How does salmonella typhimurium move

Typhimurium in host cell for up to at least 7 days in our tested condition is also striking as the bacteria inside this vacuolar compartment are likely to have restricted access to the extracellular nutrients. A Schematic diagram of S. Typhimurium lifestyles and the regulatory role of SpoT on S. Typhimurium dormancy in human epithelial cells. Typhimurium can opt for three distinct lifestyles: cytosolic, vacuolar and dormant, which exhibits discernible subcellular localization, replication rate and metabolism.

The entry of dormant state is negatively regulated by p ppGpp synthatase SpoT, while the regulatory mechanism on the dormancy exit remains to be determined.

B Schematic diagram of S. Typhimurium infection progression in the gut epithelium. Typhimurium reaches the intestinal epithelium, a portion of S. Typhimurium expresses T3SS1 purple to enter host cells and adopts various intracellular lifestyles.

Distinct S. Typhimurium lifestyles support rapid tissue colonization and gut inflammation to increase competitiveness of luminal S. Typhimurium red. Top Reactivation of dormant S. Typhimurium leads to prolonged gut inflammation that supports the continuous growth of S.

Typhimurium at gut lumen. Bottom Dormant S. Typhimurium reactivates after the eradication of gut S. Typhimurium, which serves as the reservoirs of infection relapse.

Typhimurium has been reported previously to enter dormant or persistent states within a modified SCV from a range of host cell types, including macrophages and fibroblasts [ 12 , 13 ]. Considering the presumably identical S. Typhimurium dormancy observed across the different cell models, there are substantial distinctions among the targeted host cell types in terms of the detection approaches and bacterial physiology. The first S. Typhimurium antibiotics-tolerant persisters were identified in macrophages using a dilution reporter on non-replicating S.

Typhimurium, which enters dormancy and a viable-but-not-cultivable state upon entry [ 13 ]. The dormancy is regulated by the TA system toxin, TacT that halts protein translation and induces antibiotic persistence, where the S. Typhimurium subsequently exits dormancy and activates SPI-2 [ 16 , 29 ]. Slow growing S. Typhimurium were also identified in fibroblasts as early as , where 0. Studies in fibroblasts have deciphered major genetic determinants of bacterial persistence, however the underlying mechanisms of persistence with regards to bacterial viability, antibiotics, the pathological implication have been characterized in more depth in macrophages recently [ 11 , 42 ].

In the different infected host cells intracellular S. In fibroblasts, the SCV subsequently interacts with host aggrephagy, where the majority of the S. Typhimurium residing in the SCV are eradicated whereas the remaining S.

Typhimurium were proposed to persist [ 11 ]. In epithelial cells, the reported S. Typhimurium dormancy by us is distinct from that in fibroblast and macrophage. They are different in the commence of dormancy, the capacity to replicate and the SCV microenvironment [ 13 ]. The distinct niches of dormant S.

Typhimurium may reflect cell-type specific vesicular trafficking, for example SPI-2 expression level and SCV maturation in these different target cells is not identical S4 , S8 and S9 Figs. It could also be possible that the way of entry impacts the development of dormant S. Epithelial dormant S.

Typhimurium is independent of Lon protease and is negatively regulated by SpoT, contrasting to that in macrophage that requires Lon protease, p ppGpp synthases RelA and SpoT [ 14 , 38 ]. The substantial difference between the S. Typhimurium dormancy sheds light on their potentially diverge pathophysiological implications as well as the molecular cue and mechanism that signal the establishment and exit of dormancy.

The extensive work on TA systems and S. Typhimurium physiology in fibroblasts would serve as significant groundwork for the further studies of enterocyte-borne dormant S. Typhimurium [ 42 ]. It will be interesting to investigate why these regulatory modules are differentially involved in the formation of dormant S. Typhimurium in the different cell types, and whether the distinct niche impacts their expression and implication. The p ppGpp-mediated stringent response has been closely associated with antibiotics persistence via inhibition of protein synthesis and transcription reprogramming [ 43 — 45 ].

The persistent S. In non-pathogenic E. Typhimurium models, the loss of Lon and its downstream regulated TA systems leads to a diminished antibiotics persister formation due to the inactivity of toxins [ 46 ]. Our finding contrasts the current understanding on the role of stringent response on bacterial persistence, where stringent response is activated by various stress signals and p ppGpp synthesis would act on its molecular target to achieve persistence.

Therefore, we suggest that bifunctional SpoT is required while monofunctional RelA is dispensable in S. Typhimurium dormancy in enterocytes, which echoes the previous report on the requirement of SpoT but not RelA in S.

Typhimurium invasion and colonization of an in vivo model [ 39 ]. Typhimurium dormancy could suggest that either or both the p ppGpp hydrolysis and synthase function is required, or rel A is not expressed during the course of infection.

