Biology subjects

Kjos, M.

Publications and source records attributed to Kjos, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Mild extraction preserves labile structural features of Gram-positive bacterial cell wall glycopolymers

Cell wall glycopolymers in Gram-positive bacteria are crucial for structural integrity, cell division, and stress resistance. Elucidating the structure of these glycopolymers is not only important for understanding fundamental bacterial physiology, but may facilitate vaccine development, phage therapy, antibiotic design and improvements in food fermentation safety. However, extraction protocols have so far relied on harsh chemicals that can damage these polymers, compromise their structural integrity, obscure their biological functions, and result in low yields. We present a straightforward method for isolating cell wall glycopolymers from Gram-positive bacteria that preserves essential structural features including attachment to peptidoglycan. The method was broadly applicable across phylogenetically diverse species and was not restricted to glycopolymer types. In total, we isolated 17 distinct glycopolymers from 11 species, including six with previously unreported structures, with a median yield of 49 mg per litre of culture. The simplicity and mild nature of the method, combined with the exceptional purity of the resulting products, has the potential to transform research on Gram-positive cell wall glycopolymers

microbiology↗

bioRxiv2026

Exploring the antimicrobial potential of streptococcal fratricins

Many streptococci are competent for natural transformation, during which they express a murein hydrolase that lyses and kills closely related species, thereby making homologous DNA available for uptake by competent cells. This process, termed fratricide, is mediated by enzymes known as fratricins. The most extensively studied fratricin, CbpD from Streptococcus pneumoniae, is highly specific, efficient, and rapid in its killing, highlighting the potential of fratricins as targeted antimicrobial agents. Here, we screened nine putative fratricins to evaluate their antimicrobial activity. Of these, Zoocin A from Streptococcus agalactiae, ScrM from Streptococcus dysgalactiae, and CbpD from Streptococcus vestibularis were successfully purified. Zoocin A exhibited the broadest target range, whereas ScrM and CbpD-ves showed somewhat narrower activity. All three enzymes predominantly targeted streptococci. Notably, Zoocin A was active against Streptococcus mutans and Streptococcus sobrinus, species associated with dental caries. The enzymes were also active against S. agalactiae, S. dysgalactiae, and S. pyogenes, which are associated with severe infections in humans and animals. We characterised the activity and stability of Zoocin A and ScrM and found that both retained activity under physiologically relevant conditions of pH, temperature, and ionic strength. ScrM activity was independent of divalent cations, whereas Zoocin A activity required cations and was observed with both Ca2+ and Mn2+. For both enzymes, peptidoglycan cleavage was localised to the septal region of dividing cells. Zoocin A shows promise as an antimicrobial agent against S. mutans, but its efficacy against biofilms would likely benefit from further optimisation or combination with biofilm-disrupting agents.

microbiology↗

bioRxiv2026

Cyclic di-AMP signalling affects cell division and penicillin susceptibility in Streptococcus pneumoniae.

Resistance to {beta}-lactam antibiotics in the human pathogen Streptococcus pneumoniae is mainly attributed to the acquisition of mutated versions of pbp2x, pbp2b and pbp1a, encoding functional penicillin binding proteins with reduced affinity to {beta}-lactams. This enables the bacterium to synthesise peptidoglycan required for cell division in the presence of higher {beta}-lactam concentrations. In addition, it has recently been shown that increased levels of the second messenger molecule cyclic di-AMP, resulting from mutations that compromise the cyclic di-AMP degrading phosphodiesterase Pde1, contribute to decreased {beta}-lactam susceptibility in this species. A link between cyclic di-AMP levels and beta-lactam resistance has also been reported in several other Gram-positive bacteria. Here, we further investigated this link in S. pneumoniae and present evidence supporting that cyclic di-AMP could be involved in regulation of cell division. We confirm that elevated cyclic di-AMP levels decreased penicillin susceptibility and cell size in pde1 mutants. Next, we show that low cyclic di-AMP levels had the opposite effects in which cells became more susceptible to penicillin and displayed an elongated morphology with multiple incomplete septa. Using fluorescence microscopy and bacterial two-hybrid assays, we found that proteins involved in cyclic di-AMP synthesis (CdaA, CdaR) and degradation (Pde1) were enriched around the division site and that they most probably interact with each other. Finally, we identified changes to the cell wall stem peptide composition of {Delta}pde1 cells. Together our findings indicate that cyclic di-AMP could function as a regulator of the division machinery.

