Macrophage infectivity potentiator protein, a peptidyl prolyl cis-trans isomerase, essential for Coxiella burnetii growth and pathogenesis

Abstract

Coxiella burnetii is a Gram-negative intracellular pathogen that causes the debilitating disease Q fever, which affects both animals and humans. The only available human vaccine, Q-Vax, is effective but has a high risk of severe adverse reactions, limiting its use as a countermeasure to contain outbreaks. Therefore, it is essential to identify new drug targets to treat this infection. Macrophage infectivity potentiator (Mip) proteins catalyse the folding of proline-containing proteins through their peptidyl prolyl cis-trans isomerase (PPIase) activity and have been shown to play an important role in the virulence of several pathogenic bacteria. To date the role of the Mip protein in C. burnetii pathogenesis has not been investigated. This study demonstrates that CbMip is likely to be an essential protein in C. burnetii. The pipecolic acid derived compounds, SF235 and AN296, which have shown utility in targeting other Mip proteins from pathogenic bacteria, demonstrate inhibitory activities against CbMip. These compounds were found to significantly inhibit intracellular replication of C. burnetii in both HeLa and THP-1 cells. Furthermore, SF235 and AN296 were also found to exhibit antibiotic properties against both the virulent (Phase I) and avirulent (Phase II) forms of C. burnetii Nine Mile Strain in axenic culture. Comparative proteomics, in the presence of AN296, revealed alterations in stress responses with H2O2 sensitivity assays validating that Mip inhibition increases the sensitivity of C. burnetii to oxidative stress. In addition, SF235 and AN296 were effective in vivo and significantly improved the survival of Galleria mellonella infected with C. burnetii. These results suggest that unlike in other bacteria, Mip in C. burnetii is required for replication and that the development of more potent inhibitors against CbMip is warranted and offer potential as novel therapeutics against this pathogen.

Inhibition of macrophage infectivity potentiator in Burkholderia pseudomallei suppresses pro-inflammatory responses in murine macrophages

“…We focused on the macrophage infectivity potentiator (Mip) protein, a protein folding enzyme that exhibits peptidyl-prolyl isomerases (PPIase) activity, due to its established role in B. pseudomallei pathogenesis ( Norville et al, 2011b ). Additionally, Mip has been shown to be a virulence factor in other bacterial species including, Legionella pneumophila , Neisseria gonorrhoeae , Neisseria meningitidis and Coxiella burnetii , as well as parasites Trypanosoma cruzi and Leishmania major ( Cianciotto and Fields, 1992 ; Lundemose et al, 1993 ; Moro et al., 1995 ; Leuzzi et al., 2005 ; Reimer et al., 2016 ; Iwasaki et al., 2022 ; Debowski et al., 2023 ) Since it has been shown that other PPIase proteins have immunomodulatory roles during bacterial infections ( Pandey et al., 2017 ), we investigated, for the first time, the possible involvement of Mip in the innate immune response to B. pseudomallei infection. Using small molecule inhibitors designed to inhibit Mip, we demonstrated that treatment of B. pseudomallei -infected murine macrophages with the inhibitor AN_CH_37 resulted in a decrease in secreted pro-inflammatory cytokines IL-1β, TNFα and IL-6, and an increase in MCP-1, 24 hours post-infection.…”

“…Three independent runs were performed for each protein and inhibitor/DMSO combination. The inhibition constant ( K i ) of SF235 and AN_CH_37 against Bps Mip were determined as previously described ( Iwasaki et al., 2022 ; Debowski et al., 2023 ).…”

Identification of active main metabolites of anti‐infective inhibitors of the macrophage infectivity potentiator protein by liquid chromatography using mass detection

“…Taken together, the results clearly show that the pyridinesubstituted compounds 2a-4a form N-oxides and then entirely The fact that the extent of metabolic conversion of N-oxides was much lower confirmed the hypothesis, that active metabolites, being the N-oxide, could be responsible for the long-lasting effect observed in vivo. [13] Evidence that the main metabolites of 2a, 3a, and 4a are the respective N-oxides was confirmed by comparison of the independently synthesized N-oxides [14] and the metabolites using HPLC-ultraviolet (UV) (method III) and LC/MS quadrupole time-offlight (qTOF) (method IV). With both methods, retention time and fragment spectra of both were in exact agreement (data are given in Supporting Information S1: Figure S3 and Figure S4).…”

“…[9] Starting with a lead structure 1 (Figure 1), which exhibits anti-PPIase activity particularly for Legionella pneumophila, [10] Burkholderia pseudomallei, [11] Klebsiella pneumoniae, and Neisseria meningitidis, but also for the parasite Leishmania major, [12] a series of highly potent inhibitors were developed. Compounds that gained potency due to their additional side chain, in the linker between the two carbonyl moieties, were also shown to be efficient inhibitors of the Mip of Coxiella burnetii [13] and Neisseria gonorrhea. [14] All previously described Mip inhibitors (1, 2a, 3a, 4a, 2b, 3b, and 4b (Figure 1), which were used for this study, demonstrated strong binding affinity to Burkholderia pseudomallei-Mip (BpMip) in the fluorescence polarization assay (FPA), as well as high potency in the PPIase assay.…”

“…However, a longlasting activity was observed in Galleria mellonella larvae by Debowski et al, who have shown that this type of inhibitors reduces Coxiella burnetii infections throughout the 10-day larval lifespan. [13] This is a F I G U R E 1 Chemical structures of the Mip inhibitors investigated. All inhibitors used for the metabolism study are described elsewhere and already proven to be anti-peptidyl-prolyl cis-trans isomerase (PPIase) active.…”