1AvidBiotics Corp., South San Francisco, CA, USA (E-mail: greg.govoni@avidbiotics.com); 2 Université de Sherbrooke, Sherbrooke, Quebec, Canada

Authors: Dana Gebhart1, Dean Scholl1, Chuck Vacin1, Louis-Charles Fortier2, Steve Williams1, and Gregory Govoni1

DIFFOCINS: NOVEL, HIGH-MOLECULAR WEIGHT BACTERIOCINS HIGHLY SPECIFIC FOR CLOSTRIDIUM DIFFICILE

Clostridium difficile infections (CDIs) are a major cause of antibiotic-associated colitis in and out of healthcare settings. Novel therapeutic and preventative treatments are desperately needed to help combat this disease. We have isolated from Clostridium difficile novel, high molecular weight bacteriocins, termed “Diffocins”, with unique properties that make them potential anti-C. difficile agents. Diffocins are phage tail-like particle structures similar to R-type pyocins from Pseudomonas aeruginosa. Based on the contractile structure, we believe Diffocins kill bacteria in a manner analogous to R-type pyocins, whereby the contractile tail punches a small hole in the target membrane to dissipate the membrane potential without leaking larger molecules such as DNA or toxins. Our further investigations of Diffocins have found: 1. Diffocins exhibit a narrow bactericidal spectra, highly selective for C. difficile. 2. Diffocins can be manipulated and heterologously expressed in Bacillus subtilis. 3. Diffocins are targetable; we have identified the receptor binding protein (RBP) responsible for killing specificity and can switch specificity by swapping RBPs. 4. Diffocin killing spectra can be expanded by exchanging endogenous Diffocin RBPs for RBPs from C. difficile phages/prophages. 5. Orally administered Diffocins remain active after passage through the mouse GI tract. Taken together, these properties indicate that Diffocins are excellent candidates for therapeutic and, especially, prophylactic management of CDIs without causing unintended collateral damage to the gut microbiota.

ABSTRACT FIGURE 2. Diffocin gene cluster schematic.

FIGURE 1. Electron Micrograph image of Diffocin prep containing phage tail-like (PTL) particles from C. difficile.

FIGURE 3. Schematic of Diffocin mechanism of action.

Bacteria species strain Dif

f4

Dif

f43

59

3

Dif

f16

Dif

f4-M

RB

P2

Dif

f4-M

RB

P4

C. difficile CD630 - - - - +++

C. difficile R20291 - +++ - +++ -

C. difficile 19137 +++ - - +++ -

C. difficile 19099 - - +++ - -

Bacillus subtilis BDR11 - - - - -

Bacteriodes fragilis ATCC 25285 - - - - -

Bacteriodes fragilis ATCC 23745 - - - - -

Bifidobacterium breve ATCC 15700 - - - - -

Clostridium acetobutylicum ATCC 824 - - - - -

Clostridium bifermentans ATCC 638 - - - - -

Clostridium sordellii ATCC 9714 - - - - -

Clostridium sporogenes ATCC 3584 - - - - -

Escherechia coli stx- - - - - -

Lactobacillus lactis 4356 - - - + -

Listeria ivanovvii 19119 - - - - -

Listeria monocytogenes 23074 - - - - -

Listeria innocua 33090 - - - - -

TABLE I. Sensitivity of non-Cdiff strains to heterolgously expressed, recombinant Diffocins.

Diff4_MRBP4 2h 3h 4h 5h 6h 7h 8h

AVR2-V10

Dif

f +

con

t Diff4 2h 3h 4h 5h 6h 7h 8h

1x

5x

25x

125x

2h 3h 4h 5h 6h 7h 8h

C. difficile - 19137 C. difficile – CF5 E. Coli – stx-

FIGURE 5. Diffocins with RBPs survive transit thru mouse GI tract.

Structural Proteins

13

59

13

79

13

74

13

73

13

71

13

68

13

66

13

63

CD630 ORF #

Putative Function

13

61

22kb

Phage Regulatory

Unknown Function

1366 1368 RBP1 Diff4-RBP1

N.D. Prophage (M68)

Shealth

Tape Measure

CW hydrolase RBD

Gly-Rich Protein

Holin/ endolysin

Capsid Putative Tail Fiber

1366 1368 RBP5 Diff4-RBP5

Diff4-PTF 1366 1368

RBP5

19137 19099

Spot Assay (Target Strains)

CF5

Diff4

1366 1368 ORF1374 Diff16

Diff4-16

Tape Measure

1363 Shealth CW

Hydrolase

1366 ORF1374 1368

1366 ORF1374 1368

RBD

1373 Putative Tail Fiber

FIGURE 4. Engineering Diffocin specificity by changing ORF1374/ prophage RBPs.

