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Kim, Kwak, Lee, Jung, Han, Kim, Kook, Oh, and Park: Expression of ssrA in non-pathogen-induced adaptation in the oral cavity through signal exchange with oral pathogens
Original Article
Journal of the Korean Association of Oral and Maxillofacial Surgeons

Journal of the Korean Association of Oral and Maxillofacial Surgeons 2012; 38(1): 14-19.

Published online: 29 February 2012

DOI: https://doi.org/10.5125/jkaoms.2012.38.1.14

Expression of ssrA in non-pathogen-induced adaptation in the oral cavity through signal exchange with oral pathogens

Sung-Ryoul Kim, Jae-Woo Kwak, Sung-Ka Lee, Seung-Gon Jung, Man-Seung Han, Bang-Sin Kim, Min-Suk Kook, Hee-Kyun Oh, Hong-Ju Park

Department of Oral and Maxillofacial Surgery, School of Dentistry, Dental Science Research Institute, 2nd Stage of Brain Korea 21, Chonnam National University, Gwangju, Korea.

Corresponding author: Hong-Ju Park. Department of Oral and Maxillofacial Surgery, School of Dentistry, Dental Science Research Institute, Chonnam National University, 33, Yongbong-ro, Buk-gu, Gwangju 500-757, Korea. TEL: +82-62-220-5436, FAX: +82-62-220-5437, omspark@chonnam.ac.kr

Received 25 August 2011       Revised 15 November 2011       Accepted 20 January 2012

Copyright © 2012 by The Korean Association of Oral and Maxillofacial Surgeons

(open-access):

This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Introduction

This study was conducted to evaluate ssrA expression resulting from adaptation of Escherichia coli (E. coli) to oral pathogens through signal exchange.

Materials and Methods

Human cell lines Hep2 and HT29, wild-type E. coli (WT K-12), ssrA knock-out E. coli (Δ K-12), and Scleropages aureus (S. aureus) were used. A single culture consisting of Hep2, HT29, WT K-12, and Δ K-12, and mixed cultures consisting of Hep2 and WT K-12, Hep2 and Δ K-12, WT K-12 and S. aureus , Δ K-12 and S. aureus , and Hep2, WT K-12, and S. aureus were prepared. For HT29, a mixed culture was prepared with WT K-12 and with WT K-12 and S. aureus. Total RNA was extracted from each culture with the resulting expression of ssrA, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and p53 was evaluated by Reverse transcription polymerase chain reaction (RT-PCR).

Results

The expression of ssrA in a single culture of WT K-12 was lower than that observed in the mixed culture of WT K-12 with S. aureus. Greater ssrA expression was observed in the mixed culture of WT K-12 with Hep2 than in the single culture of WT K-12. The expression of NF-κB was higher in the mixed culture of Hep2 with Δ K-12 than that in the mixed culture of Hep2 with WT K-12, and was lowest in the single culture of Hep2. The expression of ssrA was higher in the mixed culture of WT K-12 with Hep2 and S. aureus than in the mixed culture of WT K-12 with Hep2.

Conclusion

These results suggest that ssrA plays an important role in the mechanism of E. coli adaptation to a new environment.

