CRISPR-associated element analysis in Lauchelly and Attacne, novel Propionibacterium acnes Bacteriophages

Propionibacterium acnes bacteriophages display low spacer-protospacer matches and conserved, unique CRISPR-associated domains as an anti-CRISPR mechanism.

RESULTS

Evolutionary Relatedness of CRISPR-associated Proteins

Figure 7. These transmission electron micrographs were taken at CNSI at UCLA using a Philips CM120 electron microscope. Phage Lauchelly and phage Attacne exhibit a Siphoviridae morphology of an icosahedral head with a long, noncon-tractile tail.

Transmission Electron Micrograph

Figure 8. Host range analysis was conducted to determine which individual student strains Phage Lauchelly and Phage Attacne can infect. Both phages were able to infect 12 out of 16 students P. acnes clinical strains. Phage Lauchelly (B) formed clear, large, and round plaques on the A-media plates. Phage Attacne (A) formed clear, large, round plaques with colony growth in the center, potentially due to pseudolysogenic traits.

A

B

C

D

•Propionibacterium acnes (P. acnes) is associated with acne pathogenesis. It is a slow-growing, facultative anaerobe, gram-positive bacillus that can live in a multitude of environments, including the microcomedones of human skin (Farrar et al., 2007)

•Of the currently P. acnes phages sequenced thus far, all have shown lim-ited genetic diversity and broad killing capacity (Marinelli et al., 2012)

•Clustered regularly interspaced short palindromic repeats (CRISPR) have been shown to confer resistance to phage lysis in P. acnes by obtaining phage-derived sequences, known as spacers. Modifications of the spacers alter the capability of the bacteria to be resistant to phage lysis (Barrangou et al., 2007)

•

•CRISPR-associated domains conserved in Propionibacterium phages: The NCBI BlastP results show that the CRISPR-associated domains, Cas9/Csn1 and Cas4, are conserved within Propionibacterium phage endonuclease and exonuclease, respec-ctively, including Lauchelly and Attacne. •Cas4 and Cas9 CRISPR-associated genes unique to phages: The P. acnes hits from NCBI BlastP have lower query and identity values, which suggest that although the phage genes are likely distantly related to the bacterial genes, the phage genes have evolved to become unique. •Presence of phage defense mechanism: The CRISPR-associated endonuclease may have evolved over time through divergent evolution to become unique. However, the CRISPR-associated exonuclease phylogenetic trees indicate that these genes may have evolved recently via horizontal gene transfer due to the close clustering of both phage and bacterial proteins. Despite different evolu-tionary mechanisms, these genes could now be conserved to protect the phages from lysis by CRISPRs by mimicking the host P. acnes bacteria.•Widespread killing capacity with potential P. acnes strain resistance to Attacne: The high number of CRISPR spacer-protospacer mismatches suggest that the bacteria are not resistant to phage lysis via CRISPR-associated mechanisms and are thus susceptible to lysis by both Lauchelly and Attacne. This is concurrent with recent findings of broad killing capacity as both phages have the ability to lyse 12/16 clinical isolates of host bacteria in the Host Range Assay. One bacterial strain, HL110PA3, is predicted to be resistant to lysis by Attacne

We would like to thank Dr. Jordan Moberg Parker and Dr. Laura Marinelli for their academic guidance and support throughout this research project. This project was funded by the Howard Hughes Medical Institution Education Grant awarded to UCLA (No. 52006944).

17.07%17.07%

Annotated Genome Map Gene Composition

•Comparative Genomic Analysis with other Phages: Determine evolutionary relationships between other novel phage strains.•Further CRISPR analysis: Manipulate Cas genes to determine relationship between CRISPR-associated genes in phages to phage CRISPR resistance. •Clinical Trials: Determine if phage resistance predicted correlates to experimental results

1. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P. CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes. 2007. Science. 315(5819): 1709-1712 2. Farrar MD, Howson KM, Bojar RA, West D, Towler JC, Parry J, Pelton K, Holland KT Genome Sequence and Analysis of a Propionibacterium acnes Bacteriophage. 2007. Bacteriology. 189(11): 4161-67.3. Marinelli LJ, Fitz-Gibbon S, Hayes C, Bowman C, Inkeles M, Loncaric A, Russell DA, Jacobs-Sera D, Cokus S, Pellegrini M, Kim J, Miller JF, Hatfull GF, Modlin RL. Propionibacterium acnes Bacteriophages Display Limited Genetic Diversity and Broad Killing Activity against Bacterial Skin Isolates. 2012. mBio. 3(5): 00279-12.

