Letters in Applied Microbiology ISSN 0266-8254
ORIGINAL ARTICLE
TceSR two-component regulatory system of Brucella melitensis 16M is involved in invasion, intracellular survival and regulated cytotoxicity for macrophages Z. Li1, Q. Fu1, Z. Wang1, T. Li1, H. Zhang1, F. Guo2, Y. Wang2, J. Zhang3 and C. Chen1 1 College of Animal Science and Technology/Co-Innovation Center for Zoonotic Infectious Diseases in the western region, Shihezi University, Shihezi, China 2 College of Medicine, Shihezi University, Shihezi, China 3 College of Biology, Agriculture and Forestry, Tongren University, Tongren, Guizhou, China
Significance and Impact of the Study: Two-component systems (TCSs) are predominant bacterial signal transduction mechanisms. The pathogenicity of Brucella is due to its ability to adapt to the intracellular environment including low levels of acidic pH, high-salt and heat shock. TCSs are designed to sense diverse stimuli, transfer signals and enact an appropriate adaptive physiological response. Here, we show that Br. meilitensis TCS TceSR is not only involved in regulation of Br. meilitensis virulence and adaptation of environmental stresses, but also can regulate cytotoxicity in macrophages.
Keywords 16MDTceSR, Brucella melitensis, cytotoxicity, two-component regulatory system, virulence. Correspondence Chuangfu Chen, College of Animal Science and Technology, Shihezi University, Shihezi 832000, China. E-mail: [email protected] 2014/2031: received 2 October 2014, revised 21 January 2015 and accepted 17 February 2015 doi:10.1111/lam.12408
Abstract The mechanisms of invasion and intracellular survival of Brucella are still poorly understood. Previous studies showed that the two-component regulatory systems (TCSs) play an important role in the intracellular survival of Brucella. To investigate if TCSs involve in the virulence and cytotoxicity of Brucella melitensis, we introduced a mutation into one of the TCSs in chromosome II in Br. melitensis 16M strain, and generated 16MDTceSR, a mutant of Br. melitensis 16M strain. In vitro infection experiments using murine macrophage cell line (RAW 264.7) showed that the survival of 16MDTceSR mutant in macrophages decreased 091-log compared with that of wild type Br. melitensis 16M strain at 2 h postinfection, replication of 16MDTceSR mutant in macrophages was 565-log, which was much lower than that wild type strain. Results of lactate dehydrogenase cytotoxicity assays in macrophages demonstrated high dose infection with wide type strain produced high level cytotoxicity to macrophages, but 16MDTceSR mutant had very low level cytotoxicity, indicating mutation of TCSs impaired the cytotoxicity of Br. melitensis to macrophages. Animal experiments showed that the spleen colonization of 16MDTceSR was significantly reduced compared with its wild type strains. The lower levels of survival of 16MDTceSR in various stress conditions suggested that the mutation of the TCSs of Br. melitensis was the causative factor of its reduced resistance to stress conditions. Taken together, our results demonstrated TCS TceSR involves in the intracellular survival, virulence and cytotoxicity of Br. melitensis during its infection.
Introduction Brucella spp. are intracellular bacteria that cause reproductive problems in animals and febrile illness in humans (Haque et al. 2011). Brucella spp. are the aetiological
agents of brucellosis, which causes great economic losses in many developing countries. According to conventional taxonomy, Brucella melitenesis, Br. abortus and Br. suis are virulent for humans (Young 1995). The pathogenesis of Brucella resides mostly in its intracellular lifestyle,
Letters in Applied Microbiology 60, 565--571 © 2015 The Society for Applied Microbiology
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Z. Li et al.
The regulatory roles of TceSR in macrophages
Results and discussion RAW 264.7 cells invasion Invasion was analysed by infecting RAW 264.7 cells with 16M and 16MDTceSR at an multiplicity of infection (MOI) of 100 and sampling the cultures at different times postinfection. At 15 min postinfection, there was a 033-log decrease (P < 005) in the bacterial number of 16MDTceSR compared to that of 16M. By 30 min postinfection, a 055-log decrease (P < 005) was observed in the bacterial number of 16MDTceSR compared to that of 16M. At 45 min post infection, there was a 095-log decrease (P < 001) in the bacterial number of 16MDTceSR compared to that of 16M. And in 1 h postinfection, a 091-log decrease (P < 001) was observed in the bacterial number of 16MDTceSR compared to that of 16M (Fig. 1). These results showed that 16MDTceSR mutant had a decreased invasion capability in RAW 264.7 murine macrophages than 16M. Survival and intracellular multiplication in RAW 264.7 macrophages The intracellular survival of 16M and 16MDTceSR was measured at different time points in RAW 264.7 566
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being able to invade, survive and multiply in host cells (Barbier et al. 2011). Brucellae have many regulatory systems that regulated harsh intracellular conditions, such as temperature, acidic pH, high salt and so on, in order to avoid fusion with lysosomes and promote bacterial multiplication. It has been demonstrated that there are many stress-associated proteins and virulence-related protein in Brucella, but the potential mechanisms the invasion, intracellular survival and multiplication of Brucella are still unknown. Previous research showed that BvrSR mutants are avirulent in mice and reduced invasiveness in cells (Seleem et al. 2008). So, two-component regulatory system (TCS) is one of important virulence regulatory systems in Brucella. Genome sequencing has revealed 21 putative TCSs in the Brucella genus (Viadas et al. 2010). TceSR is one of TCSs of Brucella, which is located in chromosome II (Lavin et al. 2010). Given the importance of stress adaptation, the regulatory roles of the TCS TceSR could be crucial in the virulence of Brucella, as has been demonstrated for other TCSs. However, to our knowledge, no studies of the TCS TceSR have been performed in Br. melitensis. The aim of our study is to research the regulatory roles of TCS TceSR from Br. melitensis 16M in environmental stress and virulence in macrophage and mice. And then, the cytotoxicity for macrophage was detected in macrophages infected with 16MDTceSR mutant strain.
