Evaluation of the role of SsaV ‘ Salmonella pathogenicity island-2 dependent type III secretion system components on the virulence behavior of Salmonella enterica serovar Typhimurium

Salmonella is a causative agent of wide range of diseases varying from gastroenteritis to systemic typhoid fever. It uses specialized Type III secretion system (T3SS) by its two compartments to invade and intracellularly survive inside the immune cells. T3SS is expressed in two subsequent phases by two distinct Salmonella pathogenicity islands (SP) I and II. Understanding and evaluation of components T3SS-SPI1 and T3SS-SPI2 are very important, not only to evaluate the bacterial virulence but also to develop vaccines. In this study, the effect of mutation on SsaV encoding gene (one of the essential T3SS-SPI2 components) was investigated on the virulence behavior of Salmonella enterica serovar Typhimurium. We found a significant reduction in invasion capability and intracellular replication as well.


INTRODUCTION
Salmonellae are gram-negative pathogens; causing a wide range of diseases, varying from a general mild, selflimiting gastroenteritis, to typhoid fever.Several clusters of virulence genes "pathogenicity islands" are important for adhesion, invasion of enterocytes and for the triggering of symptoms.Salmonella depends on its ability to survive and replicate inside host cells, and that's why, virulence trait is linked to its ability to cause systemic infections (Hansen-Wester and Hensel, 2001).Salmonella are intracellular bacteria which require large number of genes to cope with nutritional limitations, and to avoid clearance by the host immune cells (Hensel, 2004).The Salmonella injectisome "T3SS apparatus" is considered as the most important tool to invade and survive inside immune cells.T3SS injectisome spans the inner and outer membranes of the bacterial envelope secreting translocon and effector proteins.As a consequence, translocon proteins allow effector proteins to access the host cells by forming pores in the host cell membrane, and then forming a channel-like complex which connects between the bacterium and the eukaryotic membrane (Cornelis, 2006).
Salmonella enterica encodes two distinct virulenceassociated T3SS with roles in different phases of pathogenesis.Important virulence characters of S. enterica are encoded by genes clustered within Salmonella Pathogenicity Islands (SPI).These virulence characters include the interaction with enterocytes resulting in diarrhea, the invasion of non-phagocytic cells, the ability to survive phagocytosis and to proliferate within eukaryotic host cells.Two major SPI; SPI1 and SPI2, encode T3SS that translocates bacterial effectors (Cornelis, 2006).The function of SPI2 is essential for the later hallmark of Salmonella pathogenesis; the ability to cause systemic infections and to proliferate within host organs (Hegazy and Hensel, 2012).
Salmonella translocates T3SS effector proteins into the host cell cytoplasm, mediated by either SPI1-T3SS at an early stage after entry or SPI2-T3SS from the Salmonella containing vacuole (SCV) at later stages during intracellular life (1,2; 5).Efficient immune response mainly requires T-cells induction which depends on the strength of antigen presentation.The antigen presentation strength by its role depends on the antigen-processing efficacy and antigen abundance (Kuhle and Hensel, 2004).Salmonella T3SS-mediated translocation can be used to deliver efficiently the heterologous antigens to the cytosol of antigen presenting cells (APC), leading to noticeable CD8 T cell production in orally immunized mice (Yewdell and Bennink, 1999).
Live attenuated strains of Salmonella that synthesize and secrete foreign antigens had been used successfully to develop vaccines against infectious bacterial, viral and parasitic diseases and cancer.Recent advances in the understanding the genetics of Salmonella virulence led to development of single or multiple mutations in defined virulence genes (Xu et al., 2014).SsaV is an essential component of SPI2-T3SS machinery; it is a component of SPI2-T3SS, required for secretion of most T3SS effectors (Deiwick et al., 2006;Browne et al., 2008).Mutation in ssaV gene may lead to significant attenuation, as it may affect the translocation of SPI2-effector protein affecting the intracellular survival of Salmonella.In this study we are discussing the effect of mutation in ssaV gene on the virulence behavior of Salmonella.We investigated the mutation effect on bacterial adhesion, invasion and intracellular replication.Moreover, we tested the efficacy of expression and translocation of SPI2-effector inside the containing vacuole of epithelial cells or macrophage.

Bacterial strains
Wild-type strain Salmonella enterica serovar Typhimurium NCTC 12023 (S.Typhimurium) and the mutant ∆ssaV S. Typhimurium wild strain (P2D6) were provided kindly from Hensel's lab (Germany).Bacterial strains were routinely cultured in LB broth and on LB agar plates.For selection of recombinant strains, carbenicillin (50 µg/ml) and/or kanamycin (50 µg/ml) were added.
For in-vitro analyses of promoters under control of the SsrAB regulatory system, synthetic minimal media with limiting (PCN-P media) or non-limiting (PCN media) amounts of phosphate were used (Deiwick et al., 1999).

