Like most metal micronutrients, Mn is both essential for viability and toxic in excess. Identification of bacterial Mn transporters and the capacity of eukaryotic hosts to manipulate bacterial Mn levels revealed that Mn regulation is important for bacterial virulence (Kehres and Maguire, 2003; Kehl-Fie and Skaar, 2010). Early studies established that bacteria actively acquire Mn to serve as a cofactor for diverse enzymes and provide protection against reactive oxygen species (ROS) (Kehres and Maguire, 2003). Proteins mediating Mn import were identified in the late 1990’s, followed by characterization of Mn-binding transcription factor families (DtxR and Fur variants) that regulated importer expression (Kehres and Maguire, 2003; Lisher and Giedroc, 2013; Chandrangsu et al., 2017). Thereafter, identification of Mn exporters led to characterization of a Mn-binding cis-acting RNA regulatory element (riboswitch) (Waters, 2020). Additional Mn-enzymes (Bosma et al., 2021) and regulatory mechanisms at the post-transcriptional and protein stability levels continue to be defined.

While the components of bacterial Mn homeostasis systems were being uncovered, mechanistic models of metal selectivity were developed to account for how proteins avoid mismetallation (Waldron and Robinson, 2009). In general, proteins are thought to acquire common transition metals (iron (Fe), Zn, and Mn) from distinct metal pools with differential exchangeability (e.g., a labile pool associated with small molecules such as phosphate vs. a less available pool sequestered in proteins) (Imlay, 2015; Foster et al., 2022). To prevent mismetallation, bacteria adjust intracellular metal pools so that proteins preferentially acquire their cognate metal.

Both Mn deficiency and toxicity are key problems that bacteria must avoid. How Mn deprivation occurs, particularly during pathogenesis, and its consequences for bacterial survival has been reviewed elsewhere (Juttukonda and Skaar, 2015; Sheldon and Skaar, 2019). In contrast, it is less clear how bacteria are subject to Mn toxicity during infection and what outcomes excess Mn has on physiology. Although the Mn-responsive regulatory and homeostasis machinery shared by most bacteria is well defined (Figure 1, and see (Bosma et al., 2021)), emerging regulatory mechanisms and additional specialized Mn importers relevant to pathogenesis continue to be uncovered.

Fundamental insights about bacterial physiology in pure culture have advanced understanding of the complex interplay in Mn usage and homeostasis in diverse ecosystems, and the field is ripe to examine the contribution of Mn-responsive systems during infection in the host. Here, we summarize recent advances in Mn stress responses and provide an outlook for the near future.

Beyond established modes of regulation of Mn homeostasis (Figure 1), new findings are expanding the repertoire of regulatory approaches bacteria employ to maintain optimal cellular Mn levels (Figure 2). The fact that bacteria have evolved intricate measures to control Mn usage demonstrates its importance to cellular fitness.

Although the suite of bacterial Mn transporters is well understood (Figure 1A), additional players continue to be identified. More types of Mn importers are emerging as important contributors to Mn uptake under specialized conditions, including stresses relevant to pathogenesis. These include ZIP family importers, outer membrane porins, and type VI secretion systems (T6SS).

Although primarily associated with Zn uptake, ZIP family importers can import additional metals, including Fe, Mn, and cobalt (Grass et al., 2005). Recently ZIP family members ZupT and TmpA were determined to be important under Mn stress during pathogenesis in Salmonella and Streptococcus (Yousuf et al., 2020; Puccio et al., 2022). zupT deletion in Salmonella eliminated residual Mn import in cells lacking the Mn-dedicated importers MntH and SitA and showed increased nitric oxide sensitivity (Yousuf et al., 2020). Similarly, deletion of tmpA in Streptococcus cells lacking the Mn importer SsaACB exacerbated a growth defect in serum and required higher exogenous Mn to rescue growth compared to the single ΔssaACB mutant (Puccio et al., 2022). Together, these studies demonstrate an auxiliary role for ZIP family transporters in Mn uptake during virulence across diverse bacterial phyla.

