Omp19 Enables Brucella abortus to Evade the Antimicrobial Activity From Host's Proteolytic Defense System

Pathogenic microorganisms confront several proteolytic events in the molecular interplay with their host, highlighting that proteolysis and its regulation play an important role during infection. Microbial inhibitors, along with their target endogenous/exogenous enzymes, may directly affect the host's defense mechanisms and promote infection. Omp19 is a Brucella spp. conserved lipoprotein anchored by the lipid portion in the Brucella outer membrane. Previous work demonstrated that purified unlipidated Omp19 (U-Omp19) has protease inhibitor activity against gastrointestinal and lysosomal proteases. In this work, we found that a Brucella omp19 deletion mutant is highly attenuated in mice when infecting by the oral route. This attenuation can be explained by bacterial increased susceptibility to host proteases met by the bacteria during establishment of infection. Omp19 deletion mutant has a cell division defect when exposed to pancreatic proteases that is linked to cell-cycle arrest in G1-phase, Omp25 degradation on the cell envelope and CtrA accumulation. Moreover, Omp19 deletion mutant is more susceptible to killing by macrophage derived microsomes than wt strain. Preincubation with gastrointestinal proteases led to an increased susceptibility of Omp19 deletion mutant to macrophage intracellular killing. Thus, in this work, we describe for the first time a physiological function of B. abortus Omp19. This activity enables Brucella to better thrive in the harsh gastrointestinal tract, where protection from proteolytic degradation can be a matter of life or death, and afterwards invade the host and bypass intracellular proteases to establish the chronic infection.


INTRODUCTION
The intestinal mucosa is the largest interface between the external environment and the tissues of the human body. The first line of defense in the gastrointestinal tract is in the lumen, where microorganisms are degraded in a non-specific fashion by pH and gastric, pancreatic and biliary secretions. Pathogenic microorganisms confront several proteolytic events in the molecular interplay with their host, therefore proteolysis and its regulation play an important role during infection.
Microbes synthetize protease inhibitors to control endogenous proteases. Some inhibitors can also interact with exogenous peptidases produced by other species and thus may directly affect host's defense mechanisms (1). Few works in the literature show the importance of bacterial protease inhibitors activity against host-proteases (2)(3)(4). Our hypothesis is that pathogenic bacteria synthesize protease inhibitors to evade the antimicrobial activity from host's proteases.
In our laboratory, we have been working on the use of a conserved Brucella spp. protein devoid of its lipid moiety called U-Omp19 as a vaccine against brucellosis (5)(6)(7). Omp19 has significant sequence identity with bacterial protease inhibitors from I38 family. Remarkably, recombinant U-Omp19 inhibits gastrointestinal and lysosomal proteases (8,9). However, the physiological function of Omp19 in Brucella is still unknown.
Brucellosis is a worldwide re-emerging zoonotic disease that is transmitted from domestic and wild animals to humans. The human disease, mostly caused by Brucella abortus and B. melitensis, represents an important cause of morbidity worldwide whereas animal brucellosis is associated with serious economic losses caused mainly by elicited abortions and infertility (10,11).
Oral infection is one of the principal ways of brucellosis transmission. Animals usually lick tissues from abortions or ingest contaminated pasture and humans acquire often the disease by consumption of infected, unpasteurized dairy products (10,(12)(13)(14)(15)(16). Few virulence factors required for food-borne infection by Brucella have been described: Urease and cholylglycine hydrolase that confer resistance to gastric acidity and bile salts, respectively (17,18). Once inside the host, Brucella disseminate via infected phagocytic cells to different tissues and organs, developing foci of infection, surviving intracellularly and leading to a chronic disease (19).
Digestive enzymes, primarily proteases, contribute to the non-specific host defense system exerting a toxic action on microorganisms by destruction of their cell wall (20). Omp19 is a lipoprotein anchored in the Brucella outer membrane (7). This location together with its protease inhibitor activity suggest that it may play a protective role against host proteases.
In this work, we studied if Omp19 enables Brucella to better thrive in the harsh gastrointestinal tract, where protection from proteolytic degradation can be a matter of life or death, and thus promoting host invasion and intracellular infection.

