Introduction

Multicellular organisms use various defense strategies to protect themselves from microbial infections. Production of antimicrobial peptides (AMPs) is one of these strategies. As an early component of the host response, AMPs modulate the bacterial load and prevent the establishment of an infection1. The target of these antimicrobial peptides is postulated to be the cytoplasmic membrane of Gram-positive and Gram-negative bacteria. These peptides insert into the lipid bilayer to generate voltage-gated channels, resulting in the leakage of essential cellular components and, ultimately, the death of the microbe2.

The ability of a microbe to prosper within animal host environments requires the capacity to synthesize nutrients not available from host tissues and to avoid or resist being killed by the host niche3. Brucella spp. are facultative intracellular pathogens that cause abortion and infertility in animals and severe debilitating febrile illness in humans. Brucella evolved to exist in host macrophages in the presence of a number of host-imposed stresses, including acidic pH, bactericidal compounds and low nutrient availability4. The ability of brucellae to survive and replicate within macrophages is essential for their virulence5 and many stress-associated proteins6,7,8 and virulence determinants9 essential for Brucella to infect different hosts have been described.

Previous screening of Brucella virulence determinants in our lab resulted in the identification of a gene cluster located at chromosome I of the Brucella melitensis NI genome that encodes the components of a putative ABC-type microcin C transport system. The operon consists of five genes: yejA1 (BMNI_I0010) and yejA2 (BMNI_I0009), which encode putative extracellular solute-binding proteins; yejB (BMNI_I0008) and yejE (BMNI_I0007), which encode transport system permease components; and yejF (BMNI_I0006), which encodes the ABC transporter system ATP-binding protein. Whereas the Escherichia coli and Salmonella Typhimurium genomes also contain a gene cluster that encodes the components of a putative ABC-type dipeptide/oligopeptide/nickel transport system, this putative operon consists of four genes: yejA (b2177 and STM 2216), yejB (b2178 and STM 2217), yejE (b2179 and STM 2218) and yejF (b2180 and STM2219), all of which confer resistance to antimicrobial peptides and contribute to its virulence in Salmonella10. Bacterial ABC transporter systems are associated with nutrient uptake and the export of toxins and antibiotics and they also have a potential pathogenic role during host infection11. In Brucella spp., a polysaccharide ABC transporter is required for B. abortus pathogenesis in the murine model12. In addition, a predicted ABC transporter, AbcEDCBA, of Brucella ovis promotes intracellular survival by affecting T4SS protein expression at the post-transcriptional level and, consequently, contributing to B. ovis evasion of phagosome/lysosome fusion13. It has also been reported that the yejE and yejF genes of S. enterica serovars Typhimurium and Typhi were up-regulated inside host macrophages14,15, indicating the importance of these genes inside the host cells. Apart from these studies in Salmonella, no studies of the yejABEF operon have been performed in Brucella and as a result, not much is known about the function of the yej operon in Brucella.

The objective of the present study was to investigate the possible functions of yej operon genes and determine their role in the virulence of Brucella. In this study, we show that mutants of the yej operon are more sensitive to acid stress and polymyxin B, have reduced proliferation inside macrophages and have remarkably decreased virulence in a mouse model.

Results

Products of the yejA1, A2, B, E and F genes in Brucella share amino acid sequence similarity with the peptide transporters of Salmonella

The yej operon genes of Brucella are annotated as genes encoding components of a putative ABC transporter system. To examine their potential functions, we searched for the same operon in Salmonella. Bioinformatic analysis revealed that the proteins encoded by the yej operon share high amino acid sequence identity with those in Salmonella Typhimurium (Figure S1). The proteins YejB and YejE of B. melitensis NI have relatively high sequence identities, 63.9% and 64.2%, with those in S. Typhimurium. Analysis of the B. melitensis YejB and YejE sequences revealed the presence of a BPD_transp_1motif that can be defined as a binding-protein-dependent transport system inner membrane component. In addition to containing the BPD_transp_1motif, YejE also contains an OppC_N motif, which was defined as a N-terminal TM domain of oligopeptide transport permease C, similar to that in Salmonella. The sequence identity of YejF is 55.4% and it has the typicalABC_tran (ABC transporter) motif. YejA1 and YejA2 of B. melitensis are 35.4% and 34.4% identical to the Salmonella YejA. All the YejA proteins of Brucella and Salmonella contain the SBP_bac_5 motif (Bacterial extracellular solute-binding proteins, family 5 Middle). In addition to the SBP_bac_5 motif, YejA2 also contains a TAT_signal (Twin-arginine translocation pathway signal sequence) motif. The identities of YejABEF among B. melitensis NI, B. melitensis 16 M and B. abortus 2308 are over 99.5%; the blast results were not described in detail. It was reported that the yej operon genes contribute to virulence in Salmonella spp10,16. by counteracting AMPs. Based on these facts, we hypothesized that the transporter system encoded by the yej operon might be involved in conferring virulence to Brucella and may also be involved in counteracting AMPs, similar to the transporter system encoded by the yej operon in Salmonella.

