Biology subjects

Abranches, J.

Publications and source records attributed to Abranches, J..

6 recordsLinked to original sources

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↗

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↗