Keywords: ssrA, Escherichia coli, Adaptation

I. Introduction

Infection occurs when the balance of three elements- host, environment, and microbefails to maintain homeostasis1. Among them, a microbial element means dental infection by normal flora in the mucous membrane of the oral cavity or by bacteria carried from another place or surgical wound infection.
The neonatal oral cavity is known to be in aseptic condition at birth, but oral flora is formed as it begins to come into contact with the outside2. Likewise, over 300 kinds of microorganism are reportedly found in the various oral cavity environments of normal adults3-5. Most of infection occurs due to bacteria residing in a specific region, but is often caused by the microbes residing in another place such as Escherichia coli (E. coli) or Clebsiella pneumonia6. Normal flora in the oral cavity are not the cause of infection, the bacteria are carried to the oral cavity from another place through contact of a wound or the operator's hands7.
An ssrA gene of E. coli, a small and stable mRNA-molecule, has a various functions, such as destruction of abnormal protein, growth assistance for phage, and activation control of a protein combining with DNA8-12. An ssrA gene has information for tmRNA-which has two functions, tRNA and mRNA- and rescues the ribosome that was left alone unproductively13,14. As part of the transtranslation process added to the Cterminal of a protein with peptide tag secreted, it triggers proteolysis15,16. The existence of ssrA in the prokaryotic genome means the biological existence of a transtranslation system13.
An ssrA is known as one of the genes necessary to adjust to stress in certain species. For example, E. coli ssrA mutant has variations in some control functions, and its growth speed declines in a temperature of 45℃. There are many variations in the phenotypes to phage as well13,14,17,18.
ssrA also seems to work as the various controlling elements in building proteins related to some toxic elements in pathogenic organisms. For example, a lack of ssrA function is known to cause a mutant to lose the capability to survive as a macrophage, with considerable influence on the toxicity of Salmonella enteric or Yersinia pseudotuberculosis 19,20.
Nonetheless, the expression of ssrA is known to be necessary for Bacillus subtilis to grow effectively under strong stress18. Therefore, this gene is the one that plays a role in adjusting to stress. We need to know the role of ssrA in the adaptation of nonpathogen to regions except the normal residence area.
This study was conducted to determine ssrA's role in the interaction between E. coli and Scleropages aureus (S. aureus) as host cell and pathogen, respectively, in the nonresidence regions.

II. Materials and Methods

1. Cell culture

The Hep2 cell line and HT29 cell line obtained from Korean Cell Line Bank were cultured under conditions of 37℃, 5% CO2 using Dulbeco's modified Eagle's medium (DMEM) with 10% fetal bovine serum added. Wildtype E. coli (WT K-12) and ssrA knockout E. coli (Δ K-12) were obtained from Hirosaki University in Himeno, Japan.

2. RNA isolation

To obtain total RNA for reverse transcription polymerase chain reaction (RT-PCR) and microarray, WT K-12, Δ K-12, and S. aureus were inoculated with a toothpick from a stock culture (onenight culture of colony in LB agar plate) and cultured respectively for 18 hours in a 15 mL falcon tube including 3 mL LB broth. A single culture of WT K-12 and Δ K-12 and a pre-culture of S. aureus were mixed at 30 µL each, and such mixed culture was done for 4 hours. To extract a human RNA, 3×106 Hep2 cells were cultured for 18 hours in a 60 mm culture plate using DMEM with 10% FBS added.
As a control group, Hep2 and WT K-12 were used. On the other hand, the experiment groups included a mixed culture of S. aureus and WT K-12 (inoculation of S. aureus and WT K-12 with concentration of 30 µL/3 mL), a mixed culture of S. aureus and Δ K-12 (inoculation of S. aureus and Δ K-12 with concentration of 30 µL/3 mL), a mixed culture of Hep2 and WT K-12 (inoculation of WT K-12 into the Hep2 culture with concentration of 30 µL/3 mL), a mixed culture of Hep2 and Δ K-12 (inoculation of Δ K-12 into the Hep2 culture with concentration of 30 µL/3 mL), and a mixed culture of Hep2, S. aureus, and WT K-12 (inoculation of S. aureus and Δ K-12 into the Hep2 culture with concentration of 30 µL /3 mL). The positive control group included a single culture of HT29 or a mixed culture of HT29, WT K-12 (inoculation of WT K-12 into the HT29 culture with concentration of 30 µL /3 mL), or Δ K-12 (inoculation of Δ K-12 into the HT29 culture with concentration of 30 µL/3 mL). After cell collection, the total RNAs were identified using Easy blue (iNtRON Biotechnology, Seongnam, Korea). The ratios of 28S and 18S to the total RNA samples were measured in 260/280 nm and 260/230 nm. Purity for microarray was identified using agarose gel electrophoresis.

3. RT-PCR

The RT-PCR of total RNAs was done using the RT PreMix Kit. One mg of RNA and 100 pmol of primer were put in a mixing bowl after preinoculation for 5 minutes at temperature of 70℃. The reacting dose was 20 µL cDNA synthesis was performed for 60 minutes at temperature of 45℃.
Afterward, a reverse transcriptase was inactivated for 5 minutes at temperature of 94℃. After that, 2-5 µL of RT-generated cDNA was amplified using a polymerase chain reaction (PCR) PreMix Kit. The primers used for cDNA amplification are listed in Tables 1, 2.
PCR was performed through 30 cycles of denaturation step for 30 seconds at temperature of 94℃, annealing step for 30 seconds at temperature of 55℃, and extension step for 30 seconds at temperature of 72℃. As an expected PCR output, human NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) 1 was 299 bp, p53 was 251 bp, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was 326 bp. On the other hand, 194 bp for ssrA and 300 bp for GAPDH were the expected PCR output from K-12.