Growth characteristics of yej operon gene deletion mutants

The dynamic growth profiles of the yej operon gene deletion mutants and the parent strain NI were determined in TSB and minimal medium. All yej mutants grew normally in TSB medium when compared with the parental strain over different time points (Fig. 1A). The minimal medium is a defined medium that contains only carbon and nitrogen nutrients. All these strains were able to grow in the minimal medium, indicating that the inorganic carbon and nitrogen resources provide sufficient nutrients for Brucella growth and replication. In addition, the yej operon gene deletion mutants showed similar growth profiles in minimal medium (Fig. 1B, P > 0.05), indicating that the yej operon gene deletion mutants were not compromised in their capacities to utilize limited nutrition.

Figure 1
figure 1

Growth characteristics of the yej gene deletion mutants and B. melitensis NI in TSB (A) and minimal media (B). Compared to the wild-type strain, all the yej mutants showed similar CFU/mL (TSB) and OD600 values (minimal media).

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YejABEF proteins contribute to resistance to acidic conditions

To investigate the effect of the yej operon on acid stress tolerance, brucellae survival was evaluated under a reduced pH level. After a 2-h exposure to a pH of 3.4 at 37 °C, all yej operon gene deletion mutants and the parental strain showed reduced bacterial viability. B. melitensis NI showed a 36% decrease in bacterial viability, whereas the yej operon gene deletion mutants NIΔyejA1, NIΔyejA2, NIΔyejB, NIΔyejE, NIΔyejF and NIΔyejABEF showed decreases of 58.6%, 77.2%, 72%, 91.7%, 70.5% and 91.3%, respectively. These results showed that except for NIΔyejA1, all other yej operon gene deletion mutants were more sensitive to acidic conditions than the parental NI strain (Fig. 2, P < 0.05).

Figure 2
figure 2

Survival rates of yej gene deletion mutants and B. melitensis NI under acidic conditions.

After 2 h of exposure to pH 3.4 minimal media, the survival rates of each strain were calculated. The presented values represent the means of three experiments performed in duplicate and the error bars indicate the SD. Significant differences between the strains are indicated by an asterisk (p < 0.05).

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Deletion of the yejE gene confers susceptibility to polymyxin B

Based on our hypothesis that the yej operon may confer resistance to AMP, we investigated the sensitivity of each yej gene deletion mutant to polymyxin B. Polymyxin B is a cationic peptide derived from Paenibacillus polymyxa that interacts with the outer and inner membranes of Gram-negative bacteria in a similar fashion to many AMPs17,18. The whole yej operon deletion mutant NIΔyejABEF was more sensitive to both concentrations of polymyxin B when compared with the parental NI strain (Fig. 3, P < 0.05), confirming the role of the yej operon in conferring the resistance of Brucella to AMP. The NIΔyejE mutant also showed an increased sensitivity to polymyxin B (Fig. 3, P < 0.05). Surprisingly, the mutants NIΔyejA1, NIΔyejA2, NIΔyejB and NIΔyejF did not show any increased sensitivity when compared with the parental NI strain in survival upon treatment with polymyxin B at both final concentrations (Fig. 3, P > 0.05). Introducing pBBRyejE into the mutant NIΔyejE restored the polymyxin B resistance to the level of the parental strain, suggesting that the transport system permease YejE played a key role in the AMP resistance of B. melitensis, while the proteins YejA1, YejA2, YejB and YejF are dispensable in this aspect.

