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Characterization of SEN3800-associated virulence of Salmonella enterica serovar Enteritidis phage type 8

Characterization of SEN3800-associated virulence of Salmonella enterica serovar Enteritidis phage... Ann Microbiol (2015) 65:631–637 DOI 10.1007/s13213-014-0898-8 ORIGINAL ARTICLE Characterization of SEN3800-associated virulence of Salmonella enterica serovar Enteritidis phage type 8 Daniel C. Shippy & Nicholas M. Eakley & Dareen M. Mikheil & Anna De La Cotera & Amin A. Fadl Received: 28 February 2014 /Accepted: 7 April 2014 /Published online: 30 April 2014 Springer-Verlag Berlin Heidelberg and the University of Milan 2014 Abstract Salmonella is a major public health concern due to contaminated food. Nontyphoidal Salmonella serovars, like the consumption of contaminated food. Salmonella enterica Salmonella enterica serovar Enteritidis (SE), are the leading serovar Enteritidis (SE) infection in humans is often associat- cause of hospitalization and death among the major foodborne ed with the consumption of contaminated poultry products. pathogens (Scallan et al. 2011). Therefore, the identification Binding of the bacterium to the intestinal mucosa is a major and characterization of mechanisms involved in Salmonella pathogenic mechanism of Salmonella in poultry. In this study, pathogenesis could lead to new strategies for controlling transposon mutagenesis identified SEN3800 as a potential salmonellosis. binding mutant of SE. Therefore, we hypothesize that SE outbreaks in humans are mainly associated with the SEN3800 plays a role in the colonization ability of SE in the consumption of contaminated eggs and other poultry prod- gastrointestinal tract of poultry. To test our hypothesis, we ucts. In poultry, Salmonella can colonize the intestinal tract created a mutant of SE in which SEN3800 was deleted. We and become asymptomatic carriers of the bacterium, resulting then tested the in-vitro and in-vivo binding ability of in the contamination of food (Fanelli et al. 1971; Barrow et al. ΔSEN3800 when compared to the wild-type and 1988). Eggs can then become contaminated by Salmonella complemented SE strains. Our data showed a significant penetrating the eggshell, or direct contamination of the inter- decrease in the binding ability of ΔSEN3800 to T84 intestinal nal egg during formation due to Salmonella colonizing the epithelial cells, as well as in the small intestine and cecum of reproductive tract (Okamura et al. 2001; Gantois et al. 2009). poultry. Furthermore, this binding defect correlated to a defect Over the past 25 years, the ability of SE to contaminate eggs in invasion, as evidenced by a cell culture model using T84 and other poultry products has led to a constant threat to public intestinal epithelial cells and bacterial recovery from the livers health in the United States (Braden 2006). and spleens of chickens. Overall, these studies indicate that A hallmark of Salmonella pathogenesis is its ability to SEN3800 contributes to the colonization ability of Salmonella invade intestinal epithelial cells (Patel and Galan 2005). This in the gastrointestinal tract of poultry. is a multi-step process mediated by a type 3 secretion system (T3SS) encoded within Salmonella pathogenicity island-1 . . . Keywords Salmonella Poultry SEN3800 Pathogenesis (SPI-1) (Galan 1996; Schlumberger and Hardt 2006). The initial step in the invasion process is the binding of Salmonella to the host intestinal epithelial cells. Several Introduction Salmonella virulence factors have been implicated in the binding to host cells. The best characterized are the fimbrial Salmonella is a major foodborne pathogen that frequently adhesins which include type 1, long polar, plasmid-encoded, causes gastroenteritis in humans due to the consumption of and thin aggregative fimbriae (Clegg et al. 1987; Grund and Weber 1988; Friedrich et al. 1993; Baumler and Heffron 1995). Interestingly, another study has shown that the T3SS : : : : D. C. Shippy N. M. Eakley D. M. Mikheil A. De La Cotera itself can mediate host cell binding by providing evidence that A. A. Fadl (*) SipB, SipC, and SipD are required for the intimate association Department of Animal Sciences, University of Wisconsin-Madison, of Salmonella with mammalian cells (Lara-Tejero and Galan 1675 Observatory Drive, Madison, WI 53706, USA 2009). The inhibition of Salmonella binding at the early stages e-mail: fadl@wisc.edu 632 Ann Microbiol (2015) 65:631–637 of infection is potentially the most effective strategy for con- grown and maintained in Dulbecco’s Modified Eagle trolling salmonellosis in production animals, and could lead to Medium: Nutrient Mixture F12 medium supplemented with a reduction in the contamination of our food supply 10 % fetal bovine serum, and incubated at 37 °C with 5 % (Wizemann et al. 1999). CO . In this study, we identified a novel binding mutant of SE by transposon mutagenesis. The SEN3800 deletion mutant Construction of the mutant and complemented strains (ΔSEN3800) displayed a normal growth profile when com- pared to the wild-type (WT) SE phage type (PT) 8 E2627 and The transposon binding screening that identified SEN3800 as a complemented strains (ΔSEN3800/pBRSEN3800). Binding potential binding mutant of SE is described in Shippy et al and invasion assays showed that ΔSEN3800 was deficient in (2013). The ΔSEN3800 strain was created using the lambda cell culture models of Salmonella binding and invasion. Red recombination system, as previously described (Datsenko Furthermore, ΔSEN3800 was deficient in a poultry model of and Wanner 2000). Briefly, WT SE PT8 was transformed with Salmonella binding and invasion, with the invasion defect the pKD46 plasmid, induced with arabinose, and used to most likely due to the deficiency in binding ability. Taken generate the electrocompetent cells. The kanamycin resistance together, these data indicated a role for SEN3800 in the gene (Kn ) was amplified by polymerase chain reaction (PCR) colonization of SE in the gastrointestinal tract of poultry. from the pKD4 plasmid using primer set LF/LR. The PCR product was purified and electroporated into the WT-pKD46 electrocompetent cells. After transformation, colonies growing Materials and methods on LB plates supplemented with kanamycin were selected as candidates for SEN3800 mutants of SE. To confirm deletion of Bacterial strains, plasmids, and cell lines the SEN3800 gene, the selected mutants were subjected to PCR analysis using primer sets K /K and F /R to show the pres- 3 5 2 2 The WT SE PT8 E2627 strain was isolated from an egg- ence of the Kn and the absence of SEN3800. associated outbreak in the United States and is described in The SEN3800-complemented strain was constructed by am- Buchrieser et al (1997). All Salmonella strains were grown in plifying a DNA fragment containing SEN3800 from the WT either Luria-Bertani (LB) medium (10 g casein peptone, 5 g SE strain using primer set F /R . The DNA fragment was blunt- 3 3 yeast extract, and 10 g sodium chloride per liter) or on ended using a PCR polishing kit (Stratagene, Santa Clara, CA) Salmonella-Shigella (SS) agar plates. Additionally, homoge- and ligated into the blunt-ended