Biology subjects

Korotkova, N.

Publications and source records attributed to Korotkova, N..

14 recordsLinked to original sources

bioRxiv2026

CflP couples magnesium availability to CovS signaling in Group B Streptococcus

Opportunistic pathogens must coordinate virulence-factor production with changing conditions encountered during colonization and infection. Group B Streptococcus (GBS), a common human opportunistic pathogen, uses the CovRS two-component system to regulate virulence, but how the CovS sensor kinase is tuned to environmental conditions remains unclear. Here, we identify CflP, a noncatalytic membrane protein of the CPBP family, as an accessory regulator of CovS. CflP associates with CovS and promotes CovS-dependent phosphorylation of the response regulator CovR, thereby repressing virulence-associated genes, including the {beta}-hemolysin/cytolysin operon. Loss of CflP also enhances cytotoxicity toward human immune cells. CflP acts independently of Abx1, a previously identified homologous CPBP protein that promotes CovS phosphatase activity. CflP abundance is regulated post-transcriptionally by Mg2+ availability, linking an environmental cue to CovRS signaling. Comparative genomic analysis revealed distinct patterns of natural variation in these CovS regulators: cflP is predominantly maintained as an intact gene, although specific amino acid substitutions can compromise CflP abundance, whereas abx1 is frequently disrupted by recurrent frameshifts. Together, these findings reveal an accessory regulatory layer that tunes CovS activity in response to environmental conditions and whose natural variation may contribute to strain-dependent differences in CovRS regulation. More broadly, our findings illustrate how noncatalytic membrane proteins can modulate bacterial signaling to balance virulence-factor production with adaptation to changing host environments.

microbiology↗

bioRxiv2026

Enterococcus faecalis autolysin, EpaU, binds the enterococcal polysaccharide antigen via its teichoic acid-like repeats and modulates c-di-AMP signaling

The cell wall of the Gram-positive bacterium Enterococcus faecalis is decorated with the enterococcal polysaccharide antigen (EPA), consisting of a core rhamnan backbone linked covalently with a strain-variable teichoic acid-like (TA) polymer. Current models propose that the TA decoration is a repeating polymer composed of two alternating subunits, designated TAI and TAII, which are attached to the rhamnan core via a mild-acid labile phosphodiester bond from the initiating TAI subunit. In this study, we characterize the EpaU autolysin encoded within the EPA biosynthetic gene cluster. We demonstrate that the cell wall-binding domain of EpaU associates with the intact TA domains of EPA synthesized with the aid of the glycosyltransferases EpaR and EpaX. We further show that EpaU is a potent autolysin that binds generally over the E. faecalis cell surface, suggesting that it functions as a remodeling peptidoglycan hydrolase. The absence of EpaU leads to increased ampicillin resistance and elevated intracellular levels of the second messenger c-di-AMP. These data suggest that E. faecalis possesses a mechanism that senses the integrity of the peptidoglycan meshwork and employs c-di-AMP to regulate cell turgor, potentially altering the antibiotic resistance. ImportanceEnterococcus faecalis is an important opportunistic pathogen that can cause severe nosocomial infections. Knowledge of how bacteria remodel the cell wall is key to understanding many important cellular processes, such as antibiotic resistance, cell division, biofilm formation, and stress resistance. In this study, we shed new light on the structural details of the main cell wall polysaccharide, Enterococcal Polysaccharide Antigen (EPA), and its interaction with EpaU, an autolysin that cleaves peptidoglycan during cell wall remodeling. We also report a link between EpaU and the regulation of turgor pressure via cyclic dinucleotide signaling. This work contributes to a more complete picture of E. faecalis cell wall and may provide insight into the development of antimicrobial agents based on autolysins.

microbiology↗

bioRxiv2025

A conserved glycan motif overcomes antigenic variation inducing broadly reactive antibodies against the zoonotic pathogen Streptococcus suis

