Received: 5 May 2017 Revised: 27 June 2017 Accepted: 30 June 2017

DOI: 10.1111/cmi.12766

MICROREVIEW

Intracellular Salmonella metabolism

Dirk Bumann | Joep Schothorst

Focal Area Infection Biology, Biozentrum,

University of Basel, Basel, Switzerland Abstract
Correspondence Growth of Salmonella inside infected host cells is a key aspect of their ability to cause local
Dirk Bumann, Focal Area Infection Biology, enteritis or systemic disease. This growth depends on exploitation of host nutrients through a
Biozentrum, University of Basel, CH‐4056 large Salmonella metabolism network with hundreds of metabolites and enzymes. Studies in cell
Basel, Switzerland.
culture infection models are unravelling more and more of the underlying molecular and cellular
Email: [email protected]
mechanisms but also show striking Salmonella metabolic plasticity depending on host cell line
and experimental conditions. In vivo studies have revealed a qualitatively diverse, but quantita-
tively poor, host‐Salmonella nutritional interface, which on one side makes Salmonella fitness
largely resilient against metabolic perturbations, but on the other side severely limits Salmonella
biomass generation and growth rates. This review discusses goals and techniques for studying
Salmonella intracellular metabolism, summarises main results and implications, and proposes key
issues that could be addressed in future studies.

1 | I N T RO D U CT I O N neutrophils and macrophages in the gut mucosa kill efficiently invading

Salmonella. Nevertheless, if the infectious dose is large enough (i.e.,
Salmonella enterica are Gram‐negative bacteria and close relatives of higher than 1,000 to 100,000 colony‐forming units) and competition
Escherichia coli. There are more than two 2,300 different Salmonella by resident microbiota is overcome, surviving Salmonella can over-
serovars, many of which can cause local intestinal disease (enteritis whelm these defences and cause disease in the intestine. During
and diarrhoea) in a broad range of hosts. By contrast, some serovars enteritis, Salmonella proliferates mostly in the gut lumen, although some
can also disseminate from the gut and cause systemic disease in a Salmonella invade the intestinal mucosa and proliferate in gut epithelial
host‐specific manner (enteric fever/(para)typhoid fever and cells (Wotzka et al., 2017), and this is strongly enhanced in neonate
nontyphoidal salmonellosis). Salmonella cause major mortality and mor- mice (Zhang et al., 2014). Intraepithelial Salmonella proliferation leads
bidity worldwide (Havelaar et al., 2015; Keestra‐Gounder, Tsolis, & to inflammasome activation and extrusion of infected cells (Knodler
Baumler, 2015; LaRock, Chaudhary, & Miller, 2015; Wain, Hendriksen, et al., 2014; Sellin et al., 2014; Zhang et al., 2014), which might
Mikoleit, Keddy, & Ochiai, 2015). Vaccines for prevention of human contribute to the overall inflammation response that ultimately leads
disease are available for the single serovar Typhi and have only moder- to water loss to the lumen and diarrhoea (Darwin & Miller, 1999).
ate protective efficacy. Antimicrobial chemotherapy becomes less and However, the importance of this epithelial proliferation for enteritis
less effective due to rapidly increasing multidrug resistance, and both pathology and Salmonella overall fitness and transmission is still unclear.
the U.S. Center for Disease Control and the WHO include Salmonella Salmonella serovars that can cause systemic disease on the other
among the most serious infectious disease threats for human health. hand enter intestinal Peyer's patches and solitary intestinal lymphoid
In addition to better vaccines and novel antibiotics, interference with tissue. In mouse models, Salmonella are rarely found in epithelial cells
transmission by supplying clean drinking water can dramatically reduce of adult hosts (Halle et al., 2007; Zhang et al., 2014), whereas in neo-
incidence of enteric fever, and better control of Salmonella carriage in nate mice, which largely lack differentiated M cells, Salmonella mostly
animal livestock might largely prevent diarrheal disease and invade epithelial cells and probably disseminate from there to internal
nontyphoidal salmonellosis. organs (Zhang et al., 2014). Just a few clones can successfully establish
There are two main types of Salmonella infection, causing either themselves in these tissues and disseminate systemically (Lim et al.,
enteritis or systemic disease. Both infections start with ingestion of 2014; Meynell, 1957). Within host tissues, Salmonella resides and pro-
Salmonella‐contaminated food or water. A large part of the ingested liferates predominantly in tissue macrophages but also other cell types
Salmonella is killed by stomach acid, bile, and intestinal defensins (Burton et al., 2014). Spleen and liver are major target organs, but other
(Wotzka, Nguyen, & Hardt, 2017). Neutrophils migrating through the tissues are also infected. From the liver, Salmonella can reach the gall-
gut mucosa also efficiently kill Salmonella in the gut lumen, and bladder where they proliferate in gallbladder epithelial cells (and on