ScaI restriction enzyme nates from the in-vivo experiments were incubated in digested pBR322 vector. The recombinant plasmid was trans- Selenite-F broth (BD, Sparks, MD). Nalidixic acid formed into the SEN3800 mutant by electroporation. A com- (100 μg/ml), kanamycin (50 μg/ml), tetracycline (15 μg/ml), plete list of the primers used in this study is shown in Table 2. and ampicillin (100 μg/ml) were added to the media as nec- essary. A complete list of the bacterial strains and plasmids Growth analysis used in this study is shown in Table 1. T84 intestinal epithelial cells were obtained from the American Type Culture Growth curve profiles were constructed in order to determine Collection (ATCC, Manassas, VA), and were subsequently the significance of SEN3800 on SE growth. An equal number Table 1 Strains and plasmids used in this study Strain or plasmid Relevant characteristics Source or reference Serovar Enteritidis phage Isolated from an egg-associated outbreak in the United States (Buchrieser et al. 1997) type 8 E2627 SEN3800::Tn5 Mutant of serovar Enteritidis with disruption of SEN3800 via Tn5 transposon mutagenesis This study ΔSEN3800 Mutant of serovar Enteritidis in which SEN3800 was deleted using lambda Red; Kn This study r r ΔSEN3800/pBRSEN3800 ΔSEN3800 complemented with a copy of the SEN3800 gene via pBR322; Kn,Tc This study E. coli DH5α Used for recombinant DNA methods Lab stock Plasmids pKD46 lambda Red recombinase genes; Ap (Datsenko and Wanner 2000) r r pBR322 Ap Tc Lab stock pKD4 Kn gene cassette (Datsenko and Wanner 2000) pBRSEN3800 SEN3800 gene cloned into pBR322 at the ScaIsite Thisstudy Ann Microbiol (2015) 65:631–637 633 Table 2 Sequence and purpose of primers used in this study Chicken experiments Primer name and sequence One-week-old specific-pathogen-free White Leghorn chickens LF:5′-ACTGTACATATACTGACGATAAATATCATTGTTATTCATA were obtained from Charles River (Wilmington, MA). All TGTGTAGGCTGGAGCTGCTTCG-3′ animal procedures were approved by the University of LR:5′-GCGCAAATCCGGGCCATGAGGAAGCAAAATGTATAATT Wisconsin-Madison Animal Care and Use Committee. CACATATGAATATCCTCCTTAG-3′ Groups of 11 birds were inoculated by oral crop gavage with F :5′-GGTTAGGGTAATGAATCGTC-3′ 1×10 CFU of the WTor ΔSEN3800 SE strains. An additional R :5′-GATATCGGACTTGGTCGTAA-3′ group (n=4) was inoculated by oral crop gavage with 100 μl F :5′-CGCTAAAGCTGACGTTCTCC-3′ sterile PBS to serve as a control. At 16 h and 7 days post- R :5′-GCCATGAGGAAGCAAAATGT-3′ infection, five birds from each group were euthanized using K :5′-AAAGCCACGTTGTGTCTA-3′ CO Portions of the liver, spleen, small intestine, and cecum K :5′- CGCTGAGGTCTGCCTCGT-3′ were removed from each bird. The individual organs were pooled and 1 g was homogenized in 10 ml PBS. One ml from each homogenate was removed and incubated in 10 ml of cells from the WT, ΔSEN3800,and ΔSEN3800/ Selenite-F broth at 36 °C for 18 h. Direct plating of the organ pBRSEN3800 strains were inoculated in LB and Selenite-F homogenates was done in parallel with plating from the en- broth and grown at 37 °C. The optical densities at 600 nm were richment cultures (Fernandez et al. 2002; Johny et al. 2009; recorded each hour. Johnston et al. 2012; Kollanoor-Johny et al. 2012). Enumeration of bacteria was performed by serial dilution Binding assay and plating on SS agar. The binding assay was performed as previously described Statistical analysis (Shippy et al. 2011). Briefly, 5×10 T84 intestinal epithelial cells were seeded per well in a 24-well tissue culture plate and Wherever appropriate, the data were analyzed using incubated overnight at 37 °C with 5 % CO . The following GraphPad Prism 5 software (GraphPad Software, San Diego, day, cells were infected with the WT, ΔSEN3800,and CA) and a Student’s t test. P values of ≤0.05 were considered ΔSEN3800/pBRSEN3800 strains at a multiplicity of infection significant. Unless otherwise stated, experiments were repeat- (MOI) of 10:1. The plate was briefly centrifuged at 400×g for ed two times and data were expressed as arithmetic means minutes, and incubated for 30 min at 37 °C with 5 % CO . with standard deviations. Unbound bacteria were aspirated; the wells were washed six times with phosphate buffered saline (PBS), and the cells were lysed with 0.1 % Triton X-100 (TX-100). Dilutions of the cell Results lysates were plated on SS agar for enumeration of bacteria. Characterization of the SEN3800 mutant Invasion assay The chromosomal SEN3800 gene was replaced by a The invasion assay was performed as previously described kanamycin resistance gene (Kn ) cassette using the (Shippy et al. 2011). Briefly, 5×10 T84 intestinal epithelial lambda Red recombination system. Deletion of cells were seeded per well in a 24-well plate and incubated SEN3800 from the chromosome of SE was confirmed overnight at 37 °C with 5 % CO . The cells were infected with by PCR analysis. The primer set K /K was used to 2 3 5 the WT, ΔSEN3800,and ΔSEN3800/pBRSEN3800 strains at amplify the Kn cassette, while the primer set F /R 2 2 an MOI of 10:1, and briefly centrifuged at 400×g for 5 min so was used to confirm the absence of SEN3800.Toensure that the bacterial cells would be in direct contact with the T84 correct orientation of the Kn cassette, the primer set F / cells. After incubation for 30 min at 37 °C with 5 % CO ,the K was used to amplify the upstream SEN3800 flanking 2 5 cells were washed three times with PBS and incubated for an sequence along with the Kn cassette, while R /K was 3 3 additional 45 min with gentamicin-containing medium used to amplify the downstream SEN3800 flanking se- (100 μg/ml) to kill extracellular bacteria. Following incuba- quence along with the Kn cassette. Overall, these re- tion, the gentamicin-containing medium was removed, sults indicated that a SEN3800 deletion mutant of SE the wells were washed six times with PBS, and the cells PT8 was successfully created. were lysed with 0.1 % TX-100. The lysate was diluted and Bioinformatic analysis of SEN3800 indicated an 864- plated out on SS agar plates for colony-forming unit (CFU) nucleotide gene encoding a 287-amino-acid protein. determination. Orthologs of SEN3800 are highly conserved in prokaryotes, 634 Ann Microbiol (2015) 65:631–637 (A) and SEN3800 of serovar Enteritidis is identical to orthologs in other Salmonella serovars, including Typhimurium, WT Dublin, Typhi, Paratyphi, Gallinarum, and Pullorum ΔSEN3800 (Thomson et al. 2008). The proteins encoded by ΔSEN3800/pBRSEN3800 P=0.0156 orthologs of SEN3800 in other bacterial species are characterized as members of the sulfatase superfamily, and as putative bacterial membrane proteins. The SEN3800 protein of Salmonella shares 97 % homology with a membrane-associated metal-dependent hydrolase of Escherichia coli (Nash et al. 2010). Additionally, SEN3800 contains a motif that is conserved in the sulfatase superfamily (B) WT (Thomson et al. 2008). ΔSEN3800 ΔSEN3800/pBRSEN3800 P=0.0115 SEN3800 does not affect Salmonella growth Growth curve analysis was performed for the WT, ΔSEN3800, and ΔSEN3800/pBRSEN3800 strains in order to determine the significance of SEN3800 on Salmonella growth. All three strains displayed nearly identical growth profiles, suggesting Fig. 2 