A tricyclic pyrrolobenzodiazepine produced by Klebsiella oxytoca is associated with cytotoxicity in antibiotic-associated hemorrhagic colitis

JBC Papers in Press. Published on September 26, 2017 as Manuscript M117.791558

The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M117.791558

Cytotoxins in Klebsiella oxytoca

A tricyclic pyrrolobenzodiazepine produced by Klebsiella oxytoca is associated with cytotoxicity in

antibiotic-associated hemorrhagic colitis

Herman Tse^1,2,3,4, Dan Yang⁵, Kong-Hung Sze^1,2,3,4, Ivan K. Chu⁵, Richard Y.-T. Kao^1,2,3,4, Kam-

Chung Lee^1,2,3,4, Ching-Wan Lam⁶, Qiangshuai Gu⁵, Sherlock Shing-Chiu Tai⁵, Yihong Ke¹, Elaine

Chan¹, Wan-Mui Chan¹, Jun Dai¹, Sze-Pui Leung¹, Suet-Yi Leung⁶, Kwok-Yung Yuen^1,2,3,4

From the ¹Department of Microbiology and ²Research Centre of Infection and Immunity, The University

of Hong Kong, Hong Kong,

³State Key Laboratory of Emerging Infectious Diseases, Hong Kong SAR, China,

⁴Carol Yu Centre for Infection, and Departments of ⁵Chemistry and ⁶Pathology, The University of Hong

Kong, Hong Kong

Running title: Cytotoxins in Klebsiella oxytoca

To whom correspondence should be addressed: Prof. Kwok-Yung Yuen, Department of Microbiology,

The University of Hong Kong, Pokfulam, Hong Kong; Telephone: (852) 2255 4892; Email:

kyyuen@hku.hk

Keywords:

bacterial toxin, colitis, apoptosis, infectious disease, antibiotics

we show that

a

tryptophanase-deficient,

ABSTRACT

tilivalline-negative K. oxytoca mutant induced

cytotoxicity in vitro similar to tilivalline-positive

K. oxytoca strains. Furthermore, synthetic

kleboxymycin exhibited greater than 9-fold

higher cytotoxicity than tilivalline in TC₅₀cell

culture assays. We also found that the

biosynthetic pathways for kleboxymycin and

tilivalline appear to overlap, as tilivalline is an

indole derivative of kleboxymycin. In summary,

our results indicate that tilivalline is not essential

for inducing cytotoxicity observed in K.

Cytotoxin-producing Klebsiella oxytoca is the

causative

agent

of

antibiotic-associated

hemorrhagic colitis (AAHC). Recently, the

cytotoxin associated with AAHC was identified

as

tilivalline,

a

known

(PBD)

pentacyclic

metabolite

pyrrolobenzodiazepine

produced by K. oxytoca. While this assertion of

tilivalline’s role in AAHC is supported by

evidence from animal experiments, some key

aspects of this finding appear to be incompatible

with toxicity mechanisms of known PBD toxins.

We therefore hypothesized that K. oxytoca may

produce some other uncharacterized cytotoxins.

To address this question, we investigated

whether tilivalline alone is indeed necessary and

sufficient to induce cytotoxicity or whether K.

oxytoca also produces other cytotoxins. LC/MS-

and NMR-based metabolomic analyses revealed

the presence of an abundant tricyclic PBD,

provisionally designated kleboxymycin, in the

supernatant of toxigenic K. oxytoca strains.

Moreover, by generating multiple mutants with

gene deletions affecting tilivalline biosynthesis,

oxytoca–associated

AAHC

and

that

kleboxmycin is a tilivalline-related bacterial

metabolite with even higher cytotoxicity. (224

words)

Klebsiella is a genus of non-motile Gram-

negative rod-shaped bacteria commonly

encountered in the environment. Similar to other

members of the family Enterobacteriaceae,

Klebsiella spp. are associated with a wide range

of diseases, including nosocomial pneumonia,

1

Cytotoxins in Klebsiella oxytoca

urinary tract infections, biliary tract infections,

surgical wound infections, and deep abscesses

(1). In addition, the prevalence of high-level

antibiotic resistance is increasing among clinical

isolates of Klebsiella spp., with production of

corroborated by cytotoxicity assays on the

isolates (9-14), which prevented an accurate

assessment of disease prevalence.

Arguably, the fundamental barrier to further

studies on the K. oxytoca cytotoxin, including

the development of a reliable diagnostic assay,

was uncertainty over its real identity. Early

studies have suggested that the cytotoxin is a

small molecule with a novel chemical structure

and a molecular weight (MW) of approximately

217 Da (15,16). However, this is in conflict with

recent results suggesting that the cytotoxin is

extended-spectrum

beta-lactamases

and

carbapenemases posing significant challenges to

treatment and infection control (2,3).

Among the diseases associated with Klebsiella

spp., antibiotic-associated hemorrhagic colitis

(AAHC) is a clinical entity that has gained

attention in recent years. The disease is caused

by the overgrowth of cytotoxin-producing

Klebsiella oxytoca secondary to use of

antibiotics such as penicillin or amoxicillin (4,5).

The typical colonoscopic finding of AAHC is

segmental involvement of the ascending colon

characterized by presence of diffuse mucosal

edema and hemorrhagic erosions. This

presentation is distinct from the more common

form of antibiotic-associated diarrhea caused by

toxin-producing Clostridium difficile, which

presents with watery diarrhea in mild to

moderate disease, but may resemble more severe

C. difficile infection. Besides causing human

disease, cytotoxin-producing K. oxytoca is also

suspected as the causative agent in an outbreak

of hemorrhagic colitis on a rabbit farm (6).

actually

tilivalline,

a

pentacyclic

pyrrolobenzodiazepine (PBD) with a MW of

approximately 333 Da (17,18).The association

of tilivalline with cytotoxicity and disease was

supported by multiple lines of evidence,

including mutagenesis experiments and animal

studies, imparting significantly more credibility

to the results.

In the present study, we performed an

independent investigation on the identity of the

cytotoxin. Culture supernatants from K. oxytoca

strains grown under different culture conditions

were examined, and metabolites associated with

cytotoxicity were isolated and characterized.

Surprisingly, our results revealed that

cytotoxicity is associated with a mixture of

related molecular species essentially comprising

Current knowledge on cytotoxin-producing K.

oxytoca and associated disease is severely

limited by the lack of an accessible diagnostic

test. While a differential culture medium had

been developed for isolation of K. oxytoca from

stool (7), cytotoxin detection remains a difficult

task in the clinical setting. The gold standard for

detecting cytotoxin production is the HEp-2 cell

culture assay (5,8), but it has a long turnaround

time and is impractical for most clinical

laboratories. Furthermore, the cytopathic effects

indicative of cytotoxin production are non-

specific, and confirmation by neutralization with

specific antibodies or antitoxins is currently

unavailable. Hence, several reports of K.

oxytoca-associated hemorrhagic colitis were not

a

tricyclic PBD, provisionally designated

kleboxymycin, and its derivatives, of which

tilivalline is the main species. In vitro assays

showed that both kleboxymycin and tilivalline

are correlated with cytotoxicity, but the former

has higher cytotoxicity by an order of magnitude.

The present results strongly suggest that

tilivalline is not the sole or even the main

cytotoxin produced by AAHC-associated K.

oxytoca strains.

Results

Cytotoxin production is influenced by culture

conditions. In the course of our experiments to

establish the cytotoxin production by K. oxytoca

strains, we noticed inconsistency in cytotoxin

2

Cytotoxins in Klebsiella oxytoca

production under similar conditions by the same

strain. Among various modifications of culture

conditions, it was found that culturing

temperature exceeding 37°C, significantly

reduced cytotoxicity of culture supernatants.

Bacterial growth rate, as enumerated by OD_600nm

measurement (Fig. 1) and by colonies counting

from subculturing on non-selective agar media,

was not adversely affected at culture

temperatures between 35 and 37 °C. We have

tested with tryptic soy broth (TSB), then tested

with MacConkey broth and found that

MacConkey broth could give high cytotoxicity

of K. oxytoca strain MH43-1 (64x dilution of

broth at which cytopathic effects were still

observed). Therefore, we tested which

component inside MacConkey broth was the key

component that increases the cytotoxicity of

MH43-1. Bile salt added to TSB could not

increase the cytotoxicity effect. On the other

hand, glucose and especially lactose added to

TSB could increase the cytotoxicity of MH43-1

(Table S1). MH43-1 cultured in minimal

medium only had low cytotoxicity (8x dilution

of broth at which cytopathic effects were

observed). Higher cytotoxicity was observed

with a higher glucose or lactose concentration of

up to 10 g/L (Table 1). Based on these results,

we designed a simple tryptone lactose broth

medium (see Experimental procedures) that

consistently induced high levels of cytotoxicity

in culture supernatant which facilitated

downstream metabolite identification.

found within each of the 12 samples. A toxicity

value for each of the culture media was also

performed by incubating HEp-2 cells with

samples of the media, and the toxicity was

quantified using the MTT cell viability assay.

