© 2014 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12340
APMIS 123: 252–259
Analysis of the roles of NrdR and DnaB from Streptococcus pyogenes in response to host defense YAN ZHANG,1,2 RYO OKADA,1 MASANORI ISAKA,1 ICHIRO TATSUNO,1 KEN-ICHI ISOBE2 and TADAO HASEGAWA1 1
Department of Bacteriology, Nagoya City University Graduate School of Medical Sciences, Nagoya, 2 Department of Immunology, Nagoya University Graduate School of Medicine, Nagoya, Japan
Zhang Y, Okada R, Isaka M, Tatsuno I, Isobe K-I, Hasegawa T. Analysis of the roles of NrdR and DnaB from Streptococcus pyogenes in response to host defense. APMIS 2015; 123: 252–259. Toxic shock syndrome caused by Streptococcus pyogenes (S. pyogenes) is a re-emerging infectious disease. Many virulence-associated proteins play important roles in its pathogenesis and the production of these proteins is controlled by many regulatory factors. CovS is one of the most important two-component sensor proteins in S. pyogenes, and it has been analyzed extensively. Our recent analyses revealed the existence of a transposon between covS and nrdR in several strains, and we speculated that this insertion has some importance. Hence, we examined the significances of the NrdR stand-alone regulator and DnaB, which is encoded by the gene located immediately downstream of nrdR in S. pyogenes infection. We established an nrdR-only knockout strain, and both nrdR and partial dnaB knockout strain. These established knockout strains exhibited a deteriorated response to H2O2 exposure. nrdR and partial dnaB knockout strain was more easily killed by human polynuclear blood cells, but the nrdR-only knockout strain had no significant difference compared to wild type in contrast to the combined knockout strain. In addition, the mouse infection model experiment illustrated that nrdR and partial dnaB knockout strain, but not the nrdR-only knockout strain, was less virulent compared with the parental strain. These results suggest that DnaB is involved in response to host defense. Key words: Streptococcus pyogenes; NrdR; DnaB; host defense. Tadao Hasegawa, Department of Bacteriology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi Mizuho-cho Mizuho-ku Nagoya, 467-8601 Japan. e-mail: [email protected]
Streptococcus pyogenes (S. pyogenes) is a Grampositive bacterium infecting the regions of the upper respiratory tract such as the tonsils and pharynx and it causes post-infection diseases such as rheumatic fever and glomerulonephritis. Furthermore, S. pyogenes causes other serious human diseases, including streptococcal toxic shock syndrome (STSS) (1, 2). Because of the worldwide prevalence of STSS, many studies have been conducted to identify the relevant virulence factors (3, 4). These factors include the M protein, streptococcal inhibitor of complement, streptococcal pyrogenic toxins, hemolysins, and several DNases (2). We have thus far identified several DNases (5–7) using twodimensional gel electrophoresis (2-DE) and mass analysis and also determined the significance of the Nga protein (8, 9). In addition, S. pyogenes produces many types of virulence-associated proteins in response to environmental stresses. We have Received 20 June 2014. Accepted 1 October 2014
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studied the influence of various culture conditions on the production of these exoproteins (10–13). The regulation of the expression of virulenceassociated proteins in S. pyogenes is primarily controlled by stand-alone response regulators and two-component regulatory systems (TCSs). Standalone transcriptional regulators have no identified sensory domains (14). A TCS consists of a sensor protein and a regulatory protein. CovRS (CsrRS) (15, 16), one of the 13 TCSs (17), has been most commonly analyzed. CovR was reported to influence the transcription of 15% of the S. pyogenes genome, including many genes involved in virulence (18). Mutation of CovRS was first reported to result in enhanced virulence in murine infection models (19). In addition, mutation of covS was suggested to be an extremely important step in the transition from a pharyngitis-causing strain to an invasive strain (20). Other reports also indicated a high frequency of CovRS mutations in STSS isolates (21, 22). One possible explanation is that a
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mutation within CovRS promotes resistance to neutrophil-mediated killing by altering the production of proteins such as Sda1, M protein, and hyaluronic acid capsule (23, 24). We have reported the alteration of culture supernatant protein profiles in covS mutant strains and in vivo human evidence of the significance of CovS mutation (25, 26). Thus far, we clarified the existence of some strains in which a transposon was inserted between covS and nrdR by analyzing clinically isolated group A and group G streptococcal strains ((25), data not shown). covS and nrdR are located adjacently in the S. pyogenes genome. nrdR is also located immediately upstream of dnaB and dnaI. We speculated that this insertion has some biological significance. Previously, we used a mouse infection model experiment to examine the virulence of S. pyogenes (7, 27). In addition to this mouse analysis, we also used an in vitro bactericidal assay using human blood, because we believe that the relationship between bacteria and phagocytic cells is critical in the study of the pathogenesis of virulent bacteria. The function and significance of NrdR and DnaB in the virulence of S. pyogenes are completely unknown. Hence, we attempted to reveal their significance in virulence, with special reference to the response to host defense in this study. MATERIALS AND METHODS S. pyogenes strain The S. pyogenes strain used in this study was the emm1 1529 strain isolated from a Japanese patient with STSS (11, 25). Bacteria were either cultured in brain heart infusion (Eiken Chemical, Tokyo, Japan) supplemented with 0.3% yeast extract (Becton Dickinson, Sparks, MD, USA) (BHI-YE) broth without agitation or grown on BHI-YE agar plates at 37°C.
Creation of the knockout and complemented strains Nonpolar inactivated mutants of nrdR and dnaB were constructed through a double-crossover allelic replacement in the chromosome of S. pyogenes 1529 (25). The method was virtually identical as that previously published (7, 27). All PCRs were performed using the PrimeSTAR HS DNA polymerase (Takara, Ohtsu, Japan), which yields blunt-ended DNA fragment. The DNA fragment of nrdR and dnaB was amplified with oligonucleotide primers nrdn5Sma (50 -TCCCCCGGGGTCTACAAGAGTTTCAAG GAC -30 ) and nrd-c4Spe (50 -GGACTAGTGTTTCATA CCAAACTGTAGG-30 ) (Fig. 1A, B) which have SmaI and SpeI site, respectively (fragment 1). The SmaI-SpeI digested fragment 1 was subcloned into the SmaI-SpeI site of the pFW12 vector (28) (named pTHnrd1). A second round of PCR was performed using nrd-n1Nhe (50 -GGCT AGCAAAGAGAGTTAGAGTAGCTT-30 ) and covS-c2
© 2014 APMIS. Published by John Wiley & Sons Ltd
(50 -GTAATTACATTTTGGACAAC-30 ) (fragment 2). Both the NheI digested fragment 2 and the spc1 DNA fragment containing aad9 (a promoterless spectinomycin resistance gene) obtained from a SmaI-digested fragment of pSL60-1 (28) were cloned into the NheI-SmaI site of pTHnrd1 (named pTHnrd2). Another DNA fragment of nrdR and dnaB was amplified with oligonucleotide primers nrd-n2Sma (50 -TCCCCCGGGACCAATTGATACTTTTA CAT-30 ) and nrd-c1Spe (50 -GGACTAGTGATAGCTGA TACACACTCAC-30 ) which also have SmaI and SpeI site, respectively (fragment 3). The SmaI-SpeI digested fragment 3 was subcloned into the pTHnrd2 vector (named pTHnrd3). When the pTHnrd2 vector was successfully integrated into the genome, nrdR-only was knocked out and an intact DnaB protein was expressed; however, when the pTHnrd3 vector was successfully integrated into the genome, nrdR was also knocked out and a truncated DnaB protein lacking some N-terminal amino acids was expressed, because the original start codon ATG was deleted and it is possible that alternative start codon located downstream of original ATG was used (Fig. 1B). These plasmids, pTHnrd2 and pTHnrd3, were suicide vectors for S. pyogenes. To prepare competent cells, strains were harvested at an early- to mid-log phase (OD660 = 0.4–0.5) and washed twice with 0.5 M sucrose buffer. The constructed suicide vector was transformed into the strain by electroporation. Conditions of electroporation were 1.25 kV/mm, a capacitance of 25 lF, and a resistance of 200 ohms using the Gene Pulser II (Biorad, Hercules, CA, USA). After incubation at 37°C for 3 h, competent cells were spread onto BHI-YE agar plates containing spectinomycin (final concentration, 100 lg/mL). Selected colonies on the plates were cultured and were then washed once with saline, resuspended in 10 mM Tris/ 1 mM ethylenediaminetetraacetic acid, and boiled for 15 min. Genomic DNA was obtained from the supernatant of boiled bacteria. The double-crossover replacement with genomic DNA was analyzed by PCR. Successful double-crossover replacement was further confirmed by DNA sequencing. To construct a plasmid for nrdR-only or nrdR and dnaB-complemented, the DNA fragment was amplified with the oligonucleotide primers nrd-n1Nhe/nrdc1Spe or nrd-n1Nhe/dnaB-c1(50 -TTGCTACTTCAGGA TCCGCT-30 ), respectively. The fragment was treated with T4 polynucleotide kinase and ligated into the SmaI site of the pLZ12-Km2 plasmid (29). The protocol for transformation was identical to the aforementioned protocol, except that the competent cells were spread onto BHI-YE agar plates containing spectinomycin and kanamycin (final concentrations, 100 lg/mL, respectively).
