serotypes, including Salmonella enterica
serovar Typhimurium and Salmonella enterica
serovar Enteriditis, are food-borne pathogens, and important causes of bacterial enteric disease in both humans and livestock. Infection of livestock is a common source of transmission of Salmonella
into the human food supply, and therefore interventions decreasing infection in livestock species have the potential to reduce the burden of human infection [1
]. Salmonella enterica
is an antigenically diverse species with approximately 2500 serotypes [2
]. Multiple Salmonella
serotypes can circulate concurrently on farms or production facilities resulting in antigenically distinct challenges to a single animal [3
]. However exposure to one serotype does not result in significant cross-protection against heterologous serotypes [2
]. In addition to serotype diversity, exposure of Salmonella
to diverse microenvironments within the host serves as a cue to modulate its own gene transcription leading to differences in protein expression [5
]. These changes in pathogen-expressed proteins in response to environmental cues result in skewed immune responses toward serotype-specific proteins which, in turn, contribute to the lack of cross-protection [9
]. Therefore, strategies disrupting transcriptional shifts between microenvironments could result in sustained and increased production of conserved proteins providing the means for the adaptive immune system to induce efficient cross-protection against multiple serotypes.
Oral immunization of mice with a DNA adenine methyltransferase (Dam)-deficient S.
Typhimurium (UK-1 dam
mutant) confers significant protection against challenge with homologous S.
Typhimurium, as well as heterologous challenge with serotypes Dublin and Enteritidis [13
]. Subsequent studies showed similar results in other animal models, including cattle challenged with serotypes Dublin and Newport and chickens challenged with Enteritidis and strain O6, 14, 24 [14
]. The underlying mechanism of protection has not been elucidated, but is thought to be due to de-repression of genes leading to an expanded antigenic repertoire [13
]. It is known that mutation or removal of the dam
gene from Salmonella
causes de-repression of a number of genes under non-inducing culture conditions [19
]. Genes in the SOS regulon, as well as fimbrial, conjugal transfer, bacteriophage and virulence-related genes have been reported to be up-regulated in dam
Typhimurium strains [19
]. Importantly, proteins corresponding to de-repressed genes, such as the StdA fimbrial subunit, have been detected within the outer membrane and culture supernatant, which are otherwise not present in wild-type (wt
grown in identical conditions [19
]. While these studies support the hypothesis that deficiency of Dam alters gene transcription in non-inducing culture conditions; the host environment is complex and the extent of these transcriptional alterations and their stability in host-like microenvironments is not known.
In this study, we address the hypothesis that the UK-1 dam
mutant maintains higher, yet stable, gene transcription when compared with its isogenic wt
strain, UK-1, in different host-like microenvironments. We employed a deep sequencing transcriptomics approach (RNA-Seq) to analyze gene expression by the UK-1 dam
mutant in response to growth in laboratory media under high osmolarity (HSLB) to mimic the extracellular conditions in the intestinal lumen, and in acidic minimal media with low phosphate and low magnesium concentration (LPM) conditions found intracellularly within the Salmonella
containing vacuole [22
]. We identify stably expressed and highly transcribed genes in the UK-1 dam
mutant in comparison to the parental strain when transitioning from an extracellular to an intracellular environmental condition. These findings were supported with comparison of two additional, clinically relevant Salmonella
serotypes, Dublin and Newport, allowing us to identify a core gene repertoire whose transcription may play a role in inducing cross-protective immune response.
The mechanism of protection observed utilizing an S.
Typhimurium UK-1 dam
mutant as a live attenuated vaccine is thought to be due to de-repression of gene transcription, with subsequent alteration of protein expression patterns, creating a broad repertoire of antigens that are consistently present in a variety of physiologic conditions [13
]. While de-repression of numerous genes was previously observed, whole genome transcriptional profiling enabled us to detect the full complement of stable genes with higher relative transcription, in the UK-1 dam
mutant. Our analysis revealed a complement of genes with high relative transcription the UK-1 dam
mutant compared to the wt
parent strain that were stably transcribed across host-like microenvironments (Figure 3
). Utilizing the intra- and inter-strain comparisons, a subset of 94 genes, referred to as Gene Set A (Figure 3
, Table 1
), was identified as stably up-regulated by the UK-1 dam
mutant whereas the wt
parent strain had only seven genes falling into this subset which were primarily involved in basic cellular processes (Table 2
). The genes stably up-regulated by the UK-1 dam
mutant with potential immunologic significance fall into two categories: genes encoding proteins with predicted surface exposure and genes associated with the bacteriophage ST64B (Table 1
). Other genes from this subset are not likely involved in development of an immune response as they are either SOS regulon genes, genes involved in basic cellular processes and/or not predicted to be surface expressed.