Considering that S. Typhimurium dormancy is independent of DksA and Lon, it implies that dormancy is likely to be mediated by pathways independent of DksA transcription reprogramming and Lon protease-mediated degradation. As dormant S. Typhimurium do not co-exist with S. Typhimurium of other lifestyles Fig 2F , the host cell status is also implied to serve a regulatory role on S.

Typhimurium dormancy. With the traits we uncovered in the dormant S. Typhimurium within epithelial cells, this population could represent the intestinal persister, given the close association between bacterial dormancy and antibiotic persistence. Besides the proposed antibiotic persistence and horizontal gene transfer, the physiological features of enterocyte-borne dormant S.

Typhimurium could also provide two plausible benefits to S. Typhimurium to evade cellular immunity during early invasion and to provide a sustained and extended SPI-2 expression at tissue scale, where S. Typhimurium reactivated from dormancy supports SPI-2 expression as classic vacuolar S.

Typhimurium is eradicated. The sustained SPI-2 expression fuels gut inflammation to release electron acceptors for S. Typhimurium survival benefits in the gut lumen [ 33 ]. Typhimurium resided within the intestinal tissue serves as the source of subsequent infection relapse or systemic spread, where the maximum duration of persistence and molecular cues for reactivation remain to be elucidated Fig 4B. For immunofluorescence staining, HeLa cells were seeded on UV-treated glass coverslips Marienfeld in well plates 48 hours prior to infection.

For cell sorting experiments, HeLa cells were seeded in 10 cm tissue-culture treated dishes Corning Costar at a density of 1. Bacterial strains and plasmids used in this study are listed in S1 and S2 Tables, respectively. The replication rate module, Timer bac is a generous gift from Dr. Dirk Bumann University of Basel, Switzerland [ 20 ]. Bacteria strains were streaked from glycerol stock on LB agar plates with appropriate antibiotics 2 days prior to infection. Three bacterial colonies were picked for overnight culture in LB medium supplemented with 0.

For strains harboring pSINA1. Extracellular bacteria were removed and washed with 1 x PBS 5X. At designated time points, cells were washed with 1 x PBS 1X and detached with 0. Typhimurium primary antibody and Alexaconjugated goat anti-rabbit secondary antibody [ 21 ]. The recorded events were gated according to the strategy described S2 Fig. Typhimurium populations. Infected HeLa cells were enriched by cell sorting, where infected cells were sorted for each sample. The cells were then collected by centrifugation at x g for 5 min, and subsequently lysed in 0.

The lysed cells were then serially diluted and plated on LB agar plates with appropriate antibiotics. Infected HeLa cells harboring dormant S. Typhimurium being released from dead cells. Cells were harvested at 24 h and h pi for analysis and CFU plating. The cells were harvested at 6 h pi for cell sorting and CFU plating. Typhimurium were harvested by cell sorting.

Fixed samples were washed with 1 x PBS for three times, and position of interest were defined by fluorescent light microscopy. For further sample preparation, the fixed cells washed three times by the addition of fresh 0. After three washes in 0. The resin embedded samples were removed from the dishes after gentle heating. For the correlative microscopy, the region of interest was determined thanks to landmarks printed below embedded samples.

Embedded cells were sectioned with an ultramicrotome Leica, UC7 with 70 nm thickness. Thin serial sections were collected on a single slot grid Agar scientific. Unless further specified in the figure legend, data were analyzed for statistical significance with a Mann-Whitney test using Prism 8. To gate for the basal intensity of SINA1.

Typhimurium, and harvested at 6 h pi for analysis by flow cytometry. Typhimurium after immunostaining using anti- S. The gating strategy displays a positive control sample treated with saponin. Typhimurium infected HeLa cells at 6 h pi, signal intensity of Vac - Cyt - population unpermeabilized black, negative control , permeabilized with digitonin red and saponin maroon, positive control.

HeLa cells were infected with wild type S. A Infected cells were harvested and analyzed at time intervals of 2 h, 4 h and 6 h pi. Left Timer bac profile of total cells at 2 h top , 4 h middle and 6 h bottom pi in HeLa cells. Right Fluorescence output of the localization module of infected cells at 2 h top , 4 h middle and 6 h bottom pi in HeLa cells. B-C Time-lapse microscopic acquisition of the S. Typhimurium intracellular lifestyle.

Representative images of SINA1. Typhimurium and harvested at 6 h pi for analysis by flow cytometry. Timer and Timer intensities were extracted from each event. Green:red ratios were calculated by dividing Timer by Timer values, and plotted against infected cell populations. For each population, a best-fitted line was plotted on the Timer vs Timer plot to extract the slopes for each infected cell populations.

At least a total of events of infected cells were analyzed by flow cytometry in triplicate experiments. Typhimurium, and harvested at 1 h, 2 h and 3 h pi for analysis by flow cytometry.