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gallstones, if present). Infected cells activate the inflammasome and are cause host pathology). Indeed, Salmonella mutants with severe fitness
extruded (Knodler et al., 2010). Released Salmonella then reach defects nevertheless still cause wild type‐level gut pathology in
together with bile the intestine and ultimately faeces, thus closing single‐strain infections in the mouse enteritis model (Faber et al.,
the transmission cycle. 2017; Thiennimitr et al., 2011; Winter et al., 2010). Inhibiting the
Taken together, intracellular Salmonella proliferation occurs during corresponding enzymes would thus have limited impact on disease
both diarrhoeal and systemic disease. The metabolic conditions and progression. By contrast, several metabolic mutations (galE, purA, aroA,
mechanisms that drive this intracellular growth are the focus of exten- aroCD, manA, etc.) abrogate both Salmonella in vivo fitness as well as
sive research efforts because a better overview of the Salmonella‐host systemic virulence in the mouse typhoid fever model and in human
metabolic landscape might open new windows for antimicrobial devel- volunteers (Bumann, Hueck, Aebischer, & Meyer, 2000), highlighting
opment. In this review, we discuss some aspects of this exciting field. metabolism as a key targetable aspect of systemic salmonellosis.
Additional aspects are covered in other informative recent reviews Metabolism is required not only for proliferation but also for main-
(Dandekar, Astrid, Jasmin, & Hensel, 2012; Dandekar et al., 2014; taining essential basal activities even in nondividing Salmonella cells.
Eisenreich, Dandekar, Heesemann, & Goebel, 2010; Eisenreich, Identification of such “dormancy”‐associated metabolic activities could
Heesemann, Rudel, & Goebel, 2013; Eisenreich, Heesemann, Rudel, provide a basis for specific targeting of Salmonella persisters that can
& Goebel, 2015). For Salmonella metabolism during extracellular survive even prolonged antimicrobial chemotherapy (Harms,
stages, an excellent review has recently been published (Rivera‐Chavez Maisonneuve, & Gerdes, 2016). However, such pathways seem to be
& Baumler, 2015). quite rare with the possible exception of fatty acid biosynthesis (Barat,
Steeb, Maze, & Bumann, 2012).
Finally, Salmonella metabolic enzymes might be important for
2 |M E T A B O L I S M A S A BA S I S F O R disease progression not because of their catalytic activity, but rather
SAL M ONELLA F I T NE S S , V I R U L EN C E , A N D due to unrelated so‐called moonlighting functions (Henderson, 2014).
PERSISTENCE So far, there is only one example of such a moonlighting enzyme with
a role in Salmonella virulence (the sugar transport protein and meta-
Salmonella in vivo proliferation requires de novo synthesis of biomass bolic regulator EIIAGlc that activates the second Type 3 secretion sys-
components such as proteins, carbohydrates, lipids, and nucleic acids tem encoded on Salmonella pathogenicity island 2 [SPI‐2]; Maze,
from small molecule precursors. Salmonella can obtain these precursors Glatter, & Bumann, 2014). It is possible that additional enzymes have
from internal storage such as glycogen or lipids (for a limited number of relevant moonlighting functions as is the case for other pathogens
divisions) or directly from the host micro‐environment (e.g., amino (Henderson, 2014), and this is an important caveat for “simple” meta-
acids) and/or synthesise them from a few basic host carbon (such as bolic interpretations of enzyme defect phenotypes.
acetate, glycerol, and glucose), nitrogen (e.g., ammonium), sulphur
(e.g., sulphate), and phosphor (e.g., phosphate) sources. How Salmonella
salvages these nutrients from the host is still a matter of debate and will 3 | G O A LS F O R S T U D Y I N G I N T R A C EL L U L A R
be discussed later on. Nutrient uptake and conversion into biomass SALM ONELLA M E T A B O L I S M
requires substantial energy, and finding and exploiting a suitable energy
source is the single most important metabolic activity of growing cells One of the major challenges in studying Salmonella in vivo metabolism
(Abu Kwaik & Bumann, 2015). Counter‐intuitively, Salmonella seems and identifying potential antimicrobial targets is the strong interplay
to diminish adenosine triphosphate (ATP) production under some host between Salmonella and the mammalian host cells, which provide a
conditions by direct inhibition of the ATP synthase with MgtC (Lee, very complex metabolic micro‐environment for intracellular Salmonella.
Pontes, & Groisman, 2013), a protein that is required for wild type‐level These host cells contain thousands of different metabolites and use
Salmonella fitness (Alix et al., 2008). This could be a special adaptation versatile regulatory mechanisms to modulate metabolite concentra-
to limiting magnesium availability by releasing ATP‐bound magnesium tions and metabolic fluxes. Furthermore, they can dramatically alter
for ribosome stabilisation (Pontes, Yeom, & Groisman, 2016). their metabolism in various cell differentiation and activation states,
Nutrient availability and Salmonella metabolic capabilities will which strongly influences nutrient availability and hence Salmonella
modulate overall biomass generation and proliferation (Monahan & fitness. The interplay works in both directions, with the host cell
Harry, 2016) and thus Salmonella fitness in host tissues during infec- providing nutrients for Salmonella, and Salmonella simultaneously
tion. Identifying and quantifying relevant nutrients as well as the corre- perturbing the host metabolic network by consuming certain metabo-
sponding catabolic and anabolic Salmonella pathways is therefore of lites and releasing waste products (Olive & Sassetti, 2016).
fundamental importance for understanding the disease process and In addition to these direct interactions, Salmonella releases waste
to identify opportunities for specific enzyme inhibitors as urgently products, produces stimulatory components such as lipopolysaccha-
needed novel antimicrobials to control infection. Moreover, Salmonella ride, and secretes numerous virulence factors directly into the host cell
metabolic mutants might be useful as live attenuated vaccines cytosol. These Salmonella activities modulate host cell physiology in a
(Tennant & Levine, 2015) or as cancer therapeutics (Wang, wide variety of aspects including host cell synthesis of toxic molecules
Kazmierczak, & Eisenstark, 2016). that affect Salmonella metabolism. This includes nitric oxide, which can
It is important to note that fitness (the capability to produce viable block respiration (Husain et al., 2008); itaconic acid, which inhibits the
offspring) does not always correlate with virulence (the capability to glyoxylate shunt (Michelucci et al., 2013); and superoxide/peroxide
 14625822, 2017, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cmi.12766 by Cochrane Chile, Wiley Online Library on [04/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
BUMANN AND SCHOTHORST 3 of 11