Cell culture assays using T84 intestinal epithelial cells. a Binding that SEN3800 does not play a significant role in SE growth assay and b invasion assay with the WT, ΔSEN3800,and ΔSEN3800/ (Fig. 1). Additionally, growth analysis in Selenite-F broth pBRSEN3800 strains. The actual P values are given, displaying a statis- showed no difference in growth, with minimal overall growth tically significant difference between ΔSEN3800 and the WT strain. The graphs are representative of three independent experiments observed. Deletion of SEN3800 decreases SE binding and invasion in vitro (A) Infection of T84 intestinal epithelial cells displayed a significant decrease in the binding ability of ΔSEN3800 WT (4.5 logs) to host epithelial cells when compared to the P=0.0188 ΔSEN3800 WT SE strain (6.1 logs) [P = 0.0156] (Fig. 2a). Furthermore, ΔSEN3800 displayed the same significant P=0.0155 decrease in the ability to invade T84 intestinal epithelial cells [P=0.0115] (Fig. 2b). The binding and invasive ability of ΔSEN3800 was restored after complementation, suggesting that SEN3800 plays a role in the binding and invasion of intestinal epithelial cells by SE. It is logical to 16 Hours Day 7 conclude that the invasion defect seen in ΔSEN3800 is an effect of the binding deficiency. (B) WT 2.5 WT ΔSEN3800 P=0.0035 P=0.0115 ΔSEN3800 2.0 ΔSEN3800/pBRSEN3800 1.5 1.0 0.5 0.0 16 Hours Day 7 0 2 4 6 8 10 Hour Fig. 3 Determination of in-vivo binding ability. Bacterial counts in a Fig. 1 Growth curves of the WT, ΔSEN3800,and ΔSEN3800/ small intestine and b cecum of chickens inoculated by oral crop gavage pBRSEN3800. The strains were grown in LB and the optical densities with 1×10 CFU of WT and ΔSEN3800.The actual P values are given, at 600 nm were measured each hour. The graph is representative of two displaying a statistically significant difference between ΔSEN3800 and independent experiments the WT strain O.D. 600 nm log (CFU/ml) log (CFU/ml) log (CFU/ml) log (CFU/ml) Ann Microbiol (2015) 65:631–637 635 Deletion of SEN3800 decreases SE binding and invasion and 6.1 logs for the WT and ΔSEN3800, respectively [P= in chickens 0.0069] (Fig. 4b). These data suggest that the binding deficien- cy displayed by ΔSEN3800 contributes to an overall reduction A poultry model was used to determine the role of SEN3800 in systemic infection by SE in poultry. in the binding ability of SE to the intestinal mucosa of chickens. At 16 h post-infection, the bacterial counts of WT SE from the small intestine were 7.1 logs compared to 6.2 logs Discussion [P=0.0188] for ΔSEN3800. At day 7 post-infection, bacterial counts from the small intestine were 4.1 logs versus 3.2 logs In this study, a novel binding mutant of SE was created and between the WT and ΔSEN3800, respectively [P=0.0155] characterized. Transposon mutagenesis identified SEN3800 as (Fig. 3a). For the cecum, bacterial counts from chickens a potential binding mutant of SE. A ΔSEN3800 strain of SE infected with the WT SE strain were 8.7 and 9.1 logs at the was created using the lambda Red recombination system, and 16 h and day 7 time points, respectively, compared to 6.7 and was deficient in binding both in vitro and in vivo. 7.2 logs for ΔSEN3800 [P=0.0115, P=0.0035] (Fig. 3b). Additionally, this binding defect led to a deficiency in inva- These data suggest that SEN3800 plays a role in the coloni- sion of T84 intestinal epithelial cells, and decreased overall zation ability of SE in the gastrointestinal tract of chickens. systemic infection ability in a poultry model, as evidenced by We also assayed the bacterial counts from the livers and reduced bacterial counts in the livers and spleens of chickens spleens to see if the binding deficiency displayed by ΔSEN3800 inoculated with ΔSEN3800. These data indicated that affected systemic infection in chickens. At the 16 h time point, SEN3800 plays a role in the colonization ability of SE to the there was no bacteria recovered from the livers and spleens of gastrointestinal tract of poultry. chickens infected with the WT and ΔSEN3800 strains. At the The proteins encoded by orthologs of SEN3800 in other day 7 time point, bacterial counts in the liver were 7.2 and 5.7 bacterial species are characterized as members of the sulfatase logs for the WT and ΔSEN3800, respectively [P=0.0211] superfamily. Recent work on the human gut microbiota has (Fig. 4a). Furthermore, bacterial counts in the spleen were 7.8 shown a role for sulfatase enzymes in the interaction with highly sulfated glycans on the intestinal epithelium (Benjdia et al. (A) 2011). In Bacteroides thetaiotaomicron, there are hypothesized to be 28 sulfatases with one predicted anaerobic sulfatase ma- turing enzyme, which allow bacteria to adapt and forage on host WT sulfated glycans as nutrients (Benjdia et al. 2011). Most impor- P=0.0211 ΔSEN3800 tantly, it has been shown that activation of sulfatases is essential for competitive colonization of the gut (Benjdia et al. 2011). 4 Colonization of the intestinal mucosa of production ani- mals is a key pathway for Salmonella contamination of the human food supply, but the mechanism by which Salmonella interacts with the host at the early stages of infection is still poorly understood. Fimbrial adhesins are most well-known for their role in binding, but other studies have shown that other bacterial components, like lipopolysaccharides (LPS), (B) are involved as well, however, the mechanism is not clear WT (Craven 1994; Turner et al. 1998). Furthermore, in-vivo bind- ΔSEN3800 ing studies in chickens have been complicated by the overall P=0.0069 physiology of the chicken itself. The overall behavior of bacteria in newly hatched chicks is still poorly understood, and studies have shown that Salmonella colonization of the cecum is much higher in young chicks than in older birds (Zhang-Barber et al. 1997). Newly hatched chicks without an established microbiota are particularly susceptible to Salmonella colonization, and become reservoirs of infection Fig. 4 Systemic infection ability of the WT and ΔSEN3800 SE strains. Bacterial counts in a liver and b spleen of chickens inoculated by oral that secrete large amounts of Salmonella into the environment crop gavage with 1×10 CFU of WT and ΔSEN3800. There was no over an extended period of time (Barrow et al. 1988). bacterial recovery at the 16 h time point from either the liver or spleen of Binding of Salmonella to host intestinal epithelial cells is chickens inoculated with the WT or ΔSEN3800 strain, so only the day 7 considered a precursor to invasion. It has been suggested that data is shown. The actual P values are given, displaying a statistically significant difference between ΔSEN3800 and the WT strain invasion is a consequence of engaged host cell signaling log (CFU/ml) log (CFU/ml) 636 Ann Microbiol (2015) 65:631–637 Fernandez F, Hinton M, Van Gils B (2002) Dietary mannan- pathways that leads to cytoskeletal rearrangements (Finlay oligosaccharides and their effect on chicken caecal microflora et al. 1991; Francis et al. 1992). This results in the formation in relation to Salmonella enteritidis colonization. Avian Pathol 31: of membrane ruffles that engulf the bacteria in large vesicles 49–58 known as the