Biomedical subjects

Sreedhar R Nallapareddy

Publications and source records attributed to Sreedhar R Nallapareddy.

13 recordsLinked to original sources

PubMed2006

Enterococcus faecalis adhesin, Ace, mediates attachment to particulate dentin.

An enzyme linked immunosorbent assay was developed to assess E. faecalis adhesion to particulate dentin. E. faecalis, OG1RF, which expresses the collagen binding protein (Ace+), and a derivative of OG1RF, TX5256, deficient in the collagen binding protein (Ace-) were grown at 46 degrees C, necessary for in vitro expression of Ace, and at 37 degrees C. E. faecalis binding to dentin was measured at 0, 15, 30, 60, 120, and 360 minutes. Compared to TX5256 and OG1RF grown at 37 degrees C, OG1RF grown at 46 degrees C adhered significantly better at all time points except 15 minutes (p < 0.001) exhibiting maximum binding at 120 minutes (17.4% of a positive control). Type I collagen at 100 microg/ml inhibited dentin binding by OG1RF grown at 46 degrees C in both competition (p < 0.005) and displacement assays (p < 0.046). Immunoaffinity purified anti-Ace IgG at 200 microg of protein inhibited adhesion of OG1RF grown at 46 degrees C to dentin.

Adhesins, Bacterial↗

PubMed2006

Construction of improved temperature-sensitive and mobilizable vectors and their use for constructing mutations in the adhesin-encoding acm gene of poorly transformable clinical Enterococcus faecium strains.

Inactivation by allelic exchange in clinical isolates of the emerging nosocomial pathogen Enterococcus faecium has been hindered by lack of efficient tools, and, in this study, transformation of clinical isolates was found to be particularly problematic. For this reason, a vector for allelic replacement (pTEX5500ts) was constructed that includes (i) the pWV01-based gram-positive repAts replication region, which is known to confer a high degree of temperature intolerance, (ii) Escherichia coli oriR from pUC18, (iii) two extended multiple-cloning sites located upstream and downstream of one of the marker genes for efficient cloning of flanking regions for double-crossover mutagenesis, (iv) transcriptional terminator sites to terminate undesired readthrough, and (v) a synthetic extended promoter region containing the cat gene for allelic exchange and a high-level gentamicin resistance gene, aph(2'')-Id, to distinguish double-crossover recombination, both of which are functional in gram-positive and gram-negative backgrounds. To demonstrate the functionality of this vector, the vector was used to construct an acm (encoding an adhesin to collagen from E. faecium) deletion mutant of a poorly transformable multidrug-resistant E. faecium endocarditis isolate, TX0082. The acm-deleted strain, TX6051 (TX0082Deltaacm), was shown to lack Acm on its surface, which resulted in the abolishment of the collagen adherence phenotype observed in TX0082. A mobilizable derivative (pTEX5501ts) that contains oriT of Tn916 to facilitate conjugative transfer from the transformable E. faecalis strain JH2Sm::Tn916 to E. faecium was also constructed. Using this vector, the acm gene of a nonelectroporable E. faecium wound isolate was successfully interrupted. Thus, pTEX5500ts and its mobilizable derivative demonstrated their roles as important tools by helping to create the first reported allelic replacement in E. faecium; the constructed this acm deletion mutant will be useful for assessing the role of acm in E. faecium pathogenesis using animal models.

Adhesins, Bacterial↗

PubMed2006

Ligand-signaled upregulation of Enterococcus faecalis ace transcription, a mechanism for modulating host-E. faecalis interaction.

Enterococcus faecalis, the third most frequent cause of bacterial endocarditis, appears to be equipped with diverse surface-associated proteins showing structural-fold similarity to the immunoglobulin-fold family of staphylococcal adhesins. Among the putative E. faecalis surface proteins, the previously characterized adhesin Ace, which shows specific binding to collagen and laminin, was detectable in surface protein preparations only after growth at 46 degrees C, mirroring the finding that adherence was observed in 46 degrees C, but not 37 degrees C, grown E. faecalis cultures. To elucidate the influence of different growth and host parameters on ace expression, we investigated ace expression using E. faecalis OG1RF grown in routine laboratory media (brain heart infusion) and found that ace mRNA levels were low in all growth phases. However, quantitative reverse transcription-PCR showed 18-fold-higher ace mRNA amounts in cells grown in the presence of collagen type IV compared to the controls. Similarly, a marked increase was observed when cells were either grown in the presence of collagen type I or serum but not in the presence of fibrinogen or bovine serum albumin. The production of Ace after growth in the presence of collagen type IV was demonstrated by immunofluorescence microscopy, mirroring the increased ace mRNA levels. Furthermore, increased Ace expression correlated with increased collagen and laminin adhesion. Collagen-induced Ace expression was also seen in three of three other E. faecalis strains of diverse origins tested, and thus it appears to be a common phenomenon. The observation of host matrix signal-induced adherence of E. faecalis may have important implications on our understanding of this opportunistic pathogen.

