Biology subjects

Lemos, J. A.

Publications and source records attributed to Lemos, J. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Zinc Tolerance Through Glutathione Import Strikes a Fine Balance Between Protection and Damage in Streptococcus mutans

Recently, our group showed that the dental pathogen Streptococcus mutans is inherently more tolerant to high zinc stress than other streptococci, a phenotype associated with the presence of a P-type ATPase exporter named ZccE, virtually unique to S. mutans. In addition to zccE, a previous transcriptome analysis revealed that S. mutans upregulates genes involved in glutathione uptake during initial exposure to zinc stress. Glutathione, a major supplier of organic sulfur that also plays key roles in antioxidant defense and xenobiotic detoxification, forms coordination complexes with a variety of metals, including zinc, thereby functioning as a buffer that protects cells from metal intoxication. To investigate the contribution of glutathione zinc tolerance in S. mutans, the gshT gene, which encodes the substrate-binding subunit of a glutathione transporter, was deleted in both the parent and {Delta}zccE strains and the ability of these mutants to overcome zinc stress through intracellular glutathione accumulation determined. Targeted metabolomics revealed that S. mutans accumulates glutathione in a GshT-dependent manner following zinc stress, a response that was strikingly amplified in the {Delta}zccE strain. Although glutathione supplementation had a minimal and non-significant impact on growth of either parent or mutant strains in sub-inhibitory zinc concentrations, the {Delta}gshT strain exhibited increased zinc sensitivity in a plate-based assay. However, the {Delta}zccE{Delta}gshT mutant displayed enhanced zinc tolerance compared to the {Delta}zccE single mutant. While glutathione alone did not alter zinc levels in the UA159 or {Delta}zccE strains, the combination of zinc and glutathione nearly doubled intracellular zinc levels in {Delta}zccE compared to cells grown in zinc only. We conclude that while glutathione may play a minor role in S. mutans zinc tolerance, uncontrolled glutathione uptake observed in {Delta}zccE facilitates zinc entry, as glutathione:Zn2+ complexes inadvertently promote zinc intoxication via a Trojan horse mechanism.

microbiology↗

bioRxiv2025

Metal Uptake Systems Underpin Enterococcus faecalis Virulence in Both Non-Diabetic and Diabetic Wound Infection Models

Wound infections remain an important medical problem, which is aggravated by the prevalence of multidrug resistant bacteria. Among them, Enterococcus faecalis is a major pathogen of surgical site incisional and of diabetic chronic wounds, but factors driving its colonization and persistence in wounds remain poorly understood. Iron, manganese, and zinc are essential cofactors in cellular processes, prompting the host to restrict their availability through mobilization of metal-sequestering proteins, a defense known as nutritional immunity. Previously, we showed that E. faecalis strains lacking key iron ({Delta}5Fe), manganese ({Delta}3Mn), or zinc ({Delta}2Zn) uptake systems have impaired virulence. Here, we used an excisional wound model in normoglycemic (C57Bl/6J or B6) and diabetic (C57Bl/6J lepR-/- or DB) mice to examine the role of these metal import systems in wounds. The strong upregulation of metal import genes and reduced wound colonization by {Delta}3Mn, {Delta}5Fe, and {Delta}2Zn strains in B6 mice indicate that iron, manganese, and zinc are limited during wound infection. While {Delta}2Zn and {Delta}3Mn strains showed no improved colonization in diabetic wounds, the {Delta}5Fe exhibited a temporary colonization advantage over non-diabetic mice. Quantifications of metal-sequestering proteins lactoferrin, transferrin, calprotectin and psoriasin from intact skin and infected wounds indicated that nutritional immunity, especially iron restriction, is delayed in diabetes. In conclusion, this study underscores the crucial role of trace metal acquisition in E. faecalis wound colonization and suggests differences in metal bioavailability between diabetic and non-diabetic wounds, helping to explain the increased susceptibility of diabetic wounds to chronic infection.

microbiology↗

bioRxiv2025

Zinc-Enhanced Activity of an Antimicrobial Halogenated Phenazine Against Streptococcus mutans and Other Gram-positive Bacteria