The infected cells were gated and the fluorescence profiles of vacuolar submodule P ssaG -tagBFP at 1 h black , 2 h red and 3 h blue pi were plotted as overlaying histograms. Typhimurium and harvested at 1 h and 6 h pi for analysis by flow cytometry. Typhimurium and harvested at 1 h, 6 h and 24 h pi for analysis by flow cytometry.

A Brightfield and fluorescent microscopy image of region of interest on MatTek dish. B Brightfield and fluorescent microscopy image of cells of interest harboring Vac - Cyt - S. C TEM image of cell of interest in labelled region from B. HeLa cells were infected with various S. Typhimurium strains and harvested at 6 h pi for flow cytometry analysis.

Images were taken every 15 min starting from 1 h pi. Typhimurium exhibiting Vac - Cyt - profile. We thank the members of the Dynamics of Host-Pathogen Interactions Unit for the constructive comment and discussion. We are grateful for the generous plasmid gift from D. Bumann, and we acknowledge S. Abstract Salmonella Typhimurium S.

Author summary Salmonella Typhimurium is a clinically relevant bacterial pathogen that causes Salmonellosis. Introduction Salmonella enterica serovar Typhimurium S.

Typhimurium lifestyles The distinct intracellular lifestyles of S. Download: PPT. Fig 1. SINA enables precise determination of the different Salmonella intracellular lifestyles in human epithelial cells. A novel dormant S. Fig 2. Typhimurium displays a novel inactive intracellular lifestyle in epithelial cells. Dormant S. Typhimurium resides in a unique vesicular compartment We set out to determine whether the dormant S.

Typhimurium are viable, cultivable, resume metabolism and express virulence genes in host cells Endocytic vesicles are either recycled or undergo fusion with the lysosomes for degradation.

Fig 3. Typhimurium dormancy is negatively regulated by SpoT. Typhimurium dormancy is regulated by p ppGpp biogenesis Class II toxin-antitoxin TA systems regulate the dormancy formation of non-pathogenic E. Discussion S. Fig 4. Schematic illustration of the role of p ppGpp alarmone pathway on S. Typhimurium dormancy in enterocytes and the proposed pathophysiological implication of S.

Typhimurium dormancy in enterocytes. Bacterial strains Bacterial strains and plasmids used in this study are listed in S1 and S2 Tables, respectively. Plasmid construction The replication rate module, Timer bac is a generous gift from Dr. Bacterial infections Bacteria strains were streaked from glycerol stock on LB agar plates with appropriate antibiotics 2 days prior to infection. Colony forming unit plating Infected HeLa cells were enriched by cell sorting, where infected cells were sorted for each sample.

Typhimurium persistence assay Infected HeLa cells harboring dormant S. Statistical analysis Unless further specified in the figure legend, data were analyzed for statistical significance with a Mann-Whitney test using Prism 8.

Supporting information. S1 Table. Typhimurium strains used in this study. S2 Table. Plasmids used in this study. S3 Table. Primers used for molecular cloning in this study. S4 Table. Antibodies used in this study. S1 Fig. Construction strategy of SINA1. S2 Fig. Gating strategy of SINA1. S3 Fig. With these attributes, this population of Salmonella is genetically programmed to invade new cells. The scientists observed that epithelial cells containing the hyper-replicating, invasive Salmonella are eventually pushed out of the intestinal tissue into the gut cavity, setting the Salmonella free.

The mechanism used to push these Salmonella-infected cells into the body cavity resembles the natural mechanism humans use to shed dying or dead epithelial cells from their gut. The scientists believe that Salmonella have hijacked this mechanism to facilitate their own escape. The human immune system, however, also senses that these are not normal, dying cells in the gut and triggers a response that includes release of interleukin, a small protein that sets off an inflammation cascade.

Interleukin also is prominent in chronic intestinal inflammation associated with autoimmune disorders, such as inflammatory bowel disease. The effects of interleukin release provide an explanation for the acute intestinal inflammation associated with Salmonella infections. The scientists hope their research leads to a treatment that prevents the spread of infection.

They are focusing on how this specialized population of Salmonella escapes from its membrane-bound compartment to multiply and swim freely in the cell. Note: Content may be edited for style and length. Science News. Journal Reference : L. Knodler, B. Vallance, J. For example, nitrates produced from reactive oxygen and nitrogen products within the lumen, and tetrathionate produced from reduced thiosulfate, provide alternative energy sources for Salmonella , which can be metabolized in the anaerobic gut environment Winter et al.

Salmonella -specific genes such as the tetrathionate utilization operon ttr allow the bacteria to utilize these alternate metabolites to outcompete commensal microbes Hensel et al. Although nitrate respiration is conserved among Proteobacteria , the existence of a secretion system effector that manipulates the host in order to preferentially generate nitrates has only been reported in Salmonella Lopez et al. In addition to the metabolic benefits of oxidative and nitrosative stress, Salmonella encodes redundant systems of defense against these reactive oxygen and nitrogen intermediates, acting to mitigate damage under high concentrations Vazquez-Torres et al.