Disk diffusion assays for H2O2 sensitivity An aliquot of bacterial stock solution stored frozen at 80°C was inoculated into 3 mL BHI-YE and cultured overnight at 30°C without agitation. Thirty-six microliters of the overnight culture was added to 3.6 mL Todd– Hewitt broth (Becton Dickinson) supplemented with 0.3% yeast extract (Becton Dickinson) (TH-YE) and cultured at 37°C without agitation. When bacterial cells grew to the late-exponential phase as monitored by a colorimeter (Asahi Science) (OD660 = 0.95), the turbidity was adjusted to match a 0.5 MacFarland turbidity standard using BHI. The diluted samples were inoculated onto the TH-YE agar
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A
B
Fig. 1. Strategies for the creation of knockout strains. (A) Strategies used to create the nrdR-only knockout, and nrdR and partial dnaB knockout strains. The primers used for the construction of plasmid for knockout and the deleted region of dnaB gene are shown. (B) Nucleotide and amino acid sequences around the junctions of nrdR and dnaB. The primer positions of n2-Sma, n5-Sma, c1-Spe, and c4-Spe are double underlined. The original methionine of DnaB is indicated as circled M, and a possible alternative start methionine of truncated DnaB is indicated as boxed M. In addition, putative deleted amino acids in nrdR and partial dnaB knockout strain are indicated using broken line.
plates by swabbing over the entire surface in three directions (30). Every sterile disk (diameter, 6.5 mm; cut from thin chromatography paper) was saturated with 20 lL of 275 mM H2O2, and three disks were evenly placed on every inoculated plate. After 24 h of incubation at 37°C, the diameter of the zone of inhibition was measured (31).
Lancefield bactericidal assay The Lancefield bactericidal assay (32) was performed as described previously (7, 27). In brief, approximately 5000 CFUs of the strain (OD660 = 0.75) were added to 1 mL of heparinized human whole blood and 1 ml of BHI-YE, respectively, to clarify the function of neutrophil killing and rotated at 37°C. Diluted samples of blood and BHI-YE were plated on the BHI-YE agar plates at the indicated time points to determine the number of CFUs.
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The experiments were repeated four times. The results are presented as the mean CFUs at each time point and the killing ratios at specific times were tested for significance using the Student’s t-test.
Mouse model of skin invasion and soft tissue infection The procedure was the same as that described previously (7, 27). In brief, S. pyogenes was harvested after 16 h of growth on a BHI-YE agar plate. These cells were harvested into 1 ml of PBS and centrifuged at 2000 g for 2 min. The pellets were diluted to 1 9 108 CFUs in 100 lL of PBS and injected (with a 25-gauge needle) subcutaneously into 3-week-old female Slc:ICR mice anesthetized by ketamine–xylazine injections. One hundred microliters of PBS without bacteria was injected into the control mice. A total of 36 mice were used for the © 2014 APMIS. Published by John Wiley & Sons Ltd
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experiment. The number of CFUs injected was verified for each experiment by plating bacteria on BHI-YE agar plates and counting CFUs. All animal procedures were approved by the Institutional Animal Care and Use Committee of Nagoya City University.
RESULTS Establishment of nrdR and dnaB knockout strains
We successfully established both nrdR knockout strains that expressed intact DnaB and truncated DnaB using pTHnrd2 and pTHndr3 plasmids, respectively (Fig. 1A). Truncated DnaB was considered to lose several amino acids in N-terminal helical domain (NTD), but to retain a linker domain and a RecA-core C-terminal domain (CTD) (33, 34). We unsuccessfully attempted to establish both nrdR and complete dnaB knockout strain and we did not succeed in establishing the dnaB-only knockout strain that carried the intact nrdR, either (the method and data not shown). Although nrdR and partial dnaB mutant strain exhibited delayed growth, they reached the stationary phase at the same time as the wild type and nrdR-only knockout strains (Fig. 2). The culture supernatant proteins in these established knockout strains were analyzed by SDS-PAGE; however, the production of SpeB or SIC, that are two of the important virulence-associated proteins in emm1 type S. pyogenes (25), was not significantly altered (data not shown). Analysis of sensitivities to H2O2
We performed disk diffusion analysis to test the difference of the sensitivities of the strains to H2O2. The zones of inhibition caused by H2O2 were measured, and the significance of the zone of inhibition
of each strain relative to that of the wild type and complemented strains was determined by t-test. nrdR and partial dnaB mutant strain was more sensitive to H2O2 than the wild type parental strain (Figs. 3A and 3B). nrdR- and dnaB-complemented strain displayed similar sensitivity as that of the parental strain (Fig. 3A), and nrdR-only complemented strain also displayed restored sensitivity (Fig. 3B). The nrdR-only knockout strain was a little sensitive, but the complement experiment did not work (Fig. 3C). In vitro blood bactericidal assay
To determine whether NrdR and DnaB affected the bactericidal function of human leukocytes, we performed bactericidal assays using human blood. The killing ratios suggested that nrdR and partial dnaB knockout strain was destroyed more efficiently by human blood cells than the parental strain (Figs 4A and 4B). The killing ratio of the nrdR and dnaB genes complemented strain was restored (Fig. 4A), but not nrdR-only complemented strain (Fig. 4B). In addition, there was no significant difference in the killing ratios between the nrdR-only mutant and the wild type strains (Fig. 4C). Analysis of virulence in a mouse infection model
To examine the virulence in vivo, we performed mouse infection model experiments using the parental strain 1529, and nrdR and partial dnaB knockout strain. As shown in Fig. 5A, the mice injected with the knockout strain survived longer than those injected with the parental strain (p < 0.001). The nrdR-only knockout strain did not display a significant survival difference (Fig. 5B, p > 0.05). These results suggest that DnaB plays a role in S. pyogenes infection in the mouse infection model experiments.
DISCUSSION
Fig. 2. Growth curves of streptococcal strains in the BHIYE broth. Each strain was cultured in 4 mL of BHI-YE broth, and growth was monitored using a colorimeter at OD660. © 2014 APMIS. Published by John Wiley & Sons Ltd
We previously reported that one S. pyogenes strain, MDMH from the blood and cerebrospinal fluid of patient with extremely severe STSS had a transposon between covS and nrdR (25). In addition, we recently identified some group G streptococcal strains that have the same transposon insertion. The location of nrdR is reported to be immediately upstream of dnaB and dnaI in Bacillus, Streptococcus, Lactobacillus, and Staphylococcus (35), and these regions appear to be operons. We speculated that this transposon insertion has some biological roles because of the functions of NrdR and DnaB.
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Fig. 3. Sensitivities to H2O2. Disk diffusion assays were performed using the wild type 1529 strain, its knockout strains, and their complemented strains. (A) represents the data of the wild type strain 1529, its derived nrdR and partial dnaB knockout strain, and nrdR and dnaB complemented strain; (B) represents the data of the wild type strain 1529, its derived nrdR and partial dnaB knockout strain, and nrdR-only complemented strain; (C) represents the data of the wild type strain 1529, nrdR-only knockout strain, and nrdR complemented strain.
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Fig. 4. In vitro blood bactericidal assay. (A) The wild type strain 1529, its derived nrdR and partial dnaB knockout strain, and nrdR and dnaB complemented strain; (B) the wild type strain 1529, its derived nrdR and partial dnaB knockout strain, and nrdR-only complemented strain; or (C) the wild type strain 1529, nrdR-only knockout strain, and nrdR complemented strain were added to human whole blood and BHI-YE, respectively, and after each hour, samples were plated on BHI-YE agar. The data of killing ratio, which equal the number of CFUs from human blood divided by that from BHI-YE at 3 h, are indicated.