Figure 3
figure 3

Sensitivity of yej gene deletion mutants and B. melitensis NI to different concentrations of polymyxin B.

The data are representative of three independent experiments. Significant differences between every mutant and parental strain are indicated by an asterisk (P < 0.05).

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Exposure of bacteria to high concentrations of cationic peptides in most cases results in membrane damage and bacterial death. To confirm the NIΔyejE and NIΔyejABEF mutants’ hypersensitivity to cationic peptides, we evaluated the morphology of bacteria treated with polymyxin B using a scanning electron microscope. In the case of the mutants NIΔyejE and NIΔyejABEF, we could see many damaged bacteria with membrane irregularities (box, Fig. 4) and many bacteria formed an irregular mass of debris with their extruded cytoplasm.

Figure 4
figure 4

Scanning electron microscopic images of strains B. melitensis NI (A,B), NIΔyejE (C,D) and NIΔyejABEF (E,F) treated with polymyxin B. The box indicates disrupted bacteria. Scale bar, 3 μm.

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Yej operon genes are induced by Polymyxin B

The polymyxin B sensitivity assay confirmed the role of the yej operon in conferring AMP resistance in Brucella. It is likely that Brucella encounters antimicrobial peptides within host micro-environments during in vivo infection. These peptides may contribute to environmental signals that trigger changes in bacterial gene expression. We therefore examined the cationic peptide polymyxin B to determine whether it could stimulate yej operon gene expression. A summary of the relative yej operon gene expression levels observed in different samples is presented in Fig. 5. As shown in Fig. 5, we observed that the expression of yejA1, yejA2, yejB, yejE and yejF in B. melitensis NI was induced by polymyxin B and that the expression of yejA1, yejB and yejE was significantly higher in samples treated with Polymyxin B than in samples without peptide (Fig. 5, P < 0.05). The yejA1, yejB and yejE expression levels increased by approximately 3.0, 4.0 and 3.6-fold, respectively, under polymyxin B inducement compared to the untreated control.

Figure 5
figure 5

Real-time PCR analysis of each yej operon gene expression level in B. melitensis NI under polymyxin B treatment.

16S rRNA was used as a reference gene to normalize the expression levels of the target gene. The fold change is expressed as a ratio of normalized gene expression levels under polymyxin B treatment to those under normal culture conditions. Significant differences between the gene expression levels are indicated by an asterisk (P < 0.05).

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YejE is required for the replication of B. melitensis NI in J774.A1 macrophages

The role of the yej operon in in vitro sensitivity to the acid and AMP levels that are predicted to be encountered in host macrophages prompted us to investigate the effect of the YejABEF proteins on the virulence of B. melitensis. First, we assessed the intracellular survival of the yej operon gene deletion mutants. As shown in Fig. 6, at 1 h post-infection, the macrophages infected with the NIΔyejE and NIΔyejABEF mutants showed lower bacterial loads than the macrophages infected with B. melitensis NI, NIΔyejA1, NIΔyejA2, NIΔyejB and NIΔyejF (P < 0.05), which indicated that there were significant variations in the ability of NIΔyejE and NIΔyejABEF to invade macrophages. At 4 h post-infection, the intracellular bacterial loads of each strain decreased to a different degree. The mutant NIΔyejE showed an especially sharp reduction in intracellular bacterial number. However, after 4 h post-infection, the CFUs of B. melitensis NI, NIΔyejA1, NIΔyejA2, NIΔyejB and NIΔyejF increased rapidly and the CFUs of NIΔyejE also had a trend toward recovery, while the numbers of intracellular bacterial CFUs in NIΔyejABEF-infected cells continuously decreased. At the end of the test, the number of recovered NIΔyejE and NIΔyejABEF was four orders of magnitude lower than that recovered with B. melitensis NI, NIΔyejA1, NIΔyejA2, NIΔyejB and NIΔyejF. These data showed that the NIΔyejE and NIΔyejABEF mutants have restricted invasion and replication abilities inside J774.A1 macrophages and therefore they exhibited reduced virulence in vitro. However, the NIΔyejA1, NIΔyejA2, NIΔyejB and NIΔyejF mutants did not show any such defect, which is in agreement with the results of the polymyxin B sensitivity experiments. Thus, it was clear that the yej operon confers the ability of B. melitensis to proliferate inside macrophages and more specifically, that the protein YejE played a key role in this aspect.

