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RNA-Seq reveals differences in the global transcriptome between high- and low-1
pathogenic Salmonella Enteritidis strains 2
3
4
Devendra H. Shah* 5
Department of Veterinary Microbiology and Pathology and Paul Allen School for Global 6
Animal Health, Washington State University, Pullman WA 99164 7
8
Running title: Global transcriptomics of Salmonella Enteritidis 9
10
*Author for correspondence: 11
Devendra H. Shah, DVM, PhD 12
Assistant Professor of Veterinary Bacteriology & Mycology 13
Department of Veterinary Microbiology and Pathology 14
Washington State University, Pullman WA 99164-7040 15
Phone: +1-509-335-6071 16
Fax: +1-509-335-8529 17
Email: [email protected] 18
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AEM Accepts, published online ahead of print on 22 November 2013Appl. Environ. Microbiol. doi:10.1128/AEM.02740-13Copyright © 2013, American Society for Microbiology. All Rights Reserved.
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Abstract: 20
Salmonella Enteritidis is one of the important causes of bacterial food-borne gastroenteritis 21
world-wide. Field strains of S. Enteritidis are relatively genetically homogeneous; however they 22
show extensive phenotypic diversity and differences in virulence potential. RNA sequencing 23
(RNA-Seq) was used to characterize differences in the global transcritome between several 24
genetically similar but phenotypically diverse poultry-associated field strains of S. Enteritidis 25
grown in laboratory media at avian body temperature (42°C). These S. Enteritidis strains were 26
previously characterized as high-pathogenic (HP, n=3) and low-pathogenic (LP, n=3) based on 27
both in vitro and in vivo virulence assays. Using the negative binomial distribution-based 28
statistical tools, edgeR and DESeq, 252 genes were identified as differentially expressed in LP 29
strains when compared with the HP strains (P<0.05). A majority of genes (235, 93.2%) showed 30
significantly reduced expression whereas a few genes (17, 6.8%) showed increased expression 31
in all LP strains when compared with HP strains. LP strains showed unique transcriptional 32
profile that is characterized by significantly reduced expression of several transcriptional 33
regulators, and reduced expression of genes involved in virulence (eg., SPI-1, SPI-5, fimbrial 34
and motility genes) and protection against osmotic, oxidative and other stresses such as iron 35
limiting conditions commonly encountered within the host. Several functionally uncharacterized 36
genes also showed reduced expression. This study provides a first concise view of the global 37
transcriptional differences between filed strains of S. Enteritidis with varying levels of 38
pathogenicity, providing the basis for future functional characterization of several genes with 39
potential roles in virulence or stress regulation of S. Enteritidis. 40
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Key words: Salmonella Enteritidis, transcriptome, RNA-Seq 42
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Introduction 43
Salmonellosis is one of the leading causes of diarrheal illness in humans with an estimated 1 44
million cases of food-borne illnesses resulting in 19,336 hospitalizations and 378 deaths 45
reported annually in the United States (1). Salmonella Enteritidis (S. Enteritidis) is the most 46
common non-typhoidal Salmonella serovar responsible for food-borne gastroenteritis and is 47
usually one of the top two serovars reported along with S. Typhimurium in surveys from various 48
nations around the world (2-4). S. Enteritidis primarily cause food-borne gastroenteritis, which is 49
characterized by diarrhea, fever, headache, abdominal pain, nausea and vomiting (5). In 50
addition, S. Enteritidis has been recently reported to cause increased incidence of invasive, 51
recurrent, and multiple site infections in African countries (4, 6, 7). Poultry is the major reservoir 52
of S. Enteritidis and the live poultry or poultry products such as eggs and meat are the primary 53
sources of human infection world-wide (8-13). It has been demonstrated that irrespective of the 54
phage-type or source or geographical location of isolation, field strains of S. Enteritidis are 55
relatively genetically homogeneous (14-17). Recent high-resolution genetic studies using 56
individual-gene or whole-genome sequencing have revealed that S. Enteritidis is relatively 57
genetically homogeneous with the major genetic differences between field strains of S. 58
Enteritidis occurring at the level of single nucleotide polymorphisms (SNPs) (15, 18, 19). 59
However, field strains show remarkable differences in their phenotypes and virulence potential 60
(16, 17, 19-25). For instance, it has been reported that naturally occurring strains of S. 61
Enteritidis vary in their virulence potential in murine and chicken models of infection (19, 21, 23, 62
24, 26-30). In addition, field strains of S. Enteritidis show remarkable differences in terms of 63
their epithelial cell invasiveness, motility, biofilm production, resistance to acidic and oxidative 64
stress and the ability to survive within the avian macrophages and egg-albumen (16, 19-21, 23, 65
25, 31-33). The role that these SNPs might play in differential phenotypic characteristics, 66
virulence regulation or persistence of strains within the host or environment remains elusive. 67
68
It was recently reported that certain poultry-associated field strains of S. Enteritidis show 69
impaired virulence in orally infected BALB/c mice and day-old chickens while most other strains 70
are naturally virulent (19, 21). For the purpose of this study naturally virulent strains were 71
designated as high-pathogenic (HP) whereas strains with impaired virulence as low-pathogenic 72
(LP) strains. LP strains were previously reported as low invasive in cultured human and avian 73
epithelial cells, and showed impaired survival within avian macrophages and reduced resistance 74