NrdR was first described in Streptomyces coelicolor (36), and it has been suggested to negatively regulate the transcription of nrd operons encoding ribonucleotide reductases (RNRs) in vitro (36–42). RNRs are conserved from prokaryotes to eukaryotes and they are responsible for the de novo synthesis of all four deoxyribonucleotides, the immediate precursors of DNA synthesis and repair, by reducing their respective ribonucleotides (43, 44). S. pyogenes harbors three different clusters of genes involved in ribonucleotide reduction (17). One cluster contains the genes for anaerobic class III RNR and its cognate activase (nrdDG). S. pyogenes is one of the few bacteria that possess two distinct class Ib RNR gene clusters, namely nrdHEF and nrdF*I*E*. The S. pyogenes genome also contains a second stand-alone copy of an nrdI 256
gene (nrdI2 (45)) at another location in the chromosome. The redundancy of class Ib RNR genes may be important under different environmental conditions. The expression of some RNRs is elevated by DNA damage and the inhibitors of DNA synthesis, as well as in response to oxidative stressors (46–49). Furthermore, Panosa et al. presented data illustrating that RNRs are involved in macrophage infection in Salmonella typhimurium (39). On the other hand, DnaB has a helicase activity (33, 50), and it plays an important role in the maintenance of genome stability (51). In addition, DnaB is active during DNA damage such as that caused by oxidative stress (52, 53). These studies strongly indicate that both NrdR and DnaB play important roles against oxidative stresses that cause DNA damage. We consider that the © 2014 APMIS. Published by John Wiley & Sons Ltd
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A
B
Fig. 5. Mouse infection model experiments. Survival after the subcutaneous injection with (A) the wild type strain 1529 and nrdR and partial dnaB knockout strains; (B) the wild type strain 1529 and nrdR-only knockout strains. Slc: ICR mice were injected subcutaneously with the wild type strain 1529 and knockout strains. Mice were observed daily. Survival data were assessed by Kaplan–Meier survival analysis and tested for significance by the log-rank test.
relationship between bacteria and phagocytic cells is critical in the study of the pathogenesis of virulent bacteria, and oxidative stresses that cause DNA damage are tightly associated with bacterial killing by phagocytic cells. To our knowledge, there are no reports regarding the role of NrdR and DnaB from S. pyogenes in response to host defense. Hence, we attempted to analyze the significance of these proteins. For the analyses of NrdR and DnaB, we first sought to establish several knockout strains, including nrdR-only and dnaB. However, we were unable to establish strains lacking dnaB. As DnaB is involved in the maintenance of genome stability, this failure appeared to be inevitable. Next, we used several plasmids for the creation of knockout strains and succeeded in the establishment of nrdR and partial dnaB knockout strain. The truncated DnaB protein lost some amino acids in N-terminal © 2014 APMIS. Published by John Wiley & Sons Ltd
helical domain (NTD), but it retained a linker domain and RecA-core C-terminal domain (CTD) (33, 34). Although NTD was reported to be required for helicase activity (54–56), and it is possible that the function was somewhat attenuated, the truncated DnaB in our study retained the function to survive in a normal low-stress condition from the result of growth curve (Fig. 2). Thus, we used these nrdR-only and nrdR and partial dnaB knockout strains to analyze the significance of NrdR and DnaB. As we performed oxidative stress experiment by only using H2O2, we are not sure that nrdR and partial dnaB knockout shows a higher sensitivity against general oxidative stresses. But judging from the in vitro blood bactericidal assay, and mouse infection model experiment, we concluded that NrdR and DnaB from S. pyogenes were involved in response to host defense; however, the contribution of NrdR alone was not substantial. Because the function of NrdR as a regulatory factor in S. pyogenes is not completely revealed, it is possible that not all the expressions of redundant class Ib RNR genes were changed. Hence, the deletion of only NrdR had little effect. On the other hand, as the function of DnaB was insufficient after its truncation in stress condition, its relevance became more apparent. However, as nrdR and dnaB create an operon judging from the nucleotide sequence (Fig. 1B), both proteins could play important roles in response to host defense. We did not find the apparent phenotypic differences between the strain with a transposon insertion and the nrdR- and dnaB-complemented strain, nor did we reveal the significance of transposon insertion (data not shown). Further analyses are needed to elucidate the significance of transposon insertion and the relationship between the proteins, including RNRs regulated by nrdR, DnaB, and DnaI. This study was supported by MEXT/JSPSKAKENHI Grant Number 24590531 from the Ministry of Education, Science and Culture of the Japanese government. The authors thank Enago (www.enago.jp) for the English language review.
REFERENCES 1. Cone LA, Woodard DR, Schlievert PM, Tomory GS. Clinical and bacteriologic observations of a toxic shock-like syndrome due to Streptococcus pyogenes. N Engl J Med 1987;317:146–9. 2. Cunningham MW. Pathogenesis of group A streptococcal infections. Clin Microbiol Rev 2000;13:470– 511.
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3. Hauser AR, Stevens DL, Kaplan EL, Schlievert PM. Molecular analysis of pyrogenic exotoxins from Streptococcus pyogenes isolates associated with toxic shock-like syndrome. J Clin Microbiol 1991;29: 1562–7. 4. Reichardt W, Muller-Alouf H, Alouf JE, Kohler W. Erythrogenic toxins A, B and C: occurrence of the genes and exotoxin formation from clinical Streptococcus pyogenes strains associated with streptococcal toxic shock-like syndrome. FEMS Microbiol Lett 1992;79:313–22. 5. Hasegawa T, Torii K, Hashikawa S, Iinuma Y, Ohta M. Cloning and characterization of the deoxyribonuclease sda gene from Streptococcus pyogenes. Curr Microbiol 2002;45:13–7. 6. Hasegawa T, Torii K, Hashikawa S, Iinuma Y, Ohta M. Cloning and characterization of two novel DNases from Streptococcus pyogenes. Arch Microbiol 2002;177:451–6. 7. Hasegawa T, Minami M, Okamoto A, Tatsuno I, Isaka M, Ohta M. Characterization of a virulenceassociated and cell-wall-located DNase of Streptococcus pyogenes. Microbiology 2010;156:184–90. 8. Tatsuno I, Sawai J, Okamoto A, Matsumoto M, Minami M, Isaka M, et al. Characterization of the NAD-glycohydrolase in streptococcal strains. Microbiology 2007;153:4253–60. 9. Tatsuno I, Isaka M, Minami M, Hasegawa T. NADase as a target molecule of in vivo suppression of the toxicity in the invasive M-1 group A Streptococcal isolates. BMC Microbiol 2010;10:144. 10. Nakamura T, Hasegawa T, Torii K, Hasegawa Y, Shimokata K, Ohta M. Two-dimensional gel electrophoresis analysis of the abundance of virulent exoproteins of group A streptococcus caused by environmental changes. Arch Microbiol 2004;181:74– 81. 11. Tanaka M, Hasegawa T, Okamoto A, Torii K, Ohta M. Effect of antibiotics on group A Streptococcus exoprotein production analyzed by two-dimensional gel electrophoresis. Antimicrob Agents Chemother 2005;49:88–96. 12. Sawai J, Hasegawa T, Kamimura T, Okamoto A, Ohmori D, Nosaka N, et al. Growth phase-dependent effect of clindamycin on production of exoproteins by Streptococcus pyogenes. Antimicrob Agents Chemother 2007;51:461–7. 13. Minami M, Kamimura T, Isaka M, Tatsuno I, Ohta M, Hasegawa T. Clindamycin-induced CovS-mediated regulation of the production of virulent exoproteins streptolysin O, NAD glycohydrolase, and streptokinase in Streptococcus pyogenes. Antimicrob Agents Chemother 2010;54:98–102. 14. Kreikemeyer B, McIver KS, Podbielski A. Virulence factor regulation and regulatory networks in Streptococcus pyogenes and their impact on pathogen-host interactions. Trends Microbiol 2003;11:224–32. 15. Levin JC. Wessels MR Identification of csrR/csrS, a genetic locus that regulates hyaluronic acid capsule synthesis in group A Streptococcus. Mol Microbiol 1998;30:209–19. 16. Federle MJ, McIver KS, Scott JR. A response regulator that represses transcription of several virulence operons in the group A streptococcus. J Bacteriol 1999;181:3649–57.
