Presented at LMP 2016 summer research poster day. Research project on adhesion of C. difficile spores and length of BclA exosporium protein gene.

1. Cell culture Lysis Spore purification
48h 144h 192h
Suspend all cells
and spores
Centrifuge
Wash
Vortex
one min
Dilute
Heat treat
10 mins
Measure DPA
Ability of C. Difficile Epidemic NAP1 Strain to Cause Outbreaks
Closely Related to Loss of Exosporium Protein Function
Aretha Chan , George Broukhanski . Public Health Ontario Laboratories & Department of Laboratory Medicine and Pathobiology
For additional information
please contact:
George Broukhanski
Research
Public Health Ontario
George.broukhanski@oahpp.ca
Introduction
Objective
Methods Results
Conclusion
• C. difficile infections occur in large outbreaks, which happen in hospital settings
and result in antibiotic-associated diarrhea.
• NAP1 only strain of C. diff that caused recent large outbreaks from 2000s
• Its spores are the reason for persistent, leading to outbreaks.
• NAP1 strains (north american pulsotype) spores have a very different spore
morphology than its relatives
• Its exosporium is covered in hair structures, changing its adhesive properties.#1
• Essential in the spread of C. diff, as it can be easily transferred from surface to
surface, possibly aerosolizing as well
NAP1 is the only strain that can cause outbreaks, and the
explanation for outbreaks is the spread of spores.
So how are NAP1 spores spread more easily?
• Investigate the properties and genetic makeup of BclA, the protein that makes
the hairs.
• The length is attributed to the GXX amino acid repeats in the central region of
BclA, which is the only region that varies amongst strains #2
• By comparing the gene amplicons, we can determine the origin of the hairs and
how we can control the spread of C. difficile.
• 2015 UPDATE:
• Half a million
infections
• $3 800 000 000
in costs
* Photos
1. Poster from: Centres for disease control and prevention http://www.cdc.gov/drugresistance/biggest_threats.html
2. TEM photos from Paredes-Sabja. (2014). Clostridium difficile spore biology: sporulation, germination, and spore structural proteins. Trends in microbiology.
3. Colour TEM from SPL, as seen on BBC news
4. Structures generated with RaptorX
5. Diagram from Knox, C. (n.d.). Exosporium hairs affect spore adhesion on surfaces. US Army RDECOM.
This study was funded by Public Health Ontario Special thanks to Derek Chau and Sarah McLeod for helping me in the lab
*1
Smooth exosporium
Long hair structures
630 Nap1
*2 (photos are contradictory)
Correlation between:
Length of hairs, Adhesion,
Ability to cause outbreak?
Concentration of spores and adhesion assay
DNA extraction, PCR and gel electrophoresis
Gene comparison from database genomes
• Primers were taken from a previous study on BclA1 of 630 C. diff spores
• Found in reference genomes of other strains in gen bank.
• Not present in some NAP types, and many were not annotated as BclA
• PCR of BclA1 indicated NAP1 has the shortest gene out of all NAP types
• Recent clinical isolates were typed and most matched the trends seen in confirmed NAP types
• From most common strains seen, NAP1 translates 420bp BclA1, NAP4 has ~1767bp BclA1, and
NAP7 does not have the gene.
• Study confirmed short BclA1 genes only carried on NAP1 strains, after the PCR
of over 100 clinical isolates, from few years ago to the most recent
• Short BclA gene could be a fast way to type for NAP1 strains
• Spores with lower adhesive properties are more dangerous in terms of spread.
Even though more adhesive spores may be difficult to remove, they are usually
contained in an area and not transferred.
• C. difficile is thought to be incapable of aerosolization, but aerosolization from
toilets is a real concern #3
• Cleaning thoroughly is key! Spores can be washed off but germination is possible
if it comes in contact with patients once more
Next steps and other questions
The reason for adhesion may be the full hair, so the truncated protein
impaired the ability. However, the physical reasons for adhesion need to be proven.
Possible hair structures on speculated non hair NAP types but not formed from
BclA1, so not detected by gene analysis?
Find out if the hairs are actually the speculated length from the gene.
*3
• Another band seemed to appear when ran with a lower annealing temperature.
• Possible homology with other BclA
• Alignment show primers may have amplified another ubiquitous region under the less specific
conditions.
• Amino acid comparisons of the BclA exosporium glycoprotein indicate there is no primer
homology, only structural
• From the genome database, the region the primers amplified was aligned and there seems to
be two types of BclA1 found, long and short
• Stop codon found exclusively in the NAP1 strains, which results in the production of a
truncated protein.#4 This was due to a point mutation.
• Possibly in NAP1, N terminal domain is fully functional but central region and C terminal
domain have impaired functions, decreasing adhesion.
• The figure above show the entire
BclA1 gene aligned with MUSCLE
• All NAP1 have identical sequences
• More variation in the ones with
longer BclA1 gene
• Deletions probably happened over time
• Cladogram shows 630 may not be
most historic strain
• From BclA1, all NAP1 are tightly related
in the same clade
• NAP1 spores have short hairs, NAP4 has long hairs, and NAP7 have no hairs
• More Nap1 spores were present in vortexed tubes, which meant less adhered to
the tube during vortexing
• NAP7 seems to have the strongest adhesion, as the hairless exosporium could
be more pliable and have more contact with surfaces
• CTD and CLR could be a factor that enhances adhesion in the case of hairs
# Information
1. Paredes-Sabja. (2014). Clostridium difficile spore biology: sporulation, germination, and spore structural proteins. Trends in microbiology.
2. Pizarro-Guajardo, M. (2013). Characterization of the collagen-like exosporium protein, BclA1 of Clostridium difficile spores. Anaerobe, 18-30.
3. Roberts, K. (2008). Aerial dissemination of Clostridium difficile spores. BMC infectious diseases.
4. Phetcharaburanin, J. (2014). The spore-associated protein BclA1 affects the susceptibility of animals to colonization and infection by Clostridium
difficile. Molecular microbiology, 1025–1038.
Fluorescence from terbium & DPA
Low and high adhesion on microcentrifuge tubes
0
10
20
30
40
50
60
NAP1 NAP4 NAP7
Percent(%)
NAP type
Percentage spores adhered to vortexed tubes
1st run 2nd run 3rd run
*4
630NAP1
N-terminal domain Central Region C- terminal domain
Others
NAP 1
BclA1 gene comparison
*5
Hair Orientation on B. anthrasis spore
NAP1 NAP2 NAP3 NAP4 NAP5 NAP6 NAP7 NAP8 NAP9 NAP10 NAP11 NAP12 12.232 11.443 11.276
NAP1 NAP4 NAP7
Nap: 1 1 1 1 11 1 1
Past clinical isolates plated,
incubated for 48 hours.
After exposure to oxygen,
incubated 96 hours more
2 ml of water spread on plate
to scrape off cells.
Vegetative cells were lysed
by water to release spores
Spores settle down as
pellet, supernatant
removed.
Washed spores 3 times,
centrifuging in between
Stop codon location