Calcium Rescues Streptococcus pneumoniae D39 ΔmntE Manganese-Sensitive Growth Phenotype

Abstract

Calcium (Ca²⁺) functions as a universal signal messenger in eukaryotes but in bacteria, the physiological roles for Ca²⁺ are limited. Here, we examine the role of Ca²⁺ in Streptococcus pneumoniae during manganese (Mn²⁺) intoxication. S. pneumoniae mntE mutants, lacking the Mn²⁺ efflux transporter, exhibit impaired growth due to accumulation of Mn²⁺ when exposed to elevated exogenous Mn²⁺. This Mn²⁺-sensitive growth defect is restored to wild-type growth level by exogenous Ca²⁺, in a Ca²⁺-dependent manner. Despite growth restoration of the mntE mutant to wild-type levels, cellular Mn²⁺ remains elevated in this strain. Bacterial capsule production is also increased for the mntE mutant, resulting in reduced adherence capacity to surfaces and poor biofilm formation, which is consistent with it experiencing Mn²⁺ intoxication. Ca²⁺ presence did not significantly impact bacterial capsule production or biofilm formation. Further analysis of the cell morphology demonstrates that Ca²⁺ contributes to cell division and reduces cell chain lengths. Together, these data describe the first role of Ca in S. pneumoniae that has potential implications in bacterial virulence since Ca affects cell division and likely Mn²⁺-associated cellular processes.

1. Introduction

Calcium (Ca²⁺) is an essential metal micronutrient present in the eukaryotic host environment that plays both structural and regulatory roles in a myriad of cellular processes ranging from cell division, transport, to host immune defenses against pathogens [1,2]. However, in bacteria, the physiological roles for Ca²⁺ remain elusive. A growing body of evidence indicates that Ca²⁺ may serve as a signaling molecule in regulating bacterial cellular processes from cell division, transport, stress response, competence, to lifestyle switches that include sporulation and biofilm formation [3,4,5,6,7].

More than three-quarters of all human microbial infections are due to biofilms [8,9]. These biofilms are not only associated with medical devices but also with the mucosa of the gastrointestinal and respiratory tracts. The concentration of Ca²⁺ in respiratory-associated human body fluids is 1–5 mM [10] and is up to 10,000 times higher than host cell cytosolic Ca²⁺ concentrations [11,12]. Bacterial pathogens can induce changes in host cell cytosolic Ca²⁺ concentrations via bacterial surface-associated protein or toxin interactions with host cells [13,14,15]. Such alterations in host Ca²⁺ levels have been shown to facilitate bacterial adherence, enhance cell aggregation, and increase the release of extracellular DNA, all of which contribute to strengthening biofilms formed by Streptococcus strains [16,17]. We note here that adherence is necessary for initial Streptococcus pneumoniae colonization in the nasopharynx of the human upper respiratory tract, and that biofilm formation is important for S. pneumoniae persistence and survival within the host [18].

In S. pneumoniae, Ca²⁺ is an obligatory metal micronutrient required at 150 µM for viability in laboratory medium [19]. Increasing the Ca²⁺ concentration to ≥1 mM induces two different cell states: genetic competence during exponential growth to take up DNA and cell lysis when entering the stationary phase of growth to release DNA [19,20]. It is important to note that S. pneumoniae is an extracellular pathogen and its natural environment is human body fluids in which the Ca²⁺ concentration as described above is ≥1 mM, the concentration at which S. pneumoniae competence and lysis are obtained under laboratory conditions [19].

As with host cells, the intracellular Ca²⁺ concentration range in bacteria is 80–100 nM [21,22], indicating that bacteria like their hosts also regulate Ca²⁺ storage and flux across their membranes. It is also imperative that bacterial pathogens sense Ca²⁺ levels, since Ca²⁺ concentrations differ among host infection sites and fluctuate during bacterial disease progression. To date, several Ca²⁺-sensing and Ca²⁺-dependent regulatory systems, in addition to homologs of eukaryotic Ca²⁺-leak channel and pumps, have been identified among bacteria that function in virulence and pathogenesis [10]. Much remains to be learned for Ca²⁺ homeostasis in many bacterial pathogens, including S. pneumoniae.

In comparison to Ca²⁺, Mn²⁺ is well recognized to serve a versatile role in bacteria at the host–pathogen interface, aiding in bacterial virulence, and is both essential for bacterial viability and toxic in excess [23]. As such, many bacteria, including Streptococcus pneumoniae, employ complex regulatory networks to control Mn²⁺ homeostatic levels that optimize colonization and growth within the host. Like Ca²⁺, the Mn²⁺ concentration varies across body sites and fluid ranging from 650 nM in the nasopharynx where S. pneumoniae commonly colonizes to 1100 nM Mn²⁺ in the lung; the blood contains 400–700 nM Mn²⁺ [24]. Note that these concentrations reflect total Mn²⁺ and do not represent the bioavailable free Mn²⁺ that bacteria like S. pneumoniae physically experience and use during pathogenesis in the host. As such, the topic of when and where do pathogenic bacteria experience Mn²⁺ intoxication remains obscure. It is hypothesized that Mn²⁺ levels are not stagnant in the host, and that S. pneumoniae must be equipped to quickly adapt to constant metal ion fluctuations.