The most obvious candidates for classic antigens of all identified genes include proteins encoded by the std
operon. The std
fimbrial operon is highly conserved among Salmonella
, and is present in all three human-adapted Salmonella
serotypes, Typhi and Paratyphi A and B, where it was first described [28
]. Std fimbriae are important in colonization of the bovine intestinal tract, as well as for long-term carriage in the mouse intestine [29
]. While the major fimbrial protein subunit had the highest relative transcription with a 2644-fold and 732-fold in HSLB and LPM media respectively in comparison to the wt
parent strain, it was excluded from Gene Set A as it was not stably transcribed, with a 3.18-fold increase in HSLB medium. However, high relative transcription in both culture conditions regardless of stability of the transcription supports further evaluation of this gene. The increased transcription of std
operon genes in the UK-1 dam
mutant noted here and in previous studies has been shown to be related to an increased protein production. High quantities of Std fimbrial protein, both on the cell surface and in the culture supernatant, have been detected in vitro
in a different strain of S.
Typhimurium with the dam
mutation suggesting protein expression is likely high in the UK-1 dam
mutant as both strains demonstrate high levels of transcription. [19
]. In vivo
, antibody responses have been detected in mice immunized with wild-type S.
Typhimurium, as well as to a combination of fimbrial proteins, including StdA, and a decrease in fecal shedding is noted in mice immunized with a fimbrial cocktail [31
]. The protein encoded by STMUK_3015, which is predicted to be another surface expressed member of the std
operon, has not been evaluated for antigenicity, but may prove to be a good target for future in vivo
studies. Comparison of the UK-1 dam
mutant to two other pathogenic Salmonella
serotypes identified one gene STMUK_3014, which is not predicted to be surface expressed, with higher relative transcription. Again, the stringency of our stable transcription analysis excluded stdA
, but the STMUK_3015 was also excluded as it was not present in S. Newport
. However, the high relative transcription of stdA
may still be important in cross-protection as this gene is present in both S.
Dublin and S.
Newport for reasons previously mentioned. As immune responses against an Std protein are associated with suppression of Salmonella
intestinal colonization, our evidence of increased transcription in multiple culture conditions and against multiple serotypes warrants further examination of proteins encoded by this operon [33
]. Lastly, in addition to its role as an antigen, it has also been shown that over-expression of Std fimbriae causes enhanced adherence to human colonic epithelial cells in culture, where proximity to resident dendritic cells and macrophages may facilitate antigen delivery at the mucosal surface [34
Three additional genes encoded proteins with predicted surface expression: STMUK_1011, a bacteriophage attachment and invasion protein, ydiM, a Lex-A regulated gene, and STMUK_2003, an ST64B bacteriophage portal gene. Little information is currently available for all three genes as to function or potential immunogenicity of their encoded proteins. Interestingly one gene, STMUK_1011, has higher relative transcription by the UK-1 dam mutant compared to the all wt strains and which does make it an attractive candidate for further characterization. The significance of these genes is unclear, but the transcriptional differences suggest further investigation is warranted.
We observed bacteriophage ST64B-associated genes to be consistently more highly transcribed in the UK-1 dam
mutant in comparison to the wt
parent strain in HSLB and LPM media, as well as S.
Dublin in HSLB medium. The ST64B bacteriophage is a mosaic structure incorporating different phage types, and is defective in morphogenesis, producing non-infectious tailless particles [35
]. Consistent with previous studies, the removal of the Dam enzyme de-represses prophage gene transcription, and in our study the ST64B prophage region represents the largest area of consistent expression in the UK-1 dam
mutant in both culture conditions [19
]. Not only were numerous ST64B genes up-regulated in the UK-1 dam
mutant in a previous study, but phage DNA and capsid can be found in the supernatant of the UK-1 dam
mutant bacterial cultures [36
]. Unlike the highly conserved std
operon, ST64B bacteriophage is not highly conserved in Salmonella
(it was absent from S.
Newport) and is therefore unlikely to provide cross-protective epitopes [36
]. However, ST64B may contribute to development of an immune response through immunomodulatory mechanisms. Use of bacteriophage particles in treatment of bacterial infection in humans has been described as able to modulate host immune responses [37
]. Bacteriophages are associated with normalization of inflammatory cytokines, specifically interleukin-6 and tumor necrosis factor-α, in human patients with chronic bacterial infection [38
]. There is evidence that the UK-1 dam
mutant does not induce cytokine production to the same magnitude as wild-type Salmonella
or other Salmonella
]. Bacteriophages are also shown to “prime” macrophages by increasing expression of molecules associated with antigen-presentation [41
]. A shift from M2-polarization to a more M1-polarizing milieu has also been shown, indicating the expression of phage proteins may shape a more effective immune response [41
]. Therefore, the genes encoding phage proteins could have multiple effects on the host immune response to Salmonella
proteins and promote more robust and effective protection.
Many other genes have been previously shown to be up-regulated in the UK-1 dam
mutant, most of which agree with our transcriptome data [19
]. However, a few inconsistencies with previous reports were detected. For example, spvB
was previously shown to be up-regulated in the UK-1 dam
mutant, however it is more highly transcribed in the wt
parent strain in our analysis [21
]. This likely reflects differences in media composition and incubation time utilized in different studies. In addition, because we have focused on stable genes in different environmental conditions with high relative transcription in the UK-1 dam
mutant we may have overlooked genes previously studied that did not fit our specific criteria, but which still may be biologically relevant. The wealth of data obtained from this type of sequencing platform calls for targeted analyses to begin to make meaningful conclusions, which does not preclude future analyses of other gene transcription changes.