A Sinorhizobium meliloti and Agrobacterium tumefaciens ExoR ortholog is not crucial for Brucella abortus virulence

Amanda Castillo-Zeledón; Nazareth Ruiz-Villalobos; Pamela Altamirano-Silva; Carlos Chacón-Díaz; Elías Barquero-Calvo; Esteban Chaves-Olarte; Caterina Guzmán-Verri

doi:10.1371/journal.pone.0254568

. 2021 Aug 13;16(8):e0254568. doi: 10.1371/journal.pone.0254568

A Sinorhizobium meliloti and Agrobacterium tumefaciens ExoR ortholog is not crucial for Brucella abortus virulence

Amanda Castillo-Zeledón ¹, Nazareth Ruiz-Villalobos ¹, Pamela Altamirano-Silva ², Carlos Chacón-Díaz ², Elías Barquero-Calvo ¹, Esteban Chaves-Olarte ², Caterina Guzmán-Verri ^1,^*

Editor: Roy Martin Roop II³

PMCID: PMC8362948 PMID: 34388167

Abstract

Brucella is a facultative extracellular-intracellular pathogen that belongs to the Alphaproteobacteria class. Precise sensing of environmental changes and a proper response mediated by a gene expression regulatory network are essential for this pathogen to survive. The plant-related Alphaproteobacteria Sinorhizobium meliloti and Agrobacterium tumefaciens also alternate from a free to a host-associated life, where a regulatory invasion switch is needed for this transition. This switch is composed of a two-component regulatory system (TCS) and a global inhibitor, ExoR. In B. abortus, the BvrR/BvrS TCS is essential for intracellular survival. However, the presence of a TCS inhibitor, such as ExoR, in Brucella is still unknown. In this work, we identified a genomic sequence similar to S. meliloti exoR in the B. abortus 2308W genome, constructed an exoR mutant strain, and performed its characterization through ex vivo and in vivo assays. Our findings indicate that ExoR is related to the BvrR phosphorylation state, and is related to the expression of known BvrR/BrvS gene targets, such as virB8, vjbR, and omp25 when grown in rich medium or starving conditions. Despite this, the exoR mutant strain showed no significant differences as compared to the wild-type strain, related to resistance to polymyxin B or human non-immune serum, intracellular replication, or infectivity in a mice model. ExoR in B. abortus is related to BvrR/BvrS as observed in other Rhizobiales; however, its function seems different from that observed for its orthologs described in A. tumefaciens and S. meliloti.

Introduction

Members of the Brucella genus are worldwide distributed zoonotic pathogens that belong to the Alphaproteobacteria class. Infection in humans is associated with diverse and unspecific symptoms, including fever, lassitude, general malaise, weight loss, headache, low back pain, and arthralgias [1]. Brucella abortus infects bovines, where it preferentially replicates in reproductive organs, causing abortion, infertility, decreased milk production, reproductive failure, and epididymitis [2, 3]. As a result, countries with the disease go through economic losses in the animal industry and public health [4].

B. abortus is a facultative extracellular-intracellular pathogen [4, 5], hence the transition from lifestyles requires a fine-tuned transcriptional regulatory network coordinating a bacterial response to environmental signal cues [6]. Lifestyles transitions have been linked with an invasion switch pathway for Sinorhizobium meliloti, a plant symbiont, and Agrobacterium tumefaciens, a plant pathogen [7–9]. S. meliloti invasion switch pathway is known as RSI (ExoR, ExoS, and ChvI). It involves a two-component system (TCS), ExoS/ChvI, and a periplasmic protein, ExoR. ExoS is a membrane-bound sensory protein with histidine kinase activity, and ChvI is its cognate response regulator [10, 11]. According to the canonical TCS OmpR model [12], it is inferred that ExoS is autophosphorylated upon sensing an external stimulus. Subsequently, the phosphate group is transferred to ChvI, increasing its affinity for regulatory DNA sequences, hereafter influencing the transcription of genes involved in responding to the original external stimulus. ExoS/ChvI activation is required to transition from a flagellum producing free-living cell to a succinoglycan producing host-invading cell. Constitutive exoS mutants are non-motile, do not harbor flagella, overproduce succinoglycan, and form loose biofilms [10, 13, 14]. The third member, ExoR, is a periplasm protein that contains Sel1-like repeats that mediate protein-protein associations. It is known to interact with ExoS in the periplasm, preventing ExoS/ChvI activity. Inhibition of ExoS/ChvI activity induces free-living forms with flagella and no succinoglycan production. These free-living forms cannot induce root nodulation and plant cell colonization [15–17]. ExoR undergoes proteolysis by unknown mechanisms and relieves the TCS suppression [18].

A. tumefaciens invasion switch pathway is termed RGI (ExoR, ChvG, ChvI) [9, 19]. In these bacteria, the plant cell invasion mechanism requires attachment to the host cell and biofilm formation. Without ExoR or its proteolysis induced at low pH, A. tumefaciens exhibit a non-motile, hypermucus phenotype and disabled surface adherence. Consequently, plant colonization fails [9, 20]. More than 400 differentially expressed genes have been found in mutants lacking exoR and for this reason, it is cataloged as a global regulator [20].

Phylogenetic distribution analysis of the invasion switch pathway proteins in 57 Alphaproteobacteria genomes found putative orthologs, including one in B. abortus [7]. The TCS BvrR/BvrS in B. abortus was first discovered in 1998 and is phylogenetically related to ExoS/ChvI and ChvG/ChvI [21]. BvrS, the sensory protein, does not sense a unique signal but rather an environmental cue constituted of different factors, including low pH and nutrient-limiting conditions, probably similar to those found during B. abortus trafficking through autophagosome-like compartments [22–24]. Its activation promotes the signal transference to BvrR, the regulatory protein [21]. The bvrR or bvrS mutants have increased susceptibility to polymyxin B and non-immune serum and are attenuated in mouse and cell models [25]. This phenotype was attributed to changes in cell envelope homeostasis and regulation of carbon and nitrogen metabolism [26].

Transcriptomic and proteomic analyses of the B. abortus null bvrR and bvrS mutants have shown more than 100 differentially transcribed genes and expressed proteins as compared to the wild-type [27, 28]. Direct binding of BvrR to regulatory regions of virB shows that BvrR/BvrS influences virB transcription, encoding a type IV secretion system, crucial for bacterial intracellular survival [22, 29]. For other proteins, as the outer membrane protein (OMP) Omp25 (also known as Omp3a) and the transcriptional regulator VjbR, evidence indicates that BvrR/BvrS positively affects their transcription since Omp25 and VjbR are not expressed when BvrR or BvrS are absent [29, 30]. Additionally, a direct interaction between BvrR and the upstream region of VjbR was evidenced [22, 29]. VjbR belongs to the LuxR regulator family of transcriptional activators related to quorum sensing [31–33]. Furthermore, direct regulation of VjbR over the virB promoter has been demonstrated [22, 29].

To date, ExoR has not been described for B. abortus or any other Brucella spp. Due to the homology between ExoS and BvrS, it is hypothesized that the Brucella ExoR ortholog could be functionally related to BvrR/BvrS [28]. To elucidate this hypothesis, an exoR mutant derived from the reference strain B. abortus 2308 Wisconsin was constructed and studied [34]. The results show that ExoR affects VirB, Omp25, and VjbR expression dynamics and BvrR phosphorylation, but its absence does not affect B. abortus virulence ex vivo or in vivo.