S. pneumoniae acquires Mn²⁺ via the Mn²⁺-specific ABC-type importer PsaBCA, which is negatively regulated by the Mn²⁺-dependent DtxR family transcriptional corepressor PsaR [25,26]. Excess cellular Mn²⁺ is exported out of the cell by the Mn²⁺-specific cation diffusion facilitator (CDF) transporter MntE to prevent Mn²⁺ intoxication [27]. An additional metal-dependent P_1B-type ATPase efflux transporter MgtA (formerly CaxP) is shown to function as a failsafe when cellular Mn²⁺ reaches ≥100 µM [28]. Previous characterization of the regulation of mgtA expression by a 5′ Mn/Ca²⁺-sensing yybP-ykoY family riboswitch revealed a potential intersectional role between Ca²⁺ and Mn²⁺ [28], prompting deeper examination of the Ca²⁺ impact on S. pneumoniae growth during Mn²⁺ stress.

This study is the first step in uncovering such cross-sectional roles for Ca²⁺ in Mn²⁺ physiological processes in S. pneumoniae by delving into the molecular complexities surrounding the cellular metal ion composition and its effect on bacterial virulence components.

2. Materials and Methods

2.1. Bacterial Growth Conditions

Brain heart infusion (BHI) and Todd Hewitt (TH) broth were of standard composition (BD Biosciences, San Jose, CA, USA) and prepared using ultrapure water (≈17.1 Ω). Both BHI and TH are general-purpose rich media for cultivation of fastidious microorganisms, including Streptococcus strains. Bacterial strains used in this study are listed in Table S1. All S. pneumoniae strains were grown in BHI or TH broth at 37 °C in a 5% CO₂ atmosphere. Briefly, S. pneumoniae bacterial frozen stocks were inoculated into broth, serially diluted, and propagated overnight. The next morning, exponentially growing cultures were diluted to 0.005 OD₆₂₀ in prewarmed broth supplemented with MnCl₂ and CaCl₂ as indicated, and growth was monitored over time at OD₆₂₀.

All Escherichia coli and Bacillus subtilis strains were grown in BHI broth aerobically at 37 °C with vigorous shaking. Briefly, single colonies were inoculated into broth and propagated overnight. The next morning, overnight cultures were diluted to 0.01 OD₆₂₀ in prewarmed broth. After approximately four generations of growth, exponentially growing cells were diluted to 0.005 OD₆₂₀ into fresh prewarmed broth supplemented with MnCl₂ and CaCl₂ as indicated, and growth was monitored over time at OD₆₂₀.

2.2. Dilution Drop Test

S. pneumoniae cultures were grown in BHI broth to approximately 0.40 OD₆₂₀, then serially diluted 10-fold to 10⁻⁵ in BHI broth. An aliquot of 5 µL of each dilution was spotted onto BHI agar plates containing 250 U/mL of filtered bovine liver catalase (Worthington Biochemical, Lakewood, NJ, USA) and varying concentrations of MnCl₂ and CaCl₂ as indicated [29]. All plates were incubated at 37 °C in a 5% CO₂ atmosphere for 24 h and documented photographically.

2.3. Metal Quantification

The total cell-associated metal ions for Mn²⁺, Fe²⁺, Zn²⁺, Cu²⁺, Mg²⁺, and Ca²⁺ were quantified from S. pneumoniae cultured for 3.5 h in BHI broth with MnCl₂ and CaCl₂ as indicated using slightly modified standard protocol previously described [30,31]. Briefly, cells (200 mL) were harvested by centrifugation at 4 °C, suspended in 1/200 volume of original culture with cold phosphate-buffered saline (PBS) pH 7.4, 2 mM EDTA, and washed twice with cold metal-free PBS (10 g/L chelex-100 resin was used to remove metals) pH 7.4 before storing at -80 °C. Metal quantifications of inactivated cell pellets were determined by the Center for Applied Isotope Studies (University of Georgia, Athens) using a Perkin Elmer 8300 ICP-OES (Perkin Elmer, Shelton, CT, USA). Metal quantifications were normalized to wet cell weight (wcw).

2.4. Uronic Acid Assay

S. pneumoniae was grown for 3.5 h in BHI broth with MnCl₂ and CaCl₂ as indicated prior to determining uronic acid concentrations using a standard protocol previously described [29,32]. Briefly, 10 mL culture was harvested by centrifugation after approximately 3.5 h of growth in BHI with MnCl₂ or CaCl₂ as indicated. Cell pellets were suspended in 1/20 the original culture volume with 150 mM Tris-HCl (pH 7.0)/1 mM MgSO₄, incubated with 0.1% deoxycholate, and subjected to enzymatic digestion of the cell wall (100 U mutanolysin), nucleic acids (0.1 mg/mL DNase and 0.1 mg/mL RNase), and proteins (0.1 mg/mL proteinase K). The supernatant was collected after centrifugation, mixed with a 98% (v/v) sulfuric acid–12.5 mM tetraborate solution, boiled, cooled on ice, and mixed with 0.15% (w/v) 3-phenylphenol in 0.5% NaOH. The absorbance was read at 520 nm at room temperature within 5 min. Samples incubated with 0.5% NaOH served as the background, and measurements were subtracted out prior to normalization to total protein determined using the DC protein assay (Bio-Rad, Hercules, CA, USA).

