Phase Variation of Poly-N-Acetylglucosamine Expression in Staphylococcus aureus

Polysaccharide intercellular adhesin (PIA), also known as poly-N-acetyl-β-(1–6)-glucosamine (PIA/PNAG) is an important component of Staphylococcus aureus biofilms and also contributes to resistance to phagocytosis. The proteins IcaA, IcaD, IcaB, and IcaC are encoded within the intercellular adhesin (ica) operon and synthesize PIA/PNAG. We discovered a mechanism of phase variation in PIA/PNAG expression that appears to involve slipped-strand mispairing. The process is reversible and RecA-independent, and involves the expansion and contraction of a simple tetranucleotide tandem repeat within icaC. Inactivation of IcaC results in a PIA/PNAG-negative phenotype. A PIA/PNAG-hyperproducing strain gained a fitness advantage in vitro following the icaC mutation and loss of PIA/PNAG production. The mutation was also detected in two clinical isolates, suggesting that under certain conditions, loss of PIA/PNAG production may be advantageous during infection. There was also a survival advantage for an icaC-negative strain harboring intact icaADB genes relative to an isogenicicaADBC deletion mutant. Together, these results suggest that inactivation of icaC is a mode of phase variation for PIA/PNAG expression, that high-level production of PIA/PNAG carries a fitness cost, and that icaADB may contribute to bacterial fitness, by an unknown mechanism, in the absence of an intact icaC gene and PIA/PNAG production

Genetic heterogeneity and taxonomic diversity among Gardnerella species

Gardnerella spp. are the most frequent organisms found in bacterial vaginosis (BV). However, colonization by Gardnerella spp. does not always lead to BV. Developments in molecular genetics have shed new light on the diversity of the genus Gardnerella, leading to an emended description of Gardnerella vaginalis and, for the first time, the inclusion of more species of Gardnerella, namely, Gardnerella leopoldii, Gardnerella piotii, and Gardnerella swidsinskii. While no association had been made between the novel Gardnerella species and virulence potential, there is genomic evidence of a higher prevalence of some genotypes in BV cases, as compared with colonization in women in whom BV has been clinically ruled out. Gardnerella vaginalis has been considered a pivotal player in the progression of bacterial vaginosis (BV), a condition associated with serious health complications. However, G. vaginalis is also commonly found in asymptomatic or BV-negative women. This has generated interest in the question of whether genetic differences among isolates might distinguish pathogenic from commensal isolates. G. vaginalis was the only recognized species in its genus for four decades, but recently an emended description of G. vaginalis and descriptions of three new species Gardnerella leopoldii, Gardnerella piotii, and Gardnerella swidsinskii have been proposed. This review provides background on the heterogeneity and diversity within the genus Gardnerella, highlighting the main features that distinguish species and clades, and how these features may impact BV development.J.C. and N.C. acknowledge the projects PTDC/BIA-MIC/28271/2017 (POCI-01-0145-FEDER-028271), and the strategic funding of unit UID/BIO/04469/2019, supported by the Portuguese Foundation for Science and Technology.info:eu-repo/semantics/publishedVersio

Untargeted Lipidomic Analysis to Broadly Characterize the Effects of Pathogenic and Non-Pathogenic Staphylococci on Mammalian Lipids

Modification of the host lipidome via secreted enzymes is an integral, but often overlooked aspect of bacterial pathogenesis. In the current era of prevalent antibiotic resistance, knowledge regarding critical host pathogen lipid interactions has the potential for use in developing novel antibacterial agents. While most studies to date on this matter have focused on specific lipids, or select lipid classes, this provides an incomplete picture. Modern methods of untargeted lipidomics have the capacity to overcome these gaps in knowledge and provide a comprehensive understanding of the role of lipid metabolism in the pathogenesis of infections. In an attempt to determine the role of lipid modifying enzymes produced by staphylococci, we exposed bovine heart lipids, a standardized model for the mammalian lipidome, to spent medium from staphylococcal cultures, and analyzed lipid molecular changes by MS/MSALLshotgun lipidomics. We elucidate distinct effects of different staphylococcal isolates, including 4 clinical isolates of the pathogenic species Staphylococcus aureus, a clinical isolate of the normally commensal species S. epidermidis, and the non-pathogenic species S. carnosus. Two highly virulent strains of S. aureus had a more profound effect on mammalian lipids and modified more lipid classes than the other staphylococcal strains. Our studies demonstrate the utility of the applied untargeted lipidomics methodology to profile lipid changes induced by different bacterial secretomes. Finally, we demonstrate the promise of this lipidomics approach in assessing the specificity of bacterial enzymes for mammalian lipid classes. Our data suggests that there may be a correlation between the bacterial expression of lipid-modifying enzymes and virulence, and could facilitate the guided discovery of lipid pathways required for bacterial infections caused by S. aureus and thereby provide insights into the generation of novel antibacterial agents

Introduction of plasmid DNA into Sneathia vaginalis; the first step to genetic manipulation