Western blotting
Plasmid pwsk29 PsseJ sseJ::hSurvivin was transferred to wild type and tested mutant and expression rates were analyzed (Hegazy et al., 2012).Briefly, S. enterica serovar Typhimurium wild-type or ∆ssaV mutant strains harboring plasmid of fusion proteins, consisting of SPI2 effector protein SseJ, model antigen h-survivin and C-terminal epitope tag HA under control of PsseJ promoter were grown in SPI2-inducing minimal media (PCN-P, pH 5.8).
Bacteria were gathered after 6 h of culture under suitable inducing conditions.Equal amounts of bacteria were adjusted at OD600 lysed and subjected to SDS-PAGE and Western blot analyses for the detection of HA epitope tag, which was performed.
Blots were explored with fluorescently labeled secondary antibodies and signal intensities, which were quantified using the Odyssey system (Li-Cor).The cytosolic heat shock protein DnaK was used as loading control, which was detected on the same blot and signals were quantified.The ratios of HA to DnaK signals were calculated in means and standard deviations, the experiment was performed in triplicates.

Adhesion assay
Bacterial strains were cultured in micro-titer plate, incubated at 37°C for 1 h, washed with PBS three times and fixed at 60°C for 20 min.Adhered cells were stained with equal volume of crystal violet (0.1%) for 15 min, washed 5 times with PBS and ethanol was added.Optical densities were measured at 590 nm.The assay was made in triplicate and the means and standard deviations were calculated (Vesterlund et al., 2005).

Evaluation of SPI2-T3SS translocation
In order to quantify the translocation, Salmonella wild type or ΔssaV harboring constructed plasmid, for the expression of HA tagged SPI2 effector fusion protein were used to infect HeLa cells or raw macrophage cells at MOI of 100 as previously described (Hegazy et al., 2012).
Infected HeLa cells or macrophages harboring similar numbers of intracellular Salmonella were nominated and the signal intensities for the fluorescence channel for HA tagged proteins staining were measured.The Leica laser-scanning confocal microscope was used and the intensities of fluorescence of tagged protein were measured by J-image program.The mean signal intensities and standard deviations for at least 25 infected cells per strains were calculated.

Invasion assay and intracellular replication
Gentamicin protection assay was used to determine the surveillance of S. Typhimurium strains in epithelial cells and immune cells (Holzer and Hensel, 2012).Before infection, 24 well plates were seeded either with HeLa cells at a density of 5 x 10 5 cells/ well or RAW264.7 at density of 2 x 10 5 cells/ well.S. Typhimurium strains WT and ΔssaV were sub-cultured from an overnight culture and incubated at 37 °C for 3.5 h.A master-mix of the inoculum of approximately 1 x 10 5 bacteria per well at multiplicity of infection (MOI 1) was prepared in DMEM and 300 μl were added to each well.Non-internalized Salmonella were removed by washing with PBS after 30 min.The remaining extracellular bacteria were killed by 1 h incubation with gentamicin (100 µg/ml).
Hela cells were lysed for invasion assays, with 500 μl per well 0.1% Triton X-100 10 min at room temperature.Serial dilutions of the inoculum and the lysates were plated onto Mueller Hinton (MH) plates.The percentage of invaded bacteria (1 h versus inoculum x 100) was calculated.For intracellular replication assays, the infected cells were washed twice with PBS and lysed with 500μl of 0.1% Triton-X-100 in 10 min at room temperature of 2 h and 16 h post infection.The serial dilutions of the inoculum and the lysates were plated onto MH plates.The percentage of phagocytosed cells/relative untaken cells (2 h versus inoculum x 100) and x-fold intracellular replication (16 h versus 2 h) were calculated.

Statistics
Means and standard deviations shown are deduced from one representative experiment, out of a series of three independent experiments performed in triplicates.Means and standard deviations of triplicate assays are calculated.Statistical significances were determined by Student's t-test using the Graphpad Prism 5 software which is indicated as: n.s.not significant,*P< 0.05, ** P< 0.01 and ***P< 0.001.

Generation and evaluation of SPI-2 effector expression cassettes
To test for efficacy of the live attenuated strain ∆ssaV in other to deliver SPI2-effector protein, expression cassettes contain the promoter of sseJ.Genes which encode hybrid protein and consist of SPI2-T3SS translocated effector proteins SseJ, tagged with HA was generated (Figure 1).S. enterica serovar Typhimurium wild-type or ∆ssaV mutant strains harboring the expression cassettes were tested for the expression levels of recombinant SPI2-effector.In vitro culture conditions were used to induce the expression of SsrAB regulon and the synthesis of SPI2 effector proteins.The synthesis of SPI2-effector fusion protein tagged with HA was observed in ∆ssaV mutant and wild type strains (Figure 2).
To quantify the amounts of recombinant protein produced, Western blots were analyzed using the Odyssey detection system.Signal for model antigens were normalized by the levels of constitutively synthesized control protein DnaK and the level of expression was significantly affected in ΔssaV mutant strain (p = 0.0002).

