Population heterogeneity tactics as driving force in Salmonella virulence and survival
Introduction
In 2017, Salmonella was indicated by the World Health Organisation (WHO) as one of world's most alarming pathogens and is placed 8th on the global priority list of antibiotic-resistant bacteria in need for research and development of new antibiotics. This underscores the importance of an in depth understanding of Salmonella's virulence and survival strategies to pave the way for the development of new methods to combat this pathogen or prevent its transmission. Salmonella largely owes its success to complex spatiotemporal gene expression patterns and phenotypically diverse subpopulations that facilitate its survival in the environment and mediate a strategic deployment of virulence factors throughout the infection process.
For Salmonella to successfully reach the host intestine and establish infection, the bacteria first have to survive the route through different hostile environments. Throughout evolution, Salmonella has evolved the necessary virulence tools and strategies to cope with these diverse surroundings. These tools comprise a series of integrated virulence factors that were often horizontally acquired in the form of plasmids, prophages or other mobile genetic elements (Mebrhatu, Cenens, & Aertsen, 2014; Wahl, Battesti, & Ansaldi, 2019). As the efficacy of Salmonella pathogenesis depends upon the activation of the right virulence factor at the right time, their regulation depends on a complex, hierarchal network that integrates diverse cues. The intricate coordination and cooperation of virulence factors is further achieved by extensive crosstalk between these factors. Although expression of virulence genes is tightly regulated, various factors are heterogeneously expressed at the single cell level, and often even a bifurcation into two subpopulations with the gene either in ON or OFF state occurs. This results in phenotypically distinct subpopulations that can facilitate population-level strategies, such as bet-hedging and division-of-labor, which were proven to be key for the overall success of Salmonella as a pathogen (Ackermann, 2015; Arnoldini et al., 2014; García-Pastor, Puerta-Fernández, & Casadesús, 2018; Stapels et al., 2018; Stewart, Cummings, Johnson, Berezow, & Cookson, 2011). Indeed, while bet-hedging constitutes a risk-spreading tactic that allows the pathogen to respond to sudden drastic alterations in its environment by priming a subpopulation to survive those conditions (Ackermann, 2015; de Jong, Haccou, & Kuipers, 2011), division-of-labor increases the number of specialized concerted functions the (sub)population(s) can perform (Ackermann, 2015).
Given this inherent, virulence-driven population heterogeneity, even clonal Salmonella populations should be looked upon as a collection of individuals rather than a uniformly acting population. However, although some of the heterogeneously expressed virulence factors are thought to have an influence on survival of Salmonella outside its host (Horstmann et al., 2017; MacKenzie et al., 2015), little research has been conducted to determine whether and how this differential expression among siblings affects their individual resistance against stresses encountered in the food production chain. Nevertheless, this heterogeneity could complicate straightforward predictions of a population's survival capacity, especially at smaller population sizes that are still relevant for this low infectious dose pathogen. In fact, it was most recently shown that osmotic stress-induced inactivation of Salmonella Agona populations smaller than 100 cells could not accurately be predicted based on their bulk-level inactivation kinetics, although the underlying mechanism still needs to be resolved (Aspridou, Balomenos, Tsakanikas, Manolakos, & Koutsoumanis, 2019).
This review further discusses Salmonella as a food-borne pathogen, together with its key virulence factors and their regulation. Particularly those factors related to population heterogeneity will be highlighted, together with their subsequent phenotypic consequences on population-level survival and infection dynamics.