III. Results

1. Total RNA preparation from K-12 strains following the stimulation of S. aureus for microarray

After a single culture of K-12 and a mixed culture of K-12 and S. aureus, refined RNA was detected as shown in Fig. 1 and Table 3. Highpurity RNAs were extracted in all the groups.

2. Expression of ssrA and GAPDH in K-12 strains stimulated by S. aureus

After a single culture of K-12 and a mixed culture with S. aureus, ssrA in the mixed culture was slightly more expressed than that in the single culture but was not expressed in K-12 with ssrA knock out.(Fig. 2) Meanwhile, GAPDH was expressed in all groups, showing an almost similar tendency to 5 hours after culture.(Fig. 2)

3. ssrA expression in WT K-12 strain stimulated by Host

The expression of ssrA in the K-12 culture mixed with Hep2 was analyzed using RT-PCR.
The expression of ssrA in the mixed culture with Hep2 showed greater increase than the one in a single culture of WT K-12.(Fig. 3)

4. NF-κB and p53 expression test in Hep2

The expression of NF-κB- as a factor related to inflammation- in a mixed culture with WT-K12 showed greater increase compared to the control group. Note, however, that the expression of NF-κB in a mixed culture with Δ K-12 showed much more increase. Expression of p53 was not observed in both single culture of Hep2 and mixed culture with K-12.(Fig. 4) In the case of HT29, the expression of NF-κB showed an increase in a single culture or a mixed culture with Δ K-12, not WT K-12.(Fig. 4)

5. ssrA expression in K-12 strains stimulated by the host and S. aureus

The expression of ssrA in a mixed culture of WT K-12, S. aureus, and Hep2 showed greater increase than that in a mixed culture of WT-12 and Hep2.(Fig. 5)

IV. Discussion

Human is born in aseptic condition including internal organs, and normal flora adapted to the environment of each part settles according to contact with the external environment. Normal flora in the oral cavity is known to consist of over 300 kinds2-5, which is not pathogenic in normal state but causing infection through a wound in the oral cavity or an operated part. Bacteria from other regions may travel through various route. This study was conducted to determine how such a nonnormal flora adapts to normal flora and the cells with different environment. Hep2 originating from the occipital region was used as a cell, S. aureus , as a normal flora, and K-12 cell line, E. coli, as nonnormal flora.
As a germ that normal flora of the large intestine of human and animal, E. coli becomes the cause of various diseases by leading to many infections in the other regions as well as the large intestine21. ssrA is known to be an essential element in some kinds of germs such as Coliform bacillus. E. coli with variation of ssrA shows the reduction of growth speed and abnormal control function due to nonadaptation to temperature and stress13,17,18. Since E. coli' s ssrA has information on tmRNA-, a small and stable RNA molecule, it reportedly takes a role of various functions such as tagging for degrading abnormal proteins, growth assistance of the bacteria, and activation control of proteins combined with DNA8-12. According to Muto et al.18, an ssrA gene is involved in maintaining the homeostasis of the bacteria, increasing when exposed to heat or chemical stimuli. This study sought to investigate the reaction in the host and pathogen using E. coli with ssrA (in charge of adjusting to stress) removed and normal E. coli.
In K-12 with ssrA knocked out, ssrA was not expressed in single culture and mixed culture with S. aureus. In normal E. coli, the expression of ssrA could be identified beginning 3 hours after culture. The expression of ssrA in a mixed culture of E. coli and S. aureus showed greater increase than that in a single culture of E. coli. Since ssrA was not detected in a single culture of E. coli with ssrA knocked out or in a mixed culture with S. aureus, ssrA could be said to be not expressed in S. aureus. Meanwhile, GAPDH was expressed in all the groups. This finding was consistent with the result of Muto et al.18. The expression of ssrA increases to adapt to stress for maintaining the homeostasis of a cell. As the result of Karzai et al.13, ssrA contributes to signal exchange between the bacteria.
The expression of ssrA in a mixed culture of Hep2 and K-12 showed greater increase than that in a single culture of K-12; no expression of ssrA was found in a single culture or a mixed culture in case of K-12 with ssrA knocked out. However, such result was again consistent with that of Muto et al.18 and Karzai et al.13, signal exchange between bacteria and the host. This suggested that K-12 received a certain signal from Hep2. Nonetheless, further study on such signal system will be needed in the future.
The expression of NF-κB related to inflammations in a mixed culture with WT K-12 showed greater increase than that in a single culture of Hep2, a cell line originating with the occipital region. The greatest expression was shown in a mixed culture with K-12 with ssrA knocked out. No expression of p53 was found in all the cases. This implies that ssrA is also related to the toxicity of bacteria, and that, in a variant with ssrA mutated, a nontoxic germ can be changed to a toxic one. Meanwhile, since the mixed culture with HT29, a cell line originating from the large intestine, was deemed similar to the K-12 residence region, the expression of NF-κB was similar to that in a single culture of HT29 or a mixed culture with WT K-12. In a mixed culture with K-12 with ssrA knocked out, however, the expression of NF-κB increased. Such result indirectly implies that K-12 receives more stimuli in the nonresidence region rather than in the residence region, and that, without ssrA, toxicity can occur in both regions.
The expression of ssrA in a mixed culture of K-12, Hep2, and S. aureus simultaneously showed greater increase than that in a mixed culture of K-12 and Hep2. K-12's receipt of simultaneous stimuli from Hep2 and S. aureus was believed to have led to the increase of ssrA expression.
These results suggested that ssrA gene is involved in E. coli's adaptation to a new environment in case of mixed culture by combining E. coli with S. aureus and Hep2, contributing to changing nonpathogenic bacteria in the residence region to pathogenic bacteria in the other regions. Detailed study on the mechanism related to ssrA will be necessary in the future, however.

