Controlling the bacterial load of Salmonella Typhi in an experimental mouse model by a lytic Salmonella phage STWB21: a phage therapy approach

Abstract Background Salmonella enterica serotype Typhi is one of the major pathogens causing typhoid fever and a public health burden worldwide. Recently, the increasing number of multidrug-resistant strains of Salmonella spp. has made this utmost necessary to consider bacteriophages as a potential alternative to antibiotics for S. Typhi infection treatment. Salmonella phage STWB21, isolated from environmental water, has earlier been reported to be effective as a safe biocontrol agent by our group. In this study, we evaluated the efficacy of phage STWB21 in reducing the burden of salmonellosis in a mammalian host by inhibiting Salmonella Typhi invasion into the liver and spleen tissue. Results Phage treatment significantly improved the survival percentage of infected mice. This study also demonstrated that oral administration of phage treatment could be beneficial in both preventive and therapeutic treatment of salmonellosis caused by S. Typhi. Altogether the result showed that the phage treatment could control tissue inflammation in mice before and after Salmonella infection. Conclusions To the best of our knowledge, this is the first report of phage therapy in a mouse model against a clinically isolated Salmonella Typhi strain that includes direct visualization of histopathology and ultrathin section microscopy images from the liver and spleen sections

Arabinosylated Lipoarabinomannan Skews Th2 Phenotype towards Th1 during Leishmania Infection by Chromatin Modification: Involvement of MAPK Signaling

The parasitic protozoan Leishmania donovani is the causative organism for visceral leishmaniasis (VL) which persists in the host macrophages by deactivating its signaling machinery resulting in a critical shift from proinflammatory (Th1) to an antiinflammatory (Th2) response. The severity of this disease is mainly determined by the production of IL-12 and IL-10 which could be reversed by use of effective immunoprophylactics. In this study we have evaluated the potential of Arabinosylated Lipoarabinomannan (Ara-LAM), a cell wall glycolipid isolated from non pathogenic Mycobacterium smegmatis, in regulating the host effector response via effective regulation of mitogen-activated protein kinases (MAPK) signaling cascades in Leishmania donovani infected macrophages isolated from BALB/C mice. Ara-LAM, a Toll-like receptor 2 (TLR2) specific ligand, was found to activate p38 MAPK signaling along with subsequent abrogation of extracellular signal–regulated kinase (ERKs) signaling. The use of pharmacological inhibitors of p38MAPK and ERK signaling showed the importance of these signaling pathways in the regulation of IL-10 and IL-12 in Ara-LAM pretreated parasitized macrophages. Molecular characterization of this regulation of IL-10 and IL-12 was revealed by chromatin immunoprecipitation assay (CHIP) which showed that in Ara- LAM pretreated parasitized murine macrophages there was a significant induction of IL-12 by selective phosphorylation and acetylation of histone H3 residues at its promoter region. While, IL-10 production was attenuated by Ara-LAM pretreatment via abrogation of histone H3 phosphorylation and acetylation at its promoter region. This Ara-LAM mediated antagonistic regulations in the induction of IL-10 and IL-12 genes were further correlated to changes in the transcriptional regulators Signal transducer and activator of transcription 3 (STAT3) and Suppressor of cytokine signaling 3 (SOCS3). These results demonstrate the crucial role played by Ara-LAM in regulating the MAPK signaling pathway along with subsequent changes in host effector response during VL which might provide crucial clues in understanding the Ara-LAM mediated protection during Leishmania induced pathogenesis

Hstone H3 modifications at the IL-12 promoter in Ara-LAM treated infected macrophages.

