Biology subjects

Spatafora, G. A.

Publications and source records attributed to Spatafora, G. A..

4 recordsLinked to original sources

bioRxiv2023

Small regulatory RNAs are mediators of the Streptococcus mutans SloR regulon

Dental caries is among the most prevalent chronic infectious diseases worldwide. Streptococcus mutans, the chief causative agent of caries, uses a 25 kDa manganese dependent SloR protein to coordinate the uptake of essential manganese with the transcription of its virulence attributes. Small non-coding RNAs (sRNAs) can either enhance or repress gene expression and reports in the literature ascribe an emerging role for sRNAs in the environmental stress response. Herein, we identify 18-50 nt sRNAs as mediators of the S. mutans SloR and manganese regulons. Specifically, the results of sRNA-seq revealed 56 sRNAs in S. mutans that were differentially transcribed in the SloR-proficient UA159 and SloR-deficient GMS584 strains, and 109 sRNAs that were differentially expressed in UA159 cells grown in the presence of low versus high manganese. We describe SmsR1532 and SmsR1785 as SloR- and/or manganese-responsive sRNAs that are processed from large transcripts, and that bind SloR directly in their promoter regions. The predicted targets of these sRNAs include regulators of metal ion transport, growth management via a toxin-antitoxin operon, and oxidative stress tolerance. These findings support a role for sRNAs in coordinating intracellular metal ion homeostasis with virulence gene control in an important oral cariogen. IMPORTANCESmall regulatory RNAs (sRNAs) are critical mediators of environmental signaling, particularly in bacterial cells under stress, but their role in Streptococcus mutans is poorly understood. S. mutans, the principal causative agent of dental caries, uses a 25 kDa manganese-dependent protein, called SloR, to coordinate the regulated uptake of essential metal ions with the transcription of its virulence genes. In the present study, we identified and characterize sRNAs that are both SloR- and manganese-responsive. Taken together, this research can elucidate the details of regulatory networks that engage sRNAs in an important oral pathogen, and that can enable the development of an effective anti-caries therapeutic.

microbiology↗

bioRxiv2020

Regulatory involvement of the PerR and SloR metalloregulators in the Streptococcus mutans oxidative stress response

Streptococcus mutans is a commensal of the human oral microbiome that can instigate dental caries under conditions of dysbiosis. This study investigates S. mutans metalloregulators and their involvement in mediating a response to oxidative stress. Oxidative stress in the oral cavity can derive from temporal increases in reactive oxygen species (ROS) after meal consumption, and from endogenous bacterial ROS-producers that colonize the dentition as constituents of dental plaque. We hypothesize that the PerR (SMU.593) and SloR (SMU.186) metalloregulatory proteins in S. mutans contribute to oxidative stress tolerance by regulating the expression of genes responsive to H2O2 challenge. The results of qRT-PCR experiments with S. mutans cultures exposed to 0.5mM H2O2 reveal perR transcription that is responsive to the peroxide stressor, and sloR transcription that is subject to PerR repression. The results of gel shift assays support direct binding of a PerR homolog to the S. mutans sloR promoter at Fur and PerR consensus sequences on the UA159 chromosome. In addition, transcription of the S. mutans tpx and dpr antioxidant genes is upregulated in a perR/sloR double knockout mutant, consistent with heightened resistance of the S. mutans GMS802 perR-deficient strain when challenged with H2O2. Cumulatively, these results reveal a relationship of reciprocity between the PerR and SloR metalloregulators during the S. mutans response to oxidative stress and begin to elucidate the fitness strategies that evolved to foster S. mutans survival and persistence in the transient environments of the human oral cavity. IMPORTANCEIn 2020, untreated dental caries, especially in the permanent dentition, ranked among the most prevalent infectious diseases worldwide. Moreover, caries disproportionately affects children and individuals of low socioeconomic status. Untreated caries can lead to systemic health problems and has been associated with extended school and work absences, inappropriate use of emergency departments, and an inability for military forces to deploy. In combination with public health policy, research aimed at alleviating S. mutans-induced tooth decay is important because it can improve oral health, as well as overall health, especially for underserved populations. This research is focused on the S. mutans SloR and PerR metalloregulatory proteins that can help inform the development of therapeutics aimed at alleviating and potentially preventing dental caries.

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↗

bioRxiv2018

Autoregulation of the S. mutans SloR metalloregulator is constitutive and driven by an independent promoter

Streptococcus mutans, one of [~]600 bacterial species in the human oral cavity, is among the most acidogenic constituents of the plaque biofilm. Considered to be the primary causative agent of dental caries, S. mutans harbors a 25kDa SloR metalloregulatory protein which controls metal ion transport across the bacterial cell membrane to maintain essential metal ion homeostasis. The expression of SloR derives, in part, from transcriptional readthrough of the sloABC operon which encodes a Mn2+/Fe2+ ABC transport system. Herein, we describe the details of the sloABC promoter that drives this transcription, as well as a novel independent promoter in an intergenic region (IGR) that contributes to downstream sloR expression. RT-PCR studies support sloR transcription that is independent of sloABC expression, and the results of 5' RACE revealed a sloR transcription start site in the IGR from which the -10 and -35 promoter regions were predicted. The results of gel mobility shift assays support direct SloR binding to the IGR, albeit with lower affinity than SloR binding to the sloABCR promoter. Function of the sloR promoter was validated in qRT-PCR experiments. Interestingly, sloR expression was not significantly impacted when grown in the presence of high manganese, whereas expression of the sloABC operon was repressed under these conditions. The results of in vitro transcription studies support SloR-mediated transcriptional-activation of sloR and -repression of sloABC. Taken together, these findings implicate SloR as a bifunctional regulator that represses sloABC promoter activity and encourages sloR transcription from an independent promoter.\n\nImportanceTooth decay is a ubiquitous infectious disease that is especially pervasive in underserved communities worldwide. S. mutans-induced carious lesions cause functional, physical, and/or aesthetic impairment in the vast majority of adults, and in 60-90% of schoolchildren in industrialized countries. Billions of dollars are spent annually on caries treatment, and productivity losses due to absenteeism from the workplace are significant. Research aimed at alleviating S. mutans-induced tooth decay is important because it can address the socioeconomic disparity that is associated with dental cavities and improve overall general health which is inextricably linked to oral health. Research focused on the S. mutans SloR metalloregulatory protein can guide the development of novel therapeutics and so alleviate the burden of dental cavities.

microbiology↗