Results

B. abortus 2308W genome encodes an exoR ortholog

Using Basic Local Alignment Search Tool (BLASTp) and the ExoR protein sequence from S. meliloti (GenBank Accession WP_003534542.1) [35, 36] a similar sequence (51% identity), possibly encoding a 267 amino acid residues long protein was found in the B. abortus 2308W genome (GenBank Accession ERS568782) [34]. This sequence was found under locus tag BAW1_0856, between open reading frames annotated as a predicted protein and an exodeoxyribonuclease III. Based on this information, we constructed a B. abortus 2308W mutant with an in-frame deletion of 171 internal amino acid residues. Deletion of the 515 bp was confirmed by PCR, Southern blot assays [37], and Sanger sequencing, as described in the Methods section and S1 Fig. No significant differences in growth kinetics in TSB (Fig 1A) and sugar assimilation, according to API 50 CH assay, were detected between the mutant and the wild-type strain.

Fig 1 — (A) B. *abortus* 2308W (WT) and the *exoR* mutant (*exoR*) were grown in TSB at 37°C, and the absorbance was measured at 420 nm at the indicated times. (B) The indicated strains B. *abortus* 2308W (WT), *exoR*, *bvrS*, and *bvrR* mutants, were exposed to non-immune human serum at 37°C for 90 min. These experiments are representative of at least three performed. (C) and (D) The indicated strains, B. *abortus* 2308W (WT), *exoR*, *bvrS*, and *bvrR* mutants, were exposed to increasing concentrations of polymyxin B for 48 hours at 37°C, at pH 7 and pH 6, respectively. The minimum inhibitory concentrations shown are representative of at least three experiments performed. The *bvrS* and *bvrR* mutant strains were used as susceptibility controls. *, P < 0,05 (Anova and Kruskal-Wallis test for multiple comparisons).

The exoR mutation does not affect B. abortus resistance to polymyxin B and non-immune human serum

The B. abortus LPS smooth phenotype was not altered by the exoR mutation, as judged by the acriflavine agglutination test. Exposure to non-immune human serum and increasing polymyxin B concentrations at pH 7 and pH 6 did not reveal differences between the wild-type and the exoR mutant strain (Fig 1B, 1C and 1D). As expected, the bvrR and bvrS mutants, included as controls, were sensitive to polymyxin B and non-immune human serum (Fig 1C and 1D). Both strains showed significant differences when compared to the wild-type and the exoR mutant.

ExoR impacts the expression dynamics of proteins related to BvrR/BvrS in nutrient rich conditions

We assessed the expression of BvrR and BvrS by western blot of cell lysates obtained at different moments of growth in TSB. We observed similar BvrR and BvrS expression patterns compared to the wild-type strain in the mutant strain (Fig 2). The BvrR phosphorylation state was analyzed by Phos-tag SDS-PAGE and western blot [22, 38]. As expected, two bands were recognized, corresponding to BvrR-P (upper band) and non-phosphorylated BvrR (Fig 2). BvrR-P remained constant in the wild-type strain over time, ranging from 11 to 20% of total BvrR, until 28 hours of growth where there was no phosphorylation signal. In the exoR mutant, BvrR-P constantly decreased, and its signal disappeared more radidly, going from 15 to 6%, and being undetectable at 24 hours.

Fig 2 — Western blot analysis of BvrS, BvrR, and BvrR-P expression according to growth phase in the B. *abortus exoR* mutant (*exoR*) and B. *abortus* 2308W (WT). Both strains were grown in TSB for 48 hours. Representative hours of different growth phases were analyzed (mid-, late-log, and stationary) indicated on top. For western blot, equal amounts (20 μg) of whole-bacterium lysates were separated by 12,5% SDS—PAGE. For phosphorylation analyses, samples were separated by 10% SDS-PAGE containing Phos-tag. PVDF membranes were incubated with anti-BvrS, anti-BvrR, and and reprobed with anti-omp19 antibodies indicated on the right side. After incubation, the immune complexes were detected by chemiluminescence reaction. Omp19 was used as a loading control. The blots for BvrS, BvrR and Phos-tag are from independent gels.

We also evaluated three proteins whose expression is positively regulated by BvrR/BvrS: VirB8, VjbR, and Omp25 (Fig 3). VirB8 and VjbR reached a maximum expression around late-log and mid-log in the wild-type strain, respectively, and then decreased until reaching the stationary phase, as previously reported [29]. In the exoR mutant, VirB8 and VjbR expression showed a similar trend; nevertheless, the expression declined before it occurred in the wild-type, around mid-log. Omp25 showed slight differences in expression according to the wild-type strain time of growth as reported [39]. In the exoR mutant, expression levels gradually decreased from the early log until the stationary phase of growth.

Low nutrients and pH do not significantly alter B. abortus exoR phenotype as compared to nutrient rich conditions

BvrR phosphorylation increases when exponential growing bacteria are exposed to minimal medium and pH 5.0 [22]. These conditions mimic the intracellular environment, where BvrR/BvrS is critical for survival [23, 24, 40]. A possible role for ExoR related to the activation of BvrR/BvrS was assessed using similar assays. Hence, the polymyxin B and non-immune human serum assays were carried out using bacteria after 4-hours incubation in minimal medium and pH 5.0 (Fig 4A and 4B). No significant difference was found between the wild-type and the mutant strain. As expected, the bvrR mutant, included as a control, was susceptible to polymyxin B and non-immune human serum. This strain showed significant differences when compared to the wild-type strain and the exoR mutant.

Fig 4 — (A) B. *abortus* wild-type 2308W, B. *abortus exoR*, and B. *abortus bvrR* (negative control) mutant strains were grown to exponential phase and incubated in minimal medium (MM) at pH 5.0 or rich medium (TSB) at pH 7.0 for 4 hours. After incubation, bacteria were exposed to non-immune human serum at 37°C for 45 min or (B) exposed to increasing concentrations of polymyxin B for 48 hours at 37°C at pH 7. The Anova and Kruskal-Wallis test for multiple comparisons was used. *, P < 0,05. (C) The wild-type and *exoR* mutant were grown as indicated in (A) for 6 hours, and 1 mL culture of each strain was resuspended in minimal medium, pH 5 for 0.5, 2, 4, and 6 hours. After incubation, lysates were prepared, separated by 10% SDS-PAGE and assessed using anti-VjbR and anti-VirB8 antibodies by western blotting. Omp19 detection was used as a loading control. For comparison, a bacterial sample from each strain, with no exposure to minimal medium, pH 5 was included and is labeled as “C”.

The expression of VirB8 and VjbR in the wild-type strain under these conditions was similar to the previously reported and related to BvrR transient phosphorylation [29]. The exoR mutant showed a similar expression pattern as compared to the wild-type for both proteins during the time points tested, however, their expression seems lower, particularly for VirB8, except for the 6h time point (Fig 4C).

The exoR mutant successfully infects mice and cell cultures

The ability to escape the host immune response and to survive intracellularly is critical for B. abortus pathogenicity [41, 42]. Cellular and murine models were used to evaluate the exoR mutant strain infective abilities. Fig 5A and 5B show that the exoR mutant infected HeLa and Raw macrophage cells similar to the wild-type strain. Fig 5C shows that the dynamics of infection of the wild-type strain in mice were consistent with previous results [26, 43]. We found no significant differences between the wild-type and the exoR mutant, indicating that ExoR is not required for B. abortus infection in mice or cell culture.

Fig 5 — (A, B) Murine RAW 264.7 macrophages or HeLa epithelial cells were cultivated and infected with B. *abortus* strains (2308W and *exoR* mutant) in the exponential phase. The number of intracellular viable B. *abortus* CFU was determined at different hours post-infection. Each point is the average of three independent trials. (C) Virulence assays of B. *abortus* 2308W and *exoR* mutant in mice. Mice were intraperitoneally inoculated with 10⁶ CFU of either B. *abortus* 2308W or B. *abortus* 2308W *exoR* mutant. CFU per gram of spleen was determined at 3- and 9-weeks post-infection. For these experiments, no significant difference was shown between the wild-type and the mutant (Mann-Whitney test).