to acidic and oxidative stress (19, 21). Comparative genomic hybridization microarray did not 75
reveal genetic differences between LP and HP strains that could be attributed to their 76
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phenotypes (19). Consequently, a central hypothesis for this study was that the differences in 77
virulence and phenotypic properties of LP strains of S. Enteritidis are due to the underlying 78
differences in their global transcriptional signatures. To test this hypothesis next-generation 79
mRNA sequence analysis (RNA-Seq) was performed to identify genes differentially expressed 80
by multiple HP (n=3) and LP (n=3) strains of S. Enteritidis in response to growth in laboratory 81
media at avian body temperature (42°C). Using multiple strains to obtain a comprehensive 82
image of their global transcriptional signatures, this study demonstrates that the LP strains have 83
a distinct signature that is characterized by significantly reduced expression of virulence and 84
stress-associated genes and that these differences correlate with their respective phenotypes. 85
To the best of my knowledge, this is the first study that compared transcriptional signatures of 86
multiple well-characterized S. Enteritidis strains using RNA-Seq. The deduced functions of 87
differentially regulated genes in relation to virulence and stress are also discussed. 88
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Materials and Methods 91
Bacterial strains: Six representative wild-type S. Enteritidis strains (UK, G1, BC8, C19, C45 92
and G45) isolated from poultry were analyzed in this study (Table 1). In previous studies, these 93
strains showed consistent differences in their phenotypic characteristics and virulence potential 94
in avian and murine models of infection (20, 21). Based on these characteristics, the selected 95
strains were classified in two groups: high-pathogenic (HP) and low-pathogenic (LP) groups. HP 96
group included the UK (corresponds to the sequenced phage type-4 P125109 strain), G1 97
(phage type-4) and BC8 (phage type-8) strains whereas LP group included C19 (phage type-98
13), C45 (phage type-13) and G45 (phage type-13a) strains. Frozen stocks of cultures, stored in 99
15% (v/v) glycerol at -80°C, were grown on Luria-Bertani (LB) agar for 16 h at 37°C. 100
101
Preparation of RNA samples: For all experiments, a single colony from the overnight culture 102
was inoculated into LB broth and grown at 42°C (normal body temperature of chicken) for 16 h 103
with shaking at 200 rpm. The overnight cultures were diluted 1:100 in fresh LB and incubated at 104
42°C for 4 h (exponential phase) with shaking at 180 rpm. Approximately 1 x 109 cfu of each 105
strain was pelleted (7500 rpm, 20°C, 15 min). There were no differences in the in vitro growth 106
kinetics of LP and HP strains at 42°C (data not shown). The cell pellets were processed for 107
total RNA extraction and DNase-treatment using RiboPure Bacterial Kit (Ambion, USA) 108
following the protocol supplied by the manufacturer. Total-RNA was extracted from three 109
replicates of each strain. Subsequently equal concentrations of the total-RNA samples obtained 110
from three replicates of each strain were mixed and subjected to second treatment with DNase 111
to remove any residual genomic DNA according to the protocols supplied with the RiboPure 112
Bacterial Kit. The pooled total-RNA sample from each strain was tested for gDNA contamination 113
by quantitative real-time PCR (qPCR) amplification of two genes, rpoD and sipA using following 114
primers: rpod_F 5’-acatgggtattcaggtaatggaaga-3’, rpo_R, 5’-cggtgctggttggtattttca-3’, sipA_F 5’-115
ttttaacgcctcagcgtctt-3’, sipA_R 5’-cagagaaagtgccacaacga-3’. The qPCR was performed using 116
SsoFast EvaGreen Supermix as per the manufacturer’s instructions (Bio-Rad) using the iQ5 117
iCycler (Bio-Rad, USA). Each RNA sample was tested in duplicate. All samples showed Ct 118
values >30 for both rpoD and sipA, indicating negligible levels of DNA contamination. As a final 119
step, RNA integrity was assessed using the 2100 Bioanalzyer (Agilent, Foster City, CA). The 120
total-RNA samples were stored at -80°C until further use. 121
122
RNA-Seq: The total-RNA samples were submitted to Illumina Inc, San Diego, CA, for mRNA 123
enrichment and subsequent RNA-Seq. Removal of 16S and 23S rRNA from total RNA was 124
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performed using a duplex-specific nuclease (DSN) treatment (34). The mRNA was used to 125
prepare individually barcoded (indexed) RNA-Seq libraries with the TruSeq RNA Sample Prep 126
kit (Illumina, USA). Six RNA-Seq libraries prepared from S. Enteritidis strains were sequenced 127
in one lane on HiSeq2000 instrument (Illumina Inc, USA) along with the six other unrelated 128
RNA-Seq libraries resulting in a total of 12 libraries per lane using version 3 chemistry and 129
reads were base called and quality filtered with CASAVA1.8 pipeline (Illumina Inc) to generate 130
75-bp reads. All raw data have been submitted to Geo (Accession number GSE46391). The 131
genome sequence and functional annotation information of S. Enteritidis were obtained from the 132
NCBI database (GenBank accession number AM933172). Casava 1.8 quality-filtered reads 133
were aligned to the reference genome sequence using CLC Genomics Workbench version 5.0 134
(CLC bio, USA). Mapping was based on the minimal length of 75 bp with an allowance of up to 135
two mismatches and >90% of the each read's length had to map to the reference sequence to 136
consider it as a mapped read. After mapping with CLC Workbench, total raw read counts for 137
each gene were generated. These read counts were used for further statistical analysis to 138
determine differentially expressed genes as described below. 139
140
Experimental design and data analysis: It is recommended that for RNA-Seq based 141
comparative transcritome analysis, the experimental design should include ≥3 biological 142
replicates per treatment group (35). The primary objective of this study was to identify genes 143