Within the lumen, salmonellae also encounter antimicrobial peptides secreted by epithelial cells and infiltrating immune cells, which work to disrupt membrane integrity Bevins and Salzman, Salmonella has a number of genes that modify the outer membrane to protect against this host defense activity.

Horizontally acquired genes have also been implicated in the defense to antimicrobial peptides. For example, Salmonella can inhibit antimicrobial peptide production by Paneth cells in a T3SSdependent manner Salzman et al. This is likely mediated through the modification of host cell signaling pathways induced by SPI-1 effector proteins. The attachment and invasion of Salmonella to host epithelial cells sets up conditions that eventually favor luminal colonization and systemic infection.

Epithelial cells in the gut, specifically goblet cells, produce mucins that form a physical barrier between the apical surface of these cells and the lumen McGuckin et al. Zarepour et al. Virulent Salmonella strains can bind to mucin preparations better than avirulent strains, suggesting the genetic factors involved in Salmonella virulence mediate this binding, although the interacting partners have not been identified Vimal et al.

As Salmonella approaches the epithelial barrier within the intestine, it can be endocytosed by M cells Clark et al. Entry into the lamina propria allows for TLR activation by flagellin on the basolateral surface, uptake by macrophages, and activation of B and T cells Johansson et al. In addition to luminal sampling by M cells, Salmonella can mediate uptake by host epithelial cells through a cooperative interaction among several virulence factors.

Genes on SPI-4, which encode a Type 1 secretion system and major adhesin protein SiiE, are important for initiating epithelial cell contact in the intestine Gerlach et al. The activity of SiiE is important for maximal bacterial entry into polarized epithelial cells because it allows for clusters of bacteria in close proximity to the invasion site to be pulled into the membrane ruffle at the host cell surface Lorkowski et al.

Along with the protein ShdA, which is not encoded on a genomic island, binding to surface structures like fibronectin appears to be important for intestinal persistence Kolachala et al. The activation of intestinal inflammation can increase levels of fibronectin on epithelial cells, and MisL has been suggested to bind fibronectin at areas of epithelial cell erosion, thus providing another example of the link between intestinal inflammation and efficient bacterial invasion.

The T3SSmediated invasion of Salmonella has been extremely well-studied and the specific role of SPI-1 associated effectors in this process have been reviewed elsewhere Lostroh and Lee, ; McGhie et al. The net result of effector translocation upon host cell contact is the induction of major cytoskeletal changes in the epithelial cell, leading to membrane ruffling and bacterial internalization.

The effectors involved in this process are encoded in SPI-1 and elsewhere in the genome and are coordinated at the level of gene regulation for synchronized expression. Novel functions and regulatory interactions are still being uncovered for even well-known SPI-1 effectors. For example, the phage-encoded effector, SopE, can exert an inside-out response on host epithelial cells by enhancing their expression of iNOS, which in turn liberates host-derived nitrate that luminal Salmonella can respire anaerobically Lopez et al.

The SPI-5 genomic island is another mosaic island that enables different stages of the Salmonella infection process. Recently, SPI was identified as being involved in Salmonella virulence by mediation of bacterial invasion, however the gene responsible appears to play a role in the activation of SPI-1 genes, rather than directly interacting with host processes Jiang et al.

Shortly after bacterial invasion, effectors secreted by SPI-1 control reversion of the host epithelial cell to its normal state, inducing de-ruffling of the membrane and repression of the pro-inflammatory signals. This is typically mediated by host ubiquitination pathways that target SPI-1 effector proteins for degradation. SPI-1 effectors can recruit host factors to the vacuolar membrane to redirect the vacuole in the endosomal trafficking pathway, thus limiting lysosomal fusion.

SPI-1 associated genes are also implicated in escape from the SCV in non-phagocytic cells, allowing for Salmonella survival and replication in the cytosolic compartment. This has been recently characterized in a number of epithelial cell lines Knodler et al.

The hyper-replication seen in the cytosol is associated with Salmonella induced pyroptosis of host cells, releasing primed bacteria into the lumen where they can infect other cells, or be picked up by macrophages. A subset of SCV's are subject to destabilization by SPI-1 associated genes, leading to a dual lifestyle of Salmonella within epithelial cells Knodler, Whether through bacterial mediated pyroptosis, trafficking of the SCV to the basolateral surface of epithelial cells, or by direct sampling of luminal bacteria by dendritic cells, Salmonella eventually arrives in the lamina propria where it is taken up by phagocytic cells through a combination of bacterial-mediated endocytosis and immune cell driven phagocytosis Haraga et al.

Within immune cells, several horizontally acquired virulence determinants are deployed in the creation of the SCV and the evasion of host immune defenses. The virulence genes necessary for bacterial survival within macrophages are predominantly located on the SPI-2 genomic island that encodes the T3SS-2 and two translocated effectors, SseF and SseG Ochman et al. We will not review these genes in detail as they have been comprehensively reviewed elsewhere Cirillo et al.