Multivariate analysis was performed on this data

matrix as a mean of correlating the metabolite

profiles of the culture supernatant with their

cytotoxic activities, for the objective of

identifying the potential bioactive constituents.

The data set was not transformed but was treated

with pareto scaling prior to analysis (19).

Chemical fingerprint data along with their

respective toxicity profiles were subjected to

principal component analysis (PCA) to inspect

data variation and identify potential outliers. The

data was then subjected to partial least squares

(PLS) analysis, a supervised extension of PCA,

to elucidate the covariance between quantified

metabolites as defined by their m/z and RT

values (X variables) and toxicity of the media (Y

variables) (20). The resulting score plot reveals

that the culture media metabolite profiles could

be discriminated on the basis of the strains and

culture conditions used (Fig. 2A). With the

exception of one replicate profile from the non-

toxigenic strain cultured in lactose-containing

media, all other profiles from the same sample

types clustered into discernible groups.

Interestingly, the metabolite profiles of the

toxigenic MH43-1 grown in the presence of

lactose were discriminated from the other three

sample types and located in a separate region in

the score plot. The performance of model was

assessed by examining the R²(fitness of the

model) and Q²(model predictability) values

with all values close to 1 resembling a good

model (8). In this study, the model possessed

values of R²= 0.92 and Q²= 0.8 when three

principle components were incorporated in the

regression model.

LC/MS/MS analysis on culture supernatant.

To profile the metabolite content found in the

culture supernatant, triplicate samples were

prepared for the toxigenic MH43-1 and non-

toxigenic 293206 strains of K. oxytoca cultured

in tryptone broth, with and without lactose.

Extensive profiling of the culture media was

performed using LC/MS, and 3646 unique

chemical species were identified with specific

mass to charge ratios (m/z) and retention times

(RT). These two pieces of acquired information

were used as the unique identifier pair for each

species present in the samples. The peak areas

were determined for each uniquely identified

chemical species to quantify their amounts

To identify the molecules that provided the

greatest contributions to the three principle

components used in the model, a three-

3

Cytotoxins in Klebsiella oxytoca

dimensional loading plot was generated (Fig.

2B). In this plot, most metabolites clustered in

the center indicating that their contributions

were minimal. However, three candidate

molecules were identified as significant

contributors to all three principle components.

There were two metabolites possessing m/z of

217.1 and 334.1 at the same retention time of

20.2 minutes and a third metabolite with an m/z

of 235.2 at the retention time of 15.9 minutes.

The extracted-ion chromatogram profiles of the

culture supernatants were consistent with these

findings and showed that these metabolites were

found only in the culture supernatant of

toxigenic MH43-1 strain in lactose-containing

media (Fig. 2C).

individual fractions, P1 and P2 were combined

to look for any synergistic effects; and all

isolated fractions were combined (P1+P2+O) to

assess any loss in toxicity from the separation

procedure. P1 was found to produce observable

cytotoxicity at up to 128× dilution, while P2

exhibited lower toxicity of up to 32×. No

significant synergistic effects were observed

when combining P1 and P2. The recombined

fractions demonstrated 2× reduced cytotoxicity

with respect to crude supernatant suggesting

only minimal loss during separation. These

results suggest that the main contributor to

toxicity is the metabolite present in P1 rather

than P2.

Structure elucidation of kleboxymycin (P1)

and its derivatives. The elemental compositions

of the metabolites were determined by mass

spectrometry, combined with product ion spectra,

GC-MS and NMR spectroscopy results.

Proposed structures of the compounds P1 and P2

with m/z 235 and m/z 334, respectively, are

shown in Fig. 3. Both molecules consist of a

pyrrolobenzodiazepine nucleus and differ in

their substituents at the C6 position (21).

Compound P1 has a hydroxyl group at this

position, while compound P2 has an indole

substituent and is known as tilivalline (17).

Tilivalline was among the known metabolites of

K. oxytoca (22) with cytotoxicity towards

mammalian cells. We provisionally designated

the main contributor to toxicity, compound P1,

as kleboxymycin.

To isolate these metabolites, culture supernatant

of MH43-1 in lactose-containing medium was

subjected

to

solid

phase

extraction

chromatography and eluted as three fractions as

described in the Experimental procedure. The

LC/MS profiles of each fraction were

characterized. The metabolite identified with a

m/z 235.2 at RT 15.9 min was eventually

isolated to fraction Peak 1 (P1) after a

subsequent reversed-phase chromatography step

(Supplemental Figs. S1A and S1B). This

compound was judged to be approximately 90%

pure based on GC-MS and total ion current

chromatogram profile of the fraction.

(Supplemental Figs. S2) The metabolite

identified with a m/z 334.1 at RT 20.2 min was

found in Fraction 3 (Supplemental Figs. S1C

and S1D) and was also determined to be

approximately 90% pure (Supplemental Fig. S3).

This fraction was renamed Peak 2 (P2) for

subsequent experiments. To assess the toxicity

of the remaining metabolite population, all other

fractions generated from this purification

scheme were pooled together and referred to as

Fraction O.

Kleboxymycin showed a protonated molecular

ion at m/z 235, sodium adduct of molecular ion

at m/z 257 and dimer of sodium adduct of

molecular ion at m/z 491, neutral loss of water

molecule from protonated molecular ion at m/z

217 as well as dimer of protonated ion from loss

of two water molecules at m/z 433 in positive

full scan MS mode (Fig. 4A); deprotonated

molecular ion at m/z 233, formate adduct of

molecular ion at m/z 279 from the formic acid in

the mobile phase, dimer of deprotonated

The metabolite-toxicity relationship was

assessed by cell culture toxicity assay of the

isolated fractions. In addition to testing

4

Cytotoxins in Klebsiella oxytoca

molecular ion at m/z 467 as well as neutral loss

of water molecule from deprotonated molecular

ion at m/z 215 was observed in negative full scan

MS mode (Fig. 4B). Both positive and negative

full scan MS results showed that the molecular

formula was C₁₂H₁₄N₂O₃for kleboxymycin with

the mass accuracy less than 5 ppm.

other as indicated by their COSY crosspeaks.

Their corresponding C10, C11 and C12 carbon

chemical shifts are also consistent with these

aromatic carbon chemical shifts. The position of

the H12 is confirmed by the observation of H12-

C14 crosspeak in the HMBC spectrum. H2, H3,

H4, H5 and H6 showed another spin system

linked by their COSY crosspeaks. C2, C3, C4

and C5 are having aliphatic types of carbon

chemical shifts, and their integrals, proton

chemical shifts as well as coupling constants are

typical proline side chain patterns. C6 and H6

are having exceptional high chemical shifts of

169.2 and 7.88 ppm, respectively. These indicate

that C6 is having sp²hybridization. Together

with the fact that there is no more feasible

aromatic position and aromatic ring, C6 is

assigned to be under C=N moiety. C14 has a

high carbon chemical shift of 166.4 ppm with no

proton attached. This is consistent with a

benzoic carbonyl carbon. C8 and C13 are

substituted aromatic carbons because they are

having aromatic carbon chemical shifts and have

no protons attached. C9 has a high aromatic

carbon chemical shift of 154.2 ppm, which is

consistent with the chemical shift of a phenol

carbon chemical shift. Lastly, there is a

remaining degree of unsaturation which

indicates that dehydrated-kleboxymycin should

have a three ring system to form a 7-membered

ring in the middle matching the overall chemical

structure of a tricyclic pyrrolobenzodiazepine

(23). The chemical structure of dehydrated-

kleboxymycin was shown in Fig. 3C. MS/MS

When

lyophilized

kleboxymycin

was

reconstituted in acetonitrile-d₃, multiple set of

signals were initially observed in the NMR

spectra (Supplemental Fig. S4). The signals

gradually became being dominated by one set of

signals after overnight standing at 298K. The

last traces of residual signals could be removed

by the addition of molecular sieve beads to

absorb residual water in the solvent. This

observation suggested that the kleboxymycin

was slowly dehydrated first by the acetonitrile

solvent and then by molecular sieve beads to

form a single dehydrated form. However, the

dehydrated-kleboxymycin was highly unstable

because it was readily degraded after standing in

1

13

the NMR tube for 5 days. The H and C NMR

spectrometry data of the dehydrated-

kleboxymycin in acetonitrile-d3 at 298K were

presented in Supplemental Table S2. MS

characterization indicated that dehydrated-

kleboxymycin has an protonated ion exact mass

of 217.0983 in positive ion mode (Fig. 5) and

the neutral molecule chemical formula generated

with least m/z deviation is C₁₂H₁₂N₂O₂(delta = -

2.8 ppm). No other chemical formula with 12

carbon atoms can be fit into this exact mass even

if we relaxed the m/z tolerance to 50 ppm. Fig. 6

1

showed the H 1D, COSY, HSQC and HMBC

fragmentation

analysis

on

dehydrated-

spectra of dehydrated-kleboxymycin. The

kleboxymycin was performed and summarized

in Supplemental Fig. S5.