that damages several bacterial components whose repair requires sup- tissues using flow cytometry (Becker et al., 2006; Steeb et al., 2013).
portive metabolism (Burton et al., 2014; Slauch, 2011). Importantly, Data interpretation will need to take into account the increased com-
many of these host/Salmonella interactions are highly heterogeneous plexity of whole organism metabolism of labelled nutrients. An exciting
with strong cell‐to‐cell variation on both the host cell and the Salmo- recent study tracked incorporation of deuterium‐labelled drinking water
nella side (Bumann, 2015; Helaine & Kugelberg, 2014; Knodler, 2015; of Leishmania‐infected mice into macromolecules of the parasites
Kreibich & Hardt, 2015; Mills & Avraham, 2017; Saliba & Vogel, 2017). (Kloehn, Saunders, O'Callaghan, Dagley, & McConville, 2015).
To understand how this complex and changing metabolic land- Complementary information can be obtained from enzyme
scape supports and/or limits Salmonella fitness, we need to address proteomics. A general finding from various studies has been the large
the following questions: (a) Which nutrients are available for intracellu- number and high expression levels of many metabolic enzymes indicat-
lar Salmonella, and which host cell supply routes ensure sufficient ing substantial Salmonella resource allocation into metabolism. Abso-
replacement of consumed nutrients? (b) Which pathways do Salmo- lute quantification yields enzyme copy numbers per Salmonella cell
nella employ to utilise these nutrients for energy production and bio- (Steeb et al., 2013). Combination with tabulated turnover numbers kcat
mass generation, and to what extent can Salmonella compensate for yields maximal reaction rates vmax for hundreds of metabolic conver-
perturbations of these processes by employing alternative, partially sions providing large‐scale information on feasible metabolic pathways
redundant pathways? (c) What are the consequences for host cell activities (Schubert et al., 2015; Steeb et al., 2013). Interpretation of
physiology and metabolism? (d) What is the level of heterogeneity in such data is, however, complicated by the fact that many enzymes
host and Salmonella metabolism, and are there particularly important have broader substrate spectrum besides their normally considered
subsets with unusual properties? specificities (“substrate promiscuity,” e.g., pentoses in addition to
hexoses; Khersonsky & Tawfik, 2010). In some cases, such secondary
reactions might be actually more relevant. Furthermore, a substantial
4 | M E T H O D S F O R I N V E S TI G A T I N G fraction of metabolic reactions is nonenzymatic, and protein data thus
I N T R A C E LL U L A R SALM ONELLA M E T A B O L I S M can offer only incomplete coverage of metabolic networks (Keller,
Piedrafita, & Ralser, 2015).
The most direct experimental technique to monitor host and Salmo- An additional layer of complexity is provided by the observation
nella metabolism is metabolomics, which can reveal both metabolite that the majority of metabolic reactions are dispensable for Salmonella
concentrations and metabolic fluxes (Zampieri, Sekar, Zamboni, & fitness, because alternative pathways and supplementation by host
Sauer, 2017). Infected cells contain both host and Salmonella metabo- metabolites often provide partial redundancy, or pathway products
lites, and available separation techniques are too slow compared to are not required (Becker et al., 2006; Bumann, 2009; Steeb et al.,
the turnover rates for most metabolites, making assignment difficult 2013). Several genome‐scale genetic screens and numerous more
(except for a few kingdom‐specific metabolites such as peptidoglycan focused studies have revealed a few metabolic mutations with severe
and lipid precursors, and secondary metabolites). Mass imaging tech- phenotypes. This may indicate true pathway relevance, or artefacts
niques might offer a solution, but spatial resolution is still insufficient such as toxicity of truncated gene products and polar effects on down-
for distinguishing intracellular Salmonella from surrounding host cell stream genes, or in particular accumulation of toxic upstream interme-
contents (Petras, Jarmusch, & Dorrestein, 2017). diates. Such artefacts can be minimised by clean deletion of genes
Even if we could determine metabolite concentrations in Salmo- encoding enzymes at metabolism branching points that lead into the
nella and their micro‐environment, such data would not necessarily pathway of interest (“first committed step”), with other branches buff-
reflect their relative importance. Nutrients might be present at low ering potential build‐up of toxic precursors. Access to essential bio-
concentration but still have high turnover rates with vigorous Salmo- mass precursors can be inferred from auxotrophic strains that
nella salvage and rapid host replenishment, resulting in an important depend on external supplementation. Interpretation should consider
contribution to Salmonella fitness. Instead of metabolite concentra- wasteful Salmonella degradation of such precursors (e.g., dispensable
tions, metabolic fluxes are therefore more relevant. To obtain such Salmonella lysine decarboxylase, which lowers lysine availability below
data, one can follow the fate of isotope‐labelled glucose and the incor- biomass needs; Steeb et al., 2013). Another complication could be
poration of each atom into various amino acids (that are stably retained moonlighting functions (see above). Finally, single mutants can only
as part of proteins; Eisenreich et al., 2015; Sauer, 2006). Amino acid identify nonredundant pathways. To determine the relevance of much
labelling patterns reflect their origin at various central carbon metabo- more frequent partially redundant pathways, multiple mutations must
lism intermediates, enabling the reconstruction of major fluxes in these be combined, and epistasis can provide further insights into pathway
central pathways. Interpretation of results for intracellular Salmonella architecture (Ideker & Krogan, 2012).
can be complicated due to direct transfer of labelled host amino acids into Understanding the metabolism network underlying Salmonella fit-
Salmonella (Gotz, Eylert, Eisenreich, & Goebel, 2010). Such approaches ness during infection requires genome‐scale integration of all comple-
mostly focus on incorporation of 13C and/or 15N isotope labels into Sal- mentary data sets. The consensus in silico reconstruction of Salmonella
monella biomass. In addition, it can be useful to detect waste products metabolism that was obtained in a jamboree involving more than 50
generated by fundamentally important energy conversion pathways researchers provides a suitable basis for this (Thiele et al., 2011). It
and released into the cell culture medium (Garcia‐Gutierrez et al., accounts for 1,270 metabolic genes, 2,201 metabolic reactions that
2016; Kentner et al., 2014). All these techniques might be also applica- were curated for thermodynamically feasible reversibility, and 1,110
ble for in vivo analysis as Salmonella can be purified from infected host metabolites. This reconstruction reveals all known metabolic pathways
 14625822, 2017, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cmi.12766 by Cochrane Chile, Wiley Online Library on [04/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
4 of 11 BUMANN AND SCHOTHORST