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Figure 1 Intracellular invasion capability of different Brucella strains in RAW 264.7 murine macrophages. Macrophages were infected with 16M (white bars) and 16MDTceSR (black bars) at a MOI of 100. At the indicated different time points, macrophages were lysed and the number of bacteria was calculated by plating serial dilutions on tryptone soya agar plates. The results showed that the 16MDTceSR mutant was unable to achieve the level of invasion reached by the parental strain 16M. The results are the mean of three experiments SD. Significant differences between the mutant and parent strain are indicated by *(P < 005), **(P < 001).
macrophages infected at an MOI of 100. At 0 postinfection, there are no significant differences were evident between the strains in terms of intracellular survival. But at 4 h postinfection, 16MDTceSR exhibited 214-log decrease (P < 005) in survival than 16M. By 8 h postinfection, a 428-log decrease (P < 001) was observed in the bacterial number of 16MDTceSR compared to that of 16M. At 12 h postinfection, there was a 496-log decrease (P < 001) in the bacterial number of 16MDTceSR compared to that of 16M. And in 24 h postinfection, a 565-log decrease (P < 001) was observed in the bacterial number of 16MDTceSR compared to that of 16M (Fig. 2). These results showed that 16MDTceSR mutant had a decreased survival capability in RAW 264.7 murine macrophages than 16M, indicating that 16MDTceSR was attenuated compared with Br. melitensis 16M for survival in RAW 264.7 murine macrophages. Residual virulence in the murine model To determine residual virulence in BALB/c mice, different groups of mice were inoculated i.p. with Br. melitensis strains (1 9 106 CFU). The number of Brucella in the spleen was detected 2, 4, 6, 8 and 10 weeks postinfection. Compared to 16M-infected mice, splenic CFU in 16MDTceSR-infected mice were significantly reduced (P < 001). Significantly, the 16MDTceSR was cleared in
Letters in Applied Microbiology 60, 565--571 © 2015 The Society for Applied Microbiology
Z. Li et al.
The regulatory roles of TceSR in macrophages
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Brucella melitensis 16M is cytotoxic for macrophages when infected at a high MOI
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Figure 2 Intracellular replication capability of 16MDTceSR mutant in RAW 264.7 murine macrophages. Macrophages were infected with 16M (white bars) and 16MDTceSR (black bars) at a MOI of 100. The level of initial infection was the same for the two Brucella melitensis strains. At 0, 4, 8, 12 and 24 h postinfection, infected macrophages were lysed and supernatants were diluted for CFU enumeration. The results show that the 16MDTceSR mutant was unable to achieve the level of colonization reached by the parental strain Br. melitensis M590. The results are the mean of three experiments SD. Significant differences between the mutant and parent strain are indicated by *(P < 005), **(P < 001).
TceSR-regulated cytotoxicity of Brucella melitensis for macrophages
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Lactate dehydrogenase (LDH) is a stable cytosolic enzyme and is the most widely used marker in cytotoxicity studies. To detect whether the Br. melitensis 16M is cytotoxic for macrophages, RAW 264.7 macrophages were infected with 16M at MOI of 50, 100 and 1000, and LDH release was evaluated. At 12 h postinfection, for cells infected with 16M at an MOI of 50, only 460 093% LDH was released; however, for those infected at MOI of 100 and 1000, LDH release increased to 3055 156 and 5515 202% respectively (P < 001). At 24 h postinfection, LDH release was higher than that at 12 h postinfection for MOI of 50 (863 089%), 100 (4378 196%) and 1000 (7039 214%). However, at 48 h postinfection, all cells showed high levels of LDH release and no great differences between those infected at MOI of 50 (7268 066%), 100 (7571 129%) and 1000 (7835 163%) were observed (P > 005) (Fig. 4). All results indicated that when macrophages were infected with a high MOI, cytotoxicity was observed; and when postinfection was prolonged, macrophages infected showed high levels of cytotoxicity.
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Figure 3 Clearance of different Brucella strains after infection. BABL/c mice were infected with 1 9 106 CFU of 16M (circles) or 16MDTceSR (squares). At 2, 4, 6, 8 and 10 weeks postinfection, spleens were removed aseptically and bacteria were calculated by plating serial dilutions on tryptone soya agar plates. The bacterial numbers of spleens were determined. Values are means from individual mice SD (n = 10 per time point). Differences in splenic colonization were determined via the unpaired t-test between 16MDTceSR and 16M (*P < 005, **P < 001).
spleen of mice by 10 weeks postinoculation (Fig. 3). All results demonstrated that, in vivo, the 16MDTceSR mutant was less virulent than the parental strain.
To determine whether the cytotoxicity of Br. melitensis 16M for macrophages was regulated by TCS TceSR, we used 16M and 16MDTceSR-infected RAW 264.7 macrophages at MOI of 50, 100 and 1000, and evaluated the LDH release. At different times postinfection, for cells infected at an MOI of 50, no significant differences were evident between the strains in levels of LDH release. At 12 h postinfection, for cells infected at an MOI of 100, 3055 156% of LDH was released in 16M-infected cells; but only 227 052% of LDH was released in 16MDTceSR-infected cells. Even when infected with 16MDTceSR at an MOI of 2000, only 364 081% of LDH was released (Fig. 5a). At 24 h postinfection, for cells infected at an MOI of 100, 4378 196% of LDH was released in 16M-infected cells; but only 556 153% of LDH was released in 16MDTceSR-infected cells (Fig. 5b). However, at 48 h postinfection, all cells showed high levels of LDH released in cells infected with both 16M and 16MDTceSR (Fig. 5c). All results indicated that the 16MDTceSR mutant did not induce high levels of cytotoxicity in macrophages at any MOI by 12 and 24 h postinfection. Furthermore, the cytotoxicity of strains 16MDTceSR and 16M for macrophages at 48 h postinfection may result from a nutritionally deprived environment due to the long infection time. Therefore, there
Letters in Applied Microbiology 60, 565--571 © 2015 The Society for Applied Microbiology
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Figure 4 Brucella melitenesis 16M cytotoxicity in RAW 264.7 macrophage. Macrophages were infected with 16M at MOI of 50 (white bars), 100 (black bars) and 1000 (grey bars). At the indicated time points, the supernatant was collected and the lactate dehydrogenase released by the infected cells was detected by using the CytoTox 96 nonradioactive cytotoxicity assay. The results shown are the means SD of a representative experiment that was repeated three times. Significant differences among 50 MOI, 100 MOI and 1000 MOI are indicated by ** (P < 001).