Halogenated phenazine (HP) compounds have shown promise as antimicrobial agents, particularly against biofilm-associated Gram-positive pathogens. Among these compounds, HP-29 demonstrates potent activity against methicillin-resistant Staphylococcus aureus by inducing rapid iron starvation. As maintenance of trace metals homeostasis is critical for the survival of Streptococcus mutans, this study investigated the antimicrobial efficacy of HP-29 and the impact of metal supplementation on this major oral and occasional systemic pathogen. As anticipated, HP-29 inhibited S. mutans growth in a dose-dependent manner, with iron supplementation alleviating the antimicrobial effect. Cobalt, manganese, or nickel supplementation also mitigated the inhibitory activity of HP-29 but, unexpectedly, the addition of zinc greatly enhanced HP-29 antimicrobial activity. This zinc-driven potentiation of HP-29 extended to other Gram-positive pathogens, including Enterococcus faecalis and S. aureus. Inductively coupled plasma mass spectrometry analysis revealed that intracellular iron content decreased significantly following exposure to HP-29. At the same time, exposure to HP-29 led to a slight increase in intracellular zinc, mirroring the increase observed in cells exposed to excess zinc. When combined with zinc, HP-29 triggered a 5-fold increase in intracellular zinc and reduced manganese levels by [~]50%. Transcriptome analysis showed that HP-29, with or without zinc, altered expression of genes linked to iron and manganese uptake as well as zinc efflux, suggesting broad disruption of metal ion regulation. These findings highlight HP-29 as a potent antimicrobial that broadly impairs metal homeostasis. The unexpected synergy of HP-29 with zinc points toward a promising dual-agent therapeutic strategy against Gram-positive pathogens. IMPORTANCEWidespread development of antibiotic resistance has created a constantly moving target when combating infectious microbes. Here, we further explore an antimicrobial halogenated phenazine, HP-29, which is effective against Gram-positive bacteria through disruption of intracellular trace metal equilibrium. We showed that HP-29 inhibits growth of the oral and systemic pathogen Streptococcus mutans and that its antimicrobial effect is greatly potentiated by the addition of zinc. The zinc-mediated enhancement of HP-29s efficacy was also observed in other Gram-positive pathogens, including Enterococcus faecalis and Staphylococcus aureus. Intracellular trace metal quantifications and transcriptome analysis confirmed that HP-29 treatment impairs trace metal homeostasis, an outcome that is exacerbated when S. mutans is treated with both HP-29 and zinc. The observed synergy of HP-29 with zinc supports the development of a dual-agent therapeutic strategy against Gram-positive pathogens.

microbiology↗

bioRxiv2025

Characterization of a Novel Cell Wall-Associated Nucleotidase of Enterococcus faecalis that Degrades Extracellular c-di-AMP

Enterococcus faecalis is a prolific opportunistic pathogen responsible for a range of life-threatening infections for which treatment options are increasingly limited due to the high prevalence of multidrug-resistant isolates. Cyclic di-AMP has emerged as an essential bacterial signaling molecule due to its impact on physiological processes, including osmotic adaptation, cell wall homeostasis, antibiotic tolerance, and virulence. In addition, c-di-AMP is a potent pathogen-associated molecular pattern (PAMP) molecule recognized by the host immune system to trigger protective responses. In previous work, we identified and characterized the enzymes responsible for the synthesis and degradation of intracellular c-di-AMP in E. faecalis, demonstrating that maintaining c-di-AMP homeostasis is vital for bacterial fitness and virulence. In addition to the intracellular enzymes that regulate c-di-AMP levels, a limited number of bacteria encode surface-associated nucleotidases capable of cleaving extracellular c-di-AMP, potentially facilitating immune evasion. Here, we characterize a novel and unique cell wall-anchored phosphodiesterase, termed EecP (E. faecalis extracellular c-di-AMP phosphodiesterase), which features duplicated catalytic domains and specifically degrades extracellular c-di-AMP. Deletion of eecP ({Delta}eecP) resulted in a marked accumulation of extracellular c-di-AMP. Although the {Delta}eecP strain exhibited comparable growth and behavior to the parent strain in vitro, it displayed increased susceptibility to killing by phagocytic cells. Using two murine infection models, we show that the impact of eecP deletion and the consequent buildup of extracellular c-di-AMP on E. faecalis pathogenesis may be site-specific. Notably, disseminated infection was more severe in mice infected with the {Delta}eecP strain, suggesting that extracellular c-di-AMP influences infection outcomes, likely through modulation of host immune responses. Author SummaryEnterococcus faecalis is a major opportunistic pathogen and a leading cause of several life-threatening hospital-associated infections. Cyclic di-AMP is a bacterial second messenger nucleotide that regulates essential cellular processes and plays key roles in bacterial pathogenesis and host immune activation. We previously characterized the enzymes responsible for the synthesis and degradation of c-di-AMP in E. faecalis, demonstrating that this signaling molecule is crucial for bacterial fitness and virulence. In this study, we describe the characterization of EecP, a novel cell wall-associated enzyme that degrades c-di-AMP extracellularly. Our findings identify EecP as a new virulence factor in E. faecalis, capable of modulating infection outcomes.

microbiology↗

bioRxiv2025

A Novel Heme-Degrading Enzyme that Regulates Heme and Iron Homeostasis and Promotes Virulence in Enterococcus faecalis

