Journal of Undergraduate Research
Volume 8, Issue 3 - January / February 2007

Identification of a Potential Type IV Secretion System of Porphyromonas gingivalis

Nicole Madison Lambert

ABSTRACT

Porphyromonas gingivalis, a Gram-negative oral anaerobe, is a major contributor to adult periodontal disease.  Several Gram-negative bacteria have been found to encode complex secretion systems that allow the export of molecules, such as virulence factors, from self to host cell cytosol.  This work was undertaken to investigate whether P. gingivalis uses a type IV secretion system (T4SS) to function as a pathogen.  P. gingivalis strains W83, ATCC-33277, and A7426 were utilized. Eight genes located in two different loci of the Pg W83 sequenced chromosome were found to have homologies to other T4SSs.  Pg1473-1476 is a predicted operon of PgW83 that is comprised of genes encoding conjugative transposon proteins, and Pg1758-1762 is a predicted operon of PgW83 that encodes genes functioning in ribosomal protein synthesis. The two regions analyzed appear to be co-transcribed in the PgW83 genome, and at least one predicted operon appears to be encoded and co-transcribed in the PgATCC-33277 genome.  It is uncertain whether these genes and operon regions could code for part of a type IV secretion system in P. gingivalis.

INTRODUCTION

Porphyromonas gingivalis is a Gram-negative oral anaerobe associated with the initiation and progression of severe forms of adult periodontal disease (7, 10).  This organism effectively invades, multiplies, and survives in primary gingival epithelial cells, which makes it an effective colonizer of oral tissues (5, 13). Colonization is facilitated by other microbial species that provide attachment sites, supply growth substrates, and reduce oxygen tension to levels optimal for growth. Adherence is facilitated by a variety of bacterial surface proteins, such as fimbriae, hemagglutinins, and proteinases (7).  P. gingivalis expresses a number of potential virulence factors involved in the pathogenesis of periodontitis (1).  The exact mechanism by which P. gingivalis transports these virulence factors to invade host cells has yet to be determined. 

Gram-negative bacteria have evolved complex transport mechanisms that mediate the passage of proteins and DNA across their cell envelopes and into the extracellular environment (6).  These complexes in Gram-negative bacteria require multicomponent structures that are organized into intricate machineries that often span both the inner and outer membranes (6).  An example of such an apparatus is the type IV secretion system (T4SS), which has been shown to export virulence factors across the inner and outer cell membrane.  The T4SS is a macromolecular transfer system with homology to the conjugal transfer systems of naturally occurring plasmids (3, 9).  The conjugation machinery is a multiprotein complex which facilitates the transfer of genetic material from one bacterial cell to another (6). It is thought that pathogens with T4SSs have acquired these transfer systems and use them to export toxins, effector proteins, or protein-DNA complexes to the eukaryotic cell cytosol during infection (2, 3).  A model or prototype for the T4SS has been described for Agrobacterium tumefaciens (12).  Many other bacterial species, such as Bordetella pertussis, Helicobacter pylori, Brucella suis, and Legionella pneumophila, have genes with homology to components of this T4SS (12).  Actinobacillus actinomycetemcomitans, an oral pathogen responsible for juvenile periodontal disease, harbors a T4SS (8). 

The complete 2,343,479-bp genomic sequence of P. gingivalis strain W83 has been determined (7). Our hypothesis is that P. gingivalis has a functional T4SS.  The P. gingivalis sequence database was searched through the Institute for Genomic Research Comprehensive Microbial Resource for genes presenting any homologies or similarities to any T4SS open reading frames (ORFs).  Eight genes located in two different regions of the chromosome with homologies to both known type IVA and IVB systems were identified.  Even though the W83 strain has been sequenced, we have chosen to study other strains such as ATCC-33277 and A7436 which have been shown to be more virulent and clinically relevant.  We proposed to determine by PCR if the T4SS gene homologues found in strain W83 are also present in ATCC-33277 and A7436.  To determine if the genes are expressed, mRNA from pure cultures of P. gingivalis strains W83, ATCC-33277, and A4736, and from the same cultures stimulated with supernatants of human coronary artery endothelial cells (HCAEC) culture medium, were extracted one hour and 24 hours post-infection.  RT-PCR will be performed to confirm the findings. 