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were no differences in the cytotoxicity level for the macrophages at 48 h postinfection between 16MDTceSR mutant and 16M. These results showed that the cytotoxicity of Br. melitensis 16M for macrophages was regulated by TCS TceSR. Zhong et al. and Pei et al. demonstrated that the cytotoxicity of the Brucella strain was virB-dependent (Pei et al. 2008; Zhong et al. 2009). In our current report, we found that the cytotoxicity of the Brucella strain was also regulated by TCS TceSR. This implies that the cytotoxicity phenomenon of Brucella for macrophages is not unique to T4SS-dependent.
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Mutation of TceSR affects survival of Brucella under stress conditions
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To survive in host cells, Brucella must adapt to the intracellular environment. To determine whether the mutation of TceSR affects the survival of Brucella adaptation to the intracellular environment, the survival of 16M and 16MDTceSR under various stress conditions simulating intracellular environments was compared. These stress environments include that low pH (pH 25), high salt (15 mol l1 NaCl) and high temperature (50°C). Compared to 16M, the survival of 16MDTceSR under these conditions was reduced (Fig. 6) (P < 001). Brucellae are intracellular pathogens; they must adapt to various intracellular environments and interfere with host cell signals for survival. The TCSs of Brucella have been shown to be an essential virulence determinant, which allows Brucella 568
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Figure 5 Two-component regulatory system TceSR-regulated cytotoxicity of Brucella melitenesis for macrophage. RAW 264.7 murine macrophages were infected with 16M (white bars) and 16MDTceSR (black bars) at MOI of (a) 50, (b) 100 and (c) 1000. Macrophage cytotoxicity was determined at different times postinfection using the lactate dehydrogenase release assay. The results are the means SD deviations of a representative experiment that was repeated three times with similar results. Significant differences among 50 MOI, 100 MOI and 1000 MOI are indicated by **(P < 001).
to adjust gene expression in response to environmental stimuli. Our results showed that deletion of TCS TceSR changed the environmental adaptability of Brucella, and
Letters in Applied Microbiology 60, 565--571 © 2015 The Society for Applied Microbiology
Z. Li et al.
The regulatory roles of TceSR in macrophages
mental procedures and animal care were performed in compliance with institutional animal care regulations.
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Construction of the 16MDTceSR
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The TceSR promoter region was predicted using NEURAL software (http:// NETWORK PROMOTER PREDICTION www.cbs.dtu.dk/services/Promoter/). For construction of the TceSR deletion mutant, two pairs of primers with restriction sites at 50 ends were designed for amplification of the upstream (1030 bp) and downstream (1030 bp) arms of the Br. melitensis TceSR promoter, in which the XhoI, KpnI, KpnI and SacI (underlined) sites were integrated into both PCR fragment ends. Primer sequences were as follows: TceSR-N-F, CTC GAG CTT GCC TTT TCC TTC GTC AT; TceSR-N-R, GGT ACC TCA AGC CGC ATT TGC TTG; TceSR-C-F, GGT ACC TTG AAA GAA GAC GCC CAC AT; TceSR-C-R, GAG CTC AGC ACG TCT TCC AGC TCT GC. The two arms of 16M TceSR promoter were cloned into pMD19-T Simple Vector for sequencing and then subcloned into pGEM-7Zf+ to generate the suicide plasmid pGEM-7Zf+-TceSR. One pair of primers was designed for SacB DNA fragment amplification. Primer sequences were as follows: SacB-F, GAG CTC GGG CTG GAA GAA GCA GAC CGC TA (SacI site); SacB-R, GAG CTC GCT TAT TTG TTA ACT GTT AAT TGT CC (SacI site). The 1475 bp fragment was amplified by PCR from Bacillus subtilis. The SacB fragment was subcloned into the plasmid pGEM-7Zf+-TceSR to generate the plasmid pGEM-7Zf+-TceSR-SacB. Competent 16M was electroporated with pGEM-7Zf+-TceSRSacB, and potential TceSR deletion mutant 16MDTceSR was isolated by its ampicillin and sucrose phenotype. The mutant was further confirmed by PCR amplification with primers TceSR-I-F (AAA ACA ACG GAA AAG GCT) and TceSR-I-R (TTT GCT GTT CCA TTG CCG T), which located in upstream and downstream of homologous arm of TceSR promoter respectively. PCR products were sequenced to confirm the sequence. The deletion mutant was further confirmed by PCR amplification and RT-PCR sequencing, as described previously (Wang et al. 2009).
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Figure 6 Deletion of TceSR affected on Brucella survival under stress conditions. Strains 16M (white bars) and 16MDTceSR (black bars) were first cultured in TSB to middle logarithmic phase and then subjected to the appropriate stress conditions, which were low pH (pH 25), high salt (15 mol l1 NaCl) and high temperature (50°C). After the treatment, the surviving bacteria were enumerated by plating serial dilutions onto tryptone soya agar plates. The results shown are the means SD of a representative experiment that was repeated three times. Significant differences between the mutant and parent strain are indicated by **(P < 001).