structure

elucidation

of

dehydrated-

1

13

kleboxymycin follows established methodology

of combining the information provided by peak

integral, chemical shifts, multiplicity and cross-

peak correlationships observed in the COSY,

HSQC and HMBC spectra. H10, H11 and H12

having chemical shifts between 7.08 to 7.45 ppm

are three aromatic protons neighboring to each

The H and C NMR spectrometry data of the

kleboxymycin in D₂O at 298K were presented in

Supplemental Table S3 and Fig. 7. MS/MS

characterization of kleboxymycin in positive ion

mode (Fig. 8) indicated that M-H⁺has an exact

mass of 235.10784 and the neutral chemical

formula generated with least m/z deviation is

5

Cytotoxins in Klebsiella oxytoca

C₁₂H₁₄N₂O₃(delta = 1.8 ppm). The structure

elucidation of kleboxymycin largely followed

that of dehydrated-kleboxymycin described

above except that two sets of NMR signals could

be observed in the NMR spectra based on their

integrals. The major to minor forms has an

intensity ratio of about 3:1 and we have named

the major and minor forms as Diastereomers a

and b, respectively. The substitution of the C=N

moiety with water has upfield shifted C6 carbon

chemical shift to 90.9 and 86.2 ppm for

Diastereomers a and b, respectively. These are

consistent with the formation of a hydroxylated

C6. H6 has chemical shifts of 4.99 and 5.31 ppm

for Diastereomers a and b, respectively, which

are also consistent with a hydroxyl C6. The H6

showed J coupling splitting of 9.1 and 2.3 Hz

with H5 for Diastereomers a and b, respectively.

Based on these values, the Diastereomer a

should be having a H6 proton anti to the H5

proton and Diastereomer b should be having a

H6 proton syn to the H5 proton. Note that the

present structure of kleboxymycin is further

confirmed by the association experiment of

indole with kleboxymycin to form tilivalline

described below.

The ¹H spectrum of the P2 (tilivalline) in DMSO

at 298K was presented in Supplemental Fig. S6,

which matched exactly with the reported NMR

spectrum of tilivalline (17). MS/MS spectrum of

P2 in positive mode was shown in Supplemental

Fig. S1D. It also showed a product ion at 217 as

well as many matching product ions found in the

MS/MS spectra of kleboxymycin (Fig. 4) and

dehydrated-kleboxymycin (Fig. 5) indicating

that they have common chemical skeleton. This

is consistent with the chemical structure of

tilivalline being an indole derivative of

kleboxymycin.

MH43-1 culture supernatant at 8 and 12 hours

were measured by LC-MS quantitative analysis

and summarized in Table 2. The relative molar

ratios of kleboxymycin to tilivalline were 0.54

and 0.41 at 8 and 12 hours, respectively. The

corresponding LC-MS extracted ion count plots

of kleboxymycin and tilivalline in one of the

triplicate sample set were shown in

Supplemental Fig. S7.

Gene deletion and complementation. Based on

knowledge of the proposed chemical structures

of the cytotoxins and comparison among

genome sequences of K. oxytoca strains (18), we

identified a gene cluster that is likely to be

associated with cytotoxin production. Fig. 9

shows the genetic organization of the cytotoxin

biosynthesis gene cluster, along with the

annotation of predicted gene functions and

phylogenetically closest neighbor based on

NCBI BLAST search. The organization and

annotation of the gene cluster is identical to that

described by Schneditz et al (17), except for the

presence of a putative npsC gene which encodes

a short protein with sequence similarity to OtcC,

a

post-polyketide hydroxylase/cyclase. The

sequence of kleboxymycin biosynthetic gene

cluster is sequenced and has been deposited to

Genbank and the NCBI accession number is

MF401554. Gene deletion and complementation

experiments were performed to assess the

essentiality of individual genes to cytotoxin

production. Results are summarized in Table 3.

In all cases, complementation of the deleted

gene restored cytotoxicity levels to within 2× -

4× of that of wild-type.

Surprisingly, deletion of hmoX, encoding a

putative anthranilate 3-monooxygenase, or icmX,

encoding a putative isochorismatase-like family

protein, did not affect production of

kleboxymycin or tilivalline. On the other hand,

deletion of adsX, encoding an anthranilate

Relative abundance of kleboxymycin and

tilivalline in cell culture. To evaluate the

relative abundance of kleboxymycin and

tilivalline in the cell culture, the levels of

kleboxymycin and tilivalline in K. oxytoca

synthase, clearly led to

a

non-toxigenic

phenotype as predicted. This suggests that

possible functional redundancy for parts of the

6

Cytotoxins in Klebsiella oxytoca

3-hydroxyanthranilate biosynthesis operon is

compensated by production of a functionally-

related enzyme. Alternatively, our functional

predictions based on NCBI BLAST analysis

may not be accurate, given the lack of highly

similar proteins with experimental verification

of their enzymatic activity.

only tilivalline was detected, which is consistent

with E. coli being an indole producer. Therefore,

the present results confirmed that the gene

cluster is necessary and sufficient for cytotoxin

production.

Total synthesis of kleboxymycin and

tilivalline. A mixture containing kleboxymycin

was prepared in three steps from commercially

Additionally, to further confirm that 3-

hydroxyanthranilate is a key intermediate for

kleboxymycin biosynthesis in K. oxytoca,

exogenous 3-hydroxyanthranilate was added to

the culture media at a final concentration of 0.25

g/L during the culture of the ∆adsX mutant. It

was found that presence of exogenous 3-

hydroxyanthranilate could successfully restore

cytotoxin production, similar to the effect of

available 3-hydroxyanthranilic acid

1 and

natural L-proline methyl ester 2 (Scheme I). (+)-

Tilivalline was synthesized starting from

compound 3 by following published procedures

(24) with slight modifications (Scheme II).

Scheme I. Synthesis of kleboxymycin

genetic complementation (Table

Supplemental Fig. S8).

4

and

Finally, we constructed a ∆tnpA mutant which

lacks a tryptophanase gene and hence cannot

produce indole. LC/MS/MS analysis of its

culture supernatant revealed no indole or

tilivalline, though kleboxymycin production and

cytotoxicity as measured by the cell culture

toxicity assay was not affected (Table 4 and

Supplemental Fig. S9).

Kleboxymycin, R = OH or OMe

Scheme II. Synthesis of (+)-Tilivalline

Cloning of cytotoxin gene cluster into E. coli.

As the cytotoxin gene cluster in K. oxytoca

appears to be acquired through an ancient

horizontal gene transfer event from an unknown

Actinobacteria species, an attempt was made to

clone the gene cluster into an E. coli strain to

investigate whether another species in the

Enterobacteriaceae can support cytotoxin

production. Intriguingly, the transformant E. coli

was able to produce the cytotoxins in culture

supernatant, as shown by cell culture toxicity

assay (2× dilution) and LC/MS/MS, albeit at

only low levels (Table 5 and Supplemental Fig.

S10). Kleboxymycin and tilivalline were

detected depending on the culture conditions.

Using M9 medium with limited indole

production, only kleboxymycin was detected in

the final cell culture. While using LB medium,

To a solution of 1 (1.43 g, 9.34 mmol) and L-

proline methyl ester hydrochloride 2 (7.73 g,

46.7 mmol) in anhydrous DMF (23.3 mL) were

add (PhO)₂PON₃(6.02 mL, 28.0 mmol) and

Et₃N (19.5 mL, 140 mmol) under argon

atmosphere at 0°C. Upon completion, the

resulting mixture was vigorously stirred under

this condition for ca. 107 h. The reaction

mixture was then loaded onto a silica gel column

for flash chromatography purification to provide

3 contaminated with trace amounts of impurities.

Recrystallization of this mixture from

DCM/EtOH/n-hexane afforded 1.30 g of pure 3

(60% yield) (25).

7

Cytotoxins in Klebsiella oxytoca

HRMS (EI) for C₁₉H₁₆O₃N₂(M⁺): calculated

320.1161, found 320.1147.

3: White solid, m.p. 275–277 °C; analytical TLC

(silica gel 60), EtOH/DCM = 10%, R_f= 0.39;

=520 (c 0.125, MeOH); ¹H NMR (400 MHz,

CD₃OD) δ 7.33 (dd, J = 7.8, 1.2 Hz, 1H), 7.10 (t,

J = 7.9 Hz, 1H), 7.04 (dd, J = 7.9, 1.2 Hz, 1H),

4.16 (d, J = 7.6 Hz, 1H), 3.80–3.71 (m, 1H),

3.61–3.50 (m, 1H), 2.72–2.63 (m, 1H), 2.13–

1.92 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ

172.4, 167.8, 149.2, 128.9, 126.1, 125.9, 121.5,

118.7, 58.4, 48.4, 27.1, 24.4; IR (CHCl₃) ν_max

3379, 3003, 2930, 1695, 1628, 1601 cm^-1;

LRMS (EI, 20 eV) m/z 232 (M⁺, 15), 167 (26),

149 (88), 69 (100); HRMS (EI) for C₁₂H₁₂O₃N₂

(M⁺): calculated 232.0848, found 232.0843.