Outer membrane proteins (OMPs) can also contribute to Mn transport. In Bradyrhizobium, the OMP MnoP directly mediates Mn import in proteoliposomes and is required in vivo for Mn uptake and survival during Mn scarcity (Hohle et al., 2011). In Vibrio cholerae, no dedicated Mn importers have been identified, but strains lacking the porin OmpU show decreased H₂O₂ resistance and reduced intracellular Mn (Jiang et al., 2020). Overexpression of ompU leads to increased intracellular Mn. Since ompU expression is induced by the ToxR virulence regulator, OmpU-mediated Mn import is likely important for Vibrio pathogenesis. These data suggest an underappreciated role for OMPs in Mn import and overall Mn homeostasis. A novel metal transport mechanism using T6SS effectors (TseM and TssS), together with an additional outer membrane TonB-dependent Mn transporter (MnoT), has also recently been described (Si et al., 2017; Yang et al., 2021; Zhu et al., 2021). In Burkholderia, TseM is secreted into the extracellular environment, where it specifically binds Mn and delivers it to MnoT for transport (Si et al., 2017). The small protein TssS produced by Yersinia is delivered into mammalian host cells, where it sequesters Mn. TssS-mediated Mn chelation reduces activity of the Mn-dependent enzyme cGAS, which activates the STING pathway of the innate immune response (Wang et al., 2018; Zhu et al., 2021). It is unclear if TssS also mediates Mn import into bacterial cells or if it primarily remains in the host cytoplasm in its Mn chelator role. Bacterial virulence and/or intracellular survival is reduced in both systems when the effectors or OM transporter are deleted (Si et al., 2017; Zhu et al., 2021). It will be interesting to see if additional Mn-binding proteins are found, as T6SS effector proteins are poorly characterized.

Mn influences dynamic and context-dependent processes such as bacterial virulence and biofilm formation. To do so, Mn acts in competition with other metals found within the intracellular metal milieu which is comprised of labile (freely accessible) metal ions and protein-bound (poorly accessible) metal complexes. The size and speciation of the labile metal pool is imprecisely known, especially for Mn which exists predominately in the spectroscopically hard-to-detect +2 oxidation state in cells and binds weakly to biomolecules (Irving and Williams, 1948). Nonetheless, the labile pool provides sufficient metal ions to ensure proper function of metalloenzymes, or—if perturbed—to cause mismetallation and Mn toxicity. We discuss here the interplay between Mn and other metal cations that serve as cofactors in bacterial proteins.

Fundamental studies of bacteria in pure culture have defined the strategies microbes use to acquire and remove Mn to combat Mn fluctuations, as well as the intracellular usage of Mn in enzymes and ROS detoxification. It is now commonly accepted that Mn availability, whether limited or in excess, can affect bacterial fitness. However, deciphering the functions of Mn is challenging as there is significant interplay between Mn and other metals. Many studies do not fully mimic the natural habitat where pathogenic bacteria derive Mn in hosts, but are essential for building fundamental understanding of how metals impact bacterial infection. With this knowledge, defined questions can be asked in the more complex multi-organism infection system.

In mammalian hosts, inflammation leads to rapid readjustment of the metal milieu to disrupt metal homeostasis, exploiting both essentiality and toxicity of metals to control bacterial infection. Host dietary metal uptake further influences the metal landscape (Lopez and Skaar, 2018). At the molecular level, single metal ion perturbations can significantly impact protein function. At the cellular level, metals are transported into and out of eukaryotic cells and their intracellular organelles to withhold metals or intoxicate bacteria (Figure 1B). Host siderophores and S100 proteins also sequester metal ions at the infection site. At the organismal level, host-mediated metal changes may manifest differently, depending on the site of infection and bacterial species involved (Lopez and Skaar, 2018). In a seminal study extending basic findings from pure culture into an animal model, mice fed a high-Mn diet had increased Mn levels that promoted S. aureus virulence in the heart by protecting bacteria from ROS, while not affecting other bacterial species in other niches (Juttukonda et al., 2017).

In addition to Mn deficiency imposed by nutritional immunity, it is clear that bacteria are vulnerable to excess Mn, given the ubiquity and numerous types of Mn exporters across bacteria. Pathogenic bacteria lacking Mn exporters also have reduced colonization and virulence (Johnsrude et al., 2019; Lam et al., 2020; Waters, 2020). However, it remains an open question where and how bacteria experience Mn toxicity within eukaryotic hosts. Although it is difficult to measure physiological levels of labile Mn in cells and tissues, monitoring Mn exporter expression levels during infection could provide some insight. More defined studies and new technologies are needed to elucidate Mn distribution under pathophysiological conditions. Such information will be instrumental in developing alternative antimicrobial therapies.

In summary, new modes of regulation of Mn homeostasis and intracellular management demonstrate that Mn is critical for bacterial growth and virulence. We anticipate additional post-transcriptional and post-translational regulatory mechanisms affecting Mn levels and usage to be uncovered. We have likely only begun to understand the scope of how metal balances affect physiological responses through enzymes regulated by differential metallation. We look forward to pathogenesis studies which examine how Mn factors are used by bacteria growing in different host niches to counteract host immune defenses. New technologies that can quantify labile metal pools, as well as total Mn levels, in real-time in response to changing conditions would significantly aid the endeavor to characterize the bacteria-host interaction.