258
17. Ferretti JJ, McShan WM, Ajdic D, Savic DJ, Savic G, Lyon K, et al. Complete genome sequence of an M1 strain of Streptococcus pyogenes. Proc Natl Acad Sci USA 2001;98:4658–63. 18. Graham MR, Smoot LM, Migliaccio CA, Virtaneva K, Sturdevant DE, Porcella SF, et al. Virulence control in group A Streptococcus by a two-component gene regulatory system: global expression profiling and in vivo infection modeling. Proc Natl Acad Sci USA 2002;99:13855–60. 19. Engleberg NC, Heath A, Miller A, Rivera C, DiRita VJ. Spontaneous mutations in the CsrRS twocomponent regulatory system of Streptococcus pyogenes result in enhanced virulence in a murine model of skin and soft tissue infection. J Infect Dis 2001;183:1043–54. 20. Sumby P, Whitney AR, Graviss EA, DeLeo FR, Musser JM. Genome-wide analysis of group A streptococci reveals a mutation that modulates global phenotype and disease specificity. PLoS Pathog 2006;2:e5. 21. Ato M, Ikebe T, Kawabata H, Takemori T, Watanabe H. Incompetence of neutrophils to invasive group A streptococcus is attributed to induction of plural virulence factors by dysfunction of a regulator. PLoS ONE 2008;3:e3455. 22. Ikebe T, Ato M, Matsumura T, Hasegawa H, Sata T, Kobayashi K, et al. Highly frequent mutations in negative regulators of multiple virulence genes in group A streptococcal toxic shock syndrome isolates. PLoS Pathog 2010;6:e1000832. 23. Walker MJ, Hollands A, Sanderson-Smith ML, Cole JN, Kirk JK, Henningham A, et al. DNase Sda1 provides selection pressure for a switch to invasive group A streptococcal infection. Nat Med 2007;13:981–5. 24. Cole JN, Pence MA, von Kockritz-Blickwede M, Hollands A, Gallo RL, Walker MJ, et al. M protein and hyaluronic acid capsule are essential for in vivo selection of covRS mutations characteristic of invasive serotype M1T1 group A Streptococcus. MBio 2010;1: e00191–10. 25. Hasegawa T, Okamoto A, Kamimura T, Tatsuno I, Hashikawa SN, Yabutani M, et al. Detection of invasive protein profile of Streptococcus pyogenes M1 isolates from pharyngitis patients. APMIS 2010; 118:167–78. 26. Masuno K, Okada R, Zhang Y, Isaka M, Tatsuno I, Shibata S, et al. Simultaneous isolation of emm89type Streptococcus pyogenes strains with a wild-type or mutated covS gene from a single streptococcal toxic shock syndrome patient. J Med Microbiol 2014;63:504–7. 27. Ichikawa M, Minami M, Isaka M, Tatsuno I, Hasegawa T. Analysis of two-component sensor proteins involved in the response to acid stimuli in Streptococcus pyogenes. Microbiology 2011;157:3187–94. 28. Lukomski S, Hoe NP, Abdi I, Rurangirwa J, Kordari P, Liu M, et al. Nonpolar inactivation of the hypervariable streptococcal inhibitor of complement gene (sic) in serotype M1 Streptococcus pyogenes significantly decreases mouse mucosal colonization. Infect Immun 2000;68:535–42. 29. Okada N, Tatsuno I, Hanski E, Caparon M, Sasakawa C. Streptococcus pyogenes protein F promotes invasion of HeLa cells. Microbiology 1998;144:3079–86.
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30. Mirrett S, Reller LB. Comparison of direct and standard antimicrobial disk susceptibility testing for bacteria isolated from blood. J Clin Microbiol 1979;10:482–7. 31. Hoopman TC, Liu W, Joslin SN, Pybus C, Brautigam CA. Hansen EJ Identification of gene products involved in the oxidative stress response of Moraxella catarrhalis. Infect Immun 2011;79:745–55. 32. Lancefield RC. Differentiation of group A streptococci with a common R antigen into three serological types, with special reference to the bactericidal test. J Exp Med 1957;106:525–44. 33. LeBowitz JH, McMacken R. The Escherichia coli dnaB replication protein is a DNA helicase. J Biol Chem 1986;261:4738–48. 34. Bailey S, Eliason WK, Steitz TA. The crystal structure of the Thermus aquaticus DnaB helicase monomer. Nucleic Acids Res 2007;35:4728–36. 35. Rodionov DA, Gelfand MS. Identification of a bacterial regulatory system for ribonucleotide reductases by phylogenetic profiling. Trends Genet 2005;21:385–9. 36. Borovok I, Gorovitz B, Yanku M, Schreiber R, Gust B, Chater K, et al. Alternative oxygen-dependent and oxygen-independent ribonucleotide reductases in Streptomyces: cross-regulation and physiological role in response to oxygen limitation. Mol Microbiol 2004;54:1022–35. 37. Grinberg I, Shteinberg T, Gorovitz B, Aharonowitz Y, Cohen G, Borovok I. The Streptomyces NrdR transcriptional regulator is a Zn ribbon/ATP cone protein that binds to the promoter regions of class Ia and class II ribonucleotide reductase operons. J Bacteriol 2006;188:7635–44. 38. Mowa MB, Warner DF, Kaplan G, Kana BD, Mizrahi V. Function and regulation of class I ribonucleotide reductase-encoding genes in mycobacteria. J Bacteriol 2009;191:985–95. 39. Panosa A, Roca I, Gibert I. Ribonucleotide reductases of Salmonella typhimurium: transcriptional regulation and differential role in pathogenesis. PLoS ONE 2010;5:e11328. 40. Torrents E, Grinberg I, Gorovitz-Harris B, Lundstrom H, Borovok I, Aharonowitz Y, et al. NrdR controls differential expression of the Escherichia coli ribonucleotide reductase genes. J Bacteriol 2007;189: 5012–21. 41. Grinberg I, Shteinberg T, Hassan AQ, Aharonowitz Y, Borovok I, Cohen G. Functional analysis of the Streptomyces coelicolor NrdR ATP-cone domain: role in nucleotide binding, oligomerization, and DNA interactions. J Bacteriol 2009;191:1169–79. 42. Case ED, Akers JC, Tan M. CT406 encodes a chlamydial ortholog of NrdR, a repressor of ribonucleotide reductase. J Bacteriol 2011;193:4396–404.
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43. Nordlund P, Reichard P. Ribonucleotide reductases. Annu Rev Biochem 2006;75:681–706. 44. Reichard P. From RNA to DNA, why so many ribonucleotide reductases? Science 1993;260:1773–7. 45. Roca I, Torrents E, Sahlin M, Gibert I, Sjoberg BM. NrdI essentiality for class Ib ribonucleotide reduction in Streptococcus pyogenes. J Bacteriol 2008;190:4849– 58. 46. Gibert I, Calero S, Barbe J. Measurement of in vivo expression of nrdA and nrdB genes of Escherichia coli by using lacZ gene fusions. Mol Gen Genet 1990;220:400–8. 47. Jordan A, Aragall E, Gibert I, Barbe J. Promoter identification and expression analysis of Salmonella typhimurium and Escherichia coli nrdEF operons encoding one of two class I ribonucleotide reductases present in both bacteria. Mol Microbiol 1996;19:777– 90. 48. Monje-Casas F, Jurado J, Prieto-Alamo MJ, Holmgren A, Pueyo C. Expression analysis of the nrdHIEF operon from Escherichia coli. Conditions that trigger the transcript level in vivo. J Biol Chem 2001;276: 18031–7. 49. Prieto-Alamo MJ, Jurado J, Gallardo-Madueno R, Monje-Casas F, Holmgren A, Pueyo C. Transcriptional regulation of glutaredoxin and thioredoxin pathways and related enzymes in response to oxidative stress. J Biol Chem 2000;275:13398–405. 50. West SC. DNA helicases: new breeds of translocating motors and molecular pumps. Cell 1996;86:177–80. 51. Maher RL, Branagan AM, Morrical SW. Coordination of DNA replication and recombination activities in the maintenance of genome stability. J Cell Biochem 2011;112:2672–82. 52. Indiani C, O’Donnell M. A proposal: source of single strand DNA that elicits the SOS response. Front Biosci (Landmark Ed) 2013;18:312–23. 53. Inokuchi H, Ito R, Sekiguchi T, Sekiguchi M. Search for proteins required for accurate gene expression under oxidative stress: roles of guanylate kinase and RNA polymerase. J Biol Chem 2013;288:32952–62. 54. Nakayama N, Arai N, Kaziro Y, Arai K. Structural and functional studies of the dnaB protein using limited proteolysis. Characterization of domains for DNAdependent ATP hydrolysis and for protein association in the primosome. J Biol Chem 1984;259:88–96. 55. Bird LE, Pan H, Soultanas P, Wigley DB. Mapping protein-protein interactions within a stable complex of DNA primase and DnaB helicase from Bacillus stearothermophilus. Biochemistry 2000;39:171–82. 56. Mesa P, Alonso JC, Ayora S. Bacillus subtilis bacteriophage SPP1 G40P helicase lacking the N-terminal domain unwinds DNA bidirectionally. J Mol Biol 2006;357:1077–88.