and their interconnectivity as a basis for studying the entire Salmonella Salmonella can also directly modulate host amino acid abundance.
metabolism network in different external conditions and internal Salmonella catabolism of host asparagine causes T‐cell suppression
states. in vitro but has no detectable impact in vivo (Kullas et al., 2012), sug-
The substantial progress in understanding intracellular Salmonella gesting other reasons for fitness contributions of Salmonella asparagine
metabolism is encouraging, but one crucial, previously neglected catabolism (Jelsbak et al., 2014). Salmonella induces host arginase II
aspect is just emerging. All experimental and in silico methods are expression (Lahiri, Das, & Chakravortty, 2008), in addition to “stealing”
based on population averages, but recent work has shown extensive of host arginine by ArgT‐mediated uptake (Das et al., 2010). Both pro-
heterogeneity between infected host cells and individual Salmonella cesses could deprive host cells of a precursor for generating antimicro-
cells in terms of microenvironments, stress, gene expression, overall bial nitric oxide, but Salmonella subsets experience substantial nitric
metabolic activities, growth rates, and cell fates (Bumann, 2015; oxide levels during infection (Burton et al., 2014) suggesting sufficient
Helaine & Kugelberg, 2014; Knodler, 2015; Kreibich & Hardt, 2015; arginine availability. Moreover, Salmonella arginine deiminase contrib-
Mills & Avraham, 2017; Saliba & Vogel, 2017). Differential nutrient utes to in vivo fitness without affecting host nitric oxide production
access seems to cause heterogeneous Salmonella growth rates during (Choi et al., 2012), and arginine seems to serve as Salmonella carbon/
systemic infection in vivo, with important consequences for disease nitrogen source (Steeb et al., 2013). Salmonella also induces host tryp-
progression and tolerance against antimicrobial chemotherapy (Claudi tophan catabolites with immunomodulatory properties in the blood-
et al., 2014). New approaches for Salmonella single cell and subpopula- stream of humans and mice (Blohmke et al., 2016).
tion analysis will be needed to unravel this fascinating new aspect of One major question is how the host supplies nutrients for intracel-
host‐Salmonella interactions. lular Salmonella (Figure 1b). One fascinating mechanism could be the
formation of a large membrane network called Salmonella‐induced fila-
ments (Sif; Liss et al., 2017). Salmonella can induce Sif with effector
5 | M E T A BOL I C P A TT ER N S O F proteins that it injects into the host cell cytosol using the Type 3 secre-
I N T R A C E LL U L A R SALM ONELLA I N I N V I T R O tion system encoded on SPI‐2. Sif continuously merge with endosomes
C EL L C U L T U R E M O D EL S providing a gateway between extracellular metabolites, which the host
cell takes up by pinocytosis, and the Salmonella‐containing vacuole
Salmonella metabolism in cell culture infections has been covered in (Drecktrah, Knodler, Howe, & Steele‐Mortimer, 2007; Liss et al.,
excellent recent reviews (Dandekar et al., 2014; Eisenreich et al., 2017). Indeed, this can be the major nutrient delivery pipeline for
2015), and we just summarise some findings here. Cell culture infec- driving intracellular Salmonella growth (Holzer & Hensel, 2012; Liss
tions recapitulate a central hallmark of salmonellosis, intracellular Sal- et al., 2017). An additional host cell nutrient supply pathway is
monella replication in host cells. Compared to in vivo studies, cell chaperone‐mediated autophagy providing peptides directly to the
culture infections enable detailed experimental analysis with a broad Salmonella‐containing vacuole (Singh et al., 2017), which works inde-
spectrum of techniques. Cell culture conditions such as medium com- pendently of any extracellular nutrient supply.
position and oxygen tension can be freely adjusted according to the
specific research questions, and defined pulses of isotopically labelled
nutrients enable tracking of the kinetics of metabolite conversion in 6 | FUTURE GOALS FOR IN VITRO CELL
both host and Salmonella cells. Another advantage is the focus on a CULTURE STUDIES
single host cell type simplifying analysis and interpretation of results
compared to complex in vivo situations. An important goal is to define meaningful criteria for in vitro cell cul-
The results obtained so far indicate a major role of glucose as car- ture conditions that reproduce key aspects of host‐Salmonella interac-
bon/energy source, under some, but not all, experimental conditions tions during salmonellosis. Recent studies have revealed dramatic
(Bowden, Rowley, Hinton, & Thompson, 2009; Bowden et al., 2014; differences in Salmonella metabolism and virulence mechanisms
Dandekar et al., 2014; Eisenreich et al., 2015; Garcia‐Gutierrez et al., depending on particular host cell types and experimental conditions,
2016; Holzer & Hensel, 2012; Liu et al., 2017; Popp et al., 2015; Singh and it remains often unclear, which patterns are most pertinent for
et al., 2017; Steeb et al., 2013). Peptides/amino acids (Popp et al., understanding actual disease processes.
2015; Singh et al., 2017) and other nutrients such as C3 (pyruvate Most studies used cancer cell lines that are easy to cultivate and
and/or glycerol) or C2 (acetate and/or fatty acids) metabolites (Gotz grow, and are permissive for Salmonella proliferation. However, com-
et al., 2010) are also available to intracellular Salmonella. Salmonella pared to primary cells, these cancer cells have distorted metabolism
degrades these nutrients with its central carbon metabolism pathways (respiration/fermentation, pentose pathway, lipid biosynthesis/β‐
such as glycolysis (Bowden et al., 2014; Garcia‐Gutierrez et al., 2016) oxidation, etc.; Pavlova & Thompson, 2016), which might affect the
and the tricarboxylic acid (TCA) cycle (Bowden, Ramachandran, metabolism of intracellular Salmonella. The major model for epithelial
Knudsen, Hinton, & Thompson, 2010) and uses various intermediates cell infections is HeLa cells that are cervix carcinoma cells carrying
of these pathways to synthesise several amino acids in addition to the human papilloma virus 18 genome. Salmonella infection biology in
amino acids obtained from the host cell (Gotz et al., 2010). However, HeLa cells differs in important aspects from (possibly more relevant)
results have been quite diverse depending on specific host cells and polarised epithelial cells, even when compared under identical condi-
conditions (Figure 1a). Clarification of the most relevant setting should tions (Figure 1a). This includes differential overall growth rates; rele-
be a priority of future work (see below). vance of glucose and other nutrients; divergent roles of glycolysis,
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BUMANN AND SCHOTHORST 5 of 11