Figure 6
figure 6

Multiplication of yej gene deletion mutants and B. melitensis NI in J774.A1 macrophages.

At the indicated hour p.i., the number of intracellular bacteria was measured and expressed as log10 CFU/mL. The presented values represent the means of three experiments performed in duplicate and the error bars indicate the SD. Significant differences between every mutant and parental strain are indicated by an asterisk (P < 0.05).

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YejE contributes to the virulence of B. melitensis NI in mice

Having demonstrated that yejE is necessary for the replication of Brucella in macrophages, we next investigated the virulence of the mutants NIΔyejE and NIΔyejABEF in a murine model. As shown in Fig. 7, we observed a large reduction (above a 2-log difference) in the spleen bacterial load at 1 week post-inoculation in mice inoculated by NIΔyejE and NIΔyejABEF compared to mice infected with B. melitensis NI. At 3 weeks post-infection, sharp reductions of the spleen bacterial load were observed in mice infected with NIΔyejE and NIΔyejABEF, whereas more than 105 CFUs of Brucella remained in the spleens of mice infected with B. melitensis NI. These results indicated that the mutants NIΔyejE and NIΔyejABEF were avirulent in mice, which is consistent with the results of the macrophage infection assay.

Figure 7
figure 7

Survival of yej gene deletion mutants and B. melitensis NI in mice.

Ten mice were inoculated with each strain at a dose of 106 CFU/mouse. Five mice/group were euthanized at 1 and 3 weeks post-inoculation and the virulence of each strain was determined based on the number of CFUs recovered from the spleen, which was expressed as the mean ± SD (n = 5) of individual log10 CFU/spleen values. Significant differences between every mutant and parental strain are indicated by an asterisk (P < 0.05).

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Discussion

As an essential aspect of the host’s innate immune defenses, antimicrobial peptides are largely produced by macrophages, neutrophils and mucosal epithelial cells19, which are at the front line of host defense and play critical roles both in reducing the microbial load early during infection and in linking innate to adaptive immunity. Thus, bacteria have evolved different strategies to resist AMPs, such as remodeling the bacterial outer membrane surface, exporting AMPs via multiple transferable resistance-mediated efflux pumps, secreting exoproteases for AMP degradation and releasing proteins to adsorb extracellular AMPs1. The ability of pathogenic bacteria to resist being killed by antimicrobial peptides in different host niches may therefore contribute to their virulence1. Successful pathogens, including intracellular pathogens, have evolved different mechanisms to evade the microbicidal effects of these molecules. For example, the facultative intracellular pathogen S. typhimurium harbors several proteins that enable it to resist being killed by peptides and strains with mutations in the corresponding genes are avirulent16. Two putative ATP-binding cassette (ABC) transporters encoded by the sapABCDF operon and yejABEF are required to counteract AMPs, contributing to the virulence of Salmonella10,16. In addition, it was also reported that the Sap transporter equips Haemophilus to resist AMPs by mediating the import and subsequent degradation of antimicrobial peptides20. Because the host produces AMPs to control bacterial growth, leading to bacterial clearance and bacterial AMP resistance mechanisms provide advantages to pathogens, leading to disease progression. The outcome of bacterial infection is determined by the balance between bacterial resistance mechanisms and host defense responses during infection.

In this study, the yej operon genes (yejA1, yejA2, yejB, yejE and yejF) in the Brucella genome are annotated as gene-encoding components of a putative ABC-type microcin C transport system. To better understand the role of yej genes in Brucella in resistance to being killed by antimicrobial peptides, we first characterized the regulation of yej genes in B. melitensis NI under polymyxin B treatment. The yejA1, yejA2, yejB, yejE and yejF expression levels, as measured by RT-PCR, were all increased in a medium containing polymyxin B. The up-regulation of yej genes by polymyxin B demonstrated the direct response of Brucella to the presence of antimicrobial peptides, which was similar to mig-14, a Salmonella gene strongly induced by polymyxin B21, suggesting that the expression of yej genes in B. melitensis was directly induced by polymyxin B.