However, a number of phage associated genes and genes encoded on other genomic islands are paramount to the survival of Salmonella within macrophages. SPI is a pathogenicity island relevant for immune evasion that was first linked to virulence in a calf model of S.

Typhimurium infection Hansen-Wester and Hensel, , Also present within SPI is oafA , which acetylates O-antigen on its 2-hydroxyl group to generate the O5 serotype, which confers antigenic variation beneficial for pathogenesis and immune evasion Slauch et al.

The importance of bacteriophages to the spread of virulence determinants through horizontal gene transfer is well-established Barksdale and Arden, ; Cheetham and Katz, ; Waldor, In line with this, several pathogenicity islands in the Salmonella genome are proximal to either phage-encoding genes or phage attachment sites Blanc-Potard and Groisman, ; Wood et al.

Of pathogenic importance are two prophage-like genomic regions harbored within S. Typhimurium, Gifsy-1 and 2. Both of these genetic elements influence the interactions of Salmonella with host cells, as was identified due to their transcriptional induction upon exposure to reactive oxygen species. Notably, Gifsy-2 encodes the sodC periplasmic superoxide dismutase that confers resistance to oxidative stress Figueroa-Bossi and Bossi, Variants of S.

Typhimurium that are cured of these prophage become rapidly re-lysogenized upon exposure to hydrogen peroxide, indicating the importance of these genetic elements for pathogenesis and survival in vivo Figueroa-Bossi and Bossi, ; Figueroa-Bossi et al.

Within the macrophage environment, both sequestration of essential metals as well the fusion of granules containing antimicrobial peptides to the SCV act together to restrict bacterial replication.

SPI encodes several genes that are critical for pathogenesis and survival at this stage of infection Miller et al. This pathogenicity island is present in both the Typhimurium and Typhi serovars, however it has undergone degradation to some extent within Typhimurium. Of particular importance are a set of genes that encode the envelope proteins, pagD, envE, envF Gunn et al. These genes participate in outer membrane remodeling that improves resistance to the immune defenses encountered intracellularly.

Transposon insertions within these genes have been shown to attenuate virulence and restrict survival within macrophages Miller et al. The systemic spread of Salmonella to the liver and spleen is contingent upon survival within the macrophage environment; mutants in Salmonella that cannot survive within this environment are avirulent in a systemic mouse model of infection.

Salmonella -containing macrophages enter the mesenteric lymph node, where they are shuttled to the liver and spleen Watson and Holden, Replication within macrophages is also essential for colonization at the liver and spleen, as analyses of cell types within these tissues containing Salmonella identify macrophages, and to a degree neutrophils, as the primary cells at infection foci Geddes et al. Genes encoded on SPI are important for intracellular viability Shi et al. SPI contains a cluster that encodes putative lyase, hydrolase, oxidase, arylsulphatase regulators, and deletion of this island renders Salmonella attenuated for virulence Haneda et al.

STM a homolog of acetyl-coA hydrolase is an important virulence factor contained within this island that hydrolyzes acetyl-coA to acetate in order to modify peptidoglycan as a protective mechanism against degradative enzymes found in macrophages. Additionally encoded in SPI is STM, a monoamine oxidase that converts aminoacetone degradation product of L-threonine to a peptidoglycan precursor Shi et al. Further characterizing the function of genes encoded on SPI is likely to broaden our understanding of how Salmonella modulates the intracellular environment and interacts with the host during systemic infection.

SPI is a pathogenicity island that contributes to immune evasion in the form of O-antigen variation. The gtr cluster of genes encodes proteins that add glucose residues to repeating O-antigen subunits within LPS, and ultimately confer form variation at the O antigen galactose to generate the variant.

Most commonly, this variant arises after exposure to or growth within macrophages Bogomolnaya et al. It has been demonstrated that form variation within O-antigen is critical for persistence of Salmonella infection in the murine intestine, and so SPI may play a role in the re-infection of the intestine from the gall bladder once Salmonella has established a niche in the liver in a chronic model of Salmonella infection.

Taken together, the suite of horizontally acquired virulence genes found across the SPIs act in a concerted fashion to drive infection, as Salmonella successfully outcompetes the host microbiota, colonizes the lumen, invades the intestinal epithelium, and resides within the replicative niches of neutrophils and macrophages.

At various stages of the pathogenesis process, Salmonella encounters several host defense mechanisms deployed by the immune system. However, it appears that Salmonella has evolved to integrate these immune response cues into signaling pathways leading to adaptive gene expression that, ultimately, evades these very host defenses Wong et al. However, in order for horizontally acquired genes to confer fitness advantages, their expression is subject to precise regulatory control such that they are deployed at the appropriate times to contribute beneficially to the organism.