L. pneumophila and P. gingivalis can survive and replicate inside host cells by preventing phagosome-lysosome fusion (4, 11).  It has been shown that L. pneumophila uses a T4SS encoded by the dot/icm genes to modify the autophagic pathway. Thus far, we have found some genes homologous to the dot/icm system of Legionella. Therefore, it would be possible that the T4SS of P. gingivalis is important for intracellular growth and alteration of the endocytic pathway.  The proposed project would help us to determine whether or not P. gingivalis has a T4SS and if these genes contribute to the virulence of this microorganism.

METHODS

PCR amplification of the T4SS homologous genes

Chromosomal DNA was extracted from P. gingivalis strains W83, ATCC-33277, and A7436 using the Wizard Genomic DNA Purification Kit (Promega) followed by a phenol-chloroform-isoamyl/ alcohol extraction to clean the extracted genomic DNA.  Six sets of upstream and downstream primers were designed for P.  gingivalis strain W83 (PgW83) chromosomal DNA between genes Pg1473 and Pg1486. Five sets of upstream and downstream primers were designed for PgW83 chromosomal DNA between genes Pg1758 and Pg1762.  All primers are listed in Table 1.  Each primer was used in a polymerase chain reaction (PCR) to amplify the corresponding gene segment in PgW83 as a positive control.  These primers were then used in separate PCRs to amplify the potential corresponding gene segments in P. gingivalis strains ATCC-33277 and A7436.  The PCR conditions used were 25 cycles of one min at 95°C, one min at the specified optimal annealing temperature, one min per 1000 bp at 72°C, and one cycle of 10 min at 72°C.  The samples were held after PCR completion at 4°C.  PCR reaction components used for a 100ml reaction were two units of New England Biolabs Inc. VentR DNA Polymerase, New England Biolabs Inc. 10x ThermoPol Reaction Buffer, 10mM each BioRad dNTP Mix, 10x dimethyl sulfoxide (DMSO), 100x BSA, up to 1mg DNA template, 0.5mM of downstream primer, and 0.5mM of upstream primer.  DMSO was added to facilitate separation of the DNA strands.  100x BSA was added to stabilize the DNA polymerase enzyme.

Co-transcription of the T4SS homologous genes

PgW83 was cultured on a BAP agar plate supplemented with gentamicin and left to grow for 24 hours in an anaerobic chamber.  The bacteria were then transferred into 30-40 mls of tryptic soy broth (TSB) supplemented with gentamicin into two 50 ml conical tubes with an initial OD600 of 0.1.  The culture was allowed to grow until a final OD600 of 1.0 was reached.  The OD600 was measured with a spectrophotometer.  This took approximately 10 to 12 hours.  The cultures were pelleted by centrifuging in 50 ml conical tubes at 7000 rpm for 20 minutes.  One pellet was re-suspended in 30 to 40 mls of filtered antibiotic free cell culture supernatant from HCAEC in a 1 to 1 media volume ratio, and the other pellet was re-suspended in TSB.  Both cultures were then left to grow in an anaerobic chamber for two time points of 1 hour and 24 hours.  At 1 hour and 24 hours RNAprotect Bacteria Reagent by Qiagen (cat. no. 76506) was used to stabilize the RNA.  The RNA was then isolated from the bacteria using Qiagen RNeasy Mini Kit (cat. no. 74104).  To clean up the RNA, RNase-Free DNase Set from Qiagen (cat. no. 79254) was used. In order to determine if clean RNA was obtained, a positive and negative control was run on a 1.5% agarose gel.  The negative control was a PCR product made by using a sample of RNA after the RNase kit was used to clean the RNA.  The positive control was a PCR product made by using a sample of chromosomal DNA.  Once a clean RNA sample was obtained, the RNA concentration was measured using a BioChrom GeneQuant Pro RNA/DNA Calculator and then reverse-transcription PCR (RT-PCR) was performed to obtain cDNA.  The RT-PCR profile used was obtained from Invitrogen’s SuperScript™ III Reverse Transcriptase (cat no. 18080-093).  Once cDNA had been obtained, a new PCR was run with the cDNA produced from the RT-PCR and the specific designed primers.  All primers used are listed in Table 1. 