Enterococcus faecalis, a gut commensal, is a leading cause of opportunistic infections. Its virulence is linked to its ability to thrive in hostile environments, which includes host-imposed metal starvation. We recently showed that E. faecalis evades iron starvation using five dedicated transporters that collectively scavenge iron from host tissues and other iron-deprived conditions. Interestingly, heme, the most abundant source of iron in the human body, supported growth of a strain lacking all five iron transporters ({Delta}5Fe). To release iron from heme, many bacterial pathogens utilize heme oxygenase enzymes to degrade the porphyrin that coordinates the iron ion of heme. Although E. faecalis lacks these enzymes, bioinformatics revealed a potential ortholog of the anaerobic heme-degrading enzyme anaerobilin synthase, found in Escherichia coli and a few other Gram-negative bacteria. Here, we demonstrated that deletion of OG1RF_RS05575 in E. faecalis ({Delta}RS05575) or in the {Delta}5Fe background ({Delta}5Fe{Delta}RS05575) led to intracellular heme accumulation and hypersensitivity under anaerobic conditions, suggesting RS05575 encodes an anaerobilin synthase, the first of its kind described in Gram- positive bacteria. Additionally, deletion of RS05575, either alone or in the {Delta}5Fe background, impaired E. faecalis colonization in the mouse gastrointestinal tract and virulence in mouse peritonitis and rabbit infective endocarditis models. These results reveal that RS05575 is responsible for anaerobic degradation of heme and identify this relatively new enzyme class as a novel factor in bacterial pathogenesis. Findings from this study are likely to have broad implications, as homologues of RS05575 are found in other Gram-positive facultative anaerobes. IMPORTANCEHeme is an important nutrient for bacterial pathogens, mainly for its ability to serve as an iron source during infection. While bacteria are known to release iron from heme using enzymes called heme oxygenases, a new family of anaerobic heme-degrading enzymes has been described recently in Gram-negative bacteria. Here, we report the first description of anaerobic heme degradation by a Gram-positive bacterium, the opportunistic pathogen Enterococcus faecalis, and link activity of this enzyme to their ability to colonize and infect the host. We also show that homologues of this enzyme are found in many Gram-positive facultative anaerobes, implying that the ability to degrade heme under anaerobic conditions may be an overlooked fitness and virulence factor of bacterial pathogens.

microbiology↗

bioRxiv2024

FRUCTOSE ACTIVATES A STRESS RESPONSE SHARED BY METHYLGLYOXAL AND HYDROGEN PEROXIDE IN STREPTOCOCCUS MUTANS

Fructose catabolism by Streptococcus mutans is initiated by three PTS transporters yielding fructose-1-phoshate (F-1-P) or fructose-6-phosphate. Deletion of one such F-1-P-generating PTS, fruI, was shown to reduce the cariogenicity of S. mutans in rats fed a high-sucrose diet. Moreover, a recent study linked fructose metabolism in S. mutans to a reactive electrophile species (RES) methylglyoxal. Here, we conducted a comparative transcriptomic analysis of S. mutans treated briefly with 50 mM fructose, 50 mM glucose, 5 mM methylglyoxal, or 0.5 mM hydrogen peroxide (H2O2). The results revealed a striking overlap between the fructose and methylglyoxal transcriptomes, totaling 176 genes, 61 of which were also shared with the H2O2 transcriptome. This core of 61 genes encompassed many of the same pathways affected by exposure to low pH or zinc intoxication. Consistent with these findings, fructose negatively impacted metal homeostasis of a mutant deficient in zinc expulsion and the growth of a mutant of the major oxidative stress regulator SpxA1. Importantly, fructose metabolism lowered culture pH at a faster pace, allowed better survival under acidic and nutrient-depleted conditions, and enhanced the competitiveness of S. mutans against Streptococcus sanguinis, although a moderated level of F-1-P might further boost some of these benefits. Conversely, several commensal streptococcal species displayed a greater sensitivity to fructose that may negatively affect their persistence and competitiveness in dental biofilm. In conclusion, fructose metabolism is integrated into the stress core of S. mutans and regulates critical functions required for survival and its ability to induce dysbiosis in the oral cavity. Importance.Fructose is a common monosaccharide in the biosphere, yet its overconsumption has been linked to various health problems in humans including insulin resistance, obesity, diabetes, non-alcoholic liver diseases, and even cancer. These effects are in large part attributable to the unique biochemical characteristics and metabolic responses associated with the degradation of fructose. Yet, an understanding of the effects of fructose on the physiology of bacteria and its implications to the human microbiome is severely lacking. Here we performed a series of analyses on the gene regulation of a dental pathogen Streptococcus mutans by exposing it to fructose and other important stress agents. Further supported by growth, persistence, and competition assays, our findings revealed the ability of fructose to activate a set of stress-related functions that may prove critical to the ability of the bacterium to persist and cause diseases both within and without of the oral cavity.

microbiology↗

bioRxiv2024

Genome-wide analysis of Enterococcus faecalis genes that facilitate interspecies competition with Lactobacillus crispatus