Salmonella-containing vacuole (Francis et al. Finlay BB, Ruschkowski S, Dedhar S (1991) Cytoskeletal rearrange- ments accompanying Salmonella entry into epithelial cells. J Cell 1993; Garcia-del Portillo and Finlay 1994). While the bacteria Sci 99(Pt 2):283–296 reside in the Salmonella-containing vacuole, the Salmonella Francis CL, Ryan TA, Jones BD, Smith SJ, Falkow S (1993) Ruffles cells can survive and replicate, eventually producing a sys- induced by Salmonella and other stimuli direct macropinocytosis of temic infection by colonizing the liver and spleen (Francis bacteria. Nature 364:639–642 Francis CL, Starnbach MN, Falkow S (1992) Morphological and cyto- et al. 1993). Further investigation will be needed in order to skeletal changes in epithelial cells occur immediately upon interac- understand the molecular mechanism in which the binding tion with Salmonella typhimurium grown under low-oxygen condi- deficiency of ΔSEN3800 leads to reduced systemic infection tions. Mol Microbiol 6:3077–3087 in chickens. Friedrich MJ, Kinsey NE, Vila J, Kadner RJ (1993) Nucleotide sequence of a 13.9 kb segment of the 90 kb virulence plasmid of Salmonella Additional studies will also be needed to gauge the level of typhimurium: the presence of fimbrial biosynthetic genes. Mol attenuation of the ΔSEN3800 SE strain in chickens. Microbiol 8:543–558 Depending on the outcome of these studies, further studies Galan JE (1996) Molecular genetic bases of Salmonella entry into host could be conducted to determine if ΔSEN3800 is a good cells. Mol Microbiol 20:263–271 Gantois I et al (2009) Mechanisms of egg contamination by Salmonella candidate for use in a live-attenuated vaccine or other antimi- enteritidis. FEMS Microbiol Rev 33:718–738 crobial therapies. Overall, our data suggests that SEN3800 Garcia-del Portillo F, Finlay BB (1994) Salmonella invasion of plays a role in the binding ability of Salmonella to the intes- nonphagocytic cells induces formation of macropinosomes in the tinal mucosa of chickens, and could be an important factor in host cell. Infect Immun 62:4641–4645 Grund S, Weber A (1988) A new type of fimbriae on Salmonella the early stages of Salmonella infection in poultry. typhimurium. Zentralbl Veterinarmed B 35:779–782 Johnston CE, Hartley C, Salisbury AM, Wigley P (2012) Immunological Acknowledgments We thank Dr. Charles Kaspar, University of changes at point-of-lay increase susceptibility to Salmonella Wisconsin-Madison, for providing the WT SE PT8 isolate. The help of enterica serovar Enteritidis infection in vaccinated chickens. PLoS Imaan Saeed is highly appreciated. This work was supported by a grant One 7:e48195 from the University of Wisconsin-Madison Graduate School. Johny AK et al (2009) Prophylactic supplementation of caprylic acid in feed reduces Salmonella enteritidis colonization in commercial broiler chicks. J Food Prot 72:722–727 Kollanoor-Johny A et al (2012) Reduction of Salmonella enterica serovar References Enteritidis colonization in 20-day-old broiler chickens by the plant- derived compounds trans-cinnamaldehyde and eugenol. Appl Barrow PA, Simpson JM, Lovell MA (1988) Intestinal colonisation in the Environ Microbiol 78:2981–2987 chicken by food-poisoning Salmonella serotypes; microbial charac- Lara-Tejero M, Galan JE (2009) Salmonella enterica serovar teristics associated with faecal excretion. Avian Pathol 17:571–588 Typhimurium pathogenicity island 1-encoded type III secretion Baumler AJ, Heffron F (1995) Identification and sequence analysis of system translocases mediate intimate attachment to nonphagocytic lpfABCDE, a putative fimbrial operon of Salmonella typhimurium.J cells. Infect Immun 77:2635–2642 Bacteriol 177:2087–2097 Nash JH et al (2010) Genome sequence of adherent-invasive Escherichia Benjdia A, Martens EC, Gordon JI, Berteau O (2011) Sulfatases and a coli and comparative genomic analysis with other E. coli pathotypes. radical S-adenosyl-L-methionine (AdoMet) enzyme are key for BMC Genomics 11:667 mucosal foraging and fitness of the prominent human gut symbiont, Okamura M, Kamijima Y, Miyamoto T, Tani H, Sasai K, Baba E (2001) Bacteroides thetaiotaomicron. J Biol Chem 286:25973–25982 Differences among six Salmonella serovars in abilities to colonize Braden CR (2006) Salmonella enterica serotype Enteritidis and reproductive organs and to contaminate eggs in laying hens. Avian eggs: a national epidemic in the United States. Clin Infect Dis 43: Dis 45:61–69 512–517 Patel JC, Galan JE (2005) Manipulation of the host actin cytoskeleton by Buchrieser C, Brosch R, Buchrieser O, Kristl A, Luchansky JB, Kaspar Salmonella—all in the name of entry. Curr Opin Microbiol 8:10–15 CW (1997) Genomic analyses of Salmonella enteritidis phage type Scallan E et al (2011) Foodborne illness acquired in the United States— 4 strains from Austria and phage type 8 strains from the United major pathogens. Emerg Infect Dis 17:7–15 States. Zentralbl Bakteriol 285:379–388 Schlumberger MC, Hardt WD (2006) Salmonella type III secretion Clegg S, Purcell BK, Pruckler J (1987) Characterization of genes effectors: pulling the host cell’s strings. Curr Opin Microbiol 9:46– encoding type 1 fimbriae of Klebsiella pneumoniae, Salmonella 54 typhimurium,and Serratia marcescens. Infect Immun 55:281–287 Shippy DC, Eakley NM, Bochsler PN, Chopra AK, Fadl AA (2011) Craven SE (1994) Altered colonizing ability for the ceca of broiler chicks Biological and virulence characteristics of Salmonella enterica by lipopolysaccharide-deficient mutants of Salmonella serovar Typhimurium following deletion of glucose-inhibited divi- typhimurium. Avian Dis 38:401–408 sion (gidA) gene. Microb Pathog 50:303–313 Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal Shippy DC, Eakley NM, Mikheil DM, Fadl AA (2013) Role of genes in Escherichia coli K-12 using PCR products. Proc Natl Acad the flagellar basal-body protein, FlgC, in the binding of Sci U S A 97:6640–6645 Salmonella enterica serovar Enteritidis to host cells. Curr Fanelli MJ, Sadler WW, Franti CE, Brownell JR (1971) Localization of Microbiol 68:621–628 salmonellae within the intestinal tract of chickens. Avian Dis 15: Thomson NR et al (2008) Comparative genome analysis of Salmonella 366–375 enteritidis PT4 and Salmonella gallinarum 287/91 provides insights Ann Microbiol (2015) 65:631–637 637 into evolutionary and host adaptation pathways. Genome Res 18: Wizemann TM, Adamou JE, Langermann S (1999) Adhesins as targets 1624–1637 for vaccine development. Emerg Infect Dis 5:395–403 Turner AK, Lovell MA, Hulme SD, Zhang-Barber L, Barrow PA (1998) Zhang-Barber L, Turner AK, Martin G, Frankel G, Dougan G, Barrow Identification of Salmonella typhimurium genes required for colo- PA (1997) Influence of genes encoding proton-translocating en- nization of the chicken alimentary tract and for virulence in newly zymes on suppression of Salmonella typhimurium growth and col- hatched chicks. Infect Immun 66:2099–2106 onization. J Bacteriol 179:7186–7190 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Characterization of SEN3800-associated virulence of Salmonella enterica serovar Enteritidis phage type 8

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Springer Journals
