Cyclopropanol Warhead in Malleicyprol Confers Virulence of Human‐ and Animal‐Pathogenic Burkholderia Species

Abstract Burkholderia species such as B. mallei and B. pseudomallei are bacterial pathogens causing fatal infections in humans and animals (glanders and melioidosis), yet knowledge on their virulence factors is limited. While pathogenic effects have been linked to a highly conserved gene locus (bur/mal) in the B. mallei group, the metabolite associated to the encoded polyketide synthase, burkholderic acid (syn. malleilactone), could not explain the observed phenotypes. By metabolic profiling and molecular network analyses of the model organism B. thailandensis, the primary products of the cryptic pathway were identified as unusual cyclopropanol‐substituted polyketides. First, sulfomalleicyprols were identified as inactive precursors of burkholderic acid. Furthermore, a highly reactive upstream metabolite, malleicyprol, was discovered and obtained in two stabilized forms. Cell‐based assays and a nematode infection model showed that the rare natural product confers cytotoxicity and virulence.

Burkholderia mallei and Burkholderia pseudomallei are closely related Gram-negative bacteria that have become infamous for causing human and animal diseases with high lethality.I nfections with B. mallei,t ransmitted from horses, lead to glanders,ahighly contagious zoonotic disease which shows up to 95 %m ortality when untreated, and even 50 % when treated with antibiotics. [1] Thes oil-and water-dwelling B. pseudomallei may infect various cell types and evade the immune system, causing melioidosis. [2] This life-threatening disease is an important cause of severe sepsis in Southeast Asia and Northern Australia, showing mortality rates of up to 40 %. [3] Once infected, antibiotic treatment regimes against B. pseudomallei typically last longer than five months. [4] Thus, melioidosis has been recognized as agrowing threat to global health. Because of the low infective dose needed, the possibility of infection by inhalation, and the limited preparedness in most countries, B. mallei and B. pseudomallei have been classified as potential biological warfare agents. [5] Previous studies have identified macromolecular virulence determinants [6] such as the proteinogenic toxin Burkholderia lethal factor 1(BLF1), [7] and have implicated the involvement of secondary metabolites [8] including the siderophores malleobactins [9] in pathogenesis.Y et, in light of the high biosynthetic potential of these bacteria there is ac lear gap in knowledge of small-molecule virulence factors of these notorious pathogens.Particularly enigmatic is the function of ap olyketide synthase encoded by the bur/mal gene locus ( Figure 1A), which is highly conserved in the genomes of all bacteria belonging to the B. pseudomallei group [10] and in B. contaminans,a ne merging pathogen in cystic fibrosis. [11] Deletions in the bur/mal gene cluster reduced the virulence of B. pseudomallei [12] and its low-pathogenicity model organism B. thailandensis [13] against the infection model Caenorhabditis elegans. However,t he previously identified metabolite,m alleilactone [13] syn. burkholderic acid [14] (1,F igure 1), could not explain the virulence associated to the presence of the corresponding biosynthetic gene cluster.C ompound 1 exhibited only weak antiproliferative [14] and moderate cytotoxic [13] activities in eukaryotic cell line assays,a nd no effect on the fitness of C. elegans was observed. [13] Hence,i t was questioned whether 1 was the true virulence factor associated with the bur assembly line.H erein we report the discovery of previously overlooked cyclopropanol-substituted polyketides originating from the bur-encoded pathway and show that highly reactive precursors of 1 are the actual virulence factors,asdemonstrated in the C. elegans infection model.
To identify congeners of 1 that could potentially be more active than the parent compound, we combined targeted gene inactivation in B. thailandensis with am olecular networking approach using the Global Natural Product Social Molecular Networking [15] (GNPS) platform. We retrieved MS 2 spectra from extracts of an engineered bur overexpression strain (B. thailandensis E264 Pbur) [14] and examined the main nodes of the network connected to the m/z value of 1 ( Figure 1B). Next, we scrutinized anonproducing mutant (B. thailandensis E264 PburDburJ)f or the loss of the respective m/z values compared to the overexpression strain. Through this approach we found two chromatographic peaks that correspond to an m/z of 387 in the negative ion mode in culture extracts from the overexpression strain, while the production of the same metabolites (2 and 3)w as abolished in our inactivation mutant. Thus,w ec oncluded that 2 and 3 were congeners of 1.Notably,the production of the more nonpolar congener (3)was suppressed when B. thailandensis E264 Pbur was grown at ac onstant pH of 6.5 in ab ioreactor.B y optimization of purification protocols,w es ucceeded in obtaining both compounds in pure form (2,0 .3 mg L À1 ; 3, 0.4 mg L À1 ).
