Biology subjects

Kitten, T.

Publications and source records attributed to Kitten, T..

12 recordsLinked to original sources

bioRxiv2026

Specific host translation perturbations promote TnSmu1 loss independently of the canonical ImmA regulator

Integrative and conjugative elements (ICEs) are major drivers of horizontal gene transfer and bacterial genome evolution. Although ICE-encoded regulatory circuits have been extensively characterized, the impact of host physiology on the stability of integrated ICEs remains poorly understood. Here, we identify a host-dependent pathway that links specific host translation perturbations to loss of the ICE TnSmu1 in Streptococcus mutans. Analysis of host-gene deletion mutants revealed that disruption of fmt, rnjA, or rnjB--three translation-associated host genes--reproducibly promoted TnSmu1 loss through a mechanism that bypasses the canonical ICE-encoded metalloprotease ImmA but remains dependent on the native attachment site attR. This phenotype was selective, as mutations affecting other essential cellular functions, including protein folding, tRNA modification, cell division, and fatty acid biosynthesis, failed to destabilize TnSmu1 despite undergoing the same experimental evolution and accumulating adaptive genomic changes. Preventing TnSmu1 loss in these translation-associated mutants markedly reduced bacterial growth, whereas loss of the element improved fitness, indicating that ICE elimination alleviates the cost associated with TnSmu1 retention under these conditions. Finally, we show that the relationship between host translation and TnSmu1 stability extends to a genetically distinct S. mutans clinical isolate, although with strain-dependent penetrance. Together, these findings identify host translational state as an important physiological determinant of TnSmu1 stability and reveal that bacterial hosts can influence the maintenance of integrated mobile genetic elements through mechanisms that extend beyond element-encoded regulatory circuits.

evolutionary biology↗

bioRxiv2026

A genome-wide in vivo screen reveals fitness pathways required for streptococcal infective endocarditis

Infective endocarditis (IE) is a life-threatening disease most often caused by blood-borne bacteria that infect previously damaged cardiac tissue. Despite the importance of this disease, the genetic basis for IE virulence remains poorly defined. Here, we present the first genome-wide in vivo analysis of bacterial fitness in a vertebrate model of IE. We identified 146 genes in Streptococcus sanguinis required for IE fitness, the majority of which had not previously been linked to endocarditis. These determinants cluster into conserved metabolic, cell envelope, transport, and regulatory pathways, representing a vast reservoir of potential targets for novel antimicrobial intervention. A subset of these genes was examined in Streptococcus mutans; all were found to be essential for IE fitness in this distantly related oral species as well, suggesting broad conservation. Using experimental evolution, we further show that disruption of key fitness pathways triggers reproducible compensatory "bypass" mechanisms that reveal the inherent physiological constraints of the IE fitness landscape and identify vulnerable nodes for multi-target drug strategies. Together, these findings redefine streptococcal infective endocarditis as a disease shaped by conserved bacterial fitness networks that may be exploited for therapeutic development. HighlightsO_LIA genome-wide in vivo screen identified 146 Streptococcus sanguinis genes required for infective endocarditis fitness. C_LIO_LI94% of these genes represent previously unrecognized determinants of endocarditis. C_LIO_LIMultiple pathways--including CoA biosynthesis, the shikimate pathway, and rhamnan synthesis--are required for cardiac colonization. C_LIO_LIA subset of infective endocarditis fitness factors are conserved between S. sanguinis and S. mutans, with species-specific adaptations. C_LIO_LIExperimental evolution revealed compensatory metabolic networks that buffer IE fitness defects. C_LI

genetics↗

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

Experimental evolution of gene essentiality in bacteria

Essential gene products carry out fundamental cellular activities in interaction with other components. However, the lack of essential gene mutants and appropriate methodologies to link essential gene functions with their partners poses significant challenges. Here, we have generated deletion mutants in 32 genes previously identified as essential, with 23 mutants showing extremely slow growth in the SK36 strain of Streptococcus sanguinis. The 23 genes corresponding to these mutants encode components of diverse pathways, are widely conserved among bacteria, and are essential in many other bacterial species. Whole-genome sequencing of 243 independently evolved populations of these mutants has identified >1000 spontaneous suppressor mutations in experimental evolution. Many of these mutations define new gene and pathway relationships, such as F1Fo-ATPase/V1Vo-ATPase/TrkA1-H1 that were demonstrated across multiple Streptococcus species. Patterns of spontaneous mutations occurring in essential gene mutants differed from those found in wildtype. While gene duplications occurred rarely and appeared most often at later stages of evolution, substitutions, deletions, and insertions were prevalent in evolved populations. These essential gene deletion mutants and spontaneous mutations fixed in the mutant populations during evolution establish a foundation for understanding gene essentiality and the interaction of essential genes in networks.