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APMIS 123: 252–259
Analysis of the roles of NrdR and DnaB from Streptococcus pyogenes in response to host defense YAN ZHANG,1,2 RYO OKADA,1 MASANORI ISAKA,1 ICHIRO TATSUNO,1 KEN-ICHI ISOBE2 and TADAO HASEGAWA1 1
Department of Bacteriology, Nagoya City University Graduate School of Medical Sciences, Nagoya, 2 Department of Immunology, Nagoya University Graduate School of Medicine, Nagoya, Japan
Zhang Y, Okada R, Isaka M, Tatsuno I, Isobe K-I, Hasegawa T. Analysis of the roles of NrdR and DnaB from Streptococcus pyogenes in response to host defense. APMIS 2015; 123: 252–259. Toxic shock syndrome caused by Streptococcus pyogenes (S. pyogenes) is a re-emerging infectious disease. Many virulence-associated proteins play important roles in its pathogenesis and the production of these proteins is controlled by many regulatory factors. CovS is one of the most important two-component sensor proteins in S. pyogenes, and it has been analyzed extensively. Our recent analyses revealed the existence of a transposon between covS and nrdR in several strains, and we speculated that this insertion has some importance. Hence, we examined the significances of the NrdR stand-alone regulator and DnaB, which is encoded by the gene located immediately downstream of nrdR in S. pyogenes infection. We established an nrdR-only knockout strain, and both nrdR and partial dnaB knockout strain. These established knockout strains exhibited a deteriorated response to H2O2 exposure. nrdR and partial dnaB knockout strain was more easily killed by human polynuclear blood cells, but the nrdR-only knockout strain had no significant difference compared to wild type in contrast to the combined knockout strain. In addition, the mouse infection model experiment illustrated that nrdR and partial dnaB knockout strain, but not the nrdR-only knockout strain, was less virulent compared with the parental strain. These results suggest that DnaB is involved in response to host defense. Key words: Streptococcus pyogenes; NrdR; DnaB; host defense. Tadao Hasegawa, Department of Bacteriology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi Mizuho-cho Mizuho-ku Nagoya, 467-8601 Japan. e-mail: [email protected]
Streptococcus pyogenes (S. pyogenes) is a Grampositive bacterium infecting the regions of the upper respiratory tract such as the tonsils and pharynx and it causes post-infection diseases such as rheumatic fever and glomerulonephritis. Furthermore, S. pyogenes causes other serious human diseases, including streptococcal toxic shock syndrome (STSS) (1, 2). Because of the worldwide prevalence of STSS, many studies have been conducted to identify the relevant virulence factors (3, 4). These factors include the M protein, streptococcal inhibitor of complement, streptococcal pyrogenic toxins, hemolysins, and several DNases (2). We have thus far identified several DNases (5–7) using twodimensional gel electrophoresis (2-DE) and mass analysis and also determined the significance of the Nga protein (8, 9). In addition, S. pyogenes produces many types of virulence-associated proteins in response to environmental stresses. We have Received 20 June 2014. Accepted 1 October 2014
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studied the influence of various culture conditions on the production of these exoproteins (10–13). The regulation of the expression of virulenceassociated proteins in S. pyogenes is primarily controlled by stand-alone response regulators and two-component regulatory systems (TCSs). Standalone transcriptional regulators have no identified sensory domains (14). A TCS consists of a sensor protein and a regulatory protein. CovRS (CsrRS) (15, 16), one of the 13 TCSs (17), has been most commonly analyzed. CovR was reported to influence the transcription of 15% of the S. pyogenes genome, including many genes involved in virulence (18). Mutation of CovRS was first reported to result in enhanced virulence in murine infection models (19). In addition, mutation of covS was suggested to be an extremely important step in the transition from a pharyngitis-causing strain to an invasive strain (20). Other reports also indicated a high frequency of CovRS mutations in STSS isolates (21, 22). One possible explanation is that a
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mutation within CovRS promotes resistance to neutrophil-mediated killing by altering the production of proteins such as Sda1, M protein, and hyaluronic acid capsule (23, 24). We have reported the alteration of culture supernatant protein profiles in covS mutant strains and in vivo human evidence of the significance of CovS mutation (25, 26). Thus far, we clarified the existence of some strains in which a transposon was inserted between covS and nrdR by analyzing clinically isolated group A and group G streptococcal strains ((25), data not shown). covS and nrdR are located adjacently in the S. pyogenes genome. nrdR is also located immediately upstream of dnaB and dnaI. We speculated that this insertion has some biological significance. Previously, we used a mouse infection model experiment to examine the virulence of S. pyogenes (7, 27). In addition to this mouse analysis, we also used an in vitro bactericidal assay using human blood, because we believe that the relationship between bacteria and phagocytic cells is critical in the study of the pathogenesis of virulent bacteria. The function and significance of NrdR and DnaB in the virulence of S. pyogenes are completely unknown. Hence, we attempted to reveal their significance in virulence, with special reference to the response to host defense in this study. MATERIALS AND METHODS S. pyogenes strain The S. pyogenes strain used in this study was the emm1 1529 strain isolated from a Japanese patient with STSS (11, 25). Bacteria were either cultured in brain heart infusion (Eiken Chemical, Tokyo, Japan) supplemented with 0.3% yeast extract (Becton Dickinson, Sparks, MD, USA) (BHI-YE) broth without agitation or grown on BHI-YE agar plates at 37°C.
Creation of the knockout and complemented strains Nonpolar inactivated mutants of nrdR and dnaB were constructed through a double-crossover allelic replacement in the chromosome of S. pyogenes 1529 (25). The method was virtually identical as that previously published (7, 27). All PCRs were performed using the PrimeSTAR HS DNA polymerase (Takara, Ohtsu, Japan), which yields blunt-ended DNA fragment. The DNA fragment of nrdR and dnaB was amplified with oligonucleotide primers nrdn5Sma (50 -TCCCCCGGGGTCTACAAGAGTTTCAAG GAC -30 ) and nrd-c4Spe (50 -GGACTAGTGTTTCATA CCAAACTGTAGG-30 ) (Fig. 1A, B) which have SmaI and SpeI site, respectively (fragment 1). The SmaI-SpeI digested fragment 1 was subcloned into the SmaI-SpeI site of the pFW12 vector (28) (named pTHnrd1). A second round of PCR was performed using nrd-n1Nhe (50 -GGCT AGCAAAGAGAGTTAGAGTAGCTT-30 ) and covS-c2
© 2014 APMIS. Published by John Wiley & Sons Ltd
(50 -GTAATTACATTTTGGACAAC-30 ) (fragment 2). Both the NheI digested fragment 2 and the spc1 DNA fragment containing aad9 (a promoterless spectinomycin resistance gene) obtained from a SmaI-digested fragment of pSL60-1 (28) were cloned into the NheI-SmaI site of pTHnrd1 (named pTHnrd2). Another DNA fragment of nrdR and dnaB was amplified with oligonucleotide primers nrd-n2Sma (50 -TCCCCCGGGACCAATTGATACTTTTA CAT-30 ) and nrd-c1Spe (50 -GGACTAGTGATAGCTGA TACACACTCAC-30 ) which also have SmaI and SpeI site, respectively (fragment 3). The SmaI-SpeI digested fragment 3 was subcloned into the pTHnrd2 vector (named pTHnrd3). When the pTHnrd2 vector was successfully integrated into the genome, nrdR-only was knocked out and an intact DnaB protein was expressed; however, when the pTHnrd3 vector was successfully integrated into the genome, nrdR was also knocked out and a truncated DnaB protein lacking some N-terminal amino acids was expressed, because the original start codon ATG was deleted and it is possible that alternative start codon located downstream of original ATG was used (Fig. 1B). These plasmids, pTHnrd2 and pTHnrd3, were suicide vectors for S. pyogenes. To prepare competent cells, strains were harvested at an early- to mid-log phase (OD660 = 0.4–0.5) and washed twice with 0.5 M sucrose buffer. The constructed suicide vector was transformed into the strain by electroporation. Conditions of electroporation were 1.25 kV/mm, a capacitance of 25 lF, and a resistance of 200 ohms using the Gene Pulser II (Biorad, Hercules, CA, USA). After incubation at 37°C for 3 h, competent cells were spread onto BHI-YE agar plates containing spectinomycin (final concentration, 100 lg/mL). Selected colonies on the plates were cultured and were then washed once with saline, resuspended in 10 mM Tris/ 1 mM ethylenediaminetetraacetic acid, and boiled for 15 min. Genomic DNA was obtained from the supernatant of boiled bacteria. The double-crossover replacement with genomic DNA was analyzed by PCR. Successful double-crossover replacement was further confirmed by DNA sequencing. To construct a plasmid for nrdR-only or nrdR and dnaB-complemented, the DNA fragment was amplified with the oligonucleotide primers nrd-n1Nhe/nrdc1Spe or nrd-n1Nhe/dnaB-c1(50 -TTGCTACTTCAGGA TCCGCT-30 ), respectively. The fragment was treated with T4 polynucleotide kinase and ligated into the SmaI site of the pLZ12-Km2 plasmid (29). The protocol for transformation was identical to the aforementioned protocol, except that the competent cells were spread onto BHI-YE agar plates containing spectinomycin and kanamycin (final concentrations, 100 lg/mL, respectively).
