The Mycobacterium tuberculosis mycolate flippase MmpL3 has been the proposed target for multiple inhibitors with diverse chemical scaffolds. This diversity in chemical scaffolds has made it difficult to predict compounds that inhibit MmpL3 without whole-genome sequencing of isolated resistant mutants. Here, we describe the identification of four new inhibitors that select for resistance mutations in mmpL3. Using these resistant mutants, we conducted a targeted whole-cell phenotypic screen of 163 novel M. tuberculosis growth inhibitors for differential growth inhibition of wild-type M. tuberculosis compared to the growth of a pool of 24 unique mmpL3 mutants. The screen successfully identified six additional putative MmpL3 inhibitors. The compounds were bactericidal both in vitro and against intracellular M. tuberculosis. M. tuberculosis cells treated with these compounds were shown to accumulate trehalose monomycolates, have reduced levels of trehalose dimycolate, and displace an MmpL3-specific probe, supporting MmpL3 as the target. The inhibitors were mycobacterium specific, with several also showing activity against the nontuberculous mycobacterial species M. abscessus. Cluster analysis of cross-resistance profiles generated by dose-response experiments for each combination of 13 MmpL3 inhibitors against each of the 24 mmpL3 mutants defined two clades of inhibitors and two clades of mmpL3 mutants. Pairwise combination studies of the inhibitors revealed interactions that were specific to the clades identified in the cross-resistance profiling. Additionally, modeling of resistance-conferring substitutions to the MmpL3 crystal structure revealed clade-specific localization of the residues to specific domains of MmpL3, with the clades showing differential resistance. Several compounds exhibited high solubility and stability in microsomes and low cytotoxicity in macrophages, supporting their further development. The combined study of multiple mutants and novel compounds provides new insights into structure-function interactions of MmpL3 and small-molecule inhibitors.

22combination studies of the inhibitors revealed interactions that were specific to the clades 40 identified in the cross-resistance profiling. Additionally, modeling of resistance substitutions to 41 the MmpL3 crystal structure revealed clade specific localization of the residues to specific 42 domains of MmpL3, with the clades showing differential resistance. Several compounds 43 exhibited high solubility and stability in microsomes and low cytotoxicity in macrophages, 44supporting their further development. The combined study of multiple mutants and novel 45 compounds provides new insights into structure-function interactions of MmpL3 and small 46 molecule inhibitors. 47 the last decade, several of these screens have identified MmpL3 as the proposed target for 53 diverse small molecule inhibitors including AU1235, BM212, C215, DA-5, E11, 54 indolecarboxamides, HC2091, PIPD1, Rimonabant, Spiro, THPP and 55 SQ109 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 . MmpL3 is an essential flippase responsible for transporting 56 acetylated-trehalose monomycolate (TMM) synthesized in the cytoplasm to the pseudo-57 periplasmic space 13, 14, 15, 16, 17 . These TMMs are then converted into trehalose dimycolate (TDM) 58by the Ag85 complex in the cell envelope 18 . MmpL3 is essential as evidenced by a pre-existing 59 rescue allele being required to generate an mmpL3 knockout 2, 14, 17, 19, 20, 21 , lack of mutants in 60 high-throughput transposon mutagenesis screens 22, 23 , and studies that show rapid killing in vitro 61 and in vivo in acute infection models when mmpL3 expression is conditionally inhibited 14, 19 . This 62 makes MmpL3 an attractive target for drug development, with one of its inhibitors, SQ109, 63 currently in clinical trials 24 . 64MmpL3 inhibitors fall into diverse classes of chemical scaffolds 25, 26, 27 , making it hard to 65 computationally predict potential MmpL3 inhibitors based on chemical scaffolds. However, given 66 the frequent finding of MmpL3 as a target, it is reasonable to expect that many new hits in a 67 high throughput screen (HTS) may be acting against MmpL3. MmpL3 inhibitors have been 68 identified by the isolation and sequencing of resistant mutants with single nucleotide variations 69 (SNVs) mapping to the coding region of mmpL3, which is time-consuming and costly. Efforts to 70 discover MmpL3 inhibitors using targeted approaches include generating hypomorphs, where a 71 mmpL3 knock down strain showed enhanced sensitivity to MmpL3 inhibitors, including AU1235 72 14 . However, this strain was also shown to be sensitive to isoniazid (INH) an inhibitor of InhA of 73 the FAS-II pathway involved in mycolic acid synthesis, suggesting that while a mmpL3 74 knockdown strain has robust screening potential for inhibitors of mycolic acid synthesis, 75 maturation, and transport, such strains are not specific enough to identify inhibitors that 76 selectively target MmpL3. 77An alternative approach, employed in this study, is to use a pool of mmpL3 resistant 78 mutants to discover potential MmpL3 ...