Discussion

BvrR/BvrS homologs in S. meliloti and A. tumefaciens are required to associate with plant cells [14, 36]. Similarly, B. abortus needs BvrR/BvrS to infect the host animal cell successfully [44]. However, the RSI invasion switch functioning in both plant cell microbes, where ExoR is a protagonist, seems dissimilar in B. abortus.

In the RSI invasion switch, ExoR levels are critical for host interaction and subsequent invasion in S. meliloti. exoR95:: Tn5 loss-of-function mutant overproduces succinoglycan and has a dramatic decrease of flagellar gene expression and other genes related to motility and chemotaxis [18, 36]. A similar effect was observed in an A. tumefaciens exoR null mutant [9]. In both cases, host interactions are compromised. According to our mice model result, this does not seem to be the case for B. abortus.

However, ExoR seems to be related to BvrR/BvrS. Proteins whose gene expression is under the control of BvrR/BvrS [29, 45] showed a different expression pattern in the exoR mutant as compared to the wild-type strain. The transcriptional regulator VjbR, the T4SS VirB component VirB8 (essential for intracellular survival) [23, 29, 46, 47] and Omp25 (with a potential role in virulence) [48–50], were expressed in the ΔexoR mutant at lower levels, particularly starting at late log growth phase, as compared to the wild strain. Nevertheless, the exoR mutant was able to infect and survive in cell and mice models. These results were unexpected considering the high degree of identity between the BvrR, BvrS, and ExoR homologs. If the B. abortus ExoR homolog is functioning as its counterparts, i.e., as a sensor protein inhibitor, the TCS’s upregulated genes would be overexpressed. For example, in A. tumefaciens, ChvG/ChvI upregulates the expression of an Omp25 orthologue, known as AopB [20, 38]. This protein influences the stability, permeability, and topology of the membrane, as does Omp25 and other OMPs in B. abortus [51, 52]. Since ExoR represses ChvG by direct interaction, inhibiting the signal transferred through the TCS, aopB was highly upregulated in the A. tumefaciens exoR null mutant [20]. The same has been observed for succinoglycan genes that are upregulated by ExoS/ChvI from S. meliloti [13]. Surprisingly, in B. abortus exoR mutant Omp25 was down expressed. Moreover, BvrR and BvrS did not show differences in their expression compared to the wild-type. This suggests that their expression is independent of ExoR, but possible posttranslational modifications (including phosphorylation and dephosphorylation) might not, since the BvrR phosphorylation dynamics was different as compared to the wild-type strain. The exoR mutant showed a gradual fade of the BvrR-P signal during bacterial growth, which occurs faster than in the wild-type strain. There is no direct evidence linking the phosphorylation of the response regulator and ExoR in other bacterial RSI switches; but, in A. tumefaciens and S. meliloti ExoR absence induces a phenotype similar to that of bacteria with a constitutive TCS. This does not seem to be the case in B. abortus 2308W.

In A. tumefaciens ChvG/ChvI is activated under low-pH conditions and ExoR represses it under neutral conditions. A null exoR mutant shows a phenotype at neutral pH similar to that of acid-exposed wild-type bacteria, demonstrating that ExoR is required to inactivate the system [20]. In S. meliloti, ExoR inhibits flagella production and stimulates succinoglycan production. Mutants with defective ExoR mimic non-motile succinoglycan-producing bacteria, demonstrating the need for ExoR to inhibit ExoS [18]. This inhibitory ExoR function on BvrR/BvrS was not clearly evident in our mutant under neutral pH conditions. Therefore, we also evaluated the possibility that the exoR mutation has an evident effect under starving and acid-exposed conditions, which are also relevant for BvrR phosphorylation, and induction of VjbR and VirB expression in the wild-type strain [22]. Our results show that, as in rich medium, pH 7, the exoR mutation has no effect after exposure to minimal medium and pH 5, on the B. abortus ability to resist polymyxin B or non-immune human serum treatment, and that there are subtle differences in expression of VjbR and VirB8 as compared to the wild-type strain.

Nevertheless, there seems to be a partial functional similarity between ExoR from B. abortus and its orthologs. ExoR is not required for B. abortus virulence in the analyzed models. However, there is a functional link with BvR/BvrS, clearly shown by altering BvrR phosphorylation patterns and expression levels of some of the downstream BvrR/BvrS targets. One possible interpretation is that ExoR is inhibiting, directly or indirectly, the dephosphorylation of BvrR and consequently, the expression of the analyzed genes. Unlike the studies published for S. meliloti and A. tumefaciens, we conclude B. abortus exoR mutant maintains the ability to invade and replicate inside cells and survive inside the host. Since the introduced deletion in exoR did not remove the gene entirely, we cannot exclude that the remaining protein segment kept some of the functions of the native ExoR. More studies are needed to understand the role of the conserved ExoR, not only in B. abortus but also in other members of the genus, as well as other members of the Alphaproteobacteria.

Methods

Bacterial culture

All procedures involving live B. abortus were carried out according to the “Reglamento de Bioseguridad de la CCSS 39975–0”, 2012, after the “Decreto Ejecutivo #30965-S”, 2002 and research protocol SIA 0652–19 approved by the National University, Costa Rica. B. abortus 2308W (WT Brucella strain, virulent, smooth LPS, NaI^r) [53] or B. abortus 2308W exoR mutant strain (this study) were maintained and grown at 37°C with agitation (200 rpm) in standard Tryptic Soy Broth (TSB) at pH 7.0 or minimal medium (33 mM KH₂PO₄, 60.3 mM K₂HPO₄, and 0.1% yeast extract) at pH 5.0 (adjusted with citric acid). Escherichia coli S17-1 λpir strain (sup E44, recA1, endA1, hsdR17, thi-1, gyrA96, relA1, lysogenic phage λpir) and E. coli Top10 (F- mcrA Δ (mrr-hsdRMS-mcrBC) Φ80lacZΔM15 Δ lacX74 recA1 araD139 Δ (araleu)7697 galU galK rpsL (StrR) endA1 nupG) (Invitrogen^™) used for cloning were grown in LB (37°C) (Simon et al., 1983). When needed, B. abortus and E. coli strains were supplemented with antibiotics to maintain plasmid selection.

Strain construction

B. abortus exoR mutant was constructed as reported elsewhere [54, 55]. Briefly, in-frame deletion was generated by PCR overlap using genomic DNA of B. abortus as a template. Primers were designed using the available genome sequence ERS568782, corresponding to reference strain B. abortus 2308W to perform a 515 bp internal deletion (position +172 to +687 from the first codon ATG), leaving only 35% of the coding sequence (S1 Fig). Final validation of successful deletion was done using Sanger DNA sequencing of selected fragments and Southern blot (see below and S1 Fig).

Plasmid and chromosomal DNA were extracted with QIAprep Spin Miniprep and DNeasy Blood and Tissue Kit (Qiagen).

Carbohydrate assimilation pattern

The carbohydrate assimilation pattern was performed using the API 50 CH kit (Biomerieux) according to the manufacturer’s instructions. Briefly, a bacterial suspension of both strains, mutant and wild-type, grown in TSB at pH 7, with agitation of 200 rpm to exponential phase, were used to rehydrate each of the wells. The strips were incubated at 37°C for 18 hours. During incubation, metabolism produces color changes. Positive and negative test results were annotated to obtain a profile. The profile from three different assays was compared to the wild-type strain profile.