Disk diffusion assays for H2O2 sensitivity An aliquot of bacterial stock solution stored frozen at 80°C was inoculated into 3 mL BHI-YE and cultured overnight at 30°C without agitation. Thirty-six microliters of the overnight culture was added to 3.6 mL Todd– Hewitt broth (Becton Dickinson) supplemented with 0.3% yeast extract (Becton Dickinson) (TH-YE) and cultured at 37°C without agitation. When bacterial cells grew to the late-exponential phase as monitored by a colorimeter (Asahi Science) (OD660 = 0.95), the turbidity was adjusted to match a 0.5 MacFarland turbidity standard using BHI. The diluted samples were inoculated onto the TH-YE agar
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A
B
Fig. 1. Strategies for the creation of knockout strains. (A) Strategies used to create the nrdR-only knockout, and nrdR and partial dnaB knockout strains. The primers used for the construction of plasmid for knockout and the deleted region of dnaB gene are shown. (B) Nucleotide and amino acid sequences around the junctions of nrdR and dnaB. The primer positions of n2-Sma, n5-Sma, c1-Spe, and c4-Spe are double underlined. The original methionine of DnaB is indicated as circled M, and a possible alternative start methionine of truncated DnaB is indicated as boxed M. In addition, putative deleted amino acids in nrdR and partial dnaB knockout strain are indicated using broken line.
plates by swabbing over the entire surface in three directions (30). Every sterile disk (diameter, 6.5 mm; cut from thin chromatography paper) was saturated with 20 lL of 275 mM H2O2, and three disks were evenly placed on every inoculated plate. After 24 h of incubation at 37°C, the diameter of the zone of inhibition was measured (31).
Lancefield bactericidal assay The Lancefield bactericidal assay (32) was performed as described previously (7, 27). In brief, approximately 5000 CFUs of the strain (OD660 = 0.75) were added to 1 mL of heparinized human whole blood and 1 ml of BHI-YE, respectively, to clarify the function of neutrophil killing and rotated at 37°C. Diluted samples of blood and BHI-YE were plated on the BHI-YE agar plates at the indicated time points to determine the number of CFUs.
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The experiments were repeated four times. The results are presented as the mean CFUs at each time point and the killing ratios at specific times were tested for significance using the Student’s t-test.
Mouse model of skin invasion and soft tissue infection The procedure was the same as that described previously (7, 27). In brief, S. pyogenes was harvested after 16 h of growth on a BHI-YE agar plate. These cells were harvested into 1 ml of PBS and centrifuged at 2000 g for 2 min. The pellets were diluted to 1 9 108 CFUs in 100 lL of PBS and injected (with a 25-gauge needle) subcutaneously into 3-week-old female Slc:ICR mice anesthetized by ketamine–xylazine injections. One hundred microliters of PBS without bacteria was injected into the control mice. A total of 36 mice were used for the © 2014 APMIS. Published by John Wiley & Sons Ltd
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experiment. The number of CFUs injected was verified for each experiment by plating bacteria on BHI-YE agar plates and counting CFUs. All animal procedures were approved by the Institutional Animal Care and Use Committee of Nagoya City University.
RESULTS Establishment of nrdR and dnaB knockout strains
We successfully established both nrdR knockout strains that expressed intact DnaB and truncated DnaB using pTHnrd2 and pTHndr3 plasmids, respectively (Fig. 1A). Truncated DnaB was considered to lose several amino acids in N-terminal helical domain (NTD), but to retain a linker domain and a RecA-core C-terminal domain (CTD) (33, 34). We unsuccessfully attempted to establish both nrdR and complete dnaB knockout strain and we did not succeed in establishing the dnaB-only knockout strain that carried the intact nrdR, either (the method and data not shown). Although nrdR and partial dnaB mutant strain exhibited delayed growth, they reached the stationary phase at the same time as the wild type and nrdR-only knockout strains (Fig. 2). The culture supernatant proteins in these established knockout strains were analyzed by SDS-PAGE; however, the production of SpeB or SIC, that are two of the important virulence-associated proteins in emm1 type S. pyogenes (25), was not significantly altered (data not shown). Analysis of sensitivities to H2O2
We performed disk diffusion analysis to test the difference of the sensitivities of the strains to H2O2. The zones of inhibition caused by H2O2 were measured, and the significance of the zone of inhibition
of each strain relative to that of the wild type and complemented strains was determined by t-test. nrdR and partial dnaB mutant strain was more sensitive to H2O2 than the wild type parental strain (Figs. 3A and 3B). nrdR- and dnaB-complemented strain displayed similar sensitivity as that of the parental strain (Fig. 3A), and nrdR-only complemented strain also displayed restored sensitivity (Fig. 3B). The nrdR-only knockout strain was a little sensitive, but the complement experiment did not work (Fig. 3C). In vitro blood bactericidal assay
To determine whether NrdR and DnaB affected the bactericidal function of human leukocytes, we performed bactericidal assays using human blood. The killing ratios suggested that nrdR and partial dnaB knockout strain was destroyed more efficiently by human blood cells than the parental strain (Figs 4A and 4B). The killing ratio of the nrdR and dnaB genes complemented strain was restored (Fig. 4A), but not nrdR-only complemented strain (Fig. 4B). In addition, there was no significant difference in the killing ratios between the nrdR-only mutant and the wild type strains (Fig. 4C). Analysis of virulence in a mouse infection model
To examine the virulence in vivo, we performed mouse infection model experiments using the parental strain 1529, and nrdR and partial dnaB knockout strain. As shown in Fig. 5A, the mice injected with the knockout strain survived longer than those injected with the parental strain (p < 0.001). The nrdR-only knockout strain did not display a significant survival difference (Fig. 5B, p > 0.05). These results suggest that DnaB plays a role in S. pyogenes infection in the mouse infection model experiments.
DISCUSSION
Fig. 2. Growth curves of streptococcal strains in the BHIYE broth. Each strain was cultured in 4 mL of BHI-YE broth, and growth was monitored using a colorimeter at OD660. © 2014 APMIS. Published by John Wiley & Sons Ltd
We previously reported that one S. pyogenes strain, MDMH from the blood and cerebrospinal fluid of patient with extremely severe STSS had a transposon between covS and nrdR (25). In addition, we recently identified some group G streptococcal strains that have the same transposon insertion. The location of nrdR is reported to be immediately upstream of dnaB and dnaI in Bacillus, Streptococcus, Lactobacillus, and Staphylococcus (35), and these regions appear to be operons. We speculated that this transposon insertion has some biological roles because of the functions of NrdR and DnaB.
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A
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Fig. 3. Sensitivities to H2O2. Disk diffusion assays were performed using the wild type 1529 strain, its knockout strains, and their complemented strains. (A) represents the data of the wild type strain 1529, its derived nrdR and partial dnaB knockout strain, and nrdR and dnaB complemented strain; (B) represents the data of the wild type strain 1529, its derived nrdR and partial dnaB knockout strain, and nrdR-only complemented strain; (C) represents the data of the wild type strain 1529, nrdR-only knockout strain, and nrdR complemented strain.
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Fig. 4. In vitro blood bactericidal assay. (A) The wild type strain 1529, its derived nrdR and partial dnaB knockout strain, and nrdR and dnaB complemented strain; (B) the wild type strain 1529, its derived nrdR and partial dnaB knockout strain, and nrdR-only complemented strain; or (C) the wild type strain 1529, nrdR-only knockout strain, and nrdR complemented strain were added to human whole blood and BHI-YE, respectively, and after each hour, samples were plated on BHI-YE agar. The data of killing ratio, which equal the number of CFUs from human blood divided by that from BHI-YE at 3 h, are indicated.