To a solution of 4 (961.0 mg, 3.00 mmol) in

methanol (300 mL) was added NaBH₄(567 mg,

15.0 mmol) in one portion under argon

atmosphere at 0 °C. The resulting mixture was

stirred under this condition for ca. 1 h. Then

another portion of NaBH₄(567 mg, 15.0 mmol)

was added, and the resulting mixture was stirred

under the same condition for another 1 h. Upon

completion, the reaction mixture was diluted

with methanol (2.7 L) and quenched with

NaHCO₃(3.00 g, 35.7 mmol) in water (300 mL).

The stirring was continued at room temperature

overnight, and then evaporated under reduced

pressure (20 °C water bath, water pump). The

resulting brownish aqueous mixture was

azeotroped with acetonitrile to remove most of

the water, and was subjected to solid-phase

extraction and reversed-phase chromatography

as the culture metabolites described in the

Experimental procedure. The final purified

kleboxymycin product was lyophilized and

stored as a white powder at -80 °C.

A mixture of 3 (1.25 g, 5.40 mmol) and p-

TsOH·H₂O (184.9 mg, 0.97 mmol) in

PhCH(OMe)₂(30.0 mL) was stirred at ca. 100

°C under argon atmosphere for ca. 12 hours. The

resulting mixture was loaded onto a silica gel

column for flash chromatography purification to

provide ca. 1.74 g of 4 (ca. 100% yield)

containing a small amount of impurities, which

was employed for the next step directly. A small

fraction of pure 4 was collected for structural

characterization.

4: Sticky amorphous solid; analytical TLC

(silica gel 60), EtOH/DCM = 5%, R_f= 0.54;

=444 (c 0.098, CHCl₃); ¹H NMR (400 MHz,

CDCl₃) δ 7.56 (d, J = 8.0 Hz, 1H), 7.46–7.39 (m,

2H), 7.39–7.32 (m, 3H), 7.30 (s, 1H), 7.17 (t, J =

8.0 Hz, 1H), 7.07 (d, J = 7.8 Hz, 1H), 4.18–4.10

(m, 1H), 3.78–3.64 (m, 2H), 2.92–2.81 (m, 1H),

To a mixture of 3 (0.50 g, 2.1 mmol) and

anhydrous K₂CO₃(0.59 g, 4.3 mmol) in MeCN

(10.7 mL) was added BnBr (0.26 mL, 2.2 mmol)

under argon atmosphere at room temperature.

Upon completion, the resulting mixture was

stirred at ca. 70 °C for ca. 12 h. Then the

reaction mixture was filtered through a celite

pad. The filtrate was evaporated and the residue

was purified by chromatography to afford 0.69 g

of 5 (100%) (1).

13

2.17–1.84 (m, 3H); C NMR (100 MHz, CDCl₃)

δ 169.8, 165.3, 150.8, 136.7, 130.0, 128.9, 127.2,

125.9, 125.6, 121.9, 121.5, 112.2, 94.6, 58.9,

48.0, 27.0, 23.4; IR (CHCl₃) ν_max3019, 2928,

1694, 1628, 1601 cm^-1; LRMS (EI, 20 eV) m/z

320 (M⁺, 36), 292 (24), 223 (24), 195 (100);

8

Cytotoxins in Klebsiella oxytoca

5: White solid; analytical TLC (silica gel 60),

EtOH/DCM = 5%, R_f= 0.38; =393 (c 1.00,

increased gradually from −10°C to 0°C. Then

saturated NH₄Cl aqueous solution was added to

quench the reaction. The reaction mixture was

extracted with DCM and the combined organic

layer was dried over Na₂SO₄, filtered and

evaporated. The residue was purified by silica

gel chromatography to afford 0.73 g of 7 (72%

yield).

1

CHCl₃); H NMR (400 MHz, CDCl₃) δ 7.97 (br

s, 1H), 7.59 (d, J = 7.9 Hz, 1H), 7.44–7.33 (m,

5H), 7.18 (t, J = 8.0 Hz, 1H), 7.10 (d, J = 8.1 Hz,

1H), 5.13 (s, 2H), 4.04 (d, J = 6.2 Hz, 1H),

3.86–3.77 (m, 1H), 3.66–3.56 (m, 1H), 2.82–

2.69 (m, 1H), 2.11–1.95 (m, 3H); ¹³C NMR (100

MHz, CDCl₃) δ 170.5, 165.3, 147.8, 135.6,

129.0, 128.8, 127.9, 127.6, 125.7, 124.8, 122.9,

114.6, 71.5, 57.0, 47.5, 26.4, 23.6.

A mixture of 7 (0.53 g, 1.2 mmol) and indole

(0.68 g, 5.8 mmol) in AcOH (26 mL) was

cooled to freeze under argon atmosphere. Then

the reaction flask was evacuated with an oil

pump and sealed. It was then warmed slowly

until all the solid disappeared. Argon was

introduced subsequently and the freeze-and-

thaw procedure was repeated for another two

times. Upon completion, the reaction flask was

sealed and the reaction mixture was stirred at

150°C for 5 hours. AcOH was removed by

repeatedly azeotroping with toluene for 3 times.

The residue was purified by silica gel

chromatography to afford 0.64 g of 8 (99%

yield).

To a solution of 5 (0.63 g, 1.95 mmol) in

anhydrous THF (39 mL) was added LiHMDS (1

M in THF, 2.93 mL, 2.93 mmol) dropwise at

room temperature under argon atmosphere.

Upon completion, the reaction mixture was

stirred under this condition for ca. 50 min. Then

CbzCl (418 µL, 2.93 mmol) was added under

argon atmosphere and the resulting mixture was

stirred under this condition for ca. 2 hours.

Saturated NaHCO₃aqueous solution was

subsequently added to quench the reaction, and

the mixture was extracted with DCM. The

combined organic layer was dried over

anhydrous Na₂SO₄, filtered and evaporated, and

the residue was purified by silica gel

chromatography to afford 0.83 g of 6 (93%

yield).

To a solution of 8 (0.64 g, 1.2 mmol) in

methanol (18 mL) and anhydrous THF (18 mL)

was added Pd/C (10 wt%, 0.24 g, 0.23 mmol)

under argon atmosphere at room temperature.

Then a H₂balloon was installed via a T-type

stopcock. The reaction flask was evacuated with

a water pump and refilled with H₂for several

To a solution of 6 (1.00 g, 2.2 mmol) in

anhydrous THF (136 mL) was added NaBH₄

(91.6 mg, 2.42 mmol) in one portion under

argon atmosphere at −10°C (ice-acetone bath).

The resulting mixture was stirred under this

condition for ca. 2 h while the bath temperature

9

Cytotoxins in Klebsiella oxytoca

times. Upon completion, the resulting mixture

was vigorously stirred at room temperature for

21 hours. The mixture was then filtered through

a celite pad and eluted with methanol. The

filtrate was evaporated and the residue was

purified by silica gel chromatography to afford

0.37 g of (+)-tilivalline (97% yield) with a trace

amount of impurity. The product was further

purified by recrystallization from MeCN to

afford 93.8 mg of pure (+)-tilivalline (24%

yield).

membranes exclude 7-AAD, whereas damaged

cells and dead cells are permeable to 7-AAD. In

Fig. 11, untreated cells were viable (both PE

Annexin V and 7-AAD were negative), while

cells treated with kleboxymycin were

undergoing apoptosis (PE Annexin V positive,

7-AAD negative). Camptothecin serves as a

positive control of apoptosis inducer.

Kleboxymycin can readily associate with

indole to form tilivalline. Kleboxymycin is a

labile molecule since it can be converted to the

dehydrated-kleboxymycin (Supplemental Fig.

S4) and methoxy-kleboxymycin (Supplemental

Fig. S11) readily when dissolved in acetonitrile

and methanol solvent, respectively. To test

whether kleboxymycin can associate with indole

to form tilivalline directly, we have incubated

kleboxymycin with indole at different molar

ratios. The results were summarized in

Supplemental Table S4 and Supplemental Fig.

S12. No tilivalline could be detected in the

control samples of pure indole and pure

kleboxymycin. Tilivalline could be detected in

the kleboxymycin mixture with indole and the

levels of tilivalline increased proportionally with

(+)-Tilivalline: Light pink solid; analytical TLC

(silica gel 60), EtOH/DCM = 5%, R_f=

1

214 (c 0.39, MeOH); H NMR (400

0.23;

MHz, CD₃OD) δ 7.38–7.29 (m, 3H), 7.18 (s,

1H), 7.07 (t, J = 7.6 Hz, 1H), 6.90 (t, J = 7.5 Hz,

1H), 6.85 (dd, J = 7.6, 1.0 Hz, 1H), 6.67 (t, J =

7.9 Hz, 1H), 4.72 (d, J = 9.4 Hz, 1H), 4.22–4.13

(m, 1H), 3.80–3.63 (m, 2H), 1.95–1.81 (m, 1H),

13

1.74–1.52 (m, 3H); C NMR (100 MHz, CDCl₃)

δ 170.4, 147.2, 138.4, 136.6, 126.5, 124.3, 122.9,

122.6, 121.6, 120.2, 120.2, 118.7, 117.6, 116.8,

112.6, 63.5, 61.6, 48.9, 31.2, 23.4.