Streptococcus suis is a largely neglected but emerging bacterial zoonotic pathogen of global concern for animal welfare, antibiotic resistance development and human health. No effective vaccines are currently available. Here, we identified and characterized the function and structure of two cell wall polysaccharide variants in pathogenic S. suis strains using genetic deletion and (heterologous) complementation, lectin staining, glycan composition analysis and specialized NMR spectroscopy. Both glycan variants were anionic polymers that differed in the presence of glucose in the side-chain as a result of allelic variation in a glycosyltransferase gene. Deletion of this variable glycosyltransferase revealed an identical glycan core and affected S. suis morphology and lysozyme resistance. Immunization of pigs with this core domain induced antibodies recognizing a wide range of antigenically-diverse pathogenic S. suis strains. This study provides new insights for developing next-generation glycoconjugate vaccines, whereby a single-glycan target could protect against the emerging zoonotic pathogen S. suis.

microbiology↗

bioRxiv2024

Natural variation of the streptococcal Group A carbohydrate biosynthesis genes impacts host-pathogen interaction

Streptococcus pyogenes (S. pyogenes) is a leading cause of infection-related mortality in humans globally. The characteristic cell wall-anchored Group A Carbohydrate (GAC) is expressed by all S. pyogenes strains and consists of a polyrhamnose backbone with alternating N-acetylglucosamine (GlcNAc) side chains, of which 25% are decorated with glycerol phosphate (GroP). The genes in the gacA-L cluster are critical for GAC biosynthesis with gacH-L being responsible for the characteristic GlcNAc-GroP decoration, which confers the agglutination in rapid test diagnostic assays and contributes to S. pyogenes pathogenicity. Historical research papers described S. pyogenes isolates, so-called A-variant strains, that lost the characteristic GlcNAc side chain following serial animal passage. Genomic analysis of a single viable historic parent/A-variant strain pair revealed a premature inactivating stop codon in gacI, explaining the described loss of the GlcNAc side chain. Subsequently, we analyzed the genetic variation of the 12 gacA-L genes in a collection of 2,021 S. pyogenes genome sequences. Although all gac genes (gacA-L) displayed genetic variation, we only identified 26 isolates (1.3%) with a premature stop codon in one of the gac genes. Twelve out of 26 (46%) isolates contained a premature stop codon in gacH, which encodes the enzyme responsible for the GroP modification. To study the functional consequences of the different premature stop codons for GacH function, we plasmid-expressed three gacH variants in a S. pyogenes gacH-deficient strain. Cell wall analysis confirmed GacH loss-of-function for the studied gacH variants through the significant reduction of GAC GroP, complete resistance to killing by the human bactericidal enzyme group IIA-secreted phospholipase, and susceptibility to zinc toxicity. Overall, our data provide a comprehensive overview of the genetic variation of the gacA-L cluster in a global population of S. pyogenes strains and the functional consequences of rare inactivating mutations in gacH for host interaction.

microbiology↗

bioRxiv2024

Structure and mechanism of biosynthesis of Streptococcus mutans cell wall polysaccharide

Streptococcus mutans, the causative agent of human dental caries, expresses a cell wall attached Serotype c-specific Carbohydrate (SCC) that is critical for cell viability. SCC consists of a repeating [->]3)-Rha(1[->]2)-Rha(1[->] polyrhamnose backbone, with glucose (Glc) side-chains and glycerol phosphate (GroP) decorations. This study reveals that SCC has one major and two minor Glc modifications. The major Glc modification, -Glc, attached to position 2 of 3-rhamnose, is installed by SccN and SccM glycosyltransferases and is the site of the GroP addition. The minor Glc modifications are {beta}-Glc linked to position 4 of 3-rhamnose installed by SccP and SccQ glycosyltransferases, and -Glc attached to position 4 of 2-rhamnose installed by SccN working in tandem with an unknown enzyme. Both the major and the minor {beta}-Glc modifications control bacterial morphology, but only the GroP and major Glc modifications are critical for biofilm formation.

microbiology↗

bioRxiv2024

Disorder regulates homeostasis of extracytoplasmic proteins in streptococci

Proteins harboring intrinsically disordered regions (IDRs) lacking stable secondary or tertiary structures are abundant across the three domains of life. These regions have not been systematically studied in prokaryotes. Our genome-wide analysis identifies extracytoplasmic serine/threonine-rich IDRs in several biologically important membrane-associated proteins in streptococci. We demonstrate that these IDRs are glycosylated with glucose by glycosyltransferases GtrB and PgtC2 in Streptococcus pyogenes and Streptococcus pneumoniae, and with N-acetylgalactosamine by a Pgf-dependent mechanism in Streptococcus mutans. The absence of glycosylation leads to a defect in biofilm formation under ethanol-stressed conditions in S. mutans. We link this phenotype to the C-terminal IDR of the post-translocation chaperone PrsA. Our data reveal that O-linked glycosylation protects the IDR-containing proteins from proteolytic degradation and is critical for the biological function of PrsA in biofilm formation.