NrdR was first described in Streptomyces coelicolor (36), and it has been suggested to negatively regulate the transcription of nrd operons encoding ribonucleotide reductases (RNRs) in vitro (36–42). RNRs are conserved from prokaryotes to eukaryotes and they are responsible for the de novo synthesis of all four deoxyribonucleotides, the immediate precursors of DNA synthesis and repair, by reducing their respective ribonucleotides (43, 44). S. pyogenes harbors three different clusters of genes involved in ribonucleotide reduction (17). One cluster contains the genes for anaerobic class III RNR and its cognate activase (nrdDG). S. pyogenes is one of the few bacteria that possess two distinct class Ib RNR gene clusters, namely nrdHEF and nrdF*I*E*. The S. pyogenes genome also contains a second stand-alone copy of an nrdI 256
gene (nrdI2 (45)) at another location in the chromosome. The redundancy of class Ib RNR genes may be important under different environmental conditions. The expression of some RNRs is elevated by DNA damage and the inhibitors of DNA synthesis, as well as in response to oxidative stressors (46–49). Furthermore, Panosa et al. presented data illustrating that RNRs are involved in macrophage infection in Salmonella typhimurium (39). On the other hand, DnaB has a helicase activity (33, 50), and it plays an important role in the maintenance of genome stability (51). In addition, DnaB is active during DNA damage such as that caused by oxidative stress (52, 53). These studies strongly indicate that both NrdR and DnaB play important roles against oxidative stresses that cause DNA damage. We consider that the © 2014 APMIS. Published by John Wiley & Sons Ltd
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A
B
Fig. 5. Mouse infection model experiments. Survival after the subcutaneous injection with (A) the wild type strain 1529 and nrdR and partial dnaB knockout strains; (B) the wild type strain 1529 and nrdR-only knockout strains. Slc: ICR mice were injected subcutaneously with the wild type strain 1529 and knockout strains. Mice were observed daily. Survival data were assessed by Kaplan–Meier survival analysis and tested for significance by the log-rank test.
relationship between bacteria and phagocytic cells is critical in the study of the pathogenesis of virulent bacteria, and oxidative stresses that cause DNA damage are tightly associated with bacterial killing by phagocytic cells. To our knowledge, there are no reports regarding the role of NrdR and DnaB from S. pyogenes in response to host defense. Hence, we attempted to analyze the significance of these proteins. For the analyses of NrdR and DnaB, we first sought to establish several knockout strains, including nrdR-only and dnaB. However, we were unable to establish strains lacking dnaB. As DnaB is involved in the maintenance of genome stability, this failure appeared to be inevitable. Next, we used several plasmids for the creation of knockout strains and succeeded in the establishment of nrdR and partial dnaB knockout strain. The truncated DnaB protein lost some amino acids in N-terminal © 2014 APMIS. Published by John Wiley & Sons Ltd
helical domain (NTD), but it retained a linker domain and RecA-core C-terminal domain (CTD) (33, 34). Although NTD was reported to be required for helicase activity (54–56), and it is possible that the function was somewhat attenuated, the truncated DnaB in our study retained the function to survive in a normal low-stress condition from the result of growth curve (Fig. 2). Thus, we used these nrdR-only and nrdR and partial dnaB knockout strains to analyze the significance of NrdR and DnaB. As we performed oxidative stress experiment by only using H2O2, we are not sure that nrdR and partial dnaB knockout shows a higher sensitivity against general oxidative stresses. But judging from the in vitro blood bactericidal assay, and mouse infection model experiment, we concluded that NrdR and DnaB from S. pyogenes were involved in response to host defense; however, the contribution of NrdR alone was not substantial. Because the function of NrdR as a regulatory factor in S. pyogenes is not completely revealed, it is possible that not all the expressions of redundant class Ib RNR genes were changed. Hence, the deletion of only NrdR had little effect. On the other hand, as the function of DnaB was insufficient after its truncation in stress condition, its relevance became more apparent. However, as nrdR and dnaB create an operon judging from the nucleotide sequence (Fig. 1B), both proteins could play important roles in response to host defense. We did not find the apparent phenotypic differences between the strain with a transposon insertion and the nrdR- and dnaB-complemented strain, nor did we reveal the significance of transposon insertion (data not shown). Further analyses are needed to elucidate the significance of transposon insertion and the relationship between the proteins, including RNRs regulated by nrdR, DnaB, and DnaI. This study was supported by MEXT/JSPSKAKENHI Grant Number 24590531 from the Ministry of Education, Science and Culture of the Japanese government. The authors thank Enago (www.enago.jp) for the English language review.
REFERENCES 1. Cone LA, Woodard DR, Schlievert PM, Tomory GS. Clinical and bacteriologic observations of a toxic shock-like syndrome due to Streptococcus pyogenes. N Engl J Med 1987;317:146–9. 2. Cunningham MW. Pathogenesis of group A streptococcal infections. Clin Microbiol Rev 2000;13:470– 511.
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3. Hauser AR, Stevens DL, Kaplan EL, Schlievert PM. Molecular analysis of pyrogenic exotoxins from Streptococcus pyogenes isolates associated with toxic shock-like syndrome. J Clin Microbiol 1991;29: 1562–7. 4. Reichardt W, Muller-Alouf H, Alouf JE, Kohler W. Erythrogenic toxins A, B and C: occurrence of the genes and exotoxin formation from clinical Streptococcus pyogenes strains associated with streptococcal toxic shock-like syndrome. FEMS Microbiol Lett 1992;79:313–22. 5. Hasegawa T, Torii K, Hashikawa S, Iinuma Y, Ohta M. Cloning and characterization of the deoxyribonuclease sda gene from Streptococcus pyogenes. Curr Microbiol 2002;45:13–7. 6. Hasegawa T, Torii K, Hashikawa S, Iinuma Y, Ohta M. Cloning and characterization of two novel DNases from Streptococcus pyogenes. Arch Microbiol 2002;177:451–6. 7. Hasegawa T, Minami M, Okamoto A, Tatsuno I, Isaka M, Ohta M. Characterization of a virulenceassociated and cell-wall-located DNase of Streptococcus pyogenes. Microbiology 2010;156:184–90. 8. Tatsuno I, Sawai J, Okamoto A, Matsumoto M, Minami M, Isaka M, et al. Characterization of the NAD-glycohydrolase in streptococcal strains. Microbiology 2007;153:4253–60. 9. Tatsuno I, Isaka M, Minami M, Hasegawa T. NADase as a target molecule of in vivo suppression of the toxicity in the invasive M-1 group A Streptococcal isolates. BMC Microbiol 2010;10:144. 10. Nakamura T, Hasegawa T, Torii K, Hasegawa Y, Shimokata K, Ohta M. Two-dimensional gel electrophoresis analysis of the abundance of virulent exoproteins of group A streptococcus caused by environmental changes. Arch Microbiol 2004;181:74– 81. 11. Tanaka M, Hasegawa T, Okamoto A, Torii K, Ohta M. Effect of antibiotics on group A Streptococcus exoprotein production analyzed by two-dimensional gel electrophoresis. Antimicrob Agents Chemother 2005;49:88–96. 12. Sawai J, Hasegawa T, Kamimura T, Okamoto A, Ohmori D, Nosaka N, et al. Growth phase-dependent effect of clindamycin on production of exoproteins by Streptococcus pyogenes. Antimicrob Agents Chemother 2007;51:461–7. 13. Minami M, Kamimura T, Isaka M, Tatsuno I, Ohta M, Hasegawa T. Clindamycin-induced CovS-mediated regulation of the production of virulent exoproteins streptolysin O, NAD glycohydrolase, and streptokinase in Streptococcus pyogenes. Antimicrob Agents Chemother 2010;54:98–102. 14. Kreikemeyer B, McIver KS, Podbielski A. Virulence factor regulation and regulatory networks in Streptococcus pyogenes and their impact on pathogen-host interactions. Trends Microbiol 2003;11:224–32. 15. Levin JC. Wessels MR Identification of csrR/csrS, a genetic locus that regulates hyaluronic acid capsule synthesis in group A Streptococcus. Mol Microbiol 1998;30:209–19. 16. Federle MJ, McIver KS, Scott JR. A response regulator that represses transcription of several virulence operons in the group A streptococcus. J Bacteriol 1999;181:3649–57.