Southern blot

Whole genomic DNA (0.5 μg) from Brucella strains was digested with 5U of the restriction endonucleases BamHI and PstI (Fermentas) in a reaction volume of 20 μl and according to the manufacturer’s instructions. The resulting fragments were separated in a 0.7% agarose gel. After electrophoresis, hybridization was carried out with minor variations as described previously [37]. Primers bruabI0884.3 and bruabI0884.5 were used to generate a 295 bp amplicon from B. abortus 2308W DNA (S1 Table). These primers amplify a region from position 68 bp to -363 from exoR first codon (S1 Fig), used as a probe after digoxigenin labeling (DIG DNA Labeling and detection kit, Roche). The labeled probe was denatured and added to the hybridization buffer. Hybridization was performed overnight. After exposure to ultraviolet light, the membrane was then washed, blocked, incubated with anti-DIG alkaline phosphatase-conjugated FAB-antibody, and washed according to the manufacturer’s instructions. Luminescence was recorded on X-Ray films (Kodak) for 4 hours.

Polymyxin B sensitivity assay

Polymyxin B (Sigma, USA) sensitivity assays were performed in triplicate as described with some modifications [56]. Both strains, mutant, and wild-type, were grown until exponential phase in TSB at pH 7, with the agitation of 200 rpm. Bacteria of each strain were adjusted to 5x10⁶ CFU/mL in TSB. 100 μL sample of each strain was mixed with 100 μL of different polymyxin B concentrations in a microplate (the final concentration of the first well was 250 ug/mL). After 48 h of incubation at 37°C in two different pH (6 and 7), the minimum inhibitory concentration for polymyxin was calculated.

Sensitivity to the bactericidal action of non-immune serum

Exponentially growing bacteria grown in TSB at pH 7, with agitation of 200 rpm, were adjusted to 10⁴ CFU/mL in PBS and dispensed in duplicate in microtiter plates (200 μl per well) containing fresh human serum (400 μl/well). After 90 min of incubation at 37°C, 100 μl was plated on tryptic soy agar. Results were expressed as the percentage CFU as compared to controls performed with decomplemented serum at room temperature for 30 min. To assess complement consumption, serum was homogenized with dehydrated yeast Saccharomyces cerevisiae for 1 h at 37°C. After incubation, the serum was centrifugated at 14000 rpm for 5 min [25].

Western blot and BvrR phosphorylation

Detection of BvrS, BvrR, VirB8, VjbR, Omp25, and determination of BvrR phosphorylation was performed as previously described [22]. Bacteria grown to exponential phase in TSB at pH 7, with the agitation of 200 rpm, were concentrated by centrifugation, resuspended in Laemmli sample buffer, and heated at 100°C for 20 min. The protein concentration was determined by the Bio-Rad DC method according to the manufacturer’s instructions. Equal amounts of protein (20 μg) were loaded onto a 10% gel for SDS-PAGE. Separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and probed with the indicated antibodies. To analyze the phosphorylated status of BvrR, samples were solubilized in Laemmli sample buffer without heating, and equal amounts of protein (20 μg) were loaded onto a 10% gel for SDS-PAGE containing Phos-tag (100 mM) and MnCl2 (0.2 mM). Recombinant BvrR phosphorylated with the phosphate universal donor carbamoyl phosphate was used as a positive control. The percentage of BvrR-P from total BvrR was calculated for each indicated condition by densitometry from at least three independent experiments.

For loading control each membrane was reprobed. First, the membranes were washed with PBS Tween 0,1% for 30 minutes and incubated in glycine 0.1M pH 2.5 for 1 hour. After incubation, membranes were probed with Omp19 or Bscp31 antibody.

Exposure of Brucella strains to minimal and low pH medium

Bacterial strains were grown in vitro in TSB to exponential growth phase [22]. A volume of each culture corresponding to 10⁴ CFU/mL or 5x10⁶ CFU/mL was calculated for the sensitivity to the bactericidal action of non-immune serum or the polymyxin sensitivity assays, respectively. Bacteria were centrifuged at 10 000 x g for 3 min and resuspended in minimal medium at pH 5.0 or in a nutrient-rich medium (TSB) at pH 7.0 for 4 h at 37°C at 200 rpm. After incubation, bacteria were concentrated by centrifugation at 10,000 x g for 3 min and tested as described above. For the VirB8 and VjbR protein expression assays, aliquots of 1mL of bacteria grown in TSB to exponential growth phase were centrifuged, resuspended in minimal medium, pH 5 for 0.5, 2, 4, and 6 hours. After incubation, each sample was centrifuged and tested as described above. For comparison, a 1mL aliquot with no exposure to minimal medium, pH 5 was included.

Gentamicin-protection assay and intracellular replication quantification

Murine RAW 264.7 macrophages (ATCC TIB-71) or HeLa epithelial cells (ATCC clone CCl-2) were cultivated and infected with B. abortus strains at exponential and stationary growth phase as previously described with some modifications [22]. Cells were seeded in 24-well tissue culture plates and multiplicities of infection (MOI) of 100 for macrophages and 500 for HeLa epithelial cells. The number of intracellular viable B. abortus CFU was determined at different hours post-infection. Cells were washed twice with phosphate-buffered saline (PBS) and treated with Triton X-100 (0.01%). Lysates were serially diluted and plated on tryptic soy agar dishes for the quantification of CFU.

Virulence assays in mice

BALB/c female (18–24 grams) mice were intraperitoneally (i.p.) inoculated with 10⁶ UFC of either B. abortus 2308W or B. abortus 2308W exoR mutant at exponential phase grown in TSB at pH 7, with agitation of 200 rpm. Mice were sacrificed at 3- and 9-weeks post-infection. Spleen counts were determined as described elsewhere [57]. Protocols of experimentation were revised and approved by the Welfare Commission of the Veterinary School at Universidad Nacional, Costa Rica, under protocol number FCSA-EMV_CBAB-009-2015, and agreed with the corresponding law, Ley de Bienestar de los Animales, of Costa Rica (law 7451 on animal welfare). Only certified veterinarians carried out these experiments and all efforts were made to minimize suffering.

Statistical analysis

Statistical analyses were performed using GraphPad Prism software. All results are presented as means ± SD from at least three independent experiments unless otherwise stated. Non-parametric statistics were used. Anova and Kruskal-Wallis test were used for multiple comparisons and Mann-Whitney test for the comparison of two strains.

Supporting information

S1 Fig. Mutant construction and Southern blot.