2.5. Bacteria Adherence Assay

S. pneumoniae capsule properties were assessed using a mucoviscosity assay adapted from Walker, K.A. et al., 2019 [29,33]. Briefly, S. pneumoniae strains were grown to approximately 0.20–0.40 OD₆₂₀ in BHI with MnCl₂ and CaCl₂ as indicated and harvested by centrifugation at 4 °C. Cell pellets were resuspended in 1/10 (WT strain) or 1/30 (ΔmntE strain) the original culture volume with cold PBS pH 7.4. Cell suspensions were then normalized to 1.0 OD₆₂₀/1 mL with cold PBS pH 7.4, centrifuged at 1000× g for 3 min at 4 °C, and the OD₆₂₀ of supernatant was measured. The fraction of cells remaining in solution was calculated by dividing the final OD₆₂₀ by the initial OD₆₂₀. Measurements were performed in duplicate for each independent growth condition. We note that S. pneumoniae strains expressing thicker capsules do not form tight pellets, and the supernatants therefore have higher absorbance readings [29].

2.6. Biofilm Quantification

Biofilm formation on abiotic surfaces was assessed by crystal violet staining similar to published protocols [34,35]. Briefly, S. pneumoniae was grown in TH broth to approximately 0.4 OD₆₂₀ and harvested by centrifugation at 4 °C. Cell pellets were resuspended and diluted to 0.04 OD₆₂₀ in 1 mL TH broth, TH/0.2% D-glucose, or TH/0.3% yeast extract. Each media composition was examined with or without 300 µM MnCl₂ and/or 1 mM CaCl₂. Equal aliquots of diluted cell suspension and their corresponding media composition were transferred to a sterile 96-well round-bottom plate (Fisher brand, Rockingham County, NH, USA) in triplicate. After 24 h incubation at 37 °C in a 5% CO₂ atmosphere, plates were washed with ultrapure water, stained with 0.1% crystal violet for 15 min, and air-dried before being solubilized with 95% ethanol. Absorbance was measured at 570 nm. Each media composition was used as a standard negative control for background through the process.

2.7. RNA Extraction and Differential Sequence Analysis

Total RNA was extracted using standard protocol from S. pneumoniae grown for 3.5 h in BHI with MnCl₂ and CaCl₂ as indicated. Total RNA samples devoid of DNA were sent to the Molecular Research Core Facility (Idaho State University, Pocatello) to determine RNA integrity and further preparation. Briefly, rRNA was depleted and cDNA libraries were generated using the NEBNext ultra II directional RNA library kit (New England Biolabs, Ipswich, MA, USA). Libraries were quantified by Qubit and pooled in equimolar concentrations. Sequencing was performed by the Nevada Genomics Center (University of Nevada, Reno) on one NextSeq2000 P2 SE100 (Illumina, San Diego, CA, USA) 200-cycle sequencing run.

Raw sequencing reads from mRNAseq were quality- and adapter-trimmed using Cutadapt V4.9 [36] with a minimum read length cutoff of 20 nucleotides, prior to mapping on the S. pneumoniae D39 (RefSeq NC_0085833) genome using Bowtie2 with the very sensitive option. Read counts were tabulated with HTSeq [37], and differential gene expression analysis was performed as described previously using DEseq2 [38]. Genes were defined as differentially expressed if their p-value adjusted for multiple testing (P_adj) was <0.05 and transcript level change was 1.5-fold.

2.8. Cell Morphology Measurements

Images of S. pneumoniae cells grown for 3.5 h in BHI broth with or without MnCl₂ or CaCl₂ as indicated were collected using a Leica DM6B upright brightfield microscope (Leica, Deerfield, IL, USA). The lengths and widths of approximately 100–150 cell bodies for each condition were measured using ImageJ software version 1.51m9, Java 1.8.0_101 (64-bit). The number of cell bodies were counted from 50–100 chains to determine average number of cells/chain.

2.9. Statistical Analysis

All growth and assays were independently conducted in at least triplicate. Statistical analyses were performed using GraphPad Prism (Graphpad Prism 9 Software, La Jolla, CA, USA). One-way ANOVA and unpaired t-tests were selected with 95% confidence interval. Accepted p-values are indicated in figures.

3. Results

3.1. Ca Rescues Mn²⁺-Sensitive S. pneumoniae ΔmntE Growth Phenotype

Previous reports demonstrate that S. pneumoniae lacking the Mn²⁺-specific CDF efflux transporter MntE (ΔmntE) is sensitive to elevated exogenous Mn²⁺ due to its inability to export Mn out of the cell and is thus considered Mn²⁺-stressed [27,30,39]. Likewise, we show here that the S. pneumoniae ΔmntE strain fails to reach wild-type (WT) cell densities after 8 h when cultured in rich medium with excess exogenous Mn (Figure 1). The addition of 1 mM Ca²⁺ to Mn²⁺-stressed ΔmntE permits growth patterns similar to unstressed cells, resulting in the complete rescue of its Mn²⁺-sensitive growth defect (Figure 1B,C). Ca²⁺ alone in the absence of Mn²⁺ stress had no significant effect on the growth of both the WT and ΔmntE strains (Figure 1).