microbiology↗

bioRxiv2026

Novel insights into the biosynthesis and diversity of leaderless multipeptide bacteriocins

Garvicin KS (GarKS) is a three-peptide, leaderless, broad-spectrum bacteriocin that is active against a wide range of Gram-positive bacteria, including several foodborne pathogens and antibiotic-resistant strains. This bacteriocin is considered a candidate for application in food preservation and medical treatments; however, key knowledge about the producer strain, regulation of production, and the mechanism of action is still lacking for leaderless, multipeptide bacteriocins, including GarKS. A hybrid sequencing strategy was used to obtain a high-quality closed genome assembly of the native GarKS producer Lactococcus garvieae KS1546, which showed that the bacteriocin was encoded on a 50 kb plasmid (pKS50). Comparative analysis with updated sequence databases indicates that the producer strain should be reclassified as Lactococcus petauri. The roles of the biosynthetic genes, putatively encoding a transcriptional regulator (gakR) and immunity protein (gakI), were examined using heterologous expression. Removal of gakR resulted in a 6-fold decrease in GarKS production, and expression of gakI caused a 250-fold decrease in susceptibility to GarKS, with varying degrees of cross-immunity to other multipeptide bacteriocins. Isolation and whole-genome sequencing of spontaneous GarKS mutants in L. lactis showed that resistance levels are low, but that ythA, a PspC-domain-containing protein involved in a phage stress response pathway is involved in the GarKS susceptible phenotype. To decipher conserved features involved in production of these bacteriocins, we finally used genome mining to identify 11 candidates for new multipeptide bacteriocins; four of them were obtained synthetically and confirmed to be bioactive peptides inhibiting important pathogens including Listeria monocytogenes and enterococci.

microbiology↗

bioRxiv2025

Distinct denitrification phenotypes in closely related bacteria: clues to understanding variations in nitrite accumulation among Stutzerimonas strains

Nitrite (NO2-) is a key denitrification intermediate, formed from nitrate (NO3-). Transient NO2- accumulation varies among denitrifiers, yet the underlying causes remain poorly understood, despite its potential toxicity and role in NO and N2O emissions. We profiled eighteen related Stutzerimonas strains, including the model S. perfectomarina ZoBell, and identified three phenotypic clusters (full, partial and low nitrite accumulators; FNA, PNA and LNA) based on the fraction of NO3--N transiently accumulated as NO2-. LNA strains lack or express the membrane-bound nitrate reductase (NarG) late, relying on periplasmic NapA for NO3- reduction, possibly explaining their balanced NO2- production/reduction. FNA and PNA strains possess NapA and NarG but differ in their nitrite reductase (NirS) clades. Delayed nirS transcription as FNA strains transition to NO3- respiration likely accounts for some NO2- accumulation. However, addition of NO3- halted NO2- reduction in FNA strains, suggesting additional metabolic control. This may require the cytochromes NirTB, which are only found in FNA strains. The regulator DnrE was also unique to NO2--accumulators, likely having a role in finetuning NO2- regulation. Our findings reveal diverse NO2--handling phenotypes among denitrifiers and provide insights for optimizing wastewater nitrogen removal and soil bioaugmentation strategies to mitigate N2O emissions.

microbiology↗

bioRxiv2025

The phage shock protein response of Listeria monocytogenes influences tolerance to the multipeptide bacteriocin garvicin KS

An estimated 30% of all food produced worldwide is lost or wasted every year. A considerable portion of that waste is due to perishable food products, that spoil and can become unsafe to eat relatively quickly. For some high-quality perishables, such as fresh fish and cold-smoked salmon, traditional food preservation techniques are unsuitable as they can compromise sensory qualities such as flavor, texture, and freshness. These products often support the growth of the human pathogen Listeria monocytogenes, which can be present if thermal treatment is not applied. Thus, antilisterial bacteriocins like garvicin KS (GarKS) in combination with conventional technologies like high-pressure processing (HPP) or modified atmosphere packaging (MAP) are being investigated as hurdle strategies to increase the shelf life and food safety of packaged fish products. In this study we showed that L. monocytogenes strains associated with fish food and fish processing plants are susceptible to GarKS with MIC values ranging from 20 to 275 nM. Exposure to GarKS resulted in an upregulation of genes involved in the phage shock protein response, and isolation of resistant mutants indicated a low frequency of resistance to GarKS (10{square}{square} to 10{square}11). Resistant mutants were shown to harbor disruption mutations in lmo2468, encoding a PspC-domain-containing protein. Overexpression of this gene increased susceptibility to GarKS two-fold and restored wild-type susceptibility in a disruption mutant. This study identify the phage shock protein response as a key player involved in susceptibility to GarKS.