Ethics Statement
Protocols of this study agreed with international ethical standards for animal experimentation (Helsinki Declaration and amendments, Amsterdam Protocol of welfare and animal protection and NIH guidelines for the Care and Use of Laboratory Animals). Protocols of this study were approved by the Institutional Committee for the Care and Use of Experimentation Animals from UNSAM (CICUAE-UNSAM_N • 04/2014).

Bacterial Strains, Media, and Culture Conditions
Brucella strains were derived from the wild type (wt) 2308 biovar and were: (i) smooth virulent wt B. abortus; (ii) unmarked omp19 deletion mutant ( omp19); and (iii) omp19 complemented omp19 mutant ( omp19pBBR4omp19). All strains were grown as described in Czibener and Ugalde (21). When necessary, media were supplemented with the Ampicillin (100 µg/ml) or Nalidixic acid (5 µg/ml). CFU determination from intestine containing samples were performed in medium with following antibiotics to inhibit normal flora growth: Vancomycin (20 µg/ml), Cycloheximide (100 µg/ml), Bacitracin (10 U/ml), and Nalidixic acid. All work with live Brucella was performed in BSL3-laboratories and BSL3-animal facility at UNSAM. Escherichia coli strains were grown at 37 • C in LB with Ampicillin.

Generation of Mutant Strains
Omp19 (BAB1_1930) unmarked chromosomal mutant was generated as described in Herrmann et al. (22). Briefly, two DNA fragments of ∼500 bp containing flanking regions of BAB1_1930 were amplified from B. abortus 2308 genomic DNA. Primers used to amplify omp19 ′ s upstream regions were: for downstream region. Fragments were ligated by overlapping PCR using omp19(EcoRI)_Up_Fw and omp19(BamHI)_Down_Rv. The resulting fragment was digested with EcoRI and BamHI, cloned into pK18mobSacB (23) and conjugated to B. abortus 2308 by biparental mating. Single recombinants selection, selection with sucrose, excision of plasmids, and generation of deletion mutants was performed as described previously described (21). Deletion of BAB1_1930 was confirmed by PCR and sequence analysis and western blot ( Figure S1).

(ii) Complementation of omp19 Mutant
A 1000 bp DNA fragment containing the complete gene (BAB1_1930) and its promotor was amplified using primers

Recombinant Proteins, Enzymes, and Extracts
Mouse intestine-and stomach-extracts were obtained as previously described (8). Briefly, intestines and stomachs extracts were obtained from 6 to 12 weeks old female or male Balb/c mice (n = 10). Prior to fluid preparation, mice were fasted for 2.5 h (water ad lib.) and euthanized by CO 2 inhalation. Stomachs and small intestines were resected, homogenized in PBS, and fluid separated by centrifugation (10 min, 13,200 × g at 4 • C). Pooled Intestinal or stomach fluids were snap-frozen in liquid nitrogen and stored at −80 • C. Protein concentration and proteolytic activity were determined as previously described by Ibañez et al. (8). Microsomes of J774 murine macrophages were obtained as described previously by Coria et al. (9). Pancreatin, Elastase, and Trypsin from pig and α-Chymotrypsin from bovine were from Sigma.

Infection of Mice
Six to eight-week-old female BALB/c mice were bred at IIB-UNSAM. Five mice/group were inoculated (i) wt, (ii) omp19, or (iii) omp19pBBR4omp19 Brucella strains either by gavage (i.g.) with 1 × 10 9 CFU in 0.2 ml PBS (18,26) or with 1 × 10 10 CFU directly into the oral cavity as previously described by von Bargen et al. (27). Infected mice were kept in cages within a BSL3 facility. At different times post-infection mice were euthanized by CO 2 inhalation and organs were aseptically collected, homogenized, and plated for CFU determination. Intestinal samples were plated on TSA supplemented with Vancomycin, Cycloheximide, Bacitracin, and Nalidixic acid. In some experiments tissue samples from duodenum were obtained for immunofluorescence analysis.