that are consistently differentially expressed in all LP strains when compared with all HP strains. 144
The experimental design included three wild-type S. Enteritidis strains in each treatment group: 145
HP (UK, G1 and BC8) and LP (C19, C45 and G45). Therefore, for the purpose of this study, the 146
individual strains within each group served as an independent biological replicate for that group. 147
Biological replicate within HP and LP groups demonstrated a >0.86 and >0.92 correlation 148
(Spearman rank, P <0.01), respectively when RPKM (reads mapping to the genome per 149
kilobase of transcript per million reads sequenced) normalized values were compared, 150
indicating high reproducibility of replicates. For the subsequent statistical analysis, the total raw 151
read count data for the six S. Enteritidis strains were used. Because of the high variability 152
between methods for determining differential expression using RNA-Seq data, it has been 153
recommended that more than one bioinformatics tool be used to ensure that the conservative 154
list of differentially expressed genes is obtained (36). Therefore, two well-established statistical 155
methods (DESeq and edgeR) were employed to analyze RNA-Seq data using ‘R’ software 156
(version 2.1.5.2) (37). As a first step, any gene that had zero mapped reads for all six samples 157
was removed resulting in 4418 genes mapped by CLC bio out of 4420 genes comprising the S. 158
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Enteritidis transcriptome (supplementary file 1). For DESeq analysis, the differential expression 159
testing was performed by use of the negative binomial distribution and a shrinkage estimator for 160
the distribution's variance and size-factor normalized data (38). Differential expression analysis 161
was also performed using edgeR, which also employs a negative binomial distribution-based 162
method (39-41). For edgeR analysis, the trimmed mean of the M values method (TMM; where 163
M= log2 fold change) was used to calculate the normalization factor and quantile-adjusted 164
conditional maximum likelihood (qCML) method for estimating dispersions was used to calculate 165
expression differences using an exact test with a negative binomial distribution (39-41). These 166
analyses were performed independently using the same mapping file (supplementary file 1). 167
Finally the list of genes obtained by DESeq and edgeR method was intersected to identify 168
common genes determined to be differentially expressed by both methods. The genes that were 169
determined to be significantly regulated by only one statistical method were not considered 170
further (36). 171
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Results and Discussion 173
The wild-type strains of S. Enteritidis differ in their virulence potential and other phenotypic 174
characteristics such as motility, biofilm production, survival in egg-albumen, tolerance acidic and 175
oxidative stress (16, 19-27, 30-33, 42). To explore if a genetic underpinning for the strain 176
differences that were previously characterized as high-pathogenic (HP) and low-pathogenic (LP) 177
reside within their global transcriptional signatures, a comparative transcriptomic analysis of 178
multiple well-characterized HP and LP strains of S. Enteritidis was performed using RNA-Seq. 179
In RNA-Seq analysis, sequence depth is one of the important factors that influence downstream 180
differential gene expression data analysis. For instance, Hass et al (43) showed that a 181
sequencing depth of 5-10 million non-rRNA fragments is needed for optimal profiling of vast 182
majority of transcriptional activity in diverse bacterial species such as E. coli, Mycobacterium 183
tuberculosis, Vibrio cholerae grown under diverse culture conditions (43). In this study, 184
alignment of the sequence reads to the S. Enteritidis P125109 genome (44) yielded 17.3 m 185
(BC8), 18.1 m (G1), 18.5 m (UK), 18.7 m (G45), 20.9 m (C19) and 21.4 m (C45) total mapped 186
reads, respectively. The total number of non-rRNA and non-tRNA reads that mapped uniquely 187
to the reference genome ranged from 13.8 m (G1), 15.8 m (UK), 16.4 m(BC8), 17.5 m (G45), 188
20.4 m (C45 and C19), respectively. Therefore the sequencing depth obtained in this study 189
should provide optimal coverage of the S. Enteritidis transcriptome. 190
191
As a first step in the data analyses, any genes that had zero mapped reads for all six strains 192
were removed, resulting in mapping of 4418 out of the 4420 genes comprising the S. Enteritidis 193
reference genome (44), indicating >99% coverage of the whole transcriptome of S. Enteritidis 194
(supplementary file 1). For differential expression analysis between HP and LP strains, two 195
statistical methods, edgeR (39-41, 45) and DEseq (38) were employed. Both methods are 196
based on the negative binomial distribution and have been widely accepted for modeling the 197
variation inherent between biological replicates. The DESeq method identified a total of 298 198
differentially expressed genes that showed ≥2-fold differences in the transcript abundance 199
between HP and LP group with a P value <0.05 after adjusting for multiple testing for each gene 200
(padj). In contrast, edgeR method identified 498 differentially expressed genes with ≥2-fold 201
differences in the transcript abundance between HP and LP group with a ≤5% false discovery 202
rate (FDR) and a P<0.05. From the above list of genes, all the genes from �SE20 (SEN1919A–203
SEN1966) were removed because this phage is present in S. Enteritidis PT-4 strains but absent 204
from non-PT4 strains. This resulted in the total of 253 (DESeq) and 448 (edgeR) genes that 205
were differentially expressed between HP and LP strains. Because edgeR method identified 206
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196 additional genes representing approximately 43.5% of the significant genes, a single 207
optimized dataset was constructed by integrating the results analyzed separately by DESeq and 208