molecular biology↗

bioRxiv2025

LytF contributes to pilus extrusion during natural competence in Streptococcus sanguinis SK36.

Streptococci may enter a physiological state called competence, during which they express a specific set of genes required for exogenous DNA uptake and its subsequent integration into the genome through homologous recombination. This process, termed natural transformation, facilitates the horizontal acquisition of genetic material, potentially conferring adaptive advantages that enhance bacterial survival under selective pressures. To make homologous DNA available in the surrounding environment, Streptococcus pneumoniae expresses a cell wall hydrolase (CbpD) that lyses and kills closely related species. This process has been coined fratricide, and the acting hydrolase a fratricin. A significant fraction of streptococcal species does not encode a CbpD-like protein, but another competence-induced peptidoglycan hydrolase LytF. It has been speculated that LytF serves the same purpose as CbpD, however, our investigations into the role of LytF in Streptococcus sanguinis revealed no evidence supporting LytF as a fratricin. Instead, we show that LytF is involved in natural transformation by promoting DNA uptake. An essential part of DNA uptake is the competence-induced type IV pilus, which facilitates DNA uptake by pulling nearby DNA toward the cell. By immunoblotting and microscopy imaging, we found that LytF increases the extracellular levels of the major pilus component ComGC, suggesting that LytF may modify peptidoglycan to promote pilus extrusion across the cell wall, thereby enhancing the efficiency of DNA uptake. ImportanceStreptococci are a significant cause of severe infections in both humans and animals. They are particularly adept at acquiring new genes through horizontal gene transfer as they can become competent for natural transformation. This allows them to quickly adapt to selective pressure and spread genes involved in virulence and antibiotic resistance. In Streptococcus sanguinis, the competence-induced peptidoglycan hydrolase LytF has been reported to stimulate natural transformation. Our study adds to the understanding of this process by demonstrating that LytF promotes extrusion of the transformation pilus required for DNA uptake.

microbiology↗

bioRxiv2025

Phage resistance impacts antibiotic susceptibility and virulence in Staphylococcus aureus

As antibiotic resistance continues to rise worldwide, phage therapy offers a promising solution by harnessing viruses that infect and kill bacteria. However, phage-resistance may develop, compromising therapy. Phage-resistance has primarily been linked to changes in major phage receptors that for the human pathogen Staphylococcus aureus are the cell-wall linked wall teichoic acids (WTA). To identify additional factors contributing to phage-resistance, S. aureus was evolved under selective pressure from lytic phages, including candidates from therapeutic phage cocktails, to obtain resistant clones. A third of the phage-resistant clones acquired mutations associated with cell-wall changes previously linked with phage resistance, namely in femA, involved in peptidoglycan crossbridge formation, and tagO, encoding the initiator of the WTA biosynthesis. The remaining clones had mutations in pathways not previously associated with phage-resistance, including nucleoside catabolism (deoC1), polyamine import (potAB) and DNA/RNA replication (cshA, ligA), suggesting that phage-resistance may be associated with phage-driven host takeover. Antibiotic susceptibility and virulence were diversely affected by the mutations; mutations in tagO increased {beta}-lactams sensitivity and attenuated virulence, whereas mutations in femA/ligA or cshA increased staphylococcal virulence in Galleria mellonella. This work demonstrates the involvement of several novel bacterial pathways in phage resistance, highlighting the complexity behind phage-bacterial interactions.

microbiology↗

bioRxiv2025

Cell splitting in Staphylococcus aureus is controlled by an adaptor protein facilitating degradation of a peptidoglycan hydrolase