Many bacterial pathogens are capable of secreting one or more toxins, all of which can be categorized based on their function, target, or mechanism of action. Pore-forming toxins, for instance, are characterized based on their ability to perforate the host membrane. This may result in the delivery of bacterial substances into the host cell, release of compounds from the host cell, and/or death of the host cell. Research from our lab has demonstrated that Sneathia vaginalis, a gram-negative anaerobic bacterial species that is associated with bacterial vaginosis and preterm birth, produces a pore-forming toxin referred to as cytopathogenic toxin A. CptA is capable of lysing human red blood cells and permeabilizing chorionic trophoblasts and other epithelial cells. CptA appears to contribute to the traversal of Sneathia vaginalis across fetal membranes and may therefore contribute towards preterm birth and bacterial vaginosis. In order to confirm its role in fetal membrane traversal, an isogenic mutant of Sneathia vaginalis, in which the cptA gene has been functionally deleted, is needed. To accomplish this, we tested both plasmids and linear PCR products for the ability to insertionally inactivate the cptA gene. If the mutant is no longer able to traverse fetal membranes, then this will confirm that CptA is necessary for the virulence of Sneathia vaginalis.https://scholarscompass.vcu.edu/uresposters/1405/thumbnail.jp

Quantitative analysis of initial adhesion of bacterial vaginosis-associated anaerobes to ME-180 cells

Bacterial vaginosis is the leading vaginal disorder but the transition from health to this dysbiotic condition remains poorly characterized. Our goal was to quantify the ability of BV-associated anaerobes to adhere to epithelial cells in the presence of lactobacilli. Gardnerella vaginalis outcompeted Lactobacillus crispatus and Lactobacillus iners actually enhanced its adherence.We want to generously thank to Melissa Jamerson and Guy A. Cabral, from the Department of Microbiology and Immunology (Virginia Commonwealth University), for the AMG EVOSfl fluorescence microscope usage and procedure advices. This work was supported by European Union funds (FEDER/COMPETE) and by national funds (FCT) under the project with reference FCOMP-01-0124-FEDER-008991 (PTDC/BIA-MIC/098228/2008)

Environmental regulation of the pgaABCD locus in Escherichia coli

Background : The staphylococci are a major cause of catheter-related infections, due, in part, to their ability to form a biofilm. The major constituent of the biofilm matrix is the polysaccharide PNAG, synthesized by the proteins encoded in the icaADBC locus. Recently, it has been demonstrated that the Escherichia coli locus ycdSRQP (re-named pgaABCD) has homology to the staphylococcal icaADBC locus, and also encodes proteins that direct the synthesis of a polysaccharide that is biochemically identical to PNAG. Furthermore, PNAG promotes biofilm formation in E. coli. The aim of this work was to determine environmental factors that regulate transcription of the pgaABCD locus and the production of PNAG. Methods: We cloned the promoter of the locus pgaABCD into a green fluorescent protein (GFP) reporter plasmid, and transformed the plasmid into a competent E. coli strain. The transformed cells were grown in TSB supplemented with a variety of factors, including glucose, sucrose, mannose, magnesium, manganese and ethanol. The production of GFP was detected with a fluorimeter. Factors that augmented the production of GFP were further tested for their influence on PNAG production and biofilm formation in 18 E. coli urinary tract clinical isolates by immunoblot using IgG raised against staphylococcal PNAG and by microtiter well biofilm assay, respectively. Results: Transcription of pgaABCD was augmented by glucose, manganese, potassium, and ethanol. PNAG production, as detected by immunoblot, was also increased by the same factors. However, glucose was the only factor to significantly increase biofilm formation. Conclusions: Environmental regulation of transcription of the E. coli pgaABCD locus, as determined by reporter gene activation was correlated with the production of PNAG in E. coli clinical isolates. However, biofilm formation by E.coli may not be entirely dependent on PNAG production

Effect of a single locus deletion on global genome expression : the ica locus in Staphyloccocus spp : a case study

Recent technological advances have made it possible to study global gene expression in bacteria. Microarrays have emerged as the premier tool for studying gene expression on a genomic scale and have been used in a broad range of studies1. Staphylococcus epidermidis and Staphylococcus aureus can form biofilms on medical devices. The major constituent of the biofilm matrix is the polysaccharide PNAG, synthesized by the proteins encoded in the icaADBC locus2. A knockout of the ica locus in a biofilm-forming strain will cause that strain to lose its ability to form a biofilm. However, what is not known is the effect of the loss of the icaADBC locus on the overall gene expression by the bacteria. We used a strong biofilm forming strain of S. aureus and an isogenic icaADBC knock out and grew them under the same conditions (16H, TSB with 1% glucose, 37°C, 300rpm) after which we extracted the total RNA, and converted it to cDNA. We then labeled the cDNA with 2 different dyes, and hybridized the product on a microarray slide containing the genome of S. aureus. The microarrays were analyzed using an open source software (TM4)3. Data analysis verified that the majority of the genes were equally expressed by both strains, as expected. However, approximately 60 genes were differentially expressed between the wild type and the isogenic mutant, demonstrating that a deletion mutation within a single locus can influence the expression of many other genes in the bacteria