evolutionary biology↗

bioRxiv2024

A novel infective endocarditis virulence factor related to multiple functions for bacterial survival in blood was discovered in Streptococcus sanguinis

We identified the role of a conserved hypothetical protein (SSA_0451) in S. sanguinis that is involved in the virulence of infective endocarditis. An in vitro whole blood killing assay and rabbit endocarditis model studies revealed that the SSA_0451 mutant ({Delta}SSA_0451) was significantly less virulent than the wild-type (SK36) and its complementation mutant ({Delta}SSA_0451C). The mechanism underlying the SSA_0451 mutants reduced virulence in infective endocarditis was evidentially linked to oxidative stress and environmental stress. The genes related to the survival of S. sanguinis in an oxidative stress environment were downregulated in {Delta}SSA_0451, which affected its survival in blood. Our findings suggest that SSA_0451 is a novel IE virulence factor and a new target for drug discovery against IE. Author summaryThis study focused on SSA_0451, a conserved hypothetical protein in S. sanguinis, to explore its potential role as a virulence factor. Through in vitro whole blood killing assays and rabbit IE models, it was found that the SSA_0451 mutant exhibited reduced virulence compared to the wild-type and a complemented mutant. The study linked the mutants diminished virulence in IE to heightened susceptibility to oxidative and environmental stresses, supported by downregulation of genes crucial for oxidative stress survival in S. sanguinis. These findings identify SSA_0451 as a novel virulence factor in IE and propose it as a promising target for future drug development against this condition.

microbiology↗

bioRxiv2024

Glycerol Metabolism Contributes to Competition by Oral Streptococci through Production of Hydrogen Peroxide

As a biological byproduct from both humans and microbes, glycerols contribution to microbial homeostasis in the oral cavity remains understudied. Here we examined glycerol metabolism by Streptococcus sanguinis, a commensal associated with oral health. Genetic mutants of glucose-PTS enzyme II (manL), glycerol metabolism (glp and dha pathways), and transcriptional regulators were characterized with regard to glycerol catabolism, growth, production of hydrogen peroxide (H2O2), transcription, and competition with Streptococcus mutans. Biochemical assays identified the glp pathway as a novel source of H2O2 production by S. sanguinis that is independent of pyruvate oxidase (SpxB). Genetic analysis indicated that the glp pathway requires glycerol and a transcriptional regulator, GlpR, for expression and is negatively regulated by PTS, but not the catabolite control protein, CcpA. Conversely, deletion of either manL or ccpA increased expression of spxB and a second, H2O2-non-producing glycerol metabolic pathway (dha), indicative of a mode of regulation consistent with conventional carbon catabolite repression (CCR). In a plate-based antagonism assay and competition assays performed with planktonic and biofilm-grown cells, glycerol greatly benefited the competitive fitness of S. sanguinis against S. mutans. The glp pathway appears to be conserved in several commensal streptococci and actively expressed in caries-free plaque samples. Our study suggests that glycerol metabolism plays a more significant role in the ecology of the oral cavity than previously understood. Commensal streptococci, though not able to use glycerol as a sole carbohydrate for growth, benefit from catabolism of glycerol through production of both ATP and H2O2. ImportanceGlycerol is an abundant carbohydrate found in oral cavity, both due to biological activities of humans and microbes, and as a common ingredient of foods and health care products. However, very little is understood regarding the metabolism of glycerol by some of the most abundant oral bacteria, commensal streptococci. This was in part because most streptococci cannot grow on glycerol as the sole carbon source. Here we show that Streptococcus sanguinis, an oral commensal associated with dental health, can degrade glycerol for persistence and competition through two independent pathways, one of which generates hydrogen peroxide at levels capable of inhibiting a dental pathobiont, Streptococcus mutans. Preliminary studies suggest that several other commensal streptococci are also able to catabolize glycerol, and glycerol-related genes are being actively expressed in human dental plaque samples. Our findings reveal the potential of glycerol to significantly impact microbial homeostasis which warrants further exploration.