Enterococci are opportunistic pathogens notorious for causing a variety of infections. While both Enterococcus faecalis and Lactobacillus crispatus are commensal residents of the vaginal tract, the molecular mechanisms that enable E. faecalis to outcompete L. crispatus, and consequently cause vaginal infections remains unknown. To begin to address this, we need to gain a better understanding of the competitive interactions between E. faecalis and L. crispatus. Here, we show that L. crispatus eradicates E. faecalis in a contact-independent manner. Using transposon sequencing to identify E. faecalis OG1RF transposon (Tn) mutants that are either under-represented or over-represented when co-cultured with L. crispatus, we found that Tn mutants with disruption in the dltABCD operon, that encodes the biosynthetic pathway for D-alanylation of teichoic acids, and OG1RF_11697 encoding for an uncharacterized hypothetical protein are more susceptible to killing by L. crispatus. Inversely, Tn mutants with disruption in ldh1, that encodes for L-lactate dehydrogenase, are more resistant to L. crispatus killing. Using the Galleria mellonella infection model, we show that co-injection of L. crispatus with E. faecalis OG1RF enhances larvae survival while this L. crispatus-mediated protection was lost in larvae co-infected with either L. crispatus and E. faecalis {Delta}ldh1 or {Delta}ldh1{Delta}ldh2 strains. Last, using RNA sequencing to identify E. faecalis genes that are differently expressed in the presence of L. crispatus, we found major changes in expression of genes associated with glycerophospholipid metabolism, central metabolism and general stress responses. The findings in this study provide insights on how E. faecalis mitigate assaults by L. crispatus. IMPORTANCEEnterococcus faecalis is an opportunistic pathogen notorious for causing a multitude of infections. While E. faecalis must compete with Lactobacillus crispatus to cause vaginal infections, how E. faecalis overcomes L. crispatus killing remains unknown. We show that L. crispatus eradicates E. faecalis temporally in a contact-independent manner. Using high throughput molecular approaches, we identified genetic determinants that enable E. faecalis to compete with L. crispatus. This study represents an important first step for the identification of adaptive genetic traits required for enterococci to compete with lactobacilli.

microbiology↗

bioRxiv2022

Identification of Multiple Iron Uptake Mechanisms in Enterococcus faecalis and Their Relationship to Virulence

Among the unfavorable conditions bacteria encounter within the host is restricted access to essential trace metals such as iron. To overcome iron deficiency, bacteria deploy multiple strategies to scavenge iron from host tissues with abundant examples of iron acquisition systems being implicated in bacterial pathogenesis. Yet, the mechanisms utilized by the major nosocomial pathogen Enterococcus faecalis to maintain intracellular iron balance are poorly understood. In this report, we conducted a systematic investigation to identify and characterize the iron acquisition mechanisms of E. faecalis and to determine their contribution to virulence. Bioinformatic analysis and literature surveys revealed that E. faecalis possesses three conserved iron uptake systems. Through transcriptomics, we discovered two novel ABC-type transporters that mediate iron uptake. While inactivation of a single transporter had minimal impact on the ability of E. faecalis to maintain iron homeostasis, inactivation of all five systems ({Delta}5Fe strain) disrupted intracellular iron homeostasis and considerably impaired cell growth under iron-deficiency. Virulence of the {Delta}5Fe strain was generally impaired in different animal models but showed niche-specific variations in mouse models, leading us to suspect that heme can serve as an iron source to E. faecalis during mammalian infections. Indeed, heme supplementation restored growth of {Delta}5Fe under iron-depletion and virulence in an invertebrate infection model. Collectively, this study reveals that the collective contribution of five iron transporters promotes E. faecalis virulence and that the ability to acquire and utilize heme as an iron source is critical to the systemic dissemination of E. faecalis.

microbiology↗

bioRxiv2022

Post-translational modification by the Pgf glycosylation machinery modulates Streptococcus mutans physiology and virulence

Streptococcus mutans is a keystone pathogen of dental caries, and the ability to form biofilms is essential for its pathogenicity. We identified a glycosylation machinery (Pgf) in S. mutans that post-translationally modifies two surface-associated adhesins, Cnm and WapA. The four pgf genes (pgfS, pgfM1, pgfE, and pgfM2) are part of S. mutans core genome and we hypothesized that the scope of Pgf goes beyond Cnm and WapA. By inactivating each pgf gene individually or creating a quadruple pgf mutant in S. mutans OMZ175, we showed that the Pgf machinery is important for biofilm formation. Compared to OMZ175, differences in surface charge, membrane stability, and genetic competence were also observed for most mutants. Importantly, in silico analyses and tunicamycin MIC assays suggest a functional redundancy between the Pgf machinery and the rhamnose-glucose polysaccharide synthesis pathway. Using a rat oral colonization model, we showed a 10-fold reduction in recovered CFUs for the pgf quadruple mutant compared to OMZ175. Finally, using Cnm as a model, we showed by glycoproteomics analyses that Cnm is heavily modified with N-acetyl hexosamine in OMZ175 whereas phosphorylations were observed for the pgfS mutant. Our findings indicate that the Pgf machinery participates in important aspects of S. mutans pathobiology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=164 SRC="FIGDIR/small/511621v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@146f37eorg.highwire.dtl.DTLVardef@11c0e45org.highwire.dtl.DTLVardef@ece1eaorg.highwire.dtl.DTLVardef@1e3f221_HPS_FORMAT_FIGEXP M_FIG C_FIG Abbreviated summaryIn this study, we demonstrate that the Pgf glycosylation machinery of Streptococcus mutans, a keystone pathogen of dental caries, regulates several aspects of bacterial pathophysiology that ultimately contribute to S. mutans fitness in oral colonization experiments. Using the heavily glycosylated Cnm adhesin as a model, we found that inactivation of the glycosyltransferase PgfS results in loss of Cnm glycosylation, but instead, Cnm became heavily phosphorylated, suggesting a crosstalk/competition between these two post-translational modification mechanisms.