Table Table 1
Details of designed primers between Porphyromonas gingivalis W83 gene regions Pg1467-1486 and Pg1758-1762.
Primer set Product length (bp) Product corresponds to PgW83 gene(s): Optimal annealing temperature (degrees C) Tested for
conservation in PgATCC-33277 and PgA7426 		Tested for
co-transcription in PgATCC-33277
No. 1 1858 Pg1473-1476 55.9 x  
No. 2 1495 Pg1476-1479 59.8 x  
No. 3 988 Pg1479-1481 56.1 x  
No. 4 896 Pg1481-1483 57.5 x  
No. 5 698 Pg1483-1484 59.9 x  
No. 6 828 Pg1484-1486 57.0 x  
No. 7 599 Pg1758-1759 54.4 x x
No. 8 1234 Pg1759-1761 59.1 x x
No. 9 885 Pg1761-1762 53.8 x x
No. A 338 Pg1473-1474 57.7 x x
No. B 314 Pg1474-1475 55.8 x x
No. C 336 Pg1475-1476 57.2 x x
No. D 300 Pg1476-1477 55.4   x
No. E 304 Pg1477-1478 57.9   x
No. F 291 Pg1478-1479 55.2   x
No. G 331 Pg1479-1480 54.9   x
No. H 320 Pg1480-1481 57.0   x
No. I 332 Pg1481-1482 57.7   x
No. J 345 Pg1482-1483 58.7   x
No. K 477 Pg1483-1484 58.0   x
No. L 387 Pg1484-1485 54.6   x
No. M 236 Pg1485-1486 56.2   x
No. N 433 Pg1758-1759 57.5   x
No. O 368 Pg1759-1760 57.1   x
No. P 337 Pg1760-1761 57.6   x
No. 59 1056 Pg1759 57.2 x  
No. 61 798 Pg1761 58.6 x  
No. 67 433 Pg1467 55.0 x  
No. 69 403 Pg1469 50.1 x  
No. 71 320 Pg1471 56.4 x  
No. 72 464 Pg1472 56.8 x  

 

RESULTS AND CONCLUSIONS

Gene conservation analysis

All primers designed for PgW83 chromosomal DNA were functional.  From PCR and gel electrophoresis using chromosomal DNA, PgATCC-33277 may contain genes similar but not identical to Pg1479, Pg1480, Pg1481, Pg1483, Pg1484, Pg1485, and Pg1486 of PgW83.  These results were confirmed by repeating PCR using new extracted chromosomal DNA from PgATCC-33277.  DNA sequences of the PCR products were inconclusive.  From PCR and DNA sequencing, PgATCC-33277 contains identical genes to Pg1758-Pg1762 of PgW83 (Figure 1).  These results were also confirmed by repeating PCR using new chromosomal DNA.  In addition, from PCR and gel electrophoreses using chromosomal DNA, PgA7436 contains identical genes to Pg1474-Pg1476 and Pg1758-Pg1762.  DNA sequencing was not performed from these PCR products.

Figure 1. PCR amplification displaying co-transcription results of region Pg1758-1761 in PgW83 and PgATCC-33277 using chromosomal DNA exposed to spent culture media.

Figure 1. PCR amplification displaying co-transcription results of region Pg1758-1761 in PgW83 and PgATCC-33277 using chromosomal DNA exposed to spent culture media. Wells 1 and 8 contain a 0.1-12 kbp Perfect DNA™ Marker from Novagen. Wells 2 and 3 contain amplified gene product from gene region Pg1758-1759 using primer set N and chromosomal DNA from PgATCC-33277 and PgW83, respectively. Wells 4 and 5 contain amplified gene product from gene region Pg1759-1760 using primer set O and chromosomal DNA from PgATCC-33277 and PgW83, respectively. Wells 6 and 7 contain amplified gene product from gene region Pg1760-1761 using primer set P and chromosomal DNA from PgATCC-33277 and PgW83, respectively.

Co-transcription analysis of genes Pg1473-Pg1486

PgW83 genes Pg1473-Pg1482 and Pg1483-1486 appear to be co-transcribed because the correct size products were produced from PCRs using cDNA of PgW83 and the designed primers listed in Table 1.  Some PCRs were repeated using a different sample of cDNA and these results were confirmed.  A DNA fragment showing co-transcription between Pg1482 and Pg1483 could not be amplified by PCR.  DNA sequencing was only successfully performed on the PCR product of DNA of the co-transcribed genes of Pg1481-1482 (Figure 2).