Social interaction by BV anaerobes in initial adhesion and biofilm assays

Bacterial vaginosis (BV) is the most common vaginal disorder of women of reproductive age. It is commonly accepted that the microbial switch from normal to BV state is characterized by a decrease in vaginal colonization by Lactobacillus species together with an increase in the number of Gardnerella vaginalis and others anaerobes. G. vaginalis can be found associated with normal vaginal epithelium but recent findings suggest that it is the biofilm produced by G. vaginalis that leads to establishment of BV, by allowing other anaerobes to grow. Further research into the properties of G. vaginalis and its interactions with other BV-associated anaerobes may be essential for better understanding the BV etiology. Therefore, our goal was to study competitive initial adhesion between Lactobacillus crispatus (an important vaginal species with probiotic activity) and several anaerobes. Also, this study aimed to evaluate differences in biofilm formation between a healthy G. vaginalis strain (5-1) and the other anaerobes as compared with a BV G. vaginalis strain (101). In addition to G. vaginalis strains (5-1 and 101), Prevotella bivia, Fusobacteria nucleatum, Mobiluncus mulieris and Atopobium vaginae were used in this social interaction research. First, initial adhesion assays at 100rpm for 30 min were conducted in glass 8-well slides using an equal mixture of L. crispatus 39G strain and a second anaerobe at same concentration (103 CFU/ml) in each well. Next, we used qPCR to study the biofilm social evolution (synergic, antagonist and commensal relationship) between a pre-established (24h) biofilm formed by one of the two G. vaginalis strains and a second anaerobe. Our results showed that G. vaginalis 5-1 and 101 strains had definitively the greatest initial adherence capability when in presence of the same number of L. crispatus 39G cells, followed by P. bivia, M. mulieris and A. vaginae. In addition, qPCR analysis of the two species biofilms revealed that both G. vaginalis strains established commensal relationships with all others anaerobes except with P. bivia, for which a synergic relation was found. In fact, this synergic relation between these two species had previously been noted. Importantly, G. vaginalis 101 (pathogenic strain) showed nearly a 2-fold increase in biofilm formation when compared with G. vaginalis 5-1 (healthy strain) in the presence of any other anaerobe studied

Staphylococcus aureus immunodominant surface antigen B is a cell-surface associated nucleic acid binding protein

<p>Abstract</p> <p>Background</p> <p><it>Staphylococcus aureus </it>immunodominant surface antigen B (IsaB) elicits an immune response during septicemia and is generally classified as a virulence factor, but its biological function remains completely undefined. In an attempt to identify staphylococcal RNA-binding proteins, we designed an RNA Affinity Chromatography assay and subsequently isolated IsaB.</p> <p>Results</p> <p>Western analysis indicated that IsaB was both secreted and cell-surface associated. Gel Shift analysis confirmed the RNA binding activity but revealed that IsaB bound to any nucleic acid without sequence specificity. IsaB exhibited the highest affinity for double-stranded DNA followed by single-stranded DNA and RNA. Because extracellular DNA has been shown to play a role in biofilm formation, we investigated the biofilm-forming capacity of an isogenic <it>isaB </it>deletion mutant but we found that IsaB did not contribute to biofilm formation under any conditions tested.</p> <p>Conclusion</p> <p>IsaB is an extracellular nucleic acid binding protein, with little to no sequence specificity, but its role in virulence remains unclear.</p

Use of confocal laser scan microscopy (CLSM) to evaluate S. epidermidis biofilm structure : the effect of low concentration of antibiotics : a case study

Staphylococcus epidermidis is the major organism causing nosocomial infections associated with the formation of a biofilm on medical devices. The major constituent of S. epidermidis biofilm matrix is the polysaccharide PNAG, synthesized by the proteins encoded in the icaADBC locus. CLSM low energy lasers allows the observation of living cells, and its penetration ability renders detailed tridimensional images of the biofilm. Another characteristic of CLSM associated with fluorochroms labeling is the ability to differentiate between biofilm cells and other constituents of the biofilm. In this study, differences in the structure of a S. epidermidis biofilms grown in the presence or absence of low concentrations of dicloxacillin were assessed by CLSM observations of biofilms labeled with a fluorescent lectin (wheat germ agglutinin) that binds to PNAG. Biofilms were formed in 6 wells polystyrene plates, during 48h at 37ºC and 120RPM in TSB supplemented with 1% of glucose in the presence and absence of sub-inhibitory concentration of dicloxacillin. The growth medium was carefully changed after 24h. CLSM observations were performed in a Zeiss LSM510 Meta microscope, using a single-channel analysis. The results demonstrated that biofilms formed in the presence of dicloxacillin were less thick compared with the control. Furthermore biofilms formed in the presence of dicloxacillin produced lower amounts of PNAG. The use of CLSM brought a new light into biofilm science by allowing the direct visualization of several processes that occurs inside the biofilm structure. In this particular case it was possible to demonstrate that low concentration of antibiotic alters the biofilm structure and composition