(i) Agar Disk-Diffusion Method
Brucella strains (1 × 10 8 CFU) were spread on TSA plates supplemented with Vancomycin, Cycloheximide, Bacitracin, and Nalidixic acid. Five-mm filter disks impregnated with either PBS, intestine-or stomach-extract were placed on the agar surface. After 72 h of incubation (37 • C) zones of inhibition were determined.

Bacterial Growth Analysis
Brucella strains were labeled with TRSE (Invitrogen) as previously described by Brown et al. (28). Bacteria were spotted on 1% agarose pads with 10% TSB plus PBS or pancreatin (2 mg/ml). Images were obtained before and after 24 h of culture on an Olympus IX-81 microscope with FV-1000 confocal module. Images were subtracted the background and merged using RGB format (ImageJ software). Number of total bacteria (N) and initial number of bacteria (N 0 , number of labeled or partially labeled bacteria) were enumerated using Spot Detector plugin (ICY software, Institute Pasteur). Three to nine images/condition in duplicates were evaluated (50-150 colonies/condition). Then, assuming exponential growth, the average number of cell divisions (n) was calculated: Average number of cell divisions = n = log 2 N N 0

DNA Content on Individual Bacteria
Brucella in exponential phase (5 × 10 7 CFU/ml) were incubated with or without pancreatin (2 mg/ml). After 1.5-6 h, cells were washed, fixed, incubated with RNase A and labeled with SYTOX-Green (Invitrogen). Samples were analyzed in a FACS ARIA II (BD Biosciences) and analyzed with FlowJo7.6.2 software (Tree Star).

Cell Culture and Infection Assay
J774 macrophages were maintained in RPMI 1640 supplemented with 5% fetal bovine serum (FBS) and streptomycin (50 µg/ml)penicillin (50 U/ml) (Gibco Life Technologies) in a humidify 5% CO 2 atmosphere at 37 • C. Cells (5 × 10 4 per well) were seeded on 24-well plates in antibiotic-free medium and were kept for 24 h. B. abortus infections were carried out at a multiplicity of infection (MOI) of 500:1 or 100:1. After a 1 h incubation with the bacteria, wells were washed three times with PBS and incubated with fresh medium containing 50 µg/ml of Gentamycin and 100 µg/ml streptomycin to kill non-internalized bacteria. At the indicated time points, infected cells were washed three times with PBS and lysed with 500 µl 0.1% Triton X-100 (Sigma-Aldrich). The intracellular CFU were determined by plating serial dilutions on TSA. In some experiments, prior to the infection of cell the bacteria were incubated at 5 × 10 7 CFU/ml with or without pancreatin (2 mg/ml) during 2 h. Afterwards bacteria were washed and suspended in medium to infect the cells.

Statistical Analysis
Statistical analysis and plotting were performed using Prism R 7.04 (GraphPad, Inc., USA). CFU data were logarithmically transformed. Unpaired two-tailed Student t-test was used for pairwise comparisons between means of two groups or one-way or two-way ANOVA followed by Bonferroni's posttest was used for comparing more than two means. Significance level was set at p < 0.05.