We further demonstrate that the yej operon of Brucella also confers resistance to polymyxin B. It was observed that both the whole yej operon deletion mutant NIΔyejABEF and the yejE deletion mutant NIΔyejE were more sensitive to polymyxin B (Fig. 3), revealing significantly lower survival rates compared to the parental strain NI. This observation is similar to the results of Eswarappa et al., who observed that S. Typhimurium yej mutants were sensitive to polymyxin B10. In agreement with the results of the polymyxin B sensitivity experiment, the NIΔyejABEF and NIΔyejE mutants showed restricted invasion and replication abilities inside J774.A1 macrophages, but the NIΔyejA1, NIΔyejA2, NIΔyejB and NIΔyejF mutants did not exhibit reduced virulence in vitro.

The different phenotypes of the NIΔyejA1, NIΔyejA2, NIΔyejB and NIΔyejF mutants were unexpected when compared with NIΔyejE and NIΔyejABEF, as periplasmic-binding proteins are essential for the function of bacterial importers. However, it has been reported that some histidine and maltose transporter mutants can also function independent of their periplasmic-binding proteins22. Thus, it is possible that YejA, YejB and YejF are dispensable for the function of the Yej transporter, at least in counteracting AMP and intracellular survival. Similar to the yej mutants of S. Typhimurium, the yej mutants of Brucella also did not show any discernible growth defect in TSB or minimal media.

Within host macrophages, Brucella replicate in BCVs associated with the endoplasmic reticulum. While early acidification of vacuolar compartments has been shown to be essential for the intracellular survival of Brucella23, the pH of phagocytic vacuoles has also been observed to decrease rapidly to 4.0–4.524, which indicates that Brucella must adapt to the low-pH environment. Therefore, we determined the acid resistance of yej mutants. The results indicated that yej mutants were more sensitive to acidic conditions than the parental strain.

The results indicated that the yej mutants were both sensitive to acidic stress and AMPs, but the linkage between these two phenomena has received little attention thus far. Preliminary work confirmed that asp24 was related to acid shock and the optimal expression levels of Asp24 were reached at pH values below 4.0, which indicated an active role for this protein in resistance to acidic environments25. In addition, a previous study in our lab also confirmed that a cspA mutant was sensitive to acidic stress26 and a manBA mutant was sensitive to polymyxin B (data not published). To explain the connection between acidic stress and AMP tolerance, the mutants with the respective deletions of the asp24, manBA and cspA genes previously constructed by our lab, were compared with NIΔyejE and NIΔyejABEF to evaluate their resistance to acidic and AMP stresses. The results indicated that the manBA mutant, NIΔyejE and NIΔyejABEF were all sensitive to acidic stress and polymyxin B, while the asp24 and cspA mutants were merely sensitive to acidic environments (data not shown). Thus, we inferred that the strains that were sensitive to AMPs were the most likely to be sensitive to acidic stress, while the strains that were sensitive to acidic stress were not always sensitive to polymyxin B. Notably, the mutants might also have decreased resistance to acidic stress if they were susceptible to AMPs.

To further evaluate the virulence of the yej mutants, we tested the in vivo survival of NIΔyejE and NIΔyejABEF in a mouse model. After 3 weeks of infection, there was a significant decrease in the bacterial burden in the spleens of mice infected with the NIΔyejE and NIΔyejABEF mutants compared to mice infected with the parental strain. This observation clearly indicates that the transporter encoded by the yej operon is important for in vivo infections and persistent survival. We inferred that the inability of the NIΔyejE and NIΔyejABEF mutants to replicate within host macrophages and the attenuation of their growth in mice might be due to the sensitivity of these strains to AMPs and acid stresses that would presumably be encountered by bacteria during infection. Because certain AMPs are capable of mediating changes in the gene expression profiles of bacterial virulence factors, they might influence bacterial tolerance of harsh conditions in the host. Thus, these results suggested that resistance to host defense responses is important not only in initiating infection but also in maintaining persistent infection.

As a first line of innate defense, AMPs serve to limit bacterial colonization during infection. However, bacteria, especially intracellular pathogens, have evolved different strategies to sense and resist the functions of AMPs, as in the novel finding that the yej operon is important for resistance to AMPs and the persistent survival of Brucella melitensis. This study demonstrated that resistance to host antimicrobials is a key mechanism of persistent infection for Brucella and it might be an important way for pathogens to evade the host defense system and persistently survive in vivo. This novel finding also led us to further investigate the potential correlation of antimicrobial peptide resistance with the mechanisms of immune escape and persistent infection by pathogens.