microbiology↗

bioRxiv2023

Gestational diabetes augments group B Streptococcus perinatal infection through disruptions in maternal immunity and the vaginal microbiota

Group B Streptococcus (GBS) is a pervasive perinatal pathogen, yet factors driving GBS dissemination in utero are poorly defined. Gestational diabetes mellitus (GDM), a complication marked by dysregulated immunity and maternal microbial dysbiosis, increases risk for GBS perinatal disease. We interrogated host-pathogen dynamics in a novel murine GDM model of GBS colonization and perinatal transmission. GDM mice had greater GBS in utero dissemination and subsequently worse neonatal outcomes. Dual-RNA sequencing revealed differential GBS adaptation to the GDM reproductive tract, including a putative glycosyltransferase (yfhO), and altered host responses. GDM disruption of immunity included reduced uterine natural killer cell activation, impaired recruitment to placentae, and altered vaginal cytokines. Lastly, we observed distinct vaginal microbial taxa associated with GDM status and GBS invasive disease status. Our translational model of GBS perinatal transmission in GDM hosts recapitulates several clinical aspects and enables discovery of host and bacterial drivers of GBS perinatal disease.

microbiology↗

bioRxiv2023

Heterogeneity of the group B streptococcal type VII secretion system and influence on colonization of the female genital tract

Type VIIb secretion systems (T7SSb) in Gram-positive bacteria facilitate physiology, interbacterial competition, and/or virulence via EssC ATPase-driven secretion of small [a]-helical proteins and toxins. Recently, we characterized T7SSb in group B Streptococcus (GBS), a leading cause of infection in newborns and immunocompromised adults. GBS T7SS comprises four subtypes based on variation in the C-terminus of EssC and the repertoire of downstream effectors; however, the intra-species diversity of GBS T7SS and impact on GBS-host interactions remains unknown. Bioinformatic analysis indicates that GBS T7SS loci encode subtype-specific putative effectors, which have low inter-species and inter-subtype homology but contain similar domains/motifs and therefore may serve similar functions. We further identify orphaned GBS WXG100 proteins. Functionally, we show that GBS T7SS subtype I and III strains secrete EsxA in vitro and that in subtype I strain CJB111, esxA1 appears to be differentially transcribed from the T7SS operon. Further, we observe subtype-specific effects of GBS T7SS on host colonization, as subtype I but not subtype III T7SS promotes GBS vaginal persistence. Finally, we observe that T7SS subtypes I and II are the predominant subtypes in clinical GBS isolates. This study highlights the potential impact of T7SS heterogeneity on host-GBS interactions.

microbiology↗

bioRxiv2021

PplD is a de-N-acetylase of the cell wall linkage unit of streptococcal rhamnopolysaccharides

The cell wall of the human bacterial pathogen Group A Streptococcus (GAS) consists of peptidoglycan decorated with the Lancefield group A carbohydrate (GAC). GAC is a promising target for the development of GAS vaccines. In this study, employing chemical, compositional, and NMR methods, we show that GAC is attached to peptidoglycan via glucosamine 1-phosphate. This structural feature makes the GAC-peptidoglycan linkage highly sensitive to cleavage by nitrous acid and resistant to mild acid conditions. Using this characteristic of the GAS cell wall, we identify PplD as a protein required for deacetylation of linkage N-acetylglucosamine (GlcNAc). X-ray structural analysis indicates that PplD performs catalysis via a modified acid/base mechanism. Genetic surveys in silico together with functional analysis indicate that PplD homologs deacetylate the polysaccharide linkage in many streptococcal species. We further demonstrate that introduction of positive charges to the cell wall by GlcNAc deacetylation protects GAS against host cationic antimicrobial proteins.