Figure 2. PCR amplification displaying co-transcription results of region Pg1760-1762 and Pg1481-1482 in PgW83 and PgATCC-33277

Figure 2. PCR amplification displaying co-transcription results of region Pg1760-1762 and Pg1481-1482 in PgW83 and PgATCC-33277. Well 1 contains a 0.1-12 kbp Perfect DNA™ Marker from Novagen. Wells 2 and 3 contain amplified gene product from gene region Pg1761-1762 using primer set 9 and chromosomal DNA from cultures exposed to spent culture media of PgATCC-33277 and PgW83, respectively. Well 4 contains amplified gene product from gene region Pg1761-1762 using primer set 9, and chromosomal DNA from PgW83 not exposed to spent culture media was utilized. Wells 5 and 6 contain amplified gene product from gene region Pg1481-1482 using primer set I and chromosomal DNA from cultures of PgW83 and PgATCC-33277, respectively, which were exposed to spent culture media.  Well 7 contains amplified gene product from gene region Pg1758-1759 using primer set I and chromosomal DNA from PgW83 not exposed to spent culture media. Well 8 contains amplified gene product from gene region Pg1760-1761 using chromosomal DNA from PgW83 exposed to spent culture media and primer set P.

Pg genes Pg1473-Pg1476 appear to be co-transcribed because the correct size products were produced from the PCR using cDNA of stimulated PgW83 and the designed primers A, B, and C (Figure 3).  In addition, only cultures stimulated with cell-culture media from HCAEC were used to produce the RNA used for these samples pertaining to the gene region of Pg1473-Pg1476.  PCRs were repeated using different samples of cDNA of stimulated PgW83 and primers A and C.  PgW83 genes Pg1476-Pg1477 and Pg1478-Pg1479 appear to be co-transcribed because the correct size products were produced from the PCR using cDNA of PgW83 and the designed primers D and F only if PgW83 was stimulated by HCAEC cell-culture media (Figure 4).  PgW83 genes Pg1477-1478 appear to be co-transcribed because the correct size products were produced from the PCR using cDNA of PgW83 and the designed primer E when PgW83 was stimulated by HCAEC cell-culture media and when PgW83 was not stimulated with the cell-culture media (Figure 5).  A product was formed from PCR using cDNA of stimulated PgATCC-33277 and the designed primers G, H, and I.  This corresponds to PgW83 genes Pg1479-Pg1482 (Figures 5 and 6).  This region also appears to be co-transcribed in the PgW83 genome when either stimulated or non-stimulated PgW83 is used. DNA sequencing was only successfully performed on the PCR product of DNA of the co-transcribed genes of Pg1481-1482.  PgW83 genes Pg1482-1483 were not tested for co-transcription. PgW83 genes Pg1483-1486 appear to be co-transcribed because the correct size products were produced from the PCR using cDNA of stimulated PgW83 and the designed primer K, L, and M (Figures 4, 5, and 6).  Pg1484-1485 appears to be co-transcribed even when PgW83 is not stimulated with cell-culture media.  Pg1483-1484 and Pg1484-1485 were not tested for co-transcription using non-stimulated PgW83.

Figure 3. PCR amplification displaying co-transcription results of region Pg1473-1476 in PgW83.

Figure 3. PCR amplification displaying co-transcription results of region Pg1473-1476 in PgW83.  Well 1 contains a 0.1-12kbp Perfect DNA™ Marker from Novagen. Wells 2 and 3 contain amplified gene product from gene region Pg1473-1474 using primer set A and chromosomal DNA from PgW83 exposed to spent culture media after 1 hour and 24 hours, respectively. Wells 4 and 5 contain amplified gene product from gene region Pg1474-1475 using primer set B and chromosomal DNA from PgW83 exposed to spent culture media after 1 hour and 24 hours, respectively. Wells 6 and 7 contain amplified gene product from gene region Pg1475-1476 using primer set C and chromosomal DNA from PgW83 exposed to spent culture media after 1 hour and 24 hours, respectively.

Figure 4. PCR amplification displaying co-transcription results of region Pg1484-1486 in PgW83, Pg1478-1479 in PgW83, Pg1475-1476 in PgW83, Pg1758-1759 in PgW83, and Pg1758-1759 in PgATCC-33277. 