Adhesion assay
To evaluate the mutation in ssaV gene on adhesion, the adhesion of Salmonella wild type and ∆ssaV mutant strain to abiotic surface was examined.Tested strains were cultured within micro-titer plate, incubated at 37°C for 1 h.The adherent cells were stained with crystal violet, ethanol was added and optical densities were measured at 590 nm (Figure 3).There was no significant reduction in adhesion of ∆ssaV mutant than wild type (p = 0.5).

Invasion assay
HeLa cells were infected by S. enterica serovar Typhimurium wild-type or ∆ssaV mutant strains for invasion assay.The percentage of invaded bacteria (1 h post infected HeLa cells versus inoculum x 100) was calculated (Figure 4).The invasion assay showed a significant reduction of ∆ssaV mutant capability, used to invade HeLa cells (p = 0.0073) when compared to S. enterica serovar Typhimurium wild-type.S. enterica serovar Typhimurium wild-type or ∆ssaV mutant strains harboring plasmids expressing SPI2 effector protein tagged with HA under control in-vivo inducible promoter were grown in SPI2-inducing minimal media (PCN-P, pH 5.8).The ratios of the HA to DnaK signals were calculated.
Figure 3. Adhesion assay.S. enterica serovar Typhimurium wild-type or ∆ssaV mutant strains were cultured in micro-titer plate, incubated at 37•C for 1 hour, the adhered cells were stained with crystal violet and their optical densities were measured at 590 nm.There was no significance reduction in adhesion between ∆ssaV mutant and wild type (p = 0.5).

Intracellular replication
Macrophages were infected by S. enterica serovar Typhimurium wild-type or ∆ssaV mutant strains for intracellular replication assay.The percentage of phagocytosis/relative uptake (2 h post infected macrophage versus inoculum x 100) and x-fold intracellular replication (16 h post infected macrophage cells versus 2 h) were calculated (Figure. 5).The defective secretion system apparatus ΔssaV mutant showed significant reduction (p < 0.0001) in replication inside macrophages.

Evaluation of translocation efficiency
The translocation of the HA tagged model antigen by intracellular Salmonella in HeLa cells and macrophages was analyzed.The translocation of SseJ-hsurvivin-HA fusion protein was investigated by wild type or ∆ssaV mutant strain harboring expression cassettes with PsseJ promoter.For quantification, infected HeLa cells or macrophages harboring similar numbers of intracellular Salmonella were selected and the signal intensities for the fluorescence channel for HA tagged fusion protein staining were quantified (Figure 6).The comparison of

OD 590nm
Figure 4. Invasion assay.HeLa cells were infected with S. enterica serovar Typhimurium wild-type or ∆ssaV mutant strains at an MOI of 1for HeLa cells.After incubation for 25 min at 37°C non-internalized bacteria were removed by washing and the remaining extracellular bacteria were killed by addition of medium containing 100 µg ml-1 gentamicin for 1 h.The internalized cells were counted after cell lysis.Invaded bacteria are expressed as percentage of the inoculum.. Figure 5. Intracellular replication.RAW264.7 macrophages were infected at an MOI of 1 with S. enterica serovar Typhimurium wild-type or ∆ssaV mutant strains.Infected cells were lysed 2 h and 16 h after infection and the intracellular bacteria were counted.The x-fold intracellular replication is the ratio of cell forming unit (cfu) recovered at 16 h versus 2 h after infection.expression cassette indicates a significant variation (p= 0.0002) in translocation by wild type and the ΔssaV mutant in both HeLa cells and raw macrophage.