Regulated protein degradation by Clp proteases is a highly conserved post-translational control mechanism in bacteria. In Staphylococcus aureus, the ClpXP complex targets the peptidoglycan hydrolase Sle1, maintaining a tightly regulated balance between peptidoglycan biosynthesis and hydrolysis, which is required to ensure proper cell splitting without compromising cell integrity. {beta}-lactams antibiotics disturb this balance, leading to their bactericidal effects. The mechanism underlying the specific targeting of Sle1 by the conserved ClpXP complex remains unknown. From a genome-wide screen for determinants of penicillin G susceptibility in S. aureus, we here identify the uncharacterized protein CxaR (for ClpXP-associated autolytic regulator). Growth defects, premature cell splitting, and increased cell lysis were observed in the absence of CxaR. Interestingly, these defects were mitigated by sublethal concentrations of {beta}-lactams. Through sequencing cxaR suppressor mutants, followed by immunoblotting, we show that the cxaR phenotypes are caused by excessive Sle1 accumulation. Indeed, exposure to {beta}-lactams reduces Sle1 levels, thereby rescuing the cells lacking CxaR. Furthermore, in vivo protein-protein interaction assays demonstrated that CxaR directly interacts with both ClpXP and Sle1, whereas no direct interaction was detected between Sle1 and ClpX. In line with this, CxaR was found to co-localize with ClpX adjacent to the septum. Taken together, these findings reveal that CxaR is a new regulatory factor controlling staphylococcal cell splitting by acting as an adaptor protein for controlled ClpXP-mediated degradation of Sle1.

microbiology↗

bioRxiv2025

The effect of MurM and a branched cell wall structure on penicillin resistance in Streptococcus pneumoniae

The aminoacyltransferase MurM is an important penicillin resistance determinant in Streptococcus pneumoniae. This enzyme attaches a serine or alanine to the dipeptide side chain of lipid II, resulting in branched muropeptides that can be crosslinked to stem peptides in the peptidoglycan layer by penicillin binding proteins (PBPs). Deletion of murM results in only linear muropeptides, and more importantly a significant reduction in resistance. Highly penicillin resistant pneumococci are known to express low-affinity PBPs, an altered MurM protein, and possess a highly branched cell wall structure. It has therefore been hypothesized that MurM, and thus branched muropeptides, are essential for resistance because they are better substrates for low-affinity PBPs. In this study, we found that neither the version of murM nor elevated levels of cell wall branching affected the resistance level. To further support this, we investigated whether branched muropeptide substrates compete better than linear versions with penicillin at the active site of low-affinity PBPs and quantified changes to the stem peptide composition of the resistant Pen6 strain in response to subinhibitory concentrations of penicillin. We found that the level of cell wall branching decreased during penicillin exposure. Together our results do not support the idea that elevated levels of branched muropeptides (more active MurM) are important for either the function of low-affinity PBPs or the cells response to penicillin. Nevertheless, since a functional MurM enzyme is important for resistance, we speculate that it might indirectly influence other functions related to cell wall synthesis and remodelling needed for a resistant phenotype. ImportanceA fundamental understanding of the mechanisms behind antibiotic resistance is needed to find strategies to extend the clinical relevance of existing drugs. This study explores the relationship between cell wall composition and penicillin resistance in Streptococcus pneumoniae. Here we confirm that branched peptide crosslinks in the cell wall are crucial for resistance but found no correlation between elevated branching levels and resistance. Our data suggest that the function of low-affinity penicillin binding proteins is not influenced by the lack of branched cell wall precursors. Instead, a branched cell wall might contribute to resistance via other cell wall biosynthesis and remodelling mechanisms. These insights could offer new perspectives on why a branched cell wall is important for penicillin resistance in pneumococci.

microbiology↗

bioRxiv2025

Temperature-dependent regulation of bacterial cell division hydrolases by the coordinated action of a regulatory RNA and the ClpXP protease