Animals↗

PubMed2006

Multilocus sequence typing scheme for Enterococcus faecalis reveals hospital-adapted genetic complexes in a background of high rates of recombination.

A multilocus sequence typing (MLST) scheme based on seven housekeeping genes was used to investigate the epidemiology and population structure of Enterococcus faecalis. MLST of 110 isolates from different sources and geographic locations revealed 55 different sequence types that grouped into four major clonal complexes (CC2, CC9, CC10, and CC21) by use of eBURST. Two of these clonal complexes, CC2 and CC9, are particularly fit in the hospital environment, as CC2 includes the previously described BVE clonal complex identified by an alternative MLST scheme and CC9 includes exclusively isolates from hospitalized patients. Identical alleles were found in genetically diverse isolates with no linkage disequilibrium, while the different MLST loci gave incongruent phylogenetic trees. This demonstrates that recombination is an important mechanism driving genetic variation in E. faecalis and suggests an epidemic population structure for E. faecalis. Our novel MLST scheme provides an excellent tool for investigating local and short-term epidemiology as well as global epidemiology, population structure, and genetic evolution of E. faecalis.

Alleles↗

PubMed2006

Endocarditis and biofilm-associated pili of Enterococcus faecalis.

Increasing multidrug resistance in Enterococcus faecalis, a nosocomial opportunist and common cause of bacterial endocarditis, emphasizes the need for alternative therapeutic approaches such as immunotherapy or immunoprophylaxis. In an earlier study, we demonstrated the presence of antibodies in E. faecalis endocarditis patient sera to recombinant forms of 9 E. faecalis cell wall-anchored proteins; of these, we have now characterized an in vivo-expressed locus of 3 genes and an associated sortase gene (encoding sortase C; SrtC). Here, using mutation analyses and complementation, we demonstrated that both the ebp (encoding endocarditis and biofilm-associated pili) operon and srtC are important for biofilm production of E. faecalis strain OG1RF. In addition, immunogold electron microscopy using antisera against EbpA-EbpC proteins as well as patient serum demonstrated that E. faecalis produces pleomorphic surface pili. Assembly of pili and their cell wall attachment appeared to occur via a mechanism of cross-linking of the Ebp proteins by the designated SrtC. Importantly, a nonpiliated, allelic replacement mutant was significantly attenuated in an endocarditis model. These biologically important surface pili, which are antigenic in humans during endocarditis and encoded by a ubiquitous E. faecalis operon, may be a useful immunotarget for studies aimed at prevention and/or treatment of this pathogen.

Aminoacyltransferases↗

PubMed2005

Pleiotrophic effects of 2 Enterococcus faecalis sagA-like genes, salA and salB, which encode proteins that are antigenic during human infection, on biofilm formation and binding to collagen type i and fibronectin.

BACKGROUND: We have shown previously that Enterococcus faecium SagA has broad-spectrum binding to extracellular matrix (ECM) proteins. In the present study, 2 sagA-like genes, salA and salB, were identified in Enterococcus faecalis. METHODS: We compared the salA and salB mutants; their parental strain, OG1RF; and the salB-complemented strain for binding to ECM proteins and biofilm formation. RESULTS: The salB mutant (TX5123) grew more slowly but showed greater binding (approximately 10%-20% of cells bound) to fibronectin (FN) and collagen type I (CI) than did OG1RF (approximately 1% of cells bound) (P<.001). Although TX5123 showed decreased biofilm formation in tryptic soy broth plus 0.25% glucose (TSBG) (P<.001 vs. OG1RF), a marked increase in biofilm formation was shown by TX5123 but not by OG1RF when they were grown in TSBG plus horse serum (HS) or TSBG plus FN, and the increase was coincident with increased attachment and hydrophobicity of TX5123. Complementation of the salB mutant restored the wild-type phenotypes. CONCLUSIONS: Whether SalB expression is ever sufficiently low in vivo to enhance adherence to ECM proteins or the serum-elicited increase in biofilm formation seen with the salB mutant in vitro is not currently known, but it is a potential way in which this organism could increase its adherence and biofilm formation during infection.

Bacterial Adhesion↗

PubMed2005

Fsr-independent production of protease(s) may explain the lack of attenuation of an Enterococcus faecalis fsr mutant versus a gelE-sprE mutant in induction of endocarditis.