258
17. Ferretti JJ, McShan WM, Ajdic D, Savic DJ, Savic G, Lyon K, et al. Complete genome sequence of an M1 strain of Streptococcus pyogenes. Proc Natl Acad Sci USA 2001;98:4658–63. 18. Graham MR, Smoot LM, Migliaccio CA, Virtaneva K, Sturdevant DE, Porcella SF, et al. Virulence control in group A Streptococcus by a two-component gene regulatory system: global expression profiling and in vivo infection modeling. Proc Natl Acad Sci USA 2002;99:13855–60. 19. Engleberg NC, Heath A, Miller A, Rivera C, DiRita VJ. Spontaneous mutations in the CsrRS twocomponent regulatory system of Streptococcus pyogenes result in enhanced virulence in a murine model of skin and soft tissue infection. J Infect Dis 2001;183:1043–54. 20. Sumby P, Whitney AR, Graviss EA, DeLeo FR, Musser JM. Genome-wide analysis of group A streptococci reveals a mutation that modulates global phenotype and disease specificity. PLoS Pathog 2006;2:e5. 21. Ato M, Ikebe T, Kawabata H, Takemori T, Watanabe H. Incompetence of neutrophils to invasive group A streptococcus is attributed to induction of plural virulence factors by dysfunction of a regulator. PLoS ONE 2008;3:e3455. 22. Ikebe T, Ato M, Matsumura T, Hasegawa H, Sata T, Kobayashi K, et al. Highly frequent mutations in negative regulators of multiple virulence genes in group A streptococcal toxic shock syndrome isolates. PLoS Pathog 2010;6:e1000832. 23. Walker MJ, Hollands A, Sanderson-Smith ML, Cole JN, Kirk JK, Henningham A, et al. DNase Sda1 provides selection pressure for a switch to invasive group A streptococcal infection. Nat Med 2007;13:981–5. 24. Cole JN, Pence MA, von Kockritz-Blickwede M, Hollands A, Gallo RL, Walker MJ, et al. M protein and hyaluronic acid capsule are essential for in vivo selection of covRS mutations characteristic of invasive serotype M1T1 group A Streptococcus. MBio 2010;1: e00191–10. 25. Hasegawa T, Okamoto A, Kamimura T, Tatsuno I, Hashikawa SN, Yabutani M, et al. Detection of invasive protein profile of Streptococcus pyogenes M1 isolates from pharyngitis patients. APMIS 2010; 118:167–78. 26. Masuno K, Okada R, Zhang Y, Isaka M, Tatsuno I, Shibata S, et al. Simultaneous isolation of emm89type Streptococcus pyogenes strains with a wild-type or mutated covS gene from a single streptococcal toxic shock syndrome patient. J Med Microbiol 2014;63:504–7. 27. Ichikawa M, Minami M, Isaka M, Tatsuno I, Hasegawa T. Analysis of two-component sensor proteins involved in the response to acid stimuli in Streptococcus pyogenes. Microbiology 2011;157:3187–94. 28. Lukomski S, Hoe NP, Abdi I, Rurangirwa J, Kordari P, Liu M, et al. Nonpolar inactivation of the hypervariable streptococcal inhibitor of complement gene (sic) in serotype M1 Streptococcus pyogenes significantly decreases mouse mucosal colonization. Infect Immun 2000;68:535–42. 29. Okada N, Tatsuno I, Hanski E, Caparon M, Sasakawa C. Streptococcus pyogenes protein F promotes invasion of HeLa cells. Microbiology 1998;144:3079–86.
© 2014 APMIS. Published by John Wiley & Sons Ltd
ROLES OF NRDR AND DNAB FROM S. PYOGENES
30. Mirrett S, Reller LB. Comparison of direct and standard antimicrobial disk susceptibility testing for bacteria isolated from blood. J Clin Microbiol 1979;10:482–7. 31. Hoopman TC, Liu W, Joslin SN, Pybus C, Brautigam CA. Hansen EJ Identification of gene products involved in the oxidative stress response of Moraxella catarrhalis. Infect Immun 2011;79:745–55. 32. Lancefield RC. Differentiation of group A streptococci with a common R antigen into three serological types, with special reference to the bactericidal test. J Exp Med 1957;106:525–44. 33. LeBowitz JH, McMacken R. The Escherichia coli dnaB replication protein is a DNA helicase. J Biol Chem 1986;261:4738–48. 34. Bailey S, Eliason WK, Steitz TA. The crystal structure of the Thermus aquaticus DnaB helicase monomer. Nucleic Acids Res 2007;35:4728–36. 35. Rodionov DA, Gelfand MS. Identification of a bacterial regulatory system for ribonucleotide reductases by phylogenetic profiling. Trends Genet 2005;21:385–9. 36. Borovok I, Gorovitz B, Yanku M, Schreiber R, Gust B, Chater K, et al. Alternative oxygen-dependent and oxygen-independent ribonucleotide reductases in Streptomyces: cross-regulation and physiological role in response to oxygen limitation. Mol Microbiol 2004;54:1022–35. 37. Grinberg I, Shteinberg T, Gorovitz B, Aharonowitz Y, Cohen G, Borovok I. The Streptomyces NrdR transcriptional regulator is a Zn ribbon/ATP cone protein that binds to the promoter regions of class Ia and class II ribonucleotide reductase operons. J Bacteriol 2006;188:7635–44. 38. Mowa MB, Warner DF, Kaplan G, Kana BD, Mizrahi V. Function and regulation of class I ribonucleotide reductase-encoding genes in mycobacteria. J Bacteriol 2009;191:985–95. 39. Panosa A, Roca I, Gibert I. Ribonucleotide reductases of Salmonella typhimurium: transcriptional regulation and differential role in pathogenesis. PLoS ONE 2010;5:e11328. 40. Torrents E, Grinberg I, Gorovitz-Harris B, Lundstrom H, Borovok I, Aharonowitz Y, et al. NrdR controls differential expression of the Escherichia coli ribonucleotide reductase genes. J Bacteriol 2007;189: 5012–21. 41. Grinberg I, Shteinberg T, Hassan AQ, Aharonowitz Y, Borovok I, Cohen G. Functional analysis of the Streptomyces coelicolor NrdR ATP-cone domain: role in nucleotide binding, oligomerization, and DNA interactions. J Bacteriol 2009;191:1169–79. 42. Case ED, Akers JC, Tan M. CT406 encodes a chlamydial ortholog of NrdR, a repressor of ribonucleotide reductase. J Bacteriol 2011;193:4396–404.
© 2014 APMIS. Published by John Wiley & Sons Ltd
43. Nordlund P, Reichard P. Ribonucleotide reductases. Annu Rev Biochem 2006;75:681–706. 44. Reichard P. From RNA to DNA, why so many ribonucleotide reductases? Science 1993;260:1773–7. 45. Roca I, Torrents E, Sahlin M, Gibert I, Sjoberg BM. NrdI essentiality for class Ib ribonucleotide reduction in Streptococcus pyogenes. J Bacteriol 2008;190:4849– 58. 46. Gibert I, Calero S, Barbe J. Measurement of in vivo expression of nrdA and nrdB genes of Escherichia coli by using lacZ gene fusions. Mol Gen Genet 1990;220:400–8. 47. Jordan A, Aragall E, Gibert I, Barbe J. Promoter identification and expression analysis of Salmonella typhimurium and Escherichia coli nrdEF operons encoding one of two class I ribonucleotide reductases present in both bacteria. Mol Microbiol 1996;19:777– 90. 48. Monje-Casas F, Jurado J, Prieto-Alamo MJ, Holmgren A, Pueyo C. Expression analysis of the nrdHIEF operon from Escherichia coli. Conditions that trigger the transcript level in vivo. J Biol Chem 2001;276: 18031–7. 49. Prieto-Alamo MJ, Jurado J, Gallardo-Madueno R, Monje-Casas F, Holmgren A, Pueyo C. Transcriptional regulation of glutaredoxin and thioredoxin pathways and related enzymes in response to oxidative stress. J Biol Chem 2000;275:13398–405. 50. West SC. DNA helicases: new breeds of translocating motors and molecular pumps. Cell 1996;86:177–80. 51. Maher RL, Branagan AM, Morrical SW. Coordination of DNA replication and recombination activities in the maintenance of genome stability. J Cell Biochem 2011;112:2672–82. 52. Indiani C, O’Donnell M. A proposal: source of single strand DNA that elicits the SOS response. Front Biosci (Landmark Ed) 2013;18:312–23. 53. Inokuchi H, Ito R, Sekiguchi T, Sekiguchi M. Search for proteins required for accurate gene expression under oxidative stress: roles of guanylate kinase and RNA polymerase. J Biol Chem 2013;288:32952–62. 54. Nakayama N, Arai N, Kaziro Y, Arai K. Structural and functional studies of the dnaB protein using limited proteolysis. Characterization of domains for DNAdependent ATP hydrolysis and for protein association in the primosome. J Biol Chem 1984;259:88–96. 55. Bird LE, Pan H, Soultanas P, Wigley DB. Mapping protein-protein interactions within a stable complex of DNA primase and DnaB helicase from Bacillus stearothermophilus. Biochemistry 2000;39:171–82. 56. Mesa P, Alonso JC, Ayora S. Bacillus subtilis bacteriophage SPP1 G40P helicase lacking the N-terminal domain unwinds DNA bidirectionally. J Mol Biol 2006;357:1077–88.
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