To further evaluate the ability of Ca²⁺ to rescue the ΔmntE strain Mn stress growth defect, a serial dilution spot test was performed on rich agar medium supplemented with catalase to prevent hydrogen peroxide intoxication and allow S. pneumoniae colonies to form. In comparison to the WT strain, the ΔmntE mutant showed significantly diminished growth at 500 µM Mn²⁺ and no growth was observed ≥700 µM Mn (Figure 2A, top). In the presence of 1 mM Ca²⁺, the ΔmntE mutant resembled a WT strain growth pattern up to 500 µM Mn²⁺ (Figure 2A, bottom). Residual growth up to 10⁻² dilution of the ΔmntE mutant was observed at 700 µM Mn²⁺ in the presence of Ca²⁺ (Figure 2A).

The Mn concentration was then fixed at 500 µM to examine the minimum Ca²⁺ concentration needed for the recovery of the ΔmntE mutant to the WT strain or unstressed ΔmntE mutant growth levels. During Mn²⁺ stress, residual growth was observed for the ΔmntE strain when Ca²⁺ was supplied between 100 and 500 µM. The growth density of the ΔmntE strain rose as the Ca²⁺ levels increased (Figure 2). Complete rescue of Mn²⁺-stressed ΔmntE was only visible for Ca²⁺ concentrations ≥1 mM (Figure 2B). These findings are consistent with those observed in broth cultures (Figure 1) and together demonstrate that Ca²⁺ presence is capable of impacting S. pneumoniae growth and likely has an intersecting role with Mn metabolism.

3.2. Ca²⁺ Rescue of Mn²⁺ Sensitivity Varies among Bacteria

Given the significant impact of Ca²⁺ on S. pneumoniae growth during Mn²⁺ stress and the importance of maintaining optimum intracellular Mn²⁺ concentrations among pathogenic bacteria in general, we examined the extent to which Ca²⁺ could rescue other bacteria from Mn²⁺ stress. Two individual bacteria were chosen, B. subtilis and E. coli, whose Mn²⁺ homeostatic mechanisms including import, export, and their regulation are well characterized [40,41]. We considered B. subtilis to be characteristically similar to S. pneumoniae in that both are Gram-positive organisms possessing a Mn²⁺-centric physiology, requiring relatively high Mn²⁺ levels for growth compared to other bacteria [30,40,42]. In contrast, E. coli is a Gram-negative organism with an iron-centric physiology that conditionally imports Mn²⁺ in response to oxidative stress or Fe²⁺ scarcity [43].

Akin to the S. pneumoniae WT strain (Figure 1A), the addition of Ca²⁺ or Mn²⁺ to rich growth medium did not significantly impact the growth of either the B. subtilis or E. coli WT strains (Figure 3A,D). Consistent with the published literature [41,44], B. subtilis, impaired in regulating intracellular Mn²⁺ levels (ΔmntR), and E. coli, deficient in Mn²⁺ efflux (ΔmntP), exhibited reduced growth in the presence of 50 and 500 µM Mn²⁺, respectively (Figure 3B,E). This reduced growth results from Mn²⁺ intracellular accumulation and intoxication [41,44]. The addition of 1 mM Ca²⁺ in the presence of Mn²⁺ stress did not significantly impact the growth of the B. subtilis ΔmntR strain (Figure 3B,C). We note that Mn²⁺-stressed B. subtilis ΔmntR cultures reached WT stationary cell density after 6 h incubation, independent of Ca²⁺ presence (Figure 3B). Incubation of B. subtilis ΔmntR with increased Mn²⁺ (100 µM) enhanced the growth defect, which was not further affected by Ca²⁺ presence (Figure S1).

In contrast to the B. subtilis ΔmntR strain growth, the addition of 1 mM Ca²⁺ during Mn²⁺ stress partially restored the growth of the E. coli ΔmntP mutant to half that of the WT strain cell density (Figure 3D,F). Together, these data along with those of S. pneumoniae suggest that the roles of Ca²⁺ in relation to cellular Mn²⁺ demand likely vary among bacteria and may instead depend on host environment nutrient conditions and other natural environmental selective pressures.