Omp19 Expression Is Needed for Oral Acquired B. abortus Infection
To investigate the role of Omp19 in Brucella infection, a deletion mutant ( omp19) and its complemented strain ( omp19pBBR4omp19) were constructed in the B. abortus wildtype (wt) strain 2308. Mutant and wt strains had similar growth curves, resistance to low pH and bile salts. Moreover, membrane permeability to hydrophobic substances, expression of main outer membrane proteins (Omps) (Omp1, Omp2b, Omp25, Omp10, and Omp16) and lipopolysaccharide O-antigen were similar between wt and omp19 strains (Figures S1A-F). The authenticity of the mutant was verified by PCR and immunoblot analysis on whole-cell extracts with an anti-Omp19 Mab (Figures S1E,F).
To evaluate the role of Omp19 in the establishment of B. abortus infection through the digestive tract in vivo, BALB/c mice were inoculated intragastrically (i.g.) with wt, omp19 or omp19pBBR4omp19 and 20 days post-infection bacterial loads at spleens and cervical lymph nodes (CLNs) were assessed. While wt and omp19pBBR4omp19 established infection, there were significant lower numbers of CFUs at spleens and CLNs from omp19 infected mice (p < 0.001 vs. wt) (Figures 1A,B). Upon gavage administration, initial events of bacterial invasion and onset of infection in the oral cavity may be bypassed. Thus, BALB/c mice were administered directly into the oral cavity as described in von Bargen et al. (27) with wt or omp19. Twenty days post-infection B. abortus were isolated from spleens and CLNs from wt infected mice, whereas almost no CFUs were found in these organs of omp19 infected mice (p < 0.05 and p < 0.01 vs. wt, respectively) (Figures 1C,D).
Altogether, these results demonstrate that Omp19 plays a crucial role in the establishment of infection by Brucella through the oral route in mice.

Brucella abortus Reaches Intestinal Tissues Upon Oral Infection and Requires Omp19 to Evade the Bacteriostatic Action of Intestinal Content
To evaluate if omp19 attenuation after oral infection is due to higher susceptibility to gastrointestinal content, short-term gavage infection experiments were performed. BALB/c mice were i.g. inoculated with wt or omp19 strains and at different time points post-infection the stomach and intestinal sections were analyzed. After 15 min equal numbers of bacteria were isolated from the stomachs of both groups (Figure 2A). After 1 h both strains were present in the duodenum at the lumen as well as in the epithelium ( Figure 2B).
Next, B. abortus loads in different sections of the small intestine: duodenum, jejunum, ileum and Peyer's patches were evaluated. Almost no differences in wt and omp19 CFUs were detected at 2 h post-infection with a slight but significant increase in omp19 CFUs at Ileum (Figure 2C) that may not explain the attenuation of this strain when infecting by the oral route. However, when plated undiluted (direct plating from each tissue on TSA + Antibiotics) low-density bacterial growth and small colonies were found in the drops of omp19, indicating that the intestine content impaired omp19 strain's growth. This effect was temporarily and reversible, since upon dilution it disappeared and both, wt and omp19, showed similar numbers and phenotype of colonies ( Figure 2D). These results indicate that the intestine content exerts a bacteriostatic action on omp19, suggesting that Omp19 protects Brucella from intestinal proteases.
Similar results were obtained when bacteria where inoculated directly at the oral cavity of mice. Both strains, wt and omp19, were recovered from intestinal tissues after 1 h of infection ( Figure 2E), indicating that Brucella reaches the intestine after oral infection (by gavage or oral cavity delivery) and there it is exposed to the intestinal content that exerts a bacteriostatic effect.

Omp19 Protects B. abortus Against the Action of Pancreatic Proteases
To further assess the role of Omp19 against the action of gastric and gut content, in vitro bacterial susceptibility assays were performed.
An agar disk-diffusion test indicated that omp19 is more susceptible to the action of intestine content than the wt strain (p < 0.01 vs. wt + intestine extract) (Figure 3A), whereas stomach content did not affect bacterial growth.
Incubation with intestine-extract inhibited omp19's growth and this action was bacteriostatic, since viable bacteria were recovered by dilution (Figure 3B). Viable bacteria determination over time indicated that in presence of intestine-extract omp19 was unable to grow (p < 0.001 vs. wt + intestine), whereas the wt and the complemented strains grew exponentially after 13 h of culture ( Figure 3C). Similar results were obtained using pancreatin (a pig pancreatic extract) (Figure 3D), supporting that B. abortus requires Omp19 to grow when exposed to intestinal content.
These results together demonstrate that B. abortus requires the expression of Omp19 to resist the action of intestinal proteases.