Section snippets
Salmonella as a food-borne pathogen
Salmonella pathogenicity and related clinical symptoms depend largely on the infectious serovar and the susceptibility of the infected host. The genus Salmonella contains a number of host-adapted specialists, such as the human-adapted S. Typhi. This pathogen disseminates via the lymphatics to the blood stream to ensure the systemic spread of the bacteria throughout the body (Bäumler & Fang, 2013; Gal-Mor, Boyle, & Grassl, 2014; Vazquez-Torres et al., 1999). In the latter case, a secondary
Key virulence factors during Salmonella infection and their genetic regulation
Starting at the ingestion of Salmonella, a spatiotemporally regulated expression of virulence factors is unfolded. Overall, a first important virulence program that is activated during the infection is the acid tolerance response that helps in countering the acidic pH found in the stomach of the host and allows the bacterium to travel further into the small intestine to initiate colonization (Ryan et al., 2015). This intestinal colonization of Salmonella is further promoted by the expression of
Flagella
Flagella work as molecular motors for bacterial motility, and thereby enable chemotaxis (Kojima & Blair, 2004). Moreover, flagella also play a role in adhesion and invasion (Haiko & Westerlund-Wikström, 2013), and serve as antigens that trigger the host immune response (Hayashi et al., 2001). S. Typhimurium can bear ca. 6 to 10 flagella per cell, each composed of a basal body serving as anchor, a transmembrane motor, and a large whip-like filament that is built up of either the FliC or FljB
Fimbrial adhesins
The Salmonella genome harbors various regions encoding fimbriae, which are proteinaceous, fibrillary structures that extend from the membrane into the extracellular space. Among these is the lpf operon (required for attachment to Peyer's patches) (Baumler, Tsolis, & Heffron, 1996), the pef operon (for adhesion to intestinal epithelial cells) (Bäumler et al., 1996), the std operon (for attachment to cecal epithelium) (Chessa et al., 2009; García-Pastor, Sánchez-Romero et al., 2018), and the fim
Salmonella pathogenicity island 1 (SPI-1)
The horizontal acquisition of the SPI-1 pathogenicity island has been a major deterministic event for Salmonella and is considered a phylogenetic branching point between the genera Escherichia and Salmonella from their common ancestor (Desai et al., 2013). SPI-1 comprises a ca. 40 kb region including 35 genes (Mills, Bajaj, & Lee, 1995), encoding several effector proteins and a T3SS through which effectors are secreted into the intestinal cells. These effectors trigger localized membrane
Salmonella pathogenicity island 2 (SPI-2)
SPI-2 was initially characterized in the mid 90's as a key component to survive and replicate inside epithelial cells and macrophages (Hensel et al., 1995; Ochman, Soncini, Solomon, & Groisman, 1996). The SPI-2 effector proteins are translocated across the membrane of the SCV within the infected host cell via the SPI-2 encoded T3SS-2, and are implicated in processes such as maintaining SCV integrity, positioning of the SCV, and remodeling of the host cytoskeleton (Jennings et al., 2017). SPI-2
Salmonella plasmid virulence genes (spv)
The best studied virulence plasmid of Salmonella is the plasmid of S. Typhimurium (pSLT) which contains a highly conserved 8 kb region of five genes: spvRABCD (Salmonella plasmid virulence) (overview in Fig. 5). SpvR has been established as an essential activator of the spvABCD operon (Fang, Krause, Roudier, Fierer, & Guiney, 1991), whilst the SpvB, SpvC and SpvD proteins have been proven to be virulence factors with specific roles in infected host cells (Browne, Hasegawa, Okamoto, Fierer, &
LPS modifications
The lipopolysaccharide (LPS) layer is known to stabilize the outer membrane and forms a permeability barrier that prevents unwanted molecules from entering the bacteria's cytoplasm (Raetz & Whitfield, 2002). Functional interference with this layer thus severely attenuates Salmonella virulence in multiple model systems (Hoare et al., 2006; Ilg et al., 2009; Morgan et al., 2004; Nevola, Stocker, Laux, & Cohen, 1985). A single LPS molecule consists of a lipid A anchor in the outer leaflet of the
Biofilm formation
Expression of the master regulator, CsgD, of biofilm development in S. Typhimurium also occurs in a bistable fashion (MacKenzie et al., 2015). Biofilms are organized structures of microorganisms, adhered to a biotic or abiotic surface and encased in a self-produced extracellular matrix (Steenackers, Hermans, Vanderleyden, & De Keersmaecker, 2012). It is known that Salmonella can form biofilms which consist of a three-dimensional extracellular matrix of curli fimbriae, cellulose, proteins and
myo-inositol utilization
S. Typhimurium is able to grow in minimal medium with only myo-inositol as carbon source due to the presence of the myo-inositol degradation pathway, that comprises enzymes, transporters and regulators (Kröger & Fuchs, 2009; Kröger, Stolz, & Fuchs, 2010). The expression of the pathway on solid medium supplemented with myo-inositol as sole carbon source displays a long lag time and moreover extensive heterogeneity in colony growth is observed (Kröger, Srikumar, Ellwart, & Fuchs, 2011). Kröger et
Conclusion
It is clear that virulence tactics have driven a complex cooperative heterogeneity within clonal populations of Salmonella, and other pathogens as well (Davis & Isberg, 2019). In fact, taking into account the combinatorial effect of different heterogeneously expressed virulence factors, it seems that Salmonella populations consist of highly specialized individuals rather than a uniformly acting population. However, neither the full functional heterogeneity, nor the underlying mechanisms causing
Declaration of Competing Interest
None.
Acknowledgments
This work was supported by a SB PhD fellowship (project number 1S56416N) from the Research Foundation - Flanders (FWO-Vlaanderen; to I.S.); a PhD fellowship from the Flemish Agency for Innovation by Science and Technology (IWT-Vlaanderen; to I.P.); a PhD fellowship (project number 1135116N) from the Research Foundation - Flanders (to A.C.); and a grant from the KU Leuven Research Fund (GOA/15/006).
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