3.3. S. pneumoniae ΔmntE Accumulates High Cellular Mn²⁺ Despite Growth Restoration by Ca²⁺

To investigate the mechanism by which Ca²⁺ rescues the S. pneumoniae ΔmntE strain Mn²⁺-sensitive growth phenotype, we next assessed the metal ion composition of bacterial cells. During routine growth in rich medium, the S. pneumoniae WT strain cell-associated Ca²⁺ and Mn²⁺ concentration yielded 3.8 ± 0.15 and 2.3 ± 0.07 µg/g wcw (wet cell weight), respectively (Figure 4B). Likewise, similar Ca²⁺ and Mn²⁺ levels were observed for unstressed ΔmntE cells, 4.1 ± 0.09 and 2.6 ± 0.06 µg/g wcw, respectively. Cellular Ca²⁺ increased 4.5-fold to 17.0 ± 0.05 and 18.0 ± 1.05 µg/g wcw for S. pneumoniae WT and ΔmntE strains, respectively, when cultured in the presence of 1 mM Ca²⁺ (Figure 4B), indicating that S. pneumoniae is capable of importing Ca²⁺ but the mechanism of how Ca²⁺ enters S. pneumoniae is not understood at this time. Incubation with Ca²⁺ alone did not significantly alter other cellular metals examined, including Mn²⁺, zinc (Zn²⁺), iron (Fe²⁺), and copper (Cu²⁺), for both the WT and ΔmntE strains (Figure 4A,D) but did slightly reduce magnesium (Mg²⁺) by ≈18% from approximately 89.4 ± 0.45 to 74.8 ± 0.25 µg/g for WT and 93.5 ± 1.00 to 76.0 ± 0.20 µg/g wcw for ΔmntE (Figure 4C).

Culturing S. pneumoniae WT and ΔmntE strains with 300 µM Mn yielded a 3-fold and 8-fold increase in cell-associated Mn for the WT (2.25 ± 0.70 vs. 6.71 ± 0.51 µg/g wcw) and ΔmntE (2.56 ± 0.06 vs. 16.4 ± 0.55 µg/g wcw) strains, respectively (Figure 4B). The significant accumulation of cellular Mn²⁺ is consistent with the ΔmntE mutant being incapable of efficiently exporting Mn²⁺ out of the cell [27,39]. Interestingly, the Ca²⁺ levels increased 2-fold to 9.1 ± 1.7 µg/g wcw for the Mn²⁺-stressed ΔmntE mutant but were relatively unchanged for the WT strain when compared to unstressed cells (Figure 4B). The ΔmntE mutant cell-associated Mg²⁺ levels were reduced by almost 2-fold during Mn²⁺ stress, independent of Ca²⁺ presence (Figure 4C). Cell-associated Fe²⁺ and Cu²⁺ rose 1.5-fold (from 3.5 ± 0.28 to 5.3 ± 0.12 µg/g wcw) and 5-fold (from 0.8 ± 0.04 to 4.5 ± 0.46 µg/g wcw), respectively, for the ΔmntE mutant during Mn²⁺ stress (Figure 4D), which is consistent with previous reports showing elevated Mn²⁺ interferes with the expression of Fe²⁺ and Cu²⁺ homeostatic proteins [30]. No significant change in Zn²⁺ was observed for the WT strain cultured with Mn (Figure 4D); Zn²⁺ was below the quantifiable detection for the ΔmntE mutant. We note here that a high cellular Mn²⁺ concentration can disrupt Zn²⁺ homeostasis in S. pneumoniae by possibly binding to the Zn²⁺-sensing regulatory protein SczA and antagonizing its activity to properly regulate the expression of the Zn²⁺-specific CDF exporter CzcD [45] (see Section 3.6 for further support).

When strains were cultured with 1 mM Ca²⁺ during Mn²⁺ stress, the cell-associated Mn²⁺ levels remained elevated for the ΔmntE mutant at 16.1 ± 0.30 µg/g wcw (Figure 4B), indicating that the ΔmntE strain continues to experience Mn²⁺ intoxication and that exogenous Ca²⁺ is not inhibiting Mn²⁺ import and likely not altering Mn²⁺ export through the secondary Mn²⁺ efflux transporter MgtA [28]. Fe²⁺ and Cu²⁺ were significantly reduced 5-fold and 3-fold, respectively, when Ca²⁺ was added to the Mn²⁺-stressed ΔmntE strain (Figure 4D); no significant change in Fe²⁺ or Cu²⁺ was observed for the WT strain.

3.4. Ca²⁺ Does Not Alter S. pneumoniae Capsular Polysaccharide Production

To confirm that the S. pneumonie ΔmntE strain was still physically experiencing Mn²⁺ intoxication despite growth rescue, we next evaluated the production of CPS using two different methods: uronic acid assay (a component of the S. pneumoniae D39 bacterial capsule) [32] and mucoviscosity [29,33]. During Mn²⁺ stress, elevated intracellular Mn²⁺ binds to and activates phosphoglucomutase Pgm, a Mn²⁺-requiring enzyme that is critical for the S. pnuemoniae biosynthesis of CPS [29]. As such, hyperactivation of phosphoglucomutase Pgm by Mn²⁺ leads to increased CPS production, which prevents S. pneumoniae cell adherence to surfaces [29]. For these experiments, the concentration of Mn²⁺ was reduced to 100 µM to ensure growth of the ΔmntE mutant over time during Mn²⁺ stress [29,30].