edgeR (36). This resulted in selection of overlapping gene list with a total of 252 genes that 209
were identified as differentially expressed between HP and LP group by both methods 210
(supplementary files 2 and 3). As expected, this approach resulted in elimination of 197 genes 211
that were identified as differentially expressed only by one method (supplementary file 4). For 212
this set of 197 genes, the log2 fold change values and the level of significance (P value) 213
obtained by both edgeR and DESeq were compared. Interestingly, 96% (189/197) of the genes, 214
that were originally eliminated from the analysis, showed ≥2-fold differences in transcript 215
abundance between HP and LP groups when analyzed by both methods (Fig. 1A, 216
supplementary file 5). EdgeR method revealed that these differences were statistically 217
significant with a ≤5% FDR and a p<0.05 (Fig. 1B, supplementary file 5). In contrast, none of 218
these differences achieved statistical significance (p ≥0.05) when analyzed using DESeq 219
method (Fig. 1B, supplementary file 5). Because of the discrepancy between the two statistical 220
methods this set of 197 genes was not considered for further analysis. However, it is possible 221
that some of these genes are likely to be potentially differentially expressed and therefore this 222
gene list is included (supplementary file 4). 223
224
Differentially expressed genes between LP and HP strains: The majority of differentially 225
expressed genes (93.2% or 235 out of 252 genes) showed significantly reduced expression in 226
all LP strains as compared with all HP strains (supplementary file 2). According to their cluster 227
of orthologous groups (COGs), these low-expressed genes were classified into seven broad 228
functional categories including genes encoding Salmonella pathogenicity islands (n=32), cellular 229
processes and signaling (n=65), metabolism (n=37), information storage and processing (n=13), 230
fimbriae (n=4), pseudogenes (n=5) and several functionally uncharacterized or poorly 231
characterized genes (n=79) (Fig 1). In contrast, only 17 (6.8%) genes showed significantly 232
increased expression in all LP strains when compared with all HP strains (supplementary file 3) 233
including genes involved in metabolism (n=10), cellular processes and signaling (n=4) and gene 234
with unknown functions (n=3). 235
236
Virulence-associated genes: SPI-1 encodes a type-3 secretion system (T3SS) that plays an 237
important role in invasion of intestinal epithelial cells, translocation of effector proteins in the 238
host cells, and induction of enteropathogenesis caused by Salmonella (46, 47). RNA-Seq 239
analysis revealed that all of the 38 genes located on SPI-1 showed reduced expression in LP 240
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strains (Fig. 2A, supplementary file 5). Of these, 28 showed ≥2-fold reduction in transcript 241
abundance with a ≤5% FDR and a p<0.05 (supplementary file 2). The remaining 10 genes also 242
showed reduced expression, but the differences were not statistically significant. In addition, two 243
genes (spoE and spoE2) that encode effector proteins translocated by SPI-1 T3SS, but are 244
located elsewhere in the genome showed reduced expression. Finally, three low-expressed 245
genes (pipBC, sopB) were located on SPI-5, which reportedly contributes to the intestinal 246
pathogenesis in murine, bovine and avian models (48-50). 247
248
Among the non-SPI pathogenic factors that showed reduced expression in LP strains, two 249
genes (cydA and B) encode a putative cytochrome bd oxidase and ggt encoding け-glutamyl 250
transpeptidase. Cytochrome bd oxidase performs a variety of physiological functions in 251
prokaryotes such as energy-transducing respiration, aerotolerant nitrogen fixation, protection 252
against metal toxicity and oxidative stress (51-55). This enzyme has also been implicated in the 253
virulence of S. flexneri and several Salmonella serovars such as Typhimurium, Gallinarum and 254
Dublin (56), suggesting that cytochrome bd may be particularly important for the growth and 255
survival of pathogens that encounter environments in which O2 is progressively limited. GGT 256
(EC 2.3.2.2) is reported to contribute to the virulence of H. pylori in mice (57, 58) and has been 257
implicated in inhibition of T-cell proliferation and mediation of cell apoptosis (59, 60). Another 258
down-regulated gene, yncD, encodes TonB-dependent transporters and was recently identified 259
as an in vivo induced antigen contributing to S. Typhi virulence in a murine model (61). It is 260
important to note that all of the LP strains used in this study exhibited reduced invasiveness in 261
the cultured human intestinal cells and reduced virulence in murine and avian model of infection 262
(19, 21). Therefore reduced expression of SPI-1, SPI-5 and other non-SPI related genes in LP 263
strains likely explains the mechanisms underlying their virulence attenuation. 264
265
Motility-associated genes: The flagellar regulation of Salmonella includes more than 50 genes 266
divided into three classes, class-1 (early genes), class-2 (middle genes) and class-3 (late 267
genes), according to their temporal expression after induction of the flagellar regulon (62, 63). 268
Motility and flagella are also important to gastrointestinal disease caused by Salmonella in 269
avian, rodent and bovine models (64-66). Additionally, in vitro studies using cultured epithelial 270
cells show that motility impaired S. Enteritidis mutants are defective in entering intestinal 271
epithelial cells (67-69). Interestingly, RNA-Seq analysis revealed that, irrespective of the class, 272
the vast majority of flagellar genes (n=51) showed significantly reduced expression in all LP 273
strains tested (supplementary file 2). These included the majority of genes involved in flagellar 274
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assembly (Fig. 2B, supplementary file 5) and several genes encoding known or putative methyl 275