HRMS data indicated am olecular formula of C 18 H 28 O 7 S for both compounds.Compared to 1 (C 18 H 26 O 4 ), 2 and 3 lack one double-bond equivalent, but are equipped with an additional sulfur atom and three additional oxygen atoms. Thepredicted sum formula was in full agreement with 1 Hand 13 CNMR data for both compounds.Through acomparison of the chemical shifts,a nd COSY and HMBC correlations of 2 with the data for 1, [14] it was deduced that 2 showed the same acyl side chain as 1,consisting of an a,b-unsaturated Michael acceptor system. HMBC correlations of an ester carbon center, resonating at d = 177.8 ppm, to three COSY-correlated methine protons indicated a g-lactone substructure that accounts for one of the two remaining double-bond equivalents.While 1 has apropionyl side chain, the corresponding spin system was absent in 2.Furthermore,the 13 Cspectrum of 2 showed two CH 2 groups shifted to an unusual high field at d = 10.5 and 12.1 ppm, typical for cyclopropyl moieties. [16] The remaining quaternary carbon center (d = 54.0 ppm) completed the cyclopropanol substructure,w hich was linked to the g-lactone,according to HMBC correlations.
Based on the molecular formula, we concluded that the thus far elucidated substructure bears an HSO 3 group,which could either be as ulfite or as ulfonic acid. Owing to the instability of sulfite monoesters,asulfonic acid would be the more likely candidate.Wevalidated this structure proposal by comparison of the chemical shifts of the respective sulfonatesubstituted carbon atom in 2 and its attached proton with the chemical shifts of model compounds (see Figure S4 in the Supporting Information) all showing highly similar shifts at the respective atoms.T aken together, 2 represents an unprecedented cyclopropanol-containing sulfonic acid that was named sulfomalleicyprol ( Figure 1C).
In the 13 CNMR data of the second, less polar compound (3)w en oticed as hift of the keto moiety resonating at d = 196.0 ppm in 2 to av alue of d = 168.8 in 3,i ndicative of an ester moiety.Additionally,wedetected asecond proton in the a-position to the lactone carbonyl group,w hereas the other parts of 3 showed largely similar shifts compared to the NMR data of 2.B ased on these data, we concluded that the acyl chain was not connected to the lactone ring (as in 2 and 1), but linked to the cyclopropyl moiety by an ester bond. COSY and HMBC data fully supported the proposed structure of 3, which was named iso-sulfomalleicyprol ( Figure 1C).
Thestructures of 2 and 3 suggested that both compounds could be interconverted by an intramolecular reaction. Considering that 3 was not present in cultures that were kept at aslightly acidic pH (6.5) we tested whether 3 could be formed from 2 in vitro under basic conditions,a si nc ultures growing in shaking flasks (pH 8.2 after 48 hg rowth in LB medium). By LC-HRMS monitoring we found that 3 originates from 2 when treated with phosphate buffer (pH 8.1) or K 2 CO 3 solution (pH 10.6;5m m ;F igure 1D). This transformation represents an acyl migration, which most likely proceeds through ar etro Claisen condensation with as ixmembered transition state (see Figure S2) formed between the keto and the hydroxy group of 2.I na ddition, hydrogen bonding between the sulfonate and the cyclopropanol moiety of 2 is likely to aid in the generation of the required cyclopropanolate for the reaction. Moreover,wedetected the formation of 1 when 2 was incubated in aqueous NaOH (pH 14;5 m m ;F igure 1D). This transformation could be rationalized to proceed through an E1cb mechanism (Figure 1E)toeliminate the sulfonic acid group with subsequent base-catalyzed opening of the cyclopropanol ring. These results indicated that 2 is ap recursor to both, 1 and 3. Therefore,w ei nterrogated whether 2 possessed stronger biological activity than its degradation product 1.Whentested with several cell lines,h owever, 2 did not show elevated cytotoxic/antiproliferative effects compared to 1 (see Table S5).