TCS TceSR is important for the ability of Brucella to adapt to stress conditions. In conclusion, our study provided further evidence that Br. melitensis 16M was cytotoxic for RAW 264.7 macrophages at higher levels of infection, and this cytotoxicity of the Brucella strain was also regulated by TCS TceSR. TCS TceSR mutant could not replicate and survive in RAW 264.7 macrophages and mice spleens. Furthermore, our report found that 16MDTceSR increased sensitivity to in vitro stress conditions. This result indicated that TCS TceSR is important for the ability of Brucella to adapt to intracellular environments. Therefore, it is possible that TCS TceSR regulated great changes of gene expression. The identification of more genes regulated by TCS TceSR and their functions will require further research. Materials and methods Bacterial strains, plasmids, cells and mice Brucella melitensis strain 16M was obtained from the Center of Chinese Disease Prevention and Control (Beijing, China). Brucella was cultured in tryptone soya agar (TSA) or tryptone soya broth (TSB) (Sigma, St. Louis, MO). Escherichia coli strain DH5a was grown on Luria–Bertani (LB) medium. Plasmid pGEM-7Zf+ was purchased from Promega (Madison, WI). The RAW 264.7 murine macrophage was purchased from Cell Resource Center, IBMS, CAMS/ PUMC (Beijing, China). Six-week-old BALB/c female mice were obtained from Experimental Animal Center of Academy Military Medical Science (Beijing, China). All experi-
RAW 264.7 macrophage invasion assay RAW 264.7 macrophages were infected with 16M and 16MDTceSR as previously described (Hernandez-Castro et al. 2008). RAW 264.7 cells were grown at 37°C in a 5% CO2 atmosphere in a minimal essential Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO, Waltham, MA) containing 10% heat-inactivated foetal bovine serum (FBS) (GIBCO). Macrophage invasion assays were performed as previously described. Briefly, cells were seeded (2 9 106) in six-well culture plates for 16 h at 37°C,
Letters in Applied Microbiology 60, 565--571 © 2015 The Society for Applied Microbiology
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infected with Brucella strains (16M and 16MDTceSR) at a multiplicity of infection (MOI) of 100. Culture plates were centrifuged for 5 min at 350 g at room temperature and then placed in a 5% CO2 atmosphere at 37°C (incubation point-0 min) during different time periods for invasion assays (15, 30, 45, and 60 min). Then, the cells were washed three times with phosphate buffer saline (PBS) and mono-layers of macrophages were further incubated with cell culture medium supplemented with gentamicin (100 lg ml1) (Invitrogen, Carlsbad, CA) for 1 h to kill the extracellular bacteria. Afterwards, the supernatant was discarded and the infected cells were washed three times with PBS and treated with Triton X100 (01% v/v). For bacterial counting, serial dilutions were performed and plated on TSA to determine colonyforming units (CFU). All assays were performed in triplicate and repeated three times. The results represent the means SD from at least three separate experiments. RAW 264.7 macrophage intracellular survival assay RAW 264.7 macrophages were cultured in six-well culture plates and infected with 16M and 16MDTceSR at a MOI of 100, as previously described (Pei and Ficht 2004). Culture plates were centrifuged for 5 min at 350 g at room temperature. Following 45 min incubation in a 5% CO2 atmosphere at 37°C, the cells were washed three times with PBS to remove the extracellular bacteria. Then the cells were incubated at 37°C in fresh DMEM supplemented with gentamicin (50 lg ml1) for 1 h to kill the extracellular bacteria. Subsequently, the culture was replaced with DMEM containing gentamicin (25 lg ml1) (defined as time zero). At different time points postinfection, the supernatant was discarded and cells were lysed by PBS containing Triton X100 (01% v/v). The number of CFU was obtained by plating serial dilutions of the lysates on TSA plates. All assays were performed in triplicate wells, and the results represent the means SD from at least three separate experiments. Mouse virulence assay Six-week-old female BALB/c mice were inoculated intraperitoneally (i.p.) with 200 ll PBS containing 1 9 106 CFU of 16M or 16MDTceSR. Infected mice were held in microisolator cages in biosafety level 3 facilities. Survival or persistence of the bacteria in mice was evaluated by enumerating the bacteria in the spleens at different time points postinfection. At 2, 4, 6 and 10 weeks postinoculation, mice were euthanized by CO2 asphyxiation and the spleens were removed aseptically. The spleens were collected and homogenized with 1 ml PBS containing Triton X-100 (01% v/v), 10-fold serially diluted, and plated on TSA plates. The plates were incu570
bated at 37°C, and the numbers of CFU were counted after 3 days. All assays were performed three times with similar results. Results were expressed as the means SD. All animal experiments were approved by the Academy of Military Medical Science Laboratory Animal Care and Use Committee and conducted in accordance with institutional guidelines. RAW 264.7 macrophage cytotoxicity assay RAW 264.7 cells seeded in six-well plates were infected with 16M and 16MDTceSR at MOI of 50, 100 or 1000 as described above. At different time points postinfection, the supernatant was collected, and the level of LDH was determined using the CytoTox 96 nonradioactive cytotoxicity assay as previously described (Pei and Ficht 2004). Cell death was expressed as the percentage of maximum LDH release, which was calculated using the following formula: percentage of LDH release = 100 9 (OD490 of infected cells — OD490 of uninfected cells)/(OD490 of lysed uninfected cells — OD490 of uninfected cells). The maximum release was determined following dissolution of cell monolayers using Triton X-100 (1% v/v). All assays were performed in triplicate wells, and the results represent the means SD from at least three separate experiments. In vitro environmental stress The sensitivity of Br. melitensis strains to in vitro environmental stress was determined as previously described (Ekaza et al. 2001; Kohler et al. 2002; Wang et al. 2010, 2014). Briefly, 16M and 16MDTceSR were inoculated into 10 ml of TSB medium and grown to the middle logarithmic phase (OD600 = 10) at 37°C. Then the Brucella strains subjected to different in vitro stress treatments. The in vitro stress conditions were chosen as previously described. To determine the effect of low pH stress, 16M and 16MDTceSR were inoculated into TSB medium at pH 25 for 5 min at 37°C. For high-salt stress, bacteria grown to exponential phase were incubated in 15 mol l1 NaCl for 30 min. For heat shock stress, bacteria were transferred to prewarmed 50°C tubes and incubated at 50°C for 1 h. After the treatment, the survival per cent of the bacteria was determined by plating serial dilutions of the Brucella cells on TSA plates. All the results represent the averages from at least three separate experiments. The results represent the means SD from at least three separate experiments. Statistical analysis Differences among means of the experimental groups were analysed using Student’s t-test, a P-value of
ORIGINAL ARTICLE
TceSR two-component regulatory system of Brucella melitensis 16M is involved in invasion, intracellular survival and regulated cytotoxicity for macrophages Z. Li1, Q. Fu1, Z. Wang1, T. Li1, H. Zhang1, F. Guo2, Y. Wang2, J. Zhang3 and C. Chen1 1 College of Animal Science and Technology/Co-Innovation Center for Zoonotic Infectious Diseases in the western region, Shihezi University, Shihezi, China 2 College of Medicine, Shihezi University, Shihezi, China 3 College of Biology, Agriculture and Forestry, Tongren University, Tongren, Guizhou, China
Significance and Impact of the Study: Two-component systems (TCSs) are predominant bacterial signal transduction mechanisms. The pathogenicity of Brucella is due to its ability to adapt to the intracellular environment including low levels of acidic pH, high-salt and heat shock. TCSs are designed to sense diverse stimuli, transfer signals and enact an appropriate adaptive physiological response. Here, we show that Br. meilitensis TCS TceSR is not only involved in regulation of Br. meilitensis virulence and adaptation of environmental stresses, but also can regulate cytotoxicity in macrophages.