FIGURE 1 Evidence for core Salmonella metabolic activities and nutrient supply routes. (a) Experimental evidence for Salmonella intracellular
metabolism in various cell culture models and during systemic salmonellosis in the mouse typhoid fever model. Data for intracellular growth,
glucose as a major nutrient, key metabolic pathways (EMP = Embden–Meyerhof–Parnas pathway; TCA = tricarboxylic acid cycle; Aerob
Resp = aerobic respiration), and the role of Salmonella‐induced filaments (Sif) for nutrient supply are shown (++ = strong effect; + = significant but
moderate effect; − = no detectable impact; −− = growth‐diminishing effect). Data were collected from various studies: 1(Knodler et al., 2010),
2
(Holzer & Hensel, 2012), 3(Helaine et al., 2010), 4(Claudi et al., 2014), 5(Bowden et al., 2009), 6(Bowden et al., 2014), 7(Garcia‐Gutierrez et al.,
2016), 8(Singh et al., 2017), 9(Steeb et al., 2013), 10(Bowden et al., 2010), 11(Tchawa Yimga et al., 2006), 12(Becker et al., 2006), 13(Craig et al., 2013),
14
(Popp et al., 2015), 15(Liss et al., 2017), 16(Lathrop et al., 2015), and 17(Grant et al., 2012). (b) Possible supply routes for host nutrients and toxic
molecules in Salmonella‐infected cells. Orange symbols represent various compounds