Direct measurement of uronic acid revealed the WT strain CPS production was not significantly altered by Ca²⁺ or Mn²⁺ (Figure 5A). Compared to the WT strain, the unstressed ΔmntE mutant produced 24% more uronic acid (Figure 5A). The ΔmntE strain CPS levels increased an additional 26% during Mn²⁺ stress, producing ≈45% more uronic acid than WT (Figure 5A). The addition of Ca²⁺ to Mn²⁺-stressed ΔmntE did not significantly affect CPS production. These data are consistent with elevated cell-associated Mn²⁺ (Figure 5B) and suggest that cellular Mn²⁺ actively binds to and activates Pgm, leading to thicker CPS.

Qualitative assessment of CPS thickness using mucoviscosity assay during routine growth in rich medium revealed similar mucoviscosity levels for both the WT and ΔmntE strains (Figure 5B), which are consistent with those observed by McFarland et al., 2021 [29]. Upon Mn²⁺ exposure, both the WT and ΔmntE strains showed a significant increase in likelihood of remaining in solution, up to 2-fold compared to unstressed conditions (Figure 5B). Culturing with Ca²⁺ also reduced cell adherence ability; WT and ΔmntE cells were 4- and 2-fold less likely to adhere to the surface compared to unstressed cells, respectively (Figure 5B). Similar adherence levels were observed in the presence of Ca²⁺ during Mn²⁺ stress. We note that Ca²⁺-treated cells appeared “fluffy” and that the cell pellets were easily disrupted if care was not taken. Decreasing exogenous Ca²⁺ did not change these observations and had no significant effect on S. pneumoniae cells’ adherence ability (Figure 5C). Together, these data demonstrate that exogenous Ca²⁺ does not influence CPS production in S. pneumoniae D39 but may play a role in overall extracellular bacterial surface charge possibly via the expression of adhesion molecules, molecular binding determinants of adhesion molecules, or by other unknown mechanisms.

3.5. Mn²⁺ Stress Inhibits S. pneumoniae Biofilm Formation

Like many other upper respiratory tract bacterial pathogens, S. pneumoniae forms a biofilm during asymptomatic carriage [46]. These biofilms can increase persistence in the host, as well as S. pneumoniae spread among the host population. Furthermore, initial colonization, biofilm formation, and dispersion of S. pneumoniae cells from the infection site are each influenced by multiple competing factors and environmental resources, including host nutritional immunity and viral co-infection [47,48]. Since changes in host Ca²⁺ levels have been shown to facilitate bacterial adherence [16,17] and cellular Mn²⁺ influences CPS production [29], we investigated the effect of Ca²⁺ and Mn²⁺ on biofilm formation. For these studies, S. pneumoniae was cultivated in TH broth as an alternative to BHI because of its ability to induce consistent biofilm formation [35]. The WT and ΔmntE strains showed no significant difference in biofilm formation when cultured in TH broth for 24 h (Figure 6). The addition of Ca²⁺ also did not significantly affect the biofilm formation of either strain. The WT strain biofilm formation was also not affected by Mn²⁺ or the combination of Mn²⁺/Ca²⁺ (Figure 6A). No detectable biofilm formation was observed for the ΔmntE mutant during Mn²⁺ stress independent of Ca²⁺ presence (Figure 6B).

We note that other host environmental nutrient factors, including sugar carbohydrates, can also effectively facilitate bacterial biofilm formation [34]. Sugar carbohydrate effectiveness is dependent on its concentration and the bacteria type, as well as other associated factors [49]. Glucose can inhibit initial colonization and early-stage bacterial biofilm formation but can also increase biofilm thickness at later stages of growth [50,51]. We found that supplementation of TH broth with 0.2% glucose or 0.3% yeast extract slightly increased biofilm formation by ≈20% and ≈34% for all conditions examined using the WT strain (Figure 6A); the ΔmntE mutant showed similar pattern increases (Figure 6B). Together, these data suggest that carbohydrates and Mn²⁺, but not Ca²⁺ have a role in S. pneumoniae biofilm formation. Additional studies are underway to understand the mechanism of how Mn²⁺ modulates biofilm formation.

3.6. Ca²⁺ Does Not Alter S. pneumoniae Gene Expression

Having eliminated a direct involvement of Ca²⁺ in modulating S. pneumoniae Mn²⁺ transport mechanisms, we sought to gain insight into the mechanism by which Ca²⁺ rescues the Mn²⁺-sensitive ΔmntE strain growth phenotype (Figure 1). Since Ca²⁺ is implicated in several sensor pathways in other pathogenic bacteria that regulate genes encoding virulence and resistance proteins [10], we hypothesized that Ca²⁺ might function as a signaling molecule by directly aiding the modulation of S. pneumoniae gene expression or by altering cellular protein activities by coordinating with EF-hand motifs found in calmodulin-like proteins that require Mn²⁺.