Figure 4. PCR amplification displaying co-transcription results of region Pg1484-1486 in PgW83, Pg1478-1479 in PgW83, Pg1475-1476 in PgW83, Pg1758-1759 in PgW83, and Pg1758-1759 in PgATCC-33277.  Well 1 contains a 100bp Ready-Load DNA ladder from Invitrogen. Well 2 contains amplified gene product from gene region Pg1484-1485 using chromosomal DNA from PgW83 and primer set L. Well 3 contains no amplified gene product from gene region Pg1485-1486 using chromosomal DNA from PgW83 and primer set M.  Well 4 contains amplified gene product from gene region Pg1758-1759 using chromosomal DNA from PgW83 and primer set N.  Well 5 contains amplified gene product from gene region Pg1475-1476 using chromosomal DNA from PgW83 and primer set C.  Well 6 contains amplified gene product from gene region Pg1758-1759 using chromosomal DNA from PgATCC-33277 and primer set N. Well 7 contains amplified gene product from gene region Pg1473-1474 using chromosomal DNA from PgW83 and primer set A. Well 8 contains amplified gene product from gene region Pg1478-1479 using chromosomal DNA from PgW83 and primer set F. Chromosomal DNA from PgW83 and PgATCC-33277 used was exposed to spent culture media. 

Figure 5. PCR amplification displaying co-transcription results of region Pg1477-1478, Pg1480-1482, Pg1483-1484, Pg1485-86, and Pg1758-1759.in PgW83. 

Figure 5. PCR amplification displaying co-transcription results of region Pg1477-1478, Pg1480-1482, Pg1483-1484, Pg1485-86, and Pg1758-1759.in PgW83.  Well 1 contains a 100bp Ready-Load DNA ladder from Invitrogen. Well 2 contains amplified gene product from gene region Pg1483-1484 using chromosomal DNA from PgW83 and primer set K. Well 3 contains amplified gene product from gene region Pg1758-1759 using chromosomal DNA from PgW83 and primer set 7.  Well 4 contains amplified gene product from gene region Pg1481-82 using chromosomal DNA from PgW83 and primer set I.  Well 5 contains amplified gene product from gene region Pg1477-1478 using chromosomal DNA from PgW83 and primer set E.  Well 6 contains amplified gene product from gene region Pg1480-1481 using chromosomal DNA from PgATCC-33277 and primer set H. Well 7 contains amplified gene product from gene region Pg1485-1486 using chromosomal DNA from PgW83 and primer set M. Chromosomal DNA from PgW83 used was exposed to spent culture media. 

Figure 6. PCR amplification displaying co-transcription results of region Pg1480-1481, Pg1484-1486, Pg1758-1761 using chromosomal DNA from PgW83 not exposed to spent culture media. 

Figure 6. PCR amplification displaying co-transcription results of region Pg1480-1481, Pg1484-1486, Pg1758-1761 using chromosomal DNA from PgW83 not exposed to spent culture media.  Wells 1 and 8 contain a 0.1-12 kbp Perfect DNA™ Marker from Novagen. Well 2 contains amplified gene product from gene region Pg1480-1481 using chromosomal DNA from PgW83 and primer set H. Wells 3 and 4 contain amplified gene product from gene region Pg1484-1485 and Pg1485-1486 using chromosomal DNA from PgW83 and primer set L and M, respectively. Wells 5, 6, and 7 contain amplified gene product from gene region Pg1758-1759, Pg1759-1760, and Pg1760-1761 using chromosomal DNA from PgW83 and primer set N, O, and P, respectively.

Co-transcription analysis of genes Pg1758-1762

PgW83 genes Pg1758-1761 appear to be co-transcribed because the correct size gene products were produced from a PCR using cDNA of PgW83 and the designed primers N, O, and P when PgW83 was stimulated by HCAEC cell-culture media and when PgW83 was not stimulated with the cell-culture media (Figures 6 and 7).  In addition, PCR products were formed using cDNA of PgATCC-33277 and the designed primers N, O, and P when PgW83 was stimulated by HCAEC cell-culture media, which suggests that this gene region is also co-transcribed in PgATCC-33277 (Figures 1 and 8).  Co-transcription of this gene region was not tested for in PgA7436, but from PCR and gel electrophoresis using chromosomal DNA, PgA7436 does contain this genetic region in its genome.  DNA sequencing was not performed to confirm the results, and a sample of cDNA of PgATCC-33277 not stimulated by HCAEC cell-culture media was not obtained and therefore not used in PCR amplification.