<p> <i>Murine macrophages (1×10⁶cells/mL) were treated and infected as described in the </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024141#pone-0024141-g003" target="_blank"><i>Figure 3</i></a><i> legend. After 45 min of incubation, ChIP assays were conducted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024141#s4" target="_blank">Materials and Methods</a>. Immunoprecipitations were performed using Abs specific to phosphorylated H3 (IP phospho-H3) (A) or IP acetyl-H3 (B), and conventional RT-PCR or quantitative real-time PCR was performed using primers specific to the IL-12p40 promoter. *P<.001 compared with Ara-LAM–pretreated infected macrophages. In a separate experiment peritoneal macrophages were treated and infected by Leishmania for 15, 30 min as described in </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024141#pone-0024141-g003" target="_blank"><i>Figure 3</i></a><i> legend. Cytosolic and nuclear protein extracts were analyzed for the nuclear translocation of NF-κB (C). The blot shown is a representative of experiments performed in triplicate. Band intensities were analyzed by densitometry (i,ii) (inset). Peritoneal macrophages (1×10⁶ cells/mL) were transfected with control siRNA or STAT3-specific siRNA, subsequently followed by Ara-LAM treatment (for 3 hr) and Leishmania infection for 45 min. Immunoprecipitations were conducted using NF-κB specific Abs. Conventional RT-PCR or quantitative real-time PCR was performed for amplifying the putative NF-κB binding sites of the IL-12p40 promoter. *P<.001 compared with Ara-LAM–pretreated infected macrophages.</i></p

Sequences of the PCR primers.

<p>Sequences of the PCR primers.</p

SOCS3 silencing significantly enhances host protective proinflammatory response generation by Ara-LAM in infected macrophages.

<p><i>Peritoneal macrophages (2×10⁶ cells/mL) were transfected with control siRNA or SOCS3-specific siRNA followed by Ara-LAM treatment (for 3 hr) and Leishmania infection for 24 h and assayed for the levels of TNF- α (A), IL-12 (B), and IL-10 (C) in the culture supernatant by ELISA, as described in Methods. ELISA data are expressed as means standard deviations of values from triplicate experiments that yielded similar observations. *P<.001 compared with infected macrophages, **P<.005 compared with Ara-LAM–pretreated infected macrophages. ***<.01 compared to Ara-LAM–pretreated infected macrophages. In a separate experiment macrophages were cultured and treated as described above and assayed for the levels of extracellular NO as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024141#s4" target="_blank">Materials and Methods</a> (D). *P<.001 compared with infected macrophages, **P<.005 compared with Ara-LAM–pretreated infected macrophages. In a separate set macrophages were transfected and treated with Ara-LAM as described above followed by Leishmania infection for 24 hr. Western blot analysis was performed to analyze the expression of inducible nitric oxide synthase. The blot shown is a representative of experiments performed in triplicate. Band intensities were analyzed by densitometry (E). Peritoneal macrophages were cultured and treated with Ara-LAM as described above followed by Leishmania infection for 3 hr. Changes in mRNA expression of IL-12p40, TNF- α, IL-10, NO were determined by real-time PCR analysis Results are presented as changes (n-fold) relative to uninfected control cells. The experiment was repeated 3 times, yielding similar results; data are expressed as means</i> ± <i>standard deviations. (F). *P<.001 compared with infected macrophages, **P<.005 compared with Ara-LAM–pretreated infected macrophages.</i></p

Ara-LAM mediated effector function depends on the reciprocal activation of p38MAPK and ERK–1/2.