Here, we describe the regulatory circuitry within Salmonella that has evolved to integrate newly acquired genes into the existing flexible genetic networks that mediate pathogenesis Figure 2.

Figure 2. Development of novel regulatory pathways through transcriptional rewiring. A Horizontally acquired genes in Salmonella are silenced by H-NS, and cis -regulatory elements can undergo mutations to acquire binding sites that bring these genes under the control of a core virulence regulator such as PhoP.

This counter-silencing allows for activation of virulence relevant horizontally acquired genes like pagC in infection relevant conditions. B Acquisition of the transcriptional regulator SsrB in Salmonella Typhimurium led to cis -regulatory evolution of core genes that allow them to come under the regulatory control of SsrB. This regulatory rewiring of core genes fine-tunes their expression under infection conditions with other SsrB regulated genes.

Although, the acquisition of genes through horizontal transfer has been essential to the evolution of Salmonella pathogenesis, the majority of foreign DNA is detrimental to bacteria Buckling and Rainey, ; Navarre et al. Insertion within functional genes, overexpression of energetically taxing genes, and activation of unfavorable gene products are all examples of the drawbacks to horizontal gene transfer.

Thus, the integration of these genes into core regulatory circuitry is critical for appropriate temporal gene expression and to ensure that these genes do not antagonize existing cellular functions. While this integration slowly proceeds via cis -regulatory mutation discussed later , transcriptional silencing of horizontally acquired genes, termed xenogeneic silencing, protects the bacteria from these laterally acquired genes Singh et al.

Seminal work over the past decade has identified H-NS, a DNA binding protein conserved across Gram-negative bacteria, as a broad repressor of horizontally acquired genes Lucchini et al.

The mechanism of this repression relies on the intrinsic curvature of DNA rich in adenine and thymine AT-rich; Navarre et al.

The existence of H-NS as a global sentinel of horizontally acquired genes has been speculated to contribute to the preferential retention of AT-rich horizontally acquired genes, as H-NS can mitigate the immediate harmful effects of lateral gene transfer Dorman, ; Higashi et al.

Without compensatory mutations, deletion of hns tends to be lethal in Salmonella but not in closely related bacteria like E. Given the importance of horizontally acquired genes to the host-adapted lifestyle of Salmonella , research on H-NS has focused on the protein as a regulator of the pathogenic lifestyle.

The importance of H-NS in pathoadaptation is further highlighted in in vitro evolution experiments, where mutations in hns are associated with loss of virulence determinants such as SPI-1 Ali et al. While H-NS is the most widely known silencer of virulence gene expression, it does not act alone upon horizontally acquired genes. Co-factors such as Hha and YdgT, and other nucleoid associated proteins like Fis contribute to the expression of horizontally acquired genes in Salmonella , including those that regulate virulence Schechter et al.

Hha is specific to enteric bacteria and YdgT is specific to E. Similar to H-NS, the expression of Hha facilitates acquisition of foreign genes, thus contributing to pathogenic adaptation Aznar et al.

In summary, H-NS has been well-characterized as a buffer to help the bacterial cell tolerate new horizontal gene transfer events.

Over time, these acquired genes have undergone further regulatory refinement for optimal expression in the host environment. De-repression of H-NS by environmental stimuli is one way through which horizontally acquired genes can be activated in the right environments. Changes to DNA curvature induced by osmolarity or temperature shifts can modify the bridging activity of H-NS at specific promoters, allowing for transcription to proceed Hinton et al.

However, directed activation of virulence genes predominantly occurs through regulatory rewiring of H-NS binding elements to put these genes under the control of virulence regulators, or to allow for transcription factor binding that displaces H-NS bridges from the DNA.

Examples of these methods have been identified for counter-silencing H-NS at horizontally acquired virulence genes in Salmonella , and their significance is discussed below. Given the extent of xenogeneic silencing in Salmonella , the pathogenic lifestyle is highly reliant upon the evolution of regulators to counter-silence H-NS and relieve this repression Stoebel et al. Many horizontally acquired genes become incorporated into the regulatory framework of two-component sensory systems, which are embedded in genetic networks to rapidly detect and respond to environmental cues Wallis and Galyov, The PhoP-PhoQ regulatory system is highly conserved across bacterial species and governs several aspects of the Salmonella virulence program Miller et al.

The detection of these environmental cues results in the positive regulation of PhoP-activated pag gene expression, and negative regulation of PhoP-repressed prg gene expression Groisman, The majority of genes identified to be regulated by PhoP-PhoQ have been acquired by horizontal gene transfer Groisman, and integrated into the PhoP regulon over evolutionary time.