A defining feature of bacteria is the peptidoglycan cell wall which provides structural integrity and prevents osmotic lysis. While peptidoglycan hydrolases are required for daughter cell separation, dysregulated cell wall degradation may result in cell lysis. The mechanisms allowing bacteria to control these deadly enzymes in response to environmental changes remain incompletely understood. Here, we find that in Staphylococcus aureus, temperature-dependent regulation of such hydrolases occurs by the coordinated action of a CHAP domain-specific regulatory RNA and the ClpXP protease. Using a proteomics approach, we identify a hitherto uncharacterized ClpXP controlled autolysin, CxcA, with a catalytic CHAP domain and show that it contributes to separation of daughter cells. CxcA is positively controlled by a non-coding RNA, named Rbc1 (for RNA binding to CHAP domain) transcribed from the antisense strand of cxcA. Notably, Rbc1 is capable of base pairing with RNAs encoding the CHAP domains of numerous cell wall hydrolases and we show that Rbc1 works in trans to upregulate the cell division hydrolase Sle1. Specifically, Rbc1 functions as a thermosensor allowing for upregulation of CxcA and Sle1 at low temperature where daughter cell separation is impeded. Interestingly, the Rbc1-mediated up-regulation of CxcA and Sle1 does not involve mRNA stabilization or increased translation; instead, Rbc1 depletion increases ClpXP-mediated degradation. In conclusion, we identify a novel cell division hydrolase that is highly conserved in Staphylococci and show that it is co-regulated with enzymes containing the catalytic CHAP domain via transcriptional regulation, an RNA-RNA temperature sensory mechanism and the ClpXP protease.

microbiology↗

bioRxiv2025

Tracing of streptococcal strains from infant stool across human body sites links gut-specificity to adhesins

Streptococcal species are human commensals known to colonize multiple body sites. Despite being early gut colonizers, we lack strain-level information about their origin and persistence in the gut. To gain more insight into the habitats of the streptococci present in the infant gut, we did a systematic study where mother-infant pairs were sampled from multiple body sites (stool, oral cavity, vagina, breast milk). We performed whole metagenome sequencing and isolated streptococci from 100 infant stool samples (collected at 10 days of age). To trace the streptococci at the strain level, we designed selective qPCR primers for seven streptococcal strains, and these were later utilized to screen corresponding samples from multiple body sites of the infants and their mothers. We found that two of the strains (one Streptococcus parasanguinis and one Streptococcus vestibularis) were highly prevalent in stool samples, both from infants during their first 2 years of life and from their mothers, indicating that these strains are adapted to the gut environment. Interestingly, another S. parasanguinis strain, closely related to the gut-prevalent strain, showed a completely different prevalence pattern, and was mainly detected in vaginal swabs, breast milk and oral swabs. Comparisons of their genomes revealed major differences in genes encoding adhesins, suggesting that host surface attachment could be a key factor for the observed differences in body site specificity. Together, our extensive tracing of streptococci across body sites of 100 infants and their mothers, provides strain-level information of prevalence patterns and reveals the presence of gut-specific streptococci. ImportanceStreptococci thrive on the mucosal surfaces covering the human body and colonize multiple body sites. To determine the distinct streptococcal composition in each habitat and to evaluate their presence in different habitats, strain-level characterization is crucial. We show that two closely related strains, both isolated from stool, are distributed differently across the human body, with one of them prevalent in the stool samples and the other more prevalent in other samples. This emphasizes the necessity of strain-level analysis for the identification of true colonizers of a habitat.

microbiology↗

bioRxiv2024

A programmable, selection-free CRISPR interference system in Staphylococcus aureus

Common dCas9-based CRISPR interference (CRISPRi) system for gene regulation requires antibiotic selection and exogenous inducer molecules, posing significant challenges when applied in in vivo bacterial infection models. Using Staphylococcus aureus as a model organism, we have developed a programmable, plasmid-based, but selection-free (ppsf)-CRISPRi system that is based on the pCM29- plasmid which is stable without antibiotic selection. In this ppsf-CRISPRi system, dCas9 expression is regulated by an endogenous virulence gene promoter, and sgRNA expression is driven by a constitutive promoter eliminating the need for exogenous inducer molecules. The system was programmed to silence the expression of genes encoding the virulence factor coagulase or peptidoglycan hydrolase autolysin, whenever their respective endogenous promoter was activated. The selection-free functionality was confirmed over at least 27 generations and verified by qPCR and phenotypic assays depending on the protein target, including coagulation of rabbit plasma and THP-1 macrophage cell infection in vitro as well as in vivo infection of Galleria mellonella larvae, in each case phenocopying the observations made using transposon mutant strains. The system is suitable for long-term studies of S. aureus pathogenesis in vitro or in vivo and represents a blueprint for the development of similar ppsf-CRISPRi systems in other bacterial species.