microbiology↗

bioRxiv2022

ZccE is a Novel P-type ATPase That Protects Streptococcus mutans Against Zinc Intoxication

Zinc is a trace metal that is essential to all forms of life, but that becomes toxic at high concentrations. Because it has both antimicrobial and anti-inflammatory properties and low toxicity to mammalian cells, zinc has been used as a therapeutic agent for centuries to treat a variety of infectious and non-infectious conditions. While the usefulness of zinc-based therapies in caries prevention is controversial, zinc is incorporated into toothpaste and mouthwash formulations to prevent gingivitis and halitosis. Despite this widespread use of zinc in oral healthcare, the mechanisms that allow Streptococcus mutans, a keystone pathogen in dental caries and prevalent etiological agent of infective endocarditis, to overcome zinc toxicity are largely unknown. Here, we discovered that S. mutans is inherently more tolerant to high zinc stress than all other species of streptococci tested, including commensal streptococci associated with oral health. Using a transcriptome approach, we uncovered several potential strategies utilized by S. mutans to overcome zinc toxicity. Among them, we identified a previously uncharacterized P-type ATPase transporter and cognate transcriptional regulator, which we named ZccE and ZccR respectively, as responsible for the remarkable high zinc tolerance of S. mutans. In addition to zinc, we found that ZccE, which was found to be unique to S. mutans strains, mediates tolerance to at least three additional metal ions, namely cadmium, cobalt, and copper. Loss of the ability to maintain zinc homeostasis when exposed to high zinc stress severely disturbed Zinc:Manganese ratios, leading to heightened peroxide sensitivity that was alleviated by manganese supplementation. Finally, we showed that the ability of the {Delta}zccE strain to stably colonize the rat tooth surface after topical zinc treatment was significantly impaired, providing proof of concept that ZccE and ZccR are suitable targets for the development of antimicrobial therapies specifically tailored to kill S. mutans. Author summaryDental caries is an overlooked infectious disease affecting more than 50% of the adult population. While several bacteria that reside in dental plaque have been associated with caries development and progression, Streptococcus mutans is deemed a keystone caries pathogen due to its capacity to modify the dental plaque environment in a way that is conducive with disease development. Zinc is an essential trace metal to life but very toxic when encountered at high concentrations, to the point that it has been used as an antimicrobial for centuries. Despite the widespread use of zinc in oral healthcare products, little is known about the mechanisms utilized by oral bacteria to overcome its toxic effects. In this study, we discovered that S. mutans can tolerate exposure to much higher levels of zinc than closely related streptococcal species, including species that antagonize S. mutans and are associated with oral health. In this study, we identified a new metal transporter, named ZccE, as directly responsible for the inherently high zinc tolerance of S. mutans. Because ZccE is not present in other bacteria, our findings provide a new target for the development of a zinc-based therapy specifically tailored to kill S. mutans.

microbiology↗

bioRxiv2022

Enterococcus faecalis antagonizes Pseudomonas aeruginosa growth in polymicrobial biofilms

Enterococcus faecalis is often co-isolated with Pseudomonas aeruginosa in polymicrobial biofilm-associated infections of wounds and the urinary tract. As a defense strategy, the host innately restricts iron availability at infection sites. Despite their co-prevalence, the polymicrobial interactions of these two pathogens in iron- restricted conditions, such as those found in the host, remains unexplored. Here we show that E. faecalis inhibits P. aeruginosa growth within biofilms when iron is restricted. E. faecalis lactate dehydrogenase (ldh1) gives rise to L-lactate production during fermentative growth. We find that E. faecalis ldh1 mutant fails to inhibit P. aeruginosa growth. Additionally, we demonstrate that ldh1 expression is induced in iron-restricted conditions, resulting in increased lactic acid exported and consequently, a reduction in pH. Together, our results suggest that E. faecalis synergistically inhibit P. aeruginosa growth by decreasing environmental pH and L-lactate-mediated iron chelation. Overall, this study highlights that the microenvironment in which the infection occurs is important for understanding its pathophysiology. IMPORTANCEMany infections are polymicrobial and biofilm-associated in nature. Iron is essential for many metabolic processes and plays an important role in controlling infections, where the host restricts iron as a defense mechanism against invading pathogens. However, polymicrobial interactions between pathogens are underexplored in iron- restricted conditions. Here, we explore the polymicrobial interactions between commonly co-isolated E. faecalis and P. aeruginosa within biofilms. We find that E. faecalis modulates the microenvironment by exporting lactic acid which further chelates already limited iron, and also lowers the environmental pH to antagonize P. aeruginosa growth in iron-restricted conditions. Our findings provide insights into polymicrobial interactions between pathogens in an infection-relevant condition and how manipulating the microenvironment can be taken advantage of to better control infections.