chemotaxis proteins such as SEN2995, SEN3058, SEN1374 and SEN2296 (supplementary file 276
2). In addition, other genes with ties to motility included yhjH and ycgR, which may encode 277
novel flagellar components given their ability to rescue motility defects of an H-NS mutant (70-278
72). Recent studies have revealed that several field strains of S. Enteritidis either express 279
paralyzed or a unipolar flagellum or may fail to express flagella resulting in motility impaired 280
strains that may also be virulence attenuated (21-23, 42). All of the LP strains of S. Enteritidis 281
used in this study represent a population of such naturally occurring, motility-impaired 282
phenotypes (19, 21). The motility impairment might be due to the impaired ability to secrete 283
flagellar proteins such as FljB, FlgK and FlgL (21). In addition, others have shown that a point 284
mutation (T551 s G) in motA, a gene essential for flagellar rotation may also result in naturally 285
induced motility impairment in field isolates (22). Therefore, reduced expression of entire 286
flagellar gene clusters in LP strains corroborate with previously reported motility impaired 287
phenotype among these strains and suggests that flagellar regulation is significantly impaired in 288
LP strains. 289
290
Fimbrial genes: S. Enteritidis harbors 13 fimbrial gene clusters (44). In this study, three fimbrial 291
genes (bcfA, safA and csgC) showed significantly reduced expression in LP strains. In S. 292
Typhimurium, saf fimbriae encoded on SPI-6 has been implicated in porcine ileal colonization 293
and virulence in mice (73, 74). The role of saf fimbriae in S. Enteritidis pathogenesis is not 294
known, but the safA gene in S. Enteritidis shows 81% nucleotide identity with S. Typhimurium 295
(44), suggesting that it may have similar functions. S. Typhimurium csg is important for biofim 296
formation on chicken intestinal mucosa cultured ex vivo (75). In contrast, S. Enteritidis csg (curli) 297
fimbriae has been implicated in egg contamination (76) and may also contribute to human and 298
avian epithelial cell-invasiveness (69). In addition to these known fimbrial genes, hopD gene 299
encoding putative bifunctional pre-pilin peptidase HopD showed reduced expression in LP 300
strains. The amino acid sequence of HopD showed 60% similarity with PilD peptidase protein of 301
Pseudomonas stutzeri DSM 4166. Functional PilD is required for extracellular secretion 302
(excretion) of several virulence-associated proteins such as exotoxin A, phospholipase C, and 303
elastase production in P. aeruginosa (77, 78). While the exact function of hopD in S. Enteritidis 304
has not been characterized, it would be of interest to determine whether hopD also impacts 305
secretion of virulence factors in Salmonella. 306
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Transcriptional regulators: Several known or putative transcriptional regulators showed 308
reduced expression in LP strains. These included ygaE, yncC, sdiA, ydcI, ecnR, SEN4085, 309
SEN4086, yiaG and SEN1787 (supplementary file 2). YgaE (also known as GabC) and YncC 310
belong to the GntR family of transcriptional regulators. This family of regulators has a conserved 311
N-terminal DNA binding domain and a diverse C-terminal domain involved in the effector 312
binding and/or oligomerization (79, 80). In both S. Enteritidis and E. coli, ygaE is located in the 313
gabDTPC operon. In E. coli the products of gab operon are involved in degradation of け-314
aminobutyrate (GABA) and contribute to polyamine (putrescine, spermidine, and spermine) 315
homeostasis during nitrogen-limited growth (81) and also maintain high internal glutamate 316
concentrations under stress conditions (82). Expression of the gab operon is enhanced at high 317
pH (83) and at high cell density in nitrogen-rich environments (84-86). While the exact role of 318
ygaE in S. Enteritidis has not been investigated, the simultaneous reduced expression of two 319
additional genes such as gabP (a GABA-specific permease) and patA (a putrescine-2-320
oxoglutarate aminotransferase) raises a possibility that the polyamine homeostasis in LP strains 321
may be disregulated, potentially making these strains inherently more susceptible to 322
environmental stressors. 323
324
Four genes with significantly reduced expression in LP strains (sdiA, ydcI, ecnR and SEN4085) 325
belong to LysR family of transcriptional regulators (LTTRs). Despite considerable structural and 326
functional conservation, LTTRs are known to regulate diverse set of genes, including 327
conjugation, bioluminescence, metabolism, motility and virulence gene expression (87, 88). For 328
instance, sdiA is a positive activator of ftsQAZ genes that are essential for septation and its 329
amplification results in diverse genetic effects in E. coli including mitomycin C resistance, down-330
regulation of several motility and chemotaxis related genes and up-regulation of genes involved 331
in DNA repair and replication (89). Similarly, when S. Typhimurium is grown in motility medium, 332
sdiA regulates expression of virulence plasmid-associated genes (90), Interestingly, sdiA is also 333
dually controlled by iron concentration and culture-density-derived signals and the deletion of 334
helix-turn-helix (HTH) domain of sdiA results in increased virulence of S. Typhimurium in murine 335
model of infection (91), suggesting that sdiA contributes to diverse genetic effects with possible 336
links with motility, bacterial cell septation, response to DNA-damaging agents, and virulence. 337
Similar to sdiA, ecnR is linked to motility because it negatively regulates flhDC transcription and 338
affects bacterial motility; however, the role of ydcI and SEN4085 in S. Enteritidis has not been 339
established. 340
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Apart from LTTRs, SEN4086, which belongs to the family of AraC transcriptional regulators 342
showed significantly reduced expression in LP strains. AraC transcriptional regulators control 343