microbiology↗

bioRxiv2024

An antibiotic-free antimicrobial combination of bacteriocins and a peptidoglycan hydrolase: in vitro and in vivo assessment of its efficacy

Mastitis is an inflammatory disease of the mammary gland commonly brought about by bac-terial pathogens that gain physical access to the glandular epithelium through the teat canal. In bovines, common mastitis-causing agents are environmental or pathogenic bacterial spe-cies, including staphylococci, streptococci, enterococci, and Gram-negative bacteria such as Escherichia coli. Current therapeutic strategies for bovine mastitis typically involve the ad-ministration of antibiotic formulations within the infected udder, possibly resulting in in-creased selection of antibiotic resistance and the accumulation of antibiotic residues within the milk. In this study, we sought to design an antibiotic-free antimicrobial formulation to treat bovine mastitis based on bacterial antimicrobial peptides (bacteriocins) and proteins (pepti-doglycan hydrolases). Using a combination of in vitro assays with a range of bacteriocins, we show that the combination of the thiopeptide micrococcin P1 (MP1) and the lantibiotic nisin A (NisA) is a robust antimicrobial formulation that effectively inhibits the growth of bo-vine mastitis-derived bacteria, both in planktonic and biofilm-associated growth modes. The addition of AuresinePlus (Aur, a staphylococcus-specific PGH) further increased the antimi-crobial potency against S. aureus. Furthermore, using two mouse models, a skin infection model and a mastitis model, we show that the combination MP1-NisA-Aur effectively inhibits methicillin-resistant S. aureus (MRSA) in vivo. We discuss the potential and challenges of using antibiotic-free antimicrobial combinations in the treatment of bacterial infections.

microbiology↗

bioRxiv2024

Genome-wide analysis of fitness determinants of Staphylococcus aureus during growth in milk

Staphylococcus aureus is a major concern in the dairy industry due to its significance as a pathogen causing bovine mastitis as well as a source of food poisoning. The nutrient-rich milk environment supports bacterial growth, but the specific genetic determinants that facilitate S. aureus proliferation and persistence in milk are poorly understood. In this study, we conducted a genome-wide CRISPR interference sequencing (CRISPRi-seq) screen to identify fitness determinants essential for S. aureus growth and survival in milk. We identified 282 milk-essential genes, including those with key roles in DNA replication, protein synthesis, and metabolism. Comparative analysis with brain heart infusion (BHI) as growth medium, revealed 79 genes with differential fitness, highlighting specific adaptations required for growth in milk. Notably, we found that purine biosynthesis, folate cycle pathways, and metal acquisition were particularly important in this environment. Based on this, we further demonstrate that S. aureus is more sensitive to the folate inhibitors trimethoprim-sulfamethoxazole (TMP-SMX) in milk and identify several genes whose knockdown results in hypersensitivity to TMP-SMX in milk. Additionally, our analysis showed a relatively reduced importance of cell wall components, such as teichoic acids, for S. aureus fitness in milk, which is also reflected in reduced efficiency of antimicrobials targeting teichoic acids. Together, these findings provide new insights into the genetic basis of S. aureus fitness and antibiotic susceptibility in milk, offering directions for novel treatment strategies against bovine mastitis.

microbiology↗

bioRxiv2024

Self-immunity towards a novel competence-induced streptococcal murein hydrolase is mediated by a Fem-transferase-like protein

Murein hydrolases (or peptidoglycan hydrolases) play diverse roles in bacteria, from controlled remodeling of the bacterial cell wall to lytic agents. In streptococci, a subset of these hydrolases is associated with competence-induced fratricide, a process where bacteria kill closely related cells to release DNA that can be taken up during natural transformation. Here, we characterize ScrM, a competence-induced murein hydrolase from Streptococcus dysgalactiae comprising a CHAP domain, an SH3b domain and an uncharacterized C-terminal domain (CCD). ScrM displayed lytic activity against pyogenic and salivarius group streptococci. Microscopy analysis of fluorescent fusions revealed that ScrM specifically localizes to the division zone of sensitive cells, with binding and localization mediated primarily by CCD. Upon competence induction, cells became immune to ScrM due to expression of ScrI, a Fem-transferase-like protein. We show by LC-MS/MS that ScrI incorporates Thr in place of Ala into the interpeptide bridges of peptidoglycan, which in turn prevents ScrM binding to the division zone, thereby protecting the cells from self-lysis during competence. ScrM and ScrI are conserved among pyogenic streptococcal pathogens and represent new players in the cell wall biogenesis of these bacteria that may form a platform for development of novel antimicrobial strategies.