Toxicity of kleboxymycin. The cytotoxicity of

kleboxymycin and tilivalline was evaluated by

measuring the TC₅₀of the synthetic compounds

in an MTT-based cell culture assay. As shown in

Fig. 10, the toxicity of kleboxymycin is

significantly higher than tilivalline by at least 9-

fold. As the K. oxytoca cytotoxin had been

reported to mediate its cytotoxicity through

induction of apoptosis, we sought to assess

whether kleboxymycin also mediates cell death

through a similar mechanism. Results of a PE

Annexin V apoptosis assay are shown in Fig. 11

and support that kleboxymycin is a strong

inducer of apoptosis. PE Annexin V is a

sensitive probe for identifying apoptotic cells

which binds to the membrane phospholipid

phosphatidylserine (PS). In early stage of apo

ptosis, PS is translocated from the inner to the

outer leaflet of plasma membrane, exposing it to

PE Annexin V. 7-Amino-Actinomycin (7-AAD)

is a vital dye. Viable cells with intact

the

increasing

molar

ratios

of

indole/kleboxymycin. The equilibrium was

reached rapidly since no changes on the

tilivalline levels could be observed over a period

of 4 hours. The spontaneous dissociation of

tilivalline was also tested and summarized in

Supplemental Table S5 and Supplemental Fig.

S13. Freshly prepared pure tilivalline of 0.04

mM showed no sign of dissociation to

kleboxymycin.

Detectable

level

of

kleboxymycin at 0.0016 mM was observed after

60 days incubation at 298K.

Discussion

The identity of the K. oxytoca cytotoxin has

been a topic of interest for many years, but past

studies have produced ambiguous results. The

latest results from Schneditz et al. and Darby et

al. appeared to have settled the question (17,18),

10

Cytotoxins in Klebsiella oxytoca

but our present results revealed additional

complexity. We demonstrated that

presence of an imine or carbinolamine methyl

ether at the N7-C6 position of the basic PBD

skeleton is important for DNA adduct formation.

Kleboxymycin fits nicely with the proposed

mechanism with an imine moiety at N7-C6 (in

the dehydrated form) (Fig. 3C), while the same

position is occupied by the indole substituent in

tilivalline. Schneditz et al. agreed that tilivalline

is unlikely to cause cytotoxicity via the same

mechanism (17), and the proposed interaction

between tilivalline and DNA is supported only

by indirect evidence such as induction of

apoptosis. Furthermore, in the course of our

studies, we observed in vitro degradation of

tilivalline slowly into kleboxymycin and related

molecules to a limited degree (Supplemental

Table S5). Hence, it may be possible that

kleboxymycin and tilivalline can interconvert in

vivo, with kleboxymycin being the more toxic

species directly interacting with DNA. Further

studies on the direct interactions of

kleboxymycin and tilivalline with DNA may

help to clarify their role and contribution to the

cytotoxicity.

kleboxymycin is the primary product of a

nonribosomal peptide synthesis (NRPS)

pathway in K. oxytoca. Coincidentally, since K.

oxytoca is an indole-producing bacterium, a

significant amount of kleboxymycin would react

with indole to form the stable derivative

tilivalline even in pure culture. This resulted in a

confusing situation as the cytotoxicity phenotype,

tilivalline and kleboxymycin levels would

appear to correlate experimentally, regardless of

the contribution of tilivalline and kleboxymycin

to cytotoxicity. Our construction of an indole-

negative ∆tnpA mutant of cytotoxin-producing K.

oxytoca and testing of synthetic kleboxymycin

proved that kleboxymycin alone is sufficient to

induce cytotoxicity in the HEp-2 cell culture

assay. Regrettably, as kleboxymycin can react

with indole present within HEp-2 cells to form

tilivalline, it is not possible to eliminate the

contribution of tilivalline to cytotoxicity by

conventional experiments. Similarly, any animal

or clinical experiments involving kleboxymycin

would inadvertently produce tilivalline in vivo,

due to the presence of significant amount of

indole produced by intestinal microbiota (26),

which can enter intestinal tissue (27). Hence,

experimental assessment of the independent

cytotoxicity of each molecular species would

likely require a novel and innovative approach to

circumvent this significant limitation.

Prior to the present discovery of kleboxymycin,

all known natural tricyclic PBDs are produced

by members of the Actinobacteria. As pointed

out by Schneditz et al., the kleboxymycin

biosynthetic gene cluster in K. oxytoca is not

native to the Enterobacteriaceae, and was most

likely acquired through horizontal transfer.

Bioinformatic analysis suggested that the genes

might have been acquired from Xenorhabdus

spp., which in turn had acquired them from the

Actinobacteria. In particular, the PBD

biosynthetic gene cluster in the genome of X.

nematophila F1 has an identical organization

with the kleboxymycin biosynthetic gene cluster,

whereas the PBD biosynthetic gene clusters of

Streptomyces spp. are considerably larger (≥25

kb) and contain more genes (≥18). The

differences in size and gene count of the

biosynthetic gene clusters are reflected in the

complexity of the products –kleboxymycin is

Nonetheless, fundamental consideration of the

chemical and toxicological properties of

kleboxymycin versus tilivalline argues for a

greater role of kleboxymycin than tilivalline in

causing cytotoxicity. Known PBDs are DNA

minor groove binders with covalent interactions

with base residues, and their binding can

interfere with normal cellular activity like DNA

transcription and replication. Based on the

proposed

mechanism

for

anthramycin

deoxyguanosine adduct formation and previous

structure activity studies, it is inferred that the

11

Cytotoxins in Klebsiella oxytoca

one of the two smallest natural PBDs identified

to date, and contains only a single C6 hydroxyl

substituent on the basic tricyclic PBD backbone.

A related question of interest is whether K.

oxytoca, and possibly X. nematophila, are

unique among Enterobacteriaceae to support

PBD biosynthesis, as it is generally difficult to

achieve good heterologous expression of similar

biosynthetic gene clusters, like in the case of

erythromycin biosynthesis in Escherichia coli.

Alternatively, the kleboxymycin biosynthetic

genes might have undergone adaptation for

expression in Enterobacteriaceae. Further studies

in this area may lead to discoveries that can

improve industrial-scale biosynthetic production

of complex natural products.

that correct identification of the major cytotoxic

metabolites would enable future studies into the

molecular mechanisms of actions of these toxins

and lead to applications with clinical utility.

Experimental procedures

Bacterial strains and culture conditions. K.

oxytoca MH43-1 was used as a cytotoxin-

producing control strain and was a kind gift

from Prof. Christoph Högenauer at the Medical

University of Graz, Austria. Other K. oxytoca

strains were isolated from clinical specimens in

Queen Mary Hospital, Hong Kong, and include

strains described in our previous study. Identities

of all isolates were confirmed by standard

biochemical methods and matrix-assisted laser

desorption/ionization time-of-flight MS analysis.

Unless otherwise stated, for culturing of K.

oxytoca in broth, one colony was selected from a

pure growth on horse blood agar or LB agar and

inoculated into 10ml of a tryptone-lactose-based

medium (tryptone lactose broth) with the

following composition: 17 g/L tryptone (Sigma-

Aldrich, MO), 10 g/L lactose (Sigma-Aldrich,

MO) and 2.5 g/L dipotassium hydrogen

phosphate (Sigma-Aldrich, MO). Unless

otherwise stated, the inoculated broth is then

incubated for 24 hours at 35°C and agitation at

250 rpm.

Although cytotoxin production in K. oxytoca has

only been associated with the specific syndrome

of AAHC, we speculate that the toxin might also

enhance the virulence of the organism in other

types of infections. None of the tricyclic PBDs

produced by the Actinobacteria have been

associated with human or animal disease, but

both natural and synthetic PBDs are known for

their broad-spectrum anti-neoplastic activity.

The major adverse effects associated with PBDs

in clinical trials are intestinal necrosis and

cardiotoxicity. Based on the structure-activity

relationship of tricyclic PBDs, kleboxymycin is

also predicted to be cardiotoxic, due to presence

of the C9 hydroxyl substituent. Hence, it is

possible that cytotoxin production may

exacerbate cardiovascular collapse in the setting

of septic shock and possibly lead to increased

mortality. Further study is warranted to

investigate possible linkage between cytotoxin

production and adverse clinical outcomes in K.

oxytoca infections.

E. coli with cytotoxin gene cluster culture

conditions. For culturing E. coli with cytotoxin

gene cluster, single colony was selected from a

LB agar supplemented with 12.5 µl/ml

chloramphenicol and inoculated into 3 ml M9

minimal (or LB) medium supplemented with

12.5 µl/ml chloramphenicol and 500X

CopyControl Fosmid Autoinduction Solution

(Epicentre, WI) at 37°C overnight at 250 rpm.