(A) Schematic representation of the in-frame deletion strategy used to construct the exoR mutant. Primers exoR-F1 and exoR-R2 were used to generate fragment 1, and fragment 2 was generated using exoR-F3, and exoR-R4 (S1 Table). Both fragments were ligated by PCR overlapping using nucleotides exoR-F1 and exoR-R4. The resulting deletion allele was cloned in the pCR^™ 2.1 vector (Invitrogen^™) and subcloned into the BamHI-XBaI site of the suicide plasmid pJQKm. Plasmid pJQKm containing the deleted allele was introduced in B. abortus 2308W by conjugation. Colonies corresponding to integrating the suicide vector in the chromosome were selected using Nalidixic acid (NaI, 25 μg/mL) and Kanamycin (Km, 50 μg/mL) resistance. Excision of the suicide plasmid leading to the mutant’s construction by allelic exchange was selected by 5% sucrose resistance and Km sensitivity. The resulting colonies were screened using primers exoR-F1 and exoR-R4. Mutant colonies generated a 780 bp fragment and the parental strain a 1200 bp fragment. (B) Southern blot analysis. BamH1 and PstI restriction sites were chosen according to the B. abortus 2308W genome sequence, at positions -3009 and +1552, respectively, from exoR first codon. The resulting fragments were separated in a 0.7% agarose gel. After electrophoresis, the protocol was carried out with minor variations as described in Methods. Briefly, the gel was rinsed in 0.25 M HCL, denatured (1.5 M NaCl, 0.5 M NaOH), and neutralized twice (1.5 M NaCl, 0.5M Tris HCl pH8). The DNA was then transferred to a nylon membrane (Roche) overnight in 10x SSC pH 7 (0.15 M sodium citrate, 1.5 M NaCl). Primers bruabI0884.3 and bruabI0884.5 (S1 Table) were used to generate a 295 bp amplicon from B. abortus 2308W DNA. These primers amplify a region from position 68 bp to -363 from exoR first codon. The amplicon was purified from agarose gels using QIAquick Gel Extraction Kit (Qiagen) and labeled with digoxigenin for use as a probe according to the manufacturer’s instructions (DIG DNA Labelling and detection kit, Roche). The labeled probe was denatured and added to the hybridization buffer. Hybridization was performed overnight. After exposure to ultraviolet light, the membrane was then washed, blocked, incubated with anti-DIG alkaline phosphatase-conjugated FAB-antibody, and washed according to the manufacturer’s instructions. Luminescence was recorded on X-Ray films (Kodak) for 4 hours.

(TIF)

Click here for additional data file.^{(439.6KB, tif)}

S1 Table. Primers used in this study.

(TIF)

Click here for additional data file.^{(82.9KB, tif)}

S1 Raw images. Raw images of the western blots.

(PDF)

Click here for additional data file.^{(1.8MB, pdf)}

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

This work was supported by Fondos del Sistema FEES/CONARE [02-2020, 0652-19 to C. G-V], Fondos FIDA, Universidad Nacional [SIA 0047-17 to C. G-V], Espacio Universitario de Estudios Avanzados, UCREA [B8762] from the Presidency of University of Costa Rica, and the Vice Presidency for Research, University of Costa Rica [C0456 to E.C-O]. The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0254568.r001

Decision Letter 0

Roy Martin Roop II

5 Mar 2021

PONE-D-21-03810

A Sinorhizobium meliloti and Agrobacterium tumefaciens ExoR ortholog is not crucial for Brucella abortus virulence.

PLOS ONE

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Reviewer #1: Guzman-Verri and collaborators made an exoR mutant, ExoR being a strong candidate to regulate BvrS-BvrR two component system, according to data available in S. meliloti and A. tumefaciens. Surprisingly, an exoR mutant is not sensitive to polymyxin B and exoR deletion does not affect virulence in two cellular models and a mice model of infection. These data suggest that ExoR is not a functional ortholog of ExoR from S. meliloti and A. tumefaciens, which is interesting. Surprisingly, targets of the BvrS-BvrR two component system are affected by ExoR, suggesting that ExoR plays an unexpected (and unclear) role in controlling this system.

Major comments

Fig 1B : controls with bvrR and bvrS mutants must be added, to confirm that the serum sensitivity test is performed as expected

Calling the exoR mutant is maybe not appropriate, since a fraction of the protein is probably still present. Thus the authors should rename their mutant (maybe it is not a "null" mutant) and mention, in the Discussion, that they cannot exclude that the remaining protein segment kept some of the functions of the native ExoR.

Fig 2 : the data show that exoR plays a role in the control of BvrR phosphorylation level and, consequently, on the VirB8, Omp25 and VjbR anundance. A complementation should be performed to ensure that these phenotypes are dependent on the absence of ExoR.

In the Material and Methods, the cultures used for experiments like serum/PMB sensitivity, carbohydrate assimilation patterns, infections and western blot, should be more precisely described (medium, culture phase, temperature, agitation)

Minor comments

line 24 "The evidence" is rather unclear, please be more precise

line 26 "no evident effects of" by "no evident effects of exoR deletion on"

line 35 replace "preferentially" by "it preferentially"?

Fig 1CD These experiments (according to lines 291-297) have been performed in TSB, not in minimal medium (MM), is it possible that ExoR is not needed in these conditions? Serum sensitivity assay were performed on bacteria at a pH of about 7.3 since they are in PBS, again it is not a condition in which BvrR phosphorylation should occur, according to Altamirano-Silva et al (2018). I guess that in the initial model of the authors, these conditions represent those in which absence of BvrS repression by ExoR would have been found, if it is the case, this should be indicated. In the same vein, if ExoR function is to repress BvrS, then in the absence of ExoR, the BvrS-BvrR response is constitutively active, and the mutant is not different from the wild type? In this case, the absence of phenotype does indicate that ExoR "function seems different" (line 28)

line 118 In this paragraph, sensitivity to polymyxin B and serum has been tested, but it does not confirm that envelope integrity is preserved in the exoR mutant. There may be aspects of the envelope that are altered but do not lead to either serum or polymyxin B sensitivity.

line 137 "to time growth" should become "to time of growth"

line 141 BvrR instead of BvR

Fig 2 According to Altamirano-Silva et al (2018), BvrR should be phosphorylated at acidic pH in MM, thus why is it phosphorylated in TSB at (I guess) neutral pH? In Fig 2 as well, the lanes should be annotated clearly (exoR or WT under each lane). Late log and mid-log should also be indicated in this figure. Is Omp19 really a loading control? (in other words, were all labelings performed on the same membrane? probably not, thus Omp19 is not a loading control)

Line 170 Add a "." after infection.

Line 172 Replace UFC by CFU. Same remark for Fig 3C. Also indicate that you count CFU per gram of spleen (I guess)

line 189 Replace "is" by "are"

lines 257-259 If the primers exoR-F1 and exoR-R4 are used to confirm exoR deletion, the authors distinguish these deletion events from the ectopic integration of the plasmid in the B. abortus genome. Thus Southern blot is crucial and its description (lines 272-289) should include the nature of the probe used. What fragment is amplified by the bruabI0884.3 and bruabI0884.5 primers?

line 266 replace "Biomeriux" by "Biomerieux"

Reviewer #2: The manuscript by Castillo-Zeledon and colleagues describes the identification and characterization of an ExoR ortholog in Brucella abortus. Previous literature in other members of the Alphaproteobacteria have defined the role of EoxR in the regulatory pathways of important two-component systems, and in B. abortus, the orthologous system is called BvrR/BvrS. To date, no ExoR protein has been identified and characterized in Brucella species, but the authors have identified a putative ExoR protein. Using an exoR deletion strain, the authors demonstrate that deletion of exoR does not affect normal growth of the bacteria in vitro, and the exoR deletion strain also does not exhibit any reduced survival in the presence of membrane perturbation. The authors show that deletion of exoR leads to aberrant production of important proteins, such as VirB8 and Omp25, and VjbR; however, there is no defect in the ability of the exoR deletion strain to infect and survive in cells or animals (i.e., mice).

Overall, the conclusions are well supported by the data, and the experiments were performed soundly. While he findings do not point to the importance of ExoR in virulence in a mouse model, the data still provide important insight into a previously unknown protein in Brucella. Nonetheless, there are a few minor issues the authors need to address:

-Line 35: "where preferentially" should probably be "where it preferentially"

-Line 123: bvrR and bvrS need to be italicized.

-In several places the term "Western Blot" is used, but there is no need to capitalize this term. Please see PMID: 27893304. The specific places in the manuscript using "Western Blot" are lines 129, 139-140, 146, and 150.

-The labeling of Figure 2 makes interpretation of the data very difficult. It is stated that odd lanes are the exoR mutant and even lanes are the wild-type, but the lanes are not labeled with numbers. In fact, numbes may further add to confusion, and it would help if the authors used "WT" and "exoR" to depict which lanes represent which strains.