omp19 B. abortus Stops Cell Division and Cell-Cycle Progression at G1-Phase After Incubation With Pancreatic Proteases
To evaluate if omp19's growth impairment when exposed to proteases is due to a cell division defect, Brucella's growth was studied by microscopy. Brucella Texas-red succinimidyl-ester (TRSE) labeling allows, after growth, the visualization of an unlabeled pole and subsequently unlabeled or partially labeled daughter cells. TRSE-labeled wt and omp19 were cultured on TSB-agarose pads containing buffer or pancreatin. After 24 h wt and omp19 in buffer-pads and wt in pancreatin-pads formed microcolonies with many unlabeled cells surrounding partially labeled cells. However, omp19 in pancreatin-pads formed no or small colonies (small chains) with few unlabeled sphereshaped bacteria (Figure 4A), indicating a cell division defect. Quantitative analysis of labeled (or partially labeled) cells and unlabeled cells in each image revealed a significantly lower average number of cell divisions for omp19 in pancreatin-pads (p < 0.001 vs. wt in pancreatin) ( Figure 4B).
Cell division requires critical regulation of the cell-cycle to coordinate genome replication and segmentation, therefore cellcycle progression on individual bacteria was determined. While incubation of wt with pancreatin did not alter its progression along the cell-cycle, omp19 resulted in cell-cycle arrest at G1 (Figure 4C), that was evident after 3 h of incubation by the rate of cells accumulated in G1-phase (p < 0.001 vs. wt + pancreatin, Figure 4D). Besides, expression of cell-cycle master regulator CtrA and chaperonin GroEL were evaluated upon treatment with pancreatic elastase. Pancreatic elastase treatment increased CtrA signal in omp19, whereas GroEL expression was similar in both strains exposed or not to proteases ( Figure 4E).
Together, these results reveal that omp19 exposed to pancreatic proteases has a cell division defect that is linked to impaired progression through G1-phase and CtrA accumulation.

Omp19 Protects Omp25 From Pancreatic Elastase Digestion
As cell envelope constitutes the first contact with host-proteases, cell envelope proteins were evaluated in wt and omp19 upon protease treatment. No changes between wt and omp19 were detected upon pancreatic elastase treatment in Omp1, Omp10, or Omp16. On the contrary, in both strains Omp25 presented a lower molecular weight band and reduced Omp2b intensity upon pancreatic elastase incubation that would correspond to digested Omp25 and Omp2b, respectively ( Figure 5A). While no Omp19dependent protection of Omp2b digestion was evidenced in wt strain compared to omp19 strain, the percentage of digested Omp25 was higher in omp19 (Figure 5B), highlighting     Omp19's inhibitory role of pancreatic elastase. Omp19 inhibition of pancreatic elastase digestion of Omp25 was confirmed in vitro using recombinant purified proteins. Pancreatic elastase digestion of rOmp25 was evidenced by a reduced Omp25-specific signal in western blot compared with the signal of rOmp25 without protease. This reduction was lower when U-Omp19 was added, indicating that U-Omp19 inhibits Omp25 digestion by pancreatic elastase (Figure 5C). Differences in the digestion pattern between Omp25 expressed on the Brucella membrane and recombinant Omp25, may be due to differential accessibility of pancreatic elastase cleavage sites, since in membrane associated Omp25 most cleavage sites are in predicted transmembrane regions or in loops facing the periplasm, only one cleavage site would be accessible to the protease when Omp25 is in the context of the Brucella membrane ( Figure 5D).
These results together indicate that when Omp19 is absent, pancreatic elastase gains access to the membrane following degradation of Omp25, on the contrary under physiologic condition where Omp19 is present, Brucella wt can withstand this protease activity.