overflow metabolism leading to acetate secretion, purine biosynthesis, status and source of host cells (cancer cell lines, and primary bone mar-
and chorismate biosynthesis (a key pathway for classifying biosafety of row‐derived macrophages; Figure 1a). Examples include the differen-
Salmonella mutants); and essentiality/dispensability of Sif formation tial relevance of glucose for Salmonella nutrition; divergent roles of
(Bowden et al., 2014; Garcia‐Gutierrez et al., 2016; Holzer & Hensel, Salmonella TCA cycle and overflow metabolism; and differential
2012; Liu et al., 2017; Lorkowski, Felipe‐Lopez, Danzer, Hansmeier, requirements for Sif (Bowden et al., 2009; Garcia‐Gutierrez et al.,
& Hensel, 2014; Popp et al., 2015; Singh et al., 2017). 2016; Lathrop et al., 2015; Popp et al., 2015) and extracellular small
Similarly, Salmonella metabolism and virulence mechanisms show metabolites that could be supplied via Sif (Singh et al., 2017). Interest-
remarkable differences depending on the macrophage activation ingly, one study suggested that SPI‐2 and its role in Sif formation are
 14625822, 2017, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cmi.12766 by Cochrane Chile, Wiley Online Library on [04/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
6 of 11 BUMANN AND SCHOTHORST

actually dispensable for intracellular growth in phagocytes in vivo. Salmonella subpopulation, (Knodler, 2015; Knodler et al., 2010).
Instead, SPI‐2 seems to play a major role in Salmonella cell‐to‐cell Although their differential growth rates must correspond to striking
spreading within host tissues (Grant et al., 2012), which is not the differences in biomass generation and the entire metabolism network
focus of most in vitro cell culture studies although it is an essential part (Knodler, 2015; Knodler et al., 2010; Wrande et al., 2016), and recent
of Salmonella in vivo fitness. Another particularly concerning discrep- evidence reveals distinct gene expression patterns and genetic deter-
ancy is the apparent dispensability of Salmonella respiration for growth minants for the two subpopulations (Knodler, 2015; Knodler et al.,
in macrophages in vitro (Garcia‐Gutierrez et al., 2016), whereas aerobic 2010; Wrande et al., 2016), most HeLa infection studies merely deter-
respiration is one of the key metabolic activities supporting Salmonella mined average properties. Such data initially reflect mostly phagosomal
fitness in vivo (Steeb et al., 2013; Turner et al., 2003). Salmonella, but later predominantly the overgrowing cytosolic Salmo-
Another caveat concerning in vitro Salmonella studies is the com- nella subset. Consequently, data for early time points largely ignore
mon use of cell culture media that contain nonphysiological metabolite the properties of a nevertheless particularly important Salmonella sub-
concentrations, which might affect both host cell and intracellular Sal- set (the cytosolic escapers), and differences along the time course
monella metabolism. Intestinal epithelial cells (enterocytes) that are could be misinterpreted as changing metabolic patterns of Salmonella
one important infected host cell type in vivo do, for example, not nor- cells, instead of changing contributions of two distinct subsets. It is
mally depend on glucose but rather on microbiota‐derived short‐chain possible that cytosolic Salmonella resemble cytosolic Shigella, which
fatty acids as energy source although this can change during gut rely on conversion of host‐derived pyruvate to acetate as the major
dysbiosis (Rivera‐Chavez et al., 2016). energy conversion pathway (Kentner et al., 2014). In particular, Salmo-
To deal with this complexity and to ensure that the results are nella can grow even without glycolysis in these cells, excretes large
meaningful for understanding disease mechanisms in salmonellosis, amounts of acetate, and partially depends on acetyl‐CoA to acetate
we propose to derive a couple of decisive benchmarks from in vivo conversion (Bowden et al., 2014; Garcia‐Gutierrez et al., 2016; Liu
studies and to use these benchmarks for establishment of appropriate et al., 2017).
in vitro cell culture models. As an example, Salmonella fitness in macro- Traditional bulk average read‐outs neglect this heterogeneity and
phage cell cultures should depend on respiratory pathways as it does could thus result in misleading interpretations. Single‐cell approaches