-Line 194: "identity level" sounds a bit odd, and it might be better to state "high degree of identity" instead.

-Lines 205-206 present a paradox. It is stated that "expression of these proteins is independent of ExoR," and then it is stated that "the results suggest that ExoR is related to the BvrR phosphorylation state." Perhaps clarifying which "proteins" line 205 is referring to would help alleviate the confusion, but as is, it is not clear if the authors are stating the conclusion that EoxR is or is not linked to the BvrR/BvrS systems. Some additional clarification on this point could be helpful.

**********

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PLoS One. 2021 Aug 13;16(8):e0254568. doi: 10.1371/journal.pone.0254568.r002

Author response to Decision Letter 0

8 Jun 2021

THIS INFO IS INCLUDED IN THE COVER LETTER IN A TABLE FORMAT. WE THANK THE REVIEWERS FOR THEIR VALUABLE COMMENTS.

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The funding information was removed from the Acknowledgments section.

The Funding Statement should read as follows:

“This work was supported by Fondos del Sistema FEES/CONARE [02-2020, 0652-19 to C. G-V], Fondos FIDA, Universidad Nacional [SIA 0047-17 to C. G-V], Espacio Universitario de Estudios Avanzados, UCREA [B8762] from the presidency of University of Costa Rica, and the Vice Presidency for Research, University of Costa Rica [C0456 to E.C-O].

The funders had no role in study design, data collection, and analysis, decision to

publish, or preparation of the manuscript.”

This info is in file S1_raw_image

Reviewer #1

Major comments

Fig 1B: controls with bvrR and bvrS mutants must be added, to confirm that the serum sensitivity test is performed as expected.

ANSWER: We followed the reviewer's suggestion and repeated the assays including the corresponding mutant controls. We also incubated the strains for a longer period with serum (90 min). This is now shown in Figure 1B. The figure legend was modified accordingly and includes a suggestion from the other reviewer, indicating that sensitivity to polymyxin B and serum does not confirm cell envelope integrity.

The figure legend is as follows: (page 7, line 141-151)

“Fig 1. The exoR mutation does not alter bacterial growth, non-immune human serum treatment or resistance to polymyxin B. (A) B. abortus 2308W (WT) and the exoR mutant (exoR) were grown in TSB at 37⁰C and the absorbance was measured at 420 nm at the indicated times. (B) The indicated strains B. abortus 2308W (WT), exoR, bvrS, and bvrR mutants, were exposed to non-immune human serum at 37⁰C for 90 min. These experiments are representative of at least three performed. (C) and (D) The indicated strains, B. abortus 2308W (WT), exoR, bvrS, and bvrR mutants, were exposed to increasing concentrations of polymyxin B for 48 hours at 37⁰C, at pH 7 and pH 6, respectively. The minimum inhibitory concentrations shown are representative of at least three experiments performed. The bvrS and bvrR mutant strains were used as susceptibility controls. *, P < 0,05 (Anova and Kruskal-Wallis test for multiple comparisons).

ANSWER: We agree with the reviewer and renamed the strain “B. abortus exoR mutant” instead of null mutant in the entire document.

Also, we included in the discussion the next paragraph:

(page 14, line 316-320)

“Since the introduced deletion in exoR did not remove the gene entirely, we cannot exclude that the remaining protein segment kept some of the functions of the native ExoR. More studies are needed to understand the role of the conserved ExoR, not only in B. abortus but also in other members of the genus, as well as other members of the Alphaproteobacteria.”

Fig 2: the data show that exoR plays a role in the control of BvrR phosphorylation level and, consequently, on the VirB8, Omp25 and VjbR anundance. A complementation should be performed to ensure that these phenotypes are dependent on the absence of ExoR.

ANSWER: We understand the reviewer's opinion. The results show punctual changes in BvrR phosphorylation and protein expression at specific points during bacterial growth in TSB pH 7. In this revised version, new experiments show that under conditions that promote BvrR/BvrS activation (MM and pH5.0) the trend is similar, i.e no drastic changes in protein expression as compared to the wild type, but rather subtle ones related to the protein expression dynamics. Although the idea of a complemented strain expressing exoR sounds the reasonable next step, it is our experience that complementation experiments related to gene regulation networks and involving expression of proteins related to the bacterial membrane usually do not give clear answers, but rather inconclusive ones. The lack of proper molecular biology tools proven to work in Brucella, where gene and protein expression can be accurately controlled, also precluded our intention to perform such experiments. Our opinion is that the trials that indicate a clear phenotype and relationship with bacteria virulence are the in vivo and ex vivo models using mice and cell infections, where the mutant behaved just like the wild-type strain. Therefore, we conclude that under these conditions, a complemented strain would not add any significant value to our findings.

In the Material and Methods, the cultures used for experiments like serum/PMB sensitivity, carbohydrate assimilation patterns, infections, and western blot, should be more precisely described (medium, culture phase, temperature, agitation)

ANSWER:We followed the reviewer´s suggestion and incorporated a more precise description for the indicated experiments in the methods section:

The phrase:

“B. abortus 2308W (WT Brucella strain, virulent, smooth LPS, NaIr) [50] or B. abortus 2308W ΔexoR mutant strain (this study) were maintained and grown in standard Tryptic Soy Broth (TSB) at 37⁰C.”

Was replaceD for: (page 15; line 327-330)

“B. abortus 2308W (WT Brucella strain, virulent, smooth LPS, NaIr) [52] or B. abortus 2308W exoR mutant strain (this study) were maintained and grown at 37⁰C with agitation (200 rpm) in standard Tryptic Soy Broth (TSB) at pH 7.0 or minimal medium (33 mM KH2PO4, 60.3 mM K2HPO4, and 0.1% yeast extract) at pH 5.0 (adjusted with citric acid).”

The phrase

“Essentially, exponentially growing bacteria of each strain were adjusted to 5x106 CFU/mL in TSB.

Was replaced for: (Page 17, line 375-377)

“Essentially, both strains, mutant, and wild-type, were grown until exponential phase in TSB at pH 7, with agitation of 200 rpm. Bacteria of each strain were adjusted to 5x106 CFU/mL in TSB.

The phrase:

“Exponentially growing bacteria were adjusted to 104 CFU/ml in PBS and dispensed in duplicate in microtiter plates (200 µl per well) containing fresh human serum (400 µl/well). After 45 min of incubation at 37°C, 100 µl was plated on tryptic soy agar.”

Was replaced for: (page 17, line 383-385)

“Exponentially growing bacteria grown in TSB at pH 7, with the agitation of 200 rpm, were adjusted to 104 CFU/ml in PBS and dispensed in duplicate in microtiter plates (200 µl per well) containing fresh human serum (400 µl/well). After 90 min of incubation at 37°C, 100 µl was plated on tryptic soy agar.”

The phrase:

“Bacteria were concentrated by centrifugation, resuspended in Laemmli sample buffer, and heated at 100°C for 20 min.”

Was replaced for: (page 18, line 394-396)

“Bacteria grown to exponential phase in TSB at pH 7, with the agitation of 200 rpm, were concentrated by centrifugation, resuspended in Laemmli sample buffer, and heated at 100°C for 20 min.

The phrase

“BALB/c female (18-24 grams) mice were intraperitoneally (i.p.) inoculated with 106 UFC of either B. abortus 2308W or B. abortus 2308W ΔexoR mutant and sacrificed at 3 and 9 weeks post-infection.”