To investigate this further, differential RNAseq analysis was performed to provide a global snapshot of the relative transcript levels of genes during S. pneumoniae growth. Comparison of the transcriptome expression profiles of the WT and Mn²⁺-stressed ΔmntE mutant yielded data (Figure 7) consistent with previous reports [30,42]. A total of 273 genes were significantly differentially expressed; there were 65 downregulated (Table S2) and 208 upregulated genes (Table S3). Our data show that during Mn²⁺ stress, the psaBCA genes encoding the Mn²⁺-specific ABC-type importer PsaBCA are downregulated 8- to 11-fold. The czcD gene encoding the Zn²⁺-specific CDF efflux transporter CzcD is upregulated 5-fold. The latter observations likely result from the mismetallation of the Zn²⁺ regulatory proteins AdcR and SczA by the accumulation of excess cellular Mn²⁺ [45]. piuBCAD encoding the Fe²⁺ ABC-type transporter PiuBCAD was induced 8-fold, consistent with previous work [30]. rffD (spd_0940)-encoding UDP-N-acetyl-D-mannosaminuronic acid dehydrogenase, which functions in producing sugar precursors for CPS biosynthesis, was upregulated 12-fold in addition to other genes involved in sugar transport and metabolism, which is reflective of the observed increased CPS produced by S. pneumoniae during Mn²⁺ stress (Figure 5). Despite observations of a 4.5-fold elevated cell-associated Ca²⁺ (Figure 4A), no significant difference in gene expression was measured in Mn²⁺-stressed ΔmntE cultured with and without 1 mM Ca²⁺. Several possibilities are discussed later.

3.7. Mn²⁺ Impacts S. pneumoniae Cell Division, Which Is Rescued by Ca²⁺

Accumulation of cellular Mn²⁺ can lead to dysregulation of S. pneumoniae cell division via hyperactivation of the Mn²⁺-dependent protein phosphatase PhpP [30]. Such S. pneumoniae cells show on average elongated cell bodies, some cell lysis, and increased chaining [30]. We report similar findings here in Figure 8. The average WT cell width and length was 0.99 ± 0.09 and 1.01 ± 0.16, respectively. In general, the WT cell widths and lengths were not significantly changed by Ca²⁺ and Mn²⁺ presence. The ΔmntE cells measured slightly wider than the WT cells during Mn²⁺ intoxication at 1.05 ± 0.12 µm vs. 0.98 ± 0.08 µm, respectively (Figure 8A). The addition of Ca²⁺ to the Mn²⁺-stressed ΔmntE mutant returned cell widths to the WT or unstressed ΔmntE cell sizes.

During routine growth in rich medium, ΔmntE cells were on average slightly more elongated than WT cells (1.01 ± 0.16 µm vs. 1.20 ± 0.26 µm for ΔmntE). The ΔmntE cell lengths significantly increased to 1.54 ± 0.46 µm when cultured with Mn²⁺. The increase appeared to be independent of Ca²⁺ presence, although the median cell length for ΔmntE grown with Mn²⁺/Ca²⁺ was lower than with Mn²⁺ alone and was approaching closer to the unstressed ΔmntE average cell lengths (Figure 8B). Chaining was also most prevalent for the ΔmntE mutant, with an average of 58 cells per chain observed compared to 16 for the WT strain during routine growth (Figure 8C). The addition of Mn²⁺ to the ΔmntE mutant resulted in significantly less chaining likely due to increased cell lysis and its reduced ability to divide when toxified by Mn²⁺ [30]. In all cases, the addition of Ca²⁺ led to a significant decrease in chain length; cells were mostly found in pairs or short chains of ≤8 cells per chain depending on the strain (Figure 8C). Cell lysis was not observed microscopically during Ca²⁺ treatment. Taken together with the above findings, the data suggest that Ca²⁺ likely has a role in cell division impacting Mn²⁺ physiology. Further investigation is underway to determine the actual mechanism.

4. Discussion

Unlike in eukaryotes, the role of Ca²⁺ in bacteria physiology and virulence remains elusive. Past studies are mostly correlative and have collectively suggested that bacteria may utilize Ca²⁺ as an intracellular signaling messenger whose presence or absence impacts multiple cellular processes, including biofilm formation [10,52]. The present study brings evidence for the involvement of Ca²⁺ in cellular processes requiring Mn²⁺ in S. pneumoniae D39. Our data demonstrate for the first time that Ca²⁺ is capable of rescuing S. pneumoniae ΔmntE Mn²⁺-sensitive growth phenotype via an unidentified mechanism that does not involve altering cell-associated Mn²⁺ levels or gene expression. We further demonstrate that S. pneumoniae D39 cells experiencing Mn²⁺ stress produce thicker capsules, which not only inhibit S. pneumoniae D39 adhesion to abiotic surfaces but also biofilm formation.