In light of the similarly low bioactivities of 2 and 1 it appeared plausible that yet another,c ryptic precursor could represent the true virulence factor.T herefore,w er evisited the extracts of the bur overexpression mutant. To exclude the possibility that potential congeners of 1 were overlooked in our molecular network we performed ac omplementary approach using an all-ion fragmentation (AIF) experiment in LC-HRMS with Pbur extracts.The AIF data were screened employing the extracted ion chromatogram (EIC) for highresolution m/z values of previously identified MS fragments from 1, 2,and 3.Bythis approach we identified aparent mass of m/z 611.3589 (negative ion mode) through its sole dominating fragment ion with m/z 305.1758 (see Figure S3). Subsequently,w ei solated the corresponding compounds as two diastereomers by an optimized purification protocol (4a, 1.8 mg L À1 , 4b1.5 mg L À1 ). Thededuced molecular formula of C 36 H 52 O 8 from 13 Ca nd HRMS data for 4 hinted towards ad imeric structure comprising two burkholderic acid-like subunits (C 18 H 26 O 4 ). HMBC and COSY correlations confirmed 4 to be an adduct consisting of two connected glactones,each linked to an acyl chain. Tw oadditional olefinic carbon atoms resonating at d = 157.5 and 132.0 ppm indicated at hird a,b-unsaturated double bond. HMBC correlations to aq uaternary carbon center at d = 167.9 ppm located this double bond within one of the g-lactones,whereas the second g-lactone was saturated. Ther emaining four methylene carbon atoms shifted to high field (d = 15.5, 14.8, 12.5, and 9.1 ppm) and two quaternary carbon atoms (d = 56.8 and 56.5 ppm) made up two cyclopropanol rings,e ach connected to one of the g-lactones.T hus,w ee lucidated structure 4 as ad imeric, cyclopropanol-substituted congener of 1 named bis-malleicyprol ( Figure 2A). Moreover,weinferred that this dimeric structure resulted from the conjugate addition of the two tautomeric forms, 5 and 6,o ft he corresponding monomer.S uch an on-enzymatic addition would rationalize the occurrence of 4 in various stereoisomeric forms.Notably, when incubated under basic conditions, 4a isomerizes into 4b and other isomeric forms (see Figure S5 A).
To establish ac hemical correlation between the thus far isolated congeners we subjected 4 to various reaction conditions and monitored product formation by LC-HRMS. Incubation of 4 with Na 2 SO 3 yielded the previously isolated sulfonic acid 2 (Figure 2A). Furthermore,w eo bserved the formation of 1 when subjecting 4 to basic conditions.Based on these results,weconcluded that the two proposed tautomeric monomers 5/6 coexist in ac hemical equilibrium with 4 ( Figure 2B). This chemical equilibrium enabled us to trap the reactive species 5 by conjugate addition with Na 2 SO 3 .T hus, we concluded that 4 is aprecursor to 2 and 1.Because of the chemical equilibrium between 5/6 and 4 it seems likely that 5 and 6 are the true biosynthetic outcome of the bur assembly line,w hile 4 represents ar eversibly formed product of these two highly reactive tautomers.H ence,w en amed the new compounds malleicyprol (5)and iso-malleicyprol (6).
Thechemical equilibrium between 5/6 and 4 allowed us to study the cumulated biological activity of all three species. Cytotoxic and antiproliferative assays with purified 4a on various cell lines showed ad ramatically increased molar activity,over two orders of magnitude (110-fold) higher than 1,o riginally assigned to the bur/mal assembly line (Figure 3A). Of course,b ecause of the chemical equilibrium between 4 and 5/6 the precise contribution of each species to the found biological activity cannot be shown experimentally. Yet, the high reactivity of 5/6 suggests that these two tautomers play important roles in the observed toxicity.T o validate the effects observed in the whole cell assays,w e performed atoxicity assay using the established pathogenicity model, C. elegans. As ac ontrol, 1 showed no effect on the survival of the nematodes (see Video S1) when added to the growth medium (as high as 100 mgmL À1 ). In stark contrast, when treated with 4a(50 mgmL À1 ), no viable nematodes were observed in the corresponding survival assays (Figures 3C,D; see Video S2). In addition, we performed a C. elegans liquid toxicity assay to determine the potencyo f4a (IC 50 : 0.56 mgmL À1 ;F igure 3B). Based on these results we propose that 4 and/or 5/6 represent the true virulence factors produced by the encoded bur assembly line.I ti sr emarkable that the active 6 and inactive 1 differ solely in the C3 (cyclopropanol and propanone) residues ( Figure 3E). Consequently,t he reactive cyclopropanol ring of the malleicyprols represents an important pharmacophoric moiety.Aprominent example with as imilarly strained warhead substructure is the genotoxin colibactin. [17] In conclusion, we have discovered and characterized an ew family of structurally intriguing, cyclopropanol-substituted polyketides.I ng eneral, cyclopropanol-containing natural products are exceedingly rare. [16,18] We demonstrate that these highly reactive compounds are produced by the bur/mal assembly line,w hich is correlated with virulence in the B. mallei/pseudomallei complex, and suggest alternative polyketide virulence determinants.T hus,o ur results are an important addition to the body of knowledge on smallmolecule disease mediators employed by these infamous human and animal pathogens.T his new insight may lead to abetter understanding of the molecular basis of glanders and melioidosis,a nd could facilitate the development of much needed therapeutics [19] to combat these severe diseases.