microbiology↗

bioRxiv2021

Molecular basis for recognition of the Group A Carbohydrate backbone by the PlyC streptococcal bacteriophage endolysin

Endolysins are peptidoglycan (PG) hydrolases that function as part of the bacteriophage (phage) lytic system to release progeny phage at the end of a replication cycle. Notably, endolysins alone can produce lysis without phage infection, which offers an attractive alternative to traditional antibiotics. Endolysins from phage that infect Gram-positive bacterial hosts contain at least one enzymatically active domain (EAD) responsible for hydrolysis of PG bonds and a cell wall binding domain (CBD) that binds a cell wall epitope, such as a surface carbohydrate, providing some degree of specificity for the endolysin. Whilst the EADs typically cluster into conserved mechanistic classes with well-defined active sites, relatively little is known about the nature of the CBDs and only a few binding epitopes for CBDs have been elucidated. The major cell wall components of many streptococci are the polysaccharides that contain the polyrhamnose (pRha) backbone modified with species-specific and serotype-specific glycosyl side chains. In this report, using molecular genetics, microscopy, flow cytometry and lytic activity assays, we demonstrate the interaction of PlyCB, the CBD subunit of the streptococcal PlyC endolysin, with the pRha backbone of the cell wall polysaccharides, Group A Carbohydrate (GAC) and serotype c-specific carbohydrate (SCC) expressed by the Group A Streptococcus and Streptococcus mutans, respectively.

biochemistry↗

bioRxiv2020

Modification of streptococcal cell wall polysaccharide spatially controls cell division

Bacterial cell division is driven by a tubulin homolog FtsZ, which assembles into the Z-ring structure leading to the recruitment of the cell division machinery. In ovoid-shaped Gram-positive bacteria, such as streptococci, MapZ guides Z-ring positioning at cell equators through an, as yet, unknown mechanism. The cell wall of the important dental pathogen Streptococcus mutans is composed of peptidoglycan decorated with Serotype c Carbohydrates (SCCs). Here, we show that an immature form of SCC, lacking the recently identified glycerol phosphate (GroP) modification, coordinates Z-ring positioning. Pulldown assays using S. mutans cell wall combined with binding affinity analysis identified the major cell separation autolysin, AtlA, as an SCC binding protein. Importantly, AtlA binding to mature SCC is attenuated due to GroP modification. Using fluorescently-labeled AtlA, we mapped SCC distribution on the streptococcal surface to reveal that GroP-deficient immature SCCs are exclusively present at the cell poles and equators. Moreover, the equatorial GroP-deficient SCCs co-localize with MapZ throughout the S. mutans cell cycle. Consequently, in GroP-deficient mutant bacteria, proper AtlA localization is abrogated resulting in dysregulated cellular autolysis. In addition, these mutants display morphological abnormalities associated with MapZ mislocalization leading to Z-ring misplacement. Altogether, our data support a model in which GroP-deficient immature SCCs spatially coordinate the localization of AtlA and MapZ. This mechanism ensures cell separation by AtlA at poles and Z-ring alignment with the cell equator.Competing Interest StatementThe authors have declared no competing interest.View Full Text

microbiology↗

bioRxiv2020

PE5-PPE4-EspG3 trimer structure from mycobacterial ESX-3 secretion system gives insight into cognate substrate recognition by ESX systems

Mycobacterium tuberculosis (Mtb) has evolved numerous type VII secretion (ESX) systems to secrete multiple factors important for both growth and virulence across their cell envelope. Three such systems; ESX-1, ESX-3, and ESX-5; have been shown to each secrete a unique set of substrates. A large class of these substrates secreted by these three systems are the PE and PPE families of proteins. Proper secretion of the PE-PPE proteins requires the presence of EspG, with each system encoding its own unique copy. There is no cross-talk between any of the ESX systems and how each EspG is recognizing its subset of PE-PPE proteins is currently unknown. The only current structural characterization of PE-PPE-EspG trimers is from the ESX-5 system. Here we present the crystal structure of the PE5mt-PPE4mt-EspG3mm trimer, from the ESX-3 system. Our trimer reveals that EspG3mm interacts exclusively with PPE4mt in a similar manner to EspG5, shielding the hydrophobic tip of PPE4mt from solvent. The C-terminal helical domain of EspG3mm is dynamic, alternating between an open and closed form, and this movement is likely functionally relevant in the unloading of PE-PPE heterodimers at the secretion machinery. In contrast to the previously solved ESX-5 trimers, the PE-PPE heterodimer of our ESX-3 trimer is interacting with its chaperone at a drastically different angle, and presents different faces of the PPE protein to the chaperone. We conclude that the PPE-EspG interface from each ESX system has a unique shape complementarity that allows each EspG to discriminate amongst non-cognate PE-PPE pairs.

