A rich microbiome, cellular and abiotic mucosal barriers, as well as cellular and humoral effectors of the innate and adaptive immune system may limit the colonization, growth and spread of pathogenic bacteria in mammalian hosts (Rosales and Uribe-Querol, 2017; Levinson et al., 2018; Uribe-Querol and Rosales, 2020). Hosts use metal transporters, calprotectins, siderocalins and a variety of other metal-sequestering proteins to limit the availability of Mg²⁺, Fe²⁺, Mn²⁺ and Zn²⁺ ions from bacteria, a phenomenon known as nutritional immunity (Bellamy, 2003; Loomis et al., 2014; Hennigar and McClung, 2016; Wang et al., 2018; Cunrath and Bumann, 2019; Monteith and Skaar, 2021). Bacteria display sophisticated transport systems that counteract metal restrictions imposed by hosts (Porcheron et al., 2013; Chandrangsu et al., 2017). Among the bioactive metal ions, Mn²⁺ plays a salient role in bacterial physiology and the adaptation of prokaryotic cells to stress (Papp-Wallace and Maguire, 2006). Mn²⁺ is a cofactor of enzymes involved in carbon and nucleotide metabolism, DNA replication, and protein translation (Lovley and Phillips, 1988; Martin and Imlay, 2011; Culotta and Daly, 2013; Torrents, 2014; Kaur et al., 2017; Tong et al., 2017; Daniel et al., 2018; Li and Yang, 2018; Hutfilz et al., 2019). Mn²⁺ is also a cofactor of superoxide dismutase (SOD) and catalase (KatN) family members, and this divalent metal is utilized by carbon utilization enzymes such as phosphoglycerate mutase (PGM) and fructose-1,6-bisphosphate phosphatase, or envelope stress phosphatases (Shi et al., 2001; Miriyala et al., 2012; Whittaker, 2012; Radin et al., 2019). The MntR repressor coordinates a Mn²⁺ ion and the ferric uptake regulator (Fur) can bind to Fe²⁺ or Mn²⁺ (Hiemstra et al., 1999; Kehres et al., 2002a; Ikeda et al., 2005; Waters, 2020).

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen associated with intestinal and invasive diseases in humans and animals (Galan, 2021). The success of Salmonella as a pathogen can be directly ascribed to its capacity to overcome colonization resistance by resident gut microbiota, invade epithelial cells, and establish intracellular infections within host enterocytes and macrophages (Winter and Bäumler, 2011; Lorkowski et al., 2014; Lhocine et al., 2015; Aljahdali et al., 2020; Jiang et al., 2021; Powers et al., 2021). During their associations with host cells, Salmonella are exposed to acid pH, oxidative and nitrosative stress, and nutritional deprivation (Fang et al., 2016). A variety of adaptive mechanisms are used by Salmonella to fight the hostile host environment (Bernal-Bayard and Ramos-Morales, 2018; Tanner and Kingsley, 2018; Gogoi et al., 2019), and a few reviews have examined the contribution of Mn²⁺ in Salmonella pathogenesis (Kehres and Maguire, 2003; Papp-Wallace and Maguire, 2006; Osman and Cavet, 2011). Despite these advances, many unanswered questions and occasional contradictory findings warrant a review of our current understanding of Mn²⁺-mediated stress responses in Salmonella pathogenesis. In this review, we discuss the adaptive mechanisms that allow Salmonella to compete with the host for Mn²⁺, and present ways by which this divalent metal contributes to metabolic programs associated with resistance of Salmonella to oxidative and nitrosative stress.

Hosts sequester transition metals in their fight against pathogenic organisms (Hennigar and McClung, 2016). Metal sequestration is mediated by calprotectin, lipochalin 2, metallothioneins, ferritin and transport systems including NRAMP1. Mn²⁺ limitation is mostly mediated by calprotectin and NRAMP1.