Was replaced for: (page 20, line 436-439)

“BALB/c female (18-24 grams) mice were intraperitoneally (i.p.) inoculated with 106 UFC of either B. abortus 2308W or B. abortus 2308W exoR mutant at exponential phase grown in TSB at pH 7, with the agitation of 200 rpm. Mice were sacrificed at 3- and 9-weeks post-infection.”

Minor comments

line 24 "The evidence" is rather unclear, please be more precise

ANSWER:The phrase:

“The evidence reveals that ExoR is related to the BvrR phosphorylation state, and the absence of ExoR has different impacts on known BvrR/BrvS gene targets.

was modified according to the reviewer's suggestion as follows (page 2, line 24-27):

Our findings indicate that ExoR is related to the BvrR phosphorylation state, and to the expression of known BvrR/BrvS gene targets, such as virB8, vjbR, and omp25 when grown in rich medium or starving conditions.

line 26 "no evident effects of" by "no evident effects of exoR deletion on"

ANSWER:The phrase;

“Despite this, we found no evident effects of in vitro growth, intracellular replication, or infectivity in a mice model”

was modified according to the reviewer's suggestions, including the renaming of the exoR mutant, as follows: (page 2, line 27-29).

“Despite this, the exoR mutant strain showed no significant differences as compared to the wild type strain, related to resistance to polymyxin B or human non-immune serum, intracellular replication, or infectivity in a mice model.”

line 35 replace "preferentially" by "it preferentially"?

ANSWER:The suggestion was included as follows:

(page 2, line 37) Brucella abortus infects bovines, where it preferentially replicates in reproductive organs, causing abortion, infertility, decreased milk production, reproductive failure, and epididymitis [2,3]

ANSWER: We thank the reviewer for this comment. To clarify, BvrR phosphorylation does occur under TSB and neutral pH conditions. This was shown by Altamirano-Silva et al (2018), Figure 2 where lysates from bacteria grown at pH 7 in TSB were used as BvrR-phosphorylation control, as well as in Figure 3. On the other hand, when bacteria are transferred to an acidic pH and MM, Altamirano-Silva et al (2018) observed a transient increase in BvrR phosphorylation as compared to bacteria transferred into TSB at neutral pH. This effect is shown in Altamirano-Silva et al (2018) Figure 6.

Following the reviewer´s suggestion and to assess if ExoR is not needed after exposure to minimal medium and pH 5, we performed the serum sensitivity and the polymyxin resistance assays using wild-type and exoR mutant strains grown to exponential phase and incubated in minimal medium (MM) at pH 5.0 or rich medium (TSB) at pH 7.0 for 4 hours. We also assessed the expression of VjbR and VirB8 after exposure of the exoR mutant exponentially growing cultures, to MM pH 5 for 0.5, 2, 4, and 6 hours. The results are included as a new figure, Fig 4 and modifications to the Methods, results, and discussion section were introduced accordingly (results section (page 10-11, line 206-237), Fig 4, as well as in the discussion section (page 14, line 302-308).

Regarding the second question, we are not sure if we understand the reviewer´s reasoning. As shown in fig 1, the BvrR phosphorylation pattern is a dynamic process, occurring both in the wild-type and the exoR mutant. The main difference in this pattern between both strains is that the BvrR-P signal faded faster in the exoR mutant, as compared to the wild-type strain. This is interpreted as that indeed, ExoR is related to BvrR phosphorylation dynamics and hence, to some BvrR regulation targets. The reason for this observation remains elusive and is not relevant in terms of B. abortus virulence, at least in a mice model. The phrase “function seems different” pointed out by the reviewer, is related to ExoR orthologs. To clarify those points, we made the following modifications:

Starting at line 24 of the previous version:

“The evidence reveals that ExoR is related to the BvrR phosphorylation state, and the absence of ExoR has different impacts on known BvrR/BrvS gene targets. Despite this, we found no evident effects of in vitro growth, intracellular replication, or infectivity in a mice model. ExoR in B. abortus is related to BvrR/BvrS as observed in other Rhizobiales; however, its function seems different from that observed for its orthologs described in A. tumefaciens and S. meliloti.”

It is now replaced by the following: (page 2, line: 24-31)

“Our findings indicate that ExoR is related to the BvrR phosphorylation state, and to the expression of known BvrR/BrvS gene targets, such as virB8, vjbR, and omp25 when grown in rich medium or starving conditions. Despite this, the exoR mutant strain showed no significant differences as compared to the wild-type strain, related to resistance to polymyxin B or human non-immune serum, intracellular replication, or infectivity in a mice model. ExoR in B. abortus is related to BvrR/BvrS as observed in other Rhizobiales; however, its function seems different from that observed for its orthologs described in A. tumefaciens and S. meliloti.”

Starting at line 206, Discussion section in the previous version:

“The results suggest that ExoR is related to the BvrR phosphorylation state. The data presented here show that exoR plays a role in the control of BvrR phosphorylation level. The exoR mutant showed a gradual fade of BvrR-P signal during bacterial growth, which occurs faster than in the wild-type strain. There is no direct evidence linking the phosphorylation of the response regulator and ExoR in other bacterial RSI switches; but, in A. tumefaciens and S. meliloti ExoR absence induces a phenotype similar to that of bacteria with a constitutive TCS. This does not seem to be the case in B. abortus 2308.”

It is now modified as follows (page 13, line 286-294, Discussion section):

“This suggests that their expression is independent of ExoR, but possible posttranslational modifications (including phosphorylation and dephosphorylation) might not, since the BvrR phosphorylation dynamics was different as compared to the wild-type strain. The data presented here show that exoR plays a role in the control of BvrR phosphorylation level. The exoR mutant showed a gradual fade of BvrR-P signal during bacterial growth, which occurs faster than in the wild-type strain. There is no direct evidence linking the phosphorylation of the response regulator and ExoR in other bacterial RSI switches; but, in A. tumefaciens and S. meliloti ExoR absence induces a phenotype similar to that of bacteria with a constitutive TCS. This does not seem to be the case in B. abortus 2308W.”

ANSWER:We agree with the reviewer´s comment. The subtitle “ExoR is not related to cell envelope integrity.”

was modified to (page 7, line 153-154)

“The exoR mutation does not affect B. abortus resistance to polymyxin B and non-immune human serum.”

Also, the wording of the following paragraph was amended (page 7, line 155-161).

“The B. abortus LPS smooth phenotype was not altered by the exoR mutation, as judged by the acriflavine agglutination test. Exposure to non-immune human serum and increasing polymyxin B concentrations at pH 7 and pH 6 did not reveal differences between the wild-type and the exoR mutant strain (Figs 1B, C and D). As expected, the bvrR and bvrS mutants, included as controls, were sensitive to polymyxin B and non-immune human serum (Fig 1C and D). Both strains showed significant differences when compared to the wild-type and the exoR mutant.“

As well as Figure 1 title: “Fig 1. The absence of ExoR does not alter bacterial growth nor membrane integrity.”

was modified accordingly: (page 7, line 141-142)

“Fig 1. The exoR mutation does not alter bacterial growth, resistance to non-immune human serum or polymyxin B treatment.”

line 137 "to time growth" should become "to time of growth"

ANSWER:The phrase: “Omp25 showed slight differences in expression according to time growth as reported.”

was modified according to the reviewer's suggestion as follows: (page 9, line 190-191)

“Omp25 showed slight differences in expression according to the wild-type strain time of growth as reported [39].”

line 141 BvrR instead of BvR

ANSWER:The phrase:

“As described above, the BvR expression was consistent between strains” is no longer in the manuscript due to the following modification: (page 8, line 166-167).

“We observed similar BvrR and BvrS expression patterns compared to the wild-type strain in the mutant strain (Fig 2).”