Omp19 Impairs Macrophage Microsomal Proteolytic Killing of B. abortus
Reaching the intracellular replicative niche is the next step for establishment of infection. Therefore, the ability of omp19 mutants to enter cells and replicate intracellularly was studied in professional phagocytes (Figure 6). In agreement with previous studies significant lower amounts of omp19 were found after 6, 24, and 48 h of infection in comparison to wt strain ( Figure 6A). Moreover, omp19 strain was significantly more susceptible to killing by microsomal content than wt or omp19pBBR4omp19 (p < 0.01 vs. wt + microsomes or omp19pBBR4omp19 + microsomes) (Figure 6B), suggesting that Omp19 may protect the bacteria from lysosomal proteolysis during intracellular traffic.
When infecting through the oral route Brucella will reach the intracellular compartment after facing with gastrointestinal proteases, thus omp19 and wt strains were preincubated with pancreatin or buffer for 2 h prior to infection of J774 macrophages and intracellular bacterial counts were determined after 1, 2, or 4 h of infection. Pre-incubation with pancreatin did not affect bacterial internalization, since similar amounts of intracellular bacteria of both strains were recovered after 1 h of infection. After 4 h of infection, preincubation with pancreatin led to an increased susceptibility of omp19 to intracellular killing by macrophages, compared to pancreatin pretreated wt (p < 0.0001) or buffer pretreated omp19 (p < 0.0001) (Figure 6C). These results indicate that the sequential action of intestinal proteases followed by intracellular microsome proteolytic killing has an important effect on hampering omp19 ability to establish an intracellular niche in macrophages. Altogether these results may explain the highly attenuated phenotype of this strain when infection occurs by the oral route.