An Enterococcus faecalis gelE insertion disruption mutant (TX5128), which produces neither gelatinase (GelE) nor the cotranscribed (in the wild type) serine protease (SprE), was found to be attenuated in a rat endocarditis model with a significant decrease in the endocarditis induction rate versus wild-type E. faecalis OG1RF (GelE(+), SprE(+)). TX5266, which has a nonpolar deletion in fsrB and, like TX5128, is phenotypically GelE(-) under usual conditions, was also studied; fsrB is a homologue of agrB of staphylococci and participates in regulation of gelE-sprE expression. Unexpectedly, TX5266 approximated wild-type OG1RF in the endocarditis model and was significantly less attenuated than TX5128. This is in contrast to other models which have found fsr mutants to be as or more attenuated than TX5128. Further study found that the fsrB mutant produced very low levels of gelatinase activity after prolonged incubation in vitro versus no gelatinase activity with TX5128 and did not show the extensive chaining characteristic of TX5128. Reverse transcription-PCR confirmed that gelE was expressed in TX5266 at a very low level versus wild-type OG1RF and was not expressed at all in TX5128. Possible explanations for the increased induction of endocarditis by TX5266 versus TX5128 include the production of low levels of protease(s) or some other effect(s) of the inactivation of the E. faecalis fsr regulator. The equivalent ability of OG1RF and its fsr mutant to initiate endocarditis may explain why we did not find naturally occurring fsr mutants, which account for ca. 35% of E. faecalis isolates, unrepresented in endocarditis versus fecal isolates.

Animals↗

PubMed2005

Molecular characterization of a widespread, pathogenic, and antibiotic resistance-receptive Enterococcus faecalis lineage and dissemination of its putative pathogenicity island.

Enterococcus faecalis, a common cause of endocarditis and known for its capacity to transfer antibiotic resistance to other pathogens, has recently emerged as an important, multidrug-resistant nosocomial pathogen. However, knowledge of its lineages and the potential of particular clones of this species to disseminate and cause disease is limited. Using a nine-gene multilocus sequence typing (MLST) scheme, we identified an evolving and widespread clonal complex of E. faecalis that has caused outbreaks and life-threatening infections. Moreover, this unusual clonal complex was found to contain isolates of unexpected relatedness, including the first known U.S. vancomycin-resistant enterococcus (E. faecalis strain V583), the first known penicillinase-producing (Bla(+)) E. faecalis isolate, and the previously described widespread clone of penicillinase producers, a trait found in <0.1% of E. faecalis isolates. All members of this clonal cluster (designated as BVE for Bla(+) Van(r) endocarditis) were found to contain a previously described putative pathogenicity island (PAI). Further analysis of this PAI demonstrated its dissemination worldwide, albeit with considerable variability, confirmed its association with clinical isolates, and found a common insertion site in different clonal lineages. PAI deletions, MLST, and the uncommon resistances were used to predict the evolution of the BVE clonal cluster. The finding of a virulent and highly successful clonal complex of E. faecalis with different members resistant to the primary therapies of choice, ampicillin and vancomycin, has important implications for the evolution of virulence and successful lineages and for public health monitoring and control.

Bacterial Proteins↗

PubMed2004

A family of putative MSCRAMMs from Enterococcus faecalis.

The recently published Enterococcus faecalis genome [Paulsen, I. T., Banerjei, L., Myers, G. S. & 29 other authors (2003). Science 299, 2071-2074)] was examined and 41 putative cell-wall-anchored proteins were identified. Seventeen of these proteins are predicted to contain tandemly repeated immunoglobulin-like folds characteristic of the structural organization of staphylococcal adhesins of the MSCRAMM (microbial surface component recognizing adhesive matrix molecules) type. Two of the nine proteins selected for further study appear to represent cell-wall-anchored enzymes. It is proposed that the remaining seven proteins constitute a family of structurally related proteins potentially interacting with proteins of the host. This family includes the previously identified collagen/laminin-binding MSCRAMM ACE [Rich, R. L., Kreikemeyer, B., Owens, R. T., LaBrenz, S., Narayana, S. V., Weinstock, G. M., Murray, B. E. & Hook, M. (1999). J Biol Chem 274, 26939-26945]. It is further demonstrated that genes encoding the seven putative MSCRAMMs are present in all E. faecalis strains tested and these proteins appear to be expressed during infection in humans, since sera from infected individuals contain antibodies reacting with recombinant versions of the enterococcal proteins.

Adhesins, Bacterial↗

PubMed2004

Influence of origin of isolates, especially endocarditis isolates, and various genes on biofilm formation by Enterococcus faecalis.

Endocarditis isolates of Enterococcus faecalis produced biofilm significantly more often than nonendocarditis isolates, and 39% of 79 versus 6% of 84 isolates produced strong biofilm (P < 0.0001). esp was not required, but its presence was associated with higher amounts of biofilm (P < 0.001). Mutants disrupted in dltA, efaA, ace, lsa, and six two-component regulatory systems were largely unaltered, while disruptions in epa (encoding enterococcal polysaccharide antigen), atn (encoding an autolysin), gelE (encoding gelatinase), and fsr (encoding the E. faecalis regulator) [corrected] resulted in fewer attached bacteria, as determined using phase-contrast microscopy, and less biofilm (P < 0.0001).

