The effects of cyclic peptide [R4W4] in combination with first-line therapy on the survival of Mycobacterium avium

Abstract

Background: Mycobacterium avium (M. avium) is a nontuberculous mycobacterium (NTM) that can cause pulmonary and extrapulmonary infections mostly in immunocompromised individuals, such as those with HIV and diabetes. Traditionally, rifampicin (RIF) and azithromycin (AZ) have been used for a 12-month duration as first-line antibiotics against M. avium. Due to the increased multidrug resistance, novel ways, such as enhancement of macrophages response, are needed to provide adequate immune response required to clear M. avium infection.

Methods and findings: In this study, we aim to study the effects of using THP-1 cells, which are monocyte-like cells, to induce a macrophage response and control M. avium infection when used in combination with traditional treatments such as RIF and AZ in free and liposomal forms. Traditional treatments' effects are studied when used alone and in combination therapy with cyclic peptide [R4W4] (liposomal encapsulated and liposomal combination). Colony-forming units (CFU) counts were assessed for all samples 3 hours, 4 days, and 8 days post-treatment. A significant reduction in the intracellular viability of M. avium was observed when THP-1 cells were treated with liposomal combination [R4W4]+RIF and liposomal combination [R4W4]+AZ compared to when treated with liposomal RIF or liposomal AZ alone, respectively.

Conclusion: Our findings show that liposomal combination [R4W4] is a promising adjuvant therapy to increase M. avium susceptibility to known antibiotics.

Keywords: Mycobacterium avium; antibiotics; antimicrobial; cyclic peptide; macrophages.

Figure 1

CFU counts of M. avium culture inside THP-1 cells treated with free form [R4W4]. (A) CFU/mL of M. avium treated with 2, 4, 8 µg/mL free [R4W4] at 3 h post-treatment. (B) CFU/mL of M. avium treated with 2, 4, 8 µg/mL free [R4W4] at 4 days post-treatment. M. avium was treated, then incubated at 37°C, and then terminated at 3 h and 4 days post-treatment. GraphPad Prism Software version 9.5.1 was utilized for analysis. Statistical analysis was performed using ANOVA. p-values are indicated at the top of each graph, and <0.05 (*) and <0.01 (**) were considered significant. Nonsignificant p-values are indicated as ns.

Figure 2

CFU counts M. avium culture inside THP-1 cells treated with free forms of RIF and combination [R4W4]+RIF. (A) CFU/mL of M. avium treated with 4, 8, 16 µg/mL free RIF and with 4, 8, 16 µg/mL free RIF with 2, 4, 8 µg/mL [R4W4], respectively, at 3 h post-treatment. (B) CFU/mL of M. avium treated with 4, 8, 16 µg/mL free RIF and with 4, 8, 16 µg/mL free RIF with 2, 4, 8 µg/mL [R4W4], respectively, at 4 days post-treatment. (C) CFU/mL of M. avium treated with 4, 8, 16 µg/mL free RIF and with 4, 8, 16 µg/mL free RIF with 2, 4, 8 µg/mL [R4W4], respectively, at 8 days post-treatment. GraphPad Prism Software version 9.5.1 was utilized for analysis. Statistical analysis was performed using ANOVA. p-values are indicated at the top of each graph, and <0.05 (*) and <0.01 (**) were considered significant. ****=P value <0.0001.

Figure 3

CFU counts of M. avium culture inside THP-1 cells treated with liposomal forms of RIF, liposomal combination [R4W4]+RIF (LC), and liposomal encapsulated [R4W4]+RIF (LE). (A) CFU/mL of M. avium treated with 4, 8, 16 µg/mL liposomal RIF and with 4, 8, 16 µg/mL liposomal RIF combination (LC) and encapsulated (LE) with 2, 4, 8 µg/mL [R4W4], respectively, at 3 h post-treatment. (B) CFU/mL of M. avium treated with 4, 8, 16 µg/mL liposomal RIF and with 4, 8, 16 µg/mL liposomal RIF combination (LC) and encapsulated (LE) with 2, 4, 8 µg/mL [R4W4], respectively, at 4 days post-treatment. (A–C) CFU/mL of M. avium treated with 4, 8, 16 µg/mL liposomal RIF and with 4, 8, 16 µg/mL liposomal RIF combination (LC) and encapsulated (LE) with 2, 4, 8 µg/mL [R4W4], respectively, at 8 days post-treatment. GraphPad Prism Software version 9.5.1 was utilized for analysis. Statistical analysis was performed using ANOVA. p-values are indicated at the top of each graph, and <0.05 (*), <0.01 (**), and <0.0001 (****) were considered significant.

Figure 4

(A–C) CFU counts M. avium culture inside THP-1 cells treated with free forms of AZ and unconjugated [R4W4]+AZ. (A) CFU/mL of M. avium treated with 1, 2, 4 µg/mL free AZ and with 1, 2, 4 µg/mL free AZ with 2, 4, 8 µg/mL [R4W4] (unconjugated), respectively, at 3 h post-treatment. (B) CFU/mL of M. avium treated with 1, 2, 4 µg/mL free AZ and with 1, 2, 4 µg/mL free AZ with 2, 4, 8 µg/mL [R4W4] (unconjugated), respectively, at 4 days post-treatment. (C) CFU/mL of M. avium treated with 1, 2, 4 µg/mL free AZ and with 1, 2, 4 µg/mL free AZ with 2, 4, 8 µg/mL [R4W4] (unconjugated), respectively, at 8 days post-treatment. M. avium was treated, then incubated at 37°C and then terminated at 3 h, 4 days, and 8 days post-treatment. (D–F) CFU counts of M. avium culture inside THP-1 cells treated with liposomal forms of AZ, liposomal combination [R4W4]+AZ (LC), and liposomal encapsulated [R4W4]+AZ (LE). (D) CFU/mL of M. avium treated with 1, 2, 4 µg/mL liposomal AZ and with 1, 2, 4 µg/mL liposomal AZ combination (LC) and encapsulated (LE) with 2, 4, 8 µg/mL [R4W4], respectively, at 3 h post-treatment. (E) CFU/mL of M. avium treated with 1, 2, 4 µg/mL liposomal AZ and with 1, 2, 4 µg/mL liposomal AZ combination (LC) and encapsulated (LE) with 2, 4, 8 µg/mL [R4W4], respectively, at 4 days post-treatment. (F) CFU/mL of M. avium treated with 1, 2, 4 µg/mL liposomal AZ and with 1, 2, 4 µg/mL liposomal AZ combination (LC) and encapsulated (LE) with 2, 4, 8 µg/mL [R4W4], respectively, at 8 days post-treatment. GraphPad Prism Software version 9.5.1 was utilized for analysis. Statistical analysis was performed using ANOVA. p-values are indicated at the top of each graph, and <0.05 (*), <0.01 (**), and <0.0001 (****) were considered significant.