10 µl of overnight culture was then added to 10

ml M9 minimal (or LB) medium supplemented

with 12.5 µl/ml chloramphenicol and 500X

CopyControl Fosmid Autoinduction Solution

(Epicentre, WI) for 24hr with shaking at 250

rpm.

In summary, a novel natural PBD cytotoxin has

been identified in specific K. oxytoca strains.

Importantly, our work reconciled conflicting

results from past studies, which were sometimes

confused by the effects of cytotoxin derivatives

versus that of the primary cytotoxin. It is hoped

12

Cytotoxins in Klebsiella oxytoca

Cell culture cytotoxicity assay. Cytotoxin

production in K. oxytoca was assayed by a

previously established cell-culture assay (5,8),

with modifications to the original bacterial

culture conditions. 10⁵HEp-2 cells were seeded

into each well of a 96-well plate and incubated

for 48 hr in minimal essential medium (MEM)

containing 10% fetal bovine serum, 100 U/ml

penicillin, 100 ꢀg/ml streptomycin, and 31.25

ꢀg/ml gentamicin. Broth cultures of K. oxytoca

were prepared as stated above. After

centrifugation at 20,000× g for 10 min at 4°C,

the supernatant was filtered through a membrane

filter with a pore diameter of 0.2 ꢀm (Millipore,

MA). An equal volume of MEM was mixed with

the filtrate (1:1 dilution with phosphate-buffered

saline) and inoculated onto HEp-2 cells,

followed by incubation in 5% CO₂at 37°C for

up to 48 h. Cytotoxicity was defined as presence

of cytopathic effects (CPE) manifested as cell

rounding or death that were observed with a

light microscope. When 50% or more of the

cells are seen to have rounded up or detached

under the low power field, it is defined as a

positive CPE score.

medium contained 1% FBS in concentration and

25ꢀg/ml of amikacin & meropenem. The plate

was wrapped with aluminum foil and placed in

an incubator with 5% CO₂at 37°C for 2 days.

CPE as mentioned above was recorded at 24 and

48 hr. At the end of the assay, 10ꢀl of a 5mg/ml

MTT solution was added into each well and

incubated for 4 hr, then 100ꢀl of 10% of sodium

lauryl sulfate with 0.01M hydrochloric acid was

added to solubilize the MTT crystal overnight.

MTT data was recorded at optical densities of

570 nm and 640 nm by DTX880 plate reader

(Beckman Coulter, CA).TC₅₀was calculated and

curve plotted with SigmaPlot (Systat Software,

IL).

Separation and purification of Compounds

P1 and P2. The separation and purification

strategy used to purify the top two bioactive

compounds as identified through multivariate

analysis is shown in Fig. 2. The mass to charge

ratio and retention time of the two chemical

species were used as the identifiers to trace their

location during their purification. An Oasis HLB

solid phase extraction (SPE) cartilage was first

conditioned with 12 ml of MeOH, followed by

12 ml of H₂O. 10 ml of culture media was

loaded onto the column and the metabolites were

eluted in three successive fractions. The first

fraction (F1) was eluted using 20 ml of H₂O,

fraction 2 (F2) with 10 ml of MeOH:H₂O (1:1)

and fraction 3 (F3) with 10 ml of MeOH. A

sample of each fraction was subjected to LC-

MS/MS analysis and F2 was determined to

contain compound 1. A total of 1 ml of F2 was

further resolved by HPLC-DAD on a Keystone

Hypersil C18 column (4.6 x 250 mm) using 10

injections (100 ꢀl per injection). For each

injection, isocratic elution at 10% ACN was

used to elute the fraction containing compound 1

at a retention time of 10 min (referred to as Peak

1, P1). All the eluent from the C18 column, with

exception of the fraction containing P1, was

pooled together and this was combined with F1.

This combined fraction was referred to as

TC₅₀

determination.

For

quantitative

determination of cytotoxicity of filtered culture

supernatants, purified toxin fractions or

synthetic toxins, MTT-based cytotoxicity assays

similar to a previously reported assay (17) were

performed. 1.50×10⁴HEp-2 cells were seeded

on flat bottomed, 96-well cell culture plates and

maintained at a total volume of 100 ꢀl MEM

with 10% FBS. The culture plates were placed in

an incubator with 5% CO₂at 37°C for 24 hr

until the cells had reached 70-80% conﬂuency.

Culture media was then removed, followed by

washing of cells once with 100 ꢀl plain MEM

and replenishment with 50ꢀl MEM containing

2% FBS. 50ꢀg/ml of amikacin and meropenem

was also added to the culture medium. We

performed serial 2-fold dilution of test materials

with plain MEM, transferred 50ꢀl diluted

supernatant into each well. Hence, the final

13

Cytotoxins in Klebsiella oxytoca

fraction O (other part of eluent). LC-MS/MS

analysis determined that F3 contained compound

2 with a sufficient purity that further separation

was not required and was now referred to as

Peak 2 (P2). The fractions P1, P2 and O were

subjected to rotatory evaporation to concentrate

them then diluted to a final volume of 4 ml in

H₂O. For the subsequent CPE cytotoxicity

toxicity tests where the combined fractions were

examined, P1+P2+O was generated by pooling

equal volumes of the individual fractions

together. P1+P2 was made by adding equal

volumes of P1, P2 and H₂O together. When

testing individual fractions (P1, P2 and O), one

volume of the corresponding fraction was mixed

with 2 volumes of H₂O. All the fractions were

stored at 4°C until they were examined using the

CPE cytotoxicity tests.

scans were used as the dependent scan. The two

most intense peaks picked from each EMS were

subjected to EPI with two different collision

energies (CE = 20 or 40), while CE setting was

10 in EMS. To prevent the acquisition of

redundant spectra, the same peaks in EMS

would not be selected by IDA in the following

60 seconds after 5 occurrences. The mass range

used in EMS was m/z 100 – 500 and that in EPI

was m/z 50 – 500. The linear ion trap filling

time was set to 50 ms in any of the scans with

Q0 trapping turned on. Scanning rates of EMS

and EPI were both set at 4000 amu/s. Enhanced

Resolution mode was also used to confirm the

charge state of +1. The declustering potential

(DP) was set to 50 in each of the scans.

LC-MS/MS data processing and multivariate

analysis. Markerview software 1.2.1 (AB Sciex,

CA) was used for LC-MS peak finding and peak

alignment (28). Peaks were found with the

minimum retention time set to 1.5 minutes,

minimum spectral width of 0.6 Da and noise

threshold of 1 × 10⁵counts per second. For peak

alignment, the retention time tolerance and mass

tolerance were set to 5.0% and 0.2 Da,

respectively. A 12 × 3647 (row × column) data

matrix with peak areas and toxicity values was

generated for multivariate analysis using

SIMICA-P 13.0 (Umetrics, Sweden). For each

row, it represents the four different samples,

each with three replicates. For each column, it

represents the identified LC-MS peaks depicted

with a unique pair-identifier (the mass to charge

ratio and retention time). The final column in the

data matrix represents the toxicity values

derived from the MTT assay using the culture

media. Principle component analysis (PCA)

model was used to identify outliners and a

supervised partial least square (PLS) model was

employed to correlate LC-MS profiling data

with corresponding toxicity data. The model was

generated using the autofitting option with

pareto scaling (19). Peakviewer software 2.0

(AB Sciex, CA) was used to process the high

resolution MS data and Formula finder was used

LC/MS/MS analysis. Analysis of the culture

supernatant was performed on an Agilent 1100

series HPLC system (Agilent Technologies, CA,

USA) coupled to a triple quadrupole-linear ion

trap mass spectrometer (QTRAP; AB Sciex,

Canada) with a TurboIon Spray ion source set

with positive mode. Agilent 1100 series degasser,

pump and auto sampler were used in conjunction

with an Agilent eclipse XDB C18 (150 × 2.1

mm, 3.5 µm) analytical column. 10 µl of sample

was injected by the auto sampler in each run

with a linear gradient elution at a flow rate of

200 µl/min. The mobile phase was initially run

at 100% A for 5 minutes, then the organic

content was increased linearly to 100% B within

20 minutes. The column was then flushed with

100% ACN for 14.5 minutes and back to 100%

water in 30 seconds. Finally, the column was

reconditioned using 100% water for 20 minutes.

Mobile phase A was 0.1% formic acid in water

and mobile phase B was 0.1% formic acid in

ACN.

In the electrospray ionization (ESI) setting, the

curtain gas, nebulizing gas and auxiliary gas

were set to 20, 60 and 60 units, respectively.

Auxiliary gas temperature was set at 350°C and

the ion spray voltage was 5500 V. Information

Dependent Acquisition (IDA) was implemented

and Enhanced MS (EMS) mode was used for the

survey scans. Four Enhanced Product Ion (EPI)

14

Cytotoxins in Klebsiella oxytoca

to generate the potential molecular formulas of

the identified compounds (29).

