org/10.1016/j.col surfb.2006.05.003

21. Arnoldi M, Fritz M, Bäuerlein E, Radmacher M, Sackmann E, Boulbitch A. 2000. Bacterial Turgor pressure can be measured by atomic force

microscopy. Phys Rev E Stat Phys Plasmas Fluids Relat Interdiscip Top 62:1034-1044. https://doi.org/10.1103/physreve.62.1034

22. Nasompag S, Siritongsuk P, Thammawithan S, Srichaiyapol O, Prangkio P, Camesano TA, Sinthuvanich C, Patramanon R. 2021. AFM study of nanoscale membrane perturbation induced by antimicrobial lipopeptide $C_{14}$ KYR. Membranes (Basel) 11:495. https://doi.org/10.3390/membranes11070495

23. Rossi GG, Guterres KB, da Silveira CH, Moreira KS, Burgo TAL, Iglesias BA, de Campos MMA. 2020. Peripheral tetra-cationic Pt(II) porphyrins photo-inactivating rapidly growing mycobacteria: first application in mycobacteriology. Microb Pathog 148:104455. https://doi.org/10.1016/j.micpath.2020.104455

24. Yang C-H, Chen Y-C, Peng S-Y, Tsai AP-Y, Lee TJ-F, Yen J-H, Liou J-W. 2018. An engineered arginine-rich α-helical antimicrobial peptide exhibits broad-spectrum bactericidal activity against pathogenic bacteria and reduces bacterial infections in mice. Sci Rep 8:14602. https://doi.org/10.1038/s41598-018-32981-3