Biology subjects

Kajfasz, J. K.

Publications and source records attributed to Kajfasz, J. K..

7 recordsLinked to original sources

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↗

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↗

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↗