biochemistry↗

bioRxiv2018

Genetic insight into zinc and antimicrobial toxicity uncovers a glycerol phosphate modification on streptococcal rhamnose polysaccharides

Cell wall glycopolymers on the surface of Gram-positive bacteria are fundamental to bacterial physiology and infection biology. These structures have also gained interest as vaccine antigens, in particular for the human pathogens Group A Streptococcus (GAS) and Streptococcus mutans. Streptococcal cell wall glycopolymers are considered to be functional homologues of wall teichoic acids but surprisingly lack the biologically-relevant and characteristic anionic charge. Here we identify gacH, a gene of unknown function in the GAS Group A Carbohydrate (GAC) biosynthetic cluster, in two independent transposon library screens for its ability to confer resistance to zinc and susceptibility to the bactericidal enzyme human group IIA secreted phospholipase A2. To understand the underlying mechanism of these phenotypes, we determined the structure of the extracellular domain of GacH and discover that it represents a new family of glycerol phosphate (GroP) transferases. Importantly, we demonstrate the presence of GroP in both the GAC and the homologous Serotype c Carbohydrate (SCC) from S. mutans, which is conferred by gacH and sccH products, respectively. NMR analysis of GAC released from cell wall by non-destructive methods reveals that approximately 30% of the GAC GlcNAc side-chains are modified by GroP at the C6 hydroxyl group. This previously unrecognized structural modification impacts host-pathogen interaction and has implications for vaccine design. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/337519v4_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@6d7598org.highwire.dtl.DTLVardef@435695org.highwire.dtl.DTLVardef@e9515corg.highwire.dtl.DTLVardef@1dc78b7_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2017

The molecular mechanism of N-acetylglucosamine side-chain attachment to the Lancefield group A Carbohydrate in Streptococcus pyogenes

In many Lactobacillales species (i.e. lactic acid bacteria), peptidoglycan is decorated by polyrhamnose polysaccharides that are critical for cell envelope integrity and cell shape and also represent key antigenic determinants. Despite the biological importance of these polysaccharides, their biosynthetic pathways have received limited attention. The important human pathogen, Streptococcus pyogenes, synthesizes a key antigenic surface polymer--the Lancefield group A carbohydrate (GAC). GAC is covalently attached to peptidoglycan and consists of a polyrhamnose polymer, with N-acetylglucosamine (GlcNAc) side chains, which is an essential virulence determinant. The molecular details of the mechanism of polyrhamnose modification with GlcNAc are currently unknown. In this report, using molecular genetics, analytical chemistry and mass spectrometry analysis, we demonstrated that GAC biosynthesis requires two distinct undecaprenol-linked GlcNAc-lipid intermediates: GlcNAc-pyrophosphorylundecaprenol (GlcNAc-P-P-Und) produced by the GlcNAc-phosphate transferase GacO and GlcNAc-phosphate-undecaprenol (GlcNAc-P-Und) produced by the glycosyltransferase GacI. Further investigations revealed that the GAC polyrhamnose backbone is assembled on GlcNAc-P-P-Und. Our results also suggested that a GT-C glycosyltranferase, GacL, transfers GlcNAc from GlcNAc-P-Und to polyrhamnose. Moreover, GacJ, a small membrane-associated protein, formed a complex with GacI and significantly stimulated its catalytic activity. Of note, we observed that GacI homologs perform a similar function in Streptococcus agalactiae and Enterococcus faecalis. In conclusion, the elucidation of GAC biosynthesis in S. pyogenes reported here enhances our understanding of how other Gram-positive bacteria produce essential components of their cell wall.

microbiology↗