<p><i>Peritoneal macrophages (2×10⁶cells/mL) were treated with SB203580 (SB) or PD098059 (PD) for 2 h, followed by Ara-LAM treatment for 3 hr. The cells were then infected with Leishmania parasite for 24 h and assayed for the levels of IL-12 (A) and IL-10 (C) in the culture supernatant by ELISA as described in Methods. ELISA data are expressed as means standard deviations of values from triplicate experiments that yielded similar observations. Macrophages cultured in a 24-well plate (1×10⁶ cells/mL) were pretreated and infected as described above and assayed for the levels of extracellular NO as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024141#s4" target="_blank">Materials and Methods</a> (E). Asterisks indicate statistically significant induction of nitrite generation, compared with Ara-LAM–pretreated infected macrophages. *P<.001. From a separate set of cells (2×10⁶ cells/mL) RNA was isolated and levels of mRNA expression for IL-12p40 (B), IL-10 (D), inducible nitric oxide synthase 2 (iNOS2) (F) were determined by quantitative RT-PCR. Results are presented as changes (nfold) relative to uninfected control cells. The data represent the mean values</i>±<i> standard deviation of results from 3 independent experiments that all yielded similar results. In a separate experiment, the macrophages were cultured in coverglasses treated with SB or PD for 2 h, subsequently followed by 3 hr of Ara-LAM treatment and 4 h of Leishmania infection. After indicated time of incubation intracellular parasite number were assessed as described in methods. Pretreatment with SB significantly inhibited Ara-LAM –mediated parasite killing compared with levels in corresponding Ara-LAM pretreated infected controls (G). *P<.001 for SB.</i></p

Ara-LAM mediated changes in MAPK signaling cascade in <i>Leishmania donovani</i>–infected macrophages.

<p> <i>Macrophages were pretreated with Ara-LAM for 3 hr or in some cases followed by Leishmania infection for 15, 30, 60, and 120 min. The cells were then lysed and subjected to Western blot with anti-pp38MAPK, p38MAPK and pERK1/2, ERK1/2, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024141#s4" target="_blank">Materials and Methods</a> (A). In separate experimental sets, cells were transfected with control siRNA or TLR2-specific siRNA for 24 hr washed and then treated with Ara-LAM followed by L. donovani for 30 min. Western blot analysis was performed to analyze the expression of anti-pp38MAPK, p38MAPK and pERK1/2, ERK1/2 (B).</i></p

Ara-LAM induced MHC-II expression, antigen presentation ability of infected macrophages increased under SOCS3 silenced condition.

<p><i>Peritoneal macrophages (2×10⁶ cells/mL) were transfected with control siRNA or SOCS3-specific siRNA, subsequently followed by Ara-LAM treatment (for 3 hr) and Leishmania infection for 4 hr as mentioned above. After 24 hr of incubation treated macrophages were analyzed by flow cytometry for MHCII (FL2-H) expression as described in material method (A). Data are from 1 of 3 experiments conducted in the same way with similar results. In a separate set, normal and treated macrohages either uninfected or infected were pulsed with λR_12–26, and then were incubated with T-cell hybridoma 9H 3.5. The culture supernatants were analysed for the presence of IL-2 by ELISA as described in the methods (B). Incorporation of 3H-Thymidine in the IL-2 dependent cell line HT-2 was assessed in presence of the cultured supernatant. Results are expressed as mean</i> ± <i>SD of 5 replicate experiments (C). *P<.001, **P<.005 compared with infected macrophages.</i></p

STAT3 silencing potentiate Ara-LAM mediated abrogation of SOCS3 expression in <i>Leishmania-donovani</i> infected macrophages.

<p><i>Peritoneal macrophages (2×10⁶ cells/mL) were transfected with control siRNA or STAT3-specific siRNA, subsequently followed by Ara-LAM treatment (for 3 hr) and Leishmania infection for 24 hr. The cells were then lysed and subjected to Western blot with anti-SOCS3 antibody as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024141#s4" target="_blank">Materials and Methods</a> (A). In a separate experiment macrophages were transfected with either control-siRNA or</i> STAT3 <i>specific siRNA, subsequently followed by Ara-LAM treatment for 3 hr and Leishmania infection for another 3 h. RNA was isolated and semi quantitative RT-PCR analyses for SOCS3 and GAPDH were done. Data represented here are from one of three independent experiments, all of which yielded similar results (B). Changes in expression of SOCS3 mRNA were also determined by quantitative real-time PCR. Results are presented as changes (n-fold) relative to uninfected control cells. The experiment was repeated 3 times, yielding similar results (C). *P<.001 compared with infected macrophages. The comparison of SOCS3 expression between Ara-LAM treated infected macrophages and STAT3 siRNA group did not show any statistical significance.</i></p