Methods

Ethics statement

All animal research was approved by the Beijing Association for Science and Technology. The approval ID is SYXK (Beijing) 2007–0001 and the animal research complied with the Beijing Laboratory Animal Welfare and Ethics guidelines of the Beijing Administration Committee of Laboratory Animals.

Bacterial strains and media

All bacterial strains and plasmids used in this study are listed in Table 1. Brucella strains, including the parental strain and the derived mutants, were routinely grown or incubated in TSB, tryptic soy agar (TSA) or a minimal medium that has been described previously26. The pH levels of the minimal media were adjusted with HCl. Escherichia coli strains were grown on Luria-Bertani (LB) plates overnight at 37 °C with or without supplemental ampicillin (100 mg/liter) and chloromycetin (30 mg/liter). All work with live Brucella strains was performed in the biosafety level 3 facilities of the Chinese Center for Disease Control and Prevention.

Table 1 Bacterial strains and plasmids.
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Mice

Female BALB/c mice (aged 4 to 6 weeks) were purchased from the Weitong Lihua Laboratory Animal Services Centre (Beijing, China), bred in individually ventilated cage rack systems and subsequently transferred to the biosafety level 3 facilities of the Chinese Center for Disease Control and Prevention at the beginning of the experiments. All experiments involving animals followed the regulations of the Beijing Administration Office for Laboratory Animals.

Construction of yej operon gene deletion mutants

Gene knockout constructs were used as described previously26. This method has been used successfully to knock out single genes as well as an entire operon. The primers used for each gene deletion are shown in Table S1. The gene deletion mutants were verified by PCR and sequencing analysis and are hereafter referred to as NIΔyejA1, NIΔyejA2, NIΔyejB, NIΔyejE, NIΔyejF and NIΔyejABEF.

Construction of the complemented yejE deletion mutant strain

To construct the complemented strain of NIΔyejE, the complete yejE gene and the promoter sequences were amplified from B. melitensis NI genomic DNA via PCR using the primers shown in Table S1. The resulting PCR products were digested with SmaI and PstI and then ligated into the pBBR1MCS plasmid digested with the same enzymes. The resulting recombinant vector, pBBRyejE, was subsequently electroporated into NIΔyejE to complement the function of YejE. The complementation strains, loaded with pBBRyejE, were selected on TSA plates containing chloromycetin. Finally, the selected complementation strains were verified through PCR and designated as NIΔyejEPBBRyejE.

In vitro growth characteristics in TSB and minimal medium

To monitor extracellular growth, one colony from each strain (the parental strain and all gene deletion mutants) was inoculated into 5 mL of TSB medium and grown to mid-log phase in a shaking incubator at 37 °C. The cultures were then adjusted to the same concentration (CFU/mL) and subsequently used for growth curve analysis. A 20-μL sample of each strain was inoculated into 5 mL of minimal medium or TSB, followed by incubation at 37 °C. Growth was monitored based on the CFU/mL and OD600 values. Growth characteristics were evaluated by analyzing the growth of each strain at different time points.

Acid resistance experiment

The acid challenge assay was performed as previously described with some modifications27. Exponentially growing bacteria of each strain were adjusted to 109 CFU/mL, from which a 200-μl sample of each strain was concentrated, washed and then resuspended in 200 μl of pH 4.4 minimal medium (for submitted cultures) and incubated for 2 h at 37 °C. Next, the submitted cultures were washed and resuspended in pH 3.4 minimal medium for challenge and incubated for 2 h at 37 °C. The cultures were then immediately serially diluted and plated to determine their viability post-challenge. The survival percentage was calculated by dividing the CFUs obtained 2 h post-acid challenge by those obtained prior to the acid challenge and multiplied by 100.