diverse bacterial functions including sugar catabolism, responses to stress, and virulence (92). 344
For example certain well-characterized AraC transcriptional regulators such as rstA and hilD 345
control expression of the invasion genes in S.Typhimurium (93, 94). Finally, YiaG is a putative 346
HTH type of transcriptional regulator that binds to DNA and regulates gene expression whereas 347
SEN1787 is a putative LuxR-type DNA-binding HTH domain that is activated by several different 348
mechanisms including a two-component sensory transduction system. The role of EN4086, 349
yiaG and SEN1787 in the transcriptional regulation of S. Enteritidis is currently unknown 350
although their reduced expression in all LP strains raises a possibility that these genes may play 351
a role in the regulation of virulence or they are stress-associated. 352
353
Stress-associated and iron-regulated genes: Among other genes with significantly reduced 354
expression in LP strains, many genes (yeaG, yncC, rpsV, osmE, yiaG, otsA, fbaB, talA, poxB, 355
yehY, dps, sodC, katE, bfr, wraB, yddX, ygaU, yibJ, psiF, yciF and osmY) are regulated by the 356
alternative sigma factor RpoS, which is not only required for survival of bacteria under starvation 357
or other cellular stresses, but it is also essential for Salmonella virulence (95-97). It is also 358
suggested that Salmonella lacks ectoine biosynthetic pathway and therefore cannot tolerate 359
environments with high osmolarity (98). While the functions of several of these genes have not 360
been fully characterized, a few genes such as osmY, osmC, osmE, dps, katE, talA, yciF, ygaU, 361
yjbJ, poxB, otsB, tktB, yciE, acnA and yhbO are known to be induced when E. coli is subjected 362
to osmotic stress conditions in an aerobic milieu (99-101). Additionally, two genes, SEN1557 363
and yehZ, belong to ABC superfamily of binding proteins potentially involved in glycine betaine 364
choline transport required for osmoprotection. Under stress conditions, if glycine betaine cannot 365
be imported, Salmonella produces the disaccharide trehalose, a highly effective compatible 366
solute. It has been reported that mutants defective in trehalose synthesis display an impaired 367
osmotic tolerance in minimal growth media without glycine betaine, and an impaired stationary-368
phase-induced heat tolerance (102). Trehalose accumulation also increases bacterial resistance 369
to stress such as high-salt, low-pH and -hydrogen peroxide, conditions that mimic aspects of 370
innate immunity (102, 103). Interestingly, treA needed for trehalose utilization at high osmolarity 371
showed significantly reduced expression in LP strains. In addition, the genes encoding 372
trehalose-6-phosphate synthase (otsA) and anabolic trehalose-6-phosphate phosphatase (otsB) 373
also showed reduced expression. The expression of these genes is induced both by osmotic 374
stress and by growth into the stationary phase (85). Similarly, proP encodes proline/glycine 375
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betaine transporter and protects Salmonella from inhibitory effects of high salinity, and was 376
down-regulated (98). Finally, two RpoS regulated genes, rpsV and yddX, appear to be a part of 377
seven gene operon (rpsV-yddX-osmC-SEN1494-SEN1497), all of which are transcribed in the 378
same direction. Six genes in this operon showed reduced expression in LP strains 379
(supplementary file 2). While the function of this operon is not well-characterized, osmC appears 380
to be an acid and osmotic stress response gene (99), suggesting possible link to osmotic stress. 381
Overall, significantly reduced expression of several osmotically inducible genes suggests that 382
the LP strains may have reduced tolerance to high osmolarity. 383
384
Another host defense mechanism that Salmonella encounters during infection is the production 385
of reactive oxygen species such as hydrogen peroxide (H2O2) by the phagosome NADPH 386
oxidase. Hydrogen peroxide can diffuse across bacterial membranes and damage 387
biomolecules. Several genes that play an important role in protection of microbial pathogens 388
against oxidative stress showed reduced expression in LP strains. These included katE that 389
encodes a catalase enzyme required for degradation of H2O2, and sodC that encodes a 390
periplasmic copper and zinc superoxide dismutase that protects bacteria from phagocytic 391
oxidative burst (104). Another low-expressed gene, gth (encoding glutathione S-transferase, 392
GST) not only affords protection against oxidative stress, but also plays a role in correct folding, 393
synthesis, regulation, and degradation of enzymes and multi-enzyme complexes in a large 394
number of metabolic processes (105-107). 395
396
Several genes involved in iron-regulation also showed reduced expression in LP strains. For 397
instance, the entire sufABCDSE operon, which plays a vital role in Fe-S cluster biogenesis, 398
showed reduced expression. The iron-sulfur (Fe-S) clusters are key metal cofactors of 399
metabolic, regulatory, and stress response proteins in most organisms and their assembly and 400
maturation in vivo require complex machineries. It has been reported that in E. coli, SUF system 401
is induced under adverse stress conditions such as oxidative stress or iron deprivation (108) 402
whereas in S. Typhimurium, chlorine-based oxidative stress induces expression of the SUF 403
operon (109). S. Typhimurium also possesses four ferritins: bacterioferritin (Bfr), ferritin A 404
(FtnA), ferritin B (FtnB) and DNA starvation/stationary phase protection protein (Dps). We found 405
that two ferritin genes (bfr and dps) were down-regulated in LP strains. The haem containing Bfr 406
is maximally expressed when Fe is abundant and accounts for the majority of stored Fe (110). 407
While inactivation of bfr elevates the intracellular free Fe concentration and enhances 408
susceptibility to H2O2 stress, the DNA-binding protein Dps provides protection from oxidative 409