Entry of Mn²⁺ into the periplasmic space is mostly mediated by non-specific porins on the outer membrane, whereas transporters in the cytoplasmic membrane actively influx this divalent metal cation into the bacterial cytoplasm (Figure 1A). MntH, a proton-dependent NRAMP1 homolog, actively imports Mn²⁺ with a K _m of 0.1 μM (Kehres et al., 2000). Salmonella upregulate MntH expression in response to both low Mn²⁺ concentrations and oxidative stress (Kehres et al., 2002a; Cunrath and Palmer, 2021). MntH mutant Salmonella grow poorly in mouse macrophages, but replicate rather well in mice (Boyer et al., 2002; Zaharik et al., 2004; Diaz-Ochoa et al., 2016). The slight attenuation of mntH deficient Salmonella suggests the existence of redundant transport systems. Genetic analyses revealed the presence of a Mn²⁺ responsive element in the promoter of the sitABCD operon that is encoded within the Salmonella pathogenicity island-1 gene cluster (Janakiraman and Slauch, 2000; Kehres et al., 2002b). The SitABCD transporter is a typical ABC transporter consisting of a periplasmic-binding protein SitA, an ATP-binding protein SitB and two integral membrane permeases, SitC and SitD. Initially, the SitABCD transporter was thought to function as a Fe²⁺ transport system, as this locus is regulated by Fur under iron-limiting conditions (Janakiraman and Slauch, 2000; Ikeda et al., 2005). However, metal ion uptake studies have shown that SitABCD mediates influx of Mn²⁺ with an apparent affinity of 0.1 μM, transporting Fe²⁺ with 30–100 times lower efficiency (Kehres et al., 2002b). A third transporter with broad cation specificity, ZupT, has also been implicated in Mn²⁺ transport in Salmonella during nitrosative stress (Yousuf et al., 2020).

Low Mn²⁺ concentrations activate the expression of MntH and SitABCD (Kehres and Maguire, 2003; Chandrangsu et al., 2017; Bosma et al., 2021). The mntH gene is repressed by both MntR and Fur in response to high Mn²⁺ and iron replete conditions, respectively (Kehres et al., 2000; Kehres et al., 2002a; Troxell et al., 2011; Powers et al., 2021). Expression of mntH is also induced by peroxide via H₂O₂-sensing OxyR regulatory protein (Kehres et al., 2002a) (Figure 1C). The persistence of Mn²⁺-dependent repression of mntH in ΔmntR Salmonella points to the existence of MntR-independent control. Analysis of the mntH 5′ UTR identified an Mn²⁺-sensing riboswitch that forms a Rho-independent terminator (Shi et al., 2014; Scull et al., 2020) (Figure 1C). The presence of several transcriptional breakpoints suggests dynamic expression of MntH in diverse host niches. MntR, Fur and OxyR elements are also present in the sitABCD promoter (Ikeda et al., 2005). The sitABCD operon does not contain, however, a riboswitch-like sequence at the 5′ UTR. Despite the similarities in transcriptional control and their identical affinity for Mn²⁺, MntH and SitABCD have specialized functions. MntH primarily transports Mn²⁺ at acidic pH, whereas SitABCD preferentially works at alkaline pH (Kehres et al., 2002b), suggesting that Salmonella may preferentially utilize MntH or SitABCD at different anatomical locations or times during infection (Ikeda et al., 2005).

mntH mutant Salmonella display little attenuation, whereas sitABCD mutants are attenuated in systemic models of infection (Janakiraman and Slauch, 2000; Boyer et al., 2002; Zaharik et al., 2004). Attenuation of sitABCD mutant Salmonella is contingent on the presence of host NRAMP1 (W. P. Loomis et al., 2014; Zaharik et al., 2004). Additionally, the non-specific transporter ZupT also competes with the host NRAMP1 for Mn²⁺ metal ions in phagosomes, contributing to Salmonella virulence (Karlinsey et al., 2010). By competing for Mn²⁺ with host cell calprotectin, MntH and SitABCD Mn²⁺ transport systems help Salmonella overgrow commensals in the inflamed gut (Diaz-Ochoa et al., 2016). Acquisition of Mn²⁺ also powers detoxification of ROS produced by inflammatory neutrophils in the gut mucosa (Diaz-Ochoa et al., 2016). The role of MntH and SitABCD may not be limited to extracellular bacteria, as genes encoding these Mn²⁺ transporters are transcribed in Salmonella residing in the cytosol of epithelial cells, and mntH and sitA Salmonella mutants grow poorly in the cytosol of epithelial cells (Powers et al., 2021). The acquisition of Mn²⁺ by cytosolic Salmonella may counteract oxidative stress, while facilitating utilization of sugars (Powers et al., 2021).