Bacterial Adhesion↗

PubMed2003

A potential virulence gene, hylEfm, predominates in Enterococcus faecium of clinical origin.

An open reading frame (hyl(Efm)) with homologies to previously described hyaluronidase genes has been identified in nonstool isolates of Enterococcus faecium. E. faecium isolates (n=577) from diverse sources were screened for the presence of hyl(Efm) and esp(Efm), a putative virulence gene associated with epidemic E. faecium strains. The presence of esp(Efm) was roughly twice that of hyl(Efm), but both were found primarily in vancomycin-resistant E. faecium isolates in nonstool cultures obtained from patients hospitalized in the United States. These data suggest that specific E. faecium strains may be enriched in determinants that make them more likely to cause clinical infections. Differences in the prevalence of these strains may help explain variations in the clinical importance of multiresistant E. faecium across different continents.

Anti-Bacterial Agents↗

PubMed2003

Clinical isolates of Enterococcus faecium exhibit strain-specific collagen binding mediated by Acm, a new member of the MSCRAMM family.

A collagen-binding adhesin of Enterococcus faecium, Acm, was identified. Acm shows 62% similarity to the Staphylococcus aureus collagen adhesin Cna over the entire protein and is more similar to Cna (60% and 75% similarity with Cna A and B domains respectively) than to the Enterococcus faecalis collagen-binding adhesin, Ace, which shares homology with Acm only in the A domain. Despite the detection of acm in 32 out of 32 E. faecium isolates, only 11 of these (all clinical isolates, including four vancomycin-resistant endocarditis isolates and seven other isolates) exhibited binding to collagen type I (CI). Although acm from three CI-binding vancomycin-resistant E. faecium clinical isolates showed 100% identity, analysis of acm genes and their promoter regions from six non-CI-binding strains identified deletions or mutations that introduced stop codons and/or IS elements within the gene or the promoter region in five out of six strains, suggesting that the presence of an intact functional acm gene is necessary for binding of E. faecium strains to CI. Recombinant Acm A domain showed specific and concentration-dependent binding to collagen, and this protein competed with E. faecium binding to immobilized CI. Consistent with the adherence phenotype and sequence data, probing with Acm-specific IgGs purified from anti-recombinant Acm A polyclonal rabbit serum confirmed the surface expression of Acm in three out of three collagen-binding clinical isolates of E. faecium tested, but in none of the strains with a non-functional pseudo acm gene. Introduction of a functional acm gene into two non-CI-binding natural acm mutant strains conferred a CI-binding phenotype, further confirming that native Acm is sufficient for the binding of E. faecium to CI. These results demonstrate that acm, which encodes a potential virulence factor, is functional only in certain infection-derived clinical isolates of E. faecium, and suggest that Acm is the primary adhesin responsible for the ability of E. faecium to bind collagen.

Adhesins, Bacterial↗

PubMed2002

Molecular typing of selected Enterococcus faecalis isolates: pilot study using multilocus sequence typing and pulsed-field gel electrophoresis.

The present study compared the recently developed multilocus sequence typing (MLST) approach with a well-established molecular typing technique, pulsed-field gel electrophoresis (PFGE), for subspecies differentiation of Enterococcus faecalis isolates. We sequenced intragenic regions of three E. faecalis antigen-encoding genes (ace, encoding a collagen and laminin adhesin; efaA, encoding an endocarditis antigen; and salA, encoding a cell wall associated antigen) and one housekeeping gene (pyrC) of 22 E. faecalis isolates chosen largely for their temporal and geographical diversity, but also including some outbreak isolates. MLST analysis of polymorphic regions of these four genes identified 13 distinct sequence types (STs) with different allelic profiles; the composite sequences generated from the four sequenced gene fragments of individual isolates showed 98.3 to 100% identity among the 22 isolates. We also found that the allelic profiles from two sequences, ace and salA, were sufficient to distinguish all 13 STs of this study. The 13 STs corresponded to 12 different PFGE types, with one previously designated PFGE clone (a widespread U.S. clone of beta-lactamase-producing isolates) being classified into two highly related STs which differed at 2 of 2,894 bases, both in the same allele. MLST also confirmed the clonal relationships among the isolates of two other PFGE clonal groups, including vancomycin resistant isolates. Thus, this pilot study with representative E. faecalis isolates suggests that, similar to PFGE, the sequence-based typing method may be useful for differentiating isolates of E. faecalis to the subspecies level in addition to identifying outbreak isolates.

Base Sequence↗