Keywords 16MDTceSR, Brucella melitensis, cytotoxicity, two-component regulatory system, virulence. Correspondence Chuangfu Chen, College of Animal Science and Technology, Shihezi University, Shihezi 832000, China. E-mail: [email protected] 2014/2031: received 2 October 2014, revised 21 January 2015 and accepted 17 February 2015 doi:10.1111/lam.12408
Abstract The mechanisms of invasion and intracellular survival of Brucella are still poorly understood. Previous studies showed that the two-component regulatory systems (TCSs) play an important role in the intracellular survival of Brucella. To investigate if TCSs involve in the virulence and cytotoxicity of Brucella melitensis, we introduced a mutation into one of the TCSs in chromosome II in Br. melitensis 16M strain, and generated 16MDTceSR, a mutant of Br. melitensis 16M strain. In vitro infection experiments using murine macrophage cell line (RAW 264.7) showed that the survival of 16MDTceSR mutant in macrophages decreased 091-log compared with that of wild type Br. melitensis 16M strain at 2 h postinfection, replication of 16MDTceSR mutant in macrophages was 565-log, which was much lower than that wild type strain. Results of lactate dehydrogenase cytotoxicity assays in macrophages demonstrated high dose infection with wide type strain produced high level cytotoxicity to macrophages, but 16MDTceSR mutant had very low level cytotoxicity, indicating mutation of TCSs impaired the cytotoxicity of Br. melitensis to macrophages. Animal experiments showed that the spleen colonization of 16MDTceSR was significantly reduced compared with its wild type strains. The lower levels of survival of 16MDTceSR in various stress conditions suggested that the mutation of the TCSs of Br. melitensis was the causative factor of its reduced resistance to stress conditions. Taken together, our results demonstrated TCS TceSR involves in the intracellular survival, virulence and cytotoxicity of Br. melitensis during its infection.
Introduction Brucella spp. are intracellular bacteria that cause reproductive problems in animals and febrile illness in humans (Haque et al. 2011). Brucella spp. are the aetiological
agents of brucellosis, which causes great economic losses in many developing countries. According to conventional taxonomy, Brucella melitenesis, Br. abortus and Br. suis are virulent for humans (Young 1995). The pathogenesis of Brucella resides mostly in its intracellular lifestyle,
Letters in Applied Microbiology 60, 565--571 © 2015 The Society for Applied Microbiology
565
Z. Li et al.
The regulatory roles of TceSR in macrophages
Results and discussion RAW 264.7 cells invasion Invasion was analysed by infecting RAW 264.7 cells with 16M and 16MDTceSR at an multiplicity of infection (MOI) of 100 and sampling the cultures at different times postinfection. At 15 min postinfection, there was a 033-log decrease (P < 005) in the bacterial number of 16MDTceSR compared to that of 16M. By 30 min postinfection, a 055-log decrease (P < 005) was observed in the bacterial number of 16MDTceSR compared to that of 16M. At 45 min post infection, there was a 095-log decrease (P < 001) in the bacterial number of 16MDTceSR compared to that of 16M. And in 1 h postinfection, a 091-log decrease (P < 001) was observed in the bacterial number of 16MDTceSR compared to that of 16M (Fig. 1). These results showed that 16MDTceSR mutant had a decreased invasion capability in RAW 264.7 murine macrophages than 16M. Survival and intracellular multiplication in RAW 264.7 macrophages The intracellular survival of 16M and 16MDTceSR was measured at different time points in RAW 264.7 566
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being able to invade, survive and multiply in host cells (Barbier et al. 2011). Brucellae have many regulatory systems that regulated harsh intracellular conditions, such as temperature, acidic pH, high salt and so on, in order to avoid fusion with lysosomes and promote bacterial multiplication. It has been demonstrated that there are many stress-associated proteins and virulence-related protein in Brucella, but the potential mechanisms the invasion, intracellular survival and multiplication of Brucella are still unknown. Previous research showed that BvrSR mutants are avirulent in mice and reduced invasiveness in cells (Seleem et al. 2008). So, two-component regulatory system (TCS) is one of important virulence regulatory systems in Brucella. Genome sequencing has revealed 21 putative TCSs in the Brucella genus (Viadas et al. 2010). TceSR is one of TCSs of Brucella, which is located in chromosome II (Lavin et al. 2010). Given the importance of stress adaptation, the regulatory roles of the TCS TceSR could be crucial in the virulence of Brucella, as has been demonstrated for other TCSs. However, to our knowledge, no studies of the TCS TceSR have been performed in Br. melitensis. The aim of our study is to research the regulatory roles of TCS TceSR from Br. melitensis 16M in environmental stress and virulence in macrophage and mice. And then, the cytotoxicity for macrophage was detected in macrophages infected with 16MDTceSR mutant strain.