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Copyright © 2014 by Springer-Verlag Berlin Heidelberg and the University of Milan
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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1590-4261
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1869-2044
DOI
10.1007/s13213-014-0898-8
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Abstract

Ann Microbiol (2015) 65:631–637 DOI 10.1007/s13213-014-0898-8 ORIGINAL ARTICLE Characterization of SEN3800-associated virulence of Salmonella enterica serovar Enteritidis phage type 8 Daniel C. Shippy & Nicholas M. Eakley & Dareen M. Mikheil & Anna De La Cotera & Amin A. Fadl Received: 28 February 2014 /Accepted: 7 April 2014 /Published online: 30 April 2014 Springer-Verlag Berlin Heidelberg and the University of Milan 2014 Abstract Salmonella is a major public health concern due to contaminated food. Nontyphoidal Salmonella serovars, like the consumption of contaminated food. Salmonella enterica Salmonella enterica serovar Enteritidis (SE), are the leading serovar Enteritidis (SE) infection in humans is often associat- cause of hospitalization and death among the major foodborne ed with the consumption of contaminated poultry products. pathogens (Scallan et al. 2011). Therefore, the identification Binding of the bacterium to the intestinal mucosa is a major and characterization of mechanisms involved in Salmonella pathogenic mechanism of Salmonella in poultry. In this study, pathogenesis could lead to new strategies for controlling transposon mutagenesis identified SEN3800 as a potential salmonellosis. binding mutant of SE. Therefore, we hypothesize that SE outbreaks in humans are mainly associated with the SEN3800 plays a role in the colonization ability of SE in the consumption of contaminated eggs and other poultry prod- gastrointestinal tract of poultry. To test our hypothesis, we ucts. In poultry, Salmonella can colonize the intestinal tract created a mutant of SE in which SEN3800 was deleted. We and become asymptomatic carriers of the bacterium, resulting then tested the in-vitro and in-vivo binding ability of in the contamination of food (Fanelli et al. 1971; Barrow et al. ΔSEN3800 when compared to the wild-type and 1988). Eggs can then become contaminated by Salmonella complemented SE strains. Our data showed a significant penetrating the eggshell, or direct contamination of the inter- decrease in the binding ability of ΔSEN3800 to T84 intestinal nal egg during formation due to Salmonella colonizing the epithelial cells, as well as in the small intestine and cecum of reproductive tract (Okamura et al. 2001; Gantois et al. 2009). poultry. Furthermore, this binding defect correlated to a defect Over the past 25 years, the ability of SE to contaminate eggs in invasion, as evidenced by a cell culture model using T84 and other poultry products has led to a constant threat to public intestinal epithelial cells and bacterial recovery from the livers health in the United States (Braden 2006). and spleens of chickens. Overall, these studies indicate that A hallmark of Salmonella pathogenesis is its ability to SEN3800 contributes to the colonization ability of Salmonella invade intestinal epithelial cells (Patel and Galan 2005). This in the gastrointestinal tract of poultry. is a multi-step process mediated by a type 3 secretion system (T3SS) encoded within Salmonella pathogenicity island-1 . . . Keywords Salmonella Poultry SEN3800 Pathogenesis (SPI-1) (Galan 1996; Schlumberger and Hardt 2006). The initial step in the invasion process is the binding of Salmonella to the host intestinal epithelial cells. Several Introduction Salmonella virulence factors have been implicated in the binding to host cells. The best characterized are the fimbrial Salmonella is a major foodborne pathogen that frequently adhesins which include type 1, long polar, plasmid-encoded, causes gastroenteritis in humans due to the consumption of and thin aggregative fimbriae (Clegg et al. 1987; Grund and Weber 1988; Friedrich et al. 1993; Baumler and Heffron 1995). Interestingly, another study has shown that the T3SS : : : : D. C. Shippy N. M. Eakley D. M. Mikheil A. De La Cotera itself can mediate host cell binding by providing evidence that A. A. Fadl (*) SipB, SipC, and SipD are required for the intimate association Department of Animal Sciences, University of Wisconsin-Madison, of Salmonella with mammalian cells (Lara-Tejero and Galan 1675 Observatory Drive, Madison, WI 53706, USA 2009). The inhibition of Salmonella binding at the early stages e-mail: fadl@wisc.edu 632 Ann Microbiol (2015) 65:631–637 of infection is potentially the most effective strategy for con- grown and maintained in Dulbecco’s Modified Eagle trolling salmonellosis in production animals, and could lead to Medium: Nutrient Mixture F12 medium supplemented with a reduction in the contamination of our food supply 10 % fetal bovine serum, and incubated at 37 °C with 5 % (Wizemann et al. 1999). CO . In this study, we identified a novel binding mutant of SE by transposon mutagenesis. The SEN3800 deletion mutant Construction of the mutant and complemented strains (ΔSEN3800) displayed a normal growth profile when com- pared to the wild-type (WT) SE phage type (PT) 8 E2627 and The transposon binding screening that identified SEN3800 as a complemented strains (ΔSEN3800/pBRSEN3800). Binding potential binding mutant of SE is described in Shippy et al and invasion assays showed that ΔSEN3800 was deficient in (2013). The ΔSEN3800 strain was created using the lambda cell culture models of Salmonella binding and invasion. Red recombination system, as previously described (Datsenko Furthermore, ΔSEN3800 was deficient in a poultry model of and Wanner 2000). Briefly, WT SE PT8 was transformed with Salmonella binding and invasion, with the invasion defect the pKD46 plasmid, induced with arabinose, and used to most likely due to the deficiency in binding ability. Taken generate the electrocompetent cells. The kanamycin resistance together, these data indicated a role for SEN3800 in the gene (Kn ) was amplified by polymerase chain reaction (PCR) colonization of SE in the gastrointestinal tract of poultry. from the pKD4 plasmid using primer set LF/LR. The PCR product was purified and electroporated into the WT-pKD46 electrocompetent cells. After transformation, colonies growing Materials and methods on LB plates supplemented with kanamycin were selected as candidates for SEN3800 mutants of SE. To confirm deletion of Bacterial strains, plasmids, and cell lines the SEN3800 gene, the selected mutants were subjected to PCR analysis using primer sets K /K and F /R to show the pres- 3 5 2 2 The WT SE PT8 E2627 strain was isolated from an egg- ence of the Kn and the absence of SEN3800. associated outbreak in the United States and is described in The SEN3800-complemented strain was constructed by am- Buchrieser et al (1997). All Salmonella strains were grown in plifying a DNA fragment containing SEN3800 from the WT either Luria-Bertani (LB) medium (10 g casein peptone, 5 g SE strain using primer set F /R . The DNA fragment was blunt- 3 3 yeast extract, and 10 g sodium chloride per liter) or on ended using a PCR polishing kit (Stratagene, Santa Clara, CA) Salmonella-Shigella (SS) agar plates. Additionally, homoge- and ligated into the blunt-ended ScaI restriction enzyme nates from the in-vivo experiments were incubated in digested pBR322 vector. The recombinant plasmid was trans- Selenite-F broth (BD, Sparks, MD). Nalidixic acid formed into the SEN3800 mutant by electroporation. A com- (100 μg/ml), kanamycin (50 μg/ml), tetracycline (15 μg/ml), plete list of the primers used in this study is shown in Table 2. and ampicillin (100 μg/ml) were added to the media as nec- essary. A complete list of the bacterial strains and plasmids Growth analysis used in this study is shown in Table 1. T84 intestinal epithelial cells were obtained from the American Type Culture Growth curve profiles were constructed in order to determine Collection (ATCC, Manassas, VA), and were subsequently the significance of SEN3800 on SE growth. An equal number Table 1 Strains and plasmids used in this study Strain or plasmid Relevant characteristics Source or reference Serovar Enteritidis phage Isolated from an egg-associated outbreak in the United States (Buchrieser et al. 1997) type 8 E2627 SEN3800::Tn5 Mutant of serovar Enteritidis with disruption of SEN3800 via Tn5 transposon mutagenesis This study ΔSEN3800 Mutant of serovar Enteritidis in which SEN3800 was deleted using lambda Red; Kn This study r r ΔSEN3800/pBRSEN3800 ΔSEN3800 complemented with a copy of the SEN3800 gene via pBR322; Kn,Tc This study E. coli DH5α Used for recombinant DNA methods Lab stock Plasmids pKD46 lambda Red recombinase genes; Ap (Datsenko and Wanner 2000) r r pBR322 Ap Tc Lab stock pKD4 Kn gene cassette (Datsenko and Wanner 2000) pBRSEN3800 SEN3800 gene cloned into pBR322 at the ScaIsite Thisstudy Ann Microbiol (2015) 65:631–637 633 Table 2 Sequence and purpose of primers used in this study Chicken experiments Primer name and sequence One-week-old specific-pathogen-free White Leghorn chickens LF:5′-ACTGTACATATACTGACGATAAATATCATTGTTATTCATA were obtained from Charles River (Wilmington, MA). All TGTGTAGGCTGGAGCTGCTTCG-3′ animal procedures were approved by the University of LR:5′-GCGCAAATCCGGGCCATGAGGAAGCAAAATGTATAATT Wisconsin-Madison Animal Care and Use Committee. CACATATGAATATCCTCCTTAG-3′ Groups of 11 birds were inoculated by oral crop gavage with F :5′-GGTTAGGGTAATGAATCGTC-3′ 1×10 CFU of the WTor ΔSEN3800 SE strains. An additional R :5′-GATATCGGACTTGGTCGTAA-3′ group (n=4) was inoculated by oral crop gavage with 100 μl F :5′-CGCTAAAGCTGACGTTCTCC-3′ sterile PBS to serve as a control. At 16 h and 7 days post- R :5′-GCCATGAGGAAGCAAAATGT-3′ infection, five birds from each group were euthanized using K :5′-AAAGCCACGTTGTGTCTA-3′ CO Portions of the liver, spleen, small intestine, and cecum K :5′- CGCTGAGGTCTGCCTCGT-3′ were removed from each bird. The individual organs were pooled and 1 g was homogenized in 10 ml PBS. One ml from each homogenate was removed and incubated in 10 ml of cells from the WT, ΔSEN3800,and ΔSEN3800/ Selenite-F broth at 36 °C for 18 h. Direct plating of the organ pBRSEN3800 strains were inoculated in LB and Selenite-F homogenates was done in parallel with plating from the en- broth and grown at 37 °C. The optical densities at 600 nm were richment cultures (Fernandez et al. 2002; Johny et al. 2009; recorded each hour. Johnston et al. 2012; Kollanoor-Johny et al. 2012). Enumeration of bacteria was performed by serial dilution Binding assay and plating on SS agar. The binding assay was performed as previously described Statistical analysis (Shippy et al. 2011). Briefly, 5×10 T84 intestinal epithelial cells were seeded per well in a 24-well tissue culture plate and Wherever appropriate, the data were analyzed using incubated overnight at 37 °C with 5 % CO . The following GraphPad Prism 5 software (GraphPad Software, San Diego, day, cells were infected with the WT, ΔSEN3800,and CA) and a Student’s t test. P values of ≤0.05 were considered ΔSEN3800/pBRSEN3800 strains at a multiplicity of infection significant. Unless otherwise stated, experiments were repeat- (MOI) of 10:1. The plate was briefly centrifuged at 400×g for ed two times and data were expressed as arithmetic means minutes, and incubated for 30 min at 37 °C with 5 % CO . with standard deviations. Unbound bacteria were aspirated; the wells were washed six times with phosphate buffered saline (PBS), and the cells were lysed with 0.1 % Triton X-100 (TX-100). Dilutions of the cell Results lysates were plated on SS agar for enumeration of bacteria. Characterization of the SEN3800 mutant Invasion assay The chromosomal SEN3800 gene was replaced by a The invasion assay was performed as previously described kanamycin resistance gene (Kn ) cassette using the (Shippy et al. 2011). Briefly, 5×10 T84 intestinal epithelial lambda Red recombination system. Deletion of cells were seeded per well in a 24-well plate and incubated SEN3800 from the chromosome of SE was confirmed overnight at 37 °C with 5 % CO . The cells were infected with by PCR analysis. The primer set K /K was used to 2 3 5 the WT, ΔSEN3800,and ΔSEN3800/pBRSEN3800 strains at amplify the Kn cassette, while the primer set F /R 2 2 an MOI of 10:1, and briefly centrifuged at 400×g for 5 min so was used to confirm the absence of SEN3800.Toensure that the bacterial cells would be in direct contact with the T84 correct orientation of the Kn cassette, the primer set F / cells. After incubation for 30 min at 37 °C with 5 % CO ,the K was used to amplify the upstream SEN3800 flanking 2 5 cells were washed three times with PBS and incubated for an sequence along with the Kn cassette, while R /K was 3 3 additional 45 min with gentamicin-containing medium used to amplify the downstream SEN3800 flanking se- (100 μg/ml) to kill extracellular bacteria. Following incuba- quence along with the Kn cassette. Overall, these re- tion, the gentamicin-containing medium was removed, sults indicated that a SEN3800 deletion mutant of SE the wells were washed six times with PBS, and the cells PT8 was successfully created. were lysed with 0.1 % TX-100. The lysate was diluted and Bioinformatic analysis of SEN3800 indicated an 864- plated out on SS agar plates for colony-forming unit (CFU) nucleotide gene encoding a 287-amino-acid protein. determination. Orthologs of SEN3800 are highly conserved in prokaryotes, 634 Ann Microbiol (2015) 65:631–637 (A) and SEN3800 of serovar Enteritidis is identical to orthologs in other Salmonella serovars, including Typhimurium, WT Dublin, Typhi, Paratyphi, Gallinarum, and Pullorum ΔSEN3800 (Thomson et al. 2008). The proteins encoded by ΔSEN3800/pBRSEN3800 P=0.0156 orthologs of SEN3800 in other bacterial species are characterized as members of the sulfatase superfamily, and as putative bacterial membrane proteins. The SEN3800 protein of Salmonella shares 97 % homology with a membrane-associated metal-dependent hydrolase of Escherichia coli (Nash et al. 2010). Additionally, SEN3800 contains a motif that is conserved in the sulfatase superfamily (B) WT (Thomson et al. 2008). ΔSEN3800 ΔSEN3800/pBRSEN3800 P=0.0115 SEN3800 does not affect Salmonella growth Growth curve analysis was performed for the WT, ΔSEN3800, and ΔSEN3800/pBRSEN3800 strains in order to determine the