in vivo. Glucose should contribute as a carbon and energy source, increasingly unravel the molecular differences between Salmonella
but only as one limited contribution among several nutrients (Bowden and host cell subsets, but the corresponding metabolic patterns are still
et al., 2009; Steeb et al., 2013; whereas it could be more relevant dur- largely unknown.
ing chronic infections, Eisele et al., 2013). Benchmarks for metabolic
pathways relevant for Salmonella fitness in epithelial cells in vivo are
still largely lacking, but their establishment could be straightforward 7 | SALM ONELLA M E T A B O L I S M D U R I N G
with Salmonella mutants in the mouse enteritis and/or neonate infec- SYSTEMIC INFECTIONS
tion models. In particular, the role of central metabolic pathways and
host cell processes such as Sif formation or chaperone‐activated In vivo models reflect relevant conditions during salmonellosis com-
autophagy should be clarified in vivo. Once such criteria are pared to more ambiguous cell culture conditions. For practical reasons,
established, host cell types and culture conditions could be optimised most studies used a typhoid fever model of systemic salmonellosis in
in an iterative manner until key aspects of Salmonella intracellular genetically susceptible mice. This model differs in some important
metabolism in vivo are faithfully reproduced in vitro. A final step could aspects from human systemic salmonellosis (Santos et al., 2001), but
be to reproduce the intracellular environment and Salmonella exploita- the limited available information suggests at least similarities in Salmo-
tion of accessible nutrients as well as defence against toxic molecules, nella metabolism as compared to genetically resistant mice (Steeb et al.,
in axenic cultures (without any host cell). This would be particularly use- 2013) and human volunteers immunised with attenuated S. enterica
ful for screening of novel antimicrobials under meaningful conditions. serovar Typhi (Bumann et al., 2000). A major drawback of in vivo stud-
A second focus of future studies of Salmonella in vitro cell culture ies is the severely restricted number of suitable experimental methods
infection models should be on heterogeneity. Most previous investiga- for investigating metabolism, and the much higher complexity of the
tions have determined average properties, but recent studies clearly host micro‐environment with multiple cell types and strong inflamma-
revealed striking differences between individual Salmonella cells but tion dynamics. Under these circumstances, there are at present essen-
also substantial cell‐to‐cell variation among host cells. Salmonella sub- tially only two applicable methods to study Salmonella metabolism:
sets with divergent growth rates and metabolic activities exist in proteomics to determine enzyme copy numbers and competitive infec-
infected macrophages (Claudi et al., 2014; Diacovich, Lorenzi, tions to determine fitness defects of metabolic mutants. On the other
Tomassetti, Meresse, & Gramajo, 2016; Helaine et al., 2010; Helaine, hand, contributions of many research groups over more than 3 decades
Cheverton, et al., 2014), and properties and fates of individual Salmo- have accumulated an astonishing amount of information that enabled
nella‐infected macrophages are also highly variable (Avraham et al., us to derive a genome‐scale metabolic reconstruction of Salmonella
2015; McQuate et al., 2017; Saliba et al., 2016; Thurston et al., metabolism in infected mouse spleen (Steeb et al., 2013; Figure 2).
2016). Striking heterogeneity has also been observed in epithelial HeLa Subsequent publications corrected some aspects: hydrogen oxidation
cells. Most Salmonella remain initially in phagosomes where they not required (Maier et al., 2013; Maier et al., 2014); reconsideration
slowly proliferate, but some Salmonella escape to the host cell cytosol of proline biosynthesis mutants for inferring limited proline availability
where they can vigorously proliferate and overgrow the phagosomal (Lee, Choi, & Groisman, 2014); and contribution of asparagine
 14625822, 2017, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cmi.12766 by Cochrane Chile, Wiley Online Library on [04/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
BUMANN AND SCHOTHORST 7 of 11