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Figure 1 Intracellular invasion capability of different Brucella strains in RAW 264.7 murine macrophages. Macrophages were infected with 16M (white bars) and 16MDTceSR (black bars) at a MOI of 100. At the indicated different time points, macrophages were lysed and the number of bacteria was calculated by plating serial dilutions on tryptone soya agar plates. The results showed that the 16MDTceSR mutant was unable to achieve the level of invasion reached by the parental strain 16M. The results are the mean of three experiments SD. Significant differences between the mutant and parent strain are indicated by *(P < 005), **(P < 001).
macrophages infected at an MOI of 100. At 0 postinfection, there are no significant differences were evident between the strains in terms of intracellular survival. But at 4 h postinfection, 16MDTceSR exhibited 214-log decrease (P < 005) in survival than 16M. By 8 h postinfection, a 428-log decrease (P < 001) was observed in the bacterial number of 16MDTceSR compared to that of 16M. At 12 h postinfection, there was a 496-log decrease (P < 001) in the bacterial number of 16MDTceSR compared to that of 16M. And in 24 h postinfection, a 565-log decrease (P < 001) was observed in the bacterial number of 16MDTceSR compared to that of 16M (Fig. 2). These results showed that 16MDTceSR mutant had a decreased survival capability in RAW 264.7 murine macrophages than 16M, indicating that 16MDTceSR was attenuated compared with Br. melitensis 16M for survival in RAW 264.7 murine macrophages. Residual virulence in the murine model To determine residual virulence in BALB/c mice, different groups of mice were inoculated i.p. with Br. melitensis strains (1 9 106 CFU). The number of Brucella in the spleen was detected 2, 4, 6, 8 and 10 weeks postinfection. Compared to 16M-infected mice, splenic CFU in 16MDTceSR-infected mice were significantly reduced (P < 001). Significantly, the 16MDTceSR was cleared in
Letters in Applied Microbiology 60, 565--571 © 2015 The Society for Applied Microbiology
Z. Li et al.
The regulatory roles of TceSR in macrophages
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Brucella melitensis 16M is cytotoxic for macrophages when infected at a high MOI
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Figure 2 Intracellular replication capability of 16MDTceSR mutant in RAW 264.7 murine macrophages. Macrophages were infected with 16M (white bars) and 16MDTceSR (black bars) at a MOI of 100. The level of initial infection was the same for the two Brucella melitensis strains. At 0, 4, 8, 12 and 24 h postinfection, infected macrophages were lysed and supernatants were diluted for CFU enumeration. The results show that the 16MDTceSR mutant was unable to achieve the level of colonization reached by the parental strain Br. melitensis M590. The results are the mean of three experiments SD. Significant differences between the mutant and parent strain are indicated by *(P < 005), **(P < 001).
TceSR-regulated cytotoxicity of Brucella melitensis for macrophages
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Lactate dehydrogenase (LDH) is a stable cytosolic enzyme and is the most widely used marker in cytotoxicity studies. To detect whether the Br. melitensis 16M is cytotoxic for macrophages, RAW 264.7 macrophages were infected with 16M at MOI of 50, 100 and 1000, and LDH release was evaluated. At 12 h postinfection, for cells infected with 16M at an MOI of 50, only 460 093% LDH was released; however, for those infected at MOI of 100 and 1000, LDH release increased to 3055 156 and 5515 202% respectively (P < 001). At 24 h postinfection, LDH release was higher than that at 12 h postinfection for MOI of 50 (863 089%), 100 (4378 196%) and 1000 (7039 214%). However, at 48 h postinfection, all cells showed high levels of LDH release and no great differences between those infected at MOI of 50 (7268 066%), 100 (7571 129%) and 1000 (7835 163%) were observed (P > 005) (Fig. 4). All results indicated that when macrophages were infected with a high MOI, cytotoxicity was observed; and when postinfection was prolonged, macrophages infected showed high levels of cytotoxicity.
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Figure 3 Clearance of different Brucella strains after infection. BABL/c mice were infected with 1 9 106 CFU of 16M (circles) or 16MDTceSR (squares). At 2, 4, 6, 8 and 10 weeks postinfection, spleens were removed aseptically and bacteria were calculated by plating serial dilutions on tryptone soya agar plates. The bacterial numbers of spleens were determined. Values are means from individual mice SD (n = 10 per time point). Differences in splenic colonization were determined via the unpaired t-test between 16MDTceSR and 16M (*P < 005, **P < 001).
spleen of mice by 10 weeks postinoculation (Fig. 3). All results demonstrated that, in vivo, the 16MDTceSR mutant was less virulent than the parental strain.
To determine whether the cytotoxicity of Br. melitensis 16M for macrophages was regulated by TCS TceSR, we used 16M and 16MDTceSR-infected RAW 264.7 macrophages at MOI of 50, 100 and 1000, and evaluated the LDH release. At different times postinfection, for cells infected at an MOI of 50, no significant differences were evident between the strains in levels of LDH release. At 12 h postinfection, for cells infected at an MOI of 100, 3055 156% of LDH was released in 16M-infected cells; but only 227 052% of LDH was released in 16MDTceSR-infected cells. Even when infected with 16MDTceSR at an MOI of 2000, only 364 081% of LDH was released (Fig. 5a). At 24 h postinfection, for cells infected at an MOI of 100, 4378 196% of LDH was released in 16M-infected cells; but only 556 153% of LDH was released in 16MDTceSR-infected cells (Fig. 5b). However, at 48 h postinfection, all cells showed high levels of LDH released in cells infected with both 16M and 16MDTceSR (Fig. 5c). All results indicated that the 16MDTceSR mutant did not induce high levels of cytotoxicity in macrophages at any MOI by 12 and 24 h postinfection. Furthermore, the cytotoxicity of strains 16MDTceSR and 16M for macrophages at 48 h postinfection may result from a nutritionally deprived environment due to the long infection time. Therefore, there
Letters in Applied Microbiology 60, 565--571 © 2015 The Society for Applied Microbiology
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Figure 4 Brucella melitenesis 16M cytotoxicity in RAW 264.7 macrophage. Macrophages were infected with 16M at MOI of 50 (white bars), 100 (black bars) and 1000 (grey bars). At the indicated time points, the supernatant was collected and the lactate dehydrogenase released by the infected cells was detected by using the CytoTox 96 nonradioactive cytotoxicity assay. The results shown are the means SD of a representative experiment that was repeated three times. Significant differences among 50 MOI, 100 MOI and 1000 MOI are indicated by ** (P < 001).