microbiology↗

bioRxiv2021

The AdcACB/AdcAII system is Essential for Zinc Homeostasis and an Important Contributor of Enterococcus faecalis Virulence

Bacterial pathogens require a variety of micronutrients for growth, including trace metals such as iron, manganese, and zinc (Zn). Despite their relative abundance in host environments, access to these metals is severely restricted during infection due to host-mediated defense mechanisms collectively known as nutritional immunity. Despite a growing appreciation of the importance of Zn in host-pathogen interactions, the mechanisms of Zn homeostasis and the significance of Zn to the pathophysiology of E. faecalis, a major pathogen of nosocomial and community-associated infections, have not been investigated. Here, we show that E. faecalis encoded an ABC-type transporter AdcACB and an orphan substrate-binding lipoprotein AdcAII that work cooperatively to maintain Zn homeostasis. Simultaneous inactivation of adcA and adcAII or the entire adcACB operon led to significant reduction in intracellular Zn under Zn-restricted conditions, heightened sensitivity to Zn-chelating agents including human calprotectin, aberrant cell morphology, and impaired fitness in serum ex vivo. Additionally, inactivation of adcACB and adcAII significantly reduced bacterial tolerance towards cell envelope-targeting antibiotics, which may be associated to altered fatty acid abundance and species. Lastly, we show that the AdcACB/AdcAII system contributes to E. faecalis virulence in an invertebrate (Galleria mellonella) infection model and in two catheter-associated mouse infection models that recapitulate many of the host conditions associated with enterococcal human infections. Collectively, this report reveals that high-affinity Zn import is essential for the pathogenesis of E. faecalis indicating that the surface-associated AdcA and AdcAII lipoproteins are potential therapeutic targets.

microbiology↗

bioRxiv2021

Amyloid aggregation of Streptococcus mutans Cnm influences its collagen-binding activity

The glycosylated collagen- and laminin-binding surface adhesin Cnm is present in approximately 20% of S. mutans clinical isolates and is associated with systemic infections and increased caries risk. Other surface-associated collagen-binding proteins of S. mutans such as P1 and WapA have been demonstrated to form an amyloid quaternary structure with functional implications within biofilms. In silico analysis predicted that the {beta}-sheet rich N-terminal collagen-binding domain (CBD) of Cnm has propensity for amyloid aggregation, whereas the threonine-rich C-terminal domain was predicted to be disorganized. In this study, thioflavin-T fluorescence and electron microscopy were used to show that Cnm forms amyloids either in its native glycosylated or recombinant non-glycosylated forms and that the CBD of Cnm is the main amyloidogenic unit of Cnm. We then performed a series of in vitro, ex vivo and in vivo assays to characterize the amylogenic properties of Cnm. In addition, Congo red birefringence indicated that Cnm is a major amyloidogenic protein of S. mutans biofilms. Competitive binding assays using collagen-coated microtiter plates and dental roots, a substrate rich in collagen, revealed that Cnm monomers inhibit S. mutans binding to collagenous substrates whereas Cnm amyloid aggregates lose this property. Thus, while Cnm contributes to recognition and initial binding of S. mutans to collagen-rich surfaces, Cnm amyloid aggregation appears to represent a mechanism to modulate this activity in mature biofilms. IMPORTANCEStreptococcus mutans is a keystone pathogen that promotes caries by acidifying the dental biofilm milieu. The collagen- and laminin-binding glycoprotein Cnm is a virulence factor found in about 20% of the clinical isolates of S. mutans. Expression of Cnm by S. mutans is associated with niche expansion, allowing colonization of multiple sites in the body including collagen-rich surfaces such as dentin and heart valves. Here, we demonstrate for the first time that Cnm function appears to be modulated by its aggregation status. As a monomer, its primary function is to promote attachment to collagenous substrates via its collagen binding domain (CBD). However, in later stages of biofilm maturation, the same CBD of Cnm self-assembles into amyloid fibrils, losing the ability to bind to collagen and likely becoming a component of the biofilm matrix. Our findings shed light into the role of functional amyloids in S. mutans pathobiology and ecology.