No abstract

Listeria monocytogenes (Lm) is a bacterial pathogen that causes listeriosis in immunocompromised individuals, particularly pregnant women. Several virulence factors support the intracellular lifecycle of Lm and facilitate cell-to-cell spread, allowing it to occupy multiple niches within the host and cross protective barriers, including the placenta. One family of virulence factors, internalins, contributes to Lm pathogenicity by inducing specific uptake and conferring tissue tropism. Over 25 internalins have been identified thus far, but only a few have been extensively studied. Internalins contain leucine-rich repeat (LRR) domains which enable protein-protein interactions, allowing Lm to bind host proteins. Notably, other Listeria species express internalins but cannot colonize human hosts, prompting questions regarding the evolution of internalins within the genus Listeria. Internalin P (InlP) promotes placental colonization through interaction with the host protein afadin. Though prior studies of InlP have begun to elucidate its role in Lm pathogenesis, there remains a lack of information regarding homologs in other Listeria species. Here, we have used a computational evolutionary approach to identify InlP homologs in additional Listeria species. We found that L. ivanovii londoniensis (Liv) and L. seeligeri (Ls) encode InlP homologs. We also found InlP-like homologs in L. innocua and the recently identified species L. costaricensis. All newly identified homologs lack the full-length LRR6 and LRR7 domains found in Lm InlP. These findings inform on the evolution of one key Lm virulence factor, InlP, and serve as a springboard for future evolutionary studies of Lm pathogenesis as well as mechanistic studies of Listeria internalins.

Listeria monocytogenes (Lm) is a bacterial pathogen that causes listeriosis in immunocompromised individuals, particularly pregnant women. Several virulence factors support the intracellular lifecycle of Lm and facilitate cell-to-cell spread, allowing it to occupy multiple niches within the host and cross-protective barriers, including the placenta. One family of virulence factors, internalins, contributes to Lm pathogenicity by inducing specific uptake and conferring tissue tropism. Over 25 internalins have been identified thus far, but only a few have been extensively studied. Internalins contain leucine-rich repeat (LRR) domains that enable protein-protein interactions, allowing Lm to bind host proteins. Notably, other Listeria species express internalins but cannot colonize human hosts, prompting questions regarding the evolution of internalins within the genus Listeria . Internalin P (InlP) promotes placental colonization through interaction with the host protein afadin. Although prior studies of InlP have begun to elucidate its role in Lm pathogenesis, there remains a lack of information regarding homologs in other Listeria species. Here, we have used a computational evolutionary approach to identify InlP homologs in additional Listeria species. We found that Listeria ivanovii londoniensis (Liv) and Listeria seeligeri (Ls) encode InlP homologs. We also found InlP-like homologs in Listeria innocua and the recently identified species Listeria costaricensis . All newly identified homologs lack the full-length LRR6 and LRR7 domains found in Lm’s InlP. These findings are informative regarding the evolution of one key Lm virulence factor, InlP, and serve as a springboard for future evolutionary studies of Lm pathogenesis as well as mechanistic studies of Listeria internalins.