Polymyxin B sensitivity assay

Polymyxin B (Sigma, USA) sensitivity assays were performed in triplicate essentially as described with some modification28. Essentially, exponentially growing bacteria of each strain were adjusted to 104 CFU/mL, from which a 200-μL sample of each strain was concentrated, washed and resuspended in 200 μL minimal medium and then mixed with 200 μL of different concentrations of polymyxin B (the final concentrations in the wells were 50 and 100 ug/mL). After 1 h of incubation at 37 °C, the percent survival was calculated as the initial input bacterial CFUs relative to bacterial CFUs after challenge.

yej gene expression levels in B. melitensis NI under polymyxin B treatment

The expression levels of the yej operon genes in B. melitensis NI under AMP induction were detected via RT-PCR. B. melitensis NI was grown to the exponential phase in 20 mL of TSB at 37 °C in a shaking incubator. Ten milliliters of the sample bacterial were treated with polymyxin B (100 ug/mL) for 1 h at 37 °C and another 10 mL of the sample was maintained at 37 °C for 1 h. Then, total RNA samples were prepared for the treated and untreated B. melitensis strains. DNA was removed using DNase (Ambion, Foster City, CA) and the RNA samples were reverse transcribed into cDNA using random oligonucleotide hexamers and the Fermentas First-Strand cDNA synthesis kit (Thermo Fisher Scientific, Bremen, Germany) according to the manufacturer’s protocol. The resulting cDNA samples were subjected to quantitative real-time PCR using the Power SYBR Green PCR System. The sense and anti-sense primers for each yej operon gene are shown in Table S1. The expression level of the 16S rRNA gene was used to normalize all of the obtained values.

Scanning electron microscopy

Each strain, including B. melitensis NI, NIΔyejE and NIΔyejABEF (approximately 108 CFU), was treated with 100 ug/mL polymyxin B at 37 °C for 1 h and fixed using 4% glutaraldehyde in 0.1 M phosphate buffer at 4 °C for 4 h or overnight. Then, the samples were fixed using 2% glutaraldehyde in a 0.1 M phosphate buffer at 4 °C for 1 h. The samples were subsequently washed and dehydrated in a series of ethanol washes (70% for 30 min, 90% for 30 min and 100% for 30 min twice) and air-dried prior to sputter coating with gold. The samples were then analyzed using a scanning electron microscope29.

Cell infection assay

To investigate the intracellular survival of the parental strain and each yej gene mutant, J774.A1 murine macrophage infection assays were performed as previously reported with some modifications26. Briefly explained, monolayers of cells were cultured in 24-well plates and infected with each strain at a multiplicity of infection (MOI) of 200 CFU. To synchronize the infection, the infected plates were centrifuged at 1,000 rpm for 5 min at room temperature, followed by a 20-min incubation at 37 °C in an atmosphere containing 5% (vol/vol) CO2. Then, the cells were washed three times with PBS and incubated in a medium containing gentamycin (50 μg/ml) at 37 °C under 5% CO2 until the end of the infection period. At 1 h, 4 h, 24 h and 48 h p.i., the cells were washed and lysed in sterile 0.5% (vol/vol) Tween 20 water. The number of surviving intracellular bacteria was then determined through serial dilution, followed by plating on TSA or TSA supplemented with chloromycetin. Three replicate wells for each strain were evaluated at each time point. The results presented in this paper represent the averages from at least three separate experiments.

Virulence in BALB/c mice

Ten mice were intraperitoneally inoculated with a dose of 106 CFU of NIΔyejE, NIΔyejABEF and B. melitensis NI in 0.1 ml of phosphate-buffered saline (PBS). Five infected mice from each infected group were randomly selected and euthanized via carbon dioxide asphyxiation at 1 and 3 weeks post-inoculation. At each time point, the spleens were collected aseptically, homogenized in 1 ml of PBS and then serially diluted to isolate the bacteria. The bacteria recovered from the spleens were enumerated to evaluate the survival of each strain in mice. The results are presented as the mean number of CFU per spleen±the standard deviation (SD) in each group. If no bacteria grew in the undiluted homogenized sample, the spleen was assumed to contain less than 5 bacteria, i.e., falling below the limit of detection of 5 CFU/spleen.

Statistical analysis

Student’s t-test was performed to analyze the data from the mouse virulence experiments and ANOVA was performed for the growth curve analysis, cellular infections and acid and polymyxin B sensitivity response assays. A P value of <0.05 was considered to represent a significant difference.

Additional Information

How to cite this article: Wang, Z. et al. The ABC transporter YejABEF is required for resistance to antimicrobial peptides and the virulence of Brucella melitensis. Sci. Rep. 6, 31876; doi: 10.1038/srep31876 (2016).