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were no differences in the cytotoxicity level for the macrophages at 48 h postinfection between 16MDTceSR mutant and 16M. These results showed that the cytotoxicity of Br. melitensis 16M for macrophages was regulated by TCS TceSR. Zhong et al. and Pei et al. demonstrated that the cytotoxicity of the Brucella strain was virB-dependent (Pei et al. 2008; Zhong et al. 2009). In our current report, we found that the cytotoxicity of the Brucella strain was also regulated by TCS TceSR. This implies that the cytotoxicity phenomenon of Brucella for macrophages is not unique to T4SS-dependent.
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Mutation of TceSR affects survival of Brucella under stress conditions
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To survive in host cells, Brucella must adapt to the intracellular environment. To determine whether the mutation of TceSR affects the survival of Brucella adaptation to the intracellular environment, the survival of 16M and 16MDTceSR under various stress conditions simulating intracellular environments was compared. These stress environments include that low pH (pH 25), high salt (15 mol l1 NaCl) and high temperature (50°C). Compared to 16M, the survival of 16MDTceSR under these conditions was reduced (Fig. 6) (P < 001). Brucellae are intracellular pathogens; they must adapt to various intracellular environments and interfere with host cell signals for survival. The TCSs of Brucella have been shown to be an essential virulence determinant, which allows Brucella 568
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Figure 5 Two-component regulatory system TceSR-regulated cytotoxicity of Brucella melitenesis for macrophage. RAW 264.7 murine macrophages were infected with 16M (white bars) and 16MDTceSR (black bars) at MOI of (a) 50, (b) 100 and (c) 1000. Macrophage cytotoxicity was determined at different times postinfection using the lactate dehydrogenase release assay. The results are the means SD deviations of a representative experiment that was repeated three times with similar results. Significant differences among 50 MOI, 100 MOI and 1000 MOI are indicated by **(P < 001).
to adjust gene expression in response to environmental stimuli. Our results showed that deletion of TCS TceSR changed the environmental adaptability of Brucella, and
Letters in Applied Microbiology 60, 565--571 © 2015 The Society for Applied Microbiology
Z. Li et al.
The regulatory roles of TceSR in macrophages
mental procedures and animal care were performed in compliance with institutional animal care regulations.
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Construction of the 16MDTceSR
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The TceSR promoter region was predicted using NEURAL software (http:// NETWORK PROMOTER PREDICTION www.cbs.dtu.dk/services/Promoter/). For construction of the TceSR deletion mutant, two pairs of primers with restriction sites at 50 ends were designed for amplification of the upstream (1030 bp) and downstream (1030 bp) arms of the Br. melitensis TceSR promoter, in which the XhoI, KpnI, KpnI and SacI (underlined) sites were integrated into both PCR fragment ends. Primer sequences were as follows: TceSR-N-F, CTC GAG CTT GCC TTT TCC TTC GTC AT; TceSR-N-R, GGT ACC TCA AGC CGC ATT TGC TTG; TceSR-C-F, GGT ACC TTG AAA GAA GAC GCC CAC AT; TceSR-C-R, GAG CTC AGC ACG TCT TCC AGC TCT GC. The two arms of 16M TceSR promoter were cloned into pMD19-T Simple Vector for sequencing and then subcloned into pGEM-7Zf+ to generate the suicide plasmid pGEM-7Zf+-TceSR. One pair of primers was designed for SacB DNA fragment amplification. Primer sequences were as follows: SacB-F, GAG CTC GGG CTG GAA GAA GCA GAC CGC TA (SacI site); SacB-R, GAG CTC GCT TAT TTG TTA ACT GTT AAT TGT CC (SacI site). The 1475 bp fragment was amplified by PCR from Bacillus subtilis. The SacB fragment was subcloned into the plasmid pGEM-7Zf+-TceSR to generate the plasmid pGEM-7Zf+-TceSR-SacB. Competent 16M was electroporated with pGEM-7Zf+-TceSRSacB, and potential TceSR deletion mutant 16MDTceSR was isolated by its ampicillin and sucrose phenotype. The mutant was further confirmed by PCR amplification with primers TceSR-I-F (AAA ACA ACG GAA AAG GCT) and TceSR-I-R (TTT GCT GTT CCA TTG CCG T), which located in upstream and downstream of homologous arm of TceSR promoter respectively. PCR products were sequenced to confirm the sequence. The deletion mutant was further confirmed by PCR amplification and RT-PCR sequencing, as described previously (Wang et al. 2009).
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Figure 6 Deletion of TceSR affected on Brucella survival under stress conditions. Strains 16M (white bars) and 16MDTceSR (black bars) were first cultured in TSB to middle logarithmic phase and then subjected to the appropriate stress conditions, which were low pH (pH 25), high salt (15 mol l1 NaCl) and high temperature (50°C). After the treatment, the surviving bacteria were enumerated by plating serial dilutions onto tryptone soya agar plates. The results shown are the means SD of a representative experiment that was repeated three times. Significant differences between the mutant and parent strain are indicated by **(P < 001).