This suggests that horizontally acquired genes co-evolve with the Salmonella genome to become assimilated into existing core regulatory architecture, such that the spatiotemporal expression of virulence determinants is tightly controlled. Interestingly, PhoP appears to act differentially to regulate promoters acquired by horizontal gene transfer relative to those predicted to be ancestral Will et al. A comparison of the promoter architectures between foreign and ancestral genes suggests that those that were horizontally acquired bind PhoP flexibly with high variability at a number of positions, whereas those that are part of the core genome interact with PhoP in a conserved manner at one binding site Zwir et al.

Furthermore, PhoP is capable of activating ancestral promoters directly via RNA polymerase holoenzyme interaction Will et al. The importance of PhoPQ in regulating horizontally acquired genes is also evident from the divergence in PhoP targets despite the conservation of this regulatory system across diverse taxa. For example, Yersinia pestis Grabenstein et al. These findings indicate that PhoPQ is a broadly conserved regulatory system that can flexibly integrate ancestral and acquired genes to accommodate bacterial lifestyles ranging from endosymbiosis to parasitism.

SsrB can directly bind to genes within SPI-2, and outside of this island, and activate their transcription Worley et al. This further demonstrates the cross-talk between pathogenicity islands in order to regulate the different lifestyles of Salmonella during infection. The classical definition of cis -regulatory evolution rests upon the inevitable accumulation of mutations in non-coding DNA that drift in the nearly neutral range Stone and Wray, Those mutations that generate a fitness-increasing quantitative output to alter gene expression may sweep to fixation, creating novel regulatory nodes that result in the flexible expansion of complex genetic networks Wray, Adaptation within cis -regulatory elements is proposed to contribute to genetic tunability in response to environmental cues, a critical component of host colonization.

However, up until recently, empirical evidence for this in the context of bacterial pathogenesis was largely lacking. Recent work has shown that mutations in non-coding DNA are targets for polymorphism-fixing selection to assimilate genes into the SsrB regulon, and that divergence in the regulatory patterns between S.

For example, we demonstrated that following acquisition of a new regulatory system, the promoters that regulate ancestral genes can evolve responsiveness to the new transcription factor to fine-tune fitness in the host Osborne et al.

This involves rewiring the cis -regulatory element controlling the ancestral gene that generates phenotypic diversity among the bacterial population that is selective in the host setting. Regulatory evolution explains much of the organismal diversity among closely related animals in the context of developmental evolution i.

Work by other groups has since verified and extended these findings, showing that regulatory evolution drives diverse bacterial traits including immune evasion Tuinema et al. While the contribution of horizontally acquired genes in the pathoadaptation of Salmonella has been well-studied, the role of gene loss has received somewhat less attention.

Bacterial adaptation to the host environment is understood to involve considerable genomic rearrangement and several studies have addressed the continuously fluctuating size of bacterial genomes, attributed largely to horizontal gene transfer, gene loss, and duplication Bliven and Maurelli, We have described how cis -regulatory evolution mediates the assimilation of acquired virulence determinants into existing regulatory circuitry; gene loss is another process whereby certain genes may be inactivated to permit a pathogen's newly acquired virulence factors Maurelli et al.

Several studies have suggested that an intracellular lifestyle and host range specificity facilitate genomic degradation across the Salmonella serovars, resembling that which occurs in obligate intracellular endosymbionts that are entirely dependent on their eukaryotic hosts Parkhill et al.

Within bacteria, gene loss proceeds by either genomic rearrangement or pseudogenization. We might predict that this occurs more readily within the host environment encountered by Salmonella during an infection, due to drastic population bottlenecks. These repeated reductions in population size force the random resampling of allelic variation, such that loss-of-function mutations accumulate more rapidly than expected Nilsson et al.

How is gene loss identified in bacterial genomes? Phylogenetic reconstruction is often sufficient to reveal the absence of genes when related species are compared to their last common ancestor.

The presence of segregating variants with loss-of-function mutations in a population may also signify gene loss. Exploring phylogenetic relationships with more evolutionary distance is a potential strategy to aid in the detection of reductive evolution.

For example, the distantly related tsetse fly endosymbiont S. However, an alternative method to characterize patterns of gene loss is with experimental evolution studies that mimic the intracellular environment encountered by bacteria, to measure deletion rates and identify potential fitness advantages.

Experiments of this nature are the largest contributors to our current understanding of the evolution of the salmonellae via gene loss. Investigations of long-term experimental evolution have revealed high rates of deletion in bacteria conferring fitness advantages Barrick et al. In experimentally evolved Salmonella populations simulating conditions of infection, genetic drift has been shown to drive RecA-independent gene deletions ranging from 1 to kb over a strikingly brief period of time Nilsson et al.

Others have shown that selection is also capable of driving random deletions to fixation in the Salmonella chromosome that increase fitness as measured by growth rate Koskiniemi et al. Furthermore, analyses of the more pathogenic Typhi and Paratyphi serovars have revealed low rates of purifying selection and recombination, but rather considerable loss-of-function gene mutations Holt et al.