microbiology↗

bioRxiv2021

c-di-AMP is essential for the virulence of Enterococcus faecalis

Second messenger nucleotides are produced by bacteria in response to environmental stimuli and play a major role in the regulation of processes associated with bacterial fitness, including but not limited to osmoregulation, envelope homeostasis, central metabolism, and biofilm formation. In this study, we uncovered the biological significance of c-di-AMP in the opportunistic pathogen Enterococcus faecalis by isolating and characterizing strains lacking genes responsible for c-di-AMP synthesis (cdaA) and degradation (dhhP and gdpP). Using complementary approaches, we demonstrated that either complete loss of c-di-AMP ({Delta}cdaA strain) or c-di-AMP accumulation ({Delta}dhhP, {Delta}gdpP and {Delta}dhhP{Delta}gdpP strains) drastically impaired general cell fitness and virulence of E. faecalis. In particular, the {Delta}cdaA strain was highly sensitive to envelope-targeting antibiotics, was unable to multiply and quickly lost viability in human serum or urine ex vivo, and was avirulent in an invertebrate (Galleria mellonella) and in two catheter-associated mouse infection models that recapitulate key aspects of enterococcal infections in humans. In addition to evidence linking these phenotypes to altered activity of metabolite and peptide transporters and inability to maintain osmobalance, we found that the attenuated virulence of {Delta}cdaA could be also attributed to a defect in Ebp pilus production and activity that severely impaired biofilm formation under both in vitro and in vivo conditions. Collectively, these results reveal that c-di-AMP signaling is essential for E. faecalis pathogenesis and a desirable target for drug development. IMPORTANCEEnterococcus faecalis is an opportunistic pathogen and leading cause of multidrug resistant hospital-acquired infections. During the course of an infection, bacteria encounter multiple adverse (stress) conditions and understanding the adaptive mechanisms used by pathogens to survive these stresses can facilitate the development of new antimicrobial therapies. Here, we used in vitro, ex vivo and in vivo approaches to determine the importance of the second messenger nucleotide c-di-AMP, a global regulator essential for bacterial adaptation to osmotic stress, to E. faecalis pathophysiology. We demonstrated that either accumulation of c-di-AMP or complete loss of c-di-AMP impaired cell fitness and virulence of E. faecalis. Remarkably, the strain that was unable to produce c-di-AMP was avirulent in three animal infection models indicating that c-di-AMP signaling is essential for E. faecalis pathogenesis and a suitable antimicrobial target.

microbiology↗

bioRxiv2021

Zinc Import Mediated by AdcABC is Critical for Colonization of the Dental Biofilm by Streptococcus mutans in an Animal Model

Trace metals are essential to all domains of life but toxic when found at high concentrations. While the importance of iron in host-pathogen interactions is firmly established, contemporary studies indicate that other trace metals, including manganese and zinc, are also critical to the infectious process. In this study, we sought to identify and characterize the zinc uptake system(s) of S. mutans, a keystone pathogen in dental caries and a causative agent of bacterial endocarditis. Different than other pathogenic bacteria, including several streptococci, that encode multiple zinc import systems, bioinformatic analysis indicated that the S. mutans core genome encodes a single, highly conserved, zinc importer commonly known as AdcABC. Inactivation of the genes coding for the metal-binding AdcA ({Delta}adcA) or both AdcC ATPase and AdcB permease ({Delta}adcCB) severely impaired the ability of S. mutans to grow under zinc-depleted conditions. Intracellular metal quantifications revealed that both mutants accumulated less zinc when grown in the presence of a sub-inhibitory concentration of a zinc-specific chelator. Notably, the {Delta}adcCB strain displayed a severe colonization defect in a rat oral infection model. Both {Delta}adc strains were hypersensitive to high concentrations of manganese, showed reduced peroxide tolerance, and formed less biofilm in sucrose-containing media when cultivated in the presence of the lowest amount of zinc that support their growth, but not when zinc was supplied in excess. Collectively, this study identifies AdcABC as the lone high affinity zinc importer of S. mutans and provides preliminary evidence that zinc is a growth-limiting factor within the dental biofilm.

microbiology↗

bioRxiv2020

Increased oxidative stress tolerance of a spontaneously-occurring perR gene mutation in Streptococcus mutans UA159

The ability of bacteria such as the dental pathogen Streptococcus mutans to coordinate a response against damage-inducing oxidants is a critical aspect of their pathogenicity. The oxidative stress regulator SpxA1 has been proven to be a major player in the ability of S. mutans to withstand both disulfide and peroxide stresses. While studying spontaneously-occurring variants of an S. mutans {Delta}spxA1 strain, we serendipitously discovered that our S. mutans UA159 host strain bore a single nucleotide deletion within the coding region ofperR, resulting in a premature truncation of the encoded protein. PerR is a metal-dependent transcriptional repressor that senses and responds to peroxide stress such that loss of PerR activity results in activation of oxidative stress responses. To determine the impact of loss of PerR regulation, we obtained a UA159 isolated bearing an intact perR copy and created a clean perR deletion mutant. Our findings indicate that loss of PerR activity results in a strain that is primed to tolerate oxidative stresses in the laboratory setting. Interestingly, RNA-Seq and targeted transcriptional expression analyses reveal that PerR has a minor contribution to the ability of S. mutans to orchestrate a transcriptional response to peroxide stress. Furthermore, we detected loss-of-function perR mutations in two other commonly used laboratory strains of S. mutans suggesting that this may be not be an uncommon occurrence. This report serves as a cautionary warning regarding the so-called domestication of laboratory strains and advocates for the implementation of more stringent strain authentication practices. ImportanceA resident of the human oral biofilm, Streptococcus mutans is one of the major bacterial pathogens associated with dental caries. This report highlights a spontaneously-occurring mutation within the laboratory strain S. mutans UA159, found in the coding region of perR, a gene encoding a transcriptional repressor associated with peroxide tolerance. Though perR mutant strains of S. mutans showed a distinct growth advantage and enhanced tolerance toward H2O2, a {Delta}perR deletion strain showed a small number of differentially expressed genes as compared to the parent strain, suggesting few direct regulatory targets. In addition to characterizing the role of PerR in S. mutans, our findings serve as a warning to laboratory researchers regarding bacterial adaptation to in vitro growth conditions.