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damage without affecting the steady-state intracellular free Fe concentration and it is also 410
known to be induced during oxidative stress in Burkholderia psuedomallei thereby protecting 411
this pathogen from organic hydroperoxides and aiding its intra-macrophage survival (111, 112). 412
Taken together, the reduced expression of several genes involved in resistance of Salmonella 413
to osmotic and oxidative stresses and iron-limiting conditions corroborate with the impaired 414
survival of LP strains under oxidative and acidic stress as well as reduced intra-macrophage 415
survival (19). These results suggest that LP strains are impaired in their ability to resist host 416
innate immune responses or propagate in the iron-limiting conditions encountered early during 417
infection. For example, host intestinal milieu represents an environment where Salmonella are 418
exposed to high osmolarity whereas intra-macrophage survival of Salmonella is partly 419
dependent on the ability of this bacterium to resist oxidative stress. Further studies involving 420
targeted mutations of genes with possible ties to osmotic or oxidative stress and their effects on 421
kinetics of intestinal colonization or intra-macrophage survival of S. Enteritidis may provide 422
better understanding of the colonization potential and persistence of these strains within the 423
host. 424
425
Genes involved in biofilm production and egg-contamination: Biofilm formation is important 426
for the survival of Salmonella on surfaces for increased resistance to disinfectants, and for 427
survival in the avian reproductive tract (113, 114). Several genes in S. Enteritidis genome 428
provide this bacterium a unique ability to infect reproductive organs of chickens and 429
contaminate forming eggs (115-118). Two genes (wrbA and yshA) with possible involvement 430
with biofilm formation (113, 119) and two genes (ygdl and SEN2997) that were previously 431
implicated in the survival of S. Enteritidis in the egg-albumen (116) showed significantly reduced 432
expression in LP strains. It is important to note that irrespective of their virulence potential, not 433
all S. Enteritidis strains produce biofilm or are able to survive within the egg-albumen (19, 21). 434
This is consistent with the relatively fewer differences in the expression of genes associated 435
with these phenotypes. 436
437
Functionally uncharacterized genes: Several genes (n=79) encoding functionally 438
uncharacterized proteins or proteins with putatively assigned functions showed significantly 439
reduced expression in LP strains (Fig 1). While few of these genes have been previously 440
identified as stress-regulated (supplementary file 2), the functions of most others remain elusive. 441
Of particular interest are genes encoding putative lipoproteins (SEN0081 yfbK and ygdI). 442
Bacterial lipoproteins perform various roles, including nutrient uptake, signal transduction, 443
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adhesion, conjugation, and sporulation, and participate in antibiotic resistance, transport (such 444
as ABC transporter systems), and extracytoplasmic folding of proteins (120). In the case of 445
pathogens, lipoproteins play a direct role in virulence-associated functions, such as colonization, 446
invasion, evasion of host defense, and immunomodulation (120). Therefore, it is possible that 447
the putative lipoprotein encoding genes identified here may be important in virulence or survival 448
of S. Enteritidis. Interestingly, five pseudogenes (SEN0912, SEN1154, SEN1171A, SEN1171B 449
and SEN1809) showed reduced expression in LP strains. The role of these pseudogenes in the 450
pathogenesis of Salmonella is not clearly understood, although a transposon mediated mutation 451
in pseudogenes such as SEN1154 and siiE results in attenuation in cell-invasiveness of S. 452
Enteritidis (69). In addition, others have shown that several pseudogenes are transcriptionally 453
active in S. Typhi (121), suggesting their potential misclassification as these genes in fact play a 454
role in the pathobiology of Salmonella. 455
456
Genes involved in nutrient metabolism show increased expression in LP strains: 457
Seventeen genes showed significantly increased expression in LP strains (supplementary file 458
3). These included all the genes encoded on polycistronic tdcABCDEFG operon which, under 459
anaerobic condition, are implicated in degradation of L-serine and L-threonine to acetate and 460
propionate, respectively (122). The regulatory gene tdcA, which encodes a protein homologous 461
to the LTTRs, is required for maximal induction of the tdc operon (123) and also regulates 462
expression of several genes including 50S ribosomal subunit proteins and a lipoprotein that 463
links inner and outer membranes (124) and is involved in the virulence of S. Typhimurium (125, 464
126). Interestingly, two genes (rpsC and rplV) encoding ribosomal subunit proteins and one 465
gene (SEN1797) encoding a lipoprotein were up-regulated in LP strains with potential ties to 466
tdcA expression. Finally, several genes encoding enzymes of the D-glucarate (gudP, ygcY, 467
gudD) and D-glycerate (garDLRK) pathways showed increased expression in LP strains. 468
Interestingly, genes of garDLRK operon are located immediately down-stream of tdcABCDEG 469
operon in S. Enteritidis genome (44). Exposure to H2 in S. Typhimurium leads to significant up-470
regulation of the gar and gud genes and down-regulation of several virulence-associated genes 471
(127). Hydrogen can be an important energy source for bacteria growing in an environment 472
where high-energy organic substrates are limiting (128). For instance, addition of H2 significantly 473
augments the growth of S. Typhimurium in a culture medium containing amino acids as the only 474
carbon source mainly due to the enhanced ability of the bacteria to acquire amino acids from 475