Figure 7. PCR amplification displaying co-transcription results of region Pg1480-1481, Pg1758-1761 using chromosomal DNA from PgW83 exposed to spent culture media.

Figure 7. PCR amplification displaying co-transcription results of region Pg1480-1481, Pg1758-1761 using chromosomal DNA from PgW83 exposed to spent culture media.  Wells 1 and 8 contain a 0.1-12 kbp Perfect DNA™ Marker from Novagen. Well 2 contains amplified gene product from gene region Pg1480-1481 using chromosomal DNA from PgW83 and primer set H. Wells 5 and 6 contain amplified gene product from gene region Pg1758-1759 and Pg1759-1769 using chromosomal DNA from PgW83 and primer set N and O, respectively.  Well 7 contains amplified gene product from gene region Pg1760-1761 using chromosomal DNA from PgW83 and primer set P.

Figure 8.  PCR amplification displaying co-transcription results of region Pg1480-1481, Pg1484-1486, Pg1758-1761 using chromosomal DNA from PgATCC-33277 exposed to spent culture media.

Figure 8.  PCR amplification displaying co-transcription results of region Pg1480-1481, Pg1484-1486, Pg1758-1761 using chromosomal DNA from PgATCC-33277 exposed to spent culture media.  Wells 1 and 8 contain a 0.1-12 kbp Perfect DNA™ Marker from Novagen. Well 2 contains amplified gene product from gene region Pg1480-1481 using chromosomal DNA from PgATCC-33277 and primer set H. Wells 3 and 4 contain amplified gene product from gene region Pg1484-1485 and Pg1485-1486 using chromosomal DNA from PgATCC-33277 and primer set L and M respectively.  Wells 5, 6, and 7 contain amplified gene product from gene region Pg1758-1759, Pg1759-1760, and Pg1760-1761 using chromosomal DNA from PgATCC-33277 and primer set N, O, and P, respectively.

DISCUSSION

P. gingivalis strain W83 appears to have a functional gene operon consisting of genes Pg1758-1762, which is supported by the positive test for co-transcription in this region.  Therefore, genes Pg1758-1762 appear to be co-transcribed in the P. gingivalis strain W83 genome.  These genes are thought to be involved with ribosomal protein synthesis according to the Institute for Genomic Research Comprehensive Microbial Resource.  P. gingivalis strain ATCC-33277 has identical genes to strain W83 genes Pg1758-1762, which is supported by the confirmed sequencing results from the PgATCC-33277 genome.  This genomic region in PgATCC-33277 also appears to be co-transcribed.  In addition, P. gingivalis strain A7436 seems to have this gene region, although co-transcription testing was not performed using PgA7436 chromosomal DNA.  This suggests that genes Pg1758-1762 are conserved throughout the P. gingivalis genome.  The creation of knock-out mutants in this region could help determine if these genes are involved in a Type IV secretion system.

Between genes Pg1473 and Pg1486, only two genes were confirmed to be co-transcribed, Pg1481-1482.  Due to the fact that not all the genes between Pg1473 and Pg1486 were found to be co-transcribed in PgW83, it is uncertain whether genes Pg1473-1486 comprise a gene operon.  P. gingivalis strain A7436 appears to have a similar gene region to Pg1474-1476, but due to a lack of gene sequencing these results could not be confirmed. The whole potential operon of Pg1473-1486 may only be functional if P. gingivalis is stimulated by host cells as evidenced by the results that some genes were only co-transcribed if P. gingivalis was stimulated by cell-culture media. The creation of knock-out mutants in this region could help determine if these genes are involved in a T4SS. However, the fact that transcription of these genes is turned on upon exposure of P. gingivalis to spent cell culture media suggests they are important to P. gingivalis with regard to its interactions with host cells and thus are likely related to virulence of P. gingivalis.

ACKNOWLEDGMENTS

This work was supported by the University of Florida University Scholar’s Program, the University of Florida College of Dentistry Office of Research, and the University of Florida College of Dentistry Department of Oral Biology laboratory of Dr. Ann Progulske-Fox, including Myriam Belanger, Paulo Rodrigues, Lihui Yuan, Sheila Walters, Joan Whitlock, and Eliana Vergas.

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