microbiology↗

bioRxiv2019

Manganese uptake, mediated by SloABC and MntH, is essential for the fitness of Streptococcus mutans

Early epidemiological studies implicated manganese (Mn) as a possible caries-promoting agent while laboratory studies have indicated that manganese stimulates the expression of virulence-related factors in the dental pathogen Streptococcus mutans. To better understand the importance of manganese homeostasis to S. mutans pathophysiology, we first used RNA sequencing to obtain the global transcriptional profile of S. mutans UA159 grown under Mn-restricted conditions. Among the most highly expressed genes were the entire sloABC operon, encoding a dual iron/manganese transporter, and an uncharacterized gene, herein mntH, that codes for a protein bearing strong similarity to Nramp-type transporters. While inactivation of sloC, which encodes the lipoprotein receptor of the SloABC system, or mntH alone had no major consequence on the overall fitness of S. mutans, simultaneous inactivation of sloC and mntH ({Delta}sloC{Delta}mntH) impaired growth and survival under Mn-restricted conditions, including in human saliva or in the presence of calprotectin. Further, disruption of Mn transport resulted in diminished stress tolerance and reduced biofilm formation in the presence of sucrose. These phenotypes were markedly improved when cells were provided with excess Mn. Metal quantifications revealed that the single mutant strains contain similar intracellular levels of Mn as the parent strain, whereas Mn was nearly undetectable in the {Delta}sloC{Delta}mntH strain. Collectively, these results reveal that SloABC and MntH work independently and cooperatively to promote cell growth under Mn-restricted conditions, and that mauitanence of Mn homeostasis is essential for the expression of major virulence attributes in S. mutans.\n\nIMPORTANCEAs trace biometals such as manganese (Mn) are important for all forms of life, the ability to regulate biometals availability during infection is an essential trait of successful bacterial pathogens. Here, we showed that the caries pathogen Streptococcus mutans utilizes two Mn transport systems, namely SloABC and MntH, to acquire Mn from the environment, and that the ability to maintain the cellular levels of Mn is important for the manifestation of characteristics that associate S. mutans with dental caries. Our results indicate that the development of strategies to deprive S. mutans of Mn hold promise in the combat against this important bacterial pathogen.

microbiology↗

bioRxiv2019

(p)ppGpp and CodY promote Enterococcus faecalis virulence in a murine model of catheter-associated urinary tract infection

In Firmicutes, the nutrient-sensing regulators (p)ppGpp, the effector molecule of the stringent response, and CodY work in tandem to maintain bacterial fitness during infection. Here, we tested (p)ppGpp and codY mutant strains of Enterococcus faecalis in a catheter-associated urinary tract infections (CAUTI) mouse model and used global transcriptional analysis to investigate the (p)ppGpp and CodY relationship. Absence of (p)ppGpp or single inactivation of codY led to lower bacterial loads in catheterized bladders, and diminished biofilm formation on fibrinogen-coated surfaces under in vitro and in vivo conditions. Single inactivation of the bifunctional (p)ppGpp synthetase/hydrolase rel did not affect virulence supporting previous evidence that association of (p)ppGpp with enterococcal virulence is not dependent on activation of the stringent response. Inactivation of codY in the (p)ppGpp0 strain restored E. faecalis virulence in the CAUTI model as well as the ability to form biofilms in vitro. Transcriptome analysis revealed that inactivation of codY restores, for the most part, the dysregulated metabolism of (p)ppGpp0 cells. While a clear linkage between (p)ppGpp and CodY with expression of virulence factors could not be established, targeted transcriptional analysis indicate that a possible association between (p)ppGpp and c-di-AMP signaling pathways in response to the conditions found in the bladder may plays a role in enterococcal CAUTI. Collectively, this study identifies the (p)ppGpp-CodY network as an important contributor to enterococcal virulence in catheterized mouse bladder and supports that basal (p)ppGpp pools and CodY promote virulence through maintenance of a balanced metabolism during adverse conditions.\n\nImportanceCatheter-associated urinary tract infections (CAUTI) are one of the most frequent types of infection found in the hospital setting that can develop into serious and potentially fatal bloodstream infections. One of the infectious agents that frequently cause complicated CAUTI is the bacterium Enterococcus faecalis, a leading cause of hospital-acquired infections that are often difficult to treat due to the exceptional multidrug resistance of some isolates. Understanding the mechanisms by which E. faecalis causes CAUTI will aid in the discovery of new druggable targets to treat these infections. In this study, we report the importance of two nutrient-sensing bacterial regulators, named (p)ppGpp and CodY, for the ability of E. faecalis to infect the catheterized bladder of mice.

microbiology↗