Salmonella deficient of MntH, SitA and ZupT transporters still acquire trace levels of Mn²⁺ in vitro, indicating the presence of other import systems (Karlinsey et al., 2010; Yousuf et al., 2020). The search for other modes of Mn²⁺ import into Salmonella is still underway, and examples from other bacteria may lead to the discovery of novel Mn²⁺ uptake systems in Salmonella. Burkholderia pseudomallei import Mn²⁺ via a type VI secretion system (T6SS) and the TonB cell envelope protein (Si et al., 2017; DeShazer, 2019). B. pseudomallei undergoing oxidative stress secrete the Mn²⁺-binding T6SS effector TseM, and low Mn²⁺ concentrations induce expression of the Mn²⁺ specific, TonB-dependent MnoT integral protein (Figure 1A). TseM scavenges extracellular Mn²⁺ and actively shuttles the metal via MnoT. Salmonella T6SS is activated by oxidative stress and our bioinformatics analysis has revealed a conserved locus in the Salmonella genome with high similarity to Burkholderia MnoT (Figures 1B,D) (Kroger et al., 2013). Additional T6SS substrates with Mn²⁺-scavenging capacities are still being uncovered, including the TssS micropeptide from Yersinia pseudotuberculosis that chelates Mn²⁺ and sabotages bacterial clearance by inhibiting STING-mediated innate immune response (Zhu et al., 2021).

Paradoxically, excessive Mn²⁺ evokes oxidative stress in E. coli, affecting protein stability, interfering with envelope biogenesis, disrupting iron homeostasis and diminishing both tricarboxylic acid cycle and electron transport chain functions (Kaur et al., 2017). Bacteria excrete excessive Mn²⁺ using both the LysE superfamily MntP protein, and the cation diffuser facilitator (CDF) family member MntE (Martin et al., 2015). The Xanthomonas MntP homolog has two DUF204 domains that are conserved in Salmonella MntP protein (Li et al., 2011). MntP is regulated at transcriptional and posttranscriptional levels via MntR, mismetallated Fur and a yybP-ykoY riboswitch (Dambach et al., 2015; Bosma et al., 2021). The expression of the small RNA rybA, which encodes the small protein MntS, is repressed by MntR (Waters et al., 2011; Martin et al., 2015). The accumulation of apo-MntR in Mn²⁺ starving cells activates production of MntS, which represses MntP efflux activity and thus enlarges the intracytoplasmic pool of Mn²⁺ (Martin et al., 2015). On the other hand, the yybP-ykoY riboswitch directly binds to Mn²⁺, stabilizing a secondary structure that prevents sequestration of the mntP ribosome-binding site during translation (Dambach et al., 2015). MntP mediates efflux of Mn²⁺ ions in Salmonella following nitrosative stress (Ouyang et al., 2022).

The second efflux pump MntE is widely distributed in Gram-positive bacteria (Chandrangsu et al., 2017; Lam et al., 2020). Streptococci bearing mutations in mntE harbor excessive intracellular Mn²⁺ and experience attenuation of virulence (Rosch et al., 2009). Our bioinformatic analysis shows that Salmonella FieF (Yiip) protein belonging to CDF family has around 26%–64% sequence similarity with Streptococcus MntE, although it mediates zinc and iron export (Grass et al., 2005; Huang et al., 2017).

Salmonella are exposed to ROS generated in the innate host response. Sulfur-containing cysteine and methionine amino acids are primary targets of H₂O₂ (Bin et al., 2017). In addition, ROS carbonylate arginine, lysine, proline and threonine residues, and oxidize metal prosthetic groups and histidine residues (Ortiz de Orue Lucana et al., 2012; Chang et al., 2020). Salmonella have evolved diverse mechanisms to counter ROS generation, prevent formation of hydroxy radicals, inhibit delivery of ROS into Salmonella-containing vesicles, detoxify and scavenge ROS, or repair the resultant protein and DNA modifications (Buchmeier et al., 1995; De Groote et al., 1997; Vazquez-Torres et al., 2000a; Vazquez-Torres et al., 2000b; Gallois et al., 2001; Vazquez-Torres and Fang, 2001; Waterman and Holden, 2003; Halsey et al., 2004; Aussel et al., 2011; Bogomolnaya et al., 2013; Song et al., 2013; Rhen, 2019; Bogomolnaya et al., 2020; Shome et al., 2020). Of particular interest to this review, Mn²⁺ protects Salmonella from ROS-mediated cytotoxicity by serving as a cofactor for SOD and KatN enzymes, replacing Fe²⁺ in the active sites of mononuclear iron-containing enzymes, and acting as a nonproteinaceous antioxidant (Culotta and Daly, 2013; Imlay, 2014; Ighodaro and Akinloye, 2018).

Growth of Salmonella in host cells relies on a versatile metabolism. Relevant to this review, Mn²⁺ impacts glycolysis, reductive TCA and the pentose phosphate pathway in Salmonella sustaining oxidative stress.