TABLE II. Killing spectrum of natural and heterologously expressed Diffocins from strain M68.

Diffocins are narrow spectrum, high molecular weight bacteriocins that are able to kill C. difficile. Their properties include: • Highly specific for C. difficile • Can be functionally expressed in heterologous system B. subtilis • Killing specificity (of natural Diffocins) is determined by ORF1374 variants • Killing specificity can be targeted by engineering in specific RBPs to cluster • Able to survive transit through mouse GI tract We believe Diffocins make excellent candidates for anti-Cdiff therapeutic and preventative agents.

CONCLUSIONS

The Diffocin gene cluster contains 25 predicted ORFs (1359-1379 in CD630). Many genes in the cluster have homology to known phage genes. The gene cluster is found in all sequenced strains of C. difficile.; however, the genes encoding the putative tail fiber (1373) and the receptor binding protein (1374) are highly variable between strains. We have been able to clone and express genes (1359-1376) in Bacillus subtilis and get heterologous expression of active Diffocin. Diffocin variants have been engineered that replace the C-terminal region of 1373 and all of 1374 with alternative ORF1374s or prophage RBPs (along with C-terminal region of adjacent putative tail fiber) from different C. difficile isolates.

RIBOTYPE 1 1 1 2 2 2 12 14 14 15 15 17 17 27 27 27 78 87 10

6

10

6

Strain

19

13

5

BI-

9*

LIV

24

19

09

9

19

13

1

TL1

78

CD

63

0

19

12

3

TL1

76

19

13

7

TL1

74

CF5

M6

8

19

12

6

20

06

8

R2

02

91

M1

20

43

25

5*

Liv2

2

19

10

2

CD

4

Cd

-62

CD

24

2

CH

I

19

12

1

19

11

7

19

13

4

19

09

8

Dif

foci

n p

rep

M68 (Cdiff) + + ++ ++ ++ ++ - ++ ++ ++ ++ P - ++ ++ ++ ++ + ++ ++ + ++ ++ ++ P - - +

diff4-M68_1374 (Bs) + + + - - - - - ++ + - - - + + + + - ++ ++ + ++ ++ + - - nd -

diff4-MRBP1 (Bs) - - - ++ ++ ++ - ++ - - ++ - - - - - ++ - - - ++ - - - - - nd -

diff4-MRBP2 (Bs) ++ ++ ++ - nd - - ++ - ++ - - - ++ ++ ++ - ++ - - ++ - - - - - nd -

diff4-MRBP4 (Bs) - - - - nd - ++ - - - - ++ ++ - - - - - - - - - - ++ ++ ++ ++ -

diff4-MRBP5 (Bs) - - - - nd - ++ - - - - ++ ++ - - - - - - - - - - ++ ++ ++ ++ -

Peptidoglycan

Cytoplasm ATP, K+,H+

ATP, K+,H+

Plasma

Membrane

UNBOUND

BOUND 1374/ RBP

Diffocin preparations containing PTL particles (as seen by EM) and anti-Cdiff activity were run on a SDS-PAGE gel. Silver staining was performed to identify protein bands, which were then excised and sent for mass spectrometry analysis. Excised proteins matched predicted proteins in a gene cluster of phage genes in strain CD630.

Diffocin preps from the strain M68 kill a wide number of Cdiff strains. The killing spectrum of this prep was compared to preps made from B. subtilis heterologously expressing diffocins with either ORF1374 or prophage RBPs from M68. Black boxes denote strong killing activity, grey boxes denote partial killing activity, white boxes denote no (or not determined) killing activity, and P indicates the presence of phage plaques. Sensitivity to a particular Diffocin was shared with all strains within a ribotype except for ribotypes 014 and 015.

A cocktail of Diffocins (Diff4 and Diff4_MRBP4) and Avidocins (AVR2-V10) was re-suspended in 1% Sodium Bicarbonate and orally administered to mice pre-treated with Ranitidine (100 mg/kg IP). Fecal pellets were recovered every hour post-administration, homogenized and assayed for Diffocin/Avidocin killing activity.