ABSTRACT

BACKGROUND

METHODS

FUTURE DIRECTIONS

REFERENCES

ACKNOWLEDGEMENTS

HYPOTHESIS

CONCLUSIONS

Figure 1. The annotated genome map was generated through DNAMaster. Phage Lauchelly is 29, 515 bp long, with 41 ORFs (Open Reading Frames) and a G+C content of 53.86%. Phage Attacne is 28, 876 bp long, with 42 ORFs and a G+C content of 54.7%. Gene functions were assigned by running the protein sequences of the corresponding ORFs on BLASTp, CDD, and HHpred to compare Lauchelly and Attacne ORF products to previously annotated protein sequences with high sequence identity, low E-values, and high query coverage. Genes were color-coded by grouping genes into categories of similar functions (as seen in the legend in Figure 2).

Host Range Analysis

C

Figure 2. Based on the genome map, the gene composition charts of phage Lauchelly and phage Attacne were constructed by dividing the number of genes from each grouping over the total number of genes in Lauchelly and Attacne respectively. The majority of the genes in Lauchelly and Attacne have hypothetical functions but are exclusively conserved in P. acnes bacterium. To better characterize the genes, future studies should be performed to determine the function of these genes.

DNA Replication Protein

Capsid Proteins

Tail Proteins

Conserved Hypothetical Proteins

Exclusive P. acnes Phage Hypothetical Proteins

Other

Revise “Notes” section of DNA

Master to adhere to GenBank guidelines

GeneBank Submission through DNA Master

NCBI Blast P Conserved Domain Database

Genome Maps(DNA Master)

Make functional gene calls

CRISPR ProtospacerAnalysis

Phylogenetic Trees(phylogeny.fr)

ConservedDomain Database

Lauren Echon1, Rachelle Ann Gonzales1, and Jordan Moberg Parker1

1 Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095

Figure 3. T-COFFEE from Phylogeny.fr. was used to construct phylogenetic trees in order to view the evolutionary relatedness of the phage and bacterial CRISPR-associated proteins (seen in Figure 5). These trees consist of the protein sequences from Lauchelly, Attacne, several other Propionibacterium phage, and an outlier. Bootstrap values (0-1) measure the accuracy of the branching (value of 1 indicates that branching did not occur by chance, while 0 indicates branching occurred entirely by chance). In the endonuclease tree, Lauchelly and Attacne cluster with the other Propionibacterium phages, as expected within divergent evolution. In the exonuclease tree, some Propionibacterium phage proteins, including that from Lauchelly, cluster more closely to P. acnes proteins. This could indicate that horizontal gene transfer has recently occurred from the bacteria to the phages. Although different evolutionary mechanisms could explain the presence of these proteins, these proteins may be conserved within Propionibacterium phages as an anti-CRISPR defense mechanism.

HHpred

CRISPR-associated exonuclease and

endonuclease analysis

NCBI Blast

Lauchelly Attacne

NCBI BlastP: Cas Genes Unique to Phage

Conserved CRISPR-associated Domains in Endonuclease and Exonuclease

Figure 5. To determine if the proteins containing CRISPR-associated domains are conserved across Propionibacterium phages, the endonuclease and exonuclease sequences from Lauchelly, Attacne, and several other Propionibacterium phages were analyzed using the Conserved Domain Database (CDD). The CDD results indicated that there are conserved CRISPR-associated domains within Propionibacterium phages: Cas4 in exonuclease and Cas9/Csn1 in endonuclease.

Figure 4. The Cas4 (gene 35 in both phages) and Cas9/Csn1 (gene 44 in Lauchelly and gene 43 in Attacne) sequences were put into NCBI BlastP and blasted against the Propionibacterium database in order to determine if these genes are unique to Propionibacterium phages or if they are also seen in its host P. acnes. Results for only Lauchelly are shown due to the similar patterns witnessed in Attacne. Since there are P. acnes hits with lower query and identity values than those from Propionibacterium phages, the phage genes appear to be distantly related to P. acnes bacteria, but have evolved to be unique in Propionibacterium phages. It is possible that these anti-viral genes could have been incorporated into the phages for anti-CRISPR purposes.