microbiology↗

bioRxiv2021

Contribution of metal transporters of the ABC, ZIP, and NRAMP families to manganese uptake and infective endocarditis virulence in Streptococcus sanguinis

Streptococcus sanguinis is an important cause of infective endocarditis. In strain SK36, the ABC-family manganese transporter, SsaACB, is essential for virulence. We have now identified a ZIP-family protein, TmpA, as a secondary manganese transporter. A tmpA mutant had no phenotype, but a {Delta}ssaACB {Delta}tmpA mutant was far more attenuated for serum growth and somewhat more attenuated for virulence in a rabbit model than its {Delta}ssaACB parent. The growth of both mutants was restored by supplemental manganese, but the {Delta}ssaACB {Delta}tmpA mutant required twenty-fold more and accumulated less. Although ZIP-family proteins are known for zinc and iron transport, TmpA-mediated transport of either metal was minimal. In contrast to ssaACB and tmpA, which appear ubiquitous in S. sanguinis, a mntH gene encoding an NRAMP-family transporter has been identified in relatively few strains, including VMC66. As in SK36, deletion of ssaACB greatly diminished VMC66 endocarditis virulence and serum growth, and deletion of tmpA from this mutant diminished virulence further. Virulence was not significantly altered by deletion of mntH from either VMC66 or its {Delta}ssaACB mutant. This and the accompanying paper together suggest that SsaACB is of primary importance for endocarditis virulence while secondary transporters TmpA and MntH contribute to growth under differing conditions.

microbiology↗

bioRxiv2021

Manganese transport by Streptococcus sanguinis in acidic conditions and its impact on growth in vitro and in vivo

Streptococcus sanguinis is an oral commensal and an etiological agent of infective endocarditis. Previous studies have identified the SsaACB manganese transporter as essential for endocarditis virulence; however, the significance of SsaACB in the oral environment has never been examined. Here we report that a {Delta}ssaACB mutant of strain SK36 exhibits reduced growth and manganese uptake under acidic conditions. Further studies revealed that these deficits resulted from the decreased activity of TmpA, shown in the accompanying paper to function as a ZIP-family manganese transporter. Transcriptomic analysis of fermentor-grown cultures of SK36 WT and {Delta}ssaACB strains identified pH-dependent changes related to carbon catabolite repression in both strains, though their magnitude was generally greater in the mutant. In strain VMC66, which possesses a MntH transporter, loss of SsaACB did not significantly alter growth or cellular manganese levels under the same conditions. Interestingly, there were only modest differences between SK36 and its {Delta}ssaACB mutant in competition with Streptococcus mutans in vitro and in a murine oral colonization model. Our results suggest that the heterogeneity of the oral environment may provide a rationale for the variety of manganese transporters found in S. sanguinis and point to strategies for enhancing the safety of oral probiotics. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/446192v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@a5afa7org.highwire.dtl.DTLVardef@1af3cdforg.highwire.dtl.DTLVardef@1c58693org.highwire.dtl.DTLVardef@1063980_HPS_FORMAT_FIGEXP M_FIG Depiction of methods. Streptococcal strains used are depicted at the top. The four methods illustrated are: 1. S. sanguinis cells were grown in media at pH 7.3 and pH 6.2 and quantified by plating. 2. S. sanguinis cells were grown in a fermentor vessel for RNA-sequencing and metal analysis. 3. S. sanguinis and S. mutans cells were grown in 12-well plates singly or in competition, then plated and the pH of the media measured. 4. S. sanguinis and S. mutans cells were inoculated into the mouths of mice. Oral swabs and dental biofilms from molars were assayed for bacterial composition by qPCR. (Biorender) C_FIG

microbiology↗

bioRxiv2020

Time-course analysis of Streptococcus sanguinis after manganese depletion reveals changes in glycolytic, nucleotide, and redox metabolites