DISCUSSION
Several important virulence factors of S. enterica are encoded by genes within Salmonella Pathogenicity Islands (SPI) and two important loci are termed SPI1 and SPI2 (Haraga et al., 2008).SPI1 and SPI2 genes encode distinct T3SS, which will translocate bacterial effectors during different phases of pathogenesis (Hegazy and Hensel, 2012).The invasion capability and intracellular survival are dependent on SPI1-T3SS and SPI2-T3SS machinery, respectively.As a consequence, the defect in this machinery may affect the virulence behavior (Gerlach and Hensel, 2007).Ssav is a component of SPI2-T3SS, and is required for secretion of most effectors, which enable Salmonella to cope with the nutritional limitations inside containing vacuoles (Browne et al., 2008).
In this study, we investigated the intracellular behavior of S. Typhimurium mutant ssaV strain in vitro, the invasion of HeLa cells, intracellular replication in RAW macrophages, and most importantly the expression and translocation of SPI2-effector SseJ antigen fusions.For this purpose, fusion protein SPI2-effector SseJ tagged with HA encoding gene was constructed in low copy plasmid pWSK29.As we hypothesized, the expression and translocation levels of SseJ protein fusions were significantly reduced in ∆ssaV mutant strain.The adhesion of ∆ssaV mutant was not significantly reduced in comparison to the wild type, while the invasion capability was significantly diminished.SPI2-T3SS defect by mutation in one of its essential components "SsaV protein" was enough to limit significantly, the expression.Expressions of SPI2-effectors result in significant reduction of intracellular replication of Salmonella inside raw macrophage.
Our finding proved that ssaV gene mutation affect intracellular replication significantly, in raw macrophage (10 fold) lower than wild type.Inspite ssaV gene, mutation decreased the invasion capability which is significantly (3 fold) lower than wild type.These results are matching with the fact that, SPI2-T3SS is responsible for intracellular life, but its role is not crucial in early stages of invasion in which SPI1-T3SS is the main player.Our In vitro findings were compatible with the invivo observations of other groups (Li et al., 2009a;2009b;Fierer et al., 2012;Braukmann et al., 2015).Moreover, we tested the antibiotic sensitivity of ∆ssaV mutant and wild type strains; we did not observe any change in the sensitivity pattern; the mutation in essential component of SPI2-T3SS did not affect the resistance of Salmonella in different antibiotics from different classes (Supplementary Table 1).
The effect of attenuation on ssaV gene on virulence and survival of Salmonella was also evaluated in this study.The selection of suitable mutant is crucial for designing live recombinant Salmonella vaccines.A sufficient attenuation in virulence is required to prevent undesired side effects like bacteremia, diarrhea or fever.However, the balance between attenuation and overattenuation must be considered as over-attenuation can lead to poor immunogenicity of the vaccine.
Moreover, attenuated Salmonella can be used as a carrier to deliver heterologous antigens activating both cellular and humeral responses (Hegazy and Hensel, 2012;Xu et al., 2014).Previous studies showed that T3SS-type 2 deficient S. Typhimurium were highly attenuated and conferred protection from further challenges with wild-type, by eliciting serum IgG and secretory IgA against O-antigen in C57BL/6mice (Endt et al., 2010).But ssaV mutant was found to be virulent in immune compromised C57BL/6 mice (Periaswamy et al., 2012).
The double mutant of ssaV gene and other essential SPI1-T3SS or auxotrophic genes were attenuated enough to be used as vaccines for typhoid (Kirkpatrick et al., 2006;Pati et al., 2013a;2013b), as a carrier to deliver heterologous antigens (Hegazy and Hensel, 2014;Michael et al., 2010) or to induce chemokines (Li et al., 2009b).
Finally, we can conclude that, mutation in SsaV encoding gene significantly reduced the invasion capability, intracellular replication of Salmonella.entrica Typhimurium as a direct result of decreasing the expression and translocation of SPI2-effectors.In the other side, ssaV gene mutation did not affect adhesion and microbial resistance to different antibiotics.

Figure 1 .
Figure 1.Generation of SPI-2 effector expression cassette.A fusion proteins consisting of SPI-2 effector protein SseJ model antigen hSurvivin with a C-terminal epitope tag HA was constructed on low copy number vector pWSK29.

Figure 2 .
Figure 2. Synthesis of SPI2-effector.S. enterica serovar Typhimurium wild-type or ∆ssaV mutant strains harboring plasmids expressing SPI2 effector protein tagged with HA under control in-vivo inducible promoter were grown in SPI2-inducing minimal media (PCN-P, pH 5.8).The ratios of the HA to DnaK signals were calculated.

Figure 6 .
Figure 6.Evaluation of fusion proteins translocation.S. enterica serovar Typhimurium wild-type or ∆ssaV mutant strains harboring plasmid with cassette for the expression of SPI2 fusion effector protein tagged with HA under control of PsseJ promoter were used to infect (A) HeLa cells or (B) RAW macrophages at MOI of 100.The cells were fixed 16 h after infection and processed for immune-staining.Infected cells with similar amounts of intracellular Salmonella were selected for the various conditions and the signal intensities of the Cy3 channel for the anti-HA strains were measured with identical exposure times.The mean signal intensities and standard deviations for at least 25 infected cells per strains were shown.(C) HeLa cells (D) Raw macrophages were fixed 16 h after infection and processed for immune-staining of intracellular Salmonella (red), translocated fusion protein effector-hsurvivin-HA (green) and macrophage (blue DAPI stained).