DISCUSSION
After consumption of infected milk or experimental oral infection, live Brucella are detected in fecal samples of natural host like cattle, bison, wolf and marine mammals, indicating that Brucella transits and pass the harsh environment of gastrointestinal tract (33).
Our results demonstrate that in mice, after oral infection (either by gavage or inoculation at the oral cavity) Brucella reaches the gut. After 1 h of infection brucellae were found at the lumen and epithelium of duodenum. This fast infection capacity of Brucella was shown in a calf ligated ileal-loop model, in which Brucella bacteremia was detected 30 min after intraluminal inoculation without histopathologic traces of lesions (34). Brucella may spread systemically from the digestive tract by transepithelial migration in mucosal epithelial barrier or through M cells (26,34,35).
As protease inhibitor activity against main gastrointestinal proteases was demonstrated for U-Omp19 and because of its strategic location on the outer membrane for interacting with host proteases (7-9), we speculated that Omp19 may allow Brucella to withstand the gastrointestinal proteolysis and infect orally. Omp19's protease inhibitor broad-specificity (8,9) would also be advantageous regarding the different proteases that Brucella may encounter along infection. Like broad-spectrum serine-protease inhibitor from Tannerella forsythia, that may protect it from proteases from other bacteria and from the host (3).
In this work, Omp19's role in virulence in an oral infection murine model was examined. Our results showed that Omp19 expression is needed for establishment of oral acquired B. abortus infection. In contrast to wt, omp19 was cleared from the spleens and CLNs at 20 days post infection. Remarkably after intraperitoneal infection of mice, omp19 deletion resulted in significant loss of virulence but the bacteria were not cleared (36,37), this difference highlights the importance of Omp19 for Brucella oral infection, probably due to the huge amounts of proteases encountered when infecting through this route. Attenuation upon systemic infection and intracellularly may be due to other host-proteases action, like lysosomal proteases, to which Omp19's inhibitory activity was demonstrated (9).
Intestinal content exerted a bacteriostatic action on omp19 in vivo and in vitro, revealing a protective role for Omp19 in Brucella against intestinal proteases. This is the first work demonstrating in vivo a role of a protease inhibitor in acquisition of a bacterial disease by the oral route, therefore these findings are highly relevant for foodborne infections. Interestingly, gut microbiota, that survive in this protease-rich medium, produce protease inhibitors to protect them self from exogenous proteases (38)(39)(40)(41).
In vitro experiments with purified proteases shed light into the role of individual proteases in the bacteriostatic action of intestinal content. omp19's growth is hampered by the action of pancreatic elastase, indicating that inhibition of this protease by Omp19 on B. abortus membrane is important during the initial steps of infection. Trypsin and αchymotrypsin have been shown to elicit antibacterial activities against E. coli, Proteus vulgaris, Pseudomonas aeruginosa, S. aureus, Streptococcus pyogenes, and Vibrio cholerae (42,43), but have no effect on B. abortus. This resistance is Omp19-independent, indicating that it may be mediated by other mechanism.
Pancreatic proteases induce a cell division defect in omp19 that is linked to cell-cycle arrest in G1-phase. Interestingly, G1 arrest also occurs during intracellular trafficking of B. abortus and on starvation in Sinorhizobium meliloti (44,45). Therefore, delaying initiation of DNA replication could be a common feature used by αproteobacteria in response to harsh conditions such as infection or starvation. (B) wt, omp19 or omp19pBBR4omp19 B. abortus strains (1 × 10 5 CFU/ml) were incubated with PBS or J774 derived microsomes at 37 • C. Live bacteria (CFU/ml) were determined after 8 h by serial dilutions plating (Statistical analysis was performed by one-way ANOVA followed by Bonferroni posttest to compare between the indicated groups: ns p > 0.05, **p < 0.01). (C) omp19 and wt strains were preincubated with buffer or pancreatin for 1 h prior to infection of J774 macrophages (MOI 100:1). Intracellular CFUs were determined at 1, 2, or 4 h post-infection. (Statistical analysis was performed by two-way ANOVA followed by Bonferroni posttest to compare between the indicated groups: ns p > 0.05; *p < 0.05, ***p < 0.001, ****p < 0.0001).
In Caulobacter crescentus, degradation of the CtrA cellcycle master regulator occurs at specific points in the cellcycle. Clearance of active CtrA at the G1/S transition allows the initiation of DNA replication and cell-cycle progression (30,46). Moreover, expression of a constitutively active stable CtrA derivative results in dominant G1 arrest (30). In B. abortus, the essential role of CtrA in cell division was recently confirmed (47). Thus, accumulation of CtrA in omp19 upon pancreatic protease treatment, agrees with the cell-cycle arrest in G1 induced in this strain upon treatment with proteases.
Antimicrobial functions of proteases can be due to the attack of Omps leading to loss of membrane integrity (42,43,48,49). Since outer membrane proteins are exposed on the bacterial surface, they could be targets of pancreatic elastase. Among all Omps evaluated, our results indicate that Omp10, Omp16, and Omp1 of either wt or omp19 were resistant to the action of pancreatic elastase, whereas, Omp2b and Omp25 were digested by this protease. This result is consistent with protease digestion of Omps in E. coli or P. aeruginosa, in which the major Omps, OmpA, and OmpF, respectively, were degraded, while other Omps remained not affected (48,49). Although Brucella Omp25 does not share identity with E. coli OmpA (50), topology predictions suggest that both contain similar secondary structural properties and may play a similar function (51). Notably, Omp19 expression in Brucella inhibited pancreatic elastase mediated Omp25 digestion. This role of Omp19 on inhibition of pancreatic elastase mediated Omp25 digestion was confirmed in vitro using recombinant purified proteins. Omp19 inhibition of pancreatic elastase digestion of Omp25 may explain the resistance of wt strain to the action of this protease. A similar role was described for the periplasmic protease inhibitor ecotin from E. coli, which reduces the bactericidal action of neutrophil elastase by protecting OmpA on the bacterial membrane from neutrophil elastase mediated digestion (2).
In this work, we found that a Brucella omp19 deletion mutant is highly attenuated in mice after oral infection. This attenuation can be explained by bacterial increased susceptibility to host proteases met by Brucella during establishment of infection. omp19 has a cell division defect when exposed to pancreatic proteases that is linked to cell-cycle arrest in G1-phase, Omp25 degradation on the cell envelope and CtrA accumulation. Interestingly, a link between these three molecules was found recently, in which CtrA can bind the promotor of omp25 and omp19. The same work demonstrates that CtrA controls the expression of Omp25 (47), therefore the increment in Omp25 intensity in omp19 upon pancreatic elastase treatment may be explained by the increment in CtrA expression.
Upon entry into mammalian cells, the intracellular pathogen Brucella abortus resides within a membrane-bound compartment, the Brucella-containing vacuole (BCV), the maturation of which is controlled by the bacterium to generate a replicative organelle derived from the endoplasmic reticulum (ER). BCVs traffic along the endocytic pathway and fuse with lysosomes, and such fusion events are required for further maturation of BCVs into an ER-derived replicative organelle (52). Thus, the role of Omp19 for intracellular survival was studied. In agreement with previous work (36,37), omp19 was attenuated inside macrophages. This attenuation may be due to increased susceptibility to intracellular proteases when lacking Omp19. This hypothesis is reinforced by the fact that Omp19 is able to inhibit lysosomal proteases (9) and here we demonstrated that omp19 is more susceptible to proteolytic killing by microsomes from macrophages. This increased susceptibility may explain the slight attenuation for systemic infections in mice, in which high persistence of omp19 was shown after 4 weeks of infection (36,37). An additive effect in increasing susceptibility of omp19 was observed when the strains were preincubated with pancreatic proteases prior to infection of macrophages. This increased susceptibility may account for the high attenuation of omp19 after in vivo oral infection. Therefore, Omp19 would allow Brucella spp. to bypass lysosomal destruction thus enabling Brucella to survive inside macrophages and start a chronic infection.
Overall, this study demonstrates that the protease inhibitor Omp19 confers B. abortus the ability to resist the action of proteases. Together with urease that may protect Brucella from stomach low pH (17) and cholylglycine hydrolase that confers resistance to bile salts (18), Omp19 by inhibiting intestinal and intracellular proteases contributes to the establishment of chronic infection through the oral route.