TCS TceSR is important for the ability of Brucella to adapt to stress conditions. In conclusion, our study provided further evidence that Br. melitensis 16M was cytotoxic for RAW 264.7 macrophages at higher levels of infection, and this cytotoxicity of the Brucella strain was also regulated by TCS TceSR. TCS TceSR mutant could not replicate and survive in RAW 264.7 macrophages and mice spleens. Furthermore, our report found that 16MDTceSR increased sensitivity to in vitro stress conditions. This result indicated that TCS TceSR is important for the ability of Brucella to adapt to intracellular environments. Therefore, it is possible that TCS TceSR regulated great changes of gene expression. The identification of more genes regulated by TCS TceSR and their functions will require further research. Materials and methods Bacterial strains, plasmids, cells and mice Brucella melitensis strain 16M was obtained from the Center of Chinese Disease Prevention and Control (Beijing, China). Brucella was cultured in tryptone soya agar (TSA) or tryptone soya broth (TSB) (Sigma, St. Louis, MO). Escherichia coli strain DH5a was grown on Luria–Bertani (LB) medium. Plasmid pGEM-7Zf+ was purchased from Promega (Madison, WI). The RAW 264.7 murine macrophage was purchased from Cell Resource Center, IBMS, CAMS/ PUMC (Beijing, China). Six-week-old BALB/c female mice were obtained from Experimental Animal Center of Academy Military Medical Science (Beijing, China). All experi-
RAW 264.7 macrophage invasion assay RAW 264.7 macrophages were infected with 16M and 16MDTceSR as previously described (Hernandez-Castro et al. 2008). RAW 264.7 cells were grown at 37°C in a 5% CO2 atmosphere in a minimal essential Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO, Waltham, MA) containing 10% heat-inactivated foetal bovine serum (FBS) (GIBCO). Macrophage invasion assays were performed as previously described. Briefly, cells were seeded (2 9 106) in six-well culture plates for 16 h at 37°C,
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infected with Brucella strains (16M and 16MDTceSR) at a multiplicity of infection (MOI) of 100. Culture plates were centrifuged for 5 min at 350 g at room temperature and then placed in a 5% CO2 atmosphere at 37°C (incubation point-0 min) during different time periods for invasion assays (15, 30, 45, and 60 min). Then, the cells were washed three times with phosphate buffer saline (PBS) and mono-layers of macrophages were further incubated with cell culture medium supplemented with gentamicin (100 lg ml1) (Invitrogen, Carlsbad, CA) for 1 h to kill the extracellular bacteria. Afterwards, the supernatant was discarded and the infected cells were washed three times with PBS and treated with Triton X100 (01% v/v). For bacterial counting, serial dilutions were performed and plated on TSA to determine colonyforming units (CFU). All assays were performed in triplicate and repeated three times. The results represent the means SD from at least three separate experiments. RAW 264.7 macrophage intracellular survival assay RAW 264.7 macrophages were cultured in six-well culture plates and infected with 16M and 16MDTceSR at a MOI of 100, as previously described (Pei and Ficht 2004). Culture plates were centrifuged for 5 min at 350 g at room temperature. Following 45 min incubation in a 5% CO2 atmosphere at 37°C, the cells were washed three times with PBS to remove the extracellular bacteria. Then the cells were incubated at 37°C in fresh DMEM supplemented with gentamicin (50 lg ml1) for 1 h to kill the extracellular bacteria. Subsequently, the culture was replaced with DMEM containing gentamicin (25 lg ml1) (defined as time zero). At different time points postinfection, the supernatant was discarded and cells were lysed by PBS containing Triton X100 (01% v/v). The number of CFU was obtained by plating serial dilutions of the lysates on TSA plates. All assays were performed in triplicate wells, and the results represent the means SD from at least three separate experiments. Mouse virulence assay Six-week-old female BALB/c mice were inoculated intraperitoneally (i.p.) with 200 ll PBS containing 1 9 106 CFU of 16M or 16MDTceSR. Infected mice were held in microisolator cages in biosafety level 3 facilities. Survival or persistence of the bacteria in mice was evaluated by enumerating the bacteria in the spleens at different time points postinfection. At 2, 4, 6 and 10 weeks postinoculation, mice were euthanized by CO2 asphyxiation and the spleens were removed aseptically. The spleens were collected and homogenized with 1 ml PBS containing Triton X-100 (01% v/v), 10-fold serially diluted, and plated on TSA plates. The plates were incu570
bated at 37°C, and the numbers of CFU were counted after 3 days. All assays were performed three times with similar results. Results were expressed as the means SD. All animal experiments were approved by the Academy of Military Medical Science Laboratory Animal Care and Use Committee and conducted in accordance with institutional guidelines. RAW 264.7 macrophage cytotoxicity assay RAW 264.7 cells seeded in six-well plates were infected with 16M and 16MDTceSR at MOI of 50, 100 or 1000 as described above. At different time points postinfection, the supernatant was collected, and the level of LDH was determined using the CytoTox 96 nonradioactive cytotoxicity assay as previously described (Pei and Ficht 2004). Cell death was expressed as the percentage of maximum LDH release, which was calculated using the following formula: percentage of LDH release = 100 9 (OD490 of infected cells — OD490 of uninfected cells)/(OD490 of lysed uninfected cells — OD490 of uninfected cells). The maximum release was determined following dissolution of cell monolayers using Triton X-100 (1% v/v). All assays were performed in triplicate wells, and the results represent the means SD from at least three separate experiments. In vitro environmental stress The sensitivity of Br. melitensis strains to in vitro environmental stress was determined as previously described (Ekaza et al. 2001; Kohler et al. 2002; Wang et al. 2010, 2014). Briefly, 16M and 16MDTceSR were inoculated into 10 ml of TSB medium and grown to the middle logarithmic phase (OD600 = 10) at 37°C. Then the Brucella strains subjected to different in vitro stress treatments. The in vitro stress conditions were chosen as previously described. To determine the effect of low pH stress, 16M and 16MDTceSR were inoculated into TSB medium at pH 25 for 5 min at 37°C. For high-salt stress, bacteria grown to exponential phase were incubated in 15 mol l1 NaCl for 30 min. For heat shock stress, bacteria were transferred to prewarmed 50°C tubes and incubated at 50°C for 1 h. After the treatment, the survival per cent of the bacteria was determined by plating serial dilutions of the Brucella cells on TSA plates. All the results represent the averages from at least three separate experiments. The results represent the means SD from at least three separate experiments. Statistical analysis Differences among means of the experimental groups were analysed using Student’s t-test, a P-value of