ANSWER:As mentioned above, BvrR does phosphorylate in TSB at neutral pH (Altamirano-Silva et al., 2018).

We thank the reviewer´s observation in Fig 2. Lanes are now annotated clearly and each growth phase assessed is indicated. The western blot was performed in different gels, the loading control for each gel was added to the figure. In order not to overload the figure, we divided it into two: one that evaluated the two-component system and another one that evaluates three proteins under BvrRBvrS regulation (Fig 2 and 3). Due to this new structure, the results section where the western blot is explained was modified accordingly. (page 8-9, line 163-204)

The subtitle “ExoR affects the expression of proteins related to BvrR/BvrS.”

Is now replaced for: (page 8, line 163-164)

“ExoR impacts the expression dynamics of proteins related to BvrR/BvrS in nutrient-rich conditions.”

Regarding to the loading control, labeling of the loading control was performed in the same membrane for each of the protein tested. This is clarified in the materials and methods section (page 19, line 407-409) and in Fig 2 and 3.

Line 170 Add a "." after infection.

ANSWER: The "." was added (page 12, line 255).

Line 172 Replace UFC by CFU. Same remark for Fig 3C. Also indicate that you count CFU per gram of spleen (I guess)

ANSWER:The phrase:

“Mice were intraperitoneally inoculated with 106 UFC of either B. abortus 2308W or B. abortus 2308W ΔexoR mutant. Spleen counts were determined at 3 and 9 weeks post-infection.”

Was replaced for: (page12, line 252-255, and figure 5C).

“Mice were intraperitoneally inoculated with 106 CFU of either B. abortus 2308W or B. abortus 2308W exoR mutant. CFU per gram of spleen were determined at 3 and 9 weeks post-infection.”

line 189 Replace "is" by "are"

ANSWER:The phrase:

“Proteins whose gene expression is under the control of BvrR/BvrS showed differentiated expression in the exoR mutant as compared to the wild-type strain.”

Was modified for: (page 12, line 269-271)

“Proteins whose gene expression are under the control of BvrR/BvrS [22,28] showed differentiated expression in the exoR mutant as compared to the wild-type strain.”

ANSWER:For better understanding, we included a schematic representation of the in-frame deletion strategy used to construct the exoR mutant and the Southern blot result as a supplementary figure (S1_Fig.) (Page 5, line 111-139). And wording was improved in Material and Methods (page 16-17, line 359-371).

line 266 replace "Biomeriux" by "Biomerieux"

ANSWER:The word Biomerieux was corrected (page 16, line 351).

Reviewer #2

-Line 35: "where preferentially" should probably be "where it preferentially"

ANSWER:The suggestion was included as follows:

-Line 123: bvrR and bvrS need to be italicized.

ANSWER:Both words were italicized (page 7, line 158).

-In several places, the term "Western Blot" is used, but there is no need to capitalize this term. Please see PMID: 27893304.

ANSWER:The specific places in the manuscript using "Western Blot" are lines 129, 139-140, 146, and 150. The term “Western Blot” was replaced by “western blot” in the manuscript.

ANSWER:Indeed, we agree with the reviewer. For the labeling of the figure, we followed the reviewer's suggestion. Lanes were annotated clearly, the growth phase was indicated and as the western blots were performed in different gels, the loading control for each gel was added to the figure. In order not to overload the figure, we divided it into two figures: one that evaluated the two-component system and another one that evaluates three proteins under BvrR/BvrS regulation (Fig 2 and 3).

The results section where the western blots are explained was modified accordingly (page 8-9, line 165-206).

The subtitle “ExoR affects the expression of proteins related to BvrR/BvrS.”,

was replaced for: (page 8, line 163-164)

ExoR impacts the expression dynamics of proteins related to BvrR/BvrS in nutrient rich conditions.

-Line 194: "identity level" sounds a bit odd, and it might be better to state "high degree of identity" instead.

ANSWER:The suggestion was included as follows:

(page 13, line 277-279)

Considering the high degree of identity between the BvrR, BvrS, and ExoR homologs, these results were unexpected.

ANSWER:We agreed with the reviewer´s comment, so the wording was changed as follows:

The phase:

“Therefore, the expression of these proteins is independent of ExoR. The results suggest that ExoR is related to the BvrR phosphorylation state.”

Was replaced for: (page 13, lines 287-293).

“Moreover, BvrR and BvrS did not show differences in their expression compared to the wild-type. This suggests that their expression is independent of ExoR, but possible posttranslational modifications (including phosphorylation and dephosphorylation) might not, since the BvrR phosphorylation dynamics was different as compared to the wild-type strain. The exoR mutant showed a gradual fade of BvrR-P signal during bacterial growth, which occurs faster than in the wild-type strain.”

PLoS One. doi: 10.1371/journal.pone.0254568.r003

Decision Letter 1

Roy Martin Roop II

30 Jun 2021

A Sinorhizobium meliloti and Agrobacterium tumefaciens ExoR ortholog is not crucial for Brucella abortus virulence.

PONE-D-21-03810R1

Dear Caterina,

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Reviewer #2: Yes

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6. Review Comments to the Author

Reviewer #1: The authors correctly addressed all my comments, including those related to a precise description of their experiments.

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PLoS One. doi: 10.1371/journal.pone.0254568.r004

Acceptance letter

Roy Martin Roop II

5 Aug 2021

PONE-D-21-03810R1

A Sinorhizobium meliloti and Agrobacterium tumefaciens ExoR ortholog is not crucial for Brucella abortus virulence.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Mutant construction and Southern blot.

(TIF)

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S1 Table. Primers used in this study.

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S1 Raw images. Raw images of the western blots.

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Data Availability Statement

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PERMALINK

A Sinorhizobium meliloti and Agrobacterium tumefaciens ExoR ortholog is not crucial for Brucella abortus virulence

Amanda Castillo-Zeledón

Nazareth Ruiz-Villalobos

Pamela Altamirano-Silva

Carlos Chacón-Díaz

Elías Barquero-Calvo

Esteban Chaves-Olarte

Caterina Guzmán-Verri

Roles

Abstract

Introduction

Results

B. abortus 2308W genome encodes an exoR ortholog

Fig 1. The exoR mutation does not alter bacterial growth, resistance to non-immune human serum or polymyxin B treatment.

The exoR mutation does not affect B. abortus resistance to polymyxin B and non-immune human serum

ExoR impacts the expression dynamics of proteins related to BvrR/BvrS in nutrient rich conditions

Fig 2. BvrR/BvrS expression and BvrR phosphorylation dynamics in the exoR mutant.

Fig 3. VjbR, VirB8, and Omp25 expression dynamics are different in the exoR mutant as compared to the wild-type strain.

Low nutrients and pH do not significantly alter B. abortus exoR phenotype as compared to nutrient rich conditions

Fig 4. In vitro conditions mimicking the intracellular environment do not affect B. abortus exoR resistance to non-immune human serum or polymyxin B treatment and induces expression of VjbR and VirB.

The exoR mutant successfully infects mice and cell cultures

Fig 5. The exoR mutation in B. abortus 2308W does not affect its infection capacity.

Discussion

Methods

Bacterial culture

Strain construction

Carbohydrate assimilation pattern

Southern blot

Polymyxin B sensitivity assay

Sensitivity to the bactericidal action of non-immune serum

Western blot and BvrR phosphorylation

Exposure of Brucella strains to minimal and low pH medium

Gentamicin-protection assay and intracellular replication quantification

Virulence assays in mice

Statistical analysis

Supporting information

Data Availability

Funding Statement

References

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Roy Martin Roop II

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Acceptance letter

Roy Martin Roop II

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Associated Data

Supplementary Materials

Data Availability Statement

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