The capacity for Ca²⁺ to restore growth during Mn²⁺ intoxication likely varies among bacteria, since Ca²⁺ supplementation showed some efficacy in E. coli but did not impact B. subtilis growth. Although more studies are needed to fully access the breadth of Ca²⁺ impact across bacterial pathogens, it is suspected that such differences arise from selective pressures imposed by the environmental niches for which each bacterium resides. Like Mn²⁺ and other metal ions, Ca²⁺ concentrations vary among host sites. Samples taken from the upper respiratory tract and oral cavity can reach millimolar Ca²⁺ levels, with a 10-fold difference between several sites [10]. Furthermore, elevated cellular Ca can trigger the activation of host immune responses against invading pathogens [14,53]. It has been proposed that host Ca²⁺ levels might (1) signal invading bacterial pathogens that they are entering a specific host site and (2) indicate the status of the host immune protection [10]. For example, during bacteria invasion, the EF hand containing S100 proteins like calprotectin require Ca²⁺ to interact with their targets [54]. Activated calprotectin has been shown to sequester Zn²⁺, and subsequently reduces Zn²⁺ interaction with the solute-binding proteins PsaA and MntC in S. pneumoniae and Staphylococcus aureus, respectively [55]. Sequestration of Zn²⁺ ultimately facilitates Mn²⁺ binding to the solute-binding proteins, resulting in Mn²⁺ import by bacteria. As such, it is likely advantageous for invading bacterial pathogens to recognize host cellular Ca²⁺ levels, which would predictably enhance bacterial adaptation to various host environments, leading to increased bacterial virulence and survival within the host.

Following the revelation that Ca²⁺ rescues Mn²⁺-stressed cells is not mediated through modulation of Mn²⁺ homeostatic proteins, we sought to investigate the molecular mechanism driving the growth rescue. We were somewhat surprised to find that differential RNAseq failed to report any significant changes in global gene expression patterns for the strains grown with or without 1 mM Ca²⁺ during Mn²⁺ stress. It is possible that the exposure time with Ca²⁺ relative to Mn²⁺ stress was not sufficient to observe transcriptional response differences or the relative Ca²⁺ cellular concentration remained within the unknown optimum Ca²⁺ range for S. pneumoniae growth. As such, Ca²⁺-specific transcriptional regulatory proteins would likely not be activated to alter gene expression. Note that increasing Ca²⁺, as with other metals studies, also increases the risk of contaminating metals, which may obscure results. Furthermore, the addition of Ca²⁺ concentrations >2 mM in the presence of high Mn²⁺ resulted in precipitation.

Given that Ca²⁺ is not characteristically similar to Mn²⁺ and that Ca²⁺ most often functions as a non-catalytic cofactor in proteins, we speculate that Ca²⁺-mediated rescue in S. pneumoniae occurs at the post-translational level. As such, Ca²⁺ would function as a signaling messenger that interacts with cellular proteins to modulate their activity or downstream activity of enzyme targets via post-translational modification or protein stabilization. This is not unprecedented, as several Ca²⁺-sensing two-component regulatory systems have been characterized in other bacterial pathogens [10]. There is also growing evidence showing such novel homeostatic mechanisms for other metal ions including Mn²⁺ [23], which further establishes that metals play important intricate roles in bacteria.

As previously mentioned, CPS production is a critical virulence determinant for many bacterial pathogens, including S. pneumoniae, and its production is regulated in response to environmental factors. Mn²⁺ has most recently emerged as an important regulatory component in CPS production for S. pneumoniae via the modulation of Pgm activity [29]. Our data are consistent with those reported by McFarland et al., 2021, in that elevated Mn²⁺ increases CPS production [29]. We extended these findings to S. pneumoniae D39 biofilm formation, demonstrating that thicker CPS would inhibit transition from a planktonic to sessile-community state. Although the study here is performed in-vitro, these data do reinforce the importance of Mn²⁺ in S. pneumoniae colonization and pathogenesis within the host.

Interestingly, we found that Ca²⁺ did not significantly affect biofilm formation but did influence S. pneumoniae D39 adherence to surfaces, independent of CPS thickness. Bacterial adhesion capacity is due in part to electrostatic interactions combined with cell surface structures. Gram-positive bacteria, like S. pneumoniae, have long glycan chains extending through the cell wall from the cytoplasmic membrane. These glycan chains confer a negative charge and are structurally hydrophilic in nature [56]. As such, extracellular Ca²⁺ might alter the physiochemical properties of the S. pneumoniae cell surface via cationic bridging. Direct interaction of Ca²⁺ with such charged exposed surface antigens or extracellular polymeric substances may alter the overall cell surface charge or electrostatic interactions, thereby impeding cell adhesion to surfaces. Further investigation is needed to determine the underlying mechanisms driving this phenomenon and its implication in S. pneumoniae virulence and pathogenesis.

In summary, the findings presented in this study highlight a role for Ca²⁺ in S. pneumoniae that is interconnected with Mn²⁺ physiology and its implication in virulence. Unraveling the molecular mechanism(s) surrounding the Ca²⁺ rescue of Mn²⁺ intoxication demonstrates the complex nature of how bacterial pathogens have evolved to survive in the host and possibly even hijack host mechanisms to have a selective growth advantage and evade host defenses. It will be interesting to learn how Ca²⁺ physically functions in S. pneumoniae, as well as how S. pneumoniae imports, senses, and traffics Ca²⁺. Will the Ca²⁺ trend be like that of other metals and be highly regulated to prevent the intoxication and disruption of cellular processes? Nonetheless, it is critical that we gain a better fundamental understanding of Ca²⁺ in bacterial as it will likely provide insights into the regulation of bacterial pathogenesis and aid the development of targeted therapeutics.