microbiology↗

bioRxiv2024

The Streptococcus agalactiae LytSR two-component regulatory system promotes vaginal colonisation and virulence in vivo

Streptococcus agalactiae (or Group B Streptococcus, GBS) is a leading cause of neonatal sepsis and meningitis globally. To sense and respond to variations in its environment, GBS possesses multiple two-component regulatory systems (TCSs) such as LytSR. Here, we aimed to investigate the role of LytSR in GBS pathogenicity. We generated an isogenic lytS knockout mutant in a clinical GBS isolate and used a combination of phenotypic in vitro assays and in vivo murine models to investigate the contribution of lytS to the colonisation and invasive properties of GBS. Deletion of the lytS gene in the GBS chromosome resulted in significantly higher survival rates in mice during sepsis, accompanied by reduced bacterial loads in blood, lung, spleen, kidney and brain tissue compared to infection with the wild-type strain. In a mouse model of GBS vaginal colonisation, we also observed that the lytS knockout mutant was cleared more readily from the vaginal tract compared to its wild-type counterpart. Interestingly, lower levels of proinflammatory cytokines were found in the serum of mice infected with the lytS mutant. Our results demonstrate that the LytSR TCS plays a key role in GBS tissue invasion and pathogenesis, and persistence of mucosal colonisation. ImportanceStreptococcus agalactiae (Group B strep, or GBS) is a common commensal of the female urogenital tract and one of WHOs priority pathogens. The bacterium has evolved mechanisms to adapt and survive in its host, many of which are regulated via two-component signal transduction systems (TCSs), however, the exact contributions of TCSs towards GBS pathogenicity remain largely obscure. We have constructed a TCS lytS-deficient mutant in a CC-17 hypervirulent GBS clinical isolate. Using murine models, we showed that LytSR regulatory system is essential for vaginal colonisation via promoting biofilm production. We also observed that lytS deficiency led to significantly attenuated virulence properties and lower levels of proinflammatory cytokines in blood. Our findings are of significant importance in that they unveil a previously unreported role for LytSR in GBS and pave the way towards a better understanding of its ability to transition from an innocuous commensal to a deadly pathogen.

microbiology↗

bioRxiv2024

The Spx stress regulator confers high-level β-lactam resistance and decreases susceptibility to last-line antibiotics in methicillin resistant Staphylococcus aureus

Infections caused by methicillin resistant Staphylococcus aureus (MRSA) are a leading cause of mortality worldwide. MRSA have acquired resistance to next generation {beta}-lactam antibiotics through the horizontal acquisition of the mecA resistance gene. Development of high resistance is, however, often associated with additional mutations in a set of chromosomal core genes, known as potentiators which through poorly described mechanisms enhance resistance. The yjbH gene was recently identified as a hot spot for adaptive mutations during severe infections. Here, we show that inactivation of yjbH increased {beta}-lactam MICs up to 16-folds and transformed MRSA cells with low level of resistance to being homogenously highly resistant to {beta}-lactams. The yjbH gene encodes an adaptor protein that targets the transcriptional stress regulator Spx for degradation by the ClpXP protease. Using CRISPRi to knock down spx transcription, we unambiguously linked hyper-resistance to accumulation of Spx. Spx was previously proposed to be essential, however, our data indicate that Spx is dispensable for growth at 37{degrees}C but becomes essential in the presence of antibiotics with various targets. On the other hand, high Spx levels bypassed the role of PBP4 in {beta}-lactam resistance and broadly decreased MRSA susceptibility to compounds targeting the cell wall or the cell membrane including vancomycin, daptomycin, and nisin. Strikingly, Spx potentiated resistance independently of its redox sensing switch. Collectively, our study identifies a general stress pathway that, in addition to promoting the development of high-level, broad-spectrum {beta}-lactam resistance, also decreases MRSA susceptibility to critical antibiotics of last resort.

microbiology↗