Reactive nitrogen species synthesized by inducible nitric oxide (NO) synthase are bacteriostatic against Salmonella (Vazquez-Torres et al., 2000a; Thiele et al., 2011; Henard and Vázquez-Torres, 2012; Fitzsimmons L. F. et al., 2018). RNS modify biomolecules containing radicals, heme prosthetic groups, mononuclear iron, [Fe-S] clusters or redox active thiols in cysteine residues (Mikkelsen and Wardman, 2003; Forman et al., 2004; Poole, 2005; Husain et al., 2008; Pearce et al., 2009; Crawford et al., 2016; Jones-Carson et al., 2016; Jones-Carson et al., 2020). Salmonella mutants lacking Mn²⁺ transporters are more sensitive to RNS (Frawley et al., 2018; Yousuf et al., 2020; Ouyang et al., 2022). The intracellular concentrations of Mn²⁺ increase in Salmonella undergoing nitrosative stress, likely reflecting the upregulation of Mn²⁺ importers (Richardson et al., 2011; Yousuf et al., 2020). Regulation of Mn²⁺ transport systems is under the control of the transcription factor DksA (Crawford et al., 2016). Maintaining the homeostasis of intracellular Mn²⁺ in Salmonella after exposure to NO also involves the MntP and FieF efflux pumps (Ouyang et al., 2022). It remains unknown if these Mn²⁺ efflux systems contribute to the antinitrosative defenses of Salmonella.

Transient drops in intracellular amino acids during NO stress induce RelA-catalyzed synthesis of the (p)ppGpp alarmone, and the hydrolytic activity of SpoT is essential for reestablishing ppGpp homeostasis (Richardson et al., 2011; Fitzsimmons L. F. et al., 2018). A Mn²⁺ ion is coordinated by at least two carboxylates from aspartate and glutamate residues in the hydrolase domain of SpoT (Hogg et al., 2004). Histidine along with H₂O molecules coordinate the rest of the four electrons of Mn²⁺. Studies in E. coli and Streptococcus revealed that ppGpp hydrolysis is strictly dependent on Mn²⁺ ions (Heinemeyer et al., 1978; Mechold et al., 1996). The nucleotidyltransferase domain (cd05399) of SpoT has a metal binding region dominated by aspartate and glutamate residues. Interestingly, the aspartate and glutamic acid residues of CD05399 are conserved in the synthetase domain rather than the hydrolase domain of SpoT, raising the possibility that Mn²⁺ may catalyze ppGpp synthesis rather than hydrolysis in Salmonella’s SpoT proteins. However, studies in Streptococcus revealed that intramolecular signal transmission between the two domains in the presence of ppGpp creates an allosteric shift in the synthetase domain, resulting in coordination of Mn²⁺ by an additional aspartate (Hogg et al., 2004). The additional coordination suppresses synthetase enzymatic activity. Interestingly, intracellular Salmonella lacking the non-catalytic regulatory C-terminal domain of SpoT (i.e., SpoT-ΔCTD) transcribe abnormally low levels of the Mn²⁺ importer SitABCD in macrophages (Fitzsimmons et al., 2020). SpoT-ΔCTD-expressing Salmonella are attenuated in NRAMP1⁺ C3H/HeN mice but not in NRAMP1^- C57BL/6 mice, suggesting that the SpoT-dependent regulation of SitABCD combats the Mn²⁺ restrictions associated with a functional NRAMP1 transporter (Fitzsimmons et al., 2020).

Mn²⁺ homeostasis plays a poorly understood, but vital role in Salmonella pathogenesis. The elaborate transcriptional and posttranscriptional regulation of expression of diverse Mn²⁺ uptake and efflux systems help Salmonella navigate different metal-restricted anatomical sites in the vertebrate host. Historically, Mn²⁺ has been recognized as a cofactor of critical antioxidant defenses of Salmonella. Mn²⁺-dependent SOD protects Salmonella from oxidative stress; a function for Mn²⁺-dependent catalase in Salmonella virulence remains to be determined. Recent investigations have revealed the intricate relations between Mn²⁺ homeostasis, central metabolism, and antioxidant defenses of Salmonella. Salmonella rely on Mn²⁺ independent glycolysis during their adaptations to oxidative killing, but use Mn²⁺ to power anapleurotic pentose phosphate pathway reactions involved in redox balance necessary for central metabolism and synthesis of reductive power that fuels classical antioxidant defenses, such as glutathione reductase. Many unanswered questions still exist about the involvement of Mn²⁺ in the adaptations that promote Salmonella pathogenesis, providing a myriad of opportunities for future research.