CONTRIBUTION TO THE FIELD STATEMENT
Understanding how infectious pathogens spread is critical to prevent infectious diseases. One of the principal ways in which human and animal Brucellosis is acquired, is the oral route. This implies that Brucellae must survive the harsh conditions along the gastrointestinal tract before reaching the mononuclear phagocytes to form a replicative niche. In this work, we demonstrate that Brucella has a lipoprotein, called Omp19, which is a protease inhibitor, that enables it to survive the proteolytical action of gut digestive and microsomal derived proteases. The significance of our research is in identifying a new mechanism involved in virulence in oral acquired Brucellosis, that will enhance our understanding of Brucella pathogenesis and would serve as a model for other food-borne diseases.

DATA AVAILABILITY
The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

ETHICS STATEMENT
Protocols of this study agreed with international ethical standards for animal experimentation (Helsinki Declaration and amendments, Amsterdam Protocol of welfare and animal protection and NIH guidelines for the Care and Use of Laboratory Animals). Protocols of this study were approved by the Institutional Committee for the Care and Use of Experimentation Animals from UNSAM (CICUAE-UNSAM_N • 04/2014).

AUTHOR CONTRIBUTIONS
KP, MC, and JC designed the experiments. Funding acquisition was done by JC. MC performed most laboratory assays with assistance from KP, FG, LB, DR, and LC. MR performed susceptibility to bile salts assays and some of the J774 macrophage infection assays. KP, JC, and MC performed all statistical analysis. DC provided bacterial strains, materials and, together with JC and KP contributed with their expertise on the subject. KP, MC, and JC interpreted all results. KP and JC wrote the manuscript.