GC-MS analysis. GC-MS analysis was

performed on an Agilent 7890A gas

chromatography instrument coupled to an

Agilent 5975C mass spectrometer using Agilent

ChemStation software (Agilent Technologies,

CA). Heliflex AT-5 capillary column (30 m ×

0.25 mm i.d. × 025 µm (film thickness)) with

(5% phenyl)-95% methoxpolysiloxane phase

(Grace, IL) was used for separation.

Temperature program began at 50°C, held for 5

min, then increased from 50°C to 200°C at

10°C/min and held for 5 min. Split less injection

was conducted and high purity grade helium

(99.995%) was used as carrier gas with a flow

rate of 1.0 ml/min. The MS spectrometer was

operated in electron-impact (EI) mode, the scan

range was 20-350 amu, the ionization energy

was 70 eV and the scan rate was 4.27 scans per

second. The inlet, auxiliary heater, ion source

and quadrupole temperatures were set at 180°C,

180°C, 280°C and 150°C, respectively.

High resolution LC-MS/MS analysis for

accurate mass determination. To determine

the accurate mass for compound P1 and P2,

Agilent 1200 series nano pump equipped with a

10

well-plate

auto-sampler

(Agilent

Technologies, DE) was connected to TripleTOF

5600 system fitted with a Nanospray III source

(AB SCIEX, Canada) used in positive mode.

The RP trap (150 µm i.d. × 50 mm length) and

RP nano columns (75 µm i.d. × 150 mm length)

were packed in-house with Jupiter C18 packing

materials using an ultrahigh-pressure syringe

pump operating in constant pressure mode (up to

6000 psi). 5 µg of compounds P1 and P2 were

mixed together, loaded onto RP trap column and

then eluted on the RP nano column with a binary

solvent system consisting of solvent A (H₂O

with 0.1% formic acid) and solvent B (ACN

with 0.1% formic acid) at a flow rate of 300

nl/min. The total run time was 50 min with the

following gradient conditions: 0 - 5 min, 0% B;

5.1 - 35 min, 0 - 100% B; 100% B hold for 10

min; 45.1 - 46 min, 100% - 2% B, 2% B hold for

19 min. A calibration delivery system was used

to ensure the accuracy of mass to less than 20

ppm. Parameters were the following: nebulizer

gas, 10 psi; CUR, 30 psi; heater gas, 0 psi; ISVF,

2800 V and IHT, 125°C. An IDA method was

created to acquire both MS and MS/MS data.

TOF MS spectra were acquired with the range

m/z 100-1000 Da and 0.25 s ion accumulation

time, DP at 30 V and CE at 10 V. Peak intensity,

more than 1 cps; charge state was set +1;

excluding the former target ion for 6 seconds;

excluding isotopes within 4.0 Da range;

maximum number of candidate ions to monitor

per cycle, 20; mass tolerance, 50 mDa and

dynamic background subtraction was on;

product ion spectra were acquired with the range

m/z 50 - 1000 Da and 0.25 s ion accumulation

time, DP at 30 V and CE at 30 V.

NMR spectroscopy analysis. For the NMR

measurement of dehydrated-kleboxymycin,

lyophilized kleboxymycin was reconstituted in

acetonitrile-d₃with the addition of molecular

sieve beads to remove residual water in the

sample and the solvent. The sample was

monitored with time by ZG30 1D proton NMR

experiment till the kleboxymycin was fully

dehydrated after about 24 hours. For the NMR

measurement of kleboxymycin, lyophilized

kleboxymycin was reconstituted in 100% D₂O

with 43.7 mM trimethylsilylpropanoic acid

(TMSP, Sigma). The following experiments,

ZG30, homonuclear 2D experiments COSY

(cosyesgpqfpr), TOCSY (dipsi2esgpph1) and

heteronuclear

2D

experiments

HSQC

(hsqcefgpsisp2, hsqcedetgpsisp2.3) and HMBC,

were acquired at 298K on a Bruker Avance 600

MHz NMR spectrometer (¹H and ¹³C

frequencies of 600.133 MHz and 150.916 MHz,

respectively) equipped with a 5 mm BBI probe

with Z-gradient using standard Bruker pulse

sequences. Data were acquired and analysed

15

Cytotoxins in Klebsiella oxytoca

using Topspin 3.1 (Bruker). Chemical shifts are

given on a ppm scale and coupling constants are

1

reported in Hz. All H chemical shifts were

complementation was performed by inserting

target genes into plasmid pCRII or pCR-XL

(Invitrogen, CA) by Taq-amplified cloning.

Cloning of cytotoxin biosynthesis gene cluster.

A 17.5kb DNA fragment encompassing the

complete K. oxytoca cytotoxin biosynthesis

gene cluster was first amplified by PCR using

forward primer

referenced to residual acetonitrile methyl

resonance at 1.95 ppm for dehydrated-

kleboxymycin or TMSP methyl resonance at 0

ppm for kleboxymycin sample. Chemical shifts

of ¹³C were referenced indirectly by

gyromagnetic ratio method (30). A pulse length

5’-GGCAAGATATCCGACTCGTTGTTT-3’

and reverse primer

1

of 7.97 µs and 15.94 µs were used for H and

¹³C respectively. Data of ZG30 was acquired at

64k points with spectral width of 12335.526 Hz.

Free induction decays (FIDs) were multiplied

with an exponential line broadening function of

0.3 Hz before Fourier transformation. For two-

dimensional H-¹H and H-¹³C experiments, the

sizes of F2 dimension was 2048 points and that

of F1 dimension was 512 points, which were

Fourier transformed into 2048 × 1024 points.

Spectral width of COSY was 9615.385 Hz ×

9014.423 Hz, HSQCETGPSISP was 8971.292

Hz × 25000.000 Hz, HMBC was 12019.230 Hz

× 33195.164 Hz for F2 and F1, respectively.

5’-GCAAAGGAATAAGCAAGAAGGGAA-3’.

PCR was performed in a 25 µL reaction mix

containing bacterial DNA as template, iProof

GC buffer, 200 µM of each dNTP, and 0.02U

iProof High Fidelity DNA polymerase (Bio-Rad

Laboratories, CA). Cycling conditions were as

follows: initial denaturation at 98°C for 1 minute,

followed by 40 cycles of 98°C for 10 seconds,

58°C for 30 seconds, and 72°C for 7 minutes in

an automated thermal cycler (Applied

Biosystems, CA). The PCR product was

subjected to 5’-phosphorylation using T4

polynucleotide kinase (New England Biolabs,

MA) for 30 minutes at 37°C and then cloned

into CopyControl pCC2FOS (Epicentre, WI)

according to manufacturer’s instructions. Briefly,

5’-phosphorylated PCR product was ligated into

CopyControl pCC2FOS cloning-ready vector

using Fast-Link DNA Ligase at 16°C overnight,

and the resultant plasmid was packaged into

MaxPlax Lambda Packaging Extracts, EPI300-

T1R Plating Strain. Single colonies were

selected on LB plate supplemented with 12.5

µl/ml chloramphenicol at 37°C overnight.

1

Gene deletion and complementation. Gene

deletion mutants of K. oxytoca were constructed

using the Lambda Red recombination system

(31,32). Oligonucleotide sequences used are

listed in Supplemental Table S6. Briefly, cells

harboring pSIM5 were grown to the mid-log

phase, and induced by thermal inactivation of

the λ cI857 repressor at 42°C for 15 minutes.

Cells were made electrocompetent and

transformed with PCR products containing the

kanamycin-resistance cassette flanked by 500 bp

flanking regions of the target gene.

Transformant colonies carrying the desired

modification were directly selected on

kanamycin-containing LB agar. The target

region was PCR-amplified and sequenced to

Apoptosis assay. Induction of apoptosis was

assessed using the PE Annexin V Apoptosis

Detection Kit I (BD Biosciences, NJ) according

to manufacturer’s instructions. Briefly, 2×10⁶

Jurket cells per well were seeded in 6-well plate

with RPMI1640 containing 1% fetal bovine

serum. Cells were then left untreated or treated

with one of the following: 6µM camptothecin

for 6 hours, 500 µl MH43-1 culture supernatant,

pure synthetic kleboxymycin for overnight at

confirm

site-specific

modification.

The

kanamycin cassette was then removed using the

Cre recombinase system borne on the plasmid

705-Cre(33,34). To confirm that the resultant

phenotypic changes were due to gene deletion,

16

Cytotoxins in Klebsiella oxytoca

37°C. After incubation, cells were stained with

PE Annexin V and 7-Amino-Actinomycin (7-

AAD), and analyzed by flow cytometry.

Acknowledgments

Conflict of Interest

This work was supported by donation by Mr.

Michael Tong and the Providence Foundation in

memory of Mr. and Mrs. L.H.M. Lui; the

Commissioned Research on Control of

Infectious Diseases (Phase III) (project number:

HKM-15-M06) of the Health and Medical

Research Fund under the Food and Health

Bureau, the Government of the Hong Kong

Special Administrative Region. The funders had

no role in study design, data collection and

interpretation, or the decision to submit the work

for publication.