Figures and Tables

Fig. 1
Agarose gel fraction of total RNA of K-12 strains stimulated by Scleropages aureus (A). (W: wild-type Escherichia coli [WT K-12], Δ: ssrA knock-out Escherichia coli [Δ K-12], GAPDH: glyceraldehyde 3-phosphate dehydrogenase)
jkaoms-38-14-g001
Fig. 2
Expression of ssrA and GAPDH in single and mixed cultures after stimulation by Scleropages aureus (A). (W: wild-type Escherichia coli [WT K-12], Δ: ssrA knock-out Escherichia coli [Δ K-12], GAPDH: glyceraldehyde 3-phosphate dehydrogenase)
jkaoms-38-14-g002
Fig. 3
Expression of ssrA in the mixed culture of wild-type Escherichia coli (WT K-12) with Hep2. (Con: control, W: WT K-12, Δ: ssrA knock-out E. coli [Δ K-12], GAPDH: glyceraldehyde 3-phosphate dehydrogenase)
jkaoms-38-14-g003
Fig. 4
Expression level of NF-κB and p53 from Hep2 and HT29 following K-12 treatment. (C: control, W: wild-type Escherichia coli [WT K-12], Δ: ssrA knock-out E. coli [Δ K-12], GAPDH: glyceraldehyde 3-phosphate dehydrogenase)
jkaoms-38-14-g004
Fig. 5
Expression of ssrA in wild-type Escherichia coli (WT K-12) from a mixed culture with Scleropages aureus (S. aureus). (C: control, W: WT K-12, A: S. aureus, GAPDH: glyceraldehyde 3-phosphate dehydrogenase)
jkaoms-38-14-g005
Table 1
Sequence of primers used in RT-PCR for bacterial K-12
jkaoms-38-14-i001

(RT-PCR: reverse transcription polymerase chain reaction, GAPDH: glyceraldehyde 3-phosphate dehydrogenase)

Table 2
Sequence of primers used in RT-PCR for human
jkaoms-38-14-i002

(RT-PCR: reverse transcription polymerase chain reaction, NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells, GAPDH: glyceraldehyde 3-phosphate dehydrogenase)

Table 3
Amount of total RNA extracts of K-12 strains stimulated by S. aureus (A)
jkaoms-38-14-i003

(Conc: concentration, W: wild-type Escherichia coli [WT K-12], Δ: ssrA knock-out E. coli [Δ K-12])

Notes

This study was conducted with the support of the academic research fund by the Research Institute of Clinical Medicine, Chonnam National University in 2008.