IntroductionManganese is important for the endocarditis pathogen, Streptococcus sanguinis. Little is known about why manganese is required for virulence or how it impacts the metabolome of streptococci. ObjectivesWe applied untargeted metabolomics to cells and media to understand temporal changes resulting from manganese depletion. MethodsEDTA was added to a S. sanguinis manganese-transporter mutant in aerobic fermentor conditions. Cell and media samples were collected pre- and post-EDTA treatment. Metabolomics data were generated using positive and negative modes of data acquisition on an LC-MS/MS system. Data were subjected to statistical processing using MetaboAnalyst and time-course analysis using Short Time series Expression Miner (STEM). ResultsWe observed quantitative changes in 534 and 422 metabolites in cells and media, respectively, after EDTA addition. The 173 cellular metabolites identified as significantly different indicated enrichment of purine and pyrimidine metabolism. Further multivariate analysis revealed that the top 15 cellular metabolites belonged primarily to lipids and redox metabolites. The STEM analysis revealed global changes in cells and media in comparable metabolic pathways. Products of glycolysis such as pyruvate and fructose-1,6-bisphosphate increased, suggesting that enzymes that act on them may require manganese for activity or expression. Nucleosides accumulated, possibly due to a blockage in conversion to nucleobases. Simultaneous accumulation of ortho-tyrosine and reduced glutathione suggests that cells were unable to utilize glutathione as a reductant. ConclusionDifferential analysis of metabolites revealed the activation of a number of metabolic pathways in response to manganese depletion, many of which may be connected to carbon catabolite repression.

microbiology↗

bioRxiv2020

Manganese depletion leads to multisystem changes in the transcriptome of the opportunistic pathogen Streptococcus sanguinis

1Streptococcus sanguinis is a primary tooth colonizer and is typically considered beneficial due to its antagonistic relationship with the cariogenic pathogen Streptococcus mutans. However, S. sanguinis can also act as an opportunistic pathogen should it enter the bloodstream and colonize a damaged heart valve, leading to infective endocarditis. Studies have implicated manganese acquisition as an important virulence determinant in streptococcal endocarditis. A knockout mutant lacking the primary manganese import system in S. sanguinis, SsaACB, is severely attenuated for virulence in an in vivo rabbit model. Manganese is a known cofactor for several important enzymes in S. sanguinis, including superoxide dismutase, SodA, and the aerobic ribonucleotide reductase, NrdEF. To determine the effect of manganese depletion on S. sanguinis, we performed transcriptomic analysis on a {Delta}ssaACB mutant grown in aerobic fermentor conditions after the addition of the metal chelator EDTA. Despite the broad specificity of EDTA, analysis of cellular metal content revealed a decrease in manganese, but not in other metals, that coincided with a drop in growth rate. Subsequent supplementation with manganese, but not iron, zinc, or magnesium, restored growth in the fermentor post-EDTA. Reduced activity of Mn-dependent SodA and NrdEF likely contributed to the decreased growth rate post-EDTA, but did not appear entirely responsible. With the exception of the Dps-like peroxide resistance gene, dpr, manganese depletion did not induce stress response systems. By comparing the transcriptome of {Delta}ssaACB cells pre- and post-EDTA, we determined that manganese deprivation led to altered expression of diverse systems, including ethanolamine utilization, CRISPR/Cas, and a type IV pilus. Manganese depletion also led to an apparent induction of carbon catabolite repression in a glucose-independent manner. The combined results suggest that manganese limitation produces effects in S. sanguinis that are diverse and complex, with no single protein or system appearing entirely responsible for the observed growth rate decrease. This study provides further evidence for the importance of this trace element in streptococcal biology. Future studies will focus on determining mechanisms for regulation, as the multitude of changes observed in this study indicate that multiple regulators may respond to manganese levels.

microbiology↗

bioRxiv2020

A novel sialic acid-binding adhesin present in multiple species contributes to the pathogenesis of infective endocarditis