significance of SEN3800 on Salmonella growth. All three strains displayed nearly identical growth profiles, suggesting Fig. 2 Cell culture assays using T84 intestinal epithelial cells. a Binding that SEN3800 does not play a significant role in SE growth assay and b invasion assay with the WT, ΔSEN3800,and ΔSEN3800/ (Fig. 1). Additionally, growth analysis in Selenite-F broth pBRSEN3800 strains. The actual P values are given, displaying a statis- showed no difference in growth, with minimal overall growth tically significant difference between ΔSEN3800 and the WT strain. The graphs are representative of three independent experiments observed. Deletion of SEN3800 decreases SE binding and invasion in vitro (A) Infection of T84 intestinal epithelial cells displayed a significant decrease in the binding ability of ΔSEN3800 WT (4.5 logs) to host epithelial cells when compared to the P=0.0188 ΔSEN3800 WT SE strain (6.1 logs) [P = 0.0156] (Fig. 2a). Furthermore, ΔSEN3800 displayed the same significant P=0.0155 decrease in the ability to invade T84 intestinal epithelial cells [P=0.0115] (Fig. 2b). The binding and invasive ability of ΔSEN3800 was restored after complementation, suggesting that SEN3800 plays a role in the binding and invasion of intestinal epithelial cells by SE. It is logical to 16 Hours Day 7 conclude that the invasion defect seen in ΔSEN3800 is an effect of the binding deficiency. (B) WT 2.5 WT ΔSEN3800 P=0.0035 P=0.0115 ΔSEN3800 2.0 ΔSEN3800/pBRSEN3800 1.5 1.0 0.5 0.0 16 Hours Day 7 0 2 4 6 8 10 Hour Fig. 3 Determination of in-vivo binding ability. Bacterial counts in a Fig. 1 Growth curves of the WT, ΔSEN3800,and ΔSEN3800/ small intestine and b cecum of chickens inoculated by oral crop gavage pBRSEN3800. The strains were grown in LB and the optical densities with 1×10 CFU of WT and ΔSEN3800.The actual P values are given, at 600 nm were measured each hour. The graph is representative of two displaying a statistically significant difference between ΔSEN3800 and independent experiments the WT strain O.D. 600 nm log (CFU/ml) log (CFU/ml) log (CFU/ml) log (CFU/ml) Ann Microbiol (2015) 65:631–637 635 Deletion of SEN3800 decreases SE binding and invasion and 6.1 logs for the WT and ΔSEN3800, respectively [P= in chickens 0.0069] (Fig. 4b). These data suggest that the binding deficien- cy displayed by ΔSEN3800 contributes to an overall reduction A poultry model was used to determine the role of SEN3800 in systemic infection by SE in poultry. in the binding ability of SE to the intestinal mucosa of chickens. At 16 h post-infection, the bacterial counts of WT SE from the small intestine were 7.1 logs compared to 6.2 logs Discussion [P=0.0188] for ΔSEN3800. At day 7 post-infection, bacterial counts from the small intestine were 4.1 logs versus 3.2 logs In this study, a novel binding mutant of SE was created and between the WT and ΔSEN3800, respectively [P=0.0155] characterized. Transposon mutagenesis identified SEN3800 as (Fig. 3a). For the cecum, bacterial counts from chickens a potential binding mutant of SE. A ΔSEN3800 strain of SE infected with the WT SE strain were 8.7 and 9.1 logs at the was created using the lambda Red recombination system, and 16 h and day 7 time points, respectively, compared to 6.7 and was deficient in binding both in vitro and in vivo. 7.2 logs for ΔSEN3800 [P=0.0115, P=0.0035] (Fig. 3b). Additionally, this binding defect led to a deficiency in inva- These data suggest that SEN3800 plays a role in the coloni- sion of T84 intestinal epithelial cells, and decreased overall zation ability of SE in the gastrointestinal tract of chickens. systemic infection ability in a poultry model, as evidenced by We also assayed the bacterial counts from the livers and reduced bacterial counts in the livers and spleens of chickens spleens to see if the binding deficiency displayed by ΔSEN3800 inoculated with ΔSEN3800. These data indicated that affected systemic infection in chickens. At the 16 h time point, SEN3800 plays a role in the colonization ability of SE to the there was no bacteria recovered from the livers and spleens of gastrointestinal tract of poultry. chickens infected with the WT and ΔSEN3800 strains. At the The proteins encoded by orthologs of SEN3800 in other day 7 time point, bacterial counts in the liver were 7.2 and 5.7 bacterial species are characterized as members of the sulfatase logs for the WT and ΔSEN3800, respectively [P=0.0211] superfamily. Recent work on the human gut microbiota has (Fig. 4a). Furthermore, bacterial counts in the spleen were 7.8 shown a role for sulfatase enzymes in the interaction with highly sulfated glycans on the intestinal epithelium (Benjdia et al. (A) 2011). In Bacteroides thetaiotaomicron, there are hypothesized to be 28 sulfatases with one predicted anaerobic sulfatase ma- turing enzyme, which allow bacteria to adapt and forage on host WT sulfated glycans as nutrients (Benjdia et al. 2011). Most impor- P=0.0211 ΔSEN3800 tantly, it has been shown that activation of sulfatases is essential for competitive colonization of the gut (Benjdia et al. 2011). 4 Colonization of the intestinal mucosa of production ani- mals is a key pathway for Salmonella contamination of the human food supply, but the mechanism by which Salmonella interacts with the host at the early stages of infection is still poorly understood. Fimbrial adhesins are most well-known for their role in binding, but other studies have shown that other bacterial components, like lipopolysaccharides (LPS), (B) are involved as well, however, the mechanism is not clear WT (Craven 1994; Turner et al. 1998). Furthermore, in-vivo bind- ΔSEN3800 ing studies in chickens have been complicated by the overall P=0.0069 physiology of the chicken itself. The overall behavior of bacteria in newly hatched chicks is still poorly understood, and studies have shown that Salmonella colonization of the cecum is much higher in young chicks than in older birds (Zhang-Barber et al. 1997). Newly hatched chicks without an established microbiota are particularly susceptible to Salmonella colonization, and become reservoirs of infection Fig. 4 Systemic infection ability of the WT and ΔSEN3800 SE strains. Bacterial counts in a liver and b spleen of chickens inoculated by oral that secrete large amounts of Salmonella into the environment crop gavage with 1×10 CFU of WT and ΔSEN3800. There was no over an extended period of time (Barrow et al. 1988). bacterial recovery at the 16 h time point from either the liver or spleen of Binding of Salmonella to host intestinal epithelial cells is chickens inoculated with the WT or ΔSEN3800 strain, so only the day 7 considered a precursor to invasion. It has been suggested that data is shown. The actual P values are given, displaying a statistically significant difference between ΔSEN3800 and the WT strain invasion is a consequence of engaged host cell signaling log (CFU/ml) log (CFU/ml) 636 Ann Microbiol (2015) 65:631–637 Fernandez F, Hinton M, Van Gils B (2002) Dietary mannan- pathways that leads to cytoskeletal rearrangements (Finlay oligosaccharides and their effect on chicken caecal microflora et al. 1991; Francis et al. 1992). This results in the formation in relation to Salmonella enteritidis