the medium (127). Therefore, the availability of H2 in a nutrient-limited condition such as the 476
competitive environment within the host intestinal tract could be instrumental to the survival of 477
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Salmonella. While the LP strains in this study were not grown in a nutrient-limited condition or 478
under anaerobic conditions, the fact that LP strains show increased expression of tdc, gar and 479
gud operons and reduced expression of several virulence-associated genes suggests that the 480
primary focus of these strains could be survival and cell growth through enhanced nutrient 481
acquisition rather than invasion and proliferation. 482
483
Conclusions: Using comparative RNA-Seq analysis of multiple field strains of S. Enteritidis with 484
varying phenotypic and virulence properties, this study demonstrates that the LP strains have 485
significantly impaired expression of virulence, motility and stress-associated genes. The study 486
design included a subset of low-pathogenic strains of S. Enteritidis with naturally impaired 487
motility, reduced resistance to oxidative and acidic stresses and virulence attenuation. Although 488
the strains were grown in synthetic culture media within the laboratory settings, overall the 489
transcriptional signatures of LP and HP strains grown at avian body temperature (42°C) 490
corroborated well with their phenotypic characteristics suggesting that RNA-Seq provided a 491
better understanding of the potential mechanisms underlying their differential phenotypic 492
characteristics and virulence potential. The reduced expression of several genes with 493
uncharacterized functions and transcriptional regulators demonstrates the complexity of 494
regulatory network in S. Enteritidis and may be further studied using diverse conditions that 495
better simulate in vivo infection kinetics. Differential regulation of several known and putatively 496
identified transcriptional regulators is intriguing because these genes could be involved in 497
virulence regulation or stress-response and/or environmental persistence of this important food-498
borne pathogen. For instance, LTTRs represent the most abundant type of transcriptional 499
regulator in the prokaryotic kingdom (87) and because of their abundance in the diverse variety 500
of bacterial species and relatively conserved characteristics, the detailed transcriptomic studies 501
of strains with a defined genetic background of different LTTRs under culture conditions that 502
mimic in vivo infection processes may facilitate identification of novel virulence attenuated 503
strains with the potential for use as a vaccine candidate. 504
505
While field strains of S. Enteritidis with naturally impaired motility, reduced resistance to 506
oxidative and acidic stresses and virulence attenuation have been commonly isolated (19, 21-507
23), their significance to the epidemiology of S. Enteritidis in poultry and people remains elusive. 508
It is possible that these strains may have impaired ability to infect chickens or contaminate eggs 509
or may have reduced ability to persist in the poultry environment. Nevertheless occurrence of 510
such strains in poultry or poultry-associated environment raises a possibility that these strains 511
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may represent a unique subpopulation that is adapted to a unique niche leading to intermittent 512
infection of the flock or transmission to human with a relatively mild disease that remains largely 513
unnoticed or undiagnosed. The whole genome sequences of majority of strains used in this 514
study are not available and therefore it is currently unknown if these strains also carry other 515
genomic differences such as SNPs which may potentially alter some of the phenotypes or 516
expression differences observed in this study. In the future, comprehensive phenotypic and 517
genotypic characterization of a larger set of wild-type strains of S. Enteritidis may provide some 518
clues to the epidemiological significance of these low-pathogenic strains of S. Enteritidis. 519
520
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Acknowledgements 521
Carol Casavant provided technical assistance. Dr. Douglas R. Call critically reviewed the rough 522
drafts of the manuscript. The funding for this work was provided by the Agricultural Animal 523
Health Program, College of Veterinary Medicine, Washington State University. 524
525
Availability of supporting data 526
The data sets supporting the results of this article are included within the article and its 527
additional files as follows. The raw datasets supporting the results of this article are available in 528
the Geo repository, [accession number: GSE46391]. 529
530
531
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Figure legends: 532
533
Fig. 1. Summary of functional classes of genes down-regulated in low-pathogenic strains when 534
compared with the high-pathogenic strains of Salmonella Enteritidis. 535
536
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Table 1. Phenotypic characteristics of high-pathogenic and low-pathogenic strains of S. Enteritidis used in this study (19, 21). 537
538
Group Strains Phage type
MLVA type*
Caco-2 cell invasiveness
Survival or growth characteristics in Motility Biofilm
Avian macrophages
Acidic stress (pH 2.6)
Oxidative stress
Egg-albumen
LP
C19 PT13 11 Low Low Low Low No growth Low Negative
C45 PT13 11 Low Low Low Low No growth Low Negative
G45 PT13a 4a Low Low Low Low Growth Low Negative
HP
G1 PT4 13a Medium High High High Growth High Positive
UK PT4 13a High High High High Growth High Positive
BC8 PT8 9a High High High High Growth High Mixed
* MLVA, multi-locus variable-tandem repeat analysis 539
‘Low’ for each category indicates that the difference is statistically significant (P<0.05) when compared with ‘High’ 540
‘Mixed’ biofilm phenotype indicates that this strain has mixed subpopulation of both biofilm positive and negative 541
542
543
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