Bacterial binding to platelets is a key step in the development of infective endocarditis (IE). Sialic acid, a common terminal carbohydrate on host glycans, is the major receptor for streptococci on platelets. So far, all defined interactions between streptococci and sialic acid on platelets are mediated by serine rich repeat proteins (SRRPs). However, we identified Streptococcus oralis subsp. oralis IE-isolates that bind sialic acid but lack SRRPs. In addition to binding sialic acid, some SRRP-negative isolates also bind the cryptic receptor {beta}-1,4-linked galactose through a yet unknown mechanism. Using comparative genomics, we identified a novel sialic acid-binding adhesin, here named AsaA (associated with sialic acid adhesion A), present in IE-isolates lacking SRRPs. We demonstrated that S. oralis subsp. oralis AsaA is required for binding to platelets in a sialic acid-dependent manner. AsaA comprises a non-repeat region (NRR), consisting of a FIVAR/CBM and two Siglec-like and Unique domains, followed by 31 DUF1542 domains. When recombinantly expressed, Siglec-like and Unique domains competitively inhibited binding of S. oralis subsp. oralis and directly interacted with sialic acid on platelets. We further demonstrated that AsaA impacts the pathogenesis of S. oralis subsp. oralis in a rabbit model of IE. Additionally, we found AsaA orthologues in other IE-causing species and demonstrated that the NRR of AsaA from Gemella haemolysans blocked binding of S. oralis subsp. oralis, suggesting that AsaA contributes to the pathogenesis of multiple IE-causing species. Finally, our findings provide evidence that sialic acid is a key factor for bacterial-platelets interactions in a broader range of species than previously appreciated, highlighting its potential as a therapeutic target. Authors summaryInfective endocarditis (IE) is typically a bacterial infection of the heart valves that causes high mortality. Infective endocarditis can affect people with preexisting lesions on their heart valves (Subacute-IE). These lesions contain platelets and other host factors to which bacteria can bind. Growth of bacteria and accumulation of host factors results in heart failure. Therefore, the ability of bacteria to bind platelets is key to the development of IE. Here, we identified a novel bacterial protein, AsaA, which helps bacteria bind to platelets and contributes to the development of disease. Although this virulence factor was characterized in Streptococcus oralis, a leading cause of IE, we demonstrated that AsaA is also present in several other IE-causing bacterial species and is likely relevant to their ability to cause disease. We showed that AsaA binds to sialic acid, a terminal sugar present on platelets, thereby demonstrating that sialic acid serves as a receptor for a wider range of IE-causing bacteria than previously appreciated, highlighting its potential as a therapeutic target.

microbiology↗

bioRxiv2018

Manganese acquisition is essential for virulence of Enterococcus faecalis

Manganese (Mn) is an essential micronutrient that is not readily available to pathogens during infection due to an active host defense mechanism known as nutritional immunity. To overcome this nutrient restriction, bacteria utilize high-affinity transporters that allow them to compete with host metal-binding proteins. Despite the established role of Mn in bacterial pathogenesis, little is known about the relevance of Mn in the pathophysiology of E. faecalis. Here, we identified and characterized the major Mn acquisition systems of E. faecalis. We discovered that the ABC-type permease EfaCBA and two Nramp-type transporters, named MntH1 and MntH2, work collectively to promote cell growth under Mn-restricted conditions. The simultaneous inactivation of EfaCBA, MntH1 and MntH2 ({Delta}efa{Delta}mntH1{Delta}mntH2 strain) led to drastic reductions (>95%) in intracellular Mn content, severe growth defects in body fluids (serum and urine) ex vivo, significant loss of virulence in Galleria mellonella, and virtually complete loss of virulence in rabbit endocarditis and murine catheter-associated urinary tract infection (CAUTI) models. Despite the functional redundancy of EfaCBA, MntH1 and MntH2 under in vitro or ex vivo conditions and in the invertebrate model, dual inactivation of efaCBA and mntH2 ({Delta}efa{Delta}mntH2 strain) was sufficient to prompt maximal sensitivity to calprotectin, a Mn- and Zn-chelating host antimicrobial protein, and for the loss of virulence in mammalian models. Interestingly, EfaCBA appears to play a prominent role during systemic infection, whereas MntH2 was more important during CAUTI. The different roles of EfaCBA and MntH2 in these sites could be attributed, at least in part, to the differential expression of efaA and mntH2 in cells isolated from hearts or from bladders. Collectively, this study demonstrates that Mn acquisition is essential for the pathogenesis of E. faecalis and validates Mn uptake systems as promising targets for the development of new antimicrobials.

microbiology↗