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Salmonella enterica abortusovis is able during the infection to hide itself to the host immune system using unknown mechanisms

Final Report Summary - SALMONELLA (Salmonella enterica abortusovis is able during the infection to hide itself to the host immune system using unknown mechanisms)

Salmonella enterica subsp. enterica includes many pathogenic serovars for humans and animals, Salmonella Abortusovis for example is restricted to ovine and causes abortion which is an important health problem in most Europe country. All the serovars are not dangerous in the same way, but relevant variations of virulence are observed according to pathogenicity factors shared by the different serovars.

After abortion, bacteria can be isolated from placental and tissues (liver, spleen, brain and stomach), which are the principal site of multiplication.

Comparison between adapted and non-adapted Salmonella serotypes suggests that persistence of host-adapted serotypes Salmonella Abortusovis can be based on circumvention of immune-based protective response (Montagne M, 2001) with the ability to persist at systemic sites (Uzzau 2001). Translocation of bacteria from the intestinal lumen into the lamina propria is detected by the immune system through patter-recognition receptors including TLRs (Iwasaki, Nature Imm.2004) and cytosolic nucleotide binding and oligomerization domain-like receptors (NLRs) (Franchi. Cell Micro 2008). These pattern-recognition receptors are able to recognize microbe associated molecular patterns (MAMPs) that distinguish bacteria from viral or parasitic agents (Hoebe., Nature Imm., 2004). Experimental evidence suggests that stimulation of TLR4 by LPS has an important role in the development of septic shock when S. Typhimurium is present in the blood stream (Khan,S. A. et al.1998; Engelberts, 1991; Wilson, R.P. et al.2008). Bacterial flagellin is a MAMP recognized by TLR5 (Hayashi, 2001) in a process that has been implicated in neutrophil influx in the intestinal mucosa during S. Typhimurium infection (Gewirtz, A.T. 2001; Zeng H., et al 2003).

A comparison of the diseases caused by S. Abortusovis and S. Typhimurium reveals two striking differences: (i) the absence of intestinal inflammation and diarrhea in the intestine of animals infected S. Abortusovis and (ii) the ability of S. Abortusovis to chronically persist at systemic sites of infection, which can result in abortion. Both observations are likely due to the action of S. Abortusovis specific virulence mechanisms.

Our central hypothesis is that direct contact of Salmonella Abortusovis with the host cell does not trigger an innate immune response unable to control infection before pathogens reach the uterus a cause abortion. Salmonella Abortusovis is not mobile in vitro but the flagella are express in vivo as we known in previous studies. In the current study i used mutants of Salmonella Typhimurium, previously characterized, to transduce in Salmonella Abortusovis some mutations of the flagella genes to study the changes in motility.

Phage P22 HT int-105 was used to generate generalized transducing lysates of serotype Typhimurium as previously described (Miller, J. H. 1972.). Transductants were struck for single colonies on Evans blue uridine agar (Bochner, B. R. 1984.) and light green colonies were cross-struck against P22 H5 to confirm phage sensitivity. A P22 lysate of S.Typhimurium fljB5001::MudCm, STM flgB5306::MudK, STM fliC5050::MudJ, STM flgK5396::MudJ, STM flhC5456::MudJ, STM flgM5222::MudCM was used to transduce sierotype Abortusovis to Kanr and Cmr separately, thus generating serotipe of Abortusovis with mutations of flagella proteins. The mutations were confirmed by PCR using specific primers. (these data cannot be show for publications reason )

Then we proceeded with the extraction of flagella from wild type and mutant of the two different serovars, using diverse media to observe differences. We analized our samples with SDS Page and Western Blot using Anti-Flagellin FliC (InvivoGen) to determinate the presence of flagella. Also we verified the expression of flagella by Flow Cytometry. All this different technique help us to find a media where is possible to induce the expression of flagella in Salmonella Abortusovis. (these data cannot be show for publications reason )

To verify the capacity of S. Typhimurium and S. Abortusovis to stimulate TLR5, colorectal carcinoma cell line (T84) cells were treated with purified flagellin and the IL-8 transcription in this cells line was used to verified the induction. In this work we showed for the first time that Salmonella Abortusovis flagella are expressed in specific media, and the flagella extracted from these media induced the transcription of IL-8 in T84 cells line. (these data cannot be show for publications reason ) The presence of the expression of flagella only in a particular media show for the first time that Salmonella Abortusovis is able to control this phenotype to avoide the immune response.

LPS was isolated from dried bacteria using the hot phenol/water extraction (Westphal, O., and K. Jann. 1965) . Each procedure was repeated until TLC and UV absorbance spectra showed no detectable contaminants.

The LPS sample of each samples were loaded in the Glycine/ SDS-polyacrylamide gel electrophoresis method on a 15% acrylamide gel followed by silver staining.

SDS-PAGE analysis showed a high heterogeneity with “ladder-like” patterns of slow migrating high-molecular-mass LPS species with O-polysaccharide chains of different lengths (“Smooth-type” LPS) as well as fast migrating bands of short-chain LPS molecular species with no O-chain polysaccharide attached to the core (“Rough-type” LPS). S. abortusovis displayed a larger cluster of high molecular weight smooth-type LPS bands than S. typhimurium.

Matrix-assisted laser desorption/ionization negative- ion mass spectrometry were performed on a PerSeptive Voyager-DE STR model time-of-flight mass spectrometer (Applied Biosystems) in linear mode with delayed extraction.

We check the differents of the lipid A of the two sierotype. As you know, lipid A is the hydrophobic part of LPS which anchors these molecules to the bacterial outer membrane. It plays an important role in the interaction with the immune system via Toll Like Receptors (mostly TLR4 or TLR2 depending on LPS structures) inducing strong inflammatory reactions. In this spectra we found the major different from lipid A of Salmonella Abortusovis respect Salmonella Typhimurium this data support our hypotesis (these data cannot be show for publications reason ).

In this work for the first time we were able to study the interaction of Salmonella Abortusovis LPS and Toll-like receptor 4 (TLR4). Experiments with a cell line (HEK293) transfected with human TLR4 show reduced IL-8 production by LPS of S. Abortusovis respect to the LPS of S. Typhimurium. The same result was observed using heat-killed bacteria instead of LPS. We have transfect the cell 293 with the receptor TLR4 ovine ( amplified by PCR from Ovine RNA) and we confirm our data. (these data cannot be show for publications reason )

Our results provide an important mechanistic insight into how S. Abortusovis is able to modulate host responses that distinguish abortions from infections with S. Typhimurium.

Now we are finishing to write the manuscript of the flagella and we are in the process of review for the paper about LPS story .