colonization. Avian Pathol 31: of membrane ruffles that engulf the bacteria in large vesicles 49–58 known as the Salmonella-containing vacuole (Francis et al. Finlay BB, Ruschkowski S, Dedhar S (1991) Cytoskeletal rearrange- ments accompanying Salmonella entry into epithelial cells. J Cell 1993; Garcia-del Portillo and Finlay 1994). While the bacteria Sci 99(Pt 2):283–296 reside in the Salmonella-containing vacuole, the Salmonella Francis CL, Ryan TA, Jones BD, Smith SJ, Falkow S (1993) Ruffles cells can survive and replicate, eventually producing a sys- induced by Salmonella and other stimuli direct macropinocytosis of temic infection by colonizing the liver and spleen (Francis bacteria. Nature 364:639–642 Francis CL, Starnbach MN, Falkow S (1992) Morphological and cyto- et al. 1993). Further investigation will be needed in order to skeletal changes in epithelial cells occur immediately upon interac- understand the molecular mechanism in which the binding tion with Salmonella typhimurium grown under low-oxygen condi- deficiency of ΔSEN3800 leads to reduced systemic infection tions. Mol Microbiol 6:3077–3087 in chickens. Friedrich MJ, Kinsey NE, Vila J, Kadner RJ (1993) Nucleotide sequence of a 13.9 kb segment of the 90 kb virulence plasmid of Salmonella Additional studies will also be needed to gauge the level of typhimurium: the presence of fimbrial biosynthetic genes. Mol attenuation of the ΔSEN3800 SE strain in chickens. Microbiol 8:543–558 Depending on the outcome of these studies, further studies Galan JE (1996) Molecular genetic bases of Salmonella entry into host could be conducted to determine if ΔSEN3800 is a good cells. Mol Microbiol 20:263–271 Gantois I et al (2009) Mechanisms of egg contamination by Salmonella candidate for use in a live-attenuated vaccine or other antimi- enteritidis. FEMS Microbiol Rev 33:718–738 crobial therapies. Overall, our data suggests that SEN3800 Garcia-del Portillo F, Finlay BB (1994) Salmonella invasion of plays a role in the binding ability of Salmonella to the intes- nonphagocytic cells induces formation of macropinosomes in the tinal mucosa of chickens, and could be an important factor in host cell. Infect Immun 62:4641–4645 Grund S, Weber A (1988) A new type of fimbriae on Salmonella the early stages of Salmonella infection in poultry. typhimurium. Zentralbl Veterinarmed B 35:779–782 Johnston CE, Hartley C, Salisbury AM, Wigley P (2012) Immunological Acknowledgments We thank Dr. Charles Kaspar, University of changes at point-of-lay increase susceptibility to Salmonella Wisconsin-Madison, for providing the WT SE PT8 isolate. The help of enterica serovar Enteritidis infection in vaccinated chickens. PLoS Imaan Saeed is highly appreciated. This work was supported by a grant One 7:e48195 from the University of Wisconsin-Madison Graduate School. Johny AK et al (2009) Prophylactic supplementation of caprylic acid in feed reduces Salmonella enteritidis colonization in commercial broiler chicks. J Food Prot 72:722–727 Kollanoor-Johny A et al (2012) Reduction of Salmonella enterica serovar References Enteritidis colonization in 20-day-old broiler chickens by the plant- derived compounds trans-cinnamaldehyde and eugenol. Appl Barrow PA, Simpson JM, Lovell MA (1988) Intestinal colonisation in the Environ Microbiol 78:2981–2987 chicken by food-poisoning Salmonella serotypes; microbial charac- Lara-Tejero M, Galan JE (2009) Salmonella enterica serovar teristics associated with faecal excretion. Avian Pathol 17:571–588 Typhimurium pathogenicity island 1-encoded type III secretion Baumler AJ, Heffron F (1995) Identification and sequence analysis of system translocases mediate intimate attachment to nonphagocytic lpfABCDE, a putative fimbrial operon of Salmonella typhimurium.J cells. Infect Immun 77:2635–2642 Bacteriol 177:2087–2097 Nash JH et al (2010) Genome sequence of adherent-invasive Escherichia Benjdia A, Martens EC, Gordon JI, Berteau O (2011) Sulfatases and a coli and comparative genomic analysis with other E. coli pathotypes. radical S-adenosyl-L-methionine (AdoMet) enzyme are key for BMC Genomics 11:667 mucosal foraging and fitness of the prominent human gut symbiont, Okamura M, Kamijima Y, Miyamoto T, Tani H, Sasai K, Baba E (2001) Bacteroides thetaiotaomicron. J Biol Chem 286:25973–25982 Differences among six Salmonella serovars in abilities to colonize Braden CR (2006) Salmonella enterica serotype Enteritidis and reproductive organs and to contaminate eggs in laying hens. Avian eggs: a national epidemic in the United States. Clin Infect Dis 43: Dis 45:61–69 512–517 Patel JC, Galan JE (2005) Manipulation of the host actin cytoskeleton by Buchrieser C, Brosch R, Buchrieser O, Kristl A, Luchansky JB, Kaspar Salmonella—all in the name of entry. Curr Opin Microbiol 8:10–15 CW (1997) Genomic analyses of Salmonella enteritidis phage type Scallan E et al (2011) Foodborne illness acquired in the United States— 4 strains from Austria and phage type 8 strains from the United major pathogens. Emerg Infect Dis 17:7–15 States. Zentralbl Bakteriol 285:379–388 Schlumberger MC, Hardt WD (2006) Salmonella type III secretion Clegg S, Purcell BK, Pruckler J (1987) Characterization of genes effectors: pulling the host cell’s strings. Curr Opin Microbiol 9:46– encoding type 1 fimbriae of Klebsiella pneumoniae, Salmonella 54 typhimurium,and Serratia marcescens. Infect Immun 55:281–287 Shippy DC, Eakley NM, Bochsler PN, Chopra AK, Fadl AA (2011) Craven SE (1994) Altered colonizing ability for the ceca of broiler chicks Biological and virulence characteristics of Salmonella enterica by lipopolysaccharide-deficient mutants of Salmonella serovar Typhimurium following deletion of glucose-inhibited divi- typhimurium. Avian Dis 38:401–408 sion (gidA) gene. Microb Pathog 50:303–313 Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal Shippy DC, Eakley NM, Mikheil DM, Fadl AA (2013) Role of genes in Escherichia coli K-12 using PCR products. Proc Natl Acad the flagellar basal-body protein, FlgC, in the binding of Sci U S A 97:6640–6645 Salmonella enterica serovar Enteritidis to host cells. Curr Fanelli MJ, Sadler WW, Franti CE, Brownell JR (1971) Localization of Microbiol 68:621–628 salmonellae within the intestinal tract of chickens. Avian Dis 15: Thomson NR et al (2008) Comparative genome analysis of Salmonella 366–375 enteritidis PT4 and Salmonella gallinarum 287/91 provides insights Ann Microbiol (2015) 65:631–637 637 into evolutionary and host adaptation pathways. Genome Res 18: Wizemann TM, Adamou JE, Langermann S (1999) Adhesins as targets 1624–1637 for vaccine development. Emerg Infect Dis 5:395–403 Turner AK, Lovell MA, Hulme SD, Zhang-Barber L, Barrow PA (1998) Zhang-Barber L, Turner AK, Martin G, Frankel G, Dougan G, Barrow Identification of Salmonella typhimurium genes required for colo- PA (1997) Influence of genes encoding proton-translocating en- nization of the chicken alimentary tract and for virulence in newly zymes on suppression of Salmonella typhimurium growth and col- hatched chicks. Infect Immun 66:2099–2106 onization. J Bacteriol 179:7186–7190

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Published: Apr 30, 2014

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