Why do gene loss mutations confer fitness increases in Salmonella? One hypothesis is that these readily occur in genes participating in pathways that become non-essential in new environmental conditions. As novel host adaptation occurs, neutral mutations may accumulate in these superfluous genes, mediating pseudogenization.

An example of this in Salmonella is the loss of the lacI repressor, which negatively regulates the lactose fermentation system in E. The loss of this gene has been demonstrated to confer fitness advantages, as bacteria expressing it are capable of invasion but attenuated for survival within murine macrophages.

It has been proposed that LacI was lost within S. Typhimurium to facilitate the acquisition of SPI-2, as it was found to repress virulence genes within this island and remains present in S. These findings suggest that LacI is an antivirulence gene that underwent selection for loss to promote an intracellular lifestyle. An increased emphasis on comparative genomics and transcriptomics of cells during infection has improved our ability to detect horizontal gene transfer events and identify the origins of virulence evolution Thompson et al.

The advent of RNA-sequencing RNA-Seq in particular has heralded an era of Salmonella research that has uncovered several novel aspects of innate immune evasion, as is evidenced by several recent transcriptomics experiments. To identify the gene expression changes induced by host immune pressures, RNA-Seq was performed on Salmonella grown in 22 in vitro conditions approximating those presented by the immune system over the course of infection.

Typhimurium genes Kroger et al. The regulatory nature of Salmonella -host interactions was characterized using RNA-Seq to explore the targets of 18 key transcriptional regulators within S. Typhimurium, which identified 1, genes with altered expression profiles Colgan et al.

Interestingly, single-cell RNA-Seq within macrophages identified that Salmonella undergoing active replication shifts macrophage metabolism from an M1 to an M2 polarization state, perhaps capitalizing upon the anti-inflammatory environment in M2 macrophages Saliba et al. These Salmonella -based RNA-Seq experiments have all explored gene expression profiles during intracellular survival and upon exposure to components of host immunity, as well as helped to identify the genome-wide targets of core transcription factors and how they link to horizontally acquired genes.

The interaction of Salmonella with the host during an infection requires sensing the host environment, activating genes in response to host antimicrobial defenses, and modifying the host to make it more amenable for colonization.

There are a number of core genes that contribute to pathogenesis, such as motility genes required for chemotaxis within the gut lumen, fimbrial adhesins involved in mediating host cell contact, and metabolic genes that allow for nutrient acquisition in the competitive anaerobic gut environment. However, the biology of Salmonella in the context of infection is strongly driven by the acquisition of genes through horizontal gene transfer. The identification of SPI-1, shared by all Salmonella species and subspecies, as critical for bacterial mediated endocytosis into epithelial cells initiated decades of research into the role of horizontally acquired genes in Salmonella infections.

A combination of tissue culture infections, genetic manipulation of Salmonella , and in vivo infection models have been the mainstay of research approaches to broaden our understanding of how SPIs contribute to Salmonella infection biology. This review highlighted the central contributions of the most recently identified pathogenicity islands in S. Typhimurium, and tied these findings into the overarching cellular biology of a S. Typhimurium infection.

Our summaries of these virulence determinants are not comprehensive, as their precise molecular mechanisms have yet to be fully characterized. Although a number of the SPIs and phage associated genes have been identified as playing a role in virulence or survival within host cells, much remains to be understood in how these interact with other Salmonella genes and with host processes. We have additionally discussed the complex interplay between horizontal gene transfer and cis -regulatory evolution, and described the role of these two biological processes in the promotion of patho-adaptive change.

Over evolutionary time, the virulence program of Salmonella has been shaped by the acquisition of pathogenicity islands and phage-associated genes, furthering its divergence from to its closest relative, E.

Concurrently, considerable cis -regulatory change has occurred in the Salmonella genome to integrate horizontally acquired genes and ancestral core genes into new regulatory circuitry, to control their expression such that fitness is optimized.

Evolutionary biologists have long appreciated the importance of cis -regulatory evolution in the generation of genetic variation to ultimately drive adaptive change; we have elaborated this to emphasize the impact of this process in increasing bacterial pathogenicity. The assimilation of newly acquired genes into the regulons of H-NS, PhoP-PhoQ, and SsrA-SsrB, has allowed for the expansion of flexible genetic networks that mediate bacterial pathogenesis and confer pathoadaptive fitness differences.

We have also described the genomic degradation and gene loss that occurs alongside the horizontal acquisition of novel genes, due to a combination of relaxed selection and antagonistic pleiotropy. These findings highlight the dynamic genome that underpins the evolution of bacterial pathogenesis. In the age of antibiotic resistance when even the very process of evolution itself is a potential new target Smith and Romesberg, ; Zaneveld et al.

All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication. BI was supported by the Ontario Graduate Scholarship. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

We are grateful to the many fruitful conversations with colleagues over the years that have advanced the fields of regulatory evolution and bacterial pathogenesis. Albalat, R. Evolution by gene loss. Ali, S.