The authors declare that they have no conflicts

of interest with the contents of this article.

Author contributions

HT, KHS and KYY conceived and coordinated

the study and wrote the paper. DY, and QG

produced the kleboxymycin via total synthesis.

KHS, RYTJ, KCL, CWL, SYL, YK, IKC and

SSCT

performed

LC/MS

and

NMR

characterization on the culture supernatant, and

purified and identified the toxin. RYTK, JD, EC,

WMC and SPL optimized the toxin-producing

conditions and performed cytotoxicity assays

and genetic manipulations on the Klebsiella

strains. All authors reviewed the results and

approved the final version of the manuscript.

We would like to thank Dr. Wing-Cheong Yam,

Mr. Pok Man Lai and other colleagues of the

Department of Microbiology, The University of

Hong Kong for their technical support.

17

Cytotoxins in Klebsiella oxytoca

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3

Cytotoxins in Klebsiella oxytoca

Table 1. Cytotoxicity of K. oxytoca strain MH43-1 culture supernatants from different culture conditions

as measured in a cell culture cytotoxicity assay. All cultures were incubated in a shaker at 250 rpm under

aerobic conditions for 24 hours.

Culture media

LB broth (Sigma)

Temperature Highest dilution with observable cytopathic effects

37°C

35°C

42°C

35°C

42°C

35°C

2-4×

4×

Brain-heart infusion (Sigma)

Todd-Hewitt broth

Tryptic soy broth (TSB)

TSB

4×

16×

No cytotoxicity

64×

TSB

Tryptone lactose broth (TLB)

TLB

Tryptone glucose broth

No cytotoxicity

32-64×

Table 2. Kleboxymycin and tilivalline levels in the cultures of WT K. oxytoca at 8 and 12 hours.

Kleboxymycin

Tilivalline

Kleboxymycin/Tilivalline

ratio

Strain

Time

Average STDEV Average STDEV

(µM)

3.1

(µM)

0.3

(µM)

5.7

(µM)

3.5

0.54

0.41

MH43-1

8hr

11.2

2.1

27.2

3.7

12hr

All data collected as triplicates.

4

Cytotoxins in Klebsiella oxytoca

Table 3. Cytotoxicity of culture supernatants from different deletion mutants of K. oxytoca strain MH43-

1, as measured in a cell culture cytotoxicity assay. All cultures were incubated in a shaker at 35°C and

250 rpm under aerobic conditions for 24 hours. Presence of kleboxymycin and tilivalline was assessed by

LC/MS/MS analysis.

Deletion mutant

Cytotoxicity

detected ^a

Kleboxymycin

detected

Tilivalline

detected

Within toxin gene cluster

∆mfsX

∆uvrX

∆hmoX

∆adsX

∆icmX

∆dhbX

∆aroX

∆npsA

∆thdA

∆npsB

∆npsC

∆marR

Outside toxin gene cluster

WT

ND

WT

ND

WT

Yes

No

Yes

No

Yes

No

Yes

No

Yes

∆tnpA (indole non-producing) WT

^aWT = wild-type (strain MH43-1); ND = not detected

Yes

No

Table 4. Kleboxymycin and tilivalline levels in the cultures of mutant K. oxytoca at 8 and 12

hours

Kleboxymycin

Tilivalline

Strain

Time

8hr

Average STDEV Average STDEV

(µM)

0.0

(µM)

0.0

(µM)

0.0

(µM)

0.0

ꢁadsX

ꢁadsX+3HA

ꢁtnpA

4.8

0.1

5.6

0.2

4.4

0.4

0.0

ꢁadsX

0. 0

12.1

11.0

0.0

1.5

28.3

0.0

1.5

ꢁadsX+3HA

ꢁtnpA

12hr

1.1

0.0

All data collected as triplicates. 3HA=3-hydroxyanthranilate.

5

Cytotoxins in Klebsiella oxytoca

Table 5. Kleboxymycin and tilivalline levels E. coli with cytotoxin gene cluster cultured in M9

and LB media at 24 hours.

Kleboxymycin

Tilivalline

Medium

Average STDEV Average STDEV

(µM)

0.041

0.0

(µM)

0.01

0.0

(µM)

0.0

(µM)

0.0

M9

LB

0.088

0.021

All data collected as triplicates.

6

Cytotoxins in Klebsiella oxytoca

FIGURE LEGENDS

Figure 1. MH43-1 growth curves in different culture conditions

Figure 2. Multivariate analysis of K. oxytoca culture supernatant metabolite-toxicity profiles

identifying potential bioactive compounds. (A) PLS score plot of culture supernatant metabolite-

toxicity profiles from 4 culture conditions. The profiles from each condition were prepared in triplicate

and are color coded. NT-L, non-toxigenic strain in lactose-containing media, green; NT-NL, non-

toxigenic strain in lactose-free media, grey; T-NL, toxigenic strain in lactose-free media, blue; T-L,

toxigenic strain in lactose-containing media, red. (B) Three-dimensional loadings plot for the metabolite-

toxicity profile model generated using PLS. Each green dot represents a unique chemical species

identified by its m/z and retention time (RT). Those contributing significantly to all three principle

components are labeled and highlighted in red. (C) Representative extracted-ion chromatogram of

different media conditions showing presence or absence of the metabolites m/z 235.2 at RT 15.9 min and

m/z 34.1 at RT 20.2 min. The color coding and labeling of the chromatograms are identical to those used

in (A). Cps, counts per second.

Figure 3. Proposed structures of (A) compound P1 (kleboxymycin) m/z 235 at retention time (RT) 15.8

min and (B) compound P2 (tilivalline) m/z 334 at RT 20.2 min. (C) The structure of dehydrated form of

kleboxymycin at m/z 217.

Figure 4. MS spectrum for isolated K. oxytoca cytotoxin kleboxymycin in (a) positive ionization mode

and (b) negative ionization mode.

Figure 5. Positive MS/MS spectrum for dehydrated-kleboxymycin at m/z 217.

Figure 6. Resonance assignments of dehydrated-kleboxymycin NMR spectra in acetonitrile-d3.

(A)1D ¹H zg30 spectrum of dehydrated-kleboxymycin in acetonitrile-d3 and proton assignments. (B) ¹H-

¹H COSY spectrum showing aliphatic protons (H2-H5) crosspeaks at the region 2-3.8ppm (Right top

panel). Aromatic protons (H10-H12) crosspeaks were observed at the region 6.96-7.46ppm (Right bottom

panel). H5 and H6 crosspeaks were also identified. (C) 2D ¹H-¹³C heteronuclear multiple bond correlation

(HMBC, blue) and heteronuclear single quantum coherence (HSQC, red for –CH– and pink for –CH2–)

overlaid spectrum showing the assignments of carbon without protons attached (C9, C13, C14).

Figure 7. Resonance assignment of kleboxymycin NMR spectra in D₂O. (A)1D ¹H zg30 spectrum of

kleboxymycin in D₂O and proton assignments. Kleboxymycin has two diastereomers a and b as Carbon 6

is a chiral centre after –OH addition to the double bond. (B) ¹H-¹H COSY spectrum showing aliphatic

protons (H2-H5) crosspeaks at the region 2-3.8ppm (Right top panel). Aromatic protons (H10-H12)

crosspeaks were observed at the region 6.96-7.46ppm (Right bottom panel). H5 and H6 crosspeaks were

1

also identified. (C) 2D H-¹³C heteronuclear multiple bond correlation (HMBC, blue) and heteronuclear

single quantum coherence (HSQC, red for –CH– and pink for –CH2–) overlaid spectrum showing the

assignments of carbon without protons attached (C9, C13, C14). (D) Chemical structures of diastereomers

a and b of kleboxymycin showing the stereo-isomerism on C6.

Figure 8. Positive MS/MS spectrum for kleboxymycin at m/z 235.

7

Cytotoxins in Klebsiella oxytoca

Figure 9. Gene cluster of cytotoxin synthesis. Diagram showing genetic organization of the cytotoxin

biosynthesis gene cluster, along with the annotation of predicted gene functions and phylogenetic closest

neighbor based on NCBI BLAST search. The predicted function of anthranilate 3-monooxygenase for

hmoX does not appear to be supported by our experimental results.

Figure 10. Cytotoxicity of kleboxymycin and tilivalline. (A) Results of cytotoxicity assessment (TC₅₀)

from an MTT-based cell culture assay, showing a >9-fold lower TC₅₀value of kleboxymycin relative to

tilivalline. (B) Cytopathic effects (CPE) of 1.56 ꢀM tilivalline and 1.56 ꢀM kleboxymycin on HEp-2 cells

on day 2 post-treatment. Cells exposed to tilivalline show minimal CPE relative to minimal control, but

those treated with kleboxymycin showed significant cell rounding. (20× magnification)

Figure 11. Results of a PE Annexin V apoptosis assay showing kleboxymycin as a strong inducer of

apoptosis.