FIGURE 2 Experimental evidence for nutrient supply and enzyme essentiality for Salmonella during systemic salmonellosis in the mouse typhoid
fever model. On top, nutrients are shown with font size corresponding to differential supply rates. The Salmonella metabolism network is shown
below in a schematic overview with lines (representing enzymes) connecting symbols (metabolites). Enzymes are shown in different colours that
represent enzyme relevance for Salmonella in vivo fitness. A fully annotated version of this scheme is available at http://www.biozentrum.unibas.
ch/personal/bumann/steeb_et_al/index.html

deamination and polyamine synthesis for Salmonella fitness (Jelsbak amounts, or not at all, by the host such as peptidoglycan, riboflavin,
et al., 2014). However, this did not have much impact on the overall unsaturated fatty acids, and ubiquinone (Becker et al., 2006; Bumann,
properties of the Salmonella metabolism network in vivo. 2009; Steeb et al., 2013).
One striking finding is the large complexity of the in vivo host‐ As discussed above, Salmonella has access to a wide variety of dif-
Salmonella nutritional interface (Figure 2). Salmonella has access to ferent host nutrients. However, this does not necessary imply
more than 50 diverse host nutrients comprising multiple carbon/ favourable conditions as available amounts could be still scarce. Quan-
energy and nitrogen sources, all amino acids (with limiting amounts titative simulation of the entire Salmonella metabolic network based on
for proline), many provitamins, and inorganic nutrients. Although an all available experimental data indeed suggested a severe overall nutri-
individual nutrient, glycerol, plays a key role, the broad availability of ent limitation of Salmonella growth, suggesting that although the host
many different nutrients makes Salmonella metabolism largely resilient micro‐environment is qualitatively rich (providing many different nutri-
against perturbations. This in part explains the seemingly paradoxical ents), it is quantitatively poor (nutrients are available in only scarce
finding that Salmonella invests major resources into metabolic enzymes amounts) resulting in slow growth with an average generation time
suggesting a crucial importance of metabolism for Salmonella fitness around 6 hr (Becker et al., 2006; Claudi et al., 2014).
in vivo, yet only few metabolic mutations show remarkable infection Salmonella metabolism does not only depend on nutrient access.
phenotypes (Becker et al., 2006; Bumann, 2009; Steeb et al., 2013). Host cells can also attack Salmonella with toxic molecules such as nitric
Many other microbial pathogens likely have access to similar complex oxide and reactive oxygen species (ROS), which could interfere with
host nutrients based on widespread auxotrophies for amino acids, Salmonella metabolism and consume reducing equivalents for detoxifi-
nucleosides, and (pro)vitamins (Steeb et al., 2013). cation and repair. Nitric oxide can block Salmonella respiration (Husain
Salmonella degrades many of these nutrients primarily through the et al., 2008), but exposed Salmonella upregulate the detoxifying
Embden–Meyerhof pathway (and to some extent also pentose– enzyme NO dioxygenase HmpA that lowers nitric oxide levels suffi-
phosphate and Entner–Doudoroff), followed by the TCA cycle coupled ciently to prevent fitness defects (Burton et al., 2014). Similarly, Salmo-
with aerobic respiration involving ubiquinone as the main energy pro- nella that are exposed to moderate levels of reactive oxygen species in
viding pathway. Anaerobic respiration is dispensable (Craig, Sadik, resident macrophages upregulate catalases and peroxidases that
Golubeva, Tidhar, & Slauch, 2013) although Salmonella expresses sev- together with generally expressed superoxide dismutase ensure
eral enzymes that could mediate such energy conversion pathways uncompromised fitness (Burton et al., 2014). By contrast, neutrophils
in vivo (Steeb et al., 2013). Vulnerable “Achilles heels” are rare in Sal- attack Salmonella with much higher levels of ROS that overwhelm Sal-
monella metabolism and are almost entirely restricted to biosynthesis monella defences (Burton et al., 2014; Schurmann et al., 2017). Both
of essential biomass components that are not provided in sufficient Salmonella defences against nitric oxide and ROS require reducing
 14625822, 2017, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cmi.12766 by Cochrane Chile, Wiley Online Library on [04/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
8 of 11 BUMANN AND SCHOTHORST

equivalents, but this puts only small additional demands on Salmonella vacuoles in macrophages, (b) determine nutrient access in Salmonella
metabolism (in the range of 10% of total electron flow). Activated mac- wild‐type and SPI‐2 mutants, and (c) clarify the importance of vesicular
rophages also express cis‐aconitate decarboxylase (Irg1; Michelucci trafficking and alternative mechanisms for nutrient delivery to the Sal-
et al., 2013), which produces itaconate. Itaconate could inhibit the Sal- monella‐containing vacuoles. Another very recent study proposes that
monella glyoxylate shunt, but concentrations are probably too low for host cell chaperone‐mediated autophagy provides peptides (and possi-
fitness impairment, especially because Salmonella does not depend bly other host macromolecule degradation products) directly to the
on the glyoxylate shunt during acute infections (Fang, Libby, Castor, Salmonella‐containing vacuole (Singh et al., 2017). Although this supply
& Fung, 2005; Kim, Brinsmade, Yang, Escalante‐Semerena, & Fierer, route appears to have limited relevance for fitness of wild‐type
2006). On the other hand, itaconate also inhibits host succinate dehy- Salmonella in in vitro cell culture infections (in contrast to a peptide‐
drogenase resulting in succinate accumulation, which increases inflam- dependent mutant), this mechanism might still play a role in vivo.
matory responses (Cordes et al., 2016) and modulates host fatty acid Methods that have been established to purify and analyse the
metabolism and ROS production (Hall et al., 2013). phagosomal membrane from in vitro infected cells (Herweg et al.,
As might be expected from the striking variation among both Sal- 2015; Vorwerk, Krieger, Deiwick, Hensel, & Hansmeier, 2015) might
monella and infected host cells in vitro (see above), Salmonella growth be applicable to in vivo conditions to obtain informative comprehen-
and metabolism is also highly heterogeneous in vivo (Bumann, 2015; sive data on suitable marker proteins (in addition to immunohisto-
Claudi et al., 2014; Helaine, Cheverton, et al., 2014). This is in part a chemistry data) to address these issues.
consequence of differential stress conditions and Salmonella toxin
expression, but varying access to host nutrients and inhomogeneous ACKNOWLEDGEMENTS
activities across the entire Salmonella metabolism network are also We thank past and present lab members for their contributions and
involved. The individual relevance of the many metabolic differences Max–Planck–Gesellschaft, Deutsche Forschungsgemeinschaft,
is still largely unclear, but growth patterns of a purine auxotrophic Schweizerischer Nationalfonds, and SystemsX.ch for funding our work
mutant indicate divergent Salmonella access to purines in vivo. Inter- on pathogen metabolism.
estingly, nutrient access seems to be rather homogeneous within one
Salmonella microcolony in one host cell but can be very different in CONFLIC T OF IN TE RE ST
neighbouring host cells suggesting that host cell properties might
We declare to have no conflicts of interest.
modulate nutrient supply (Claudi et al., 2014).

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