Endonuclease

Exonuclease

CRISPR Spacer/Protospacer Mismatches indicate P. acnes Strain Resistance or Susceptibility

putative_endonuclease_Propionibacterium_phage_Lauchellyputative_endonuclease_Propionibacterium_phage_PHL037M02Gp48_Propionibacterium_phage_PAD20gp48_Propionibacterium_phage_PA6putative_endonuclease_Propionibacterium_phage_AttacneMULTISPECIES_hypothetical_protein_PropionibacteriumHNH_endonuclease_Propionibacterium_acnesHNH_endonuclease_Mycobacterium_phage_Validus

0

0

0.420

0.78

putative_exonuclease_Propionibacterium_phage_Attacnehypothetical_protein_P141_36_Propionibacterium_phage_P14.4putative_exonuclease_Propionibacterium_phage_Lauchellyhypothetical_protein_partial_Propionibacterium_acnesgp37_Propionibacterium_phage_PA6hypothetical_protein_Propionibacterium_acnesputative_exonuclease_Propionibacterium_phage_PHL037M02RecB-like_protein_Mycobacterium_phage_PattyP

0

0.79

0.770.79

0.16

Endonuclease Exonuclease

12

34

56

78

910

1112

1314

1516

1718

1920

21

2223

2425

2627

2829

3031

3233

3435

3637

3839

4041

42

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

12

34

56

78

910

1112

1314

1516

1718

1920

21

2223

2425

2627

2829

3031

3233

3435

3637

3839

40

41

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

Terminase subunits

PortalProtein

Major tail protein

Tapemeasure protein

Collagen-like protein

Endolysin Sigma factor

CRISPR-associatedExonuclease

DnaB-likehelicase

DNA primase

CRISPR-associated EndonucleaseMinor tail

protein

Lauchelly

Attacne

Hollidayjunctionresolvase

AttacneLauchelly

38.09%

14.29%

16.67%14.29%

11.90%

4.76%39.02%

12.20%

17.07%17.07%

9.76%

4.88%

Lauchelly

Attacne

Predicted resistance to phage lysis One pair contains 0 mismatches Figure 6. Previously sequenced P. acnes CRISPR spacers from several bacterial strains were obtained and aligned with Lauchelly and Attacne separately in BlastN to find paired CRISPR protospacers in phage. Pairs containing 2 mismatches or less have the potential to confer resistance to phage lysis in P. acnes, while those greater than 5 mismatches likely do not. In Lauchelly, none of the P. acnes strains have a majority of pairs with less than two mismatches. Thus, it is predicted that there will be no resistance to phage via CRISPR mechanisms and that all represented bacterial strains will be susceptible to phage lysis by Lauchelly. In Attacne, one of the P. acnes strains has a majority of pairs with less than two mismatches as shown in dark blue. As a result, it is predicted that HL110PA3 will be resistant to lysis by Attacne, whereas all other strains will be susceptible to phage lysis.

In order to expand the currently existing knowledge of Propionibacterium acnes phages in the growing field of phage biology, this study was aimed to further characterize the many hypothetical genes conserved in Propionibacterium phages through the comparative genomic analysis of two novel Propionibacte-rium phages, Lauchelly and Attacne. Upon further analysis of these genomes, it was discovered that both the endonuclease and exonuclease within both phages contained CRISPR-associated domains, Cas9/Csn1 and Cas4, respec-tively. Upon analysis within the Conserved Domain Database, it was deter-mined that these domains are conserved within the currently sequenced Propi-onibacterium phages. The CRISPR-associated protein sequences appear to be unique to Propionibacterium phages, with higher query cover and identity to Propionibacterium phages over P. acnes proteins. Phylogenetic trees were gen-erated for both CRISPR-associated proteins utilizing Lauchelly, Attacne, other Propionibacterium phages, and an outlier. In the endonuclease tree, Propioni-bacterium phages clustered together, whereas within the exonuclease tree, some Propionibacterium phages were more closely related to P. acnes proteins than to other Propionibacterium phage strains. Bacterial CRISPR spacer se-quences were obtained and aligned with both Lauchelly and Attacne to find spacer-protospacer pairs. Only one strain has a majority of pairs less than two mismatches within phage Attacne. These results contribute to the growing field of phage biology and could possibly contribute to a phage defense mechanism against prokaryotic anti-viral CRISPRs.