References

1. Isselbacher KJ, Braunwald E, Wilson JD, Martin JB, Fauci AS, Kasper DL. Harrison's principles of internal medicine. 1997. 13th edition. New York: McGraw-Hill.
2. Frisken KW, Higgins T, Palmer JM. The incidence of periodontopathic microorganisms in young children. Oral Microbiol Immunol. 1990. 5:43–45.
crossref
3. Slots J, Genco RJ. Black-pigmented Bacteroides species, Capnocytophaga species, and Actinobacillus actinomycetemcomitans in human periodontal disease: virulence factors in colonization, survival, and tissue destruction. J Dent Res. 1984. 63:412–421.
crossref
4. Moore WE. Microbiology of periodontal disease. J Periodontal Res. 1987. 22:335–341.
crossref
5. Genco RJ, Zambon JJ, Christersson LA. The origin of periodontal infections. Adv Dent Res. 1988. 2:245–259.
crossref
6. Braiteh F, Golden MP. Cryptogenic invasive Klebsiella pneumoniae liver abscess syndrome. Int J Infect Dis. 2007. 11:16–22.
crossref
7. Socransky SS, Haffajee AD. Periodontal microbial ecology. Periodontol 2000. 2005. 38:135–187.
crossref
8. Kirby JE, Trempy JE, Gottesman S. Excision of a P4-like cryptic prophage leads to Alp protease expression in Escherichia coli. J Bacteriol. 1994. 176:2068–2081.
crossref
9. Keiler KC, Waller PR, Sauer RT. Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA. Science. 1996. 271:990–993.
crossref
10. Retallack DM, Johnson LL, Friedman DI. Role for 10Sa RNA in the growth of lambda-P22 hybrid phage. J Bacteriol. 1994. 176:2082–2089.
crossref
11. Withey J, Friedman D. Analysis of the role of trans-translation in the requirement of tmRNA for lambdaimmP22 growth in Escherichia coli. J Bacteriol. 1999. 181:2148–2157.
crossref
12. Retallack DM, Friedman DI. A role for a small stable RNA in modulating the activity of DNA-binding proteins. Cell. 1995. 83:227–235.
crossref
13. Karzai AW, Roche ED, Sauer RT. The SsrA -SmpB system for protein tagging, directed degradation and ribosome rescue. Nat Struct Biol. 2000. 7:449–455.
crossref
14. Withey JH, Friedman DI. A salvage pathway for protein structures: tmRNA and trans-translation. Annu Rev Microbiol. 2003. 57:101–123.
15. Fujihara A, Tomatsu H, Inagaki S, Tadaki T, Ushida C, Himeno H, et al. Detection of tmRNA-mediated trans-translation products in Bacillus subtilis. Genes Cells. 2002. 7:343–350.
crossref
16. Wiegert T, Schumann W. SsrA -mediated tagging in Bacillus subtilis. J Bacteriol. 2001. 183:3885–3889.
17. Komine Y, Kitabatake M, Yokogawa T, Nishikawa K, Inokuchi H. A tRNA-like structure is present in 10Sa RNA, a small stable RNA from Escherichia coli. Proc Natl Acad Sci U S A. 1994. 91:9223–9227.
crossref
18. Muto A, Fujihara A, Ito KI, Matsuno J, Ushida C, Himeno H. Requirement of transfer-messenger RNA for the growth of Bacillus subtilis under stresses. Genes Cells. 2000. 5:627–635.
crossref
19. Baumler AJ, Kusters JG, Stojiljkovic I, Heffron F. Salmonella typhimurium loci involved in survival within macrophages. Infect Immun. 1994. 62:1623–1630.
20. Julio SM, Heithoff DM, Mahan MJ. ssrA (tmRNA) plays a role in Salmonella enterica serovar Typhimurium pathogenesis. J Bacteriol. 2000. 182:1558–1563.
crossref
21. Kovach ME, Shaffer MD, Peterson KM. A putative integrase gene defines the distal end of a large cluster of ToxR-regulated colonization genes in Vibrio cholerae. Microbiology. 1996. 142:2165–2174.
crossref
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